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. 2013 Jan;6(1):177-88.
doi: 10.1038/mi.2012.61. Epub 2012 Aug 1.

CX(3)CR1(+) macrophages support IL-22 production by innate lymphoid cells during infection with Citrobacter rodentium

C Manta  1 , E Heupel, K Radulovic, V Rossini, N Garbi, C U Riedel, J H Niess
Affiliations
Free PMC article

CX(3)CR1(+) macrophages support IL-22 production by innate lymphoid cells during infection with Citrobacter rodentium

C Manta et al. Mucosal Immunol. 2013 Jan.
Free PMC article

Abstract

Innate immune cells, such as intestinal epithelial cells, dendritic cells (DCs), macrophages, granulocytes, and innate lymphoid cells provide a first line of defence to enteric pathogens. To study the role of CX(3)CR1(+) DCs and macrophages in host defence, we infected CX(3)CR1-GFP animals with Citrobacter rodentium. When transgenic CX(3)CR1-GFP animals are infected with the natural mouse pathogen C. rodentium, CX(3)CR1(-/-) animals showed a delayed clearance of C. rodentium as compared with (age- and sex-matched) wild-type B6 animals. The delayed clearance of C. rodentium is associated with reduced interleukin (IL)-22 expression. In C. rodentium-infected CX(3)CR1-GFP animals, IL-22 producing lymphoid-tissue inducer cells (LTi cells) were selectively reduced in the absence of CX(3)CR1. The reduced IL-22 expression correlates with decreased expression of the antimicrobial peptides RegIIIβ and RegIIIγ. The depletion of CX(3)CR1(+) cells by diphtheria toxin injection in CX(3)CR1-GFP × CD11c.DOG animals confirmed the role of CX(3)CR1(+) phagocytes in establishing IL-22 production, supporting the clearance of a C. rodentium infection.

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Figures

Figure 1
Figure 1
Delayed clearance of Citrobacter rodentium in CX3CR1-GFP animals. (a) C. rodentium counts in fecal samples from wt B6 animals and (age- and sex-matched) heterozygous and homozygous CX3CR1-GFP animals were determined by collecting fecal pellets from each animal every 2–3 days over the course of the infection. Pellets were weighted and resuspended in 1 ml of phosphate-buffered saline (PBS), plated in serial dilutions, and bacterial load was calculated as cfu g−1 feces. P-values were calculated with a nonparametric Student's t-test; P<0.05 was considered statistically significant. (b) Histopathological scores of colon sections taken from control or infected B6, CX3CR1GFP/+, and CX3CR1GFP/GFP. In the nonparametric Student's t-test, P<0.05 was considered statistically significant. (c) At the end of the experiment, colons were removed and representative colons of the indicated groups are shown. (d) The colon weight and length was determined and expressed as colon weight/length ratios. In the nonparametric Student's t-test, P<0.05 was considered statistically significant. (e) Colony-forming units (CFU) from plates spotted with homogenates from liver, spleen, and mesenteric lymph nodes (MLN) of the indicated groups were determined. The numbers of animals per group of each experiment is given within the figure. In the nonparametric Student's t-test, P<0.05 was considered statistically significant.
Figure 2
Figure 2
Citrobacter rodentium can be located in CX3CR1+ macrophages. (a) Homozygous CX3CR1-GFP animals were infected with C. rodentium mutants expressing the red fluorescent protein mRuby. Twelve days post infection, living intestinal tissues from the proximal colon was analyzed by ex vivo microscopy. (b) CX3CR1+CD11c+ cells were isolated from homozygous CX3CR1-GFP animals infected with the C. rodentium mutant ICC 169 expressing the red fluorescent protein mRuby. Colonic lamina propria isolates were stained for CD11c and analyzed by flow cytometry. Histograms were obtained by gating on CX3CR1-GFP+CD11c+ cells. Gray areas represent isolates obtained from animals infected with C. rodentium ICC 169; open histograms represent isolates obtained from animals infected with mRuby expressing C. rodentium mutants. (c) CX3CR1+ cells were defined as area of interest and scatter diagrams obtained. The percentage of C. rodentium located in CX3CR1+ macrophages was determined. (d) Mean (±s.e.m.) percentage of C. rodentium located in CX3CR1+ macrophages from the indicated mice is shown. P-values were calculated with a nonparametric Student's t-test; P<0.05 was considered statistically significant.
Figure 3
Figure 3
Interleukin (IL)-22 expression is reduced in Citrobacter rodentium-infected CX3CR1GFP/GFP animals. (a) From the proximal colon of noninfected and C. rodentium-infected CX3CR1GFP/GFP and wild-type littermate controls mRNA was isolated. cDNA was prepared by reverse transcription and quantitative real-time PCR was performed with cyber green and the indicated primers. β-actin was used as a housekeeping gene to normalize cDNA input between samples. Normalized ct-values of the untreated samples (baseline) are set to 1 and values are plotted as fold expression of the baseline. The assays were carried out in triplicates. As per indicated, group 4 animals were analyzed. (b) IL-22 expression by colonic lamina propria (cLP) cell isolates from noninfected and C. rodentium-infected B6, (age- and sex-matched) and CX3CR1GFP/+ and CX3CR1GFP/GFP animals was analyzed by multicolor flow cytometry. Numbers indicate the percentage of IL-22-positive cells. (c) cLP IL22+ cells from noninfected and C. rodentium-infected B6, (age- and sex-matched), and CX3CR1GFP/+ and CX3CR1GFP/GFP animals were stained for CD3ɛ and CD4. Numbers indicate the percentage of CD3ɛ+, CD3ɛ+CD4+, CD4+, and CD3ɛ−CD4− cells within the IL-22+ cell population. Data from an individual representative mouse per group (of five to seven individual mice analyzed per group) are shown. (d) cLP IL-22+ CD3ɛ−CD4+ cells were (intracellular) stained for related orphan receptor gamma-t (RORγt) or surface stained for CD25, CD127, and CD117, and analyzed by multi-color flow cytometry. Opened curves represent the respective negative controls. Seven mice were analyzed and the data from a representative individual mouse are presented. Numbers represent the percentage of cells that are positive for the indicated antigen. (e) Mean (±s.e.m.) percentage of IL-22+ cells isolated from the cLP of C. rodentium-infected B6, (age- and sex-matched) and CX3CR1GFP/+ and CX3CR1GFP/GFP animals is shown. In the nonparametric Student's t-test, P<0.05 was considered statistically significant (*P<0.05). (f) Mean (±s.e.m.) percentage of CD3ɛ−CD4+ lymphoid-tissue inducer cells within the IL-22+ cells from cLP isolates of B6, (age- and sex-matched) and heterozygous and homozygous CX3CR1-GFP animals is shown. P-values were calculated with a nonparametric Student's t-test; P<0.05 was considered statistically significant. (g) Total RNA isolated from the colonic tissues of noninfected and C. rodentium-infected B6, (age- and sex-matched), and CX3CR1GFP/+ and CX3CR1GFP/GFP animals were reverse transcribed to cDNA and RegIIIγ, and (h) RegIIIβ expression was analyzed by qRT-PCR. The number of animals per group of each experiment is given within the figure. In the nonparametric Student's t-test, P<0.05 was considered statistically significant.
Figure 4
Figure 4
Recombination-activating gene (RAG)−/− mice lacking CX3CR1 develop a rapid Citrobacter rodentium infection. (a) RAG−/− × CX3CR1GFP/GFP and RAG−/− mice were infected with 2 × 109 C. rodentium. Mean±s.e.m. loss of body weight (%) per group is shown for the indicated animals. P-values were calculated with a nonparametric Student's t-test; P<0.05 was considered statistically significant. (b) Survival of RAG−/− × CX3CR1GFP/GFP and RAG−/− infected with 2 × 109 C. rodentium is shown. (c) Colons were removed at the end of the experiment and representative colons of the indicated groups are shown. (d) The colon weight and length was determined and expressed as colon weight/length ratios. In the nonparametric Student's t-test, P<0.05 was considered statistically significant. (e) Interleukin (IL)-22 expression in the proximal colon of infected RAG−/− and RAG−/− × CX3CR1GFP/GFP animals was analyzed by quantitative real-time PCR. β-actin was used as housekeeping gene to normalize cDNA input between samples. Normalized ct-values of the noninfected samples (baseline) are set to 1 and values are plotted as fold expression of the baseline (f) IL-22 expression by colonic lamina propria (cLP) cell isolates from C. rodentium-infected RAG−/−, (age- and sex-matched) and RAG−/− × CX3CR1GFP/GFP animals was analyzed by multicolor flow cytometry. Numbers indicate the percentage of IL-22-positive cells. (g) Mean (±s.e.m.) percentage of IL-22+ cells isolated from the cLP of C. rodentium-infected B6, (age- and sex-matched), and CX3CR1GFP/+ and CX3CR1GFP/GFP animals is shown. In the nonparametric Student's t-test, P<0.05 was considered statistically significant (*P<0.05).
Figure 5
Figure 5
The depletion of CX3CR1+CD11c+ macrophages results in an accelerated infection. (a) CX3CR1-GFP animals were crossed with CD11c.DOG animals to obtain CX3CR1-GFP × CD11c.DOG mice. CX3CR1-GFP × CD11c.DOG mice were intraperitoneally injected with 30 ng bwg−1 diphtheria toxin (DT) every third day. Isolates were obtained from spleen, mesenteric lymph nodes (MLN), and colonic lamina propria (cLP), surface stained for CD11c, and analyzed by flow cytometry at the indicated time points. The numbers indicate the percentage of cells located in the respective area of the dot blots. (b) Colons were removed from nontreated and DT-treated CX3CR1-GFP × CD11c.DOG animals, fixed in 4% paraformaldehyde sectioned on a microtome, mounted on slides, and analyzed by fluorescence microscopy Representative images from one individual mouse per group (from five individual mice per group analyzed) are shown. (c) Nontreated CD11c.DOG, DT-treated CD11c.DOG, CX3CR1GFP/+ × CD11c.DOG, and CX3CR1GFP/GFP × CD11c.DOG were infected with 2 × 109 Citrobacter rodentium. Mean±s.e.m. loss of body weight (%) per group is shown for the indicated groups. In the nonparametric Student's t-test, P<0.05 was considered statistically significant (*P<0.05). (d) Survival of nontreated CD11c.DOG, DT-treated CD11c.DOG, CX3CR1GFP/+ × CD11c.DOG, and CX3CR1GFP/GFP × CD11c.DOG infected with 2 × 109 C. rodentium is shown. At day 18 post infection, cLP cell isolates were obtained, stained for intracellular interleukin (IL)-22, and analyzed by flow cytometry. Numbers indicated the percentages of cells in the respective quadrants of the dot blots. (e) IL-22+ cLP isolates from DT-treated CX3CR1-GFP × CD11c.DOG animals were stained for CD3ɛ and CD4 at day 18 post infection and analyzed by multicolor flow cytometry. Numbers indicate the percentage of CD3ɛ+, CD3ɛ+CD4+, CD4+, and CD3ɛ−CD4− cells within the IL-22+ cell population. (f) Mean (±s.e.m.) percentage of IL-22+ cells isolated from the cLP of nontreated CD11c.DOG and DT-treated (age- and sex-matched) CX3CR1GFP/+ × CD11c.DOG and CX3CR1GFP/GFP × CD11c.DOG animals is shown. In the nonparametric Student's t-test, P<0.05 was considered statistically significant. (g) Mean (±s.e.m.) percentage of CD3ɛ−CD4+ lymphoid-tissue inducer cells within the IL22+ cells from cLP isolates obtained from nontreated CD11c.DOG and DT-treated (age- and sex-matched) CX3CR1GFP/+ × CD11c.DOG and CX3CR1GFP/GFP × CD11c.DOG animals is shown at day 18 post infection. P-values were calculated with a nonparametric Student's t-test; P<0.05 was considered statistically significant. (h) Mean±s.e.m. of colony forming units (CFUs) from plates spotted with homogenates from liver, spleen, and MLN of the indicated groups is presented. In the nonparametric Student's t-test, P<0.05 was considered statistically significant.
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