Fig 2: Epithelial cell priming by the proinflammatory cytokine TNF A549 cells with the indicated KOs were treated with 10 ng/ml of individual recombinant human cytokines for 8 h. MDA5 (IFIH1) and RIG‐I (RIGI) expression were determined by qRT‐PCR. Values were normalized to GAPDH and expressed relative to mock‐treated cells.Indicated cell lines were treated with 10 ng/ml IL‐1β or TNF, and MDA5 (IFIH1) and RIG‐I (RIGI) expression were determined as in (A).A549 cells were treated with TNF (10 ng/ml) or with IFN‐β (1× IC50, 170 IU/ml) for 16 h. Expression of the indicated IFN‐stimulated genes (ISG) was determined by qRT‐PCR.Western blot analysis of MDA5 in A549 cells treated with recombinant human TNF at the indicated concentrations (or mock‐treated) for 4, 8, or 24 h. The blot is representative of two biological replicates.A549ACE2/TMPRSS2 cells were mock‐treated or primed with TNF at the indicated concentrations for 8 h followed by infection with SARS‐CoV‐2 (MOI 1) for 24 h. IFN‐β (IFNB1) gene expression was determined by qRT‐PCR.A549ACE2/TMPRSS2 wild‐type (NT) cells or with the indicated KO (R/MDKO: RIG‐I and MDA5 double KO) were mock‐treated or primed with TNF (10 ng/ml) for 8 h followed by SARS‐CoV‐2 infection (MOI 1). At 24 h post infection, induction of IFN‐β (IFNB1) expression was determined by qRT‐PCR. Data information: In (A–C, E, and F) bars represent means ± SD, n = 3 (biological replicates, individual experiments shown as dots). (B, E and F) Statistical significance was tested by an unpaired one‐tailed t‐test. *P < 0.05, ***P < 0.001, no asterisk: not significant. Source data are available online for this figure.

Fig 3: Antiviral priming of epithelial cells by immune–epithelial cell crosstalk through cytokinesscRNA‐Seq of nasal swabs from healthy children (4–16 years, n = 18) and adults (≥ 24 years, n = 23) (Loske et al, 2022).Assignment of single cell types and states based on their transcriptional profile (Loske et al, 2022). Similar types were grouped into more general classes (“epithelial cells,” “T‐cells”). The relative abundance of immune cell types was determined and compared between children and adults. Figure EV3A shows a detailed immune cell composition.Cell interactivity was determined in children and adults based on the expression of corresponding pairs of signaling ligands (on immune cells) and receptors (on epithelial cells). The bar graph (left) shows the total number of inferred interactions. The interaction plot (right) shows interactions between specific immune and epithelial cell types. Line thickness indicates the number of ligand‐receptor interactions (yellow = higher numbers in children, blue = in adults). Outer circle segments: blue marks epithelial cells, pink immune cells. See also Fig EV3B.Isolated human PBMCs were exposed to live Gram‐negative bacteria (Yersinia enterocolitica, Ye), killed after 1 h by gentamicin treatment. Upon further incubation for 24 h, culture supernatants were harvested and analyzed.A549 NT (wild‐type control), IFNAR1KO and type I, II, III and IFN receptor triple KO (IFNRTKO) were incubated with collected PBMC supernatants for 8 h. Expression levels of MDA5 (IFIH1) and RIG‐I (RIGI) were determined by qRT‐PCR. Values were normalized to GAPDH levels and expressed relative to wild‐type A549 incubated with supernatants from mock‐stimulated PBMC.The cytokine content of the supernatants of mock‐treated or Ye‐exposed PBMCs was analyzed by an electroluminescent multiplex assay.ScRNA‐Seq data (see A) were analyzed for the expression of cytokines (see E) across all immune cell types and states. Values displayed as violin plots with the average per cell expression level of each donor (n = 18 children, n = 23 adults) shown as dots.ScRNA‐Seq data were analyzed for ligand–receptor interactions between cell types (cell type identified by color; see panel B) specifically for IFN‐γ (left) and TNF (right) signaling in children and adult samples. Interaction strength is coded by line thickness; direction (ligand⇾receptor) indicated by arrowheads; arrows are color‐coded by cell type of origin. See also Fig EV3. Data information: In (D and E) bars represent means ± SD, n = 3 (biological replicates, individual experiments shown as dots). The statistical significance between mock and stimulated samples was tested by an unpaired, one‐tailed t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, no asterisk: not significant. In (F) statistical significance was tested using the Kolmogorov–Smirnov test with a Benjamini‐Hochberg correction. **P < 0.01, *P < 0.05. Source data are available online for this figure.

Fig 4: TNF priming induces the expression of MDA5 and RIG‐I via IRF1 and is sufficient to respond to SARS‐CoV‐2 A549 cells with the indicated KOs (NT: nontargeting control) were treated with 10 ng/ml of the indicated recombinant human cytokines. After 8 h, cells were lysed, and MDA5 and RIG‐I protein levels were determined by immunoblotting. The blot is representative of two biological replicates.MDA5 and RIG‐I protein expression in A549 cells (KOs as indicated) treated with 10 ng/ml IL1‐β or TNF for 8 h, as described in (A). The blot is representative of two biological replicates.IRF1 expression in nasal swabs of children and adults across all cell types.A549ACE2/TMPRSS2 cells with the indicated KOs were mock treated or primed with TNF (10 ng/ml) for 8 h, followed by SARS‐CoV‐2 infection (MOI 1). At 24 h post infection, culture supernatants were harvested and IFN‐β levels were measured.A549ACE2/TMPRSS2 cells with the indicated KOs were mock treated or primed with TNF (10 ng/ml) for 8 h, followed by SARS‐CoV‐2 infection (MOI 1) for 24 h. Induction of IFN‐β expression was determined by qRT‐PCR. Values were normalized to 45S rRNA. Data information: (C) Notch marks medians (n = 18 children, n = 23 adults). (E) Bars represent means ± SD, n = 3 (biological replicates, individual experiments shown as dots). Statistical significance was tested by an unpaired, one‐tailed t‐test. ****P < 0.0001. (D) n = 2 (biological replicates, individual experiments shown as dots).