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. 2016 Mar;99(3):475-82.
doi: 10.1189/jlb.4A0115-003RR. Epub 2015 Oct 14.

Lack of the Programmed death-1 Receptor Renders Host Susceptible to Enteric Microbial Infection Through Impairing the Production of the Mucosal Natural Killer Cell Effector Molecules

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Free PMC article

Lack of the Programmed death-1 Receptor Renders Host Susceptible to Enteric Microbial Infection Through Impairing the Production of the Mucosal Natural Killer Cell Effector Molecules

Shahram Solaymani-Mohammadi et al. J Leukoc Biol. .
Free PMC article

Abstract

The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.

Keywords: Citrobacter rodentium; PD-1; attaching/effacing bacteria; granzyme B; perforin.

Figures

Figure 1
Figure 1
NK cell accumulation in the colon and the intracellular production of GrzB and perforin by colonic NK cells are impaired in the absence of intact PD‐1. LP lymphocytes were isolated from sex‐ and age‐matched naïve WT and PD‐1−/− mice by enzymatic digestion. (A) Flow cytometric analysis of NK cells isolated from the colon LP of WT and PD‐1−/−. Data presented as mean and sem, n = 5 mice per group. Data are representative of 2 independent experiments. (B) Gating strategy for flow cytometry analysis of NK cells defined as CD3NK1.1+ cells. FSC and SSC represent forward and side scatter, respectively. (C) Intracellular expression of GrzB and perforin in WT (left) and PD‐1−/− (right) mice. Data are representative of 3 pooled independent experiments (n = 4–5 mice per experiment). (D) Flow cytometric analysis of NK1.1+GrzB+ and NK1.1+, perforin‐positive cells isolated from colon LP. Fluorescence minus one controls were used to set the gates. Data are mean and sem. *P < 0.05; **P < 0.01 (Mann‐Whitney U test). Numbers indicate percentage of cells in each gate.
Figure 2
Figure 2
C. rodentium infection in PD‐1−/− and anti‐PD‐1–treated mice. Sex‐ and age‐matched mice (n = 5–7 per group) were infected with 5 × 108 CFU of C. rodentium by oral gavage. Fecal samples were collected at the indicated times after infection, and C. rodentium CFU/g feces was determined in WT C57BL/6J and PD‐1−/− (A) and in WT mice (B) receiving either anti‐PD‐1 or isotype control antibody. Dotted lines represent the detection limit of the culture method. Data are representative of ≥3 independent experiments. Data are presented as mean ± sem. *P < 0.05; **P < 0.01 (Mann‐Whitney U test).
Figure 3
Figure 3
Systemic spread of intestinal infection with C. rodentium in PD‐1−/− and WT mice treated with anti‐PD‐1. (A) Bacterial translocation into liver, spleen, and MLNs of infected WT and PD‐1−/− mice were detected 3 d p.i (n = 4 mice per group). (B) Immunolocalization of C. rodentium in distal colon of WT and PD‐1−/− mice. Actin filaments stained green, C. rodentium stained red, nuclei stained blue. (C) Bacterial translocation into liver, spleen, and MLNs of infected WT mice receiving control rat IgG2a (rIgG2a) or rat IgG2a anti‐PD‐1 (α‐PD‐1) (n = 9 mice per group). (D) Immunolocalization of C. rodentium in distal colon of WT mice treated with control rIgG2a or α‐PD‐1. Similar data were obtained from 2 repeated experiments. Data are presented as mean ± sem. *P < 0.05; **P < 0.01 (Mann‐Whitney U test or Wilcoxon rank sum test).
Figure 4
Figure 4
PD‐1 expression in the colon increased after C. rodentium infection. (A) mRNA expression of PD‐1 in distal colon of WT mice infected with 5 × 108 CFU of C. rodentium was measured at d 0 and 3 p.i. using RT‐PCR. Data are presented as mean and sem (n = 3–7 mice per group). Data are representative of 2 independent experiments. (B) Sex‐ and age‐matched WT C57BL/6J mice (n = 4 per time point) were infected with 5 × 108 CFU of C. rodentium by oral gavage on d 0. MLNs and spleen were removed on d 0 and 3 p.i. PD‐1 expressed on conventional NK cells (i.e., NK1.1+CD3) from the MLNs and spleen was assessed by flow cytometry on d 0 and 3 p.i. Fluorescence minus one controls were used to set the gates. Numbers indicate percentage of NK1.1+PD‐1+ cells. **P < 0.01 (Mann‐Whitney U test). Data are representative of 2 independent experiments (n = 4 mice per experiment). Numbers indicate percentage of cells in each gate.
Figure 5
Figure 5
Increased C. rodentium infection in anti‐NK1.1–treated mice. Efficacy of the α‐NK1.1 antibody treatment in depleting NK cells in mice (A and B). Sex‐ and age‐matched WT mice (n = 7–8 per group) receiving depleting anti‐NK1.1 or mouse IgG2a control were infected with 5 × 108 CFU of C. rodentium by oral gavage on d 0. Fecal samples and spleens were collected 3 d p.i., and C. rodentium CFU/g feces and spleen (C) were determined. Dotted lines indicate the detection limit of the culture method. (C) Immunostaining of C. rodentium in distal colon of WT mice receiving an anti‐NK1.1 antibody or mouse IgG2a control. **P < 0.01; ***P < 0.001. Mann‐Whitney U test (C, left) or Wilcoxon rank sum test (C, right). Data are presented as mean ± sem.
Figure 6
Figure 6
GrzB‐ and perforin‐producing conventional NK cells in C. rodentium infected WT and PD‐1−/− mice. Single cells from the colon LP of WT and PD‐1−/− mice infected with 5 × 108 CFU of C. rodentium by oral gavage were isolated on d 0 and 3 p.i. and restimulated with PMA/ionomycin, as described previously. Flow cytometric analysis of intracellular expression of GrzB (A) and perforin (B) by conventional NK cells isolated from the colon LP of PD‐1−/− and WT controls. Data are representative of 2 pooled independent experiments (n = 4–8 mice per experiment). Fluorescence minus one controls were used to set the gates. Numbers indicate the percentage of cells in each gate.
Figure 7
Figure 7
Increased susceptibility to C. rodentium in perforin‐deficient (Prf−/−) mice. Sex‐ and age‐matched WT and Prf−/− mice (n = 5 per genotype) were infected with 5 × 108 CFU of C. rodentium by oral gavage on d 0. Fecal samples and spleens were collected 3 d p.i., and C. rodentium CFU/g feces and CFU/g spleen were determined (A). Dotted lines indicate the detection limit of the culture method. Data presented as mean ± sem. *P < 0.05; **P < 0.01 (Mann‐Whitney U test or Wilcoxon rank sum test). (B) Immunostaining of C. rodentium in distal colon of sex‐ and age‐matched WT and Prf−/− mice infected with 5 × 108 CFU of C. rodentium. Data are representative of 2 independent experiments.

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