Inhibiting Bruton's tyrosine kinase rescues mice from lethal influenza-induced acute lung injury

Abstract

Infection with seasonal influenza A virus (IAV) leads to lung inflammation and respiratory failure, a main cause of death in influenza-infected patients. Previous experiments in our laboratory indicate that Bruton's tyrosine kinase (Btk) plays a substantial role in regulating inflammation in the respiratory region during acute lung injury in mice; therefore, we sought to determine if blocking Btk activity has a protective effect in the lung during influenza-induced inflammation. The Btk inhibitor ibrutinib (also known as PCI-32765) was administered intranasally to mice starting 72 h after lethal infection with IAV. Our data indicate that treatment with the Btk inhibitor not only reduced weight loss and led to survival, but also had a dramatic effect on morphological changes to the lungs, in IAV-infected mice. Attenuation of lung inflammation indicative of acute lung injury, such as alveolar hemorrhage, interstitial thickening, and the presence of alveolar exudate, together with reduced levels of the inflammatory mediators TNFα, IL-1β, IL-6, KC, and MCP-1, strongly suggests amelioration of the pathological immune response in the lungs to promote resolution of the infection. Finally, we observed that blocking Btk specifically in the alveolar compartment led to significant attenuation of neutrophil extracellular traps released into the lung in vivo and neutrophil extracellular trap formation in vitro. Our innovative findings suggest that Btk may be a new drug target for influenza-induced lung injury, and, in general, that immunomodulatory treatment may be key in treating lung dysfunction driven by excessive inflammation.

Keywords: Bruton’s tyrosine kinase; acute lung injury; acute respiratory distress syndrome; influenza; neutrophil.

Fig. 1.

Survival (A) and weight loss (B) of C57BL/6 mice infected intranasally with influenza A virus (A/PR/8/34). Starting 3 days after infection, mice were treated daily with PBS (n = 7) or Bruton’s tyrosine kinase (Btk) inhibitor (Inh, n = 10) administered intranasally. Animals were monitored until death (4 mice) or weight loss of >30%, at which point they were euthanized and counted as dead (3 mice). Values for weight loss are means ± SE. Statistical significance of weight loss for days 4–9 of PBS-treated mice (n = 7) and Btk inhibitor-treated mice (n = 10) was determined by 2-way repeated-measures ANOVA with post hoc Bonferroni’s t-test: **P < 0.01.

Fig. 2.

A: representative hematoxylin-eosin-stained lung sections (left and middle) and CT images (right) from mice 7 days after influenza A virus (Flu) infection [n = 10 and 5 mice for PBS- and Btk inhibitor (Inh)-treated groups, respectively]. B: representative images of Bruton’s tyrosine kinase (Btk) in lungs of influenza A virus-infected mice. Tissue sections were analyzed by immunofluorescent staining for Btk and a PMN marker (Ly6G 1A8). White arrows indicate Ly6G/Btk double-positive cells; note differences in Btk staining between PBS- and Btk inhibitor-treated groups. C: lung volume derived from CT data (n = 10 and 5 mice for PBS- and Btk inhibitor-treated groups, respectively). Values are means ± SD; n = 3 mice in each group. *P < 0.05 (by Mann-Whitney rank sum test). D: average intensity of Btk staining expressed as fold change over control. Values are means ± SD; n = 3 mice in each group.

Fig. 3.

A: bronchoalveolar lavage (BAL) protein concentration, total white blood cell (WBC) count, and number of neutrophils in BAL fluids at 7 days after influenza A virus (Flu) infection. Values are means ± SD; n = 10 and 4 mice for PBS- and Bruton’s tyrosine kinase (Btk) inhibitor (Inh)-treated groups, respectively, for BAL protein concentration and 10 and 5 mice for PBS- and Btk inhibitor-treated groups, respectively, for cell counts. **P < 0.01 (by Mann-Whitney rank sum test); ***P < 0.001 (by Student’s t-test). B: inflammatory cytokine/chemokine concentrations in lung homogenates from mice at 7 days after influenza A virus infection. Values are means ± SD; n = 10 and 5 mice for PBS- and Btk inhibitor-treated groups, respectively. *P < 0.001 (by Student’s t-test).

Fig. 4.

A: representative images of neutrophil extracellular traps (NETs) in influenza virus (Flu)-infected mice. Lung tissue sections were analyzed by immunofluorescent staining for markers of NETs. White circles indicate Ly6G⁺ cells of interest; white arrows indicate extracellular proteins of interest. Background is included to demonstrate the presence of tissue that is otherwise absent in colored images configured for specific staining to differentiate between specific and nonspecific tissue staining. Note differences in prevalence of NETs between siRNA-Bruton’s tyrosine kinase (Btk)- and siRNA-control-treated groups. For each group, n = 3. CitH-3, citrullinated histone-3; MMP-9, matrix metalloproteinase 9. B: NET formation in purified mouse bone marrow neutrophils stimulated with influenza. Cells were stimulated with influenza or influenza + Btk inhibitor (Inh) for 2 h at 37°C. Media served as a negative control/baseline. NET release was minimal within 2 h; expression of MMP-9 and CitH-3 increased in cells exposed to influenza A virus, and this effect was abrogated in cells pretreated with Btk inhibitor. Purified neutrophils from 6 C57BL/6 mice were pooled for analysis, and images of 130–240 cells per group were captured and evaluated. For A and B, markers of NETs were MMP-9 (red), CitH-3 (green), and DNA (blue); Ly6G (Gr-1, 1A8; magenta) served as a neutrophil-specific marker. C: histone citrullination in purified human blood neutrophils stimulated with influenza or influenza + Btk inhibitor for 2 h. An increase in CitH-3 was detected in cells exposed to influenza A virus, and this effect was abrogated in cells pretreated with the Btk inhibitor. Images represent results from 1 of 3 experiments with 3 separate blood donors.