Fig 2: TNF-induced necroptosis of uninfected ATII cells in Patient 1(A) Graphic. TNF, downstream necroptosis pathway components and NINJ1 are initially expressed in latticework bodies. Activation of the pathway generates a porous latticework comprised of darker staining pathway component ring membranes surrounding cores 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.TNF. (B-F) Brown-stained TNF+ cells and structures. Hematoxylin counterstain. (B) TNF+ cell with densely stained latticework. Exiting cell contents visible as blurred lighter staining at the cell’s perimeter. (C) 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. (D) Arrow points to lightly stained porous latticework emptied from the disrupted cell at the top. (E) Trinucleate cell fused to a cell from which emptying of contents leaves residual cell membranes traced by the arrowheads. (F) Rectangle encloses residual TNF+ cell membranes lining or detached from alveolar walls.RIPK3. (G-J) Red-stained RIPK3+ cells and structures. Hematoxylin counterstain. (G1–3) Progressive emptying of RIPK3+ latticework RSBs reveals partially and largely empty pores. (H) Fused RIPK3+ cells with visible porous latticework lining and detaching from alveolar wall. Red arrowhead points to pores with visible staining of the RIPK3+ cores; white outlined arrowhead points to largely emptied cores. (I) RIPK3+ porous latticework and empty pores from disrupted cells in adjacent alveolar space. (J) Largely emptied pores and lightly staining RIPK3+ cell remnants lining and detached from the alveolar wall.MLKL-p. (K, L) Red-stained MLKL-p+ cells and structures. Hematoxylin counterstain. (K) Rectangle encloses an alveolus lined by fused MLKL-p+ cells. Arrow points to porous latticework emptied from disrupted cells. (L) Porous latticework in fused cells lining and lying within alveolar space. Lines connect to magnifier views of partially and largely emptied pores.NINJ1. (M-P) Brown-stained cells and structures. Hematoxylin counterstain. (M1) Darkly stained NINJ1+ latticework RSBs and blurred staining of exiting contents at the perimeter. (M2) NINJ1+ porous latticework in a cell in which an intact nuclear membrane and clumped chromatin impart an “owl’s eye” like appearance to the nucleus. (M3) Arrowheads point to porous latticework shared by fused contiguous cells. (N) Fused cells lining and lying within alveoli. Arrowheads trace the emptying of the latticework leaving residual cell membranes lining the alveolar wall. (O) Enlarged fused cells with porous latticework and residual membranes from emptied cells (arrowheads). (P) Rectangle encloses alveolar space with NINJ1+ porous latticework from disrupted cells. Arrowheads trace residual cell membranes. Circle encloses cells at an early stage of emptying (blurred staining).

Fig 3: TNF-induced necroptosis of uninfected ATII cells in Patient 4TNF. (A-C) Brown-stained TNF+ cells and structures. Hematoxylin counterstain. (A1) TNF+ cell with densely stained latticework bodies. (A2) Line delineates TNF+ latticework RSBs with partially emptied lighter staining cores. (A3) Multinucleated cell with dark staining latticework bodies and pores of varying size and staining intensity commensurate with extent of emptying. (B) Fused cells lining and within alveolar space in which the intensity of the TNF staining decreases with emptying of the porous latticework. Arrows point to multinucleated cells. The arrowheads point to a nucleus with owl’s eye-like appearance and two nuclei in a more advanced state of emptying in the trinucleate cell. The outlined arrow points to residual cell membrane attached to the alveolar wall. (C) Emptying of the porous latticework into alveolar space and reduction of fused cells to thin residual cell membranes lining or detached from alveolar walls. Rectangle encloses porous latticework emptying into alveolar space. Residual cell contents are enclosed by thin membrane or in detached fragments (arrows). Deconvoluted image shown at the right. TNF, green; nuclei and cell contents, blue. White rectangle encloses porous latticework. White arrowhead points to pores.RIPK3. (D-G) Red-stained RIPK3+ cells and structures. Hematoxylin counterstain. (D) Alveolar space lined and filled by fused RIPK3+ cells. Line traces a RIPK3+ syncytium in which the latticework RSB pores retain RIPK3 staining (red arrowhead) or are largely emptied (outlined white arrowhead) The black arrowhead points to residual cell membranes. (E) Fused and disrupted RIPK3+ cells with partially and largely emptied pores in the cells and adjacent alveolar space. (F) Arrowheads trace residual cell membranes of fused cells. (G) Arrowheads trace narrowing and residual membranes of one of three fused cells.MLKL-p. (H-J) Red-stained MKLK-p+ cells and structures. Hematoxylin counterstain. (H) MLKL-p+ cell with densely stained latticework bodies. Emptying cell contents visible as blurred lighter staining at the cell’s perimeter. (I) Fused MLKL-p+ giant cells with owls-eye like nuclei detaching from an alveolar wall. (J) Red arrowheads point to MLKL-p+ pores in cells or in the latticework from disrupted cells in alveolar space.NINJ1. (K-M) Brown-stained NINJ1+ cells and structures. Hematoxylin counterstain. (K1) Deeply stained NINJ+ bodies and latticework with blurred lighter staining of emptying contents at the cell’s perimeter (arrowhead). (K2) Partially emptied visible porous latticework with blurred lighter staining of emptying contents at the cell’s perimeter (arrowhead). (K3) Enlarged cell with “owl’s-eye” like appearance of the nucleus. Arrow points to disrupted region of the cell. (L) Trinucleate giant cell with latticework at various stages from early darkly staining latticework RSBs to later stages in which the intensity of staining decreases as NINJ1 and cell content empty. (M) Fused NINJ1+ cells lining an alveolar wall. Arrowheads trace formation of residual cell membranes in largely emptied cells.

Fig 4: TNF-induced necroptosis in SARS-CoV-2 infected and uninfected ATII cells(A) Graphic of TNF-induced necroptosis. SARS-CoV-2 infected and uninfected ATII cells express TNF and downstream components of the necroptotic pathway, including NINJ1, in latticework bodies that also contain SARS-CoV-2 RNA and virus in infected cells. Activation of the pathway generates a porous latticework through which cell contents and virus are emptied, leaving residual cell membranes lining alveolar walls.TNF± SARS-CoV-2 RNA. (B-E) TNF+ cells and structures, brown; SARS-CoV-2 viral RNA, red. Hematoxylin counterstain.(B) Stages of porous latticework formation. (B1) 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). (B2) Dark 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. (B3) Red arrow points to vRNA emptying from a TNF+ pore.(C) Fused TNF+ cells detached from the alveolar wall. Red arrowheads point to vRNA in a TNF+ porous latticework.(D) 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 comprised of the RSBs in Figures 1, 2, now seen as a TNF+ ring surrounding a vRNA core.(E) Remnants of fused cells lining an alveolar wall. The red double-headed arrow points to TNF+ vRNA+ pores of varying size.RIPK3. (F, G) RIPK3+ cells and structures, red. Hematoxylin counterstain. (F1) Black arrow points to barely discernible large RIPK3+ bodies at the cell perimeter. Lavender arrow points to blurred staining of RIPK3+ content emptying from the cell. (F2) Brown arrow points to RIPK3+ body; pink arrow points to blurred staining of RIPK3+ contents exiting the cell. The pink arrow points to pores at the cell’s perimeter. (F3) RIPK3+ latticework at various stages of emptying.(G) Fused RIPK3+ cells and porous latticework remnants.MLKL-p. (H, I) MLKL-p+ cells and structures, red. (H) Detached porous latticework comprised of MLKL-p rings surrounding MLKL-p cores. (I) Purple arrowheads trace MLKL-p+ residual cell membranes lining alveolar walls. Red arrowhead points to MLKL-p+ pores emptying into alveolar space.NINJ1. (J-M) NINJ1+ cells and structures, brown. Hematoxylin counterstain. (J1) Densely stained NINJ1+ bodies, blurred staining indicative of NINJ1 released from the cell (arrowheads). (J2) NINJ1+ porous latticework of darker-staining NINJ1+ rings surrounding lighter staining NINJ1+ cores. (K) Porous latticework in fused cells. Brown arrowheads point to release of NINJ1 at cell margins. (L) Alveolar walls lined by fused NINJ1+ residual cell membranes. Large dark brown double-headed arrow and orange arrow show magnified views of the margins where NINJ1 is visibly released into alveolar space. The white-outlined double-headed arrow shows the NINJ1+ porous latticework. (M) Orange arrowheads trace the residual cell membranes of fused cells lining the alveolar wall and the emptying (blurred staining) of NINJ1 into alveolar space.