Jak1 Integrates Cytokine Sensing to Regulate Hematopoietic Stem Cell Function and Stress Hematopoiesis

Abstract

JAK1 is a critical effector of pro-inflammatory cytokine signaling and plays important roles in immune function, while abnormal JAK1 activity has been linked to immunological and neoplastic diseases. Specific functions of JAK1 in the context of hematopoiesis, and specifically within hematopoietic stem cells (HSCs), have not clearly been delineated. Here, we show that conditional Jak1 loss in HSCs reduces their self-renewal and markedly alters lymphoid/myeloid differentiation in vivo. Jak1-deficient HSCs exhibit decreased competitiveness in vivo and are unable to rescue hematopoiesis in the setting of myelosuppression. They exhibit increased quiescence, an inability to enter the cell cycle in response to hematopoietic stress, and a marked reduction in cytokine sensing, including in response to type I interferons and IL-3. Moreover, Jak1 loss is not fully rescued by expression of a constitutively active Jak2 allele. Together, these data highlight an essential role for Jak1 in HSC homeostasis and stress responses.

Keywords: Jak1; cytokine signaling; hematopoietic stem cells; stress hematopoiesis.

Figure 1. Generation of a Conditional Jak1 Allele and Characterization of Mice with Jak1 Loss in the Hematopoietic System

(A) Design of a conditional targeting vector. (B) Confirmation of gene and protein deletion in the bone marrow. (C) Deletion of Jak1 in the hematopoietic system leads to reduced activation of Stat3 in peripheral blood cells. WT, wild-type; het, heterozygous deletion; ko, homozygous deletion. (D) Jak1 KO mice feature reduced white blood cell (WBC) counts (mean ± SEM). n = 11–13. See also Figure S1. (E) Jak1 KO mice feature reduced spleen (n = 14) and thymus weights (n = 18). Mean ± SEM. *p < 0.05. (F) Bone marrow SCAFFoLD map for Jak1 KO and Mx1-Cre-negative control mice. Red nodes: manually gated landmark populations using Jak1 wild-type as reference sample. Blue nodes: unsupervised cell clusters. n = 5. (G) Median frequencies of bone marrow cell populations from Jak1 wild-type and KO mice defined by conventional criteria. n = 5. HSPC, hematopoietic stem and progenitor cell; cDC, conventional dendritic cell; NK, natural killer cell; NKT, natural killer T cell; pDC, plasmacytoid dendritic cell. See also Figure S2.

Figure 2. Jak1 Signaling Is Required for Normal HSC Function

(A) Phenotypic landscape of lineage-negative (lin^–) cells. Clusters are colored according to genotype (Jak1 wild-type: red, Jak1 KO: green) and sized by the number of cells in each cluster as a percentage of the total number of lin^– cells in the bone marrow. n = 5. LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP, multipotent progenitors; CMP, common myeloid progenitor; MEP, megakaryocyte-erythroid progenitor; GMP, granulocyte-monocyte progenitor. See also Figure S3. (B) Manual analysis by traditional criteria confirmed that Jak1 KO mice have a significant reduction of total LSKs in the bone marrow. (C) Frequency of LT-HSCs (CD34^–CD135/Flk2^– LSK) and ST-HSCs (CD34⁺CD135/Flk2^– LSK) in the bone marrow of Jak1 wild-type (Cre^–) and KO mice (Cre⁺). (D) Bar graph showing the number of LSK stem cell fractions subdivided according to SLAM markers. n = 11 mice per group. (E) Relative frequency of progenitor cells in the bone marrow of Jak1 KO (Cre⁺) and wild-type mice (Cre^–). n = 6. (F) Representative image of methylcellulose colony plate 10 days after plating of whole bone marrow from KO and control mice. Images are representative of a total of six mice per group. (G) Bar graph displaying total number of scored colonies. Data shown are representative of three independent experiments with a total of six mice per group. (H) Number of colonies formed 10 days after plating of whole bone marrow cells from wild-type mice with or without 500 nM JAK1 inhibitor GLPG0634. Data are representative of three independent experiments. All data displayed as bar graphs represent mean ± SEM. *p < 0.05.

Figure 3. Jak1 Is Required for HSC Stress Responses In Vivo

(A) Schematic diagram showing competitive bone marrow transplant design. Wks, weeks. (B) Percentage (mean ± SEM) of CD45.1 versus CD45.2 chimerism in the PB of recipients. Excision occurred 3 weeks prior to transplantation. n = 5. *p < 0.05. (C) Flow cytometric analysis of total chimerism in the periphery 16 weeks post-transplantation. A representative flow plot of a total of five mice per group is shown. (D) Schematic diagram showing competitive bone marrow transplant design. Wks, weeks. (E) Percentage (mean ± SEM) of CD45.1 versus CD45.2 chimerism in the PB of recipients at 1, 2, 3, and 4 months after gene deletion in recipient mice. n = 5. *p < 0.05. Data from Mx1-Cre and ERT2-Cre are shown. (F) Donor chimerism in the spleen and bone marrow of recipient mice at 16 weeks post-poly(I:C) (Mx1-Cre) or tamoxifen (ERT2-Cre) injection. n = 5. *p < 0.05. (G) Donor chimerism within the LSK compartment of recipient bone marrow (Mx1-Cre). n = 5. See also Figure S4. (H) Kaplan-Meier analysis of survival of Jak1 KO (Mx1-Cre⁺) and wild-type (Mx1-Cre^–) mice following serial injections of 5-FU (on days 1, 10, and 20). *p < 0.001. (I) Kaplan-Meier analysis of survival of Jak1 KO (Cre⁺) and wild-type (Cre^–) mice following lethal irradiation (ERT2-Cre). *p < 0.05.

Figure 4. Jak1 KO Stem Cells Display Loss of Interferon-Regulated Gene Programs

(A) Contour plots showing level IdU incorporation in LSKs. Representative data from two independent experiments with n = 5 mice are shown. See also Figure S5. (B) Representative cell-cycle profiles of LSKs from Jak1 KO (Cre⁺) and wild-type (Cre^–) mice. The percentage of cells in the different cell-cycle stages is presented. n = 4. (C) Hierarchical clustering of differentially expressed genes. (D) Heatmap of selected genes identified to be differentially expressed in LT-HSCs of Jak1 KO mice to controls. Data shown are from two independent experiments with a total of six mice per group. See also Figure S5. (E) GSEA enrichment plots of differentially expressed interferon-related genes associated with loss of Jak1 signaling in LT-HSCs. FDR, false discovery rate; NES, normalized enrichment score.(legend continued on next page) (F) The networks illustrate the results of the pathway enrichment analysis contrasting Jak1 KO and wild-type HSCs. Each node represents a pathway and the size of each node reflects the number of genes included in the network. The node colors represent the strength and direction of the enrichment. (G) Heatmap of known cell-cycle regulators identified to be downregulated in LT-HSCs of Jak1 KO mice compared to controls. (H) Number of colonies formed 10 days after plating of whole bone marrow cells from wild-type mice with or without Cdk4/6 inhibitor PD332991. Data are representative of three independent experiments. Mean ± SEM. *p < 0.05.

Figure 5. Jak1 KO Cells Are Insensitive to Type I Interferons

(A) Histogram showing the intensity of phospo-Stat1 and phospo-Stat5 versus cell count for LSKs isolated from JAK1 KO and wild-type mice analyzed at baseline (unstim) and in response to increasing IFN-α concentrations. Data are representative of three independent experiments. See also Figure S6. (B) Flow cytometry histogram showing fluorescence intensity versus cell count for stem cell surface marker Sca1 (Ly6A/E). Representative analysis of viable lin/ckit⁺ bone marrow cells is shown. Analysis was performed 24 hr after injection of the mice with poly(I:C) or PBS (control). n = 2 mice per condition. (C) Representative cell-cycle profiles of LSKs from poly(I:C) or PBS-treated mice using DAPI and Ki-67. n = 4. (D) Contour plot showing LSK stem cell fractions subdivided according to CD135 and SLAM markers. MPP-F⁺, CD135⁺ multipotent progenitors; MPP-F^–, CD135^–negative multipotent progenitors. (E) Bar graph depicting the frequency (% of total viable cells) of bone marrow LSKs upon culture ex vivo for 48 hr in the presence or absence of type I and II interferons. Mean ± SEM. Data are representative of two independent experiments and each experiment was performed in triplicate. *p < 0.05. (F) Donor chimerism in the spleen and bone marrow of recipient mice at 16 weeks post-transplantation of whole bone marrow from Irf7 KO or wild-type mice. SEM. *p < 0.05 n = 5.

Figure 6. IL-3 Signals through Jak1 in HSCs and Regulates the Balance between Quiescence and Expansion

For a Figure360 author presentation of Figure 6, see the figure legend at https://doi.org/10.1016/j.stem.2017.08.011. (A) Bar graph depicting the total number of viable LSKs upon culture ex vivo for 48 hr in the presence of indicated cytokines. Mean ± SEM. Data are representative of two independent experiments and each experiment was performed in triplicate. *p < 0.05. (B) Histogram showing the intensity of phospho-Stat5 versus cell count for LSKs isolated from Jak1 KO and wild-type mice (ERT2-Cre) analyzed at baseline (unstim) and in response to increasing IL-3 concentrations. See also Figure S6. (C and D) Effect of IL-3 (C) and IL-6 (D) on cell proliferation of LT-HSCs from Jak1 KO and wild-type mice. Analysis of proliferation of single cells in microwells was measured as average number of cells in experimental wells at each indicated time point. Basal condition shown in panels (C) and (D) are identical. Data are representative of two independent experiments with about n ~ 30 experimental wells per condition analyzed. Mean ± SEM. *p < 0.001. (E) Jak1 KO and wild-type LSKs (ERT2-Cre) were seeded out in cytokine-free methylcellulose medium supplemented with IL-3 (10 ng/mL). Bar graph displaying total number of scored colonies. *p < 0.05. (F) Bar graph depicting the total number of viable LSKs upon culture ex vivo for 48 hr in the presence or absence of Jak1 inhibitor (500 nM). Mean ± SEM. Data are representative of two independent experiments, and each experiment was performed in triplicate. *p < 0.05.

Figure 7. Non-redundant Role of Jak1 Signaling in HSCs

(A) White blood cell (WBC) counts of recipient mice transplanted with whole bone marrow cells. *p < 0.05. n = 5. (B) Schematic diagram showing competitive bone marrow transplant design. (C) Percentage (mean ± SEM) of CD45.1 versus CD45.2 chimerism in the PB of recipients at 1, 2, 3, and 4 months after poly(I:C) administration. Pre-poly(I:C) indicates the ratio of CD45.1 to CD45.2 bone marrow cells in recipient mice before gene deletion. n = 5. *p < 0.05. (D) Donor chimerism within the LSK compartment in the bone marrow of recipient mice at 16 weeks post-poly(I:C) injection. n = 5. *p < 0.05. See also Figure S7. (E and F) Percentage (mean ± SEM) of donor-derived total CD45.2 cells in the PB of recipient animals. (E) Secondary transplant, (F) tertiary transplants. n = 5 for each genotype. *p < 0.05. ns, not statistical significant. Only genotypes Jak2V617F and Jak1fl/fl,Jak2V617F were serially transplanted into tertiary recipients.