Increased expression of microRNA-155-5p by alveolar type II cells contributes to development of lethal ARDS in H1N1 influenza A virus-infected mice

Abstract

Alveolar type II (ATII) cells are essential to lung function and a primary site of influenza A virus (IAV) replication. Effects of IAV infection on ATII cell microRNA (miR) expression have not been comprehensively investigated. Infection of C57BL/6 mice with 10,000 or 100 pfu/mouse of IAV A/WSN/33 (H1N1) significantly altered expression of 73 out of 1908 mature murine miRs in ATII cells at 2 days post-infection (d.p.i.) and 253 miRs at 6 d.p.i. miR-155-5p (miR-155) showed the greatest increase in expression within ATII cells at both timepoints and the magnitude of this increase correlated with inoculum size and pulmonary edema severity. Influenza-induced lung injury was attenuated in C57BL/6-congenic miR-155-knockout mice without affecting viral replication. Attenuation of lung injury was dependent on deletion of miR-155 from stromal cells and was recapitulated in ATII cell-specific miR-155-knockout mice. These data suggest that ATII cell miR-155 is a potential therapeutic target for IAV-induced ARDS.

Keywords: Acute respiratory distress syndrome; Alveolar type II cell; Influenza A virus; Mouse; microRNA.

Figure 1.. Infection of C57BL/6 mice with IAV induces significant alterations in the ATII cell miRNome.

(a) ATII cell yields in uninfected C57BL/6 mice, C57BL/6 mice infected with H1N1 influenza A/WSN/33 (10,000 pfu/mouse) for 2 and 6 days, and C57BL/6 mice mock-infected for 6 days; (b): Number of ATII cell miRs for which level of expression increased (black bars) or decreased (white bars) > 2-fold at 2 and/or 6 days post-infection (d.p.i.), relative to ATII cells from uninfected control mice; (c) Post-infection fold increases in expression of miRs that were most elevated at 6 d.p.i.; and (d) Post-infection fold decreases in expression of miRs that were most reduced at 6 d.p.i.. n=3 per timepoint. Broken horizontal line indicates 2-fold cut-off for significance.

Figure 2.. miR-155 expression in ATII cells is dependent on inoculum size and correlates with severity of hypoxemia and pulmonary edema.

Effects of intranasal infection of C57BL/6 mice with 10,000 or 100 pfu/mouse of influenza A/WSN/33 (H1N1) virus on: (a) Body weight (BWT; % change from day 0; n>9 per group); (b) Carotid arterial oxygen saturation (% S_aO₂; n>9 group); (c) Lung water content (wet:dry weight ratio; n=5-6 per group); and (d) miR-155-5p expression (fold change from day 0; n=3-4 per group). All data were analyzed by ANOVA with a post hoc Tukey-Kramer multiple comparison post-test and are presented as mean ± SEM. *P<0.05, ^#P<0.001, vs. WT mice infected with 10,000 pfu/mouse WSN virus at 6 d.p.i.

Figure 3.. Influenza severity at 6 d.p.i. is most attenuated in influenza-infected mice lacking miR-155 in stromal cells.

Effects of intranasal mock and IAV infection (with 10,000 pfu/mouse WSN virus) of wild-type (WT) C57BL/6 mice, C57BL/6-congenic miR-155-knockout (155-KO) mice, or mice that have undergone reciprocal bone marrow transfer on: (a) Body weight (BWT; % change from day 0; n=10-20 per group); (b) Carotid arterial oxygen saturation (S_aO₂; n=10-20 per group); (c) Heart rate (beats per minute; n=10-20 per group); and (d) Lung water content (wet:dry weight ratio; n=4-6 per group). All data were analyzed by ANOVA with a post hoc Tukey-Kramer multiple comparison post-test and are presented as mean ± SEM. *P<0.05, **P<0.005, ^#P<0.001, vs. WSN-infected WT mice.

Figure 4.. Severity of IAV-induced lung dysfunction and cellular inflammation at 6 d.p.i. is dependent on expression of miR-155 in stromal cells.

Effects of intranasal mock and IAV infection (with 10,000 pfu/mouse WSN virus) of wild-type (WT) C57BL/6 mice, C57BL/6-congenic global 155-KO mice, or mice that have undergone reciprocal bone marrow transfer on: (a) Static lung compliance (C_ST; ml/cmH₂O, x 10); (b) Baseline total lung resistance (R_BASAL; cmH₂O.s/ml); (c) Bronchoalveolar lavage fluid (BALF) alveolar macrophage (AM) counts (x 10⁶/ml); and (d) BALF neutrophil (PMN) counts (x 10⁶/ml). n>5 per group. No neutrophils were found in BALF from mock-infected WT mice. All data were analyzed by ANOVA with a post hoc Tukey-Kramer multiple comparison post-test and are presented as mean ± SEM. *P<0.05, **P<0.005, ^#P<0.001, vs. WSN-infected WT mice. †P<0.05, ‡P<0.005, vs. WSN-infected 155-KO mice.

Figure 5.. BALF inflammatory mediator levels are altered in influenza-infected 155-KO mice at 6 d.p.i.

Effects of intranasal IAV infection of wild-type (WT) C57BL/6 mice, C57BL/6-congenic global 155-KO mice, or mice that have undergone reciprocal bone marrow transfer on bronchoalveolar lavage fluid levels of: (a) Interferon-γ (IFN-γ; ng/ml); (b) Interleukin-6 (IL-6; ng/ml); (c) p40 subunit of interleukin-12 (IL-12 [p40]; ng/ml); and (d) Keratinocyte cytokine/CXCL-1 (KC; ng/ml). n>5 per group. All data were analyzed by ANOVA with a post hoc Tukey-Kramer multiple comparison post-test and are presented as mean ± SEM. *P<0.05, **P<0.005, ^#P<0.001, vs. WSN-infected WT mice. †P<0.05, ¶P<0.001, vs. WSN-infected 155-KO mice.

Figure 6.. Influenza severity is reduced in ATII cell-specific 155-KO mice.

Effects of intranasal mock and IAV infection of wild-type (WT) C57BL/6 mice and C57BL/6-congenic, ATII cell-specific 155-KO (155-KO^ATII) mice for 6 days on: (a) Carotid arterial oxygen saturation (% S_aO₂); (b) Lung water content (wet:dry weight ratio); (c) Static lung compliance (C_ST; ml/cmH₂O, x 10); and (d) Baseline total lung resistance (R_BASAL; cmH₂O.s/ml). n>10 per group. Broken horizontal lines indicate values in uninfected WT mice. All data were analyzed by ANOVA with a post hoc Tukey-Kramer multiple comparison post-test and are presented as mean ± SEM. ^#P<0.001, vs. WSN-infected WT mice.

Figure 7.. ATII cell-specific deletion of mir-155 does not attenuate influenza-induced pulmonary inflammation at 6 d.p.i.

Effects of intranasal infection of wild-type (WT) C57BL/6 mice and 155-KO^ATII mice with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1) virus on bronchoalveolar lavage fluid: (a) Alveolar macrophage (AM) and neutrophil (PMNs) counts; and (b) Interferon-γ (IFN-γ; ng/ml), interleukin-6 (IL-6; ng/ml), and keratinocyte cytokine (KC; ng/ml). n>6 per group. All data were analyzed by ANOVA with a post hoc Tukey-Kramer multiple comparison post-test and are presented as mean ± SEM.