Fig 1: 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 2: 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.
Fig 3: BTK-induced pyroptosis of uninfected ATII cells in Patient 3(A) Graphic. 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. Stages labeled 1–4 show an initial stage 1 of a largely intact cell and latticework 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.BTK. (B-E) Red-stained BTK+ cells and structures. Hematoxylin counterstain. Numbers correspond to stages shown in the graphic. (B) Alveolar space filled with fused BTK+ cells. (C) Cavities and walls with cells at an earlier stage numbered to correspond to BTK+ cells at the stages shown in the graphic. (D) Arrows point to remnants of BTK+ cells in cavities; arrowheads to residual cell membranes that constitute cavity walls. (E) Honeycombed space with BTK+ cells at earlier stages of pyroptosis fused to cell remnants and membranes of BTK+ “ghosts.”CASP1. (F, G) Brown-stained CASP1 cells and structures, immunohistochemical staining, hematoxylin counterstain (F1, G1). CASP1+ cells and structures are red and hematoxylin is blue in deconvoluted images (F2, G2). (F, G) Arrows point to cell remnants in cavities; arrowheads point to residual cell membranes in the cavity walls. LW= latticework. The loss of CASP1 and cell contents is reflected in the loss of detectable staining in the deconvoluted images.IL-1ß. (H-K) Red-stained IL-1ß+ cells and structures. Hematoxylin counterstain. (H) Box encloses IL-1ß+ cells lining cavities. (I) Magnifier 2X view showing porous latticework (arrow) and residual membranes (arrowheads). (J) Arrowheads point to residual cell membranes in fused cells created by emptying (blurred staining) of IL-1ß. (K) Arrow points to emptying IL-1ß + cell fused to remnants of cells and latticework.GSD. (L-O) Red-stained GSD+ cells and structures. Hematoxylin counterstain. (L) Arrows point to two early stage 1 fused GSD+ cells. (M) Formation of cavities by emptying and disruption of GSD+ cells and latticework (arrows M1, 2) leaving walls of residual cell membranes (arrowheads in M2). (N 1–3) GSD+ cell and latticework remnants of fused cells in cavity walls. Porous remnants in (N2) shown at 2x magnification in (N3). (O1) Honeycombed space with cavities and walls with GSD+ cell and latticework remnants. (O2) Deconvoluted image, GSD is red, hematoxylin is blue. Boxes in O1 and O2 correlate the darker staining blue-black latticework bodies and pores with a hematoxylin-stained component.NINJ1. (P-S) Brown-stained cells and structures. Hematoxylin counterstain. (P) Box encloses cavities, cell remnants and residual membranes. Arrow points to NINJ+ disrupted cell. (Q) Box encloses cavity with NINJ1+ cells/latticework. Arrow points to emptying porous latticework. (R) Arrowheads point to NINJ+ residual cell membranes. (S) Contrasting cytopathology of an enlarged necroptotic (ncpt) cell with largely intact NINJ1+ porous latticework and owl’s-eye like nucleus surrounded by NINJ1+ largely disrupted cells, cell remnants, latticework bodies, pores, and residual membranes.
Fig 4: 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 5: NG2-specific Ninj1 KO reduces re-epithelialization of wound skin during healing. (a) Representative H&E images on day-5 skin wounds of control 1 and NG2-specific Ninj1-KO mice. Bars = 500 µm. (b) Wound length, wound closure, and granulation tissue area are evaluated in control and NG2-specific Ninj1-KO mice. (c, d) Representative images of inflammatory cell infiltration on day-5 wound skin sections of Ninj1-KO and control 1 mouse. (c) F4/80+ macrophages and (d) MPO+ neutrophils were costained with CD31 to evaluate inflammatory cell infiltration in the wound margin. Data are expressed as mean ± SEM. **P < 0.01; n = 9 (for a and b) and 4 (c and d). KO, knockout; MPO, myeloperoxidase; Ninj1, Ninjurin-1; n.s., not significant; WE, wound epithelium.
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