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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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Cxcr3−/− mice are susceptible to severe intestinal pathology following oral T. gondii infection.WT and CXCR3-deficient mice were orally inoculated with 30 ME49 cysts (A) or 50 cysts (B) of T. gondii and monitored for survival. In another set of experiments (C–J), mice were orally inoculated with 30 ME49 cysts, and tissues were collected at Day 10 post-infection. (C) Gross intestinal lesions in representative WT and CXCR3 KO mice. (D) Average lengths of noninfected (NI) and infected (INF) WT and Cxcr3−/− small intestines (NI WT, n = 5; NI KO, n = 3; INF WT, n = 8, INF KO: n = 7). E–H, H&E stained sections of small intestines from infected WT (E and G) and KO (F and H) mice. In panel G, the arrow points to an area of inflammatory cell influx. In panel H, the yellow arrow indicates an area of vascular congestion, and the red arrow indicates a necrotic villus. Blind scoring was performed on H&E stained intestine sections for inflammation (I) and damage (J) criteria (WT: n = 14; KO: n = 13; * p<0.05, ** p<0.01, *** p<0.001). Pooled data are represented as mean +/− SEM.
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ppat-1003706-g002: Cxcr3−/− mice are susceptible to severe intestinal pathology following oral T. gondii infection.WT and CXCR3-deficient mice were orally inoculated with 30 ME49 cysts (A) or 50 cysts (B) of T. gondii and monitored for survival. In another set of experiments (C–J), mice were orally inoculated with 30 ME49 cysts, and tissues were collected at Day 10 post-infection. (C) Gross intestinal lesions in representative WT and CXCR3 KO mice. (D) Average lengths of noninfected (NI) and infected (INF) WT and Cxcr3−/− small intestines (NI WT, n = 5; NI KO, n = 3; INF WT, n = 8, INF KO: n = 7). E–H, H&E stained sections of small intestines from infected WT (E and G) and KO (F and H) mice. In panel G, the arrow points to an area of inflammatory cell influx. In panel H, the yellow arrow indicates an area of vascular congestion, and the red arrow indicates a necrotic villus. Blind scoring was performed on H&E stained intestine sections for inflammation (I) and damage (J) criteria (WT: n = 14; KO: n = 13; * p<0.05, ** p<0.01, *** p<0.001). Pooled data are represented as mean +/− SEM.

Mentions: To further examine the role of CXCR3 during T. gondii infection, mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts, and the outcome of infection was monitored. While all wild-type (WT) mice survived acute infection with 30 cysts, Cxcr3−/− animals displayed increased susceptibility with nearly 75% of mice dying by 2 weeks post-infection (Fig. 2A). When the cyst dose was increased to 50, all CXCR3 knockout (KO) mice rapidly succumbed to infection, but some WT mice also died (Fig. 2B). Interestingly, when WT and KO mice were infected by intraperitoneal injection, lack of CXCR3 did not affect survival, indicating that the effect of CXCR3 is specific to the mucosal response (Fig. S3A). To further examine the overall response in orally infected mice, we examined the gross appearance of the small intestine of WT and Cxcr3−/− mice after 30-cyst infection. The small intestines of the KO mice were strikingly damaged as demonstrated by massive hemorrhage compared to WT (Fig. 2C). Consistent with intestinal shortening associated with increased damage [24]–[26], the length of the small intestine was reduced in the KO mice during infection (Fig. 2D). Increased damage was further confirmed by H&E staining of small intestinal sections. WT mice displayed minor villus blunting accompanied by moderate to severe inflammatory cell recruitment in the submucosa (Fig. 2E and G). In contrast, Cxcr3−/− mice displayed severe villus blunting, fusion, epithelial necrosis, sloughing of villus tips, and vascular congestion and hemorrhage (Fig. 2F and H). Blind scoring of H&E sections revealed a significant decrease in inflammation scores in the absence of CXCR3 (Fig. 2I), but when parameters of intestinal damage were quantitated, Cxcr3−/− mice scored significantly higher than WT counterparts (Fig. 2J). This damage was infection-dependent as intestines from non-infected WT and Cxcr3−/− mice both had normal architecture with few inflammatory cells (Fig. S3B). Increased epithelial damage in the absence of CXCR3 was further verified by loss of epithelial surface-associated Muc1 compared to infected WT animals, suggesting epithelial cell sloughing (Fig. S3C). Despite the overall decreased inflammatory score, Cxcr3−/− mice consistently displayed an influx of neutrophils into the LP compartment compared to WT mice, suggesting a role for these cells in causing damage, as argued by others [13], [14], [27] (Fig. S3D and E).


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)

Cxcr3−/− mice are susceptible to severe intestinal pathology following oral T. gondii infection.WT and CXCR3-deficient mice were orally inoculated with 30 ME49 cysts (A) or 50 cysts (B) of T. gondii and monitored for survival. In another set of experiments (C–J), mice were orally inoculated with 30 ME49 cysts, and tissues were collected at Day 10 post-infection. (C) Gross intestinal lesions in representative WT and CXCR3 KO mice. (D) Average lengths of noninfected (NI) and infected (INF) WT and Cxcr3−/− small intestines (NI WT, n = 5; NI KO, n = 3; INF WT, n = 8, INF KO: n = 7). E–H, H&E stained sections of small intestines from infected WT (E and G) and KO (F and H) mice. In panel G, the arrow points to an area of inflammatory cell influx. In panel H, the yellow arrow indicates an area of vascular congestion, and the red arrow indicates a necrotic villus. Blind scoring was performed on H&E stained intestine sections for inflammation (I) and damage (J) criteria (WT: n = 14; KO: n = 13; * p<0.05, ** p<0.01, *** p<0.001). Pooled data are represented as mean +/− SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795032&req=5

ppat-1003706-g002: Cxcr3−/− mice are susceptible to severe intestinal pathology following oral T. gondii infection.WT and CXCR3-deficient mice were orally inoculated with 30 ME49 cysts (A) or 50 cysts (B) of T. gondii and monitored for survival. In another set of experiments (C–J), mice were orally inoculated with 30 ME49 cysts, and tissues were collected at Day 10 post-infection. (C) Gross intestinal lesions in representative WT and CXCR3 KO mice. (D) Average lengths of noninfected (NI) and infected (INF) WT and Cxcr3−/− small intestines (NI WT, n = 5; NI KO, n = 3; INF WT, n = 8, INF KO: n = 7). E–H, H&E stained sections of small intestines from infected WT (E and G) and KO (F and H) mice. In panel G, the arrow points to an area of inflammatory cell influx. In panel H, the yellow arrow indicates an area of vascular congestion, and the red arrow indicates a necrotic villus. Blind scoring was performed on H&E stained intestine sections for inflammation (I) and damage (J) criteria (WT: n = 14; KO: n = 13; * p<0.05, ** p<0.01, *** p<0.001). Pooled data are represented as mean +/− SEM.
Mentions: To further examine the role of CXCR3 during T. gondii infection, mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts, and the outcome of infection was monitored. While all wild-type (WT) mice survived acute infection with 30 cysts, Cxcr3−/− animals displayed increased susceptibility with nearly 75% of mice dying by 2 weeks post-infection (Fig. 2A). When the cyst dose was increased to 50, all CXCR3 knockout (KO) mice rapidly succumbed to infection, but some WT mice also died (Fig. 2B). Interestingly, when WT and KO mice were infected by intraperitoneal injection, lack of CXCR3 did not affect survival, indicating that the effect of CXCR3 is specific to the mucosal response (Fig. S3A). To further examine the overall response in orally infected mice, we examined the gross appearance of the small intestine of WT and Cxcr3−/− mice after 30-cyst infection. The small intestines of the KO mice were strikingly damaged as demonstrated by massive hemorrhage compared to WT (Fig. 2C). Consistent with intestinal shortening associated with increased damage [24]–[26], the length of the small intestine was reduced in the KO mice during infection (Fig. 2D). Increased damage was further confirmed by H&E staining of small intestinal sections. WT mice displayed minor villus blunting accompanied by moderate to severe inflammatory cell recruitment in the submucosa (Fig. 2E and G). In contrast, Cxcr3−/− mice displayed severe villus blunting, fusion, epithelial necrosis, sloughing of villus tips, and vascular congestion and hemorrhage (Fig. 2F and H). Blind scoring of H&E sections revealed a significant decrease in inflammation scores in the absence of CXCR3 (Fig. 2I), but when parameters of intestinal damage were quantitated, Cxcr3−/− mice scored significantly higher than WT counterparts (Fig. 2J). This damage was infection-dependent as intestines from non-infected WT and Cxcr3−/− mice both had normal architecture with few inflammatory cells (Fig. S3B). Increased epithelial damage in the absence of CXCR3 was further verified by loss of epithelial surface-associated Muc1 compared to infected WT animals, suggesting epithelial cell sloughing (Fig. S3C). Despite the overall decreased inflammatory score, Cxcr3−/− mice consistently displayed an influx of neutrophils into the LP compartment compared to WT mice, suggesting a role for these cells in causing damage, as argued by others [13], [14], [27] (Fig. S3D and E).

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