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. 2014 Jun;88(12):6862-72.
doi: 10.1128/JVI.03364-13. Epub 2014 Apr 2.

CCR2 deficiency promotes exacerbated chronic erosive neutrophil-dominated chikungunya virus arthritis

Yee Suan Poo  1 , Helder Nakaya  2 , Joy Gardner  3 , Thibaut Larcher  4 , Wayne A Schroder  3 , Thuy T Le  3 , Lee D Major  3 , Andreas Suhrbier  5
Affiliations

CCR2 deficiency promotes exacerbated chronic erosive neutrophil-dominated chikungunya virus arthritis

Yee Suan Poo et al. J Virol. 2014 Jun.

Abstract

Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1β [IL-1β], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides.

Importance: Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.

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Figures

FIG 1
FIG 1
Disease, virus replication, and RNA persistence. (A) Mean percent increase in foot swelling over time for wild-type (WT) (n = 16 feet; 8 mice) and CCR2−/− (n = 14 feet; 7 mice) mice. Asterisks indicate significant differences (P < 0.01) by the t test or Kolmogorov-Smirnov test; the choice of test at each time point was dependent on the skewness, kurtosis, and variance of the data being compared (see Materials and Methods). (A single experiment, representative of 3 repeat experiments, is shown.) (B) Images of WT and CCR2−/− feet on day 0 and day 6 (peak arthritis). (C) Peripheral blood viremia (n = 7 for CCR2−/− mice; n = 8 to 14 for WT mice). (D) Virus titers in the indicated tissues at the indicated times postinfection (n = 3 to 6 mice per time point) (NT, not tested). (E) qRT-PCR analyses of CHIKV RNA in feet. Feet were taken at the indicated times (n = 3 to 6), and the level of CHIKV positive-strand RNA was determined by using qRT-PCR analyses and primers specific for E1 (a major CHIKV structural protein); data were normalized to RPL13A mRNA levels.
FIG 2
FIG 2
Histology and immunohistochemistry of rheumatic lesions in CHIKV-infected WT and CCR2−/− mice. (A to G) WT mice. (H to N) CCR2−/− mice. (A and H) H&E staining of muscle from uninfected mice. (B and I) H&E staining of muscle at day 6 postinfection. (C and J) High magnification of H&E staining of muscle at day 6 postinfection. (D and K) F4/80 antibody (monocyte/macrophage) staining of connective tissue at day 6 postinfection. (E and L) Leder staining (neutrophils) of connective tissue at day 6 postinfection. (F and M) ApoTag staining (apoptotic cells) at day 6 postinfection (two images for panel F and three images for panel M). (G and N) Chromotrope2R staining (eosinophils) of connective tissue at day 21 postinfection.
FIG 3
FIG 3
Histology quantitation. (A) Image analysis (strong brown) of F4/80 staining of whole foot sections from WT and CCR2−/− mice at the indicated times postinfection. The dotted line indicates the upper limit of nonspecific staining in control slides (n = 3 to 5 feet from different mice per time point and mouse strain; 2 sections per foot were analyzed). Statistics were determined by the Kolmogorov-Smirnov test. (B) Image analysis (strong red) of Leder staining of whole foot sections of WT and CCR2−/− mice at the indicated times postinfection (same numbers of feet as described above for panel A). Statistics were determined by Kolmogorov-Smirnov, Mann-Whitney U, or t tests. (C) Image analysis (strong brown) of ApoTag staining of whole foot sections of WT and CCR2−/− mice at the indicated times postinfection (same numbers of feet as described above for panel A). Statistics were determined by Kolmogorov-Smirnov tests. (D) Image analysis (strong red) of Chromotrope2R staining of whole foot sections of WT and CCR2−/− mice at the indicated times postinfection (same numbers of feet as described above for panel A). Statistics were determined by Kolmogorov-Smirnov or Mann-Whitney U tests. (E) WT mice were treated with the CCR2 antagonist MK0815 or diluent (control) after CHIKV infection, and feet were stained with anti-Ly6G (a neutrophil-specific marker), followed by image analysis. Statistics were determined by a Kolmogorov-Smirnov test (n = 4 to 6 feet per group).
FIG 4
FIG 4
Cartilage damage in CCR2−/− feet. (A) H&E staining of joint from a WT mouse on day 45 postinfection. SS, synovial space. (B) H&E staining of a joint from a CCR2−/− mouse on day 45 postinfection. Arrows indicate empty chondrocytic lacunae within the hyaline cartilage. Arrowheads show cartilage damage at the articular surface. (C) Safranin O staining (cartilage) of a joint from a WT mouse on day 45 postinfection. (D) Safranin O staining (cartilage) of a joint from a CCR2−/− mouse on day 45 postinfection. Arrows show a loss of red collagen staining (replaced by blue staining) at the articular surfaces.
FIG 5
FIG 5
Immune and inflammatory mediator responses. (A) CHIKV-specific IgG2c and IgG1 titers at 7 weeks postinfection as determined by ELISA. Statistics were determined by a Mann-Whitney U test (n = 5 mice per group, assayed in duplicate). (B) qRT-PCR analysis of T-bet (Th1 T cell transcription factor) and RORγT (Th17 transcription factor) at the indicated times postinfection. The key is the same as the one shown in panel A. Statistics were determined by Kolmogorov-Smirnov tests (n = 4 to 5 mice and feet per group and time point, assayed in duplicate). (C) qRT-PCR analysis of the indicated species at the indicated times postinfection. The key is the same as the one shown in panel A. Statistics were determined by Kolmogorov-Smirnov or t tests (n = 4 to 5 mice and feet per group and time point, assayed in duplicate).
FIG 6
FIG 6
Microarray analysis of arthritic feet from WT and CCR2−/− mice. (A) Genes differentially expressed on day 7 postinfection (compared to day 0) in WT and CCR2−/− feet were determined by microarray analysis. A total of 2,406 differentially expressed genes were up- or downregulated in WT and/or CCR2−/− feet (primary data available upon request). For 181 of these genes, the ratio of the fold change in CCR2−/− mice to the fold change in WT mice was >1.5 (top left) (for 603 DEGs, this ratio was >1). For 920 of these genes, the ratio of the fold change in WT mice to the fold change in CCR2−/− mice was >1.5 (bottom right) (for 1,794 genes, this ratio was >1, and for 9 genes, this ratio was 1). IPAs of the 181 genes more induced in CCR2−/− feet (top right) and the 920 genes less induced in CCR2−/− feet (bottom left) are shown, with pathways grouped into themes. Pathway details are shown in Tables S3 and S4 in the supplemental material. (B) Comparison of gene signatures of chikungunya virus (CHIKV) arthritis in WT and CCR2−/− mice with a gene signature from RA patients. Gene set enrichment analysis showed statistically significant enrichment of upregulated (UP) genes (163 genes in CCR2−/− mice and 341 genes in WT mice in “core enrichment”; overlap, 150) and downregulated (DOWN) genes (82 genes in CCR2−/− mice and 76 genes in WT mice in “core enrichment”) from CHIKV arthritis in the up- and downregulated genes, respectively, identified in synovial biopsy specimens from RA patients.
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