Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis

Abstract

Invasive candidiasis is the 4(th) leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1(lo) to Ccr1(high) at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1(+/+) and Ccr1(-/-) donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1(+/+) recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1(+/+) cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.

Figure 1. Phagocyte-targeted chemokines and their receptors are induced in a mouse model of invasive candidiasis.

(A) Mortality of mice intravenously injected with Candida albicans is inoculum-dependent. The inocula tested in this study are given in the upper right hand corner of the figure as CFU. (B–D) Transcriptional profile of (B) cytokines, (C) chemokine receptors and (D) chemokines in a mouse model of invasive candidiasis. D1, day 1; D4, day 4; D7, day 7 post-infection. Color code is indicated to the right of each panel. Black color denotes either 0.5–2.0-fold change in gene expression over uninfected control mice, or a larger change that did not reach statistical significance. Inoculum, 2.5×10⁵ CFU. Data are from one experiment with three animals per time point. (E) Ccr1 and (F) its ligands Ccl3, Ccl5, Ccl6, Ccl7, Ccl8 and Ccl9 are induced after Candida infection, predominantly in the kidney. Inoculum, 1.25×10⁵ CFU. Data are from one experiment with four animals per time point.

Figure 2. Lack of Ccr1 improves survival without affecting tissue fungal burden in a mouse model of invasive candidiasis.

(A) Ccr1^−/− mice have 3-fold better survival compared to Ccr1^+/+ mice. P = 0.0008. Data are from three independent experiments with a total of thirty-two Ccr1^+/+ and thirty-three Ccr1^−/− mice. (B) Ccr1^−/− mice develop less severe weight loss compared to Ccr1^+/+ mice after Candida infection. ^* P<0.05. Data are from three independent experiments using twenty-four Ccr1^+/+ and twenty-one Ccr1^−/− mice. (C–F) Ccr1 deficiency does not affect tissue fungal burden in kidney (C), spleen (D), liver (E) or brain (F). Data are from four independent experiments using twenty-four Ccr1^+/+ and twenty-one Ccr1^−/− mice. Inoculum, 1.25×10⁵ CFU.

Figure 3. Ccr1 deficiency results in decreased kidney tissue damage.

(A) Representative gross pathology pictures of Ccr1^+/+ and Ccr1^−/− kidneys at day 9 post-infection. Ccr1^+/+ kidneys appear more swollen and pale compared to Ccr1^−/− kidneys. (B) Weight of Ccr1^−/− kidneys is significantly less compared to Ccr1^+/+ kidneys at day 9 post-infection. ^* P = 0.01. Data are from four independent experiments with twenty-four Ccr1^+/+ and twenty-one Ccr1^−/− mice. (C) Representative histopathology cross-sections of Ccr1^+/+ and Ccr1^−/− kidneys at day 9 post-infection. Ccr1^+/+ kidneys sustain significantly greater inflammatory changes compared to Ccr1^−/− kidneys. Original magnifications: 20× (top row) and 400× (bottom row). Images are representative of eight Ccr1^+/+ and eight Ccr1^−/− mice from three independent experiments. (D) 7/4 IHC staining of Ccr1^+/+ and Ccr1^−/− kidneys at day 9 post-infection. Original magnifications: 20× (top row) and 400× (bottom row). Images are representative of six mice from two independent experiments. (E–F) Kidney failure is significantly more severe in Ccr1^+/+ compared to Ccr1^−/− mice at day 9 post-infection as shown by (E) BUN and (F) creatinine measurements in mouse serum. ^* P<0.001. ^** P<0.01. Data are from two independent experiments using eight Ccr1^+/+ and eight Ccr1^−/− mice. (G) Relative mRNA expression of KIM-1 is greater in Ccr1^+/+ kidneys compared to Ccr1^−/− kidneys at day 9 post-infection. Data are from one experiment with four Ccr1^+/+ and four Ccr1^−/− mice per time point. ^* P = 0.01.

Figure 4. Ccr1 mediates neutrophil accumulation in the kidney at late time-points post-infection.

(A) Accumulation of CD45⁺ leukocytes in Ccr1^+/+ and Ccr1^−/− kidneys post-Candida infection. ^* P = 0.01. ^** P<0.01. (B) Representative FACS plots of Ly6c^intLy6G⁺CD11b⁺ neutrophils in Ccr1^+/+ and Ccr1^−/− kidneys at different time-points after Candida infection. (C) Accumulation of Ly6c^intLy6G⁺CD11b⁺ neutrophils is Ccr1-mediated late (days 9 and 12) but not early (days 3 and 6) post-infection. ^* P<0.01. ^** P = 0.01. (D) Percent of Ly6c^intLy6G⁺CD11b⁺ neutrophils in total CD45⁺ leukocytes accumulating in Ccr1^+/+ and Ccr1^−/− kidneys at different time-points after Candida infection. ^* P<0.01. ^** P = 0.02.

Figure 5. Ccr1 deficiency does not affect kidney neutrophil survival or efflux of neutrophils into the urine.

(A) Representative FACS plots of annexin V and 7-AAD staining in kidney neutrophils at day 9 post-infection. Percent of (B) live annexin V⁻ 7-AAD⁻, (C) apoptotic annexin V⁺ 7-AAD⁻, and (D) dead annexin V⁺ 7-AAD⁺ kidney neutrophils is similar in Ccr1^+/+ and Ccr1^−/− mice at days 6 and 9 post-infection. Data are shown from one of two independent experiments with similar pattern of results using a total of seven Ccr1^+/+ and seven Ccr1^−/− mice per time-point. (E) Accumulation of neutrophils in the urine of Ccr1^+/+ and Ccr1^−/− mice is similar at days 6 and 9 post-infection. Data are from two experiments with eight to ten Ccr1^+/+ and eight to ten Ccr1^−/− animals per time-point.

Figure 6. Ccr1 expression increases late after Candida infection in blood and kidney neutrophils.

(A) Representative FACS histograms of Ccr1 staining on blood neutrophils at days 3 and 9 post-infection. (B) Ccr1 expression on blood neutrophils is significantly increased at day 9 compared to days 3 and 6 post-infection. ^* P<0.01. ^** P = 0.03. Data are from two independent experiments using four to ten Ccr1^+/+ mice per time-point. (C) Relative Ccr1 mRNA expression in MACS-sorted kidney neutrophils increases late post-infection. Data are from two independent experiments using six to eight Ccr1^+/+ mice per time-point. ^* P = 0.02, ^** P = 0.003 (D) Representative FACS histograms of Ccr1 staining on kidney neutrophils at days 3 and 9 post-infection. (E) Ccr1 expression on kidney neutrophils is significantly increased at days 6 and 9 compared to day 3 post-infection. ^* P<0.0001. Data are from three independent experiments using six to eight Ccr1^+/+ mice per time-point.

Figure 7. Ccr1 ligands are induced after Candida infection and are chemotactic for kidney neutrophils ex vivo.

(A) Relative mRNA expression of Ccl3, Ccl5, Ccl6, Ccl7, Ccl8 and Ccl9 in Ccr1^+/+ and Ccr1^−/− kidneys after infection. Data are shown from one of two independent experiments with similar pattern of results using a total of seven to nine Ccr1^+/+ and seven to eight Ccr1^−/− mice per time-point. ^* P = 0.03 (B) Protein levels of CCL3, CCL5, CCL6, CCL7, CCL8 and CCL9 in Ccr1^+/+ and Ccr1^−/− kidneys after infection. Data are from one experiment using four Ccr1^+/+ and four Ccr1^−/− mice per time-point. (C) Chemotaxis of MACS-sorted kidney neutrophils recovered from Ccr1^+/+ mice at day 9 post-infection to CCL3, CCL5, CCL6, CCL7, CCL8 and CCL9. Data are from one experiment using twelve pooled kidneys from six Ccr1^+/+ mice. (D) Relative mRNA expression of Ccl3, Ccl6 and Ccl9 in MACS-sorted Ccr1^+/+ kidney neutrophils after Candida infection. Data are shown from one of two independent experiments with similar pattern of results using a total of six to eight Ccr1^+/+ mice per time-point. Data on relative mRNA expression of the ligands in whole kidney homogenates (right panel) are excerpted from Figure 7A for comparison.

Figure 8. Ccr1 mediates neutrophil trafficking from the blood to the kidney post-infection.

Ly6c^intLy6G⁺CD11b⁺ neutrophils from Ccr1^+/+ and Ccr1^−/− mice were differentially-labeled, mixed at a 1∶1 ratio, and analyzed prior to injection of Ccr1^+/+ recipient mice (A and B, left panels). The mixed donor cells were then injected into Ccr1^+/+ recipients that had been infected 9 days earlier with Candida. The distribution of labeled Ccr1^+/+ and Ccr1^−/− donor neutrophils in Candida-infected Ccr1^+/+ recipient kidneys four hours post-injection is represented as the ratio of Ccr1^+/+ to Ccr1^−/− neutrophils in the kidney (A and B, right panels). (C) Labeling of neutrophils does not differentially affect cell survival. Similar percentages of differentially labeled Ccr1^+/+ and Ccr1^−/− neutrophils are dead in the kidney four hours after injection. Data are from one experiment using sixteen Ccr1^+/+ recipient mice.

Figure 9. Ccr1 deficiency does not decrease the expression of other neutrophil-targeted chemotactic factors or adhesion molecules in Candida-infected kidneys.

(A) Relative mRNA expression of neutrophil-targeted chemoattractant receptors in Ccr1^+/+ and Ccr1^−/− kidneys at day 9 after infection. (B) Relative mRNA expression of neutrophil-targeted ELR CXC chemokines in Ccr1^+/+ and Ccr1^−/− kidneys at day 9 after infection. (C) Relative mRNA expression of neutrophil-targeted adhesion molecules in Ccr1^+/+ and Ccr1^−/− kidneys at day 9 after infection. Data are from two independent experiments using nine Ccr1^+/+ and eight Ccr1^−/− mice.