Feili Gong

Extracellular hmgb1 functions as an innate immune-mediator implicated in murine cardiac allograft acute rejection.

Hmgb1, an evolutionarily conserved chromosomal protein, was recently re‐discovered to be an innate immune‐mediator contributing to both innate and adaptive immune responses. Here, we show a pivotal role for Hmgb1 in acute allograft rejection in a murine cardiac transplantation model. Extracellular Hmgb1 was found to be a potent stimulator for adaptive immune responses. Hmgb1 can be either passively released from damaged cells after organ harvest and ischemia/reperfusion insults, or actively secreted by allograft infiltrated immune cells. After transplantation, allografts show a significant temporal up‐regulation of Hmgb1 expression accompanied by inflammatory infiltration, a consequence of graft destruction. These data suggest the involvement of Hmgb1 in acute allograft rejection. In line with these observations, treatment of recipients with rA‐box, a specific blockade for endogenous Hmgb1, significantly prolonged cardiac allograft survival as compared to those recipients treated with either rGST or control vehicle. The enhanced graft survival is associated with reduced allograft expression of TNFα, IFNγ and Hmgb1 and impaired Th1 immune response.

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.

Extracellular High-Mobility Group Box 1 Acts as an Innate Immune Mediator to Enhance Autoimmune Progression and Diabetes Onset in NOD Mice

OBJECTIVE—The implication of innate immunity in type 1 diabetes development has long been proposed. High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was recently recognized to be a potent innate inflammatory mediator when released extracellularly. We sought to test the hypothesis that HMGB1 acts as an innate immune mediator implicated in type 1 diabetes pathogenesis. RESEARCH DESIGN AND METHODS—Eight- and 12-week-old NOD mice were treated with an HMGB1 neutralizing antibody once a week until 25 weeks of age and monitored for insulitis progression and diabetes onset. The underlying mechanisms of HMGB1 regulation of autoimmune response were further explored. RESULTS—During autoimmunity, HMGB1 can be passively released from damaged pancreatic β-cells and actively secreted by islet infiltrated immune cells. Extracellular HMGB1 is potent in inducing NOD dendritic cell maturation and stimulating macrophage activation. Blockade of HMGB1 significantly inhibited insulitis progression and diabetes development in both 8- and 12-week-old NOD mice. HMGB1 antibody treatment decreased the number and maturation of pancreatic lymph node (PLN) CD11c++CD11b+ dendritic cells, a subset of dendritic cells probably associated with autoantigen presentation to naïve T-cells, but increased the number for PLN CD4+Foxp3+ regulatory T-cells. Blockade of HMGB1 also decreased splenic dendritic cell allo-stimulatory capability associated with increased tolergenic CD11c+CD8a+ dendritic cells. Interestingly, the number of CD8+interferon-γ+ (Tc1) T-cells was increased in the PLNs and spleen after blockade of HMGB1, which could be associated with retarded migration of activated autoreactive T-cells into the pancreatic islets. CONCLUSIONS—Extracellular HMGB1 functions as a potent innate immune mediator contributing to insulitis progression and diabetes onset.

Gene Silencing of NALP3 Protects Against Liver Ischemia–Reperfusion Injury in Mice

Liver ischemia-reperfusion (I/R) injury is a multifactorial process that affects graft function after liver transplantation. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18, have been shown to play key roles in the pathophysiology of liver I/R injury. Studies have indicated that NALP3 (NACHT domain, leucine-rich repeat [LRR] domain, and pyrin domain [PYD]-containing protein-3) inflammasome is pivotal in the processing and releasing of IL-1β and IL-18. The aim of this study was to test whether NALP3 silencing has a protective effect in murine liver I/R injury. Using a partial lobar liver warm ischemia model, mice were hydrodynamically injected with pNALP3shRNA, pshRNANC, or saline 48 hr before ischemia. Those mice pretreated with pNALP3shRNA showed decreased serum alanine aminotransferase levels; inhibited production of proinflammatory cytokines such as IL-1β, IL-18, TNF-α, and IL-6 by downregulation of caspase-1 activation and NF-κB activity; as well as decreased release of HMGB1 (high-mobility group box-1) and inflammatory cell infiltration, leading to the prevention of liver I/R injury, when compared with controls. Histology revealed that pretreatment with pNALP3shRNA significantly ameliorated hepatocellular damage after I/R. Thus, by using a small hairpin RNA approach, our study confirms that NALP3 signaling is involved in liver I/R and that silencing of NALP3 can protect the liver from I/R injury by reducing IL-1β, IL-18, TNF-α, IL-6, and HMGB1 release through downregulation of caspase-1 activation and NF-κB activity.

Heme oxygenase-1 upregulation significantly inhibits TNF-α and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice

The present study was designed to investigate whether administration of CoPPIX, an HO-1 inducer, could significantly inhibit TNF-alpha and Hmgb1 expression and thus attenuate the acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. Acute lung injury was induced successfully by intratracheal administration of LPS (0.5 mg/kg) in male BALB/c mice. CoPPIX or ZnPPIX (an HO-1 inhibitor) was administered to mice 24 h prior to LPS exposure. It was found that CoPPIX (5, 10 mg/kg, i.p.) caused a significant reduction in the total cells and neutrophils in BALF, a significant reduction in the W/D ratio and EBA leakage at 24 h after LPS challenge. Furthermore, the histopathologic findings indicated that alveolitis with leukocyte infiltration in the alveolar space was less severe in the CoPPIX-treated mice than in the mice treated with LPS alone. In addition, CoPPIX was also believed to have down-regulated the expression of LPS-induced proinflammatory cytokines, including early proinflammatory cytokine TNF-a, and late proinflammatory cytokine Hmgb1. In contrast, no obvious difference was observed between the ZnPPIX group and the LPS group. These findings demonstrate the significant protection of CoPPIX against LPS-induced ALI, and the effect mechanism of CoPPIX was associated with decreasing the expression of TNF-a and Hmgb1.

High-mobility group box 1 promotes early acute allograft rejection by enhancing IL-6-dependent Th17 alloreactive response

Previously, we reported that extracellular high-mobility group box 1 (HMGB1) functions as an innate alarmin implicated in cardiac allograft acute rejection. We now present evidence suggesting that HMGB1 is pivotal in inducing interleukin-17 (IL-17)-producing alloreactive T cells by stimulating dendritic cells secretion of IL-6. Those IL-17+ T cells are likely to be the major effector cells responsible for the early stage of cardiac allograft rejection through mediating an influx of neutrophils into allografts, and therefore, blockade of IL-17A significantly prolonged murine cardiac allograft survival. In contrast to the classical model for a dominant role of IFN-γ+-Th1 cells have in acute allograft rejection, our data suggest that IFN-γ+-Th1 cells are responsible for the late stage of graft destruction by inducing monocyte infiltration when IL-17+ T-cell response recedes. Blockade of HMGB1 significantly decreased splenic alloreactive Th17 cells and IFN-γ-producing CD8+ T cells in the recipients, leading to less infiltration of neutrophils along with lower IL-6 and IL-17 expression levels in the grafts as well as prolongation of cardiac allograft survival. Together, these data support a novel model in which HMGB1 induces IL-17-producing alloreactive T cells to mediate early stage of allograft rejection, whereas IFN-γ-producing alloreactive Th1 cells provoke graft destruction after Th17 response recedes.

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.

IL-33 prolongs murine cardiac allograft survival through induction of TH2-type immune deviation.

Background. In Th (T helper) 1/Th2 balance in response to signals given during donor antigen presentation, induction of allograft prolongation is more often related to Th2-type than with Th1-type immunity. Here, we examined the effect of interleukin (IL)-33, a novel member of the IL-1 family, on cardiac allograft survival in mice. Methods. Mice heterotopic cardiac transplants were performed with sequential recipient sacrifice at anticipated time points to examine the immunoregulatory action of IL-33 in recipient mice. Results. In vitro Th1-polarized CD4+ T cells did not express ST2L; however, most CD4+ T cells became ST2L+ on repeated stimulation under Th2-polarizing conditions. Similarly, we found that IL-33 was able to enhance the expression of Th2-associated cytokines (IL-5 and IL-13) but not interferon (IFN)-&ggr;. Treatment of recipient mice with IL-33 results in the improvement of allograft survival (more than 20 days) when compared with phosphate-buffered saline- or glutathione S-transferase-treated groups (all less than 9 days). Intracellular cytokine staining in CD4+ splenocytes confirmed an increase in the percentage of IL-4+ cells and a decrease in the percentage of IFN-&ggr;+ cells in IL-33 treated mice. In addition, IL-33 significantly enhanced the gene expression of Th2-type cytokines IL-4 and IL-5 but suppressed the Th1-type cytokine IFN-&ggr; mRNA levels in both allograft and recipient spleen. Conclusion. These data demonstrate that IL-33 serves as a potent inducer of Th2 immune response and can markedly contribute to the prolongation of cardiac allograft survival.

Loss of Jak2 selectively suppresses DC-mediated innate immune response and protects mice from lethal dose of LPS-induced septic shock.

Given the importance of Jak2 in cell signaling, a critical role for Jak2 in immune cells especially dendritic cells (DCs) has long been proposed. The exact function for Jak2 in DCs, however, remained poorly understood as Jak2 deficiency leads to embryonic lethality. Here we established Jak2 deficiency in adult Cre+/+Jak2fl/fl mice by tamoxifen induction. Loss of Jak2 significantly impaired DC development as manifested by reduced BMDC yield, smaller spleen size and reduced percentage of DCs in total splenocytes. Jak2 was also crucial for the capacity of DCs to mediate innate immune response. Jak2−/− DCs were less potent in response to inflammatory stimuli and showed reduced capacity to secrete proinflammatory cytokines such as TNFα and IL-12. As a result, Jak2−/− mice were defective for the early clearance of Listeria after infection. However, their potency to mediate adaptive immune response was not affected. Unlike DCs, Jak2−/− macrophages showed similar capacity secretion of proinflammatory cytokines, suggesting that Jak2 selectively modulates innate immune response in a DC-dependent manner. Consistent with these results, Jak2−/− mice were remarkably resistant to lethal dose of LPS-induced septic shock, a deadly sepsis characterized by the excessive innate immune response, and adoptive transfer of normal DCs restored their susceptibility to LPS-induced septic shock. Mechanistic studies revealed that Jak2/SATA5 signaling is pivotal for DC development and maturation, while the capacity for DCs secretion of proinflammatory cytokines is regulated by both Jak2/STAT5 and Jak2/STAT6 signaling.

A limited course of soluble CD83 delays acute cellular rejection of MHC-mismatched mouse skin allografts.

CD83 is a surface marker expressed on matured dendritic cells (DCs). It plays a pivotal role in the mediation of DC/T cell interaction and induction of T‐cell activation. Previous studies have suggested that a soluble form of CD83 could suppress DC maturation and inhibit T‐cell activation and, as a result, it can prevent paralysis associated with experimental autoimmune encephalomyelitis. Here, we explored its potential effect on allograft rejection in a fully major histocompatibility complex‐mismatched murine skin transplantation model. A form of mouse soluble CD83 (CD83‐Ig) fused the extracellular domain of murine CD83 with human IgG1α Fc tail was purified from transfected COS‐7 cell. It was found that the treatment of recipient mice with CD83‐Ig significantly delayed allograft rejection. Especially, when T cells originated from recipients treated with CD83‐Ig re‐stimulated with donor‐specific splenocytes, they showed a significant reduced responding capability as compared with that of originated from control recipients. In line with these results, a reduction for serum IFN‐γ and IL‐2 and a decreased mRNA expression of IFN‐γ and IL‐2 in allograft infiltrated immune cells were also observed. Our results suggest that CD83‐Ig could be useful for the treatment of allograft rejection in combination with other therapeutic strategies.