Clonorchis sinensis aggravated liver fibrosis by activating PARP-1 signaling to induce parthanatos via DNA damage.

Clonorchis sinensis is an important food-borne zoonotic parasite that is highly associated with liver fibrosis and cholangiocarcinoma. Further understanding of the pathogenesis of C. sinensis, especially liver fibrosis, could help us develop novel strategies for controlling clonorchiasis. Poly (ADP-ribose) polymerase-1 (PARP-1) can induce cellular parthanatos which is reported to be involved in liver fibrosis. Currently, whether C. sinensis could activate PARP-1 signaling to induce parthanatos or whether parthanatos play a role in C. sinensis-induced liver fibrosis is not clear. In the present study, the expression of PARP-1 and parthanatos indicators were detected in C. sinensis-infected mouse liver and in human intrahepatic biliary epithelial cells (HiBEpiCs) incubated with excretory/secretory products (ESPs) of C. sinensis. To explore the role of PARP-1 in C. sinensis infection, PARP-1 inhibitor NMS-P118 was used to block PARP-1 expression in vivo and vitro. The mortality rate, body weight, worm load, liver and bile duct lesions as well as PARP-1 and parthanatos indicators in C57BL/6 mice infected with C. sinensis, or in HiBEpiCs incubated with C. sinensis ESPs and NMS-P118 were analyzed and compared to the group without NMS-P118. The results showed that C. sinensis infection induced the activation of PARP-1 signaling as well as the translocation of AIF and MIF into the nucleus in mouse liver. ESPs of C. sinensis could induce PARP-1 up-regulation, ATP depletion and DNA damage in HiBEpiCs, indicating that C. sinensis could induce parthanatos. Inhibiting PARP-1 with NMS-P118 significantly reduced liver fibrosis and the number of larvae, increased the survival rate and body weight gain of the mice infected with C. sinensis. In addition, NMS-P118 decreased the expression of PARP-1 and alleviated ATP depletion as well as DNA damage in HiBEpiCs incubated with ESPs of C. sinensis. Our data indicated that C. sinensis and its ESPs could activate PARP-1 signaling to induce cellular parthanatos. NMS-P118 treatment alleviated liver fibrosis and promoted survival of the mice by inhibiting PARP-1, which suggested that PARP-1 could be used as a potential therapeutic target against clonorchiasis.

The effects of anti-lung cancer in nude mice by a fully human single-chain antibody against associated antigen Ts7TMR between A549 cells and Trichinella spiralis.

Lung cancer is a dangerous disease that is lacking in an ideal therapy. Here, we evaluated the anti-lung cancer effect in nude mice of a fully human single-chain antibody (scFv) against the associated antigen 7 transmembrane receptor (Ts7TMR), which is also called G protein-coupled receptor, between A549 cells and Trichinella spiralis (T. spiralis). Our data showed that anti-Ts7TMR scFv could inhibit lung cancer growth in a dose-dependent manner, with a tumour inhibition rate of 59.1%. HE staining did not reveal any obvious tissue damage. Mechanistically, immunohistochemical staining revealed that the scFv down-regulated the expression of PCNA and VEGF in tumour tissues. Overall, this study found that anti-Ts7TMR scFv could inhibit A549 lung cancer growth by suppressing cell proliferation and angiogenesis, which may provide a new strategy for treating lung cancer.

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis.

BACKGROUND: The unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported. METHODS: To examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1ß secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. RESULTS: VSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1ß secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated. CONCLUSIONS: Overall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.

Rapid, sensitive, and visual detection of Clonorchis sinensis with an RPA-CRISPR/Cas12a-based dual readout portable platform.

Clonorchis sinensis is a food-borne parasite that parasitizes the liver and bile ducts of humans and many animals. This parasite exerts a high burden due to diverse hepatobiliary morbidities (e.g., cholangitis, cholecystitis, cholelithiasis, and cholangiocarcinoma), and an effective detection strategy is urgently needed. CRISPR/Cas12a exhibits nonspecific trans-cleavage activity upon binding to its specific target and has been widely used for nucleic acid detection. In this study, an RPA-CRISPR/Cas12a-based dual readout portable detection platform was established, which shows high sensitivity (one copy/µl) and specificity (no cross-reactivity with common pathogens) by rapid preamplification and combines lateral flow strips and visual fluorescence for visualization of results by the naked eye within 1 h. Moreover, 50 human fecal swabs and 50 fish flesh samples were detected by this platform and nested PCR. The CRISPR/Cas12a-based dual readout portable platform showed 10.0 % (5/50) C. sinensis-positive samples in human fecal swabs and 28.0 % (14/50) in fish flesh, which was consistent with the results of nested PCR. The results demonstrate that our portable platform has the advantages of stability, sensitivity, accuracy, and low equipment requirements. Furthermore, we provide novel point-of-care testing (POCT) for clinical use in remote rural and resource-constrained areas.

TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis.

Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) in C. sinensis infection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined in C. sinensis-infected wildtype (WT) and TLR3-/- mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treat C. sinensis infections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-ß1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviated C. sinensis-induced liver fibrosis. Furthermore, C. sinensis extracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF in TLR3-/- BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation of C. sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.

The NLRP3 inflammasome recognizes alpha-2 and alpha-7.3 giardins and decreases the pathogenicity of Giardia duodenalis in mice.

BACKGROUND: Giardia duodenalis is a parasitic organism that can cause giardiasis, an intestinal infection, particularly prevalent in young children, with clinical symptoms of diarrhea. We previously reported that extracellular G. duodenalis triggers intracellular nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs). However, the exact pathogen-associated molecular patterns in G. duodenalis EVs (GEVs) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated. METHODS: Recombinant eukaryotic expression plasmids of pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEVs were constructed, transfected into primary mouse peritoneal macrophages and screened by measuring the expression levels of the inflammasome target molecule caspase-1 p20. The preliminary identification of G. duodenalis alpha-2 and alpha-7.3 giardins was further verified by measuring the protein expression levels of key molecules of the NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1ß], pro-caspase-1, and caspase-1 p20), the secretion levels of IL-1ß, the level of apoptosis speck-like protein (ASC) oligomerization and the immunofluorescence localization of NLRP3 and ASC. The roles of the NLRP3 inflammasome in G. duodenalis pathogenicity were then evaluated using mice in which NLRP3 activation was blocked (NLRP3-blocked mice), and body weight, parasite burden in the duodenum and histopathological changes in the duodenum were monitored. In addition, we explored whether alpha-2 and alpha-7.3 giardins triggered IL-1ß secretion in vivo through the NLRP3 inflammasome and determined the roles of these molecules in G. duodenalis pathogenicity in mice. RESULTS: Alpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vitro. This led to caspase-1 p20 activation, upregulation of the protein expression levels of NLRP3, pro-IL-1ß and pro-caspase-1, significant enhancement of IL-1ß secretion, ASC speck formation in the cytoplasm and also induction of ASC oligomerization. Deletion of the NLRP3 inflammasome aggravated G. duodenalis pathogenicity in mice. Compared to wild-type mice gavaged with cysts, mice gavaged with cysts in NLRP3-blocked mice displayed increased trophozoite loads and severe duodenal villus damage, characterized by necrotic crypts with atrophy and branching. In vivo assays revealed that alpha-2 and alpha-7.3 giardins could induce IL-1ß secretion through the NLRP3 inflammasome and that immunization with alpha-2 and alpha-7.3 giardins decreased G. duodenalis pathogenicity in mice. CONCLUSIONS: Overall, the results of the present study revealed that alpha-2 and alpha-7.3 giardins trigger host NLRP3 inflammasome activation and decrease G. duodenalis infection ability in mice, which are promising targets for the prevention of giardiasis.

Cryptosporidium parvum maintains intracellular survival by activating the host cellular EGFR-PI3K/Akt signaling pathway.

Autophagy is a critical cellular mechanism in helping infected cells remove intracellular pathogens and is countered by pathogens maintaining intracellular survival by regulating autophagy through the manipulation of the host cellular signal transduction pathway. Cryptosporidium parvum is a zoonotic intracellular but extracytoplasmic protozoon that causes diarrhea in infants and young children worldwide. However, it is still unclear how Cryptosporidium adapts to intracellular survival. In the present study, we demonstrated that C. parvum could activate the EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells. The western blot results showed that C. parvum induced EGFR and Akt phosphorylation in HCT-8 cells. The EGFR inhibitor AG1478 decreased EGFR and Akt phosphorylation, and the PI3K inhibitor LY294002 impaired Akt phosphorylation induced by C. parvum in HCT-8 cells. Inhibition of EGFR or Akt decreased the number of intracellular parasites. Second, low-dose infection of C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells. The expressions of mTOR and p62 were decreased, and the expressions of LC3 and Beclin1 were increased in C. parvum-infected HCT-8 cells. Transfection with siBeclin1 or siATG7 reduced LC3 accumulation, while lysosome inhibitor E64d+pepA increased LC3 accumulation induced by C. parvum in HCT-8 cells. Intracellular parasite proliferation was decreased when treated with autophagy inducer rapamycin, whereas autophagy inhibitor 3-MA, E64d+pep A, siBeclin1 or siATG7 increased intracellular parasites. Third, C. parvum inhibited autophagy killing to promote its own intracellular survival by activating EGFR-Akt signaling pathway. The EGFR inhibitor AG1478 enhanced autophagic flux, and Akt inhibitor IV increased LC3 accumulation and inhibited C. parvum proliferation in HCT-8 cells. Akt inhibitor IV-inhibited C. parvum proliferation was attenuated by E64d+pepA. In summary, C. parvum could maintain intracellular survival by inhibiting autophagy via EGFR-PI3K/Akt pathway. These results revealed a new mechanism for the interaction of C. parvum with host cells.

Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways.

Clonorchis sinensis is an important food-borne zoonotic parasite which has been linked to biliary fibrosis and cholangiocarcinoma. However, the details of the pathogenesis of C. sinensis were unclear. To explore the role and regulatory mechanism of toll-like receptor 2 (TLR2) in C. sinensis-induced biliary fibrosis, we established the C. sinensis-infected C57BL/6 mouse model with TLR2-/- and wild type (WT) mice. The mortality rate, liver lesions, TLR2 and TGF-ß1 expression, phosphorylation of Smad2/3, AKT, p38, ERK and p65, and cytokine productions were analyzed. Furthermore, similar parameters were examined in mouse biliary epithelial cells (BECs) co-cultured with C. sinensis excretory/secretory proteins (ESPs). The results showed that TLR2 expression was enhanced significantly in C. sinensis-infected WT mice and mouse BECs. C. sinensis-infected TLR2-/- mice exhibited an increased weight and a decreased mortality rate; significantly alleviated liver lesions and biliary fibrosis, reduced numbers of myofibroblasts; decreased expression of TGF-ß1 and phosphorylation level of AKT, p38 and Smad2/3; significantly decreased production of IL-6, TNF-α and IL-4, while increased production of IFN-γ compared with C. sinensis-infected WT mice. Furthermore, C. sinensis ESPs could activate TLR2-mediated AKT and p38 pathways to increase the production of IL-6 in mouse BECs. In conclusion, these data indicate that C. sinensis infection activated TGF-ß1-Smad2/3 through TLR2-mediated AKT and p38 pathways to promote IL-6 production, which resulted in myofibroblast activation and aggravating biliary fibrosis in mice.

Antitumor effect of invasive Lactobacillus plantarum delivering associated antigen gene sHSP between Trichinella spiralis and Lewis lung cancer cells.

Cancer is a frequent disease that seriously harms human health, but there are no ideal therapies for it. Currently, some food-grade microorganisms such as Lactobacillus plantarum have shown better anti-tumor effects. Here, recombinant Lactobacillus plantarum lives vector vaccine NC8-sHSP was generated by using the invasive Lactobacillus plantarum NC8 expressing FnBPA to deliver the associated antigen gene sHSP between trichinella spiralis and Lewis lung cancer cells (LLC) to host cells. NC8-sHSP colonized the mouse intestine to deliver plasmids to intestinal epithelial cells and controlled the growth of LLC by inducing humoral, cellular, and mucosal immunity. The tumor inhibition rates were 62.36% and 68.37% in the prophylactic assay and 40.76% and 44.22% in the treatment assay, respectively. Recombination of Lactobacillus plantarum did not cause significant damage. In conclusion, the recombinant invasive Lactobacillus plantarum constructed in this study has better anti-Lewis lung cancer effects in mice, which will provide new ideas for the application of food-grade microorganisms in anti-tumor and the development of oral tumor vaccines.

Extracellular vesicles of Clonorchis sinensis promote IL-6 and TNF-α secretion via the Toll-like receptor 9-mediated ERK pathway in biliary epithelial cells.

Clonorchis sinensis is closely associated with cholangitis, cholecystitis, biliary fibrosis and cholangiocarcinoma. The present study elucidated the role of extracellular vesicles of C. sinensis (CsEVs) in activating Toll-like receptor 9 (TLR9) and regulating inflammatory responses. The results showed that TLR9 expression was increased in the livers of C. sinensis-infected mice. CsEVs were cup-shaped or saucer-shaped and 80-120 nm in diameter. CsEVs activated TLR9 and promoted IL-6 and TNF-α expression in mouse biliary epithelial cells (BECs), and TLR9 siRNA interference reduced the secretion of the two cytokines. CsEV stimulation promoted the phosphorylation of ERK, p38, AKT, and p65, and TLR9 siRNA interference regulated the phosphorylated ERK, AKT and p65 levels. The ERK inhibitor decreased the CsEVs-induced IL-6 and TNF-α secretion. The present study elucidated for the first time that CsEVs induced IL-6 and TNF-α production in BECs via the TLR9-mediated ERK pathway.

Changes of gut microbiota in colorectal cancer patients with Pentatrichomonas hominis infection.

Pentatrichomonas hominis is a parasitic trichomonads protozoa that parasitizes in the colon and cecum of humans and other animals. Our previous studies have demonstrated that infection with P. hominis is associated with the incidence of colon cancer (37.93%). However, the mechanism by which P. hominis infections increase the incidence of colon cancer remains unclear. Previous studies have suggested that certain parasites promote colon cancer by regulating gut microbiota. This study aimed to elucidate whether the association between P. hominis infections and the increased incidence of colon cancer is related to changes in gut microbiota. Therefore, the gut microbiota patients with colon cancer who were infected with P. hominis and uninfected patients with colon cancer were analyzed by 16S rRNA high-throughput sequencing. The results demonstrated that patients with colon cancer who were not infected with P. hominis showed increased gut bacterial diversity, a higher relative abundance of Alcaligenes sp., Leucobacter sp., Paraprevotella sp., Ruminococcaceae UCG-002, and a significant reduction in the abundance of Veillonella sp., compared to individuals without colon cancer. Additionally, the relative abundance of the Ruminococcaceae UCG-002 and the Eubacterium eligens groups was reduced, while the relative abundance of bacteria associated with colon cancer, including Flavonifractor sp., Lachnoclostridium sp., and the Ruminococcus gnavus group, increased significantly in patients with colon cancer who were infected with P. hominis, compared to those of uninfected patients with colon cancer. In conclusion, these results suggested that P. hominis infections may aggravate the development of colon cancer and the findings provide new insights for subsequent in-depth studies on the pathogenesis, diagnosis, and prevention of colon cancer.

Giardia lamblia regulates the production of proinflammatory cytokines through activating the NOD2-Rip2-ROS signaling pathway in mouse macrophages.

Giardia lamblia is a zoonotic protozoan that causes the diarrheal illness giardiasis, with the highest prevalence reported in the tropics and subtropics. Giardia is currently the most frequently identified pathogen in waterborne outbreaks in the United States. Nucleotide oligomerization domain (NOD) 1 and NOD2, intracellular NOD-like receptors, recognize pathogens to induce proinflammatory and antimicrobial responses. However, the roles of NOD1 and NOD2 signaling in Giardia infection have not yet been investigated. In the present study, the activation of NOD1 and NOD2 signaling pathways and the production of proinflammatory cytokines, reactive oxygen species (ROS) and nitric oxide in mouse macrophages stimulated with G. lamblia or parasite excretory-secretory products (ESPs) were examined. The results showed that G. lamblia and ESPs activated NOD2 and its downstream adaptor protein kinase, Receptor-interacting protein 2 (Rip2), in mouse macrophages. Blocking NOD2-Rip2 signaling significantly reduced the production of ROS and subsequently decreased the phosphorylation of nuclear factor-κB p65 and extracellular signal-regulated kinase, which in turn inhibited the production of four proinflammatory cytokines, namely, interleukin (IL)-1ß, IL-6, IL-12p40 and tumor necrosis factor-α. In summary, our results indicate that the NOD2-Rip2 signal, which is activated by G. lamblia, contributes to the production of proinflammatory cytokines and ROS in mouse macrophages.

Anti-osteosarcoma effect of antiserum against cross antigen TPD52 between osteosarcoma and Trichinella spiralis.

BACKGROUND: Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. METHODS: To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. RESULTS: Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 µg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. CONCLUSIONS: Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage.

Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice.

Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2-/- mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3-/- mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2-/- and TLR3-/- mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2-/- and TLR3-/- mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2-/-, and TLR3-/- mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.

Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways.

Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase-quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)-free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis-stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis-induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.

Trypanosoma evansi evades host innate immunity by releasing extracellular vesicles to activate TLR2-AKT signaling pathway.

Surra, one of the most important animal diseases with economic consequences in Asia and South America, is caused by Trypanosoma evansi. However, the mechanism of immune evasion by T. evansi has not been extensively studied. In the present study, T. evansi extracellular vesicles (TeEVs) were characterized and the role of TeEVs in T. evansi infection were examined. The results showed that T. evansi and TeEVs could activate TLR2-AKT pathway to inhibit the secretions of IL-12p40, IL-6, and TNF-α in mouse BMDMs. TLR2-/- mice and mice with a blocked AKT pathway were more resistant to T. evansi infection than wild type (WT) mice, with a significantly lower infection rate, longer survival time and less parasite load, as well as an increased secretion level of IL-12p40 and IFN-γ. Kinetoplastid membrane protein-11 (KMP-11) of TeEVs could activate AKT pathway and inhibit the productions of IL-12p40, TNF-α, and IL-6 in vitro. TeEVs and KMP-11 could inhibit the productions of IL-12p40 and IFN-γ, promote T. evansi proliferation and shorten the survival time of infected mice in vivo. In conclusion, T. evansi could escape host immune response through inhibiting the productions of inflammatory cytokines via secreting TeEVs to activate TLR2-AKT pathway. KMP-11 in TeEVs was involved in promoting T. evansi infection.Extracellular vesicles (EVs) secreted by Trypanosoma evansi (T. evansi) activate the TLR2-AKT signaling pathway to inhibit the production of inflammatory cytokines, thereby escaping the host's immune response. Kinetoplastid membrane protein-11 (KMP-11) in EVs is related to the promotion of T.evansi infection via AKT pathway.

Giardia duodenalis extracellular vesicles regulate the proinflammatory immune response in mouse macrophages in vitro via the MAPK, AKT and NF-κB pathways.

BACKGROUND: Giardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host's innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored. METHODS: In this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs. RESULTS: The results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1ß, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects. CONCLUSIONS: The results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis-host interactions.

ROS-mediated NLRP3 inflammasome activation participates in the response against Neospora caninum infection.

BACKGROUND: Neospora caninum is an obligate intracellular protozoan that causes neosporosis, N. caninum infection is a major cause of abortion in cattle worldwide. Currently, specific treatment for neosporosis is not available. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a cytoplasmic protein complex that plays an important role in host defense against N. caninum infection, but the underlying mechanisms are poorly understood. METHODS: The reactive oxygen species (ROS) inhibitor and the ROS inducer, wild-type (WT) and NLRP3-deficient peritoneal macrophages or mice were used to investigate the role of ROS in NLRP3 inflammasome activation and controlling parasite burdens. ROS production, cell death and cell viability, production of inflammasome-mediated IL-1ß or IL-18, cleavage of caspase-1 and NLRP3 expression, as well as parasite burdens were detected. RESULTS: In vitro, N. caninum induced ROS generation in a dose-dependent manner in peritoneal macrophages. The pretreatment of ROS inhibitor N-acetyl-L-cysteine (NAC) significantly attenuated N. caninum-induced ROS production, LDH release, IL-1ß secretion and NLRP3 expression, whereas N. caninum proliferation was notably increased. In contrary, the ROS inducer pyrogallol (PG) significantly enhanced ROS production and NLRP3 inflammasome activity and decreased the parasite burden in N. caninum-infected peritoneal macrophages. NADPH-dependent ROS-mediated NLRP3 inflammasome activation induced by N. caninum can also be confirmed by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). However, the NAC or DPI pre-treatment or PG treatment did not significantly alter N. caninum-induced inflammasome activities and parasite proliferation in Nlrp3-/- peritoneal macrophages. In vivo, IL-18 releases in serum and parasite burdens in peritoneal exudate cells were significantly increased in PG-treated WT mice after infection with N. caninum; however, IL-18 productions and parasite burdens were not changed in PG-treated Nlrp3-/- mice. Furthermore, PG treatment in WT mice infected with N. caninum significantly decreased the mortality, weight loss and parasite burdens in tissues and histopathological lesions. CONCLUSIONS: Neospora caninum-induced NADPH-dependent ROS generation plays an important role in NLRP3 inflammasome activation and controlling parasites. The ROS inducer PG can control N. caninum infection mainly by promoting NLRP3 inflammasome activation. ROS-mediated NLRP3 inflammasome axis can be a potential therapeutic target for neosporosis.

A Novel Anti-Microbial Peptide from Pseudomonas, REDLK Induced Growth Inhibition of Leishmania tarentolae Promastigote In Vitro.

Leishmaniasis is a prevalent cause of death and animal morbidity in underdeveloped countries of endemic area. However, there is few vaccine and effective drugs. Antimicrobial peptides are involved in the innate immune response in many organisms and are being developed as novel drugs against parasitic infections. In the present study, we synthesized a 5-amino acid peptide REDLK, which mutated the C-terminus of Pseudomonas exotoxin, to identify its effect on the Leishmania tarentolae. Promastigotes were incubated with different concentration of REDLK peptide, and the viability of parasite was assessed using MTT and Trypan blue dye. Morphologic damage of Leishmania was analyzed by light and electron microscopy. Cellular apoptosis was observed using the annexin V-FITC/PI apoptosis detection kit, mitochondrial membrane potential assay kit and flow cytometry. Our results showed that Leishmania tarentolae was susceptible to REDLK in a dose-dependent manner, disrupt the surface membrane integrity and caused parasite apoptosis. In our study, we demonstrated the leishmanicidal activity of an antimicrobial peptide REDLK from Pseudomonas aeruginosa against Leishmania tarentolae in vitro and present a foundation for further research of anti-leishmanial drugs.