key: cord-0781943-3fnur0dj
authors: Ramos da Silva, Suzane; Ju, Enguo; Meng, Wen; Paniz Mondolfi, Alberto E; Dacic, Sanja; Green, Anthony; Bryce, Clare; Grimes, Zachary; Fowkes, Mary; Sordillo, Emilia M; Cordon-Cardo, Carlos; Guo, Haitao; Gao, Shou-Jiang
title: Broad SARS-CoV-2 cell tropism and immunopathology in lung tissues from fatal COVID-19
date: 2021-04-10
journal: J Infect Dis
DOI: 10.1093/infdis/jiab195
sha: ffe5c18a63f9f944dabbc94c7d041d90f3fb3a48
doc_id: 781943
cord_uid: 3fnur0dj

BACKGROUND: COVID-19 patients manifest with pulmonary symptoms reflected by diffuse alveolar damage (DAD), excessive inflammation, and thromboembolism. The mechanisms mediating these processes remain unclear. METHODS: We performed multicolor staining for SARS-CoV-2 proteins and lineage markers to define viral tropism and lung pathobiology in 5 autopsy cases. RESULTS: Lung parenchyma showed severe DAD with thromboemboli. Viral infection was found in an extensive range of cells including pneumocyte type II, ciliated, goblet, club-like and endothelial cells. Over 90% infiltrating immune cells were positive for viral proteins including macrophages, monocytes, neutrophils, and natural killer (NK), B and T cells. Most but not all infected cells were ACE2-positive. The numbers of infected and ACE2-positive cells are associated with extensive tissue damage. Infected tissues exhibited high inflammatory cells including macrophages, monocytes, neutrophils and NK cells, and low B- but abundant T-cells consisting of mainly T helper cells, few cytotoxic T cells, and no T regulatory cell. Robust interleukin-6 expression was present in most cells, with or without infection. CONCLUSIONS: In fatal COVID-19 lungs, there are broad SARS-CoV-2 cell tropisms, extensive infiltrated innate immune cells, and activation and depletion of adaptive immune cells, contributing to severe tissue damage, thromboemboli, excess inflammation and compromised immune responses.

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

Single cell RNA sequencing (scRNA-seq) analysis of lung tissues from healthy subjects have revealed that many cell types express SARS-CoV-2 entry receptors and cofactors, including angiotensin-converting enzyme-2 (ACE2), transmembrane serine protease 2 (TMPRSS2), and furin, suggesting susceptibility of these cells to infection [4] [5] [6] [7] .

Furthermore, scRNA-seq analysis of bronchoalveolar lavage fluid, blood, oropharyngeal or lung tissues from COVID-19 patients has identified different types of SARS-CoV-2-infected cells, including macrophages, neutrophils, type II pneumocytes (AT2), and ciliated and endothelial cells [8] [9] [10] . However, in general, these studies detected low numbers of infected cells, which harbored low counts of viral genomes and transcripts [8] [9] [10] . The reason for the discrepancy between high numbers of cells expressing viral entry receptors/cofactors and low numbers of infected cells even in COVID-19 patients with severe pulmonary disease remains unclear. Interestingly, the expression of ACE2, TMPRSS2 and furin is upregulated in macrophages, neutrophils, AT2 and ciliated cells in COVID-19 patients compared to healthy controls, and that type 1 interferons (IFNs) induce the expression of ACE2 in epithelial cells, hence increasing their susceptibility to infection [11] . However, another study showed that type 1 IFNs only induced an ACE variant expression but not ACE2 involved in viral entry [12] . Although immunohistochemistry (IHC) staining of lung tissues detected SARS-CoV-2 spike (S1) or nucleocapsid (NC) protein in macrophages (CD68 + and CD183 + ), and AT2, ciliated, goblet, club and endothelial progenitor cells, infected cells were often observed at low numbers, and the exact identity of many infected cells remain unknown [13- A c c e p t e d M a n u s c r i p t 5 15] . Questions remain regarding the SARS-CoV-2 targeted cell types and percentages of infected cells, and whether the extent of infection is correlated with expression of viral entry factors and disease status.

Interleukin-6 (IL6), among others including IL1, IL10, tumor necrosis factor- (TNF-), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN--induced protein 10 (IP10), IL17, and IL1 receptor antagonist (IL1RA), is one of the most abundant cytokines detected in patients with severe COVID-19, and its expression is correlated with patient prognosis [16] [17] [18] [19] [20] . Although treatments with IL6 antagonists have been shown to improve the survival and shortened the recovery time [21, 22] , other studies argued that IL6 is beneficial for infection [23, 24] . The cell types responsible for the increased IL6 expression in the lungs are poorly defined, and consequently the relationship between IL6 expression and the extent of SARS-CoV-2 infection, as well as disease severity has not been clearly defined.

We found broad SARS-CoV-2 infection in lungs of these patients, and more infected cells were observed in cases with more extensive pathology. Infected immune cell types were comprised of monocytes and macrophages (CD68 + or CD163 + ), neutrophils (ELA-2 + ), and natural killer (NK) (CD56 + ), B (CD20 + ), and T (CD3ε + , CD4 + and CD8 + ) cells, including Table 1 . Autopsies were performed with written consent from the legal next-of-kin, and specimens were obtained per the Autopsy Service protocol.

Anonymized lung specimens from five adults with other lung conditions but without 

We analyzed the expression of S1 and NC in postmortem lung tissues from five fatal COVID-19 patients. We performed multicolor IF for SARS-CoV-2 proteins, ACE2 protein and lineage-restricted cell markers. Postmortem biopsies were fixed with 10% neutral buffered formalin and embedded in paraffin. Slides were stained with H&E for histological analyses.

For IHC single staining (CD3, CD4, CD8, CD45, CD19, CD20 and FOXP3), the slides were deparaffinized at 60ºC for 30 min and rehydrated using a standard histology protocol of 3 changes of xylene of 5 min each followed by 3 changes of 100% ethanol, 2 with 95% ethanol and 1 with 70% ethanol for 1 min each, then rinsed in distilled water. Antigen retrieval was performed using citrate buffer (Agilent Dako, Santa Clara, CA, USA) in Decloaking chamber at 123°C for 2 min. The slides were stained using an Autostainer Plus (Agilent Dako) platform with TBS-T rinse buffer (Agilent Dako). The IHC slides were treated with 3% hydrogen peroxide for 10 min. The primary antibodies were applied at room temperature for 30 min, followed by 30 min of secondary antibodies Envision + Dual Link (Agilent Dako) HRP polymer at room temperature. Slides were exposed to 3,3, Diaminobenzidine+ (Agilent Dako) for 5 min, and counterstained with Hematoxylin (Agilent Dako).

For IF, slides were deparaffinized at 95°C for 10 min, followed by 3 washes of xylene for 5 min. Dehydration was performed with step-wise 10 min incubation of ethanol at 100%, 95% and 75%, followed by water. Antigen retrieval used citrate buffer at pH 6.0 on microwave for 3 min at maximum potency, followed by 15 min with 30% potency, and cooled down for 30 min at room temperature. Slides were treated for 1 h with 5% bovine serum albumin (BSA) solution. Primary antibodies were incubated overnight at 4°C, and secondary A c c e p t e d M a n u s c r i p t 8 antibodies were incubated for 1 h at room temperature. Slides were treated with Vector TrueVIEW™ autofluorescence quenching (Vector Laboratories, Burlingame, CA, USA) for 5 min followed by incubation with 4',6-diamidino-2-phenylindole (DAPI) for 10 min.

Supplementary Table 2 summarizes all antibodies and dilutions used in the study.

Lung tissues from all five cases showed various combinations of diffuse alveolar damage (DAD), pulmonary thromboemboli and pulmonary consolidation ( Figure 1A , Table 1 and Supplementary Figure 1A) . Case 4 had the most extensive and severe pathologic changes, including early exudative phase of DAD, vascular congestion and rare hyaline membranes. Air-spaces filled with blood were noted in cases 1, 2 and 4. The least dramatic changes were cases 2 and 3; both had incidental anthracosis. These findings are consistent with previous descriptions of lung pathology in COVID-19 patients [25] [26] [27] [28] .

Evidence of SARS-CoV-2 infection was detected by IHC with antibodies against the S1 protein receptor binding domain (RBD) and NC protein. cases compared to controls using CD68 as a monocyte, pan-macrophage or M1 marker, and CD163 as a M2 cell marker ( Figure 1D and Supplementary Figure 4A , 5A). CD68 + cells were more abundant than CD163 + cells in COVID-19 cases.

We identified B cells by staining for CD19 and CD20. Almost no B cells were detected in controls (Supplementary Figure 5B ). However, a few CD19 + cells were detected in COVID-19 tissues, and cases 1 and 2 showed pockets of infiltrating CD20 + cells, which appeared to be surrounding venous structures ( Figure 1D and Supplementary Figure 4B) .

Infiltration by T cell receptor (TCR) CD3ε + cells, predominantly T CD4 + helper, and fewer T CD8 + cytotoxic cells, was detected in COVID-19 cases but with no obvious difference from controls ( Figure 1D 

Since we observed extensive damage to lung tissues ( Figure 1A 

Since we detected vast infiltrations by innate immune response cells, we examined SARS-CoV-2 infection in these cells. Monocytes and macrophages (CD68 + or CD163 + ) were widely infected by SARS-CoV-2 ( Figure 3A , B, E and Supplementary Figure 9A, B) . Neutrophils, positive for elastase-2 (ELA-2 + ) protein, were extensively infected by SARS-CoV-2 ( Figure   3C , E and Supplementary Figure 9C ). The extent of neutrophil infection was positively correlated with ACE2 protein expression in all cases except for case 5, for which 96% of cells expressed ACE2, but only 19% had detectable S1 protein ( Figure 3E ). We detected S1 protein in NK cells (CD56 + ) ( Figure 3D and Supplementary Figure 9D) ; however the percentages of infected cells were much smaller than other cell types examined, ranging from 0 to 40% ( Figure 3E ).

Among the adaptive immune cells, B cells (CD20 + ) were found in low numbers in the lung specimens, but ACE2 protein expression and SARS-CoV-2 infection were positively Figure 11D) . Interestingly, CD4 + , CD8 + or CD3ε + T cells presented either as a membrane-associated pattern or as a dot-like organization pattern, possibly as the result of membrane rupture following SARS- Figure 11E) .

To exclude the possibility that the detection of S1 and NC proteins in different cells were not due to any unspecific events such as engulfment, we stained for NSP8 and NSP13 proteins.

Both proteins are not associated with virions and expressed only during viral replication.

Indeed, we detected wide expression of both proteins in COVID-19 cases with NSP8 being more abundant than NSP13 protein (Supplementary Figure 12 ). Dual staining with CD68 revealed NSP8 and NSP13 expression in monocytes and macrophages. These results provided clear evidence of SARS-CoV-2 replication in these lung tissues.

IL6 is one of the most abundant cytokines detected in COVID-19 patients and its level is correlated with prognosis [16, [20] [21] [22] . Upregulation of IL6 in lungs of COVID-19 patients have been reported [30] . In all cases, we found IL6 expression in most cells examined, with or without SARS-CoV-2 infection ( Figure 5A and Supplementary Figure 13 ). Tissue regions with higher numbers of infected cells also had more cells expressing IL-6 (Supplementary Figure 13 ). Compared to controls, COVID-19 cases generally had higher numbers of IL6positive cells albeit strong staining in some areas was observed with control 2, which could A c c e p t e d M a n u s c r i p t 13 be due to its specific lung condition (Supplementary Figure 13, 14) . Dual staining of IL6 and CD20, CD68 or CD163 in COVID-19 cases revealed that B cells, monocytes and macrophages were strongly positive for IL6 ( Figure 5B ).

Respiratory symptoms are a prominent complaint during most SARS-CoV-2 infections, and progressive respiratory dysfunction is a major feature of fatal COVID-19 [1, 3] .

Our results present a direct visualization of multiple cell types infected by SARS-CoV-2 from patients who died of COVID-19, and offer insights into the pathogenesis of the overwhelming damage found in lung tissues.

The detection of S1 or NC protein revealed widespread SARS-CoV-2 infection in lung tissues, including multiple lung parenchymal cell types and multiple cell types involved in immune responses. These viral proteins were most abundant in specimens with the most histologic evidence of tissue damage. As expected, the extent of infection was positively correlated with ACE2-positive cells numbers. However, we also found SARS-CoV-2 infection in ACE2-negative cells, supporting a role for other possible receptors for viral entry into ACE-negative cells. It is important to note that there may be multiple factors that might influence ACE2 expression during SARS-CoV-2 infection and should be considered as confounders. For example, ACE2 expression can be stimulated by IFNs [7, 11] . Furthermore, ACE2 may have a role in protection against severe acute lung failure, as has been reported in severe COVID-19 patients [31] .

We obtained direct evidence for widespread ACE2 expression and extensive SARS- [32] ; and pneumocytes, ciliated, secretory and lymphomononuclear cells by IHC [15] in lung tissues from COVID-19 patients. In ex-vivo culture, SARS-CoV-2 infects type I pneumocytes, ciliated, goblet and club cells as well as conjunctival mucosa [14] .

We detected ACE2 protein expression in different immune cells including CD68 + and CD163 + monocytes and macrophages, ELA-2 + neutrophils, CD56 + NK cells, and B-and Tcells; these findings are consistent with previous reports based on scRNA-seq studies [4, 6, 7, 33] . ACE2 expression detected by flow cytometry in T cells from lung tissues from COVID-19 patients has been reported [34] . Notably, we found rates of SARS-CoV-2 infection approaching 100% for most types of immune cells, in contrast to a much lower infection rate in NK cells ( Figure 3E, 4F) . Although SARS-CoV-2 proteins have been detected in macrophages by IHC [35] , to our knowledge, our study is the first to demonstrate and quantify SARS-CoV-2 infection in different types of T cells and also in neutrophils. Our observation of SARS-CoV-2 infection of neutrophils was in contrast to the results of a study, which failed to detect any infected neutrophils [34] . The observed immune responses in lungs of fatal COVID-19 patients are different from acute infection of respiratory syncytial virus, which has strong neutrophil response positively correlated with disease severity and mediated by IL8, dendritic cells migration to lungs as the primary antigen-presenting cells, and an initial systemic T-cell lymphopenia followed by a pulmonary CD8+ T-cell response to mediate viral clearance [36] . SARS-CoV-2 infection caused compromised immune response by dysregulating recruitment of immune cells [39] . Decreased levels of CD4 + and CD8 + T cells were associated with worsening COVID-19 outcomes [39, 40] , and there was evidence of activation of CD8 + T and NK cells as well as depletion of T cells in the lung tissues from COVID-19 patients [41] [42] [43] , all of which could contribute to the increased proinflammatory or anti-inflammatory cytokines. We found that these immune cells were effectively infected by SARS-CoV-2, and noted a low level of CD20 + B-cells, and a lower level of CD8 + T as compared to CD4 + T cells. These results suggested immunosuppression in the lungs of COVID-19 patients. Most of the inflammatory infiltrates were characterized as CD68 + , CD163 + and CD45 + cells. We did not detect an increase of FOXP3 + Treg cells, which potentially support the T cell exhaustion theory [41] [42] [43] , and the lack of Treg cells as a mechanism leading to failed control of excess inflammation observed in COVID-19 patients.

The inflammatory cytokine IL6 is highly expressed in COVID-19 patients, and an elevated IL6 level is associated with poor prognosis [16, [20] [21] [22] . However, the source of IL6 in COVID-19 patients remains unclear. We detected a broad, increased IL6 expression in all cell types in the lung specimens from all the cases. Furthermore, IL6 expression was associated with the detection of SARS-CoV-2 proteins, as well as with the degree of tissue damage. Of note, our findings were consistent with previous studies reporting that patients with a high level of IL6 and a poor prognosis also had decreased CD8 + T, NK and Treg cells [41] . protein (pseudo color red) and SARS-CoV-2 S1 protein (RBD, pseudo color green) in case 4. B, Alveolar epithelial type II / pneumocytes type II cells (AT2) (pseudo color green), ACE2

(pseudo color red) and SARS-CoV-2 S1 protein (RBD, pseudo color white) in case 5. C, Tyrα-tubulin (pseudo color red) and SARS-CoV-2 NC protein (pseudo color green) in case 5. D, M a n u s c r i p t 23 MUC5AC (goblet cells, pseudo color green), ACE2 (pseudo color red) and SARS-CoV-2 S1 protein (RBD, pseudo color white) in case 4. E, MUC5B (club-like cells, pseudo color green), ACE2 (red) and SARS-CoV-2 S1 protein (RBD, pseudo color white) in case 4. F, CD34

(pseudo color green), ACE2 (red) and SARS-CoV-2 NC protein (pseudo color white) in case 5. G, CD31 (pseudo color red) and SARS-CoV-2 NC protein (pseudo color green) in case 1.

Nuclei were stained with DAPI (pseudo color blue). Scale bar represents 20 µm. (pseudo color red) and S1 protein (pseudo color white) in case 4. B, CD163, a macrophage M2 marker (pseudo color green), ACE2 (pseudo color red) and S1 protein (pseudo color white) in case 5. C, Elastase 2 (ELA-2), a neutrophil marker (pseudo color green), ACE2

(pseudo color red) and S1 protein (pseudo color white) in case 4. D, CD56, a NK cell marker (pseudo color red) and S1 protein (pseudo color green) in case 5. For A to D, nuclei were stained with DAPI (pseudo color blue). Scale bar represents 20 µm. E, Quantification of CD68 + , CD163 + , ELA-2 + and CD56 + cells in five different fields in each lung sample from all COVID-19 cases (Supplementary Figure 9 ). S1-positive and/or ACE2 + cells were counted in the same fields and shown as percentages of positive cells. Statistical significance was determined using one-way analysis of variance (ANOVA) and Tukey's test for post hoc analysis. P values indicated when p < 0.05. CoV-2 S1 protein (RBD), and representative cellular markers in lung tissues from COVID-19 patients. A, IL6 (pseudo color red) and S1 protein (pseudo color green) in case 4. B, IL6 (pseudo color green), and cellular markers CD20 (pseudo color red), CD68 (pseudo color red) or CD163 (pseudo color red) in case 1. Nuclei were stained with DAPI (pseudo color blue). Scale bar represents 20 µm.

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