Type III interferons disrupt the lung epithelial barrier upon viral recognition

Abstract

Viral infections of the lower respiratory tract are a leading cause of mortality. Mounting evidence indicates that most severe cases are characterized by aberrant immune responses and do not depend on viral burden. In this study, we assessed how type III interferons (IFN-λ) contribute to the pathogenesis induced by RNA viruses. We report that IFN-λ is present in the lower, but not upper, airways of patients with coronavirus disease 2019 (COVID-19). In mice, we demonstrate that IFN-λ produced by lung dendritic cells in response to a synthetic viral RNA induces barrier damage, causing susceptibility to lethal bacterial superinfections. These findings provide a strong rationale for rethinking the pathophysiological role of IFN-λ and its possible use in clinical practice against endemic viruses, such as influenza virus as well as the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Fig. 1. Morbidity correlates with the high expression of type I IFN and IFN-λ in the lung of COVID-19 patient BALF and of poly (I:C)–treated mice.

(A to E) IFNL2,3, IFNL1 (A), IFNB (B), IFNA2 (C), IL1B (D), and IL6 (E) mRNA expression was evaluated in naso-oropharyngeal swabs from SARS-CoV-2–positive (Swab CoV⁺) and –negative (Swab CoV⁻) participants and from the BALF of intensive care unit (ICU)–hospitalized SARS-CoV-2–positive patients (BALF CoV⁺) (five participants per group). GAPDH, glyceraldehyde phosphate dehydrogenase; ND, not detectable. (F to J) Mice were intratracheally (i.t.) administered 2.5 mg of poly (I:C) per kilogram of body weight, 2.5 mg of R848 per kilogram of body weight, or saline daily for 6 days. (F) Body temperatures of the treated mice measured over time. (G) Amount of total protein in the BALF measured after 6 days of poly (I:C) treatment. (H to J) Ifnl2,3 (H), Ifnb1 (I), and Il1b (J) mRNA expression was assessed in total lung lysate harvested 6 days after treatment. (K and L) Mice treated as in (F) to (J) were infected at day 6 with 5 × 10⁷ colony-forming units (CFU) of S. aureus administered i.t. and were monitored for survival (K). Bacterial loads in the lungs of the treated mice normalized to lung weight were assessed 12 hours postinfection (hpi) (L). Mice were i.t. administered R848 (2.5 mg/kg) or a combination of R848 and IFN-λ (50 μg/kg) daily for 6 days and were then infected as in (K). Lung bacterial burdens (M) and body temperatures (N) before and after S. aureus infection are shown [(G) to (J), (L) to (N)]. Each symbol represents one mouse. The median and range are represented. (F) Means ± SDs of five mice per group are represented. (G to J) Four, (L and M) five, and (N) six mice per group are represented, and median and range are shown. (K) Survival plot of five mice per group. (F to N) Representative data of three independent experiments. Statistics: ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ****P < 0.0001. Two-way analysis of variance (ANOVA) [(F) and (N)], one-way ANOVA [(G) to (J), (L)], or two-tailed t test (M) was performed. Logarithmic values were fitted when evaluating bacterial load [(L) and (M)]. Log-rank (Mantel-Cox) test, corrected for multiple comparisons, was performed to evaluate survival (K).

Fig. 2. IFN-λ is necessary to increase susceptibility to bacterial infection induced by antiviral immunity.

(A and B) WT and Ifnlr1^−/− mice were i.t. treated with 2.5 mg/kg poly (I:C) or saline daily for 6 days. (A) Body temperatures of poly (I:C)–treated WT and Ifnlr1^−/− mice were recorded on day 6. (B) On day 6, mice were i.t. treated with fluorescein isothiocyanate (FITC)–dextran (10 μg per mouse). Barrier permeability was measured as relative fluorescent units (RFU) of FITC-dextran leaked in plasma 1 hour after injection. (C to F) WT and Ifnlr1^−/− mice i.t. treated with 2.5 mg/kg poly (I:C) or saline for 6 days were i.t. infected with 5 × 10⁷ CFU of S. aureus and monitored for survival (C). Lung bacterial burdens normalized by lung weight (D), body temperature (E), and barrier permeability (F) [as in (B)] were assessed 12 hpi. (G and H) Lethally irradiated WT or Ifnlr1^−/− recipients were reconstituted with donor bone marrow (Ifnlr1^−/− or WT) for 6 weeks and were then treated as in (C) to (F). Resulting chimeric mice were defective for IFN-λ signaling in either the hematopoietic compartment (Ifnlr1^−/− → WT) or in the stromal compartment (WT → Ifnlr1^−/−). Ifnlr1^−/− → Ifnlr1^−/− and WT → WT chimeras were used as controls. (G) Barrier permeability [as in (B)] and (H) lung bacterial burdens were evaluated 12 hpi. Each symbol represents one mouse. The median and range are represented. (C) Survival plot of five mice per group. (A to H) Representative data of three independent experiments. (A, E, G, and H) Four, (B) 14, and (D and F) 10 mice per group; median and range are represented. Statistics: ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Two-way ANOVA [(B), (D), (F), (H)], one-way ANOVA (G), or two-tailed t test [(A) and (E)] was performed. Logarithmic values were fitted when evaluating bacterial load [(D) and (H)]. Log-rank (Mantel-Cox) test, corrected for multiple comparisons, was performed to evaluate survival (C).

Fig. 3. IFN-λ signaling directly inhibits lung epithelia proliferation and impairs repair upon viral recognition.

(A to C) Targeted transcriptome sequencing was performed on lung epithelial cells isolated on day 6 from WT and Ifnlr1^−/− mice i.t. treated with 2.5 mg/kg poly (I:C) daily for 6 days. (A) Volcano plot of differentially expressed genes (DEGs) between WT and Ifnlr1^−/−. DEGs (P < 0.005) with a fold change >1.5 (or <−1.5) are indicated in red; DEGs with a fold change <1.5 (or >−1.5) are in blue. Nonsignificant DEGs (P > 0.005) and genes not differentially expressed are indicated in green and gray, respectively. (B and C) Dot plot visualization of gene set enrichment analysis for pathways enriched in (B) WT epithelial cells compared to Ifnlr1^−/− and (C) Ifnlr1^−/− epithelial cells compared to WT. The color of the dots represents the adjusted P value (significance) for each enriched pathway; dot size represents the gene set size. FDR, false discovery rate. (D and E) Epithelial cell proliferation was assessed as 5-ethynyl-2′-deoxyuridine (EdU) incorporation in (D) lung epithelial cells (CD45⁻CD31⁻EPCAM⁺) in WT and Ifnlr1^−/− mice treated as in (A) to (C) or (E) treated as in (A) to (C) and i.t. infected on day 6 with 5 × 10⁷ CFU S. aureus for 12 hours. (F) Mean fluorescence intensity (MFI) of Ki67 in CD45⁻CD31⁻EPCAM⁺ cells of WT and Ifnlr1^−/− mice treated as in (A) to (C). (G) EdU incorporation in lung epithelial cells of WT or Ifnlr1^−/− chimeric mice reconstituted with Ifnlr1^−/− or WT bone marrow treated as in (E). (H and I) p21 levels in lung epithelial cells (CD45⁻CD31⁻EPCAM⁺) from WT and Ifnlr1^−/− mice treated as in (A) to (C). Representative histogram (H) and MFI (I) are depicted. (A to C) Four mice per genotype. (D and E) Five and (F to I) four mice per group; median and range are represented. (D to I) Representative data of three independent experiments. Statistics: ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001. (D and E) Two-way ANOVA, (G) one-way ANOVA, and (F and I) and two-tailed t tests were performed.

Fig. 4. Lung-resident DCs produce IFN-λ downstream of TLR3 upon viral recognition.

(A) Ifnl2,3 relative mRNA expression in lung epithelial cells (EC), resident DCs (resDCs), monocyte-derived DCs (moDCs), and alveolar macrophages (aMacs) sorted from WT mice i.t. treated with 2.5 mg/kg poly (I:C) or saline daily for 6 days measured on day 6. (B and C) CD11c-DTR mice were injected with diphtheria toxin (DTx) to deplete the CD11c⁺ cells in vivo. Relative Ifnl2,3 mRNA (B) and IFN-λ protein levels (C) from lung homogenates were evaluated on day 6. NT, no toxin. (D) DCs differentiated from bone marrow cells in the presence of FMS-like tyrosine kinase 3 ligand (Flt3l) for 9 days from WT, Ticam1^−/−, or Mavs^−/− mice were treated with 50 μg/ml poly (I:C), 1 μg transfected poly (I:C) per 10⁶ cells, or 50 μg/ml R848 for 3 hours. Relative Ifnl2,3 mRNA expression was evaluated by quantitative polymerase chain reaction. (E and F) Ifnl2,3 (E) and Ifnb1 (F) relative mRNA expression in lung EC, resDCs, and moDCs sorted from WT and Ticam1^−/− mice treated as in (A) was measured on day 6. (G to I) WT and Ticam1^−/− mice were treated with poly (I:C) as in (A) and subsequently i.t. infected with 5 × 10⁷ CFU of S. aureus on day 6 for 12 hours. Lung bacterial burden normalized by lung weight (G), Ifnl2,3 (H), and Ifnb1 (I) relative mRNA expression were evaluated. (J to L) WT chimeric mice reconstituted with Ticam1^−/− bone marrow (Ticam1^−/− → WT) or WT bone marrow (WT → WT) were treated as in (G) to (I). Lung bacterial burden normalized by lung weight (J) and Ifnl2,3 (K) and Ifnb1 (L) relative mRNA expression 12 hpi were evaluated. Representative data of three independent experiments are shown. Statistics: ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.001 (two-way ANOVA). Four mice per group; median and range are depicted [(A) to (C), (E) to (L)]. Means ± SEMs of four mice [(A) to (C), (E), and (F)] and of three independent experiments (D) are depicted.