Fig 2: CD9 Macs provide a niche for intracellular S.Tm replication during the second phase of ELID(A and B) Spleens of E2+/+ (WT; n = 3) and E2−/− (n = 4) were harvested and analyzed by flow cytometry using antibodies to mark the indicated populations (A) and presented as the ratio of the population from WT mice (B).(C and D) WT (n = 5) and E2−/− mice (n = 5) were infected with S.Tm. Twenty-four hpi spleens were harvested and analyzed by flow cytometry using antibodies to mark the indicated populations (C) and presented as the ratio of the population from WT mice (D).(E) WT and E2−/− mice were infected with 1,000 CFU of S.Tm. Three days post-infection, spleen were harvested and plated for CFU (n = 5 per condition).(F and G) WT and E2−/− mice were infected with 1,000 CFU of S.Tm (n = 7 per condition). Mice were weighed (F) and monitored for survival for 5 days, presented on a Kaplan-Meier plot (G).Results are representative of three independent experiments in all panels. (B and D) Error bars indicate standard deviation. (F) Error bars indicate standard error. (B) ∗p < 0.05 Student’s t test. (D)∗p < 0.05, Mann-Whitney U test. (E) p < 0.01, Mann-Whitney U test. (F) ∗∗p > 0.01 two-way analysis of variance. (G) ∗∗p < 0.01 log-rank test.

Fig 3: Functional analysis reveals distinct inflammatory programs of iNOS Macs and reducing oxidized lipids programs of CD9 Macs(A) Monocytes from Ms4a3cre-RosaTdT chimera were transferred to naive mice and 16 h (top panel) or 4 days (bottom panel) post-transfer mice were infected with S.Tm or PBS (n = 5, per group per time point). Twenty-four hpi spleens were harvested and analyzed by FACS. Presented are calculated proportions of tdTomato+ of the mononuclear populations.(B) Mice were infected with S.Tm and 24 hpi spleens were harvested and analyzed by flow cytometry using CD11b, F4/80, CD9, and Ly6C antibodies. Presented are contour plots of iNOS Macs and CD9 Macs from the gated Lin–CD11b+F4/80+ population.(C–E) Mice were infected as in (B), and CM or NCM from naive mice (n = 5) or bystander or infected CD9 Macs and iNOS Macs from S.Tm challenged mice (n = 4) were sorted by FACS and analyzed by bulk RNA-seq. Presented are PCA plots (C) and a heatmap (D) of the significant differentially expressed genes between all groups and log2 fold expression changes of selected genes (E).Results are representative of two (A and C–E) or four (B) independent experiments. See also Figure S3 and Tables S1 and S2.

Fig 4: scRNA-seq reveals three concomitant splenic macrophage populations during ELID of S.Tm(A) Naive mice or mice infected i.v. with WT or ΔSPI-2 S.Tm were harvested 24 hpi and analyzed by flow cytometry using CD11b and F4/80 antibodies.(B) Single cells from gated populations in (A) were sorted by flow cytometry and analyzed by scRNA-seq. Shown is a forced layout map of single cells (dots) and MCs (large circles), with infection conditions indicated (naive mice [white, n = 631 cells], mice infected with WT [black, n = 643 cells], or a ΔSPI-2 mutant [gray, n = 658 cells]). Cell-type annotations are indicated by outline color of the MC; infection status of the MC by fill color and similarity between MCs are indicated by connecting nodes.(C) Marker gene annotations presented as the percentage of cells expressing the indicated genes in each MC (shown as the size of the circle) and the relative log2 fold change of the gene expression in each MC (shown as the color of the circle).(D) Expression analysis across all MCs of genes used as markers for iNOS Macs (Ly6c2, Nos2) and CD9 Macs (Cd9, Ly6i).(E) kNN classification of cell types presented as percentage of infected cells classified to cell types in the naive sample. The color bar indicates percentage of cells of each cell type in the infected mice classified to cell types from the naive mice.See also Figure S2.

Fig 5: Loss of NR2F6 alters the expression of genes involved in phagocytosis in RPMs. A) Volcano plot representation of differentially expressed genes (DEGs) between wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient (Nr2f6−/− ) RPMs. DEG are selected by DESeq2 (adjusted p‐value (padj)< 0.05). B) Heatmap of DEG between wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient RPMs were defined by adjusted p‐value (padj) < 0.05 and z‐score normalized. C) Heatmap of DEG involved in phagocytic responses. A padj‐value < 0.05 was considered statistically significant. * padj < 0.05,** padj < 0.01. D) Histogram and E) quantification of Sirpα (CD172a) MFI of wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient splenic macrophages. F) Representative dot‐plots of CD11bint/F4/80+ splenic macrophages, iNOS, and CD9+ macrophages out of splenic CD11b+/F4/80int cells of wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient (Nr2f6−/− ) Salmonella Typhimurium infected mice after 22 h. G) Quantification of frequencies of splenic CD9+ and H) iNOS macrophages out of CD45+ cells of wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient (Nr2f6−/− ) mice. I) Histogram and J) quantification of Sirpα (CD172a) MFI of wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient (Nr2f6−/− ) splenic CD9+ macrophages 22 h post‐infection. K) Quantification of frequencies of CD11bint/F4/80+ splenic macrophages out of CD45+ cells of wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient mice. L) Histogram and M) quantification of Sirpα (CD172a) MFI of wild‐type (Nr2f6+/+ ) or Nr2f6‐deficient splenic macrophages 22 h post‐infection. Data shown are from one RNA‐Seq sort experiment with n = 3/2 (Nr2f6+/+ )/(Nr2f6‐/‐ ); flow cytometric data was generated from at least two experiments, with n = 2‐3 per group and experiment, total n = 4/5 (for CD172a) under healthy conditions and following 22 h after Salmonella Typhimurium infection (Nr2f6+/+ )/(Nr2f6‐/‐ ). Each dot represents the data from one individual mouse. Results are shown as median ± IQR with whiskers from min. to max. The Shapiro‐Wilk test evaluated the normality of data. Asterisks indicate statistically significant differences between genotypes calculated using the Student's t‐test or Mann‐Whitney U test for non‐parametric data. A p‐value < 0.05 was considered statistically significant. *0.05,**0.01, ***0.001 (See also Figure S6, Supporting Information).