Linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection

Abstract

Influenza A virus (IAV) is among the most common causes of pneumonia-related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection result from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L, and HOIP, is a critical regulator of NF-κB-dependent inflammation. Using mice with lung epithelial-specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial-driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and noninfected cells triggered by the activation of type I IFN receptor and mediated by IRF1, which was maladaptive and contributed to hyperinflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.

Keywords: Cellular immune response; Cytokines; Inflammation; Influenza; Pulmonology.

Figure 1. Linear ubiquitin chains modulate the NF-κB response to IAV downstream of RIG-I signaling in vitro.

(A) Simplified signaling pathway. (B–G) A549 cells were infected with WSN (1 MOI, 16 hours) after transfection with nontargeting siRNA (siControl) or siRNA against either HOIL-1L or HOIP. (B–E) Representative immunoblots of RIG-I activation (n = 4) (B–C), linear ubiquitination of NEMO (n = 3) (D), and p-IkBα, total IkBα, pIRF3, and total IRF3 (n = 4) (E). Means ± SD overlaid with individual data points representing replicates are depicted; *P < 0.05, ****P < 0.0001 (1-way ANOVA, Bonferroni post hoc test).

Figure 2. Loss of HOIL-1L from the alveolar epithelium improves survival and reduces lung injury in mice infected with IAV.

(A) Survival of WT and SPC^Cre/HOIL-1L^fl/fl mice infected with a low (n = 18, dashed line) or lethal (n = 30, solid line) dose of WSN. (B and C) BALF was collected at 0 (n = 5), 3, 5, and 7 d.p.i. (n = 18) with a lethal dose of WSN and analyzed for total protein concentration (B) and cellular infiltration (C). (D) Quantification of viral titers in WT and SPC^Cre/HOIL-1L^fl/fl mice at 3, 5, and 7 d.p.i. (n = 9). Means ± SD overlaid with individual data points representing replicates are depicted; **P < 0.01, ***P < 0.005, ****P < 0.0001 (A, 2-tailed log-rank Mantel-Cox test; B–D, 1-way ANOVA, Bonferroni post hoc test).

Figure 3. Alveolar epithelial loss of HOIL-1L reduces the inflammatory response in IAV-infected mice.

WT and SPC^Cre/HOIL-1L^fl/fl mice were infected with a lethal dose of WSN. (A–C) BALF at 0 (n = 5), 3, 5, and 7 d.p.i. (n = 9) was analyzed by ELISA for IL-6 (A), IFN-α (B), and IFN-β (C). (D–H) Lung immune cell populations at 0 (n = 7), 3, 5, and 7 (n = 10) d.p.i. were analyzed by flow cytometry for CD11b^hiMHCII^loLy6C^hi classical monocytes (D), CD11b^hiMHCII^hiCD24^loCD64^hi inflammatory macrophages (E), NK1.1⁺CD11b^hiCD24^hi natural killer cells (F), CD44⁺CD62L^–CD8⁺ T cells (G), and CD4⁺CD25⁺Foxp3⁺ Tregs (H). Means ± SD overlaid with individual data points representing replicates are depicted; *P < 0.05, ***P < 0.005, ****P < 0.0001 (1-way ANOVA, Bonferroni post hoc test).

Figure 4. HOIL-1L is upregulated during IAV infection through a type I IFN receptor signaling axis.

(A and B) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. (A) HOIL-1L mRNA (n = 5). (B) Representative HOIL-1L immunoblot and its quantification (n = 4). (C) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN (n = 3). (D) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA (n = 8). (E–I) Representative HOIL-1L immunoblots and quantification. (E) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) (n = 4). (F) A549 cells treated with recombinant IFN-α (n = 4). (G) AT2 cells isolated from WT and IFNAR1^–/– mice treated in vitro with CM (n = 3). (H) AT2 cells isolated from WT and IFNAR1^–/– mice treated in vitro with WSN (n = 3). (I) AT2 cells isolated from WT and CCR2^–/– mice treated in vitro with WSN (n = 4). (J) Quantitative reverse transcriptase PCR quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells (n = 4). (K) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM (n = 4). (L) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; **P < 0.01, ***P < 0.005 (1-way ANOVA, Bonferroni post hoc test).

Figure 5. Loss of HOIP from the alveolar epithelium decreases survival and enhances lung injury in mice infected with IAV.

(A) Survival of WT and SPC^Cre/HOIP^fl/fl mice infected with a low (n = 18, dashed line) or lethal (n = 13, solid line) dose of WSN. (B and C) BALF was analyzed at 0, 3, 5, and 7 d.p.i. (n = 9) for total protein concentrations (B) and cellular infiltration (C). (D) Viral titers in WT and SPC^Cre/HOIP^fl/fl mice at 3, 5, and 7 d.p.i. (n = 9). Means ± SD overlaid with individual data points representing replicates are depicted; *P < 0.05, **P < 0.01, ***P < 0.005 (A, 2-tailed log-rank Mantel-Cox test; B–D, 1-way ANOVA, Bonferroni post hoc test).

Figure 6. Loss of HOIP from the alveolar epithelium inhibits alveolar epithelial-driven inflammatory response to IAV infection.

(A–G) WT and SPC^Cre/HOIP^fl/fl mice were infected with a lethal dose of WSN. (A–C) AT2 cells at 0 and 4 d.p.i. (n = 9) analyzed for Il6 mRNA (A), Ifnb mRNA (B), and Mcp1 mRNA (C). (D–F) BALF analyzed by ELISA at 0, 3, 5, and 7 d.p.i. (n = 9) for IL-6 (D), MCP-1 (E), and IFN-β (F). (G–I) Lung immune cell populations at 0, 5, and 7 (n = 9) d.p.i. analyzed by flow cytometry for Ly6G⁺CD11b⁺CD24⁺ neutrophils (G), NK1.1⁺CD11b^hiCD24^hi natural killer cells (H), and CD44⁺CD62L^–CD8⁺ T cells (I). Means ± SD overlaid with individual data points representing replicates are depicted; *P < 0.05, **P < 0.01, ****P < 0.0001 (1-way ANOVA, Bonferroni post hoc test).

Figure 7. Restoration of NF-κB pathway in SPC^Cre/HOIP^fl/fl mice improves survival in response to IAV infection.

(A) Survival curve of SPC^Cre/HOIP^fl/fl mice given either AdNull (n = 11, solid line) or AdIKKβ-CA (n = 10, dashed line) followed by low-dose WSN. (B) Immunoblot of AT2 cell lysates showing overexpression of IKKβ (n = 3). (C–E) BALF from SPC^Cre/HOIP^fl/fl mice given either AdNull or AdIKKβ-CA collected at 0 (n = 3) and 7 d.p.i. (n = 7) and low-dose WSN and analyzed for total protein concentration (C), cellular infiltration (D), and IL-6 (E). Means ± SD overlaid with individual data points representing replicates are depicted; *P < 0.05, ****P < 0.0001 (A, 2-tailed log-rank Mantel-Cox test; C–E, 1-way ANOVA, Bonferroni post hoc test).

Figure 8. Illustration representing HOIL-1L upregulation via IRF1 in AT2 cells.

Upon IAV infection, LUBAC-dependent activation of NF-κB and IRF3 pathways occurs downstream of RIG-I. Subsequent release of proinflammatory cytokines and type I IFNs recruits immune cells to the airspace, contributing to the growing cytokine storm. In noninfected AT2 cells, IFN secreted by either epithelial cells or the recruited immune cells binds to IFNAR1, triggering a signal cascade that upregulates HOIL-1L via IRF1. Newly synthesized HOIL-1L may contribute to either an increased number of LUBAC complexes or a change in LUBAC stoichiometry to include a higher ratio of HOIL-1L within the complex. Both situations result in increased stability of LUBAC and exaggerated production of cytokines, which contributes to the morbidity and mortality observed in severe cases of influenza infection.