A non-classical monocyte-derived macrophage subset provides a splenic replication niche for intracellular Salmonella

Abstract

Interactions between intracellular bacteria and mononuclear phagocytes give rise to diverse cellular phenotypes that may determine the outcome of infection. Recent advances in single-cell RNA sequencing (scRNA-seq) have identified multiple subsets within the mononuclear population, but implications to their function during infection are limited. Here, we surveyed the mononuclear niche of intracellular Salmonella Typhimurium (S.Tm) during early systemic infection in mice. We described eclipse-like growth kinetics in the spleen, with a first phase of bacterial control mediated by tissue-resident red-pulp macrophages. A second phase involved extensive bacterial replication within a macrophage population characterized by CD9 expression. We demonstrated that CD9⁺ macrophages induced pathways for detoxificating oxidized lipids, that may be utilized by intracellular S.Tm. We established that CD9⁺ macrophages originated from non-classical monocytes (NCM), and NCM-depleted mice were more resistant to S.Tm infection. Our study defines macrophage subset-specific host-pathogen interactions that determine early infection dynamics and infection outcome of the entire organism.

Keywords: Salmonella; host-pathogen interactions; in-vivo infection; macrophages; non-classical monocytes; single-cell RNA-seq.

Graphical abstract

Figure 1

Early systemic infection with S.Tm follows ELID (A and B) Mice were infected i.v. with S.Tm, and CFU was measured from spleens at 1, 4, 8, 24, and 48 hpi (A); n = 2 per time point). Lines represents the median of two replicates. Using flow cytometry, cells were gated to exclude CD19, CD3e, NK1.1, and Ly6G⁺ positive cells (Lin^–) and analyzed for CD11b and F4/80 expression (B). (C and D) Mice were infected i.v. with S.Tm-GFP and spleens were harvested at 4 hpi (n = 4) and 24 hpi (n = 4). Infected cells were analyzed by flow cytometry using the GFP signal of the bacteria (C). Calculated ratios of GFP⁺ cells between F4/80⁺ and Cd11b⁺ F4/80^– populations are shown (D). (E) Mice were infected i.v. with either WT (black) or ΔSPI-2 (gray) S.Tm strains, and CFU was measured from spleens at 4, 8, and 24 hpi (n = 2 mice per condition). (F) Mice were infected i.v. with either WT or ΔSPI-2 GFP-expressing strains. Twenty-four hpi, spleens were harvested and analyzed by flow cytometry and CD11b⁺F4/80⁺ macrophages were sorted and individually plated on LB agar (n = 94 in WT, n = 34 in ΔSPI-2) to determine the number of CFU per cell. Results are representative of four (A) or three (B–F) independent experiments. (E) ^∗∗p < 0.01 using two-way ANOVA. See also Figure S1.

Figure 2

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.

Figure 3

Functional analysis reveals distinct inflammatory programs of iNOS Macs and reducing oxidized lipids programs of CD9 Macs (A) Monocytes from Ms4a3^cre-Rosa^TdT 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.

Figure 4

ELID is mirrored by a decline of Rp Macs and increase of CD9 Macs (A and B) Mice were infected with S.Tm-GFP (n = 3 per time point). At the indicated time points, spleens were harvested and analyzed by flow cytometry using antibodies to the indicated populations or the GFP signal of the bacteria (A). Bar graphs (left y axis) indicates mean ratio from the population of F4/80, and dots (right y axis) indicate cells containing intracellular bacteria (presented as ratio from F4/80⁺ GFP⁺ population) (B). (C) Mice were infected as in (A), 24 hpi single GFP⁺ CD9 Macs and iNOS Macs were sorted, and scCFU was measured (n = 494, n = 553 respectively). Presented are the proportions of cells according to number of bacteria per cell. (D and E) Mice were infected as in (A). Twenty-four hpi spleens were harvested, fixed, stained, and imaged with CD9 and F4/80 antibodies, DAPI, and GFP for intracellular bacteria (D). An example of CD9 Macs containing multiple bacteria. Number of intracellular bacteria were quantified across F4/80⁺CD9⁺ or F4/80⁺CD9^– cells (n = 94 cells from 3 mice) (E). Results are representative of four (A and B) or three (C–E) independent experiments. See also Figure S4.

Figure 5

During the first phase of ELID, S.Tm growth is restricted by Rp Macs (A and B) Mice expressing diphtheria toxin receptor under Siglec-1 promoter were treated with diphtheria toxin (DTx; n = 3) or PBS (vehicle; n = 3). Twenty-four hours later, mice were infected with S.Tm, and 8 hpi spleens were harvested. Spleens were analyzed by flow cytometry using antibodies to mark the indicated populations (A) and presented as the ratio of the population from vehicle-treated mice (B). (C) Mice were treated as in (A), spleens were harvested at the indicated time points, and CFU was measured (n = 2 per treatment in each time point). (D–F) Mice were injected intravenously with either NEC-1 (n = 5) or vehicle (n = 4). One hour later, mice were infected with S.Tm, and 12 hpi spleens were harvested, and analyzed by flow cytometry using antibodies to mark the indicated populations (D), presented as the ratio of the population from vehicle-treated mice (E) and plated for CFU (n = 4 per treatment) (F). (G–I) Mice were injected intraperitoneally with αCCR2 antibodies or vehicle (n = 4 per treatment). Twenty-four hours later, mice were infected with S.Tm, and 24 hpi spleens were harvested. Spleens were analyzed by flow cytometry using antibodies to mark the indicated populations (G), presented as the ratio of the population from vehicle-treated mice (H), and plated for CFU (I). Results are representative of four (A)–(C) or three (D)–(I) independent experiments. (B, E, and H) Error bars indicate standard deviation. (B) ^∗p < 0.05, using Student’s t test. (E, F, and H) ^∗p < 0.05, using Mann-Whitney U test. (C) ^∗∗p < 0.01 using two-way analysis of variance. See also Figure S5.

Figure 6

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.