Fig 2: Influence of N. brasiliensis exposure on uncolonized FGT, increase in FGT eosinophils following Nb exposure(A) Female mice were hormone-synchronized 7 days prior to Nb infection.(B) At day 9 post-Nb infection (Nb 9dpi), levels of IL-33, IL-4, and IL-5 in FGT homogenates or lavages were assessed by ELISA or Luminex. ELISA. Dotted line represents lower limit of quantification (LLOQ) for Luminex analysis. Vaginal tissue was analyzed by (C and D) (C) hematoxylin and eosin (H&E) and (D) Sirius red staining. Representative images (n = 5) were taken at ×200, ×400, and ×1,000 magnification. Black arrows identify eosinophils.(E) Frequencies (mean ± SEM) and numbers (×103) of neutrophils (CD11b+Ly-6G+), Ly-6Chi monocytes (CD11b+Ly-6Chi), macrophages (CD11b+F480+), and eosinophils (CD11b+Siglec-F+SSChi) in the FGT of naive and Nb-infected mice.(F) Mean fluorescence intensity (MFI) of Siglec-F, CD11b, Gr-1, CD62L, and CD49d on lung (green, blue) and FGT (pink) eosinophils at Nb 9 dpi. Dotted line represents the MFI of CD45+ FGT cells. Data are representative of two independent experiments with 4–5 mice per group (mean ± SEM). Statistical significance was calculated by Mann-Whitney t test. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 3: Nb-exacerbated HSV-2 pathology and FGT eosinophil infiltration is Il4ra independentWT and Il4ra-/- mice were infected with HSV-2 following Nb exposure as previously described.(A) HSV-2 progression was determined by daily pathology scoring.(B) Viral shedding was measured by plaque assay of day 6 vaginal washes.(C) Genital levels of IL-5 and IFN-? at day 2 post-HSV-2 infection, determined by Luminex and ELISA, respectively. Dotted line represents LLOQ of Luminex analysis. At 6 dpi, vaginal tissue was analyzed by H&E staining.(D) Representative sections (n = 3–4), displaying ulceration and inflammation of vaginal tissue. Images were taken at ×50 magnification. HSV-2-ulcerated vaginal epithelium is indicated by black dotted lines and qualified as percentage (%) of ulcerated epithelium.(E) Representative Sirius-red-stained sections (n = 3–4) of virus-induced (Ei) epithelial ulcers and (Eii) stromal inflammation. Black arrows indicate eosinophil presence in the vaginal epithelial layer. Images were taken at ×400 and ×1,000 magnification.(F) Numbers (×103) of FGT eosinophils in WT and Il4ra-/- co-infected mice compared with HSV-2-only controls. Data are representative of two independent experiments with 3–6 mice per group (mean ± SEM). Statistical significance was calculated by two-way ANOVA with Bonferroni correction for multiple comparisons. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 4: Prior Nb exposure results in elevated inflammation, eosinophil infiltration, and ILC2 presence in genital tissue, following HSV-2 vaginal infection(A) At day 9 post-Nb infection (Nb only) and day 3 post-HSV-2 infection (HSV-2 only and Nb + HSV-2), numbers of FGT myeloid cells were analyzed by flow cytometry.(B) Vaginal tissue was analyzed by Sirius red staining. Representative images (n = 4) of virus-induced ulcers were taken at ×400 magnification.(C) Black boxes indicate magnified sections of (Ci) vaginal stroma (St) and (Cii) epithelium (Ep), taken at ×1,000 magnification. White arrows indicate eosinophilic cell infiltration and migration in ulcerated vaginal tissue. Black arrowheads indicate eosinophil presence in vaginal epithelial layer.(D) At day 2 post-HSV-2 infection, levels of MBP (25 kDa) and GAPDH (37 kDa) were measured in HSV-2-only and Nb + HSV-2 FGT homogenates. Density of MBP was measured relative to that of GAPDH.(E) MFI of Siglec-F, CD11b, Ly6G, Ly6C, CD62L, and CD49d on FGT eosinophils in Nb 9 dpi and Nb + HSV-2 mice.(F) Levels of IL-5 in vaginal lavages at day 2 post-virus infection, determined by Luminex. Dotted line represents LLOQ.(G) Frequencies (mean ± SEM) and numbers of Lin-IL-7Ra+ICOS+ST2+ cells (ILC2s) in the FGT of Nb 9 dpi, HSV-2-only and Nb + HSV-2 mice.(H and I) Frequency and number of (H) Lin-IL-7Ra+ICOS+ cells and (I) Ly6Chi monocytes that are IL-5+ in the FGT, determined by flow cytometry. Data are representative of two independent experiments with 5–6 mice per group (mean ± SEM).(J) Nb-infected mice were treated with 20 µg a-IL-5 or isotype control, on day -2, 0, and 2 post-HSV-2 infection. Viral progression was determined by daily pathology scoring and HSV-2 ulcerated epithelium was qualified as percentage (%) of ulcerated epithelium. At day 6 post-HSV-2 infection, numbers of eosinophils in the iLN were determined by flow cytometry. Data are representative of two independent experiments with 4 mice per group (mean ± SEM). Statistical significance was calculated by two-way ANOVA with Bonferroni correction for multiple comparisons and Mann-Whitney t test. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.