key: cord-1055372-v4vbtpkq
authors: Upadhyay, Amit A.; Hoang, Timothy N.; Pino, Maria; Boddapati, Arun K.; Viox, Elise G.; Lee, Michelle Y.H.; Corry, Jacqueline; Strongin, Zachary; Cowan, David A.; Beagle, Elizabeth N.; Horton, Tristan R.; Hamilton, Sydney; Aoued, Hadj; Harper, Justin L.; Nguyen, Kevin; Pellegrini, Kathryn L.; Tharp, Gregory K.; Piantadosi, Anne; Levit, Rebecca D.; Amara, Rama R.; Barratt-Boyes, Simon M.; Ribeiro, Susan P.; Sekaly, Rafick P.; Vanderford, Thomas H.; Schinazi, Raymond F.; Paiardini, Mirko; Bosinger, Steven E.
title: TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques
date: 2021-10-05
journal: bioRxiv
DOI: 10.1101/2021.10.05.463212
sha: ef35aa4c6e9ddccc43882781d8e6662a4300671b
doc_id: 1055372
cord_uid: v4vbtpkq

The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1-population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant®), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection. One sentence summary Multi-omic analyses of hyperacute SARS-CoV-2 infection in rhesus macaques identified two population of infiltrating macrophages, as the primary orchestrators of inflammation in the lower airway that can be successfully treated with baricitinib

The COVID-19 pandemic began with a series of reports of localized outbreaks of pneumonia caused by a novel coronavirus, SARS-CoV-2, in Wuhan, China in December 2019 1, 2 . As of late 2021, there have been over 200,000,000 documented infections, and over 4,500,000 fatalities attributed to sequelae of COVID-19. The rapid development and availability of effective vaccines [3] [4] [5] against SARS-CoV-2 infection has provided much needed optimism that infection rates will decline and that the containment of the virus at the population level is possible. Despite these landmark achievements, continued research efforts are essential to safeguard against potential breakthrough variants, to develop therapies for those afflicted while the vaccine rollout continues, and to prevent or minimize the impact of future viral outbreaks. In this light, basic research into the innate and adaptive immune responses to SARS-CoV-2 continues to be critical for informing vaccine and therapeutic approaches directed at ending the COVID-19 pandemic or at decreasing mortality.

Since the emergence of the COVID-19 pandemic, research into the virology, immune responses and pathogenesis of SARS-CoV-2 infection has amassed at an unprecedented rate, and numerous hypotheses have arisen to explain the underlying mechanisms of severe COVID-19. Of these, the concepts that have accumulated the most supporting evidence are: (i) evasion or impairment of early Type I interferon (IFN) responses 6 , (ii) vascular complications arising from hypercoagulability syndromes 7 , and (iii) perturbations of the granulocyte and myeloid compartments in the lower airway and blood manifesting in inflammatory cytokine production 8,9 . Immunologically, severe disease in COVID-19 patients has been associated with a widespread increase in levels of inflammatory mediators (e.g. CXCL10, IL-6, and TNFα) in plasma and bronchoalveolar lavage (BAL) fluid in what is commonly referred to as a "cytokine storm" 10 , and an expansion of macrophages, neutrophils and lymphocytes in the lower airway 8 . Despite this impressive accruement of data, the precise early immunological events and immune cell infiltration that drive inflammation in the lower airway remain uncharacterized.

African green monkeys (AGMs)) have proven to be critical tools, primarily due to the ability to examine early events after infection longitudinally and in tissues not available in most human studies 11 . NHPs support high levels of viral replication in the upper and lower airway [12] [13] [14] , share tissue distribution of ACE2 and TMPRSS2 with humans 15 , and have been invaluable pre-clinical models of vaccines [16] [17] [18] and therapeutics 19, 20 . Additionally, mild to moderate COVID-19 has been shown to be recapitulated in SARS-CoV-2-infected NHPs 11 that typically resolve by 10-15 days post infection (dpi) 11, 20, 21 . Mechanistic studies of SARS-CoV-2 infection in NHPs have utilized a variety of high-throughput techniques and have reported (i) Type I IFN responses are robustly induced in blood and the lower airway very early after infection 20, 22 , (ii) elevated proinflammatory cytokines consistent with the "cytokine storm" seen in humans are detectable in plasma and BAL 23 , (iii) vascular pathology and gene expression consistent with hypercoagulability are evident in the lower airways 22 , and (iv) increased production of inflammatory cytokines by myeloid origin cells 20, 24 .

In the current study, we used SARS-CoV-2 infected rhesus macaque (RM) to investigate the early inflammatory events occurring in the blood and lower airway using high dimensional flow cytometry, multi-analyte cytokine detection, and bulk and single-cell RNA-Seq (sc-RNA-Seq).

To dissect the role of discrete immune subsets within the myeloid fraction in SARS-CoV-2driven inflammation, we used two different strategies, employing scRNA-Seq and bulk-RNA-Seq reference datasets to classify the macrophage/monocyte population. With this approach, we identified the main subsets of pro-inflammatory macrophages that expand after SARS-CoV2 infection and are the predominant source of inflammatory cytokines in the airway. We also observed an early induction of plasmacytoid dendritic cells (pDCs) in blood and the lower airway that coincided with the peak of the IFN signaling. Finally, we described that treatment of SARS-CoV-2 infected RMs with baricitinib, a JAK1/2 inhibitor recently demonstrated to reduce hospitalization time and mortality for severe COVID-19 patients 25 , suppressed airway inflammation by abrogating the infiltration of pro-inflammatory macrophages to the alveolar space. Collectively, this study defines the early kinetics of pDC recruitment and Type I IFN responses, and identifies discrete subsets of infiltrating macrophages as the predominant source of pro-inflammatory cytokine production in SARS-CoV-2 infection.

An overview of the study design is shown in Fig. 1a . Eight RMs (mean age 14 years old; range 11-17 years old) were inoculated intranasally and intratracheally with 1.1 x 10 6 plaque-forming units (PFU) of SARS-CoV-2 (2019-nCoV/USA-WA1/202). These animals were previously reported in a study evaluating the therapeutic efficacy of baricitinib in SARS-CoV-2 infection 20 .

At 2dpi, four of the eight animals started receiving baricitinib 20 . For this study, pre-infection baseline and hyperacute time points (1-2dpi) include n = 8 RMs, all untreated, and the remaining longitudinal time-points assessed to determine the pathogenesis of SARS-CoV-2 infection are comprised of n = 4 of the RMs that remained untreated. Inoculation with SARS-CoV-2 led to reproducibly high viral titers detectable in the upper and lower airways by genomic and subgenomic qPCR assays (Fig. 1b) . The peak of viremia in the nasal passage, throat and BAL was at 2-4dpi (Fig. 1b) .

To characterize the innate immune response following SARS-CoV-2 infection, we analyzed changes in innate populations using multi-parametric flow cytometry in blood and BAL samples in the first 2dpi, or "hyperacute" phase of infection ( Fig. 1c-e, Fig. S1 ), and over the full course of infection (Fig. S2) . In blood, we did not observe a significant increase in the proportion of classical monocytes (CD14+CD16-) at 2dpi (Fig. 1c) nor at extended time-points (Fig. S2a) .

Similar to reports in humans 9 , we observed a rapid, but transient, increase in blood CD14-CD16+ and CD14+CD16+ monocytes (Fig. 1c, Fig. S2c ). Using these conventional markers for blood monocyte subsets, we did not observe any significant changes in CD14-CD16+, CD14+CD16+, nor CD14-CD16+ within the BAL (Fig. 1c, Fig. S2c ).

We observed a significantly elevated level of pDCs in blood at 2dpi and similarly, a trend of elevated pDCs in BAL samples (Fig. 1d, Fig. S2c ). This expansion was transient, as pDC numbers returned to baseline by 4dpi. While the overall frequencies of natural killer cells (NK) were not changed in blood or BAL (Fig. S2b) , the fraction of Granzyme B+ NK cells increased significantly at 2dpi in blood, from 4% to 25% (Fig. 1e) and remained elevated throughout the course of infection (Fig. S2) . Similarly, increases in NK cell activation were also observed in the BAL, rising from 12% to 33% at 2dpi (Fig. 1e) , and persisting at this level until the study termination at 10/11dpi (Fig. S2b) . Collectively, these data indicate that during the hyperacute phase of SARS-CoV-2 infection, there is a significant mobilization of innate immune cells capable of initiating and orchestrating effector responses of the Type I IFN system.

To understand the extent of immunological perturbations induced by SARS-CoV-2 infection, we performed extensive gene expression profiling of PBMC and BAL samples. During the hyperacute phase, the BAL had widespread induction of pathways associated with innate immunity and inflammation (Fig. 2a) . Notably, we observed a rapid and robust induction of interferon-stimulated genes (ISGs) in the PBMC and BAL compartments starting at 1 or 2dpi ( Fig. 2b, Fig. S3a ). The ISG response, although widespread, had largely returned to baseline by 10/11dpi (Fig 2b, Fig. S3a) . We also detected a trend of elevated IFNα protein in 4/6 and 5/8 animals in BAL and plasma, respectively (Fig. 2c,d) and a significant increase in RNA-Seq read counts mapping to IFNA genes at 2dpi in BAL (Fig. 2e) , which coincided with the expansion of pDCs in the airway and blood (Fig. 1d) . A significant enrichment of genes representing NK cell cytotoxicity ( Fig. 2a) was observed at 2dpi in BAL, consistent with our observation of elevated Granzyme B+ NK cells by flow cytometry (Fig. 1e) . Taken together, these data demonstrate the presence of primary cells able to produce Type I IFNs (i.e., pDCs), coincident with detectable IFNA transcripts and protein, and with downstream IFN-induced effector functions (ISGs, NK cell activation) following SAR-CoV-2 infection, and that these responses were transient, having largely subsided by 10/11dpi.

We observed that SARS-CoV-2 infection induced significant enrichment of several inflammatory cytokine signaling pathways, namely IFNA, IL4, IL6, IL10, IL12, IL23 and TNF, and the chemokine pathways CXCR4 and CXCR3, in both PBMCs and BAL of RMs, with higher magnitude in the BAL (Fig. 2a, Supplementary Files 1&2) . For many of these pathways, we were able to quantify significant increases in the upstream regulator at either the protein, or mRNA level, or both: IL6 protein levels were significantly increased in the BAL fluid (BALF) ( Fig. 2c) , as were RNA transcripts in BAL (Fig. S3b) . Similarly, the induction of CXCR3 pathways signaling was consistent with detection of increased IP10/CXCL10 protein in BALF and RNA at 2dpi in BAL (Fig. 2c, Fig. S3b ). The appearance of inflammatory pathways in the blood and airway have been reported in a multitude of human studies (reviewed in 26 ). However, we noted that SARS-CoV-2 infection also drove early expression of several immunoregulatory/immunosuppressive pathways in the BAL, namely: PD1 and CTLA4 signaling, and negative regulators of MAP kinase and DDX58/RIG-I signaling (Fig. 2a) .

Previously, we reported that the myeloid fraction in BAL was primarily responsible for the production of pro-inflammatory mediators, however the specific immunophenotypes were not defined. To further investigate the presence of different macrophage subsets within the lower airway after SARS-CoV-2 infection, we performed GSEA on bulk BAL data using AM gene signature (obtained from SingleR 27 ) specific for RM pulmonary macrophages. We observed that genes specific for alveolar macrophages (AMs) were significantly enriched at baseline (-5dpi) relative to 4dpi, indicating a downregulation of this gene set after SARS-CoV-2 infection (Fig.   2f) . Collectively, these bulk RNA-Seq data indicate a rapid and significant shift in the balance of macrophage populations in the lower airway following SARS-CoV-2 infection.

In our prior work in RMs, we demonstrated that cells of myeloid origin were the predominant subset responsible for production of inflammatory cytokines in the lower airway following SARS-CoV-2 infection 20 . While our prior sc-RNA-seq analyses determined the majority of cells in the BAL after infection to be of monocyte/macrophage origin, with relatively few neutrophils or granulocytes, the precise immunophenotypes of the myeloid cells driving inflammation in the lower airway have not been precisely delineated.

Cell classification based on cell-surface marker genes is typically problematic in sc-RNA-seq data due to gene dropouts inherent to the technology. Accurate classification is further complicated in the rhesus model system, in which genomic references have incomplete annotation, and markers from other model species may not phenocopy. Several significant advances have been made recently elucidating the resident tissue macrophage subsets in the lung and their function during viral infection and inflammation [28] [29] [30] [31] . However, analysis of sc-RNA-Seq data from RM lung suspensions and BAL during steady state condition indicated that several key markers used to differentiate macrophages in the murine lung (e.g. Lyve1) were not expressed at levels sufficient to distinguish populations in the rhesus pulmonary myeloid populations (Fig. S4) . Therefore, we used two overlapping alternative strategies to accurately classify tissue macrophages and monocyte-derived/infiltrating macrophages in the RM airway after SARS-Cov-2 infection in our sc-RNA-Seq data. The first strategy was based on using existing lung scRNA-Seq data from uninfected RMs as a reference to map and annotate the BAL cells. We processed lung 10X data from three uninfected RMs (NCBI GEO: GSE149758) 32 through Seurat pipeline 33 and reproduced the four reported macrophage/monocyte subsets: CD163+MRC1+, resembling alveolar macrophages; CD163+MRC1+TREM2+ macrophages, similar to infiltrating monocytes; CD163+MRC1-, similar to interstitial macrophages; and CD16+ non-classical monocytes (Fig S4 a-d) . We used Seurat to map BAL macrophages/monocytes from SARS-CoV2 infected RMs and transfer annotations from the lung reference. The second strategy involved using bulk RNA-Seq on sorted AM and IM from the lungs of three uninfected RMs, according to the phenotype defined by Cai et al 34 , based on expression of CD206/MRC1 and CD163, to annotate cells using SingleR 27 Fig. S4e) . 2069 genes were found to be differentially expressed between IMs and AMs (FDR< 0.05, fold-change>2) (Fig. S4f) . Of note, CX3CR1 was highly expressed in the IMs, consistent with both murine and human definitions of this subset (Fig. S4g) . APOBEC3A, an RNA-editing cytidine deaminase, was also highly expressed in IMs along with PTGS2, a pro-inflammatory COX-2 cyclooxygenase enzyme, TIMP1, which enables migration of cells via the breakdown of connective tissue, VCAN, an immunosuppressive regulator, and PDE4B, which regulates expression of TNFα (Fig. S4g) . We annotated the lung macrophage/monocyte subsets using the bulk sorted AM and IM datasets and found that almost all of CD163+MRC1+ cluster and some CD163+MRC1+TREM2+ cells were annotated as AM and the remaining as IM (Fig S4h) . Thus, benchmarking our lung sc-RNA-seq based reference against rudimentary bulk transcriptomic signatures demonstrated their accuracy in resolving the AM phenotype from non-AM in steady state conditions. We next analyzed changes in the myeloid populations within the BAL of RMs after SARS-CoV-2 infection. Using our lung/sc-RNA-seq reference, we found that most of the BAL macrophages/monocytes belonged to the AM-like CD163+MRC1+ macrophage subset at -5dpi along with some cells from the CD163+MRC1+TREM2+ macrophage subset (Fig. 3ab) . At 4dpi, there was an influx of both CD163+MRC1+TREM2+ macrophages and the IM-like CD163+MRC1-macrophages with few cells annotated as CD16+ non-classical monocytes. The expression of gene markers such as MARCO, FABP4 and CHIT1 further supported the cell subset annotations (Fig 3c) . We also observed a similar increase in APOBEC3A and decreases in MARCO and CHIT1 expression in bulk BAL samples (Fig 3d) . The percentage of CD163+MRC1+ macrophages reduced overall from 70% to 29% of all macrophages/monocytes in BAL (Fig 3e) . Similarly, we saw an overall increase in the percentage of CD163+MRC1+TREM2+ macrophages from 5% to 21% and the IM-like CD163+MRC1macrophages from 0.1% to 4%. Thus, COVID-19 infection resulted in an influx of monocytederived and IM-like macrophages in BAL at 4dpi. We also found that these infiltrating macrophages expressed higher levels of several pro-inflammatory cytokines and chemokines compared to the CD163+MRC1+ AM-like macrophages (Fig 3f-h, Supplementary File 4) .

To further validate our cell classification and support the observation that it is the infiltrating cells that increase in numbers and predominantly producing inflammatory mediators, we used the second strategy of using gene expression of bulk sorted AM and IM cells to classify the BAL macrophages/monocytes. Using this definition, we confirmed that there is an increase in the percentage of non-AM population with a corresponding decrease in the AM population (Fig S5b,   d) . The non-AM population was also found to show higher expression of pro-inflammatory cytokines (Fig S5e, Supplementary File 4) .

Differential expression analysis of 4dpi and -5dpi BAL macrophages/monocytes showed that CHIT1, MARCO and MRC1 were among the top-ranking genes exhibiting downregulation in the BAL, while genes such as ADAMDEC1 35 and S100A8 36 that are associated with monocytederived macrophages were among the most upregulated (Supplementary File 4) . These data demonstrate that our observation of an influx of infiltrating macrophages into the BAL at 4dpi was consistent across multiple definitions of this phenotype.

Given our observation of the dynamics of pulmonary macrophages within the alveolar space during early SARS-CoV-2 infection, we characterized the transcriptional changes in each macrophage/monocyte population. Several chemokines (CCL4L1, CCL3, CXCL3, CCL2), multiple ISGs, NFKB1A, S100A8, and GZMB were among the most upregulated genes at 4dpi in BAL populations (Supplementary File 4) . Elevated expression of multiple inflammatory genes, including IL6, TNF, IL10, IFNB1 and IL1B, were observed in the CD163+MRC1+TREM2 Mac and CD163+MRC1-subsets (Fig. 3f, Fig. S6 ) after infection. The infiltrating macrophages were also observed to upregulate multiple chemokines, including those specific for recruiting neutrophils (CXCL3, CXCL8), macrophages (CCL2, CCL3, CCL5, CCL4L1), and activated T cells (CXCL10) as well as multiple ISGs (Fig. 3g-h, Fig. S6 ). When we examined CD163+ MRC1+ macrophages, many of the same inflammatory cytokines and gene sets seen in the infiltrating macrophages were elevated at 4dpi, albeit at much lower magnitude ( Fig. 3f-h, Fig. S6 ). Having observed a significantly higher average expression of inflammatory cytokines in infiltrating macrophages compared to CD163+MRC1+ macrophages, we compared the fractions of sequencing reads detected from each of the subsets to assess the overall contribution to inflammatory cytokine production (Fig. 3i) . At 4dpi, we observed that the CD163+MRC1+TREM2+ macrophages accounted for 55% of IL6, 57% of TNF, 86% of IL10 and 66% of IFNB1 expression while the CD163+MRC1-macrophages accounted for 20% of IL6, 21% of TNF, 6% of IL10 and 19% of IFNB1 expression. We also found that CD163+MRC1-macrophages expressed higher levels of several of these genes (Fig. S6) . Thus, infiltrating macrophages are responsible for the majority of lower airway inflammatory cytokine production during acute SARS-CoV-2 infection.

Baricitinib is a JAK1/2 inhibitor approved for the treatment of active rheumatoid arthritis that was recently granted emergency use authorization for the treatment of hospitalized COVID-19 patients, and reported to reduce mortality when administered as monotherapy 37 or in combination with remdesivir 25 . In our earlier study, we found that baricitinib was able to suppress the expression of pro-inflammatory cytokines in BAL of RMs infected with SARS-CoV-2 20 . Here, we extended this study to further characterize the impact of baricitinib on the myeloid populations in the airway from five RMs before infection (-5) and at 4dpi, with three RMs that remained untreated and two that received baricitinib). We found that two days of baricitinib administration virtually abrogated the influx of infiltrating macrophages into the alveolar space at 4dpi (Fig. 4a-c) . This observation was consistent using classifications of macrophages either based on mapping to 10X lung reference or using bulk sorted AM/IM cells (Fig. 4a-c, Fig. S5ad) . In addition to preventing the influx of infiltrating macrophages, baricitinib treatment also resulted in significantly lower expression of inflammatory cytokines and chemokines, but the ISG expression remained comparable to untreated animals ( Fig. 4d-f, Fig. S5e) . In summary, these data further elucidate the mechanism of action by which baricitinib treatment abrogates airway inflammation in SARS-CoV-2 infection 20 , by demonstrating its ability to block infiltration of discrete pro-inflammatory macrophage populations into the alveolar compartment.

The mechanisms by which SARS-CoV-2 infection establishes severe disease remain largely unknown, but remain a key priority for reducing the toll of the COVID-19 pandemic. As the appearance of symptoms range from 2-14 days after SARS-CoV-2 infection, characterization of the early immunological events using clinical samples is challenging. Here, we utilized the RM model of SARS-CoV-2 infection and an integrated systems analysis to dissect the immune response during hyper-acute infection. Our findings were: (i) SARS-CoV-2 infection initiated a robust Type I IFN response in the blood and lower airway apparent at 1-2dpi; (ii) SARS-CoV-2 induced a rapid influx of two infiltrating macrophage populations, into the bronchoalveolar space, which produced the majority of inflammatory cytokine production; and (iii) the mechanism of action of baricitinib, a drug recently authorized for emergency use in the treatment of severe COVID-19, is to abrogate infiltration of these inflammatory cells into the airway. Our data present, to date, the most comprehensive analysis of the immunopathological events occurring during hyperacute SARS-CoV-2 infection.

Using our reference datasets of RM lung macrophages, we identified two myeloid cell subsets, both clearly distinct from alveolar macrophages, infiltrating the airway after SARS-CoV-2 infection, that were the main producers of lower airway inflammatory cytokines and chemokines.

One population, defined as CD163+MRC+TREM2+ cells, were highly similar to murine definitions of infiltrating CCR2+ monocytes. The second, CD163+MRC-, largely resembled interstitial macrophages. Our data are consistent with a recent observation of a rapid (3dpi) increase of IMs in the BAL of RMs using flow cytometry 21 . Similarly, an accumulation of non-AMs (defined as CD16+CD206-HLA-DR+/CD11b+), and reciprocal reduction of AMs, has been observed in the BAL and lungs of infected RMs and AGMs 23 . Lastly, our data are consistent with our recent findings in the murine model, in which SARS-CoV-2 elicited recruitment of circulating monocytes to the lung parenchyma, but was significantly abrogated in CCR2-deficient mice 38 . The CD163+MRC1+ Mac/AM-like subset also contributed to the inflammatory milieu, producing IL6, TNF, and IL10, albeit in significantly lower quantities.

It is important to note that our observations were during hyperacute infection, and that our animals did not develop severe disease, so although our data indicate that these infiltrating populations orchestrate early inflammation and may contribute to airway pathogenesis, we cannot formally make this link. However, this model is consistent with recent data by Ren et al 39 ., who observed a significant loss of MARCO expression in BAL-resident myeloid populations of patients with severe COVID-19 relative to those with moderate disease, similar to our observations, in which the appearance of infiltrating macrophages diluted the population of MARCO+ macrophages 40 . Those observations, taken together with our data, suggest that the inflammatory macrophage phenotype we identify here may be preferentially retained in the lower airway of patients with severe COVID-19. Additionally, we demonstrated that in vivo treatment with the JAK1/2 inhibitor baricitinib, which has demonstrated efficacy in reducing severe disease, was able to virtually abrogate the recruitment of these inflammatory macrophages into the airway, providing an additional mechanistic link with the development of COVID-19related pathogenesis.

In addition to inflammatory cytokines, we observed that the infiltrating macrophage subsets produced high levels of IL10, and were enriched in IL10 signaling pathways. Lung IM's are considered to be a "professional IL10-producing cell" producing IL10 at both a steady state and in response to innate stimuli (LPS, unmethylated CpGs) 41 . The majority of data to date has demonstrated an immunoregulatory, protective role for IMs in murine models of asthma, lung fibrosis, and allergen induced inflammation 29 . However, while the pro-inflammatory potential of IMs has been relatively understudied, they have been demonstrated to be efficient at producing IL6 and TNF in response to TLR ligands 42 . Given our observations of high IL10 production in infiltrating macrophages, we cannot exclude a potential immunoregulatory role for this subset, and indeed, it presents an interesting hypothesis in which the balance of infiltrating IM vs TREM2+ macrophages into the bronchoalveolar space determines the pathogenic outcome of SARS-CoV-2 infection. Lastly, recent publications have reported that lung IMs may be comprised of two, or even three, functionally distinct populations, defined by an axis of expression of Lyve1, MHC, CD169, and CD11c 28-31 . We did not observe separate clustering amongst BAL IMs, nor differential expression amongst these markers, and further work is needed to understand the congruency of macaque macrophage subsets with those identified in the murine model.

We observed a very rapid and robust induction of the Type I IFN pathway at 1-2dpi, characterized by elevated pDCs in the airway and blood, IFNA and IFNB transcripts and protein, Our multiparametric analyses demonstrated an increase of pDCs at 2dpi that coincided with the peak of ISG production, IFNA/B detection, and NK cell activation, thus implicating pDCs as the primary cell orchestrating the IFN response in the lower airway. We had previously observed a reduction in peripheral blood pDCs frequencies and activity in human SARS-CoV-2 infection 52 , and other have reported signatures of pDCs apoptosis that predicted lower IFN-I responses 53 .

Taken in the context of these clinical findings, our observation of pDC accumulation in the BAL indicates that they undergo rapid mobilization from the blood to the lower airway, and this suggests they likely drive early protective innate immune responses. However, pDCs may also contribute to pathological inflammation; and future interventional studies targeting the pDC/IFN axis in animal models will be necessary to test these hypotheses.

In our prior study, we demonstrated the ability of baricitinib to block airway pro-inflammatory cytokine production in SARS-CoV-2-infected RMs while preserving Type I IFN responses 20 .

Here, we extended these findings to demonstrate that baricitinib blocked the influx of inflammatory macrophages into the bronchoalveolar space. These data adds to our mechanistic understanding of the action of baricitinib, and provide a potential explanation for the disparity of baricitinib's impact on IFN vs IL6/TNF signaling when considering the timing of the drug administration. We administered baricitinib at 2dpi, after the peak influx of pDCs, but before the likely appearance of the APOBEC3A+ inflammatory macrophages at 3-4dpi. The ongoing ISG response, and suppressed TNF/IL6 response, suggest that the primary mechanism by which baricitinib protects the airway is by blocking recruitment of inflammatory cells to the bronchoalveolar space. In regards to guiding future clinical application of baricitinib, our data suggest that timing is critical, and would favor earlier drug administration.

Our study had some limitations; first, while the RM/SARS-CoV-2 model has rapidly been adopted by several groups for pre-clinical testing of anti-COVID drugs and vaccines, no group has demonstrated overt, reproducible symptomatic disease 11 . Thus, linking early immunological events to the development of severe COVID-19 requires validation in human studies, such as the observations of reduced MARCO expression in the airway myeloid populations of severe COVID-19 patients noted above 40 . Another drawback was the relatively low power of our study.

While our observations at 0, 1, and 2dpi were n = 8, we were limited to n = 4 for day 4-10 observations. However, it should be noted that there was no lack of statistical power for our key observations.

While the global vaccine rollout has made great strides to reduce the transmission and severity of SARS-CoV-2 infection, millions of people remain vulnerable. Understanding the early events of SARS-CoV-2 infection, and the mechanisms by which clinically approved drugs afford protection, remains a global priority. In this study, we have identified a novel population of inflammatory myeloid cells that are responsible for the preponderance of airway inflammation in acute SARS-CoV-2 infection. We also demonstrated that treatment with the emergency authorized JAK1/2 inhibitor baricitinib in combination with remdesivir, blocked infiltration of these inflammatory cells into the alveolar space. These data identify both a key druggable target (airway infiltrating macrophages), and an efficacious mechanism by which to lower airway inflammation, and should prove useful for identifying additional drugs to reduce the incidence and mortality of severe COVID-19 disease.

The animal care facilities at YNPRC are accredited by the Association for Assessment and CoV-2 (see SI for details), as previously described 20 and were maintained in the ABSL3 at YNPRC. The processing of nasopharyngeal swabs, BAL and mononuclear cells was performed as described previously 20 .

The SARS-CoV-2 genomic and sub-genomic RNA was quantified in nasopharyngeal swabs, throat swabs, and BAL as previously described 18, 20 (see SI for details).

23-parameter flow cytometric analysis was perform on fresh PBMCs and BAL mononuclear cells from SARS-CoV-2 infected RMs as described previously 20 . For purification of CD163 + CD206 + (AM), CD163 + CD206 -(IM) cells, cryopreserved single-cell lung suspensions from RMs were stained with BD CD163(GHI/61), CD206(19.2)(BD Biosciences), and purified using BD FACSAria in the Regional Biocontainment Laboratory at the University of Pittsburgh.

The data for -5dpi, 2dpi and 4dpi for bulk BAL samples was obtained from our previous study 20 .

Here we expanded our study to include 7dpi and 10dpi/11dpi samples for BAL and -5dpi, 1dpi, 2dpi, 4dpi, 6dpi, 7dpi, 8dpi and 10/11dpi for PBMC. Cell suspensions were prepared in BSL3, for bulk RNA-Seq, 250,000 cells (PBMCs) or 100,000 cells (BAL) were lysed directly into 700 ul of QIAzol reagent. Libraries were prepared as described previously 20 

The filtered count matrices for BAL were obtained from 20 . The Seurat library v4.0.4 33 was used to perform the analysis (see SI for details). The macrophages/monocytes from BAL samples were annotated into subsets using two approaches -(i) mapping to macrophages/monocytes from lung reference using Seurat and (ii) using bulk sorted cells as reference with SingleR 27 . The 10X lung scRNA-seq data from three uninfected macaque was obtained from a published study (NCBI GEO: GSE149758) 32 . The SingleR library was used for cell classification with the BluePrintEncodeData reference. Regularized log counts obtained from DESeq2 for bulk sorted IM and AM were used as reference to annotate the cells from BAL samples using SingleR with default parameters. Bulk RNA-Seq analysis was performed as previously described 20 .

U-PLEX assays (Meso Scale MULTI-ARRAY Technology) were used for plasma and BALF cytokine detection according to manufacturer's instructions, using 25 microliters as input.

Source data supporting this work are available from the corresponding author upon reasonable request.

The scripts used for analysis are available at https://github.com/BosingerLab/NHP_COVID-19_2. SingleR) when comparing bulk BAL RNA-Seq samples from 4dpi to -5dpi. The red bar represents the mean. Statistical analysis was performed using one-tailed Wilcoxon signed-rank test comparing each timepoint to -5dpi. * p-value < 0.05, ** p-value < 0.01.

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Animal models for COVID-19

Respiratory disease in rhesus macaques inoculated with SARS-CoV-2

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model

SARS-CoV-2 infection protects against rechallenge in rhesus macaques

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques

Modified Vaccinia Ankara Vector-Based Vaccine Protects Macaques from SARS-CoV-2 Infection, Immune Pathology and Dysfunction in the Lung

Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2

Baricitinib treatment resolves lower-airway macrophage inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets

Vascular Disease and Thrombosis in SARS-CoV-2-Infected Rhesus Macaques

Cellular events of acute, resolving or progressive COVID-19 in SARS-CoV-2 infected non-human primates

Single-cell RNA sequencing reveals SARS-CoV-2 infection dynamics in lungs of African green monkeys

Baricitinib plus Remdesivir for Hospitalized Adults with Covid-19

COVID-19 and the human innate immune system

Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage

Three Unique Interstitial Macrophages in the Murine Lung at Steady State

Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches

Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties

Non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung

The immune landscape in tuberculosis reveals populations linked to disease and latency

Integrated analysis of multimodal single-cell data

In vivo characterization of alveolar and interstitial lung macrophages in rhesus macaques: implications for understanding lung disease in humans

ADAMDEC1 and its role in inflammatory disease and cancer

Baricitinib restrains the immune dysregulation in severe COVID-19 patients

CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection. bioRxiv

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas

COVID-19 immune features revealed by a large-scale single cell transcriptome atlas

The interstitial macrophage: A long-neglected piece in the puzzle of lung immunity

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

X-linked recessive TLR7 deficiency in ~1% of men under 60 years old with life-threatening COVID-19

Autoantibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70 years old and account for ~20% of COVID-19 deaths

Preexisting autoantibodies to type I IFNs underlie critical COVID-19 pneumonia in patients with APS-1

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

Early nasal type I IFN immunity against SARS-CoV-2 is compromised in patients with autoantibodies against type I IFNs

Pre-existing Autoantibodies Neutralizing High Concentrations of Type I Interferons in Almost 10% of COVID-19 Patients Admitted to Intensive Care in Barcelona

Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19

Systems biological assessment of immunity to mild versus severe COVID-19 infection in humans

Time-resolved systems immunology reveals a late juncture linked to fatal COVID-19

Writing: -Original Draft

represent the mean. Statistical analysis was performed using one-tailed Wilcoxon signed-rank test comparing each timepoint to -5dpi. * p-value < 0.05, ** p-value < 0.01. Figure 3. Influx of pro-inflammatory macrophages in BAL (a) Projection of single-cell macrophages/monocytes from -5dpi (green) and 4dpi (magenta) 10X BAL samples obtained from three SARS-CoV2 infected rhesus macaques onto the reference UMAP of lung macrophage/monocytes from uninfected rhesus macaques (NCBI GEO : GSE149758). (b) by time of sample collection. (c) DotPlots showing the expression of marker genes for the different macrophage/monocyte subsets in SARS-CoV2 infected BAL samples (d) Log2 fold-changes compared to -5dpi for APOBEC3A, CHIT1 and MARCO in bulk BAL RNA-Seq data. (e) Percentage of a given subset out of all macrophage/monocyte subsets at -5dpi and 4dpi from all three animals (f,g,h) FeaturePlots showing the expression of selected proinflammatory cytokines (f), chemokines (g) and ISG (h) in different macrophage/monocyte subsets at 4 dpi. (i) Contribution of each macrophage/monocyte subset towards the production of the pro-inflammatory genes and ISG. The percentage contribution was calculated by dividing the

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Competing interests: R.F.S. has served as an unpaid consultant for Eli Lilly whose drugs are being evaluated in the research described in this paper, and owns shares in Eli Lilly. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies. All other authors do not have any conflicts to declare.