RANKL-responsive epigenetic mechanism reprograms macrophages into bone-resorbing osteoclasts.

Monocyte/macrophage lineage cells are highly plastic and can differentiate into various cells under different environmental stimuli. Bone-resorbing osteoclasts are derived from the monocyte/macrophage lineage in response to receptor activator of NF-κB ligand (RANKL). However, the epigenetic signature contributing to the fate commitment of monocyte/macrophage lineage differentiation into human osteoclasts is largely unknown. In this study, we identified RANKL-responsive human osteoclast-specific superenhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) by integrating data obtained from ChIP-seq, ATAC-seq, nuclear RNA-seq and PRO-seq analyses. RANKL induced the formation of 200 SEs, which are large clusters of enhancers, while suppressing 148 SEs in macrophages. RANKL-responsive SEs were strongly correlated with genes in the osteoclastogenic program and were selectively increased in human osteoclasts but marginally presented in osteoblasts, CD4+ T cells, and CD34+ cells. In addition to the major transcription factors identified in osteoclasts, we found that BATF binding motifs were highly enriched in RANKL-responsive SEs. The depletion of BATF1/3 inhibited RANKL-induced osteoclast differentiation. Furthermore, we found increased chromatin accessibility in SE regions, where RNA polymerase II was significantly recruited to induce the extragenic transcription of SE-eRNAs, in human osteoclasts. Knocking down SE-eRNAs in the vicinity of the NFATc1 gene diminished the expression of NFATc1, a major regulator of osteoclasts, and osteoclast differentiation. Inhibiting BET proteins suppressed the formation of some RANKL-responsive SEs and NFATc1-associated SEs, and the expression of SE-eRNA:NFATc1. Moreover, SE-eRNA:NFATc1 was highly expressed in the synovial macrophages of rheumatoid arthritis patients exhibiting high-osteoclastogenic potential. Our genome-wide analysis revealed RANKL-inducible SEs and SE-eRNAs as osteoclast-specific signatures, which may contribute to the development of osteoclast-specific therapeutic interventions.

MYC-mediated early glycolysis negatively regulates proinflammatory responses by controlling IRF4 in inflammatory macrophages.

MYC activates different metabolic programs in a cell-type- and cell-status-dependent manner. However, the role of MYC in inflammatory macrophages has not yet been determined. Metabolic and molecular analyses reveal that MYC, but not hypoxia inducible factor 1 (HIF1), is involved in enhancing early glycolytic flux during inflammatory macrophage polarization. Ablation of MYC decreases lactate production by regulating lactate dehydrogenase (LDH) activity and causes increased inflammatory cytokines by regulating interferon regulatory factor 4 (IRF4) in response to lipopolysaccharide. Moreover, myeloid-specific deletion of MYC and pharmacological inhibition of the MYC/LDH axis enhance inflammation and the bacterial clearance in vivo. These results elucidate the potential role of the MYC/LDH/IRF4 axis in inflammatory macrophages by connecting early glycolysis with inflammatory responses and suggest that modulating early glycolytic flux mediated by the MYC/LDH axis can be used to open avenues for the therapeutic modulation of macrophage polarization to fight against bacterial infection.

Sexual Dimorphism in Differentiating Osteoclast Precursors Demonstrates Enhanced Inflammatory Pathway Activation in Female Cells.

Sexual dimorphism of the skeleton is well documented. At maturity, the male skeleton is typically larger and has a higher bone density than the female skeleton. However, the underlying mechanisms for these differences are not completely understood. In this study, we examined sexual dimorphism in the formation of osteoclasts between cells from female and male mice. We found that the number of osteoclasts in bones was greater in females. Similarly, in vitro osteoclast differentiation was accelerated in female osteoclast precursor (OCP) cells. To further characterize sex differences between female and male osteoclasts, we performed gene expression profiling of cultured, highly purified, murine bone marrow OCPs that had been treated for 3 days with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that 125 genes were differentially regulated in a sex-dependent manner. In addition to genes that are contained on sex chromosomes, transcriptional sexual dimorphism was found to be mediated by genes involved in innate immune and inflammatory response pathways. Furthermore, the NF-κB-NFATc1 axis was activated earlier in female differentiating OCPs, which partially explains the differences in transcriptomic sexual dimorphism in these cells. Collectively, these findings identify multigenic sex-dependent intrinsic difference in differentiating OCPs, which results from an altered response to osteoclastogenic stimulation. In humans, these differences could contribute to the lower peak bone mass and increased risk of osteoporosis that females demonstrate relative to males. © 2021 American Society for Bone and Mineral Research (ASBMR).

MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS.

Osteoporosis is a metabolic bone disease with dysregulated coupling between bone resorption and bone formation, which results in decreased bone mineral density. The MEF2C locus, which encodes the transcription factor MADS box transcription enhancer factor 2, polypeptide C (MEF2C), is strongly associated with adult osteoporosis and osteoporotic fractures. Although the role of MEF2C in bone and cartilage formation by osteoblasts, osteocytes, and chondrocytes has been studied, the role of MEF2C in osteoclasts, which mediate bone resorption, remains unclear. In this study, we identified MEF2C as a positive regulator of human and mouse osteoclast differentiation. While decreased MEF2C expression resulted in diminished osteoclastogenesis, ectopic expression of MEF2C enhanced osteoclast generation. Using transcriptomic and bioinformatic approaches, we found that MEF2C promotes the RANKL-mediated induction of the transcription factors c-FOS and NFATc1, which play a key role in osteoclastogenesis. Mechanistically, MEF2C binds to FOS regulatory regions to induce c-FOS expression, leading to the activation of NFATC1 and downstream osteoclastogenesis. Inducible deletion of Mef2c in mice resulted in increased bone mass under physiological conditions and protected mice from bone erosion by diminishing osteoclast formation in K/BxN serum induced arthritis, a murine model of inflammatory arthritis. Our findings reveal direct regulation of osteoclasts by MEF2C, thus adding osteoclasts as a cell type in which altered MEF2C expression or function can contribute to pathological bone remodeling.

NRF2 Is an Upstream Regulator of MYC-Mediated Osteoclastogenesis and Pathological Bone Erosion.

Osteoclasts are the sole bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathogenic bone destruction such as inflammatory arthritis. Pharmacologically targeting osteoclasts has been a promising approach to alleviating bone disease, but there remains room for improvement in mitigating drug side effects and enhancing cell specificity. Recently, we demonstrated the crucial role of MYC and its downstream effectors in driving osteoclast differentiation. Despite these advances, upstream regulators of MYC have not been well defined. In this study, we identify nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor known to regulate the expression of phase II antioxidant enzymes, as a novel upstream regulator of MYC. NRF2 negatively regulates receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis through the ERK and p38 signaling-mediated suppression of MYC transcription. Furthermore, the ablation of MYC in osteoclasts reverses the enhanced osteoclast differentiation and activity in NRF2 deficiency in vivo and in vitro in addition to protecting NRF2-deficient mice from pathological bone loss in a murine model of inflammatory arthritis. Our findings indicate that this novel NRF2-MYC axis could be instrumental for the fine-tuning of osteoclast formation and provides additional ways in which osteoclasts could be therapeutically targeted to prevent pathological bone erosion.

Nuclear receptors in osteoclasts.

Osteoclasts are bone-resorbing cells that play an essential role in the remodeling of bone under physiological conditions and numerous pathological conditions, such as osteoporosis, bone metastasis, and inflammatory bone erosion. Nuclear receptors are crucial to various physiological processes, including metabolism, development and inflammation, and function as transcription factors to activate target genes. Synthetic ligands of nuclear receptors are also available for the treatment of metabolic and inflammatory diseases. However, dysregulated bone phenotypes have been documented in patients who take synthetic nuclear receptor ligands as a therapy. Therefore, the effect of nuclear receptors on bone cells has become an important area of exploration; additionally, the molecular mechanisms underlying the action of nuclear receptors in osteoclasts have not been completely understood. Here, we cover the recent progress in our understanding of the roles of nuclear receptors in osteoclasts.

IFN-γ selectively suppresses a subset of TLR4-activated genes and enhancers to potentiate macrophage activation.

Activation of macrophage proinflammatory and antimicrobial phenotypes is regulated by IFN-γ and LPS via synergistic induction of canonical, inflammatory NF-κB target genes. However, whether IFN-γ negatively regulates components of the LPS response, and how this may affect macrophage activation, is still unclear. Here we use combined transcriptomic and epigenomic approaches to find that IFN-γ selectively abrogates LPS-induced feedback and alters macrophage metabolic pathways by suppressing TLR4-mediated gene activation. In contrast to superinduction of inflammatory genes via enhancers that bind IRF1 and STAT1, IFN-γ represses target enhancers that bind STAT3. TLR4-activated but IFN-γ-suppressed enhancers comprise two subsets discernable by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin. Our findings thus show that IFN-γ suppresses feedback inhibitory and metabolic components of TLR responses to enhance macrophage activation; they also provide insights for IFN-γ-mediated selective inhibition of TLR4-induced transcription. Such inhibition can contribute to severe and sustained inflammatory responses.

The enhanced tumor inhibitory effects of gefitinib and L-ascorbic acid combination therapy in non-small cell lung cancer cells.

Despite documentation of successful therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with lung cancer, the response rate of patients treated with this therapy remains low. The present study investigated whether L-ascorbic acid serves an adjuvant role in vitro when combined with the EGFR tyrosine kinase inhibitor gefitinib (Iressa®) in lung cancer cell lines. A total of three human lung cancer cell lines were used. The antiproliferative effects and changes in the cell cycle and expression of intracellular signaling molecules, including extracellular signal-regulated kinases (Erk), signal transducer and activator of transcription 3 (Stat3) and protein kinase B (Akt), were measured in cells treated with gefitinib and/or L-ascorbic acid at various concentrations. When combined with gefitinib, L-ascorbic acid exhibited an additive effect on cell proliferation in all gefitinib-sensitive and gefitinib-resistant cell lines. A decrement of ~40% was observed with a low dose 0.5 mM L-ascorbic acid and gefitinib in the relatively gefitinib-resistant A549 cell line (85.6±5.4% with gefitinib alone vs. 52.7±7.3% with combination therapy; P=0.046). The downregulation of intracellular signaling cascades, including EGFR, Akt, Erk and Stat3, was also observed. L-Ascorbic acid serves an adjuvant role when administered in combination with gefitinib; however, the degree of inhibition of cell proliferation differs between lung cancer cell lines.

Hypoxia-Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis.

Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction.

MYC-dependent oxidative metabolism regulates osteoclastogenesis via nuclear receptor ERRα.

Osteoporosis is a metabolic bone disorder associated with compromised bone strength and an increased risk of fracture. Inhibition of the differentiation of bone-resorbing osteoclasts is an effective strategy for the treatment of osteoporosis. Prior work by our laboratory and others has shown that MYC promotes osteoclastogenesis in vitro, but the underlying mechanisms are not well understood. In addition, the in vivo importance of osteoclast-expressed MYC in physiological and pathological bone loss is not known. Here, we have demonstrated that deletion of Myc in osteoclasts increases bone mass and protects mice from ovariectomy-induced (OVX-induced) osteoporosis. Transcriptomic analysis revealed that MYC drives metabolic reprogramming during osteoclast differentiation and functions as a metabolic switch to an oxidative state. We identified a role for MYC action in the transcriptional induction of estrogen receptor-related receptor α (ERRα), a nuclear receptor that cooperates with the transcription factor nuclear factor of activated T cells, c1 (NFATc1) to drive osteoclastogenesis. Accordingly, pharmacological inhibition of ERRα attenuated OVX-induced bone loss in mice. Our findings highlight a MYC/ERRα pathway that contributes to physiological and pathological bone loss by integrating the MYC/ERRα axis to drive metabolic reprogramming during osteoclast differentiation.

Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment.

Adoptive cell transfer utilizing tumour-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against haematological malignancies, but significant clinical success has not yet been achieved in solid tumours due in part to the strong immunosuppressive tumour microenvironment. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is in part mediated by TGFß-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Highly selective optical control of Ca2+ signalling in adoptively transferred CTLs enhances T cell activation and IFN-γ production in vitro, leading to a significant reduction in tumour growth in mice. Altogether, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses at the tumour sites.

Intravenous Immunoglobulin (IVIG) Attenuates TNF-Induced Pathologic Bone Resorption and Suppresses Osteoclastogenesis by Inducing A20 Expression.

Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody and immune complex-mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone-resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast-mediated pathologic bone resorption. IVIG or cross-linking of Fcγ receptors with plate-bound IgG suppressed receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteoclastogenesis and expression of osteoclast-related genes such as integrin ß3 and cathepsin K in a dose-dependent manner. Mechanistically, IVIG or plate-bound IgG suppressed osteoclastogenesis by downregulating RANKL-induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL-induced NF-κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)-induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders.

Direct Interaction of CD40 on Tumor Cells with CD40L on T Cells Increases the Proliferation of Tumor Cells by Enhancing TGF-ß Production and Th17 Differentiation.

It has recently been reported that the CD40-CD40 ligand (CD40L) interaction is important in Th17 development. In addition, transforming growth factor-beta (TGF-ß) promotes tumorigenesis as an immunosuppressive cytokine and is crucial in the development of Th17 cells. This study investigated the role of CD40 in breast cancer cells and its role in immunosuppressive function and tumor progression. CD40 was highly expressed in the breast cancer cell line MDA-MB231, and its stimulation with CD40 antibodies caused the up-regulation of TGF-ß. Direct CD40-CD40L interaction between MDA-MB231 cells and activated T cells also increased TGF-ß production and induced the production of IL-17, which accelerated the proliferation of MDA-MB231 cells through the activation of STAT3. Taken together, the direct CD40-CD40L interaction of breast tumor cells and activated T cells increases TGF-ß production and the differentiation of Th17 cells, which promotes the proliferation of breast cancer cells.

Vitamin C Deficiency Causes Severe Defects in the Development of the Neonatal Cerebellum and in the Motor Behaviors of Gulo(-/-) Mice.

AIMS: The developing brain of a neonate is particularly susceptible to damage by vitamin C deficiency because of its rapid growth and immature antioxidant system. Cognitive impairment and sensory motor deficits are found in the adult brain upon vitamin C deficiency. Therefore, the aim of this study was to clarify the role of vitamin C in its own right and its related mechanisms in Gulo(-/-) mice incapable of synthesizing vitamin C. RESULTS: When vitamin C supplementation was ceased for 2 weeks until delivery, stillbirths and a significant reduction in neonatal mice were observed and the growth of neonates was remarkably decreased. In addition, intraparenchymal hemorrhages were found in most of the brains, especially in the stillborn neonates. In addition, the levels of malondialdehyde (MDA) and 8-isoprostanes were increased and structural abnormalities were found in the cortex, hippocampus, and cerebellum. Especially, vitamin C deficiency caused the failure of or a delay in the formation of cerebellar fissures accompanied by abnormal foliation and altered Purkinje cell alignment. In the developed adult brains from vitamin C-deficient Gulo(-/-) mice, the levels of glutathione, MDA, nitrate, IL-6, TNF-α, and Bax were increased and the expression of the GABRA6 and calbindin-28k was decreased. Due to atrophy of the granule and Purkinje cells, the motor behavior of vitamin C-deficient Gulo(-/-) mice declined. INNOVATION AND CONCLUSION: Vitamin C deficiency during gestation induces intraparenchymal hemorrhages and severe defects in the development of the cerebellum. In fully developed brains, it induces the functional impairment by altering the cellular composition in the cerebellum.

Optogenetic control of chemokine receptor signal and T-cell migration.

Adoptive cell transfer of ex vivo-generated immune-promoting or tolerogenic T cells to either enhance immunity or promote tolerance in patients has been used with some success. However, effective trafficking of the transferred cells to the target tissue sites is the main barrier to achieving successful clinical outcomes. Here we developed a strategy for optically controlling T-cell trafficking using a photoactivatable (PA) chemokine receptor. Photoactivatable-chemokine C-X-C motif receptor 4 (PA-CXCR4) transmitted intracellular CXCR4 signals in response to 505-nm light. Localized activation of PA-CXCR4 induced T-cell polarization and directional migration (phototaxis) both in vitro and in vivo. Directing light onto the melanoma was sufficient to recruit PA-CXCR4-expressing tumor-targeting cytotoxic T cells and improved the efficacy of adoptive T-cell transfer immunotherapy, with a significant reduction in tumor growth in mice. These findings suggest that the use of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with outstanding spatial resolution in tissues and may be feasible in other cell transfer therapies.

Vitamin C prevents stress-induced damage on the heart caused by the death of cardiomyocytes, through down-regulation of the excessive production of catecholamine, TNF-α, and ROS production in Gulo(-/-)Vit C-Insufficient mice.

It is thought that vitamin C has protective roles on stress-induced heart damage and the development of cardiovascular diseases, but its precise role and mechanisms are unclear. In the present study, we investigated the specific mechanisms by which vitamin C leads to protecting the heart from stress-induced damage in the Gulo(-/-) mice which cannot synthesize vitamin C like humans. By exposure to stress (1h/day), the heartbeat and cardiac output in vitamin C-insufficient Gulo(-/-) mice were definitely decreased, despite a significant increase of adrenaline (ADR) and noradrenaline (NA) production. A change of cardiac structure caused by the death of cardiomyocytes and an increased expression of matrix metalloprotease (MMP)-2 and -9 were also found. Moreover, lipid peroxidation and the production of tumor necrosis factor-alpha (TNF-α) in the heart were increased. Finally, all vitamin C-insufficient Gulo(-/-) mice were expired within 2 weeks. Interestingly, all of the findings in vitamin C-insufficient Gulo(-/-) mice were completely prevented by the supplementation of a sufficient amount of vitamin C. Taken together, vitamin C insufficiency increases the risk of stress-induced cardiac damage with structural and functional changes arising from the apoptosis of cardiomyocytes.

Vitamin C Is an Essential Factor on the Anti-viral Immune Responses through the Production of Interferon-α/ß at the Initial Stage of Influenza A Virus (H3N2) Infection.

L-ascorbic acid (vitamin C) is one of the well-known anti-viral agents, especially to influenza virus. Since the in vivo anti-viral effect is still controversial, we investigated whether vitamin C could regulate influenza virus infection in vivo by using Gulo (-/-) mice, which cannot synthesize vitamin C like humans. First, we found that vitamin C-insufficient Gulo (-/-) mice expired within 1 week after intranasal inoculation of influenza virus (H3N2/Hongkong). Viral titers in the lung of vitamin C-insufficient Gulo (-/-) mice were definitely increased but production of anti-viral cytokine, interferon (IFN)-α/ß, was decreased. On the contrary, the infiltration of inflammatory cells into the lung and production of pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-α/ß, were increased in the lung. Taken together, vitamin C shows in vivo anti-viral immune responses at the early time of infection, especially against influenza virus, through increased production of IFN-α/ß.

In vivo consequence of vitamin C insufficiency in liver injury: vitamin C ameliorates T-cell-mediated acute liver injury in gulo(-/-) mice.

AIM: l-ascorbic acid (vitamin C) insufficiency is considered one of the major risk factors for the development of liver disease. However, its specific effects and related mechanisms in vivo are largely unknown. The objective of this study was to investigate the in vivo protective role of vitamin C and its related mechanisms in liver injury with Gulo(-/-) mice that cannot synthesize vitamin C like humans due to the lack of l-gulonolactone-γ-oxidase (Gulo), an essential enzyme for vitamin C synthesis. RESULTS: When liver injury was induced in Gulo(-/-) mice by injection of concanavalin A (Con A), there was greater extensive liver damage accompanied by an increased number of apoptotic hepatocytes in vitamin C-insufficient Gulo(-/-) mice. Additionally, the plasma and hepatic levels of the proinflammatory cytokines, such as TNF-α and IFN-γ, were much higher in the vitamin C-insufficient Gulo(-/-) mice than in the control mice. Moreover, increased numbers of liver-infiltrating T-cells in the vitamin C-insufficient Gulo(-/-) mice were related to the increased hepatic levels of IFN-inducible factor (IP-10). Although the vitamin C-insufficient Gulo(-/-) mice had higher amounts of interleukin-22 (IL-22), a hepatoprotective cytokine, a defect in IL-22Rα expression and its downstream STAT3 activation in hepatocytes were found. INNOVATION: We first demonstrate the novel in vivo action mechanisms of vitamin C on the prevention of disease development in the liver, through the regulation of excessive immune activation and maintenance of the IL-22Rα signaling pathways. CONCLUSION: These results suggest that severe liver damage induced by inflammation could be prevented by sufficient supplementation with vitamin C.

The effect of alloferon on the enhancement of NK cell cytotoxicity against cancer via the up-regulation of perforin/granzyme B secretion.

Alloferon is a novel immunomodulatory peptide originally isolated from infected insects. It has anti-viral and anti-tumor effects via the activation of NK cells. However, specific mechanisms leading to NK cell activation and anti-tumor responses yet to be clarified. In this study, we demonstrate that alloferon increases killing activity of NK cells to cancer cells via the up-regulation of the expression of NK-activating receptors, 2B4. In addition, the production of IFN-γ and TNF-α and granule exocytosis from NK cells against cancer cell were increased by alloferon. Lastly, the anti-tumor effect of alloferon was confirmed in vivo to demonstrate effective retardation of tumor growth in the human-to-mouse xenograft model. All taken together, these results suggest that alloferon has anti-tumor effects through up-regulation of NK-activating receptor 2B4 and the enhancement of granule exocytosis from NK cells.

The anti-inflammatory effect of alloferon on UVB-induced skin inflammation through the down-regulation of pro-inflammatory cytokines.

UVB irradiation can induce biological changes in the skin, modulate immune responses and activate inflammatory reactions leading to skin damage. Alloferon, which is isolated from the blood of an experimentally infected insect, the blow fly Calliphora vicina, is known for its anti-viral and anti-tumor activities in mice model. However, the effect of alloferon against UVB irradiation and its specific mechanism are still unknown. In this study, we investigated the effect of alloferon on UVB-induced cutaneous inflammation in a human keratinocyte cell line, HaCaT. RPA and ELISA data showed that alloferon decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-1α, IL-1ß, IL-6 and IL-18, both on the mRNA and protein level. Western blot analysis was done to determine if alloferon regulates the MAPK signaling pathway since the MAPK signaling pathway is activated by numerous inflammatory mediators and environmental stresses including UVB irradiation. Alloferon inhibited the activation of p38 mitogen-activated protein kinase (MAPK) induced by UVB irradiation. Furthermore, the topical application of alloferon on the UVB exposed skin of hairless mice showed that alloferon treatment significantly inhibited an increase in epithelial thickness in chronic UVB-irradiated mouse skin. These findings suggest that alloferon has significant anti-inflammatory effects not only on UVB-induced inflammation in the human keratinocyte cell line, HaCaT, but also on mouse skin.