The Role Of PGRN In The Pathogenesis Of Experimental Colitis And Potential Mechanism

Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is characterized by inflammation of the gastrointestinal tract. IBD results from a continuum of complex interactions between a quartet of host-derived and external elements that involve various aspects of the intestinal microbiota, the immune system, the genetic composition of the host, and specific environmental factors. Nearly4million people worldwide are affected with either ulcerative colitis or Crohn’s disease, and about1.4million of these cases occur in the United States, and the incidence and prevalence of IBD increasing in our country recently. It is well established that TNFa is of crucial importance in the pathogenesis of IBD. TNF acts as a transmembrane or soluble protein by transducing signals ranging from cellular activation and proliferation to cytotoxicity and apoptosis through two distinct TNF receptors, TNFR1(p55) and TNFR2(p75). Tnf△ARE mice overproduce TNF and develop CD-like deep transmural intestinal inflammation. TNF also plays an important role in other models of intestinal inflammation, like TNBS-induced models and in IL-10-/-mice. In the CD45RBhigh transfer model of colitis, continuous anti-TNF antibody treatment was required to decrease disease severity. TNF-blockers represent a well-accepted therapeutic option in the treatment of these diseases. It is very important to investigate new cytokine target or other TNF-a substitutes in IBD since TNF-blockers can risk cancer and exhibit its effect on50-70%IBD patients.Progranulin (PGRN), also known as granulin-epithelin precursor(GEP, PC-cell -derived growth factor (PCDGF), or acrogranin, was first purified as a growth factor from conditioned tissue culture media. It is composed of seven and one half copies of the cysteine-rich granulin-epithelin motif, separated by linker regions. Progranulin is translated as a593amino acid,68.5kDa cysteine-rich protein, that is heavily glycosylated and appears as an approximately90-kDa protein on SDS-PAGE. Elastase and other extracellular proteases can cleave PGRN within the linker regions to generate GRNs, and granulins also can modulate cell growth. Progranulin is expressed in a variety of tissues throughout the body, particularly in epithelial and hematopoietic cells. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Although PGRN plays crucial roles in multiple physiological and pathological conditions, in the past two decades efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). In2012, Dr. Liu from New York University reported that PGRN binds to TNF receptors (TNFR) and inhibits the inflammation response of TNF-a and the progression of inflammatory arthritis.At present, the role of PGRN in IBD is not well understand. In this study, we intend to explore the role and mechanism underlying PGRN signaling in the pathogenesis of IBD. We believe that our studies will not only discern the immune regulatory mechanism of PGRN in the pathogenesis of IBD, but will encourage the application of PGRN to other immune-related disease models as well.1. The role of PGRN in the pathogenesis of experimental colitisTo determine the requirement of PGRN in the development of immune cell populations in wild-type and PGRN-deficient mice. Flow cytometry method were used to analyze the percentage of MHCII+CD11c+, CD3-CD19-mPDCA-1+and CD4+Foxp3+cells of spleen and mesenteric lymph nodes from WT and PGRN-deficient mice. Our results demonstrate that no significant differences in the distribution of major cellular components and no evidence of spontaneous colitis and immune system activation were found in PGRN-/-mice.To determine whether PGRN is expressed in the intestinal tissue and induced during inflammatory conditions. Immunohistochemistry method was used to detect the expression of PGRN in IBD patients intestinal tissue samples and mouse intestinal tissue after DSS induction. Real-time PCR results suggest that PGRN gene is highly expressed in CDllb+cells and Ly6G+cells compared to other immune cells including T cells and B cells. Immunohistochemistry results demonstrate that PGRN is stronger expression in IBD conditions in intestinal colonic tissue including epithelial cells and inflammatory cells infiltrating areas. Western blot results show that recombinant PGRN can stimulate the phosphorylation of Erk pathway in human Caco-2cells. These finding suggest PGRN is induced in the IBD inflammatory conditions and functional on the intestinal cells.To further study the contribution of endogenous PGRN to the protection of colitis development, we first assessed the susceptibility of age-and sex-matched wild-type and PGRN-/-mice after oral administration of3%DSS in drinking water. The results demonstrate that PGRN-/-mice are more susceptible to DSS induction than WT mice, displaying significantly body weight loss, higher stool scores and bleeding scores. Colons of PGRN-/-mice were on average20%shorter than those of wild-type mice treated with DSS (P<0.03). HE staining of colonic sections of DSS-fed PGRN-/-mice show that marked histopathological changes in characterized by severe transmural inflammation with focal areas of extensive ulceration and necrotic lesions, marked infiltration of inflammatory cells. Similar results were found in TNBS model which resembles Crohn’s disease was established through intrarectal administration of150mg/kg2,4,6-trinitrobenzenesulf-onic acid (TNBS) in ethanol. In conclusion, these results suggest that PGRN plays a protective role in chemical-induced acute colitis.To determine whether immune cells-derived PGRN is play a role in DSS-induced colitis model, two groups of PGRN bone marrow chimera were generated by reconstituting irradiated wild-type mice with bone marrow cells from KO/WT donors and subjected them to3%DSS solution for5days. Clinical parameters and histopathological parameters demonstrate that wild-type mice transplanted with PGRN-/-bone marrow were more sensitive to DSS-induced colitis, suggesting that immune cells-derived PGRN can protect colitis development.The composition of intestinal bacteria was further examined using real-time PCR and intestinal permeability using FITC-dextran method in naive and DSS-treated wild-type and PGRN-/-mice. The results suggest that PGRN deficiency does not affects intestinal bacteria overgrowth and alter the FITC-dextran level in serum. These finding suggest that the severity of colitis in PGRN-/-mice is not due to cause by bacteria overgrowth or alter the composition of intestinal microbiota and affect intestinal permeability.We also established TNBS colits model in TNFR1-and TNFR2-deficient mice, and injected same dose of PGRN with wild-type mice. The results demonstrated that PGRN lost its therapeutic effect in these mice.The results show that recombinant PGRN can ameliorate colitis syndrome, and PGRN treatment significantly increased the IL-10release from colonic explant cultures in DSS colitic mice compare with PBS treatment (P<0.01). To determine whether IL-10signaling is required for PGRN-mediated protection from experimental colitis, IL-10signaling were blocked with a specific anti-IL-10receptor antibody and TNBS model was established in IL-10-deficient mice. Anti-IL-10R group mice suffered from significant body weight loss, HE staining of colonic sections displaying marked inflammatory cells infiltration, colonic ucleration and changes of crypt architecture. The results suggest that anti-lOR mAb can offset protection of PGRN against body weight loss caused by DSS induction. PGRN’s therapeutic effect was lost in IL-10-/-mice, at least in part. Anyway, these results suggest that PGRN upregulates the expression of IL-10, and IL-10signaling is required for PGRN-mediated protection from intestinal inflammation.In conclusion, PGRN is expressed in multiple cells and regulates intestinal homeostasis in acute experimental colitis in mice. PGRN deficiency accelerates the progression of experimental colitis and recombinant PGRN decrease the severity of colitis syndrome, suugesting that PGRN plays a protective role in acute colitis. The results of chimeric mice model demonstrated that immune cells-derived PGRN plays a protective role in acute colitis. Further investigation suggest that the mechanism of PGRN’s therapeutic effect is associated with upregulation of IL-10level. IL-10as a important microenvironment regulatory factor involved in gut repair. Anyway, our results demonstrated that PGRN plays a crucial role in regulation of intestinal inflammation, these findings are helpful for the treatment of IBD patients.2. The function of PGRN in CD4+T cells in vitro and in vivoTo determine the role of PGRN in the development of CD4+Foxp3+regulatory T cells, we examined the CD4+Foxp3+T cells in normal wild-type1,3and6weeks old C57BL/6mice and in comparable PGRN-/-mice using flow cytometry method. Our results demonstrate that no significant differences in the percentage of CD4+Foxp3+T cells in thymus, spleen and lymph nodes were found in WT and PGRN mice. The results suggest that PGRN is not required for the development of natural regulatory T cells (nTreg) in vivo.To further investigate whether PGRN plays a redundant or additive role regarding the development of inducible regulatory T cells (iTreg). CD4+CD25-T cells were stimulated with plate-bound CD3Ab and solute CD28Ab and cultured the cells for3days in the presence or absence of TGF-β and PGRN. The results demonstrate that PGRN increased, in a dose-dependent manner, the conversion to Foxp3+cells induced by a low concentration (0.1ng/ml) of TGF-β. Even in the absence of TGF-β, PGRN (1μg/ml) could induce Foxp3expression in7.51%of the cells, as compared with0.19%Foxp3cells without PGRN. The results demonstrated that PGRN enhances the development of inducible regulatory cells.To evaluate the role of PGRN signaling in regulation of Treg function, in vitro suppression assay were also performed.5×105CFSE-labeling Teff cells were stimulated for72hours with CD3antibody (5μg/ml) in the presence of1×105APC cells and varying ratios of FACS purified WT or PGRN-/-Tregs. The results demonstrate that PGRN-/-Tregs significantly decreased the ability to suppress Teff cells proliferation in vitro at ratios of1:2and1:1(P<0.05), compared with WT Treg cells.To further study the molecular mechanism of functional difference between WT and PGRN-/-Treg cells, the expression of BrdU and Wnt family gene were detected in WT and PGRN-/-Treg by FACS method and real-time PCR respectively. The findings suggest that PGRN deficiency does not affect the proliferation of Treg cells, and wnt receptor gene Fzd2significantly upregulates in PGRN-/-Treg cells (P<0.01).To determine the role of PGRN signaling in CD4+T cells in vivo, CD4+CD25-Rbhi T cells from WT and PGRN-/-mice were transferred into Ragl-/-mice. These results demonstrate that transfer of CD4+CD25-Rbhi T cells from PGRN-/-donor mice into Ragl-/-recipient mice lead to an accelerated onset of body weight loss. The results of the histopathological analysis of the colons from Ragl-/-recipient of PGRN-/-CD4+CD25-Rbhi T cells demonstrated severe alterations of the colon, including extensive inflammatory cell infiltration, loss of globet cells, epithelial erosion and crypt abscesses. The enhanced colitogenic potential of the transferred PGRN-/-CD4+CD25-Rbhi T cells may be explained by a reduced activation-induced cell death, leading to an enhanced accumulation of CD4+T cells.In conclusion, the results demonstrated that the role of PGRN in the development of nTreg and iTreg cells. PGRN alone and coordinated with TGF-β both enhance the conversion of CD4+CD25-T cells into iTreg cells. PGRN deficiency decreases the ability of Treg cells to suppress the proliferation of Teff cells. Further investigation suggest that the mechanism of PGRN’s regulatory effect is associated with upregulation of Fzd2expression. In vivo results suggest that PGRN deficiency accerlerates the severity and progression of T cell transfer colitis in mice. Anyway, our results demonstrated that PGRN plays a important role in T cells, especially in Treg cells, these findings are helpful for the investigation of immunological mechanism of IBD and treatment of IBD patients.Taken together, these observations that PGRN is induced in multiple cell types during intestinal inflammation and regulated intestinal homeostasis in several experimental colitis models. PGRN deficiency accelerates the severity and progression of chemical-induced colitis and T cell transfer colitis in mice. And immune cells-derived PGRN plays a protective role in DSS-induced colitis in mice. Recombinant PGRN decreases the colits severity of DSS-induced mice. We concluded that IL-10is a important regulator involved in PGRN-mediated therapeutic effect that favors resolution of intestinal inflammation. Further study demonstrated that PGRN regulates the development of iTreg cells and the function of Treg cells, and the role of PGRN in Treg function is associated with upregulation of Fzd2expression. In summary, our results demonstrate that the importance of PGRN in modulating intestinal immune response, these finding will help to investigate the immunological mechanism of IBD and design a new strategy in the treatment of intestinal inflammation.