The Metabolic Sensor GPR43 Receptor Plays a Role in the Control of Klebsiella pneumoniae Infection in the Lung

Abstract

Pneumonia is one of the leading causes of death and mortality worldwide. The inflammatory responses that follow respiratory infections are protective leading to pathogen clearance but can also be deleterious if unregulated. The microbiota is known to be an important protective barrier against infections, mediating both direct inhibitory effects against the potential pathogen and also regulating the immune responses contributing to a proper clearance of the pathogen and return to homeostasis. GPR43 is one receptor for acetate, a microbiota metabolite shown to induce and to regulate important immune functions. Here, we addressed the role of GPR43 signaling during pulmonary bacterial infections. We have shown for the first time that the absence of GPR43 leads to increased susceptibility to Klebsiella pneumoniae infection, which was associated to both uncontrolled proliferation of bacteria and to increased inflammatory response. Mechanistically, we showed that GPR43 expression especially in neutrophils and alveolar macrophages is important for bacterial phagocytosis and killing. In addition, treatment with the GPR43 ligand, acetate, is protective during bacterial lung infection. This was associated to reduction in the number of bacteria in the airways and to the control of the inflammatory responses. Altogether, GPR43 plays an important role in the "gut-lung axis" as a sensor of the host gut microbiota activity through acetate binding promoting a proper immune response in the lungs.

Keywords: GPR43; inflammation; lung infection; microbiota; pneumonia; short-chain fatty acids.

Figure 1

Gpr43^−/− mice are more susceptible to Gram-negative [Klebsiella pneumoniae (Kp)] lung infection. (A) Survival rate of mice were observed during 7 days following intratracheal Kp (1 × 10⁶ CFU). (B) Weight loss infection post-Kp infection. (C) Bacterial burden in the airways was assessed in bronchoalveolar fluid [bronchoalveolar lavage (BAL)] 48 h post-Kp infection. (D) Representative photographs of H&E-stained sections of lung from wild-type mice control [WT phosphate-buffered saline (PBS)], wild-type mice Kp infected (WT INF), Gpr43^−/− mice control (Gpr43^−/− PBS) and Gpr43^−/− mice infected (Gpr43^−/− INF). Scale bar—400 mm. Results are expressed as mean ± SEM of 8–10 mice per group. *Statistical difference (P < 0.05) when comparing to WT and Gpr43^−/− mice. ***Statistical difference (P < 0.001) when comparing to WT and Gpr43^−/− mice. Results representative of three independent experiments and the statistical (ANOVA—Posttest Newman–Keuls) was used.

Figure 2

The onset of inflammatory cells in the lung of Gpr43^−/− mice 48 h after Klebsiella pneumoniae infection. (A) Number of total cells infiltration, (B) neutrophils, and (C) mononuclear cells in airway spaces recovered using bronchoalveolar lavage (BAL) were counted at different time points (0–48 h) after intratracheal inoculation of 1 × 10⁶ CFU of Kp. (D) Myeloperoxidase activity in homogenized lungs harvested from wild-type and Gpr43^−/− mice. (E) IL-1β levels and (F) TNF-α levels were measured by ELISA in BAL supernatant harvested from wild-type and Gpr43^−/− mice 48 h after Kp infection. Results are presented as mean ± SEM (n = 6). *Statistical difference (P < 0.05) when comparing to WT and Gpr43^−/− mice. ***Statistical difference (P < 0.001) when comparing to WT and Gpr43^−/− mice. Results representative of three independent experiments and the statistical (ANOVA—Posttest Newman–Keuls) was used.

Figure 3

Oral Treatment with the Gpr43 metabolic ligand, acetate in the lung 48 h after Klebsiella pneumoniae (Kp) infection. (A) Survival rate of mice pretreated for 5 days with acetate were observed during 7 days following intratracheal Kp (1 × 10⁶ CFU). (B) Bacterial burden in the airways was assessed in lung 48 h post-Kp infection. (C) Myeloperoxidase activity in homogenized lungs harvested from wild-type and wild-type pretreated for 5 days with acetate. (D) Number of total infiltrated cells, (E) neutrophils, and (F) mononuclear cells in airway spaces recovered using bronchoalveolar lavage were counted at 48 h post-Kp infection. (G) Representative photographs of H&E-stained sections of lung from wild-type mice control [phosphate-buffered saline (PBS)], wild-type mice Kp infected, and wild-type pretreated 5 days with acetate Kp infected (acetate). Scale bar—400 mm. (H) Number of neutrophils from posttreatment mice with acetate (150 mM) by gavage 24 h after Kp infection. Results are expressed as mean ± SEM of 8–10 mice per group. *Statistical difference (P < 0.05) when comparing to WT and WT + acetate. ***Statistical difference (P < 0.001) when comparing to WT and WT + acetate. Results representative of three independent experiments and the statistical (ANOVA—Posttest Newman–Keuls) was used.

Figure 4

Macrophages from Gpr43^−/− had impairment in phagocytosis and alveolar macrophages (AM) transference in vivo restore protective phenotype. (A) Macrophages from wild-type and Gpr43^−/− were incubated with opsonized-pHRodo-marked Kp (MOI 1:1) for 60 min. Samples were analyzed by flow cytometer, and the mean fluorescence of bacteria within the cells were analyzed. (B) Bacterial burden in the airways was assessed in bronchoalveolar fluid 24 h post-Kp infection. Results are presented as mean ± SEM (n=6). *Statistical difference (P < 0.05) when comparing to WT control (ANOVA—Posttest Newman–Keuls). ^#Statistical difference (P < 0.05) when compared with WT infected (ANOVA—Posttest Newman–Keuls). Results representative of three independent experiments.

Figure 5

Neutrophils from Gpr43^−/− mice present less chemotaxis. Peritoneal macrophage (MF) harvest from wild-type mice treated or not with 150 mM of acetate was stimulated with lipopolysaccharides (LPS) for 24 h. The supernatant MF culture was used as a chemoattractant to bone marrow neutrophils elicited from wild-type and Gpr43^−/− mice. Results are presented as mean ± SEM (n=6). *Statistical difference (P < 0.05) when compared with WT (ANOVA—Posttest Newman–Keuls). ^#Statistical difference (P < 0.05) when compared with WT + Acetate (ANOVA—Posttest Newman–Keuls). Results representative of three independent experiments.

Figure 6

In vitro neutrophils analyses of phagocytosis and cytokines production after Klebsiella pneumoniae (Kp) exposure. (A) Bone marrow neutrophils from wild type and Gpr43^−/− were incubated with opsonized-pHRodo-marked Kp (MOI 1:1) and acetate (1 mM) for 120 min. Samples were analyzed by flow cytometer, and the mean fluorescence of bacteria within the cells were analyzed. (B) IL-10 and (C) IL-1β were measured in supernatant from neutrophils by ELISA. Results are presented as mean ± SEM (n=6). C− = without bacteria C+ = with bacteria. *Statistical difference (P < 0.05) when compared with WT control (ANOVA—Posttest Newman–Keuls). **Statistical difference (P < 0.01) when compared with WT control without bacteria (ANOVA—Posttest Sidak’s). Results are representative of three independent experiments.