Libo Zheng

Immunopathology of inflammatory bowel disease

Inflammatory bowel disease (IBD) results from a complex series of interactions between susceptibility genes, the environment, and the immune system. The host microbiome, as well as viruses and fungi, play important roles in the development of IBD either by causing inflammation directly or indirectly through an altered immune system. New technologies have allowed researchers to be able to quantify the various components of the microbiome, which will allow for future developments in the etiology of IBD. Various components of the mucosal immune system are implicated in the pathogenesis of IBD and include intestinal epithelial cells, innate lymphoid cells, cells of the innate (macrophages/monocytes, neutrophils, and dendritic cells) and adaptive (T-cells and B-cells) immune system, and their secreted mediators (cytokines and chemokines). Either a mucosal susceptibility or defect in sampling of gut luminal antigen, possibly through the process of autophagy, leads to activation of innate immune response that may be mediated by enhanced toll-like receptor activity. The antigen presenting cells then mediate the differentiation of naïve T-cells into effector T helper (Th) cells, including Th1, Th2, and Th17, which alter gut homeostasis and lead to IBD. In this review, the effects of these components in the immunopathogenesis of IBD will be discussed.

Inhibition of a novel fibrogenic factor Tl1a reverses established colonic fibrosis

Intestinal fibrostenosis is among the hallmarks of severe Crohn’s disease. Patients with certain TNFSF15 (gene name for TL1A) variants over-express TL1A and have a higher risk of developing strictures in the small intestine. In addition, sustained Tl1a expression in mice leads to small and large intestinal fibrostenosis under colitogenic conditions. The aim of this study was to determine whether established murine colonic fibrosis could be reversed with Tl1a antibody (Ab). Treatment with neutralizing Tl1a Ab reversed colonic fibrosis back to the original pre-inflamed levels, potentially as a result of lowered expression of connective tissue growth factor, Il31Ra, transforming growth factor β1 and insulin-like growth factor-1. In addition, blocking Tl1a function by either neutralizing Tl1a Ab or deletion of death domain receptor 3 (Dr3) reduced the number of fibroblasts and myofibroblasts, the primary cell types that mediate tissue fibrosis. Primary intestinal myofibroblasts expressed Dr3 and functionally responded to direct Tl1a signaling by increasing collagen and Il31Ra expression. These data demonstrated a direct role for TL1A–DR3 signaling in tissue fibrosis and that modulation of TL1A–DR3 signaling could inhibit gut fibrosis.

Differential expression of PTEN in hepatic tissue and hepatic stellate cells during rat liver fibrosis and its reversal

To evaluate the change in phosphatase and tensin homology deleted on chromosome ten (PTEN) expression in liver fibrogenesis, particularly the reversal of fibrogenic liver tissues, and to investigate the relation with the proliferation and apoptosis of hepatic stellate cells (HSCs) in vivo, a rat model of hepatic fibrosis was established by hypodermic injection of carbon tetrachloride (CCl4) mixed with olive oil at the concentration of 40% for 5 weeks (2 ml/kg, twice a week). Reversal of fibrosis was achieved with normal feedings for 4 weeks after CCl4 injection for 5 weeks. The expression of PTEN was measured by immunofluorescence, western blot analysis and real-time PCR. Co-expression of α-smooth muscle actin (α-SMA) with PTEN and α-SMA with terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) were assessed by confocal laser scanning microscopy. The results displayed that the expression of PTEN was reduced with fibrosis in both rat liver tissues and activated HSCs. By contrast, PTEN expression was increased with the reversal of liver fibrosis. Compared to the fibrogenic state, there were increased numbers of apoptotic activated HSCs during reversal of fibrosis. These data suggest that the dynamic expression of PTEN in rat liver tissues is negatively correlated with liver fibrosis and activated HSCs and is positively correlated with reversal of fibrosis and apoptotic activated HSCs. Modulation of PTEN expression may be an effective and novel method for the treatment of liver fibrosis.

Down-regulation of focal adhesion kinase by short hairpin RNA increased apoptosis of rat hepatic stellate cells

An JY, Zheng LB, Xie SR, Dun ZN, Hao LS, Yao DM, Shih DQ, Zhang XL. Down‐regulation of focal adhesion kinase by short hairpin RNA increased apoptosis of rat hepatic stellate cells. APMIS 2011; 119: 319–29.

Myeloid ATG16L1 Facilitates Host–Bacteria Interactions in Maintaining Intestinal Homeostasis

Intact ATG16L1 plays an essential role in Paneth cell function and intestinal homeostasis. However, the functional consequences of ATG16L1 deficiency in myeloid cells, particularly macrophages, are not fully characterized. We generated mice with Atg16l1 deficiency in myeloid and dendritic cells and showed that mice with myeloid Atg16l1 deficiency had exacerbated colitis in two acute and one chronic model of colitis with increased proinflammatory to anti-inflammatory macrophage ratios, production of proinflammatory cytokines, and numbers of IgA-coated intestinal microbes. Mechanistic analyses using primary murine macrophages showed that Atg16l1 deficiency led to increased reactive oxygen species production, impaired mitophagy, reduced microbial killing, impaired processing of MHC class II Ags, and altered intracellular trafficking to the lysosomal compartments. Increased production of reactive oxygen species and reduced microbial killing may be general features of the myeloid compartment, as they were also observed in Atg16l1-deficient primary murine neutrophils. A missense polymorphism (Thr300Ala) in the essential autophagy gene ATG16L1 is associated with Crohn disease (CD). Previous studies showed that this polymorphism leads to enhanced cleavage of ATG16L1 T300A protein and thus reduced autophagy. Similar findings were shown in primary human macrophages from controls and a population of CD patients carrying the Atg16l1 T300A risk variant and who were controlled for NOD2 CD-associated variants. This study revealed that ATG16L1 deficiency led to alterations in macrophage function that contribute to the severity of CD.

The protection role of Atg16l1 in CD11c+dendritic cells in murine colitis

The autophagy-related 16-like 1 gene (Atg16l1) is associated with inflammatory bowel disease (IBD) and has been shown to play an essential role in paneth cell function and intestinal homeostasis. However, the functional consequences of Atg16l1 deficiency in myeloid cells, particularly in dendritic cells (DCs), are not fully characterized. The aim of this study is to investigate the functional consequence of Atg16l1 in CD11c+DCs in murine colitis. We generated mice deficient in Atg16l1 in CD11c+DCs. Dextran Sulfate Sodium (DSS) and S. typhimurium infection induced colitis was used to assess the role of DCs specific Atg16l1 deficiency in vivo in murine colitis. Bone marrow derived dendritic cells (BMDC) were isolated and autophagy function was assessed with microtubule-associated protein 1 light chain 3β (Map1lc3b or LC3) by western blot. Uptake of Salmonella enteric serovar typhimurium (S. typhimurium) was assessed by flow cytometry and transmission electron microscopy (TEM). The production of reactive oxygen species (ROS) and intracellular S. typhimurium killing in BMDCs were assessed. We showed worsened colonic inflammation in Atg16l1 deficiency mice in DSS induced murine colitis with increased proinflammatory cytokines of IL-1β and TNF-α. Mechanistic studies performed in primary murine BMDCs showed that Atg16l1 deficiency increased ROS production, reduced microbial killing and impaired antigen processing for altered intracellular trafficking. Together, these results indicate impaired CD11c+DCs function with Atg16l1 deficiency contributes to the severity of murine colitis.

Regulatory Effects and Mechanism of Adenovirus-Mediated PTEN Gene on Hepatic Stellate Cells

BackgroundTension homology deleted on chromosome ten (PTEN) is important in liver fibrosis.AimsThe purpose of this study was to evaluate the PTEN gene effects and mechanism of action on hepatic stellate cells (HSCs).MethodsThe rat primary HSCs and human LX-2 cells were transfected by an adenovirus containing cDNA constructs encoding the wild-type PTEN (Ad-PTEN), the PTEN mutant G129E gene (Ad-G129E) and RNA interference targeting the PTEN sequence PTEN short hairpin RNA (PTEN shRNA), to up-regulate and down-regulate PTEN expression, respectively. The HSCs were assayed with a fluorescent microscope, real time PCR, Western blot, MTT, flow cytometry and Terminal-deoxynucleoitidyl transferase mediated nick end labeling. In addition, the CCl4 induced rat hepatic fibrosis model was also established to check the in vivo effects of the recombinant adenovirus with various levels of PTEN expression.ResultsThe data have shown that the over-expressed PTEN gene led to reduced HSCs activation and viability, caspase-3 activity and cell cycle arrest in the G0/G1 and G2/M phases, as well as negative regulation of the PI3K/Akt and FAK/ERK signaling pathways in vitro. The over-expressed PTEN gene improved liver function, inhibited proliferation and promoted apoptosis of HSCs both in vitro and in vivo.ConclusionsThese data have shown that gene therapy using the recombinant adenovirus encoding wild-type PTEN inhibits proliferation and induces apoptosis of HSCs, which is a potential treatment option for hepatic fibrosis.

783 TL1A Modulates the Differential Effect of IL-17 Blockade on Mucosal Inflammation

Background: IL-17 has been thought to be pathogenic in IBD. However, a recent clinical trial using IL-17 blockade unexpectedly worsened IBD (Hueber, et al. Gut. 2012). A proportion of trial patients who responded to IL-17 blockade were found to carry a risk TL1A IBD polymorphism that predicted elevated expression of TL1A. The mechanism of IL-17 and its interaction with TL1A on mucosal inflammation has not been found. Aim: To determine the mechanism of TL1A mediated differential effects of IL-17 on mucosal inflammation Methods: Naive (CD4+CD45RBhi) T-cells were isolated from WT, Tl1a-Tg, Il-17a-/-, and Tl1a-Tg with deficiency in Il-17a (Tl1a-Tg/Il-17a-/-) mice and adoptively transferred into Rag-/mice. Naive (CD4+CD25loCD44loCD62hi) T-cells were differentiated in vitro into T helper (Th) effector cells. Intestinal inflammation was evaluated by disease activity index (DAI) and histological analyses. Flow cytometry was used to determine CD4+ T-cell infiltration, activation, and cytokine expression in the LPMC from the colon. Results: Consistent with the human IL-17 trial, we found that mice that received Il-17a-/Naive cells had worsened colitis (increased DAI scores and cecal inflammation) as compared to WT. Comparable results were seen with Tl1a-Tg Naive cells. Similar to the human IL-17 trial, Il-17a deficiency under Tl1a driven conditions (Tl1a-Tg/Il-17a-/-) ameliorated colitis (reduced DAI and cecal inflammation). Mucosal CD4+ T-cell infiltration and activation (CD69 and CD44) were increased in both Il-17a-/and Tl1a-Tg, but reduced with Tl1a-Tg/Il-17a-/-. Analysis of Th effector pathways demonstrated a shift towards Th-1 (increased Ifn-γ) and Th-9 (increased Il-9), and reduced regulatory cytokine Il-10 production with both Il-17a-/and Tl1a-Tg. In contrast, Il-17 deficiency under Tl1a driven conditions (Tl1a-Tg/Il-17a-/-) had a reduction in Th1 (reduced Ifn-γ) and Th9 (reduced Il-9) responses, and increased regulatory responses (increased Il-10). To assess whether intrinsic cellular differences exist in Naive T-cells between WT, Il-17a-/-, Tl1a-Tg, and Tl1a-Tg/Il-17a-/-, in vitro differentiation assays were performed. Consistently, in vitro differentiated Naive Il-17a-/and Tl1a-Tg T-cells expressed increased Ifn-γ and Il-9, whereas Tl1a-Tg/Il17a-/T-cells expressed lower Il-9 but higher Il-10 levels. Conclusion: IL-17 blockade induces mucosal inflammation by enhancing Th1 and Th9 effector pathways and reducing regulatory response. However under TL1A driven conditions, inhibiting IL-17 may be beneficial by reducing Th1 and Th9 effector responses and enhancing regulatory responses. This may be one mechanism of why the subset of IBD patients with TL1A polymorphisms improved with IL-17 blockade, while most trial patients did not. This highlights the importance of understanding pathway-pathway interactions in designing clinical trials. Table 1: Inflammatory Markers

In vivo hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells: Therapeutic effect on liver fibrosis/cirrhosis

AIM To investigate the hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and to evaluate their therapeutic effect on liver fibrosis/cirrhosis. METHODS A CCl4-induced liver fibrotic/cirrhotic rat model was used to assess the effect of hUC-MSCs. Histopathology was assessed by hematoxylin and eosin (H&E), Masson trichrome and Sirius red staining. The liver biochemical profile was measured using a Beckman Coulter analyzer. Expression analysis was performed using immunofluorescent staining, immunohistochemistry, Western blot, and real-time PCR. RESULTS We demonstrated that the infused hUC-MSCs could differentiate into hepatocytes in vivo. Functionally, the transplantation of hUC-MSCs to CCl4-treated rats improved liver transaminases and synthetic function, reduced liver histopathology and reversed hepatobiliary fibrosis. The reversal of hepatobiliary fibrosis was likely due to the reduced activation state of hepatic stellate cells, decreased collagen deposition, and enhanced extracellular matrix remodeling via the up-regulation of MMP-13 and down-regulation of TIMP-1. CONCLUSION Transplanted hUC-MSCs could differentiate into functional hepatocytes that improved both the biochemical and histopathologic changes in a CCl4-induced rat liver fibrosis model. hUC-MSCs may offer therapeutic opportunities for treating hepatobiliary diseases, including cirrhosis.

Effects and mechanism of adenovirus-mediated phosphatase and tension homologue deleted on chromosome ten gene on collagen deposition in rat liver fibrosis

AIM To evaluate the effects of phosphatase and tension homologue deleted on chromosome ten (PTEN) gene on collagen metabolism in hepatic fibrosis and the underlying mechanisms. METHODS Rat primary hepatic stellate cells (HSCs) and human LX-2 cells were transfected with adenovirus containing cDNA constructs encoding wild-type PTEN (Ad-PTEN), PTEN mutant G129E gene (Ad-G129E), and RNA interference constructs targeting the PTEN sequence PTEN short hairpin RNA to up-regulate and down-regulate the expression of PTEN. HSCs were assayed using fluorescent microscopy, real-time polymerase chain reaction, and western blotting. Moreover, a CCl4-induced rat hepatic fibrosis model was established to investigate the in vivo effects. Hematoxylin and eosin, and Masson’s trichrome were used to assess the histological changes. The expression of collagen I and III was assessed using immunohistochemistry and western blot analysis. RESULTS Elevated expression of PTEN gene reduced serum levels of alanine transaminase and aspartate transaminase, decreased collagen deposition in the liver, and reduced hepatocyte necrosis. In contrast, knockdown of PTEN expression had an opposite effect, such as increased collagen deposition in the liver, and was molecularly characterized by the increased expression of matrix metalloproteinase (MMP)-13 (P < 0.01) and MMP-2 (P < 0.01), as well as decreased expression of the tissue inhibitor of metalloproteinase (TIMP)-1 (P < 0.01) and TIMP-2 (P < 0.01). CONCLUSION These data indicated that gene therapy using recombinant adenovirus encoding PTEN might be a novel way of treating hepatic fibrosis.