key: cord-343330-wuzts3mt
authors: Ramos da Silva, S.; Ju, E.; Meng, W.; Paniz Mondolfi, A. E.; Dacic, S.; Green, A.; Bryce, C.; Grimes, Z.; Fowkes, M. E.; Sordillo, E. M.; Cordon-Cardo, C.; Guo, H.; Gao, S.-J.
title: Broad SARS-CoV-2 cell tropism and immunopathology in lung tissues from fatal COVID-19
date: 2020-09-29
journal: nan
DOI: 10.1101/2020.09.25.20195818
sha: 
doc_id: 343330
cord_uid: wuzts3mt

Background Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection in patients with Coronavirus Disease 2019 (COVID-19) prominently manifests with pulmonary symptoms histologically reflected by diffuse alveolar damage (DAD), excess inflammation, pneumocyte hyperplasia and proliferation, and formation of platelet aggregates or thromboemboli. However, the mechanisms mediating these processes remain unclear. Methods We performed multicolor staining for viral proteins, and lineage cell markers to identify SARS-CoV-2 tropism and to define the lung pathobiology in postmortem tissues from five patients with fatal SARS-CoV-2 infections. Findings The lung parenchyma showed severe DAD with thromboemboli in all cases. SARS-CoV-2 infection was found in an extensive range of cells including alveolar epithelial type II/pneumocyte type II (AT2) cells (HT2-280), ciliated cells (tyr--tubulin), goblet cells (MUC5AC), club-like cells (MUC5B) and endothelial cells (CD31 and CD34). Greater than 90% of infiltrating immune cells were positive for viral proteins including macrophages and monocytes (CD68 and CD163), neutrophils (ELA-2), natural killer (NK) cells (CD56), B-cells (CD19 and CD20), and T-cells (CD3{varepsilon}). Most but not all infected cells were positive for the viral entry receptor angiotensin-converting enzyme-2 (ACE2). The numbers of infected and ACE2-positive cells correlated with the extent of tissue damage. The infected tissues exhibited low numbers of B-cells and abundant CD3{varepsilon}+ T-cells consisting of mainly T helper cells (CD4), few cytotoxic T cells (CTL, CD8), and no T regulatory cell (FOXP3). Antigen presenting molecule HLA-DR of B and T cells was abundant in all cases. Robust interleukin-6 (IL-6) expression was present in most uninfected and infected cells, with higher expression levels observed in cases with more tissue damage. Interpretation In lung tissues from severely affected COVID-19 patients, there is evidence for broad SARS-CoV-2 cell tropisms, activation of immune cells, and clearance of immunosuppressive cells, which could contribute to severe tissue damage, thromboemboli, excess inflammation and compromised adaptive immune responses.

Evidence before this study 73

Pulmonary symptoms reflected by diffuse alveolar damage (DAD), excess inflammation, 74 pneumocyte hyperplasia and proliferation, formation of platelet aggregates, and 75 thromboemboli are the pathological features of COVID-19. However, the mechanisms 76 mediating these processes have not been elucidated. We searched PubMed up to 77

September 15, 2020 using the keywords "coronavirus disease 2019", "COVID-19", 78 "SARS-CoV-2", "cell tropism", "cell markers", "inflammation", "interleukin 6", "immune 79 response", "immune suppression", "immunofluorescence" and "immunohistochemistry", 

In lung tissues from severely affected COVID-19 patients, there is evidence for broad 107 SARS-CoV-2 cell tropisms, hyperactive immune cells, and clearance of immune cells 108 including immunosuppressive cells, which could contribute to severe tissue damage, 109 thromboemboli, excess inflammation and compromised adaptive immune responses. 110

These results have implications for development of treatments. 111 112 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

Coronavirus Disease 2019 (COVID-19) is a complex disease caused by Severe 114

Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. 1, 2 Multiple organs 115 are affected, and severe lung damage is a prominent finding in fatal cases. 3-6 Although 116 dysregulated immune responses and excess inflammation are commonly observed in 117 the lung tissues from these patients, the precise mechanism underlying the pulmonary 118 pathology remains unclear. 4 119

Single cell RNA sequencing (scRNA-seq) analysis of lung tissues from healthy 120 subjects have revealed that many cell types express SARS-CoV-2 entry receptor and 121 cofactors including angiotensin-converting enzyme-2 (ACE2), transmembrane serine 122 protease 2 (TMPRSS2), and furin, that are involved in viral entry, suggesting 123 susceptibility of these cells to infection. 7-10 Furthermore, scRNA-seq analysis of 124 bronchoalveolar lavage fluid (BALF), blood, oropharyngeal or lung tissues from COVID-125 19 patients has identified different types of SARS-CoV-2-infected cells, including 126 macrophages, neutrophils, type II pneumocytes (AT2), and ciliated and endothelial 127 cells. 11-14 However, in general, these studies detected very low numbers of infected 128 cells, which harbored low counts of viral genomes and transcripts. 11-16 The reason for 129 the discrepancy between the high numbers of cells expressing viral entry 130 receptors/cofactors and the low numbers of infected cells detected even in COVID-19 131 patients with severe pulmonary disease remains unclear. It has been reported that the 132 expression of ACE2, TMPRSS2 and furin is upregulated in macrophages, neutrophils, 133 AT2 and ciliated cells in COVID-19 patients compared to healthy controls, and that type 134 1 interferons (IFNs) induce the expression of ACE2 in epithelial cells, hence increasing 135 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 29, 2020. antibodies detected SARS-CoV-2 spike (S1) protein or nucleocapsid (NC) protein in 139 macrophages (cluster of differentiation 68 + , CD68 + and CD183 + ), and AT2, ciliated, Interleukin-6 (IL-6) is one of the most abundant cytokines detected in COVID-19 146 patients. 25 The expression level of IL-6 has been correlated with patient prognosis. 26-28 147 Treatment with IL-6 antagonists improved the survival and shortened the recovery 148 time. 29-33 However, the cell types responsible for increased IL-6 expression in the lung 149 are poorly defined, and understanding the relationship among IL-6 expression, the 150 extent of SARS-CoV-2 infection, and disease severity is incomplete. 151

In this study, we analyzed the expression of SARS-CoV-2 S1 and NC proteins in 152 postmortem lung tissues from five severe COVID-19 patients with various degrees of 153 lung damage. We performed multicolor immunofluorescence staining (IF) for the SARS-154

CoV-2 proteins, ACE2 protein as well as for lineage-restricted cell markers. We found 155 broad and extensive SARS-CoV-2 infection in the lungs of these patients, and more 156 infected cells were observed in more severe cases. Infected immune cell types were 157 comprised of monocytes and macrophages (CD68 + or CD163 + ), neutrophils (ELA-2 + ), 158 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Hematoxylin (#K8018, Agilent Dako). For IF, slides were deparaffinized at 95°C for 10 203 min, followed by 3 washes of xylene for 5 min. Dehydration was performed with step-204 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 29, 2020. . https://doi.org/10.1101/2020.09.25.20195818 doi: medRxiv preprint wise 10 min incubation of ethanol 100%, 95% and 75%, followed by water. Antigen 205 retrieval used citrate buffer pH 6.0 on microwave for 3 min at maximum potency, 206 followed by 15 min with 30% potency, and cooled down for 30 min at room temperature. 207

Slides were treated for 1 h with 5% bovine serum albumin (BSA) solution. Primary 208 antibodies were incubated overnight at 4°C, and secondary antibodies were incubated 209 for 1 h at room temperature. Slides were treated with Vector TrueVIEW™ 210 autofluorescence quenching (#SP-8400, Vector Laboratories) for 5 min followed by 211 incubation with 4',6-diamidino-2-phenylindole (DAPI) for 10 min. Table S2 summarizes 212 all antibodies and dilutions used in the study. 213 214

All five cases showed various combinations of DAD, pulmonary thromboemboli 216 and pulmonary consolidation (table S1, figure 1A and S1A). Case 4 had the most 217 extensive and severe pathologic changes, including early exudative phase of DAD, 218 vascular congestion and rare hyaline membranes. Air-spaces filled with blood were 219 noted in cases 1, 2 and 4. The least dramatic changes were found in cases 2 and 3; 220 both had incidental anthracosis. These findings are in agreement with previous 221 descriptions of lung pathology in COVID-19 cases. 6,21,34 222

Evidence of SARS-CoV-2 infection was detected by IHC with antibodies against 223 the S1 protein receptor binding domain (RBD) and NC protein. All 5 cases were positive 224 for SARS-CoV-2 proteins (figure 1B and S1B) with the widest distribution of infected 225 cells observed in tissues from case 4 followed by cases 1 and 5. The fewest infected 226 cells were observed in lung tissues from cases 2 and 3. Tissue damage was 227 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. macrophages were detected in all cases using CD68 as a monocyte, pan-macrophage 240 or M1 marker, and CD163 as a M2 cell marker. CD68 + cells were more abundant than 241 CD163 + cells ( figure 1D and S2A) . 242

We identified B cells by staining for CD19 and CD20. Although there was a 243 paucity of CD19 + cells, lung tissues from cases 1 and 2 showed pockets of infiltrating 244 CD20 + cells, which appeared to be surrounding venous structures ( figure 1D and S2B) . 245

Infiltration by T cell receptor (TCR) CD3ε + cells, predominantly T CD4 + helper, and 246 fewer T CD8 + cytotoxic cells, was detected in all cases ( figure 1D and S2C) . Interesting, 247 all cases were negative for FOXP3, a marker for natural T regulatory (Treg) cells ( figure  248   1D and S2C) . 249 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 29, 2020. . https://doi.org/10.1101/2020.09.25.20195818 doi: medRxiv preprint Since ACE2 protein expression was correlated with SARS-CoV-2 infection (figure 250

1B-C, and S1B-C), we performed dual IF staining for ACE2 and SARS-CoV-2 S1 251 protein ( figure 2A and S3 ). Most infected cells expressed ACE2 protein but we also 252 observed some ACE2-negative infected cells, which could be due to low expression 

( figure 4E and F, and S8D) . Interestingly, CD4 + , CD8 + or CD3ε + T cells presented either 299 as a membrane-associated pattern or as a dot-like organization pattern, possibly as the 300 result of membrane rupture following SARS-CoV-2 infection ( figure S8E) . We detected ACE2 protein expression in different immune cells including CD68 + 339 and CD163 + monocytes and macrophages, ELA-2 + neutrophils, CD56 + NK cells, and B-340

and T-cells; these findings are consistent with previous reports based on scRNA-seq 341 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. that decreased levels of CD4 + and CD8 + T cells were associated with worsening 363 COVID-19 outcomes, 48,50-52 and there was evidence of activation of CD8 + T and NK 364 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 29, 2020. . https://doi.org/10.1101/2020.09.25.20195818 doi: medRxiv preprint cells as well as exhaustion of T cells in the lung tissues from COVID-19 patients, 53-56 all 365 of which could contribute to the increased proinflammatory or anti-inflammatory 366 cytokines. In the lung tissues examined in this study, we noted a low level of CD20 + B-367 cells, and a lower level of CD8 + T as compared to CD4 + T cells. These results 368 suggested a general immunosuppression in the lungs of COVID-19 patients. Most of the 369 inflammatory infiltrates were characterized as CD68 + , CD163 + and CD45 + cells. By 370 contrast, we did not detect any FOXP3 + Treg cells, potentially supporting the T cell 371 exhaustion theory, 54-56 and the lack of Treg cells as a mechanism leading to failed 372 control of inflammatory cells and the excess inflammation observed in COVID-19 373

The inflammatory cytokine IL-6 is highly expressed in COVID-19 patients, and 375 elevated IL-6 levels have been associated with poor prognosis. 25-28 However, the 376 source of IL-6 in COVID-19 patients remains unclear. We detected a broad, increased Research Pathology/Pitt Biospecimen Core shared resource, which is supported in part 408 by award P30CA047904. This study was supported by UPMC Hillman Cancer Center 409 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Shown are ACE2 protein (pseudo color red) and SARS-CoV-2 S1 protein (RBD, pseudo 439 color green) in case 4 (Panel A); Alveolar epithelial type II / pneumocytes type II cells 440 (AT2) (pseudo color green), ACE2 (pseudo color red) and SARS-CoV-2 S1 protein 441 Shown are CD68, a monocytic lineage marker (pseudo color green), ACE2 (pseudo 455 color red) and S1 protein (pseudo color white) in case 4 (Panel A); CD163, a 456 macrophage M2 marker (pseudo color green), ACE2 (pseudo color red) and S1 protein 457 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 29, 2020. . https://doi.org/10.1101/2020.09.25.20195818 doi: medRxiv preprint (pseudo color white) in case 5 (Panel B); Elastase 2 (ELA-2), a neutrophil marker 458 (pseudo color green), ACE2 (pseudo color red) and S1 protein (pseudo color white) in 459 case 4 (Panel C); CD56, a NK cell marker (pseudo color red) and S1 protein (pseudo 460 color green) in case 5 (Panel D). Nuclei were stained with DAPI (pseudo color blue); 461 and quantification of CD68 + , CD163 + , ELA-2 + and CD56 + cells in five different fields in 462 each lung sample from all COVID-19 cases ( figure S6 ). S1-positive and/or ACE2 + cells 463 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 29, 2020. . https://doi.org/10.1101/2020.09.25.20195818 doi: medRxiv preprint 481 Figure 5 : Representative images of multicolor immunofluorescence staining of IL-482 6, SARS-CoV-2 S1 protein (RBD), and cellular markers in lung tissues from 483

Shown are IL-6 (pseudo color red) and S1 protein (pseudo color green) in case 4 (Panel 485 A); and IL-6 (pseudo color green), and cellular markers CD20 (pseudo color red), CD68 486 (pseudo color red) or CD163 (pseudo color red) in case 1 (Panel B). Nuclei were 487 stained with DAPI (pseudo color blue). 488 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 29, 2020. . https://doi.org/10.1101/2020.09.25.20195818 doi: medRxiv preprint

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