Effect of Interleukin-36β on Activating Autophagy of CD4+CD25+ Regulatory T cells and Its Immune Regulation in Sepsis

Abstract

Background

CD4⁺CD25⁺ regulatory T cells (Tregs) play an essential role in sepsis-induced immunosuppression. How, the effects of interleukin 36 (IL-36) cytokines on CD4⁺CD25⁺ Tregs and their underlying mechanism(s) in sepsis remain unknown.

Methods

Our study was designed to investigate the impacts of IL-36 cytokines on murine CD4⁺CD25⁺ Tregs in presence of lipopolysaccharide (LPS) and in a mouse model of sepsis induced by cecal ligation and puncture (CLP). IL-36–activated autophagy was evaluated by autophagy markers, autophagosome formation, and autophagic flux.

Results

IL-36α, IL-36β, and IL-36γ were expressed in murine CD4⁺CD25⁺ Tregs. Stimulation of CD4⁺CD25⁺ Tregs with LPS markedly up-regulated the expression of these cytokines, particularly IL-36β. IL-36β strongly suppressed CD4⁺CD25⁺ Tregs under LPS stimulation and in septic mice challenged with CLP, resulting in the amplification of T-helper 1 response and the proliferation of effector T cells. Mechanistic studies revealed that IL-36β triggered autophagy of CD4⁺CD25⁺ Tregs. These effects were significantly attenuated in the presence of the autophagy inhibitor 3-methyladenine or Beclin1 knockdown. In addition, early IL-36β administration reduced the mortality rate in mice subjected to CLP. Depletion of CD4⁺CD25⁺ Tregs before the onset of sepsis obviously abrogated IL-36β–mediated protection against sepsis.

Conclusions

These findings suggest that IL-36β diminishes the immunosuppressive activity of CD4⁺CD25⁺ Tregs by activating the autophagic process, thereby contributing to improvement of the host immune response and prognosis in sepsis.

Sepsis is a major cause of death among critically ill patients, and treatments have been limited to date [1]. The essential characteristic of sepsis is persistent immunoparalysis, which is strongly associated with poor prognosis [2–6]. A disordered immune response contributes to the pathogenesis and progression of this lethal complication, according to the new definition of sepsis, although few interventional measures are available, because the specific mechanism(s) have not been clarified.

Emerging evidence has been demonstrated that sepsis-mediated immune dysfunction involves CD4⁺CD25⁺ regulatory T cells (Tregs), which exert dominant negative effect on various autoimmune and infectious diseases [7–9]. Of note, Tregs are elevated in animals and patients with sepsis and cause a decrease in the number and activity of effector T cells [10–13]. It is well known that Tregs express forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). Tregs also produce interleukin 10 (IL-10), interleukin 35, and transforming growth factor (TGF) β1, which inhibit effector T cells [14–17]. Tregs are themselves regulated by several cytokines, and interleukin 33 (also known as interleukin 1F11 [IL-1F11]) is a member of the interleukin 1 (IL-1) family and plays a key role in Treg expansion after sepsis onset [18, 19].

Other cytokines of the IL-1 family that may regulate function of Tregs during sepsis are IL-36α (IL-1F6), IL-36β (IL-1F8), and IL-36γ (IL-1F9) [20, 21]. Collectively referred to as IL-36, these cytokines and their receptor (IL-36R) are extensively expressed by various cells and tissues, and they orchestrate diverse biologic and immune roles in various contexts. It was noted that they were expressed predominantly in murine CD4⁺ T lymphocytes and bone marrow–derived dendritic cells [20–23], and IL-36 triggered the production of proinflammatory cytokines, such as interferon (IFN) γ, interleukin 4 (IL-4), and interleukin 17, and drove the T-helper (Th) 1 polarization of effector T cells. IL-36β was reported to up-regulate the expression of molecular markers of bone marrow–derived dendritic cells, in turn amplifying Th1 response [24]. Stimulating human keratinocytes with IL-1β or tumor necrosis factor α enhanced IL-36α and IL-36γ expression [25–27], and IL-36 levels were increased in chronic kidney disease and human rheumatoid synovial tissues [20, 23] and in the serum of septic mice [28]. Interestingly, the outcomes of these mice were correlated with IL-36 levels.

These results led us to question whether and how IL-36 influences the activity of CD4⁺CD25⁺ Tregs in the development of sepsis. In the current study, we attempted to investigate the potential role and underlying mechanisms of IL-36 in activating autophagy of CD4⁺CD25⁺ Tregs during sepsis because cytokine-dependent autophagy was critically involved in infection, cancer, atherosclerosis, and other diseases [29–33].

METHODS

Animals

Male C57BL/6J mice (6–8 weeks old; 18–22 g) and mice deficient in Beclin1 (Beclin1^+/−; B6.129X1-Becn1tm1Blev/J) were used in our study. All experiments and procedures were conducted according to the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and with the approval of the Scientific Investigation Board of the Chinese PLA General Hospital (see Supplementary Data).

Purification and Culture of CD4⁺ CD25⁺ Tregs and CD4⁺ CD25⁻ T Cells

CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ T cells were isolated from murine splenocytes using a CD4⁺CD25⁺ T-cell isolation kit (Miltenyi Biotec) by strictly after the manufacturer’s instructions (see Supplementary Data). Sorted cells were cultured separately in Roswell Park Memorial Institute 1640 medium. Cells were stimulated with 2 μg/mL soluble anti-CD28 monoclonal antibody and 2 μg/mL soluble anti-CD3 monoclonal antibody (eBioscience). In some experiments, cells were also stimulated with 5 μg/mL LPS (Sigma-Aldrich). After 2 hours, IL-36β (R&D Systems) was added to the medium at concentrations of 50, 100, or 200 ng/mL, and the cultures were incubated for 48 hours. These CD4⁺CD25⁺ Treg cultures were then used for suppressive activity assays and molecular marker measurements (see Supplementary Data). Culture supernatants were harvested for the measurement of cytokines.

Carboxyfluorescein Succinimidyl Ester Staining

The proliferation of sorted CD4⁺CD25⁻ T cells was analyzed using carboxyfluorescein succinimidyl ester (CFSE) staining (Invitrogen), according to the manufacturer’s recommendations (see Supplementary Data).

Suppressive Activity Assay

CD4⁺CD25⁺ Tregs (1 × 10⁵) that had been stimulated with LPS and IL-36β, as described above, were mixed with CD4⁺CD25⁻ T cells (1 × 10⁵) in U-bottom 96-well plates. Mixed cells were suspended in Roswell Park Memorial Institute 1640 medium and treated with 2 μg/mL soluble anti-CD28 monoclonal antibody and 2 μg/mL soluble anti-CD3 monoclonal antibody. These cells were cocultured for 72 hours, and the supernatants were collected for enzyme-linked immunosorbent assay. Then, cells were harvested and determined using flow cytometry. The proliferation of CFSE-labeled CD4⁺CD25⁻ T cells was analyzed using a FlowJo system (BD Bioscience).

Flow Cytometry

Cultured murine CD4⁺CD25⁺ Tregs were examined using a FACSCalibur (BD Bioscience) (see Supplementary Data).

Enzyme-Linked Immunosorbent Assay

IL-10, TGF-β1, IFN-γ, interleukin 2 (IL-2), and IL-4 levels were measured using commercial kits according to the manufacturer’s instructions (ExCell Biology).

Laser Scanning Confocal Microscopy

CD4⁺CD25⁺ Tregs treated in different ways were stained with 4′6-diamidino-2-phenylindole (DAPI) and imaged using a laser scanning confocal microscope (Leica) (see Supplementary Data).

Quantitative Reverse-Transcription Polymerase Chain Reaction

Total messenger RNA of cells was extracted using an RNeasy Mini Kit (Qiagen) and then reverse-transcribed using an iScript kit (Bio-Rad) according to the manufacturer’s instructions. Polymerase chain reaction was performed using a CFX96TM Real-Time PCR Detection System (Bio-Rad) (see Supplementary Data).

Western Blot Analysis

Cells were used to determine the protein expression of LC3, Beclin1, p62, and β-actin (Cell Signaling Technology), IL-36α, IL-36β, and IL-36γ (R&D Systems) (see Supplementary Data).

Transmission Electron Microscopy

CD4⁺CD25⁺ Tregs were fixed with 2.5% glutaraldehyde and stored at 4°C. Samples were dehydrated, embedded, cured, and stained as recommended by the electron microscope manufacturer. Autophagic vacuoles were observed under an electron microscope (JEOL).

Mouse Model of Sepsis Induced by Cecal Ligation and Puncture

Sepsis model was induced by cecal ligation and puncture (CLP), including severe CLP or nonsevere CLP, as described elsewhere [28]. Mice in the CLP group were injected intraperitoneally with 1 μg of IL-36β (R&D Systems) 2 hours before or 2 hours after CLP (severe model). In some experiments, animals were administered 0.25, 1, or 4 μg of IL-36β per animal at 2 hours before CLP (severe model), and other animals were injected intraperitoneally with IL-36R antagonist (IL-36ra; 4 μg per mouse) (R&D Systems) immediately after CLP (nonsevere model) to block IL-36β activity. Control mice received phosphate-buffered saline (PBS) instead of IL-36β.

In Vivo Depletion of CD4⁺CD25⁺ Tregs

Mice were injected intraperitoneally with 200 µg of anti-CD25 antibody (PC61) or rat immunoglobulin G1 (Biolegend) in 200 µL of PBS 48 hours before the CLP procedure (see Supplementary Material).

Statistical Analyses

All data were presented as the mean (with standard deviation) of ≥3 experiments. Statistical analysis was performed using SPSS software (IBM). Differences between groups were assessed for significance using the Brown-Forsythe test or, when appropriate, 1-way analysis of variance followed by the Dunnett test. Survival was analyzed using Kaplan-Meier survival curves, and results for different groups were compared using the log-rank test. Differences were considered significant at P < .05.

Ethics Statement

All experiments and procedures were conducted according to the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and with the approval of the Scientific Investigation Board of the Chinese PLA General Hospital, Beijing, China.

RESULTS

IL-36 Expression in Murine CD4⁺CD25⁺ Tregs After LPS Stimulation

In the present study, we found that IL-36R, IL-36α, IL-36β, and IL-36γ were positively expressed in CD4⁺CD25⁺ Tregs. Stimulation with 5 μg/mL LPS for 48 hours obviously enhanced the expression of 3 subtypes of cytokines, especially IL-36β, relative to the expression in controls treated with PBS (Supplementary Figure 1C–1H). IL-36β expression did not significantly increase after a 12-hour stimulation with 5 μg/mL LPS or a 48-hour stimulation with 0.5 μg/mL LPS (Supplementary Figure 1A and 1B).

Benefits of IL-36β Treatment on the Survival Rate in Septic Mice

To examine whether the effects of IL-36β on CD4⁺CD25⁺ Tregs were positive or negative for septic outcomes, we further investigated the influence of IL-36β on the survival rate of mice subjected to CLP (lethal model). The results showed that IL-36β significantly reduced the CLP-induced mortality rate when administered 2 hours before CLP (Figure 1A) but not when administered 2 hours after it (Figure 1B). These results suggest that IL-36β, when delivered early, can exert therapeutic benefits against septic challenge. In addition, to evaluate the potential detrimental impacts of blocking IL-36β on sepsis, we established a nonsevere model of CLP followed by IL-36ra administration, revealing that the CLP-associated mortality rate was higher after treatment with IL-36ra (Figure 1C).

CD4⁺CD25⁺ Tregs Required for IL-36β–Mediated Protection Against Sepsis

We further assessed the role of CD4⁺CD25⁺ Tregs in IL-36β–mediated protection against sepsis in vivo. Anti-CD25 antibody (PC61) treatment resulted in a dramatic reduction in splenic proportion of CD4⁺CD25⁺Tregs (Figure 2A and 2B). Meanwhile, blocking CD4⁺CD25⁺ Tregs with PC61 obviously aggravated the inhibition of CD4⁺CD25⁺ Tregs via IL-36β in the CLP group (Figure 2C-2J). Notably, depletion of CD4⁺CD25⁺ Tregs markedly worsened the survival of septic mice pretreated with IL-36β (Figure 2K), indicating that CD4⁺CD25⁺ Tregs were required for IL-36β–mediated protection against septic challenge.

Effects of IL-36β on the Immune Activity of CD4⁺CD25⁺ Tregs

Foxp3 and CTLA-4 are essential biomarkers of Tregs. Treatment of CD4⁺CD25⁺ Tregs with IL-36β at concentrations of 100–200 ng/mL for 24–72 hours markedly reduced the expression of these biomarkers, whereas treatment with a concentration of 50 ng/mL had little effect on their expressions (Figure 3A, 3B, and 3E). The greatest down-regulation of Foxp3 and CTLA-4 was observed after 48-hour incubation with 100 ng/mL IL-36β. Conversely, LPS stimulation enhanced CTLA-4 and Foxp3 expressions in CD4⁺CD25⁺ Tregs (Figure 3C and 3F). A less prominent up-regulation was observed when cells were incubated with LPS at 5 μg/mL in combination with IL-36β at 100 or 200 ng/mL (Figure 3C and 3F). Mice pretreated with IL-36β before CLP showed significantly lower levels of CTLA-4 and Foxp3 in CD4⁺CD25⁺ Tregs at 48 hours after CLP than mice without IL-36β treatment (Figure 3D and 3G).

Tregs can produce potent anti-inflammatory activities, including TGF-β1 and IL-10; thus, we determined the release of these cytokines after exposure to IL-36β. Treatment of CD4⁺CD25⁺ Tregs with IL-36β at 100 or 200 ng/mL decreased levels of TGF-β1 and IL-10 at 24, 48, and 72 hours (Figure 3H, 3I, 3K, and 3L). LPS stimulation strongly increased TGF-β1 and IL-10 production at 48 hours, and the release of both cytokines was lower when Tregs were treated with IL-36β and LPS (Figure 3J and 3M). Consistent with these in vitro results, CLP per se elevated TGF-β1 and IL-10 levels in mice (Figure 3N and 3O), whereas lower levels of TGF-β1 and IL-10 were observed after IL-36β treatment in sepsis (Figure 3N and 3O).

Regulation of IL-36β in CD4⁺CD25⁺ Tregs in Mediating Immunosuppression

CD4⁺CD25⁺ Tregs were pretreated with IL-36β for 48 hours and then mixed with CD4⁺CD25⁻ T cells at a ratio of 1:1. The cells were cocultured for 72 hours, after which CD4⁺CD25⁻ T-cell proliferation was measured by CFSE staining; CD4⁺CD25⁻ T-cell proliferation was obviously inhibited. In contrast, coculture of T lymphocytes with CD4⁺CD25⁺ Tregs pretreated with IL-36β at 100 or 200 ng/mL induced greater lymphocyte proliferation than the coculture of T lymphocytes with CD4⁺CD25⁺ Tregs pretreated with PBS (Figure 4A and 4D). LPS stimulation (5 μg/mL for 48 hours) augmented the immunosuppressive activity of CD4⁺CD25⁺ Tregs, and these effects were dampened when CD4⁺CD25⁺ Tregs were treated with IL-36β (100 ng/mL) (Figure 4B and 4E). Consistent with these in vitro results, we found that CD4⁺CD25⁺ Tregs isolated from mice in the CLP group treated preoperatively with IL-36β promoted CD4⁺CD25⁻ T-cell proliferation (Figure 4C and 4F).

Cytokine levels (IL-2, IL-4, and IFN-γ) in the supernatant of coculture of CD4⁺CD25⁺ Tregs (pretreated with IL-36β) with CD4⁺CD25⁻ T cells were determined at 72 hours. IL-2 levels were substantially higher after treatment with 100 or 200 ng/mL IL-36β, but not 50 ng/mL (Figure 4G). Similarly, IL-36β at 100 or 200 ng/mL significantly reduced the ratio of IL-4 to IFN-γ, which indicated a change in T-cell polarization (Figure 4H). In addition, we noted that the coculture of LPS-stimulated CD4⁺CD25⁺ Tregs with T cells decreased IL-2 levels and increased the ratio of IL-4 to IFN-γ in T cells, indicating a predominant Th2 response (Figure 4I and 4J). However, treatment with IL-36β markedly reversed these effects, promoting a Th1 response in T lymphocytes. Similar changes in IL-2 levels and the IL-4/IFN-γ ratio were observed in mice in the CLP group after IL-36β administration (Figure 4K and 4L).

Effect of IL-36β Blockade the Ability of IL-36β to Inhibit CD4⁺CD25⁺ Tregs

To confirm the impact of IL-36β on the immunosuppressive activity of CD4⁺CD25⁺ Tregs in sepsis, we stimulated cells with LPS, followed by treatment with IL-36β and IL-36ra (a natural IL-36β inhibitor). IL-36ra treatment prevented IL-36β–mediated suppressive activity of CD4⁺CD25⁺ Tregs, based on the analysis of CTLA-4 and Foxp3 expression, the production of IL-10, TGF-β1, IL-2, IL-4, and IFN-γ, and the proliferation of CD4⁺CD25⁻T cells cocultured with CD4⁺CD25⁺ Tregs (Figure 5A, 5C, 5D, 5G, 5H, 5K, 5M, and 5O). Similarly, injection of IL-36ra into mice subjected to CLP remarkably diminished the ability of IL-36β to inhibit the activity of CD4⁺CD25⁺ Tregs (Figure 5B, 5E, 5F, 5I, 5J, 5L, 5N, and 5P).

Activation of Autophagy by IL-36β in Murine CD4⁺CD25⁺ Tregs

CD4⁺CD25⁺ Tregs were exposed to IL-36β at 50, 100, and 200 ng/mL for 24, 48, and 72 hours, and the expression levels of the autophagy markers LC3-II, Beclin1, and p62 were assessed. IL-36β obviously increased the levels of all these markers (Figure 6A and 6B), and this up-regulation was associated with more numerous LC3 puncta and higher Beclin1 fluorescence in cells under confocal microscopy (Figure 6F and 6G). Moreover, IL-36β colocalized with Beclin1 in LPS-stimulated CD4⁺CD25⁺ Tregs (Figure 6H). Autophagy was confirmed by imaging autophagosomes and autophagic vacuoles under transmission electron microscopy (Figure 6I). These data indicated that IL-36β could activate autophagy in CD4⁺CD25⁺ Tregs, which likely explained at least in part how the cytokine inhibited the activity of Tregs in the septic response.

Next, we examined whether IL-36β affected autophagic flux in CD4⁺CD25⁺ Tregs. Cells were treated with bafilomycin A1, which inhibited fusion between autophagosomes and lysosomes. CD4⁺CD25⁺ Tregs treated with bafilomycin A1 and IL-36β showed higher expression of LC3-II, Beclin1, and p62 than cells treated with IL-36β alone (Figure 6C–6G). Elevated p62 indicates enhanced autophagic flux because p62 delivers cytoplasmic cargo to LC3 and targets them for degradation by autophagy.

Blockade of Autophagy and the Ability of IL-36β to Inhibit CD4⁺CD25⁺ Tregs

In support of our hypothesis that IL-36β regulates Tregs by activating autophagy, we noticed that blocking autophagy in CD4⁺CD25⁺ Tregs with 3-methyladenine obviously attenuated the inhibition of CD4⁺CD25⁺ Tregs by IL-36β (Supplementary Figure 2). These results were obtained in CD4⁺CD25⁺ Tregs stimulated with LPS and in mice subjected to CLP.

As a complementary method to inhibit autophagy and further verify our hypothesis, we examined the inhibition of CD4⁺CD25⁺ Tregs by IL-36β in mice deficient in Beclin1 (Beclin1^+/−), which was needed for efficient autophagy activation. IL-36β suppressed the activity of wild-type CD4⁺CD25⁺ Tregs to a greater extent than that of Beclin1^+/− CD4⁺CD25⁺ Tregs based on in vitro and in vivo experiments (Figure 7). These results were consistent with the notion that IL-36β inhibited the activity of CD4⁺CD25⁺ Tregs, at least in part by inducing autophagy.

DISCUSSION

Sepsis is defined as life-threatening organ dysfunction due to a dysregulated host response to infection [1], and the World Health Organization has declared it an essential medical disorder of the 21st century because of the limited available treatments and poor prognosis [34]. Tailored immunotherapies have been increasingly deemed a promising approach for the treatment of sepsis. Sepsis is essentially triggered by microbial infection, leading to an aberrant host response distinct from immune dysfunction or immunoparalysis. Notably, Tregs play a central role in sepsis-driven immunosuppression. The numbers of Tregs were remarkably increased, and these Tregs reduced the number of effector T cells, resulting in immune disorder [35–38]. In the current study, we identified the novel cytokine IL-36β as a potent immune regulator in sepsis that mitigated CD4⁺CD25⁺ Treg-mediated immunosuppression (Supplementary Figure 3).

IL-36, the most recently characterized cytokine, is abundantly expressed in various tissues and cells after stimulation with cytokines, bacteria, Toll-like receptor agonists, rhinovirus or other pathogens [39–45], although IL-36 expression in CD4⁺CD25⁺ Tregs remains unexplored thus far. However, there has been growing interest in studying IL-36, which has emerged as a potent regulator in numerous inflammatory and autoimmune diseases. Tao et al [28] recently reported that IL-36 release was obviously increased in sepsis and was tightly associated with prognosis, suggesting that it might be involved in the pathogenesis of sepsis. Nevertheless, the potential role and underlying mechanisms of IL-36 in the mediated host immune response remain to be elucidated in the context of sepsis.

In the present study, our findings of IL-36 expression in CD4⁺CD25⁺ Tregs extend the list of cells mentioned above. In addition, we noted that LPS stimulation of CD4⁺CD25⁺ Tregs significantly up-regulated all 3 IL-36 forms, with the greatest increase in IL-36β. Moreover, it was revealed that IL-36β could inhibit the immunosuppressive activity of CD4⁺CD25⁺ Tregs and augment the proliferation of effector T cells, which was in accordance with reports showing that IL-36α promoted phagocytosis and the killing of macrophages, helped clear bacteria, and mitigated tissue inflammation and damage [28]. We further confirmed that all of these effects of IL-36β could be reversed by treatment with the natural IL-36β inhibitor IL-36ra, thus providing novel evidence for the suppressive response of IL-36β on CD4⁺CD25⁺ Tregs.

What are the underlying mechanisms by which IL-36β modulates the immune function of CD4⁺CD25⁺ Tregs in sepsis? Our results suggested that autophagy induction in CD4⁺CD25⁺ Tregs might be a major mechanism by which IL-36β regulates these cells and improves the outcome of sepsis. It is now evident that autophagy plays an essential role in the growth, development, and function of T lymphocytes, Tregs, and B lymphocytes [46–48]. Autophagy is likely critically involved in immunosuppression during the pathophysiological alterations associated with sepsis [49], in which autophagy can play a harmful or beneficial role for the patient depending on the context [50].

On the one hand, autophagy can preserve immune homeostasis, increase survival of immune cells and decrease multiple organ injuries. On the other hand, extensive autophagy or impaired autophagic flux can lead to type II programmed cell death. Accumulating evidence has indicated that autophagy is activated by diverse stimuli, such as cytokines, LPS, high-mobility group box-1 protein, starvation, and other physiological or pathological conditions. For example, IFN-γ was shown to trigger autophagy to eliminate invading pathogens. Subsequent research found that tumor necrosis factor α, IL-1, interleukin 6, and interleukin 17 were autophagic inducers, whereas IL-4, IL-10, interleukin 13, and interleukin 33 could block autophagy [33].

Herein, we revealed that IL-36β markedly triggered autophagy in CD4⁺CD25⁺ Tregs, which was closely associated with the enhanced expression of key autophagy markers, LC3-II, Beclin1, and p62, as well as elevated autophagic flux. To our knowledge, we are the first to report that IL-36β can serve as an important inducer to activate autophagy subsequent to sepsis. In addition, we demonstrated that the effects of IL-36β on CD4⁺CD25⁺ Tregs were reversed by chemical (3-methyladenine) or genetic (Beclin1 knockdown) blockage of autophagy. These data suggest that IL-36β negatively regulates the suppressive property ofCD4⁺CD25⁺ Tregs via autophagy induction in sepsis. More importantly, the present study shed light on the fact that treatment with IL-36β was associated with a higher survival rate in mice subjected to polymicrobial sepsis, suggesting that IL-36β–induced autophagy in CD4⁺CD25⁺ Tregs might exert a beneficial impact on the prognosis of septic complications. In addition, it revealed a novel role of IL-36β in regulating immune functions of CD4⁺CD25⁺ Tregs and CD4⁺CD25⁺ Tregs were required for IL-36β–mediated protection against septic challenge, which might provide a potential therapeutic target for the treatment of sepsis.

The present study has several limitations. We verified the potential influence of IL-36β by IL-36ra, but the effectiveness of IL-36β knockdown should be evaluated in septic mice. Instead of an anti-CD25 antibody, the role of conditional Foxp3 knockout should be explored in septic mice treated with IL-36β. In fact, our results here with murine CD4⁺CD25⁺ Tregs need to be confirmed in human Tregs. Future studies are warranted to assess the impact and mechanism of IL-36β on other potential targets of CD4⁺CD25⁺ Tregs (eg, lymphocyte activation gene-3, perforin, and granzymes), and other T-cell subsets.

In conclusion, our studies of murine CD4⁺CD25⁺Treg cultures in vitro and mice subjected to CLP as a sepsis model in vivo suggest that IL-36 is positively expressed in CD4⁺CD25⁺ Tregs and down-regulates the immunosuppressive activity of CD4⁺CD25⁺ Tregs by inducing the autophagic response. These effects of IL-36β can be reversed by blocking IL-36β or autophagy. Importantly, early treatment with IL-36β can significantly improve the survival rate of mice with sepsis, and CD4⁺CD25⁺ Tregs seem to be required for the beneficial effect of IL-36β.

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

Author contributions. Y. G. and Y. M. Y. and designed the study. Y. G., M. H., and N. D. performed the experiments and analyzed the results. Y. G. and M. H. wrote the first draft of the manuscript, which Y. M. Y. revised. All authors read and approved the final manuscript.

Financial support. This work was supported by the National Natural Science Foundation of China (grants 81873946, 81974293, and 81730057), the Key Project of Military Medical Innovation Program of Chinese PLA (grant 18CXZ026), the National Key Research and Development Program of China (grant 2017YFC1103302), and the Natural Science Foundation of Zhejiang Province (grant LY20H150011).

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