Coinfection with influenza virus and non-typeable Haemophilus influenzae aggregates inflammatory lung injury and alters gut microbiota in COPD mice

Abstract

Background: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is associated with high mortality rates. Viral and bacterial coinfection is the primary cause of AECOPD. How coinfection with these microbes influences host inflammatory response and the gut microbiota composition is not entirely understood.

Methods: We developed a mouse model of AECOPD by cigarette smoke exposure and sequential infection with influenza H1N1 virus and non-typeable Haemophilus influenzae (NTHi). Viral and bacterial titer was determined using MDCK cells and chocolate agar plates, respectively. The levels of cytokines, adhesion molecules, and inflammatory cells in the lungs were measured using Bio-Plex and flow cytometry assays. Gut microbiota was analyzed using 16S rRNA gene sequencing. Correlations between cytokines and gut microbiota were determined using Spearman's rank correlation coefficient test.

Results: Coinfection with H1N1 and NTHi resulted in more severe lung injury, higher mortality, declined lung function in COPD mice. H1N1 enhanced NTHi growth in the lungs, but NTHi had no effect on H1N1. In addition, coinfection increased the levels of cytokines and adhesion molecules, as well as immune cells including total and M1 macrophages, neutrophils, monocytes, NK cells, and CD4 + T cells. In contrast, alveolar macrophages were depleted. Furthermore, coinfection caused a decline in the diversity of gut bacteria. Muribaculaceae, Lactobacillus, Akkermansia, Lachnospiraceae, and Rikenella were further found to be negatively correlated with cytokine levels, whereas Bacteroides was positively correlated.

Conclusion: Coinfection with H1N1 and NTHi causes a deterioration in COPD mice due to increased lung inflammation, which is correlated with dysbiosis of the gut microbiota.

Keywords: COPD; inflammation; influenza; influenzae; mice; microbiota.

FIGURE 1

Experimental flow chart. Mice were exposed to one cigarette per day for 14 weeks and then intranasally inoculated with non-lethal dose of PR8 or PBS. At 3 days post-infection (dpi), mice were challenged with NTHi or PBS. After detecting invasive lung function at 24 h post-NTHi challenge, mice were sacrificed and samples were collected for further analysis, including histopathology, lung index, replication of pathogens, inflammatory mediators, immune cells, and gut microbiota. Survival rate was calculated at day 15 after PR8 infection.

FIGURE 2

Lung function and body weight changes in conscious mice during CS exposure. Inspiratory time (Ti) (A), tidal volume (TV) (B), expiratory volume (EV) (C), frequency (F) (D), and body weight changes (E) in control (n = 21) and CS-exposed (n = 16) group. Data was expressed as means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with air exposure group.

FIGURE 3

Lethal synergistic effect of PR8-NTHi coinfection in COPD mice. Three days following intranasal infection with 1 LD₅₀ of PR8, COPD mice were challenged with 10⁵ CFU of NTHi. Survival (n = 5) was recorded for 15 days. **p < 0.01 compared with CS-exposed group.

FIGURE 4

Coinfection with PR8 and NTHi impaired invasive lung function in AECOPD mice. At 24 h after NTHi challenge, invasive lung function was measured by forced expiratory volume in 0.1 s/forced vital capacity (FEV0.1/FVC) (A), respiratory resistance (Rrs) (B), tissue damping (G) (C), and respiratory system compliance (Crs) (D). Data are expressed as mean ± SD (n = 10–15). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

Replication of PR8 and NTHi in AECOPD mice. At 24 h post-NTHi infection, lung homogenates were added to chocolate agar plates containing vancomycin to determine bacterial CFU (A), and the supernatants of lung homogenates were added to MDCK cells to determine viral TCID₅₀ (B). Data are expressed as mean ± SD (n = 6). ***p < 0.001.

FIGURE 6

Coinfection aggravates lung injury in AECOPD mice. Pathological changes of control group (A,B), CS-exposed group (C,D), CS + NTHi group (E,F), CS + PR8 group (G,H), and CS + PR8 + NTHi group (I,J) were observed in H and E-stained sections. Original magnification, × 4 and × 20. Lung index was calculated as lung weight/body weight × 100 (K). Collagen fibers of control (L), CS-exposed (M), CS + NTHi (N), CS + PR8 (O), and CS + PR8 + NTHi (P) groups were stained with Masson’s trichrome and quantified using ImageJ (Q). Data are representative images (n = 3 in each group) or expressed as mean ± SD (n = 12 for lung index, n = 3 for percentage of collagen fibers). ^**p < 0.01 and ^***p < 0.001.

FIGURE 7

Cytokine and adhesion molecule expression in lung homogenates of AECOPD mice. The levels of CRP (A), IL-1β (B), IL-6 (C), TNF-α (D), IL-22 (E), IL-17 (F), KC (G), MIG (H), ICAM-1 (I), CEACAM-1 (J), and Fn (K) were measured by the Bio-Plex Mouse Cytokines assay or ELISA. Data are expressed as mean ± SD (n = 6 in each group). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 8

Flow cytometric analysis of innate and adaptive immune cells in AECOPD mice lungs. Following digestion of lungs, the collected cells were analyzed by flow cytometry for monocyte-derived macrophages (A), M1 macrophages (B), M2 macrophages (C), M1 macrophages to M2 macrophages ratio (D), alveolar macrophages (E), neutrophils (F), monocytes (G), NK cells (H), T cells (I), CD4 + T cells (J), CD8 + T cells (K), and B cells (L). Data are expressed as mean ± SD (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 9

Coinfection alters gut microbiota in COPD mice. Alpha diversity indices, including observed OTUs (A) and Chao 1 (B), for control group, CS group, CS + NTHi group (CN), CS + PR8 group (CP), and CS + PR8 + NTHi group (CPN). (C) PCoA of weighted UniFrac distances, with R and p-values calculated by Anosim. The average relative abundance of the gut microbiota at phylum (D) and genus (E) levels analyzed by Bar-plot. (F) Comparison of the relative abundance at the genus levels. (G) Correlations between cytokines and gut microbiota identified by (CCA), with mental test displayed on the lop-left corner. (H) Correlation between each bacteria genus and cytokine analyzed by spearman correlation tests. *p < 0.05 and **p < 0.01.