Tumor promoting effect of PDLIM2 downregulation involves mitochondrial ROS, oncometabolite accumulations and HIF-1α activation.

BACKGROUND: Cancer is characterized by dysregulated cellular metabolism. Thus, understanding the mechanisms underlying these metabolic alterations is important for developing targeted therapies. In this study, we investigated the pro-tumoral effect of PDZ and LIM domain 2 (PDLIM2) downregulation in lung cancer growth and its association with the accumulation of mitochondrial ROS, oncometabolites and the activation of hypoxia-inducible factor-1 (HIF-1) α in the process. METHODS: Databases and human cancer tissue samples were analyzed to investigate the roles of PDLIM2 and HIF-1α in cancer growth. DNA microarray and gene ontology enrichment analyses were performed to determine the cellular functions of PDLIM2. Seahorse assay, flow cytometric analysis, and confocal microscopic analysis were employed to study mitochondrial functions. Oncometabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS). A Lewis lung carcinoma (LLC) mouse model was established to assess the in vivo function of PDLIM2 and HIF-1α. RESULTS: The expression of PDLIM2 was downregulated in lung cancer, and this downregulation correlated with poor prognosis in patients. PDLIM2 highly regulated genes associated with mitochondrial functions. Mechanistically, PDLIM2 downregulation resulted in NF-κB activation, impaired expression of tricarboxylic acid (TCA) cycle genes particularly the succinate dehydrogenase (SDH) genes, and mitochondrial dysfunction. This disturbance contributed to the accumulation of succinate and other oncometabolites, as well as the buildup of mitochondrial reactive oxygen species (mtROS), leading to the activation of hypoxia-inducible factor 1α (HIF-1α). Furthermore, the expression of HIF-1α was increased in all stages of lung cancer. The expression of PDLIM2 and HIF-1α was reversely correlated in lung cancer patients. In the animal study, the orally administered HIF-1α inhibitor, PX-478, significantly reduces PDLIM2 knockdown-promoted tumor growth. CONCLUSION: These findings shed light on the complex action of PDLIM2 on mitochondria and HIF-1α activities in lung cancer, emphasizing the role of HIF-1α in the tumor-promoting effect of PDLIM2 downregulation. Additionally, they provide new insights into a strategy for precise targeted treatment by suggesting that HIF-1α inhibitors may serve as therapy for lung cancer patients with PDLIM2 downregulation.

Omentin-1 ameliorates the progress of osteoarthritis by promoting IL-4-dependent anti-inflammatory responses and M2 macrophage polarization.

Osteoarthritis (OA) is a prevalent joint disease commonly associated with aging and obesity, which can lead to pain, stiffness, joint dysfunction, and disability. Omentin-1 (also called intelectin-1) is a newly discovered adipokine, which plays a protective role in suppressing the secretion of pro-inflammatory cytokines. Based on data from the Gene Expression Omnibus (GEO) dataset and clinical samples obtained at our institution revealed, determined that omentin-1 and IL-4 (an anti-inflammatory cytokine) levels were significantly lower in OA patients than in normal controls. Omentin-1 was shown to induce IL-4-depedent anti-inflammatory responses and M2 macrophage polarization in OA synovial fibroblasts via the PI3K, ERK, and AMPK pathways. Administering omentin-1 was shown to block cartilage degradation and bone erosion resulting from anterior cruciate ligament transection by inhibiting the production of pro-inflammatory cytokines and promoting M2 macrophage polarization in vivo. Our findings indicate omentin-1 as a promising therapeutic avenue for the treatment for OA.

Glutamine metabolism controls amphiregulin-facilitated chemoresistance to cisplatin in human chondrosarcoma.

Chondrosarcoma is the second most common type of bone cancer. At present, the most effective clinical course of action is surgical resection. Cisplatin is the chemotherapeutic medication most widely used for the treatment of chondrosarcoma; however, its effectiveness is severely hampered by drug resistance. In the current study, we compared cisplatin-resistant chondrosarcoma SW1353 cells with their parental cells via RNA sequencing. Our analysis revealed that glutamine metabolism is highly activated in resistant cells but glucose metabolism is not. Amphiregulin (AR), a ligand of the epidermal growth factor receptor, enhances glutamine metabolism and supports cisplatin resistance in human chondrosarcoma by promoting NADPH production and inhibiting reactive oxygen species (ROS) accumulation. The MEK, ERK, and NrF2 signaling pathways were shown to regulate AR-mediated alanine-serine-cysteine transporter 2 (ASCT2; also called SLC1A5) and glutaminase (GLS) expression as well as glutamine metabolism in cisplatin-resistant chondrosarcoma. The knockdown of AR expression in cisplatin-resistant chondrosarcoma cells was shown to reduce the expression of SLC1A5 and GLS in vivo. These results indicate that AR and glutamine metabolism are worth pursuing as therapeutic targets in dealing with cisplatin-resistant human chondrosarcoma.

In Vitro Assessment of the Antioxidant and Anticancer Properties of Flammulina velutipes Stipe Extracts.

BACKGROUND/AIM: Flammulina velutipes (FV), also known as the golden needle mushroom, is an edible and medicinal fungus that contains bioactive substances regulating various physiological functions. While the fruiting bodies of FV are commonly consumed, their stipes are often discarded despite containing polysaccharides. In this study, the biological functions of FV stipes (FV-S) were investigated to reduce waste and pollution while increasing their value. MATERIALS AND METHODS: The antioxidant activity of FV was evaluated using three methods: the DPPH radical-scavenging capacity assay, ferrous ion chelating assay, and reducing power analysis. The anti-cancer potential was assessed through MTT viability and immunoblotting analyses. RESULTS: Results showed that FV-S had higher polysaccharide and total phenolic contents and greater antioxidant abilities, particularly in ethanolic extracts. FV-S also exhibited significant anticancer properties, specifically in hot water extracts with high polysaccharide contents, and suppressed prostate cancer cell viability by inhibiting androgen receptor and PCa-specific antigen mRNA expression while inducing caspase-3/7 activation. CONCLUSION: FV-S is rich in bioactive components, possesses higher antioxidant and anticancer abilities, and has potential as an anticancer agent, which could enhance the value of FV.

Melatonin inhibits chondrosarcoma cell proliferation and metastasis by enhancing miR-520f-3p production and suppressing MMP7 expression.

Chondrosarcoma has a high propensity to metastasize and responds poorly to chemotherapy and radiation treatment. The enzymatic activity of matrix metalloproteinases (MMPs) is very important in chondrosarcoma metastasis. Melatonin exhibits anticarcinogenic activity in many types of cancers by suppressing the expression of certain MMP family members, but this has not yet been clearly determined in chondrosarcoma. Our study demonstrates that MMP7 plays an essential role in chondrosarcoma cell proliferation, migration, and anoikis resistance. We also found that MMP7 is highly expressed in chondrosarcomas. Our in vitro and in vivo investigations show that melatonin strongly inhibits chondrosarcoma cell proliferation, migration, and anoikis resistance by directly suppressing MMP7 expression. Melatonin reduced MMP7 synthesis by promoting levels of miR-520f-3p expression, which were downregulated in human chondrosarcoma tissue samples. Pharmacological inhibition of miR-520f-3p markedly reversed the effects of melatonin upon chondrosarcoma proliferation and metastasis. Thus, our study suggests that melatonin has therapeutic potential for reducing the tumorigenesis and metastatic potential of chondrosarcoma via the miR-520f-3p/MMP7 axis.

Melatonin Inhibits VEGF-Induced Endothelial Progenitor Cell Angiogenesis in Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (AMD) is described as abnormal angiogenesis in the retina and the leaking of fluid and blood that generates a huge, dark, blind spot in the center of the visual field, causing severe vision loss in over 90% of patients. Bone marrow-derived endothelial progenitor cells (EPCs) contribute to pathologic angiogenesis. Gene expression profiles downloaded from the eyeIntegration v1.0 database for healthy retinas and retinas from patients with neovascular AMD identified significantly higher levels of EPC-specific markers (CD34, CD133) and blood vessel markers (CD31, VEGF) in the neovascular AMD retinas compared with healthy retinas. Melatonin is a hormone that is mainly secreted by the pineal gland, and is also produced in the retina. Whether melatonin affects vascular endothelial growth factor (VEGF)-induced EPC angiogenesis in neovascular AMD is unknown. Our study revealed that melatonin inhibits VEGF-induced stimulation of EPC migration and tube formation. By directly binding with the VEGFR2 extracellular domain, melatonin significantly and dose-dependently inhibited VEGF-induced PDGF-BB expression and angiogenesis in EPCs via c-Src and FAK, NF-κB and AP-1 signaling. The corneal alkali burn model demonstrated that melatonin markedly inhibited EPC angiogenesis and neovascular AMD. Melatonin appears promising for reducing EPC angiogenesis in neovascular AMD.

IOX-1 suppresses metastasis of osteosarcoma by upregulating histone H3 lysine trimethylation.

New therapeutic approaches are needed for metastatic osteosarcoma (OS), as survival rates remain low despite surgery and chemotherapy. Epigenetic changes, such as histone H3 methylation, play key roles in many cancers including OS, although the underlying mechanisms are not clear. In this study, human OS tissue and OS cell lines displayed lower levels of histone H3 lysine trimethylation compared with normal bone tissue and osteoblast cells. Treating OS cells with the histone lysine demethylase inhibitor 5-carboxy-8-hydroxyquinoline (IOX-1) dose-dependently increased histone H3 methylation and inhibited cellular migratory and invasive capabilities, suppressed matrix metalloproteinase expression, reversed epithelial-to-mesenchymal transition by increasing levels of epithelial markers E-cadherin and ZO-1 and decreasing the expression of mesenchymal markers N-cadherin, vimentin, and TWIST, and also reduced stemness properties. An analysis of cultivated MG63 cisplatin-resistant (MG63-CR) cells revealed lower histone H3 lysine trimethylation levels compared with levels in MG63 cells. Exposing MG63-CR cells to IOX-1 increased histone H3 trimethylation and ATP-binding cassette transporter expression, potentially sensitizing MG63-CR cells to cisplatin. In conclusion, our study suggests that histone H3 lysine trimethylation is associated with metastatic OS and that IOX-1 or other epigenetic modulators present promising strategies to inhibit metastatic OS progression.

Apelin promotes osteosarcoma metastasis by upregulating PLOD2 expression via the Hippo signaling pathway and hsa_circ_0000004/miR-1303 axis.

Osteosarcoma is a highly mortal bone tumor, with a high metastatic potential, promoted in part by the enzyme procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2). Increasing level of PLOD2 in osteosarcoma tissue correlates with lymphatic and distant metastasis. The adipokine apelin (APLN) is also found in different cancers and APLN upregulation promotes angiogenesis and metastasis, but its effects on osteosarcoma metastasis are uncertain. We explored APLN functioning in metastatic osteosarcoma. An analysis of records from the Gene Expression Omnibus (GEO) database showed higher levels of APLN expression in osteosarcoma tissue than in normal tissue. Similarly, levels of APLN and PLOD2 mRNA synthesis were upregulated in osteosarcoma tissue. Levels of APLN and PLOD2 protein correlated positively with osteosarcoma clinical stages. APLN increased PLOD2 expression in human osteosarcoma cell lines and cell migration via the mammalian Sterile 20-like kinase 1 (MST1), monopolar spindle-one-binder protein (MOB)1, and YAP cascades, and through hsa_circ_0000004 functioning as a sponge of miR-1303. We also found that knockdown of APLN antagonized lung metastasis in mice with osteosarcoma. APLN may be a therapeutic target in osteosarcoma metastasis.

IL-17 Facilitates VCAM-1 Production and Monocyte Adhesion in Osteoarthritis Synovial Fibroblasts by Suppressing miR-5701 Synthesis.

Osteoarthritis (OA) is characterized by the infiltration and adhesion of monocytes into the inflamed joint synovium. Interleukin (IL)-17 is a critical inflammatory mediator that participates in the progression of OA, although the mechanisms linking IL-17 and monocyte infiltration are not well understood. Our analysis of synovial tissue samples retrieved from the Gene Expression Omnibus (GEO) dataset exhibited higher monocyte marker (CD11b) and vascular cell adhesion molecule 1 (VCAM-1) levels in OA samples than in normal, healthy samples. The stimulation of human OA synovial fibroblasts (OASFs) with IL-17 increased VCAM-1 production and subsequently enhanced monocyte adhesion. IL-17 affected VCAM-1-dependent monocyte adhesion by reducing miR-5701 expression through the protein kinase C (PKC)-α and c-Jun N-terminal kinase (JNK) signaling cascades. Our findings improve our understanding about the effect of IL-17 on OA progression and, in particular, VCAM-1 production and monocyte adhesion, which may help with the design of more effective OA treatments.

Single-cell RNA sequencing uncovers the individual alteration of intestinal mucosal immunocytes in Dusp6 knockout mice.

Single-cell RNA sequencing (scRNA-seq) approach can broadly and specifically evaluate the individual cells with minimum detection bias. To explore the individual compositional and transcriptional alteration of intestinal leukocytes in the Dual Specificity Phosphatase six knockout (D6KO) mice, we performed a scRNA-seq followed by the cell type annotation based on ImmGen database. Composition assessments found that D6KO-derived intestinal leukocytes tend to stay inactivate or immature status. The enrichment analysis showed that D6KO-derived intestinal leukocytes are less sensitive to microbes. The mod PhEA phenotypic analysis showed that the D6KO leukocytes may link to not only immune-associated but also diverse previously non-immune-related diseases. Integrating our dataset with the published dataset GSE124880 generated a comprehensive dataset for exploring intestinal immunity. Down-regulation of Ccl17 gene was found in the D6KO-derived dendritic cells. Our results demonstrated the advantage of applying scRNA-seq for dissecting the individual alteration of intestinal leukocytes, particularly in the D6KO mice at a naive state.

Type I Interferon Signaling Accelerates Liver Regeneration by Metabolic Modulation in Noninfectious Conditions.

Type I interferon (IFN-I) has a well-known function in controlling viral infections, but its contribution in hepatocyte proliferation and hepatocellular carcinoma (HCC) formation remains unclear. Mice deficient in IFN-α receptor expression in whole mice or only in hepatocytes (Ifnar-/- and IfnarΔliver) were used to investigate the role of IFN-I signaling in cell proliferation and cancer formation in the liver. Ifnar-/- mice were resistant to chemical-induced HCC formation in the absence of infection. The results show that low grade of IFN-I and interferon-stimulated gene were expressed substantially in naïve mouse liver. The low level of IFN-I activation is constantly present in mouse liver after weaning and negatively modulates forkhead box O hepatic expression. The IFN-I signaling can be partially blocked by the clearance of lipopolysaccharide. Mice lacking IFN-I signaling have lower basal proliferation activity and delayed liver regeneration processes after two-thirds partial hepatectomy. The activation of IFN-I signaling on hepatocyte controls glucose homeostasis and lipid metabolism to support proliferation potency and long-term tumorigenesis. Our results reveal a positive role of low-grade IFN-I singling to hepatocyte proliferation and HCC formation by modulating glucose homeostasis and lipid metabolism.

Toll-Like Receptor 21 of Chicken and Duck Recognize a Broad Array of Immunostimulatory CpG-oligodeoxynucleotide Sequences.

CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the function of microbial CpG-dideoxynucleotides containing DNA (CpG-DNA) are potent immune stimuli. The immunostimulatory activity and the species-specific activities of a CpG-ODN depend on its nucleotide sequence properties, including CpG-hexamer motif types, spacing between motifs, nucleotide sequence, and length. Toll-like receptor (TLR) 9 is the cellular receptor for CpG-ODNs in mammalian species, while TLR21 is the receptor in avian species. Mammalian cells lack TLR21, and avian cells lack TLR9; however, both TLRs are expressed in fish cells. While nucleotide sequence properties required for a CpG-ODN to strongly activate mammalian TLR9 and its species-specific activities to different mammalian TLR9s are better studied, CpG-ODN activation of TLR21 is not yet well investigated. Here we characterized chicken and duck TLR21s and investigated their activation by CpG-ODNs. Chicken and duck TLR21s contain 972 and 976 amino acid residues, respectively, and differ from TLR9s as they do not have an undefined region in their ectodomain. Cell-based TLR21 activation assays were established to investigate TLR21 activation by different CpG-ODNs. Unlike grouper TLR21, which was preferentially activated by CpG-ODN with a GTCGTT hexamer motif, chicken and duck TLR21s do not distinguish among different CpG-hexamer motifs. Additionally, these two poultry TLR21s were activated by CpG-ODNs with lengths ranging from 15 to 31 nucleotides and with different spacing between CpG-hexamer motifs. These suggested that compared to mammalian TLR9 and grouper TLR21, chicken and duck TLR21s have a broad CpG-ODN sequence recognition profile. Thus, they could also recognize a wide array of DNA-associated molecular patterns from microbes. Moreover, CpG-ODNs are being investigated as antimicrobial agents and as vaccine adjuvants for different species. This study revealed that there are more optimized CpG-ODNs that can be used in poultry farming as anti-infection agents compared to CpG-ODN choices available for other species.

Epigenetic Silencing of Ubiquitin Specific Protease 4 by Snail1 Contributes to Macrophage-Dependent Inflammation and Therapeutic Resistance in Lung Cancer.

There is a positive feedback loop driving tumorigenesis and tumor growth through coordinated regulation of epigenetics, inflammation, and stemness. Nevertheless, the molecular mechanism linking these processes is not well understood. In this study, we analyzed the correlation of de-ubiquitinases (DUBs) expression with survival data from the OncoLnc database. Among the DUBs analyzed, ubiquitin specific protease 4 (USP4) had the lowest negative Cox coefficient. Low expression of USP4 was associated with poor survival among lung cancer patients and was inversely correlated with expression of stemness and inflammation markers. Expression of USP4 were reduced at more advanced stages of lung cancer. Mechanistically, expression of USP4 was downregulated in snail1-overexpressing and stemness-enriched lung cancer cells. Snail1 was induced in lung cancer cells by interaction with macrophages, and epigenetically suppressed USP4 expression by promoter methylation. Stable knockdown of USP4 in lung cancer cells enhanced inflammatory responses, stemness properties, chemotherapy resistance, and the expression of molecules allowing escape from immunosurveillance. Further, mice injected with USP4 knockdown lung cancer cells demonstrated enhanced tumorigenesis and tumor growth. These results reveal that the Snail1-mediated suppression of USP4 is a potential mechanism to orchestrate epigenetic regulation, inflammation and stemness for macrophage-promoted tumor progression.

Correction: USP17 mediates macrophage-promoted inflammation and stemness in lung cancer cells by regulating TRAF2/TRAF3 complex formation.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Immunostimulatory Activities of CpG-Oligodeoxynucleotides in Teleosts: Toll-Like Receptors 9 and 21.

Toll-like receptors (TLRs) are pattern-recognition receptors that detect a wide variety of microbial pathogens for the initiation of host defense immunological responses. Thirteen TLRs have been identified in mammals, and teleosts contain 22 mammalian or non-mammalian TLRs. Of these, TLR9 and TLR21 are the cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) recognition TLRs in teleosts. TLR9 is a mammalian TLR expressed in teleost but not in the avian species. TLR21 is a non-mammalian TLR expressed in both teleost and the avian species. Synthetic CpG-ODNs are potent immunostimulants that are being studied for their application against tumors, allergies, and infectious diseases, and as a vaccine adjuvant in humans. The immunostimulatory effects of CpG-ODNs as vaccine adjuvants and their antimicrobial function in domestic animals and teleosts are also being investigated. Most of our current knowledge about the molecular basis for the immunostimulatory activity of CpG-ODNs comes from earlier studies of the interaction between CpG-ODN and TLR9. More recent studies indicate that in addition to TLR9, TLR21 is another receptor for CpG-ODN recognition in teleosts to initiate immune responses. Whether these two receptors have differential functions in mediating the immunostimulatory activity of CpG-ODN in teleost has not been well-studied. Nevertheless, the existence of two recognition TLRs suggests that the molecular basis for the immunostimulatory activity of CpG-ODN in teleosts is different and more complex than in mammals. This article reviews the current knowledge of TLR9 and TLR21 activation by CpG-ODNs. The key points that need to be considered for CpG-ODNs as immunostimulants with maximum effectiveness in activation of immune responses in teleosts are discussed. This includes the structure/activity relationship of CpG-ODN activities for TLR9 and TLR21, the structure/functional relationship of these two TLRs, and differential expression levels and tissue distributions for these two TLRs.

USP17 mediates macrophage-promoted inflammation and stemness in lung cancer cells by regulating TRAF2/TRAF3 complex formation.

Macrophage accumulation and inflammation in the lung owing to stresses and diseases is a cause of lung cancer development. However, molecular mechanisms underlying the interaction between macrophages and cancer cells, which drive inflammation and stemness in cancers, are poorly understood. In this study, we investigated the expression of ubiquitin-specific peptidase 17 (USP17) in lung cancers, and role of elevated USP17 in the interaction between macrophages and lung cancer cells. USP17 expression in lung cancers was associated with poor prognosis, macrophage, and inflammatory marker expressions. Macrophages promoted USP17 expression in cancer cells. TNFR-associated factor (TRAF) 2-binding and TRAF3-binding motifs were identified in USP17, through which it interacted with and disrupted the TRAF2/TRAF3 complex. This stabilized its client proteins, enhanced inflammation and stemness in cancer cells, and promoted macrophage recruitment. In different animal studies, co-injection of macrophages with cancer cells promoted USP17 expression in tumors and tumor growth. Conversely, depletion of macrophages in host animals by clodronate liposomes reduced USP17 expression and tumor growth. In addition, overexpression of USP17 in cancer cells promoted tumor growth and inflammation-associated and stemness-associated gene expressions in tumors. These results suggested that USP17 drives a positive-feedback interaction between macrophages and cancer cells to enhance inflammation and stemness in cancer cells, and promotes lung cancer growth.

Establishment of a mouse model for the complete mosquito-mediated transmission cycle of Zika virus.

Zika virus (ZIKV) is primarily transmitted by Aedes mosquitoes in the subgenus Stegomyia but can also be transmitted sexually and vertically in humans. STAT1 is an important downstream factor that mediates type I and II interferon signaling. In the current study, we showed that mice with STAT1 knockout (Stat1-/-) were highly susceptible to ZIKV infection. As low as 5 plaque-forming units of ZIKV could cause viremia and death in Stat1-/- mice. ZIKV replication was initially detected in the spleen but subsequently spread to the brain with concomitant reduction of the virus in the spleen in the infected mice. Furthermore, ZIKV could be transmitted from mosquitoes to Stat1-/- mice back to mosquitoes and then to naïve Stat1-/- mice. The 50% mosquito infectious dose of viremic Stat1-/- mouse blood was close to 810 focus-forming units (ffu)/ml. Our further studies indicated that the activation of macrophages and conventional dendritic cells were likely critical for the resolution of ZIKV infection. The newly developed mouse and mosquito transmission models for ZIKV infection will be useful for the evaluation of antiviral drugs targeting the virus, vector, and host.

Involvement of M1 Macrophage Polarization in Endosomal Toll-Like Receptors Activated Psoriatic Inflammation.

Psoriasis is a chronic inflammatory skin disorder that affects ~2%-3% of the worldwide population. Inappropriate and excessive activation of endosomal Toll-like receptors 7, 8, and 9 (TLRs 7-9) at the psoriatic site has been shown to play a pathogenic role in the onset of psoriasis. Macrophage is a major inflammatory cell type that can be differentiated into phenotypes M1 and M2. M1 macrophages produce proinflammatory cytokines, and M2 macrophages produce anti-inflammatory cytokines. The balance between these two types of macrophages determines the progression of various inflammatory diseases; however, whether macrophage polarization plays a role in psoriatic inflammation activated by endosomal TLRs has not been investigated. In this study, we investigated the function and mechanism of macrophages related to the pathogenic role of TLRs 7-9 in the progression of psoriasis. Analysis of clinical data in database revealed significantly increased expression of macrophage markers and inflammatory cytokines in psoriatic tissues over those in normal tissues. In animal studies, depletion of macrophages in mice ameliorated imiquimod, a TLR 7 agonist-induced psoriatic response. Imiquimod induced expression of genes and cytokines that are signature of M1 macrophage in the psoriatic lesions. In addition, treatment with this TLR 7 agonist shifted macrophages in the psoriatic lesions to a higher M1/M2 ratio. Both of the exogenous and endogenous TLR 7-9 ligands activated M1 macrophage polarization. M1 macrophages expressed higher levels of proinflammatory cytokines and TLRs 7-9 than M2 macrophages. These results suggest that by rendering macrophages into a more inflammatory status and capable of response to their ligands in the psoriatic sites, TLR 7-9 activation drives them to participate in endosomal TLR-activated psoriatic inflammation, resulting in an amplified inflammatory response. Our results also suggest that blocking M1 macrophage polarization could be a strategy which enables inhibition of psoriatic inflammation activated by these TLRs.

CpG-oligodeoxynucleotides developed for grouper toll-like receptor (TLR) 21s effectively activate mouse and human TLR9s mediated immune responses.

Synthetic phosphorothiolate-modified CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli. Toll-like receptor (TLR) 9 and TLR21 are their cellular receptors in different species. The structural requirements for CpG-ODN to strongly activate TLR9 have been relatively well studied, but studies on TLR21 are in their infancy. Therefore, in this study, we investigated the interaction between CpG-ODNs and TLR21s from groupers (Epinephelus spp.), which are economically important fish species. We cloned the cDNA of giant grouper (E. lanceolatus) TLR21, and compared its sequence with orange-spotted grouper (E. coioides) TLR21A and TLR21B. These three receptors were activated by CpG-ODNs containing the GTCGTT motif but not by those containing the GACGTT motif. We developed two CpG-ODNs that contained 19 phosphorothiolated deoxynucleotides with one or two GTCGTT motifs. These CpG-ODNs had better activity on grouper TLR21s than currently developed CpG-ODNs, and produced similar immune stimulatory profiles when applied to cells isolated from orange-spotted grouper. The developed CpG-ODNs also effectively activated both human and mouse TLR9-mediated NF-κB activation and cytokine productions. These findings suggest that the GTCGTT motif is required for CpG-ODNs to activate grouper TLR21s, and that the CpG-ODNs that were developed for grouper TLR21s contain structures that effectively activate human and mouse TLR9s.

Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation.

Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.