CCR2 Signaling Restricts SARS-CoV-2 Infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45⁺ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45⁺ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Keywords: SARS-CoV-2; innate immunity; lung inflammation; monocytes; mouse model.

FIG 1

MA-SARS-CoV-2 and B.1.351 replicate in the respiratory tract. C57BL/6J mice were infected intranasally with 5 × 10⁵ PFU of MA-SARS-CoV-2 or an equal volume of PBS for mock mice. (A) Percentage of initial weight for mock- and MA-SARS-CoV-2-infected mice over 8 days. (B) Quantification of MA-SARS-CoV-2 titers from lung tissue at the indicated day postinfection as measured by a plaque assay (left) or qRT-PCR (right). Threshold cycle (C_T) values are represented as relative fold changes over mock (log₁₀). (C) In situ hybridization was performed using a probe for MA-SARS-CoV-2 spike protein RNA. Representative images of lung slices from mock or day 4 p.i. are shown. (D) The fold change over mock for the indicated gene was plotted against the corresponding MA-SARS-CoV-2 RNA for each sample, and linear regression was used to determine correlation. (E) Quantification of viral titers from lung tissue by a plaque assay at day 2 p.i. from mice infected with MA-SARS-CoV-2 or human variant B.1.351 (5 × 10⁵ PFU/mouse). On the right is the quantification of the RNA-dependent RNA polymerase (RdRp) fold change over mock. (F) Gene expression measured via qRT-PCR for the indicated genes from lungs infected with MA-SARS-CoV-2 or B.1.351 at 2 days p.i. Results are representative of data from 2 independent experiments with 5 mice per group. Statistical significance was determined using unpaired Student’s t tests or linear regression. *, P < 0.05; ****, P < 0.0001.

FIG 2

MA-SARS-CoV-2 induces hyperinflammatory monocytes and macrophages in the lung. C57BL/6 mice were infected with MA-SARS-CoV-2, and lungs were harvested at days 0 and 4 p.i., processed to a single-cell suspension, captured in droplets on a 10× chromium controller, and analyzed via scRNA-Seq (n = 4 per group). (A) UMAP plot illustrating the different cellular subsets identified in the lung. (B) UMAP distribution of cells from mock- or MA-SARS-CoV-2-infected mice. On the right is the frequency of mock versus infected cells that make up each subset defined by UMAP analysis. (C) Feature plots displaying average expression in normalized read count (NRC) of the indicated gene from mock and infected lungs. (D) GSEA of inflammatory monocytes using the hallmark database from MSigDB for the indicated gene set. (E) Heat map analysis of top-scoring DEGs in alveolar macrophages from mock- or SARS-CoV-2-infected lungs. (F) GSEA plots of the indicated gene set from Liao et al. (6) in alveolar macrophages from mock and infected lungs.

FIG 3

Monocytes and monocyte-derived cells rapidly infiltrate the lung parenchyma in response to MA-SARS-CoV-2 infection. C57BL/6 mice were infected with MA-SARS-CoV-2, and lung tissue was harvested at 0, 2, and 4 days p.i. and analyzed via flow cytometry. (A) Five minutes prior to euthanization, mice were intravitally labeled with CD45:PE. Representative gating of in vivo-labeled CD45⁺ cells used to identify lung circulating (CD45⁺ in vivo) or lung parenchymal (CD45⁻ in vivo) cells is shown. The total number of CD45⁺ ex vivo cells is quantified on the right. (B) Counts of neutrophils (lineage negative CD11b⁺ Ly6G⁺) over the course of infection. (C) Counts of macrophages at days 0, 2, and 4 p.i. (lineage negative Ly6G⁻ CD64⁺ F4/80⁺). (D) Representative flow gating for alveolar (Siglec-F⁺ CD11c⁺), interstitial (Siglec-F⁻ CD11c⁻ Ly6C⁻), or transitional (Siglec-F⁻ CD11c⁻ Ly6C⁺) macrophages from day 4 p.i. Quantified on the right are the counts of each population. (E) Quantification of cDCs (lineage negative Ly6G⁻ CD64⁻ MHC-II⁺ CD11c⁺ CD26⁺) or moDCs (lineage negative Ly6G⁻ MHC-II⁺ CD11b⁺ CD11c⁺) at the indicated time points. (F) Total monocyte (lineage negative Ly6G⁻ MHC-II⁻ CD11c⁻ CD64 low) counts for circulating or lung parenchymal cells at days 0, 2, and 4 p.i. (G) Representative gating strategy demonstrating forward-scatter height (FSC-H) against Ly6C to identify different monocyte subsets (Ly6C high, Ly6C intermediate [Int], and Ly6C low), with quantification on the right. Results are representative of data from two independent experiments with 5 mice per group. Statistical significance was determined using unpaired one- or two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

Expansion of monocyte-derived cells in the lung during MA-SARS-CoV-2 infection is CCR2 dependent. C57BL/6 and Ccr2^−/− mice were infected with MA-SARS-CoV-2, and lung tissue was harvested at 0 and 4 days p.i. and analyzed via flow cytometry. Circulating (Circ.) versus parenchymal (Par.) cells were distinguished as described in the legend of Fig. 2. (A) Number of total monocytes in the lung circulation or parenchyma. (B) Representative gating identifying Ly6C-high, -intermediate (Int), or -low monocytes from WT and Ccr2^−/− lung parenchyma. Counts for each subset are quantified to the right. (C) Quantification of moDCs at day 4 p.i. (D) Total numbers of cDCs and parenchymal cDC subsets (right). (E) MFIs for CD86 (left) and MHC-I (right) expression on monocyte subsets from the lung parenchyma. (F) Quantification of the total macrophage numbers at day 4 p.i. (G) Representative flow plots illustrating interstitial or transitional macrophage populations from WT and Ccr2^−/− lung-infiltrating cells. Counts of macrophage subsets are quantified on the right. (H) Representative histograms of the MFIs for CD86 (left) and MHC-I (right) for each macrophage subset at 4 days p.i. from WT, Ccr2^−/−, or WT mock lung-infiltrating cells. Results are representative of data from two independent experiments with 5 mice per group. Statistical significance was determined using unpaired one- or two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

CCR2 restricts MA-SARS-CoV-2 burden and inflammatory cytokines in the lung. C57BL/6 or Ccr2^−/− mice were infected with MA-SARS-CoV-2, and lung tissue was collected at day 4 p.i. (A) Infectious virus at day 4 p.i. as quantified via plaque assays. (B) qRT-PCR for SARS-CoV-2 RdRp. (C) qRT-PCR was performed to probe for the indicated IFN signaling (left) or inflammatory (right) transcripts. (D) Representative images of in situ hybridization to visualize MA-SARS-CoV-2 RNA in lung tissue slices from 0 and 4 days p.i. in both WT and Ccr2^−/− mice. (E) WT or Ccr2^−/− mice were infected with B.1.351, and lungs were harvested at day 4 p.i. Virus was quantified via a plaque assay (top) or qRT-PCR (bottom). (F) WT and Ccr2^−/− mice were monitored over 12 days after infection with B.1.351 for survival (top) and weight loss (bottom). Results are representative of data from two independent experiments with 5 mice per group. Statistical significance was determined using unpaired Student’s t test, one-way ANOVA, or Kaplan-Meier survival curve analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.