Fig 2: The microbiota does not control inflammatory lung phenotypes in epg5-/- mice. (A,B) Flow cytometric analysis was performed on lung tissue from Epg5+/- and epg5-/- SPF, GF, or GF+FT littermates, and the percentage of neutrophils (PTPRC/CD45+ Ly6G+ ITGAM/CD11b+) (A) as well as the ITGAM/CD11b-positivity of alveolar macrophages (pre-gated on PTPRC/CD45+ SIGLECF+ ITGAX/CD11c+) (B) was assessed. (C) Histological analysis of Epg5+/- and epg5-/- SPF, GF, or GF+FT littermate lungs, stained for SQSTM1 with DAPI counterstaining. Representative photomicrographs are shown. (D) QPCR was used to analyze expression of Il1b and Tnf in lungs from these mice. N = 6–8 mice per group over two independent experiments. Statistical analyses performed using Mann-Whitney test.

Fig 3: The PP adjuvant combination induces high nAb responses in mice, dependent on the presence of functional macrophages. a–f C57BL/6 mice were immunized twice at 14-day interval with DENV-2, EDIII alone or combined with AL or PP, and immune response analyzed as shown in a. b Log10 EDIII-specific IgG titers, (c) log10 PRNT50 titers, and (d) proportion of EDIII-specific IgM, IgG1, IgG2b, IgG2c, and IgG3 in the serum of the indicated mice at 28 days post vaccination (n = 9-10 mice per group from two independent experiments). Isotyping was done on pooled samples for each group. e, f Seven days post initial immunization, spleens were harvested from two mice per group and GCs detected by IFA. Representative images are shown in Supplementary Fig. 9. e Quantification of GCs in the entire spleen section for two mice per group. f Quantification of IgG+ cells (PBs) in two 1 mm2 areas for each spleen section. Data shown are the mean ± SEM. g–h C57BL/6 mice were treated with PBS-L or CL-L before immunization with EDIII + PP as shown in g. Cell depletion after CL-L treatment is shown in Supplementary Fig. 10. h Log10 PRNT50 titers at 28 days post vaccination (n = 9–10 mice per group from two independent experiments). i, j Conditional IFNAR knockout mice CD11c-Cre IFNARfl/fl and LysM-Cre IFNARfl/fl were immunized with EDIII + AL or EDIII + PP as shown in i. Viremia was analyzed by plaque assay in the serum 4 days after DENV-2 challenge in CD11c-Cre IFNARfl/fl (i) and in LysM-Cre IFNARfl/fl mice (j) (n = 8–10 mice per group from two independent experiments). Data shown are the mean ± SEM. Significant differences are determined by unpaired two-tailed t test. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.005

Fig 4: MCPIP1 suppresses the activation of macrophages and, indirectly, Th17 lymphocytes and B-cell subsets. (A) The total number of CD11c+ dendritic cells and F4/80+ macrophages and their activation in spleens was quantified by flow cytometry in both investigated genotypes as described in the Materials and Methods. Data represent means±s.e.m. from at least eight mice per group. **P<0.01, ***P<0.001 versus control mice. (B) Spleen sections were stained for CD3+ T cells and quantified with image software as described in the Materials and Methods (magnification ×100). Data are means±s.e.m. from at least eight mice per group; ***P<0.001. RNA was isolated from spleens, and mRNA levels in spleen were analyzed for relevant transcripts. (C) Expression levels of Il2 were quantified by real-time PCR. Data are shown as means of the ratio of the specific mRNA versus that of Gapdh mRNA; *P<0.05. (D,E) The total numbers of the CD4+ T-cell subset (D) and Th17/Treg ratio (E) were quantified by flow cytometry in both investigated genotypes as described in the Materials and Methods. Data represent means±s.e.m. from at least eight mice per group. *P<0.05 versus control mice. (F) mRNA levels of T-cell-relevant transcription factors in spleens were quantified by real-time PCR. Data are shown as means of the ratio of the specific mRNA versus that of Gapdh mRNA; *P<0.05, ***P<0.001 versus control mice. (G) Spleen sections were stained for CD19+ B cells and quantified with image software as described in the Materials and Methods (magnification ×200). Data are means±s.e.m. from at least eight mice per group; ***P<0.001. The total numbers of spleen B220+/CD19+/MHCII+ cells were quantified in 6-month-old mice by flow cytometry. Data are means±s.e.m. of at least eight mice per group; **P<0.01, ***P<0.001 versus control mice. (H) mRNA levels in the spleen were analyzed for B-cell survival and stimulatory factors. Expression levels were quantified by real-time PCR. Data are shown as means of the ratio of the specific mRNA versus that of Gapdh mRNA; *P<0.05, ***P<0.001. (I) Mature B cells and kappa light chain+ CD138+ (also known as Sdc1+) plasma cells were quantified in 6-month-old mice by flow cytometry. Data are means±s.e.m. of at least eight mice per group; **P<0.01 versus control mice. n.s., not significant.

Fig 5: Gating strategy for identification of respiratory lymphocyte subsets and Linnegleukocytes. Respiratory B and T lymphocytes were identified according to low side scatter characteristics, CD19 and CD3e surface expression. This gating strategy resulted also in exclusion of autofluorescent respiratory macrophages. Out of the CD3e-positive population CD4+ T helper, CD4+CD25+ T helper and CD8 T cells were gated according to staining with the respective mAbs and ?d T cells were identified by using a ?d TCR mAb. Please note that the CD4+CD25+ T cells are not CD25high. A more detailed gating strategy of Treg subsets including isotpye controls is shown in Figure 8 . Natural killer cells were identified according to CD49b+Cd3eneg expression. Respiratory lineage-negative leukocytes were identified by absence of CD3e, CD8a, CD11b, CD11c, CD19, B220, TER-119, FceR1 (Lineage cocktail) and CD4 expression and subsequently separated into CD90+ and CD90neg cells.