Abstract

Infections affect hematopoietic stem cell (HSC) biology, with significant implications in ageing, cancer development, and bone marrow failure syndromes. We previously showed that some infections induce HSCs to proliferate through a cell autonomous response to interferon gamma (IFNg). Given that interactions of HSCs with their bone marrow niche are a critical factor in maintaining quiescence, we considered how IFNg affects the interaction between HSCs and the niche. We hypothesized that infection would displace HSCs from the niche and reduce homing in an IFNg-dependent manner, thereby contributing to activation.

We used a Mycobacterium avium model of infection to characterize the effects of infection on HSC homing. In this model HSCs are activated to divide and differentiate via an IFNg-dependent mechanism with depletion of HSCs after several months of infection. We compared the homing of hematopoietic stem and progenitor cells (HSPCs) from M. avium infected versus non-infected animals by IV administration of CSFE-labeled cKit+ cells into lethally irradiated recipient mice. Homing was determined by the frequency with which labeled cells could be found in the bone marrow 17 hours later, relative to a DDAO-stained internal control. Surprisingly, HSPCs from M. avium -infected animals displayed improved homing compared to control HSPCs. A similar phenotype was seen for cKit+ cells isolated from mice treated with recombinant IFNg, whereas cKit+ cells from Ifngr1-/- mice showed decreased homing compared to wildtype. Using transplantation assays, we found decreased engraftment of Ifngr1-deficient whole bone marrow when cells were administered intravenously; but engraftment was normal when cells were infused intrafemorally. Altogether, these findings show that basal IFNg signaling contributes to normal HSPC homing and that IFNg induced during M. avium infection increases homing to the bone marrow.

The above studies reflect homing of HSPCs to the bone marrow, not cellular interactions within the niche. Since proximity to Cxcl12-expressing cells in the perivascular niche can affect HSC activation status, we used 3-D intravital imaging to study the effects of IFNg on HSC-niche interactions. We injected CMTMR-stained HSCs into Cxcl12-GFP transgenic mice and measured the distance of the HSCs from quiescence-associated Cxcl12-abundant reticular (CAR) cells. CMTMR-stained HSCs were seen moving upon IFNg treatment, and 24hrs post-IFNg HSCs were located significantly further away from CAR cells compared to untreated or TGF-b treated controls. These results suggest that IFNg can induce movement away from CAR cells, thereby allowing HSC proliferation during inflammatory conditions.

To define the mechanism by which IFNg causes HSC relocalization, we performed gene expression profiling of HSCs from control or IFNg-treated mice. Gene set enrichment analysis of differentially regulated genes showed modulation of the expected immune and cytokine response pathways, but also genes related to adhesion and extracellular matrix remodeling. Of note, the glycoprotein Bst2 (Tetherin) was upregulated in HSCs following IFNg treatment. Bst2 has been shown to bind to E-selectin, and localization to E-selectin+ endothelial cells is associated with increased HSC proliferation. Thus we hypothesized that IFNg-induced upregulation of surface Bst2 may facilitate proliferation by promoting HSC binding to E-selectin. We confirmed induction of Bst2 mRNA and protein by qPCR and flow cytometry, and we found that Bst2 was upregulated following M. avium infection via an IFNg-dependent mechanism. In vitro binding assays showed increased HSPC binding to recombinant E-selectin following IFNg treatment. Thus, IFNg-dependent cell surface expression of Bst2 may facilitate the displacement of HSCs from the quiescence-enforcing CAR niche to an active E-selectin-positive niche. Because E-selectin expression along blood vessel walls is also important for adhesion and transendothelial migration of HSCs, increased Bst2 on HSCs may also contribute to more efficient homing. In summary, IFNg improves HSPC homing but displaces HSCs from quiescence-enforcing CAR cells in the niche, potentially by inducing Bst2-mediated movement to the E-selectin rich proliferative niche. Future studies will ascertain whether blocking Bst2 is a feasible method to protect HSCs from the deleterious effects of chronic inflammation.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.