Loss of T cell and B cell quiescence precedes the onset of microbial flora-dependent wasting disease and intestinal inflammation in Gimap5-deficient mice

Abstract

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea-induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4(+) T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-kappaB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4(+) T cells adopt a CD44(high)CD62L(low)CD69(low) phenotype and show reduced IL-7ralpha expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor-induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

FIGURE 1

Identification of sphinx, an ENU germline mutant exhibiting severe lymphopenia and hepatic extramedullary hematopoiesis. A, CD8⁺ T cell and NK cell cytotoxicity were tested in ENU mutagenized mice that had been previously immunized with irradiated act-mOVA cells. Cytotoxic function was assessed by the ability to remove β2m^−/− (CFSE^int) NK cell or SIINFEKL-loaded (CFSE^high) CD8⁺ T cell target cells. C57BL/6J control cells (CFSE^low) were used as a reference population. The percentage of CD3⁺ T cells and NK1.1⁺ NK cells (B), and CD4⁺ and CD8⁺ T cells (C) were quantified among splenocytes from 6-wk-old mice. D, Gross liver morphology in 15-d-old (upper) and 40-d-old (lower) mice. E, H&E staining of liver sections from 40-d-old mice (original magnification ×100). Circulating concentrations of hemoglobin (F) and lymphocytes (G) were measured in the blood of mice of various ages. SD is shown and statistical analyses were performed for each time point. n = 5; **p < 0.01; ***p < 0.001. H, Gimap5 protein expression was measured in lysates of total bone marrow and purified splenic CD19⁺ B cells. I, Gimap5 RNA levels in purified splenic CD19⁺ B cells isolated from C57BL/6J and Gimap5^sph/sph mice were measured by RT-PCR. All analyses were performed at least three times.

FIGURE 2

Gimap5^sph/sph mice develop wasting disease, colitis and aberrant hematopoiesis. A, Cohoused C57BL/6J and Gimap5^sph/sph males were weighed at indicated ages. SEMis shown for each time point (n = 6) and statistical analysis was performed using a two-tailed paired t test. B, H&E-stained sections from the colon of 10-wk-old cohoused mice were assessed for intestinal inflammation (original magnification ×100). C, Hematopoietic cell egress from the neonatal liver was monitored by examining H&E-stained sections from livers obtained at indicated developmental time-points (original magnification ×200). At least three livers were analyzed for each time point, and representative sections are shown.

FIGURE 3

Hematopoietic defects in the adult and fetal liver of Gimap5^sph/sph mice. A, HSCs and hematopoietic precursor cells in the liver of 6-wk-old mice were analyzed by flow cytometry. The mean number of cells for each subset is indicated along with SEM. n = 3; *p < 0.05; **p < 0.01. B, Irradiated (1000 rad γ-radiation) Rag2^−/− Il2rγ^−/− recipient mice were reconstituted with ED 19 fetal liver cells. Six weeks later, splenocyte populations in chimeric mice were quantified by flow cytometry. The mean total number of each cell type in the spleen is shown along with SD. n = 3; **p < 0.01; ***p < 0.001.

FIGURE 4

T cell development and function in Gimap5^sph/sph mice. To assess positive and negative selection, thymocyte (A) and splenocyte (B) populations from H-Y TCR⁺ transgenic mice of both genders were quantified and the average percentage of cells in each quadrant is shown. C, Expression of terminal thymocyte maturation markers—CD5, CD24, CD69, and IL-7Rα—was compared between C57BL/6J (shaded, gray) and Gimap5^sph/sph (open, dark line) TCRβ⁺ CD4 SP and CD8 SP thymocytes. D, Surface expression of IL-7rα was measured in splenic CD4⁺ T cells. E, Proliferation of CFSE-labeled CD4⁺ T cells isolated from 8-wk-old mice was assessed 96 h after stimulation with anti-CD3/CD28, with or without exogenous IL-2. F, Expression of CD62L and CD69 was measured on splenic CD4⁺ T cells from 3-, 4-, 6- and 10-wk-old mice. Numbers represent mean ± SD. All analyses were performed at least twice.

FIGURE 5

Reduced iNKT cell survival and aberrant αGal-Cer-induced cytokine responses in Gimap5^sph/sph mice. A, iNKT cells in the spleen of 6-wk-old Gimap5^sph/sph mice were identified by αGal-Cer-loaded CD1d–tetramer binding and quantified by flow cytometry. B, Cytokine production was measured 90 min after injection of the iNKT cell agonist αGal-Cer on a per cell basis by intracellular staining for IFN-γ, TNF-α, IL-4, or IL-13 expression among αGal-Cer-loaded CD1d-tetramer⁺ iNKT cells (blue, unstimulated C57BL/6J; red, stimulated C57BL/6J; green, stimulated Gimap5^sph/sph). C, Serum cytokine concentrations of IFN-γ and IL-4 were also measured by ELISA 90 min after injection of αGal-Cer. SD is shown. n = 3; ***p < 0.001.

FIGURE 6

Impaired BCR-dependent proliferation in Gimap5^sph/sph B cells. A–C, Among CD19⁺ splenocytes from 6-wk-old mice, the percentage of IgM^high IgD^int naive and IgM^low IgD^high mature B cells (A) and the percentage of CD21^high CD23^low marginal zone (B) and CD21^high CD23^high follicular B cells (C) were determined. Six-week-old mice were immunized with 50 µg NP36-CGG with alum and LPS or 50 µg of NP50-Ficoll to assess T-dependent (D) or T-independent (E) Ab responses. NP-specific serum Abs were measured 14 d after immunization by ELISA. F, To assess B cell proliferation, CFSE-labeled CD19⁺ splenocytes were cultured in vitro for 90 h with media alone or with anti-IgM-Fab (10 µg/ml), LPS (2 µg/ml), anti-CD40 (10 µg/ml) plus IL-4 (10 ng/ml), or PMA (50 ng/ml) and ionomycin (500 ng/ml). G, Cell cycle progression of B cells cultured in the presence or absence of LPS was measured by propidium iodide staining after 48 h of incubation.

FIGURE 7

Antibiotic treatment abrogates colitis but not lymphopenia and hepatic extramedullary hematopoiesis in Gimap5^sph/sph mice. The percentage of NKp46⁺, CD8⁺, CD4⁺, and CD11b⁺ splenocytes (A) and splenic CD44^high CD62L^low CD4⁺ T cells (B) in 9-wk-old C57BL/6J, Gimap5^sph/sph, and antibiotic-treated Gimap5^sph/sph mice was determined. Bars represent mean values ± SEM. n = 3 per group; ***p < 0.001. C, H&E-stained sections from the colons and livers of 9-wk-old cohoused mice were assessed for extramedullary hematopoiesis and intestinal inflammation (original magnification ×100). Representative histology from each group is shown. n = 3.

FIGURE 8

Prevention of wasting disease by adoptive transfer of splenocytes. A, Twenty-five- to-35-d-old Gimap5^sph/sph mice were injected with 1 × 10⁷ splenocytes from C57BL/6J or Rag2^−/− donors. The survival of recipient mice was monitored for up to 9 mo (n ≥ 6 per group) and differences were statistically analyzed using the Gehan-Breslow-Wilcoxon test. B, Gimap5^sph/sph mice injected with C57BL/6J splenocytes did not develop wasting disease. Representative 15-wk-old mice are shown. C and D, In 30-d-old Gimap5^sph/sph mice injected with congenically marked splenocytes, the percentages of transferred (CD45.1) and endogenous (CD45.2) CD8⁺ T cells (C) and CD4⁺ T cells (D) were determined at the indicated time points after transfer. E, Expression of CD62L and CD44 on splenic CD4⁺ T cells from 13-wk-old C57BL/6 mice, Gimap5^sph/sph mice, and Gimap5^sph/sph mice injected with CD45.1⁺ congenic splenocytes (only cells of Gimap5^sph/sph origin are shown). Lin myeloid precursor cells were quantified in the liver (F) and bone marrow (G) of 15-wk-old mice.