Regulation Of Immune Responses In Humans and in Experimental Animals

纤维化是炎症性疾病的重要继发性表现，但也可能是器官功能障碍的主要来源。因此，在克罗恩病中，慢性肠道炎症引起的纤维化是肠梗阻和瘘管形成的原因。纤维化和相关结缔组织异常也可能是疾病的原发表现。系统性硬化症（SSc/硬皮病）就是这种情况，这是一种结缔组织的复杂疾病，其特征是皮肤和各种内脏器官中细胞外基质的积累。最近有研究表明，成纤维细胞中Friend白血病整合-1 （Fli1）和kruppel样因子5 （KLF5）通过高甲基化下调，概括了SSc的所有特征。然而，这种下调与纤维化之间关系的机制尚不明确，此外，尚不清楚Fli1和/或KLF5是否以及如何影响继发性纤维化，如发生在克罗恩病中的纤维化。

Fibrosis is an important secondary manifestation of inflammatory disorders that may nevertheless be the main source of organ dysfunction. Thus, in Crohns disease, fibrosis resulting from chronic intestinal inflammation is the cause of intestinal obstruction and fistula formation. Fibrosis and related connective tissue abnormalities may also be a primary disease manifestation. This is the case in systemic sclerosis (SSc/scleroderma), a complex disorder of connective tissue characterized by accumulation of extra-cellular matrix in the skin and various internal organs. Recently it has been shown that down-regulation of Friend leukemia integration-1 (Fli1) as well as Kruppel-Like Factor 5 (KLF5) in fibroblasts via hypermethylation recapitulates all of the features of SSc. However, the mechanism underlying the relation of such down-regulation to fibrosis is not yet defined and, in addition, it is not known if and how Fli1 and/or KLF5 influence secondary fibrosis such as that occurring in Crohns disease. In initial studies we conducted an extensive analysis of Fli1 regulation of fibrosis in Fli1 KO fibroblasts generated by CRISPR/CAS9 system. In performing Cas9 gene editing we chose a paired guideRNA/nickase system to reduce off-target effect and to enhance gene editing specificity and fidelity. Accordingly, paired gRNAs targeting Fli1 (designed by the ZiFiT program) along with a Csy4-nickase Cas9 cassette were mounted in one lentiviral vector by modifying pLenti-CRISPRv2. The latter was then transfected into NIH 3T3 cells and colonies with vector integration were selected by culture with puromycin. Confirmation of the efficiency of gene knockout in selected colonies was determined by T7 assay, sequencing, and Western blot analysis. With these Fli1-deficient cells in hand, we evaluated collagen and profibrotic cytokine production in the absence of Fli1. To this end, we measured altered function of Fli1 KO cells (compared to WT cells) at the mRNA level using RNA extracted from cultured cells, reverse-transcribed and then quantitated with real-time PCR using Taqman Gene Expression assays. In multiple studies we conducted assays of Col1a1, Col1a2, Tgfb1, CTGF, Il6, Fn1, Il11, Igf1, Igfbp3, Igfbp5, Csf2 mRNA production of cells cultured alone or in the presence of various antibodies and inhibitors. The latter included anti-TGF-1,2,3 Ab (1D11), TGF-1 receptor (ALK5) inhibitor SB-431542, anti-mouse IL-6 Ab and IL-6R Ab, Anti-IGF1-R Ab, mouse rIL-11, Erk1/2 inhibitor U0126, PI3K inhibitor LY294002, dual PI3K/mTOR inhibitor BEZ235, GSK-3 inhibitor SB216763, mTORC1/2 inhibitor PP242, and BET bromodomain inhibitor JQ1. We found that Fli1 KO fibroblasts exhibited numerous positive and negative changes in function compared to WT cells. Most notably this included a 3-5 folds increase in Col1a1 and Col1a2 synthesis that was indicative of previously reported Fli-1 inhibition of collagen synthesis. In addition it included increased synthesis of Tgf1, IL-6, Fn1, Igf1, and Csf2. Conversely, it included decreased IL-11, Igfbp3, and Igfbp5 synthesis. Thus, antibody neutralization of TGF-1, IL-6/IL6R, IGF-1, singly or in combination failed to suppress increased collagen production by Fli1KO cells; in addition, autocrine production of TGF-1 or IL-6 by these cells was not the main mechanism for increased synthesis of collagen or other extracellular matrix components by these cells. A very surprising and potentially important finding was that both Col1a1 and Col1a2 mRNA synthesis by Fli1 KO cells was dramatically inhibited by a PI3K inhibitor, LY294002, strongly suggesting that a PI3K-Akt signaling pathway was regulating collagen synthesis in KO cells. Since LY294002 is also an inhibitor of bromodomain-containing protein 4 (BRD4), in further studies we determined if a specific inhibitor of BRD4, JQ1, also inhibited collagen synthesis in Fli1 KO cells. we found that, indeed, JQ1 also inhibited collagen synthesis in Fli1 KO cells and that JQ1 in combination with a dual inhibitor of PI3K/mTOR, BEZ235, caused virtually complete inhibition of collagen synthesis in these cells. An additional finding is that BRD4 production is increased 4-fold in Fli-1 deficient cells. The discovery that PI3K and BRD4 are important regulators of collagen synthesis in the face of Fli1-deficiency opened a new avenue of research into the mechanism of Fli1 function and its regulation of fibrosis in fibrotic diseases. In current studies we determined that Gli-2 signaling, a component of the Hedgehog signaling pathway, is activated in Fli1-deficient fibroblasts and that inhibition of GLi-2 signaling by GANT61, causes down-regulation of increased collagen synthesis by the deficient cells. In addition, we found that Fli1 deficient cells secrete a factor that interacts with a fibroblast surface receptor to initiate PI3K signaling. Thus, it became apparent that Fli1 deficiency acts via GLi-2 to induce a factor that, in turn, induces collagen synthesis via PI3K. Indeed we found that supernatants obtained from culture FliKO cells contain a factor that enhances collagen synthesis that is inhibited by blockade of the PI3K signaling pathway. We further studies we conducted an RNAseq analysis of RNA extracted from Fli1KO cells that had been cultured in the presence of GANT61 or not so cultured. Our reasoning here was that cells exposed to a GLI-2 inhibitor would not synthesize a factor the acts through the PI3K signaling system to induce collagen synthesis whereas cells not so exposed would synthesize such a factor; hence differences in the genes activated under these different conditions would include the gene encoding the sought-after collagen enhancing factor. The results of this gene expression study was informative inasmuch as it identified IGF1 and/or IGF2 as possible factors inducing collagen in Fli1KO cells. On the basis of these findings we embarked on extensive studies in which we used various inhibitors of IGF1 or IGF2 (or their receptors) to determine if one or both of these factors was indeed signaling via the PI3K pathway to induce collagen synthesis. Because of the complexity of the IGF1 and IGF2 signaling system in fibroblasts and other cells these studies proved difficult and confusing. However, we have now been successful in establishing that IGF2 signaling through the insulin homodimer receptor as the main pathway in FliKO cells that results in increased collagen synthesis. In parallel in vivo studies we are investigating if treating Fli1KO mice with fibrosis with a compound that simultaneously inhibits the PI3K and BRD4 signaling pathways ameliorates colitis in the mice. These studies being conducted in collaboration with Dr. Donald Durden at the Moores Cancer Center of the Univ. of California at San Diego who has developed a compound, known as SF2523 which has such blocking properties and is theoretically able to inhibit the IGF2 signaling shown to induce collagen in cells lacking Fli1. This possibility is supported by in vitro studies that show that culture of Fli1 cells in the presence of SF2523 does greatly inhibit collagen synthesis by such cells. We have obtained preliminary data showing that a particular regimen of SF2523 administration ameliorates pulmonary fibrosis induced in Fli1KO by administration of intra-tracheal bleomycin.