Aveolar Macrophage Activation and Cytokine Storm in the Pathogenesis of Severe COVID-19

The coronavirus disease-19 (COVID-19) caused by SARS-CoV-2 infection can lead to a series of clinical settings from non-symptomatic viral carriers/spreaders to severe illness characterized by acute respiratory distress syndrome (ARDS)1,2. A sizable part of patients with COVID-19 have mild clinical symptoms at the early stage of infection, but the disease progression may become quite rapid in the later stage with ARDS as the common manifestation and followed by critical multiple organ failure, causing a high mortality rate of 7-10% in the elderly population with underlying chronic disease1-3. The pathological investigation in the lungs and other organs of fatal cases is fundamental for the mechanistic understanding of severe COVID-19 and the development of specic therapy in these cases. Gross anatomy and molecular markers allowed us to identify, in two fatal patients subject to necropsy, the main pathological features such as exudation and hemorrhage, epithelium injuries, inltration of macrophages and brosis in the lungs. The mucous plug with brinous exudate in the alveoli and the activation of alveolar macrophages were characteristic abnormalities. These ndings shed new insights into the pathogenesis of COVID-19 and justify the use of interleukin 6 (IL6) receptor antagonists and convalescent plasma with neutralizing antibodies against SARS-CoV-2 for severe patients. equally to this work. Authors Hematoxylin and eosin (HE) staining of the slides of 10% neutral formaldehyde-xed, paran-embedded tissues was performed and carefully reviewed on each patient. Alcian blue/periodic acid-Schiff (AB-PAS) staining and Masson staining were carried out for the examinations of mucus, brin and collagen ber in lung tissues. Immunohistochemical staining was performed on the slides of lung tissues from two patients. A panel of primary antibodies were used, including the macrophage marker CD68 (Monoclonal mouse anti-human CD68, clone KP1; 1:100; Dako Omnis, Agilent); T-lymphocyte marker CD3 (Monoclonal rabbit anti-human CD3, clone SP7; ready-to-use; Dako Omnis, Agilent), CD4 (Monoclonal mouse anti-human CD4, clone 4B12; ready-to-use; Dako Omnis, Agilent) and CD8 (Monoclonal mouse anti-human CD8, clone C8/144B; ready-to-use); B-lymphocyte markers CD20 (Monoclonal mouse anti-human CD20cy, clone L26; ready-to-use); natural killer cell/T cell (NK/T cell) marker CD56 (Monoclonal mouse anti-human CD56, clone 123C3; ready-to- use), and the markers of Programmed Cell Death–1 (PD–1 Monoclonal mouse anti-human PD- 1, clone UMAB199; ready-to-use) and Programmed Cell Death-Ligand 1 (PD-L1 Monoclonal mouse anti-human PD-L1, clone 22C3; ready-to-use). Antibodies specic for chemokine and inammatory cytokines were also used, including interleukin 6 (polyclonal rabbit anti-IL–6 human; 1:250; abcam), interleukin


Introduction
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Results And Discussion
The two body donors of fatal cases with COVID-19 were a 53 years old female and a 62 years old male, respectively. Both patients had progressively decreased lymphocytes with elevated serum IL-6 and C reactive protein (CRP) levels in the late stage of disease (Supplementary Table 1), which was consistent with recent report1,2. The gross anatomy of the lung showed moderate bilateral pleural effusion and pleural adhesion in the two patients. The hepatization of lung tissues was observed on the cut-surface of the collapsed and consolidated lungs. The microscopic manifestation of the lung injury was consistent with diffuse alveolar damage (DAD). Alveolar cavities were lled with a large number of macrophages with scattered neutrophils and lymphocytes (Fig. 1a). The massive serous (Fig. 1b) and brinoid exudate in the alveolar spaces were shown by the Masson staining (Fig. 1c, d). The acidic mucopolysaccharides from a large amount of mucinous secretion were observed by the Alcian blue-periodic acid-Schiff (AB-PAS) staining in the bronchi and bronchioles, terminal bronchioles and pulmonary alveoli (Fig. 1e, f). A lot of mucus in the distal respiratory tract lined by mucous cells were shown, reminiscent of the morphology of mucoid adenocarcinoma (Fig. 1g). The peribronchiolar metaplasia (PBM) with interstitial brous hyperplasia but without invasive growth of atypical cells was observed. The mucous plug with brinous exudate in the alveoli and terminal bronchioles formed the cribriform pattern. The bronchial phlegm combined with epithelial detachment and brinoid exudate was visible (Fig. 1h).
The hyaline membranes and widened alveolar walls with collagen bers proliferation and lymphocyte in ltration were observed in alveoli occasionally (Extended Data Fig. 1a, b). Focal or patchy hemorrhage with brinous exudate were seen in the alveolar cavities and interstitial tissues (Extended Data Fig. 1c, d).
The broken alveolar walls ushed by huge hemorrhagic effusion formed the "blood lake". The endothelial cells of small pulmonary arteries were swollen and shed (Extended Data Fig. 1e). Mixed thrombi were present in small veins (Extended Data Fig. 1f).
Intensive loss of bronchiole and alveolar epithelial cells was remarkable (Fig. 2a, b) while abundant swollen and degenerated alveolar cells desquamated in the alveoli (Fig. 2c, d). Patchy type II pneumocytes proliferated with atypical changes, including enlarged nuclei, clearing of nuclear chromatin, prominent nucleoli and suspected viral inclusions (Fig. 2e, f). The notable proliferation of type alveolar epithelial cells resembled the morphological changes of atypical adenomatous hyperplasia, in situ adenocarcinomas, or even invasive adenocarcinoma. Thickened alveolar walls and widened interstitial tissues were accompanied by lymphocyte in ltration and broblast proliferation (Fig. 2g, h).
Notably, the alveolar macrophages signi cantly increased and lled in a part of the alveolar cavities with scattered neutrophils and lymphocytes. CD68, one of the scavenger receptors, is a well-documented speci c surface marker of macrophages4. The alveolar macrophages were presented in diverse forms, including aggregation in small clusters (Fig. 3a, b), diffused distribution (Fig. 3c), single macrophage exhibiting intracytoplasmic phagocytosis, spherical acidophilic hyaline bodies or hemophagocytic phenomenon (Fig. 3d, e), and multinucleated giant cells (Fig. 3f). Furthermore, using immunohistochemistry approach, we examined several chemokine and in ammatory cytokines secreted by alveolar macrophages including IL-6, IL-10 and TNFα with speci c antibodies. IL-6 and TNFα were moderately expressed in macrophages (Fig. 3g, i), while the expression of IL-10 was strong (Fig. 3h). Besides, extensive and strong expression of Programmed Death-Ligand 1 (PD-L1) was observed (Fig. 3j).
In general, the degree of in ltration of lymphocytes into the pulmonary tissues was much inferior to that of macrophages, although some focal lymphocyte in ltrations were present in lungs (Extended Data We carefully examined the heart and kidney in the two donor bodies. No obvious gross abnormalities were observed. Nevertheless, microscopical abnormalities were observable in both organs. Multifocal myocardial degeneration was present in the heart, together with myocardial atrophy and interstitial brous tissue hyperplasia (Extended Data Fig.3a). A few CD20-positive B cells and CD3-positive T cells were scattered (Extended Data Fig. 3b, c). In the kidneys, normal renal structures were retained. However, the brotic glomeruli and edematous tubular epitheliums (Extended Data Fig.3d) were focally present with a small amount of in ltrating B (Extended Data Fig.3e) and T lymphocytes (Extended Data Fig.3f). It is worth noting that no viral particles were found in parenchymal cells in both heart and kidney.
Next, we examined lymph nodes and other lymphoid organs. Notably, lymph nodes in the pulmonary hilum were swollen whereas splenic volume was slightly reduced with shrunken capsule in the two cases. Morphological changes of the pulmonary hilum lymph nodes were characterized by an obvious dilation of cortical sinuses with numerous macrophages (Extended Data Fig.4e). In the spleen, the lymphocytes in the white pulp were slightly reduced with in ltration of macrophages in the red pulp (Extended Data However, the pathology of lungs with SARS-CoV-2 infection also exhibited some distinct features as compared to that found in SARS patients. The hyaline membranes in alveoli, which constituted major anatomical abnormalities leading to gas exchange obstruction in SARS, were uncommon in COVID-19. On the other hand, we observed mucous plugs in all respiratory tracts, terminal bronchioles and pulmonary alveoli in COVID-19, and this was neither described in SARS5,7-11 nor in the recently reported autopsy studies on COVID-19 patients12,13. Another unique feature of COVID-19 was the excessive mucus secretion with serous and brinous exudation, which could aggravate the dysfunction of ventilation. These ndings suggested the existence of different pathogenic mechanisms responsible for the hypoxemia between COVID-19 and SARS patients. We found the hyperplasia and peribronchiolar metaplasia of mucosal epithelium, a phenomenon which might result from the in ammation-induced pulmonary tissue reparatory processes or even proliferative reaction of cells originated from bronchioles and terminal bronchioles. We assume that the mucus aggregation in distal respiratory tracts by peribronchiolar metaplasia of mucosal epithelium as a result of in ammation-induced reparatory changes should play a part in the sputum suction failure in very severe COVID-19 patients as previously reported12. Of particular note, we found the alveolar macrophages with SARS-CoV-2 infection were expressing ACE2, a well-established receptor for both SARS-CoV and SARS-CoV-2 (Extended Data Fig.5). It was reported that SARS-CoV could occasionally be identi ed in the alveolar macrophages9. In COVID-19 patients, the extraordinary aggregation and activation of these macrophages could occupy a central position in pathogenesis of the very severe "in ammatory factor storm" or "cytokine storm". Therefore, the spectacular in ltration and activation of alveolar macrophages in COVID-19, especially among patients with severe and critical stages of ARDS, might represent the shift of classically activated phenotype (M1) to alternatively activated phenotype (M2) of alveolar macrophages, whereas this shifted property of alveolar macrophages could contribute to the in ammatory injuries and brosis of respiratory tracts14. To further address the issue of accumulation of macrophages in lung tissues and to explore the potential function of macrophages in response to SARS-CoV-2, we incubated puri ed and Fc-tagged spike proteins (S protein), which contains the receptor binding domain (RBD) responsible for the entry of SARS-CoV-2 into the host cells15, with white blood cell samples from six healthy donors. The possible location of the S protein on the surface of these white blood cells was examined by ow cytometry analysis. To our surprise, the S protein interacted with CD68-expression monocytes/macrophages but not with T or B lymphocytes, suggesting a direct viral infection of the macrophage/monocytes. We then determined the expression of ACE2 on the surface of macrophages. Indeed, an expression pattern similar to the binding of S protein by monocytes/macrophages was observed (Fig. 4b). These ndings highlighted the role of macrophages as direct host cells of SARS-CoV-2 and potential drivers of "cytokine storm syndrome" in COVID-19.
Additionally, an elevated serum IL-6 was observed in the two cases in this study and also in some other very recent reports16. These features were similar to the pathogenesis of "cytokine storm syndrome" in patients with hemophagocytic lymphohistocytosis (HLH) or macrophage activation syndrome (MAS)17,18. The blockage of cytokine storm using anti-IL-6 or IL-6R antibody, such as Tocilizumab, has promising therapeutic effects and clinical practice in the treatment of MAS or HLH. Therefore, our data are in support of the bene cial use of anti-IL-6/IL-6R antibody for the inhibition of alveolar macrophage activation as well as in ammatory injuries in COVID-19 patients. Recently, the Tocilizumab therapy has been recommended in the Guideline of Diagnosis and Treatment of COVID-19 (version 7) by the National Health Commission.
The fact that the known ACE2-exressing cells19-21, including type II alveolar epithelial cells, alveolar macrophages, intestinal epithelial cells and spermatogenic cells, were all found infected by SARS-CoV-2 infection suggests the necessarily of clinical tests of SARS-CoV-2 in feces samples and the blockade of possible fecal-oral transmission22. Infected submucosa ganglion cells in small intestine were never reported before. Whether it could be the host cells for long-term coexistence of virus or not remains to be investigated. It is worth noting that remarkable viral infection persisted even at the end stage of COVID-19, when the viremia was well passed in the great majority of patients. Under this circumstance, use of speci c anti-viral therapy should be encouraged. Recently, our group identi ed the convalescent plasma (CP) from recovered COVID-19 to be a speci c and effective therapy for this disease, especially in severe cases, since the overwhelming majority of patients presented high neutralizing antibody titers against SARS-CoV-2 and the preliminary results of a phase I trial of CP showed very promising effects (Duan K, ZHANG XX, YANG XM et al, submitted).
Still, some issues remain to be addressed in future studies: rst, what are the molecular and cellular mechanism underlying the infection of alveolar macrophages of SARS-CoV-2 should be illustrated so that a deeper understanding of the pathogenic role of viral infection and the mechanism for its escape from immune reaction can be achieved. These studies may accelerate smart drug and vaccine design targeting vulnerabilities of viral proliferation; Second, in the two cases studied here and in some other recent reports, there is a remarkable reduction of both CD4 and CD8 cells in the peripheral blood in COVID-19 patients. A graded decrease of T cells was found with increase clinical severity of COVID-19.
Intriguingly, there is a negative correlation between the extent of T lymphocytopenia and increased IL-6 and Il-8 levels in the serum. The causal relationship between these two phenomena should be addressed; Third, in this study, no ACE2-expression was found on the surface of T cells, which may eliminate the possibility of a direct toxic effect of SARS-CoV-2 on distinct subsets of T cell population. However, only a small number of T lymphocytes were observed in the in ammatory lung tissues. This situation seems to be a paradox to the initial assumption that the severe T cell reduction could be ascribed to a tremendous in ltration of T cells into damaged lung tissues in response to the effect of IL-6 and other cytokines. The detailed mechanism of T cell depletion in severe COVID-19 certainly requires in-depth study in the future either among patients or in experimental animal models. Declarations obtained. This study was approved by the Medical Ethics Committee of the National Health Commission of China. The autopsy procedures were performed in the negative pressure-ventilation P3 Laboratory.

Histological, histochemical and immunohistochemical staining
Hematoxylin and eosin (HE) staining of the slides of 10% neutral formaldehyde-xed, para n-embedded tissues was performed and carefully reviewed on each patient. Alcian blue/periodic acid-Schiff (AB-PAS) staining and Masson staining were carried out for the examinations of mucus, brin and collagen ber in lung tissues. Immunohistochemical staining was performed on the slides of lung tissues from two patients. A panel of primary antibodies were used, including the macrophage marker CD68 (Monoclonal mouse anti-human CD68, clone KP1; 1:100; Dako Omnis, Agilent); T-lymphocyte marker CD3 (Monoclonal rabbit anti-human CD3, clone SP7; ready-to-use; Dako Omnis, Agilent), CD4 (Monoclonal mouse anti-antibodies, according to manufactures' instructions. BD LSRFortessa™ X-20 was used for ow cytometry analysis.