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GPR18 Controls Reconstitution of Mouse Small Intestine Intraepithelial Lymphocytes following Bone Marrow Transplantation - PubMed

  • ️Thu Jan 01 2015

GPR18 Controls Reconstitution of Mouse Small Intestine Intraepithelial Lymphocytes following Bone Marrow Transplantation

Amy M Becker et al. PLoS One. 2015.

Abstract

Specific G protein coupled receptors (GPRs) regulate the proper positioning, function, and development of immune lineage subsets. Here, we demonstrate that GPR18 regulates the reconstitution of intraepithelial lymphocytes (IELs) of the small intestine following bone marrow transplantation. Through analysis of transcriptional microarray data, we find that GPR18 is highly expressed in IELs, lymphoid progenitors, and mature follicular B cells. To establish the physiological role of this largely uncharacterized GPR, we generated Gpr18-/- mice. Despite high levels of GPR18 expression in specific hematopoietic progenitors, Gpr18-/- mice have no defects in lymphopoiesis or myelopoiesis. Moreover, antibody responses following immunization with hapten-protein conjugates or infection with West Nile virus are normal in Gpr18-/- mice. Steady-state numbers of IELs are also normal in Gpr18-/- mice. However, competitive bone marrow reconstitution experiments demonstrate that GPR18 is cell-intrinsically required for the optimal restoration of small intestine TCRγδ+ and TCRαβ+ CD8αα+ IELs. In contrast, GPR18 is dispensable for the reconstitution of large intestine IELs. Moreover, Gpr18-/- bone marrow reconstitutes small intestine IELs similarly to controls in athymic recipients. Gpr18-/- chimeras show no changes in susceptibility to intestinal insults such as Citrobacter rodentium infections or graft versus host disease. These data reveal highly specific requirements for GPR18 in the development and reconstitution of thymus-derived intestinal IEL subsets in the steady-state and after bone marrow transplantation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. GPR18 is highly expressed by intestinal IELs.

(A) Microarray expression data obtained from the Immunological Genome Project. (B) Targeting strategy for Gpr18-deficient alleles. For reporter assays, the neomycin cassette was deleted by crossing Gpr18 +/- mice to a CMV-Cre-expressing strain. (C) Quantitative RT-PCR analysis of GPR18 expression in Gpr18 +/+ (black bar), Gpr18 +/- (gray bar), and Gpr18 -/- splenocytes (not detectable, so not visible on graph). Reactions were performed in technical triplicates and data are normalized to β-actin expression. Mean values ± SEM are shown. (D) Flow cytometry analysis of GPR18 expression through β-galactosidase expression in small intestine TCRγδ+ and TCRαβ+ IELs. Gating strategies are shown in S1 Fig. Cells from heterozygous Gpr18 +/- CMV-Cre+ (solid lines) and control Gpr18 +/+ (filled histogram) mice were stained with the β-galactosidase substrate fluorescein di-β-galactopyranoside. HSC, hematopoietic stem cell; FO, follicular B cell; MZ, marginal zone B cell; GC, germinal center B cell; 4DC, CD4+ splenic dendritic cell; 8DC, CD8+ splenic dendritic cell; pDC, plasmacytoid dendritic cell; RP Mθ, red pulp macrophage; Mono, bone marrow Ly6c- monocyte; Gran, bone marrow granulocyte; NK, splenic natural killer cell; CD4 T, CD4+ T cell; CD8 T, CD8+ T cell, γδ T, splenic TCRγδ T cell; γδ IEL, TCRγδ+ intestinal IEL.

Fig 2
Fig 2. GPR18 is not required for steady state intestinal IELs.

(A) H&E sections of small intestine from Gpr18 +/+ and Gpr18 -/- mice. White arrows depict examples of IELs. Insets show relevant portions at higher magnification. Sections were obtained from duodenum of small intestine, and are representative of 2 mice. (B) Numerical analysis of small intestine IELs from Gpr18 +/+, Gpr18 +/-, and Gpr18 -/- mice. Frequencies of TCRγδ (B) and TCRαβ (C) IELs were determined by flow cytometry, and IEL absolute numbers were calculated using the total recovered cell number from the small intestine. Mean values ± SEM are shown. Each symbol represents one mouse. n.s., not significant using 1-way ANOVA.

Fig 3
Fig 3. GPR18 is essential for optimal IEL reconstitution following bone marrow transplantation.

Donor chimerism analysis of T cells in the intestine and spleen. Equal numbers of wild type CD45.1+ and either Gpr18 +/+ or Gpr18 -/- CD45.2+ bone marrow cells were transplanted into 800cGy-irradiated CD45.1+ recipients. Eight weeks after reconstitution, small (A, C) or both small and large (B) intestines were harvested and donor CD45.2+ chimerism in TCRγδ and TCRαβ (A, B) and specific IEL subsets (C) were analyzed by flow cytometry. (D) Total splenic T cell chimerism is shown. Gating strategies are shown in S1 Fig. Mean values ± SEM are shown. *p<0.05 using student's 2-tailed unpaired t-test.

Fig 4
Fig 4. GPR18 is essential for competitive reconstitution of thymus-derived IELs irrespective or irradiation.

Equal numbers of wild type CD45.1+ and either Gpr18 +/+ or Gpr18 -/- CD45.2+ bone marrow cells were transplanted into irradiated athymic nude mice. Eight weeks post-transplantation, CD45.2+ donor chimerism of CD8αα+ TCRγδ+ IELs were analyzed in the small intestine by flow cytometry. Mean values ± SEM are shown. *p<0.05 using student's 2-tailed unpaired t-test.

Fig 5
Fig 5. GPR18 is dispensable for resistance to C. rodentium infections and graft versus host disease.

(A) 5 x 106 bone marrow cells from Gpr18 -/- or Gpr18 +/+ mice were transplanted into sublethally irradiated wild type CD45.1 recipients. Intestinal IEL chimerism was measured 8 weeks post-transplantation. Mean values ± SEM are shown. *p<0.05 using student's 2-tailed unpaired t-test. (B) Gpr18 -/- or Gpr18 +/+ bone marrow chimeras were orally gavaged with C. rodentium. Weight after infection was quantified for 37 days. Mean values ± SEM are shown, n = 10 for each group. No statistically significant differences were observed at any timepoint by students' 2-tailed t test. (C) Lethally-irradiated (900 cGy) Balb/c mice were transplanted with 5 x 106 T cell-depleted bone marrow cells from a CD45.1+ C57Bl/6 mice with or without 5 x 105 splenic pan T cells from CD45.2+ Gpr18 +/+ or Gpr18 -/- littermates. Survival was measured over the course of 50 days. Two recipients of T cell-depleted bone marrow only, 10 recipients of Gpr18 -/- splenic pan T cells, and 5 recipients of Gpr18 +/+ splenic pan T cells were assessed. No significant differences (p = 0.49) in survival were observed using log-rank Mantel-Cox test.

Fig 6
Fig 6. GPR18 is preferentially expressed by lymphoid vs. myeloid progenitors.

(A) Microarray expression analysis of GPR18 in all lymphoid progenitors (ALP) and Flk2+ common myeloid progenitors (CMP). Mean values ± SEM are shown; n = 3 microarrays each, derived from previously published work [65, 66]. (B) Quantitative RT-PCR analysis of GPR18 in ALP and Flk2+ common myeloid progenitors CMP. Mean values ± SEM are shown. Gating strategies are shown in S2 Fig. Data are normalized to Gapdh expression levels. *p<0.05 using students 2-tailed unpaired t-test.

Fig 7
Fig 7. GPR18 is not required for lymphopoiesis or myelopoiesis.

(A) Hematopoietic stem cell and progenitor numbers were quantified in Gpr18 +/+, Gpr18 +/-, and Gpr18 -/- littermates. Mean values ± SEM are shown. No significant differences were observed between any genotype in any progenitor analyzed using 1-way ANOVA. Data are cumulative of 3 independent experiments. Gating strategies are shown in S2 Fig. (B) Mature splenic B and T cells, monocytes, and granulocytes were quantified in Gpr18 +/+, Gpr18 +/-, and Gpr18 -/- mice. No significant differences were observed between any genotype in any progenitor analyzed using 1-way ANOVA. Gating strategies are shown in S3 Fig. (C) Donor chimerism analysis of bone marrow progenitors. Equal numbers of wild type CD45.1+ and Gpr18 +/+ or Gpr18 -/- CD45.2+ bone marrow cells were transplanted into 800cGy-irradiated CD45.1+ recipients. Eight weeks after reconstitution, CD45.2+ chimerism was quantified in progenitors as gated in (A) followed by analysis of CD45.1 and CD45.2 expression. Each unique symbol connected by a line represents chimerism within an individual mouse. *p<0.05 using student's 2-tailed paired t-test. No other significant differences were observed in downstream progenitors relative to upstream MPPs. HSC, hematopoietic stem cell; MPP, multipotent progenitor; ALP, all-lymphoid progenitor; BLP, B lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte macrophage progenitor; MDP, monocyte dendritic cell progenitor; CDP, common dendritic cell progenitor.

Fig 8
Fig 8. GPR18 is not required for antibody responses.

(A) Quantitative RT-PCR analysis of GPR18 expression in B cell progenitors and mature subsets. Data are normalized to Gapdh expression. Mean values ± SEM are shown; n = 3. Data are representative of 2 independent experiments. Gating strategies are shown in S2 and S3 Figs. (B) Serum ELISA analysis of NP-specific IgG1b antibodies at 2, 4, and 8 week post-immunization with NP-CGG. Each data point represents one chimeric animal. No significant differences were observed by Mann-Whitney non-parametric test. (C) ELISA measurements of relative affinity of NP-specific serum antibodies. Data were obtained by quantifying the ratio of high affinity (NP4-binding) to total (NP16-binding) NP-specific antibodies at 4 and 8 weeks post-NP-CGG immunization. No significant differences were observed by Mann-Whitney non-parametric test. (D) Serum ELISA analysis of WNV envelope-specific antibody titers 8 weeks post-infection. Each data point represents Gpr18 +/+ or Gpr18 -/- animal. Mean values ± SEM are shown. No significant differences were observed by Mann-Whitney non-parametric test. (E) Weight of Gpr18 +/+ and Gpr18 -/- bone marrow chimeras following infection with influenza A virus. Mean values ± SEM are shown, n = 9 mice. No significant differences using 2-tailed students' unpaired t-test were observed. ALP, all-lymphoid progenitor; BLP, B lymphoid progenitor; A-E, Hardy fractions A, B, C, D, and E; FO, follicular B cell; TR, transitional B cell; SW, isotype switched B cell.

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