Regulatory effects of Trichinella spiralis serpin-type serine protease inhibitor on endoplasmic reticulum stress and oxidative stress in host intestinal epithelial cells.

Endoplasmic reticulum stress (ERS) and oxidative stress (OS) are adaptive responses of the body to stressor stimulation. Although it has been verified that Trichinella spiralis (T. spiralis) can induce ERS and OS in the host, their association is still unclear. Therefore, this study explored whether T. spiralis-secreted serpin-type serine protease inhibitor (TsAdSPI) is involved in regulating the relationship between ERS and OS in the host intestine. In this study, mice jejunum and porcine small intestinal epithelial cells (IECs) were detected using qPCR, western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and detection kits. The results showed that ERS- and OS-related indexes changed significantly after TsAdSPI stimulation, and Bip was located in IECs, indicating that TsAdSPI could induce ERS and OS in IECs. After the use of an ERS inhibitor, OS-related indexes were inhibited, suggesting that TsAdSPI-induced OS depends on ERS. When the three ERS signalling pathways, ATF6, IRE1, and PERK, were sequentially suppressed, OS was only regulated by the PERK pathway, and the PERK-eif2α-CHOP-ERO1α axis played a key role. Similarly, the expression of ERS-related indexes and the level of intracellular Ca2+ were inhibited after adding the OS inhibitor, and the expression of ERS-related indexes decreased significantly after inhibiting calcium transfer. This finding indicated that TsAdSPI-induced OS could affect ERS by promoting Ca2+ efflux from the endoplasmic reticulum. The detection of the ERS and OS sequences revealed that OS occurred before ERS. Finally, changes in apoptosis-related indexes were detected, and the results indicated that TsAdSPI-induced ERS and OS could regulate IEC apoptosis. In conclusion, TsAdSPI induced OS after entering IECs, OS promoted ERS by enhancing Ca2+ efflux, and ERS subsequently strengthened OS by activating the PERK-eif2α-CHOP-ERO1α axis. ERS and OS induced by TsAdSPI synergistically promoted IEC apoptosis. This study provides a foundation for exploring the invasion mechanism of T. spiralis and the pathogenesis of host intestinal dysfunction after invasion.

Trichinella spiralis galectin binding to toll-like receptor 4 induces intestinal inflammation and mediates larval invasion of gut mucosa.

Previous studies showed that Trichinella spiralis galectin (Tsgal) facilitates larval invasion of intestinal epithelium cells (IECs). However, IEC proteins binding with Tsgal were not identified, and the mechanism by which Tsgal promotes larval invasion is not clear. Toll-like receptors (TLRs) are protein receptors responsible for recognition of pathogens. The aim of this study was to investigate whether recombinant Tsgal (rTsgal) binds to TLR-4, activates inflammatory pathway in gut epithelium and mediates T. spiralis invasion. Indirect immunofluorescence (IIF), GST pull-down and co-immunoprecipitation (Co-IP) assays confirmed specific binding between rTsgal and TLR-4 in Caco-2 cells. qPCR and Western blotting showed that binding of rTsgal with TLR-4 up-regulated the TLR-4 transcription and expression in Caco-2 cells, and activated p-NF-κB p65 and p-ERK1/2. Activation of inflammatory pathway TLR-4/MAPK-NF-κB by rTsgal up-regulated pro-inflammatory cytokines (IL-1ß and IL-6) and down-regulated anti-inflammatory cytokine TGF-ß in Caco-2 cells, and induced intestinal inflammation. TAK-242 (TLR-4 inhibitor) and PDTC (NF-κB inhibitor) significantly inhibited the activation of TLR-4 and MAPK-NF-κB pathway. Moreover, the two inhibitors also inhibited IL-1ß and IL-6 expression, and increased TGF-ß expression in Caco-2 cells. In T. spiralis infected mice, the two inhibitors also inhibited the activation of TLR-4/MAPK-NF-κB pathway, ameliorated intestinal inflammation, impeded larval invasion of gut mucosa and reduced intestinal adult burdens. The results showed that rTsgal binding to TLR-4 in gut epithelium activated MAPK-NF-κB signaling pathway, induced the expression of TLR-4 and pro-inflammatory cytokines, and mediated larval invasion. Tsgal might be regarded as a candidate molecular target of vaccine against T. spiralis enteral invasive stage.

Binding of Trichinella spiralis C-type lectin with syndecan-1 on intestinal epithelial cells mediates larval invasion of intestinal epithelium.

C-type lectin (CTL) is a protein that binds to saccharides and plays an important role in parasite adhesion, host cell invasion and immune evasion. Previous studies showed that recombinant T. spiralis C-type lectin (rTsCTL) promotes larval invasion of intestinal epithelium cells (IEC), whereas anti-rTsCTL antibodies inhibits larval invasion. Syndecan-1 (SDC-1) is a member of the heparan sulfate proteoglycan family which is mainly expressed on the surface of IEC and in extracellular matrices where they interact with a plethora of ligands. SDC-1 has a principal role in maintaining cell morphogenesis, establishing cell-cell adhesions, and regulating the gut mucosal barrier. The aim of this study was to investigate whether rTsCTL binds to SDC-1 on IEC, and the binding of rTsCTL with SDC-1 promotes larval invasion and its mechanism. IFA results show that rTsCTL and SDC-1 co-localized on Caco-2 cell membrane. GST pull-down and Co-IP verified the direct interaction between rTsCTL and SDC-1 on Caco-2 cells. qPCR and Western blotting revealed that rTsCTL binding to SDC-1 increased the expression of SDC-1 and claudin-2, and reduced the expression of occludin and claudin-1 in Caco-2 cells incubated with rTsCTL via the STAT3 pathway. ß-Xyloside (a syndecan-1 synthesis inhibitor) and Stattic (a STAT3 inhibitor) significantly inhibited rTsCTL binding to syndecan-1 in Caco-2 cells and activation of the STAT3 pathway, abrogated the effects of rTsCTL on the expression of gut tight junctions, and impeded larval invasion. The results demonstrate that binding of rTsCTL to SDC-1 on Caco-2 cells activated the STAT3 pathway, decreased gut tight junction expression, damaged the integrity of the gut epithelial barrier, and mediated T. spiralis invasion of the gut mucosa. TsCTL might be regarded as a candidate vaccine target against T. spiralis invasion and infection.

Assessment of the potential prophylactic and therapeutic effects of kaempferol on experimental Trichinella spiralis infection.

Currently, no effective treatment is available for trichinellosis, a zoonotic parasitic disease caused by infection with the genus Trichinella. Kaempferol (KPF), a dietary flavonoid, has been documented to have anti-parasitic effects and various medicinal uses. Thus, this study aimed to investigate the efficacy of KPF in preventing and treating the intestinal and muscular phases of trichinellosis in mice compared with albendazole (ABZ). To achieve this, mice were divided into six groups: negative control; positive control; KPF prophylaxis; KPF treatment; ABZ treatment; and a combination of ABZ and KPF. Parasitological, histopathological and immunohistochemical evaluations were conducted to assess the effectiveness of the treatments. The parasitological assessment involved counting small intestinal adult worms and encysted muscle larvae. Additionally, the histopathological evaluation used the haematoxylin and eosin staining method for intestinal and muscular sections and picrosirius red stain for muscular sections. Moreover, the immunohistochemical expression of the intestinal NOD-like receptor-pyrin domain containing 3 (NLRP3) was evaluated. The group treated with combined drugs demonstrated a statistically significant reduction in the count of adults and encysted larvae (P < 0.05), a remarkable improvement in the inflammation of the intestines and muscles and a decrease in the thickness of the larvae's capsular layer. Additionally, the highest reduction in NLRP3 expression was observed in this group. Based on this study, KPF shows promise as an anti-trichinellosis medication that, when taken with ABZ, has a synergistic impact by modulating inflammation and larval capsule formation.

Proteases secreted by Trichinella spiralis intestinal infective larvae damage the junctions of the intestinal epithelial cell monolayer and mediate larval invasion.

The intestinal epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism of larval invasion of the gut epithelium is not fully elucidated. The aim of this study was to investigate whether the excretory/secretory proteins (ESPs) of T. spiralis intestinal infective larvae (IIL) degrade tight junction (TJ) proteins, to assess the main ESP proteases hydrolysing TJ proteins using various enzyme inhibitors and to define the key invasive factors in IIL invasion of the gut epithelium. The results of immunofluorescence, Western blot and Transwell assays showed that serine proteases and cysteine proteases in the ESPs played main roles in hydrolysing occludin, claudin-1 and E-cad and upregulating claudin-2 expression. Challenge infection results showed that IIL expulsion from the gut at 12 hpi was significantly higher in mice which were infected with muscle larvae (ML) treated with a single inhibitor (PMSF, E-64, 1,10-Phe or pepstatin) or various mixtures containing PMSF and E-64 than in mice in the PBS group or the groups treated with an inhibitor mixture not containing PMSF and E-64 (P < 0.0001). At 6 days post-infection, mice which were infected with ML treated with PMSF, E-64, 1,10-Phe or pepstatin exhibited 56.30, 64.91, 26.42 and 31.85% reductions in intestinal adult worms compared to mice in the PBS group (P < 0.0001). The results indicate that serine proteases and cysteine proteases play key roles in T. spiralis IIL invasion, growth and survival in the host and that they may be main candidate target molecules for vaccines against larval invasion and development.

Effects of exosomes derived from Trichinella spiralis infective larvae on intestinal epithelial barrier function.

Muscle larvae of Trichinella spiralis parasitize the host intestinal epithelium. The mechanisms of exosomes participating in the invasion of T. spiralis muscle larvae are unclear. Hence, the purpose of this study was to explore the effect of exosomes derived from T. spiralis infective larvae (TsExos) on the barrier function of porcine small intestinal epithelial cells (IPEC-J2). First, TsExos were successfully obtained, and their ingestion by epithelial cells was validated. Furthermore, the optimal induction condition was determined by the CCK8 kit, and we found that exposure to 150 µg/mL TsExos for 12/24 h decreased the viability of IPEC-J2 cells by 30%. Based on this outcome, the effects of TsExos on cell biological processes and tight junctions were studied. After coincubation of TsExos and IPEC-J2 cells, the results showed a significant increase in the content of FITC-dextran and in the levels of lactate dehydrogenase (LDH) and reactive oxygen species (ROS). The rate of apoptosis increased by 12.57%, and nuclear pyknosis and nuclear rupture were observed. After the cells were induced by TsExos, the expression of IL-1 was upregulated, but the expression of IL-10, TGF-ß, TLR-5, MUC-1 and MUC-2 was downregulated. TsExo induction also led to a decrease in the levels of ZO-1, CLDN-3, and OCLN. In conclusion, TsExos are involved in several cellular biological processes, and they function by disrupting physiological and biochemical processes, hyperactivating innate immunity, and damaging tight junctions.

Modulation of lymphocyte subpopulations in the small intestine of mice treated with probiotic bacterial strains and infected with Trichinella spiralis.

AIMS: To study the local intestinal lymphocyte immunity in mice with trichinellosis affected by probiotic bacteria. METHODS AND RESULTS: Enterococcus faecium CCM8558, Enterococcus durans ED26E/7, Limosilactobacillus fermentum CCM7421 and Lactiplantibacillus plantarum 17 L/1 were administered daily (109  CFU ml-1 ) and mice were infected with Trichinella spiralis (400 larvae) on the 7th day of treatment. T. spiralis infection significantly inhibited lymphocyte subpopulations from 5 to 25 days postinfection (dpi). L. fermentum CCM7421 and L. plantarum 17 L/1 restored the CD4+ T cell numbers in the epithelium and lamina propria at the control level from 11 dpi. All strains stimulated the CD8+ T cells numbers in infected mice, which were restored in the lamina propria on 11 dpi and in the epithelium only on 32 dpi. B cells (CD19+ ) inhibition after T. spiralis infection was not affected by treatment till 25 dpi. CONCLUSIONS: The strain-specific immunomodulatory effect of tested bacteria was confirmed. L. fermentum CCM7421 and L. plantarum 17 L/1 showed the greatest immunomodulatory potential on CD4+ and CD8+ T lymphocytes in trichinellosis. E. faecium CCM8558 and E. durans ED26E/7 activated only CD8+ T cells in the lamina propria. SIGNIFICANCE AND IMPACT OF THE STUDY: Positive modulation of the gut lymphocyte immunity in T. spiralis infection with bacterial strains showed their beneficial effect with the host's antiparasitic defence.

Biological properties and roles of a Trichinella spiralis inorganic pyrophosphatase in molting and developmental process of intestinal larval stages.

Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg2+, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.

RNAi-mediated silencing of Trichinella spiralis glutaminase results in reduced muscle larval infectivity.

Trichinella spiralis is an important foodborne parasitic nematode distributed worldwide that infects humans and animals. Glutaminase (GLS) is an important gene in the glutamine-dependent acid resistance (AR) system; however, its role in T. spiralis muscle larvae (ML) remains unclear. The present study aimed to characterize T. spiralis GLS (TsGLS) and assess its function in T. spiralis ML AR both in vitro and in vivo using RNA interference. The results indicated that native TsGLS (72 kDa) was recognized by anti-rTsGLS serum at the muscle larvae stage; moreover, an immunofluorescence assay confirmed that TsGLS was located in the epidermis of ML. After silencing the TsGLS gene, the relative expression of TsGLS mRNA and the survival rate of T. spiralis ML were reduced by 60.11% and 16.55%, respectively, compared to those in the PBS and control groups. In vivo AR assays revealed that the worm numbers at 7 and 35 days post-infection (dpi) decreased by 61.64% and 66.71%, respectively, compared to those in the PBS group. The relative expression of TsGLS mRNA in F1 generation T. spiralis ML was reduced by 42.52%, compared to that in the PBS group. To the best of our knowledge, this is the first study to report the presence of the glutamine-dependent AR system in T. spiralis. Our results indicate that TsGLS plays a crucial role in the T. spiralis AR system; thus, it could be used as a potential candidate target molecule for producing vaccines against T. spiralis infection.

Characterization of a novel cysteine protease in Trichinella spiralis and its role in larval intrusion, development and fecundity.

The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in Trichinella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 peptidase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofluorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifically bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specific dsRNA, TsGSCP expression and enzymatic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were significantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection.

Regulation of human THP-1 macrophage polarization by Trichinella spiralis.

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1ß and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1ß and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1ß, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

Niosomal versus nano-crystalline ivermectin against different stages of Trichinella spiralis infection in mice.

Ivermectin (IVM) is one of the competitive treatments used for trichinellosis. However, several studies linked its efficacy with early diagnosis and administration to tackle the intestinal phase with limited activity being recorded against encysted larvae. The aim of this study was to employ niosomes for enhancing effectiveness of oral IVM against different stages of Trichinella spiralis (T. spiralis) infection with reference to nano-crystalline IVM. Mice were randomized into four groups: group Ι, 15 uninfected controls; group ΙΙ, 30 infected untreated controls; group ΙΙΙ, 30 infected nano-crystalline IVM treated, and group ΙV, 30 infected niosomal IVM treated. All groups were equally subdivided into 3 subgroups; (a) treated on the 1st day post infection (dpi), (b) treated on the 10th dpi, and (c) treated on the 30th dpi. Assessment was done by counting adult worms and larvae plus histopathological examination of jejunum and diaphragm. Biochemical assessment of oxidant/antioxidant status, angiogenic, and inflammatory biomarkers in intestinal and muscle tissues was also performed. Both niosomes and nano-crystals resulted in significant reduction in adult and larval counts compared to the infected untreated control with superior activity of niosomal IVM. The superiority of niosomes was expressed further by reduction of inflammation in both jejunal and muscle homogenates. Biochemical parameters showed highly significant differences in all treated mice compared to infected untreated control at different stages with highly significant effect of niosomal IVM. In conclusion, niosomal IVM efficacy exceeded the nano-crystalline IVM in treatment of different phases of trichinellosis.

Comparative expression profiles of host circulating miRNAs in response to Trichinella spiralis infection.

Trichinellosis is an important food-borne parasitic zoonosis throughout the world. At present, the mechanisms of Trichinella spiralis infection remain unclear. Acquiring detailed information on the host-Trichinella interaction would be beneficial for the development of new strategies for trichinellosis control. Circulating miRNAs are stably detectable in the blood of humans and animals infected with parasites. Circulating miRNAs might regulate the expression of target genes in pathological responses during infection and might be novel potential biomarkers of parasitic diseases. In the present study, a total of ten differentially expressed circulating mouse miRNAs with |log2(fold change)| ≥ 1.0 and FDR < 0.01 were found during T. spiralis infection, of which five were upregulated and five were downregulated. GO and KEGG analyses showed that the target genes of the ten miRNAs were enriched in many signalling pathways, especially focal adhesion, MAPK pathway, and so on. The results of qRT-PCR showed that among the five upregulated miRNAs, mmu-miR-467a-3p and mmu-miR-467d-3p expression in mouse serum reached a peak at 30 days post-infection (dpi). The expression of mmu-miR-376b-3p and mmu-miR-664-3p increased significantly at 18 dpi and then decreased at 30 dpi. The expression of mmu-miR-292a-5p gradually decreased from 12 to 30 dpi. Among the 5 downregulated miRNAs, mmu-miR-199a-5p expression was significantly downregulated at 30 dpi, while the expression levels of the other four miRNAs (mmu-miR-455-5p, mmu-miR-125b-5p, mmu-miR-125a-5p, and mmu-miR-615-3p) were significantly lower compared with the control, showing a steady downregulation at different phases of infection. These findings will help to further understand the host-Trichinella interaction and provide promising serum biomarkers for trichinellosis.

Primary characterization of the immune response in pigs infected with Trichinella spiralis.

Trichinellosis, which is caused by Trichinella spiralis (T. spiralis), is a serious zoonosis. Pigs play an important role in the transmission of human trichinellosis. Characterizing the immune response to T. spiralis infection is key to elucidating host-parasite interactions. However, most studies on the immune response to T. spiralis infection have employed murine models. In this study, we investigated the immune response to T. spiralis infection in pigs. The results showed that the average numbers of larvae per gram (lpg) for the 100-muscle larvae (ML), 1000-ML, and 10 000-ML groups were 1.502, 35.947, and 398.811, respectively. The percentages of CD3+ T cells, B cells, CD4+ T cells, Treg cells, and Th17 cells were elevated in the infection groups compared to the control animals. In contrast, CD8+ T cell percentages were reduced after infection in the low-dose group. The number of neutrophils was increased at 3-17 days post-infection (dpi). Th1 cytokine IL-2 levels were significantly decreased at 7 dpi, and Th2 cytokine IL-4 levels were significantly elevated at 3 dpi. Treg cytokine IL-10 levels were significantly elevated between 7 dpi and 30 dpi. Th17 cytokine IL-17A levels were significantly increased beginning at 11 dpi. These results confirmed that pigs infected with T. spiralis predominantly induced Th2 and Treg immune responses, which suppress the Th1 immune responses. This study provides novel insights into the immune response of pigs infected with T. spiralis.

The cysteine protease ATG4B of Trichinella spiralis promotes larval invasion into the intestine of the host.

The cysteine proteases of parasites are vital contributors that induce parasite migration to and invasion of host tissue. In this study, we analysed the cysteine protease ATG4B of Trichinella spiralis (TsATG4B) isolated from the soluble proteins of Trichinella spiralis (T. spiralis) adult worms to ascertain its biochemical properties and functions during invasion into the intestine of the host. The 43 kDa recombinant cysteine protease ATG4B protein (rTsATG4B) consists of a conserved peptidase_C54 domain and was expressed in Escherichia coli. Gelatine zymography showed that rTsATG4B could hydrolyse gelatine and that the hydrolytic activity was prevented by the cysteine protease inhibitor E-64 (pH 5.2). Immunofluorescence assays showed that TsATG4B is expressed at different stages and is localized at the cuticles and stichosomes of worms. Far-Western blotting and confocal microscopy revealed that rTsATG4B interacts with intestinal epithelial cells (IECs) and that it was subcellularly localized to the membrane and cytoplasm in IECs. Real­time quantitative PCR (qPCR) results indicated that the transcription level of the TsATG4B gene was the higher in 6-day-old adult worms (6 days AW) than in any other stage. An in vitro larval invasion assay verified that rTsATG4B promoted larval invasion and that invasion was inhibited when rTsATG4B was pre-incubated with E-64, whereas anti-rTsATG4B serum inhibited larval invasion in a dose-dependent manner. Collectively, these results suggested that the enzymatic activity of TsATG4B significantly influences the hydrolysis process, which is necessary for larval invasion of the host intestinal epithelium.

Characterization of a Trichinella spiralis aminopeptidase and its participation in invasion, development and fecundity.

A Trichinella spiralis aminopeptidase (TsAP) has been identified in intestinal infectious larvae (IIL) and adult worms (AW), but its biological function in the T. spiralis life cycle is unknown. The aim of this study was to characterize TsAP and ascertain its functions in the invasion, development and fecundity of T. spiralis. Recombinant TsAP (rTsAP) was expressed and purified. rTsAP has strong immunogenicity. qPCR and western blotting show that TsAP was transcribed and expressed at all T. spiralis lifecycle stages, but the expression level of TsAP mRNA and proteins at IIL and AW stages was obviously higher than those in muscle larvae (ML) and newborn larvae (NBL). The IFT results reveal that TsAP was principally located at the cuticle and the intrauterine embryos of this nematode. rTsAP had the enzymatic activity of natural aminopeptidase to hydrolyze the substrate Leu-pNA with an optimal temperature of 50 °C and optimal pH of 8.0. rTsAP promoted the larval penetration into intestinal epithelial cells, whereas anti-rTsAP antibodies suppressed the larval intrusion; the promotion and suppression was dose-dependently related to rTsAP or anti-rTsAP antibodies. TsAP protein expression level and enzymatic activity were reduced by 50.90 and 49.72% through silencing of the TsAP gene by specific siRNA 842. Intestinal AW and muscle larval burdens, worm length and female reproductive capacity were significantly declined in mice infected with siRNA-transfected ML compared to the control siRNA and PBS group. These results indicate that TsAP participates in the invasion, development and fecundity of T. spiralis and it might be a candidate target for anti-Trichinella vaccines.

NLRP3 played a role in Trichinella spiralis-triggered Th2 and regulatory T cells response.

Trichinella spiralis maintains chronic infections within its host. Muscle larvae excretory-secretory products (MLES) typically induce parasite-specific immune responses such as the Th2 response and regulatory T cells (Tregs) by modulating dendritic cell (DC) phenotype via the recognition of pattern recognition receptors (PRRs), such as Nod-like receptors (NLRs). We aimed to investigate the role of NLRP3 in T. spiralis-triggered immune response. We found that larvae burden was increased in NLRP3-/- mice compared to wild type (WT) mice. Administration of MLES induced higher levels of IL-4, IL-10, TGF-ß and population of Tregs in WT mice than in NLRP3-/- mice. In vitro, we showed that increased expression of CD40 on the surface of MLES-treated DCs was inhibited after NLRP3 knockout. Increased production of IL-1ß, IL-18, IL-10 and TGF-ß, but not IL-12p70, was significantly diminished in the absence of NLRP3. Furthermore, our results demonstrated that MLES-treated DCs induced higher levels of IL-4, IL-10 and TGF-ß and populations of Tregs in vitro. These inductions were abolished by NLRP3 deficiency in DCs, suggesting that NLRP3 in MLES-treated DCs plays a role in promoting the Th2 and Treg response. Taken together, we identified for the first time the involvement of NLRP3 in host defences against T. spiralis.

RNAi-mediated silencing of Trichinella spiralis serpin-type serine protease inhibitors results in a reduction in larval infectivity.

Trichinella spiralis serpin-type serine protease inhibitors (TsSPIs) are expressed in adult worms (AW), newborn larvae (NBL) and muscle larvae (ML) of T. spiralis, with the ML stage demonstrating the highest expression level. This study aims to determine TsSPI functions in larval viability and invasion of intestinal epithelial cells in vitro, as well as their development, survival, and fecundity in vivo via RNAi. TsSPI-specific siRNAs and dsRNA were transfected into ML by incubation. The silencing effect of TsSPI transcription and expression was determined using qPCR and western blot, respectively. After incubation in 60 ng/µL dsRNA-TsSPI for 3 days, larval TsSPI mRNA and protein expression levels were reduced by 68.7% and 68.4% (P < 0.05), respectively. dsRNA-mediated silencing of TsSPI significantly impacted larval invasion into intestinal epithelial cells in vitro but did not affect the survival rate of larvae. After challenge with dsRNA-TsSPI-treated ML, mice exhibited a 56.0% reduction in intestinal AW burden and 56.9% reduction in ML burden (P < 0.05), but NBL production of female AW remained the same (P > 0.05). Our results revealed that RNAi-mediated silencing of TsSPI expression in T. spiralis significantly reduced larval infectivity and survival in the host but had no effect on the survival rate and fecundity. Furthermore, TsSPIs have no effect on the growth and reproduction of parasites but may be directly involved in regulating the interaction of T. spiralis and the host. Therefore, TsSPIs are crucial in the process of T. spiralis larval invasion and parasite survival in the host.

Protective immunity in mice vaccinated with a novel elastase-1 significantly decreases Trichinella spiralis fecundity and infection.

Trichinella spiralis is an important foodborne parasitic nematode that represents an enormous threat to the food safety of pork meat. The development of a preventive vaccine is valuable for the prevention and control of Trichinella infection in domestic pigs to ensure pork safety. Elastase is a trypsin-like serine protease that hydrolyzes the host's diverse tissue components and participates in parasite penetration, and it might be a novel vaccine target molecule. The aim of this study was to assess the protective immunity produced by vaccination with a novel Trichinella spiralis elastase-1 (TsE) in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsE elicited a systemic humoral response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and significant local enteral mucosal sIgA responses. Anti-rTsE IgG recognized the native TsE at the cuticle, stichosome of intestinal infective larvae and adult worm (AW), and intrauterine embryos of female AW. The rTsE vaccination also produced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10) after spleen, mesenteric lymph node and Peyer's patch cells from immunized mice were stimulated with rTsE. The immunized mice exhibited a 52.19% reduction in enteral AW and a 64.06% reduction in muscle larvae after challenge infection. The immune response triggered by rTsE vaccination protected enteral mucosa from larval intrusion, suppressed larval development and reduced female fecundity. The results indicate that TsE may represent a novel target molecule for anti-T. spiralis vaccines.

Effects of TLR agonists on immune responses in Trichinella spiralis infected mice.

Human trichinellosis is acquired by eating raw or undercooked meats carrying muscle larvae of Trichinella spp. Toll-like receptors (TLRs) are essential components of the innate immune system. However, little is known about the potential application of TLR agonists for immunotherapy against Trichinella spiralis (T. spiralis) infection. Here, we evaluated the effects of four TLR agonists (i.e., TLR3, TLR4, TLR8, and TLR9 agonists) on T. spiralis infection in mice. The reduction rate of worm burden showed that TLR3 agonist poly(I:C) significantly reduced T. spiralis infection rather than TLR4, TLR8, and TLR9 agonists (p < 0.05). Moreover, TLR3 showed a continuous high-level of expression during 6-35 days post infection (dpi). The levels of interferon-gamma (IFN-γ), interleukin (IL)-2, and IL-6 increased significantly in mice serum compared with control group after treatment with TLR3 agonist at 0, 3, 6, 9, 12, 15, 18, 21, 28, and 35 dpi (p < 0.05). A significant decreasing trend was also detected in levels of IL-10 and IL-4 after treatment with TLR3 agonist compared with control group at 0, 3, 6, 9, 12, 15, 18, 21, 28, and 35 dpi (p < 0.05). Overall, this study suggested that TLR3-targeted therapies might be effective on worm burden reduction by regulation of the cytokine levels in the mice infected with T. spiralis.