JNJ-26366821 Attenuates Radiation-Induced Pro-Inflammatory Cytokines and miRNAs and Triggers TR/RXR Signaling Pathway

Abstract

JNJ-26366821, a novel thrombopoietin mimetic peptide (TPOm), is shown to increase platelets (PLTs) transiently in peripheral blood. We hypothesized that increases in PLT counts may involve stimulation of hematopoiesis via induction of cytokines, growth factors, and microRNAs. Hence, we measured various cytokines, chemokines, and growth factors in serum. Time-course analysis of G-CSF, IL-5, IL-6, IL-9, IL-10, TNFα, IL-1α, and IL-1β expression was significantly altered in the control group at 9.5 Gy compared to a lower non-lethal dose of 7 Gy on days 7 to 15 post-exposure. TPOm pre-treatment significantly ameliorated the changes in expression of these pro-inflammatory cytokines and growth factors. Additionally, we show that TPOm differentially modulates the miRNA expression profiles in the spleen of irradiated mice compared to controls at both early times as well as later times after irradiation. These results suggest a possible role of TPOm in protecting animals from radiation-induced thrombocytopenia and lethality by attenuating radiation-induced inflammatory cytokines and miRNAs.

1. Introduction

The use of nuclear energy and materials is prevalent in the modern world, and continued development of nuclear weapons and acts of terrorism have posed the threat of nuclear accidents/incidents and the resultant potential for the exposure of people to ionizing radiation (IR). Following exposure to IR, a decline in peripheral blood cells and bone marrow hematopoietic stem and progenitor cells is expected, which is described as hematopoietic acute radiation syndrome (H-ARS) [1]. In addition to pancytopenia, H-ARS is characterized by an inflammatory response in the body manifested by infiltration of immune cells and release of various cytokines, chemokines, growth factors, adhesion molecules, and coagulation factors, leading to a self-amplified cascade [1]. These inflammatory response cascades result in vascular damage and coagulopathy, including hemorrhage and microvascular thrombosis [2,3,4]. In addition to the hematopoietic system, the vascular endothelium also plays an integral role in the tissue response following stress, such as exposure to IR, and controls the initiation and resolution of inflammatory responses through the regulation of chemotaxis and activation of leukocytes in the periphery [4].

Appropriate preventive strategies and prophylactic medical countermeasures are critically important to protect first responders and war fighters from the deleterious effects of exposure to radiation. To date, several medical countermeasures (MCMs) and biosimilars have been approved by the U.S. Food and Drug Administration (FDA) as radiation mitigators for H-ARS [5,6,7,8]. In addition to these drugs, it is necessary to have drugs that could be used as a prophylactic should a need arise in a radiological or nuclear event. A promising group of MCMs that are currently being tested for the mitigation of effects of exposure to IR include thrombopoietin (TPO) receptor agonists.

Since the first identification of the role of TPO in the 1950s [9], isolation of the hormone followed by characterization of its receptor c-Mpl was accomplished in the early 1990s [10]. Its role as a hematopoietic cytokine and the primary regulator of platelet production and proliferation of immature hematopoietic stem cells has been investigated [11]. Studies on the receptor agonists followed soon after identification of the receptor [11]. Previously, clinical trials with recombinant thrombopoietin have failed due to adverse immune responses and necessitated alternative compounds. A new strategy targeting the receptor, c-Mpl, was developed by generating the thrombopoietin mimetic (TPOm) compounds. JNJ-26366821, a novel TPO mimetic peptide (TPOm), stimulates platelet production without developing neutralizing antibodies or causing any adverse effects [11]. We have shown that a single dose of JNJ-26366821 administered 24, 12, or 2 h pre-radiation resulted in 100% survival from a lethal dose of total body irradiation (TBI) with a dose reduction factor of 1.36 [12]. Accelerated recovery from radiation-induced peripheral blood cytopenia and depletion of bone narrow progenitor cells was observed in mice exposed to a non-lethal dose (7 Gy) [13,14] of radiation with JNJ-26366821 pre-treatment [12]. These findings indicate that JNJ-26366821 is a promising prophylactic radiation countermeasure for hematopoietic acute radiation syndrome.

Radiation causes cellular DNA damage leading to pro-inflammatory and immune reactions in target cells [1]. The interleukin (IL)-1 family of cytokines are linked closely to the innate immune response and as the first line of host defense against stress-induced acute and chronic inflammation [15]. In healthy humans, IL-1β induces production of secondary inflammatory factors IL-6, IL-8, tumor necrosis factor-alpha (TNFα), granulocyte colony-stimulating factor (G-CSF), and granulocyte–macrophage colony-stimulating factor (GM-CSF) [16]. We have demonstrated that pre-administration of TPOm alleviated radiation-induced soluble markers of bone marrow aplasia and endothelial damage in mice exposed to either non-lethal or lethal doses of radiation [12]. These effects support the hypothesis that a TPO agonist could effectively stimulate vascular endothelial repair and protect mice from radiation-induced lethality by modulating cytokines and growth factors.

MicroRNAs (miRNAs) are small, evolutionarily conserved non-coding RNAs that regulate gene expression post-transcriptionally by targeting messenger RNAs (mRNAs), thereby influencing key biological processes, such as apoptosis, cell cycle regulation, and immune responses [17,18,19]. Increasing evidence suggests that radiation exposure disrupts miRNA expression in a dose- and time-dependent manner across various cell types and tissues [20,21]. Recently, miRNAs have gained significant attention in evaluating the efficacy of therapeutic agents, particularly in determining whether specific drugs can reverse or modulate these radiation-induced changes [22,23,24,25]. By mapping drug-induced alterations in miRNA expression and linking them to downstream signaling pathways, this approach offers a mechanistic framework to better understand therapeutic efficacy and safety. We have previously reported on the regulation of the mode of action of various MCMs by miRNAs [21,26].

In this study, we examined the cytokine profiles in mouse serum to test whether TPOm differentially regulated radiation-induced hematopoietic cytokines, chemokines, and growth factors. We measured various cytokines in serum at different times in non-lethally and lethally irradiated as well as unirradiated animals using multiplex Luminex assays. Our results demonstrate that TPOm induced G-CSF and macrophage inflammatory proteins (MIP)-1α and MIP-1β significantly in peripheral blood between 12 and 48 h post-administration of TPOm. Pre-administration of the drug also modulated production of pro-inflammatory cytokines and chemokines (IL-1α, IL-1β, IL-5, IL-6, IL-9, IL-10, and TNF-α) in blood following radiation exposure.

2. Results

2.1. Prophylactic Administration of TPOm Protects Mice from Lethal Gamma Radiation Exposure by Attenuating Pro-Inflammatory Cytokines, Chemokines, and Growth Factors

We analyzed the serum from irradiated mice, either pre-treated with TPOm or vehicle-treated with saline, for various pro-inflammatory cytokines, chemokines, and growth factors using a 23-plex cytokine panel. This panel included G-CSF, various interleukins (IL-1α, IL-1β, IL-5, IL-6, IL-9, and IL-10), TNF-α, and MIP-1α and MIP-1β. Baseline cytokine levels were established using blood samples from a non-irradiated cohort that received saline (vehicle) instead of TPOm. We determined statistical significance using a two-way analysis of variance (ANOVA), with a p-value of less than 0.05 indicating a notable effect. All data are tabulated in Supplementary Tables S1–S3.

2.1.1. Induction of Specific Pro-Inflammatory Cytokines Following TPOm Administration in Healthy Mice

At 12 h post-injection, non-irradiated animals administered 1.0 mg/kg TPOm exhibited significantly elevated levels of G-CSF compared to those given 0.3 mg/kg TPOm (p = 0.0005) or saline (p = 0.0007; Figure 1A). This trend continued at 72 h post-injection, with the 1.0 mg/kg TPOm group showing significantly higher G-CSF levels compared to the vehicle-treated group (p = 0.028).

Inflammatory marker IL-6 was significantly higher in animals administered 1.0 mg/kg TPOm at both 48 h and 72 h post-injection compared to control animals receiving vehicle (p < 0.0001). Animals treated with 0.3 mg/kg TPOm also displayed significantly higher IL-6 levels at these time points relative to the vehicle group (p < 0.0001; Figure 1B).

For MIP-1α, vehicle-treated animals showed significantly lower levels at 48 h post-injection compared to animals given 0.3 mg/kg TPOm (p < 0.0001) and 1.0 mg/kg TPOm (p < 0.0001). At 72 h post-injection, animals treated with 0.3 mg/kg TPOm had significantly higher MIP-1α levels than vehicle-treated animals (p < 0.0001; Figure 1C).

Finally, MIP-1β levels at 48 h post-injection were significantly higher in both the 0.3 mg/kg (p = 0.0017) and 1.0 mg/kg (p < 0.0001) TPOm groups compared to vehicle-treated animals. Similarly, at 72 h post-injection, MIP-1β levels remained significantly higher in the 0.3 mg/kg (p = 0.013) and 1.0 mg/kg (p = 0.0006) TPOm groups (Figure 1D).

2.1.2. Dose-Dependent Differentially Regulated Pro-Inflammatory Cytokines, Chemokines, and Growth Factors with Pre-Administration of TPOm

In mice irradiated at 7 Gy, G-CSF levels were significantly higher (Figure 2A) in vehicle-treated animals on day 3 (p = 0.018), day 7 (p = 0.001), and day 15 (p < 0.001) compared to the 0.3 mg/kg TPOm group and the 1.0 mg/kg TPOm group on day 7. At 9.5 Gy, on days 3 and 7, all irradiated groups exhibited higher G-CSF levels compared to the non-irradiated vehicle group. The difference between vehicle- and TPOm-treated groups was significant (p < 0.001) on day 15 (Figure 2A).

The effect on IL-1α (Figure 2B) appeared biphasic. Initially on day 1, the levels lowered significantly in all irradiated groups (both at 7 and 9.5 Gy) when compared to the non-irradiated control and increased closer to normal levels at 7 Gy in TPOm groups by day 3. At 7 Gy and 9.5 Gy on day 15, the IL-1α level in the vehicle group was significantly higher (7 Gy: p ≤ 0.002; 9.5 Gy: p < 0.001) compared to the levels in 0.3 mg/kg and 1.0 mg/kg TPOm groups. For IL-1β (Figure 2C), no significant differences were observed at 7 Gy. However, at 9.5 Gy on day 15, IL-1β levels were significantly higher (p < 0.001) in vehicle-treated irradiated animals compared to 0.3 mg/kg and 1.0 mg/kg TPOm-treated animals. In irradiated vehicle-treated animals, IL-5 levels were significantly higher (p = 0.02 to <0.001) than TPOm-treated animals on days 3, 7, and 15, indicating protection by TPOm (Figure 2D). All groups returned to non-irradiated baseline levels by day 30. Regarding IL-6 (Figure 2E), at early time points (days 1 and 3), irradiated TPOm-treated animals showed significantly higher levels (at 1.0 mg/kg TPOm, p = 0.007 and p < 0.001, respectively) than the vehicle group at both 7 and 9.5 Gy. At 7 Gy, IL-6 levels were significantly lower (p < 0.001) in vehicle-treated irradiated animals on days 1 and 3. However, on day 7, IL-6 levels were inverted, with vehicle having significantly higher levels compared to 0.3 mg/kg or 1.0 mg/kg TPOm, which had reached normal levels similar to non-irradiated, vehicle-treated animals. At 9.5 Gy, IL-6 levels were significantly lower (p = 0.014) in vehicle-treated irradiated animals on day 3 versus 0.3 mg/kg TPOm and 1.0 mg/kg TPOm. However, on day 15, the trend reversed, with significantly higher levels (p < 0.001) in vehicle-treated irradiated animals versus 0.3 mg/kg TPOm and 1.0 mg/kg TPOm.

Non-irradiated, vehicle-treated animals had a baseline IL-9 level of 32.82 ± 6.08 pg/mL (Figure 2F). At 7 Gy, vehicle-treated irradiated animals showed significantly lower (p ≤ 0.007) IL-9 on days 1 and 3 compared to 1.0 mg/kg TPOm. However, by day 7, IL-9 levels were significantly higher (p < 0.001) in vehicle-treated irradiated animals versus 0.3 mg/kg and 1.0 mg/kg TPOm. At 9.5 Gy, IL-9 levels in vehicle-treated irradiated animals on days 7 and 15 were higher compared to 0.3 mg/kg TPOm and 1.0 mg/kg TPOm.

For IL-10 (Figure 2G), there were no significant differences between animals treated with vehicle or TPOm (0.3 mg/kg and 1.0 mg/kg) at 7 Gy. At 9.5 Gy, on days 7 and 15, IL-10 levels were significantly higher in vehicle-treated irradiated animals compared to 0.3 mg/kg TPOm (p = 0.007 and <0.001, respectively) and 1.0 mg/kg TPOm (p = 0.006 and <0.001, respectively).

At 7 Gy on day 1, 0.3 mg/kg TPOm-treated irradiated animals had higher TNF-α levels than 1.0 mg/kg TPOm-treated or vehicle-treated animals (Figure 2H). At 9.5 Gy, TNF-α levels in vehicle-treated irradiated animals on days 7 (p = 0.003) and 15 (p < 0.001) were much higher compared to 0.3 mg/kg TPOm and 1.0 mg/kg TPOm groups.

On days 1 and 3, for both chemokines MIP-1α (Figure 2I) and MIP-1β (Figure 2J), the irradiated groups with TPOm (7 and 9.5 Gy) had significantly higher (p < 0.001) levels compared to irradiated or non-irradiated vehicle-treated animals. At 7 Gy, MIP-1α levels in vehicle-treated irradiated animals on days 1 and 3 were significantly lower than 0.3 mg/kg (p < 0.001) or 1.0 mg/kg (p < 0.001) TPOm-treated animals. Similarly, at 9.5 Gy, MIP-1α levels in vehicle-treated irradiated animals on days 1 and 3 were significantly lower than 0.3 mg/kg (p <0.001) or 1.0 mg/kg (p <0.001) TPOm-treated animals. As for MIP-1β, at 7 Gy, MIP-1β levels in vehicle-treated irradiated animals on days 1 and 3 were significantly lower (p < 0.001) than 0.3 mg/kg or 1.0 mg/kg TPOm-treated animals. Similarly, at 9.5 Gy, MIP-1β levels in vehicle-treated irradiated animals on days 1 and 3 were significantly lower than 0.3 mg/kg (p =0.014) or 1.0 mg/kg (p <0.001) TPOm-treated animals.

2.2. Temporal Regulation of Signaling Pathways in Irradiated Mice in Response to Radiation and TPOm Pre-Treatment

To identify the molecular pathways modulated by TPOm pre-treatment in irradiated mice, we conducted a time-course pathway enrichment analysis of differentially expressed miRNAs in spleen samples using ingenuity pathway analysis (IPA). Canonical pathway analysis revealed temporal and treatment-specific alterations, with the thyroid hormone receptor/retinoid X receptor (TR/RXR) activation pathway being the most significantly affected across multiple time points. As shown in Figure 3, inhibition of the TR/RXR activation pathway (negative activation z-score) was observed in the vehicle-treated group at day 1 post-irradiation (Vehicle_D1), indicating a strong downregulation of this pathway shortly after radiation exposure. No significant changes were detected at later time points (days 7, 15, and 30) in the vehicle-treated groups. In contrast, mice pre-treated with TPOm showed inhibition of the TR/RXR activation pathway at days 1 and 30 post-irradiation, suggesting a delayed and distinct regulatory effect of TPOm on this signaling axis. Notably, no significant modulation of this pathway was detected at time points (days 7 and 15) in the TPOm-treated groups. Other canonical pathways, including the HOTAIR regulatory pathway, Th1 and Th2 activation pathway, and regulation of the epithelial mesenchymal transition by growth factors pathway, were not significantly altered in either treatment group at any of the examined time points. These findings highlight a unique temporal pattern of TR/RXR pathway inhibition in response to irradiation, which is modulated by TPOm pre-treatment, suggesting a potential mechanistic link between TPOm intervention and delayed transcriptional regulatory responses post-radiation.

2.3. Integration of TPOm-Regulated miRNAs and Cytokine Networks Reveals Temporal Remodeling of Inflammatory and Hematopoietic Signaling

To further elucidate the mechanistic relationships between cytokine modulation and miRNA expression following TPOm pre-treatment, integrated miRNA–cytokine interaction networks were constructed for differentially expressed miRNAs in spleen samples of control and TPOm-treated animals at days 15 and 30 post-irradiation using IPA (Figure 4). These networks provide a system-level view of post-transcriptional regulatory interactions linking inflammatory and hematopoietic recovery pathways.

On day 15, the vehicle-treated group network showed moderate interaction density with IL-6, IL-10, TNF-α, and MIP-1β serving as key hubs connecting to multiple regulatory miRNAs (Figure 4A). In contrast, the TPOm-treated network demonstrated enhanced complexity and connectivity, particularly among IL-6, TNF-α, MIP-1α, and IL-1β, which interacted with miR-125a-5p, miR-126a-5p, and miR-342-3p (Figure 4B). These miRNAs are known to influence hematopoietic differentiation and cytokine signaling, suggesting a coordinated post-transcriptional regulation of cytokine output during early hematopoietic recovery. The emergence of the MPL-THPO-TPOm subnetwork indicates that thrombopoietin receptor activation directly engages miRNA-mediated cytokine feedback control.

By day 30, the cytokine-miRNA network displayed greater consolidation of pro-inflammatory and hematopoietic signaling nodes. The vehicle-treated group maintained a relatively simple network dominated by IL-6, IL-10, and TNF-α, whereas the TPOm-treated group exhibited a markedly expanded network linking IL-6, IL-1β, and TNF-α with miR-125a-3p, miR-17, miR-31, and miR-150 (Figure 4C,D). These interactions indicate sustained engagement of miRNA regulatory loops modulating cytokine signaling, likely contributing to immune rebalancing and vascular recovery. Together, these findings demonstrate that TPOm treatment progressively remodels cytokine–miRNA interaction topology, reinforcing a dynamic, time-dependent adaptation that supports hematopoietic regeneration following radiation exposure.

2.4. TPOm Modulates Upstream Signaling Molecules in Irradiated Mice

To identify key regulators responsible for the observed alterations in gene expression, we performed upstream regulator analyses on differentially expressed miRNAs from spleen samples of the vehicle- and TPOm-treated groups. Our findings reveal dynamic changes in transcriptional regulatory networks in response to radiation, with several upstream regulators exhibiting a trend toward activation or inhibition on days 15 and 30. Notably, pre-treatment with TPOm led to a reversal or modulation of these regulatory patterns (Figure 5). On day 15 post-radiation, the vehicle-treated group exhibited a trend toward activation of EZH2 and IFNB1, and a trend toward inhibition of MTDH (Figure 5A). In contrast, the TPOm-treated group showed no change in EZH2, a stronger trend of activation of IFNB1, and a reversal to activation of MTDH (Figure 5B). By day 30, radiation alone in the vehicle-treated group induced a trend toward activation of LATS2, along with a trend toward inhibition of TGFB1 and MYC (Figure 5C). However, TPOm pre-treatment appeared to suppress LATS2 activation, enhance MYC inhibition, and reverse TGFB1 activity toward activation (Figure 5D). These results collectively indicate that TPOm alters the trajectory of upstream regulatory signaling in the spleen, potentially contributing to a delayed or modified transcriptional response following radiation exposure.

2.5. TPOm Modulates Diseases and Cellular Functions in Irradiated Mice

Subsequently, to investigate the biological processes and disease functions influenced by radiation and TPOm pre-treatment, we conducted IPA on the differentially expressed miRNAs identified in spleen samples from the vehicle- and TPOm-treated groups. Our analysis revealed that radiation exposure induced significant alterations in multiple cellular functions in the spleen on days 15 and 30 post-irradiation, with several functions exhibiting either activation or inhibition. Notably, pre-treatment with TPOm was able to reverse these radiation-induced functional changes (Figure 6).

Exposure to radiation led to a trend toward inhibition of growth of organism, survival of organism, invasion of lung cancer cell lines, and digestive system cancer in the control vehicle-treated samples after 15 days post-TBI (Vehicle_D15; Figure 6A). However, TPOm pre-treatment attenuated these alterations, restoring functional activities toward baseline levels and suggesting a protective effect against radiation-induced cellular dysfunction (TPOm_D15; Figure 6B). Furthermore, functions such as colony formation and cell proliferation of cervical cancer cell lines were inhibited, while myelopoiesis of leukocytes was activated following TPOm pre-treatment—changes not observed in the spleens of the vehicle-treated group on day 15.

Thirty days after exposure to radiation, a trend toward activation of pathways related to migration of adenocarcinoma cell lines, migration of lung cancer cell lines, proliferation of lung cancer cell lines, survival of organism, and cytostasis of tumor cell lines in the control vehicle-treated samples (Vehicle_D30) was observed. Concurrently, the TPOm pre-treatment inhibited several processes, including solid tumor growth, the neoplasia of both tumors as well as other cells, reactive oxygen species synthesis, squamous cell carcinoma cell migration, and stem cell proliferation. Additionally, it activated lymphocytes and helped maintain normal blood cell counts (Figure 6C). Importantly, TPOm administration (TPOm_D30) prior to radiation exposure reversed many of these effects, mitigating the activation of pro-tumorigenic pathways and restoring immune-related functions (Figure 6D).

Together, these results demonstrate that pre-treatment with TPOm modulates miRNA expression in spleen tissues following radiation exposure, thereby influencing disease pathways and cellular functions critical for survival and recovery after irradiation.

3. Discussion

Various cell types, including endothelial cells, monocytes, fibroblasts, and T-lymphocytes, are known to synthesize glycoprotein hematopoietic growth factors [1,16]. G-CSF is one of these factors essential for the differentiation of progenitor cells into neutrophils in bone marrow. Our results showed that animals pre-treated with TPOm have significantly faster recovery and reduced nadir of the different white blood cell types, red blood cells, as well as platelets compared to vehicle-treated animals from exposure to a lethal dose of ionizing radiation (Figure S1). In addition to the effect of TPOm on peripheral blood cells, clonogenic assays based on colony-forming unit (CFU) assays indicate either enhanced preservation or faster recovery of functional BMCs (Figure S2). Animals pre-treated with TPOm exhibited faster recovery of the total colony-forming units by day 15 post-TBI compared to the vehicle control after significant depletion noted on days 1 through 7 in both irradiated groups at 7 Gy [12] and 9.5 Gy (Figure S2). A non-lethal dose of 7 Gy was selected to induce significant hematopoietic injury in the absence of confounding mortality over a 30-day window [13,14]. These results are further corroborated by histological examinations of the sternal bone marrow, which showed significantly higher cellularity and megakaryocyte counts in animals treated with TPOm 24 h pre-TBI compared to vehicle-treated animals at 7 Gy [12] and 9.5 Gy (Figure S3). Macrophage inflammatory protein-1α (MIP-1α) and MIP-1β are highly related members of the CC chemokine subfamily and have important roles in the development of inflammatory responses during infection by recruiting mononuclear cells via modulating cytokine production [27]. Elevated levels of MIP-1α and MIP-1β were detected within 24 h of sepsis in patients due to endotoxemia and dropped in parallel with TNF-α and IL-6 [27]. Since MIP-1α and MIP-1β increase during acute inflammation, we examined the MIP-1α and MIP-1β levels in serum inducing the release of inflammatory cytokines. Our data show that both MIP-1α and MIP-1β levels are higher in TPOm-treated groups, which decrease at later times post-radiation, suggesting reduced inflammation in animals during the recovery phase from hematopoietic damage.

In recent years, miRNAs have gained significant prominence in the assessment of drug efficacy, giving rise to a new interdisciplinary field known as miRNA pharmacogenomics. This approach focuses on understanding how drug administration influences miRNA expression and its downstream effects on target genes and regulatory pathways [22,23,24,25]. Drugs and small molecules can influence miRNA biogenesis and expression at multiple stages, before, during, and after transcription, highlighting their potential as therapeutic modulators of post-transcriptional gene regulation [28,29,30].

In this study, we investigated the therapeutic potential of TPOm, a promising prophylactic radiation countermeasure, with a focus on miRNA-mediated regulatory mechanisms in spleen tissues of mice exposed to TBI. Pro-inflammatory cytokine–radiation-induced miRNA integrated networks provide novel insights into the multi-layered regulatory mechanisms underlying the radioprotective efficacy of TPOm. Our findings reveal that TPOm not only activates the canonical thrombopoietin receptor (c-Mpl) signaling cascade but also reprograms cytokine expression through miRNA-mediated post-transcriptional control, thereby maintaining hematopoietic homeostasis under radiation stress. On day 15, the increased connectivity between miR-125a, miR-150, miR-126a, and cytokines, such as IL-6 and TNF-α, suggests early hematopoietic activation supported by miRNA-guided cytokine regulation. miR-125a enhances hematopoietic stem cell survival and stress resistance, while miR-150 promotes megakaryocyte and erythroid lineage differentiation [31,32]. miR-126a, predominantly expressed in endothelial and hematopoietic progenitor cells, regulates vascular integrity and cytokine-mediated hematopoietic signaling [33]. The association of these miRNAs with IL-6 and TNF-α, both central to hematopoietic recovery and inflammation, implies that TPOm induces a fine-tuned feedback mechanism balancing regeneration with inflammatory control. By day 30, the persistence of strong radiation-induced miRNA–pro-inflammatory cytokine connectivity in the TPOm-treated group, particularly between IL-6, IL-1β, and TNF-α, reflects long-term adaptation of immune and hematopoietic signaling. The inclusion of miR-17 and miR-31, known regulators of NF-κB and MAPK pathways [34,35], suggests that TPOm promotes resolution of inflammation and stabilization of hematopoietic recovery through negative feedback loops. This is consistent with our cytokine data demonstrating TPOm-mediated normalization of IL-1, IL-6, IL-9, IL-10, and TNF-α, which collectively regulate post-radiation inflammatory tone and bone marrow regeneration. The sustained MPL-THPO-TPOm signaling node across both time points highlights continued receptor activation and downstream modulation of hematopoietic and endothelial repair genes.

Importantly, several of the radiation-responsive miRNAs identified in our integrated network analysis, including miR-125a-5p/3p, miR-126a-5p, miR-342-3p, miR-17, miR-31, and miR-150, have been previously implicated in radiation injury, hematopoietic stress, endothelial dysfunction, and inflammatory regulation in independent experimental models. Members of the miR-125 family (miR-125a-5p and miR-125a-3p) are established regulators of hematopoietic stem cell (HSC) self-renewal and inflammatory signaling [36,37]. Altered expression of miR-125 family members has been observed in radiation-induced hematopoietic stress models [38]. miR-126a-5p plays a critical role in endothelial homeostasis and vascular integrity [33,39], processes that are central to bone marrow niche maintenance and are highly susceptible to ionizing radiation injury [40]. Similarly, miR-342-3p has been linked to inflammatory pathway modulation and NF-κB-associated signaling, pathways known to be activated following radiation exposure [35,41]. The miR-17 family is a key regulator of cell cycle progression and progenitor expansion [42], and radiation-induced proliferative stress has been associated with modulation of miR-17-related networks [43]. miR-31 has been implicated in DNA damage response signaling and cellular radiosensitivity [44]. Additionally, miR-150 is a well-characterized regulator of lymphocyte differentiation and immune reconstitution, with documented downregulation following hematopoietic injury and radiation exposure [32,45,46]. Collectively, the concordance between our findings and previously published radiation-responsive miRNAs supports the robustness and translational relevance of our profiling dataset.

Strikingly, our canonical pathway analysis revealed that the TR/RXR activation pathway was the most significantly impacted across multiple time points. The TR/RXR activation pathway is a critical nuclear receptor signaling axis involved in regulating cellular metabolism, development, differentiation, and apoptosis [47,48]. TRs (thyroid hormone receptors) and RXRs (retinoid X receptors) form heterodimers that bind to specific DNA response elements to regulate gene transcription in a ligand-dependent manner. These receptors influence a broad range of physiological processes through transcriptional control of genes associated with lipid metabolism, mitochondrial function, oxidative stress, and immune signaling. In the context of radiation exposure, the TR/RXR pathway plays a significant regulatory role in the cellular stress response. Ionizing radiation induces oxidative damage, inflammation, and mitochondrial dysfunction, processes tightly linked to metabolic demand and redox homeostasis. Because TR signaling enhances mitochondrial biogenesis, oxidative phosphorylation, and the basal metabolic rate, sustained TR/RXR activation following radiation could theoretically exacerbate metabolic stress in damaged tissues. Elevated mitochondrial respiration during radiation-induced DNA and membrane injury may increase electron transport chain instability, thereby amplifying reactive oxygen species (ROS) generation. During the early or “latent” phase of radiation injury, when subcellular damage persists despite a lack of clinical tissue failure, transient suppression of TR/RXR signaling may serve an adaptive function. By reducing metabolic throughput and mitochondrial oxidative activity, inhibition of this pathway could limit secondary ROS production, decrease oxidative burden, and prevent the propagation of DNA and lipid damage. In this framework, early TR/RXR inhibition may represent a protective metabolic downscaling response aimed at preserving cellular viability. Conversely, prolonged or excessive suppression of TR/RXR activity may eventually become maladaptive.

Downregulation or inhibition of this pathway after radiation exposure has been associated with impaired DNA repair, reduced mitochondrial efficiency, and increased cellular apoptosis or senescence, especially in sensitive tissues like the spleen, liver, and bone marrow. Radiation-induced suppression of the TR/RXR axis may also disrupt thyroid hormone homeostasis, which is critical for hematopoietic and immune recovery following injury [49]. Thus, the biological consequence of TR/RXR inhibition likely depends on its magnitude and temporal dynamics, and acute suppression may mitigate oxidative stress, whereas sustained inhibition may impair recovery mechanisms. Furthermore, RXR partners with multiple other nuclear receptors (e.g., PPARs, LXR, and VDR), serving as a central hub in nuclear signaling pathways [50]. Disruption of RXR function may, therefore, contribute to broader transcriptomic disturbances following radiation. In the present study, inhibition of the TR/RXR activation pathway was observed in spleen tissues from irradiated mice, particularly at early time points, indicating acute suppression of this regulatory mechanism. Notably, pre-treatment with TPOm altered the temporal pattern of TR/RXR pathway inhibition, suggesting a delayed or modulatory effect on this signaling axis. This finding implies that TPOm may exert radioprotective effects in part by regulating nuclear receptor activity, potentially restoring transcriptional balance in immune-related and stress response genes. Together, these observations highlight the TR/RXR pathway as a key mediator of radiation-induced transcriptional responses, and a potential target for radiomitigation strategies aimed at promoting recovery and tissue homeostasis post-irradiation.

In addition to pathway enrichment, we conducted upstream regulator analysis to identify key transcriptional regulators influenced by radiation and TPOm pre-treatment. On day 15 post-irradiation, radiation alone (RV group) led to a trend toward activation of EZH2 and IFNB1, alongside inhibition of MTDH. Notably, TPOm pre-treatment altered this pattern by abolishing EZH2 activation, enhancing IFNB1 activation, and reversing MTDH inhibition into activation. By day 30, radiation exposure resulted in activation of LATS2 and inhibition of TGFB1 and MYC. However, TPOm pre-treatment modulated this regulatory landscape, suppressing LATS2, further inhibiting MYC, and reversing TGFB1 to an activated state. These changes reflect TPOm’s capacity to reshape upstream regulatory signaling in a manner that may promote recovery and tissue regeneration after radiation injury. The biological relevance of these upstream regulators is notable. EZH2, a histone methyltransferase, is commonly overexpressed following radiation and contributes to radioresistance through chromatin remodeling and enhanced DNA repair [51]. Its suppression by TPOm, via regulation of EZH2-targeting miRNAs, may sensitize cells to appropriate DNA repair without supporting unchecked survival or genomic instability. IFNB1, encoding interferon-beta, plays a central role in the innate immune response to radiation [52,53]. While beneficial in acute phases, prolonged activation may drive chronic inflammation and tissue injury. TPOm’s enhancement of IFNB1 activation, likely through miRNA modulation, suggests a calibrated immune response that avoids pathological inflammation. MTDH, a known promoter of cell survival and oxidative stress resistance, was inhibited by radiation but activated with TPOm treatment. This suggests that TPOm supports cell survival mechanisms under stress via miRNA-mediated restoration of MTDH activity. LATS2, a tumor suppressor in the Hippo pathway, governs cell cycle arrest and apoptosis following DNA damage [39,54,55]. Radiation-induced LATS2 activation may impair tissue regeneration. TPOm appears to recalibrate this balance by suppressing LATS2-associated miRNAs, promoting recovery while preserving necessary damage responses. TGFB1, a key regulator of fibrosis and immune suppression, when chronically activated, contributes to radiation-induced fibrotic remodeling [56,57]. Interestingly, radiation suppressed TGFB1 in our model, and TPOm restored its activity, possibly to promote controlled tissue repair without driving fibrosis, as reflected in associated miRNA expression patterns. MYC, a transcription factor with dual roles in promoting proliferation and apoptosis, is highly sensitive to stress [56]. Radiation-induced inhibition of MYC may reduce the risk of proliferation under genomic instability, and TPOm’s further inhibition may reinforce this protective mechanism by modulating MYC-targeting miRNAs. Collectively, these insights underscore that TPOm not only modulates miRNA expression but also reprograms upstream transcriptional regulators and canonical pathways, including TR/RXR signaling and regulators, such as EZH2, IFNB1, TGFB1, and MYC. These coordinated molecular changes reflect a shift from damage response toward regeneration and functional restoration.

To further assess the biological functions and disease processes affected by TPOm, we evaluated functional annotations associated with the differentially expressed miRNAs. At day 15 post-irradiation, the vehicle-treated group displayed a trend toward inhibition of organism growth, survival, invasion of cancer cell lines, and several digestive system cancer-related functions. Strikingly, TPOm pre-treatment reversed these trends, suggesting a protective effect against radiation-induced suppression of vital cellular processes. Additionally, TPOm uniquely promoted functions such as myelopoiesis of leukocytes and proliferation of cervical cancer cell lines, responses that were not evident in the vehicle-treated group. These effects may reflect TPOm’s known role in stimulating hematopoiesis and supporting immune cell recovery [58].

On day 30 post-irradiation, radiation exposure led to a complex profile: activation of cancer-related processes (e.g., migration and proliferation of adenocarcinoma and lung cancer cell lines and cytostasis of tumor cells) and simultaneous inhibition of functions including fibrosis, lymphocyte quantity, solid tumor growth, neoplasia, reactive oxygen species synthesis, and stem cell proliferation. Pre-treatment with TPOm mitigated many of these alterations, reversing pro-tumorigenic activity while restoring immune-related and regenerative functions. The modulation of both immune suppression and cancer-related processes by TPOm suggests a dual role in immune recovery and tumor surveillance.

4. Materials and Methods

4.1. Animals

Male CD2F1 mice (11–12 weeks old) were purchased from Envigo (Indianapolis, IN, USA). The mice were housed in the in the Uniformed Services University of the Health and Sciences Laboratory Animal Medicine (LAM) facility (Bethesda, MD, USA). After receiving the animals at the facility, they were acclimated for 5 days.

Animals were identified by tail tattoos and housed in groups of up to 5 in Allentown NexGen cage systems. Mice were provided Harlan Teklad Global Rodent Diet 8604 (Envigo, Indianapolis, IN, USA), and acidified water, pH ~2.5, ad libitum. All mice were kept in cages and rooms were ventilated at 10–15 air changes per hour, maintained at a temperature between 68 °F and 79 °F (20 °C and 26 °C) and humidity of between 30% and 70%. Animals were kept in a 12:12 h light/dark schedule.

Appropriate animal enrichment and veterinary care were provided throughout the course of the study. All animal procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) using the principles and procedures outlined in the National Research Council’s Guide for the Care and Use of Laboratory Animals.

4.2. Drug Preparation

TPOm (JNJ-26366821) is a novel PEGylated TPO mimetic peptide developed by Janssen Research and Development, LLC. (Raritan, NJ, USA). TPOm was supplied to AFRRI in a powder form, and it was formulated in normal sterile vehicle (0.9% NaCl) before use and protected from light. Either drug or its vehicle was injected subcutaneously (SC) at the nape of the neck, 24 h pre-irradiation.

4.3. Total Body Irradiation (TBI) Studies

Mice were total body irradiated (TBI) bilaterally (simultaneously) at an estimated dose rate of 0.6 Gy/min in the Cobalt-60 (Co-60) gamma-irradiation facility at the Armed Forces Radiobiology Research Institute, Bethesda, MD. These animals were placed in well-ventilated plexiglass chambers made specifically for irradiating mice. An alanine/electron spin resonance (ESR) dosimetry system (in accordance with American Society for Testing and Material ISO/ASTM Standard 51607:2004(E)) [59] was used to measure the dose rates in the cores of acrylic phantoms (3 inches long and 1 inch in diameter) located in all empty slots of the exposure rack in the Lucite restraint boxes. ESR signals were measured with a calibration curve based on standard calibration dosimeters provided by the National Institute of Standard and Technology (NIST, Gaithersburg, MD, USA). The calibration curve was verified by intercomparison with the National Physical Laboratory (NPL) in the United Kingdom. The corrections applied to the measured dose rates in phantoms were for decay of the Co-60 source and for a small difference in mass-energy absorption coefficients for water and soft tissue at the Co-60 energy level. The radiation field was previously reported to be uniform within ±2% [12].

4.4. Housing and Care of Animals After Irradiation

After irradiation animals were returned to their cages and monitored three to four times daily. Any sick animals were monitored closely and their health status scored in accordance with pre-defined criteria, as described in the IACUC protocol. Clinical signs of pain and distress were assessed using a scoring system adapted from Koch et al. [60], which included criteria such as unresponsiveness, abnormal posture, unkempt appearance, immobility, and ataxia. The animals that were found moribund were euthanized according to American Veterinary Medical Association (AVMA) guidelines.

4.5. Harvesting Blood and Tissues for Various Molecular Assays

CD2F1 male mice were administered either vehicle or TPOm (0.3 mg/kg or 1 mg/kg, SC, 24 h pre-TBI) and irradiated at either 7 or 9.5 Gy (at a dose rate of ~0.6 Gy/min in the AFRRI Co-60 gamma radiation facility). Animals were euthanized under anesthesia for tissue collection. Tissue collection (femoral bone marrow, sternum, spleen, thymus, liver, kidney, intestine, brain, heart, and lungs) and blood collection (n = 6 for unirradiated groups and n = 8 for irradiated groups) were carried out on days 1, 3, 7, 9, 15, and 30 post-irradiation (day 0 is the day of irradiation).

Whole blood was collected, and serum was separated by centrifugation at 5000× g for 10 min in serum collection tubes. The supernatant was aspirated and stored for subsequent analysis. Serum was used for measuring various cytokines and biomarkers using ELISA, miRNA array, and Luminex assays. Sternal sections were fixed in formalin for megakaryocyte and crypt cell counting. Spleen tissues were snap frozen and stored at −80 °C for later use.

4.6. Analyses of Inflammatory Cytokines and Growth Factors

The Bio-Plex Pro Mouse Cytokine 23-plex assay kit (BioRad, Hercules, CA, USA) was used to analyze serum preserved at −80 °C, as per the vendor’s protocol. Fifty microliters of magnetic beads provided in the kit were used with 50 µL of standards or diluted (1:4) serum samples. A group of inflammatory cytokines, chemokines, and growth factors (Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (P40), IL-12 (P70), IL-13, IL-17A, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α) was assessed in serum. The magnetic plate washer, Bio-Plex Pro, was used for the recommended number of washes. The quantity of the biotinylated detection antibody cocktail and streptavidin-phycoerythrin was as per the vendor’s protocol. The plate was then immediately read on Luminex 200 (BioRad, Hercules, CA, USA) and the Bio-Plex Manager 6.1.1 was used for data acquisition.

4.7. Murine microRNA Analysis from Spleen

Spleens were harvested from mice at the indicated time points and immediately snap frozen in liquid nitrogen and stored at 80 °C until use. Approximately 50 mg of the frozen tissues were homogenized by brief sonication on ice, and total RNA enriched for small RNAs was extracted using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, Frederick, MD, USA) following the manufacturer’s protocol. RNA yield and quality were analyzed on a NanoDrop spectrophotometer ND-1000 (Thermo Fisher Scientific Inc., Rockville, MD, USA) with all samples showing A260/A280 absorbance ratios between 1.8 and 2.0, indicative of high-quality RNA. RNA integrity and small RNA enrichment were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples displaying intact small RNA profiles and minimal degradation were included for miRNA expression analysis. Samples failing quality control criteria were excluded prior to downstream processing.

4.8. MicroRNA (miRNA) Expression Profiling

Global miRNA expression profiling was performed using TaqMan^® Low-Density Rodent MicroRNA Arrays v2.0 (Applied Biosystems, Thermo Fisher Scientific, Frederick, MD, USA), which enable simultaneous quantification of hundreds of murine miRNAs. For each sample, 100 ng of total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), following the manufacturer’s instructions. The resulting cDNA was pre-amplified using Megaplex™ PreAmp Primers and TaqMan PreAmp Master Mix to enhance detection sensitivity, particularly for low-abundance miRNAs.

Pre-amplified products were diluted and used to prepare PCR reactions with TaqMan^® Universal PCR Master Mix (No AmpErase^® UNG). Reactions were loaded onto the microRNA array cards and quantitative PCR was performed on an ABI 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using default thermal cycling conditions recommended by the manufacturer. Each experimental group consisted of independent biological replicates, and miRNA profiling was conducted using identical reaction conditions across all samples to minimize technical variability.

4.9. MiRNA Data Preprocessing, Normalization, and Pathway Analysis

Raw cycle threshold (Ct) values were extracted using Expression Suite Software v1.0.3 (Applied Biosystems, Thermo Fisher Cloud). miRNAs with Ct values >35 or those undetected in more than 50% of samples within a given group were excluded from further analysis to reduce background noise and technical artifacts.

Relative miRNA expression levels were calculated using the 2^−ΔΔCt method. To account for inter-sample technical variation and differences in RNA input, global mean normalization was applied across all detected miRNAs within each array. This normalization strategy was selected because it provides robust performance in large-scale miRNA profiling studies and avoids reliance on single endogenous reference miRNAs, which may be altered by radiation exposure.

Differentially expressed miRNAs were identified using a fold-change threshold of >0.5 or <1.2 by comparing mice treated with vehicle (Saline) or TPOm 24 h prior to TBI at corresponding time points relative to non-irradiated vehicle (saline)-treated controls. This threshold was selected based on the regulatory nature of miRNAs, which often exert biologically significant effects through modest but coordinated changes in expression rather than large amplitude shifts. Unlike mRNA profiling, miRNA-mediated regulation frequently involves subtle expression changes that can collectively produce substantial downstream effects on signaling pathways and cellular functions. In addition, the use of a qPCR-based TaqMan Low-Density Array platform provides high analytical sensitivity and low technical variability, enabling reliable detection of small fold changes. This thresholding approach has been widely adopted in miRNA pharmacogenomic and radiation biology studies to capture pathway-level regulatory dynamics rather than isolated miRNA effects.

In silico pathway and network analyses of differentially expressed miRNAs were conducted using ingenuity pathway analysis (IPA; Qiagen: https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) (accessed on 1 January 2025). Canonical pathways, upstream regulators, and disease- and function-associated networks were identified based on experimentally validated and predicted miRNA–target interactions curated within the IPA knowledge base.

4.10. Statistical Analysis

The expression levels of miRNAs in each group at different time points were compared using Student’s t-test. To control for multiple hypothesis testing inherent to high-dimensional miRNA datasets, p-values were adjusted using the Benjamini–Hochberg false discovery rate (FDR) correction. miRNAs with an adjusted p-value < 0.05 were considered statistically significant. For pathway analyses, Fisher’s exact test was used to evaluate the enrichment of differentially expressed miRNAs within specific biological pathways. A significance threshold of p < 0.05 was applied unless otherwise stated.

A minimum of 6 animals per group was considered sufficient to yield a power of 80% [61]. Two-sample t-tests were used to identify differentially expressed miRNAs that were significantly up- or down-regulated. MicroRNAs that exhibited significant changes in expression (>2-fold), with p < 0.05 or p < 0.01 and an FDR of 10%, were considered for further analyses [61]. Means and standard errors were reported for all other data using GraphPad Prism 7 software. Analysis of variance (ANOVA) was used to determine if there was a significant difference among different groups.

5. Conclusions

Collectively, our data demonstrated that TPOm pre-treatment exerts a regulatory influence on radiation-induced miRNA expression, modulating both upstream transcriptional networks and downstream biological functions in a time-dependent manner (Figure 7). This modulation includes suppression or reversal of radiation-induced pro-inflammatory and tumorigenic signaling, restoration of immune functions, and support of hematopoietic and regenerative processes. These findings highlight the potential of miRNA pharmacogenomics as a powerful approach to evaluate and optimize radiation countermeasures and underscore the therapeutic promise of TPOm in mitigating delayed radiation-induced tissue injury.

Importantly, the observed cytokine-miRNA regulatory signature was not merely molecularly descriptive but functionally consequential. The coordinated normalization of inflammatory cytokines and hematopoietic-associated miRNAs paralleled improvements in survival and hematologic recovery in irradiated mice, key outcome measures of hematopoietic acute radiation syndrome (H-ARS). Thus, the identified miRNA-cytokine signature may serve both as a mechanistic biomarker of TPOm efficacy and as a translational indicator of improved H-ARS outcomes. Establishing this molecular-to-phenotypic linkage strengthens the clinical relevance of our findings and supports the potential utility of these signatures for monitoring the therapeutic response in radiation countermeasure development.

Future studies are warranted to validate these findings in additional tissue types and to investigate the direct targets of TPOm-regulated miRNAs, which could offer deeper mechanistic insights into its radioprotective and immunomodulatory functions. Further validation in additional models is necessary to enhance the translational relevance of this study. Additionally, it would be beneficial to explore the efficacy of TPOm under delayed administration regimens. Understanding the full scope of miRNA-mediated pathways influenced by TPOm will be instrumental in advancing it as a candidate for clinical application in radiation injury and related syndromes.

A limitation of the present study is the exclusive use of male mice to minimize hormonal variability during initial miRNA profiling. As sex-specific differences may influence radiation responses and miRNA regulation, future investigations incorporating female cohorts will be essential to evaluate potential sex-dimorphic effects and improve translational applicability.