Inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine - PubMed (original) (raw)

Inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine

Patrick A McLaughlin et al. PLoS Pathog. 2019.

Abstract

Salmonella exploit host-derived nitrate for growth in the lumen of the inflamed intestine. The generation of host-derived nitrate is dependent on Nos2, which encodes inducible nitric oxide synthase (iNOS), an enzyme that catalyzes nitric oxide (NO) production. However, the cellular sources of iNOS and, therefore, NO-derived nitrate used by Salmonella for growth in the lumen of the inflamed intestine remain unidentified. Here, we show that iNOS-producing inflammatory monocytes infiltrate ceca of mice infected with Salmonella. In addition, we show that inactivation of type-three secretion system (T3SS)-1 and T3SS-2 renders Salmonella unable to induce CC- chemokine receptor-2- and CC-chemokine ligand-2-dependent inflammatory monocyte recruitment. Furthermore, we show that the severity of the pathology of Salmonella- induced colitis as well as the nitrate-dependent growth of Salmonella in the lumen of the inflamed intestine are reduced in mice that lack Ccr2 and, therefore, inflammatory monocytes in the tissues. Thus, inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. iNOS-producing IM infiltrate ceca of mice infected with STm.

(A-C) Effects of Sm pretreatment and STm infection on C57BL/6J mice (n = 3–7 per group). Samples were harvested on day 4 after infection. Data show mean with SEM (A and B) or mean with SEM and individual data points (C), and were analyzed by one-way ANOVA with Sidak post hoc test. (A) Relative fold induction of Ccl2 expression in cecal tissues, normalized to Gapdh. (B) Percentage of IM among total gated cells from cecal tissues. (C) Number of STm CFU recovered from cecal contents, normalized to weight. (D-E) Four-day time course tracking IM recruitment into cecal tissues from C57BL/6J mice (n = 4–9 per group) treated with Sm prior to inoculation with STm. Mice treated with Sm prior to inoculation with PBS (uninfected, UI) were used as a control. Data show mean with SEM (D) or mean with SEM and individual data points (E), and were analyzed by one-way ANOVA with Fisher’s LSD post hoc test. (D) Percentage of IM among total gated cells from cecal tissues. (E) Number of STm CFU recovered from cecal contents, normalized to weight. (F) Percentage of IM among live cells in cecal contents from C57BL/6J mice (n = 6 per group) treated with Sm prior to inoculation with PBS (uninfected, UI) or STm. Cecal contents were collected on day 4 after inoculation. Data show mean with SEM and were analyzed by Student’s _t_-test. (G) Level of iNOS expressed by IM and neutrophils purified and pooled from cecal tissues of C57BL/6J mice (n = 5–6 per group) treated with Sm prior to inoculation with STm. Ceca were harvested on day 4 after inoculation. β-Actin was used as a loading control for Western blotting. Data are representative of two independent experiments. Also see S1 Fig.

Fig 2

Fig 2. Inactivation of T3SS-1 and T3SS-2 renders STm unable to induce _Ccr2_- and _Ccl2_-dependent IM recruitment.

(A) Percentage of IM among total gated cells in cecal tissues from C57BL/6J mice (n = 3 per group) treated with Sm prior to inoculation with PBS (uninfected; UI) or wild-type (WT) STm, or invA (T3SS-1), ssaD (T3SS-2), or invA ssaD (T3SS-1 T3SS-2) mutant STm. Ceca were harvested on day 4 after inoculation. Data show mean with SEM and were analyzed by one-way ANOVA with Sidak post hoc test. (B) Corresponding number of STm CFU recovered from cecal contents, normalized to weight. Data show mean with SEM and individual data points, and were analyzed by one-way ANOVA with Sidak post hoc test. (C) Percentage of IM among total gated cells in cecal tissues from C57BL/6J (WT), _Ccr2_-/-, and _Ccl2_-/- mice (n = 6–9 per group) treated with Sm prior to inoculation with PBS (uninfected, UI) or STm. Ceca were harvested on day 4 after inoculation. Data show mean with SEM and were analyzed by one-way ANOVA with Sidak post hoc test. (D) Corresponding number of STm CFU recovered from cecal contents, normalized to weight. Data show mean with SEM and individual data points, and were analyzed by one-way ANOVA with Sidak post hoc test. Also see S2 Fig.

Fig 3

Fig 3. IM contribute to the severity of the pathology of STm-induced colitis.

(A) Representative hematoxylin- and eosin-stained cecal sections from C57BL/6J (WT) and _Ccr2_-/- mice (n = 5–7 per group) treated with Sm prior to inoculation with PBS or STm. Ceca were harvested on day 4 after inoculation. Top left: WT mouse treated with Sm prior to inoculation with PBS. Top right: WT mouse treated with Sm prior to inoculation with STm. Bottom left: WT mouse treated with Sm prior to inoculation with STm. Bottom right: _Ccr2_-/- mouse treated with Sm prior to inoculation with STm. Arrows point to cecal lymphoid tissue. Arrowheads delineate the cecal wall. Asterisk indicates accumulation of inflammatory exudate within the cecal lumen. (B) Blinded, semi-quantitative histopathological analysis of cecal tissues from C57BL/6J (WT) and _Ccr2_-/- mice (n = 4–7 per group) left untreated or treated with Sm prior to inoculation with PBS or STm. Ceca were harvested on day 4 after inoculation. Data show mean with SEM and were analyzed by one-way ANOVA with Sidak post hoc test. (C-H) Relative fold induction of Il1b (C), Tnfa (D), Ifng (E), Il22 (F), Cxcl1 (G), and Cxcl2 (H) expression in cecal tissues from C57BL/6J (WT) and _Ccr2_-/- mice (n = 5–7 per group) treated with Sm prior to inoculation with PBS (uninfected, UI) or STm. Ceca were harvested on day 4 after inoculation. Relative target gene expression was normalized to Gapdh. Data show mean with SEM and were analyzed by one-way ANOVA with Sidak post hoc test.

Fig 4

Fig 4. IM contribute to the STm-induced generation of nitrate in the cecal mucosa.

(A) Relative fold induction of Nos2 expression in cecal tissues from C57BL/6J (WT) and _Ccr2_-/- mice (n = 5–7 per group) treated with Sm prior to inoculation with PBS (uninfected, UI) or STm. Ceca were harvested on day 4 after inoculation. Relative Nos2 expression was normalized to Gapdh. Data show mean with SEM and were analyzed by one-way ANOVA with Sidak post hoc test. (B) Relative level of iNOS expressed in cecal tissues from C57BL/6J (WT) and _Ccr2_-/- mice (n = 6–9 per group) treated with Sm prior to inoculation with PBS (uninfected, UI) or STm. Ceca were harvested on day 4 after inoculation. β-Actin was used as a loading control for Western blotting. Data show mean with SEM and were analyzed by one-way ANOVA with Sidak post hoc test. (C) Concentration of nitrate in cecal mucus from C57BL/6J (WT) and _Ccr2_-/- mice (n = 5–7 per group) left untreated or treated with Sm prior to inoculation with PBS or STm. Ceca were harvested on day 4 after inoculation. Data show mean with SEM and individual data points, and were analyzed by one-way ANOVA with Sidak post hoc test. Also see S3 Fig.

Fig 5

Fig 5. IM contribute to the generation of host-derived nitrate used by STm for growth in the lumen of the inflamed intestine.

(A-B) Competitive indices of WT and napA mutant STm CFU in cecal contents (A) and fecal pellets (B) from C57BL/6J (WT) and _Ccr2_-/- mice (n = 16 per group) treated with Sm prior to inoculation with a 1:1 mixture of differentially marked WT and napA mutant STm. Cecal contents were collected on day 4 after inoculation. Fecal pellets were collected daily up to day 4 after inoculation. Competitive indices were calculated by dividing the number of napA mutant STm CFU by the number of WT STm CFU, and normalized to the input ratio. Data show mean with SEM from three independent experiments and were analyzed by one-way ANOVA (A) or two-way ANOVA (B) with Sidak post hoc test.

Fig 6

Fig 6. Model of how IM promote nitrate-dependent STm expansion in the lumen of the inflamed intestine.

We postulate that IM contribute both directly and indirectly to the generation of host- derived nitrate used by STm for growth in the lumen of the inflamed intestine. IM may contribute directly to the generation of host-derived nitrate used by STm for growth in the lumen of the inflamed intestine by expressing Nos2 and indirectly by potentiating Nos2 expression by other cells such as IEC. See the text for details.

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