Pyruvate-GPR31 axis induces LysoDC dendrite protrusion to M-cell pockets for effective immune responses

Abstract

Peyer's patches (PPs) are sites of antigen entry and immunoinduction in the small intestine. In PPs, pathogens are transferred through microfold (M) cells; however, the mechanisms of antigen capture by mononuclear phagocytes beneath M cells remain unclear. Here, we demonstrate that bacterial metabolite pyruvate acted on lysozyme-expressing dendritic cells (LysoDCs), a monocyte-derived phagocyte subset, and induced protrusion of dendrites particularly with "balloon" shapes into basolateral M-cell pockets via its receptor, G-protein coupled receptor 31 (GPR31). Pyruvate administration in wild-type but not Gpr31b-deficient mice increased LysoDC uptake of orally infected Listeria monocytogenes. GPR31 signaling boosted antigen processing and altered gene expression. It also increased LysoDC migration to the interfollicular region, thereby promoting production of pathogen-specific Th1 cells as well as cytotoxic T cells, and effector T cell migration to the lamina propria. Furthermore, oral pyruvate administration conferred high resistance to a virulent L. monocytogenes strain in a GPR31-dependent manner. Collectively, the pyruvate - GPR31 axis plays critical roles in orchestrating intestinal protective immunity.

Keywords: GPCR: dendritic cell; Listeria monocytogenes; Peyer’s patch; commensal metabolite.

Graphical abstract

Figure 1.

Gpr31b is highly expressed in LysoDCs. (a) Gating strategies of MNPs in PPs for cell sorting. Leukocytes were collected from PPs of Gpr31b^+/+ Cx3cr1^gfp/+ mice, and CD11c⁺ cells were magnetically enriched. The LysoDC and LysoMac subsets were separated by the expression of MHC class II, CD4, and Tim-4. (b) Quantitative PCR analysis of Gpr31b in PP phagocytes. Expression levels were normalized to gapdh (n = 3). (c) UMAP analysis for the expression of Gpr31b and PP phagocyte markers in PP CD11c⁺ MHC class II⁺ cell populations from a publicly available dataset. (d) Gpr31b expression in LysoDCs of Gpr31b^+/+ Cx3cr1^gfp/gfp mice. (b,d) Data represent the mean ± standard deviation from three mice.

Figure 2.

Pyruvate – GPR31 axis enhances dendrite protrusion of LysoDCs into M-cell pockets. (a–f) Gpr31b^+/+ Cx3cr1^gfp/+, Gpr31b^-/- Cx3cr1^gfp/+, and Gpr31b^+/+ Cx3cr1^gfp/gfp mice were treated with oral pyruvate administration. PP tissue from each mouse was stained with anti-GP2 mAb (red) and treated with the optical tissue clearing solvent ScaleS4D25. The morphology of CX3CR1⁺ cell dendrites (green) in PPs were observed using two-photon microscopy. Arrowheads indicate CX3CR1⁺ cell dendrites protruding into M-cell pockets (a). Total dendrites in M-cell pockets (b), finger-like dendrites in M-cell pockets (d), and spherical dendrites in M-cell pockets (f) per FAE area. (c,e) volume rendering-based 3D reconstruction of LysoDCs with a finger-like dendrite (c) or a spherical dendrite (e). The white line delineates a single CX3CR1⁺ cell or M cell. The yellow line indicates a leukocyte in a M-cell pocket as judged by the autofluorescence. Bars in (a, c, and e) represent 20 µm. Each symbol represents an individual PP (n = 4–7 tissues from three mice). Data represent the mean ± standard deviation. *p < 0.05, ns: not significant.

Figure 3.

Pyruvate–GPR31 signaling enhances L. monocytogenes uptake by LysoDCs. (a and b) L. monocytogenes uptake by PP phagocytes. Gpr31b^+/+ or Gpr31b^−/−mice were orally infected with 1 × 10⁹ CFU of L. monocytogenes EGDe strain. Representative dot plot (a) and the percentage (b) of L. monocytogenes⁺ LysoDCs (left), LysoMacs (middle), and cDCs (right) at 1 dpi (water, n = 9 mice; pyruvate,n = 6 mice). *P < 0.05. ns: not significant.

Figure 4.

GPR31 signaling enhances bacterial antigen processing with altered gene expression profiles. (a–c) RNA-seq analysis of LysoDCs, sorted from Gpr31b^+/+ and Gpr31b^-/- mice with or without L. monocytogenes infection at 2 dpi (1 × 10 CFU/mice; n = 3 mice). (a) Principal component (PC) analysis. (b) Enriched gene ontology (GO) biological process terms between Gpr31b^+/+ and Gpr31b^-/- mice upon infection. (c) Differentially expressed genes mapped to antigen processing and presentation in Kyoto encyclopedia of genes and genomes (KEGG) pathways (left) and their expression in Gpr31b^-/- LysoDCs is shown (right). Log₂ Fold changes with p-adjusted value (Padj) < 0.1 were considered significant. (d) Phagosome acidification activity of LysoDCs. PP cells from L. monocytogenes-infected Gpr31b^+/+ or Gpr31b^-/- mice at 2 dpi (1 × 10 CFU/mice; n = 4 mice) were incubated with FITC or pHrodo Red -conjugated L. monocytogenes for 1 h. The mean fluorescence intensities (MFI) of FITC and pHrodo Red were measured using flow cytometry. (e) PP cells from L. monocytogenes-infected Gpr31b^+/+ or Gpr31b^-/- mice at 2 dpi (1 × 10 CFU/mice; n = 4 mice) were incubated with Alexa Fluor 647-conjugated OVA, unlabeled-OVA, or BSA for 6 h. Frequencies of LysoDCs positive for Alexa Fluor 647 or MHC-I SIINFEKL peptide-loading complex were measured using flow cytometry. *p < 0.05, ns: not significant.

Figure 5.

GPR31 signaling enhances LysoDC migration to the IFR in PPs. (a) Chemokine binding of LysoDCs. Mice were orally infected with 1 × 10 CFU of L. monocytogenes EGDe strain (n = 3 mice). PP LysoDCs were analyzed for CCL19 and CXCL12 binding at 2 dpi. (b) Chemotactic responses of LysoDCs to CCL19. PP cells prepared from Gpr31b^+/+ Cx3cr1^gfp/+ or Gpr31b^-/-Cx3cr1^gfp/+ mice were applied to the upper wells of Transwells, and LysoDC migration toward CCL19 was evaluated using flow cytometry (n = 3 wells). (c) Quantification of CX3CR1⁺ cells in the IFR of PPs collected from Gpr31b^+/+ Cx3cr1^gfp/+ or Gpr31b^-/- Cx3cr1^gfp/+ mice. The percentage of CX3CR1⁺ area (green) per IFR area (dashed line) was quantified using image processing software. The IFR area was defined as the CD4⁺ area (red). Higher magnification views within the square of the left panels are shown in the right panels. Each symbol represents an individual section (n = 6 sections from three mice). Bars represent 100 µm. *p < 0.05, ns: not significant.

Figure 6.

GPR31 signaling enhances T cell responses to L. monocytogenes in PPs and the LP. (a and b) frequency of L. monocytogenes-specific T cell populations in PPs and the LP from Gpr31b^+/+ or Gpr31^-/- mice at 7 dpi. PP and LP cells were secondarily stimulated with L. monocytogenes listeriolysin O_190–201 peptide (a) or cell wall surface peptide LMON_0576 (b) in vitro. CD4⁺ cells producing IFN-γ or IL-17 ((a); PP, n = 11 mice; LP, n = 7 mice) and CD8⁺ cells producing IFN-γ ((b); PP, n = 3–4 mice; LP, n = 4 mice) were analyzed. (c) T cell migration from PPs to the LP after L. monocytogenes infection. PPs of Gpr31b^+/+ KikGR mice (n = 9) and Gpr31b^-/- KikGR mice (n = 6) were labeled using violet light irradiation at 5 dpi. One day after photoconversion, KikGR-Red⁺ T cells in PPs and the LP were analyzed using flow cytometry. Effector CD4/CD8⁺ T cell populations were gated as TCRβ⁺ CD62L⁻ CD44⁺ cells. *p < 0.05, ns: not significant.

Figure 7.

GPR31 signaling enhances resistance to L. monocytogenes infection. (a and b) titers of EGDe and 10403S strains in PPs and the LP (a) and body weight loss of Gpr31b^+/+ mice infected with each strain (b) at 4 dpi (1 × 10 CFU/mice; EGDe, n = 3 mice; 10403S, n = 4 mice). The dashed line in (a) indicates the detection limit. Mann–Whitney U-test (a) was performed for the statistical analysis. (c and d) effects of immunization with the EGDe strain on infection with the invasive 10403S strain. Mice were orally administered pyruvate for 2 weeks before being infected with the EGDe strain (1 × 10 CFU/mice). Two weeks later, mice were further treated with the 10403S strain (1 × 10 CFU/mice). Body weight loss (c) and survival rate (d) after 10403S infection were monitored (n = 13–15 mice). Abx; streptomycin and clindamycin. Log-rank test (d) was performed for statistical analysis. *p < 0.05, ns: not significant.