Bidirectional Regulation Between CD4~+T Cell Homeostasis And Hepatic Stellate Cells During Hepatitis B-induced Liver Fibrosis

BackgroundHepatic stellate cells (HSCs) play a central role in liver fibrosis (LF), and are recently reported to act as antigen presenting cells (APC) and modulate CD4+T cell response. CD4+T cells with different lineages could exert diverse immune regulation on LF progression, essentially an immunological and inflammatory process in response to liver injury. The discovery that the differentiation of both T helper cell type 17 (Th17) and regulatory T cells (Treg), new members of CD4+T cell family with opposite functions to modify immune response, requires transforming growth factor-β(TGFβ) provided the first evidence that these subsets might be developmentally linked. Besides, when encountering antigen in an inflammatory milieu, Treg cells could acquire the functions of Th17 cells. Nowadays, many studies have proposed the concept of ’Treg/Th17 homeostasis’ like old Th1/Th2 homeostasis, simply as constrains between Treg and Th17 in cell number and function, to make better understanding of disease progression mechanisms involving both Treg and Th17.Elevated frequency of both Treg and Th17 cells had already been detected in chronic hepatitis B (CHB) but exact role of Treg, Th17 even Treg/Th17 homeostasis when CHB progressing to hepatitis B-induced liver cirrhosis (LC) is unclear and debatable. In addition, activated HSCs secret critical cytokines such as TGFβ,IL-6 and retinoic acid (RA) to control development and maintain function of both Treg and Th17, we suppose HSCs might participate in regulating Treg/Th17 homeostasis and the outcome of the regulation, modified Treg/Th17 homeostasis, might vice verse influence the pro-fibrotic features of HSCs.Methods1. CD4+T cell homeostasis evaluation during hepatitis B-induced liver fibrosisClinical parameters, blood and liver biopsy specimens of patients at different stages of HBV-related chronic liver diseases (CHB and LC) were collected. Peripheral blood mononuclear cells (PBMC) were isolated. Frequency, phenotype of four major CD4+T cell subsets (Thl,Th2,Treg and Th17) and CD4+T cell homeostasis (mainly Thl/Th2 homeostasis and Treg/Th17 homeostasis) in PBMC were analyzed by multi-color flowcytometry using antibodies (CD4, CD25, CD28, CCR6, CTLA-4, Foxp3, IL-10, TGF-β, IL-17, IFN-y and IL-4). Critical transcription factors (Foxp3, RORA, RORC, T-bet, GATA3, SATA3, STAT4 and STAT5a) that control the development of CD4+T cell subsets were evaluated by Real-time PCR. Hepatic expression of RORyt, Foxp3 and a-SMA in CHB patients was assessed by immunohistochemistry to elucidate hepatic Treg/Th17 homeostasis.2. In vitro study of HSCs’ regulation on Treg/Thl7 homeostasisStimulants such as TGFβ1, IL-17 and lipopolysaccharide (LPS) were used to stimulate human hepatic stellate cell line LX-2 to verify whether Treg/Th17-associated genes (TGFβ1, IL-21, IL-23 and RARa) expressed in LX-2 cells could be influenced by stimuli from microenvironment. LX-2 cells pre-stimulated by TGFβ1 were co-cultured with peripheral CD4+T cells from LC patients to evaluate LX-2 cells’ influence on the expression of lineages-specific transcriptional factors (T-bet, GATA3, Foxp3 and RORA) in CD4+T cells. CD4+T cell homeostasis (Foxp3/IL-17 ratio and IFN-y/IL-4 ratio in total CD4+T cells) of in vitro co-culture system was analyzed by flowcytometry and cell proliferation of CD4+T cells in this system was detected by continuous monitoring using Cell-IQ machine.3. In vitro study of Treg/Th17 homeostasis on HSCs’ pro-fibrotic functionPeripheral CD4+T, CD4+CD25+T and CD4+CD25-T cells in PBMC from LC patients were sorted using flowcytometry. We created different modes of Treg/Th17 homeostasis using CD4+cells, CD4+CD25+cells, CD4+CD25-cells, recombinant human cytokine IL-17 and neutralizing antibody to human IL-17. Then diverse modes of Treg/Th17 homeostasis were used to perform co-culture experiments with LX-2 cells. After co-culture, fibrosis-associated genes (TGF-β1, Smad3, MMP2, TIMP2, CollagenⅠand CollagenⅢ) expressed in LX-2 cells were analyzed by Real-time PCR while cell apoptosis by flowcytometry and cell proliferation of LX-2 cells by Cell-IQ.Results1. CD4+T cell homeostasis evaluation during hepatitis B-induced liver fibrosisPeripheral Foxp3/CD4, IL-17/CD4 and IL-4/CD4 ratio, respectively the proportion of Treg, Th17 and Th2 cells in total CD4+T cells, significantly increased during liver fibrosis progression. And Treg is the only subset of the major four CD4+T lineages whose proportion kept continuous elevation during CHB-LC process. Besides, relative mRNA level of Foxp3(Treg), RORA(Th17)、GATA3(Th2) and STAT5a(Treg) in PBMC also manifested dramatic elevation during this process, of which Foxp3 and STAT5a mRNA kept continuous elevation which was in coincidence with the results from flowcytometry.Peripheral Foxp3/IL-17 ratio kept elevated significantly during CHB-LC process, which indicated that Treg/Th17 imbalance exhibited Treg dominance. And Th1/Th2 imbalance was not as dramatic as Treg/Th17 imbalance duing this process. In CHB patients, peripheral Treg dominance aggravated with high serum HBV load while in LC patients with deteriorated Child stages classifying liver function. Both peripheral Treg dominance and hepatic Treg dominance, manifesting as elevated hepatic Foxp3/RORyt ratio, were much more evident in CHB patients with advanced stages/grades classifying hepatic inflammation and fibrosis degree in chronic hepatitis.2. In vitro study of HSCs’ regulation on Treg/Th17 homeostasisLX-2 cells could promote the expression of RORA gene and inhibit Foxp3 gene in CD4+T cells in cell-cell contact and cell number-dependent manner. Each of the following inflammatory stimulants:TGF-β1, IL-17 and LPS could promote TGF-β1, IL-21, IL-23 mRNA expression and inhibit RARa mRNA in LX-2 cells. Peripheral Foxp3/IL-17 ratio was dramatically down-regulated by TGF-β1-prestimulated LX-2 cells and this regulation was dependent on the expression of IL-21 and IL-23 in LX-2 cells. Besides, LX-2 could maintain CD4+T cell survival in vitro.3. In vitro study of Treg/Th17 homeostasis on HSCs’ pro-fibrotic functionTreg/Th17 imbalance exhibiting as Th17 dominance could promote the expression of pro-fibrotic genes (TGF-β1, Smad3, CollagenⅢand MMP2) in LX-2 cells while Treg dominance could inhibit these genes. The above regulation on HSCs’ fibrosis-associated genes might depend on the expression of IL-8 and IL-10 in CD4+T cells, respectively. Treg/Th17 imbalance had no influence on apoptosis of LX-2 cells. Besides, IL-17 (the most important functional cytokine secreted by Th17 cells) could enhance pro-fibrotic TGF-β1 and inhibit anti-fibrotic IL-10 secretion in LX-2 cells, while CD4+CD25+cells (approximately representative of Treg cells) exerted opposite function on the secretion of above cytokines.Conclusions Peripheral and hepatic Treg/Th17 imbalance exists in the process of hepatitis B-induced liver fibrosis which manifests as Treg dominance and positively correlates to serum HBV load, grades/stages of chronic hepatitis and child stage of liver function. Activated human HSCs cell line LX-2 could promote Th17 cells and inhibit Treg cells to down-regulate Foxp3/IL-17 ratio in vitro co-culture systems. And modified Treg/Th17 homeostasis by LX-2 cells vice versa could promote the pro-fibrotic function of LX-2 cells.