GPR43/FFAR2 gene-deficient (Gpr43^−/−) mice were produced as previously described (11) backcrossed onto the C57BL/6 background for 10 generations, and maintained in the animal facilities at Universidade Federal de Minas Gerais (UFMG; Belo Horizonte, Brazil). Wild-type male C57BL/6 mice (8–12 weeks) were obtained from the centre of animal care (CEBIO) from UFMG. Mice were maintained in pathogen-free and co-housing conditions with free access to filtered water and food. This study was carried out in accordance with the recommendations of the local animal ethics committee at Universidade Federal de Minas Gerais (CETEA/UFMG 219/2014).

Klebsiella pneumoniae (ATCC 27736) lung infection was made as previously described (22). Briefly, animals were anesthetized i.p. with 0.2 mL of a solution containing xylazine (02 mg/mL), ketamine (50 mg/mL), and saline in a proposition of 1:0.5:3. The trachea was exposed and 25 µL of a suspension containing 1 × 10⁶ CFU of Kp or saline was administered with a 26-gauge needle. After the infection, the animals were euthanized at different time points.

Streptococcus pneumoniae (ATCC 6303 serotype 3) (Sp) infection was made as previously described (7). Briefly, mice were instilled intranasally with 10⁵ or 10⁶ CFU of Sp in 40 µL and were euthanized at 48 h post infection. The data were not shown, and it is only mentioned in the Section “Result.”

Mice were given sodium acetate (Sigma-Aldrich, St. Louis MO, USA) for 5 days before the infection until they were killed. Acetate was added in the sterile drinking water of mice ad libitum at 150 mM. The acetate dose was chosen according previous data (11, 13), and pH was adjusted as needed. The water solution was changed every day to avoid contamination and significant changes.

Mice were euthanized with a lethal solution of ketamine/xylazine (180 and 12 mg/kg, respectively), and bronchoalveolar lavage (BAL) was performed by inserting and collecting three times 1 mL aliquots of phosphate-buffered saline (PBS), through a 1.7-mm catheter in a 1-mL syringe to quantify leukocytes recruited to the airways and bacteria counts. Total counts were performed by Neubauer chamber count. Differential counts were obtained from cytospin preparations. Lungs were collected for quantification of myeloperoxidase to assess neutrophil infiltration, cytokines determination, and histological examination of lung injury.

Tissues were collected and homogenized in PBS containing anti-proteases (22). Samples were centrifuged, and the supernatant was used for cytokine concentrations, in accordance with the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

For histopathological analysis, lungs were removed and fixed for 24 h in 10% neutral buffered formalin (Sigma-Aldrich, St. Louis MO, USA). Samples were washed with PBS and dehydrated. After embedding in paraffin, 3-mm sections were obtained and stained with hematoxylin and eosin to allow assessment of inflammation in the lung. All lung sections, H&E-stained and at 100× magnification (11 mm²/field), were evaluated in a blinded manner. Two parameters were measured: severity of inflammation (0–3: none, slight, moderate, severe) and presence of edema (0–2: none, one-third damaged, and two-thirds damaged). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (1, 0–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%) and added to a sum. The maximum possible score is 20.

The phagocytosis assay was performed as previously described (23). Briefly, MF and neutrophils isolated from the bone marrow by gradient percoll separation were incubated in the presence of acetate (1 mM) and opsonized-pHRodo-marked Kp (MOI1:1). Cells [0.5 × 10⁶ for neutrophils and 0.5 × 10⁵ alveolar macrophage (AM)] were incubated in RPMI 1640 medium without antibiotics supplemented with 10% heat-inactivated FBS for 60 (AM) or 120 min (neutrophils). Cells were then washed, resuspended in 100 µL PBS, and incubated with anti-F4/80 (APC anti-mouse F4/80 Clone BM8, BioLegend, San Diego, CA, USA) and −CD45 (Pe-Cy7 anti-mouse CD45, BioLegend) or anti-Ly-6G (PE anti-mouse Ly-6G Clone 1A8, BioLegend). Trypan blue at a final concentration of 0.5% was added to quench the fluorescence of non-internalized bacteria. Negative controls consisting of cells incubated without Kp were used to set the fluorescence. Samples (10,000 events) were analyzed by flow cytometry (Gallios, Beckman Coulter, Brea, CA, USA). Both the percentage of cells that internalized bacteria and the mean fluorescence of bacteria within the cells were analyzed.

Alveolar macrophage (0.5 × 10⁵) were incubated in the presence of acetate (1 mM) and opsonized Kp. Samples were incubated in RPMI 1640 medium without antibiotics supplemented with 10% heat-inactivated FBS for 30 or 120 min. Cells were centrifuged (1,500 rpm, 10 min, 4°C), and the supernatant was collected, serially diluted in sterile PBS (1/10, 1/1,000, and 1/100,000) and plated in triplicate at MacConkey agar. A 10-µL volume of undiluted bacteria and bacterial incubated in the absence of cells (total bacteria) were also plated as positive control. The plates were incubated in incubator at 37°C for 24 h. Colonies were then counted and the total number of CFU was obtained considering the dilution of the sample. The number of CFU was normalized considering the viable bacteria counted in the control condition (WT without treatment).

Bone marrow isolated neutrophils (0.5 × 10⁶) and BMDM (1.5 × 10⁵) were incubated in medium RPMI 1640 without antibiotics and supplemented with 10% FBS in the presence or absence of Kp (MOI: 1:1) and acetate (1 mM) for 120 min. The supernatant of the cultures was collected and then assayed for cytokines (TNF-α, IL-1β, and IL-10) as described above.

Residents peritoneal MF were harvested from wild-type and Gpr43^−/− mice pretreated for 5 days or not with oral acetate (150 mM). Macrophages (1 × 10⁶ cells/well) were plated and stimulated with lipopolysaccharides (LPS) (1 µg/mL) for 24 h, and the supernatant was collected. Thioglycollate elicited neutrophils from wild-type and Gpr43^−/− were collected 18 h after intraperitoneal administration. Neutrophils were plated (1 × 10⁵ cells/well) on top of the filter membrane in a transwell insert and incubated for 10 min at 37°C and 5% CO₂ to allow the cells to settle down. Macrophage supernatant was used as a chemoattractant into the bottom of the lower chamber in a 24-well plate. Cells were incubated for 30 min. The quantification of migrated cells was performed by count the migrated cells dropped into the media in the lower chamber. The number of migrated cells was counted by using flow cytometer and the confirmation by cytospin.

Wild-type mice were pretreated for 5 days with oral acetate (150 mM). Mice were euthanized with a lethal solution of ketamine/xylazine and BAL was performed by inserting and collecting three times 1 mL aliquots of PBS, through a 1.7-mm catheter in a 1-mL syringe. The isolation of AMs were performed as previously described (24). Briefly, AM were harvested from BAL and isolated by adherent cells to plastic for 1 h at 37 C in 5% CO₂. The purity was confirmed by morphological analysis by cyotspin (92% of AM). For adoptive in vivo transfer AM were recovered by cold PBS and 2 × 10⁵ cells were instilled intranasally in wild-type and Gpr43^−/− mice 1 h before the Kp infection. 24 h after infection, mice were euthanized, and the BAL was performed. CFU and number of neutrophils were analyzed.

All results are presented as the means ± SEM. The analyses of the difference between two groups were performed by Student’s t-test. Normalized data of three or more groups were analyzed by one-way and two-way ANOVA, and differences between groups were assessed using the Newman–Keuls and Sidak’s posttest respectively. Results were considered significant when P ≤ 0.05. Calculations were performed using Prism 5.0 software for Windows (GraphPad Software, La Jolla, CA, USA).

Gpr43 is a receptor that binds to SCFA, particularly acetate, and is highly expressed on neutrophils and MF (11). To better understand the role of Gpr43 and its effects during infection, wild-type and Gpr43^−/− mice were infected with Gram-negative bacteria, Kp, by intratracheal challenge (1 × 10⁶ CFU). Mice weight and lethality were monitored for 7 days. Interestingly, Gpr43^−/− mice exhibited an increased susceptibility to infection, as observed by the higher mortality (Figure 1A) and by weight loss (Figure 1B) when compared to wild-type infected animals. These factors were associated to an increased burden of bacteria in the airways of Gpr43^−/− mice (Figure 1C). Histological analyses and score (WT PBS: 0; WT INF: 15.6 ± 1.15; GPR43^−/− PBS:0; GPR43^−/− INF:13.3 ± 2.3) showed a higher level of lung injury, which was observed by increased pulmonary edema and inflammatory cell infiltration in lung parenchyma and airways after 48 h of Kp infection in both Gpr43^−/− and wild-type mice (Figure 1D). Toward these results, we investigated whether the protective role of GPR43 was also evident during a Gram-positive bacterial infection. For that, Gpr43^−/− and wild-type mice were infected with Sp. After 48 h of pneumococcal infection, we observed an equally high susceptibility of Gpr43^−/− mice to infection (sixth day: 40% mortality in WT and 100% mortality in Gpr43^−/− mice—data not shown). All together, these data suggest that GPR43 is important to control bacterial infection in the lungs.

Next, aiming to evaluate the inflammatory response that follows Kp infection, Gpr43^−/− and wild-type mice were injected with 1 × 10⁶ CFU intratracheally, and inflammation was evaluated at different time point. As previously shown (22), Kp infection leads to an increased infiltration of leukocytes into the airways and our results showed that there was marked airway inflammatory cells infiltration during the outcome of the infection (Figure 2). There is no difference between Gpr43^−/− and wild-type mice at 24 h regarding total cells infiltration (Figure 2A) and neutrophils (Figure 2B). Although wild-type mice seems to exhibited less mononuclear cells compared to GPR43^−/− mice at 24 h as observed in (Figure 2C), also, there was no significant difference between them. At 48 h post-Kp infection, Gpr43^−/− exhibited more total cells infiltration (Figure 2A) and neutrophils (Figure 2B) and less mononuclear cells (Figure 2C) when compared to wild-type mice. However, there were no significant differences in the neutrophils that migrated into the lung tissue measured by the activity of myeloperoxidase (Figure 2D) at 48 h post Kp infection. In accordance with marked airway inflammation, the levels of the pro-inflammatory cytokines IL-1β and TNF-α were increased in the BAL when compared to wild-type infected mice (Figures 2E,F). Taken together, these results indicate that inflammation persists longer in Gpr43^−/− airspaces, thus Gpr43 plays an important role in the control of inflammation induced by Kp infection.

Because acetate is the mostly abundant microbial metabolite produced which is also able to reach systemic levels and to bind to Gpr43, we pretreated mice with acetate in drinking water (150 mM) for 5 days and then infected with Kp. We observed that acetate treatment was able to reduce the mortality and bacterial counts (Figures 3A,B). Mononuclear cells were increased in the airway space in acetate-treated mice 48 h post-Kp infection (Figure 3F). Neutrophil infiltration is a sign of inflammation, and neutrophil clearance is the most important marker of recovery from inflammation. Accordingly to this, we observed a reduction in myeloperoxidase activity in the lungs and neutrophils in the airway space (Figures 3C–E) with no alteration in the total number of cells after acetate treatment. Acetate was able to protect lungs from pathological injury observed by reduced pulmonary inflammatory cell infiltration in lung parenchyma (Figure 3G). Furthermore, we also treated mice with acetate (150 mM—oral gavage) 24 h after infection and it was also able to reduce neutrophils in the airway space (Figure 3H). In accordance with previous results, oral treatment with acetate reduces TNF-α and IL-1β levels in lungs 48 h after Kp infection (Table 1). These data corroborate with the Gpr43^−/− result, suggesting the role of acetate/Gpr43 activation in the control of inflammation induced by Kp infection.

To gain further insight into the possible mechanism by which Gpr43 are involved in the protection of Kp infection, we analyzed the capacity of AMs to phagocytose bacteria. Resident AMs are the first line of lung defense against respiratory pathogens without evoking a measurable systemic inflammatory response. Because MF have been shown to be important during Klebsiella-induced pneumonia (22), we decided to investigate the contribution of Gpr43^−/− MF into their susceptible phenotype. We observed that MF from Gpr43^−/− mice had impaired phagocytosis when compared to the wild type (Figure 4A). Therefore, this might be a possible mechanism of increased bacterial burden in Gpr43^−/− mice. To prove this hypothesis, we transferred in vivo AM from wild-type mice to Gpr43^−/− mice, intranasally, 1 h before the Kp infection. The AMs from wild-type mice restored protective phenotype on Gpr43^−/−, showing a reduction of bacterial burden in the airways. Interestingly, AM recovered from acetate-treated WT mice reduced bacteria numbers on WT mice. Our results suggest the importance of Gpr43 in the clearance of bacteria (Figure 4B).

It was observed that Gpr43^−/− mice present a marked lung inflammatory neutrophil infiltration; however, the role of neutrophils during Kp infection was not yet fully investigated. In order to investigate the role of Gpr43^−/− in neutrophils, resident MF from wild-type and Gpr43^−/− pretreated or not with oral acetate were stimulated with LPS for 24 h. The supernatant was used as a chemoattractant to the elicited bone marrow neutrophils. Neutrophils from Gpr43^−/− mice showed reduced migration when compared to the wild-type mice and the treatment with acetate was able to increase neutrophils migration from WT mice, which was not observed in GPR43^−/− mice (Figure 5). This result show that GPR43 receptor expressed on neutrophil’s surface played an important role in neutrophil activity state and subsequent migration. In order to better understand the role of GPR43 in neutrophils, we analyzed their capacity to phagocytose bacteria. As observed in MF, neutrophils from Gpr43^−/− mice also show reduced capacity to phagocytose bacteria when compared to the wild type (Figure 6A). These results are accompanied with no significant difference in cytokine levels between wild-type and Gpr43^−/− mice (Figures 6B,C).