TY - JOUR
AU - Kovacs, Elizabeth, J
AB - Abstract Pulmonary and systemic insults from inhalation injury can complicate the care of burn patients and contribute to significant morbidity and mortality. However, recent progress in diagnosis and treatment of inhalation injury has not kept pace with the care of cutaneous thermal injury. There are many challenges unique to inhalation injury that have slowed advancement, including deficiencies in our understanding of its pathophysiology, the relative difficulty and subjectivity of bronchoscopic diagnosis, the lack of diagnostic biomarkers, the necessarily urgent manner in which decisions are made about intubation, and the lack of universal recommendations for the application of mucolytics, anticoagulants, bronchodilators, modified ventilator strategies, and other measures. This review represents a summary of critical shortcomings in our understanding and management of inhalation injury identified by the American Burn Association’s working group on Cutaneous Thermal Injury and Inhalation Injury in 2018. It addresses our current understanding of the diagnosis, pathophysiology, and treatment of inhalation injury and highlights topics in need of additional research, including 1) airway repair mechanisms; 2) the airway microbiome in health and after injury; and 3) candidate biomarkers of inhalation injury. A review of the National Burn Repository shows us that some degree of inhalation injury is present in 10 to 20% of all burn admissions and that morbidity and mortality from burn injury increase significantly in the presence of inhalation injury.1 Mortality, specifically, increases by more than 10-fold, a fact which has not changed in recent decades. Despite meaningful advances in the overall care of cutaneous burn wounds and resuscitation, strategies to mitigate damage from inhalation injury, including inhaled bronchodilators, mucolytics, anticoagulants, and modified ventilator strategies, have not evolved significantly and are not applied consistently. This may be explained by the lack of uniform and objective criteria for diagnosis and stratification of inhalation injury, as well as gaps in our understanding of its systemic and long-term effects and underlying pathophysiology. Inhalation injury is particularly challenging to manage because, unlike cutaneous burn wounds which can be excised and grafted, injured airway tissue must be treated supportively and protected from secondary damage. This report represents a synopsis from a recent meeting of the working group on Cutaneous Thermal Injury and Inhalation Injury, administered by the American Burn Association, on August 27, 2018 and August 28, 2018. Its objective is to identify unanswered questions and unmet clinical needs in this field, in order to guide future research and improve patient care. To this end, we focus on three areas in need of further investigation: 1) airway repair mechanisms; 2) the airway microbiome; and 3) the search for a biomarker of inhalation injury. To begin, it is worth reviewing our current understanding of inhalation injury, with respect to diagnosis, pathophysiology, and treatment, and its shortcomings. DIAGNOSIS OF INHALATION INJURY AND ITS LIMITATIONS Clinicians maintain a high index of suspicion for inhalation injury because significant airway compromise from severe edema, bronchospasm, or mucous plugging/cast formation can occur minutes to days after the initial insult and failing to protect an at-risk airway can have devastating consequences. Early suspicion for inhalation injury in the field or trauma bay is often prompted by the circumstances of an injury. Burns in enclosed spaces, loss of consciousness, disability or extremes of age, and prolonged extrication times are associated with increased risk.2,3 Inhalation injury is also predicted by “soft” signs, including singed nasal hairs, burns to the face or neck, nausea/emesis, or carbonaceous debris in sputum or nasal secretions, and “hard” signs, including reduced oxygen saturation, cyanosis, stridor, or visible mucosal changes in the upper airway.4 There are several diagnostic modalities which can help to confirm inhalation injury. Flexible fiberoptic bronchoscopy remains the de facto standard since it allows direct visualization of the upper and proximal lower respiratory tract. Moreover, a useful, albeit subjective, bronchoscopic grading scheme for inhalation injury based on the Abbreviated Injury Score is in use (Table 1) and has been shown to correlate with mortality in some studies.5–7 However, such schemes have limitations beyond their subjectivity. Bronchoscopy cannot evaluate the distal lower respiratory tract (respiratory bronchioles) and alveoli where impaired gas exchange and altered hemostasis can have dramatic effects. It also fails to account for heterogeneity in the inflammatory response of individual patients, which may lead to disparate clinical presentations. Furthermore, repeated bronchoscopies to assess evolution of inhalation injury or gauge response to therapy can be time- and resource-intensive and expose the patient to additional risks. These limitations may help to explain why some studies have demonstrated inconsistent correlation between bronchoscopic evaluation and clinical outcomes.8 Other modalities include serum carboxyhemoglobin measurement, chest computed tomography (looking for increased interstitial markings, ground glass opacities, and consolidation,9 or increased bronchial wall thickness10), pulmonary function testing, or even radionuclide imaging with 133Xenon,4 but most have low sensitivity or specificity. Table 1. Current bronchoscopic grading of inhalation injury by Abbreviated Injury Score. Grade AIS1 Class Description 0 No Injury No carbonaceous deposits, erythema, edema, bronchorrhea, or obstruction 1 Mild Injury Minor or patchy areas of erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction present 2 Moderate Injury Moderate erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction 3 Severe Injury Severe Inflammation with friability, copious carbonaceous deposits, bronchorrhea, or obstruction 4 Massive Injury Mucosal sloughing, necrosis, or endoluminal obliteration Grade AIS1 Class Description 0 No Injury No carbonaceous deposits, erythema, edema, bronchorrhea, or obstruction 1 Mild Injury Minor or patchy areas of erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction present 2 Moderate Injury Moderate erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction 3 Severe Injury Severe Inflammation with friability, copious carbonaceous deposits, bronchorrhea, or obstruction 4 Massive Injury Mucosal sloughing, necrosis, or endoluminal obliteration 1Abbreviated Injury Score View Large Table 1. Current bronchoscopic grading of inhalation injury by Abbreviated Injury Score. Grade AIS1 Class Description 0 No Injury No carbonaceous deposits, erythema, edema, bronchorrhea, or obstruction 1 Mild Injury Minor or patchy areas of erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction present 2 Moderate Injury Moderate erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction 3 Severe Injury Severe Inflammation with friability, copious carbonaceous deposits, bronchorrhea, or obstruction 4 Massive Injury Mucosal sloughing, necrosis, or endoluminal obliteration Grade AIS1 Class Description 0 No Injury No carbonaceous deposits, erythema, edema, bronchorrhea, or obstruction 1 Mild Injury Minor or patchy areas of erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction present 2 Moderate Injury Moderate erythema, carbonaceous deposits, bronchorrhea, or bronchial obstruction 3 Severe Injury Severe Inflammation with friability, copious carbonaceous deposits, bronchorrhea, or obstruction 4 Massive Injury Mucosal sloughing, necrosis, or endoluminal obliteration 1Abbreviated Injury Score View Large A prospective study by Hassan et al demonstrated that the PaO2/FiO2 or P/F ratio (the ratio of arterial oxygen partial pressure to the fraction of oxygen in inspired gas) can predict survival in adults with inhalation injury.11 However, the P/F ratio is known to vary as a function of fluid resuscitation, barometric pressure (ie, altitude), and mechanical ventilator mode, so it may be more useful when trending inhalation injury than grading it outright. Ultimately, a multimodal approach to the diagnosis and grading of inhalation injury, incorporating more objective imaging, putative biomarkers, and relevant clinical data, may be best. As early as 1987, Shirani et al proposed a simple risk stratification scheme wherein morbidity and mortality were highest in patients with evidence of inhalation injury on bronchoscopy, intermediate with evidence of inhalation injury on 133Xenon scan only, and lowest without evidence of inhalation injury by either modality.12 In a retrospective review of the U.S. Army Institute of Surgical Research Burn Center between 2002 and 2008, Oh et al found that detection of inhalation injury on both admission computed tomography (CT) scan and bronchoscopy better predicted a composite outcome of pneumonia, acute respiratory distress syndrome (ARDS), and death when compared with bronchoscopy alone.9 Of note, a multicenter clinical trial to validate an inhalational injury scoring system based on demographics, bronchoscopy, CT imaging, and inflammatory markers (ClinicalTrials.gov Identifier: NCT01194024) was just completed in December 2018, with results pending. We recognize that a biomarker or set of biomarkers for inhalation injury, measured in blood or sputum, could be very useful as an adjunct to bronchoscopy and noninvasive imaging. It could even help inform the decision to intubate or extubate a patient. The potential role of an inhalation injury biomarker and specific candidates are discussed in greater depth later in this paper. PATHOPHYSIOLOGY OF INHALATION INJURY AND CURRENT GAPS IN KNOWLEDGE Smoke is a colloidal product of combustion; it is a mixture of gases and aerosolized solid/liquid particles whose composition depends on specific combustible materials involved in a fire.13 A great variety of compounds have been identified in smoke, but some are more common than others (Table 2).14 High temperature fires in high-oxygen environments produce less smoke, predominantly consisting of carbon and nitrogen oxides. When relatively less oxygen is available, combustion is incomplete, generating a much greater diversity of compounds, such as ammonia and hydrogen cyanide.13 Of the known and measurable compounds in smoke, carbon monoxide and hydrogen cyanide likely have the greatest impact on mortality. Table 2. Selected compounds found in smoke, examples of source materials, and pathophysiology Compound Source material Mechanism of action Effects Aldehydes (eg, formaldehyde) Acrylics (textiles paint, windows, wallpaper, adhesives) Cellulose (wood, paper, cotton) Propene (carpet, upholstery, containers) Protein and RNA denaturation Necrosis of airway mucosa* Hydrogen cyanide Fire retardants (insulation, upholstery) Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting, car parts) Polyamine resins (household and kitchen goods) Polyacrylonitrile (clothing, blankets, appliances) Polyurethane (insulation, upholstery, electronics) Histotoxic hypoxia—cells unable to create ATP due to inhibition of mitochondrial cytochrome C oxidase26 Lactic acidosis, tissue hypoxia, depressed consciousness, cardiac arrest and multiorgan failure, death with high concentrations Hydrogen sulfide Rubber (tires) Silk, wool (clothing, furniture, fabrics) Binds iron in mitochondrial cytochrome enzymes, preventing cellular respiration and ATP generation; combines with alkali in moist tissues to form caustic sodium sulfide104 Airway and eye irritation, bronchospasm, pulmonary edema Nitric oxide Most combustible materials Free radical damage, vasodilation Worsened ventilation– perfusion mismatch and hypoxia; may have anti-inflammatory effect105 Carbon monoxide Most combustible materials Binds hemoglobin, creating carboxyhemoglobin, reducing oxygen carrying capacity and delivery Tissue hypoxia, multiorgan failure, death with high concentrations Carbon dioxide Most combustible materials Asphyxia; increases PaCO2* Increased respiratory drive Acrolein (propenal) Acrylics (textiles, windows, wallpaper) Cellulose (wood, paper, cotton) Polypropylene (carpet, upholstery, kitchen goods) Protein denaturation Irritation and necrosis of airway mucosa, death with high concentrations22 Ammonia Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting) Polyamine resins (household and kitchen goods) Polyacrylonitrile (appliances, plastics) Polyurethane (insulation, upholstery) Household cleaners Generates caustic ammonium hydroxide in aqueous environments Irritation and necrosis of airway mucosa, alveolar edema* Sulfur dioxide Rubber (tires) Chemical irritant properties (exact mechanism of action unknown); asthmatics more susceptible106 Potent ocular and airway irritant, bronchoconstriction, pulmonary edema Chlorine Cl Bleach/sanitation products Industrial solvents Water soluble—diffuses into respiratory epithelial lining fluid, hydrolyzes to acids which generate reactive chlorine and oxygen species Necrosis of proximal, superficial airway mucosa, pulmonary edema, airway hyperreactivity107 Phosgene Polyvinyl chloride (flooring, furniture, upholstery, piping) Fat soluble—generates caustic hydrochloric acid and chlorine. Increases pulmonary vascular permeability through effects on arachidonic acid metabolism108 Deep delayed damage/ necrosis to distal airway mucosa (fat- soluble, penetrates), alveolar cell death, delayed pulmonary edema, ARDS109 Hydrogen chloride Polyester (clothing, fabrics) Polyvinyl chloride (flooring, furniture, upholstery, piping) Water soluble—direct caustic effects of hydrochloric acid in the upper airway Irritation and necrosis of airway mucosa, airway edema, acute bronchitis, ARDS Aeromatic hydrocarbons (eg, benzene) Fossil fuels Paints, dyes, adhesives, wax Detergents Greater density than air— asphyxiation in enclosed environments, bone marrow (hematopoiesis) suppression Irritation of mucous membranes, systemic toxicity, drowsiness. Long term—carcinogenicity, anemia. Effects worsened by concurrent alcohol intoxication22 Compound Source material Mechanism of action Effects Aldehydes (eg, formaldehyde) Acrylics (textiles paint, windows, wallpaper, adhesives) Cellulose (wood, paper, cotton) Propene (carpet, upholstery, containers) Protein and RNA denaturation Necrosis of airway mucosa* Hydrogen cyanide Fire retardants (insulation, upholstery) Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting, car parts) Polyamine resins (household and kitchen goods) Polyacrylonitrile (clothing, blankets, appliances) Polyurethane (insulation, upholstery, electronics) Histotoxic hypoxia—cells unable to create ATP due to inhibition of mitochondrial cytochrome C oxidase26 Lactic acidosis, tissue hypoxia, depressed consciousness, cardiac arrest and multiorgan failure, death with high concentrations Hydrogen sulfide Rubber (tires) Silk, wool (clothing, furniture, fabrics) Binds iron in mitochondrial cytochrome enzymes, preventing cellular respiration and ATP generation; combines with alkali in moist tissues to form caustic sodium sulfide104 Airway and eye irritation, bronchospasm, pulmonary edema Nitric oxide Most combustible materials Free radical damage, vasodilation Worsened ventilation– perfusion mismatch and hypoxia; may have anti-inflammatory effect105 Carbon monoxide Most combustible materials Binds hemoglobin, creating carboxyhemoglobin, reducing oxygen carrying capacity and delivery Tissue hypoxia, multiorgan failure, death with high concentrations Carbon dioxide Most combustible materials Asphyxia; increases PaCO2* Increased respiratory drive Acrolein (propenal) Acrylics (textiles, windows, wallpaper) Cellulose (wood, paper, cotton) Polypropylene (carpet, upholstery, kitchen goods) Protein denaturation Irritation and necrosis of airway mucosa, death with high concentrations22 Ammonia Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting) Polyamine resins (household and kitchen goods) Polyacrylonitrile (appliances, plastics) Polyurethane (insulation, upholstery) Household cleaners Generates caustic ammonium hydroxide in aqueous environments Irritation and necrosis of airway mucosa, alveolar edema* Sulfur dioxide Rubber (tires) Chemical irritant properties (exact mechanism of action unknown); asthmatics more susceptible106 Potent ocular and airway irritant, bronchoconstriction, pulmonary edema Chlorine Cl Bleach/sanitation products Industrial solvents Water soluble—diffuses into respiratory epithelial lining fluid, hydrolyzes to acids which generate reactive chlorine and oxygen species Necrosis of proximal, superficial airway mucosa, pulmonary edema, airway hyperreactivity107 Phosgene Polyvinyl chloride (flooring, furniture, upholstery, piping) Fat soluble—generates caustic hydrochloric acid and chlorine. Increases pulmonary vascular permeability through effects on arachidonic acid metabolism108 Deep delayed damage/ necrosis to distal airway mucosa (fat- soluble, penetrates), alveolar cell death, delayed pulmonary edema, ARDS109 Hydrogen chloride Polyester (clothing, fabrics) Polyvinyl chloride (flooring, furniture, upholstery, piping) Water soluble—direct caustic effects of hydrochloric acid in the upper airway Irritation and necrosis of airway mucosa, airway edema, acute bronchitis, ARDS Aeromatic hydrocarbons (eg, benzene) Fossil fuels Paints, dyes, adhesives, wax Detergents Greater density than air— asphyxiation in enclosed environments, bone marrow (hematopoiesis) suppression Irritation of mucous membranes, systemic toxicity, drowsiness. Long term—carcinogenicity, anemia. Effects worsened by concurrent alcohol intoxication22 ARDS, acute respiratory distress syndrome; ATP, adenosine triphosphate; RNA, ribonucleic acid. *Arterial carbon monoxide partial pressure. View Large Table 2. Selected compounds found in smoke, examples of source materials, and pathophysiology Compound Source material Mechanism of action Effects Aldehydes (eg, formaldehyde) Acrylics (textiles paint, windows, wallpaper, adhesives) Cellulose (wood, paper, cotton) Propene (carpet, upholstery, containers) Protein and RNA denaturation Necrosis of airway mucosa* Hydrogen cyanide Fire retardants (insulation, upholstery) Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting, car parts) Polyamine resins (household and kitchen goods) Polyacrylonitrile (clothing, blankets, appliances) Polyurethane (insulation, upholstery, electronics) Histotoxic hypoxia—cells unable to create ATP due to inhibition of mitochondrial cytochrome C oxidase26 Lactic acidosis, tissue hypoxia, depressed consciousness, cardiac arrest and multiorgan failure, death with high concentrations Hydrogen sulfide Rubber (tires) Silk, wool (clothing, furniture, fabrics) Binds iron in mitochondrial cytochrome enzymes, preventing cellular respiration and ATP generation; combines with alkali in moist tissues to form caustic sodium sulfide104 Airway and eye irritation, bronchospasm, pulmonary edema Nitric oxide Most combustible materials Free radical damage, vasodilation Worsened ventilation– perfusion mismatch and hypoxia; may have anti-inflammatory effect105 Carbon monoxide Most combustible materials Binds hemoglobin, creating carboxyhemoglobin, reducing oxygen carrying capacity and delivery Tissue hypoxia, multiorgan failure, death with high concentrations Carbon dioxide Most combustible materials Asphyxia; increases PaCO2* Increased respiratory drive Acrolein (propenal) Acrylics (textiles, windows, wallpaper) Cellulose (wood, paper, cotton) Polypropylene (carpet, upholstery, kitchen goods) Protein denaturation Irritation and necrosis of airway mucosa, death with high concentrations22 Ammonia Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting) Polyamine resins (household and kitchen goods) Polyacrylonitrile (appliances, plastics) Polyurethane (insulation, upholstery) Household cleaners Generates caustic ammonium hydroxide in aqueous environments Irritation and necrosis of airway mucosa, alveolar edema* Sulfur dioxide Rubber (tires) Chemical irritant properties (exact mechanism of action unknown); asthmatics more susceptible106 Potent ocular and airway irritant, bronchoconstriction, pulmonary edema Chlorine Cl Bleach/sanitation products Industrial solvents Water soluble—diffuses into respiratory epithelial lining fluid, hydrolyzes to acids which generate reactive chlorine and oxygen species Necrosis of proximal, superficial airway mucosa, pulmonary edema, airway hyperreactivity107 Phosgene Polyvinyl chloride (flooring, furniture, upholstery, piping) Fat soluble—generates caustic hydrochloric acid and chlorine. Increases pulmonary vascular permeability through effects on arachidonic acid metabolism108 Deep delayed damage/ necrosis to distal airway mucosa (fat- soluble, penetrates), alveolar cell death, delayed pulmonary edema, ARDS109 Hydrogen chloride Polyester (clothing, fabrics) Polyvinyl chloride (flooring, furniture, upholstery, piping) Water soluble—direct caustic effects of hydrochloric acid in the upper airway Irritation and necrosis of airway mucosa, airway edema, acute bronchitis, ARDS Aeromatic hydrocarbons (eg, benzene) Fossil fuels Paints, dyes, adhesives, wax Detergents Greater density than air— asphyxiation in enclosed environments, bone marrow (hematopoiesis) suppression Irritation of mucous membranes, systemic toxicity, drowsiness. Long term—carcinogenicity, anemia. Effects worsened by concurrent alcohol intoxication22 Compound Source material Mechanism of action Effects Aldehydes (eg, formaldehyde) Acrylics (textiles paint, windows, wallpaper, adhesives) Cellulose (wood, paper, cotton) Propene (carpet, upholstery, containers) Protein and RNA denaturation Necrosis of airway mucosa* Hydrogen cyanide Fire retardants (insulation, upholstery) Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting, car parts) Polyamine resins (household and kitchen goods) Polyacrylonitrile (clothing, blankets, appliances) Polyurethane (insulation, upholstery, electronics) Histotoxic hypoxia—cells unable to create ATP due to inhibition of mitochondrial cytochrome C oxidase26 Lactic acidosis, tissue hypoxia, depressed consciousness, cardiac arrest and multiorgan failure, death with high concentrations Hydrogen sulfide Rubber (tires) Silk, wool (clothing, furniture, fabrics) Binds iron in mitochondrial cytochrome enzymes, preventing cellular respiration and ATP generation; combines with alkali in moist tissues to form caustic sodium sulfide104 Airway and eye irritation, bronchospasm, pulmonary edema Nitric oxide Most combustible materials Free radical damage, vasodilation Worsened ventilation– perfusion mismatch and hypoxia; may have anti-inflammatory effect105 Carbon monoxide Most combustible materials Binds hemoglobin, creating carboxyhemoglobin, reducing oxygen carrying capacity and delivery Tissue hypoxia, multiorgan failure, death with high concentrations Carbon dioxide Most combustible materials Asphyxia; increases PaCO2* Increased respiratory drive Acrolein (propenal) Acrylics (textiles, windows, wallpaper) Cellulose (wood, paper, cotton) Polypropylene (carpet, upholstery, kitchen goods) Protein denaturation Irritation and necrosis of airway mucosa, death with high concentrations22 Ammonia Silk, wool (clothing, furniture, fabrics) Polyamides (clothing, carpeting) Polyamine resins (household and kitchen goods) Polyacrylonitrile (appliances, plastics) Polyurethane (insulation, upholstery) Household cleaners Generates caustic ammonium hydroxide in aqueous environments Irritation and necrosis of airway mucosa, alveolar edema* Sulfur dioxide Rubber (tires) Chemical irritant properties (exact mechanism of action unknown); asthmatics more susceptible106 Potent ocular and airway irritant, bronchoconstriction, pulmonary edema Chlorine Cl Bleach/sanitation products Industrial solvents Water soluble—diffuses into respiratory epithelial lining fluid, hydrolyzes to acids which generate reactive chlorine and oxygen species Necrosis of proximal, superficial airway mucosa, pulmonary edema, airway hyperreactivity107 Phosgene Polyvinyl chloride (flooring, furniture, upholstery, piping) Fat soluble—generates caustic hydrochloric acid and chlorine. Increases pulmonary vascular permeability through effects on arachidonic acid metabolism108 Deep delayed damage/ necrosis to distal airway mucosa (fat- soluble, penetrates), alveolar cell death, delayed pulmonary edema, ARDS109 Hydrogen chloride Polyester (clothing, fabrics) Polyvinyl chloride (flooring, furniture, upholstery, piping) Water soluble—direct caustic effects of hydrochloric acid in the upper airway Irritation and necrosis of airway mucosa, airway edema, acute bronchitis, ARDS Aeromatic hydrocarbons (eg, benzene) Fossil fuels Paints, dyes, adhesives, wax Detergents Greater density than air— asphyxiation in enclosed environments, bone marrow (hematopoiesis) suppression Irritation of mucous membranes, systemic toxicity, drowsiness. Long term—carcinogenicity, anemia. Effects worsened by concurrent alcohol intoxication22 ARDS, acute respiratory distress syndrome; ATP, adenosine triphosphate; RNA, ribonucleic acid. *Arterial carbon monoxide partial pressure. View Large Exposure to fire and smoke causes injury via direct thermal effects as well as deposition of toxic particulate matter and respiratory irritants. These effects are pathologic at several distinct anatomic levels: the upper (supraglottic) airway, lower (infraglottic) airway or tracheobronchial system, lung parenchyma/alveoli, and remote organ systems (Figure 1). Direct thermal injury below the vocal cords is less common; it predominantly affects the nasal cavity and oropharyngeal mucosa due to efficient heat exchange in this region.13 An exception is exposure to large volumes of steam or superheated vapors in industrial settings, though the resulting severe glottic edema is often fatal.13 Chemical injury via particulate deposition depends largely on particle size and solubility. Under normal circumstances, particles over 5 µm in diameter rarely pass the vocal cords due to entrapment in mucoid secretions and robust ciliary clearance in the nasopharynx.15 However, in the presence of fire and smoke, damaged cilia and mouth-breathing due to nasopharyngeal irritation can send large particles into the lower airway. Small, hydrophobic particles like phosgene tend to deposit distally and deeper in the respiratory mucosa. Figure 1. View largeDownload slide Known respiratory and systemic effects of inhalation injury. Figure 1. View largeDownload slide Known respiratory and systemic effects of inhalation injury. Thermal injury to the upper airway manifests as erythema, edema, bronchorrhea, and ulceration. Work of breathing increases due to increased airway resistance from edema, obstruction, reduced airway compliance, and hypermetabolism causing increased minute ventilation requirements. These changes are due, in part, to protein denaturation and destruction of the epithelial layer, which trigger the complement cascade and release of damaging reactive oxygen species (ROS) and histamine.16 Histamine in turn stimulates nitric oxide (NO) formation by endothelial cells. The combination of ROS and NO increases endothelial permeability to proteins, leading to airway edema, sometimes exacerbated by aggressive fluid resuscitation.17 Further release of cytokines and chemokines, such as interleukin (IL)-8,5 recruits polymorphonuclear leukocytes to the area, ramping up the inflammatory response. Damage-associated molecular patterns (DAMPs) are molecular signals derived from mitochondria and other exposed intracellular structures of injured or hypoxic cells that may also play a key role in initiating and propagating inflammation after lung injury.18 Examples include double-stranded DNA (dsDNA), hyaluronan, S100 proteins, heat-shock protein 70, and high-mobility group box 1.19 Maile et al found that elevation in levels of dsDNA, hyaluronic acid, and IL-10, within 72 hours, in the bronchioalveolar lavage fluid of patients admitted with inhalation injury were associated with development of pneumonia over the subsequent 14 days.20 An analogous process is seen in ventilator-induced barotrauma, where DAMPs promote inflammation in noninfectious conditions via action on toll-like receptors.21 Finally, increased viscous mucoid airway secretions further obstruct the upper airway, particularly in the setting of impaired ciliary clearance. In the lower airway, chemical injury to the tracheobronchial system stimulates release of neuropeptides from sensory and motor nerve endings, which are potent bronchoconstrictors and vasodilators.3,22 Neuropeptides also act as chemoattractants for neutrophils, thereby generating more ROS, which react with NO to form reactive nitrogen species (RNS). Both ROS and RNS can damage DNA through oxidation and nitrosylation, or modify key enzymes and lipids, triggering apoptosis pathways.23 As in the upper airway, mucosal damage can act as a trigger for leukocyte activation and scar formation, and also promotes edema and production of excessive amounts of mucus. When mixed with soot, denuded airway epithelium, and fibrin deposits, this can form dreaded “casts” (rubbery bronchial plugs) that can cause catastrophic airway obstruction. Parenchymal or alveolar injury is characterized by atelectasis from decreased and dysfunctional surfactant and loss of hypoxic pulmonary vasoconstriction, which leads to ventilation–perfusion mismatch and hypoxemia. This mismatch is worsened by the formation of nitric oxide, which causes vasodilation, increasing blood flow to poorly ventilated bronchioles. As in more proximal structures, the alveolar capillary membrane damage from inhalation injury triggers a cascade of inflammatory mediators and generation of highly reactive free radicals, causing direct DNA damage and tissue necrosis.4 This inflammatory milieu, combined with impaired bacterial clearance by airway cilia and pulmonary edema, predisposes patients to developing ARDS and pneumonia, two of the most common pulmonary complications of inhalation injury. In addition, inhalation injury can cause a marked imbalance in alveolar hemostasis, characterized by increased levels of fibrinogen and decreased antifibrinolytic activity.4,24 This imbalance can accelerate airway cast formation. The long-term effects of inhalation injury are still poorly understood, but insights could be drawn from chronic exposure in smokers, which is associated with pulmonary interstitial fibrosis and emphysema.25 Likewise, a better understanding of airway repair mechanisms following inhalation injury is needed. These subjects are addressed in detail below. The systemic effects of inhalation injury are often delayed and partly a result of reduced P/F ratio and cellular asphyxiation, which decreases oxygen delivery to end organs. Its effects can also be attributed to circulation of lung-derived inflammatory mediators that trigger a systemic hypermetabolic state. In this state, blood is shunted away from visceral organs such as the intestines and pancreas, potentially leading to organ dysfunction.22 Certain toxins in smoke exert more specific systemic effects. For instance, hydrogen cyanide causes histotoxic hypoxia, wherein cellular adenosine triphosphate (ATP) generation is impaired due to inhibition of mitochondrial cytochrome C oxidase.26 Phosgene increases pulmonary vascular permeability through effects on arachidonic acid metabolism.27 The human body has mechanisms to convert many of these molecules into useful organic compounds once they reach systemic circulation (eg, ammonia metabolites incorporated into amino acids) or excreted in the urine. However, in high concentrations, inhaled toxins can overwhelm normal clearance mechanisms and result in significant harm. Recent studies have also identified diffuse blood–brain barrier dysfunction following smoke inhalation injury. Randolph et al demonstrated congested and dilated blood vessels with basement membrane damage in the frontal cortex, basal ganglia, amygdala, hippocampus, pons, cerebellum, and pituitary gland of sheep after smoke inhalation injury alone and in combination with full-thickness cutaneous burn, independent of changes in hemodynamics and PaO2.28 There is still much that we do not understand about the pathogenesis of inhalation injury and its systemic effects. For instance, the relative effects of smoke and heat exposure on the airway microbiome and the timeline for microbiome recovery are also in need of investigation. These questions are discussed further in a subsequent section. Another subject in need of further research is the influence of lung-derived cytokines/chemokines and DAMPs on other organ systems once they reach systemic circulation, as well as the role of skin-derived DAMPs, generated after cutaneous burn injury, on pulmonary inflammation. Finally, identifying the most appropriate animal model for inhalation injury and refining exposure techniques remains an ongoing effort. Disregarding primate models, guinea pigs and larger mammals such as sheep and pigs have airways with the greatest macro- and epithelial micro-structure to humans.29,30 However, ease of handling, ethical concerns, and cost have to be considered. TREATMENT OF INHALATION INJURY AND UNMET CLINICAL NEEDS The treatment of inhalation injury is less consistent than that of cutaneous thermal wounds, perhaps because diagnosis and grading is not as standardized. In essence, inhalation injury is treated by supporting normal host repair mechanisms and protecting the airway from further injury from infection, mechanical ventilation, or over-resuscitation with crystalloids.31 Supportive respiratory care includes chest physiotherapy and postural drainage of excessive secretions, airway suctioning/therapeutic bronchoscopy, humidified oxygen by face mask, and early ambulation.32 Inhaled bronchodilators can also be effective and are used routinely. Treatments used inconsistently in current practice include mucolytic agents, aerosolized anticoagulants/heparin sulfate, N-acetylcysteine,33 nonconventional ventilator modes, and prone positioning. The potential role of steroids and other anti-inflammatory agents is also an area of active research. Ventilation modes in use include high frequency percussive ventilation and lung-protective strategies derived from National Heart, Lung, and Blood Institute ARDS Network data (generally tidal volumes < 7 ml/kg, plateau pressures < 30 cm water, and high positive end-expiratory pressure).32 The use of much more invasive measures, including extracorporeal membrane oxygenation and selective pulmonary flow ligation (to lessen the effect of ventilation–perfusion mismatch) remains under preliminary investigation. Some specific identifiable toxicities from smoke inhalation have well-defined treatment protocols or antidotes. Carbon monoxide poisoning is largely treated with simple normobaric oxygen; hyperbaric oxygen therapy is rarely used. Patients may also be intubated. In the United States, cyanide poisoning is most often treated with hydroxocobalamin, which is employed in “Cyanokit” antidotes. Alternatively, kits that first employ a relatively small dose of inhaled amyl nitrite, followed by intravenous sodium nitrite, then intravenous sodium thiosulfate are also available. Nevertheless, there is significant room for improvement in current therapies and several questions remain unanswered. For example, how should inhalation injury influence fluid resuscitation and mortality predictions? Many burn centers take inhalation injury into account when estimating total burn area, which in turn influences calculated resuscitation requirements, but these techniques rarely discriminate between injury grades. Likewise, the revised Baux score predicts mortality after burn injury and takes the presence of inhalation injury into account, as follows, but not the grade of injury. Revised Baux Score = % TBSA + Age+ X where X = 17 in presence of inhalation injury; total body surface area (TBSA). One could similarly ask whether grading of inhalation injury should take area of luminal involvement into account instead of simply localized severity. Lastly, are certain subgroups of patients (eg, children, the elderly, those who present with coincident alcohol intoxication, or those with a history of pulmonary disease) affected differently by inhalation injury than others? We know that older burn-injured patients have a much greater general risk of morbidity and mortality and that specific proinflammatory cytokines and chemokines are elevated in burn patients >65 years old who meet intubation criteria in the serum—interleukin 1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, granulocyte colony-stimulating factor (G-CSF), interferon-gamma (IFN-γ), induced protein 10 (IP-10), and monocyte chemoattractant protein 1 (MCP-1), as well as bronchoalveolar lavage fluid—MCP-1.34 Moreover, changes in these systemic immune mediators appear to vary as a function of inhalation injury grade.35 We also know that acute alcohol intoxication at the time of burn injury is associated with increased carboxyhemoglobin levels.36 AIRWAY REPAIR MECHANISMS AND EVOLUTION OF INHALATION INJURY OVER TIME The literature surrounding airway repair mechanisms specifically following inhalation injury is relatively sparse. As a result, it may be useful to look for insights from more mature fields, including embryology/lung development and lung transplant biology. We can also look to other mechanisms of injury, including mechanical trauma and chronic smoking-related lung injury, where similar cellular damage pathways and repair mechanisms may be at play. Many of the same toxic compounds found in smoke generated from burning buildings and vehicles are found in tobacco smoke, though the effects are altered by chronic exposure. In general, human airway tissues maintain a low steady-state level of cellular turnover but can mount a vigorous response to injury through the action of progenitor cell populations. The epithelial lining of the respiratory tract is derived from ventral foregut endoderm, which differentiates to produce a continuous layer, spanning the trachea to individual alveoli, with varying properties along the proximal-distal axis.37,38 Mucin-producing goblet cells predominate in the pseudostratified ciliated columnar epithelium of larger airways and glycosaminoglycan-producing club cells predominate in smaller airways. Alveoli are lined with a simple monolayer composed of squamous (type 1) or cuboidal (type 2) cells; they share a thin basal lamina with endothelial cells, allowing for efficient gas exchange. Basal secretory cells and neuroendocrine cells are scattered throughout the airway. In contrast, most of the lung parenchyma, comprised of cartilage, vascular smooth muscle, myofibroblasts, and pericytes, is derived from mesoderm.39 Importantly, basal cells in the mucociliary epithelium of larger airways, club cells, and type 2 alveolar epithelial cells are all known to act as adult/long-term stem cells, with the ability to self-renew and give rise to other cell types, though they display varying degrees of differentiation and specialization in function.39 This suggests that pulmonary progenitor cells may undergo dedifferentiation or trans-differentiation following injury. One critical component in airway wound healing appears to be tissue factor (TF), which is most recognizable in its role in the conversion of prothrombin to thrombin in the extrinsic coagulation cascade. TF is released by cells in response to inflammatory mediators or damage to vascular endothelium. Davis et al found that injured human airway epithelial cells release TF after mechanical injury in vitro in response to proinflammatory stimuli, including lipopolysaccharide.40 Furthermore, exposure to an exogenous tissue factor pathway inhibitor decreased cellular proliferation by 60%, suggesting an important role for tissue factor in recovery after lung injury. In addition to in vitro studies, rodent models of lung injury, such as pneumonectomy, have shed additional light on remodeling and regrowth at the level of alveoli. Removal of the single left lobe in mice (leaving four right lobes intact) results in a dramatic increase of new alveoli through septation of existing units, especially at the lung periphery,41 and production of surfactant.39 Note that new lobes are not added. This process is thought to be modulated by mechanical cues, such as the availability of new space for growth and differential strain between the peri-hilar region and periphery.42 Mechano-transduction likely plays a critical role in all aspects of pulmonary cell differentiation.43,44 Several groups have attempted to generate pulmonary cell types from pluripotent stem cells by replicating the sequence of growth factors and other autocrine/paracrine factors these cells are exposed to in early lung development. Seminal studies have identified signal transduction pathways of particular importance downstream of TGF-β, Wnt, Notch, various fibroblast growth factors, and bone morphogenetic protein.39,45–48 This is clearly a nascent field and we are still years, if not decades, away from effective stem cell therapies for lung repair and regeneration. Many questions involving lung repair after inhalation injury remain unanswered. What is the general timeline for repair/regeneration of respiratory tract epithelium in humans? Are there exogenous factors which could accelerate scar remodeling and alleviation of airway strictures? What role does the immune system and airway microbiome play in lung repair? To what degree do variations or errors in regeneration (eg, mucus hyperplasia or squamous metaplasia) contribute to chronic pulmonary diseases? THE AIRWAY MICROBIOME Starting in the 1880s, early medical literature portrayed the respiratory tract as a sterile environment, except in cases of disease.49,50 We now know that healthy human airway mucosa is populated by a diverse array of bacteria, collectively known as the microbiome. The airway microbiome can vary greatly, even in a healthy state, with respect to bacterial number and diversity, both between individuals and at different levels of the same respiratory tree.51 For instance, bacterial flora of the upper airway are heavily influenced by microaspirations from the gastrointestinal tract. Some data also suggest that the microbiome can vary as a function of an individuals’ age.52,53 However, not all variation is normal and many disease states are characterized by altered pulmonary bacterial flora. Specific variations in the airway microbiome have been linked to asthma and pneumonia,54 cancer,55 chronic obstructive pulmonary disorder,56 and cystic fibrosis.57 The lungs of chronic smokers also exhibit altered microbiota.58 Depending on the disease, there can be changes in the relative abundance of particular bacterial genera and/or an overall decrease in the bacterial diversity.59 These changes disrupt the molecular crosstalk between bacteria and host, an essential component of the health of local tissues and the organism as a whole.60,61 The most well-studied microbiome is that of the small and large intestines. This likely stems from their abundant bacterial biomass, the ease of sampling, and clear implications for nutrition, infections, and inflammatory bowel disease. Drawing parallels to the intestinal microbiome, certain bacterial phenotypes in the lung may be more resistant to postburn complications, including respiratory tract infections.62,63 With respect to inhalation injury, several physiologic changes occur in the airway that can likely lead to profound alterations in the lung microbiome (Figure 2). Depending on the extent of the inhalation injury, a spectrum of inflammation and mucosal edema can develop.64,65 Byproducts of this inflammation include reactive nitrogen species, which exhibit some antimicrobial functions. Ironically, some facultative anaerobes actually thrive on the increase in extracellular nitrate, allowing for their proliferation and a general state of dysbiosis.66 Similarly, compromised mucociliary function after inhalation injury can lead to ineffective clearance of pathogenic bacteria.67 In patients requiring intubation and antibiotics, the initial intubation process itself may alter the lung microbiome by directly introducing upper respiratory organisms into the infraglottic airway. The increased presence of specific upper respiratory bacteria within the lung of healthy patients has been linked to increased lung inflammation.68 Meanwhile, Segal et al demonstrated that in a ventilator-dependent patient, there is a decrease in the diversity of the lung microbiome over time,69 which correlates with respiratory tract infection.70 Another consideration is that certain bacteria, such as pseudomonas species, can preferentially colonize the endotracheal tube and further alter the lung microbiome.71,72 Figure 2. View largeDownload slide Potential mechanisms for lung microbiome dysbiosis mediated by inhalation injury. After lung inhalation injury, the lung microbiome may be influenced by intubation, colonization of the endotracheal tube, antibiotic administration, and increased bacterial metabolism of inflammatory byproducts. Figure 2. View largeDownload slide Potential mechanisms for lung microbiome dysbiosis mediated by inhalation injury. After lung inhalation injury, the lung microbiome may be influenced by intubation, colonization of the endotracheal tube, antibiotic administration, and increased bacterial metabolism of inflammatory byproducts. Beyond the initial mischaracterization of the airway as a sterile environment, there are several reasons why lung microbiome research has lagged behind that of other microbiomes and has only come to the forefront in the past 10 years.73 Lung microbiome sampling requires the use of invasive procedures, such as a bronchoalveolar lavage (BAL) or lung tissue biopsy, since sputum samples are not representative of the lung microbiome. Another consideration is that the lower respiratory tract has a low microbial biomass relative to the sizable bacterial populations residing in the upper airway and care must be taken to avoid large-scale contamination. On the other hand, the upper airway microbiome of healthy patients somewhat resembles that of the whole lung microbiome, suggesting that the upper airway microbiome may act as a reservoir, replenishing the lower airway microbiome through small translocations.50,74 Using 16s rRNA sequencing, studies have revealed that a healthy lung microbiome, as well as that of the upper airway, appears to be composed primarily of bacteria from the Bacteroidetes, Firmicutes, and Proteobacteria phyla.50,75 With renewed focus on the role of the airway microbiome in health and disease and the advent of widespread sequencing technologies for enhanced detection of bacteria from low biomass microbiomes, an improved understanding of microbial influences on inhalation injury is likely on the horizon. Future studies will need to characterize specific changes in bacterial populations after injury, relative effects of heat and smoke exposure, the timeline for microbiome recovery, and properties unique to the pediatric and geriatric airway microbiomes. Mechanisms of microbial-host crosstalk and its role in immune function will also need to be elucidated. It is not unreasonable to expect that dysregulation of the airway microbiome following inhalation injury may influence leukocyte infiltration and release of inflammatory chemokines and cytokines, though data on this topic is still sparse. Ultimately, investigators will need to determine whether the microbiome can be manipulated to improve clinical outcomes following inhalation injury. BIOMARKERS OF INHALATION INJURY As outlined above, current techniques used to diagnose and grade inhalation injury have many limitations. The gold standard remains flexible fiberoptic bronchoscopy; however, the Abbreviated Injury Score used alongside bronchoscopy to grade inhalation injury is inherently subjective, as it requires the operator to assess the extent of airway damage based on gestalt. Furthermore, decisions to intubate a patient with suspected inhalation injury are often made before bronchoscopy can be performed. In fact, many patients arriving at a burn center are intubated prophylactically, resulting in a high rate of unnecessary intubation, as evidenced by frequent extubation within 48 hours of arrival.76,77 The Denver Criteria, a modification of the 2011 American Burn Association intubation guidelines with the addition of singed facial hair and suspected inhalation injury, were shown to have increased sensitivity but decreased specificity for appropriate intubations.78 Alternative techniques have their own problems. Clinical signs and symptoms commonly associated with inhalation injury discriminate poorly between those patients who have inhalation injury and those who do not and are often inconsistent with bronchoscopic findings.79,80 Given these issues, there is considerable interest in identifying a biomarker, or biomarkers, capable of not only early diagnosis of inhalation injury and discrimination of severity, but also prognostication of patient outcomes. Ideal biomarkers would also be measured rapidly and could thus inform the decision to intubate a patient with suspected inhalation injury. Potential biomarkers studied so far in adults can be divided further into cytokines, larger proteins, and DNA/RNA. Each group can be further separated by sample type—namely tracheobronchial or bronchoalveolar lavage fluid (BALF) and plasma. This section reviews putative biomarkers and their role in the pathogenesis of inhalation injury, with a focus on adult human studies. We also examine pediatric studies, which have largely focused on cytokines, as well as animal studies, which cover a much broader array of biomarkers, though not all lend themselves to rapid clinical assays. Candidate biomarkers are summarized in Table 3. Table 3. Summary of studies reporting on potential biomarkers Authors Type Subjects Specimens Controls Results Davis et al81 BALF 60 adults Bronchoalveolar lavage within 14 h of injury No control group; controlled for effects of age and %TBSA by comparing subjects with adjusted Baux scores > 69.5 Nonsurvivors had fewer leukocytes in BALF and these were hyporesponsive to LPS stimulation, producing fewer cytokines Albright et al5 BALF 60 adults Bronchoalveolar lavage within 14 h of injury 6 healthy volunteers Immunomodulator concentrations increased with worsening inhalation injury grade Frankel et al34 BALF and Plasma 104 adults Bronchoalveolar lavage and blood within 15 h of injury No control group; cohorts stratified by age BALF MCP-1 and plasma IL-1RA were increased in patients >65 y old; MCP-1 was associated with mortality while IL-1RA was the strongest predictor of mortality Kurzius–Spencer et al82 BALF 21 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects divided by P:F 200 (lowest value in 72 h postinjury) Increasing initial IL-8 values correlated with increasing P:F ratios Jones et al83 BALF 40 adults Mainstem bronchial washings taken within 72 h of injury No control group, subjects divided by P:F 200 (lowest value in 2 wk postinjury) Increased IL-10 in patients with P:F <200, increased IL-12p70 in patients with P:F >200 Davis et al35 Plasma 72 adults Blood at the time of bronchoscopy, within 15 h of injury 17 healthy adults, subjects divided by survival status IL-1RA was most strongly associated with mortality after controlling for age, %TBSA, and grade of inhalation injury Shelhamer et al84 Plasma 62 adults (24 with inhalation injury) Blood taken at days 0, 3, and 7 No control group, subjects divided by survival status Nonsurvivors had increased levels of IL-6 and IL-8 7 days after admission; IL-8 was associated with VAP or death with AUROC 0.82 at all time points Albright et al85 BALF 28 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Concentration of 26S proteasome and specific proteasome activity was decreased in patients and further decreased in those with VAP Baker et al86 BALF 51 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Ubiquitin negatively correlated with grade of inhalation injury, revised Baux score, IVF requirements, and positively with P:F ratio Kurzius–Spencer et al87 BALF 29 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects stratified by %TBSA and survival status AAT and A2M14 were reduced in subjects with >35% TBSA, and high initial levels of A2M with >35% TBSA burn were correlated with mortality Backes et al88 BALF and Plasma 11 adults Nondirected lung lavage and blood taken at admission and then days 3 and 5 15 ventilated subjects without burn, inhalation injury, or ARDS Pulmonary suPAR was increased compared to controls and was predictive of inhalation injury; plasma suPAR could predict ventilation duration and ICU LOS Maile et al20 BALF 72 adults Serial bronchial washings; first taken within 72 h of injury No control group, subjects divided by presence of positive bronchial bacterial culture Subjects who developed pulmonary infection within 2 wk postinjury had elevated initial hyaluronic acid and dsDNA levels Afshar et al89 Plasma 113 adults Blood taken within 24 h of injury No control group, subjects divided by presence of ARDS vWF-A2 had the greatest odds ratio for ARDS development, with an optimal cut-point of 1025 ng/ml Tokarik et al90 Plasma 22 adults (8 with inhalation injury) Blood taken at days 2 and 7 ProANP values compared to normal; subjects stratified by SOFA score No differences in ProANP based on inhalation injury status at days 2 and 7, but association between ProANP and SOFA score at day 7 Joyner et al91 BALF 6 children Bronchoalveolar lavage with 24 h of admission Subjects compared to case-matched adults as well as bronchiectasis and pneumonia cohorts dsDNA correlated with IL-6, IL-8, and TGF-β1 but not inhalation injury grade; similar dsDNA levels in adults and bronchiectasis cohort Finnerty et al94 Plasma 42 children Blood taken at admission and again at 5–7 d 15 healthy children On admission, IL-7 was higher and IL-12p70 was lower in subjects without inhalation injury Gauglitz et al95 Plasma 28 children Blood taken at admission and again at 5–7 d Normal children; subjects divided by survival status On admission, IL-4, IL-6, IL-10, and IL-13 were higher in nonsurvivors; by 5–7 days, IL-6, IL-7, & IL-10 fell; IL-6, IL-7, IL-10 were most predictive of mortality Abali et al96 BALF and plasma 48 Sprague- Dawley rats Lung tissue and plasma taken at 24 h postinjury Sham group of eight rats CD68+, IL-6 producing macrophages were sequestered in the lungs in full thickness and combined burn and inhalation injuries; less lung neutrophils in combined injury compared to burn alone Xiao et al97 BALF and plasma 42 Wistar rats Lung tissue and plasma taken after euthanasia at days 1, 2, 3, 7, 14, and 28 postinjury Control group of six rats Plasma IL-6 and BALF TNF-α and IL-10 reached a second peak 14 d postinjury; 25 pulmonary miRNAs were differentially regulated at day 1 postinjury Ye et al98 BALF 24 Wistar rats BALF and lung tissue taken at hours 6 and 24 postinjury Control group of eight rats 10 circRNAs were differentially regulated at hour 24 postinjury Sood et al99 Plasma 324 adults Blood taken within 7 days of No control group, subjects divided by age, presence of inhalation injury, and %TBSA There were no differentially expressed gene probe sets associated with inhalation injury Zhang et al100 BALF and plasma 32 Wistar rats BALF, lung tissue, and blood taken at hours 6 and 24 postinjury Control group of 10 rats Ratios of Th1/Th2 and Th17/Treg decreased and increased, respectively, following inhalation injury Authors Type Subjects Specimens Controls Results Davis et al81 BALF 60 adults Bronchoalveolar lavage within 14 h of injury No control group; controlled for effects of age and %TBSA by comparing subjects with adjusted Baux scores > 69.5 Nonsurvivors had fewer leukocytes in BALF and these were hyporesponsive to LPS stimulation, producing fewer cytokines Albright et al5 BALF 60 adults Bronchoalveolar lavage within 14 h of injury 6 healthy volunteers Immunomodulator concentrations increased with worsening inhalation injury grade Frankel et al34 BALF and Plasma 104 adults Bronchoalveolar lavage and blood within 15 h of injury No control group; cohorts stratified by age BALF MCP-1 and plasma IL-1RA were increased in patients >65 y old; MCP-1 was associated with mortality while IL-1RA was the strongest predictor of mortality Kurzius–Spencer et al82 BALF 21 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects divided by P:F 200 (lowest value in 72 h postinjury) Increasing initial IL-8 values correlated with increasing P:F ratios Jones et al83 BALF 40 adults Mainstem bronchial washings taken within 72 h of injury No control group, subjects divided by P:F 200 (lowest value in 2 wk postinjury) Increased IL-10 in patients with P:F <200, increased IL-12p70 in patients with P:F >200 Davis et al35 Plasma 72 adults Blood at the time of bronchoscopy, within 15 h of injury 17 healthy adults, subjects divided by survival status IL-1RA was most strongly associated with mortality after controlling for age, %TBSA, and grade of inhalation injury Shelhamer et al84 Plasma 62 adults (24 with inhalation injury) Blood taken at days 0, 3, and 7 No control group, subjects divided by survival status Nonsurvivors had increased levels of IL-6 and IL-8 7 days after admission; IL-8 was associated with VAP or death with AUROC 0.82 at all time points Albright et al85 BALF 28 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Concentration of 26S proteasome and specific proteasome activity was decreased in patients and further decreased in those with VAP Baker et al86 BALF 51 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Ubiquitin negatively correlated with grade of inhalation injury, revised Baux score, IVF requirements, and positively with P:F ratio Kurzius–Spencer et al87 BALF 29 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects stratified by %TBSA and survival status AAT and A2M14 were reduced in subjects with >35% TBSA, and high initial levels of A2M with >35% TBSA burn were correlated with mortality Backes et al88 BALF and Plasma 11 adults Nondirected lung lavage and blood taken at admission and then days 3 and 5 15 ventilated subjects without burn, inhalation injury, or ARDS Pulmonary suPAR was increased compared to controls and was predictive of inhalation injury; plasma suPAR could predict ventilation duration and ICU LOS Maile et al20 BALF 72 adults Serial bronchial washings; first taken within 72 h of injury No control group, subjects divided by presence of positive bronchial bacterial culture Subjects who developed pulmonary infection within 2 wk postinjury had elevated initial hyaluronic acid and dsDNA levels Afshar et al89 Plasma 113 adults Blood taken within 24 h of injury No control group, subjects divided by presence of ARDS vWF-A2 had the greatest odds ratio for ARDS development, with an optimal cut-point of 1025 ng/ml Tokarik et al90 Plasma 22 adults (8 with inhalation injury) Blood taken at days 2 and 7 ProANP values compared to normal; subjects stratified by SOFA score No differences in ProANP based on inhalation injury status at days 2 and 7, but association between ProANP and SOFA score at day 7 Joyner et al91 BALF 6 children Bronchoalveolar lavage with 24 h of admission Subjects compared to case-matched adults as well as bronchiectasis and pneumonia cohorts dsDNA correlated with IL-6, IL-8, and TGF-β1 but not inhalation injury grade; similar dsDNA levels in adults and bronchiectasis cohort Finnerty et al94 Plasma 42 children Blood taken at admission and again at 5–7 d 15 healthy children On admission, IL-7 was higher and IL-12p70 was lower in subjects without inhalation injury Gauglitz et al95 Plasma 28 children Blood taken at admission and again at 5–7 d Normal children; subjects divided by survival status On admission, IL-4, IL-6, IL-10, and IL-13 were higher in nonsurvivors; by 5–7 days, IL-6, IL-7, & IL-10 fell; IL-6, IL-7, IL-10 were most predictive of mortality Abali et al96 BALF and plasma 48 Sprague- Dawley rats Lung tissue and plasma taken at 24 h postinjury Sham group of eight rats CD68+, IL-6 producing macrophages were sequestered in the lungs in full thickness and combined burn and inhalation injuries; less lung neutrophils in combined injury compared to burn alone Xiao et al97 BALF and plasma 42 Wistar rats Lung tissue and plasma taken after euthanasia at days 1, 2, 3, 7, 14, and 28 postinjury Control group of six rats Plasma IL-6 and BALF TNF-α and IL-10 reached a second peak 14 d postinjury; 25 pulmonary miRNAs were differentially regulated at day 1 postinjury Ye et al98 BALF 24 Wistar rats BALF and lung tissue taken at hours 6 and 24 postinjury Control group of eight rats 10 circRNAs were differentially regulated at hour 24 postinjury Sood et al99 Plasma 324 adults Blood taken within 7 days of No control group, subjects divided by age, presence of inhalation injury, and %TBSA There were no differentially expressed gene probe sets associated with inhalation injury Zhang et al100 BALF and plasma 32 Wistar rats BALF, lung tissue, and blood taken at hours 6 and 24 postinjury Control group of 10 rats Ratios of Th1/Th2 and Th17/Treg decreased and increased, respectively, following inhalation injury AAT, alpha-1 antitrypsin; A2M, alpha-2-macroglobulin; ARDS, acute respiratory distress syndrome; AUROC, area under receiver operating characteristic curve; BALF, bronchoalveolar lavage fluid; circRNA, circular ribonucleic acid; dsDNA, double-stranded DNA; IL-1RA, interleukin 1 receptor antagonist; IVF, intravenous fluid; LPS, lipopolysaccharide; MCP-1, monocyte Chemoattractant Protein-1; miRNA, micro-ribonucleic acid; P:F, PaO2:FiO2 (ratio of arterial oxygen partial pressure to the fraction of oxygen in inspired gas); ProANP, proatrial natriuretic peptide; SOFA, sequential organ failure assessment; suPAR, soluble urokinase plasminogen activator receptor; %TBSA, percent total BSA (of burn injury); TGF-β1, transforming growth factor-beta 1; TNF-α, tumor necrosis factor-alpha; VAP, ventilator-associated pneumonia; vWF, Von Willebrand factor. View Large Table 3. Summary of studies reporting on potential biomarkers Authors Type Subjects Specimens Controls Results Davis et al81 BALF 60 adults Bronchoalveolar lavage within 14 h of injury No control group; controlled for effects of age and %TBSA by comparing subjects with adjusted Baux scores > 69.5 Nonsurvivors had fewer leukocytes in BALF and these were hyporesponsive to LPS stimulation, producing fewer cytokines Albright et al5 BALF 60 adults Bronchoalveolar lavage within 14 h of injury 6 healthy volunteers Immunomodulator concentrations increased with worsening inhalation injury grade Frankel et al34 BALF and Plasma 104 adults Bronchoalveolar lavage and blood within 15 h of injury No control group; cohorts stratified by age BALF MCP-1 and plasma IL-1RA were increased in patients >65 y old; MCP-1 was associated with mortality while IL-1RA was the strongest predictor of mortality Kurzius–Spencer et al82 BALF 21 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects divided by P:F 200 (lowest value in 72 h postinjury) Increasing initial IL-8 values correlated with increasing P:F ratios Jones et al83 BALF 40 adults Mainstem bronchial washings taken within 72 h of injury No control group, subjects divided by P:F 200 (lowest value in 2 wk postinjury) Increased IL-10 in patients with P:F <200, increased IL-12p70 in patients with P:F >200 Davis et al35 Plasma 72 adults Blood at the time of bronchoscopy, within 15 h of injury 17 healthy adults, subjects divided by survival status IL-1RA was most strongly associated with mortality after controlling for age, %TBSA, and grade of inhalation injury Shelhamer et al84 Plasma 62 adults (24 with inhalation injury) Blood taken at days 0, 3, and 7 No control group, subjects divided by survival status Nonsurvivors had increased levels of IL-6 and IL-8 7 days after admission; IL-8 was associated with VAP or death with AUROC 0.82 at all time points Albright et al85 BALF 28 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Concentration of 26S proteasome and specific proteasome activity was decreased in patients and further decreased in those with VAP Baker et al86 BALF 51 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Ubiquitin negatively correlated with grade of inhalation injury, revised Baux score, IVF requirements, and positively with P:F ratio Kurzius–Spencer et al87 BALF 29 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects stratified by %TBSA and survival status AAT and A2M14 were reduced in subjects with >35% TBSA, and high initial levels of A2M with >35% TBSA burn were correlated with mortality Backes et al88 BALF and Plasma 11 adults Nondirected lung lavage and blood taken at admission and then days 3 and 5 15 ventilated subjects without burn, inhalation injury, or ARDS Pulmonary suPAR was increased compared to controls and was predictive of inhalation injury; plasma suPAR could predict ventilation duration and ICU LOS Maile et al20 BALF 72 adults Serial bronchial washings; first taken within 72 h of injury No control group, subjects divided by presence of positive bronchial bacterial culture Subjects who developed pulmonary infection within 2 wk postinjury had elevated initial hyaluronic acid and dsDNA levels Afshar et al89 Plasma 113 adults Blood taken within 24 h of injury No control group, subjects divided by presence of ARDS vWF-A2 had the greatest odds ratio for ARDS development, with an optimal cut-point of 1025 ng/ml Tokarik et al90 Plasma 22 adults (8 with inhalation injury) Blood taken at days 2 and 7 ProANP values compared to normal; subjects stratified by SOFA score No differences in ProANP based on inhalation injury status at days 2 and 7, but association between ProANP and SOFA score at day 7 Joyner et al91 BALF 6 children Bronchoalveolar lavage with 24 h of admission Subjects compared to case-matched adults as well as bronchiectasis and pneumonia cohorts dsDNA correlated with IL-6, IL-8, and TGF-β1 but not inhalation injury grade; similar dsDNA levels in adults and bronchiectasis cohort Finnerty et al94 Plasma 42 children Blood taken at admission and again at 5–7 d 15 healthy children On admission, IL-7 was higher and IL-12p70 was lower in subjects without inhalation injury Gauglitz et al95 Plasma 28 children Blood taken at admission and again at 5–7 d Normal children; subjects divided by survival status On admission, IL-4, IL-6, IL-10, and IL-13 were higher in nonsurvivors; by 5–7 days, IL-6, IL-7, & IL-10 fell; IL-6, IL-7, IL-10 were most predictive of mortality Abali et al96 BALF and plasma 48 Sprague- Dawley rats Lung tissue and plasma taken at 24 h postinjury Sham group of eight rats CD68+, IL-6 producing macrophages were sequestered in the lungs in full thickness and combined burn and inhalation injuries; less lung neutrophils in combined injury compared to burn alone Xiao et al97 BALF and plasma 42 Wistar rats Lung tissue and plasma taken after euthanasia at days 1, 2, 3, 7, 14, and 28 postinjury Control group of six rats Plasma IL-6 and BALF TNF-α and IL-10 reached a second peak 14 d postinjury; 25 pulmonary miRNAs were differentially regulated at day 1 postinjury Ye et al98 BALF 24 Wistar rats BALF and lung tissue taken at hours 6 and 24 postinjury Control group of eight rats 10 circRNAs were differentially regulated at hour 24 postinjury Sood et al99 Plasma 324 adults Blood taken within 7 days of No control group, subjects divided by age, presence of inhalation injury, and %TBSA There were no differentially expressed gene probe sets associated with inhalation injury Zhang et al100 BALF and plasma 32 Wistar rats BALF, lung tissue, and blood taken at hours 6 and 24 postinjury Control group of 10 rats Ratios of Th1/Th2 and Th17/Treg decreased and increased, respectively, following inhalation injury Authors Type Subjects Specimens Controls Results Davis et al81 BALF 60 adults Bronchoalveolar lavage within 14 h of injury No control group; controlled for effects of age and %TBSA by comparing subjects with adjusted Baux scores > 69.5 Nonsurvivors had fewer leukocytes in BALF and these were hyporesponsive to LPS stimulation, producing fewer cytokines Albright et al5 BALF 60 adults Bronchoalveolar lavage within 14 h of injury 6 healthy volunteers Immunomodulator concentrations increased with worsening inhalation injury grade Frankel et al34 BALF and Plasma 104 adults Bronchoalveolar lavage and blood within 15 h of injury No control group; cohorts stratified by age BALF MCP-1 and plasma IL-1RA were increased in patients >65 y old; MCP-1 was associated with mortality while IL-1RA was the strongest predictor of mortality Kurzius–Spencer et al82 BALF 21 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects divided by P:F 200 (lowest value in 72 h postinjury) Increasing initial IL-8 values correlated with increasing P:F ratios Jones et al83 BALF 40 adults Mainstem bronchial washings taken within 72 h of injury No control group, subjects divided by P:F 200 (lowest value in 2 wk postinjury) Increased IL-10 in patients with P:F <200, increased IL-12p70 in patients with P:F >200 Davis et al35 Plasma 72 adults Blood at the time of bronchoscopy, within 15 h of injury 17 healthy adults, subjects divided by survival status IL-1RA was most strongly associated with mortality after controlling for age, %TBSA, and grade of inhalation injury Shelhamer et al84 Plasma 62 adults (24 with inhalation injury) Blood taken at days 0, 3, and 7 No control group, subjects divided by survival status Nonsurvivors had increased levels of IL-6 and IL-8 7 days after admission; IL-8 was associated with VAP or death with AUROC 0.82 at all time points Albright et al85 BALF 28 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Concentration of 26S proteasome and specific proteasome activity was decreased in patients and further decreased in those with VAP Baker et al86 BALF 51 adults Bronchoalveolar lavage within 14 h of injury 10 healthy volunteers Ubiquitin negatively correlated with grade of inhalation injury, revised Baux score, IVF requirements, and positively with P:F ratio Kurzius–Spencer et al87 BALF 29 adults Tracheobronchial washings: first within 6.5 h, then q2 hours for 36 h No control group, subjects stratified by %TBSA and survival status AAT and A2M14 were reduced in subjects with >35% TBSA, and high initial levels of A2M with >35% TBSA burn were correlated with mortality Backes et al88 BALF and Plasma 11 adults Nondirected lung lavage and blood taken at admission and then days 3 and 5 15 ventilated subjects without burn, inhalation injury, or ARDS Pulmonary suPAR was increased compared to controls and was predictive of inhalation injury; plasma suPAR could predict ventilation duration and ICU LOS Maile et al20 BALF 72 adults Serial bronchial washings; first taken within 72 h of injury No control group, subjects divided by presence of positive bronchial bacterial culture Subjects who developed pulmonary infection within 2 wk postinjury had elevated initial hyaluronic acid and dsDNA levels Afshar et al89 Plasma 113 adults Blood taken within 24 h of injury No control group, subjects divided by presence of ARDS vWF-A2 had the greatest odds ratio for ARDS development, with an optimal cut-point of 1025 ng/ml Tokarik et al90 Plasma 22 adults (8 with inhalation injury) Blood taken at days 2 and 7 ProANP values compared to normal; subjects stratified by SOFA score No differences in ProANP based on inhalation injury status at days 2 and 7, but association between ProANP and SOFA score at day 7 Joyner et al91 BALF 6 children Bronchoalveolar lavage with 24 h of admission Subjects compared to case-matched adults as well as bronchiectasis and pneumonia cohorts dsDNA correlated with IL-6, IL-8, and TGF-β1 but not inhalation injury grade; similar dsDNA levels in adults and bronchiectasis cohort Finnerty et al94 Plasma 42 children Blood taken at admission and again at 5–7 d 15 healthy children On admission, IL-7 was higher and IL-12p70 was lower in subjects without inhalation injury Gauglitz et al95 Plasma 28 children Blood taken at admission and again at 5–7 d Normal children; subjects divided by survival status On admission, IL-4, IL-6, IL-10, and IL-13 were higher in nonsurvivors; by 5–7 days, IL-6, IL-7, & IL-10 fell; IL-6, IL-7, IL-10 were most predictive of mortality Abali et al96 BALF and plasma 48 Sprague- Dawley rats Lung tissue and plasma taken at 24 h postinjury Sham group of eight rats CD68+, IL-6 producing macrophages were sequestered in the lungs in full thickness and combined burn and inhalation injuries; less lung neutrophils in combined injury compared to burn alone Xiao et al97 BALF and plasma 42 Wistar rats Lung tissue and plasma taken after euthanasia at days 1, 2, 3, 7, 14, and 28 postinjury Control group of six rats Plasma IL-6 and BALF TNF-α and IL-10 reached a second peak 14 d postinjury; 25 pulmonary miRNAs were differentially regulated at day 1 postinjury Ye et al98 BALF 24 Wistar rats BALF and lung tissue taken at hours 6 and 24 postinjury Control group of eight rats 10 circRNAs were differentially regulated at hour 24 postinjury Sood et al99 Plasma 324 adults Blood taken within 7 days of No control group, subjects divided by age, presence of inhalation injury, and %TBSA There were no differentially expressed gene probe sets associated with inhalation injury Zhang et al100 BALF and plasma 32 Wistar rats BALF, lung tissue, and blood taken at hours 6 and 24 postinjury Control group of 10 rats Ratios of Th1/Th2 and Th17/Treg decreased and increased, respectively, following inhalation injury AAT, alpha-1 antitrypsin; A2M, alpha-2-macroglobulin; ARDS, acute respiratory distress syndrome; AUROC, area under receiver operating characteristic curve; BALF, bronchoalveolar lavage fluid; circRNA, circular ribonucleic acid; dsDNA, double-stranded DNA; IL-1RA, interleukin 1 receptor antagonist; IVF, intravenous fluid; LPS, lipopolysaccharide; MCP-1, monocyte Chemoattractant Protein-1; miRNA, micro-ribonucleic acid; P:F, PaO2:FiO2 (ratio of arterial oxygen partial pressure to the fraction of oxygen in inspired gas); ProANP, proatrial natriuretic peptide; SOFA, sequential organ failure assessment; suPAR, soluble urokinase plasminogen activator receptor; %TBSA, percent total BSA (of burn injury); TGF-β1, transforming growth factor-beta 1; TNF-α, tumor necrosis factor-alpha; VAP, ventilator-associated pneumonia; vWF, Von Willebrand factor. View Large Adult Studies Cytokine Biomarkers in BALF Davis et al81 reported on a prospective series of 60 patients with burn and suspected smoke inhalation injuries who underwent bronchoscopy and BALF collection within 14 hours of injury. They compared BALF cytokine levels between survivors and nonsurvivors, partly controlling for the effects of age and %TBSA on mortality by limiting analysis to patients high Baux scores (>69.5). Levels of IL-1β, IL-1RA, IL-2, IL-4, IL-8, IL-10, G-CSF, IFN-γ, and macrophage inflammatory protein-1β (MIP-1β) were significantly lower in nonsurvivors, as were total white blood cell counts in BALF. Furthermore, production of all measured cytokines by isolated lipopolysaccharide-stimulated pulmonary leukocytes was blunted in nonsurvivors, with the exceptions of IL-1β, IL-4, IL-7, and IL-12. Overall, the data suggest that early pulmonary immune hyporesponsiveness, predicted by a decrease in specific cytokines, may contribute to mortality in patients with inhalation injury. Interestingly, Albright et al5 reporting on the same patient population as Davis et al81 plus six healthy volunteers, found that cytokine concentrations in BALF generally increased as a function of inhalation injury grade. Patients were divided into lower (Abbreviated Injury Score of 1–2) and higher (Abbreviated Injury Score of 3–4) severity cohorts. Per volume of BALF, IL-1β, IL-1RA, IL-6, IL-8, IL-9, G-CSF, IFN-γ, MCP-1, MIP-1β, platelet-derived growth factor, and regulated on activation, normal T-cell expressed and secreted (RANTES) were increased over both controls and grade 0 patients. Similarly, concentrations of proinflammatory IL-4, IL-6, IL-9, IL-15, granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, and MCP-1 were increased in higher grade injuries versus lower grade injuries. Importantly, in this study population, there were no significant differences in mortality when patients were divided by inhalation injury grade (0–5) or severity (lower or higher grade), suggesting that any differences seen were due to changes in the concentration of the cytokines studied. Frankel et al34 examined the influence of age on BALF cytokine profiles in a population of 104 patients admitted with burn and inhalation injury. Patients were stratified into age groups (<50, 50–64, and ≥ 65). MCP-1 was the only cytokine significantly different in the > 65 cohort, with a 3-fold elevation compared with the < 50 cohort. When adjusting for differences in sex, race, %TBSA, and inhalation injury grade, the difference was nearly significant (P = .06). There was also a significant association with in-hospital mortality (odds ratio [OR] 1.16, 95% confidence interval [CI] 1.01–1.32 per each increase of 100 pg/mg of admission MCP-1). MCP-1 is a regulator of monocyte and macrophage migration and it is possible that in the elderly population, recruited cells are unable to mount an adequate immune response. There have been conflicting reports regarding the relationship between specific pulmonary cytokines and clinical respiratory status. Kurzius–Spencer et al82 reported on 21 intubated patients who underwent serial tracheobronchial washings, which have the advantage of being less invasive than bronchoalveolar lavage. Of the seven cytokines measured in these washings (IL-1β, IL-8, tumor necrosis factor-α [TNF-α] transforming growth factor-β [TGF-β], soluble Fas ligand (sFasL), complement component 5a [C5a], and IL-10), only IL-8 was positively associated with P:F ratio, with each unit increase in initial log concentration of IL-8 associated with an increase in P:F ratio of 47.8 over the first 72 hours. This relationship remained true after adjusting for positive end-expiratory pressure and %TBSA and after excluding deaths. One reason for the positive association between IL-8 and oxygenation was thought to be the increased binding of molecules like α-2-macroglobulin (A2M) or autoantibodies to IL-8 in more severe injury. Conversely, Jones et al83 reported that of 11 pulmonary cytokines studied in bronchial washings from 40 intubated patients over 72 hours, only the anti-inflammatory IL-10 and IL-12p70 were significant predictors of P:F ratio within 2 weeks of injury. An increased IL-10 concentration (per volume of BALF) was found in patients with P:F ratio <200, whereas an increased IL-12p70 concentration was found in patients with P:F ratio >200. No association between IL-8 and P:F ratio was found. IL-10 may result in immunosuppression which leads to an increase in Gram-negative infections, resulting to poor respiratory function. Cytokine Biomarkers in Plasma Davis et al35 reported on the concentrations of plasma cytokines in 72 patients with suspected inhalation injury undergoing bronchoscopy within 15 hours of injury. Plasma IL-1RA, IL-6, IL-8, G-CSF, MCP-1 increased with worsening inhalation injury severity after controlling for age and %TBSA, similar to the findings of Albright et al5 in BALF. With respect to mortality, after controlling for age, %TBSA, and inhalation injury grade only plasma IL-1RA demonstrated a significant difference between survivors and nonsurvivors. Specifically, an increase in IL-1RA was observed in nonsurvivors (OR 3.12, 95% CI 1.03–9.44). This relationship persisted when cytokine levels were correlated to mortality, with the anti-inflammatory IL-1RA having the strongest correlation (r = .453, P < .001). This may account for the pulmonary immune hyporesponsiveness previously identified in nonsurvivors. During their investigation into the age-dependent response to inhalation injury, Frankel et al34 also analyzed plasma cytokines. Similar to Davis et al,35 they found that IL-1RA, IL-6, G-CSF, IP-10, and MCP-1 were significantly increased in the ≥ 65 year-old cohort versus < 50 after adjustments for age, sex, race, %TBSA, and inhalation injury severity. When plotted on a receiver operating characteristic curve, IL-1RA was the strongest predictor of in-hospital death with an area under the receiver operating characteristic curve (AUROC) of 0.84 which improved to 0.87 with addition of %TBSA. Further confirming the work of Davis et al,35 Shelhamer et al84 reported that in 62 burn patients with respiratory failure, with or without bronchoscopic evidence of inhalation injury, nonsurvivors had higher plasma concentrations of IL-6 and IL-8 at 0, 3, and 7 days postintubation. The difference was significant at day 7. A logistic regression model analyzing the 33 patients who met criteria showed that only age, injury severity score, and IL-8 reached significance at all time points to predict death or ventilator-associated pneumonia (VAP). Specifically, elevated IL-8 was associated with a significantly increased risk of death or VAP (OR 7.9, 95% CI 1.9–33.4, 26, 95% CI 4.0–178.7, and 7.3, 95% CI 1.9–28.4 on days 0, 3, 7, respectively). Large Protein Biomarkers in BALF Albright et al85 compared concentrations of proteasomes in BALF from 28 patients suspected of having inhalation injury who underwent bronchoscopy within 14 hours of injury to 10 healthy volunteers. Patients had less 26S proteasome per mg of BALF protein and globally decreased proteasome activity per ng of 20S proteasome compared with volunteers. In a subgroup analysis of BALF from patients who developed VAP, the 26S proteasome concentration and overall proteasome activity were similarly decreased compared with BALF from patients who did not develop VAP. Importantly, these findings were independent of the presence of a cutaneous burn, and, in fact, the concentration of 26S proteasomes per mg of BALF inversely correlated to grade of inhalation injury (r = −.410, P = .013). Thus, depressed proteasome activity likely propagates inhalation injury by delaying clearance of proteins accumulating in the alveolar space, increasing the risk of infection. Furthermore, the finding of the 26S proteasome in BALF was novel, even more so because it was assayed in the absence of ATP/Mg2+, which was thought to be required to prevent dissociation into its subunits. These findings make the 26S proteasome in BALF a promising biomarker of inhalation injury which may also be used to predict the development of VAP. Baker et al86 added to this work by investigating the C-X-C chemokine receptor 4 (CXC4) ligands ubiquitin and stromal-cell-derived factor-1α (SDF-1α) in BALF collected from 51 patients with inhalation injury as well as 10 healthy volunteers. Normalized to total BALF protein, ubiquitin concentrations increased versus controls and SDF-1α concentrations decreased to nearly undetectable levels. Ubiquitin concentrations correlated inversely with grade of inhalation injury, revised Baux score, and resuscitation volumes in the first 24 and 48 hours, and positively with P:F ratio. Although ubiquitin functions as an endogenous anti-inflammatory immune modulator, it also plays a role as a signaling peptide which guides proteins marked for destruction to the 26S proteasome. Thus, diminished proteasome activity and ubiquitin concentrations likely have an additive pathophysiological effect in higher inhalation injury grades. The absence of BALF SDF-1α could also act as an indicator of inhalation injury. Kurzius–Spencer et al87 reported that in 29 burn patients undergoing serial tracheobronchial washings, those with more severe burns (>35% TBSA) had significantly lower initial levels of the protease inhibitors α-1-antitrypsin and A2M compared with those with less severe burns, likely due to increased overall protease activity. This finding also suggests that binding of A2M to IL-8 is unlikely to explain the correlation between IL-8 and P:F ratios reported earlier by this group. The relative risk of death was 6.0 (95% CI 1.81–19.9) in those patients with > 35% TBSA burn and initial A2M concentration greater than the median, most likely due to increased pulmonary permeability and massive leakage into the airways from more extensive inhalation injury. In this study, initial secretory leukocyte peptidase inhibitor (SLPI) was a negative predictor of ventilator peak airway pressure requirements. None of these proteins were predictive of P:F ratios. Backes et al88 found that soluble urokinase plasminogen activator receptor (suPAR), a proposed marker of fibrinolysis and inflammation, was significantly elevated in nondirected pulmonary lavage samples from 11 ventilated patients with inhalation injury compared with 15 unburned but ventilated controls. At a cut-off of 6.1 ng/ml, pulmonary suPAR had an AUROC of 0.84 (95% CI 0.67–1.0, P = .003) to discriminate between the presence or absence of inhalation injury. There was no significant difference in plasma levels between groups, but systemic levels of suPAR greater than 9.5 ng/ml were associated with longer duration of mechanical ventilation (AUROC 0.93, 95% CI 0.73–1.0, sensitivity 100%, specificity 80%) and ICU length of stay (AUROC 0.93, 95% CI 0.78–1.0, sensitivity 100%, specificity 80%) in the burn patient group. This relationship held true even after accounting for surgical intervention during the first week, which may confound the decision to extubate. As noted earlier, DAMPs also show promise as biomarkers of inhalation injury. Maile et al20 reported that in 72 patients with suspected inhalation injury, who underwent bronchoscopy with bronchial washings within 72 hours of admission, there was a significant association between elevated hyaluronic acid and dsDNA levels and bacterial respiratory infection, defined as at least one positive bacterial bronchial culture within the first 2 weeks postinjury. No such association was found with other DAMPs, including heat-shock protein 70 and high-mobility group box 1. Patients who developed early respiratory infections within the first 72 hours had significantly elevated hyaluronic acid levels but not dsDNA levels. Large Protein Biomarkers in Plasma Afshar et al89 examined 113 burn patients to identify associations between plasma biomarkers measured within the first 24 hours after injury and ARDS. Of 5 biomarkers studied, von Willebrand Factor A2 best predicted the development of ARDS (OR 7.72, 95% CI 1.6–20.2, P = .01). At a value of 1025 ng/ml, von Willebrand Factor A2 had a positive predictive value of 78.4% and a negative predictive value of 91.2%. A three-factor model including %TBSA, bronchoscopic evidence of inhalation injury, and elevated von Willebrand Factor A2 was the strongest predictor of ARDS development (AUC 0.90, 95% CI 0.84–0.95). Similarly, Tokarik et al90 assessed plasma proatrial natriuretic peptide (proANP) as a marker for ARDS in burn patients. There were no significant differences in proANP levels on postburn day 2 and 7 among patients with or without inhalation injury. However, proANP may yet be a useful biomarker of inhalation injury in burn patients as it correlated strongly to sequential organ failure assessment (SOFA) scores ≥ 2 and was a stronger predictor of mortality in a logistic regression analysis. Pediatric Studies Cytokine Biomarkers in BALF In a small series, Joyner et al91 measured BALF levels of IL-6, IL-8, TGF-β1, and dsDNA in six pediatric patients with inhalation injury and compared them to both case-matched adults and pediatric patients with chronic bronchiectasis and recurrent pneumonia. BALF dsDNA was initially elevated but began to decline by postburn day 2, although there was no significant correlation between dsDNA levels and inhalation injury grade. The levels of dsDNA in the pediatric burn group were similar to levels in the bronchiectasis group as well as in case-matched adults, and these were significantly increased compared with dsDNA levels in the pneumonia group. Finally, in both pediatric and adult patients with inhalation injury, dsDNA levels correlated with IL-6, IL-8, and TGF-β1 (r2 = .416–.742, P < .05 for all). Although the results lend credence to the therapeutic strategy of treating pediatric inhalation injury with DNase, the small sample size limits generalizability. Furthermore, these results stand in contrast to the findings of Maile et al,20 who reported an association between increased BALF dsDNA levels and pneumonia. Cytokine Biomarkers in Plasma Finnerty et al92 compared levels of serum cytokines between severely burned (≥40% TBSA) patients less than 16 years of age with and without inhalation injury and also between 15 unburned, healthy controls. In an extension of this work, and mirroring Davis’ examination of mortality, Gauglitz et al93 compared cytokine levels between 13 nonsurviving and 15 surviving burn patients with inhalation injury. On admission, all cytokines studied (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, GM-CSF, G-CSF, IFN-γ, MCP-1, MIP-1β, and TNF-α) were increased in burned patients compared with controls, but specific cytokines exhibited differences with respect to the presence of inhalation injury and/or mortality. IL-7 was significantly higher on admission in patients without inhalation injury. While IL-7 rose in survivors (and, on average, in all patients with inhalation injury) by 5–7 days postadmission, it decreased by nearly 3-fold in nonsurvivors. In contrast, there were no differences in IL-6 and IL-10 levels between those with and without inhalation injury. In terms of mortality, nonsurvivors had an approximately 10-fold increase in admission levels of IL-6 and IL-10 compared with survivors, and although levels in both groups decreased by 5 to 7 days postadmission, levels in nonsurvivors remained significantly higher than in survivors. Admission levels of IL-12p70 were decreased in patients without inhalation injury compared with those with inhalation injury, but there were no differences by survival status. IL-4 and IL-13 demonstrated no differences by inhalation injury status, but admission levels were increased in nonsurvivors by 3- and 4-fold, respectively. When analyzed in a multiple regression model, admission levels of IL-10 and postadmission days 5 to 7 levels of IL-6 and IL-7 were strongly correlated with mortality. There were no other differences in the other cytokines studied. Several conclusions can be drawn from these studies. First, as the cytokine profile of patients with and without inhalation injury remained largely similar, both at admission and 5 to 7 days postadmission, the pathophysiology of inhalation injury in pediatric patients is unlikely to be due to an augmented, hyperimmune systemic inflammatory response. In fact, T-cell-mediated immunity may be suppressed, given the changes in the levels of IL-7 and IL-12p70. The majority of cytokines studied may not be useful as specific biomarkers of inhalation injury, but IL-10 and IL-6, through their early inhibition of macrophage-derived proinflammatory mediators and late stimulation of neutrophil-mediated hyperinflammation, respectively, could be used to determine survivability. Although the authors posit that the overall decrease in serum levels of cytokines is due to increased pulmonary production and consumption, this has not been shown to be true in adult studies, except in the case of IL-8.34,35,81 Furthermore, although children had greater %TBSA burns than adults in these studies, the pattern of cytokine alterations does not appear to be consistent across age groups. Overall, it is likely that the inflammatory response to burn and inhalation injury in children is different from the response in adults. Animal Studies Cytokine Biomarkers in Plasma Abali et al94 examined the effects of different burn depths, with or without the presence of inhalation injury, on levels of circulating cytokines over 24 hours. Forty-eight Sprague-Dawley rats were divided by burn depth and inhalation injury (via exposure to 17°C cotton smoke) into sham, partial thickness, full thickness, inhalation injury, partial-thickness with inhalation injury, and full thickness with inhalation injury groups. IL-6 was increased in all experimental groups except sham-injured animals, but CD68+ macrophages, the main source of IL-6, were sequestered in the pulmonary tissues in rats with full thickness burns, as well as rats with both partial thickness and full thickness burns with inhalation injury. This suggests that in severe burns alone or in the presence of inhalation injury, the lungs are the major source of serum IL-6. Although the authors suggested that a significant increase in IFN-γ in rats with partial thickness and full thickness burns with inhalation injury over corresponding groups without inhalation injury may allow it to be used as a biomarker of inhalation injury, it is unclear if this can be translated to humans. Davis et al40 found no significant differences in IFN-γ levels between patients with and without inhalation injury, even when controlling for age and %TBSA. However, Abali et al did find that the largest neutrophilic pulmonary infiltrate occurred in the full-thickness group. Fewer neutrophils were found in the full-thickness burn with inhalation injury group, echoing Davis’ finding that BALF from nonsurvivors with inhalation injury had a lower total WBC count. RNA Biomarkers in Plasma There has been considerable interest in RNAs as biomarkers of inhalation injury. Xiao et al95 exposed 42 Wistar rats to 37°C wood smoke containing 700 ppm of carbon monoxide for 15 minutes and performed histological, protein, and RNA analyses over 28 days. They found that plasma IL-6 and BALF TNF-α and IL-10, after peaking 1 to 2 days postburn, decreased and peaked again 14 days postburn. This corresponded to their observation that pulmonary fibrosis, indicated by collagen deposition, appeared in pulmonary tissues 14 days postinjury. Twenty-five distinct BALF micro-RNAs (miRNA), which are single-stranded, noncoding RNAs that regulate gene expression via mRNA changes, were found to be significantly differentially regulated on day 1 postburn. Two of these, miR-125b and miR-205, have been associated with ARDS in previous studies.96,97 Seven miRNAs with sequence homology to humans were validated using RT-qPCR. Three of these, miR-34c-5p, miR-34b-3p, and let-7c-5p, have been shown to be involved in pulmonary fibrosis,98 endothelial cell regulation,99 and apoptosis,100 respectively. Ye et al101 exposed 24 Wistar rats to smoke for 15 minutes and analyzed the expression of circular RNAs (circRNA) 1 day postburn. CircRNAs have recently been shown to regulate miRNA and could act as biomarkers themselves. The investigators identified 10 differentially regulated circRNAs and validated 8 of these with qPCR. All four of the circRNAs selected for confirmation were found to indeed be in circular form via RT-PCR using primers specific for amplification of circRNA. Further work is needed to clarify the functions and underlying molecular pathways of these novel circRNAs. These studies must be taken in the context of a report by Sood et al102 analyzing the RNA expression of whole-blood leukocytes within 7 days of injury in 324 adult patients with >20% TBSA burn. No differentially expressed probe sets associated with inhalation injury were found. However, the authors analyzed the entire transcriptome and did not focus specifically on miRNA or circRNA. Lymphocyte Patterns in Inhalation Injury Zhang et al103 compared lymphocytes in BALF and serum in 30 Wistar rats exposed to 26.6°C gunpowder smoke containing approximately 300 ppm of carbon monoxide for 8 minutes. Using flow cytometry, they found that the concentrations of various CD4+ T-cell populations and their related cytokines in BALF and serum followed similar, significant trends over the 24-hour study period. Overall, CD4+ cells increased compared with controls. Th1 cells and IFN-γ decreased while Th2 cells and IL-4 increased. Th17 cells and the cytokines IL-6, IL-17A, TGF-β, and IL-23 increased while Treg cells and the cytokines IL-10, IL-2, and IL-35 decreased. Consequently, the ratios of Th1/Th2 and Th17/Treg decreased and increased, respectively. The increase in CD4+ cells was thought to be due to the decrease in the anti-inflammatory Treg lymphocytes, which in turn was caused by a decrease in IL-2 and an increase in IL-6 and Th2 cells. Furthermore, in combination with IL-6, TGF-β promotes differentiation of the proinflammatory Th17 cells, which are involved in neutrophil recruitment and maintained by IL-23. Thus, specific alterations in T-cell effector functions likely contribute to the pathophysiology of inhalation injury; furthermore, unique changes in the ratios of T-cell subsets may differentiate inhalation injury from other pulmonary pathologies. DISCUSSION In this review, we identified significant limitations in our current understanding of inhalation injury as well as diagnostic and treatment paradigms now in use. This underpinned a discussion of yet unmet clinical needs and specific areas of research effort that could help to address them. Our ultimate aim is to provide investigators with a framework to guide future research and spark further interest in these topics. With respect to airway repair mechanisms, we encourage researchers to draw insight from other forms of lung injury, including ventilator barotrauma, ARDS, and chronic tobacco smoke exposure, where similar cellular damage pathways and repair mechanisms may be at play. It is clear that a population of progenitor cells, namely basal cells in the mucociliary epithelium, club cells, and type 2 alveolar epithelial cells, are maintained at distinct levels in the airway and respond to injury through both dedifferentiation or trans-differentiation. Improving our understanding of how pluripotent stem cells differentiate and how simple basal and luminal cells regenerate damaged airway segments will be a crucial part of efforts to develop the “holy grail” of autologous bioengineered respiratory tissues. The role of the airway microbiome in maintaining a healthy respiratory mucosa and its response to inhalation injury would is another area ripe for further investigation. A better understanding of these processes could improve the diagnostic utility of bronchoalveolar lavage or even yield new interventions aimed at restoring healthy airway flora in patients following dysbiosis. Regarding diagnosis of inhalation injury, bronchoscopy remains the gold standard, but undoubtedly has shortcomings which could be addressed by a multimodality approach that incorporates rapidly assayable biomarkers. For instance, evidence presented here suggests that serum and pulmonary cytokines patterns early after heat and smoke exposure may reflect the degree of inhalation injury and even predict development of ARDS or, ultimately, mortality rates. Importantly, they could also help to account for heterogeneity in the inflammatory response of individual patients, which may be contributing to disparate clinical presentations with similar bronchoscopic findings. Many challenges remain, as evidenced by the fact that levels of cytokines appear to differ across age groups after similar injuries. More work is also needed to elucidate the role of novel biomolecules like circular RNAs and to determine whether specific candidate biomarkers lend themselves to rapid clinical assays. The unmet clinical needs discussed here are by no means comprehensive. Future efforts should also focus on developing algorithms for consistent application of existing techniques and treatments as well as verifying the efficacy of treatments currently in clinical use without strong evidence, including specific lung-protective ventilatory strategies in inhalation injury populations, inhaled mucolytics, and anticoagulants. Future studies must also follow patients longitudinally to clarify the long-term implications of inhalation injury. Funding sources include NIH R01 AG018859 (E.J.K.), R01 GM115257 (E.J.K.), R01 GM128242 (M.A.C.), F30 AA027442 (P.V.K.), and Underwriters Laboratory (Standards in Biologic Lesions: Cutaneous Thermal Injury and Inhalation Injury Working Group – American Burn Association). Conflict of interest statement. None declared. REFERENCES 1. Mosier MJ , Bernal N , Faraklas IH et al. National burn repository . Chicago (IL) : American Burn Association ; 2017 . Google Preview WorldCat COPAC 2. Gupta K , Mehrotra M , Kumar P , Gogia AR , Prasad A , Fisher JA . Smoke inhalation injury: etiopathogenesis-, diagnosis, and management . Indian J Crit Care Med. 2018 ; 22 : 180 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Rehberg S , Maybauer MO , Enkhbaatar P , Maybauer DM , Yamamoto Y , Traber DL . Pathophysiology, management and treatment of smoke inhalation injury . 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Google Scholar Crossref Search ADS PubMed WorldCat © American Burn Association 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Inhalation Injury: Unmet Clinical Needs and Future Research
JF - Journal of Burn Care & Research
DO - 10.1093/jbcr/irz055
DA - 2019-08-14
UR - https://www.deepdyve.com/lp/oxford-university-press/inhalation-injury-unmet-clinical-needs-and-future-research-pzs629irr7
SP - 570
VL - 40
IS - 5
DP - DeepDyve
ER -