Eosinophils enhance granuloma-mediated control of persistent Salmonella infection in mice

Abstract

Salmonella enterica can persist asymptomatically within tissues for extended periods. This is achieved through intricate host-pathogen interactions in immune cell aggregates called granulomas, wherein Salmonella establish favourable cellular niches to exploit while the host limits its expansion and tissue dissemination. Here, using a mouse model of persistent Salmonella infection, we identify a host-protective role for eosinophils in the control of Salmonella Typhimurium (STm) infection within the mesenteric lymph nodes, the main lymphoid tissue of STm persistence. Combining spatial transcriptomics and experimental manipulations, we found that monocytes and macrophages responding to STm infection recruited eosinophils in a C-C motif chemokine ligand 11 (CCL11)-dependent manner and enhanced their activation. The protein major basic protein, primarily expressed by eosinophils, was associated with altered macrophage polarization and bacterial control. Thus, eosinophils play a vital role in restraining Salmonella exploitation of granuloma macrophages at a key site of bacterial persistence.

Fig. 1. Cytokine and spatial transcriptomics analyses identify CCL11 and macrophage populations that correlate with STm persistence.

129×1/SvJ mice were orally infected with STm SL1344 (10⁸ c.f.u.s) and monitored for 8 weeks. a, Heat map of significantly regulated soluble mediators in MLN supernatants at 1, 2 and 4 w.p.i. compared to uninfected controls (pooled samples from 5 mice per group across 2 experiments; n = 10 per condition). b, ELISA of CCL11 and CCL24 in MLN supernatants at 1, 2, 4 and 8 w.p.i. compared to uninfected controls (n = 8–10 mice per time point, 2 experiments). c, Bacterial burden in the MLN at 1, 2, 4 and 8 w.p.i. (n = 14 mice per time point). d, Correlation matrix between MLN bacterial levels and MLN tissue levels of CCL11 and CCL24. Data from mice at 1, 2, 4 and 8 w.p.i. (n = 10 mice per time point). e, Schematic representation of Xenium single-cell in situ analysis of MLN tissue sections from uninfected controls and mice at 4 w.p.i (n = 2–8 mice per group, 3 experiments). f, UMAP plot of MLN immune cells from Xenium single-cell in situ analysis in uninfected controls and mice at 4 w.p.i.: cluster 0 (salmon), cluster 1 (beige), cluster 2 (grey), cluster 3 (olive green), cluster 4 (high green), cluster 5 (orange), cluster 6 (light blue), cluster 7 (purple), cluster 8 (pink), cluster 9 (magenta) and cluster 10 (cyan). g, UMAP plot of clusters 0–10 represented in uninfected controls and mice at 4 w.p.i. h, Heat map of top 1–5 differentially expressed genes within each cluster in f (cut-off: log₂FC = 1.41, P < 0.05). i, UMAP plots showing total Ccl11 and Ccl24 expression in uninfected controls and mice at 4 w.p.i. j, Ccl11 expression levels in the MLN of uninfected controls and mice at 4 w.p.i. Ccl11 enriched regions are outlined, segmented cells are pseudocoloured according to clusters in f, and yellow symbols indicate Ccl11 transcripts.Data are presented as mean ± s.e.m. All analyses are two-tailed and data were analysed using Student’s t-test (a), Kruskal–Wallis with Dunn’s post hoc test (b), Pearson correlation (d) and Wilcoxon rank-sum test (h). Exact P values in Supplementary Table 1.**P < 0.01, ***P < 0.001. Source data

Fig. 2. Targeted CCL11 neutralization depletes eosinophils and increases bacterial burdens in MLN.

a, Schematic of the CCL11-neutralization approach. Mice were orally infected with STm (10⁸ c.f.u.s) and treated with either anti-CCL11 antibodies or anti-IgG₂ antibodies starting at day 3 post infection and continuing every 3 days until sacrifice at day 28 (n = 6–8 mice per group, 2 experiments). b, Representative FACS plots of neutrophils and eosinophils from MLN in mice treated with anti-IgG₂ or anti-CCL11 at 4 w.p.i. compared to uninfected controls. c, Quantification of frequency (left) and absolute numbers (right) of MLN eosinophils in mice treated with anti-IgG₂ (purple closed circles) or anti-CCL11 (purple open circles) at 4 w.p.i. compared to uninfected controls (black open circles). d, Quantification of frequency of live neutrophils, macrophages (Mϕ), monocytes, resident dendritic cells (DCs), migratory DCs, T cells and B cells from MLN in mice treated with anti-IgG₂ or anti-CCL11 at 4 w.p.i. compared to uninfected controls. e, Percentage of body weight variation (day 28 vs day 0) in mice treated with anti-IgG₂ or anti-CCL11 at 4 w.p.i. compared to uninfected controls. f, Quantification of bacterial levels in MLN, spleen, liver and faeces in mice treated with anti-IgG₂ or anti-CCL11 at 4 w.p.i. Data are presented as mean ± s.e.m. All analyses are two-tailed and data were analysed using Kruskal–Wallis with Dunn’s post hoc test (c–e) or Mann–Whitney U-test (f). Exact P values in Supplementary Table 1. Uninf., uninfected. NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Fig. 3. MLN eosinophils are activated and localize to the periphery of STm-containing granuloma structures.

129×1/SvJ mice were orally infected with STm (10⁸ c.f.u.s) and monitored for up to 8 w.p.i. a, Quantification of frequency (left) and absolute numbers (right) of live eosinophils at 1, 2, 4 and 8 w.p.i. compared to uninfected controls (n = 7–12 mice per time point, 2–4 experiments). b, Representative immunofluorescence image of MLN sections from mice at 4 w.p.i showing eosinophils (EPX, cyan) localized at the periphery of CD11b⁺ (magenta) and iNOS⁺ (grey) granulomas containing STm (green). Arrows highlight eosinophils (n = 5 mice, 2 tissue sections per mouse). c, Quantification of distance between eosinophils and granuloma structures (n = 5 mice, 2 experiments). d, Representative image from Xenium analysis showing Prg2 transcripts (white symbols) in eosinophils at the periphery of granuloma macrophages (gran-Mϕ). Segmented cells are pseudocoloured according to UMAP cluster in Fig. 1f. e, Quantification of total Prg2 transcripts within 50 μm of granuloma structures and in the MLN parenchyma (n = 8 mice, 3 experiments). f, Representative FACS histogram for activation markers on MLN eosinophils from uninfected controls (black) and mice at 4 w.p.i (orange). Fluorescence minus one control (FMO) is shown as dashed black line. g, Quantification of MFI of activation markers: SSC-A, Siglec F, CD11b and CD63 on MLN eosinophils from uninfected controls and mice at 4 w.p.i. (n = 8–12 mice per group, 3 experiments). h, Representative FACS plots showing PD-L1 and CD80 on MLN eosinophils from uninfected controls and mice at 4 w.p.i. i, Quantification of the frequency of CD80⁺ PD-L1⁺ eosinophils from MLN in uninfected controls and mice at 4 w.p.i. (8–12 mice per group, 3 experiments). j, Quantification of PD-L1 and CD80 MFI on MLN eosinophils in uninfected controls and mice at 4 w.p.i. (8–12 mice per group, 3 experiments). k, Immunofluorescence image of infected MLN at 4 w.p.i showing CD11b⁺Siglec F⁺PD-L1⁺ positive eosinophils (n = 3 mice, 2 experiments). Data are presented as mean ± s.e.m. All analyses are two-tailed and data were analysed using Kruskal–Wallis with Dunn’s post hoc test (a), Wilcoxon signed-rank test (e) or Mann–Whitney U-test (g,i,j). Exact P values in Supplementary Table 1. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Fig. 4. Eosinophil-deficient mice are more susceptible to persistent STm infection.

a, Schematic of transient eosinophil depletion using anti-Siglec F antibodies. Mice were orally infected with STm for 4 weeks (10⁸ c.f.u.s) before receiving 7 doses of either anti-IgG₂ or anti-Siglec F antibodies every 2 days before sacrifice on day 41 (n = 15 mice per group, 3 experiments). b, Quantification of the frequency of live MLN eosinophils in infected mice treated with anti-IgG₂ (blue filled squares) or anti-Siglec F (blue open squares) at day 41 (n = 10 mice per group, 2 experiments; FACS plots in Extended Data Fig. 5a). c, Quantification of bacterial levels in MLN, spleen, liver and faeces in infected mice treated with anti-IgG₂ or anti-Siglec-F at day 41. d, Percentage of body weight variation (day 41 vs day 28) in infected mice treated with anti-IgG₂ or anti-Siglec F antibodies. e, Kaplan–Meier survival curve (left) comparing infected 129 WT mice (orange) and genetically eosinophil-deficient 129^ΔdblGATA1 mice (grey) over 8 weeks along with body weight variation (right) in the same groups (n = 14–16 mice per group, 4 experiments). f, Quantification of bacterial levels in MLN, spleen, liver and faeces in 129 WT and eosinophil-deficient 129^ΔdblGATA1 mice at 4 w.p.i. g, Schematic of transient eosinophilia by rIL-5 treatment. 129X1/SvJ mice were infected for 4 weeks before receiving 3 doses of rIL-5 or PBS every 2 days and sacrifice on day 35 (n = 11–12 mice per group, 3 experiments). h, Quantification of the frequency of live MLN eosinophils in infected mice treated with PBS (closed teal triangles) or rIL-5 (open teal triangles) at day 31. FACS plots in Extended Data Fig. 5g. i, Quantification of bacterial levels in MLN, spleen, liver and faeces in infected mice treated with PBS or rIL-5 at day 35. j, Percentage of body weight variation (day 35 vs day 28) in infected mice treated with PBS or rIL-5. Data are presented as mean ± s.e.m. All analyses are two-tailed and data were analysed using Mann–Whitney U-test (b–d,h–j), mixed effects analysis with Šidák’s multiple comparisons test (e) or Mantel–Cox analysis (e). Exact P values in Supplementary Table 1. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Fig. 5. Eosinophil-deficient mice have altered myeloid responses and higher intracellular STm burdens.

129 WT mice and eosinophil-deficient 129^ΔdblGATA1 mice were orally infected with STm (10⁸ c.f.u.s) for 4 weeks, after which immune populations in the MLN were analysed. See also Extended Data Figs. 6–8. a, Representative FACS plots of MLN macrophages (Mϕ), neutrophils and eosinophils in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i. and in uninfected controls. b, Frequencies (top) and absolute numbers (bottom) of Mϕ, neutrophils and eosinophils in the MLN of 129 WT (orange) and 129^ΔdblGATA1 mice (grey) at 4 w.p.i and in uninfected controls (black open circles) (n = 4–13 mice per group, 2–3 experiments). c, UMAP plot of myeloid subsets from scRNA-seq in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i.: Mϕ (salmon), PMN (cyan), monocytes (green), proliferating Mϕ (prof-Mϕ, beige), cDC1 (light blue) and pDC (magenta); pooled samples from 3–5 mice, n = 2 mice per group, 1 experiment. d, Heat map of top 3–5 differentially expressed genes defining different myeloid clusters. e, Myeloid cluster proportions in 129 WT and 129^ΔdblGATA1 mice. f, Volcano plot of differentially expressed genes in 129 WT and 129^ΔdblGATA1 mice (cut-off: log₂FC > 1, adjusted P = 0.05). Up in WT, orange; up in 129^ΔdblGATA1, blue. g, Representative Xenium images of granuloma-associated transcript levels (Itgam, Nos2, Il-1b, Il-1rn and Tlr2) in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i. (n = 6–8 mice per group, 3 experiments). h, Quantification of average granuloma size (top) and numbers (bottom) in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i (defined by CD11b⁺iNOS⁺ foci, by immunofluorescence) (n = 8–10 mice per group, 3 experiments). i, Representative Xenium images of granuloma clusters in MLN tissue from 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i. Segmented cells are pseudocoloured according to UMAP clusters in Extended Data Fig. 8b (n = 6–8 mice per group, 3 experiments). j, Granuloma cluster proportions in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i. Data are presented as mean ± s.e.m. or means (e,j). All analyses are two-tailed and data were analysed using Kruskal–Wallis with Dunn’s post hoc test (b), Wilcoxon rank-sum test with Bonferroni-adjusted P values (f) or Mann–Whitney U-test (h). Exact P values in Supplementary Table 1. *P < 0.05, ** P < 0.01, *** P < 0.001. Source data

Fig. 6. Eosinophil-derived MBPs influence macrophage polarization and bacterial control.

Eosinophil-deficient 129^ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing STm (10⁸ c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a, Representative FACS plots of tdTomato-STm in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i. b, Frequency of tdTomato-STm⁺ immune cells in 129 WT and 129^ΔdblGATA1 mice (n = 7–8 mice per group, 2 experiments). c, Representative FACS histogram of tdTomato-STm⁺ macrophages (Mϕ) in 129 WT (orange) and 129^ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d, MFI of tdTomato-STm within Mϕ in 129 WT and 129^ΔdblGATA1 mice. e, Frequency of tdTomato-STm⁺ iNOS⁺ or tdTomato-STm⁺ CD206⁺ Mϕ in 129 WT and 129^ΔdblGATA1 mice (n = 7–8 mice per group, 2 experiments). f, MFI of tdTomato-STm within iNOS⁺, CD206⁺ and iNOS⁻CD206⁻ Mϕ in 129 WT and 129^ΔdblGATA1 mice (n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. 7k). g–i, Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with recombinant EPX (100 ng ml⁻¹), MBP (100 ng ml⁻¹), MBP and EPX (100 ng ml⁻¹ each), LPS (100 ng ml⁻¹) or IL-4 (20 ng ml⁻¹) (g); dose-dependent MBP effects (1–100 ng ml⁻¹) (h); kinetics of MBP effects (10 ng ml⁻¹, 1–24 h) (i). n = 3 biological replicates (see also Extended Data Fig. 9). j, Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml⁻¹) and LPS (100 ng ml⁻¹) (n = 3 biological replicates). k, Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l, Fold-replication of STm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml⁻¹), EPX (10 ng ml⁻¹), LPS (100 ng ml⁻¹) or IL-4 (20 ng ml⁻¹) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m–p, Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with STm for 4 weeks and then given 5 daily doses of anti-IgG₂ or anti-MBP antibodies before sacrifice on day 33 (m); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG₂ or anti-MBP at 33 days post infection (n); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG₂-treated mice and uninfected controls (n = 3–9 mice per group, 2 experiments) (o); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice (n = 9 mice per group, 2 experiments) (p). q, Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in STm-infected MLN and release MBP which leads to STm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U-test (b,d,e,p); two-way ANOVA with uncorrected Fisher’s LSD test (f), two-way ANOVA with Tukey correction (j), one-way ANOVA with Tukey correction (l) or Kruskal–Wallis with Dunn’s post hoc test (o). Exact P values in Supplementary Table 1. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 1. STm trigger a CCL11 response in the MLN.

129×1/SvJ mice were orally infected with STm (10⁸ CFUs) for up to 8 weeks to investigate the immune landscape in the MLN (a) Representative cytokine array dot blots of MLN supernatants highlighting CCL11 (purple box), supporting main Fig. 1a. (Pooled samples from n = 10 mice/time point, 2 experiments) (b) Kinetic of Log₁₀ absolute bacterial burden in MLN tissue homogenates (n = 14 mice per time point from 3 experiments). (c) Quantification of CCL11 and CCL24 levels from splenic cell lysates (n = 5 mice per group from 2 experiments) (d) Quantification of Log₁₀ bacterial burden per gram and Log₁₀ absolute bacterial burden in spleen tissue homogenates (n = 10 mice/time point, 2 experiments). (e) Correlation matrix between splenic CCL11, CCL24 and Log₁₀ c.f.u.s g⁻¹ from infected mice at 4 w.p.i. (f) Overlay of transcript levels in uninfected controls and infected mice based on seurat-clusters identified by Xenium in situ transcriptomic analysis in main Fig. 1h. (g) Pseudocolored macrophage and monocyte clusters identified by Xenium in situ transcriptomic analysis. (h) Representative Ifng transcript levels and overlay of Ifng transcripts on top of segmented cells using Xenium in situ transcriptomic analysis, segmented cells are pseudocolored according to UMAP cluster identities from Fig. 1f. Data are presented as Means ± SEM. All analyses are two-tailed and the data were analyzed by Mann-Whitney U-test (c) or Pearson correlation (d). Exact P-values in Supplementary Table 1. ns = not significant. Source data

Extended Data Fig. 2. Monocyte/macrophage-dependent CCL11 release.

129×1/SvJ mice were orally infected with STm (10⁸ CFUs) for 4 weeks to investigate immune cells influence CCL11-mediated eosinophil-recruitment. (a) Quantification of normalized Ccl11 and Ccl24 transcripts by Xenium in situ transcriptomic analysis (n = 2-8 mice/group, 3 experiments). (b) Normalized Ccl11 across Seurat-clusters identified in main Fig. 1h. (n = 10 mice, 2 uninfected, 8 infected, 3 experiments). (c) Representative FACS histogram of intracellular CCL11 in infected MLN. (d) Frequencies of CCL11⁺ cells and CCL11 MFI in immune subsets (n = 10 mice from 2 experiments). (e) Immunofluorescence images of CD11b (magenta), CD11c (yellow), CCL11 (green) and DAPI (blue) in infected mice at 4 w.p.i. (f) Schematic of transient anti-CSF1 neutralization. Mice were infected for 4 weeks before receiving 3 doses of anti-IgG₂ or anti-CSF1 antibodies every other day before sacrifice on day 33 (n = 4-10 mice/group, 2 experiments). (g) Frequencies of macrophages and monocytes in uninfected controls, or infected mice treated with anti-IgG₂ or anti-CSF1 at day 33. (h) Representative FACS histogram of CCL11 in uninfected controls, or infected mice treated with anti-IgG₂ or anti-CSF1 at day 33. (i) Frequency and absolute number of CCL11 producing cells in uninfected controls, or infected mice treated with anti-IgG₂ or anti-CSF1 at day 33 (j) Quantification of CCL11 and CCL24 levels in MLN supernatants in controls, or infected mice treated with anti-IgG₂ or anti-CSF1 at day 33. (k) Quantification of the percentage of eosinophils in uninfected controls, or infected mice treated with anti-IgG₂ or anti-CSF1 at day 33. (l) Quantification of bacterial burden MLN, spleen, liver and feces in infected mice treated with anti-IgG₂ or anti-CSF1 at day 33. Data are presented as Means ± SEM. All analyses are two-tailed and the data and were analyzed by Mann-Whitney U-test (a, k) or Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction (b–j). Exact P-values in Supplementary Table 1. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 3. Effects of CCL11 neutralization in the MLN and spleen.

(a) Gating strategy to identify different myeloid cells, including neutrophils, eosinophils, macrophages, monocytes, resident dendritic cells (DCs) and migratory DCs isolated from infected MLN. (b) Absolute number of neutrophils, macrophages (Mφ), monocytes, resident DCs, migratory DCs, B cells and T cells in MLN of infected mice treated with anti-IgG₂ (purple closed circles) or anti-CCL11 (purple open circles) compared to uninfected controls (black open circles). In support of main Fig. 2d (n = 6-8 mice/group, 2 experiments). (c) Frequencies of eosinophils, neutrophils, macrophages (Mφ), monocytes, resident DCs, migratory DCs, B cells and T cells isolated from spleens of infected mice treated with anti-IgG₂ or anti-CCL11 compared to uninfected controls (n = 6-8 mice/group, 2 experiments). Data are presented as Means ± SEMs. All analyses are two-tailed and the data were analyzed using Kruskal Wallis with uncorrected Dunn’s test. ns = non-significant, * = p < 0.05, ** = p < 0.01. Source data

Extended Data Fig. 4. Organ eosinophil phenotype.

129×1/SvJ mice were orally infected with STm (10⁸ CFUs) for 4 weeks to investigate eosinophil phenotypes in different organs. (a) Alternative FACS gating strategy for eosinophils (MLN, 4 w.p.i) (b) Representative FACS plots showing eosinophils in uninfected controls (uninf.) and mice at 4 w.p.i isolated from small intestinal lamina propria (SiLP), colon lamina propria (cLP), mesenteric lymph node (MLN), blood and spleen. (c) Frequency of eosinophils in SiLP, CLP, blood and spleen (n = 4-10 mice per group, from 2-3 experiments). (d) Representative FACS histograms showing eosinophils activation markers in SiLP, CLP, MLN, blood and spleen eosinophils. (e) Quantification of mean fluorescence intensity (MFI) of activation markers on SiLP, CLP, MLN, blood and spleen eosinophils (n = 9 mice per group from 3 experiments). Data are presented as Means ± SEMs. All analyses are two-tailed, and were analyzed using Mann-Whitney U-test (c) or Kurskal-Wallis test with Dunn’s correction (e). Exact P-values in Supplementary Table 1. ns = non-significant, *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 5. Eosinophils are dispensable during acute STm infection in C57BL/6 mice.

(a) FACS plots showing efficacy of eosinophil depletion using anti-Siglec F antibodies in MLN and spleen tissue in support of Main Fig. 4a, b. (b) Kaplan-Mayer plot showing survival and line graph showing body weight variation in orally infected (STm 10⁸ c.f.u.s) C57BL/6 WT mice (orange) and eosinophil-deficient ΔdblGATA1 mice (black) (n = 10 mice/group, 2 independent experiments). (c) Systemic bacterial burden in C57BL/6J and eosinophil-deficient ΔdlbGATA1 mice at day 7 post infection (n = 10 mice per group). (d) Representative histology of cecum and colon tissue in 129×1/SvJ WT and 129^ΔdlbGATA1 mice (n = 4 mice/group, 2 experiments). (e) STm-burden in cecal tissue and total bacterial burden in blood in 129×1/SvJ WT and 129^ΔdlbGATA1 mice (n = 7–8 mice/group, 2 experiments) (f) Histology of MLN tissue showing granuloma size differences in 129×1/SvJ WT and 129^ΔdlbGATA1 mice (n = 4 mice/group, 2 experiments). (g) Representative FACS plots showing efficacy of recombinant IL-5 mediated eosinophilia in MLN and spleen tissue in support of Main Fig. 4h. Data are presented as Means ± SEMs. All analyses are two-tailed, and the data were analyzed by Mantel-Cox (b) Kruskal Wallis with uncorrected Dunn’s test (b) or Mann-Whitney U-test (c, e). Exact P-values in Supplementary Table 1. ns = not significant, *P < 0.05, **P < 0.01. Source data

Extended Data Fig. 6. MLN and splenic myeloid and lymphocyte responses in 129 WT and 129^ΔdblGATA1 mice.

Eosinophil-deficient 129^ΔdblGATA1 mice and 129 WT mice were orally infected with STm (10⁸ CFUs) for 4 weeks to investigate eosinophils impact on lymphocyte populations in the MLN. (a) Gating strategy for B cells and different T cell subsets (CD4⁺ or CD8⁺) isolated from the MLN of STm-infected mice. (b) Representative FACS plots and quantification of T_H1 cells (Tbet⁺), T_H2 cells (GATA3⁺) and Tregs (FOXP3⁺) in the MLN of infected 129 WT and eosinophil-deficient 129^ΔdblGATA1 mice at 4 w.p.i. compared to uninfected controls. (c, d) Frequency and absolute number of live monocytes, resident DCs and migratory DCs isolated from the MLN of STm-infected 129 WT and 129^ΔdblGATA1 mice compared to uninfected controls (n = 3 – 10 mice/group, 2-3 experiments). (e) Frequencies of different myeloid cells - including eosinophils, neutrophils, monocytes, macrophages and DC populations in the spleens of infected 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i. compared to uninfected controls (n = 3-10 mice/group, 2 experiments). Data are presented as Means ± SEMs. All analyses are two-tailed, and the data were analyzed by Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction. Exact P-values in Supplementary Table 1. ns = non-significant, *P < 0.05, ** P < 0.01. Source data

Extended Data Fig. 7. Eosinophils influence myeloid cells in infected MLN.

Eosinophil-deficient 129^ΔdblGATA1 mice and 129 WT mice were orally infected with STm (10⁸ CFUs) for 4 weeks to investigate how eosinophils impact on myeloid populations in the MLN. (a, b) iNOS⁺ and CD206⁺ macrophages in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i compared to uninfected controls (c) Quantification of the frequency of iNOS⁺ and CD206⁺ macrophages in 129 WT (orange) and 129^ΔdblGATA1 (grey) mice at 4 w.p.i., compared to uninfected controls (black open circles) (n = 6-14 mice/group, 3 experiments). (d) FACS histograms showing additional markers expressed by iNOS⁺ CD206⁺ macrophages in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i. (e) UMAP plot with sample overlay of 129 WT (orange) and 129^ΔdblGATA1 (blue) samples on myeloid clusters from in main Fig. 5c. (f) UMAP plot of macrophage subsets (Adgre1⁺ (FC > 1.41), Ly6g⁻ Sglech⁻ and Xcr1⁻). (g) UMAP plot of macrophage clusters represented in 129 WT and 129^ΔdblGATA1 samples at 4 w.p.i. (Pooled samples from 3-5 mice, n = 2 mice/group, 1 experiment). (h) Heatmap showing significant genes defining macrophage clusters in (f). (i) Heatmap showing differentially expressed genes in macrophage subsets in 129 WT and 129^ΔdblGATA1 mice (FC > 1.41, Adjusted p-value < 0.05). (j) Gene-ontology analysis showing molecular functions in macrophages in 129 WT and 129^ΔdblGATA1 mice. Data are presented as Means ± SEMs. All analyses are two-tailed, and the data were analyzed by Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction (c). Exact P-values in Supplementary Table 1. ns = non-significant, *P < 0.05, **P < 0.01, ***P 0.001. Source data

Extended Data Fig. 8. Eosinophil mediated granuloma control.

Eosinophil-deficient 129^ΔdblGATA1 mice and 129 WT mice were orally infected with STm (10⁸ CFUs) for 4 weeks to assess eosinophils impact on MLN granulomas. (a) UMAP plot showing granuloma associated cells within 50 μm of granuloma macrophages in 129 WT and 129^ΔdblGATA1 mice at 4 w.p.i by Xenium single cell in situ analysis (n = 6-8 mice/group, 2 granulomas per section, from 3 experiments). (b) Heatmap showing significant genes defining granuloma associated cells in 129 WT and 129^ΔdblGATA1 mice. (c) UMAP plot of granuloma associated cells represented in 129 WT and 129^ΔdblGATA1 samples. (d) Volcano plot of differentially expressed genes in granuloma associated cells in 129 WT (orange) or 129^ΔdblGATA1 mice (grey) (Cut-off = Log₂FC = 1, p < 0.05). (e) Heatmap of granuloma associated transcripts identified in Fig. 1f in 129 WT and 129^ΔdblGATA1 at 4 w.p.i. (f) Quantification of main granuloma transcripts normalized per number of granuloma cell in 129 WT (orange) or 129^ΔdblGATA1 mice (grey) (n = 6-8 mice/group, 3 experiments). (g, h) Immunofluorescence (IF) images of granulomas (CD11b = magenta, CD14 = green, iNOS = white) (g); Quantification of granuloma size in mice treated with anti-Siglec F or IgG₂ isotype at day 41, per the protocol in main Fig. 4a (n = 5 mice/group, 1 experiment, 2 tissues/mouse). (i, j) IF images of granulomas (CD11b = magenta, CD14 = green, iNOS = white) (i); Quantification of granuloma size in mice treated with recombinant (r)IL-5 or PBS on day 33, per the protocol in main Fig. 4g. (n = 5 mice/ group, 1 experiment, 2 tissues/mouse). (k) IF images showing STm (green) in granulomas (CD11b⁺ (magenta) CD14⁺ (cyan) iNOS⁺ (grey)) or in the subcapsular sinus (SCS) (CD11b⁺ (magenta) CD206⁺ (blue) iNOS (grey)). Orange arrows highlight localization of STm staining. Scale-bar = 50 μm (n = 4 mice/group, 2 experiments). (l) Representative FACS plots of tdTomato-STm in iNOS⁺ or CD206⁺ macrophages in support of Fig. 6e, f. Data are presented as Means ± SEMs. All analyses are two-tailed and the data were analyzed by Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction (f), Wilcoxon rank-sum test (d) or Mann-Whitney U-test (h, j). Exact P-values in Supplementary Table 1. ns = not significant, *P < 0.05. Source data

Extended Data Fig. 9. Major basic protein influences macrophage polarization.

(a) Representative immunofluorescence image of MLN tissue sections from mice at 4 w.p.i. showing CD11b (magenta), iNOS (Grey), EPX (Blue) and MBP (Green). Scale bar = 50 μm (n = 5 mice, 2 sections each). (b) STm growth in media containing either PBS (Ctrl), EPX (0.155-1.55 μg/ml) or MBP (90-900 ng/ml) for 18 h (n = 3 biological repeats). (c) Representative FACS plots showing iNOS and Arginase-1 in MBP (100 ng/ml), EPX (100 ng/ml) MBP and EPX (100 ng/ml each), LPS (100 ng/ml) or IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice. (d) Quantification of percentage of iNOS+ and Arginase-1+ cells in MBP (100 ng/ml), EPX (100 ng/ml) MPB and EPX (100 ng/ml each), LPS (100 ng/ml) and IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice (n = BMDMs from 5 mice across two experiments). (e) Quantification of iNOS levels by western blots in main Fig. 6 (n = 3 biological repeats). (f) Representative western blot showing iNOS expression in murine (mMBP), human (hMBP) (10 ng/ml each) or LPS treated BMDMs from 129×1/SvJ mice (n = 2 biological repeats). (g) Quantification of cell death as determined by SYTOX green positive cells (n = 3 biological repeats) (h) Frequencies of eosinophils, neutrophils, mast cells and, basophils in the MLN of 129×1/SvJ mice. Basophils are gated as Live, CD45⁺ CD200R3⁺ c-Kit⁻. Mast cells are gated as Live, CD45⁺ CD200R3⁺ c-Kit⁺. Data are presented as Means ± SEM. All analyses are two-tailed and analyzed by Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction (b) or one-way ANOVA (d, e). Exact P-values in Supplementary Table 1. ns = non-significant, *P < 0.05, **P < 0.01. ***P < 0.001. Source data