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CXCR3-dependent CD4⁺ T cells are required to activate inflammatory monocytes for defense against intestinal infection.

Cohen SB, Maurer KJ, Egan CE, Oghumu S, Satoskar AR, Denkers EY - PLoS Pathog. (2013)

Bottom Line: CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation.Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3.Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4⁺ and CD8⁺ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3⁻/⁻ mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3⁻/⁻ mice. Strikingly, adoptive transfer of wild-type but not Ifnγ⁻/⁻ CD4⁺ T lymphocytes into Cxcr3⁻/⁻ animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

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CD4+ T-cell recruitment, but not the presence of inflammatory monocytes, is impaired in the small intestine in the absence of CXCR3.(A) Frozen sections of intestines from infected WT mice were co-stained for Ly6C/G (Gr-1) (green) and iNOS (red) to confirm the presence of inflammatory monocytes in the mucosa of Day 6-infected animals. (B and C) Small intestinal LP cells were isolated from CXCR3-GFP reporters and Cxcr3−/− mice 6 days following oral infection. In the CXCR3 reporter mice, inflammatory monocytes (B, blue line) and CD4+ T cells (C, red line) were assessed for GFP expression by flow cytometry as compared to Cxcr3−/− cells (gray shaded in both histograms). (D) Total numbers of lamina propria inflammatory monocytes 6 days after infection. Neutrophils were excluded by gating on Ly6G-negative cells. (E) Total numbers of CD4+CXCR3-GFP+ T cells, CD4+CXCR3-GFP− T cells, inflammatory monocytes, and NK cells in the lamina propria of WT and Cxcr3−/− mice 4 and 7 days post-infection. Statistical comparisons were made between time points of respective cell types, where * p<0.05 and ** p<0.01. In panels F–G, WT (F) and Cxcr3−/− (G) intestinal frozen sections were stained with anti-CD4 antibody followed by anti-rat Alexa-647. Sections were visualized by immunofluorescence microscopy. (H) To quantify CD4+ T-cell infiltration, the ratio of Alexa-647 over DAPI fluorescence was calculated (WT: n = 3; KO: n = 3; 6–12 fields/mouse; p<0.01). Pooled ratios are represented as mean +/− SEM.
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ppat-1003706-g004: CD4+ T-cell recruitment, but not the presence of inflammatory monocytes, is impaired in the small intestine in the absence of CXCR3.(A) Frozen sections of intestines from infected WT mice were co-stained for Ly6C/G (Gr-1) (green) and iNOS (red) to confirm the presence of inflammatory monocytes in the mucosa of Day 6-infected animals. (B and C) Small intestinal LP cells were isolated from CXCR3-GFP reporters and Cxcr3−/− mice 6 days following oral infection. In the CXCR3 reporter mice, inflammatory monocytes (B, blue line) and CD4+ T cells (C, red line) were assessed for GFP expression by flow cytometry as compared to Cxcr3−/− cells (gray shaded in both histograms). (D) Total numbers of lamina propria inflammatory monocytes 6 days after infection. Neutrophils were excluded by gating on Ly6G-negative cells. (E) Total numbers of CD4+CXCR3-GFP+ T cells, CD4+CXCR3-GFP− T cells, inflammatory monocytes, and NK cells in the lamina propria of WT and Cxcr3−/− mice 4 and 7 days post-infection. Statistical comparisons were made between time points of respective cell types, where * p<0.05 and ** p<0.01. In panels F–G, WT (F) and Cxcr3−/− (G) intestinal frozen sections were stained with anti-CD4 antibody followed by anti-rat Alexa-647. Sections were visualized by immunofluorescence microscopy. (H) To quantify CD4+ T-cell infiltration, the ratio of Alexa-647 over DAPI fluorescence was calculated (WT: n = 3; KO: n = 3; 6–12 fields/mouse; p<0.01). Pooled ratios are represented as mean +/− SEM.

Mentions: The dominant effector cells required for elimination of T. gondii following oral infection are inflammatory monocytes. These cells express Ly6C/G (Gr-1), produce TNF-α, IL-12, and are likely to kill parasites via activation of IFN-γ-inducible p47 GTPases that assemble at the parasitophorous vacuole membrane and mediate its destruction [8], [11]. Consistent with others [11], we observed these cells in the LP of infected mice (Fig. 4A). Inflammatory monocytes are dependent upon CCR2 for exit from the bone marrow, but we wondered whether CXCR3 might be involved in recruiting these cells to the LP in response to T. gondii. Therefore, we examined CXCR3-GFP expression by intestinal inflammatory monocytes during infection. Inflammatory monocytes in the small intestinal LP of infected reporter mice did not express any GFP as compared to inflammatory monocytes isolated from infected Cxcr3−/− mice (Fig. 4B). In stark contrast, approximately 50% of LP CD4+ T cells expressed high levels of GFP (Fig. 4C). Furthermore, Cxcr3−/− mice displayed unaltered total numbers of LP inflammatory monocytes compared to wild-type controls (defined as CD11b+Ly6C+Ly6G−) (Fig. 4D). We next assessed the kinetics by which CD4+ T cells and inflammatory monocytes were recruited to the lamina propria during infection. Between days 4 and 7 of infection, there was a significant increase in the total numbers of CD4+CXCR3−GFP+ T cells and inflammatory monocytes (Fig. 4E). However, the total number of CD4+CXCR3−GFP− cells remained unchanged, further indicating that infection promotes the recruitment of CD4+CXCR3+ T cells (Fig. 4E). Few NK cells were observed in the lamina propria, but there was a small increase in their number during infection. This was attributable to an increase in CXCR3− NK cells (data not shown). Consistent with these results, there was an influx of CD4+ T cells in WT small intestines that was diminished in Cxcr3−/− mice (Fig. 4F–H). These findings demonstrate that CD4+ T cells fail to effectively traffic to the intestinal compartment in the absence of CXCR3, but the presence of LP inflammatory monocytes does not require this chemokine receptor.


CXCR3-dependent CD4⁺ T cells are required to activate inflammatory monocytes for defense against intestinal infection.

Cohen SB, Maurer KJ, Egan CE, Oghumu S, Satoskar AR, Denkers EY - PLoS Pathog. (2013)

CD4+ T-cell recruitment, but not the presence of inflammatory monocytes, is impaired in the small intestine in the absence of CXCR3.(A) Frozen sections of intestines from infected WT mice were co-stained for Ly6C/G (Gr-1) (green) and iNOS (red) to confirm the presence of inflammatory monocytes in the mucosa of Day 6-infected animals. (B and C) Small intestinal LP cells were isolated from CXCR3-GFP reporters and Cxcr3−/− mice 6 days following oral infection. In the CXCR3 reporter mice, inflammatory monocytes (B, blue line) and CD4+ T cells (C, red line) were assessed for GFP expression by flow cytometry as compared to Cxcr3−/− cells (gray shaded in both histograms). (D) Total numbers of lamina propria inflammatory monocytes 6 days after infection. Neutrophils were excluded by gating on Ly6G-negative cells. (E) Total numbers of CD4+CXCR3-GFP+ T cells, CD4+CXCR3-GFP− T cells, inflammatory monocytes, and NK cells in the lamina propria of WT and Cxcr3−/− mice 4 and 7 days post-infection. Statistical comparisons were made between time points of respective cell types, where * p<0.05 and ** p<0.01. In panels F–G, WT (F) and Cxcr3−/− (G) intestinal frozen sections were stained with anti-CD4 antibody followed by anti-rat Alexa-647. Sections were visualized by immunofluorescence microscopy. (H) To quantify CD4+ T-cell infiltration, the ratio of Alexa-647 over DAPI fluorescence was calculated (WT: n = 3; KO: n = 3; 6–12 fields/mouse; p<0.01). Pooled ratios are represented as mean +/− SEM.
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Related In: Results  -  Collection

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ppat-1003706-g004: CD4+ T-cell recruitment, but not the presence of inflammatory monocytes, is impaired in the small intestine in the absence of CXCR3.(A) Frozen sections of intestines from infected WT mice were co-stained for Ly6C/G (Gr-1) (green) and iNOS (red) to confirm the presence of inflammatory monocytes in the mucosa of Day 6-infected animals. (B and C) Small intestinal LP cells were isolated from CXCR3-GFP reporters and Cxcr3−/− mice 6 days following oral infection. In the CXCR3 reporter mice, inflammatory monocytes (B, blue line) and CD4+ T cells (C, red line) were assessed for GFP expression by flow cytometry as compared to Cxcr3−/− cells (gray shaded in both histograms). (D) Total numbers of lamina propria inflammatory monocytes 6 days after infection. Neutrophils were excluded by gating on Ly6G-negative cells. (E) Total numbers of CD4+CXCR3-GFP+ T cells, CD4+CXCR3-GFP− T cells, inflammatory monocytes, and NK cells in the lamina propria of WT and Cxcr3−/− mice 4 and 7 days post-infection. Statistical comparisons were made between time points of respective cell types, where * p<0.05 and ** p<0.01. In panels F–G, WT (F) and Cxcr3−/− (G) intestinal frozen sections were stained with anti-CD4 antibody followed by anti-rat Alexa-647. Sections were visualized by immunofluorescence microscopy. (H) To quantify CD4+ T-cell infiltration, the ratio of Alexa-647 over DAPI fluorescence was calculated (WT: n = 3; KO: n = 3; 6–12 fields/mouse; p<0.01). Pooled ratios are represented as mean +/− SEM.
Mentions: The dominant effector cells required for elimination of T. gondii following oral infection are inflammatory monocytes. These cells express Ly6C/G (Gr-1), produce TNF-α, IL-12, and are likely to kill parasites via activation of IFN-γ-inducible p47 GTPases that assemble at the parasitophorous vacuole membrane and mediate its destruction [8], [11]. Consistent with others [11], we observed these cells in the LP of infected mice (Fig. 4A). Inflammatory monocytes are dependent upon CCR2 for exit from the bone marrow, but we wondered whether CXCR3 might be involved in recruiting these cells to the LP in response to T. gondii. Therefore, we examined CXCR3-GFP expression by intestinal inflammatory monocytes during infection. Inflammatory monocytes in the small intestinal LP of infected reporter mice did not express any GFP as compared to inflammatory monocytes isolated from infected Cxcr3−/− mice (Fig. 4B). In stark contrast, approximately 50% of LP CD4+ T cells expressed high levels of GFP (Fig. 4C). Furthermore, Cxcr3−/− mice displayed unaltered total numbers of LP inflammatory monocytes compared to wild-type controls (defined as CD11b+Ly6C+Ly6G−) (Fig. 4D). We next assessed the kinetics by which CD4+ T cells and inflammatory monocytes were recruited to the lamina propria during infection. Between days 4 and 7 of infection, there was a significant increase in the total numbers of CD4+CXCR3−GFP+ T cells and inflammatory monocytes (Fig. 4E). However, the total number of CD4+CXCR3−GFP− cells remained unchanged, further indicating that infection promotes the recruitment of CD4+CXCR3+ T cells (Fig. 4E). Few NK cells were observed in the lamina propria, but there was a small increase in their number during infection. This was attributable to an increase in CXCR3− NK cells (data not shown). Consistent with these results, there was an influx of CD4+ T cells in WT small intestines that was diminished in Cxcr3−/− mice (Fig. 4F–H). These findings demonstrate that CD4+ T cells fail to effectively traffic to the intestinal compartment in the absence of CXCR3, but the presence of LP inflammatory monocytes does not require this chemokine receptor.

Bottom Line: CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation.Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3.Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4⁺ and CD8⁺ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3⁻/⁻ mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3⁻/⁻ mice. Strikingly, adoptive transfer of wild-type but not Ifnγ⁻/⁻ CD4⁺ T lymphocytes into Cxcr3⁻/⁻ animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.

Show MeSH
Related in: MedlinePlus