Interleukin 38 Protects Against Lethal Sepsis

Interleukin 38 Protects Against Lethal Sepsis Abstract Background Interleukin 38 (IL-38) is the most recently characterized cytokine of the interleukin 1 family. However, its role in sepsis remains unknown. Methods Circulating IL-38 levels were measured in 2 cohorts of adult and pediatric patients with sepsis. Using 2 murine models of lipopolysaccharide (LPS)–induced endotoxemia and cecal ligation and puncture (CLP)–induced sepsis, the effects of IL-38 on survival, inflammation, tissue injury, and bacterial clearance were assessed. Results Serum IL-38 concentrations were significantly elevated in adult and pediatric patients with sepsis relative to corresponding healthy adult and pediatric controls, respectively. An increased IL-38 level negatively correlated with the number of blood leukocytes and with the level of inflammatory cytokines, including interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in clinical sepsis. Anti–IL-38 antibody impaired survival and while recombinant IL-38 improved survival in the 2 murine models of LPS-induced endotoxemia and CLP-induced sepsis. IL-38 administration decreased the inflammatory response, as reflected by lower levels of cytokines and chemokines (including IL-6, TNF-α, interleukin 10, interleukin 17, interleukin 27, CXCL1, and CCL2), and less damage to tissues (including lung, liver, and kidney) in CLP-induced sepsis. Furthermore, IL-38 augmented bacterial clearance in CLP-induced polymicrobial sepsis. Conclusions These findings suggest that IL-38 attenuates sepsis by decreasing inflammation and increasing bacterial clearance, thus providing a novel tool for antisepsis therapy. Interleukin 38, sepsis, inflammation, infection Despite the development of modern intensive care and new antimicrobial agents, the morbidity and mortality of patients with sepsis remain high [1]. Sepsis is newly defined as a life-threatening organ dysfunction that is caused by a dysregulated host response to infection [2]. In sepsis, the host immune response that is triggered by microbial infection fails to return to normal homeostasis, resulting in an aberrant inflammatory response and subsequent multiple organ failure [3]. Strategies to successfully treat sepsis should contain infection and aberrant inflammation [4]. Interleukin 38 (IL-38; also known as “interleukin 1 family member 10” [IL-1F10]) is the most recently characterized cytokine of the IL-1 family [5]. The IL-38 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsils, and it has also been found to be expressed in the spleen, heart, lung, placenta, fetal liver, and thymus [6, 7]. IL-38 shares 41% homology with IL-1 receptor antagonist (IL-1Ra) and 43% homology with interleukin 36 receptor antagonist (IL-36Ra), and it has a 3-dimensional structure similar to that of IL-1Ra [5, 6]. There is growing evidence suggesting that IL-38 is involved in the pathogenesis of various inflammatory diseases, including rheumatoid arthritis [8], psoriatic arthritis [9], systemic lupus erythematosus [10], asthma [11], and liver injury [12]. In a genome-wide association study of 66185 subjects with elevated C-reactive protein (CRP) levels, IL-38 was identified as one of only 18 markers associated with elevated CRP levels [13]. However, there are no data on the expression or function of IL-38 in sepsis. In the present study, we first determined the production of IL-38 in 2 well-characterized cohorts of patients with sepsis. In addition, we identified a novel function of IL-38 and its associated mechanism in 2 animal models, CLP-induced polymicrobial sepsis and lipopolysaccharide (LPS)–induced endotoxemia. MATERIALS AND METHODS Patients and Healthy Controls Forty adult patients who met the clinical criteria of the Third International Consensus Definitions for Sepsis and Septic Shock were screened for eligibility within the first 24 hours after they were admitted to the intensive care unit (ICU) or the department of infectious diseases of the First Affiliated Hospital of Chongqing Medical University. 26 pediatric patients were recruited from Children’s Hospital of Chongqing Medical University. Patients who had malignancy, who were pregnancy, who underwent organ transplantation, who had human immunodeficiency virus infection, and who were receiving immunosuppressive agents in the past 4 weeks were excluded from this study. Clinical data, including Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score, the counts of white blood cell count, CRP level, microbial culture results, the length of ICU stay and hospital stay, or mortality during the 28-day study period, were recorded. Control samples were obtained from healthy donors with no medical problems in the medical examination center of the First Affiliated Hospital of Chongqing Medical University or Children’s Hospital of Chongqing Medical University. This protocol was approved by the Clinical Research Ethics Committee of Chongqing Medical University, and informed consent was obtained from all participants according to the Declaration of Helsinki. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) and Mouse Leukocytes PBMCs were isolated from fresh blood of healthy volunteers by density gradient centrifugation (Histopaque, Sigma). Isolated human cells were resuspended and stimulated with LPS (Sigma-Aldrich) or heat-killed Escherichia coli in the presence or absence of recombinant human IL-38 (Adipogen International). Primary mouse peritoneal leukocytes were obtained by peritoneal lavage with ice-cold apyrogenic phosphate-buffered saline (PBS; Sigma). Isolated mouse cells were stimulated with heat-killed E. coli and recombinant murine IL-38 (Adipogen International) in the presence or absence of anti–IL-38 antibodies (R&D Systems). At the indicated time after culture, cell-free supernatants were stored at −80°C until used. Sepsis Models CLP was performed as previously described [14, 15]. Briefly, mice were anesthetized with pentothal sodium (50 mg/kg intraperitoneally), and the cecum was exposed, ligated, and punctured with a 21-gauge needle. The cecum was returned to the peritoneal cavity, and incisions were closed. Sham-operated (control) animals underwent identical laparotomy, and the cecum was exposed but not ligated or punctured and was then replaced in the peritoneal cavity. Mice received saline (5 mL per 100 g body weight) subcutaneously for resuscitation. For the LPS-induced endotoxemia model, mice were injected intraperitoneally with LPS from Salmonella abortus equi (Sigma-Aldrich) at a dose of 100 μg. All experiments involving animals adhered to guidelines and received the approval of the Institutional Review Committee for Animal Care and Use at Chongqing Medical University. Tissue Specimen Histological Analysis Mice were subjected to CLP or sham surgery. The mice were euthanized 24 hours after surgery, after which their lungs, livers, and kidneys were fixed, sectioned, and stained with hematoxylin and eosin for morphological analysis. Pathology Score Assessment Mouse lungs, livers, and kidneys were harvested 24 hours after CLP or sham surgery, fixed in 10% buffered formalin, and embedded in paraffin. Four-micrometer-thick sections were stained with hematoxylin and eosin and analyzed by a pathologist blinded to the study groups. Serum Biochemical Analysis Cardiac puncture was performed, and blood specimens were collected in tubes containing heparin. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine levels were quantified according to the protocols of the International Federation of Clinical Chemistry by spectrophotometry (modular DP; Roche-Hitachi). Determination of Bacterial Colony-Forming Units (CFU) Peritoneal fluid (PLF) was obtained by peritoneal lavage with 5 mL of sterile PBS (Sigma). Serial dilutions of peripheral blood specimens or peritoneal lavage fluid from mice were plated on blood-agar plates. CFU were counted after 24 hours of incubation at 37°C. Measurement of Cytokine and Chemokine Levels IL-38 levels were determined by a murine IL-38 enzyme-linked immunosorbent assay (ELISA) kit (RayBiotech) or a human IL-38 Quantikine ELISA kit (R&D Systems). Assessment of inflammatory cytokine and chemokine levels, including tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 17 (IL-17), interleukin 1β (IL-1β), interleukin 10 (IL-10), CXCL1, and CCL2 was performed using ELISA kits (Biolegend), and IL-27 levels were measured using ELISA kits (R&D Systems). All analyses were performed according to the manufacturers’ instructions. In Vivo Administration of Recombinant Murine IL-38 Female C57BL/6 mice aged 8–12 weeks were purchased from the Animal Center of Chongqing Medial University and allowed to acclimatize at a specific-pathogen-free facility for 1 week. Recombinant murine IL-38 protein (1 μg; Adipogen International) was injected 2 hours before CLP, for preventive treatment, and 1 dose of IL-38 was injected 2 hours after CLP or LPS challenge, for therapeutic treatment. PBS was delivered in a similar fashion as control vehicle. In Vivo Blockade of IL-38 To block IL-38 during experimental sepsis, 50 μg of rat anti-mouse IL-38 antibody (R&D systems) was administered intraperitoneally in 100 μL of PBS 2 hours after CLP or LPS challenge, followed by a booster dose of 50 µg 24 hours later after CLP or LPS challenge. As a control, rat immunoglobulin G2a (IgG2a) control antibody was used. Macrophage/Neutrophil Phagocytosis Assays For isolation of peritoneal macrophages, mice were injected with 5 mL of PBS. Macrophages were isolated from peritoneal lavage by plastic adherence. For isolation of granulocytes, mice were injected intraperitoneally with 1.5 mL of sterile thioglycollate (3%). Elicited cells were harvested 4 hours later by peritoneal lavage with 5 mL of cold PBS, and neutrophils were further purified from peritoneal lavage fluid by magnetic cell sorting (Miltenyi Biotec). Fluorescein isothiocyanate (FITC)–labeled E. coli was prepared by incubation with 0.5 mg/mL FITC (Sigma) for 20 minutes at 37°C. Peritoneal macrophages (1 × 105 cells) or neutrophils (1 × 106 cells) were incubated with FITC-labeled bacteria at a multiplicity of infection of 100 for 30 minutes at 37°C. After washing steps, cell nuclei were stained with DAPI (Invitrogen), followed by visualization using confocal laser scanning microscopy (LSM 510; Zeiss). The ratio of engulfed bacteria (as determined by overlay of green bacteria) was quantified by an independent researcher from 300 counted cells/well. In some experiments, peritoneal macrophages and neutrophils were pretreated with recombinant murine IL-38 (100 ng/mL; R&D Systems) before infection by FITC-labeled E. coli. Macrophage/Neutrophil Bacterial Killing Assays Peritoneal macrophages (1 × 105 cells) were infected with live E. coli (multiplicity of infection, 10) at 37°C for 1 hour. Infected macrophages were washed with buffer containing tobramycin (100 µg/mL) to remove extracellular bacteria and then lysed with lysis buffer (Promega). Live intracellular bacteria were quantified by culture of lysates for determination of bacterial uptake (at t = 0 hours) and intracellular killing (at t = 2 hours). Killing was calculated from the percentage of colonies present at t = 2 hours as compared to t = 0 hours, as follows: 100 − [number of CFU at t = 2 hours]/[number of CFU at t = 0 hours]. In another experiment, peritoneal neutrophils (1 × 106 cells) were infected with E. coli at a multiplicity of infection ratio of 1:100 at 37°C for 30 minutes. Infected neutrophils were resuspended in medium containing 100 μg/mL tobramycin to remove extracellular bacteria and then lysed in PBS containing 0.1% Triton 100 for assessment of uptake (t = 0 hours). Additional samples were incubated for 1 additional hour (t = 1 hour) to assess bacterial killing as described above. In some experiments, peritoneal macrophages and neutrophils were pretreated with recombinant murine IL-38 (100 ng/mL; R&D Systems) before infection with live E. coli. Statistical Analysis Human data were expressed as scatter dot plots with medians. Mice data were expressed as box plots, showing the smallest observation, the lower quartile, the median value, the upper quartile, and the largest observation, or as median values with interquartile ranges. Comparisons between groups were tested using the Mann-Whitney U test. For survival studies, Kaplan-Meier analyses followed by log-rank tests were performed. For correlation studies, the Spearman rank correlation test was used. All analyses were done using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA). P values of <.05 were considered statistically significant. RESULTS Circulating IL-38 Levels Were Elevated in Adult Patients With Sepsis First, IL-38 was quantified in 40 serum samples from adult patients with sepsis and 29 serum samples from healthy controls. The general characteristics of the study groups were summarized in Supplementary Table 1. Adult patients with sepsis displayed significantly elevated serum IL-38 concentrations, compared with healthy individuals (Figure 1A). The serum concentrations of IL-38 in septic nonsurvivors were lower than those in septic survivors, but it did not reach statistical significance. There was no apparent correlation between serum IL-38 levels and the severity of disease: serum IL-38 levels did not correlate with either the APACHE II score or the SOFA score (data not shown). However, serum concentration of IL-38 negatively correlated with the number of blood leukocytes and with levels of inflammatory cytokines, including IL-6 and TNF-α (Figure 1B). Figure 1. View largeDownload slide The circulating interleukin 38 (IL-38) level was elevated in patients with sepsis. A, IL-38 concentrations were measured by enzyme-linked immunosorbent assays (ELISAs) in serum samples collected from 40 adult patients with sepsis and from 29 healthy control subjects. Horizontal bars represent median values, and dots represent individual participants. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). B, Correlations between IL-38 levels and blood leukocyte counts, interleukin 6 (IL-6) levels, and tumor necrosis factor α (TNF-α) levels in adult patients with sepsis. Correlation was determined by the nonparametric Spearman correlation test. C, IL-38 concentrations were measured by ELISAs in serum samples collected from 26 pediatric patients with sepsis and from 20 healthy control subjects. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). D, Correlations between IL-38 levels and blood leukocyte counts, IL-6 levels, and TNF-α levels in pediatric patients with sepsis. Figure 1. View largeDownload slide The circulating interleukin 38 (IL-38) level was elevated in patients with sepsis. A, IL-38 concentrations were measured by enzyme-linked immunosorbent assays (ELISAs) in serum samples collected from 40 adult patients with sepsis and from 29 healthy control subjects. Horizontal bars represent median values, and dots represent individual participants. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). B, Correlations between IL-38 levels and blood leukocyte counts, interleukin 6 (IL-6) levels, and tumor necrosis factor α (TNF-α) levels in adult patients with sepsis. Correlation was determined by the nonparametric Spearman correlation test. C, IL-38 concentrations were measured by ELISAs in serum samples collected from 26 pediatric patients with sepsis and from 20 healthy control subjects. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). D, Correlations between IL-38 levels and blood leukocyte counts, IL-6 levels, and TNF-α levels in pediatric patients with sepsis. To validate our findings in adult patients with sepsis, we measured IL-38 concentrations in an independent cohort of pediatric patients with sepsis. The general characteristics of the study groups are summarized in Supplementary Table 2. IL-38 levels in pediatric patients with sepsis were also significantly elevated, compared with those in healthy control subjects (Figure 1C). Similarly, the serum concentration of IL-38 negatively correlated with the number of blood leukocytes and with levels of inflammatory cytokines, including IL-6 and TNF-α, in all pediatric patients with sepsis (Figure 1D). To determine whether LPS or heat-killed E. coli could induce IL-38 release in human cells in vitro, PBMCs were isolated and incubated with LPS or heat-killed E. coli. As with IL-6 and TNF-α levels, the IL-38 level was significantly elevated in PBMCs 24 hours after stimulation with LPS or heat-killed E. coli (Supplementary Figure 1). IL-38 Production Was Enhanced in Experimental Sepsis We next analyzed local, systemic, and organ IL-38 concentrations, using our well-established CLP-induced polymicrobial sepsis model [14, 15]. In experimental sepsis, IL-38 levels in the lung, spleen, PLF, and blood were significantly enhanced 24 or 48 hours after CLP (Figure 2). Figure 2. View largeDownload slide Local and systemic interleukin 38 (IL-38) production in mice after cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice (6/group) were subjected to sham surgery or CLP. Organs were removed at the indicated time points, blood specimens were collected by cardiac puncture, and peritoneal fluid was obtained by washing the peritoneal cavity with 5 mL of sterile phosphate-buffered saline. Samples were assayed for IL-38 content by enzyme-linked immunosorbent assays. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with sham control mice. Figure 2. View largeDownload slide Local and systemic interleukin 38 (IL-38) production in mice after cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice (6/group) were subjected to sham surgery or CLP. Organs were removed at the indicated time points, blood specimens were collected by cardiac puncture, and peritoneal fluid was obtained by washing the peritoneal cavity with 5 mL of sterile phosphate-buffered saline. Samples were assayed for IL-38 content by enzyme-linked immunosorbent assays. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with sham control mice. IL-38 Blockade Aggravated CLP-Induced Sepsis and LPS-Induced Endotoxemia Having observed that the IL-38 level was elevated in clinical and experimental sepsis, we first used an IL-38 blocking mouse monoclonal antibody (mouse IL-38/IL-1F10 antibody; clone 798036) to define the role of IL-38 in sepsis. In vitro studies showed that this antibody significantly inhibited the IL-38–induced decrease of IL-6 and TNF-α levels upon stimulation with LPS (Supplementary Figure 2). Subsequently, we induced sepsis in C57BL/6 mice by CLP, and anti–IL-38 antibody treatment was administered after surgery. The survival rate in septic mice treated with anti–IL-38 blocking antibodies was significantly lower than that in the IgG-treated control group (Figure 3A). The protein concentrations in both bronchoalveolar lavage fluid (BALF) and PLF, reflecting vascular permeability, were significantly increased in septic mice treated with anti–IL-38 antibodies (Figure 3B). Serum concentrations of ALT and AST, markers for hepatocellular injury, LDH, a marker for general cellular injury, and creatinine, a marker for renal failure, were significantly increased in mice treated with anti–IL-38 antibodies (Figure 3C). Lung, liver, and kidney damage was also confirmed by histological detection of alveolar hemorrhage, centrilobular necrosis, and tubular epithelial necrosis, respectively (Supplementary Figure 3A). In addition, blockade of IL-38 significantly increased the pathology scores for lungs, livers, and kidneys after sepsis (Supplementary Figure 3B). Furthermore, the exacerbated mortality of septic mice treated with anti–IL-38 was associated with higher levels of bacteria (primarily E. coli and Enterococcus), as assessed by comparing the number of CFU in the peritoneum and blood (Figure 3D). Figure 3. View largeDownload slide Interleukin 38 (IL-38) blockade aggravated cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice were subjected to CLP. Two hours after CLP, mice were administered 50 µg of anti–IL-38 blocking antibodies, followed by a booster dose of 50 µg 24 hours after CLP. Rat immunoglobulin G2a (IgG2a) served as control antibody. A, Survival of septic mice (12/group) following IL-38 blockade after CLP with anti–IL-38 antibodies. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with isotypical IgG control. B, Vascular permeability was validated 24 hours after CLP by determining the protein concentration in bronchoalveolar fluid (BALF) and peritoneal fluid (PLF) from 5 mice with CLP-induced sepsis treated with or without anti–IL-38 blocking antibodies. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without anti–IL-38 blocking antibodies. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units was determined. *P < .05, by the Mann-Whitney U test, compared with mice with CLP-induced sepsis treated with isotypical IgG control. Figure 3. View largeDownload slide Interleukin 38 (IL-38) blockade aggravated cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice were subjected to CLP. Two hours after CLP, mice were administered 50 µg of anti–IL-38 blocking antibodies, followed by a booster dose of 50 µg 24 hours after CLP. Rat immunoglobulin G2a (IgG2a) served as control antibody. A, Survival of septic mice (12/group) following IL-38 blockade after CLP with anti–IL-38 antibodies. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with isotypical IgG control. B, Vascular permeability was validated 24 hours after CLP by determining the protein concentration in bronchoalveolar fluid (BALF) and peritoneal fluid (PLF) from 5 mice with CLP-induced sepsis treated with or without anti–IL-38 blocking antibodies. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without anti–IL-38 blocking antibodies. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units was determined. *P < .05, by the Mann-Whitney U test, compared with mice with CLP-induced sepsis treated with isotypical IgG control. The effect of IL-38 blockade in the LPS-induced endotoxemia mouse model was also evaluated. IL-38 blocking increased the incidence of fatal endotoxemia in mice challenged with LPS (Supplementary Figure 4A), and it significantly enhanced vascular permeability, as reflected by significantly increased protein concentrations in both BALF and PLF from LPS-challenged mice treated with IL-38 antibodies (Supplementary Figure 4B). IL-38 Blockade Increased Cytokine and Chemokine Levels During CLP-Induced Sepsis The effect of IL-38 blockade on CLP-induced levels of cytokines and chemokines in PLF, BALF, and serum was evaluated 24 hours after CLP. Treatment with anti–IL-38 antibodies upregulated the cytokine and chemokine response in the CLP model and resulted in a significant increase in levels of IL-6, TNF-α, IL-10, IL-17, IL-27, CXCL1, and CCL2 but not IL-1β after CLP (Figure 4). Figure 4. View largeDownload slide Interleukin 38 (IL-38) blockade upregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PFL), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without anti–IL-38 blocking antibodies were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical immunoglobulin G control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. Figure 4. View largeDownload slide Interleukin 38 (IL-38) blockade upregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PFL), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without anti–IL-38 blocking antibodies were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical immunoglobulin G control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. IL-38 Treatment Protected Against CLP-Induced Sepsis and LPS-Induced Endotoxemia Because the administration of antibody against IL-38 was found to aggravate sepsis in mice, we performed the reverse experiment and examined the effect of treatment with recombinant mouse IL-38 on experimental sepsis. Prophylactic administration of IL-38 at 2 hours before the onset of sepsis significantly improved survival, compared with survival among vehicle-treated control animals, and IL-38 administration 2 hours after CLP also significantly improved survival, compared with vehicle-treated control animals (Figure 5A), indicating both preventive and therapeutic effects of IL-38 on sepsis. Mice treated with IL-38 at 2 hours after CLP had significantly lower protein concentrations in both BALF and PLF (Figure 5B). Mice with therapeutic IL-38 administration displayed lower serum concentrations of ALT, AST, LDH, and creatinine (Figure 5C). Lung, liver, and kidney damage was also confirmed by histological detection of alveolar hemorrhage, centrilobular necrosis, and tubular epithelial necrosis, respectively (Supplementary Figure 5A), and therapeutic IL-38 treatment significantly decreased the pathology scores of lung, liver, and kidney after CLP (Supplementary Figure 5B). Furthermore, bacterial titers were significantly lower in PLF and blood specimens from mice that received IL-38 therapeutic treatment (Figure 5D). Figure 5. View largeDownload slide Interleukin 38 (IL-38) administration attenuated cecal ligation and puncture (CLP)–induced sepsis. A, Survival of septic mice (12/group) following IL-38 supplementation. Recombinant murine IL-38 (1 μg/injection) was given 2 hours before CLP, for preventive treatment; 1 dose of IL-38 (1 μg/injection) was given at the time of CLP; and 1 dose of IL-38 (1 μg/injection) was given 2 hours after CLP challenge, for therapeutic treatment. Phosphate-buffered saline (PBS) was delivered in a similar fashion as control vehicle. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with recombinant IL-38. B, Vascular permeability 24 hours after CLP was validated by determining the protein concentrations in bronchoalveolar fluid and peritoneal fluid (PLF) from 5 mice that underwent CLP and were treated with or without recombinant IL-38 therapeutically. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) therapeutically. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units (CFU) was counted. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38 therapeutically. Figure 5. View largeDownload slide Interleukin 38 (IL-38) administration attenuated cecal ligation and puncture (CLP)–induced sepsis. A, Survival of septic mice (12/group) following IL-38 supplementation. Recombinant murine IL-38 (1 μg/injection) was given 2 hours before CLP, for preventive treatment; 1 dose of IL-38 (1 μg/injection) was given at the time of CLP; and 1 dose of IL-38 (1 μg/injection) was given 2 hours after CLP challenge, for therapeutic treatment. Phosphate-buffered saline (PBS) was delivered in a similar fashion as control vehicle. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with recombinant IL-38. B, Vascular permeability 24 hours after CLP was validated by determining the protein concentrations in bronchoalveolar fluid and peritoneal fluid (PLF) from 5 mice that underwent CLP and were treated with or without recombinant IL-38 therapeutically. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) therapeutically. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units (CFU) was counted. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38 therapeutically. In a LPS-induced endotoxemia mouse model, therapeutic administration of IL-38 at 2 hours after LPS challenge also significantly enhanced survival in mice (Supplementary Figure 6A), which was associated with significantly lower vascular permeability (Supplementary Figure 6B). IL-38 Administration Ablated the Production of Cytokines and Chemokines During CLP-Induced Sepsis As shown in Figure 6, the concentrations of inflammatory cytokines and chemokines, including IL-6, TNF-α, IL-10, IL-17, IL-27, CXCL1, and CCL2 but not IL-1β, in PLF, BALF, and blood specimens obtained 24 hours after CLP were significantly downregulated in septic mice that received therapeutic IL-38 treatment. Figure 6. View largeDownload slide Interleukin 38 (IL-38) administration downregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PLF), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with phosphate-buffered saline control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. Figure 6. View largeDownload slide Interleukin 38 (IL-38) administration downregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PLF), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with phosphate-buffered saline control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. IL-38 Did Not Influence Bacterial Phagocytosis and Killing by Phagocytes To investigate whether IL-38 influenced intrinsic antibacterial functions of phagocytes, we studied the bacterial uptake and killing capacities of peritoneal macrophages and neutrophils. Preincubation with recombinant IL-38 did not have direct effects on phagocytosis and intracellular killing of E. coli by macrophages (Supplementary Figure 7A) or neutrophils (Supplementary Figure 7B). DISCUSSION Since 2015, there has been increasing interest in studying IL-38, and IL-38 has emerged as an important regulatory mediator in some inflammatory diseases [5, 6]. In the present report, we identified IL-38 to be potentially involved in the pathogenesis of clinical and experimental sepsis. We made the following key observations: (1) both adult and pediatric patients with sepsis had increased circulating IL-38 levels, which negatively correlated with the number of blood leukocytes and levels of inflammatory cytokines, including IL-6 and TNF-α; (2) IL-38 blockade aggravated experimental sepsis by increasing inflammation and decreasing bacterial clearance; and (3) IL-38 administration protected mice from experimental sepsis by suppressing inflammation and decreasing bacterial loading. IL-38 production has been found to be elevated in childhood asthma, and it was negatively correlated with IL-10 secretion [11]. Elevated IL-38 levels in the blood have also been shown to be associated with the severity of chronic hepatitis B [16]. In addition, IL-38 expression was enhanced in the synovium of patients with rheumatoid arthritis and in the colon of patients with Crohn disease [17]. It was also found at significantly higher concentrations in patients with systemic lupus erythematosus, which correlated with the severity of the disease [10]. To the best of our knowledge, this is the first study demonstrating that circulating IL-38 levels were elevated in 2 cohorts of adult and pediatric patients with sepsis. Our present work on the serum levels of IL-38 is related to previous studies on serum IL-38 concentrations [11, 16], which revealed detectable levels of IL-38 in healthy subjects that could be upregulated during some inflammatory conditions. IL-38 concentrations did not correlate with the severity of sepsis, as reflected by APACHE II and SOFA scores, or with mortality. Although the levels of blood leukocytes, IL-6, and TNF-α were elevated in clinical sepsis, elevated IL-38 levels negatively correlated with the blood leukocyte count, IL-6 level, and TNF-α level in patients with sepsis, suggesting the antiinflammatory potential of IL-38 in clinical sepsis. IL-38 has been shown to exert antiinflammatory properties in several animal models of pathology. Articular injection of an adeno-associated virus (AAV) 2/8 encoding IL-38 has been shown to significantly decrease clinical inflammatory scores in joints of mice with collagen-induced arthritis and K/BxN serum transfer-induced arthritis, and this induced IL-38 overexpression could significantly reduce the expression of T-helper type 17 (Th17) cytokines (ie, IL-17A, interleukin 23p19 [IL-23p19], and interleukin 22 [IL-22]), CXCL1, and receptor activator of nuclear factor kappa-B ligand [18]. Intravenous administration of murine recombinant IL-38 into MRL/lpr mice ameliorated lupus-like clinical symptoms, and serum concentrations of IL-6, IL-17, IL-22, and CXCL10 were also decreased by treatment with recombinant IL-38 [19]. Furthermore, expression of exogenous human IL-38 significantly reduced hepatic toxicity, and serum levels of ALT, AST, and proinflammatory cytokines, including TNF-α, interferon γ (IFN-γ), IL-6, IL-17, and IL-22, in a model of concanavalin A–induced liver injury [12]. In this study of CLP-induced sepsis, we further found that the survival benefit obtained with recombinant IL-38 was associated with a reduction of the local and systemic inflammatory response, as shown by a significant decrease of the concentrations of IL-6, TNF-α, IL-10, IL-17, IL-27, CXCL1, and CCL2 in PLF, BALF, and blood specimens, but IL-1β secretion was not affected. Findings of this in vivo study are therefore consistent with previous in vitro work showing that IL-38 inhibited the production of cytokines such as IL-6 and TNF-α by THP-1 monocytic cells but did not alter IL-1β expression [18]. Furthermore, blockade of IL-38 activity was associated with enhanced local and systemic inflammatory response in CLP-induced sepsis. In fact, organ injury observed in sepsis is due to widespread inflammation, which is caused by the explosive release of cytokines and chemokines [20]. Therefore, IL-38–induced protection against experimental sepsis was associated with decreased inflammation. Our data also demonstrated that one mechanism of improved survival in sepsis with IL-38 treatment included increased bacterial clearance. We found that IL-38 might play an important role in controlling both bacterial outgrowth and inflammatory responses. Increased bacterial loads, which are critical inflammatory stimulus, may be able to further enhance inflammation and tissue damage, resulting in increased mortality of septic mice [21, 22]. Therefore, the improved capacities of IL-38 to clear bacterial infection and to downregulate host inflammatory reactions are both responsible for the decreased mortality observed in septic mice treated with recombinant IL-38. Our study includes some limitations. First, the number of patients with sepsis in this study was relatively small. The clinical value of the IL-38 level as a sepsis biomarker remains to be established in a larger-sized clinical trial in regarding to clinical state, infectious source, pathogen type, and other possible factors. Second, although in vivo findings showed that IL-38 mediated bacterial clearance during sepsis, in vitro studies demonstrated that IL-38 did not directly influence bacterial phagocytosis and killing by macrophages and neutrophils. Therefore, the mechanisms by which IL-38 regulated the clearance of bacteria require further studies. Currently, there are no specific therapeutic agents available to clinicians [22]. The present study is the first to document that IL-38 release occurs in clinical and experimental sepsis and that IL-38 could improve survival in experimental sepsis by decreasing inflammation and increasing bacterial clearance. As a novel cytokine-targeted sepsis treatment, IL-38 could be combined with other immunoadjuvants that function via other different mechanisms, such as progranulin [14], interleukin 5 [23], interleukin 7 [24], interleukin 30 [25], and interleukin 33 [26], to further enhance antisepsis therapy. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This work was supported by the National Natural Science Foundation of China (grants 81722001 and 81572038 to J. C.) and the Chongqing Science and Technology Commission (Distinguished Young Scholars of Chongqing grant cstc2014jcyjjq10002 to J. C.). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Rhodes A, Evans LE, Alhazzani W, et al.   Surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016. Crit Care Med   2017; 45: 486– 552. Google Scholar CrossRef Search ADS PubMed  2. Seymour CW, Liu VX, Iwashyna TJ, et al.   Assessment of clinical criteria for sepsis: for the third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA   2016; 315: 762– 74. Google Scholar CrossRef Search ADS PubMed  3. van der Poll T, van de Veerdonk FL, Scicluna BP, Netea MG. The immunopathology of sepsis and potential therapeutic targets. Nat Rev Immunol   2017; 17: 407– 20. Google Scholar CrossRef Search ADS PubMed  4. Cohen J, Vincent JL, Adhikari NK, et al.   Sepsis: a roadmap for future research. Lancet Infect Dis   2015; 15: 581– 614. Google Scholar CrossRef Search ADS PubMed  5. Garlanda C, Dinarello CA, Mantovani A. The interleukin-1 family: back to the future. Immunity   2013; 39: 1003– 18. Google Scholar CrossRef Search ADS PubMed  6. Garraud T, Harel M, Boutet MA, Le Goff B, Blanchard F. The enigmatic role of IL-38 in inflammatory diseases. Cytokine Growth Factor Rev   2018; 39: 26– 35. Google Scholar CrossRef Search ADS PubMed  7. Tominaga M, Okamoto M, Kawayama T, et al.   Overexpression of IL-38 protein in anticancer drug-induced lung injury and acute exacerbation of idiopathic pulmonary fibrosis. Respir Investig   2017; 55: 293– 9. Google Scholar CrossRef Search ADS PubMed  8. Takenaka SI, Kaieda S, Kawayama T, et al.   IL-38: a new factor in rheumatoid arthritis. Biochem Biophys Rep   2015; 4: 386– 91. Google Scholar PubMed  9. Li J, Liu L, Rui W, et al.   New interleukins in psoriasis and psoriatic arthritis patients: the possible roles of Interleukin-33 to Interleukin-38 in disease activities and bone erosions. Dermatology   2017; 233: 37– 46. Google Scholar CrossRef Search ADS PubMed  10. Rudloff I, Godsell J, Nold-Petry CA, et al.   Brief report: interleukin-38 exerts antiinflammatory functions and is associated with disease activity in systemic lupus erythematosus. Arthritis Rheumatol   2015; 67: 3219– 25. Google Scholar CrossRef Search ADS PubMed  11. Chu M, Chu IM, Yung EC, et al.   Aberrant expression of novel cytokine IL-38 and regulatory T lymphocytes in childhood asthma. Molecules   2016; 21:doi: https://doi.org/10.3390/molecules21070933. 12. Yuan X, Li Y, Pan X, et al.   IL-38 alleviates concanavalin A-induced liver injury in mice. Int Immunopharmacol   2016; 40: 452– 7. Google Scholar CrossRef Search ADS PubMed  13. Dehghan A, Dupuis J, Barbalic M, et al.   Meta-analysis of genome-wide association studies in >80000 subjects identifies multiple loci for C-reactive protein levels. Circulation   2011; 123: 731– 8. Google Scholar CrossRef Search ADS PubMed  14. Song Z, Zhang X, Zhang L, et al.   Progranulin plays a central role in host defense during sepsis by promoting macrophage recruitment. Am J Respir Crit Care Med   2016; 194: 1219– 32. Google Scholar CrossRef Search ADS PubMed  15. Tao X, Song Z, Wang C, et al.   Interleukin 36α attenuates sepsis by enhancing antibacterial functions of macrophages. J Infect Dis   2017; 215: 321– 32. Google Scholar PubMed  16. Wang HJ, Jiang YF, Wang XR, Zhang ML, Gao PJ. Elevated serum interleukin-38 level at baseline predicts virological response in telbivudine-treated patients with chronic hepatitis B. World J Gastroenterol   2016; 22: 4529– 37. Google Scholar CrossRef Search ADS PubMed  17. Boutet MA, Bart G, Penhoat M, et al.   Distinct expression of interleukin (IL)-36α, β and γ, their antagonist IL-36Ra and IL-38 in psoriasis, rheumatoid arthritis and Crohn’s disease. Clin Exp Immunol   2016; 184: 159– 73. Google Scholar CrossRef Search ADS PubMed  18. Boutet MA, Najm A, Bart G, et al.   IL-38 overexpression induces anti-inflammatory effects in mice arthritis models and in human macrophages in vitro. Ann Rheum Dis   2017; 76: 1304– 12. Google Scholar CrossRef Search ADS PubMed  19. Chu M, Tam LS, Zhu J, et al.   In vivo anti-inflammatory activities of novel cytokine IL-38 in murphy roths large (MRL)/lpr mice. Immunobiology   2017; 222: 483– 93. Google Scholar CrossRef Search ADS PubMed  20. Angus DC, Seymour CW, Coopersmith CM, et al.   A framework for the development and interpretation of different sepsis definitions and clinical criteria. Crit Care Med   2016; 44: e113– 21. Google Scholar CrossRef Search ADS PubMed  21. Ward PA. New approaches to the study of sepsis. EMBO Mol Med   2012; 4: 1234– 43. Google Scholar CrossRef Search ADS PubMed  22. Fink MP, Warren HS. Strategies to improve drug development for sepsis. Nat Rev Drug Discov   2014; 13: 741– 58. Google Scholar CrossRef Search ADS PubMed  23. Linch SN, Danielson ET, Kelly AM, Tamakawa RA, Lee JJ, Gold JA. Interleukin 5 is protective during sepsis in an eosinophil-independent manner. Am J Respir Crit Care Med   2012; 186: 246– 54. Google Scholar CrossRef Search ADS PubMed  24. Venet F, Demaret J, Blaise BJ, et al.   IL-7 restores T lymphocyte immunometabolic failure in septic shock patients through mTOR activation. J Immunol   2017; 199: 1606– 15. Google Scholar CrossRef Search ADS PubMed  25. Yan J, Mitra A, Hu J, et al.   Interleukin-30 (IL27p28) alleviates experimental sepsis by modulating cytokine profile in NKT cells. J Hepatol   2016; 64: 1128– 36. Google Scholar CrossRef Search ADS PubMed  26. Alves-Filho JC, Sônego F, Souto FO, et al.   Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection. Nat Med   2010; 16: 708– 12. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, 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/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Interleukin 38 Protects Against Lethal Sepsis

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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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1537-6613
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10.1093/infdis/jiy289
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Abstract

Abstract Background Interleukin 38 (IL-38) is the most recently characterized cytokine of the interleukin 1 family. However, its role in sepsis remains unknown. Methods Circulating IL-38 levels were measured in 2 cohorts of adult and pediatric patients with sepsis. Using 2 murine models of lipopolysaccharide (LPS)–induced endotoxemia and cecal ligation and puncture (CLP)–induced sepsis, the effects of IL-38 on survival, inflammation, tissue injury, and bacterial clearance were assessed. Results Serum IL-38 concentrations were significantly elevated in adult and pediatric patients with sepsis relative to corresponding healthy adult and pediatric controls, respectively. An increased IL-38 level negatively correlated with the number of blood leukocytes and with the level of inflammatory cytokines, including interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in clinical sepsis. Anti–IL-38 antibody impaired survival and while recombinant IL-38 improved survival in the 2 murine models of LPS-induced endotoxemia and CLP-induced sepsis. IL-38 administration decreased the inflammatory response, as reflected by lower levels of cytokines and chemokines (including IL-6, TNF-α, interleukin 10, interleukin 17, interleukin 27, CXCL1, and CCL2), and less damage to tissues (including lung, liver, and kidney) in CLP-induced sepsis. Furthermore, IL-38 augmented bacterial clearance in CLP-induced polymicrobial sepsis. Conclusions These findings suggest that IL-38 attenuates sepsis by decreasing inflammation and increasing bacterial clearance, thus providing a novel tool for antisepsis therapy. Interleukin 38, sepsis, inflammation, infection Despite the development of modern intensive care and new antimicrobial agents, the morbidity and mortality of patients with sepsis remain high [1]. Sepsis is newly defined as a life-threatening organ dysfunction that is caused by a dysregulated host response to infection [2]. In sepsis, the host immune response that is triggered by microbial infection fails to return to normal homeostasis, resulting in an aberrant inflammatory response and subsequent multiple organ failure [3]. Strategies to successfully treat sepsis should contain infection and aberrant inflammation [4]. Interleukin 38 (IL-38; also known as “interleukin 1 family member 10” [IL-1F10]) is the most recently characterized cytokine of the IL-1 family [5]. The IL-38 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsils, and it has also been found to be expressed in the spleen, heart, lung, placenta, fetal liver, and thymus [6, 7]. IL-38 shares 41% homology with IL-1 receptor antagonist (IL-1Ra) and 43% homology with interleukin 36 receptor antagonist (IL-36Ra), and it has a 3-dimensional structure similar to that of IL-1Ra [5, 6]. There is growing evidence suggesting that IL-38 is involved in the pathogenesis of various inflammatory diseases, including rheumatoid arthritis [8], psoriatic arthritis [9], systemic lupus erythematosus [10], asthma [11], and liver injury [12]. In a genome-wide association study of 66185 subjects with elevated C-reactive protein (CRP) levels, IL-38 was identified as one of only 18 markers associated with elevated CRP levels [13]. However, there are no data on the expression or function of IL-38 in sepsis. In the present study, we first determined the production of IL-38 in 2 well-characterized cohorts of patients with sepsis. In addition, we identified a novel function of IL-38 and its associated mechanism in 2 animal models, CLP-induced polymicrobial sepsis and lipopolysaccharide (LPS)–induced endotoxemia. MATERIALS AND METHODS Patients and Healthy Controls Forty adult patients who met the clinical criteria of the Third International Consensus Definitions for Sepsis and Septic Shock were screened for eligibility within the first 24 hours after they were admitted to the intensive care unit (ICU) or the department of infectious diseases of the First Affiliated Hospital of Chongqing Medical University. 26 pediatric patients were recruited from Children’s Hospital of Chongqing Medical University. Patients who had malignancy, who were pregnancy, who underwent organ transplantation, who had human immunodeficiency virus infection, and who were receiving immunosuppressive agents in the past 4 weeks were excluded from this study. Clinical data, including Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score, the counts of white blood cell count, CRP level, microbial culture results, the length of ICU stay and hospital stay, or mortality during the 28-day study period, were recorded. Control samples were obtained from healthy donors with no medical problems in the medical examination center of the First Affiliated Hospital of Chongqing Medical University or Children’s Hospital of Chongqing Medical University. This protocol was approved by the Clinical Research Ethics Committee of Chongqing Medical University, and informed consent was obtained from all participants according to the Declaration of Helsinki. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) and Mouse Leukocytes PBMCs were isolated from fresh blood of healthy volunteers by density gradient centrifugation (Histopaque, Sigma). Isolated human cells were resuspended and stimulated with LPS (Sigma-Aldrich) or heat-killed Escherichia coli in the presence or absence of recombinant human IL-38 (Adipogen International). Primary mouse peritoneal leukocytes were obtained by peritoneal lavage with ice-cold apyrogenic phosphate-buffered saline (PBS; Sigma). Isolated mouse cells were stimulated with heat-killed E. coli and recombinant murine IL-38 (Adipogen International) in the presence or absence of anti–IL-38 antibodies (R&D Systems). At the indicated time after culture, cell-free supernatants were stored at −80°C until used. Sepsis Models CLP was performed as previously described [14, 15]. Briefly, mice were anesthetized with pentothal sodium (50 mg/kg intraperitoneally), and the cecum was exposed, ligated, and punctured with a 21-gauge needle. The cecum was returned to the peritoneal cavity, and incisions were closed. Sham-operated (control) animals underwent identical laparotomy, and the cecum was exposed but not ligated or punctured and was then replaced in the peritoneal cavity. Mice received saline (5 mL per 100 g body weight) subcutaneously for resuscitation. For the LPS-induced endotoxemia model, mice were injected intraperitoneally with LPS from Salmonella abortus equi (Sigma-Aldrich) at a dose of 100 μg. All experiments involving animals adhered to guidelines and received the approval of the Institutional Review Committee for Animal Care and Use at Chongqing Medical University. Tissue Specimen Histological Analysis Mice were subjected to CLP or sham surgery. The mice were euthanized 24 hours after surgery, after which their lungs, livers, and kidneys were fixed, sectioned, and stained with hematoxylin and eosin for morphological analysis. Pathology Score Assessment Mouse lungs, livers, and kidneys were harvested 24 hours after CLP or sham surgery, fixed in 10% buffered formalin, and embedded in paraffin. Four-micrometer-thick sections were stained with hematoxylin and eosin and analyzed by a pathologist blinded to the study groups. Serum Biochemical Analysis Cardiac puncture was performed, and blood specimens were collected in tubes containing heparin. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine levels were quantified according to the protocols of the International Federation of Clinical Chemistry by spectrophotometry (modular DP; Roche-Hitachi). Determination of Bacterial Colony-Forming Units (CFU) Peritoneal fluid (PLF) was obtained by peritoneal lavage with 5 mL of sterile PBS (Sigma). Serial dilutions of peripheral blood specimens or peritoneal lavage fluid from mice were plated on blood-agar plates. CFU were counted after 24 hours of incubation at 37°C. Measurement of Cytokine and Chemokine Levels IL-38 levels were determined by a murine IL-38 enzyme-linked immunosorbent assay (ELISA) kit (RayBiotech) or a human IL-38 Quantikine ELISA kit (R&D Systems). Assessment of inflammatory cytokine and chemokine levels, including tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 17 (IL-17), interleukin 1β (IL-1β), interleukin 10 (IL-10), CXCL1, and CCL2 was performed using ELISA kits (Biolegend), and IL-27 levels were measured using ELISA kits (R&D Systems). All analyses were performed according to the manufacturers’ instructions. In Vivo Administration of Recombinant Murine IL-38 Female C57BL/6 mice aged 8–12 weeks were purchased from the Animal Center of Chongqing Medial University and allowed to acclimatize at a specific-pathogen-free facility for 1 week. Recombinant murine IL-38 protein (1 μg; Adipogen International) was injected 2 hours before CLP, for preventive treatment, and 1 dose of IL-38 was injected 2 hours after CLP or LPS challenge, for therapeutic treatment. PBS was delivered in a similar fashion as control vehicle. In Vivo Blockade of IL-38 To block IL-38 during experimental sepsis, 50 μg of rat anti-mouse IL-38 antibody (R&D systems) was administered intraperitoneally in 100 μL of PBS 2 hours after CLP or LPS challenge, followed by a booster dose of 50 µg 24 hours later after CLP or LPS challenge. As a control, rat immunoglobulin G2a (IgG2a) control antibody was used. Macrophage/Neutrophil Phagocytosis Assays For isolation of peritoneal macrophages, mice were injected with 5 mL of PBS. Macrophages were isolated from peritoneal lavage by plastic adherence. For isolation of granulocytes, mice were injected intraperitoneally with 1.5 mL of sterile thioglycollate (3%). Elicited cells were harvested 4 hours later by peritoneal lavage with 5 mL of cold PBS, and neutrophils were further purified from peritoneal lavage fluid by magnetic cell sorting (Miltenyi Biotec). Fluorescein isothiocyanate (FITC)–labeled E. coli was prepared by incubation with 0.5 mg/mL FITC (Sigma) for 20 minutes at 37°C. Peritoneal macrophages (1 × 105 cells) or neutrophils (1 × 106 cells) were incubated with FITC-labeled bacteria at a multiplicity of infection of 100 for 30 minutes at 37°C. After washing steps, cell nuclei were stained with DAPI (Invitrogen), followed by visualization using confocal laser scanning microscopy (LSM 510; Zeiss). The ratio of engulfed bacteria (as determined by overlay of green bacteria) was quantified by an independent researcher from 300 counted cells/well. In some experiments, peritoneal macrophages and neutrophils were pretreated with recombinant murine IL-38 (100 ng/mL; R&D Systems) before infection by FITC-labeled E. coli. Macrophage/Neutrophil Bacterial Killing Assays Peritoneal macrophages (1 × 105 cells) were infected with live E. coli (multiplicity of infection, 10) at 37°C for 1 hour. Infected macrophages were washed with buffer containing tobramycin (100 µg/mL) to remove extracellular bacteria and then lysed with lysis buffer (Promega). Live intracellular bacteria were quantified by culture of lysates for determination of bacterial uptake (at t = 0 hours) and intracellular killing (at t = 2 hours). Killing was calculated from the percentage of colonies present at t = 2 hours as compared to t = 0 hours, as follows: 100 − [number of CFU at t = 2 hours]/[number of CFU at t = 0 hours]. In another experiment, peritoneal neutrophils (1 × 106 cells) were infected with E. coli at a multiplicity of infection ratio of 1:100 at 37°C for 30 minutes. Infected neutrophils were resuspended in medium containing 100 μg/mL tobramycin to remove extracellular bacteria and then lysed in PBS containing 0.1% Triton 100 for assessment of uptake (t = 0 hours). Additional samples were incubated for 1 additional hour (t = 1 hour) to assess bacterial killing as described above. In some experiments, peritoneal macrophages and neutrophils were pretreated with recombinant murine IL-38 (100 ng/mL; R&D Systems) before infection with live E. coli. Statistical Analysis Human data were expressed as scatter dot plots with medians. Mice data were expressed as box plots, showing the smallest observation, the lower quartile, the median value, the upper quartile, and the largest observation, or as median values with interquartile ranges. Comparisons between groups were tested using the Mann-Whitney U test. For survival studies, Kaplan-Meier analyses followed by log-rank tests were performed. For correlation studies, the Spearman rank correlation test was used. All analyses were done using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA). P values of <.05 were considered statistically significant. RESULTS Circulating IL-38 Levels Were Elevated in Adult Patients With Sepsis First, IL-38 was quantified in 40 serum samples from adult patients with sepsis and 29 serum samples from healthy controls. The general characteristics of the study groups were summarized in Supplementary Table 1. Adult patients with sepsis displayed significantly elevated serum IL-38 concentrations, compared with healthy individuals (Figure 1A). The serum concentrations of IL-38 in septic nonsurvivors were lower than those in septic survivors, but it did not reach statistical significance. There was no apparent correlation between serum IL-38 levels and the severity of disease: serum IL-38 levels did not correlate with either the APACHE II score or the SOFA score (data not shown). However, serum concentration of IL-38 negatively correlated with the number of blood leukocytes and with levels of inflammatory cytokines, including IL-6 and TNF-α (Figure 1B). Figure 1. View largeDownload slide The circulating interleukin 38 (IL-38) level was elevated in patients with sepsis. A, IL-38 concentrations were measured by enzyme-linked immunosorbent assays (ELISAs) in serum samples collected from 40 adult patients with sepsis and from 29 healthy control subjects. Horizontal bars represent median values, and dots represent individual participants. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). B, Correlations between IL-38 levels and blood leukocyte counts, interleukin 6 (IL-6) levels, and tumor necrosis factor α (TNF-α) levels in adult patients with sepsis. Correlation was determined by the nonparametric Spearman correlation test. C, IL-38 concentrations were measured by ELISAs in serum samples collected from 26 pediatric patients with sepsis and from 20 healthy control subjects. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). D, Correlations between IL-38 levels and blood leukocyte counts, IL-6 levels, and TNF-α levels in pediatric patients with sepsis. Figure 1. View largeDownload slide The circulating interleukin 38 (IL-38) level was elevated in patients with sepsis. A, IL-38 concentrations were measured by enzyme-linked immunosorbent assays (ELISAs) in serum samples collected from 40 adult patients with sepsis and from 29 healthy control subjects. Horizontal bars represent median values, and dots represent individual participants. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). B, Correlations between IL-38 levels and blood leukocyte counts, interleukin 6 (IL-6) levels, and tumor necrosis factor α (TNF-α) levels in adult patients with sepsis. Correlation was determined by the nonparametric Spearman correlation test. C, IL-38 concentrations were measured by ELISAs in serum samples collected from 26 pediatric patients with sepsis and from 20 healthy control subjects. ***P < .001, by the Mann-Whitney U test, for between-group comparison (denoted by horizontal bracket). D, Correlations between IL-38 levels and blood leukocyte counts, IL-6 levels, and TNF-α levels in pediatric patients with sepsis. To validate our findings in adult patients with sepsis, we measured IL-38 concentrations in an independent cohort of pediatric patients with sepsis. The general characteristics of the study groups are summarized in Supplementary Table 2. IL-38 levels in pediatric patients with sepsis were also significantly elevated, compared with those in healthy control subjects (Figure 1C). Similarly, the serum concentration of IL-38 negatively correlated with the number of blood leukocytes and with levels of inflammatory cytokines, including IL-6 and TNF-α, in all pediatric patients with sepsis (Figure 1D). To determine whether LPS or heat-killed E. coli could induce IL-38 release in human cells in vitro, PBMCs were isolated and incubated with LPS or heat-killed E. coli. As with IL-6 and TNF-α levels, the IL-38 level was significantly elevated in PBMCs 24 hours after stimulation with LPS or heat-killed E. coli (Supplementary Figure 1). IL-38 Production Was Enhanced in Experimental Sepsis We next analyzed local, systemic, and organ IL-38 concentrations, using our well-established CLP-induced polymicrobial sepsis model [14, 15]. In experimental sepsis, IL-38 levels in the lung, spleen, PLF, and blood were significantly enhanced 24 or 48 hours after CLP (Figure 2). Figure 2. View largeDownload slide Local and systemic interleukin 38 (IL-38) production in mice after cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice (6/group) were subjected to sham surgery or CLP. Organs were removed at the indicated time points, blood specimens were collected by cardiac puncture, and peritoneal fluid was obtained by washing the peritoneal cavity with 5 mL of sterile phosphate-buffered saline. Samples were assayed for IL-38 content by enzyme-linked immunosorbent assays. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with sham control mice. Figure 2. View largeDownload slide Local and systemic interleukin 38 (IL-38) production in mice after cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice (6/group) were subjected to sham surgery or CLP. Organs were removed at the indicated time points, blood specimens were collected by cardiac puncture, and peritoneal fluid was obtained by washing the peritoneal cavity with 5 mL of sterile phosphate-buffered saline. Samples were assayed for IL-38 content by enzyme-linked immunosorbent assays. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with sham control mice. IL-38 Blockade Aggravated CLP-Induced Sepsis and LPS-Induced Endotoxemia Having observed that the IL-38 level was elevated in clinical and experimental sepsis, we first used an IL-38 blocking mouse monoclonal antibody (mouse IL-38/IL-1F10 antibody; clone 798036) to define the role of IL-38 in sepsis. In vitro studies showed that this antibody significantly inhibited the IL-38–induced decrease of IL-6 and TNF-α levels upon stimulation with LPS (Supplementary Figure 2). Subsequently, we induced sepsis in C57BL/6 mice by CLP, and anti–IL-38 antibody treatment was administered after surgery. The survival rate in septic mice treated with anti–IL-38 blocking antibodies was significantly lower than that in the IgG-treated control group (Figure 3A). The protein concentrations in both bronchoalveolar lavage fluid (BALF) and PLF, reflecting vascular permeability, were significantly increased in septic mice treated with anti–IL-38 antibodies (Figure 3B). Serum concentrations of ALT and AST, markers for hepatocellular injury, LDH, a marker for general cellular injury, and creatinine, a marker for renal failure, were significantly increased in mice treated with anti–IL-38 antibodies (Figure 3C). Lung, liver, and kidney damage was also confirmed by histological detection of alveolar hemorrhage, centrilobular necrosis, and tubular epithelial necrosis, respectively (Supplementary Figure 3A). In addition, blockade of IL-38 significantly increased the pathology scores for lungs, livers, and kidneys after sepsis (Supplementary Figure 3B). Furthermore, the exacerbated mortality of septic mice treated with anti–IL-38 was associated with higher levels of bacteria (primarily E. coli and Enterococcus), as assessed by comparing the number of CFU in the peritoneum and blood (Figure 3D). Figure 3. View largeDownload slide Interleukin 38 (IL-38) blockade aggravated cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice were subjected to CLP. Two hours after CLP, mice were administered 50 µg of anti–IL-38 blocking antibodies, followed by a booster dose of 50 µg 24 hours after CLP. Rat immunoglobulin G2a (IgG2a) served as control antibody. A, Survival of septic mice (12/group) following IL-38 blockade after CLP with anti–IL-38 antibodies. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with isotypical IgG control. B, Vascular permeability was validated 24 hours after CLP by determining the protein concentration in bronchoalveolar fluid (BALF) and peritoneal fluid (PLF) from 5 mice with CLP-induced sepsis treated with or without anti–IL-38 blocking antibodies. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without anti–IL-38 blocking antibodies. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units was determined. *P < .05, by the Mann-Whitney U test, compared with mice with CLP-induced sepsis treated with isotypical IgG control. Figure 3. View largeDownload slide Interleukin 38 (IL-38) blockade aggravated cecal ligation and puncture (CLP)–induced sepsis. C57BL/6 mice were subjected to CLP. Two hours after CLP, mice were administered 50 µg of anti–IL-38 blocking antibodies, followed by a booster dose of 50 µg 24 hours after CLP. Rat immunoglobulin G2a (IgG2a) served as control antibody. A, Survival of septic mice (12/group) following IL-38 blockade after CLP with anti–IL-38 antibodies. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with isotypical IgG control. B, Vascular permeability was validated 24 hours after CLP by determining the protein concentration in bronchoalveolar fluid (BALF) and peritoneal fluid (PLF) from 5 mice with CLP-induced sepsis treated with or without anti–IL-38 blocking antibodies. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without anti–IL-38 blocking antibodies. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with isotypical IgG control. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units was determined. *P < .05, by the Mann-Whitney U test, compared with mice with CLP-induced sepsis treated with isotypical IgG control. The effect of IL-38 blockade in the LPS-induced endotoxemia mouse model was also evaluated. IL-38 blocking increased the incidence of fatal endotoxemia in mice challenged with LPS (Supplementary Figure 4A), and it significantly enhanced vascular permeability, as reflected by significantly increased protein concentrations in both BALF and PLF from LPS-challenged mice treated with IL-38 antibodies (Supplementary Figure 4B). IL-38 Blockade Increased Cytokine and Chemokine Levels During CLP-Induced Sepsis The effect of IL-38 blockade on CLP-induced levels of cytokines and chemokines in PLF, BALF, and serum was evaluated 24 hours after CLP. Treatment with anti–IL-38 antibodies upregulated the cytokine and chemokine response in the CLP model and resulted in a significant increase in levels of IL-6, TNF-α, IL-10, IL-17, IL-27, CXCL1, and CCL2 but not IL-1β after CLP (Figure 4). Figure 4. View largeDownload slide Interleukin 38 (IL-38) blockade upregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PFL), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without anti–IL-38 blocking antibodies were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical immunoglobulin G control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. Figure 4. View largeDownload slide Interleukin 38 (IL-38) blockade upregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PFL), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without anti–IL-38 blocking antibodies were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with isotypical immunoglobulin G control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. IL-38 Treatment Protected Against CLP-Induced Sepsis and LPS-Induced Endotoxemia Because the administration of antibody against IL-38 was found to aggravate sepsis in mice, we performed the reverse experiment and examined the effect of treatment with recombinant mouse IL-38 on experimental sepsis. Prophylactic administration of IL-38 at 2 hours before the onset of sepsis significantly improved survival, compared with survival among vehicle-treated control animals, and IL-38 administration 2 hours after CLP also significantly improved survival, compared with vehicle-treated control animals (Figure 5A), indicating both preventive and therapeutic effects of IL-38 on sepsis. Mice treated with IL-38 at 2 hours after CLP had significantly lower protein concentrations in both BALF and PLF (Figure 5B). Mice with therapeutic IL-38 administration displayed lower serum concentrations of ALT, AST, LDH, and creatinine (Figure 5C). Lung, liver, and kidney damage was also confirmed by histological detection of alveolar hemorrhage, centrilobular necrosis, and tubular epithelial necrosis, respectively (Supplementary Figure 5A), and therapeutic IL-38 treatment significantly decreased the pathology scores of lung, liver, and kidney after CLP (Supplementary Figure 5B). Furthermore, bacterial titers were significantly lower in PLF and blood specimens from mice that received IL-38 therapeutic treatment (Figure 5D). Figure 5. View largeDownload slide Interleukin 38 (IL-38) administration attenuated cecal ligation and puncture (CLP)–induced sepsis. A, Survival of septic mice (12/group) following IL-38 supplementation. Recombinant murine IL-38 (1 μg/injection) was given 2 hours before CLP, for preventive treatment; 1 dose of IL-38 (1 μg/injection) was given at the time of CLP; and 1 dose of IL-38 (1 μg/injection) was given 2 hours after CLP challenge, for therapeutic treatment. Phosphate-buffered saline (PBS) was delivered in a similar fashion as control vehicle. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with recombinant IL-38. B, Vascular permeability 24 hours after CLP was validated by determining the protein concentrations in bronchoalveolar fluid and peritoneal fluid (PLF) from 5 mice that underwent CLP and were treated with or without recombinant IL-38 therapeutically. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) therapeutically. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units (CFU) was counted. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38 therapeutically. Figure 5. View largeDownload slide Interleukin 38 (IL-38) administration attenuated cecal ligation and puncture (CLP)–induced sepsis. A, Survival of septic mice (12/group) following IL-38 supplementation. Recombinant murine IL-38 (1 μg/injection) was given 2 hours before CLP, for preventive treatment; 1 dose of IL-38 (1 μg/injection) was given at the time of CLP; and 1 dose of IL-38 (1 μg/injection) was given 2 hours after CLP challenge, for therapeutic treatment. Phosphate-buffered saline (PBS) was delivered in a similar fashion as control vehicle. ***P < .001, by Kaplan-Meier analysis followed by log-rank tests, compared with septic mice treated with recombinant IL-38. B, Vascular permeability 24 hours after CLP was validated by determining the protein concentrations in bronchoalveolar fluid and peritoneal fluid (PLF) from 5 mice that underwent CLP and were treated with or without recombinant IL-38 therapeutically. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. C, Levels of serological markers of organ injury, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine, 24 hours after CLP in 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) therapeutically. *P < .05, **P < .01, and ***P < .001, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38. D, Dilutions of PLF and blood specimens obtained from 5 septic mice 24 hours after CLP were cultured on blood agar plates, and the number of bacterial colony-forming units (CFU) was counted. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with recombinant IL-38 therapeutically. In a LPS-induced endotoxemia mouse model, therapeutic administration of IL-38 at 2 hours after LPS challenge also significantly enhanced survival in mice (Supplementary Figure 6A), which was associated with significantly lower vascular permeability (Supplementary Figure 6B). IL-38 Administration Ablated the Production of Cytokines and Chemokines During CLP-Induced Sepsis As shown in Figure 6, the concentrations of inflammatory cytokines and chemokines, including IL-6, TNF-α, IL-10, IL-17, IL-27, CXCL1, and CCL2 but not IL-1β, in PLF, BALF, and blood specimens obtained 24 hours after CLP were significantly downregulated in septic mice that received therapeutic IL-38 treatment. Figure 6. View largeDownload slide Interleukin 38 (IL-38) administration downregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PLF), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with phosphate-buffered saline control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. Figure 6. View largeDownload slide Interleukin 38 (IL-38) administration downregulated the production of cytokines and chemokines during cecal ligation and puncture (CLP)–induced sepsis. Cytokine and chemokine concentrations in peritoneal fluid (PLF), bronchoalveolar lavage fluid (BALF), and blood specimens from 5 septic mice treated with or without recombinant IL-38 (1 μg/injection) were determined by enzyme-linked immunosorbent assays 24 hours after CLP. *P < .05, by the Mann-Whitney U test, compared with septic mice treated with phosphate-buffered saline control. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-17, interleukin 17; IL-27, interleukin 27. IL-38 Did Not Influence Bacterial Phagocytosis and Killing by Phagocytes To investigate whether IL-38 influenced intrinsic antibacterial functions of phagocytes, we studied the bacterial uptake and killing capacities of peritoneal macrophages and neutrophils. Preincubation with recombinant IL-38 did not have direct effects on phagocytosis and intracellular killing of E. coli by macrophages (Supplementary Figure 7A) or neutrophils (Supplementary Figure 7B). DISCUSSION Since 2015, there has been increasing interest in studying IL-38, and IL-38 has emerged as an important regulatory mediator in some inflammatory diseases [5, 6]. In the present report, we identified IL-38 to be potentially involved in the pathogenesis of clinical and experimental sepsis. We made the following key observations: (1) both adult and pediatric patients with sepsis had increased circulating IL-38 levels, which negatively correlated with the number of blood leukocytes and levels of inflammatory cytokines, including IL-6 and TNF-α; (2) IL-38 blockade aggravated experimental sepsis by increasing inflammation and decreasing bacterial clearance; and (3) IL-38 administration protected mice from experimental sepsis by suppressing inflammation and decreasing bacterial loading. IL-38 production has been found to be elevated in childhood asthma, and it was negatively correlated with IL-10 secretion [11]. Elevated IL-38 levels in the blood have also been shown to be associated with the severity of chronic hepatitis B [16]. In addition, IL-38 expression was enhanced in the synovium of patients with rheumatoid arthritis and in the colon of patients with Crohn disease [17]. It was also found at significantly higher concentrations in patients with systemic lupus erythematosus, which correlated with the severity of the disease [10]. To the best of our knowledge, this is the first study demonstrating that circulating IL-38 levels were elevated in 2 cohorts of adult and pediatric patients with sepsis. Our present work on the serum levels of IL-38 is related to previous studies on serum IL-38 concentrations [11, 16], which revealed detectable levels of IL-38 in healthy subjects that could be upregulated during some inflammatory conditions. IL-38 concentrations did not correlate with the severity of sepsis, as reflected by APACHE II and SOFA scores, or with mortality. Although the levels of blood leukocytes, IL-6, and TNF-α were elevated in clinical sepsis, elevated IL-38 levels negatively correlated with the blood leukocyte count, IL-6 level, and TNF-α level in patients with sepsis, suggesting the antiinflammatory potential of IL-38 in clinical sepsis. IL-38 has been shown to exert antiinflammatory properties in several animal models of pathology. Articular injection of an adeno-associated virus (AAV) 2/8 encoding IL-38 has been shown to significantly decrease clinical inflammatory scores in joints of mice with collagen-induced arthritis and K/BxN serum transfer-induced arthritis, and this induced IL-38 overexpression could significantly reduce the expression of T-helper type 17 (Th17) cytokines (ie, IL-17A, interleukin 23p19 [IL-23p19], and interleukin 22 [IL-22]), CXCL1, and receptor activator of nuclear factor kappa-B ligand [18]. Intravenous administration of murine recombinant IL-38 into MRL/lpr mice ameliorated lupus-like clinical symptoms, and serum concentrations of IL-6, IL-17, IL-22, and CXCL10 were also decreased by treatment with recombinant IL-38 [19]. Furthermore, expression of exogenous human IL-38 significantly reduced hepatic toxicity, and serum levels of ALT, AST, and proinflammatory cytokines, including TNF-α, interferon γ (IFN-γ), IL-6, IL-17, and IL-22, in a model of concanavalin A–induced liver injury [12]. In this study of CLP-induced sepsis, we further found that the survival benefit obtained with recombinant IL-38 was associated with a reduction of the local and systemic inflammatory response, as shown by a significant decrease of the concentrations of IL-6, TNF-α, IL-10, IL-17, IL-27, CXCL1, and CCL2 in PLF, BALF, and blood specimens, but IL-1β secretion was not affected. Findings of this in vivo study are therefore consistent with previous in vitro work showing that IL-38 inhibited the production of cytokines such as IL-6 and TNF-α by THP-1 monocytic cells but did not alter IL-1β expression [18]. Furthermore, blockade of IL-38 activity was associated with enhanced local and systemic inflammatory response in CLP-induced sepsis. In fact, organ injury observed in sepsis is due to widespread inflammation, which is caused by the explosive release of cytokines and chemokines [20]. Therefore, IL-38–induced protection against experimental sepsis was associated with decreased inflammation. Our data also demonstrated that one mechanism of improved survival in sepsis with IL-38 treatment included increased bacterial clearance. We found that IL-38 might play an important role in controlling both bacterial outgrowth and inflammatory responses. Increased bacterial loads, which are critical inflammatory stimulus, may be able to further enhance inflammation and tissue damage, resulting in increased mortality of septic mice [21, 22]. Therefore, the improved capacities of IL-38 to clear bacterial infection and to downregulate host inflammatory reactions are both responsible for the decreased mortality observed in septic mice treated with recombinant IL-38. Our study includes some limitations. First, the number of patients with sepsis in this study was relatively small. The clinical value of the IL-38 level as a sepsis biomarker remains to be established in a larger-sized clinical trial in regarding to clinical state, infectious source, pathogen type, and other possible factors. Second, although in vivo findings showed that IL-38 mediated bacterial clearance during sepsis, in vitro studies demonstrated that IL-38 did not directly influence bacterial phagocytosis and killing by macrophages and neutrophils. Therefore, the mechanisms by which IL-38 regulated the clearance of bacteria require further studies. Currently, there are no specific therapeutic agents available to clinicians [22]. The present study is the first to document that IL-38 release occurs in clinical and experimental sepsis and that IL-38 could improve survival in experimental sepsis by decreasing inflammation and increasing bacterial clearance. As a novel cytokine-targeted sepsis treatment, IL-38 could be combined with other immunoadjuvants that function via other different mechanisms, such as progranulin [14], interleukin 5 [23], interleukin 7 [24], interleukin 30 [25], and interleukin 33 [26], to further enhance antisepsis therapy. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This work was supported by the National Natural Science Foundation of China (grants 81722001 and 81572038 to J. C.) and the Chongqing Science and Technology Commission (Distinguished Young Scholars of Chongqing grant cstc2014jcyjjq10002 to J. C.). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Rhodes A, Evans LE, Alhazzani W, et al.   Surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016. Crit Care Med   2017; 45: 486– 552. 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The Journal of Infectious DiseasesOxford University Press

Published: May 12, 2018

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