Active secretion of IL-33 from astrocytes is dependent on TMED10 and promotes central nervous system homeostasis.

Interleukin-33 (IL-33), secreted by astrocytes, regulates the synapse development in the spinal cord and hippocampus and suppresses autoimmune disease in the central nervous system (CNS). However, the mechanism of unconventional protein secretion of this cytokine remains unclear. In this study, we found that IFN-γ promotes the active secretion of IL-33 from astrocytes, and the active secretion of IL-33 from cytoplasm to extracellular space was dependent on interaction with transmembrane emp24 domain 10 (TMED10) via the IL-1 like cytokine domain in astrocytes. Knockout of Il-33 or its receptor St2 induced hippocampal astrocyte activation and depressive-like disorder in naive mice, as well as increased spinal cord astrocyte activation and polarization to a neurotoxic reactive subtype and aggravated passive experimental autoimmune encephalomyelitis (EAE). Our results have identified that IL-33 is actively secreted by astrocytes through the unconventional protein secretion pathway facilitated by TMED10 channels. This process helps maintain CNS homeostasis by inhibiting astrocyte activation.

Local Stimulation of Liver Sinusoidal Endothelial Cells with a NOD1 Agonist Activates T Cells and Suppresses Hepatitis B Virus Replication in Mice.

Functional maturation of liver sinusoidal endothelial cells (LSECs) induced by a NOD1 ligand (diaminopimelic acid [DAP]) during viral infection has not been well defined. Thus, we investigated the role of DAP-stimulated LSEC maturation during hepatitis B virus (HBV) infection and its potential mechanism in a hydrodynamic injection (HI) mouse model. Primary LSECs were isolated from wild-type C57BL/6 mice and stimulated with DAP in vitro and in vivo and assessed for the expression of surface markers as well as for their ability to promote T cell responses via flow cytometry. The effects of LSEC maturation on HBV replication and expression and the role of LSECs in the regulation of other immune cells were also investigated. Pretreatment of LSECs with DAP induced T cell activation in vitro. HI-administered DAP induced LSEC maturation and subsequently enhanced T cell responses, which was accompanied by an increased production of intrahepatic cytokines, chemokines, and T cell markers in the liver. The HI of DAP significantly reduced the HBsAg and HBV DNA levels in the mice. Importantly, the DAP-induced anti-HBV effect was impaired in the LSEC-depleted mice, which indicated that LSEC activation and T cell recruitment into the liver were essential for the antiviral function mediated by DAP application. Taken together, the results showed that the Ag-presenting ability of LSECs was enhanced by DAP application, which resulted in enhanced T cell responses and inhibited HBV replication in a mouse model.

Toll-like receptor 2 deficiency promotes the generation of alloreactive Th17 cells after cardiac transplantation in mice.

The emergence of alloreactive Th17 cells that mediate allograft rejection has provided an impetus to understand the factors affecting the generation of Th17 cells in allograft transplantation. How toll-like receptor 2 (TLR2) signalling regulates the generation of Th17 cells upon alloantigen stimuli remains unclear. In this study, we used a mouse model of cardiac allograft transplantation to investigate whether TLR2 signalling influences the development of Th17 cells. Here, we demonstrate that the TLR2-deficient recipient mice show high Th17 cells, both in spleens and allografts, as well as higher infiltrating inflammatory leukocytes in cardiac allografts compared to wild-type control recipient mice. mRNA expression of IL-17, IL-6, TNF-α, CCR6 and CCL20 within the allografts is markedly increased in TLR2-deficient recipient mice compared to wild-type recipient mice. In addition, TLR2 deficiency leads to upregulation of Signal transducer and activator of transcription 3 (STAT3) phosphorylation in both spleens and allografts. In an in vitro experiment, a mixed lymphocyte reaction was assessed, which further confirmed that TLR2 deficiency leads to a significant increase in the generation of Th17 cells compared with wild-type controls. Furthermore, IL-6 secreted by the dendritic cells of TLR2-deficient mice contributes to driving the generation of these Th17 cells. These results suggest that TLR2 signalling is important in regulating the development of Th17 cells after cardiac allograft transplantation.

CD8+ T cell/IL-33/ILC2 axis exacerbates the liver injury in Con A-induced hepatitis in T cell-transferred Rag2-deficient mice.

BACKGROUND: Previous studies showed that CD4+ T cells play a critical role in Con A-induced hepatitis in wild-type mice. However, the role of CD8+ T cells in the setting of Con A-induced hepatitis is enigmatic. The aim of study is to investigate the function of CD8+ T cells in the context of Con-A-induced hepatitis. MATERIALS AND SUBJECTS: Two different mouse models of Con A-induced hepatitis, T cell-transferred Rag2-/- mice and wild-type C57BL/6 mice, were used in the present study. IL-33 gene knockout mice were used to confirm the role of alarmin in Con A-induced hepatitis. RESULTS: Opposing to the previous results obtained in wild-type mice, transferred CD4+ T cells alone into Rag2-knockout mice cannot cause hepatitis upon Con A challenge. In stark contrast, transferred CD8+ T cells play an important role in the pathogenesis of Con A-induced liver injury in T cell-transferred Rag2-deficient mice. Furthermore, we found that hepatocytes injured by perforin-based CD8+ T cell cytotoxicity release the alarmin IL-33. This cytokine promotes ST2+ ILC2 development and the secretion of cytokines IL-5 and IL-13 to mediate liver inflammation triggered by Con A challenge. In addition, these type 2 cytokines, including those originated from CD4+ T cells, result in eosinophils accumulation in liver to exacerbate the liver injury after Con A administration. CONCLUSION: Our data for the first time revealed that CD8+ T cells play an indispensable role in the pathogenesis of Con A-induced liver injury in T cell-transferred Rag2-deficient mice. Therefore, the CD8+ T cell/IL-33/ILC2 axis is a potential therapeutic target for acute immune-mediated liver injury.

Kupffer-cell-expressed transmembrane TNF-α is a major contributor to lipopolysaccharide and D-galactosamine-induced liver injury.

Tumor necrosis factor (TNF)-α exists in two bioactive forms, a 26-kDa transmembrane form (tmTNF-α) and a 17-kDa soluble form (sTNF-α). sTNF-α has been recognized as a key regulator of hepatitis; however, serum sTNF-α disappears in mice during the development of severe liver injury, and high levels of serum sTNF-α do not necessarily result in liver damage. Interestingly, in a mouse model of acute hepatitis, we have found that tmTNF-α expression on Kupffer cells (KCs) significantly increases when mice develop severe liver injury caused by lipopolysaccharide (LPS)/D-galactosamine (D-gal), and the level of tmTNF-α expression is positively related to the activity of serum transaminases. Therefore, we hypothesized that KC-expressed tmTNF-α constitutes a pathomechanism in hepatitis and have explored the role of tmTNF-α in this disease model. Here, we have compared the impact of KCs(tmTNFlow) and KCs(tmTNFhigh) on acute hepatitis in vivo and ex vivo and have further demonstrated that KCs(tmTNFhigh), rather than KCs(tmTNFlow), not only exhibit an imbalance in secretion of pro- and anti-inflammatory cytokines, favoring inflammatory response and exacerbating liver injury, but also induce hepatocellular apoptosis via tmTNF-α and the expression of another pro-apoptotic factor, Fas ligand. Our data suggest that KC(tmTNFhigh) is a major contributor to liver injury in LPS/D-gal-induced hepatitis.

Basophil activation through ASGM1 stimulation triggers PAF release and anaphylaxis-like shock in mice.

Basophils have been shown to contribute to anaphylaxis through either an IgE-FcεRI-dependent pathway or an IgG-FcγR pathway. However, it remains largely unclear whether basophils can be activated to promote anaphylaxis via a non-FcR pathway as well. The glycolipid receptor ASGM1 (Asialoganglioside gangliotetraosylceramide), which has an exposed GalNAcß1-4Gal moiety and serves as a receptor for pathogen associated molecular patterns such as flagellin, was recently found to be expressed on basophils. Here, we demonstrate that stimulation of basophils with anti-ASGM1 antibodies promotes platelet-activating factor (PAF) secretion in vitro and in vivo. Moreover, we found that ASGM1 stimulation triggers basophil- and PAF-dependent anaphylactic shock in pertussis toxin (PTX)-pretreated mice. Thus, ASGM1 has a crucial role in basophil activation and basophil-mediated anaphylaxis-like shock in mice, especially when the vascular permeability is increased by PTX treatment. Our findings describe a novel anaphylaxis-associated pathway that is antigen-, antibody-, and FcR-independent.

Preliminary study of high mobility group box chromosomal protein 1(HMGB1) in ankylosing spondylitis patients.

OBJECTIVES: To compare the serum levels of high mobility group box chromosomal protein 1 (HMGB1) between patients with AS and healthy controls, and evaluate its association with disease activities and functional abilities; to investigate the cell surface receptors related to HMGB1 in AS patients. METHODS: The HMGB1 serum levels from71 previously untreated AS patients and 40 healthy controls were detected by ELISA method. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Ankylosing Spondylitis Disease Activity Score (ASDAS), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocytesedimentationrate (ESR), and C-reactive protein (CRP) levels were assessed on these participants. The mRNA expression of HMGB1 and its relevant cell surface receptors RAGE, TLR2, TLR4, and IL-1Racp complex were analysed by RT-PCR. RESULTS: The HMGB1 serum levels from AS patients were significantly higher than those from healthy controls and remarkably positive correlated with BASDAI, ASDAS, BASFI, CRP, and ESR. ASDAS showed more correlated to HMGB1 serum levels than BASDAI. Besides, the expression of TLR2, TLR4, and IL-1Racp from PBMCs revealed significant correlations with the expression of HMGB1. CONCLUSIONS: HMGB1 might be a good laboratory index for the evaluation of disease activities and disease severity in AS patients. Further, extracellular HMGB1 play its inflammatory role mainly via the expression of cell surface receptors TLR2, TLR4 and IL-1RAcP complex.

Carbon monoxide potently prevents ischemia-induced high-mobility group box 1 translocation and release and protects against lethal renal ischemia-reperfusion injury.

High-mobility group box 1 (HMGB1) is a chromatin-binding nuclear molecule that has potent proinflammatory effects once released by damaged cells. In some disease models, carbon monoxide (CO) exhibits anti-inflammatory and protective properties. Here, we investigated whether the protective effect of CO on renal ischemia-reperfusion injury is associated with the inhibition of HMGB1 translocation and release. A renal ischemia-reperfusion injury model was established with a 100% mortality rate in untreated mice. Pretreatment with the CO-releasing molecule-2 (CORM-2) resulted in 100% survival, maximal preservation of renal function, a marked reduction in pathological damage, and blunted upregulation of TLR4, RAGE, TNF-α, IL-1ß, IL-6, and MCP1 mRNA. Interestingly, CORM-2 pretreatment almost completely inhibited ischemia-induced HMGB1 nucleocytoplasmic shuttling and release. This inhibition was associated with a decrease in nuclear histone acetyltransferase activity. Indeed, CORM-2 pretreatment inhibited the acetylation and release of HMGB1 during hypoxic culture of primary mouse renal tubular epithelia cells in vitro. Using the same renal ischemia-reperfusion injury model, neutralization of HMGB1 was protective, and administration of exogenous HMGB1 largely reversed the protective effect of CORM-2 on kidney ischemia-reperfusion injury. Thus, CORM-2-delivered CO protects against lethal renal ischemia-reperfusion injury. This protection is correlated with the prevention of HMGB1 nuclear-cytoplasmic translocation and release.

Suppressor of cytokine signaling 3 (SOCS3) gene transfer prolongs the survival of the murine cardiac allograft by attenuating interleukin-17-producing alloreactive T-cell responses.

BACKGROUND: Suppressor of cytokine signaling 3 (SOCS3) is the main negative feedback regulator of cytokine signals. We investigated the hypothesis that overexpression of SOCS3 in a murine cardiac allograft transplantation model may result in a survival advantage of the allograft by attenuating alloreactive T-cell responses. METHODS: With the use of BALB/c (H-2(d) ) donor mice and C57Bl/6j (H-2(b) ) recipient mice, the murine cardiac transplantation model was established. Recipient mice received a tail intravenous injection with eukaryotic expression plasmid pEF-FLAG-I/mSOCS3 before and after transplantation. The heart beat of the grafts and immune responses were monitored. RESULTS: Overexpression of SOCS3 within grafts and spleens can prolong the survival time of cardiac allografts by attenuating infiltration of inflammatory cells such as T cells and macrophages in the grafts, decreasing the number of CD4(+) IL-17(+) cells and CD8(+) IL-17(+) cells in spleens, as well as reducing the expression of STAT3 in grafts and phosphorylation of STAT3 in both grafts and spleens. CONCLUSIONS: Overexpression of SOCS3 significantly delays cardiac allograft acute rejection, which is associated with reduced allograft proinflammatory leukocyte infiltration and impaired alloreactive IL-17(+) T cell immunity.

MBD2 regulates TH17 differentiation and experimental autoimmune encephalomyelitis by controlling the homeostasis of T-bet/Hlx axis.

Unlike genetic alterations, epigenetic modifications are reversible and amenable to pharmacological interventions, which make them appealing targets for clinical therapy. However, little is known about epigenetic regulation in experimental autoimmune encephalomyelitis (EAE). Here we demonstrated that methyl-CpG-binding domain protein 2 (MBD2), an epigenetic regulator, controls autoimmunity and EAE through T-bet/Hlx. Tbx21 and Hlx underwent a DNA methylation turnover upon polarizations and a unique methylation pattern was essential for TH17 development. Loss of Mbd2 resulted in a defect for reading the information encoded by this methylation turnover, which disrupted the homeostasis of T-bet/Hlx axis and suppressed TH17 differentiation. DNA demethylation induced similar effect on helper T cell differentiation. Therefore, Mbd2(-/-) mice were completely protected from EAE. Pathogenic splenocytes isolated from wild-type mice challenged with MOG35-55 could adoptively transfer disease to Mbd2(-/-) mice. In addition, Mbd2(-/-) mice reconstituted with unstimulated wild-type splenocytes developed EAE as wild-type mice did. These data would provide novel insights into epigenetic regulation of EAE.

In vitro differentiation of human adipose tissue-derived stem cells into islet-like clusters promoted by islet neogenesis-associated protein pentadecapeptide.

Human adipose tissue-derived stem cells (hASCs) are considered an ideal tool for the supply of insulin-producing cells to treat diabetes mellitus, with high differentiation efficiency. Islet neogenesis-associated protein (INGAP) is an initiator of islet neogenesis, and the peptide sequence comprising amino acids 104-118, named INGAP pentadecapeptide (INGAP-PP), has been shown to increase ß-cell mass in animals and human pathological states. Here, we report a novel 4-step method to promote hASCs to differentiate into islet-like clusters (ILCs) more efficiently by adding INGAP-PP. The hASCs were isolated, purified and differentiated using a 4-step protocol including trichostatin A, INGAP-PP/scrambled peptide (Scrambled-P), dexamethasone, nicotinamide, glucagon-like peptide-1, transforming growth factor ß1 and exendin-4. Results showed that ILCs in the INGAP-PP group were more similar to the fresh islets with regard to both size and morphology and expressed significantly higher levels of both insulin and C-peptide than those in the Scrambled-P group. Moreover, the ILCs from the INGAP-PP group secreted higher levels of insulin and C-peptide than those from the Scrambled-P group in response to both a low (5.6 mM) and high (25 mM) glucose challenge and secreted 6 times more hormones under the high-glucose challenge. Real-time PCR and immunocytochemistry showed that ILCs of the INGAP-PP group expressed human pancreatic endocrine hormones and transcription factors. Transplantation of ILCs into diabetic rats partially reversed diabetes and prolonged their life span. In conclusion, the INGAP-PP protocol can efficiently induce hASCs to differentiate into ILCs in vitro, and thus hASCs could be a promising source of cells for transplantation to treat diabetes mellitus.

High-mobility group box 1 accelerates early acute allograft rejection via enhancing IL-17+ γδ T-cell response.

Th17 and γδ T cells are the dominant IL-17-producing cell. We previously reported that high-mobility group box 1 (HMGB1) is critical in inducing IL-17-producing alloreactive T cells during early stage of acute allograft rejection. However, the role of γδ T cells during this process and its implication in HMGB1-mediated allograft rejection are not fully understood. Here, we use a murine model of cardiac allograft transplantation to further study the role of HMGB1 and IL-17-producing γδ T cells in acute allograft rejection. It was found that the expression of HMGB1 was increased in allograft, while blockade of HMGB1 suppressed IL-17(+) γδ T-cell response and inhibited the gene transcription of IL-23 and IL-1ß. Furthermore, in vitro HMGB1 indirectly promoted the development of IL-17(+) γδ T cells by stimulating dendritic cells to produce IL-23 and IL-1ß, meanwhile depletion of γδ T cells in vivo prolonged allograft survival and reduced the level of IL-17 in serum. In conclusion, our findings inferred that increased HMGB1 expression could enhance IL-17(+) γδ T-cell response by promoting the secretion of IL-23 and IL-1ß, while IL-17(+) γδ T cells contribute to the early stage of acute allograft rejection.

Extracellular acidosis modulates the endocytosis and maturation of macrophages.

Extracellular acidosis is involved in various pathological situations of central nervous system and the effects are largely mediated by acid sensing ion channels (ASICs). However, it remains unclear whether extracellular acidosis affects immune cells. Macrophages are immune cells that play important role in immune reactions. In this study we investigated the impact of extracellular acidosis on the function of bone marrow derived macrophages (BMMs). The results showed that extracellular acidosis upregulated the endocytosis, surface molecular expression and interleukin-10 secretion of BMMs, in which the expression of ASIC1 and ASIC3 was detected. Notably, extracellular acidosis stimulated endocytosis and upregulation of surface molecules expression in BMMs could be abolished by amiloride, a blocker of ASICs, and nonsteroid anti-inflammatory drugs. Our findings provide new insight into the role of extracellular acidosis in the regulation of immune function and suggest ASICs as new targets for the modulation of immune response.

The role of high mobility group box chromosomal protein 1 in rheumatoid arthritis.

High mobility group box chromosomal protein 1 (HMGB1) is a ubiquitous highly conserved single polypeptide in all mammal eukaryotic cells. HMGB1 exists mainly within the nucleus and acts as a DNA chaperone. When passively released from necrotic cells or actively secreted into the extracellular milieu in response to appropriate signal stimulation, HMGB1 binds to related cell signal transduction receptors, such as RAGE, TLR2, TLR4 and TLR9, and becomes a proinflammatory cytokine that participates in the development and progression of many diseases, such as arthritis, acute lung injury, graft rejection immune response, ischaemia reperfusion injury and autoimmune liver damage. Only a small amount of HMGB1 release occurs during apoptosis, which undergoes oxidative modification on Cys106 and delivers tolerogenic signals to suppress immune activity. This review focuses on the important role of HMGB1 in the pathogenesis of RA, mainly manifested as the aberrant expression of HMGB1 in the serum, SF and synovial tissues; overexpression of signal transduction receptors; abnormal regulation of osteoclastogenesis and bone remodelling leading to the destruction of cartilage and bones. Intervention with HMGB1 may ameliorate the pathogenic conditions and attenuate disease progression of RA. Therefore administration of an HMGB1 inhibitor may represent a promising clinical approach for the treatment of RA.

Expression and significance of HMGB1, TLR4 and NF-κB p65 in human epidermal tumors.

BACKGROUND: High mobility group protein box 1 (HMGB1) is a DNA binding protein located in nucleus. It is released into extracellular fluid where it acts as a novel proinflammatory cytokine which interacts with Toll like receptor 4 (TLR4) to activate nuclear factor-κB (NF-κB). This sequence of events is involved in tumor growth and progression. However, the effects of HMGB1, TLR4 and NF-κB on epidermal tumors remain unclear. METHODS: Human epidermal tumor specimens were obtained from 96 patients. Immunohistochemistry was used to detect expression of HMGB1, TLR4 and NF-κB p65 in human epidermal tumor and normal skin specimens. Western blot analysis was used to detect the expression of NF-κB p65 in epithelial cell nuclei in human epidermal tumor and normal tissues. RESULTS: Immunohistochemistry and western blot analysis indicated a progressive but statistically significant increase in p65 expression in epithelial nuclei in benign seborrheic keratosis (SK), precancerous lesions (PCL), low malignancy basal cell carcinoma (BCC) and high malignancy squamous cell carcinoma (SCC) (P <0.01). The level of extracellular HMGB1 in SK was significantly higher than in normal skin (NS) (P <0.01), and was higher than in SCC but without statistical significance. The level of TLR4 on epithelial membranes of SCC cells was significantly higher than in SK, PCL, BCC and NS (P <0.01). There was a significant positive correlation between p65 expression in the epithelial nuclei and TLR4 expression on the epithelial cell membranes (r = 0.3212, P <0.01). CONCLUSIONS: These findings indicate that inflammation is intensified in parallel with increasing malignancy. They also indicate that the TLR4 signaling pathway, rather than HMGB1, may be the principal mediator of inflammation in high-grade malignant epidermal tumors. Combined detection of p65 in the epithelial nuclei and TLR4 on the epithelial membranes may assist the accurate diagnosis of malignant epidermal tumors.

Acid-sensing ion channels contribute to the effect of acidosis on the function of dendritic cells.

As an H(+)-gated subgroup of the degenerin/epithelial Na(+) channel family, acid-sensing ion channels (ASICs) were reported to be involved in various physiological and pathological processes in neurons. However, little is known about the role of ASICs in the function of dendritic cells (DCs). In this study, we investigated the expression of ASICs in mouse bone marrow-derived DCs and their possible role in the function of DCs. We found that ASIC1, ASIC2, and ASIC3 are expressed in DCs at the mRNA and protein levels, and extracellular acid can evoke ASIC-like currents in DCs. We also demonstrated that acidosis upregulated the expression of CD11c, MHC class II, CD80, and CD86 and enhanced the Ag-presenting ability of DCs via ASICs. Moreover, the effect of acidosis on DCs can be abolished by the nonsteroidal anti-inflammatory drugs ibuprofen and diclofenac. These results suggest that ASICs are involved in the acidosis-mediated effect on DC function.

The role of IL-33 in rheumatic diseases.

Interleukin-33 (IL-33), a novel member of IL-1 family, has been recently implicated in several inflammatory and autoimmune diseases. IL-33 can be produced by various types of tissues and cells and induce gene expression of Th2-associated cytokines via binding to the orphan receptor ST2. By promoting Th2 type immune response, IL-33 plays important roles in the allergy, whereas its function in autoimmune diseases attracts more attention. Recent studies reported the correlation of IL-33 with rheumatic diseases, and most of them found that the IL-33 expression levels were consistent with disease activity and development. Furthermore, evidence has indicated that IL-33-related treatment may ameliorate the pathogenic conditions and attenuate disease progression of those rheumatic diseases. Therefore, elucidation of the roles of IL-33 in rheumatic diseases would be beneficial to understand the pathogenesis and therapy of these diseases. In this paper, we will summarize the roles of IL-33 in the rheumatic diseases.

Native soluble carcinoembryonic antigen is not involved in the impaired activity of CD56 natural killer cells in malignant pleural effusion.

BACKGROUND: Natural killer (NK) cells are lymphocytes of the innate immune system that play a crucial role in tumor immune surveillance. Accumulated data indicated that NK cells in the tumor microenvironment often display a suppressed function. However, the mechanism is not clear. OBJECTIVE: In this study, the effects and relative mechanisms of malignant pleural effusion (MPE) from patients with lung cancer on NK cells were researched. METHODS: MPE and peripheral blood (PB) samples were collected from patients with lung cancer. The cytotoxic activity of CD56(dim) NK cells in PB and MPE mononuclear cells was analyzed by flow cytometry. RESULTS: It was observed that the percentages of total NK cells and a CD56(dim) NK subset in MPE reduced accompanying impaired cytotoxic activity compared with that in paired PB. Cell-free MPE treatment reduced both the proportion and cytotoxic activity of CD56(dim) NK cells in PB from healthy donors. The suppression effects were not based on soluble carcinoembryonic antigen and the inhibitory cytokines interleukin-10 and transforming growth factor-ß1, but were dependent on the factor with a molecular weight >100 kDa. CONCLUSIONS: These results demonstrated that native soluble carcinoembryonic antigen does not suppress the activity of NK cells, and an unknown factor with a molecular weight >100 kDa plays a critical role in the impairment of CD56(dim) NK cells in MPE, which might lead to tumor progression.

Interleukin-33 ameliorates experimental colitis through promoting Th2/Foxp3⁺ regulatory T-cell responses in mice.

Crohn's disease (CD) is characterized by the activation of Th1 and Th17 cells and deficiency of regulatory T cells (Tregs), leading to intestine tissue injury and destruction. As a novel cytokine of the interleukin (IL)-1 family, the role and underlying mechanisms of IL-33 in CD remain poorly understood. Here, we assess the effects and mechanisms of IL-33 on the trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis that mimics human CD. We found that IL-33 levels were increased in the TNBS-treated mice, whereas recombinant IL-33 (rIL-33) administration substantially ameliorated TNBS-mediated colonic tissue injury and clinical symptoms of colitis. The protective effect of rIL-33 was partly associated with the markedly increased induction of Th2-type cytokines. Importantly, rIL-33 treatment resulted in prominently upregulated Foxp3 expression in the TNBS-treated mice, and depletion of Tregs significantly abrogated the impact of IL-33 on reducing the development of colitis. Notably, the level of CD103⁺ dendritic cells (DCs), which promotes development of Tregs, is also increased in mesenteric lymph node and lamina propria of rIL-33-treated mice. The impact of rIL-33 on CD103⁺ DC induction was the result of indirectly upregulating intestine epithelial cells that produce thymic stromal lymphopoietin and retinoic acid but do not directly act on DCs. In conclusion, our data provide clear evidence that IL-33 plays a protective role in TNBS-induced colitis, which is closely related to a Th1-to-Th2/Treg switch. Thus, IL-33 is a promising candidate for the development of new treatments for CD.

Perforin is recaptured by natural killer cells following target cells stimulation for cytotoxicity.

When encountering target cells, NK (natural killer) cells exocytose Pfn (perforin) and granzyme B to kill challengers. We previously reported that granzyme B is recycled and reused by NK cells via clathrin-dependent endocytosis. However, whether Pfn, a main secretory vesicle content, indispensible to granzyme B killing, undergoes endocytosis remains unknown. We demonstrate that Pfn is recaptured by early endosomes of NK cells via a clathrin-dependent endocytosis after target cell stimulation. Inhibition of clathrin-dependent endocytosis significantly attenuated the cytotoxicity of NK cells. The data suggest that the recovery of Pfn contributes to the cytotoxicity of NK cells. The assay of endocytosis of lytic molecule presents a particular focus for exploring the mechanism of abnormal cytotoxicity of NK cells.