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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

IL-18 enhances the recruitment of NK cells into the infected cecum LP thereby stimulating early cecal inflammation.(a) Il18-/- mice and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h (n = 3–4 per group). RNA-Seq was performed on RNA extracted from complete cecum tissue. RNA-Seq analysis: The Volcano plot shows the induction (log2 fold change) versus the -log10 p-value for all chemokines. Chemokines able to induce NK cell recruitment are highlighted in red. (b) C57BL/6 WT mice were Sm-pretreated and either uninfected or infected orally with 5x107 CFU S.Tm for 12h (n = 4 per group). Cxcl9, Cxcl10, Cxcl11, Ccl3 and Ccl4 mRNA levels in whole cecum tissue were measured by RT-qPCR. Results are presented relative to the expression of Actb. (c) Same as (b) but comparing cecum tissues from infected Il18-/- mice vs infected littermate controls. (d and e) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x107 CFU S.Tm (n = 5 per group). Single live cells were gated on CD45+ lymphocytes. (d) Representative dot plots and (e) quantification of NK1.1+ CD3- cells. (f and g) Flow cytometric analysis of isolated cecal LP cells from Il18-/- mice and littermates, Sm-pretreated and orally infected with 5x107 CFU S.Tm for 12h (n = 5–6 per group). Single live cells were gated on CD45+ lymphocytes. (f) Representative dot plots and (g) quantification of NK1.1+ CD3- cells. (h) C57BL/6 mice were infected for 12h with 5x107 CFU S.Tm and isolated cecal LP cells of two mice were pooled for staining and isotype control staining; data are shown for one out of three independent experiments. CD45+ NK1.1+ CD3- cells were characterized according to their surface expression of KLRG1, NKp46, CD122, TCRγδ and Thy1. (i) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm. Isolated cecal LP cells of two mice each were pooled for fluorescence activated cell sorting. Live CD45+ CD3- cells were further sorted according to their expression of NK1.1. Tbx21, Eomes, Rorc, Gata3, Prf1 and Sell transcripts were analyzed by RT-qPCR. Results are presented relative to the expression of Actb. (j and k) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated (j) Casp1/11-/- or (k) Casp11-/- mice. Single live cells were gated on CD45+ lymphocytes and NK1.1+ CD3- cells were quantified. (l and m) C57BL/6 WT mice were injected intraperitoneally with anti-asialo GM1 antiserum (50μL antiserum/mouse; three consecutive days), anti-NK1.1 (10mg/kg; 2 consecutive days) or PBS and mice were infected orally with 5x107 CFU S.Tm for 12h (n = 8–10 per group). (l) HE-stained cryosections of anti-asialo GM1 -treated or control mice, (m) pathological score; arrows indicate mice of representative HE-stained cryosections; SE = submucosal edema, L = lumen; scale bar = 100μm. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01).
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ppat.1005723.g003: IL-18 enhances the recruitment of NK cells into the infected cecum LP thereby stimulating early cecal inflammation.(a) Il18-/- mice and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h (n = 3–4 per group). RNA-Seq was performed on RNA extracted from complete cecum tissue. RNA-Seq analysis: The Volcano plot shows the induction (log2 fold change) versus the -log10 p-value for all chemokines. Chemokines able to induce NK cell recruitment are highlighted in red. (b) C57BL/6 WT mice were Sm-pretreated and either uninfected or infected orally with 5x107 CFU S.Tm for 12h (n = 4 per group). Cxcl9, Cxcl10, Cxcl11, Ccl3 and Ccl4 mRNA levels in whole cecum tissue were measured by RT-qPCR. Results are presented relative to the expression of Actb. (c) Same as (b) but comparing cecum tissues from infected Il18-/- mice vs infected littermate controls. (d and e) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x107 CFU S.Tm (n = 5 per group). Single live cells were gated on CD45+ lymphocytes. (d) Representative dot plots and (e) quantification of NK1.1+ CD3- cells. (f and g) Flow cytometric analysis of isolated cecal LP cells from Il18-/- mice and littermates, Sm-pretreated and orally infected with 5x107 CFU S.Tm for 12h (n = 5–6 per group). Single live cells were gated on CD45+ lymphocytes. (f) Representative dot plots and (g) quantification of NK1.1+ CD3- cells. (h) C57BL/6 mice were infected for 12h with 5x107 CFU S.Tm and isolated cecal LP cells of two mice were pooled for staining and isotype control staining; data are shown for one out of three independent experiments. CD45+ NK1.1+ CD3- cells were characterized according to their surface expression of KLRG1, NKp46, CD122, TCRγδ and Thy1. (i) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm. Isolated cecal LP cells of two mice each were pooled for fluorescence activated cell sorting. Live CD45+ CD3- cells were further sorted according to their expression of NK1.1. Tbx21, Eomes, Rorc, Gata3, Prf1 and Sell transcripts were analyzed by RT-qPCR. Results are presented relative to the expression of Actb. (j and k) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated (j) Casp1/11-/- or (k) Casp11-/- mice. Single live cells were gated on CD45+ lymphocytes and NK1.1+ CD3- cells were quantified. (l and m) C57BL/6 WT mice were injected intraperitoneally with anti-asialo GM1 antiserum (50μL antiserum/mouse; three consecutive days), anti-NK1.1 (10mg/kg; 2 consecutive days) or PBS and mice were infected orally with 5x107 CFU S.Tm for 12h (n = 8–10 per group). (l) HE-stained cryosections of anti-asialo GM1 -treated or control mice, (m) pathological score; arrows indicate mice of representative HE-stained cryosections; SE = submucosal edema, L = lumen; scale bar = 100μm. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01).

Mentions: In addition to Neutrophil recruiting chemokines, a second group of chemokines was significantly differentially expressed in our RNA-Seq dataset, which is known to coordinate the recruitment of NK cells to sites of infection (Fig 3a, depicted in red;[40]). NK cells are early effectors in the mucosal defense against several viruses, bacteria, protozoa and fungi [41]. They respond to a wide range of chemokines, are rapidly mobilized in response to danger signals and therefore quickly recruited to sites of inflammation and disease [42]. There, NK cells are activated either indirectly by cytokines or directly via the recognition of stressed and infected cells [43–45]. During systemic bacterial infections, activated NK cells can limit tissue infection and prevent systemic spread of the pathogen through direct lysis of target cells or by releasing GM-CSF, TNF and IFNγ to orchestrate further responses [41]. The secretion of such pro-inflammatory cytokines by NK cells and other early effector cells orchestrates and amplifies the local immune response, inducing a full-blown mucosal inflammation to boost pathogen elimination. Our findings provided a first hint that NK cells might be important coordinators initiating gut inflammation.


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)

IL-18 enhances the recruitment of NK cells into the infected cecum LP thereby stimulating early cecal inflammation.(a) Il18-/- mice and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h (n = 3–4 per group). RNA-Seq was performed on RNA extracted from complete cecum tissue. RNA-Seq analysis: The Volcano plot shows the induction (log2 fold change) versus the -log10 p-value for all chemokines. Chemokines able to induce NK cell recruitment are highlighted in red. (b) C57BL/6 WT mice were Sm-pretreated and either uninfected or infected orally with 5x107 CFU S.Tm for 12h (n = 4 per group). Cxcl9, Cxcl10, Cxcl11, Ccl3 and Ccl4 mRNA levels in whole cecum tissue were measured by RT-qPCR. Results are presented relative to the expression of Actb. (c) Same as (b) but comparing cecum tissues from infected Il18-/- mice vs infected littermate controls. (d and e) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x107 CFU S.Tm (n = 5 per group). Single live cells were gated on CD45+ lymphocytes. (d) Representative dot plots and (e) quantification of NK1.1+ CD3- cells. (f and g) Flow cytometric analysis of isolated cecal LP cells from Il18-/- mice and littermates, Sm-pretreated and orally infected with 5x107 CFU S.Tm for 12h (n = 5–6 per group). Single live cells were gated on CD45+ lymphocytes. (f) Representative dot plots and (g) quantification of NK1.1+ CD3- cells. (h) C57BL/6 mice were infected for 12h with 5x107 CFU S.Tm and isolated cecal LP cells of two mice were pooled for staining and isotype control staining; data are shown for one out of three independent experiments. CD45+ NK1.1+ CD3- cells were characterized according to their surface expression of KLRG1, NKp46, CD122, TCRγδ and Thy1. (i) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm. Isolated cecal LP cells of two mice each were pooled for fluorescence activated cell sorting. Live CD45+ CD3- cells were further sorted according to their expression of NK1.1. Tbx21, Eomes, Rorc, Gata3, Prf1 and Sell transcripts were analyzed by RT-qPCR. Results are presented relative to the expression of Actb. (j and k) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated (j) Casp1/11-/- or (k) Casp11-/- mice. Single live cells were gated on CD45+ lymphocytes and NK1.1+ CD3- cells were quantified. (l and m) C57BL/6 WT mice were injected intraperitoneally with anti-asialo GM1 antiserum (50μL antiserum/mouse; three consecutive days), anti-NK1.1 (10mg/kg; 2 consecutive days) or PBS and mice were infected orally with 5x107 CFU S.Tm for 12h (n = 8–10 per group). (l) HE-stained cryosections of anti-asialo GM1 -treated or control mice, (m) pathological score; arrows indicate mice of representative HE-stained cryosections; SE = submucosal edema, L = lumen; scale bar = 100μm. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01).
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ppat.1005723.g003: IL-18 enhances the recruitment of NK cells into the infected cecum LP thereby stimulating early cecal inflammation.(a) Il18-/- mice and littermate controls were Sm-pretreated, infected orally with 5x107 CFU S.Tm for 12h (n = 3–4 per group). RNA-Seq was performed on RNA extracted from complete cecum tissue. RNA-Seq analysis: The Volcano plot shows the induction (log2 fold change) versus the -log10 p-value for all chemokines. Chemokines able to induce NK cell recruitment are highlighted in red. (b) C57BL/6 WT mice were Sm-pretreated and either uninfected or infected orally with 5x107 CFU S.Tm for 12h (n = 4 per group). Cxcl9, Cxcl10, Cxcl11, Ccl3 and Ccl4 mRNA levels in whole cecum tissue were measured by RT-qPCR. Results are presented relative to the expression of Actb. (c) Same as (b) but comparing cecum tissues from infected Il18-/- mice vs infected littermate controls. (d and e) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated C57BL/6 WT mice, either uninfected or infected with 5x107 CFU S.Tm (n = 5 per group). Single live cells were gated on CD45+ lymphocytes. (d) Representative dot plots and (e) quantification of NK1.1+ CD3- cells. (f and g) Flow cytometric analysis of isolated cecal LP cells from Il18-/- mice and littermates, Sm-pretreated and orally infected with 5x107 CFU S.Tm for 12h (n = 5–6 per group). Single live cells were gated on CD45+ lymphocytes. (f) Representative dot plots and (g) quantification of NK1.1+ CD3- cells. (h) C57BL/6 mice were infected for 12h with 5x107 CFU S.Tm and isolated cecal LP cells of two mice were pooled for staining and isotype control staining; data are shown for one out of three independent experiments. CD45+ NK1.1+ CD3- cells were characterized according to their surface expression of KLRG1, NKp46, CD122, TCRγδ and Thy1. (i) C57BL/6 mice were infected for 18h with 5x107 CFU S.Tm. Isolated cecal LP cells of two mice each were pooled for fluorescence activated cell sorting. Live CD45+ CD3- cells were further sorted according to their expression of NK1.1. Tbx21, Eomes, Rorc, Gata3, Prf1 and Sell transcripts were analyzed by RT-qPCR. Results are presented relative to the expression of Actb. (j and k) Flow cytometric analysis of isolated cecal LP cells from Sm-pretreated (j) Casp1/11-/- or (k) Casp11-/- mice. Single live cells were gated on CD45+ lymphocytes and NK1.1+ CD3- cells were quantified. (l and m) C57BL/6 WT mice were injected intraperitoneally with anti-asialo GM1 antiserum (50μL antiserum/mouse; three consecutive days), anti-NK1.1 (10mg/kg; 2 consecutive days) or PBS and mice were infected orally with 5x107 CFU S.Tm for 12h (n = 8–10 per group). (l) HE-stained cryosections of anti-asialo GM1 -treated or control mice, (m) pathological score; arrows indicate mice of representative HE-stained cryosections; SE = submucosal edema, L = lumen; scale bar = 100μm. Statistical analysis was performed using the Mann-Whitney-U test (ns = not significant, * = p<0.05; ** = p<0.01).
Mentions: In addition to Neutrophil recruiting chemokines, a second group of chemokines was significantly differentially expressed in our RNA-Seq dataset, which is known to coordinate the recruitment of NK cells to sites of infection (Fig 3a, depicted in red;[40]). NK cells are early effectors in the mucosal defense against several viruses, bacteria, protozoa and fungi [41]. They respond to a wide range of chemokines, are rapidly mobilized in response to danger signals and therefore quickly recruited to sites of inflammation and disease [42]. There, NK cells are activated either indirectly by cytokines or directly via the recognition of stressed and infected cells [43–45]. During systemic bacterial infections, activated NK cells can limit tissue infection and prevent systemic spread of the pathogen through direct lysis of target cells or by releasing GM-CSF, TNF and IFNγ to orchestrate further responses [41]. The secretion of such pro-inflammatory cytokines by NK cells and other early effector cells orchestrates and amplifies the local immune response, inducing a full-blown mucosal inflammation to boost pathogen elimination. Our findings provided a first hint that NK cells might be important coordinators initiating gut inflammation.

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