The Defenders of the Alveolus Succumb in COVID-19 Pneumonia to SARS-CoV-2 and Necroptosis, Pyroptosis, and PANoptosis

Abstract

Alveolar type II (ATII) pneumocytes as defenders of the alveolus are critical to repairing lung injury. We investigated the ATII reparative response in coronavirus disease 2019 (COVID-19) pneumonia, because the initial proliferation of ATII cells in this reparative process should provide large numbers of target cells to amplify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus production and cytopathological effects to compromise lung repair. We show that both infected and uninfected ATII cells succumb to tumor necrosis factor-α (TNF)-induced necroptosis, Bruton tyrosine kinase (BTK)-induced pyroptosis, and a new PANoptotic hybrid form of inflammatory cell death mediated by a PANoptosomal latticework that generates distinctive COVID-19 pathologies in contiguous ATII cells. Identifying TNF and BTK as the initiators of programmed cell death and SARS-CoV-2 cytopathic effects provides a rationale for early antiviral treatment combined with inhibitors of TNF and BTK to preserve ATII cell populations, reduce programmed cell death and associated hyperinflammation, and restore functioning alveoli in COVID-19 pneumonia.

Keywords: BTK; COVID-19 pneumonia; PANoptosis; SARS-CoV-2; TNF; lung repair; necroptosis; pyroptosis; type II pneumocytes.

Figure 1.

SARS-CoV-2 replication and cytopathic effects in bronchiolar epithelium and ATI and ATII pneumocytes. RNAscope in situ hybridization on autopsy lung tissue: red, SARS-CoV-2 viral RNA, and hematoxylin counterstain. A, SARS-CoV-2 RNA in fused cells in a bifurcation of terminal bronchiolar epithelium. Viral RNA is concentrated in small RBs. B, Alveoli lined by thin fused SARS-CoV-2 RNA⁺ ATI cells. Arrow points to a SARS-CoV-2 RNA⁺ cell, identified morphologically as an ATII cell. C, Syncytial clusters of fused SARS-CoV-2 RNA⁺ ATII cells with viral RNA in dark-staining RBs. D, Large desquamated syncytial mat with viral RNA in RBs. Abbreviations: AT, alveolar type; RB, round body; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2.

SARS-CoV-2 spread to and replication in the lung. Individual SARS-CoV-2 virions detected by in situ hybridization with ELF-97 substrate appear green and are approximately 0.25 μm. Nuclei stained blue with DAPI. Large numbers of virions are amassed in RBs of varying size. A, Virus production and spread in bronchiolar epithelium leave a visible trace of the hub and spoke mode of spread from branching terminal bronchioles into the lung parenchyma (encircled). B, Virus is concentrated in RBs in fused lysed ATI cells lining alveolar walls. C, Virus spread and fusion of spatially contiguous ATII cells generate focal clusters of virus-producing cells. D, Syncytial mat of virus RBs in lysed epithelium. Abbreviations: AT, alveolar type; RB, round body; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3.

SARS-CoV-2 replication and spread in type I interferon-negative ATII pneumocytes. A, Red cells are viral RNA⁺; green cells are napsin A⁺ ATII cells; red-green cells are infected ATII cells; nuclei stained blue with DAPI. The viral RNA⁺ ATII cells in numbered regions 1–5 are spatially contiguous, consistent with spread of infection to susceptible type II cells in close spatial proximity. Region 6 shows a napsin A⁻ viral RNA⁺ cell overlying lysed viral RNA⁺ epithelial syncytium. Region 7 shows a viral RNA⁺ napsin A⁺ and napsin A⁻ cell conjugate, consistent with acquisition of viral RNA in macrophages by phagocytosis. B, SARS-CoV-2 virions and intracellular RNA, green. Virus spread and fusion of spatially contiguous ATII cells generates focal clusters of virus-producing cells. C, SARS-CoV-2 RNA⁺ cells, red; type I interferon, green; nuclei stained blue with DAPI. Viral RNA⁺ cells are type I interferon⁻, and interferon⁺ cells are viral RNA⁻. Abbreviations: AT, alveolar type; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 4.

TNF-induced necroptosis in SARS-CoV-2–infected ATII cells. A, Graphic of TNF-induced necroptosis. SARS-CoV-2–infected ATII cells express TNF and downstream components of the necroptotic pathway, including NINJ1, in a latticework of RBs that also contain SARS-CoV-2 RNA and virus. Activation of the TNF necroptotic pathway generates a porous latticework through which cell contents and virus are emptied, leaving residual cell membranes lining alveolar walls, and disrupted cells and porous latticework in alveoli. B–D, TNF⁺ cells and structures: brown, SARS-CoV-2 viral RNA; red, hematoxylin counterstain. B, Stages of porous latticework formation. Top, densely stained large TNF⁺ vRNA⁺ bodies in a cell in which emptying of cell contents blurs the red-brown staining at the cell margins (orange arrowheads). Middle, brown arrowheads point to the cell border lined by darkly stained TNF⁺ bodies. Red arrowhead points to TNF⁺ vRNA⁺ pores emerging from the disrupted cell margins. Lower, red arrow points to vRNA emptying from a TNF⁺ pore. C, Syncytial mass of TNF⁺ vRNA⁺ cells lining alveolar space. Orange arrowheads point to blurred staining of vRNA and TNF emptying from the porous latticework. The porous latticework within the cells is composed of the RBs in Figure 1 and Figure 2, now seen as a TNF pore surrounding vRNA. D, Remnants of fused cells lining an alveolar wall. The red double-headed arrow points to TNF⁺ vRNA⁺ pores of varying size. Abbreviations: AT, alveolar type; RB, round body; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF, tumor necrosis factor; vRNA, viral RNA.

Figure 5.

TNF-induced necroptosis of uninfected ATII cells. A, Graphic showing TNF, downstream necroptosis pathway components, and NINJ1 are initially expressed in latticework bodies. Activation of the pathway generates a porous latticework composed initially of darker staining latticework bodies that stain with decreasing intensity as pathway and cell contents are progressively emptied. In the process, the cells enlarge and are eventually disrupted, releasing the pores and porous latticework into alveolar space. Because ATII cells proliferate in the reparative response to lung injury, alveoli are lined and filled by contiguous ATII cells. Activation of the necroptotic pathway fuses adjacent cells to amplify and shape the cytopathology, giving rise to multinucleated giant cells, syncytia, and residual cell membranes lining and detached from alveolar walls. B, Brown-stained TNF⁺ cells and structures, and hematoxylin counterstain. Fused syncytial mass of TNF⁺ cells lining alveolar wall (traced). Arrow points to a trinucleate cell with “owl's eye” appearance created by residual clumped chromatin in a largely emptied nucleus. Asynchronous emptying results in variable clearing with the most extensive clearing in the nucleus at the top of the cell. C, Red-stained RIPK3⁺ cells and structures and hematoxylin counterstain. Alveolar space lined and filled by fused RIPK3⁺ cells. Line traces a RIPK3⁺ syncytium in which the latticework pores retain RIPK3 staining (red arrowhead) or are largely emptied (outlined white arrowhead) The black arrowhead points to residual cell membranes. D, Brown-stained cells and structures and hematoxylin counterstain. Trinucleate cell with latticework at various stages of emptying from early darkly staining latticework to later stages in which the intensity of staining decreases as NINJ1 and cell content empty. Abbreviations: AT, alveolar type; TNF, tumor necrosis factor; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 6.

BTK-induced pyroptosis of uninfected ATII cells. A, Graphic showing expression of BTK and downstream pyroptotic/NLRP3 inflammasome pathway components in latticework bodies leads to cell fusion and subsequent disruption and emptying of the cells and latticework. In Supplementary Figure 4B and C, stages labeled 1–4 show an initial stage 1 of a largely intact cell and latticework RBs with cell disruption and progressive emptying of the latticework in stages 2 and 3 to create cavities and walls in stage 4, formed from cell remnants and residual cell membranes. This asynchronous process imparts a characteristic honeycombed and moth-eaten appearance to foci composed of cavities with cell remnants, lined by residual cell membranes and cells at earlier stages of pyroptosis. B, Red-stained BTK⁺ cells and structures and hematoxylin counterstain. Arrows point to remnants of BTK⁺ cells in cavities and arrowheads to residual cell membranes that constitute cavity walls. C, Brown-stained CASP1⁺ cells and structures, immunohistochemical staining, and hematoxylin counterstain. Arrows point to cell remnants in cavities and arrowheads point to residual cell membranes in the cavity walls. D, Red-stained gasdermin D (GSD)⁺ cells and structures, and hematoxylin counterstain. In the deconvoluted image, GSD is red and hematoxylin is blue. Boxes correlate the appearance of latticework bodies and pores of the images with or without deconvolution to reveal a hematoxylin-stained component in nuclear pores. Abbreviations: AT, alveolar type; BTK, Bruton tyrosine kinase; CASP1, caspase 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 7.

Apoptosis and PANoptosis of uninfected ATII cells. A, Graphic showing intrinsic pathway apoptosis components CASP9 and CASP3 are expressed in the same latticework LBs and pores as described for necroptosis and pyroptosis pathways. In individual cells undergoing apoptosis, the latticework bodies and pores and cell membranes are largely intact. In PANoptosis, (1) the latticework may independently express apoptotic and necroptotic or pyroptotic pathways with necroptotic and pyroptotic cytopathology dominant; or (2) apoptotic and necroptotic pathways may be coexpressed in latticework bodies and pores with disruption of the pores and necroptotic cytopathology dominant. B and C, Red-stained CASP3⁺ cells and structures and hematoxylin counterstain. B, Apoptosis arrow points to largely intact CASP3⁺ latticework pores in a binucleate cell. The necroptosis arrow points to a largely CASP^– necroptosis-appearing porous latticework enclosed by the line. In necroptosis + pyroptosis, coexpression of the necroptotic and apoptotic pathway in the same latticework disrupts apoptotic pores, resulting in blurred staining of released CASP3 traced by a line in the multinucleated cell at the bottom of the panel. C, Pyroptotic cell remnants and residual membranes with superimposed intact CASP3⁺ latticework bodies and pores (red arrowhead). Red arrow points to blurred CASP3 staining in latticework in which the pyroptotic pathway is coexpressed. D, Green-stained CASP3⁺ cells and structures, red-stained RIPK3⁺ cells and structures, and DAPI blue-stained nuclei. Green arrows point to predominantly CASP3⁺ apoptotic latticework; green-outlined red arrow points to separate CASP3⁺ and RIPK3⁺ latticework and pores; and green-outlined orange arrow points to porous latticework expressing both CASP3 and RIPK3. Abbreviations: AT, alveolar type; CASP, caspase; DAPI, 4′,6-diamidino-2-phenylindole; PANoptosis, pyroptosis, apoptosis, necroptosis.