Interleukin 36α Attenuates Sepsis by Enhancing Antibacterial Functions of Macrophages

Sepsis is now an important cause of death and is projected to account for >5 million deaths worldwide [1]. Despite considerable efforts to improve outcomes, the mortality rate associated with sepsis remains higher than 25%–30%, and even 40%–50% when shock is present [2]. At present, no effective antisepsis treatments exist to improve patient survival [3].

Sepsis is newly defined as life-threatening organ dysfunction caused by a dysregulated host response to infection [4]. Autopsy results have shown that most patients with sepsis had unresolved septic foci at post mortem [5], indicating that sepsis-induced death is closely associated with the failure of the host's immunity to eradicate invading pathogens [6, 7]. Therefore, potential therapies that improve protective anti-infective immunity while limiting sepsis-induced tissue injury might increase survival in sepsis.

Cytokines play important roles in anti-infective immunity, and a detailed understanding of local cytokine function during infections may provide hope for potential immunostimulatory therapies in sepsis [8–10]. Recently, knowledge about the role of interleukin 36 (IL-36) family members, new members of the interleukin 1 (IL-1) superfamily, has begun to emerge. The IL-36 subfamily comprises the proinflammatory molecules IL-36α, IL-36β, and IL-36γ and a natural inhibitor, IL-36Ra receptor antagonist (IL-36Ra). IL-36 signals by binding to a heterodimeric receptor consisting of the IL-36 receptor (IL-36R) subunit and the IL-1R accessory protein [11]. IL-36 and IL-36R are widely expressed in several tissues, including skin, lung, and gut, and IL-36 signaling regulates inflammation and immunity in these tissues [12–14]. However, a functional role for IL-36 in the pathogenesis of sepsis is still unknown. We therefore investigated the role of IL-36 in experimental sepsis.

MATERIALS AND METHODS

Ethics Statement

All animal experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China. The protocol was approved by the Chongqing Medical University Institutional Animal Care and Use Committee (assurance No. SYXK-2012-0001).

Animals and Sepsis Model

Female C57BL/6 mice aged 6–8 weeks were obtained from and raised at Chongqing Medical University. Mice were bred and maintained under specific pathogen-free conditions. Cecal ligation and puncture (CLP) was performed to establish a model of polymicrobial sepsis. Briefly, mice were anesthetized intraperitoneally with a mixture of xylazine (4.5 mg/kg) and ketamine (90 mg/kg), and the cecum was exposed, ligatured at its external third, and punctured through with a 26-gauge needle (nonsevere CLP) or a 22-gauge needle (severe CLP). The cecum was then returned to the peritoneal cavity, and incisions were closed. Mice subjected to sham CLP underwent the same procedure, except for ligation and puncture of the cecum. For survival analysis, mice were monitored twice daily for 14 days. In an additional series of experiments, mice were injected intranasally with live Staphylococcus aureus.

Assay of Inflammatory Mediators

Inflammatory cytokines and chemokines—including IL-36α, IL-36β, IL-36γ, interleukin 6, 1β, and 10 (IL-6, IL-1β, and IL-10), tumor necrosis factor (TNF) α, interferon (IFN) γ, CXCL1, and CXCL10—were assessed using enzyme-linked immunosorbent assay kits (R&D systems), according to the manufacturers' instructions.

Serum Biochemistry Serum

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine were determined with commercial available kits (Sigma-Aldrich), using a Hitachi analyzer (Boehringer Mannheim) according to the manufacturers' instructions.

Tissue Histology

Lungs, livers, kidneys, and spleens were harvested and fixed in 4% formalin, stained with hematoxylin-eosin, and examined with light microscopy. Pathology scores were determined and analyzed by a pathologist blinded for groups.

Determination of Bacterial Colony-Forming Units

Serial dilutions of peripheral blood, peritoneal lavage fluid (PLF), or spleens of mice were plated on blood-agar plates. Colony-forming unit (CFU) counts were then determined after 24-hour culture.

Apoptosis Analysis

The TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) assay was performed with paraffin-embedded tissue sections from spleen, liver, or kidney, which were first deparaffinized, as described elsewhere [3]. The sections were then permeabilized with Triton X-100 and flooded with TUNEL reagent. The percentage of apoptotic (TUNEL-positive) cells was then determined by counting 500 total cells under a light microscope.

Determination of Inflammatory Cells

Peritoneal cell suspension was pelleted and resuspended. Cell counts and viability were determined using Trypan blue exclusion counting on a hemacytometer. Cytospin slides were prepared and stained with a Wright-Giemsa stain. Peritoneal cells were also characterized accordingly with monoclonal antibodies against CD45, CD3, CD4, CD11b, Ly6G, and F4/80 (BD Pharmingen). At least 5 × 10³ cells were collected and analyzed with a FACScan flow cytometer (Becton Dickinson).

Administration of IL-36α

For in vivo cytokine treatment, each mouse was injected intraperitoneally with 1.0 µg of recombinant mouse IL-36α (R&D Systems), 2 hours before or after CLP. Phosphate-buffered saline (PBS) was delivered in a similar fashion as a control.

Antibody-Mediated Neutralization of IL-36α

For in vivo neutralization of cytokine, each mouse was injected intraperitoneally with 50 µg of anti–IL-36α antibodies (R&D Systems) at the time of CLP, followed by booster doses of 25 µg 24 hours later after CLP. Goat immunoglobulin G was delivered in a similar fashion as a control.

Isolation of Macrophages and Neutrophils

To isolate peritoneal macrophages, mice were injected with 5 mL of PBS. Macrophages were isolated from peritoneal lavage by plastic adherence. To isolate peritoneal neutrophils, mice were injected intraperitoneally with 1 mL of 3% thioglycollate broth (Sigma-Aldrich). Cells were harvested 4 hours later by peritoneal lavage with 5 mL of PBS, and neutrophils were then purified by discontinuous Percoll gradient centrifugation followed by magnetic cell sorting (Miltenyi Biotec), as described elsewhere [10]. Peritoneal macrophages and neutrophils were then treated with recombinant mouse IL-36α, IFN-γ (R&D systems), ultrapurified lipopolysaccharide (LPS) from Escherichia coli K12 strain without any contamination by lipoprotein (InvivoGen), or peptidoglycan (PGN) for Toll-like receptor (TLR) 2 from Fluka Chemie GmbH.

3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide (MTT) Assay

Cells (1 × 10⁵ cells/well) were inoculated into a 96-well plate. At the indicated times, the treated or untreated cells were incubated with 10 µL of MTT solution (Sigma) for another 2 hours. Cells were then collected by centrifugation and lysed with 200 µL of dimethyl sulfoxide. The absorbance at an optical density of 550 nm was assayed to quantify the viable cells.

Phagocytosis Assay

peritoneal macrophages or neutrophils (1 × 10⁵) were incubated with fluorescein isothiocyanate–labeled E. coli at a multiplicity of infection (MOI) of 100 for 30 minutes at 37°C. Cells were then washed and stained with 4′,6-diamidino-2-phenylindole (Invitrogen), followed by visualization using confocal laser scanning microscopy (LSM 510, Zeiss).

Bacterial Killing Assays

For determination of bacterial killing by macrophages, peritoneal macrophages (1 × 10⁵ cells) were infected with E. coli (MOI, 10) at 37°C for 2 hours, and then cells were washed with buffer containing tobramycin (100 µg/mL) to remove extracellular bacteria and lysed in PBS containing 0.1% Triton X-100. Live intracellular bacteria were determined by culture of lysates for quantitation of bacterial killing (t = 2 hours). Killing was calculated from the percentage of colonies present at t = 2 hours compared with t = 0 hour, as follows: 100 − (No. of CFUs at t = 2 hours/No. of CFUs at t = 0 hour). For determination of bacterial killing by neutrophils, peritoneal neutrophils (1 × 10⁵ cells) were infected with E. coli at an MOI ratio of 1:100 at 37°C for 1 hour, and cells were washed and lysed. Neutrophil killing was calculated from the percentage of colonies present at t = 1 hour compared with t = 0 hour, as described above.

Macrophage Depletion

The clodronate-encapsulated liposomes and PBS-encapsulated liposomes were prepared as described elsewhere [9]. The mice were injected intraperitoneally with 200 µL of a suspension of clodronate-encapsulated liposomes or PBS-encapsulated liposomes 48 hours before CLP. The macrophage depletion analysis was performed 48 hours after treatment by flow cytometry based on expression of F4/80.

Statistical Analysis

Data from mice experiments were expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation or as medians with interquartile ranges. Comparisons between groups were tested using the Mann–Whitney U test. Survival studies were performed by Kaplan–Meier analyses followed by log–rank tests. All analyses were done using GraphPad Prism software, version 5.01 (GraphPad Software). Differences were considered statistically significant at P < .05.

RESULTS

Up-regulation of IL-36α Production During CLP-Induced Polymicrobial Sepsis

To investigate whether IL-36 family member expression is altered in sepsis, we first examined protein expression levels of IL-36α, IL-36β, and IL-36γ during intra-abdominal sepsis in mice by performing CLP, a well-described model of sepsis. Through this analysis we found that IL-36α protein was significantly and strongly elevated in the blood, lungs and PLF after CLP (Figure 1A). Although IL-36γ production was somewhat increased in the blood, no differences in its protein levels were found in the lungs or PLF. In contrast, IL-36β elevation was not detected in any tissues. We further confirmed these findings in a pneumonia-induced sepsis model caused by S. aureus, a major cause of sepsis in the clinic. We also detected a strong up-regulation of IL-36α protein in the blood and lungs after S. aureus–induced sepsis (Figure 1B). Together, these data demonstrated that IL-36α production was prominently enhanced during sepsis, suggesting that IL-36α might be involved in the pathogenesis of sepsis.

Figure 1.

Local and systemic interleukin 36α (IL-36α) production in mice with sepsis. A, C57BL/6 mice (n = 5 per group) were subjected to sham or nonsevere cecal ligation and puncture (CLP) with a 26-gauge needle. Organs were removed at 24 hours after CLP, blood was collected by cardiac puncture and peritoneal lavage fluid (PLF) was obtained by washing the peritoneal cavity with 1 mL of sterile phosphate-buffered saline (PBS). Samples were assayed for IL-36α, IL-36β, and IL-36γ content by specific sandwich enzyme-linked immunosorbent assay (ELISA). *P < .05; ^†P < .001 (vs sham controls; Mann–Whitney U test). B, C57BL/6 mice (n = 5 per group) were intranasally infected with Staphylococcus aureus (2 × 10⁸ colony-forming units), and organs were removed 24 hours after infection. Samples were finally assayed for IL-36α, IL-36β, and IL-36γ content by specific sandwich ELISA. ^†P < .001 (vs PBS controls; Mann–Whitney U test).

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Protective Effect of IL-36α in Polymicrobial Sepsis

To investigate whether IL-36α was protective or pathogenic in sepsis, we examined the effects of IL-36α administration using CLP-induced severe sepsis (lethal) model. We initially injected the mice intraperitoneally with recombinant IL-36α (1 µg) 2 hours before CLP. The IL-36α–treated mice showed significantly reduced the mortality rate compared with PBS-treated controls (Figure 2A). To establish the use of IL-36α as a therapeutic modality in CLP, we further delayed the time to assess the therapeutic benefits of IL-36α administration. Our subsequent experiments were performed in CLP mice using recombinant IL-36α (0.5 µg) beginning 2 hours after CLP, followed by booster doses of 0.25 µg at both 24 and 48 hours after CLP. IL-36α administration after CLP also significantly improved survival compared with PBS-treated control mice (Figure 2B). Bacteria colony counts in PLF and spleens did not differ significantly between IL-36α– and PBS-treated mice at 6 hours after CLP. However, IL-36α–treated mice displayed a significant decrease in bacterial loads from peritoneum, spleen, and blood compared with PBS-treated mice at 24 hours after CLP (Figure 2C). These data indicate that IL-36α administration is a viable therapeutic modality to improve survival in sepsis.

Figure 2.

Interleukin 36α (IL-36α) treatment afforded protection against sepsis. A, C57BL/6 mice were treated intraperitoneally with 1 µg of recombinant mouse IL-36α 2 hours before severe cecal ligation and puncture (CLP) with a 22-gauge needle, and survival was monitored. Comparison between groups was done by Kaplan–Meier analysis followed by log–rank tests. ^†P < .01 (vs mice treated with phosphate-buffered saline [PBS] control). B, C57BL/6 mice (n = 12 per group) were injected intraperitoneally with 0.5 µg of recombinant mouse IL-36α at 2 hours after severe CLP with a 22-gauge needle and then 0.25 µg intraperitoneally at both 24 and 48 hours after surgery, after which survival was monitored. ^†P < .01 (vs mice treated with PBS control). C, Bacterial counts in peritoneal lavage fluid (PLF), spleens, or blood from mice (n = 5 per group) treated with or without recombinant IL-36α, as in B, at 6 or 24 hours after severe CLP. *P < .05 (vs mice treated with PBS control; Mann–Whitney U test). D, Survival in mice following IL-36α neutralization after nonsevere CLP with a 26-gauge needle (n = 10 per group). ^†P < .01 (vs mice treated with immunoglobulin [Ig] G controls). All results are representative of ≥3 independent experiments.

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In addition, a sublethal model of CLP was established to determine the potential detrimental effects of neutralizing the biological function of IL-36α in vivo. We induced nonsevere sepsis (sublethal) by CLP followed by treatment with neutralizing antibodies against IL-36α. The survival rate in mice treated with anti–IL-36α antibodies was significantly lower than that in mice treated with immunoglobulin G control (Figure 2D), showing that blockade of IL-36α could exacerbate sepsis.

IL-36α Inhibition of Vital Organ Inflammation and Immune Cell Apoptosis in Sepsis

Sepsis is characterized by life-threatening organ dysfunction [2]. At 6 and 24 hours after CLP, inflammation of lung, liver, and kidney was reduced by the administration of IL-36α (Figure 3A), which was reflected by significantly lower pathology scores compared with PBS-treated mice (Figure 3B). IL-36α–treated mice showed significantly decreased serum levels of ALT and AST, markers for hepatocellular injury (Figure 3C). The serum levels of creatinine, a marker for renal failure, were also significantly decreased in IL-36α–treated mice (Figure 3C). In addition, IL-36α treatment significantly decreased cell apoptosis in the spleen, liver, and kidney, as measured by TUNEL assay (Figure 3D and 3E).

Figure 3.

Effects of interleukin 36α (IL-36α) on sepsis–induced inflammation of vital organs and immune cell apoptosis. A, Representative examples of hematoxylin-eosin–stained lung, spleen, kidney, and liver tissues from mice (n = 5 per group) treated with or without recombinant IL-36α, as in Figure 2B, at 6 and 24 hours after severe cecal ligation and puncture (CLP) with a 22-gauge needle. PBS, phosphate-buffered saline. B, Pathology scores for lung, spleen, kidney, and liver tissues from mice (n = 5 per group) treated with or without recombinant IL-36α, as in Figure 2B, at 6 and 24 hours after severe CLP. *P < .05 (vs mice treated with PBS control; Mann–Whitney U test). C, Serological markers of organ injury including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine in mice (n = 5 per group), treated with or without recombinant IL-36α, as in Figure 2B, at 24 hours after severe CLP. *P < .05 (vs mice treated with PBS control; Mann–Whitney U test).

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Figure 3 continued. D, Spleen, liver and kidney samples from mice (n = 5 per group) treated with or without recombinant IL-36α, as in Figure 2B, at 6 and 24 hours after severe CLP, were subjected to DNA fragmentation analysis (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling [TUNEL]). Representative examples are shown. E, TUNEL-positive cells counts (n = 5 per group). *P < .05 (vs mice treated with PBS control; Mann–Whitney U test). All results are representative of ≥3 independent experiments.

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In a sublethal model of CLP, mice treated with anti–IL-36α antibodies showed significantly increased serum levels of ALT, AST and creatinine (Supplementary Figure 1A). Anti–IL-36α treatment also significantly increased cell apoptosis in the spleen, liver, and kidney (Supplementary Figure 1B). These results confirmed a protective role of IL-36α in sepsis-induced tissue injury and cell apoptosis.

Effect of IL-36α on TNF-α, IFN-γ, IL-1β, and CXCL1 Levels in Sepsis

The effect of IL-36α on CLP-induced proinflammatory cytokines and chemokines was evaluated 6 and 24 hours after CLP. CLP induced dramatic increases in TNF-α, IL-6, IFN-γ, IL-1β, IL-10, CXCL1, and CXCL10 levels (Table 1). Although no differences in levels of IL-6, IFN-γ, and CXCL10 were noted, IL-36α treatment blunted serum TNF-α and IL-1β at 6 hours after CLP and significantly decreased peritoneal TNF-α and CXCL1, as well as serum IL-1β and CXCL1, at 24 hours after CLP. Likewise, neutralization of IL-36α led to a significant increase in peritoneal TNF-α and CXCL1, as well as serum IL-1β and CXCL1, at 24 hours after CLP (Supplementary Figure 2).

Impact of IL-36α on Leukocyte Recruitment in Sepsis

We further investigated whether IL-36α modulates leukocyte influx in sepsis. Leukocyte counts and differentials including macrophages, neutrophils, and lymphocytes were similar in PLF samples obtained from IL-36α– and PBS-treated mice at 6 and 24 hours after CLP (Table 2 and Supplementary Figure 3).

IL-36α Enhancement of Bacterial Phagocytosis and Killing by Macrophages

Because IL-36α could enhance bacterial clearance (Figure 2), which resulted in decreased inflammatory responses (Figure 3 and Table 1), we therefore investigated whether it would modulate the intrinsic antibacterial functions of phagocytes. Preincubation with recombinant IL-36α significantly enhanced phagocytosis and intracellular killing of live E. coli by peritoneal macrophages (Figure 4A and 4B). Next, we tested IL-36α for its ability to induce proinflammatory cytokines in macrophages activated by various Toll ligands relevant to sepsis, including PGN (TLR2 ligand) and LPS (TLR4 ligand). IL-36α had little effect on PGN- and LPS-mediated production of IL-6, TNF-α, and IL-1β in macrophages (Figure 4C).

Figure 4.

Effects of interleukin 36α (IL-36α) on bacterial phagocytosis and killing by macrophages and neutrophils. A, Peritoneal mouse macrophages were stimulated with or without recombinant IL-36α at the concentration of 50 ng/mL for 48 hours and infected with fluorescein isothiocyanate (FITC)–labeled Escherichia coli for 30 minutes at 37°C. Arrows indicate engulfed bacteria (as determined by overlay of green bacteria) by macrophages. Representative examples are shown. PBS, phosphate-buffered saline. B, Peritoneal mouse macrophages were pretreated with or without recombinant IL-36α (50 ng/mL) for 48 hours, and then infected with E. coli (multiplicity of infection, 10) for 30 minutes. Cells were then washed in PBS and resuspended for 30 minutes in medium containing 100 µg/mL tobramycin to remove extracellular bacteria. Cells were lysed in PBS containing 0.1% Triton X-100 for assessment of phagocytosis (t = 0), and additional samples were incubated for 2 additional hour (t = 2 hours) to assess bacterial killing. Intracellular killing (t = 2 hours) was determined as described in Materials and Methods. CFUs, colony-forming units. *P < .05 (vs macrophages pretreated with PBS control; Mann–Whitney U test). C, Macrophages were stimulated with peptidoglycan (PGN; 1 µg/mL) or lipopolysaccharide (LPS; 1 µg/mL) in the presence or absence of IL-36α (50 ng/mL) for 24 hours, and release of cytokines in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). IL-1β, interleukin 1β; IL-6, interleukin 6; TNF, tumor necrosis factor. D, Peritoneal mouse neutrophils were stimulated with or without recombinant IL-36α at the concentration of 50 ng/mL for 12 hours and infected with FITC-labeled E. coli for 30 minutes at 37°C. Arrows indicate engulfed bacteria (as determined by overlay of green bacteria) by macrophages. Representative examples are shown. E, Peritoneal mouse neutrophils were pretreated with or without recombinant IL-36α (50 ng/mL) for 12 hours, and then infected with E. coli (multiplicity of infection, 100) for 30 minutes. Cells were then washed in PBS and resuspended for 30 minutes in medium containing 100 µg/mL tobramycin to remove extracellular bacteria. Cells were lysed in PBS containing 0.1% Triton X-100 for assessment of phagocytosis (t = 0), and additional samples were incubated for 1 additional hour (t = 1 hour) to assess bacterial killing. Intracellular killing (t = 1 hour) was determined, as described in Materials and Methods. F, Neutrophils were stimulated with PGN (1 µg/mL) or LPS (1 µg/mL) in the presence or absence of IL-36α (50 ng/mL) for 24 hours, and release of cytokines in the culture supernatant was determined by ELISA. All results are representative of ≥5 independent experiments.

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In neutrophils, preincubation with recombinant IL-36α did not influence bacterial phagocytosis and killing in neutrophils on live E. coli infection (Figure 4D and 4E). As a positive control, IFN-γ could significantly enhance bacterial phagocytosis and killing by mouse neutrophils (Supplementary Figure 4). As observed in macrophages, IL-36α did not modulate the production of IL-6, TNF-α, and IL-1β in neutrophils activated by PGN and LPS (Figure 4F).

In these experiments in vitro, IL-36α had no effect on survival of primary mouse macrophages (the percentage of viable macrophages was about 85% 48 hours after treatment with 50 ng/mL IL-36α) and neutrophils in culture (the percentage of viable neutrophils was about 78% 12 hours after treatment with 50 ng/mL IL-36α).

Role of Macrophages in the Protection Against Sepsis Elicited by IL-36α

Having observed that IL-36α could enhance bacterial phagocytosis and killing by macrophages in vitro, we further investigated the role of macrophages in IL-36α–mediated protection against sepsis in vivo. We depleted macrophages by administration of clodronate coupled with liposomes. Clodronate liposome treatment resulted in a 10-fold reduction in spleen macrophage numbers (Figure 5A). Depleting macrophages dramatically impaired the survival of septic mice treated with IL-36α (Figure 5B), suggesting that macrophages were required for IL-36α–mediated protection in sepsis.

Figure 5.

Effects of macrophage depletion on the protection against sepsis elicited by interleukin 36α (IL-36α). A, Flow cytometry plots showed F4/80⁺ macrophages in the circulation isolated from spleens (n = 5 per group), at 48 hours after clodronate-encapsulated liposome treatment. ^†P < .001 (vs mice treated with phosphate-buffered saline [PBS]–encapsulated liposomes as a control; Mann–Whitney U test). B, C57BL/6 mice depleted of macrophages by clodronate liposomes were treated with or without recombinant mouse IL-36α, as in Figure 2B, after severe cecal ligation and puncture (CLP) with a 22-gauge needle, and survival was monitored. Comparison between groups was done by Kaplan–Meier analysis followed by log–rank tests. ^†P < .001 (vs mice treated with PBS liposomes as a control).

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DISCUSSION

Although clinical outcomes from sepsis have greatly improved over the past 2 decades, we still lack any specific pharmacotherapies for sepsis. Manipulation of the immune system has been considered a promising approach for the development of an effective therapeutic strategy for sepsis [15]. In the current study, we identified the cytokine IL-36α as a novel protective factor in sepsis that enhanced bacterial phagocytosis and killing by macrophages, resulting in increased bacterial clearance, as well as decreased tissue inflammation/damage and immune cell apoptosis. More importantly, the ability of IL-36α administration after the onset of sepsis to significantly reduce the sepsis-induced mortality rate in mice suggests a therapeutic potential of IL-36α in severe sepsis. Furthermore, IL-36α blockade exacerbated mortality rate in a model of nonsevere sepsis, providing evidence for an important role of IL-36α in antiseptic activity.

IL-36 cytokines include 3 agonistic cytokines, namely, IL-36α, IL-36β, and IL-36γ, and a natural inhibitor, IL-36Ra. Expression of IL-36 cytokines is found at low levels in various tissues, such as the skin, esophagus, tonsil, lung, gut, and brain. IL-36 expression could be up-regulated in epithelial cells, keratinocytes, monocytes/macrophages, and T cells in response to a number of stimuli, including cytokines, TLR agonists, bacteria, rhinovirus infection, smoke, or other pathologic conditions [11, 16]. The messenger RNA expression of IL-36α and IL-36γ, but not of IL-36β, was enhanced in the inflamed mucosa of patients with inflammatory bowel disease, in particular, in ulcerative colitis [17]. During imiquimod-induced mouse skin inflammation and in human psoriasis, IL-36α and IL-36γ was induced, but not IL-36β [18].

It has been demonstrated elsewhere that IL-36γ was induced by live Aspergillus fumigatus conidia and Mycobacterium tuberculosis [19, 20]. We showed that IL-36α production was prominently up-regulated and thus focused on its role in the pathogenesis of sepsis. In contrast to other reports demonstrating that IL-36 did not affect the development of inflammatory arthritis and M. tuberculosis–induced lethality [11, 21], our findings showed that replenishment of IL-36α could attenuate sepsis, and neutralization of IL-36α could worsen it.

What underlying mechanisms mediate the ability of IL-36α to attenuate pathologic effects in sepsis? Our data suggest a novel role of IL-36α in regulating antibacterial function of macrophages, and macrophages were required for the protection against sepsis elicited by IL-36α. Impaired anti-infective function of macrophages has been reported to be associated with increased mortality rates and higher bacterial burden during sepsis [22, 23]. Recently, Song et al [24] demonstrated that progranulin protected against sepsis by promoting macrophage recruitment. In addition, macrophages were required for IL-5–mediated protection in polymicrobial sepsis [9], and the improved outcomes after leukocyte cell-derived chemotaxin 2 treatment of sepsis were also mediated mainly by macrophages [25], whereas mast cells could aggravate sepsis by inhibiting peritoneal macrophage phagocytosis [26]. Although neutrophils play an important role in protection against bacterial sepsis [8, 27], we found that IL-36α did not influence bacterial phagocytosis and killing by neutrophils, cytokine secretion from neutrophils, or neutrophil recruitment; one possible explanation is that neutrophils showed no expression of IL-36R and failed to respond to IL-36 treatment [12].

Death due to sepsis is closely associated with tissue injuries and immune cell apoptosis, and prevention of cell apoptosis and tissue injury could improve survival rates [3]. The present study identified IL-36α as a negative regulator of CLP-induced inflammation, and IL-36α decreased CLP-induced apoptosis and tissue injuries. Although no differences in levels of IL-6, IFN-γ, and CXCL10 were noted, IL-36α treatment blunted systemic TNF-α and IL-1β at 6 hours after CLP and significantly decreased local TNF-α and CXCL1, as well as systemic IL-1β and CXCL1, at 24 hours after CLP. The reduced TNF-α, IFN-γ, IL-1β, and CXCL1 concentrations in IL-36α–treated mice after CLP were the consequence of IL-36α–mediated bacterial clearance by macrophages, which resulted in lower bacterial burden, providing a less potent proinflammatory stimulus, leading to reduced tissue inflammation, apoptosis, and injuries and thereby improving survival with sepsis.

Although IL-36 has been demonstrated to act on keratinocytes and other immune cells to induce the expression of inflammatory mediators [11, 12, 27–31], we found that IL-36α did not modulate the production of proinflammatory cytokines in macrophages and neutrophils activated by various Toll ligands relevant to sepsis, including TLR2 and TLR4 ligands. The effects of IL-36α on inflammatory reactions based on other cell or animal models might be different from ours seen with a CLP-induced sepsis model. In any case, the detailed regulatory impact of IL-36α on the expression of cytokines and chemokines during infection and immunity requires further studies.

Collectively, IL-36α provided protection from sepsis-induced lethality after microbial infection via enhanced bacterial clearance, improved organ injury, and decreased apoptosis of immune cells. Our data provide new insight into the IL-36α–driven regulation of anti-infective function by macrophages, which might offer a novel therapeutic target for the treatment of sepsis or other infectious diseases.

Notes

Financial support. This work was supported by the National Natural Science Foundation of China (grants 81572038 and 81370110 to J. C.), Chongqing Science and Technology Commission (grant cstc2014jcyjjq10002 for Distinguished Young Scholars of Chongqing to J. C.), the Chongqing Medical University training program (research grant 201409 to J. C.), and Chongqing Youth Top-notch Talent Support Program (J. C.).

Potential conflicts of interest. All authors: No reported conflicts. 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

Author notes