IL-33 ameliorates experimental colitis involving regulation of autophagy of macrophages in mice
Previously, we have demonstrated that IL-33 administration protecting TNBS-induced experimental colitis is associated with facilitation of Th2/Tregs responses in mice. However, whether IL-33 regulates autophagy to ameliorate experimental colitis is unclear.
IL-33 administration (2 μg/day, intraperitoneal injection), while facilitating Th2/Tregs responses, also enhances the autophagy in mice with TNBS-induced colitis as well as macrophages. In the meantime, we observed that inhibition of the autophagy with 3-methyladenine (3-MA) (24 mg/kg, intraperitoneal injection) in mice exacerbates TNBS-induced experimental colitis. On the contrary, administration of rapamycin (2 mg/kg,intragastric administration), an autophagy-enhancer, alleviates the colitis in mice. In vivo, Immunofluorescence analysis revealed that TNBS combined with IL-33 enhanced the autophagy of macrophages in the inflammatory gut tissue. In vitro, treatment with IL-33 promoted the autophagy of macrophages generated from bone marrow cells in dose-dependant manner. Furthermore, the effect of autophagy-enhancement by IL-33 is TLR4 signaling pathway dependant. Our notion was further confirmed by IL-33-deficient bone marrow-derived macrophages cells.
IL-33 regulates the autophagy is a new immunoregulatory property on TNBS-induced experimental colitis in mice.
KeywordsIL-33 TNBS-induced experimental colitis Autophagy Macrophages TLR4
Interleukin (IL)-33 is a member of the IL-1 family of cytokines. IL-33 is a nuclear protein that is also released into the extracellular space, and thus acts as a dual-function molecule, as does IL-1α. Extracellular IL-33 binds to the cell-surface receptor ST2, leading to the activation of intracellular signaling pathways similar to those used by IL-1. Unlike conventional cytokines, IL-33 might be secreted via unconventional pathways, and can be released upon cell injury as an alarmin . Moreover, like HMGB1 (high-mobility group box 1), IL-33 has been suggested to act as an “alarmin” that amplifies immune responses during tissue injury. In contrast to HMGB1, however, the precise roles of IL-33 in those settings are poorly understood [2, 3].
Autophagy is an evolutionarily conserved lysosomal mechanism that enables cells to conserve and maintain cellular biomass quality and quantity by targeting damaged or unused proteins and even organelles for degradation [4, 5, 6]. Previously, we and others have demonstrated that IL-33 ameliorates experimental colitis through promoting Th2/Foxp3 + regulatory T Cell responses in the experimental colitis in mice [7, 8, 9, 10], and IL-33 expression actually increased in the inflamed mucosa of IBD patients, in particular in UC patients. Furthermore, colonic subepithelial myofibroblasts (SEMFs) is a source of IL-33 in the human colonic mucosa . However, whether IL-33 ameliorates experimental colitis through regulating autophagy is unknown. In this study, we show that TNBS-induced colitis in mice was exacerbated when autophagy was inhibited by 3-MA. On the contrary, administration of TNBS combined with rapamycin, an autophagy-enhancer, the mice displayed less severe colitis as compared with treatment with TNBS alone. Similarly, we demonstrate that IL-33 ameliorates TNBS-induced colitis via enhancing the autophagy. Moreover, IL-33 enhances the autophagy via regulates the TLR4 signaling pathway.
Materials and methods
Male BALB/c mice of 6–8 weeks old were purchased from the Institute of Experimental Animal, Chinese Academy of Medical Sciences (Beijing, China). Male C57/BL6 mice 5–6 weeks of age were purchased from Centers for Disease Control (CDC), Wuhan (Hubei, China). C57BL/10ScNJNju (TLR4−/−) mice were purchased from biomedical research institute (Nanjing, China). IL-33 gene knockout mice with C57BL/6 background were obtained from the laboratory of Dr. Fang Zheng in Department of Immunology, Tongji Medical College, HUST (China). The mice were housed in the SPF facility at the Tongji Medical College for at least 1 week before inclusion in experiments. All of the studies were performed in accordance with the Tongji Medical College Animal Care and Use Committee guidelines.
Reagents and antibodies
2,4,6-trinitrobenzenesulfonic acid (TNBS), 3-methyladenine (3-MA), starvation medium EBSS (Earle’s balanced salt solution), chloroquine and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse IL-33 was purchased from Pepro Tech Inc (London, UK). Antibodies against Beclin-1, LC3B I/II, P62 and β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Induction of TNBS-colitis and treatment with 3-MA or rapamycin
Colitis was induced by TNBS in male BALB/c mice as described elsewhere . In brief, 2.5 mg of TNBS (Sigma-Aldrich Corp., St. Louis, MO) dissolved in 50% ethanol (EtoH) (total volume, 100 μl) was administered intrarectally to lightly anesthetized (methoxyflurane) mice through a polyurethane catheter (Becton–Dickinson, San Jose, CA, USA) equipped with a 1-ml syringe. Control mice received 50% EtoH using the same technique. The body weight and disease activity of each mouse were evaluated every day after TNBS or EtoH administration. All mice were euthanized on 4th day after induction of colitis. Mice were randomly assigned to five groups: the EtoH + PBS group, EtoH + TNBS group, TNBS + 3-MA group (24 mg/kg, intraperitoneal injection), TNBS + IL-33 (2 μg/mouse/day, intraperitoneal injection) and TNBS + Rapamycin group (2 mg/kg,intragastric administration). Each group was consisted of 3–6 mice.
The mice were sacrificed by cervical dislocation. Macroscopic assessment of inflammation was scored as described in previous study . Subsequently, samples of colon tissues were prepared for tissue sections, and then stained with hematoxylin and eosin. Histological analysis was performed as described in previous report .
The formation of autophagosomes in colonic macrophages after TNBS induction with or without IL-33 administration was compared by immunofluorescence assay. Briefly, the sections were deparaffinized, rehydrated and washed in 1% PBS Tween. Then they were treated with 3% hydrogen peroxide, blocked with 5% bovine serum albumin (BSA) and incubated simultaneously with Beclin-1 (1:500, Sigma) and F4/80 (1:500, Abcam) overnight. After staining with the- secondary antibody, the slides were then counter-stained with DAPI for 5 min. Images were acquired by a fluorescence microscope (Olympus, Lake Success, NY). Settings for image acquisition were identical for control and experimental tissues.
Generation of mouse BMs and incubated with IL-33, IL-33 plus chloroquine as well as rapamycin respectively
Mouse bone marrow derived macrophages (BMs) were propagated from bone marrow cells as described previously . Recombinant cytokine M-CSF in vitro experiments were obtained from Peprotech (London, UK). BMs were incubated with IL-33 (100 ng/ml) for 24 h. Experimental groups were designed as follows: 1) PBS control group, 2) IL-33 alone group, 3) IL-33 combined with chloroquine (50 μM). Thereafter, the proteins were extracted for western blotting analysis. Macrophages were treated with rapamycin (500 ng/ml) for 24 h. Thereafter, the cells were lysed for western blot examination.
Colonic samples were homogenized in ice-cold lysis buffer with a protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO). Specimens were then purified by centrifugation at 10,000g for 10 min at 4 °C. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membrane ((EMD Millipore, Billerica, MA). The membrane was incubated overnight in blocking buffer with primary LC3BI/II,P62, Beclin-1 and β-actin antibodies at 4 °C.
The data are presented as mean ± SD. Statistical differences were determined by Student’s t test. Two-sided probability (p) values less than 0.05 were considered significant.
TNBS-induced experimental colitis enhanced the autophagy in intestine
Inhibition of autophagy exacerbated the TNBS-induced experimental colitis
IL-33 attenuated the TNBS-induced experimental colitis through enhancing autophagy
TNBS combined with IL-33 promotes the autophagy of macrophages in inflammatory gut tissue
Treatment of macrophages with IL-33 enhances autophagy of macrophages
IL-33 enhanced the autophagy via regulating TLR4 signaling pathway
Deficiency of IL-33 of bone marrow-derived macrophages displayed dramatically change of autophagy induced by rapamycin treatment
In the present study, we investigated the effects and mechanisms of IL-33 on experimental colitis induced by TNBS in mouse. Consistent with our previous observation that treatment with IL-33 ameliorated the severity of colitis, improved the loss of animal body weight, relieved the activity of illness, and decreased the infiltration of inflammatory leukocytes. A novel finding is that IL-33 ameliorates experimental colitis through enhancing autophagy in mice.
There is a growing interest in the role of autophagy in normal health and in various disease states [15, 16, 17, 18]. Modulation of autophagy is a novel therapeutic strategy for diseases including HIV . What are the possible mechanisms by which IL-33 enhancing autophagy alleviate TNBS-induced experimental colitis? IL-33 localizes to intestinal epithelial cells, and its full-length form is released upon epithelial damage, acting as an “alarmin” and triggering wound healing at the mucosa . Increased mucosal IL-33 in human UC and murine colitis may be a homeostatic response to limit inflammation, potentially through effects on epithelial barrier function . We can hypothesize that that activation of the autophagy of intestinal epithelial cells confers these cells to be resistant to inflammatory insult elicited by TNBS treatment. Furthermore, macrophage is one of the largest population of macrophages resides in the gastrointestinal tract . IL-33 is expressed by CD14+ macrophages , and plays a therapeutic role in autoimmune CNS disease by switching a predominantly pathogenic Th17/Th1 response to Th2 activity, and by polarization of anti-inflammatory M2 macrophage . Likewise, the autophagy-enhanced macrophages by IL-33 could be more polarized to M2 against the TNBS-induced Th1 inflammatory injury in the setting of colitis.
Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL-13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease. It has been shown that autophagy is essential for airway mucus secretion in a type 2, IL-13-dependent immune disease process and the regulation of autophagy by Th2 cytokines is cell-context dependent . In support to our notion, deletion of the IL-33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in IL-33(−/−) mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive life span was shortened to two-thirds of that for IL-33(±) littermates. IL-33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues . However, IL-33 treatment apparently suppressed the expression of pro-inflammation cytokines IL-1β and TNF-α, evidently increased Bcl-2 but decreased cleaved-caspase-3, and obviously decreased the levels of autophagy-associated proteins LC3-II and Beclin-1 but maintained P62 at high level after ICH. On the contrary, treatment with sST2, a decoy receptor of IL-33, exacerbated ICH-induced brain damage and neurological dysfunction by promoting apoptosis, and enhancing autophagic activity . This observation indicates that the regulation of autophagy by IL-33 is also cell-context dependent.
Besides LC3, levels of other autophagy substrates can be used to monitor autophagic flux. p62 (also known as SQSTM1/sequestome 1) is selectively incorporated into autophagosomes through direct binding to LC3 and is efficiently degraded by autophagy . To evaluate the modulation of autophagy by IL-33, we observed that LC3 expression as an indicator of autophagy is better than that of p62 because LC3 is more sensitive to the treatment with IL-33 (Fig. 6). Moreover, there appears a requirement for the concentration of IL-33 to modulate the autophagy of macrophages (Fig. 5).
It has previously been demonstrated that IL-33 increases the expression of the LPS receptor components MD2 (myeloid differentiation protein 2) and TLR-4, the soluble form of CD14 and the MyD88 adaptor molecule. In addition, IL-33 pretreatment of macrophages enhances the cytokine response to TLR-2 but not to TLR-3 ligands. Thus, IL-33 treatment preferentially affects the MyD88-dependent pathway activated by the TLR . In line with these findings, we show that IL-33 also activates the autophagy of macrophages through regulating TLR4 signaling pathway, because TLR4 deficiency abolishes the enhancement of the autophagy of macrophages by IL-33 treatment. Nevertheless,further studies will be required to delineate the specific molecules as well as cells of intestine modulated by IL-33 in the setting of TNBS-induced experimental colitis in mice.
In conclusion, we presented strong evidences to support that IL-33 regulating autophagy plays a role, at least in part, in alleviating the inflammation in the context of TNBS-induced experimental colitis. Therefore, modulation of the autophagy in intestine might have therapeutic implications in colitis.
ZW, LS, SH, CQ performed experiments and analyzed data. MF designed experiments and wrote the paper. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (91542110, 81373167 to M. Fang). We thank Dr. Fang Zheng kindly provided the IL-33 gene knockout mice.
The authors declare that they have no competing interests.
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The study was approved by the Institutional Review Board of Huazhong University of Science and Technology, China.
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