Regulatory Roles Of Soluble DC-SIGN In Th17 Cells Differentiation And HBV Infection

Backgrounds and objectsThe increase of Th17 in patients with hepatitis B virus(HBV)infection is the main factor of HBV induced cirrhosis.Therefore,how to regulate the differentiation of Th17 cells in patients with HBV infection may be a key point in the treatment of chronic HBV infection.DC-SIGN plays a crucial role in the differentiation process of Na(?)ve T cells into Th17 cells to anti-infection.Additionally,the differentiation of Th17 cells could not be induced by inflamamatory stimulation in the DC-SIGN deficient or gene knockout mice.SDC-SIGN is a soluble form of DC-SIGN,which has not been reported in relation with Th17 cell differentiation and HBV infection.Our previous study has found that the concentrations of sDC-SIGN are lower in patients with HBV infection than those in healthy volunteers.However,whether sDC-SIGN plays a role in HBV infection by regulating Th17 cell differentiation still remains unclear.In the present study,we developed a sandwich ELISA method for sDC-SIGN measurement and analyzed the relationships between sDC-SIGN and Th17 cell in patients with HBV infection,to explore the regulatory role of sDC-SIGN on Th17 cell differentiation and the underlying molecular mechanisms,and to elucidate the role of sDC-SIGN in HBV infection.At the same time,the relationship between nanol and DC-SIGN and its inhibitory effect on tumor cells were also studied.MethodsRecombinant sDC-SIGN protein was obtained via constructed sDC-SIGN prokaryotic expression vector and monoclonal/polyclonal antibodies against sDC-SIGN protein were prepared.A double antibody sandwich ELISA was developed and evaluated for detecting human sDC-SIGN.We measured the expression of DC-SIGN and sDC-SIGN in HBV infective mice and then analyzed the relationships between levels of sDC-SIGN,IL-17a,TGF-β1,IL-10,IL-6,IFN-γ,HBsAg,HBV DNA,ALT and TBIL.SDC-SIGN and HepG2.2.15 cells were co-cultured in vitro to observe its direct effects on HBV virus.In vitro,CD3+T culture system(anti-CD3/CD28 presence),Th17 polarize system and DC-T mixed lymphocyte reaction system were co-cultured with sDC-SIGN,to observe its regulatory role in T cell proliferation,differentiation,secretion of cytokines and phosphorylation levels of JAK2-STAT3 molecules.In addition,the concentration of sDC-SIGN in peripheral blood of patients with hepatocellular carcinoma was analyzed.And a new zinc complex,nano1 was co-cultured with human leukemia cell line ARH-77 to analyze its anti-cancer activity and its relationship with sDC-SIGN.Results1.Development of ELISA detection systemRecombinant sDC-SIGN protein and monoclonal/polyclonal antibodies against sDC-SIGN were prepared.The double antibody sandwich ELISA for detecting human sDC-SIGN were developed and evaluated,with a sensitivity of 0.2 ng/ml and a wide linear range.The correlation coefficient R2 was as high as 0.98.The intra-assay variation was 10.4%and inter-assay variation was 13.8%,respectively.SDC-SIGN concentrations of 172 normal individuals were positively skewed,ranging from 0 to 319 ng/ml with a median of 27.14 ng/ml.The plasma concentrations of sDC-SIGN in HBV infected patients were significantly lower than those in healthy volunteers.Plasma from a healthy volunteer with high sDC-SIGN concentration was collected,purified,and then subjected to mass spectrometry analysis,which demonstrated that native sDC-SIGN is highly homologous to CD209 by the mass-to-charge ratio(M/Z)of fragmentation for the first time.2.The relationships between sDC-SIGN and Th17 and its significance in HBV infection.The maturation of DCs were blocked in HBV infection mice,mainly due to the phagocytic functions of the phagocytes,such as the weakened capacity of secreting cytokines,the weakened T cell differentiation ability,and significantly downregulated mDC-SIGN expression.The results of clinical samples analysis showed that sDC-SIGN were negative correlated with HBV DNA,HBsAg,ALT,IL-17a and Th17 cells,and positively correlated with TGF-β1 and Treg cells.In vitro,neither HBV DNA replication nor HBsAg expression in HepG2.2.15 cells can be directly inhibited by sDC-SIGN.3.The regulatory of sDC-SIGN in Th17 cells differentiation and the underlying mechanisms.In vitro,studies in CD3+ T cells showed that sDC-SIGN inhibited the secretion of IL-17a,IFN-γ,IL-10 and IL-2 in presence of anti-CD3 and anti-CD28,but stimulated the secretion of IL-6.Besides,the proliferation of T cells was suppressed by sDC-SIGN on dose-dependent manner.Th17 polarization could be inhibited by sDC-SIGN on dose-dependent manner,with the expression of CD69,CD25 and transcription factor RORyt.And the secretion of IL-17a,IL-21 and other cytokines secreting in Th17 cells were inhibited by sDC-SIGN on a doses-dependent and time-dependent manner.The phosphorylation of ERK and JAK2-STAT3 were also inhibited by sDC-SIGN.DC-T mixing lymphocyte test showed that sDC-SIGN inhibited the secretion of IFN-y and proliferation in T cells.4.Relationship between nanol and sDC-SIGN and its inhibitory effect on tumor cellsSoluble DC-SIGN was low expressed in patients with hepatocellular carcinoma.Nano1 can inhibit the proliferation,migration and invasion of ARH-77 cells,and may also affect the expression of soluble DC-SIGN.ConclusionsThe maturation of DCs were blocked due to HBV infection,and mDC-SIGN expressions were downregulated,resulting in decreased sDC-SIGN secretion,and sDC-SIGN is negatively correlated with HBV DNA,HBsAg,IL-17a,and Th17.Meanwhile,sDC-SIGN can inhibit the differentiation of Th17 cells and then affect the process of HBV infection.The potential mechanisms were that the competition with DC-SIGN for T cells surface ligands interferes with the formation of immune synapses to inhibit Th17 cell differentiation or inhibit IL-6 signaling pathway-related molecules phosphorylation,downregulate the expression of transcription factor ROR y t and inhibit the differentiation of Th17 cells directly.Nano1,a new anti-leukemia chemical,can inhibit the proliferation,migration and invasion of ARH-77 cells.However,whether Nano1 can inhibit tumor growth by regulating the expression of DC-SIGN needs further research.Innovation and significance:1.The study revealed the relationships between HBV-sDC-SIGN-Th17 first time.It indicated that HBV infection induces DC immature,resulting in decreased expressions of mDC-SIGN and sDC-SIGN.And at the same time sDC-SIGN may regulate Th17 cell differentiation to influence HBV infection process.This study will provide a new perspestive for clarifying persistent chronic HBV infection,and also shed new light on the prevention and therapy strategies in HBV infection.2.This study,for the first time,attempts to elucidate the mechanism of sDC-SIGN regulating Th17 cells differentiation.The potential mechanism may be that the competition with DC-SIGN for T cell surface ligands interferes with the formation of immune synapses and thereby inhibits Th17 cell differentiation.SDC-SIGN directly inhibits IL-6 signaling pathway-related molecules phosphorylation,and then downregulates the expression of transcription factor RORyt and inhibits the differentiation of Th17 cells may be another potential mechanism of regulate on Th17 cells differentiation by sDC-SIGN.3.For the first time,this study demonstrated that natural sDC-SIGN is highly homologous to CD209 by the mass-to-charge ratio(M/Z)of fragmentation.Serum from a healthy volunteer with high sDC-SIGN concentration was collected,purified for mass spectrometry analysis and the results showed that native sDC-SIGN were highly homologous to CD209.It provided fundamental ground for further study of the structure and function of sDC-SIGN.4.This study attempts to explore the effect of chemical drugs on immune regulation and expand the new ideas of developing chemical anti-tumor drugs.