NLRP6 Induces Lung Injury and Inflammation Early in Brucella and Influenza Coinfection

Abstract

(1) Background: With the resurgence of brucellosis epidemics in China in recent years, the chances of a brucella coinfection with other common respiratory pathogens, such as the influenza virus, have increased dramatically. However, little is known about the pathogenicity or the mechanisms of brucella and influenza coinfections. (2) Methods: To clarify the interventions in the early stages of lung damage due to brucella and influenza coinfections, we evaluated the effect of the coinfection on disease progression and mortality using a coinfection model in WT mice and NLRP6^-/- mice, and we verified the function of NLRP6 in infection and proinflammation. (3) Results: The coinfection induced significant respiratory symptoms, weight loss, and a high mortality rate in WT mice. Influenza in the coinfection group significantly increased brucella proliferation in a synergistic manner. Meanwhile, a histological examination showed severe lung tissue destruction and excessive inflammatory responses in coinfected WT animals, and the expression of NLRP6 and IL-18 was dramatically increased in the lung tissues. Furthermore, NLRP6 deletion attenuated lung injuries and inflammation, a reduced bacterial load, and decreased IL-18 protein expression. (4) Conclusions: Our findings indicated that NLRP6 plays a critical role and might be a promising potential therapeutic target for brucella-influenza coinfections.

Keywords: Brucella; IL-18; Influenza A virus; NLRP6; coinfection.

Figure 1

Coinfection caused massive Brucella proliferation and lethal lung tissue damage. (A) Experimental flow chart. Mice were infected with Brucella and Influenza through the respiratory tract. Mice were sacrificed, and lungs were collected via sterile dissection 3 days after infection. Lung pathology, viral/bacterial load, and inflammation-associated molecules and cytokines were detected for analysis of the proinflammatory mechanism of coinfection. Finally, the function of NLRP6 using NLRP6^−/− mice was validated. (B) Weight changes in mice. Results expressed as mean ± standard deviation. The data were analyzed using repeated measures ANOVA. (C) Coinfection mice mortality rate increased: comparison of survival curve analysis. (D,E) The Brucella load (log₁₀) increased in the coinfected group, while the viral load decreased when compared to single infection. Two-tailed unpaired Student’s t-test for two groups was performed. (F) Coinfection caused massive inflammation and severe tissue damage. Pathological sections of lung tissue were observed under a light microscope at 3 days post infection. (With magnification of 10× and 40×). (G) Graph showing pathology score of the lung tissues after the infection. Two-tailed unpaired Student’s t-test for two groups. (H) The TNF-α cytokine was measured with ELISA. Results expressed as mean ± standard; * p < 0.05; *** p < 0.001; and **** p < 0.0001.

Figure 2

The expression levels of NLRP6, GSDMD, IL-1β, and IL-18 in WT mice. (A–D) The overexpression of NLRP6 and GSDMD was caused by coinfection in the lungs. The mRNA expression was measured with qPCR, and the protein expression was detected with Western blot. Two-tailed unpaired Student’s t-test was used to compare two groups, and one-way ANOVA was used to compare three groups or more. (E–H) The expression of IL-1β and the overexpression of IL-18 were caused by coinfection in the lungs. The mRNA expression was measured with qPCR, and the cytokines were measured with ELISA. Two-tailed unpaired Student’s t-test were used to compare two groups, and one-way ANOVA was used to compare three groups or more. Results expressed as mean ± standard; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 3

NLRP6^−/− mice were more resistant to coinfection than WT mice. (A) The Brucella count (log₁₀) of NLRP6^−/− was more significantly decreased than that of WT mice. Two-tailed unpaired Student’s t-test was used to compare two groups. (B) The levels of inflammation and tissue damage in NLRP6^−/− mice were milder than those in WT mice. Pathological sections of lung tissue were observed under a light microscope 3 days post infection. (With magnification of 10× and 40×). (C) Graph showing pathology score of the lung tissues after the infection. Two-tailed unpaired Student’s t-test was used to compare two groups. Results expressed as mean ± standard; * p < 0.05; *** p < 0.001.

Figure 4

The expression levels of IL-1β and IL-18 in NLRP6 deleted mice. (A,B) The mRNA of IL-1β was not differentially expressed. The cytokine IL-1β was downregulated in NLRP6^−/− mice. Two-tailed unpaired Student’s t-test was used to compare two groups. (C,D) The mRNA and protein of IL-18 were differentially expressed. Two-tailed unpaired Student’s t-test was used to compare two groups. Results expressed as mean ± standard; * p < 0.05; ** p < 0.01; *** p < 0.001.