key: cord-0910901-4dfrjlu8 authors: Rosa, Bruce A.; Ahmed, Mushtaq; Singh, Dhiraj K.; Choreño-Parra, José Alberto; Cole, Journey; Jiménez-Álvarez, Luis Armando; Rodríguez-Reyna, Tatiana Sofía; Singh, Bindu; Gonzalez, Olga; Carrion, Ricardo; Schlesinger, Larry S.; Martin, John; Zúñiga, Joaquín; Mitreva, Makedonka; Khader, Shabaana A.; Kaushal, Deepak title: IFN signaling and neutrophil degranulation transcriptional signatures are induced during SARS-CoV-2 infection date: 2020-08-06 journal: bioRxiv DOI: 10.1101/2020.08.06.239798 sha: d161a934b69bf60a5d72bbe4b0b259f9c0aaa2c4 doc_id: 910901 cord_uid: 4dfrjlu8 The novel virus SARS-CoV-2 has infected more than 14 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Limited information on the underlying immune mechanisms that drive disease or protection during COVID-19 severely hamper development of therapeutics and vaccines. Thus, the establishment of relevant animal models that mimic the pathobiology of the disease is urgent. Rhesus macaques infected with SARS-CoV-2 exhibit disease pathobiology similar to human COVID-19, thus serving as a relevant animal model. In the current study, we have characterized the transcriptional signatures induced in the lungs of juvenile and old rhesus macaques following SARS-CoV-2 infection. We show that genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. We demonstrate that Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. In contrast, pathways involving VEGF are downregulated in lungs of old infected macaques. Using samples from humans with SARS-CoV-2 infection and COVID-19, we validate a subset of our findings. Finally, neutrophil degranulation, innate immune system and IFN gamma signaling pathways are upregulated in both tuberculosis and COVID-19, two pulmonary diseases where neutrophils are associated with increased severity. Together, our transcriptomic studies have delineated disease pathways to improve our understanding of the immunopathogenesis of COVID-19 to facilitate the design of new therapeutics for COVID-19. ≤ 0.01) up-regulated in response to infection, while 1,109 genes were significantly downregulated 205 ( Figure 1B) . Expression, annotation and differential expression data for all genes is available in 206 Table S2 . Complete lists of differentially expressed genes for each comparison of interest 207 (described below) ranked by P value, with Z-scores for expression visualization are available in 208 Table S3 , and significant pathway enrichment (Reactome(21), KEGG(23) and Gene 209 Ontology(24)) for all comparisons is shown in Table S4 . CD36 and CD58, in comparison to expression in uninfected macaque lungs (Figure 2A) . 214 Cathepsin G is a serine protease prominently found in neutrophilic granules. IFNgR1 associates 215 with IFNgR2 to form a receptor for the cytokine interferon gamma (IFNg)(26-29), and required for 216 activation of antiviral responses, such as IRF3 (IFN regulatory factor-3), nuclear factor KB (NF-217 KB) and JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signaling 218 pathways (30). Reactome pathway analysis on up-and down-regulated genes in the lungs of 219 SARS-CoV-2 infected rhesus macaques showed that genes significantly up-regulated by 220 infection, included pathway enrichment for genes involved in "Neutrophil degranulation", "Innate 221 Immune system", "Immune system" and "IFN signaling" ( Table 1; Table S4A ). The up-regulation 222 of CD36 during COVID-19 in lungs is in conformity with these enriched pathways, since CD36, a 223 scavenger receptor expressed in multiple cell types, mediates lipid uptake, immunological 224 recognition, inflammation, molecular adhesion, and apoptosis (31), and is a Matrix 225 Metalloproteinase-9 substrate that induces neutrophil apoptosis. CD58 molecule (lymphocyte 226 function-associated antigen-3) is expressed on human hematopoietic and non-hematopoietic 227 cells, including dendritic cells, macrophages and endothelial cells (32-35), and interacts with its 228 receptor CD2 molecule (36, 37) on CD8 + cytotoxic T lymphocytes and NK cells to mediate 229 cytotoxic reactions (38-40). The complete ranked list of the 1,026 genes upregulated during 230 COVID-19 is shown in Table S3A . 231 ATP6AP2 was the most significantly up-regulated of the 65 genes upregulated within the enriched 232 "neutrophil degranulation" (R-HSA-6798695) pathway (Table S3B) for ARDS(44). Expression of 162 genes belonging to the "immune system" (R-HSA-168256) 245 pathway was upregulated in SARS-CoV-2 infected macaques (Table S3C) . These included 246 LAMP-2(Lysosomal Associated Membrane Protein 2), and ATG7 (Autophagy Related 7), key 247 genes involved in autophagy. LAMP-2 is known to influence phagosomal maturation in neutrophil 248 (45). The IFN response constitutes the major first line of defense against viruses. Consistent with 249 this, we found up-regulation of genes associated with the IFN signaling pathways, specifically 250 Interferon Induced Protein with Tetratricopeptide Repeats 1 (IFIT3), IFN alpha receptor 1 251 (IFNAR1), IFN gamma receptor 1 (IFNGR1) and OAS 1 protein (2'-5'-252 Oligoadenylate Synthetase 1). Together, these results suggest that upregulation of neutrophil 253 degranulation, Type I IFN signaling, and innate immune system is a characteristic feature of host 254 responses to SARS-CoV-2 infection. 255 It is thought that up to 40% of patients with COVID-19 develop ARDS, and 20% of ARDS cases 259 are severe (46). A well-documented sequela of ARDS is the development of fibrotic disease (47, 260 48). We found that the 1,109 genes downegulated in SARS-CoV-2-infected macaques were 261 significantly enriched for collagen degradation, regulation and formation ( Figure 2B ; Table 2 ; 262 Table S3D; Table S4B ). For example, among the "collagen degradation" (R-HSA-1442490) 263 enriched pathway (Table S3E) comprised of "collagen degradation", "collagen chain trimerization", "degradation of extracellular 270 matrix" and "collagen formation" ( Table 2) . Increased collagen degradation is essential for the 271 prevention of fibrosis, a sequelae of COVID-19 and ARDS. Therefore, regulation of collagen 272 degradation and extracellular matrix modeling suggest that this may be a feature of SARS-CoV-273 2 infection of rhesus macaques being a self-limiting model with early and robust anamnestic 274 responses. TGFβ (Transforming Growth Factor Beta 1) is involved in normal tissue repair 275 following lung injury, and in mediating fibrotic tissue remodeling by increasing the production and 276 decreasing the degradation of connective tissue (49). Our results indicate a downregulation of 277 genes associated with TGFβ signaling (Table 2) , including the genes PARD3 (par-3 family cell 278 polarity regulator) and PARD6A (par-6 family cell polarity regulator alpha), which are involved in 279 regulating epithelial cell apico-basolateral polarization, SMURF (SMAD specific E3 ubiquitin 280 protein ligase 1), a negative regulator of TGFβ pathway, and FURIN, which is a TGFβ converting enzyme (Table S3F) . While the interaction of the genes within these pathways is complex, our 282 results project a broad downregulation of mechanisms that contribute to lung repair and 283 remodeling in animals with anamnestic control of SARS-CoV-2 infection. 284 285 Type I interferon signaling and Notch signaling pathways are upregulated in young 286 Age is a significant risk factor for increased morbidity and mortality in COVID-19 disease (11). In 288 order to identify the differential immune responses associated with SARS-CoV-2 infection in old 289 macaques, we carried out differential expression analysis between the groups; namely between 290 juvenile (n=3) vs naive (n=4), and old (n=5) vs naive (n=4). In order for a gene to be considered 291 to be differentially expressed only in the juvenile macaques, we required a stringent P value for 292 significance ≤ 0.01 in the juvenile COVID-19 vs naive, and a P value for significance ≥ 0.1 in the 293 old COVID-19 vs naive comparison. This approach identified 86 genes significantly up-regulated 294 ( Figure 3A ; Table S3G ) and 96 genes significantly down-regulated ( Figure 3B ; Table S3H ) with 295 COVID-19 disease only in juveniles. Note that no genes were significantly upregulated in juveniles 296 and significantly downregulated in old, and vice-versa. Of these genes, the top 30 most 297 significantly differential between juvenile and old are shown for up-regulated genes in Figure 4A 298 and for down-regulated genes in Figure 4B . No pathways were found to be significantly enriched 299 among the 96 genes significantly downregulated only in juveniles, but the Reactome and KEGG 300 pathways significantly enriched among the 86 genes upregulated only in juveniles are shown in 301 Table 3 . Complete gene lists per pathway, and all significant pathways enrichment results 302 including for Gene Ontology (GO) are available in Table S4C . 303 The genes with significantly upregulated expression in SARS-CoV-2 infected juvenile but not old 304 macaques included MX1 (MX Dynamin Like GTPase 1), MX2 (MX Dynamin Like GTPase 2) and 305 USP18 (Ubiquitin Specific Peptidase 18) (Figure 5) . This is consistent with and highlights the role 306 of the Reactome pathway "interferon alpha/beta signaling" being enriched in juvenile macaques during SARS-CoV-2 infection ( Table 3, Table S4C ). Other genes in this pathway which exhibited 308 increased expression included IFIT1 and IFIT2. Additionally, by KEGG analysis, the Notch 309 signaling pathway was observed to be significantly upregulated in juvenile infected macaques 310 when compared with old infected macaques. ADAM17 (ADAM Metallopeptidase Domain 17), a 311 key component of the Notch signaling pathways is known to be involved in shedding of the surface 312 protein ACE2 (Angiotensin converting enzyme 2) (50). Therefore, it is interesting that a linear 313 correlation in the expression of ACE2 and ADAM17 exists in infected macaques ( Figure 4C) . 314 Note that we also see a significant upregulation of ACE2 across all samples (4.2-fold, P = 4.9x10 -315 3 ), and a substantially larger upregulation among the juvenile samples (7.1-fold, P = 3.4x10 -4 ). 316 Additionally, the induction of DLL4, a Notch ligand, was increased in the infected juvenile 317 macaques. Finally, the differential induction of DTX3L (Deltex E3 Ubiquitin Ligase 3L) in juvenile 318 infected macaques compared to old infected macaques is important because Deltex stabilizes 319 the receptor in the endocytic compartment allowing signal transduction to proceed in Notch 320 signaling(52). Of the Hepatitis-induced pathway genes that are upregulated in juvenile COVID-19 321 diseased lungs, CXCL-10 (C-X-C Motif Chemokine Ligand 10) is a chemokine associated with 322 severe disease in COVID-19 in humans (53), but can also be involved in recruitment of CXCR3 323 (C-X-C Motif Chemokine Receptor 3) expressing immune cells. 14-3-3 (otherwise called YWHAG) 324 interacts with MDA5 (melanoma differentiation-associated protein 5), which belongs to the RIG-I-325 like receptor family and drive anti-viral immunity. Together, these results suggest that specific 326 pathways including Type I IFN and Notch signaling are highly induced in juvenile macaques 327 during SARS-CoV-2 infection, when compared to similarly infected old macaques. 328 329 Genes related to VEGF signaling are downregulated in old macaques but not juvenile 330 macaques during COVID-19-disease 331 Using the same approach as for the juvenile macaque-specific differentially regulated genes, we 332 identified 97 genes significantly up-regulated ( Figure 3A ; Table S3I ) and 160 genes significantly 333 down-regulated ( Figure 3B ; Table S3J ) with COVID-19 disease only in infected old macaques, 334 and not infected juveniles. Pathway enrichment analysis only identified significant functional 335 enrichment among the down-regulated gene set ( Table 4; Table S4D ). Our results show that in 336 the lungs of old macaques, the only Reactome pathways enriched among genes downregulated 337 during COVID-19 included genes involved in the "VEGF-VEGFR2 Pathway" and "Signaling by 338 VEGF" (Figure 6, 7) . Vascular endothelial growth factor (VEGF) is a signaling protein that 339 promotes angiogenesis, and is a key factor that promotes ARDS. Previous research showed that 340 ACE2 antagonizes and down-regulates VEGFA(54), improving lung function following acute lung 341 injury (55). Here, we observe both a significant increase in ACE2 in response to COVID-19 and 342 a significant decrease in VEGF pathways in old macaques, which may be due to this antagonistic Overall, despite juvenile and old macaques having a comparable clinical course with resolution, 348 our data suggest that there are significant differenes in signaling pathways, especially those 349 related to VEGF signaling that may ultimately result in differences is long term outcomes. Thus, 350 our results suggest that down-regulation of VEGF pathways is associated with increasing age, in 351 a macaque cohort of self-limiting disease model, and protect from serious lung injury during 352 COVID-19 disease. 353 To further address if our findings were relevant in the human setting of SARS-CoV-2 infection, we 356 stratified COVID-19 patients into aged group (>60 years) and a group of COVID-19 patients <60 357 years (Table S5) . We found that with increasing age, there were increased association of disease 358 parameters and comorbidities (Table S5) . We measured the levels of human plasma proteins 359 levels for IFN-α, IFN-b and IFN-g. While levels of plasma IFN-α, and IFN-b were below the levels 360 of reliable detection, we found that the COVID-19 patients who were <60 years expressed 361 significantly higher plasma IFN-g levels when compared to levels in plasma of healthy controls 362 (Fig. 8A) . Although plasma levels of IFN-g protein was also increased in aged 363 COVID-19 patient group, levels were not significantly different from healthy controls (Fig. 8A) . 364 This was in contrast to plasma protein levels of VEGF, which was significantly higher in aged 365 individuals with COVID-19 disease when compared with levels in individuals with COVID-19 366 disease who were <60 years old (Fig. 8B) . The increased levels of VEGF in aged COVID-19 367 patients coincided with significantly increased peripheral neutrophil counts as well as increased 368 peripheral neutrophil to lymphocyte ratios, when compared with both healthy controls and COVID-369 19 group <60 years old (Fig. 8C,D) . These results show that plasma protein levels of VEGF and 370 accumulation of peripheral neutrophils is increased in aged individuals with COVID-19 disease, 371 when compared to younger individuals with COVID-19 disease. 372 Tuberculosis (TB) is a pulmonary granulomatous disease caused by infection with Mycobacterium 374 tuberculosis. TB disease in humans and macaques is associated with a neutrophil and IFN 375 signature(13). Thus, we next compared and contrasted the transcriptional profile of genes and 376 pathways that are shared by the two diseases, and those that are unique to COVID-19.There was 377 not a substantial overlap between differentially expressed genes in response to COVID-19 and 378 TB. However, of the 97 genes that were commonly upregulated in TB and COVID-19 ( Figure 379 9A, Table S3K ), the Reactome pathway enrichment was well featured in "Neutrophil degranulation", "Innate immune response", and "Interferon gamma signaling" (Figure 9B , Table 381 S4E). Nearly as many genes (76) had opposite differential expression patterns (upregulated in 382 COVID-19, downregulated in TB), as genes upregulated in both ( Figure 10A, Table S3L ). These 383 genes were associated with blood vessel morphogenesis and angiogenesis including leptin 384 receptor (LEPR), TGFb2 ( Figure 10B, Table S4F ). These results suggest that both TB and 385 COVID-19 share features of neutrophil accumulation of IFN signaling, but that COVID-19 disease 386 immunopathogenesis uniquely features vascularization of the lung. from the unique perceptive of age as a contributing factor. 407 As we learn more about the pathophysiology of COVID-19, it is becoming clear that disease 408 severity is associated with hyperinflammation which in turn induces lung and multiorgan injury 409 and mortality via a cytokine storm (1, 2, 56) . While therapeutic options that focus on 410 immunomodulatory agents such as corticosteroids are being considered and used, a risk exits 411 that immunomodulators may also inhibit protective pathways. Therefore, a thorough ARDS, it is predicted that a similar outcome of fibrosis will be associated with COVID-19. Also, 497 since the risk factors associated with COVID-19 including increasing age, male and associated 498 co-morbidities coincide with IPF risk factors, it is expected that COVID-19 patients will experience 499 fibrotic lung disease. Despite these associations, there is no evidence currently that "scarring of 500 the lung" experienced by COVID-19 patients is fibrotic or progressive and an outcome of COVID-501 19 disease post recovery. Therefore, our results provide unique insights into the role of fibrosis 502 during SARS-CoV-2 infection. Most notably, we find significant downregulation of collagen 503 degradation pathways, as well as pathways associated with collagen formation, collagen 504 trimerization and assembly. Furthermore, the role for TGF-b and ECM degradation is well 505 documented in fibrosis. Indeed, the genes associated with these pathways are also significantly 506 down-regulated. These results for the first time provide novel insights into the early pathological 507 events occurring during COVID-19 in the lungs with relevance to underlying immune mechanisms 508 associated with canonical fibrosis pathways. While long term consequences of the pulmonary 509 COVID-19 such as fibrosis remain to be determined, our results on down-regulation of collagen 510 degradation and TGF-b pathways may represent important early events on the lungs of SARS-511 CoV-2 infected individuals. We speculate that such events may protect individuals from 512 progression to ARDS and fibrosis, while it is possible that in individuals with early activation of 513 collagen degradation progress more severe outcomes may ensue. 514 Finally, we provide novel insights into the transcriptional regulation of immune pathways that are to two weeks, and it was initially thought that this virus causes acute infection. However, details 526 are now emerging from both animal models(12) (in review) and patients, that the virus can persist 527 for longer periods, leading to persistent shedding from tissues, and exhaustion of adaptive 528 responses. While innate and T cell responses are comparable between juvenile and old 529 macaques following infection, SARS-CoV-2 specific antibody is generated at significantly higher 530 levels in the plasma of juveniles, relative to old macaques(12) (in review). Since Notch signaling 531 regulates multiple stages of B-cell differentiation and shapes the antibody repertoire(74), higher 532 expression of many of the Notch pathway member genes in juvenile macaques may be 533 responsible for the development of stronger antibody responses in these animals, impacting 534 disease progression. Alternatively, it is possible that the differences in Notch signaling and 535 production of virus-specific antibody between jouvenile and old macaques may impact disease 536 progression over a longer period of time, or be particularly relevant in models of co-morbidity, 537 such as diabetes. Similarly, Type I IFN responses are critical for the downstream breadth of 538 antibody production and recognition (75-77). Thus, while T cell responses are comparable in 539 juvenile and old macaques, differences in critical signaling pathways uncovered by our RNA-seq 540 analysis potentially explain why juvenile macaques mount significantly stronger antibody 541 responses, and consequently why younger subjects have reduced susceptibility to COVID-19. 542 While this has not been recapitulated in the macaque model, older patients of COVID-19 are more 543 susceptible to progression. This is consistent with increased disease progression when COVID-544 19 patients were stratified based on age. A previous study found that peripheral VEGF 545 concentrations were significantly higher in COVID-19 patients than in healthy controls(81). We 546 also find this effect in our human samples ( Figure 8B) where people with COVID-19 that are older 547 than 60 years of age have more VEGF protein in their peripheral blood. 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The role of notch signaling in the development of a normal 782 B-cell repertoire B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of 784 Tolerance and the Development of Autoreactive B Cells Role of type I interferons 786 in the activation of autoreactive B cells Type I IFN sets the stringency of 788 B cell repertoire selection in the bone marrow CD36, a scavenger receptor involved in immunity, 790 metabolism, angiogenesis, and behavior Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis 792 in Covid-19 CoV-2 infection: The role of cytokines in COVID-19 disease Table S1 : Read processing and mapping statistics, and download accessions for all RNA-seq 989 samples. 990 991 Table S2 : Fragment counts, relative gene expression levels, gene annotations, and differential 992 expression data for every macaque gene.