CD11c+ Cells Control Platelet Homeostasis in a Murine Bone Marrow Chimeric Atherosclerosis Model

TL;DR

This study shows that CD11c+ cells help control platelet levels and inflammation in a mouse model of atherosclerosis.

Contribution

The study identifies a novel CD11c+ cell–TPO–platelet axis linking immune regulation to platelet homeostasis in atherosclerosis.

Findings

Chronic CD11c+ cell depletion increases circulating platelet counts in ApoE−/− mice.

CD11c+ cell loss elevates serum thrombopoietin (TPO) and pro-inflammatory cytokines.

Findings reveal a pro-thrombopoietic shift and systemic inflammatory changes linked to CD11c+ cell depletion.

Abstract

Background/Objectives: Dendritic cells (DCs) are key regulators of immune responses in cardiovascular disease, yet their role in platelet homeostasis and thrombopoiesis remains incompletely understood. We previously demonstrated that chronic depletion of CD11c+ cells accelerates atherosclerotic plaque development. The objective of this study was to determine whether sustained loss of CD11c+ cells alters platelet production and systemic inflammatory signaling under atherogenic conditions. Methods: CD11c-DTR bone marrow chimeric mice on ApoE−/− background were generated and fed a high-cholesterol diet. CD11c+ cells were depleted by repeated diphtheria toxin administration over six weeks. Circulating platelet counts were quantified by automated hematology analysis. Systemic inflammatory changes were assessed using serum cytokine and chemokine profiling, and serum thrombopoietin (TPO) levels were measured by ELISA. Results: Chronic CD11c+ cell depletion resulted in a significant increase in circulating platelet counts in ApoE−/− mice. Serum cytokine profiling revealed broad inflammatory remodeling, including increased levels of cytokines associated with megakaryopoiesis and platelet activation, such as IL-4, MCP-1, CXCL9, IL-16, and IL-1α. In parallel, serum TPO levels were significantly elevated following CD11c+ cell depletion. Conclusions: In the specific context of hyperlipidemic CD11c-DTR bone marrow chimeric mice, these findings demonstrate that loss of CD11c+ cells is associated with a pro-thrombopoietic shift, elevated platelet counts, and systemic inflammatory changes. Our data identify a CD11c+ cell–TPO–platelet axis linking immune regulation to platelet homeostasis and thrombo-inflammatory signaling under these specific atherogenic conditions.

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Full text

1. Introduction

Dendritic cells (DCs) serve as professional antigen-presenting cells that orchestrate systemic and local immune responses, balancing pro- and anti-inflammatory signals. In chronic inflammatory settings, such as atherosclerosis [1,2], DCs represent a critical cell population that shapes immune response by orchestrating T cell priming, lipid antigen presentation, and local cytokine composition. Given their pivotal role in regulating inflammation, the systemic consequences of DC loss on hematopoiesis and thrombopoiesis have not been systematically examined. While our previous work demonstrated that CD11c^+^ cell depletion accelerates inflammation in a model of atherosclerosis [3], the direct effect of CD11c^+^ cells on platelet production and thrombotic potential in this context remains unknown.

CD11c^+^ cells, primarily representing conventional dendritic cells (DCs), serve as professional antigen-presenting cells that orchestrate systemic and local immune responses by balancing pro- and anti-inflammatory signals [3,4]. In chronic inflammatory settings, such as atherosclerosis, CD11c^+^ cells represent a critical cell population that shapes the immune response by orchestrating T cell priming, lipid antigen presentation, and local cytokine composition [5,6,7]. Beyond their classical role in lymphoid tissues, CD11c^+^ cells have emerged as key regulators of hematopoiesis, with specific subsets acting as homeostatic sensors in the bone marrow to monitor megakaryocytes [8,9].

Platelets have emerged as immune–effector cells that promote leukocyte recruitment, endothelial activation, NLRP3 inflammasome signaling, and plaque growth [10,11]. That shows that beyond their classical role in thrombosis, they contribute to vascular inflammation by recruiting leukocytes and activating endothelial cells [12]. Elevated platelet counts and activation are associated with increased cardiovascular events [13,14]. The regulation of thrombopoiesis and platelet activation by immune cells such as DCs remains poorly defined.

Previous studies have described DC–bone marrow crosstalk in the context of inflammatory myelopoiesis [15]. Recently, plasmacytoid dendritic cells (pDCs) were identified as homeostatic sensors in the bone marrow that monitor exhausted megakaryocytes and regulate megakaryopoiesis via interferon-alpha signaling [8]. Given the central endocrine role of thrombopoietin (TPO) as the master driver of megakaryopoiesis [16], we hypothesized that CD11c^+^ cell depletion shifts hematopoietic output toward a thrombopoietic phenotype driven by altered cytokine and TPO signaling. To test this, we depleted CD11c^+^ DCs in bone marrow chimeric ApoE^−^/^−^ mice under high-cholesterol diet feeding and quantified platelet counts, inflammatory cytokines, and serum TPO. ApoE^−^/^−^ mice were utilized as they represent a gold-standard model for chronic hyperlipidemia and spontaneous atherosclerosis development, providing the inflammatory environment necessary to study this axis. The use of bone marrow chimeras was necessary to circumvent the systemic lethality associated with the germline CD11c-DTR (Jung) model—while effective for short-term studies, repeated diphtheria toxin (DT) administration in whole-body transgenic mice (typically >3 injections) leads to death due to ectopic DTR expression [17,18]. By utilizing donor-derived CD11c-DTR bone marrow, we restricted DTR expression to the hematopoietic compartment, enabling the sustained depletion required for our chronic atherosclerosis study. Upon administration, diphtheria toxin (DT) binds with high affinity to the primate DTR—which is not natively expressed in mice—leading to the inhibition of protein synthesis and selective apoptosis of CD11c^+^ cells [19]. We and other groups have shown, that CD11c^+^ positive cells are specifically depleted [3,6].

With this work, we aimed to show a role of CD11c^+^ cells in mediating alterations in platelet counts as found during inflammatory diseases like atherosclerosis, which also might offer approaches for therapeutic intervention.

2. Materials and Methods

2.1. Mice and Bone Marrow Transplantation

Eight-week-old female ApoE^−/−^ mice were lethally irradiated (9 Gy split dose) and transplanted intravenously with 1 × 10^7^ bone marrow cells from CD11c-DTR donor mice as previously described in detail [3]. This chimeric approach is a standard methodology utilized here to circumvent the inherent lethal limitations of the CD11c-DTR strain during chronic DT administration.

2.2. CD11c+ Cell Depletion

Diphtheria toxin (DT; 200 ng/mouse) was administered intraperitoneally three times weekly during high-cholesterol diet feeding for 6 weeks to maintain CD11c^+^ DC depletion, following the established protocol [3]. The donor marrow was derived from CD11c-DTR mice on a C57Bl6 genetic background to ensure specificity of depletion to the CD11c^+^ hematopoietic compartment. The CD11c-DTR system facilitates targeted depletion through the expression of the primate diphtheria toxin receptor. DT administration induces selective apoptosis in CD11c^+^ cells. Validation of this chimeric model was already performed in our previous paper [3]: this protocol achieves a depletion efficiency of about 90% in hematopoietic tissues even after a depletion period of up to six weeks.

2.3. High-Cholesterol Diet

Mice were fed a high-cholesterol diet containing 1.25% cholesterol and 21% fat (Ssniff) during DC depletion.

2.4. Platelet Counts

Peripheral blood was collected by retro-orbital puncture into EDTA tubes. Platelet counts were determined using an automated hematology analyzer (Sysmex Germany, Hamburg, Germany).

2.5. Serum Cytokine Profiling

Serum was isolated by centrifugation and analyzed using the Mouse Cytokine Array Panel A (R&D Systems, Minneapolis, MN, USA) according to manufacturer instructions. Signal intensities were quantified and normalized. Data from the CD11c^+^ cell-depleted group were then calculated as a percentage of the mean control group values, with the control mean defined as 100%.

2.6. Thrombopoietin ELISA

Serum was isolated by centrifugation and analyzed using the Thrombopoietin (TPO) ELISA Kit from R&D Systems (Quantikine Mouse Thrombopoietin ELISA Kit, MTP00; R&D Systems, Minneapolis, MN, USA) according to manufacturer instructions.

2.7. Statistical Analysis

Data are presented as mean ± SEM. Statistical significance was assessed using unpaired two-tailed Student’s t-tests or Mann–Whitney tests as appropriate. Analyses were performed with GraphPad Prism (Version 8.0.2). A p-value < 0.05 was considered significant.

3. Results

3.1. Chronic CD11c+ Cell Depletion Increases Platelet Counts in ApoE−/− Mice

To investigate the effect of CD11c^+^ cell depletion on platelet homeostasis, we generated bone marrow chimeras by transplanting CD11c-DTR donor marrow into lethally irradiated ApoE^−/−^ recipients. After 8 weeks of reconstitution, mice were fed a high-cholesterol diet and injected with diphtheria toxin (200 ng, thrice weekly) for 6 weeks to maintain DC depletion.

Automated hematology analysis revealed a significant increase in circulating platelet counts in CD11c^+^ cell-depleted mice compared to controls in ApoE^−/−^ mice (p < 0.01) (Figure 1). Platelet increases were consistent across independent experiments and absent in controls. These findings indicate a conserved role of CD11c^+^ cells in restraining platelet production. Importantly, as demonstrated in our previous validation of this model, the administration of DT to ApoE^−/−^ mice lacking the DTR transgene does not alter platelet counts, systemic inflammatory parameters, or atherosclerotic burden [3]. These data confirm that the observed phenotype is a biological consequence of CD11c^+^ cell depletion rather than a non-specific effect of DT injection.

3.2. Chronic CD11c+ Cell Loss Triggers a Pro-Thrombopoietic Systemic Cytokine Shift

To evaluate the systemic inflammatory changes induced by chronic CD11c^+^ cell depletion, we performed a comprehensive cytokine and chemokine array on serum collected at the experimental endpoint. This profiling revealed a coordinated systemic response characterized by the significant upregulation of mediators closely linked to hematopoiesis and vascular inflammation (Figure 2A,B). Specifically, CD11c^+^ cell-depleted mice displayed marked increases in cytokines involved in megakaryocyte differentiation and platelet production, such as G-CSF, GM-CSF, IL-3, IL-4, IL-6, and IL-13, with IL-16 exhibiting the most prominent induction among all measured factors. In parallel, multiple cytokines linked to platelet activation and thrombo-inflammatory signaling, including IL-1α, IL-1β, TNF-α, and members of the MIP family—specifically MIP-1α, MIP-1β, and MIP-2—were significantly elevated. This shift was accompanied by a reorganization of the chemokine profile, where mediators implicated in leukocyte recruitment and vascular inflammation, such as CXCL9, CXCL11, IP-10, CCL17, and RANTES/CCL5, were significantly increased, whereas a subset of markers including SDF-1 and CXCL1 was conversely reduced following depletion. Taken together, these findings demonstrate that the loss of CD11c^+^ cells dismantles systemic immune equilibrium and establishes a pro-thrombotic cytokine environment that drives both accelerated thrombopoiesis and platelet-mediated vascular inflammation.

3.3. Serum TPO Levels Are Increased After CD11c+ Cell Depletion

To determine whether enhanced platelet output was associated with altered thrombopoietin levels, we quantified serum TPO by ELISA (Quantikine Mouse Thrombopoietin ELISA Kit, R&D Systems, MTP00) (Figure 3). TPO was significantly increased in CD11c^+^ cell -depleted ApoE^−/−^ chimeric mice compared to non-depleted controls (control 353.96 pg/mL vs. DC-depleted 576.33 pg/mL, n = 5/group, p = 0.03). This direct increase in the canonical master regulator of megakaryopoiesis mechanistically links CD11c^+^ cell loss to enhanced thrombopoiesis.

4. Discussion

This study uncovers a novel immunohematologic axis linking CD11c^+^ dendritic cells (DCs), thrombopoiesis, and platelet activation in cardiovascular disease. Chronic loss of CD11c^+^ DCs increases circulating platelet counts in specifically hyperlipidemic ApoE^−/−^ chimeric mice, accompanied by upregulation of cytokines known to enhance megakaryopoiesis and platelet function.

Our data identify a DC–TPO–platelet axis as a regulator of thrombo-inflammation in atherosclerosis. CD11c^+^ cell depletion increased platelet counts and induced a cytokine constellation—including MCP-1, CXCL9, IL-1α, and IL-16—linked to megakaryopoiesis and platelet priming [11]. Strikingly, serum thrombopoietin (TPO) was significantly elevated, providing a direct endocrine driver linking DC loss to enhanced megakaryocyte output [16].

A key mechanistic insight from our study is the identification of a CD11c^+^ cell–TPO–platelet axis. The observed elevation in serum TPO provides a direct endocrine driver linking DC loss to enhanced megakaryocyte output [20,21]. This increase is likely fueled by the significant upregulation of IL-6, which we identified in our cytokine profiling as a key factor in the megakaryopoietic drive, particularly in inflammatory states. IL-6 is a well-known potent stimulator of hepatic TPO production [21]. Furthermore, the induction of IL-16 and GM-CSF—factors known to promote myeloid progenitor expansion and megakaryocyte maturation—suggests a multi-layered stimulation of the thrombopoietic niche [22,23]. While the full causal pathway remains to be dissected, our data demonstrate that CD11c^+^ cell depletion is associated with a systemic cytokine shift—specifically increases in IL-6, IL-16, and GM-CSF—favoring pro-inflammatory and pro-thrombopoietic mediators. This shift establishes a pro-thrombotic environment that likely drives the observed acceleration in platelet formation. Our findings suggest that CD11c^+^ cells serve as an immunological buffer, preventing excessive platelet production; their depletion triggers a surge in pro-thrombopoietic cytokines—specifically IL-6 and IL-16—which in turn raises circulating TPO levels to accelerate platelet formation. This positions CD11c^+^ cells not only as regulators of adaptive immunity but as an essential immunologic checkpoint restraining the TPO-driven thrombopoietic shift that exacerbates atherosclerosis.

Elevated platelet numbers and activation can enhance platelet–leukocyte aggregate formation, increasing delivery of platelet-derived chemokines (like RANTES/CCL5, PF4) to monocytes and endothelial cells. This mechanism was elegantly demonstrated in ApoE^−/−^ mice: infusion of activated platelets (but not P-selectin-deficient platelets) increased monocyte adhesion to plaques, promoting atherogenesis [24]. Our data suggest that CD11c^+^ cell loss primes this cycle by enhancing platelet formation and systemic pro-inflammatory mediators.

Our cytokine array reveals that CD11c^+^ cell depletion induces a comprehensive cytokine and chemokine reorganization that strongly favors thrombopoiesis and platelet activation. The observed increases in GM-CSF, G-CSF, IL-3, IL-4, IL-6, and IL-13 are highly consistent with a megakaryopoietic drive, supporting our platelet-count findings. GM-CSF and IL-4 in particular have been shown to enhance megakaryocyte progenitor expansion and increase platelet output, while IL-16—our strongest hit—has emerging roles in hematopoietic niche activation. The parallel rise in platelet-activating cytokines such as IL-1α, IL-1β, TNF-α, and MIP-1α further suggests that both platelet quantity and platelet activation potential are enhanced. This aligns with seminal work demonstrating that inflammatory cytokines potentiate platelet reactivity, promote platelet–leukocyte aggregates, and accelerate atherosclerosis [25,26,27,28].

Mechanistically, these observations are aligned with recent work showing that inflammatory tissue signals can redirect bone marrow progenitors toward megakaryopoiesis [29] and with emerging recognition that platelets are not late “downstream markers”, but causal immunomodulators accelerating plaque progression [24,30,31]. Thus, DCs function as a previously unappreciated immunologic checkpoint restraining thrombopoiesis. Loss of this restraint induces a TPO-driven thrombopoietic shift that amplifies platelet-mediated plaque inflammation—providing a mechanistic bridge between DC dysfunction and exacerbated atherosclerosis.

Together, these data demonstrate that DCs restrain thrombopoiesis and platelet activation not only through cell–cell interactions but also by maintaining a balanced systemic cytokine environment. Loss of DCs dismantles this equilibrium and drives the system toward a pro-thrombotic, pro-inflammatory state. The pro-thrombotic state induced by DC depletion likely contributes to accelerated plaque formation, highlighting the clinical relevance of this DC–platelet axis. In patients, impaired DC function—due to aging, metabolic inflammation, or chronic immune perturbations—may predispose to thrombocytosis, platelet hyper-reactivity, and increased risk of atherothrombotic events [32].

In summary, our findings reveal a novel DC–TPO–platelet axis in which DCs restrain thrombopoiesis and platelet activation, thereby protecting against a pro-thrombotic, pro-atherogenic state. Loss of CD11c^+^ DCs disrupts this homeostatic balance, triggering a cascade of cytokine-mediated changes, platelet expansion, and enhanced vascular inflammation. These results extend the paradigm of cardiovascular immunology, positioning DCs not only as regulators of adaptive immunity but also as critical controllers of hematopoiesis and platelet biology. Therapeutic targeting of this axis may offer new opportunities to mitigate thrombo-inflammatory risk in atherosclerotic cardiovascular disease.

This study has some limitations. DC depletion was achieved using the CD11c-DTR system, which may affect additional CD11c-expressing cell populations beyond classical DCs. Moreover, this study primarily assessed systemic platelet counts and inflammatory mediators without directly interrogating megakaryocyte dynamics or platelet functional responses. Future studies employing DC subset-specific targeting strategies and functional platelet assays will be required to further define the mechanisms by which DCs regulate thrombopoiesis and thrombo-inflammatory signaling. Additionally, while the bone marrow chimera model effectively prevents the systemic lethality associated with repeated DT administration in germline CD11c-DTR mice [17,18], it may not fully account for the potential role of radio-resistant CD11c^+^ populations.