Fig 2: scRNAseq of infected bladders maps myeloid interactome of type 17 immune cells(A) UMAP plot showing integrated analysis of 5,566 T cells isolated from the mouse bladders treated with PBS (n = 10; 3,557 cells) or with UTI89 (n = 10; 2,009 cells). Canonical T cell markers have been identified in each of the populations, showing expression levels in the dot plot. Tem, effector memory T cells; Teff, T effector T cells; Tnaive, naive T cells; Th1, T helper 1; ILC, innate lymphoid cells; NK, natural killer cells; NKT, natural killer T cells; T.Prolif., Proliferating T cells; Treg, regulatory T cells; and Th17, T helper 17 cells.(B) Dot plot showing expression patterns of genes characteristic of ILC3, Th17, and ?dT cells.(C) UMAP plot identifying two subpopulations within ILC3s (group 1 in red and group 2 in blue). Cells expressing Ncr1 were indicated by the gradient blue color in the feature plot.(D) UMAP plot showing cells from control (CTRL) or UTI mouse bladders in the ILC3s. Cells expressing proliferation markers Ki67, Top2a, or Birc5 were highlighted in red.(E) Feature plots showing expression levels of the characteristic ILC3 markers Il17a, Il22, Ifng, and Ltb.(F) UMAP plot showing integrated analysis of monocytes, macrophages, and neutrophils isolated from the mouse bladders treated with PBS or UTI89. Each population was characterized by a group of canonical markers, shown in the dot plot.(G) Heatmap showing unique ligand-receptor interactions between T cells and myeloid cells in the mouse bladders, treated with or without UTI89. Point size and color correspond to the scaled mean of ligand and receptor genes, analyzed by CellPhoneDB (https://www.cellphonedb.org). Interactions with statistical significance have been highlighted by the red circles.(H) Heatmap of Ltb and Ltbr transcripts from data in Figures 1A and Figure 5D (upper and lower panels, respectively). Representative confocal image (n = 2) of infected bladder from Rorc?tGFP mouse at 40× demonstrating co-staining of (I) Rorc?tGFP (green); CD3 (red) and Ifn? (pink) and (J) Rorc?tGFP (green); CD3 (red) and Ltb (pink).(I and J) White arrows in (I) and (J) denote ILC3s. *p<0.05, **p < 0.01, ***p<0.001 by two-way ANOVA with Šídák’s multiple comparisons test (H).

Fig 3: ILC depletion in Rag2-/- mice increases the severity of cystitis(A) Schematic of experimental design.(B) Colony-forming units per bladder 24 h after infection with UTI89 in Rag2-/- + isotype (gray) and Rag2-/- + anti-Thy1 (red) mice (left panel) (Table S1) and corresponding image of bacterial growth on agar plates; 1:400 dilution (right panel). N = 7-8 mice per group.(C and D) Corresponding qPCR of Th17 cytokines (C) and selected AMPs (D) in Rag2-/- + isotype (gray) and Rag2-/- + anti-Thy1 (red) bladders 24 h post infection (n = 6 per group). Results relative to Rag2-/- bladders. Data are representative of three independent experiments.(E) Quantification of absolute cell counts in Rag2-/- + isotype (gray) and Rag2-/- + anti-Thy1 (red) bladders 24 h post infection (n = 7-8 per group) for the indicated subsets.(F) Bladder “monocyte waterfall” subset quantification by flow cytometry 24 h post infection with UTI89 in Rag2-/- + isotype and Rag2-/- + anti-Thy1 bladders (n = 7-8 per group). Flow plots of CD45+Ly6G-CD11b+CX3CR1+ waterfall subsets (left) and quantification of absolute cell counts for the indicated subsets (right) are shown.(G) Heatmap of Th17 cytokines from RNA sequencing of bladders infected with UTI89 in Rag2-/- + isotype (n = 4) and Rag2-/- + anti-Thy1 (n = 4) female mice 24 h after challenge. Data represent four biological replicates per group (isotype and anti-Thy1).(H) Heatmap of selected IL22-dependent AMPs from (G).(I) Gene set enrichment analysis of the differential expression from (G) against hallmarks pathways. Only significant pathways (FDR q value < 0.05) are plotted. Red dots indicate positive enrichment and blue negative, the size of the dot is inversely correlated with the FDR q value and the position indicates the normalized enrichment score (NES).(J) Heatmap of cellular deconvolution of data in (G) using xCell (https://xcell.ucsf.edu). Scaled enrichment score is plotted (blue-red) with greatest enrichment in red.(K) Heatmap of scaled enrichment scores from single-sample gene set enrichment analysis (ssGSEA, https://www.genepattern.org/modules/docs/ssGSEAProjection/4) of data in (G) for IL17a and GM-CSF signatures (up-regulated genes, p < 0.05, LFC>1.5). IL17a and GM-CSF signatures are derived from GEO: GSE20087 (Zhang et al, 2010) and GEO: GSE95404 (Zhang et al, 2010), respectively. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann-Whitney test (B, D-E), one-way ANOVA with Dunn’s multiple comparisons test (C, F) and two-way ANOVA with Šídák’s multiple comparisons test (H, J, and K). All bladders used were from female mice unless otherwise stated.

Fig 4: Induction of type 17 immunity during bladder infection(A) Volcano plot of up- and down-regulated genes in murine bladders 24 h after UTI. RNA-seq was performed on C57BL/6J bladders catheterized with either PBS (n = 3) or UTI89 (n = 4) and euthanized after 24 h.(B) STRING analysis (https://string-db.org) of top 100 up-regulated genes following UTI ranked by LFC. Th17 immunity and neutrophil recruitment nodes highlighted in yellow and green, respectively.(C) Heatmap of top 11 differentially expressed cytokines following UTI.(D) GSEA of IL1b (top panel) and IL17a/IL22 (bottom panel) response signatures in UTI from (A) (IL1b response: GO:0071347; IL17/22 response: M303, msigdb.org).(E) qPCR of Th17 cytokines in C57BL/6J bladders day 1 (blue) and day 2 post UTI (red) (n = 4-6 per group) relative to uncatheterized bladders (gray).(F) qPCR of Th17 cytokines in C57BL/6J (blue) and Rag-/- (red) bladders day 1 post UTI (n = 4-6 per group) relative to uncatheterized bladders (gray). Data are representative of two independent experiments. Each point represents a single mouse bladder.(G) Heatmap of selected IL22-dependent AMPs from data in (A).(H and I) Intravital images of naïve RorcGFP murine bladder following intravenous Qtracker (blood vessels, red) and 3 kDa dextran-TMR (bladder lumen, cyan) labeling, with collagen in gray and ROR?tGFP cells green, showing location of ROR?tGFP cells (H) in submucosa in the x-z plane and (I) near blood vessels at different z-depths from submucosa (Z1) to bladder lumen (Z4).(J) Quantification of mean speed of ROR?tGFP cells in uninfected (black) and UPEC-infected (red) murine bladder, each point representing one cell track. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Mann-Whitney test (H), one-way ANOVA with Dunn’s multiple comparisons test (E-F), and two-way ANOVA with Šídák’s multiple comparisons test (C and G). All bladders used were from female mice unless otherwise stated.

Fig 5: Bladder macrophages produce ILC3-stimulating cytokines during infection(A) Representative confocal image of naive murine bladder at 40× (upper panel) and whole mount (lower panel) (cyan, F4/80; green, CD11c; red, phalloidin).(B) Confocal image of naive bladder from Rorc?tGFP mouse at 40× (blue, F4/80; green, Rorc?tGFP; red, CD3).(C) Heatmap of selected cytokines from data in Figure 1A.(D and E) Rank log fold change in expression of cytokines (D) and chemokines (E) in UTI compared with control sorted bladder macrophages. Red bars indicate absolute log fold change of greater than 2.(G) Raw counts of selected cytokines in control bladder macrophages.(F) Correlation of Il17a (left) and Il23a (right) expression with Il1b in murine bladders challenged with PBS (black) or UTI89—24 (blue), 48 (red) and 72 (gray) hours post infection.(H) Efficiency of A647-labeled UPEC phagocytosis by murine bone marrow-derived macrophages with and without prior stimulation with Il17a for 24 h. Flow cytometry gating strategy for macrophages—Live/CD45+/CD64+/F4/80+. Each circle represents a technical replicate (n = 4–6). The 4°C negative control is denoted in blue. Data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Šídák’s multiple comparisons test (C, H) and linear regression analysis (F). All bladders used were from female mice unless otherwise stated.