TGF-β-induced IL-6 prevents development of acute lung injury in influenza A virus-infected F508del CFTR-heterozygous mice

Abstract

As the eighth leading cause of annual mortality in the USA, influenza A viruses are a major public health concern. In 20% of patients, severe influenza progresses to acute lung injury (ALI). However, pathophysiological mechanisms underlying ALI development are poorly defined. We reported that, unlike wild-type (WT) C57BL/6 controls, influenza A virus-infected mice that are heterozygous for the F508del mutation in the cystic fibrosis transmembrane conductance regulator (HETs) did not develop ALI. This effect was associated with higher IL-6 and alveolar macrophages (AMs) at 6 days postinfection (d.p.i.) in HET bronchoalveolar lavage fluid (BALF). In the present study, we found that HET AMs were an important source of IL-6 at 6 d.p.i. Infection also induced TGF-β production by HET but not WT mice at 2 d.p.i. TGF-β neutralization at 2 d.p.i. (TGF-N) significantly reduced BALF IL-6 in HETs at 6 d.p.i. Neither TGF-N nor IL-6 neutralization at 4 d.p.i. (IL-6-N) altered postinfection weight loss or viral replication in either mouse strain. However, both treatments increased influenza A virus-induced hypoxemia, pulmonary edema, and lung dysfunction in HETs to WT levels at 6 d.p.i. TGF-N and IL-6-N did not affect BALF AM and neutrophil numbers but attenuated the CXCL-1/keratinocyte chemokine response in both strains and reduced IFN-γ production in WT mice. Finally, bone marrow transfer experiments showed that HET stromal and myeloid cells are both required for protection from ALI in HETs. These findings indicate that TGF-β-dependent production of IL-6 by AMs later in infection prevents ALI development in influenza A virus-infected HET mice.

Keywords: acute lung injury; cystic fibrosis transmembrane conductance regulator; influenza; interleukin-6; transforming growth factor-β.

Fig. 1.

Alveolar macrophages (AMs) from influenza A virus-infected mice that are heterozygous for the F508del mutation in the cystic fibrosis transmembrane conductance regulator (HET) produce more IL-6 than wild-type (WT) AMs in response to influenza A virus infection. A: representative immunohistochemistry for IL-6 at 6 days postinfection (d.p.i.) in formalin-fixed, paraffin-embedded lung tissue from a WT mouse intranasally infected with influenza A/WSN/33 [10,000 plaque-forming units (pfu)/mouse; original objective lens magnification ×40]. B: representative immunohistochemistry for IL-6 in formalin-fixed, paraffin-embedded lung tissue from a HET mouse at 6 d.p.i. (original objective lens magnification ×40; inset: tissue stained with nonspecific polyclonal goat antibody). C: fold change in il-6 gene expression in AMs isolated from WT mice (n = 11) and HET mice (n = 13) at 6 d.p.i., relative to uninfected WT mice (n = 4) and HET mice (n = 3). P < 0.005, vs. WT mice. Data are presented as means ± SE.

Fig. 2.

The exaggerated IL-6 response of HETs to influenza infection is TGF-β dependent. A: effect of influenza A/WSN/33 infection on bronchoalveolar lavage fluid (BALF) TGF-β in WT controls (n = 10–14) and HETs (n = 10–12). B: effect of intraperitoneal treatment with nonspecific IgG (IgG; 100 μg/mouse), treatment with a neutralizing antibody to TGF-β at 2 d.p.i. (TGF-N; 100 μg/mouse), or treatment with a neutralizing antibody to IL-6 at 4 d.p.i. (IL-6-N; 0.5 μg/mouse) on BALF IL-6 in WT mice (n = 8) and HETs (n = 8) at 6 d.p.i. UNTx: untreated animals. ‡P < 0.005, vs. WT mice at the same time point. **P < 0.005, #P < 0.001, vs. WT mice in the same treatment group. Data are presented as means ± SE.

Fig. 3.

Neutralization of TGF-β or IL-6 increases severity of cardiopulmonary dysfunction in influenza-infected HETs without significantly impacting viral replication. Effects of systemic treatment with nonspecific IgG, TGF-N, and IL-6-N on body weight (BWT; % change from day 0; n > 10 per group) (A), viral titers in lung homogenates at 6 d.p.i. (log pfu/g; n = 5–8 per group) (B), carotid arterial oxygen saturation (S_aO₂) at 6 d.p.i. (n > 10 per group) (C), and heart rate at 6 d.p.i. (n > 10 per group) (D). Dashed line indicates mean value for each parameter in uninfected WT mice. *P < 0.05, **P < 0.005, #P < 0.001, vs. WT mice in the same treatment group. Data are presented as means ± SE.

Fig. 4.

Neutralization of TGF-β or IL-6 in HETs increases severity of influenza A virus-induced pulmonary edema to WT levels. Effects of systemic treatment with nonspecific IgG, TGF-N, and IL-6-N on lung water content (wet:dry weight ratio) at 6 d.p.i. Dashed line indicates mean value for each parameter in uninfected WT mice; n ≥ 8 per group. *P < 0.05, #P < 0.001, vs. WT mice in the same treatment group. Data are presented as means ± SE.

Fig. 5.

TGF-β and IL-6 are necessary to maintain normal lung function in influenza-infected HETs. Effects of systemic treatment with nonspecific IgG, TGF-N, and IL-6-N on static lung compliance (C_ST; ml/cmH₂O, × 10) at 6 d.p.i. (A) and baseline total lung resistance (R_BASAL; cmH₂O·s⁻¹·ml⁻¹) at 6 d.p.i. (B). Dashed line indicates mean value for each parameter in uninfected WT mice. n = 6–8 per group. #P < 0.001, vs. WT mice in the same treatment group. Data are presented as means ± SE.

Fig. 6.

Neutralization of TGF-β or IL-6 does not impact leukocyte infiltration of the lung in response to influenza infection. Effects of systemic treatment with nonspecific IgG, TGF-N, and IL-6-N on BALF AMs (× 10⁶) at 6 d.p.i. (A) and BALF neutrophil counts (polymorphonuclear leukocytes, PMNs; × 10⁶) at 6 d.p.i. (B); n = 5–7 per group. **P < 0.005, #P < 0.001, vs. WT mice in the same treatment group. Data are presented as means ± SE.

Fig. 7.

Both stromal and myeloid cells from HET mice are necessary for protection from acute lung injury and exaggerated secretion of IL-6. Effects of reciprocal bone marrow transfer on lung water content (wet:dry weight ratio) at 6 d.p.i. (A), static lung compliance (C_ST; ml/cmH₂O, × 10) at 6 d.p.i. (B), BALF IL-6 (ng/ml) at 6 d.p.i. (C), and viral titers in lung homogenates at 6 d.p.i. (log pfu/g) (D). WT TO WT: transfer of WT donor bone marrow to WT recipient mice. WT TO HET: transfer of WT donor bone marrow to HET recipient mice. HET TO WT: transfer of HET donor bone marrow to WT recipient mice. Dashed line indicates mean value for each parameter in uninfected WT mice; n = 5–7 per group. **P < 0.005, #P < 0.001, vs. WT mice. Data are presented as means ± SE.