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. 2020 Oct 24:2020:5291714.
doi: 10.1155/2020/5291714. eCollection 2020.

Innate Immune Effectors Play Essential Roles in Acute Respiratory Infection Caused by Klebsiella pneumoniae

Dong Liu  1 , Zhifu Chen  1 , Yue Yuan  1 , Haiming Jing  1 , Jintao Zou  1 , Xiaoli Zhang  2 , Xi Zeng  1 , Weijun Zhang  1 , Quanming Zou  1 , Jinyong Zhang  1
Affiliations

Innate Immune Effectors Play Essential Roles in Acute Respiratory Infection Caused by Klebsiella pneumoniae

Dong Liu et al. J Immunol Res. .

Abstract

Innate immune effectors constitute the first line of host defense against pathogens. However, the roles of these effectors are not clearly defined during Klebsiella pneumoniae (K. pneumoniae) respiratory infection. In the current study, we established an acute pneumonia model of K. pneumoniae respiratory infection in mice and confirmed that the injury was most severe 48 h post infection. Flow cytometric assay demonstrated that alveolar macrophages were the predominant cells in BALF before infection, and neutrophils were quickly recruited after infection, and this was in consistent with the kinetics of chemokine expression. Further, we depleted neutrophils, macrophages, and complement pathways in vivo and challenged these mice with a sublethal dose of K. pneumonia, the result showed that 80%, 60%, and 40% of mice were died in these groups, respectively, while no deaths occurred in the control group. Besides, innate immune effector depleted mice showed higher bacterial burdens in lungs and blood, companied with more severe lung damage and increased levels of cytokine/chemokine expression. These results demonstrated that the innate immune effectors are critical in the early controlling of K. pneumoniae infection, and neutrophils are the most important. Thus, alternative strategies targeting these innate immune effectors may be effective in controlling of K. pneumoniae respiratory infection.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The virulence of different strains of K. pneumoniae in mice. Mice (n = 10 per group, data were collected from 2 separate experiments) were intratracheally administrated with 1 × 106 CFUs, 5 × 106 CFUs, 1 × 107 CFUs, and 1 × 108 CFUs of K. pneumoniae strain YBQ, YYD, HXT, and 700721, respectively. All mice were monitored for 7 days for survival rate calculation.
Figure 2
Figure 2
Bacteria burdens and histopathology in pneumonia model of K. pneumoniae lung infection. Mice (n = 5) were infected intratracheally with 5 × 104 CFUs of strain YBQ, blood, and lung tissue were collected at 24 h, 48 h, and 72 h, respectively. (a) Bodyweight curve of mice was monitored for 7 days. (b) Bacterial burdens in the lungs and blood were counted on Luria-Bertani (LB) plates. (c) Lung sections were stained with hematoxylin-eosin, and histopathology of lung samples was evaluated (magnification = 200×). Statistical analyses were performed by Student's t test. P < 0.05 (∗), P < 0.01 (∗∗), and P < 0.001 (∗∗∗) compared to each other.
Figure 3
Figure 3
Inflammatory cell infiltration and cytokine/chemokine expression in pneumonia model of K. pneumoniae infection. Mice (n = 5) were infected intratracheally with 5 × 104 CFUs of strain YBQ, blood, lungs, and bronchoalveolar lavage fluid (BALF) samples were collected at 0 h, 4 h, 24 h, 48 h, and 72 h, respectively. (a) Total cells, macrophages, and neutrophils in BALF samples were quantified through flow cytometry analysis. (b) The levels of IL-1β, IL-6, and TNF-α in serum and lung samples were detected by ELISA. (c) The levels of MCP-1, MIP-2, and CXCL-1 in lung samples were quantified by ELISA. These assays were performed in triplicate, and representative data from one experiment were expressed as means ± SEM. Statistical analyses were performed by Student's t test. P < 0.05 (∗), P < 0.01 (∗∗), P < 0.001 (∗∗∗), and P < 0.0001 (∗∗∗∗) compared to each other.
Figure 4
Figure 4
Susceptibility of K. pneumoniae to complement-mediated killing. 50 μl of the bacteria suspension (1 × 104 CFUs) was mixed with 50 μl of either active or heat-inactivated rabbit serum in 96-well microplates and incubated at 37°C for 1 h, and viable bacteria were quantified after culture overnight. Complement mediated killing was calculated as follows: (CFUHI − CFUA)/CFUHI, CFUHI represents heat-inactivated number, and CFUA indicates a complement-active number. This experiment was performed in triplicate, and representative data from one experiment were expressed as means ± SEM. Statistical analyses were performed by Student's t test. P < 0.01 (∗∗) compared to each other.
Figure 5
Figure 5
Depletion of innate immune effectors exacerbated K. pneumoniae lung infection. Mice (n = 5) were infected intratracheally with 1 × 106 CFUs of strain YBQ and blood and lungs were collected 24 h after infection. (a) Survival curves for neutrophil, macrophage, and complement depleted mice were monitored for 7 days. (b) Bacterial burdens in the lungs and blood for different groups were counted on Luria-Bertani (LB) plates. (c) 24 hours after infection, lung tissues in each group were collected, and lung sections were stained with hematoxylin-eosin, and histopathology of different groups was evaluated (magnification = 200×). Statistical analyses were performed by Student's t test. P < 0.05 (∗) and P < 0.01 (∗∗) compared to control mice.
Figure 6
Figure 6
Depletion of innate immune effectors resulted in increased levels of cytokines/chemokines in K. pneumoniae lung infection. (a) The levels of IL-1β, IL-6, and TNF-α in the lung were detected by ELISA. (b) The levels of IL-1β, IL-6, and TNF-α in blood samples were detected by ELISA. (c) The levels of MCP-1, MIP-2, and CXCL-1 in lung samples were measured by ELISA. These assays were performed in triplicate, and representative data from one experiment were expressed as means ± SEM. Statistical analyses were performed by Student's t test. P < 0.05 (∗), P < 0.01 (∗∗), and P < 0.001 (∗∗∗) compared to each other.
Figure 7
Figure 7
Innate immune effectors exhibited synergy effect in K. pneumoniae lung infection. Three pathways were simultaneously disrupted, and mice (n = 5) were intratracheally administrated with 5 × 105 CFUs of strain YBQ 48 h later. The mice were monitored for 7 days to calculate the survival rate. Statistical analyses were analyzed using the log-rank test. P < 0.01 (∗∗) compared to control mice.
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