TY - JOUR
AU - PhD, Mashkoor A. Choudhry,
AB - Abstract Intestinal inflammation has been linked with multiorgan failure in patients with burn and other traumatic injuries. We hypothesized that markers of intestinal inflammation are detectible noninvasively. Fecal samples were collected from seven severely burned patients and 15 control patients for the measurement of inflammatory cytokines using a multiplex assay kit. In addition, fecal levels of myeloperoxidase (MPO) and elastase were measured using standard procedures. Compared with a control group, levels of inflammatory cytokines were significantly increased in the burn group. Interleukin (IL)-6 increased to a mean (± SEM) of 2.16 ± 0.61 to 3.81 ± 0.49 pg/mg (P < .05), as did IL-8 (3.32 ± 0.76 to 20.51 ± 6.65 pg/mg; P < .05), IL-12 (6.23±0.98 to 8.11±0.95pg/mg; P=0.01), IL-13 (3.86 ± 0.32 to 11.83 ± 1.47 pg/mg; P < .05), monocyte chemoattractant protein-1 (2.78 ± 2.61 to 6.5 ± 3.97 pg/mg; P < .05), MPO (13.41 ± 1.40 to 24.52 ± 4.31 units/mg protein; P < .05), and elastase (2.46 ± 0.38 to 5.08 ± 0.72 pg/mL; P < .05). Our results suggest that markers of intestinal inflammation are measurable by noninvasive means and are increased after burn injury compared with controls. Of note, increased IL-8 correlated with increased MPO and elastase activity, suggesting a role for neutrophil activation in burn-mediated intestinal inflammation. Thus, these inflammatory cytokine profiles may be valuable biomarkers of intestinal inflammation after burn injury. Sepsis and multiorgan dysfunction syndrome (MODS) remain an overwhelming cause of mortality after burn injury. It is well known that burn injury can lead to marked organ and immune dysfunction that is associated with a distinct increase in the circulatory levels of proinflammatory cytokines and chemokines. Although the injury itself is a major insult, several lines of evidence suggest that in the postburn period, the gastrointestinal tract is actually playing a central role in driving organ and immune dysfunction and that it may be the primary source of the mediators of inflammation found in systemic circulation.1,–8 Previous findings from our laboratory have shown that in an animal model of burn injury, an elevation in cytokines, chemokines and neutrophil infiltration exists in the gut.9,–12 Furthermore, we found that an elevation in these inflammatory mediators correlates well with actual damage to the tissues of the gut itself.9,–12 The human colon is the major reservoir of bacteria within the body. Under healthy conditions, it maintains an effective barrier against luminal bacteria; however, it is known that this barrier is disrupted after burn injury.1,3,12,–14 This compromised barrier may play a key part in the process that leads to organ and immune dysfunction after burn, but the exact role is unknown. There is evidence that it is not the actual bacteria themselves that translocate and lead to this immune dysfunction; instead it is the cytokine milieu induced by our immune system's interaction with these bacteria that seems to be the culprit. Thus the situation that is created by the burn injury is one where we have a permeable gut loaded with intestinal bacteria, and this sets the stage for an abnormal immune interaction leading to the production of mediators that can fuel organ and immune dysfunction. Inflammation of the gut is historically difficult to assess by noninvasive means. Over the last three decades there has been developing interest in fecal markers and their role in inflammatory bowel diseases.15,16 Specifically, calprotectin and lactoferrin have been used to successfully predict mucosal disease, healing, response to therapy, and relapse. There are new data to suggest a role for fecal markers in the prediction of necrotizing enterocolitis in neonates.15,16 Because of the significant evidence for gut dysfunction after burn injury, we hypothesized an observable change in inflammatory mediators in the feces of burn victims, and we are hopeful that a noninvasive method of monitoring this process will serve as a window into what is actually happening in the gut, increasing our understanding of the pathophysiology regarding these injuries. METHODS Sample Group This study was approved by our institutional review board for human subjects and informed consent was obtained from all participants. Samples were collected from burn patients admitted to the burn unit at Loyola University Health Sciences Campus, Maywood, Illinois, from December 2010 to November 2011. During the care of severely injured burn patients, we routinely place a fecal management system. This system facilitates routine care of the patient by allowing the egress of feces into a collection device rather than onto the patient. From December 2010 to November 2011, samples of this effluent were collected from seven individuals who suffered burn injury over various periods during their admission. Patients selected for observation met the following criteria: they were adult men and women older than age 18 with superficial or full-thickness burn injury, >10% TBSA; they also had no preexisting clinical infection or preexisting clinical or historical evidence of gastrointestinal illness, such as ulcerative colitis or Crohn's disease, or gastrointestinal infection, such as Clostridium difficile or cytomegalovirus colitis. They were not taking antibiotics (other than surgical prophylaxis) or immune-suppressing medications; had no perforated viscera, peritonitis, or any form of stoma; were not transplant recipients; and did not have AIDS, known immunodeficiency, or disseminated cancer. Control Group To examine the feces of burn-injured patients we needed comparable samples from otherwise healthy patients. Patients with “physiologically insignificant” burns, for example, superficial or partial-thickness burns <10% TBSA that are located in sensitive areas such as the hands and face, also are routinely hospitalized for care. These patients do not require the use of a fecal management system and instead use the toilet as needed. We used this patient population as a control for comparison with those with significant burn injury. During the aforementioned time period, samples from 15 of these patients were collected at a minimum of two time points during the course of their hospitalization. These patients also were subject to the above inclusion and exclusion criteria. This study was approved by the Loyola University Medical Center Institutional Review Board, and informed consent was obtained from all study subjects. Sample Handling/Processing Patient samples were collected roughly once every 7 days of hospitalization via sterile sample cups and immediately frozen at −80°C. Samples of 50 mg were sonicated in phosphate-buffered saline with protease inhibitor (Roche Diagnostics, Indianapolis, IN) for two rounds of 15 seconds each and then centrifuged at 10,000 rpm for 15 minutes at 4°C. Supernatant was collected for the measurement of inflammatory mediators using a Bio-Plex Pro Human Cytokine 17-plex Assay (Bio-Rad, Hercules, CA), according the manufacturer's specifications. Myeloperoxidase (MPO) activity in fecal samples was measured by incubating fecal homogenates (10 μL) in a 96-well plate with 290 μL of 50 mM phosphate buffer, 3 μL of substrate solution containing 20 mg/ml o-dianisidine hydrochloride, and 3 μL of 20 mM H2O2. The reaction was stopped by adding 3 μL of 30% sodium azide. Plates were read at 460 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT). For the measurement of elastase, fecal lysates (25 μL) were incubated in a 96-well plate at room temperature for 60 minutes with 1 mM methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide (Sigma-Aldrich, St. Louis, MO), 0.1 M HEPES, and 0.5 M NaCl (pH 7.5) in a total volume of 150 μL. Absorbance was measured at 405 nm. These methods have been used previously by our lab.9,11,17 The protein content of each aliquot of stool sample was measured by standard Bio-Rad assay to which all cytokines and chemokines were normalized, and the values are expressed per milligram protein. RESULTS Table 1 shows a comparison of specific patient characteristics. A total of seven patients met the inclusion and exclusion criteria. Samples from these patients were collected at various time points, with at least one sample in each week after injury. Samples from three separate time points during hospitalization were taken from four patients; one or two samples were taken from the remaining three patients. This results in a burn group of seven patients in total but yielding 14 to 17 fecal samples. The mean age of these patients is 58 ± 13 years, ranging from 31 to 81 years. Types of burn consisted of flame and chemical, with flame being the predominant type (86%). The mean TBSA was 36 ± 14 and ranged from 17 to 57%. One patient suffered concomitant orthopedic trauma. Four patients developed subsequent septic complications as a result of injury, and six patients (86%) died as a result of injury or withdrawal of physiologic support after the family's decision to withdraw care. Our control group consisted of 15 patients with physiologically insignificant burns, yielding a total of 23 fecal samples. Table 1. Characteristics of burn injured patients View Large Table 1. Characteristics of burn injured patients View Large As described in the Methods section, we used a Bio-Plex Pro Human Cytokine 17-plex Assay, which detects interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, interferon (INF)-γ, macrophage inflammatory protein-1, and monocyte chemoattractant protein (MCP)-1β. Of those, only the following cytokines were observed to be measurable at a detectable level in the stool samples: IL-6, IL-7, IL-8, IL-12, IL-13, G-CSF, TNF-α, and INF-γ. Among these, levels of IL-6, IL-12, and IL-13 were significantly increased in the burn group compared with the control. As demonstrated in Figure 1, IL-6 increased a mean (± SEM) of 2.16 ± 0.61 to 3.81 ± 0.49 pg/mg (P < .05), as were IL-12 (6.23±0.98 to 8.11±0.95pg/mg; P=0.01) and IL-13 (3.86 ± 0.32 to 11.83 ± 1.47 pg/mg; P < .05). Figure 1. View largeDownload slide Fecal levels of cytokines after burn injury. Feces collected from patients at various time points after burn injury was used to determine the levels of cytokines using multiplex kit. Interleukin (IL)-6 (A), IL-12 (B), and IL-13 (C) are increased in the burn group compared with the control group. Data are shown as mean ± SEM. *P < .05 compared with control group. Figure 1. View largeDownload slide Fecal levels of cytokines after burn injury. Feces collected from patients at various time points after burn injury was used to determine the levels of cytokines using multiplex kit. Interleukin (IL)-6 (A), IL-12 (B), and IL-13 (C) are increased in the burn group compared with the control group. Data are shown as mean ± SEM. *P < .05 compared with control group. TNF-α and G-CSF were increased from 512 ± 246 to 933 ± 274 pg/mg and from 32.52 ± 9.62 to 155.34 ± 58.36 pg/mg, respectively, in the burn group compared with the control group, but this difference was not statistically significant. INF-γ also was increased in the burn compared with control group (70.85 ± 12.99 vs 46.67 ± 12.92 pg/mg protein), but, again, this difference was not statistically significant. Figure 2 shows chemokine levels after burn injury. Similar to cytokines, the levels of IL-8 and MCP-1 were increased significantly in the feces from burn patients compared with controls: IL-8 increased from 3.32 ± 0.76 to 20.51 ± 6.65 pg/mg (P < .05) and MCP−1 from 2.78 ± 0.55 to 6.5 ± 0.99 pg/mg protein (P < .05). Figure 2. View largeDownload slide Fecal levels of chemokines interleukin (IL)-8 (A) and monocyte chemoattractant protein (MCP)-1 (B) after burn injury. Inflammatory chemokines IL-8 and MCP-1 also were increased in the burn group compared with the control group. Data are shown as mean ± SEM. *P < .05 compared with control group. Figure 2. View largeDownload slide Fecal levels of chemokines interleukin (IL)-8 (A) and monocyte chemoattractant protein (MCP)-1 (B) after burn injury. Inflammatory chemokines IL-8 and MCP-1 also were increased in the burn group compared with the control group. Data are shown as mean ± SEM. *P < .05 compared with control group. Figure 3 demonstrates the neutrophil markers MPO and elastase. Both were found to be significantly elevated in samples harvested from burn patients compared those from controls. The levels of MPO and elastase were elevated from 13.41 ± 1.40 to 24.52 ± 4.31 units/mg protein (P < .05) and 2.46 ± 0.38 to 5.08 ± 0.72 pg/ml (P < .05), respectively. Figure 3. View largeDownload slide Fecal levels of neutrophil markers myeloperoxidase (MPO) (A) and elastase (B) were increased in the burn sample compared with the control sample. Data are shown as mean ± SEM. *P < .05 compared with control group. Figure 3. View largeDownload slide Fecal levels of neutrophil markers myeloperoxidase (MPO) (A) and elastase (B) were increased in the burn sample compared with the control sample. Data are shown as mean ± SEM. *P < .05 compared with control group. DISCUSSION The role of the gastrointestinal tract in burn morbidity and mortality has long been recognized. Over the past several decades many advances have been made in burn care such that morbidity and mortality have fallen precipitously. Sepsis remains an overwhelming cause of death and morbidity in burn and other traumatic injuries, and the role of the gut in inflammation and immunomodulation after burn has been well described. Our results show an increase in IL-6, IL-8, IL-12, IL-13, MCP-1, elastase, and MPO in the fecal samples of burned patients compared with samples from those without physiologically significant burn injury. We now know that cutaneous burn injury causes gut mucosal atrophy, alters mucosal integrity, and leads to a breakdown in mucosal defense mechanisms.18,–24 This breakdown has been blamed for the translocation of indigenous gut bacteria and the occurrence of sepsis and multiple organ failure in burn patients.1,–8 Burn injury also is associated with mesenteric vasoconstriction, which results in gut mucosal damage and an increase in bacterial translocation.25 The gut origin hypothesis of MODS, or essentially what is termed bacterial translocation, initially was based on the concept that failure of the gut barrier and translocation of intestinally derived bacteria, endotoxins, or both to the bloodstream and systemic tissues triggered a septic state and promoted the development of MODS. However, conflicting data from human studies indicated that translocating bacteria themselves may not be responsible for the development of gut-induced MODS.5 A predominant explanation to resolve this notion is the possibility that gut-derived factors contributing to systemic inflammation and organ injury were reaching the systemic circulation via the mesenteric lymphatics rather than the portal venous system. This gut–lymph hypothesis is supported by previous experimental studies indicating that many gut-derived factors, including bacteria, exit the intestine via the intestinal lymphatics rather than the portal blood and that gut origin bacteremia rarely was observed in animal models unless they were highly and rapidly lethal.13 An important conceptual consequence of the gut–lymph hypothesis is that the lung rather than the liver would be the first major vascular bed to be exposed to mesenteric lymph because mesenteric lymph reaches the systemic circulation via the thoracic duct, which empties into the subclavian vein and hence the pulmonary circulation. This concept that gut-derived toxic and inflammatory factors are reaching the systemic circulation via the intestinal lymphatics, with the lung being the first organ exposed to these lymph factors, is consistent with extensive evidence documenting a strong link between gut ischemia/injury and the subsequent development of acute lung injury.13,26 Noninvasive assessment of intestinal inflammation was first investigated by those in the field of inflammatory bowel disease; these researchers saw tremendous clinical utility in a simple, reliable, reproducible, and noninvasive test with the ability to differentiate inflammatory bowel disease (IBD) from other gastrointestinal conditions, such as irritable bowel syndrome, and to monitor disease activity.15,16 Fecal markers are a noninvasive way of objectively measuring intestinal inflammation, with the potential to play a primary or adjunctive role in the assessment of disease activity; as such, they have been evaluated in IBD. To our knowledge, it has not been determined whether intestinal inflammation could be observed noninvasively in burned patients before this study. A simple, noninvasive way of determining the extent of intestinal inflammation in the burn-injured patient could represent an important adjunct to the existing complement of clinical observational methods available. Based on prior research, we know that intestinal inflammation after burn injury involves various cell types such as dendritic cells, epithelial cells, macrophages, T cells, and neutrophils. While the source of increased fecal cytokines in burned patients is not specifically identified, changes in one or more of the above cell types may contribute to the observed increase. Our data offer substantial and statistically significant evidence that the inflammatory cytokine profile of fecal effluent in burn patients differs from that of controls. In addition, the cytokine and chemokine profiles of the burn samples indicate the presence of increased intestinal inflammation. Specifically increased are neutrophil activity and the products of neutrophilic granules MPO and elastase. Neutrophilia and its implication on intestinal permeability is a well-recognized finding in the intestine of burned-injured animals.11,12,14,26 We are encouraged that these findings indicate comparable evidence of acute inflammation in human burn patients. One potential concern is the possibility of the fecal management system itself causing local inflammation in our sample, which may confound our results. However in clinical practice the fecal management system is routinely used, and the majority have no complications. Short of erosion, bleeding, and necrosis, any degree of inflammatory change specifically due to the indwelling rectal tube should be negligible and not influence the local inflammatory response. An additional concern could be that the patients in the burn sample received diets that were largely administered through tubes as well as stool softening regimens, as opposed to the general diets of the control population. Whether such differential dietary intake affects the fecal cytokine levels in burn patients compared with controls is beyond the scope of this study and remains to be established. We set out to determine whether a noninvasive method could be used to monitor intestinal inflammation in the setting of burn injury. Although we were able to observe changes in cytokine profiles, these can offer only a speculative idea of what is occurring without direct histologic or radiographic evidence of intestinal injury. As such, the potential for this method represents an adjunct marker that could be used to evaluate a clinical picture overall, rather than a specific diagnostic test. An additional goal was and is to apply our method in such a way that the severity of disturbance in cytokine profile could be correlated with severity of injury. Unfortunately, these data did not provide enough support to answer that question. We also hoped to characterize a hypothetical correlation between the time course of injury (in this case based on when each fecal sample was collected) and the level of observed inflammatory change. We were unable to find such a correlation. We feel that this failure unquestionably comes from the small sample size of our burn group. We are confident that as we increase the complement of sample patients, we will have sufficient numbers to address these questions properly. An additional issue that arises is the significance of the high rate of mortality seen in the burn population and whether the findings of probable intestinal inflammation are in some way related. It is notable that four of our seven burn patients with intestinal inflammation, as assumed by the presence of higher levels of inflammatory cytokines in the feces, developed infectious complications within 1 month after the samples were collected. This in turn raises the question of whether intestinal inflammation in the setting of burn injury predisposes patients to or is perhaps reflective of an impending immune perturbation. A larger retrospective study might be aimed at correlating (if there is correlation to be found) intestinal inflammation as measured by fecal cytokines with downstream complications. CONCLUSION Sepsis and multiorgan dysfunction are an overwhelming cause of morbidity and mortality in burn-injured patients. Intestinal inflammation is a well-recognized mediator of systemic inflammation and bacterial translocation in burn and traumatic injury. Noninvasive assessment of intestinal inflammation has been used successfully in the IBD population, but to date no one had investigated the utility of this method in the burn-injured patient. We hypothesized that noninvasive assessment of the cytokine profile of fecal effluent samples in burn-injured patients would show evidence of inflammation and serve as an important clinical adjunct to existing methods. Our data show that fecal samples of burned patients indeed show increased markers of inflammation. While more studies are needed to confirm this observation within a large burn patient population, the findings suggest that fecal levels of inflammatory markers may be considered for determining gut inflammation after burn injury. ACKNOWLEDGMENTS We thank the physicians and other nurses, especially Marcia Halerz, BSN, MBA, and Melissa Mounts, BSN, at Loyola University Burn Center and the Burn Shock Trauma Institute, for their ongoing contributions, without which this study could not have been performed. REFERENCES 1. Baron P, Traber LD, Traber DL, et al. Gut failure and translocation following burn and sepsis. J Surg Res. 1994;57:197–204. 2. Baue AE, Durham R, Faist E. Systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF): are we winning the battle? Shock. 1998;10:79–89. 3. Choudhry MA, Fazal N, Namak SY, Haque F, Ravindranath T, Sayeed MM. PGE2 suppresses intestinal T cell function in thermal injury: a cause of enhanced bacterial translocation. Shock. 2001;16:183–8. 4. Choudhry MA, Rana SN, Kavanaugh MJ, Kovacs EJ, Gamelli RL, Sayeed MM. 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The role of the gastrointestinal tract in postinjury multiple organ failure. Am J Surg. 1999;178:449–53. 22. Ravin HA, Fine J. Biological implications of intestinal endotoxins. Fed Proc. 1962;21:65–8. 23. Schweinburg FB, Seligman AM, Fine J. Transmural migration of intestinal bacteria; a study based on the use of radioactive Escherichia coli. N Engl J Med. 1950;242:747–51. 24. Sori AJ, Rush BF Jr, Lysz TW, Smith S, Machiedo GW. The gut as source of sepsis after hemorrhagic shock. Am J Surg. 1988;155:187–92. 25. Tokyay R, Zeigler ST, Traber DL, et al. Postburn gastrointestinal vasoconstriction increases bacterial and endotoxin translocation. J Appl Physiol. 1993;74:1521–7. 26. Sayeed MM. Exuberant Ca(2+) signaling in neutrophils: a cause for concern. News Physiol Sci. 2000;15:130–6. Footnotes 1 This study is supported by National Institute of Health grants R01AA015731 and F30AA020167 (JLR) and the Dr. Ralph and Marian C. Falk Medical Research Trust (MS and JLR). Copyright © 2013 by the American Burn Association
TI - Noninvasive Measurement of Intestinal Inflammation After Burn Injury
JF - Journal of Burn Care & Research
DO - 10.1097/BCR.0b013e318280e2f8
DA - 2013-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/noninvasive-measurement-of-intestinal-inflammation-after-burn-injury-2ODBT1D27J
SP - 633
EP - 638
VL - 34
IS - 6
DP - DeepDyve
ER -