Irgm1 protects hematopoietic stem cells by negative regulation of IFN signaling

Abstract

The IFN-inducible immunity-related p47 GTPase Irgm1 has been linked to Crohn disease as well as susceptibility to tuberculosis. Previously we demonstrated that HSC quiescence and function are aberrant in mice lacking Irgm1. To investigate the molecular basis for these defects, we conducted microarray expression profiling of Irgm1-deficient HSCs. Cell-cycle and IFN-response genes are up-regulated in Irgm1(-/-) HSCs, consistent with dysregulated IFN signaling. To test the hypothesis that Irgm1 normally down-regulates IFN signaling in HSCs, we generated Irgm1(-/-)Ifngr1(-/-) and Irgm1(-/-)Stat1(-/-) double-knockout animals. Strikingly, hyperproliferation, self-renewal, and autophagy defects in Irgm1(-/-) HSCs were normalized in double-knockout animals. These defects were also abolished in Irgm1(-/-)Irgm3(-/-) double-knockout animals, indicating that Irgm1 may regulate Irgm3 activity. Furthermore, the number of HSCs was reduced in aged Irgm1(-/-) animals, suggesting that negative feedback inhibition of IFN signaling by Irgm1 is necessary to prevent hyperproliferation and depletion of the stem cell compartment. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFN-dependent stimulation in HSCs, with an essential role in preserving HSC number and function. The deleterious effects of excessive IFN signaling may explain how hematologic abnormalities arise in patients with inflammatory conditions.

Figure 1

The expression profile for Irgm1^−/− HSCs shows enrichment of cell proliferation and immune-related GO categories; IFNγ levels are increased. (A) Genes that were differentially expressed in Irgm1^−/− HSCs were subjected to an analysis of GO category enrichment by use of the FatiGO webtool (Babelomics 4.2). This tool compared genes differentially regulated in KO HSCs (black bars) against all other genes (unchanged genes: gray bars); these lists were analyzed for assortment into GO categories. Genes from cell-cycle/M-phase and immune categories were overrepresented in the list of differentially expressed genes. Only GO categories that showed a significant difference (P < .05) between the list of genes differentially expressed and those that were not changed in KO HSCs are shown. (B) IFNγ levels in serum from WT and Irgm1-deficient mice were quantified by ELISA. n = 10. Box and whiskers plot represents the 25th-75th percentile and 95% confidence intervals. **P < .01.

Figure 2

Mutations in Ifngr1 and Stat1 rescue phenotypic and functional defects in Irgm1-deficient HSCs. (A) Gene expression of Ccnb1 and Ccl5 in HSCs (CD150⁺ SP^KLS) from WT, Irgm1^−/−, Ifngr1^−/−, Irgm1^−/−Ifngr1^−/−, Stat1^−/−, and Irgm1^−/−Stat1^−/− was analyzed by the use of quantitative RT-PCR. Bars represent n = 3. (B) Noncompetitive BM transplants were performed by the use of WBM from CD45.2 WT, Irgm1^−/−, Irgm1^−/−Ifngr1^−/−, or Irgm1^−/−Stat1^−/− donors at a dose of 2 × 10⁶ cells. Engraftment was monitored by flow cytometric analysis of peripheral blood chimerism at the indicated time points. n = 3-5 for each bar. (C) HSC proliferation status was assessed by in vivo BrdU labeling. CD150⁺ SP^KLS cells were purified from mice after 3 days of exposure to BrdU. The percentage of CD150⁺ SP^KLS cells that incorporated BrdU during the labeling period is indicated. Bars indicate the mean and SEM and are representative of 2 independent experiments, each performed in triplicate. *P < .05, **P < .01, ***P < .001.

Figure 3

Irgm1-deficient HSCs exhibit excessive IFN-induced autophagy. (A) LC3-GFP HSCs (CD150⁺ SP^KLS) were isolated and treated with IFNγ in vitro. Representative examples as well as proportions of cells with aggregation are shown for all experiments, each repeated 2 or 3 times with at least 40 cells visualized per cohort. (B) HSCs (CD150⁺ SP^KLS) were isolated from WT, Irgm1^−/−, Irgm1^−/−Ifngr1^−/−, and Irgm1^−/−Stat1^−/− mice and analyzed for LC3 aggregation by immunofluorescence. Data are representative of 2 independent experiments. *Indicates that the proportions are statistically different with 95% confidence according to a 2-sample test of proportions with P < .05.

Figure 4

Irgm1^−/− HSC function is rescued by the addition of homozygous disruption of Irgm3. (A) The absolute number of SP^L cells per mouse is indicated. Data are representative of 2 experiments, each performed in triplicate. (B) Competitive BM were performed with the use of 2 × 10⁶ WBM cells from CD45.2 WT, Irgm1^−/−, Irgm3^−/−, or Irgm1^−/−Irgm3^−/− donors admixed with 2 × 10⁶ WBM competitor cells from CD45.1 WT mice. Data are representative of 2 independent experiments, each with 5-6 recipients per cohort. (C) LC3 aggregation was determined by immunohistochemistry in HSCs (CD150⁺ SP^KLS) of WT, Irgm1^−/−, Irgm3^−/−, or Irgm1^−/−Irgm3^−/− mice. A minimum of 40 cells was assessed per genotype, and the percentage of cells with LC3 aggregation is shown in the bar graph. Data are representative of 2 independent experiments. The proportions are statistically different with 95% confidence according to a 2-sample test of proportions. ***P < .001.

Figure 5

The absolute number of HSCs is depleted in the BM of aged Irgm1^−/− mice. The absolute number of HSCs (SP^LSK) per bone is indicated. Mice were either 8-10 weeks (young) or 12 months of age (old) for all genotypes. For each bar, n = 3. Data are representative of 2 independent experiments. *P < .05.