Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection
Patricia L. Brazee, Luisa Morales-Nebreda, Natalia D. Magnani, Joe G.N. Garcia, Alexander V. Misharin, Karen M. Ridge, G.R. Scott Budinger, Kazuhiro Iwai, Laura A. Dada, Jacob I. Sznajder
Patricia L. Brazee, Luisa Morales-Nebreda, Natalia D. Magnani, Joe G.N. Garcia, Alexander V. Misharin, Karen M. Ridge, G.R. Scott Budinger, Kazuhiro Iwai, Laura A. Dada, Jacob I. Sznajder
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Research Article Inflammation Pulmonology

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.

Authors

Patricia L. Brazee, Luisa Morales-Nebreda, Natalia D. Magnani, Joe G.N. Garcia, Alexander V. Misharin, Karen M. Ridge, G.R. Scott Budinger, Kazuhiro Iwai, Laura A. Dada, Jacob I. Sznajder

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Figure 4

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

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HOIL-1L is upregulated during IAV infection through a type I IFN recepto...
(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).