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An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection.

Müller AA, Dolowschiak T, Sellin ME, Felmy B, Verbree C, Gadient S, Westermann AJ, Vogel J, LeibundGut-Landmann S, Hardt WD - PLoS Pathog. (2016)

Bottom Line: IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells.NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation.This may have broad relevance for mucosal defense against microbial pathogens.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Zürich, Switzerland.

ABSTRACT
Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.

No MeSH data available.


Related in: MedlinePlus

IFNγ expression by NK cells is IL-18-dependent but not required for mounting tissue inflammation during the first 12h of the infection.(a) Volcano plot of all cytokines differentially expressed in the cecal mucosa of the Il18-/- mice and littermate shown in Fig 2A. We plotted log2 (fold change) against -log10 (p-value). NK cell effector cytokines are highlighted in red. (b-d) Il18-/- mice and littermate controls were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h (b) or 18h (c-d). (b) Ifng, Iigp1 and Cxcl10 transcripts in whole cecum tissue were analyzed by RT-qPCR. Results are presented relative to the expression of Actb (n = 8–9 per group). (c) IFNγ protein concentration in whole cecum tissue lysates (n = 5 per group) as measured by CBA; dashed line = detection limit. (d) Quantification of IFNγ-producing cells by flow cytometric analysis of isolated cecal LP cells (n = 6 per group). (e and f) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm and cecal LP cells were isolated for staining. Data are shown from one out of three independent experiments. (e) Representative dot plot of IFNγ-expressing cells, pre-gated on single live lymphocytes. (f) FACS-analysis of CD3 and NK1.1 surface marker expression by IFNγ+ cell populations. (g) Ifng-/-, Ifngr1-/- and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h and pathological scores were assessed (n = 6–8 per group). (h) Mesenteric lymph node loads as determined by plating of organs from (left) Ifng-/- and (right) Il18-/- mice and their littermate controls. Mice were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h or 72h. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).
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ppat.1005723.g006: IFNγ expression by NK cells is IL-18-dependent but not required for mounting tissue inflammation during the first 12h of the infection.(a) Volcano plot of all cytokines differentially expressed in the cecal mucosa of the Il18-/- mice and littermate shown in Fig 2A. We plotted log2 (fold change) against -log10 (p-value). NK cell effector cytokines are highlighted in red. (b-d) Il18-/- mice and littermate controls were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h (b) or 18h (c-d). (b) Ifng, Iigp1 and Cxcl10 transcripts in whole cecum tissue were analyzed by RT-qPCR. Results are presented relative to the expression of Actb (n = 8–9 per group). (c) IFNγ protein concentration in whole cecum tissue lysates (n = 5 per group) as measured by CBA; dashed line = detection limit. (d) Quantification of IFNγ-producing cells by flow cytometric analysis of isolated cecal LP cells (n = 6 per group). (e and f) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm and cecal LP cells were isolated for staining. Data are shown from one out of three independent experiments. (e) Representative dot plot of IFNγ-expressing cells, pre-gated on single live lymphocytes. (f) FACS-analysis of CD3 and NK1.1 surface marker expression by IFNγ+ cell populations. (g) Ifng-/-, Ifngr1-/- and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h and pathological scores were assessed (n = 6–8 per group). (h) Mesenteric lymph node loads as determined by plating of organs from (left) Ifng-/- and (right) Il18-/- mice and their littermate controls. Mice were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h or 72h. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).

Mentions: As our previous analysis has revealed that the accumulated NK cells in the infected cecal LP are phenotypically mature, we next wanted to address the NK cell effector function, contributing to the onset of cecal inflammation. NK cells can affect defense via (at least) two different mechanisms, i.e. their cytotoxic function and the production of effector cytokines that boost antimicrobial defenses of other cell types [41, 52]. Our RNA-Seq data suggested a decreased expression of the three major NK cell-derived effector cytokines TNF, GM-CSF and IFNγ (Fig 6a, depicted in red). Therefore, we investigated the potential contribution of those three cytokines in the induction of early mucosal pathology after S.Tm infection. Although RNA-Seq analysis had shown a clear downregulation of GM-CSF transcripts, GM-CSF protein was not yet detectable in the cecal mucosa at 12h p.i. (S5a Fig) rendering it an unlikely candidate for promoting pathology at this initial phase of the infection. Other than GM-CSF, TNF protein levels were induced in the cecal LP by 12h p.i. and markedly reduced in IL-18-deficient mice (S5b and S5c Fig). However, flow cytometric analysis of TNF-producing cells in the cecal LP uncovered that the protein was not produced by NK cells but rather by cells from the myeloid compartment, at least at this early stage of the infection (S5d Fig). This excluded TNF as a likely NK cell effector cytokine and prompted us to focus on IFNγ.


An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection.

Müller AA, Dolowschiak T, Sellin ME, Felmy B, Verbree C, Gadient S, Westermann AJ, Vogel J, LeibundGut-Landmann S, Hardt WD - PLoS Pathog. (2016)

IFNγ expression by NK cells is IL-18-dependent but not required for mounting tissue inflammation during the first 12h of the infection.(a) Volcano plot of all cytokines differentially expressed in the cecal mucosa of the Il18-/- mice and littermate shown in Fig 2A. We plotted log2 (fold change) against -log10 (p-value). NK cell effector cytokines are highlighted in red. (b-d) Il18-/- mice and littermate controls were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h (b) or 18h (c-d). (b) Ifng, Iigp1 and Cxcl10 transcripts in whole cecum tissue were analyzed by RT-qPCR. Results are presented relative to the expression of Actb (n = 8–9 per group). (c) IFNγ protein concentration in whole cecum tissue lysates (n = 5 per group) as measured by CBA; dashed line = detection limit. (d) Quantification of IFNγ-producing cells by flow cytometric analysis of isolated cecal LP cells (n = 6 per group). (e and f) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm and cecal LP cells were isolated for staining. Data are shown from one out of three independent experiments. (e) Representative dot plot of IFNγ-expressing cells, pre-gated on single live lymphocytes. (f) FACS-analysis of CD3 and NK1.1 surface marker expression by IFNγ+ cell populations. (g) Ifng-/-, Ifngr1-/- and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h and pathological scores were assessed (n = 6–8 per group). (h) Mesenteric lymph node loads as determined by plating of organs from (left) Ifng-/- and (right) Il18-/- mice and their littermate controls. Mice were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h or 72h. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920399&req=5

ppat.1005723.g006: IFNγ expression by NK cells is IL-18-dependent but not required for mounting tissue inflammation during the first 12h of the infection.(a) Volcano plot of all cytokines differentially expressed in the cecal mucosa of the Il18-/- mice and littermate shown in Fig 2A. We plotted log2 (fold change) against -log10 (p-value). NK cell effector cytokines are highlighted in red. (b-d) Il18-/- mice and littermate controls were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h (b) or 18h (c-d). (b) Ifng, Iigp1 and Cxcl10 transcripts in whole cecum tissue were analyzed by RT-qPCR. Results are presented relative to the expression of Actb (n = 8–9 per group). (c) IFNγ protein concentration in whole cecum tissue lysates (n = 5 per group) as measured by CBA; dashed line = detection limit. (d) Quantification of IFNγ-producing cells by flow cytometric analysis of isolated cecal LP cells (n = 6 per group). (e and f) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm and cecal LP cells were isolated for staining. Data are shown from one out of three independent experiments. (e) Representative dot plot of IFNγ-expressing cells, pre-gated on single live lymphocytes. (f) FACS-analysis of CD3 and NK1.1 surface marker expression by IFNγ+ cell populations. (g) Ifng-/-, Ifngr1-/- and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h and pathological scores were assessed (n = 6–8 per group). (h) Mesenteric lymph node loads as determined by plating of organs from (left) Ifng-/- and (right) Il18-/- mice and their littermate controls. Mice were Sm-pretreated and infected orally with 5x107 CFU S.Tm for 12h or 72h. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01; *** = p<0.001).
Mentions: As our previous analysis has revealed that the accumulated NK cells in the infected cecal LP are phenotypically mature, we next wanted to address the NK cell effector function, contributing to the onset of cecal inflammation. NK cells can affect defense via (at least) two different mechanisms, i.e. their cytotoxic function and the production of effector cytokines that boost antimicrobial defenses of other cell types [41, 52]. Our RNA-Seq data suggested a decreased expression of the three major NK cell-derived effector cytokines TNF, GM-CSF and IFNγ (Fig 6a, depicted in red). Therefore, we investigated the potential contribution of those three cytokines in the induction of early mucosal pathology after S.Tm infection. Although RNA-Seq analysis had shown a clear downregulation of GM-CSF transcripts, GM-CSF protein was not yet detectable in the cecal mucosa at 12h p.i. (S5a Fig) rendering it an unlikely candidate for promoting pathology at this initial phase of the infection. Other than GM-CSF, TNF protein levels were induced in the cecal LP by 12h p.i. and markedly reduced in IL-18-deficient mice (S5b and S5c Fig). However, flow cytometric analysis of TNF-producing cells in the cecal LP uncovered that the protein was not produced by NK cells but rather by cells from the myeloid compartment, at least at this early stage of the infection (S5d Fig). This excluded TNF as a likely NK cell effector cytokine and prompted us to focus on IFNγ.

Bottom Line: IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells.NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation.This may have broad relevance for mucosal defense against microbial pathogens.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Zürich, Switzerland.

ABSTRACT
Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.

No MeSH data available.


Related in: MedlinePlus