Differential roles of CCL2 and CCR2 in host defense to coronavirus infection

Abstract

The CC chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1) is important in coordinating the immune response following microbial infection by regulating T cell polarization as well as leukocyte migration and accumulation within infected tissues. The present study examines the consequences of mouse hepatitis virus (MHV) infection in mice lacking CCL2 (CCL2(-/-)) in order to determine if signaling by this chemokine is relevant in host defense. Intracerebral infection of CCL2(-/-) mice with MHV did not result in increased morbidity or mortality as compared to either wild type or CCR2(-/-) mice and CCL2(-/-) mice cleared replicating virus from the brain. In contrast, CCR2(-/-) mice displayed an impaired ability to clear virus from the brain that was accompanied by a reduction in the numbers of antigen-specific T cells as compared to both CCL2(-/-) and wild-type mice. The paucity in T cell accumulation within the central nervous system (CNS) of MHV-infected CCR2(-/-) mice was not the result of either a deficiency in antigen-presenting cell (APC) accumulation within draining cervical lymph nodes (CLN) or the generation of virus-specific T cells within this compartment. A similar reduction in macrophage infiltration into the CNS was observed in both CCL2(-/-) and CCR2(-/-) mice when compared to wild-type mice, indicating that both CCL2 and CC chemokine receptor 2 (CCR2) contribute to macrophage migration and accumulation within the CNS following MHV infection. Together, these data demonstrate that CCR2, but not CCL2, is important in host defense following viral infection of the CNS, and CCR2 ligand(s), other than CCL2, participates in generating a protective response.

Fig. 1

Morbidity and mortality following MHV infection of the CNS. Wild type, CCL2^−/−, and CCR2^−/− mice (all on the C57BL/6 background) were infected ic with 10 PFU of MHV and disease severity recorded. CCR2^−/− mice exhibited an overall increase in the severity of clinical disease progression (A) as compared to wild type and CCL2^−/− mice, and this correlated with a more rapid onset and overall increase in mortality (B). Results presented were from five separate experiments. Wild-type mice, n = 54; CCR2^−/− mice, n = 28; CCL2^−/− mice, n = 29. Data were presented as the mean ± SEM. Clinical disease severity was significantly (*P ≤ 0.005) worse in CCR2^−/− mice when compared to wild type and CCL2^−/− mice.

Fig. 2

Characterization of T cell infiltration into the CNS of MHV-infected mice. Wild type, CCL2^−/−, and CCR2^−/− mice were infected ic with 10 PFU of MHV and T cell infiltration into the CNS determined at days 5 and 7 pi. In order to determine the frequency and numbers of virus-specific T cells present within the brains, mononuclear cells were surface stained for either CD4 or CD8 and IFN-γ (intracellular) expression evaluated following stimulation with either the defined CD4 epitope M133–147 or CD8 epitope S598–605. Total numbers of infiltrating CD4⁺ and CD8⁺ T cells are indicated in the left-hand y-axis while numbers of antigen-specific CD4⁺ and CD8⁺ T cells are indicated in right-hand y-axis (A and B, respectively). Data are presented as the mean ± SEM. The frequency of infiltrating CD4⁺ and CD8⁺ T cells present within the brains of MHV-infected mice is indicated in the left-hand y-axis while the frequency of antigen-specific CD4⁺ and CD8⁺ T cells is indicated in the right-hand y-axis (C and D, respectively). Data are presented as the average of frequencies. Results presented were from two separate experiments; n = 7 for MHV-infected wild type and CCL2^−/− mice, n = 4 for MHV-infected CCR2^−/− mice. *P ≤ 0.01 as compared to infected wild type at the corresponding time point. **P ≤ 0.005 to infected wild type at the corresponding time point and not significant as compared to that of CCL2^−/− mice. ***P ≤ 0.005 as compared to infected wild type at the corresponding time point and P ≤ 0.05 as compared to infected CCL2^−/− mice. ^aP ≤ 0.05 as compared to antigen-specific CD4⁺ T cells from infected wild type at day 7 pi. Not significant as compared to that of CCL2^−/− mice. ^bP ≤ 0.01 as compared to antigen-specific CD8⁺ T cells from infected wild type at day 7 pi. Not significant as compared to that of CCL2^−/− mice. ^cP ≤ 0.001 as compared to antigen-specific CD4⁺ T cells from infected wild type at day 7 pi. Not significant as compared to that of CCL2^−/− mice. ^dP ≤ 0.04 as compared to antigen-specific CD8⁺ T cells from infected wild-type mice at day 7 pi. P ≤ 0.05 as compared to antigen-specific CD8⁺ T cells from infected CCL2^−/− mice at day 7 pi.

Fig. 3

Macrophage infiltration into the CNS of MHV-infected mice. Wild type, CCL2^−/−, and CCR2^−/− mice were infected with 10 PFU of MHV and macrophage infiltration determined. Total cells were isolated from the brains of infected mice and sham (noninfected) mice at days 5 and 7 pi, and F4/80⁺CD45^high cells determined by flow cytometry using FITC-F4/80- and PE-CD45-conjugated antibodies. Numbers presented indicate the total number of dual-positive cells within the gated population. Data are presented as the mean ± SEM. Results presented were from two separate experiments; n = 7 for MHV-infected wild type and CCL2^−/− mice, n = 4 for MHV-infected CCR2^−/− mice. *P ≤ 0.004 as compared to infected wild type at day 5 pi.

Fig. 4

Expression of non-ELR CXC chemokine ligands 9 and 10. (A) Total RNA was isolated from the brains of MHV-infected mice at days 5 and 7 pi and subjected to RPA to assess transcript levels of CXCL9 and CXCL10. Sham (noninfected) mice were included as control. Each lane indicates an individual mouse at the indicated time pi. An L32 probe was included to verify consistency in RNA and assay performance. (B) Densitometric analysis of RPA autoradiography. Data are presented as normalized units representing the ratio of signal intensity of chemokine transcript to the internal L32 included in the probe set. Values were obtained from the scanned autoradiograph using NIH image software. Data represent the mean ± SEM.

Fig. 5

Characterization of inflammatory infiltrate in the CLN of MHV-infected mice. Percent CD11c⁺ cells within the CLN was determined by flow cytometry using a FITC-conjugated CD11c antibody. Data are presented as the mean ± SEM. Results presented were from four separate experiments; 3–6 mice were used from each group at an experimental time point.

Fig. 6

Analysis of MHV-specific T cells within the CLN of MHV-infected mice. CCR2^−/−, CCL2^−/−, and wild-type mice were infected ic with 10 PFU of MHV and CLN isolated at day 5 pi, and the T cell response to virus was determined. Harvested cells from experimental mice were pooled (three mice per group) and stained for either CD4 or CD8 (FITC conjugated), and IFN-γ (PE conjugated) expression evaluated following stimulation with the CD4 epitope M133–147 or CD8 epitope S598–605. The percent of responding T cells from MHV-infected mice is shown. Data are presented as the mean ± SEM and representative of three separate experiments.