Megakaryocyte derived immune-stimulating cells regulate host-defense immunity against bacterial pathogens

Megakaryocytes (MKs) continuously produce platelets to support hemostasis and form a niche for hematopoietic stem cell maintenance in the bone marrow. MKs are also involved in inflammation responses; however, the mechanism remains poorly understood. Here, using single-cell sequencing we identified an MK-derived immune-stimulating cell (MDIC) population exhibiting both MK-specific and immune characteristics, which highly expresses CXCR4 and immune response genes to participate in host-protective response against bacteria. MDICs interact with myeloid cells to promote their migration and stimulate the bacterial phagocytosis of macrophages and neutrophils by producing TNFα and IL-6. CXCR4high MDICs egress circulation and infiltrate into the spleen, liver, and lung upon bacterial infection. Ablation of MKs suppresses the innate immune response and T cell activation to impair the anti-bacterial effects in mice under the Listeria monocytogenes challenge. Using hematopoietic stem/progenitor cell lineage-tracing mouse line, we show that MDICs are generated from infection-induced emergency megakaryopoiesis in response to bacterial infection. Overall, we identify MDICs as an MK subpopulation, which regulates host-defense immune response against bacterial infection.


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Megakaryocytes (MKs) are large and rare hematopoietic cells in the bone marrow, which 19 continually produce platelets to support hemostasis and thrombosis (Deutsch and Tomer,20 2006). MK progenitors undergo multiple rounds of endomitosis during maturation to 21 achieve polyploid (Chang et al., 2007;Deutsch and Tomer, 2013;Machlus and Italiano, 22 2013; Nagata et al., 1997;Patel et al., 2005). MKs and their progenitors also migrate 23 observed mean distance of myeloid cells to CXCR4 low MKs (25.62 μm) is not different 1 from random simulations [27.76 μm, p (μ<25.62) = 0.14] ( Figure 2C). We also noticed that 2 bone marrow myeloid cells were preferably adjacent to the CXCR4 high MK-blood vessel  To explore how CXCR4 high MKs regulate myeloid cells, we interestingly found that 7 CXCR4 high MKs more effectively promoted myeloid cell mobilization than CXCR4 low 8 MKs in our transwell assays ( Figure 2D). Furthermore, we asked whether CXCR4 high MKs 9 regulate myeloid cell function against pathogens. To this aim, we incubated purified 10 CXCR4 low MKs and CXCR4 high MKs with neutrophils or macrophages for bacterial 11 phagocytosis analysis. We found that CXCR4 high MKs more efficiently enhanced the 12 bacterial phagocytosis of neutrophils and macrophages than CXCR4 low MKs ( Figure 2E-13 H). Overall, our data show that CXCR4 high might be a potential marker to identify a 14 functional immune-modulating MK subpopulation. We, therefore, referred CXCR4 high 15 MKs as MK-derived immune-stimulating cells (MDICs). 16 Our scRNA-seq also exhibited that the high expression of Cxcr4 was positively 17 correlated with immune cell-stimulating cytokines, such as Ccl6, Tnf, and Il6 (Li et al.,   Figure 3C). 11 We also found that MK ablation reduced the number of myeloid cells, including monocytes, 12 macrophages, dendritic cells (DCs), and neutrophils, in the liver and spleen ( Figure 3D, E), 13 suggesting the role of MKs in promoting myeloid cells against pathogens. We further 14 investigated how MKs regulate adaptative immunity against pathogen infection. To this 15 aim, we challenged Pf4-cre; iDTR mice with ovalbumin (OVA)-expressing recombinant 16 microbe (L. monocytogenes-OVA). Seven days after L. monocytogenes-OVA infection, 17 splenocytes from control or MK ablated mice were re-stimulated with OVA peptide in vitro 18 to assess OVA-specific T cell activation ( Figure 3F). Notably, MK ablation dramatically 19 reduced the number of CD4 + IFNγ + Th1, CD4 + IL4 + Th2, and CD8 + cytotoxic T 20 lymphocytes but did not impact the total number of CD4 + T cells and CD8 + T cells ( Figure   21 3G). These observations demonstrated that MKs regulate innate and adaptive immunity 22 against L. monocytogenes infection. To explore whether MDICs contribute to the immune 23 response against bacterial pathogens, we infused the purified MDICs (CXCR4 high MKs) 1 and CXCR4 low MKs into MK ablation mice during L. monocytogenes infection. Notably, 2 we found that the infusion with MDICs but not with CXCR4 low MKs partially rescued the 3 bacterial clearance defect in MK ablation mice ( Figure 3H, I). High Cxcr4 expression indicated that MDICs might migrate between bone marrow 7 microenvironment and circulation in response to infection (Suraneni et al., 2018). In line 8 with this, our spatial distribution analysis showed that ~80% of MKs directly contacted 9 blood vessels three days after L. monocytogenes infection, which was much higher than in

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To further explore the dynamic migration of MKs upon pathogen infection, we 19 developed a live imaging method to trace MK migration in the bone marrow (seen in the 20 method). Using Pf4-cre; tdTomato mice and in vivo live imaging approach, we observed 21 that small tdTomato + MKs rapidly migrated into sinusoids without rupture or platelet 14 To further explore how MKs migrate in organs during bacterial infection in vivo, we 15 employed Pf4-cre; Rosa26-cell membrane-localized tdTomato cell membrane-localized 16 EGFP (R26R mT/mG ) mice in which cell membrane-localized EGFP (mGFP) expresses 17 exclusively in MK lineage (Tiedt et al., 2007). We found that mGFP + MKs increased 18 dramatically in the liver (10.4-fold increased) and spleen (2.33-fold increased) three days 19 after L. monocytogenes infection ( Figure 4H, I). To further confirm the tissue infiltration 20 of MKs upon infection, we intravenously injected membrane-localized tdTomato 21 (mtdTomato) expressing bone marrow cells from R26R mT/mG mice into control recipients 22 or recipients infected with L. monocytogenes one day before mtdTomato + cell perfusion 23 ( Figure 4J). We found that two days after mtdTomato + cell perfusion, engrafted 1 mtdTomato + CXCR4 high MDICs more efficiently infiltrated into the liver (92.1%) and 2 spleen (92.5%); by contrast, mtdTomato + CXCR4 low MKs (66.7%) migrated to the bone 3 marrow ( Figure 4K).  we observed that tdTomato + HSPCs derived tdTomato + CXCR4 high MDICs rapidly 6 increased in the bone marrow, similar to the platelet-generating MKs (tdTomato + 7 CXCR4 low MKs) ( Figure 5F), without a noticeable rise of hematopoietic progenitors 8 ( Figure 5G). Overall, our observations indicated that MDICs might be generated by 9 emergency megakaryopoiesis to stimulate pathogen defense.  , 2021;Pariser et al., 2021;Sun et al., 2021;Wang et al., 2021); however, the mechanism 20 that MKs regulate immune response remains elusive. Here, we identified that MK5 has 21 both MK and immune cell characteristics, producing platelets and enriching immune 22 response genes. We also demonstrated the role of this cell type, a CXCR4 high MK 23 subpopulation (MDICs) in recruiting and stimulating innate myeloid cells by producing 1 TNFα and IL-6, for bacterial phagocytosis.
2 Normal HSC to MK development takes 11-12 days in humans and 4 days in mice; 3 However, emergency megakaryopoiesis takes less than a day to generate MKs upon 4 inflammation stress (Couldwell and Machlus, 2019;Liu et al., 2021;Sun et al., 2021) 5 ( Figure 5D). Previously, researchers believed that the emergency megakaryopoiesis mainly 6 contributes to the replenishment of damaged platelets upon acute inflammation (Haas et 7 al., 2015). Here, for the first time, we found that emergency megakaryopoiesis also quickly 8 generated MDICs to facilitate immune responses against bacterial infection.

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A recent report showed that the lung is a reservoir of MKs for platelet production   bodyweight into Pf4-cre + ; iDTR +/mice and their cre negative counterparts to induce 23 megakaryocyte ablation as indicated.

Materials and Methods
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BrdU incorporation assay for 15 minutes at room temperature in dark, and then 300 μl Annexin V binding buffer was 18 added to each tube. Cells were analyzed by a flow cytometer.

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Cells were then fixed by cold methanol for 15 minutes and blocked with 10% BSA 10 overnight, followed by incubation with F4/80 (BM8, eBioscience; 1:100) for two hours at 11 room temperature before being quantified using a spinning disk confocal microscope 12 (Dragonfly, Andor).

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For neutrophil phagocytosis, CD11b + Gr1 + Ly6cneutrophils were sorted from the spleen proplatelet formation were measured on day three or day five post-cultured, respectively, 10 using Nikon NIS-Elements BR.

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For in vivo CXCR4 high MK function assay in MK ablation mice, DT was 23 intraperitoneally injected every day for five days. On the second and fourth days, 2 × 10 5 1 sorted wild type CXCR4 high MKs or CXCR4 low MKs were intravenously injected into 2 indicated groups. PBS or 2500 CFUs of L. monocytogenes as previously described were 3 injected intravenously on the third day. Spleen and liver were harvested three days after 4 infection to determine the bacterial burdens as described.  Data are presented as means ± s.e.m except for phagocytosis assays and MK size 22 measurement, which are presented as means ± first and third quartiles. For phagocytosis 23 assay and MK size measurement, data were analyzed by a one-dimensional KS test. The scRNA-seq data generated in this study are deposited in GEO (GSE168224,