key: cord-0801213-iyr86kkk
authors: Vanderheiden, Abigail; Thomas, Jeronay; Soung, Allison L.; Davis-Gardner, Meredith E.; Floyd, Katharine; Jin, Fengzhi; Cowan, David A.; Pellegrini, Kathryn; Creanga, Adrian; Pegu, Amarendra; Derrien-Colemyn, Alexandrine; Shi, Pei-Yong; Grakoui, Arash; Klein, Robyn S.; Bosinger, Steven E.; Kohlmeier, Jacob E.; Menachery, Vineet D.; Suthar, Mehul S.
title: CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection
date: 2021-05-04
journal: bioRxiv
DOI: 10.1101/2021.05.03.442538
sha: 3715fdbf51bc8048e9be4c3fff5ec962c2472e60
doc_id: 801213
cord_uid: iyr86kkk

SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests 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 viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. 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. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and 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 CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Granulocyte numbers were elevated in circulation at 2 and 4 days p.i. and infiltrated into the lung 142 by day 2 p.i., with a 100-fold increase in parenchymal neutrophils. (Fig. 3B, Supplemental Fig.  143 3A). Circulating macrophage numbers were unchanged by infection, however beginning at day 2 p.i., parenchymal macrophage numbers decreased as compared to mock-infected mice (Fig. 3C) . 145

This downward trend appeared to be due to a sequential loss of alveolar macrophages at day 2 146 and 4 p.i. (Siglec-F+ CD11c+), while interstitial macrophages (CD11c-, SiglecF-, Ly6C-) were 147 unaffected ( Fig. 3D ). At day 4 p.i., we observed a 100-fold increase in cells that expressed both 148 macrophage markers, CD64 and F4/80, and monocyte markers, Ly6C and CD11b, which we 149 designated 'transitional macrophages' (Fig. 3D ). All macrophages upregulated MHC-I in response 150 to MA-SARS-CoV-2, although the effect was more pronounced in transitional macrophages, 151 which also upregulated CD86 (Supplemental Fig. 3B -C). Together these data identify a shift in 152 the lung macrophage composition, with decreased numbers of alveolar macrophages and 153 increased numbers of activated transitional macrophages during MA-SARS-CoV-2 infection. 154

We next examined the role of dendritic cells during MA-SARS-CoV-2 infection. Plasmacytoid 156 dendritic cell (pDC) numbers did not change in the circulation or parenchyma at 2-4 days p.i. 157 (Supplemental Fig. 3D ). Conventional dendritic cell (cDC) populations remained steady in 158 circulation, but increased 10-fold in the lung parenchyma at day 4 p.i. that was primarily due to an 159 increase in cDC Type 2 cells (cDC2s) (Fig. 3E, Supplemental Fig. 3E ). Lung parenchymal cDCs 160 increased expression of MHC-I and CD86 in response to MA-SARS-CoV-2 at day 4 p.i 161 (Supplemental Fig. 3F ). moDCs had slightly increased numbers in circulation and a 10-fold 162 increase in lung parenchymal populations at day 4 p.i. (Fig. 3E ). Parenchymal moDCs also 163 upregulated expression of MHC-I and CD86 at day 4 p.i. as compared to uninfected controls 164 (Supplemental Fig. 3G ). Thus, cDCs and moDCs undergo expansion and activation in response 165 to MA-SARS-CoV-2 infection in the lung. 166

Investigation of monocyte dynamics during MA-SARS-CoV-2 infection observed a 5-fold increase 168 in circulating monocytes and a 20-fold increase in lung parenchymal monocytes by day 2 p.i. that 169 remained high through day 4 p.i. (Fig. 3F ). Ly6C high monocytes drove monocytic infiltration to 170 the lung, as their numbers increased 100-fold at days 2 and 4 p.i., but the numbers of Ly6C low 171 monocytes remained constant (Fig. 3G) in Ccr2 -/mice at day 4 p.i. (Fig. 4C ). cDC numbers in circulation were similar between WT and 191

Ccr2 -/mice at day 4 p.i., but lung parenchymal cDC numbers at day 4 p.i. were 5-fold lower than inflammatory cytokine levels and disease severity during COVID-19 6 . In accordance, we found 237 that SARS-CoV-2 infected mice had a pro-inflammatory cytokine profile in the lung containing 238 pyrogens (Il6, Tnf), chemoattractants for monocytes (Ccl2) and T cells (Cxcl10), ISGs (Irf7 and  239 Isg15), alarmins (S100a8), and matrix metalloproteinases (Mmp14) 6,20-23 . Interestingly, inflammatory gene expression in the lung was MA-SARS-CoV-2 viral load dependent, as most 241 cytokine transcripts surveyed by qPCR positively correlated with viral RNA. Similar phenomenon 242 have been noted in human subjects, with one study describing an association between SARS-243

CoV-2 viral burden, IL-6 levels, and increased risk of death 24 . Similar to studies of post-mortem 244 lung tissue or BALs from patients suffering from COVID-19, we observed a significant increase in 245 the numbers of S100a8+ granulocytes in the lung parenchyma 4,6-8 . S100a8 CD86 expression on parenchymal moDCs at day 0 and 4 p.i., with the quantification on the right. 408

Representative histograms for H) MHC-I or I) CD86 expression on lung parenchymal monocyte 409 subsets and the corresponding quantification on the right. Results are representative of two 410 independent experiments. Statistical significance was determined using unpaired one or two-way 411 ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. 412 413 Log-normalized and scaled gene expression data. Clustering was conducted using 555

FindNeighbors and FindClusters functions, with resolution parameters between 0.5-1.4. Overall, 556 23 clusters were identified and FindAllMarkers function utilized to identify DEGs, from which 557 marker genes for cluster cell annotation was conducted. After annotating cells, DEGs were 558 determined based on sub-clusters or experimental group. Gene set enrichment analysis (GSEA) 559 was conducted using ranked gene list produced with Seurat FindMarkers function (comparing 560

CoV2 samples with mock samples) and Genelist were obtained from: MsigDB (hallmarks) and 561 https://www.nature.com/articles/s41422-020-00455-9#MOESM1 (macrophage suppressive and 562 hyperinflammatory). GSEA was conducted using Broad (4.0.3) and plotted in R. 563 564

Single cell RNA sequencing data will be publicly accessible through Gene Expression Omnibus 566 following acceptance of this article. 567

A pneumonia outbreak associated with a new coronavirus of probable bat 593 origin

A Novel Coronavirus from Patients with Pneumonia in China

MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung 597

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Type I and Type III Interferons Restrict SARS-CoV-2 Infection of 602

Single-cell landscape of bronchoalveolar immune cells in patients with 605

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Longitudinal profiling of respiratory and systemic immune responses 610 reveals myeloid cell-driven lung inflammation in severe COVID-19

Blood monocytes consist of two principal 613 subsets with distinct migratory properties

The Inflammatory versus Constitutive Trafficking of Mononuclear 616

Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their 617

CCR2+ monocyte-620 derived dendritic cells and exudate macrophages produce influenza-induced pulmonary 621 immune pathology and mortality

Type I interferon signaling regulates Ly6C(hi) monocytes and 624 neutrophils during acute viral pneumonia in mice

Rapid differentiation of monocytes into type I IFN-producing myeloid 627 dendritic cells as an antiviral strategy against influenza virus infection

An Infectious cDNA Clone of SARS-CoV-2

Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in 635 the Respiratory Tract

Detection of a SARS-CoV-2 variant of concern in South Africa

Induction of alarmin S100A8/A9 mediates activation of aberrant 639 neutrophils in the pathogenesis of COVID-19

Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone 642 marrow and recruitment to inflammatory sites

COVID-19 severity correlates with airway epithelium-immune cell 645 interactions identified by single-cell analysis

Severe COVID-19 Is Marked by a Dysregulated Myeloid 648

Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate 650

Severe from Mild COVID-19

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SARS-CoV-2 viral load is associated with increased disease severity 656 and mortality

Chemokine receptor 2 serves an early and essential role in resistance 659 to Mycobacterium tuberculosis

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(http://www.yerkes.emory.edu/nhp_genomics_core/) . Analysis was conducted using R (v4) and 548 Seurat (v4). Cell Ranger (v6) was used for demultiplexing, aligning barcodes, mapping to the 549 genome (mm10) and quantifying UMIs. Filtered Cell Ranger matrices were processed with 550Read10x function in Seurat for preprocessing and cluster analysis. Data was filtered to remove 551 cells with less than 200 genes, abnormally high gene counts (feature counts >5000) and greater 552 than 5% mitochondrial genes. After qualify control, there were 9,399 mock cells and 10,982