MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2

McCarthy, Mary K.; Reynoso, Glennys V.; Winkler, Emma S.; Mack, Matthias; Diamond, Michael S.; Hickman, Heather D.; Morrison, Thomas E.

Myeloid cells can suppress immune responses by a variety of mechanisms, including through the production of nitric oxide (NO) via the action of inducible nitric oxide synthase (iNOS, encoded by Nos2) [33] . Moreover, superoxide derived from the activity of the phagocyte NADPH oxidase (NOX2, encoded by gp91phox, also known as Cybb) can combine with NO to form the reactive nitrogen species peroxynitrite (ONOO -), which also can suppress immune responses [34, 35] . Based on this knowledge, we hypothesized that monocytes and neutrophils infiltrating the dLN disrupt lymphocyte accumulation and tissue organization through the actions of iNOS and NOX2. Consistent with the lack of effect of monocyte and neutrophil depletion on accumulation of lymphocytes in the dLN following attenuated CHIKV infection (S3 Fig) , total cell numbers in the dLN were unchanged in Nos2 -/and Nox2 -/mice infected PFU of CHIKV AF15561 in the left footpad. Representative flow cytometry plots of CD11b + Ly6C hi monocytes or CD11b hi Ly6C + Ly6G + neutrophils in the (A) blood or (B) dLN. At 5 dpi, the dLN was analyzed for (C) total cells, (D) CD19 + B220 + B cells, (E) TCRβ + CD4 + T cells, and (F) TCRβ + CD8 + T cells. (G-L) C57BL/6 mice were treated with 50 μg of IgG2b isotype control mAb or anti-CCR2 mAb via intraperitoneal injection one day prior to inoculation with 10 3 PFU of CHIKV AF15561 in the left footpad. Representative flow cytometry plots of CD11b + Ly6C hi monocytes or CD11b h iLy6C + Ly6G + neutrophils in the (G) blood or (H) dLN. At 5 dpi, the dLN was analyzed for (I) total cells, (J) CD19 + B220 + B cells, (K) TCRβ + CD4 + T cells, and (L) TCRβ + CD8 + T cells. Errors bars represent mean ± SEM. Data are combined from two independent experiments (n = 6-9 per group). Statistical significance was determined by Student's t-test (n.s., not significant).