Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43

Abstract

The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases. One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in 'healthy' microbiota such as Bifidobacterium and Bacteriodes, and a concurrent reduction in SCFAs. Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2), and here we show that SCFA-GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient (Gpr43(-/-)) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43(-/-) immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.

Figure 1. Exacerbated colitis in germ-free mice is ameliorated by acetate

a, Germ-free (open squares) and CNV (closed triangles) mice were given DSS colitis (4%), n = 7 (experimental groups). Dashed lines, control mice; solid lines, DSS-treated mice. The percentage weight change (left), DAI (middle) and haematocrit (right) were measured. b, Germ-free mice were fed acetate (grey squares; 150 mM; n = 3) in the drinking water or water only (black squares; n = 5), 5 days before and during DSS administration. Control fed denotes no DSS (open squares). Daily activity score (left), colon length (middle) and colonic MPO (right) were determined. c, MIP1α and TNFα levels in acetate-fed mice. Data are median ± s.e.m., representative of two independent experiments.

Figure 2. GPR43 expression and role in inflammatory responses

a, Immune expression signature of genes encoding cellular receptors across a large panel of leukocyte subsets. Clustering of receptor genes exhibiting enriched expression in neutrophils and eosinophils reveals GPR43, along with other receptors important for innate immunity and chemoattractant-induced responses. Correlation analysis across a wider set of genes in this immune panel identified a rank-ordered list of the top 150 genes (N₁₅₀) in the co-expression neighbourhood of GPR43. b–e, Comparison of wild-type (WT) and Gpr43^−/− bone marrow neutrophils with respect to acetate-induced Ca²⁺ flux (b; MFI, mean fluorescence intensity), chemotaxis (c, left panel), ROS production (d), and phagocytosis of fluorescently labelled S. aureus (e). The right panel of c shows the GPR43 synthetic agonist phenylacetamide 1 in human neutrophil chemotaxis. f, DSS colitis (2.5% (w/v)) in wild-type and Gpr43^−/− mice, fed with acetate or control water (Ctrl). Shown are colon length, the DAI, histology score and colon MPO levels. NS, not significant. The far right panels show representative histological sections from wild-type or Gpr43^−/− mice as indicated (scale bar, 50 μm). g, Chronic DSS-induced colitis (n = 7 per group, median ± s.e.m.). The inset shows the percentage morbidity. Shown are the percentage change in weight, colon weight per cm of colon, colonic MPO, and histological score, for wild-type and Gpr43^−/− mice.

Figure 3. Inflammatory arthritis and allergic airway disease and GPR43 deficiency

a, Inflammatory arthritis (K/BxN serum injection on day 0 and 2) in Gpr43^−/− mice (n = 5) versus wild-type littermates (n ≥ 3). Scores shown are mean ± s.e.m. for each time point, representative of three independent experiments. Wild-type mice are represented with closed squares, Gpr43^−/− mice with open squares, controls with dashed lines, and arthritic mice with solid lines. Change in ankle thickness (top) and measurement of MPO in the peripheral blood (bottom) showed that both naive and arthritic Gpr43^−/− mice had higher MPO production when stimulated with phorbol 12-myristate 13-acetate (PMA; bottom), indicating greater neutrophil activation (P < 0.001 Gpr43^−/− control compared to wild-type control, P = 0.0019 Gpr43^−/− compared to wild-type arthritic). Histological assessment at day 18 (right) (scale bars, 50 μm). b, OVA-induced allergic airway inflammation. BAL fluid cell counts (left), eosinophil peroxidase (EPO) activity in lung tissue (middle), and inflammation as scored by histology (right). The bottom panel shows representative haematoxylin-and-eosin-stained lung sections from wild-type and Gpr43^−/− mice, and control (no OVA) mice. Scale bar, 50 μm.

Figure 4. GPR43 signalling and immune cell functions

a, Protein expression analysis using Kinex antibody microarrays. Z-score-transformed values reflecting positive or negative shifts in differential protein expression fold-changes after acetate treatment of neutrophils from wild-type mice compared to that from Gpr43^−/− mice. Proteins highlighted in red or green indicate those with Z-scores above +1.5 or below −1.5, respectively. b, Apoptosis in wild-type and Gpr43^−/− bone marrow cells, with or without acetate stimulation (apoptotic cells are annexin V and propidium iodide (PI) double positive). c, Chemotactic response to fMLP and C5a by wild-type and Gpr43^−/− bone marrow granulocytes. d, Recruitment of neutrophils and macrophages to the peritoneum in wild-type and Gpr43^−/− mice, injected with 1 × 10⁶ heat-inactivated S. aureus particles. e, Reactive oxygen species production by peripheral blood granulocytes.