Functional roles of circular RNAs in lung injury.

Lung injury leads to respiratory dysfunction, low quality of life, and even life-threatening conditions. Circular RNAs (circRNAs) are endogenous RNAs produced by selective RNA splicing. Studies have reported their involvement in the progression of lung injury. Understanding the roles of circRNAs in lung injury may aid in elucidating the underlying mechanisms and provide new therapeutic targets. Thus, in this review, we aimed to summarize and discuss the characteristics and biological functions of circRNAs, and their roles in lung injury from existing research, to provide a theoretical basis for the use of circRNAs as a diagnostic and therapeutic target for lung injury.

NLRP3 regulates CIITA/MHC II axis and interferon-γ-inducible chemokines in Malassezia globosa-infected keratinocytes.

CIITA, a member of NOD-like receptor (NLR) family, is the major MHC II trans-activator and mediator of Th1 immunity, but its function and interaction with NLRP3 have been little studied. We found activation of NLRP3 inflammasome, increased expression of CIITA, CBP, pSTAT1, STAT1, MHC II, IFN-γ and IFN-γ-inducible chemokines (CCL1 and CXCL8), and colocalisation of NLRP3 with CIITA in Malassezia folliculitis lesions, Malassezia globosa-infected HaCaT cells and mouse skin. CoIP with anti-CIITA or anti-NLRP3 antibody pulled down NLRP3 or both CIITA and ASC. NLRP3 silencing or knockout caused CIITA downexpression and their colocalisation disappearance in HaCaT cells and mouse skin of Nlrp3-/- mice, while CIITA knockdown had no effect on NLRP3, ASC, IL-1ß and IL-18 expression. NLRP3 inflammasome inhibitors and knockdown significantly suppressed IFN-γ, CCL1, CXCL8 and CXCL10 levels in M. globosa-infected HaCaT cells. CCL1 and CXCL8 expression was elevated in Malassezia folliculitis lesions and reduced in Nlrp3-/- mice. These results demonstrate that M. globosa can activate NLRP3 inflammasome, CIITA/MHC II signalling and IFN-γ-inducible chemokines in human keratinocytes and mouse skin. NLRP3 may regulate CIITA by their binding and trigger Th1 immunity by secreting CCL1 and CXCL8/IL-8, contributing to the pathogenesis of Malassezia-associated skin diseases.

Toxoplasma gondii Induces Pyroptosis in Human Placental Trophoblast and Amniotic Cells by Inducing ROS Production and Activation of Cathepsin B and NLRP1/NLRP3/NLRC4/AIM2 Inflammasome.

Toxoplasma gondii infection in pregnant women may cause fetal anomalies; however, the underlying mechanisms remain unclear. The current study investigated whether T. gondii induces pyroptosis in human placental cells and the underlying mechanisms. Human placental trophoblast (BeWo and HTR-8/SVneo) and amniotic (WISH) cells were infected with T. gondii, and then reactive oxygen species (ROS) production, cathepsin B (CatB) release, inflammasome activation, and pyroptosis induction were evaluated. The molecular mechanisms of these effects were investigated by treating the cells with ROS scavengers, a CatB inhibitor, or inflammasome-specific siRNA. T. gondii infection induced ROS generation and CatB release into the cytosol in placental cells but decreased mitochondrial membrane potential. T. gondii-infected human placental cells and villi exhibited NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation and subsequent pyroptosis induction, as evidenced by increased expression of ASC, cleaved caspase-1, and mature IL-1ß and gasdermin D cleavage. In addition to inflammasome activation and pyroptosis induction, adverse pregnancy outcome was shown in a T. gondii-infected pregnant mouse model. Administration of ROS scavengers, CatB inhibitor, or inflammasome-specific siRNA into T. gondii-infected cells reversed these effects. Collectively, these findings show that T. gondii induces NLRP1/NLRP3/NLRC4/AIM2 inflammasome-dependent caspase-1-mediated pyroptosis via induction of ROS production and CatB activation in placental cells. This mechanism may play an important role in inducing cell injury in congenital toxoplasmosis.

Trichomonas vaginalis induces apoptosis via ROS and ER stress response through ER-mitochondria crosstalk in SiHa cells.

BACKGROUND: Trichomonas vaginalis causes lesions on the cervicovaginal mucosa in women; however, its pathogenesis remains unclear. We have investigated the involvement of the endoplasmic reticulum (ER) in the induction of apoptosis by T. vaginalis and its molecular mechanisms in human cervical cancer SiHa cells. METHODS: Apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), ER stress response and Bcl-2 family protein expression were evaluated using immunocytochemistry, flow cytometry, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide dye staining and western blotting. RESULTS: Trichomonas vaginalis induced mitochondrial ROS production, apoptosis, the ER stress response and mitochondrial dysfunction, such as MMP depolarization and an imbalance in Bcl-2 family proteins, in SiHa cells in a parasite burden- and infection time-dependent manner. Pretreatment with N-acetyl cysteine (ROS scavenger) or 4-phenylbutyric acid (4-PBA; ER stress inhibitor) significantly alleviated apoptosis, mitochondrial ROS production, mitochondrial dysfunction and ER stress response in a dose-dependent manner. In addition, T. vaginalis induced the phosphorylation of apoptosis signal regulating kinase 1 (ASK1) and c-Jun N-terminal kinases (JNK) in SiHa cells, whereas 4-PBA or SP600125 (JNK inhibitor) pretreatment significantly attenuated ASK1/JNK phosphorylation, mitochondrial dysfunction, apoptosis and ER stress response in SiHa cells, in a dose-dependent manner. Furthermore, T. vaginalis excretory/secretory products also induced mitochondrial ROS production, apoptosis and the ER stress response in SiHa cells, in a time-dependent manner. CONCLUSIONS: Trichomonas vaginalis induces apoptosis through mitochondrial ROS and ER stress responses, and also promotes ER stress-mediated mitochondrial apoptosis via the IRE1/ASK1/JNK/Bcl-2 family protein pathways in SiHa cells. These data suggest that T. vaginalis-induced apoptosis is affected by ROS and ER stress response via ER-mitochondria crosstalk.

FAF1 downregulation by Toxoplasma gondii enables host IRF3 mobilization and promotes parasite growth.

Fas-associated factor 1 (FAF1) has gained a reputation as a member of the FAS death-inducing signalling complex. However, the role of FAF1 in the immunity response is not fully understood. Here, we report that, in the human retinal pigment epithelial (RPE) cell line ARPE-19 cells, FAF1 expression level was downregulated by Toxoplasma gondii infection, and PI3K/AKT inhibitors reversed T. gondii-induced FAF1 downregulation. In silico analysis for the FAF1 promoter sequence showed the presence of a FOXO response element (FRE), which is a conserved binding site for FOXO1 transcription factor. In accordance with the finding, FOXO1 overexpression potentiated, whereas FOXO1 depletion inhibited intracellular FAF1 expression level. We also found that FAF1 downregulation by T. gondii is correlated with enhanced IRF3 transcription activity. Inhibition of PI3K/AKT pathway with specific inhibitors had no effect on the level of T. gondii-induced IRF3 phosphorylation but blocked IRF3 nuclear import and ISGs transcription. These results suggest that T. gondii can downregulate host FAF1 in PI3K/AKT/FOXO1-dependent manner, and the event is essential for IRF3 nuclear translocation to active the transcription of ISGs and thereby T. gondii proliferation.

Silver nanoparticles induce apoptosis via NOX4-derived mitochondrial reactive oxygen species and endoplasmic reticulum stress in colorectal cancer cells.

Aim: To investigate the anticancer mechanisms of silver nanoparticles (AgNPs) in colorectal cancer. Methods: Anticancer effects of AgNPs were determined in colorectal cancer HCT116 cells and xenograft mice using cellular and molecular methods. Results: AgNPs induced mitochondrial reactive oxygen species production, mitochondrial dysfunction and endoplasmic reticulum (ER) stress responses through NOX4 and led to HCT116 cell apoptosis. Pretreatment with DPI or 4-PBA significantly inhibited mitochondrial reactive oxygen species production, apoptosis, ER stress response, NOX4 expression and mitochondrial dysfunction in AgNP-treated HCT116 cells. AgNPs also significantly suppressed HCT116 cell-based xenograft tumor growth in nude mice by inducing apoptosis and ER stress responses. Conclusion: AgNPs exert anticancer effects against colorectal cancer via ROS- and ER stress-related mitochondrial apoptosis pathways.

Expression profiles of NOD-like receptors and regulation of NLRP3 inflammasome activation in Toxoplasma gondii-infected human small intestinal epithelial cells.

BACKGROUND: Toxoplasma gondii is a parasite that primarily infects through the oral route. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play crucial roles in the immune responses generated during parasitic infection and also drive the inflammatory response against invading parasites. However, little is known about the regulation of NLRs and inflammasome activation in T. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. METHODS: FHs 74 Int cells infected with T. gondii were subsequently evaluated for morphological changes, cytotoxicity, expression profiles of NLRs, inflammasome components, caspase-cleaved interleukins (ILs), and the mechanisms of NLRP3 and NLRP6 inflammasome activation. Immunocytochemistry, lactate dehydrogenase assay, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and western blotting techniques were utilized for analysis. RESULTS: Under normal and T. gondii-infected conditions, members of the NLRs, inflammasome components and caspase-cleaved ILs were expressed in the FHs Int 74 cells, except for NLRC3, NLRP5, and NLRP9. Among the NLRs, mRNA expression of NOD2, NLRP3, NLRP6, and NAIP1 was significantly increased in T. gondii-infected cells, whereas that of NLRP2, NLRP7, and CIITA mRNAs decreased significantly in a time-dependent manner. In addition, T. gondii infection induced NLRP3, NLRP6 and NLRC4 inflammasome activation and production of IL-1ß, IL-18, and IL-33 in FHs 74 Int cells. T. gondii-induced NLRP3 inflammasome activation was strongly associated with the phosphorylation of p38 MAPK; however, JNK1/2 had a weak effect. NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells. CONCLUSIONS: This study highlighted the expression profiles of NLRs and unraveled the underlying mechanisms of NLRP3 inflammasome activation in T. gondii-infected FHs 74 Int cells. These findings may contribute to understanding of the mucosal and innate immune responses induced by the NLRs and inflammasomes during T. gondii infection in FHs 74 Int cells.

Involvement of endoplasmic reticulum stress response and IRE1-mediated ASK1/JNK/Mcl-1 pathways in silver nanoparticle-induced apoptosis of human retinal pigment epithelial cells.

Silver nanoparticles (AgNPs) have cytotoxic effects on various human cell types. The endoplasmic reticulum (ER) is very sensitive to cytotoxic damage. Retina tissue is easily affected by internal and external stimuli. However, the effect of AgNPs on human retinal cells is not known. This study examined the effect of AgNPs on ER stress induction and their mechanism of action in human retinal pigment epithelium (RPE) ARPE-19 cells. We found that AgNPs significantly increased ARPE-19 cell cytotoxicity and stimulated caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage, as well as mitochondrial membrane potential (MMP) depolarization, in ARPE-19 cells in a dose-dependent manner (0.2-5 µg/mL for 18 h). AgNPs (5 µg/mL for 18 h) induced several signature ER stress markers, as indicated by the upregulated expressions of CCAAT/enhancer-binding protein-homologous protein (CHOP), phosphorylated protein kinase RNA-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), and inositol-requiring protein 1 (IRE1), and cleaved activating transcription factor 6 (ATF6). AgNPs also activated ASK1 and JNK in ARPE-19 cells, and induced increases in Bax and Puma expressions, as well as a decrease in Mcl-1 expression. However, inhibition of the ER stress response by pretreatment with 4-PBA included apparently and dose-dependently reduced levels of p-PERK, p-IRE1, CHOP, cleaved ATF6, p-ASK1, p-JNK, cleaved caspase-3, procaspase-12, and MMP depolarization in AgNP-treated ARPE-19 cells; it also led to significantly increased Mcl-1 protein levels in a dose-dependent manner in ARPE-19 cells. Pretreatment with JNK inhibitor SP600125 significantly attenuated caspase-3 cleavage and MMP depolarization and increased Mcl-1 protein levels in AgNPs-treated ARPE-19 cells in a dose-dependent manner. Hence, our study demonstrated that AgNPs induced apoptosis in human RPE ARPE-19 cells by ER stress response and ER stress-dependent mitochondrial apoptosis via the IRE1/ASK1/JNK/Mcl-1 pathways.

Silver Nanoparticle-Induced Apoptosis in ARPE-19 Cells Is Inhibited by Toxoplasma gondii Pre-Infection Through Suppression of NOX4-Dependent ROS Generation.

PURPOSE: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity. MATERIALS AND METHODS: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4-/ - mice. RESULTS: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed. CONCLUSION: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.

VEGF Production Is Regulated by the AKT/ERK1/2 Signaling Pathway and Controls the Proliferation of Toxoplasma gondii in ARPE-19 Cells.

The retina is the primary site of Toxoplasma gondii infection in the eye, and choroidal neovascularization in ocular toxoplasmosis is one of the most important causes of visual impairment. Vascular endothelial growth factor (VEGF) is one of the key regulators of blood vessel development, however, little is known about the mechanisms of T. gondii-induced VEGF production in ocular toxoplasmosis. Here, we investigate the effect of T. gondii on VEGF production regulation in human retinal pigment epithelium ARPE-19 cells and attempted to unveil the underlying mechanism of this event by focusing on the interaction between parasite and the selected host intracellular signaling pathways. T. gondii infection increased the expression of VEGF mRNA and protein in ARPE-19 cells in parasite burden- and infection time-dependent manner. The proportional increase of VEGF upstream regulators, HIF-1α and HO-1, was also observed. T. gondii induced the activation of host p-AKT, p-ERK1/2, and p-p38 MAPK in ARPE-19 cells in a parasite-burden dependent manner. However, VEGF expression decreased after the pre-treatment with PI3K inhibitors (LY294002 and GDC-0941), ERK1/2 inhibitor (PD098059), and p38 MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125), in a dose-dependent manner. The anti-VEGF agent bevacizumab or VEGF siRNA transfection prominently inhibited the activation of p-AKT and p-ERK1/2, but not p-p38 MAPK and JNK1/2 in T. gondii-infected ARPE-19 cells. Bevacizumab treatment or VEGF siRNA transfection significantly inhibited the proliferation of T. gondii tachyzoites in the host cell, dose-dependently, but not invasion of parasites. VEGF-receptor 2 (VEGF-R2) antagonist, SU5416, attenuated VEGF production and tachyzoite proliferation in T. gondii-infected ARPE-19 cells in a dose-dependent manner. Collectively, T. gondii prominently induces VEGF production in ARPE-19 cells, and VEGF and AKT/ERK1/2 signaling pathways mutually regulate each other in T. gondii-infected ARPE-19 cells, but not p38 MAPK and JNK1/2 signaling pathways. VEGF and VEGF-R2 control the parasite proliferation in T. gondii-infected ARPE-19 cells. From this study, we revealed the putative mechanisms for VEGF induction as well as the existence of positive feedback between VEGF and PI3K/MAPK signaling pathways in T. gondii-infected retinal pigment epithelium.

Evaluation of Protective Immune Response Induced by a DNA Vaccine Encoding GRA8 against Acute Toxoplasmosis in a Murine Model.

Toxoplasma gondii is an apicomplexan zoonotic protozoan parasite that infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicate a need for the development of an effective vaccine. T. gondii GRA8 is a member of the dense granules protein family and is used as a marker of acute infection. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pDsRed2-GRA8, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with the pDsRed2-GRA8 plasmid and then challenged by infection with the highly virulent GFP-RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii of this vaccine were analyzed by measuring cytokine and serum antibody titers, splenocyte proliferation assays, and the survival times of mice after challenge. Our results showed that mice immunized with pDsRed2-GRA8 demonstrated specific humoral and cellular responses, induced higher IgG antibody titers with predominant IgG2a production; increased levels of IL-10, IL-12 (p70), IFN-γ, TNF-α, and splenocyte proliferation; and prolonged survival times compared to those of control mice. The present study showed that DNA immunization with pDsRed2-GRA8 induced humoral and cellular immune responses, and all immunized mice showed greater Th1-type immune responses and longer survival times than those of control mice. These results indicated that T. gondii GRA8 DNA immunization induces a partial protective effect against acute toxoplasmosis.

FoxO1 enhances differentiation and apoptosis in human primary keratinocytes.

Forkhead box-O1 (FoxO1) is a key nutrient- and growth factor-dependent regulator of metabolism, but its functional role in human primary keratinocytes (HPKs) is less known. To investigate the role of FoxO1 in HPKs and effect of insulin-like growth factor 1 (IGF-1) and isotretinoin on FoxO1 expression, HPKs were treated with 1.2 mmol/L calcium chloride, 1-20 ng/mL IGF-1 and 0.1-10 µmol/L isotretinoin. Recombinant adenovirus expressing FoxO1 or FKHR shRNA lentivirus transfection was introduced to upregulate or silence FoxO1 expression. Epidermal FoxO1 immunostaining was lower in acne lesion than in normal skin. FoxO1 overexpression induced involucrin expression, G2/M arrest and apoptosis but suppressed proliferation, while FoxO1 silencing decreased involucrin expression but increased proliferation, S phase and viable cells in HPKs. IGF-1 downregulated FoxO1 and involucrin but upregulated p-Akt expression in HPKs, which was blocked by pretreatment with LY294002. Isotretinoin enhanced FoxO1, p53 and p21 but inhibited p-FoxO1 and involucrin expression in HPKs. These results demonstrate that FoxO1 promotes differentiation and apoptosis in HPKs. IGF-1 may reduce keratinocyte differentiation through PI3K/Akt/FoxO1 pathway, while isotretinoin can reinforce FoxO1 expression. FoxO1 may be involved in acne pathogenesis and could serve as a potential therapeutic target.

Differentiation Model Establishment and Differentiation-Related Protein Screening in Primary Cultured Human Sebocytes.

Sebocyte differentiation is a continuous process, but its potential molecular mechanism remains unclear. We aimed to establish a novel sebocyte differentiation model using human primary sebocytes and to identify the expression profiles of differentiation-associated proteins. Primary human sebocytes were cultured on Sebomed medium supplemented with 2% serum for 7 days. Flow cytometry showed that S phase cells were decreased time-dependently, while G1 and subG1 (apoptosis) phase cells increased under serum starvation. Transmission electron microscopy and Oil Red O staining revealed a gradual increase of intracellular lipid accumulation. Expression of proliferation marker was diminished, while expression of differentiation, apoptosis, and lipogenic markers elevated gradually during 7-day culture. iTRAQ analysis identified 3582 expressed proteins in this differentiation model. Compared with day 0, number of differentially expressed proteins was 132, 54, 321, and 96 at days 1, 3, 5, and 7, respectively. Two overexpressed proteins (S100 calcium binding protein P and ferredoxin reductase) and 2 downexpressed proteins (adenosine deaminase and keratin 10) were further confirmed by Western blot and immunohistochemistry.

Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway.

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2ß, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

P2X7 receptor mediates NLRP3-dependent IL-1ß secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells.

BACKGROUND: Toxoplasma gondii can invade and replicate in all nucleated cells in a wide range of host species, and infection induces IL-1ß production. IL-1ß plays central roles in the stimulation of the innate immune system and inflammation. However, little is known of the innate immune responses in human fetal small intestinal epithelial cells (FHs 74 Int cells) after T. gondii infection. METHODS: FHs 74 Int cells were infected with the T. gondii GFP-RH strain. Then, IL-1ß production and its mechanisms of action were evaluated using ELISA, MTT cell viability assays, Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and gene-specific small interfering RNA (siRNA) transfection. RESULTS: Infection of FHs 74 Int cells by T. gondii triggered significant time- and dose-dependent IL-1ß production. Although T. gondii activated NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in FHs 74 Int cells, NLRP3 levels were consistently and significantly time-dependently increased, while the other inflammasomes were not. Transfection with siRNA targeting NLRP3, cleaved caspase-1 (Casp-1) or ASC significantly reduced T. gondii-induced IL-1ß production, whereas T. gondii proliferation was markedly increased. Toxoplasma gondii infection activated P2X7 receptor (P2X7R) levels in FHs 74 Int cells in a time-dependent manner; however, transfection with siRNA targeting P2X7R significantly reduced T. gondii-induced IL-1ß secretion and substantially increased T. gondii proliferation, which is mediated by decreased protein expression levels of NLRP3, cleaved Casp-1 and ASC. Collectively, NLRP3-dependent IL-1ß secretion is mediated by P2X7R in small intestinal epithelial cells in response to T. gondii infection, thereby controlling parasite proliferation. CONCLUSIONS: This study revealed that the P2X7R/NLRP3 pathway plays important roles in IL-1ß secretion and inhibition of T. gondii proliferation in small intestinal epithelial cells. These results not only contribute to our understanding of the mucosal immune mechanisms of T. gondii infection but also offer new insight into the identification of innate resistance in the gut epithelium.

Fasciola hepatica: Infection Status of Freshwater Snails Collected from Gangwon-do (Province), Korea.

Fasciola hepatica is a trematode that causes zoonosis, mainly in cattle and sheep, and occasionally in humans. Few recent studies have determined the infection status of this fluke in Korea. In August 2015, we collected 402 samples of freshwater snails at Hoenggye-ri (upper stream) and Suha-ri (lower stream) of Song-cheon (stream) in Daegwalnyeong-myeon, Pyeongchang-gun in Gangwon-do (Province) near many large cattle or sheep farms. F. hepatica infection was determined using PCR on the nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 402 samples, F. hepatica 1TS-2 marker was detected in 6 freshwater snails; thus, the overall prevalence in freshwater snails was 1.5%. The prevalence varied between collection areas, ranging from 0.0% at Hoenggye-ri to 2.9% at Suha-ri. However, F. gigantica ITS-2 was not detected in the 6 F. hepatica-positive samples by PCR. The nucleotide sequences of the 6 F. hepatica ITS-2 PCR-positive samples were 99.4% identical to the F. hepatica ITS-2 sequences in GenBank, whereas they were 98.4% similar to F. gigantica ITS-2 sequences. These results indicated that the prevalence of F. hepatica in snail intermediate hosts was 1.5% in Gangwon-do, Korea; however the prevalence varied between collection areas. These results may help us to understand F. hepatica infection status in natural environments.

Toxoplasma gondii induces autophagy and apoptosis in human umbilical cord mesenchymal stem cells via downregulation of Mcl-1.

Autophagy and apoptosis are critical for controlling Toxoplasma gondii (T. gondii) infection. T. gondii infection during pregnancy can damage the fetus and cause birth defects; however, the molecular mechanisms of this process are poorly understood. This study aims to determine the activities of autophagy and apoptosis as well as their regulatory mechanisms during T. gondii infection by using human umbilical cord mesenchymal stem cells (hUC-MSCs) as a model of congenital diseases. LC3B, a hallmark protein of autophagy was incrementally upregulated with the infection duration, whereas p62 was downregulated in T. gondii-infected hUC-MSCs. Concurrent to this result, the invasion of T. gondii into hUC-MSCs increased in a time-dependent manner. The expression levels of Bcl-2 family proteins including Bcl-2, Bcl-xL, Bim, Bax, Bid and Bak were not altered; however, Mcl-1 levels in hUC-MSCs were dramatically decreased upon T. gondii infection. In addition, at 24 h post-infection, cleaved PARP and cleaved caspase-3 protein levels were elevated in hUC-MSCs. Importantly, Mcl-1 overexpression reduced the levels of autophagy- and apoptosis-related proteins in T. gondii-infected hUC-MSCs. Mcl-1 proteins were primarily expressed in the fraction containing mitochondria and strongly interacted with Beclin-1 under normal conditions; however, these interactions were remarkably attenuated by T. gondii infection. These results suggest that mitochondrial Mcl-1 is an essential signaling mediator regulating the activation of autophagy and apoptosis during T. gondii infection.

Sox9 facilitates proliferation, differentiation and lipogenesis in primary cultured human sebocytes.

BACKGROUND: The transcription factor Sox9 is pivotal in the morphogenesis of hair follicles, but its role in sebocytes is poorly understood. OBJECTIVE: We investigated the effect of Sox9 on human sebocyte proliferation, differentiation and lipogenesis. METHODS: Sox9 expression was detected by immunohistochemistry in normal skin and acne lesion. Primary cultured human sebocytes were transfected with adenovirus expressing GFP-Sox9 or Sox9 microRNA. Sox9 and peroxisome proliferator-activated receptor (PPAR)γ expression in sebocytes was detected by quantitative real-time PCR, Western blot and immunocytofluorescence; cell proliferation was measured by MTS and [3H]-thymidine incorporation assays; cell cycle distribution and apoptosis were evaluated by propidium iodide staining-based flow cytometry; and intracellular lipid levels were assessed by Oil Red O stain. RESULTS: Sox9 immunostaining was increased in mature sebocytes of acne lesion compared with normal skin. Expression of Sox9 mRNA and protein and PPARγ protein was elevated with cell confluent levels in sebocytes. Sox9 overexpression enhanced proliferation, differentiation, proportion of S and G2/M cells, lipogenesis and PPARγ expression in sebocytes, while Sox9 silencing caused inhibition of differentiation, lipogenesis and PPARγ expression, and increase of G1 and sub-G1 (apoptotic) cell fraction. The suppression of Sox9 knockdown on sebocyte growth was observed using [3H]-thymidine incorporation but not MTS assay. CONCLUSION: These results demonstrate that Sox9 can reinforce sebocyte proliferation, differentiation and lipogenesis. The G1/S transition arrest and apoptotic induction might contribute to inhibitory effect of Sox9 silencing on sebocyte proliferation.

Trichomonas vaginalis Induces SiHa Cell Apoptosis by NF-κB Inactivation via Reactive Oxygen Species.

Trichomonas vaginalis induces apoptosis in host cells through various mechanisms; however, little is known about the relationship between apoptosis, reactive oxygen species (ROS), and NF-κB signaling pathways in the cervical mucosal epithelium. Here, we evaluated apoptotic events, ROS production, and NF-κB activity in T. vaginalis-treated cervical mucosal epithelial SiHa cells, with or without specific inhibitors, using fluorescence microscopy, DNA fragmentation assays, subcellular fractionation, western blotting, and luciferase reporter assay. SiHa cells treated with live T. vaginalis at a multiplicity of infection of 5 (MOI 5) for 4 h produced intracellular and mitochondrial ROS in a parasite-load-dependent manner. Incubation with T. vaginalis caused DNA fragmentation, cleavage of caspase 3 and PARP, and release of cytochrome c into the cytoplasm. T. vaginalis-treated SiHa cells showed transient early NF-κB p65 nuclear translocation, which dramatically dropped at 4 h after treatment. Suppression of NF-κB activity was dependent on parasite burden. However, treatment with the ROS scavenger, N-acetyl-C-cysteine (NAC), reversed the effect of T. vaginalis on apoptosis and NF-κB inactivation in SiHa cells. Taken together, T. vaginalis induces apoptosis in human cervical mucosal epithelial cells by parasite-dose-dependent ROS production through an NF-κB-regulated, mitochondria-mediated pathway.