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.

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.

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.

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.

A preliminary study of the innate immune memory of Kupffer cells induced by PEGylated nanoemulsions.

The accelerated blood clearance (ABC) phenomenon describes a dilemma of polyethylene glycol (PEG) applied in drug delivery system (DDS) caused by its immunogenicity, that results in the enhanced blood clearance rate and increased hepatic and splenic accumulation after secondary injection of PEGylated nanocarriers. However, the ABC index, as the judgement of ABC phenomenon, only describes the accelerated blood clearance rate, but ignores the enhanced hepatic and splenic accumulation. Therefore, we proposed the hepatic accumulation (HA) index and the splenic accumulation (SA) index as supplements for assessing the ABC phenomenon, to emphasize the contribution of liver and spleen, especially the liver, possessing the most population of tissue resident macrophages. By altering the first injection site from the tail vein to the liver portal vein, there was no impact on anti-PEG IgM production, and the secondary hepatic accumulation of PEGylated nanoemulsions (PE) was observed to be proportionate to the first PE stimulation strength on the liver. We also determined that Kupffer cells (KCs) were the main contributor to this enhancement. On this basis, we revealed a definite phenomenon that PE could induce innate immune memory in KCs, by enhancing the phagocytosis of KCs toward PE during the secondary stimulation. The PE-stimulated KCs could carry this memory to the naïve rats through adoptive transfer, resulting in increased hepatic accumulation in the recipient rats without antibody production. Studies examining the phagocytosis of KCs in vivo, ex vivo and in vitro revealed that the memory of KCs against PE triggered by first-stimulated PE could be maintained independently of other cells or components until 21 days after the first stimulation, and possessing specificity to PEG, which was invalid to long-circulating GE (GM1 modified nanoemulsions). The discovery of immune memory in KCs induced by PE highlights the importance of focusing on the relationship between the innate immune system and PEGylated nanocarriers during the development of DDS to improve medication safety in the clinic.

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.

Sialic acid-conjugate modified liposomes targeting neutrophils for improved tumour therapy.

Neutrophils are the most abundant white blood cells in humans. Many tumor-treatment methods that are related to tissue infiltration and the activation of neutrophils have been developed. In particular, one strategy, which aims to improve tumor treatment, involves the exploitation or targeting of activated neutrophils. Peripheral blood neutrophils (PBNs) from tumor-bearing mice display high expression of l-selectin, which is well known to be targeted by the sialic acid (SA) ligand. Hence, in this research, we developed a drug delivery platform involving liposomes modified with an SA conjugate that targets activated PBNs. The uptake of doxorubicin (DOX)-loaded liposomes by PBNs did not alter their activation and transmigration. Furthermore, in tumor-bearing mice, SA-modified liposomes displayed a greater tumor-targeting ability and stronger tumor treatment efficacy, which were mediated by the neutrophil infiltration induced by inflammatory factors released from the tumor microenvironment. In conclusion, SA-modified liposomal DOX was shown to be an effective neutrophil-mediated drug delivery system for tumor therapy.

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.

Neutrophil-Mediated Delivery of Dexamethasone Palmitate-Loaded Liposomes Decorated with a Sialic Acid Conjugate for Rheumatoid Arthritis Treatment.

PURPOSE: The aim of this research was to design dexamethasone palmitate (DP) loaded sialic acid modified liposomes, with the eventual goal of using peripheral blood neutrophils (PBNs) that carried drug-loaded liposomes to improve the therapeutic capacity for rheumatoid arthritis (RA). METHODS: A sialic acid - cholesterol conjugate (SA-CH) was synthesized and anchored on the surface of liposomal dexamethasone palmitate (DP-SAL). The physicochemical characteristics and in vitro cytotoxicity of liposomes were evaluated. Flow cytometry and confocal laser scanning microscopy were utilized to investigate the accumulation of liposomes in PBNs. The adjuvant-induced arthritis was adopted to investigate the targeting ability and anti-inflammatory effect of DP loaded liposomes. RESULTS: Both DP-CL and DP-SAL existed an average size less than 200 nm with remarkably high encapsulation efficiencies more than 90%. In vitro and in vivo experiments manifested SA-modified liposomes provided a reinforced accumulation of DP in PBNs. As well, DP-SAL displayed a greater degree of accumulation in the joints and a stronger anti-inflammatory effect in terms of RA suppression. CONCLUSIONS: SA-modified liposomal DP was a promising candidate for RA-targeting treatment through the neutrophil-mediated drug delivery system.

Dual-Ligand Modification of PEGylated Liposomes Used for Targeted Doxorubicin Delivery to Enhance Anticancer Efficacy.

Mannose receptor (CD206) and E-selectin are selectively expressed in M2-like tumor-associated macrophages (M2-TAMs) and activated endothelial cells of vessels surrounding tumor tissues. With the knowledge that D-mannose is the natural ligand of mannose receptors and L-fucose is the key calcium chelator for tumor-associated carbohydrate antigens (TACAs) binding to E-selectin, herein, we firstly reported D-mannose polyethylene glycol (PEG) conjugates (Man-PEG) and L-fucose PEG conjugates (Fuc-PEG) co-modified liposomal doxorubicin (DOX-MFPL) to improve tumor-targeting ability. The dual-ligand modified PEGylated liposomes (DOX-MFPL) were assessed by both in vitro and in vivo trials. Compared with the single-ligand D-mannose- or L-fucose-modified liposomes (DOX-MPL or DOX-FPL), DOX-MFPL achieved an increased distribution of DOX in tumor tissues. The antitumor study based on S180 tumor-bearing mice was conducted and the superior tumor inhibitory rate was shown with DOX-MFPL, probably owing to the superior tumor-targeting effect of DOX-MFPL and the modulation of the tumor microenvironment with the exhaustion of TAMs. In general, the dual-ligand drug delivery systems are expected to be promising in the development of specific and efficient methods for tumor treatment.

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.

Effect of Kupffer cells depletion on ABC phenomenon induced by Kupffer cells-targeted liposomes.

Accelerated blood clearance (ABC) phenomenon is common in many PEGylated nanocarriers, whose mechanism has not been completely elucidated yet. In this study, the correlation between Kupffer cells (KCs) and ABC phenomenon has been studied by KCs-targeted liposomes inducing ABC phenomenon and KCs depletion. In other words, the 4-aminophenyl-α-D-mannopyranoside (APM) lipid derivative DSPE-PEG2000-APM (DPM), and 4-aminophenyl-ß-L-fucopyranoside (APF) lipid derivative DSPE-PEG2000-APF (DPF) were conjugated and modified on alendronate sodium (AD) liposomes to specifically target and deplete KCs. The dual-ligand modified PEGylated liposomes (MFPL) showed stronger ability to damage KCs in vitro and in vivo, which also could indirectly illustrate that dual-ligand modification could better target KCs. Besides, the hepatic biodistribution and pharmacokinetics could directly prove that MFPL had a stronger targeting ability to KCs. In addition, in depletion rats, plasma concentration and splenic biodistribution of MFPL and PEGylated liposomes (PL) were significantly elevated and hepatic biodistribution was significantly reduced, which demonstrated that KCs played an important role on elimination of nanoparticles. What's more, ABC phenomenon of the secondary injection of PL was stronger in KCs depletion rats than that in normal rats, which indicated that depletion of KCs prolonged the circulation of PL in the first injection repeatedly stimulating B-cells in the marginal region of the spleen and causing it to secrete more IgM antibodies. This could also illustrate that anti-PEG IgM takes up a major station compared with KCs. Most important of all, KCs-targeted liposomes could induce a stronger ABC phenomenon than PL in normal rats, which declared that based on the same IgM concentration, the more the KCs were stimulated, the stronger ABC phenomenon was induced. However, in depletion rats, this difference of ABC phenomenon between PL and MFPL could no more exist, further demonstrating that KCs could participate and play a certain role in the ABC phenomenon.