The Role Of Lipoxin A4in Tumor Immunity By Targeting Regulatory B Cells

Objective:Lipoxin A4(LXA4) plays an important role in the regulation of inflammation. LXA4also has anti-tumor effect. In our previous study, LXA4limited hepatocarcinoma cells growth by remodeling tumor microenvironment and inhibiting tumor angiogenesis. However, the underlying immunological mechanism remains unclear. Regulatory B cells, are defined as IL-10producing B cells, have been proved to be critical for the regulation of inflammation, autoimmune disease and cancer. Recent study showed that LXA4receptor expressed on human and mouse B cells. Our study aims to elucidate the immunological mechanism by which LXA4inhibits tumor growth through examining the effect of LXA4on regulatory B cells.Methods:(1) Different tumor cells were inoculated subcutaneously (s.c.) into mice. Mice were randomly divided into two groups and one group were i.p. injected with BML-111(1mg/kg) once per two days. The tumor growth was measured. Moreover, H22tumor cells were labeled with CFSE. After24hours, cells were harvested and analyzed by flow cytometry.(2) To explore the mechanism by which LXA4limits tumor growth, H22tumor cells were inoculated s.c. into SCID mice. Mice were divided into four groups, control group, LXA4treatment group, CD3+T cells adoptive transfer group and adoptive transfer plus LXA4treatment group. The tumor growth was measured.(3) To study the effect of LXA4on regulatory B cells. Splenocytes of naive mice were stimulated with PMA, ionomycin, LPS and monesin in the absence or presence of LXA4for5hours. The number of IL-10-producing Breg cells was determined by flow cytometry. On the other hand, splenocytes were isolated from LXA4-treated or PBS-treated tumor-bearing mice, the induction of IL-10-producing Breg cells was determined by flow cytometry. To study the effects of LXA4on other functions of B cells. CFSE staining, flow cytometry and HE staining were employed to examine the role of LXA4in B cells proliferation, plasma cells differentiation and germinal center formation.(4) H22tumor cells were inoculated s.c. into nude mice. The mice were i.p. injected with LXA4once per two days. The rumor growth was measured. The number of regulatory B cells was determined by flow cytometry and the expression of IL-6, TNF-α, IL-1β in serum and spleen was determined by ELISA.(5) Purified CD4+T cells were labeled with CFSE and the effect of LXA4on their proliferative ability was determined. Purified CD8+T cells were stimulated with PMA and ionomycin, the effect of LXA4on their IFN-y production was determined.(6) H22tumor cells were inoculated s.c. into Balb/c mice. The mice were i.p. injected with LXA4once per two days. On day15, mice were sacrificed and cells isolated from spleen, draining lymph nodes and tumor tissue. The number of regulatory T cells and the cytotoxic ability of CD8+T cells were determined by flow cytometry.(7) CD4positive and CD25negative T cells were isolated by MACS and stimulated by CD3and CD28in the presence of cytokines TGF-β and IL-2.72hours later, the role of LXA4in the induction of Treg cells was determined by flow cytometry. Moreover, Purified CD4+T cells co-culture with purified B cells at a1:1ratio and were stimulated with anti-CD3and LPS.72hours later, cells were harvested for Treg cells analysis by flow cytometry.(8) To explore the molecular basis through which LXA4inhibited the generation of regulatory B cells. Splenocytes or B cells were isolated from LXA4-treated or PBS-treated tumor-bearing mice for protein extraction. The phosphorylation levels of STAT3and ERK were determined by Western blot. Regulatory B cells were induced with ERK inhibitor or STAT3inhibitor treatment for5hours. IL-10production was analyzed by flow cytometry.Results:(1) LXA4analogue BML-111inhibited various tumor cells growth in vivo. Moreover, LXA4didn’t have direct effect on tumor cells.(2) In H22tumor-bearing SCID mice, the treatment of LXA4did not suppress tumor growth and even favored it. Although anti-tumor effect enhanced after CD3+T cells were adoptive transferred into them, tumor growth was also not influenced by LXA4treatment.(3) LXA4 suppressed the generation of regulatory B cells in vivo and in vitro. However, LXA4did not influence B cells proliferation, germinal center formation and plasma cells differentiation.(4) LXA4significantly suppressed regulatory B cells generation as well as the expression of inflammatory cytokines IL-6, TNF-a and IL-1β. However, LXA4did not cause tumor regression in nude mice.(5) LXA4did not influence the proliferation of CD4+T cells and the IFN-γ production by CD8+T cells.(6) LXA4decreased the number of Treg cells and enhanced the cytotoxic ability in the draining lymph nodes and tumor tissue. However, LXA4did not influence these two cells in the spleen. IHC staining showed that LXA4promoted the infiltration of CD3+T cells into tumor.(7) Although LXA4did not directly affect the induction of Treg cells, the addition of LXA4blocked the induction of Treg cells by either resting or activated B cells in vitro.(8) LXA4treatment inhibited the phosphorylation of ERK and STAT3in splenic B cells but not total splenocytes. The ERK and STAT3inhibitors effectively decreased the number of regulatory B cells. This indicated that the phosphorylation of these two transcriptional factors was responsible for the generation of regulatory B cells.Conclusions:LXA4can effectively inhibit various tumor cells growth; LXA4cannot directly affect the proliferation of tumor cells and can inhibit tumor growth by suppressing the generation of regulatory B cells; LXA4decreases the number of regulatory T cells and promots the cytotoxic ability of CD8+T cells in both draining lymph nodes and tumor microenvironment; LXA4suppress IL-10production of B cells by decreasing the phosphorylation levels of ERK and STAT3.