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IAS VISITING FELLOWSHIP REPORT - LINDA FOGELSTRAND, MD, PHD

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Interleukin-10 (IL-10) is an immunosuppressive cytokine produced by B and T cells and monocytes/macrophages. It exerts strong inhibitory effects on the innate immune system by down-regulating expression of proinflammatory cytokines in macrophages and decreasing production of reactive oxygen species in polymorphonuclear cells. It has also been shown to be protective against atherosclerosis. Accumulating data also support an atheroprotective effect of natural antibodies. These antibodies are mostly germ-line encoded IgM with broad antigen specificity. They are called the humoral arc of innate immunity, and in uninfected mice, the vast majority of plasma IgM are such natural antibodies. In mice, they are generated in large part by a special subset of primordial B cells, termed B-1 cells, by a process that is independent of cognate T cell help. However, a variety of cytokines were thought to influence B-1 cell function in a generalized manner. Based on the atheroprotective effect of IL-10, and previous literature suggesting that IL-10 was necessary for the B-1 cell compartment, we postulated that IL-10 might positively regulate the production of natural antibodies from B-1 cells.

B-1 cells reside in large part in the peritoneal cavity, allowing for isolation of a pure population. Surprisingly, we found that IL-10 had inhibitory effects on the production of IgM from B-1 cells isolated from the peritoneal cavity of mice. Both TLR-4 and TLR-2 induced IgM secretion was inhibited by IL-10. In parallel with IgM, TLR-activated B-1 cells secreted IL-10. We also found that B-1 cells expressed the receptor for IL-10, IL-10R. An anti-IL-10Rα antibody blocked the inhibitory effect of exogenous IL-10 and caused an increase in IgM compared with TLR4 agonist alone. Thus, IL-10 may regulate activation/IgM production from B-1 cells in an autocrine fashion. [³H]-Thymidine incorporation assay and CFSE staining showed decreased proliferation when TLR4-activated cells were cultured in the presence of IL-10. No effect of IL-10 was seen on the differentiation of B-1 cells to plasma cells or on apoptosis, as judged from staining with anti-CD138 and Annexin-V. Thus, the inhibitory effect of IL-10 on IgM production was due to an inhibition of proliferation.

Three in vivo findings support our in vitro data: 1) Injection of IL-10 together with a TLR4 agonist intraperitoneally into C57BL/6 mice caused decreased B-1 cell numbers in the peritoneum compared with Lipid A alone, 2) mice overexpressing IL-10 (Il-10^Tg) (IL-10 transgene under the IL-2 promoter) displayed lower titers of total plasma IgM and lower titers of IgM against CuOx-LDL and MDA-LDL (prominent epitopes for natural antibodies) compared with wild type mice, and 3) bone marrow transplantation of Il-10^Tg bone marrow into Ldlr^-/-Rag1^-/- mice, which have no endogenous antibodies, caused a dramatic decrease in circulating IgM against CuOx-LDL and MDA-LDL compared with transplantation of wild type bone marrow. Atherosclerosis measurements in these mice showed no difference between wild type and Il-10^Tg bone marrow. We therefore hypothesize that the lack of effect of IL-10 in this system (Ldlr^-/-Rag1^-/- mice as recipients) could be at least in part due to very low levels of presumably atheroprotective IgM.

In conclusion, my research has uncovered a previously unrecognized autoregulatory loop whereby activation of B-1 cells leads to the secretion of IL-10, which in turn exerts immunosuppressive effects on B-1 cells as on other cells of innate immunity. This appears to be the first such autoregulatory loop described for control of B-1 cell function. This proposed autocrine regulatory loop may be involved in vivo in the regulation of B-1 cells and their generation of natural antibodies.

• 3-Month Visiting Fellowship Award: $5,000
• 6-Month Visiting Fellowship Award: $8,000

• Improve skills and knowledge in the field of cardiovascular disease
• Learn new research techniques in the field of cardiovascular disease
• Implement new techniques or initiate new programs in atherosclerosis and cardiovascular disease in home country

• Member of a constituent society of the IAS

  Actively engaged in research in the field of cardiovascular disease
• Individuals only MD/PhD or equivalent degree required
• Age 40 or less