Type I IFNs drive hematopoietic stem and progenitor cell collapse via impaired proliferation and increased RIPK1-dependent cell death during shock-like ehrlichial infection.

Type I interferons (IFNα/β) regulate diverse aspects of host defense, but their impact on hematopoietic stem and progenitor cells (HSC/HSPCs) during infection remains unclear. Hematologic impairment can occur in severe infections, thus we sought to investigate the impact of type I IFNs on hematopoiesis in a tick-borne infection with a virulent ehrlichial pathogen that causes shock-like disease. During infection, IFNα/β induced severe bone marrow (BM) loss, blunted infection-induced emergency myelopoiesis, and reduced phenotypic HSPCs and HSCs. In the absence of type I IFN signaling, BM and splenic hematopoiesis were increased, and HSCs derived from Ifnar1-deficient mice were functionally superior in competitive BM transplants. Type I IFNs impaired hematopoiesis during infection by both limiting HSC/HSPC proliferation and increasing HSPC death. Using mixed BM chimeras we determined that type I IFNs restricted proliferation indirectly, whereas HSPC death occurred via direct IFNαR -mediated signaling. IFNαR-dependent signals resulted in reduced caspase 8 expression and activity, and reduced cleavage of RIPK1 and RIPK3, relative to Ifnar1-deficient mice. RIPK1 antagonism with Necrostatin-1s rescued HSPC and HSC numbers during infection. Early antibiotic treatment is required for mouse survival, however antibiotic-treated survivors had severely reduced HSPCs and HSCs. Combination therapy with antibiotics and Necrostatin-1s improved HSPC and HSC numbers in surviving mice, compared to antibiotic treatment alone. We reveal two mechanisms whereby type I IFNs drive hematopoietic collapse during severe infection: direct sensitization of HSPCs to undergo cell death and enhanced HSC quiescence. Our studies reveal a strategy to ameliorate the type I IFN-dependent loss of HSCs and HSPCs during infection, which may be relevant to other infections wherein type I IFNs cause hematopoietic dysfunction

Summary model.

Ixodes ovatus ehrlichia results in lethal shock-like infection and hematopoietic collapse. During infection of WT mice (left panel), type I IFN levels were elevated in the bone marrow, inducing hematopoietic stem cell (HSC) and progenitor (HSPC) loss. Type I IFNs restricted entry into the cell cycle, and type I IFN receptor (IFNαR) stimulation induced HSPC cell death. IFNαR signals limited caspase 8 expression and activation, blocked RIPK1 and RIPK3 cleavage, and caused RIPK1-kinase dependent cell death. In the absence of IFNαR signaling (right panel), HSCs and HSPCs were more proliferative and exhibited improved survival. IFNαR-deficiency results in increased pro-caspase 8 (C8FL), cleaved caspase products (C8p43 and C8p18), and increased cleavage of RIPK1 and RIPK3, resulting in improved HSC/HSPC numbers and function.</p

IFNα/β impair infection-induced proliferation of HSCs and HSPCs and indirectly restrict cycling.

(A) 4-hour BrdU incorporation by HSPCs and HSCs in IOE-infected WT and Ifnar1-/- mice 7 d.p.i. (B) Representative plots of Ki67 and DAPI staining, gated on HSCs from infected WT and Ifnar1-/- mice 6 d.p.i. Numbers indicate quadrant percentages. (C) Percent cells in G0 among HSPCs and HSCs in mock and IOE-infected WT and Ifnar1-/- mice 6 d.p.i. (D) The percent of WT and Ifnar1-/- cells in G0 in mock- or IOE-infected WT: Ifnar1-/- chimeric mice (as described in Fig 2D) at 6 and 8 d.p.i. *P P < 0.001.</p

IFNα/β reduce phenotypic and functional HSCs and HSPCs via direct and indirect mechanisms.

<p><b>(A)</b> HSCs, analyzed using the strategy in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007234#ppat.1007234.g001" target="_blank">Fig 1C</a> over time in WT and <i>Ifnar1</i>^-/- mice, n = 4–14 mice/group, <i>P</i> < 0.0001 for <i>Ifnar1</i>^-/- vs. WT by 2-way ANOVA. <b>(B)</b> Transplantation strategy used to test HSC function in IOE-infected WT and <i>Ifnar1</i>^-/- mice. Equal numbers of HSCs (50) were sort purified and competitively transferred to irradiated recipients. <b>(C)</b> Repopulation of total peripheral blood mononuclear cells (PBMCs) by WT and <i>Ifnar1</i>^-/- HSCs 16 weeks post-transplant. n = 6 recipient mice. <b>(D)</b> Generation of radiation-induced WT:<i>Ifnar1</i>^-/- mixed BM chimeric mice. (<b>E-F</b>) Fold change in proportions of WT to <i>Ifnar1</i>^<i>-/-</i> HSPCs and HSCs in infected WT:<i>Ifnar1</i>^<i>-/-</i> chimeric mice 6–8 d.p.i., normalized to baseline chimerism. n = 3 mice/time point.</p

IFNαR-dependent regulation of caspase 8 during IOE infection.

<p><b>(A)</b> Detection of full length (C8), and cleaved caspase 8 (CC8) and β-actin in BM cell lysates from mock and infected WT and <i>Ifnar1</i>^-/- mice 7d.p.i. Lanes represent individual mice. <b>(B)</b> Relative amounts of C8 and CC8 p41/43 and CC8 p18 in WT and <i>Ifnar1</i>^-/- mock and IOE–infected mice 7 d.p.i., normalized to β-actin. <b>(C)</b> HSPCs and HSCs in mock and vehicle (veh) control- or zVAD-FMK-treated infected WT and <i>Ifnar1</i>^-/- mice 7 d.p.i. n = 5–13 mice/group. <b>(D)</b> Vehicle-, zVAD-FMK-treated, or zVAD-FMK- and Necrostatin-1s treated <i>Ifnar1</i>^-/- mice day 7 p.i. n = 3–5 mice/group. *<i>P</i> < 0.05, **<i>P</i> < 0.001, ***<i>P</i> < 0.0001.</p

Targeting RIPK1 improves hematologic recovery in IOE infection survivors.

<p>(<b>A</b>) Mice were mock- or IOE-infected mice and treated with vehicle (Veh), Nec-1s, doxycycline (Doxy)-, or Doxy+ Nec-1s, as shown. (<b>B</b>) Survival of mock- (black line) or IOE-infected mice treated with vehicle (gray line), Nec1s (blue line), Doxy (orange line), or Doxy + Nec1s (purple line). (<b>C</b>) Surviving mice were evaluated at 15 d.p.i. for frequencies of BM HSPCs and HSCs, n = 5 mice/group, 4 mice/mock group. (<b>D</b>) Numbers of HSPCs and HSCs in mock and Veh-, Doxy-, and Doxy + Nec-1s-treated mice 15 d.p.i (<b>E</b>) Bacterial burden in the spleens of Doxy-, and Doxy + Nec-1s-treated mice 7 and 15 d.p.i., represented as <i>Dsb</i> copy/50ng total DNA. n = 3–5 mice/group. Dashed line represents the limit of detection. * <i>P</i> < 0.05, ** <i>P</i> < 0.001.</p

Targeting RIPK3 kinase activity protects HSPCs from IOE infection-induced depletion in mixed BM chimeric mice.

<p><b>(A-C)</b> BM cellularity (A), HSPCs (B), and HSCs (C) in mock or infected WT and <i>Ripk3</i>^Δintron2 mice 7 d.p.i. n = 4–5 mice/infected group; n = 2–4 mice/mock group. (<b>D</b>) Generation and treatment of <i>Ripk3</i>^Δintron2:WT mixed BM chimeric mice, and numbers of HSPCs and HSCs in mock and IOE-infected chimeric mice 7 d.p.i. (<b>E</b>) Numbers of WT (white bars) and <i>Ripk3</i>^Δintron2 (green bars) HSPCs and HSCs in mock and IOE-infected chimeric mice 7 d.p.i. n = 5–6 mice/group.*<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Type I IFNs induce caspase-independent HSPC death during IOE infection.

<p><b>(A)</b> Fold change in dead/dying cell frequency within the CD48^- HSPC population of WT and <i>Ifnar1</i>^-/- mice 7 d.p.i. normalized to WT mock levels. n = 5–9 mice/group. <b>(B)</b> Fold change in the proportion of live HSPCs with active caspases 8 or 3/7 over mock mice. n = 3–6 mice/group. <b>(C)</b> Frequency of apoptotic (Annexin V+ 7AAD-), dying (Annexin V+ 7AAD+), and dead (Annexin V- 7AAD+) cells and (<b>D</b>) caspase 8 or 3/7-active cells (gated on live, 7AAD- cells) among WT and <i>Ifnar1</i>^-/- HSPCs in mixed BM chimeric mice 7 d.p.i. n = 3 mice. (<b>E</b>) Detection of full length RIP1, cleaved RIP1, full length RIP3, cleaved RIP3, and actin in BM lysates of WT and <i>Ifnar1</i>^-/- mock and IOE–infected mice 7 d.p.i. Lanes represent individual mice. (<b>F</b>) Fold change of cleaved RIP1 to full length RIP1 and cleaved RIP3 to full length RIP3 in WT and <i>Ifnar1</i>^-/- mock (m) and IOE–infected mice, 7 d.p.i. *<i>P</i> < 0.05, **<i>P</i> < 0.001, ***<i>P</i> < 0.0001.</p

RIPK1 antagonism during infection rescues HSPCs and HSCs but does not impact bacterial burden or survival.

<p><b>(A)</b> Schematic of IOE infection and treatment with Necrostatin 1 (Nec1s), which was administered twice/day. <b>(B-D)</b> BM cellularity (B), HSPCs (D) and HSCs (E) in vehicle (Veh)- or Nec1s-treated mock- or infected WT mice 7 d.p.i. n = 4–5 mice/group. *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.001, ****<i>P</i> < 0.0001. <b>(E)</b> Survival of IOE-infected WT mice treated with Veh or Nec-1s, as depicted in (A), n = 8 mice/group. <b>(F)</b> Bacterial burden in the spleen of Veh- and Nec-1s-treated IOE-infected WT mice at 7 d.p.i., represented as <i>Dsb</i> copy/50ng total DNA.</p