Fig 2: 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 3: ROR?t-specific ablation of Tfam leads to small intestine tissue remodeling with enhanced tuft cell–ILC2 circuit.Ctr included Tfam+/+Rorc-cre and Tfamfl/+Rorc-cre mice in a, b, f. KO indicated Tfamfl/flRorc-cre mice. a Differential gene expression by RNA-seq analysis of small intestine tissues of Ctr (n = 3) and KO mice (n = 3) at 12-week-old age (fold change =1.5, q value =0.05). b Heatmap of indicated gene expression from RNA-seq analysis of small intestine tissues of Ctr and KO mice. Each sample represented one individual mouse. c Analysis of tuft cell-related gene expression by qRT-PCR in the small intestine tissues of 12-week-old Ctr and Tfamfl/flRorc-cre mice (n = 3) (Dclk1, ****P < 0.0001; Trpm5, ****P < 0.0001; Gnat3, ***P = 0.0002; Plcb2, **P = 0.0023; Il25, **P = 0.0030). Representative data of three independent experiments. Ctr included Tfam+/+, Tfamfl/+, Tfamfl/fl, Tfam+/+Rorc-cre, and Tfamfl/+Rorc-cre mice in c, e, g–o. d Confocal analysis of tuft cells by DCLK1 staining in the small intestine (ileum) of 12-week-old mice with indicated genotypes. Red, DCLK1 staining. Blue, DAPI nuclear staining. Scale bar, 200 µm. Representative data from three independent experiments. e Quantification of tuft cells (DCLK1+) in each small intestine villus viewed by confocal microscopy (n = 9 villi for each group) (****P < 0.0001). Compiled data from two independent experiments. f Heatmap of indicated gene expression from RNA-seq analysis of small intestine tissues of Ctr and KO mice. g Analysis of Il4, Il5, Il13, and Areg expression by qRT-PCR in small intestine tissues of 12-week-old Ctr and Tfamfl/flRorc-cre mice (n = 3) (Il4, ***P = 0.0001; Il5, ****P < 0.0001; Il13, **P = 0.0066; Areg, **P = 0.0023). Representative data of three independent experiments. h IL-4, IL-5, and IL-13 expression in ILC2s or CD4+ T cells by flow cytometry from the small intestine draining lymph nodes (siLN) of Ctr (n = 5) and Tfamfl/flRorc-cre (n = 4) mice at 12-week-old age (ILC2: IL-4, **P = 0.0082; IL-5, **P = 0.0064; IL-13, **P = 0.0097; CD4+ T cells: IL-4, P = 0.0929; IL-5, P = 0.0652; IL-13, *P = 0.0194). Compiled data from two independent experiments. i Confocal analysis of ILC2s (CD3–KLRG1+) in the small intestine (ileum) of 12-week-old mice with indicated genotypes. Red, CD3 staining. Green, KLRG1 staining. White, EpCAM staining. Scale bar, 200 µm. Representative data of three independent experiments. j Quantification of ILC2s (CD3–KLRG1+) in each small intestine villus viewed by confocal microscopy (****P < 0.0001). Ctr, n = 21, Tfamfl/flRorc-cre, n = 22. Compiled data from two independent experiments. k Small intestine length in Ctr or Tfamfl/flRorc-cre mice treated with anti-Thy1.2 neutralizing antibody for 3 weeks (n = 3 for each group) (Ctr vs. Tfamfl/flRorc-cre, **P = 0.0098; Tfamfl/flRorc-cre vs. Tfamfl/flRorc-cre + anti-Thy1.2, *P = 0.0312). Compiled data from two independent experiments. l Picture of small intestines in mice of indicated genotypes treated with anti-IL-4 antibody (100 µg/mouse) or anti-IL-13 antibody (50 µg/mouse) twice a week from 6-week-old age for 3 weeks by intraperitoneal cavity injection. Representative data of two independent experiments. m Small intestine length. Compiled data from two independent experiments (Ctr vs. Tfamfl/flRorc-cre, ****P < 0.0001; Ctr vs. Tfamfl/flRorc-cre + anti-IL-4, ***P = 0.0009; Tfamfl/flRorc-cre vs. Tfamfl/flRorc-cre + anti-IL-4, ***P = 0.0006; Tfamfl/flRorc-cre vs. Tfamfl/flRorc-cre + anti-IL-13, ****P < 0.0001; Tfamfl/flRorc-cre + anti-IL-4 vs. Tfamfl/flRorc-cre + anti-IL-13, **P = 0.0022). n = 5 for Ctr, n = 4 for Tfamfl/flRorc-cre mice; n = 3 for Tfamfl/flRorc-cre mice treated with anti-IL-4; n = 4 for Tfamfl/flRorc-cre mice treated with anti-IL-13. n Analysis of Relmb expression by qRT-PCR in the small intestine tissues of 12-week-old Ctr and Tfamfl/flRorc-cre mice (n = 3) mice at 14 days after Hpb infection (****P < 0.0001). Representative data of three independent experiments. o Left panel, Hpb worm burden in small intestine lumen of Ctr (n = 8) and Tfamfl/flRorc-cre mice (n = 7) at 14 days after Hpb infection (****P < 0.0001). Right panel, Hpb egg counts per gram feces in Ctr (n = 8) and Tfamfl/flRorc-cre mice (n = 7) mice at 14 days after Hpb infection (***P = 0.0008). Compiled data from three independent experiments. Data are shown as mean ± SD in c, e, g, h, j, k, m–o. *, **, ***, **** indicated P values =0.05, 0.01, 0.001, 0.0001, respectively.

Fig 4: 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 5: GM-CSF programs Ly6Chi monocyte-derived cells to respond to low-dose LPS and contributes toward the prevention of Th2 cell development to HDM + LPS(A–D) B6 mice were sensitized and treated intraperitoneally (i.p.) with or without GM-CSF. Frequencies (A) and numbers (B) of Ly6ChiCD11b+ monocytic cells and moDCs (CD11c+MHCII+ or CD11c+CD64hi cells). Frequencies (C) and numbers (D) of TNFa+ monocytic cells.(E–G) Sorted Ly6Chi monocytes were stimulated with or without GM-CSF for 3 h, and RNA-seq was performed (three replicates). Volcano plot highlighting DEG (E) from a total of 2,431 genes, with 1,177 upregulated and 1,254 downregulated (false discovery rate <0.01, =2 fold change; see Table S1). Blue box displays gene members of PathCard TLR-IFNa/ß signaling that are downregulated in GM-CSF versus Ctrl monocytes. Red box displays gene members of PathCard TLR4 signaling that are upregulated in GM-CSF versus Ctrl monocytes. GSEA for the indicated gene signatures (F and G).(H and I) Fresh and GM-CSF pre-treated monocytes were stimulated with LPS ± GM-CSF. Expression of CD11c, MHCII (H), and intracellular TNFa (I). Values in triplicate.(J and K) Frequencies (J) and numbers (K) of IL-12p70+ mDC2s.(L–N) B6 mice were treated as shown (L). Frequencies (M) and numbers (N) of IL-13+IL-5+ CD4+ T cells in the lungs.Data are representative of three independent experiments (mean ± SD, n = 4, two-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See Figure S10 and Table S1.