Masanobu Tsuda

Deletion of interleukin‐12p40 suppresses autoimmune cholangitis in dominant negative transforming growth factor β receptor type II mice

Our laboratory has reported that mice that express a dominant negative form of transforming growth factor β receptor restricted to T cells (dnTGFβRII) develop an inflammatory biliary ductular disease with elevated serum levels of interleukin (IL)‐12p40 and other proinflammatory cytokines and antimitochondrial autoantibodies (AMAs) closely resembling human primary biliary cirrhosis (PBC). We have used this mouse model to address the potential mechanisms of immunomodulation of liver disease by creating two unique genetic strains: IL‐12p40 knockout (KO)‐dnTGFβRII mice and IFN‐γ KO‐dnTGFβRII mice. The two colonies of genetically modified mice—and, for purposes of controls, the dnTGFβRII mice—were monitored for liver immunopathology, AMAs, and intrahepatic cytokine production. Disease expression in the IFN‐γ KO‐dnTGFβRII mice, including liver immunopathology, were similar to those of dnTGFβRII mice, whereas the IL‐12p40 KO‐dnTGFβRII mice had a dramatic reduction in histological autoimmune cholangitis and significant decreases in levels of intrahepatic proinflammatory cytokines, but similar levels of AMAs compared with dnTGFβRII controls. Conclusion: These data indicate that in this mouse model of PBC, signaling by way of IL‐12p40 is an essential requirement for the development of autoimmune cholangitis. The results of these studies will play an important role in identifying pathways and reagents that will selectively inhibit IL‐12 signaling for the outlining of future therapeutic strategies for human PBC. (HEPATOLOGY 2009.)

Biochemical and immunologic effects of rituximab in patients with primary biliary cirrhosis and an incomplete response to ursodeoxycholic acid.

The aim of this study was to determine the safety and potential efficacy of B‐cell depletion with the anti‐CD20 monoclonal antibody rituximab in patients with primary biliary cirrhosis (PBC) and an incomplete response to ursodeoxycholic acid (UDCA). This open‐label study enrolled six patients with PBC and incomplete responses to UDCA to be treated with 2 doses of 1000 mg rituximab separated by 2 weeks and followed for 52 weeks. The primary endpoints were safety and changes in B‐cell function. Two patients received only 1 dose of rituximab, one due to activation of latent varicella and the other due to a viral upper respiratory infection. Serum levels of total IgG, IgM, and IgA as well as anti‐mitochondrial autoantibodies (AMAs) IgA and IgM decreased significantly from baseline by 16 weeks and returned to baseline levels by 36 weeks. Stimulation of B cells with CpG produced significantly less IgM at 52 weeks after treatment compared with B cells at baseline. In addition, transient decreases in memory B‐cell and T‐cell frequencies and an increase in CD25high CD4+ T cells were observed after treatment. These changes were associated with significant increases in mRNA levels of FoxP3 and transforming growth factor‐β (TGF‐β) and a decrease in tumor necrosis factor‐α (TNF‐α) in CD4+ T cells. Notably, serum alkaline phosphatase levels were significantly reduced up to 36 weeks following rituximab treatment. Conclusion: These data suggest that depletion of B cells influences the induction, maintenance, and activation of both B and T cells and provides a potential mechanism for treatment of patients with PBC with an incomplete response to UDCA. (HEPATOLOGY 2012)

The spectrum of autoantibodies in IPEX syndrome is broad and includes anti-mitochondrial autoantibodies

IPEX syndrome is a congenital disorder of immune regulation caused by mutations in the FOXP3 gene, which is required for the suppressive function of naturally arising CD4 + CD25 + regulatory T cells. In this case series we evaluated serum samples from 12 patients with IPEX syndrome for the presence of common autoantibodies associated with a broad range of autoimmune disorders. We note that 75% of patients (9/12) had 1 or more autoantibodies, an incidence far above the cumulative rate observed in the general population. The range of autoantibodies differed between patients and there was no predominant autoantibody or pattern of autoantibodies present in this cohort. Surprisingly, one patient had high-titer anti-mitochondrial antibodies (AMA) typically associated with primary biliary cirrhosis (PBC) although the patient had no signs of cholestasis. PBC is a well-characterized autoimmune disease that occurs primarily in women and includes the serological hallmarks of serum AMA and elevated IgM which were both present in this patient. PBC is virtually absent in children with the exception of one reported child with interleukin 2 receptor α (CD25) deficiency which is associated with an IPEX-like regulatory T cell dysfunction. Based on the present data and the available literature we suggest a direct role for CD4 + CD25 + regulatory T cells in restraining B cell autoantibody production and that defects in regulatory T cells may be crucial to the development of PBC.

Deletion of interleukin (IL)‐12p35 induces liver fibrosis in dominant‐negative TGFβ receptor type II mice

Mice with a dominant‐negative transforming growth factor β receptor restricted to T cells (dnTGFβRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)‐12p40 resulted in a strain (IL‐12p40−/−dnTGFβRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL‐12 cytokine family in dnTGFβRII autoimmune biliary disease, we deleted the gene encoding the IL‐12p35 subunit from dnTGFβRII mice, resulting in an IL‐12p35−/− dnTGFβRII strain which is deficient in two members of the IL‐12 family, IL‐12 and IL‐35. In contrast to IL‐12p40−/− mice, the IL‐12p35−/−mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFβRII mice. The p35−/− mice also demonstrated a distinct cytokine profile characterized by a shift from a T‐helper 1 (Th1) to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL‐12p35−/− mice. In conclusion, IL‐12p35−/− dnTGFβRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC. (HEPATOLOGY 2013;)

Subcutaneous adipose tissue–derived stem cells facilitate colonic mucosal recovery from 2,4,6‐trinitrobenzene sulfonic acid (TNBS)–induced colitis in rats

Background: Adipose tissue–derived stem cells (ADSCs) can be easily obtained from subcutaneous adipose tissue, and ADSCs can be demonstrated to display multilineage developmental plasticity. In this study, using TNBS‐induced colitis rats, we show the feasibility of repairing injured intestinal mucosa with adipose tissue–derived stem cells. Methods: The subcutaneous adipose tissue of F344 rats was obtained and digested by collagenase. The digested tissue was cultured in DMEM containing 10% FBS for 1 month. ADSCs were confirmed to differentiate under appropriate conditions into various lineages of cells, including bone, neural cells, adipocytes, and epithelial cells. HGF, VEGF, TGF‐&bgr;, and adiponectin in the culture supernatants of ADSCs were determined by ELISA. ADSCs (107 cells) were injected into the submucosa of the colon to examine their capacity to repair intestinal mucosa injured by TNBS. Results: In the experimental colitis model, the injection of ADSCs facilitated colonic mucosal repair and reduced the infiltration of inflammatory cells. High levels of HGF, VEGF, and adiponectin were detected in the culture supernatants of ADSCs. Moreover, injected ADSCs distributed to several layers of the colon, and some of them differentiated into mesodermal lineage cells. Conclusions: ADSCs can accelerate the regeneration of injured regions in experimental colitis. HGF, VEGF, and adiponectin might be responsible for the regeneration of injured regions in the colon.

Identification of Potential Cytokine Pathways for Therapeutic Intervention in Murine Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is considered a model autoimmune disease, with the most highly directed and specific autoantibody in both murine and human autoimmunity, the anti-mitochondrial autoantibody (AMA). However, therapeutic advances in this disease have lagged behind. Herein we have taken advantage of our unique model of murine PBC in which mice immunized with 2-octynoic acid coupled to BSA (2OA-BSA), a compound identified by quantitative structure activity relationships (QSAR) of human AMA binding, develop an intense inflammatory cholangitis with striking similarities to humans with PBC. In particular, we have constructed several unique gene-deleted mice, including mice deleted of IL-12p40, IL-12p35, IFN-γ, IL-23p19, IL-17A, IL-17F and IL-22, immunized these animals with 2OA-BSA and followed the natural history of immunopathology to identify key pathways that might provide clues for successful therapy. Our data indicate that whereas both IL-12/Th1 and IL-23/Th17 are involved in cholangitis, it is the IL-12/Th1 signaling pathway that elicits pathology. In fact, deletion of IFN-γ prevents disease and suppresses autoantibodies. Importantly, deletion of the Th17 cytokines IL-17A and IL-22, but not IL-17F, reduces biliary damage; IL-17A-knockout mice have reduced levels of anti-mitochondrial antibody. We further demonstrate that the production of IFN-γ is significantly decreased in the liver of IL-23p19−/−, IL-17A−/− and IL-22−/− mice compared with controls. However, the ability of T cells to produce IFN-γ was not affected in Th17 cytokine-deficient mice. Our data indicate that a deficient Th17 pathway suppresses the accumulation of IFN-γ producing cells in liver during the early phase of cholangitis. In conclusion, whereas IFN-γ has a pivotal role in the early events involved in the pathogenesis of autoimmune cholangitis induced by 2OA-BSA, the IL-23/Th17 pathway potentiates the effects of IL-12/IFN-γ-mediated immunopathology.

Prevention of graft-versus-host disease by intra-bone marrow injection of donor T cells.

We have recently found that intra‐bone marrow‐bone marrow transplantation (IBM‐BMT) can be used to prevent graft‐versus‐host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM‐BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM‐BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV‐DLI]). GvHD in recipients treated with allogeneic IBM‐BMT plus IBM‐DLI was far milder than in those treated with allogeneic IBM‐BMT plus IV‐DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4+CD25+ and CD4+Foxp3+ cells was significantly higher in recipients treated with IBM‐BMT plus IBM‐DLI than in those treated with IBM‐BMT plus IV‐DLI. Donor‐derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM‐BMT plus IBM‐DLI, whereas Th1 cells were dominant in recipients treated with IBM‐BMT plus IV‐DLI. Furthermore, the production of transforming growth factor‐β and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM‐DLI. Thus, IBM‐BMT plus IBM‐DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD.

Induction of Senile Osteoporosis in Normal Mice by Intra‐Bone Marrow‐Bone Marrow Transplantation from Osteoporosis‐Prone Mice

A P6 substrain of the senescence accelerated mouse (SAMP6) spontaneously develops osteoporosis early in life. These mice show the clinical signs of osteoporosis, such as elevated levels of urinary deoxypyridinoline (Dpd), decreased bone mineral density (BMD), and a significant loss of trabecular and cortical bone thickness at 12 months of age. Here, we describe the transfer of osteoporosis to a normal strain by the injection of bone marrow cells from SAMP6 donors directly into the bone marrow cavity (intra‐bone marrow‐bone marrow transplantation [IBM‐BMT]). More than 1 month after IBM‐BMT, hematolymphoid cells were completely reconstituted by donor‐derived cells, and bone marrow stromal cells that could differentiate into osteocytes were also found to be of donor origin. In addition, the recipient C57BL/6 mouse showed the features of osteoporosis in the trabecular bone. Decreases in BMD and increases in urinary Dpd were also observed. When the message levels of cytokines (interleukin [IL]‐11, IL‐6, receptor activator of NF‐κB ligand [RANKL], osteoprotegerin, macrophage–colony‐stimulating factor, and insulin‐like growth factor‐1) were examined by reverse transcription‐polymerase chain reaction (RT‐PCR) and real‐time RT‐PCR analysis, IL‐6 and IL‐11 were reduced to a level similar to that in SAMP6 mice, whereas that of RANKL was increased. These findings indicate that not only the hemopoietic system but also the bone marrow microenvironment are reconstituted as a result of IBM‐BMT, and suggest that the development of senile osteoporosis might be attributable to “stem cell disorders.”

Deletion of Interleukin-6 in Mice With the Dominant Negative Form of Transforming Growth Factor β Receptor II Improves Colitis but Exacerbates Autoimmune Cholangitis

The role of interleukin‐6 (IL‐6) in autoimmunity attracts attention because of the clinical usage of monoclonal antibodies to IL‐6 receptor (IL‐6R), designed to block IL‐6 pathways. In autoimmune liver disease, activation of the hepatocyte IL‐6/STAT3 (signal transducer and activator of transcription 3) pathway is associated with modulating pathology in acute liver failure, in liver regeneration, and in the murine model of concanavalin A–induced liver inflammation. We have reported that mice expressing a dominant negative form of transforming growth factor β receptor II (dnTGFβRII) under control of the CD4 promoter develop both colitis and autoimmune cholangitis with elevated serum levels of IL‐6. Based on this observation, we generated IL‐6–deficient mice on a dnTGF‐βRII background (dnTGFβRII IL‐6−/−) and examined for the presence of antimitochondrial antibodies, levels of cytokines, histopathology, and immunohistochemistry of liver and colon tissues. As expected, based on reports of the use of anti–IL‐6R in inflammatory bowel disease, dnTGFβRII IL‐6−/− mice manifest a dramatic improvement in their inflammatory bowel disease, including reduced diarrhea and significant reduction in intestinal lymphocytic infiltrates. Importantly, however, autoimmune cholangitis in dnTGFβRII IL‐6−/− mice was significantly exacerbated, including elevated inflammatory cytokines, increased numbers of activated T cells, and worsening hepatic pathology. Conclusion: The data from these observations emphasize that there are distinct mechanisms involved in inducing pathology in inflammatory bowel disease compared to autoimmune cholangitis. These data also suggest that patients with inflammatory bowel disease may not be the best candidates for treatment with anti–IL‐6R if they have accompanying autoimmune liver disease and emphasize caution for therapeutic use of anti–IL‐6R antibody. HEPATOLOGY 2010

Fine phenotypic and functional characterization of effector cluster of differentiation 8 positive T cells in human patients with primary biliary cirrhosis

In primary biliary cirrhosis (PBC), patients develop a multilineage response to a highly restricted peptide of the E2 component of pyruvate dehydrogenase (PDC‐E2) involving autoantibody and autoreactive cluster of differentiation (CD)4+ and CD8+ T‐cell responses. Recent data from murine models have suggested that liver‐infiltrating CD8+ cells play a critical role in biliary destruction in PBC. We hypothesized that chronic antigen stimulation of CD8+ T cells alters effector memory T cell (TEM) frequency and function similar to that seen with chronic viral infections, including failure to terminally differentiate and relative resistance to apoptosis. We have rigorously phenotyped CD8+ T‐cell subpopulations from 132 subjects, including 76 patients with PBC and 56 controls, and report a higher frequency of TEM cells characterized as CD45ROhighCD57+CD8high, but expressing the gut homing integrin, α4β7, in peripheral blood mononuclear cells of PBC. These CD8high TEM cells have reduced expression of Annexin V after TCR stimulation. Consistent with a TEM phenotype, CD45ROhighCD57+CD8high T cells express higher levels of granzyme A, granzyme B, perforin, CCR5 and α4β7, and lower levels of CCR7 and CD28 than other CD8high T cells. Furthermore, interleukin (IL)‐5 produced by CD8+CD57+ T lymphocytes upon in vitro T‐cell receptor stimulation are increased in PBC. Histologically, CD8+CD57+ T cells accumulate around the portal area in PBC. Moreover, CD8+CD57+ T cells respond specifically to the major histocompatibility class I epitope of PDC‐E2. Conclusion: In conclusion, our data demonstrate that CD45ROhighCD57+CD8high T cells are a subset of terminally differentiated cytotoxic TEM cells, which could play a critical role in the progressive destruction of biliary epithelial cells. (HEPATOLOGY 2011;54:1293–1302)