Protective role of Mincle in bacterial pneumonia by regulation of neutrophil mediated phagocytosis and extracellular trap formation

Abstract

Background: Nosocomial infections with Klebsiella pneumoniae are a frequent cause of Gram-negative bacterial sepsis. To understand the functioning of host innate immune components in this disorder, we examined a previously uninvestigated role of the C-type lectin receptor Mincle in pneumonic sepsis caused by K. pneumoniae.

Methods: Disease progression in wild-type and Mincle(-/-) mice undergoing pulmonary infection with K. pneumoniae was compared.

Results: Whereas the wild-type mice infected with a sublethal dose of bacteria could resolve the infection with bacterial clearance and regulated host response, the Mincle(-/-) mice were highly susceptible with a progressive increase in bacterial burden, despite their ability to mount an inflammatory response that turned to an exaggerated hyperinflammation with the onset of severe pneumonia. This correlated with severe lung pathology with a massive accumulation of neutrophils in their lungs. Importantly, Mincle(-/-) neutrophils displayed a defective ability to phagocytize nonopsonic bacteria and an impaired ability to form extracellular traps (NETs), an important neutrophil function against invading pathogens, including K. pneumoniae.

Conclusion: Our results demonstrate protective role of Mincle in host defense against K. pneumoniae pneumonia by coordinating bacterial clearance mechanisms of neutrophils. A novel role for Mincle in the regulation of neutrophil NET formation may have implications in chronic disease conditions characterized by deregulated NET formation.

Keywords: Clec4e; Klebsiella; Mincle; NETs; neutrophils; phagocytosis; pneumonia; sepsis.

Figure 1.

Mincle is highly induced in the lungs of Klebsiella pneumoniae infected pneumonic mice, and Mincle deficiency increases susceptibility to the infection. A, Total RNA was extracted by the Trizol method from the lungs of K. pneumoniae–infected wild-type (WT) C57/BL6 mice, harvested at the indicated times after infection. The messenger RNA levels of Mincle were analyzed by real-time polymerase chain reaction as described in Methods and are expressed as fold changes over the levels in mock control mice, calculated as 2^−(ΔΔCt). Data are the averages of 6–8 mice per group in 2 independent experiments. B, Mincle expression was examined by flow cytometry on infiltrating lung cells harvested from K. pneumoniae–infected WT C57/BL6 mice. Cells were harvested 3 days after infection and stained, using a rat anti-mouse Mincle antibody followed by goat anti-rat secondary antibody labeled with Alexa-488. Mincle-positive cells were gated and further analyzed for expression of CD11b and Ly6G as mentioned in Methods. The dot plots are representative of 3 independent experiments with 3 mice each. C. Fifteen each of WT and Mincle^−/− mice were intranasally infected with 2.5 × 10⁴ colony-forming units of K. pneumonia in 20 µL of sterile phosphate-buffered saline and were assessed daily for disease severity. Survival was monitored for 2 weeks. Statistical comparison of susceptibility was done by Kaplan-Meier survival curve statistical analysis (P < .001).

Figure 2.

Mincle^−/− mice display increased bacterial burden and systemic dissemination during pneumonic Klebsiella pneumoniae infection. Wild-type (WT) and Mincle^−/− mice were intranasally infected with 2.5 × 10⁴ colony-forming units of K. pneumoniae. At the indicated times after infection, the mice were euthanized, and systemic organs were isolated, homogenized, and plated as described in Methods. The bacterial burden was enumerated after incubating the plates overnight at 37°C. Data are from 3 independent experiments with 3–5 mice at each time point per experiment. Significant differences in bacterial burden (determined using the nonparametric Mann–Whitney U test) in WT and Mincle^−/− mice are denoted by asterisks (*P < .05; **P < .005, and ***P < .001).

Figure 3.

Pneumonic Mincle^−/− mice exhibit a hyperinflammatory response. The lungs from mock control and Klebsiella pneumoniae–infected wild-type (WT) and Mincle^−/− mice were harvested at the indicated time points after infection, homogenized in phosphate-buffered saline with protease inhibitors, and analyzed commercially for host immune mediators, by a rodent multianalyte profile (Myriad RBM, Austin, TX). Results are average values for the infected and mock control groups (3–4 mice per group) in 3 independent experiments. The amounts of mediators shown were significantly higher (***P < .001) in K. pneumoniae–infected Mincle^−/− mice 3 and 5 days after infection, compared with their levels in infected WT mice at the time points tested. Abbreviations: IFN-γ, interferon γ; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-10, interleukin 10; TNF-α, tumor necrosis factor α.

Figure 4.

Pneumonic Mincle^−/− mice exhibit severe lung pathology characterized by massive accumulation of neutrophils. Hematoxylin and eosin staining of lung cryosections from mock control and Klebsiella pneumoniae–infected wild-type (WT) and Mincle^−/− mice isolated at the indicated times after infection (original magnification, 100×). Inset shows a highly magnified area (original magnification, 1000×) of a lesion in an infected lung from a Mincle^−/− mouse that depicts neutrophils, as indicated by characteristic multilobed nuclear morphology.

Figure 5.

Increased neutrophil accumulation coincides with elevated expression of neutrophil chemoattractant and activation markers in lungs of Klebsiella pneumoniae–infected Mincle^−/− mice. A, Flow cytometry of Ly6G⁺CD11b⁺ neutrophils in mock control and K. pneumoniae–infected wild-type (WT) and Mincle^−/− mice. Total lung cells were isolated from mice by collagenase treatment 3 days after infection. The cells were stained with anti-Ly6G-APC and anti-CD11b-Pacific blue antibodies as markers for neutrophils. The bar graph shows the average of the total number of neutrophils in the lungs of 2–3 mock control and 3–4 K. pneumoniae–infected WT and Mincle^−/− mice from 3 independent experiments. Dot plots shown on the right are from 1 representative experiment. Statistical significance is denoted by asterisks (***P < .001). B, The lungs from mock control and K. pneumoniae–infected WT and Mincle^−/− mice were harvested at the indicated time points after infection and analyzed commercially for host immune mediators by a rodent multianalyte profile (Myriad RBM, Austin, TX). Levels of neutrophil chemoattractants (CXCL2, CXCL6, and granulocyte macrophage colony-stimulating factor [GM-CSF]) and activation markers (matrix metalloproteinase 9 [MMP-9] and myeloperoxidase [MPO]) are averages for infected and mock control mice (3–4 per group) from 3 independent experiments. Amounts of mediators shown were significantly higher (***P < .001) in Mincle^−/− mice 3 and 5 days after infection, compared with levels in WT mice at the time points tested.

Figure 6.

Mincle deficiency impairs neutrophil phagocytosis of nonopsonized bacteria. Peritoneal neutrophils from wild-type (WT) and Mincle^−/− mice were incubated with green fluorescent protein–labeled Klebsiella pneumoniae with (opsonized) or without (nonopsonized) 10% normal mouse serum for 1 hour, followed by quantitation of phagocytosis by flow cytometry. The results are expressed as the percentage of cells positive for fluorescent bacteria. Significant differences are denoted by asterisks (**P < .005).

Figure 7.

Mincle deficiency causes a defect in formation of neutrophil extracellular traps (NETs) in the lungs of mice upon pneumonic Klebsiella pneumoniae infection. A, Enumeration of CD11b⁺Ly6G⁺ neutrophils by flow cytometry in bronchoalveolar lavage isolated from K. pneumoniae–infected wild-type (WT) and Mincle^−/− mice. B, Quantitation of neutrophils, showing NETs in bronchoalveolar lavage isolated from WT and Mincle^−/− mice infected with K. pneumoniae (***P < .001). C, Representative fluorescence images of neutrophils that were isolated from bronchoalveolar lavage fluid of WT (upper panel) and Mincle^−/− (lower panel) mice infected with K. pneumoniae and then stained with Sytox Green, to label DNA (green), and a rabbit anti-neutrophil elastase (NE) polyclonal antibody followed by goat anti-rabbit Alexa546 (red). The neutrophils from WT mice showed web-like structures that stained positive for NE and Sytox green (white arrow), whereas the Mincle^−/− neutrophils appeared inactive and displayed occasional small DNA fibers that lacked the typical web-like appearance of NETs (blue arrow). The experiment was repeated 3 times with 3–4 mice in each group (original magnification, 400×).