key: cord-1018037-bqax0k3b authors: Zhang, Fan; Mears, Joseph R.; Shakib, Lorien; Beynor, Jessica I.; Shanaj, Sara; Korsunsky, Ilya; Nathan, Aparna; Donlin, Laura T.; Raychaudhuri, Soumya title: IFN-γ and TNF-α drive a CXCL10+ CCL2+ macrophage phenotype expanded in severe COVID-19 and other diseases with tissue inflammation date: 2020-08-05 journal: bioRxiv DOI: 10.1101/2020.08.05.238360 sha: 20b5e389d7202e1897268280d942b2061ec35ac8 doc_id: 1018037 cord_uid: bqax0k3b Immunosuppressive and anti-cytokine treatment may have a protective effect for patients with COVID-19. Understanding the immune cell states shared between COVID-19 and other inflammatory diseases with established therapies may help nominate immunomodulatory therapies. Using an integrative strategy, we built a reference by meta-analyzing > 300,000 immune cells from COVID-19 and 5 inflammatory diseases including rheumatoid arthritis (RA), Crohn’s disease (CD), ulcerative colitis (UC), lupus, and interstitial lung disease. Our cross-disease analysis revealed that an FCN1+ inflammatory macrophage state is common to COVID-19 bronchoalveolar lavage samples, RA synovium, CD ileum, and UC colon. We also observed that a CXCL10+ CCL2+ inflammatory macrophage state is abundant in severe COVID-19, inflamed CD and RA, and expresses inflammatory genes such as GBP1, STAT1, and IL1B. We found that the CXCL10+ CCL2+ macrophages are transcriptionally similar to blood-derived macrophages stimulated with TNF-α and IFN-γ ex vivo. Our findings suggest that IFN-γ, alongside TNF-α, might be a key driver of this abundant inflammatory macrophage phenotype in severe COVID-19 and other inflammatory diseases, which may be targeted by existing immunomodulatory therapies. Tissue inflammation is a unifying feature across diseases. While tissue-and disease-specific 51 factors shape distinct inflammatory microenvironments, seemingly unrelated diseases can 52 7 observed that applying Harmony increased mixing among donors (LISI increasing from mean 160 2.9 to 6.1) and tissue sources (LISI increasing from mean 1.0 to 1.8, Supplementary Figure 161 Figure 1c) . Furthermore, we observed 174 that the majority of variance (>60% in PC1 and PC2) derived from gene expression patterns are 175 explained by major cell types (Figure 1f, Supplementary Figure 1d ). In contrast, variables 176 such as tissue type, technology, or donor sample accounted for <1% of the variation in PC1 and 177 PC2 after batch effect correction. We note that prior to Harmony batch effect correction, the 178 same cell types from different tissues fail to integrate together (Supplementary Figure 2b) . Macrophages represented a dominant cell type across all affected target tissues 12,14,22-25 . 195 Therefore, we performed a fine clustering analysis on these cells to define shared and distinct 196 macrophage states and phenotypes across these inflammatory diseases and COVID-19 197 (Figure 2a) . To this end, we applied the same integrative pipeline on 74,373 macrophages and 198 monocytes from synovium, ileum, colon, lung, and BALF from 108 individuals (Supplementary 199 Table 2 ). We identified a total of four states: CXCL10+ CCL2+ CD14+ FCGR3A+ inflammatory 200 macrophages, FCN1+ CD14+ FCGR3A+ inflammatory macrophages, M2-like anti-inflammatory 201 MRC1+ FABP4+ macrophages, and non-inflammatory macrophages (Figure 2a Notably, in this clustering, previously described inflammatory macrophages identified in inflamed 205 RA synovium and in inflamed UC and CD intestinal tissue clustered along with the majority of 206 the severe COVID-19 macrophages, which spanned across these two inflammatory CXCL10+ 207 CCL2+ and FCN1+ states (Figure 2c, Supplementary Figure 3b-c) . The LISI score that 208 evaluates dataset mixing decreased with respect to previously described macrophage 209 annotations, and increased with respect to donor-and tissue-specific effects after batch driven primarily by macrophage biology-related gene expression patterns rather than tissue or 212 donor source. 213 To further explore how the CXCL10+ CCL2+ and FCN1+ macrophages are involved in tissue 215 inflammation, we examined key inflammatory features 14 that were expressed in these two 216 states. A high proportion of inflammatory macrophages in severe COVID-19, RA, UC, and CD 217 expressed inflammation-associated factors including CXCL10, CXCL11, CCL2, CCL3, STAT1, 218 IFNGR1, IFNGR2, NFKB1, TGFB1, and IL1B (Figure 2d, Supplementary Figure 4a) . The 219 gene signature for the CXCL10+ CCL2+ inflammatory macrophage state was found in a higher 220 proportion of macrophages in severe COVID-19 than in the other inflamed tissues (Figure 2d ). 221 Applying PCA to the two inflammatory macrophage states, we found that PC1 captured a 222 gradient from the FCN1+ state to the CXCL10+ CCL2+ state (Figure 2e) , suggesting a potential 223 continuum with intermediates between the inflammatory FCN1+ and CXCL10+ CCL2+ states. 224 While the majority of inflammatory macrophages in RA, UC, and CD align more closely with the 225 To elucidate cell states that were phenotypically associated, we tested the association of each 311 cluster with severe COVID-19 compared to healthy BALF using a logistic regression model 312 accounting for technical cell-level and donor-level effects 30 (Methods). We observed two clusters abundant in severe COVID-19 compared to healthy BALF: CXCL10+ CCL2+ (cluster 314 1), which is transcriptionally similar to the TNF-" and IFN-! induced phenotype and cluster 4, 315 which most closely matches a TNF-" with fibroblasts induced phenotype (Figure 4e) . The 316 CXCL10+ CCL2+ inflammatory macrophages are significantly more abundant in severe COVID-317 19 (23.7%) compared to healthy BALF (3.7%), and express high levels of the genes that 318 synergistically respond to TNF-" and IFN-! stimulation (Figure 4d-e, Supplementary Figure 319 8d-e). We examined other diseases also, and observed that the CXCL10+ CCL2+ inflammatory 320 macrophages are expanded in inflamed CD compared to non-inflamed CD, RA compared to 321 non-inflammatory OA, and inflamed UC compared to healthy colon, respectively (Figure 4f) . 322 Taken together, these results indicate that TNF-" and IFN-! drive the synergistic inflammatory 323 response in the CXCL10+ CCL2+ inflammatory macrophage phenotype that is expanded not 324 only in COVID-19, but also in inflamed tissues from other diseases, which suggests this 325 inflammation-associated macrophage state may present a viable target for these diseases. 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