Trichinella spiralis dipeptidyl peptidase 1 suppressed macrophage cytotoxicity by promoting M2 polarization via the STAT6/PPARγ pathway.

Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1), or cysteine cathepsin C, is a secretory protein that is highly expressed during the infective larvae and adult worm stages in the intestines. The aim of this study was to investigate the mechanism by which recombinant TsDPP1 (rTsDPP1) activates macrophages M2 polarization and decreases macrophage cytotoxicity to kill newborn larvae via ADCC. RAW264.7 macrophages and murine peritoneal macrophages were used in this study. The results of the immunofluorescence test (IFT) and confocal microscopy showed that rTsDPP1 specifically bound to macrophages, and the binding site was localized on the cell membrane. rTsDPP1 activated macrophage M2 polarization, as demonstrated by high expression levels of Arg1 (M2 marker) and M2-related genes (IL-10, TGF-ß, CD206 and Arg1) and high numbers of CD206+ macrophages. Furthermore, the expression levels of p-STAT6, STAT6 and PPARγ were obviously increased in rTsDPP1-treated macrophages, which were evidently abrogated by using a STAT6 inhibitor (AS1517499) and PPARγ antagonist (GW9662). The results indicated that rTsDPP1 promoted macrophage M2 polarization through the STAT6/PPARγ pathway. Griess reaction results revealed that rTsDPP1 suppressed LPS-induced NO production in macrophages. qPCR and flow cytometry results showed that rTsDPP1 downregulated the expression of FcγR I (CD64) in macrophages. The ability of ADCC to kill newborn larvae was significantly decreased in rTsDPP1-treated macrophages, but AS1517499 and GW9662 restored its killing capacity. Our results demonstrated that rTsDPP1 induced macrophage M2 polarization, upregulated the expression of anti-inflammatory cytokines, and inhibited macrophage-mediated ADCC via activation of the STAT6/PPARγ pathway, which is beneficial to the parasitism and immune evasion of this nematode.

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.

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis.

Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 µM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.

A novel C-type lectin from Trichinella spiralis mediates larval invasion of host intestinal epithelial cells.

The aim of this study was to investigate the characteristics of a novel type C lectin from Trichinella spiralis (TsCTL) and its role in larval invasion of intestinal epithelial cells (IECs). TsCTL has a carbohydrate recognition domain (CRD) of C-type lectin. The full-length TsCTL cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of qPCR, Western blotting and immunofluorescence assays (IFAs) showed that TsCTL was a surface and secretory protein that was highly expressed at the T. spiralis intestinal infective larva (IIL) stages and primarily located at the cuticle, stichosome and embryos of the parasite. rTsCTL could specifically bind with IECs, and the binding site was localized in the IEC nucleus and cytoplasm. The IFA results showed that natural TsCTL was secreted and bound to the enteral epithelium at the intestinal stage of T. spiralis infection. The rTsCTL had a haemagglutinating effect on murine erythrocytes, while mannose was able to inhibit the rTsCTL agglutinating effect for mouse erythrocytes. rTsCTL accelerated larval intrusion into the IECs, whereas anti-rTsCTL antibodies and mannose significantly impeded larval intrusion in a dose-dependent manner. The results indicated that TsCTL specifically binds to IECs and promotes larval invasion of intestinal epithelium, and it might be a potential target of vaccines against T. spiralis enteral stages.

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.

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.

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.

Molecular cloning and characterization of a novel peptidase from Trichinella spiralis and protective immunity elicited by the peptidase in BALB/c mice.

In our previous studies, a novel T. spiralis peptidase (TsP) was identified among the excretory/secretory (ES) proteins of T. spiralis intestinal infective larvae (IIL) and T. spiralis at the adult worm (AW) stage using immunoproteomics, but the biological function of TsP in the life cycle of T. spiralis is not clear. The objective of this study was to investigate the biological properties and functions of TsP in larval intrusion and protective immunity induced by immunization with rTsP. The complete TsP cDNA sequence was cloned and expressed. The results of RT-PCR, indirect immunofluorescence assay (IIFA) and western blotting revealed that TsP is a surface and secretory protein expressed in T. spiralis at different stages (muscle larvae, IIL, AWs and newborn larvae) that is principally localized at the epicuticle of the nematode. rTsP facilitated the larval intrusion of intestinal epithelial cells (IECs) and intestinal mucosa, whereas anti-rTsP antibodies suppressed larval intrusion; these facilitative and suppressive roles were dose-dependently related to rTsP or anti-rTsP antibodies. Immunization of mice with rTsP triggered an obvious humoral immune response (high levels of IgG, IgG1/IgG2a, and sIgA) and also elicited systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune responses, as demonstrated by an evident increase in the cytokines IFN-γ and IL-4. Immunization of mice with rTsP reduced the numbers of intestinal adult worms by 38.6% and muscle larvae by 41.93%. These results demonstrate that TsP plays a vital role in the intrusion, development and survival of T. spiralis in hosts and is a promising candidate target molecule for anti-Trichinella vaccines.

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.

Intranasal immunization with recombinant Trichinella spiralis serine protease elicits protective immunity in BALB/c mice.

The aim of this study was to observe the intestinal mucosal/systemic responses triggered by intranasal vaccination using recombinant Trichinella spiralis serine protease (rTsSP) and its capacity to elicit immune protection against larva challenge in a murine model. rTsSP coupled with cholera toxin B subunit (CTB) was used to vaccinate mice via intranasal route. The results revealed that intranasal vaccination with rTsSP plus CTB elicited significantly intestinal local sIgA response and a TsSP-specific systemic antibody response in vaccinated mice. Furthermore, more goblet cells/acidic mucins and IgA-secreting cells were observed in jejunum from vaccinated mice. Anti-rTsSP immune serum strongly recognized the cuticle of various worm stages (muscle larva, intestinal infective larva and adult worm). The level of IFN-γ, IL-4 and IL-10 of rTsSP-vaccinated mice was significantly elevated relative to CTB and PBS control groups. The vaccinated mice exhibited a 71.10% adult reduction at 9 days pi and a 62.10% muscle larva reduction at 42 days pi following larva challenge. Additionally, vaccination with rTsSP also dampened intestinal T. spiralis development and decreased the female fecundity. Our results showed that intranasal vaccination using rTsSP adjuvanted with CTB triggered significantly local sIgA response and systemic concurrent Th1/Th2 response that induced an obvious protection against Trichinella infection.

In vitro silencing of a serine protease inhibitor suppresses Trichinella spiralis invasion, development, and fecundity.

In a previous study, immunoproteomics was used to identify a serine protease inhibitor (TsSPI) of T. spiralis excretory/secretory (ES) proteins that exhibited an inhibitory effect on trypsin enzymatic activity, but the precise role of TsSPI on worm infection and development in its host is not well understood. The objective of the present study was to use RNA interference to ascertain the function of TsSPI in larval invasion and growth. TsSPI-specific small interference RNAs (siRNAs) were delivered to muscle larvae (ML) to silence TsSPI expression by electroporation. Four days after electroporation, the ML transfected with 2 µM siRNA-653 exhibited a 75.75% decrease in TsSPI transcription and a 69.23% decrease in TsSPI expression compared with control ML. Although the silencing of TsSPI expression did not decrease worm viability, it significantly suppressed the larval invasion of intestinal epithelium cells (IEC) (P < 0.01), and the suppression was siRNA dose-dependent (r = 0.981). The infection of mice with siRNA-653-treated ML produced a 63.71% reduction of adult worms and a 72.38% reduction of muscle larvae. In addition, the length of the adults, newborn larvae, and ML and the fecundity of female T. spiralis from mice infected with siRNA-treated ML were obviously reduced relative to those in the control siRNA or PBS groups. These results indicated that the silencing of TsSPI by RNAi suppressed larval invasion and development and decreased female fecundity, further confirming that TsSPI plays a crucial role during the T. spiralis lifecycle and is a promising molecular target for anti-Trichinella vaccines.

Oral vaccination with Trichinella spiralis DNase II DNA vaccine delivered by attenuated Salmonella induces a protective immunity in BALB/c mice.

Trichinellosis is one of the most serious foodborne parasitic zoonosis with worldwide distribution, and it is necessary to develop a vaccine to interrupt transmission from animals to humans. Trichinella spiralis adult-specific DNase II-1 (TsDNase II) were identified by immunoproteomics in surface or excretory/secretory proteins of adult worms (AW) and intestinal infective larvae (IIL). The aim of this study was to investigate the systemic, mucosal responses and immune protection elicited by oral vaccination with TsDNase II DNA vaccine delivered by attenuated Salmonella typhimurium strain⊿cyaSL1344. Oral vaccination with TsDNase II DNA vaccine triggered an obvious mucosal sIgA response and a systemic IgG response in mice, and IgG1 was predominant. Th1 (IFN-γ) and Th2 (IL-4, 10) cytokines were distinctly increased in the spleen and mesenteric lymph node (MLN) cells of vaccinated mice. An indirect immunofluorescent test revealed that native TsDNase II is present at the cuticle of this nematode after the 2nd molting, further confirming that TsDNase II is adult-specific and expressed at AW and pre-adult stages. Oral immunization of mice with TsDNase II exhibited a 53.85% reduction in AW and a 59.26% reduction in ML after larval challenge. The in vitro NBL production of adult females from TsDNase II-vaccinated mice was also reduced in comparison with pcDNA3.1 or the PBS control group (P < 0.01). Our results show that oral immunization of mice with TsDNase II produced an intestinal and systematic concurrent Th1/Th2 immune response, and a significant immune protection against challenge.

Characterization of a putative glutathione S-transferase of the parasitic nematode Trichinella spiralis.

The aim of this study was to identify the biological characteristics and functions of a putative Trichinella spiralis glutathione S-transferase (TspGST). The results of real-time PCR and immunofluorescent test (IFT) showed that the TspGST gene was expressed at all of T. spiralis different developmental stages (muscle larvae, intestinal infective larvae, adult worms and newborn larvae). When anti-rTspGST serum, mouse infection serum, and pre-immune serum were added to the medium, the inhibition rate of the larvae penetrated into the intestinal epithelial cells (IECs) was 25.72%, 49.55%, and 4.51%, respectively (P < 0.01). The inhibition of anti-rTspGST serum on larval invasion of IECs was dose-dependent (P < 0.05). Anti-rTspGST antibodies killed T. spiralis newborn larvae by an ADCC-mediated mechanism. Our results showed that the TspGST seemed to be an indispensable protein for T. spiralis invasion, growth and survival in host.

DsRNA-mediated silencing of Nudix hydrolase in Trichinella spiralis inhibits the larval invasion and survival in mice.

The aim of this study was to investigate the functions of Trichinella spiralis Nudix hydrolase (TsNd) during the larval invasion of intestinal epithelial cells (IECs), development and survival in host by RNAi. The TsNd-specific double-stranded RNA (dsRNA) was designed to silence the expression of TsNd in T. spiralis larvae. DsRNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with dsRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. After being soaked with 40 ng/µl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 65.8% and 56.4%, respectively. After being electroporated with 40 ng/µl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 74.2% and 58.2%, respectively. Silencing TsNd expression by both soaking and electroporation inhibited significantly the larval invasion of IECs in a dose-dependent manner (r1 = -0.96798, r2 = -0.98707). Compared with the mice inoculated with untreated larvae, mice inoculated with larvae soaked with TsNd dsRNA displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larvae, while mice inoculated with larvae electroporated with TsNd dsRNA displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larvae, indicating that electroporation has a higher efficiency than soaking in inhibiting the larval development and survival in mice. Our results showed that silencing TsNd expression in T. spiralis inhibited significantly the larval invasion and survival in host.

Characterization and functional analysis of Trichinella spiralis Nudix hydrolase.

Trichinella spiralis Nudix hydrolase (TsNd) was identified by screening a T7 phage display cDNA library from T. spiralis intestinal infective larvae (IIL), and vaccination of mice with recombinant TsNd protein (rTsNd) or TsNd DNA vaccine produced a partial protective immunity. The aim of this study was to identify the characteristics and biological functions of TsNd in the process of invasion and development of T. spiralis larvae. Transcription and expression of TsNd gene at all developmental stages of T. spiralis were observed by qPCR and immunofluorescent test (IFT). The rTsNd had the Nd enzymatic activity to dGTP, NAD, NADP and CoA. Its kinetic properties on the preferred substrate dGTP were calculated, and the Vmax, Km, and kcat/Km values at pH 8.0 were 3.19 µM min(-1) µg(-1), 370 µM, and 144 s(-1) M(-1), respectively, in reaction matrix containing 5 mM Zn(2+) and 2 mM DTT. The rTsNd was active from 25 °C to 50 °C, with optimal activity at 37 °C. rTsNd was able to bind specifically to mouse intestinal epithelial cells (IECs) and promoted the larval invasion of IECs, whereas anti-rTsNd antibodies inhibited the larval invasion of IECs in a dose-dependent manner. Anti-rTsNd antibodies could kill T. spiralis infective larvae by an ADCC-mediated mechanism. Our results showed that the rTsNd protein was able to interact with host IECs, had the Nudix hydrolasing activity and the enzymatic activity appeared to be essential indispensable for the T. spiralis larval invasion, development and survival in host.

Oral vaccination of mice with Trichinella spiralis nudix hydrolase DNA vaccine delivered by attenuated Salmonella elicited protective immunity.

We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity against challenge infection in mice. In this study, the full-length cDNA sequence of TsNd gene was cloned into the eukaryotic expression plasmid pcDNA3.1, and the recombinant TsNd DNA was transformed into attenuated Salmonella typhimurium strain ⊿cyaSL1344. Oral immunization of mice with TsNd/S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice upon stimulation with the rTsNd. The oral immunization of mice with TsNd/S. typhimurium displayed a statistically significant 73.32% reduction in adult worm burden and a 49.5% reduction in muscle larvae after challenge with T. spiralis muscle larvae, compared with PBS control group. Our results demonstrated that TsNd DNA delivered by attenuated live S. typhimurium elicited a local IgA response and a mixed Th1/Th2 immune response, and produced a partial protection against T. spiralis infection in mice.

The siRNA-mediated silencing of Trichinella spiralis nudix hydrolase results in reduction of larval infectivity.

Previous studies showed that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and vaccination of mice with rTsNd or TsNd DNA produced a partial protective immunity against T. spiralis infection. In this study, three TsNd specific small interfering RNA (siRNA) were designed to silence the expression of TsNd in T. spiralis larvae. SiRNAs were delivered to the larvae by electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of the larvae treated with siRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. The results showed that siRNAs were efficiently delivered into T. spiralis larvae through electroporation. Real-time PCR and Western blotting showed that transcription and expression level of TsNd gene was inhibited 73.3 and 76.7 %, respectively, after being electroporated with 2 µM of siRNA-275 for 1 day. Silencing TsNd expression inhibited significantly the larval invasion of IECs (P < 0.01) and was in a dose-dependent manner (r = -0.97941). The mice with infected larvae treated with TsNd siRNA displayed a 63.6 % reduction in intestinal adult worms and 68.8 % reduction in muscle larval burden compared with mice infected with control siRNA-treated larvae. Our results showed that silencing TsNd expression in T. spiralis significantly reduced the larval infectivity and survival in host.

Trichinella-derived protein ameliorates colitis by altering the gut microbiome and improving intestinal barrier function.

BACKGROUND: Inflammatory bowel disease (IBD) encompasses Crohn's Disease and Ulcerative Colitis. Reports have highlighted the potential use of helminths or their byproducts as a possible treatment for IBD; however, the mechanisms underlying their ability to modulate inflammation remain incompletely understood. In the present study, we analyze the possible mechanism of a serine protease inhibitor from adult T. spiralis excretion-secretion products (rTsSPI) on the improvement of colitis. METHODS: The immune protective effect of rTsSPI was studied by using DSS or Salmonella-induced colitis in female C56BL/6 mice. The effect of rTsSPI on the immune and inflammatory responses, gut microbiota, permeability of colon epithelium and junction proteins was analyzed. RESULTS: Treating mice with rTsSPI induced type 2 immunity and significantly attenuated clinical symptoms, macroscopical and histological features of DSS or bacteria-induced colonic inflammation. This was accompanied by decreasing neutrophil recruitment in the colonic lamina propria, and reducing TNF-α mRNA levels in the colon; in contrast, the recruitment of M2 macrophages, the expression level of IL-10 and adhesion molecules increased in the colon tissue. Moreover, treatment with rTsSPI led to an improvement in gut microbiota diversity, as well as an increase in the abundance of the bacterial genera Bifidobacterium and Ruminclostridium 5. CONCLUSIONS: Collective findings suggest that pretreatment with rTsSPI can ameliorate colitis in mice by inducing a Th2-type response with M2 macrophages. Data also indicate that immunotherapy with rTsSPI represents an additional strategy to ameliorate inflammatory processes in IBD by enhancing probiotic colonization and maintaining intestinal epithelial barrier function.

Characterization of a novel dipeptidyl peptidase 1 of Trichinella spiralis and its participation in larval invasion.

The research aimed to describe a new Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1) and investigate its functions in the larval invasion of intestinal epithelial cells (IECs). The gene TsDPP1 was successfully replicated and produced in Escherichia coli BL21 (DE3), showing a strong immune response. TsDPP1 was detected in diverse stages of T. spiralis and showed significant expression in the intestine infective larvae (IIL) and adult worms at 6 days post infection, as confirmed by qPCR and Western blot analysis. The primary localization of TsDPP1 in this parasite was observed in cuticles, stichosomes, and embryos by using the indirect immunofluorescence assay (IIFA). rTsDPP1 exhibited the enzymatic function of natural dipeptidyl peptidase and showed specific binding to IECs, and the binding site was found to be localized on cell membrane. Following transfection with dsRNA-TsDPP1, the expression of TsDPP1 mRNA and protein in muscle larvae (ML) were decreased by approximately 63.52 % and 58.68 %, correspondingly. The activity of TsDPP1 in the ML and IIL treated with dsRNA-TsDPP1 was reduced by 42.98 % and 45.07 %, respectively. The acceleration of larval invasion of IECs was observed with rTsDPP1, while the invasion was suppressed by anti-rTsDPP1 serum. The ability of the larvae treated with dsRNA-TsDPP1 to invade IECs was hindered by 31.23 %. In mice infected with dsRNA-treated ML, the intestinal IIL, and adults experienced a significant decrease in worm burdens and a noticeable reduction in adult female length and fecundity compared to the PBS group. These findings indicated that TsDPP1 significantly impedes the invasion, growth, and reproductive capacity of T. spiralis in intestines, suggesting its potential as a target for anti-Trichinella vaccines.

Galactomannan inhibits Trichinella spiralis invasion of intestinal epithelium cells and enhances antibody-dependent cellular cytotoxicity related killing of larvae by driving macrophage polarization.

Previous studies have shown that recombinant Trichinella spiralis galectin (rTsgal) is characterized by a carbohydrate recognition domain sequence motif binding to beta-galactoside, and that rTsgal promotes larval invasion of intestinal epithelial cells. Galactomannan is an immunostimulatory polysaccharide composed of a mannan backbone with galactose residues. The aim of this study was to investigate whether galactomannan inhibits larval intrusion of intestinal epithelial cells and enhances antibody-dependent cellular cytotoxicity (ADCC), killing newborn larvae by polarizing macrophages to the M1 phenotype. The results showed that galactomannan specially binds to rTsgal, and abrogated rTsgal facilitation of larval invasion of intestinal epithelial cells. The results of qPCR, Western blotting, and flow cytometry showed that galactomannan and rTsgal activated macrophage M1 polarization, as demonstrated by high expression of iNOS (M1 marker) and M1 related genes (IL-1ß, IL-6, and TNF-α), and increased CD86+ macrophages. Galactomannan and rTsgal also increased NO production. The killing ability of macrophage-mediated ADCC on larvae was also significantly enhanced in galactomannan- and rTsgal-treated macrophages. The results demonstrated that Tsgal may be considered a potential vaccine target molecule against T. spiralis invasion, and galactomannan may be a novel adjuvant therapeutic agent and potential vaccine adjuvant against T. spiralis infection.

Title: Le galactomannane inhibe l'invasion par Trichinella spiralis des cellules de l'épithélium intestinal et améliore la cytotoxicité cellulaire dépendante des anticorps tuant les larves en activant la polarisation des macrophages. Abstract: Des études antérieures ont montré que la galectine recombinante de Trichinella spiralis (rTsgal) est caractérisée par un motif de séquence de domaines de reconnaissance des glucides se liant au bêta-galactoside, et que la rTsgal favorise l'invasion larvaire des cellules épithéliales intestinales. Le galactomannane est un polysaccharide immunostimulateur composé d'un squelette mannane avec des résidus galactose. Le but de cette étude était de déterminer si le galactomannane inhibe l'intrusion larvaire des cellules épithéliales intestinales et améliore la cytotoxicité cellulaire dépendante des anticorps (CCDA) tuant les larves nouvelles-nées en polarisant les macrophages au phénotype M1. Les résultats ont montré que le galactomannane se liait spécialement au rTsgal et supprimait la facilitation du rTsgal sur l'invasion larvaire des cellules épithéliales intestinales. Les résultats de la qPCR, du Western blot et de la cytométrie en flux ont montré que le galactomannane et le rTsgal activaient la polarisation des macrophages M1, comme le démontre la forte expression de l'iNOS (marqueur de M1) et des gènes liés à M1 (IL-1ß, IL-6 et TNF-α), et l'augmentation des macrophages CD86+. Le galactomannane et le rTsgal ont également augmenté la production de NO. La capacité de destruction de la CCDA médiée par les macrophages sur les larves était également significativement améliorée dans les macrophages traités au galactomannane et au rTsgal. Les résultats ont démontré que Tsgal pourrait être considéré comme une molécule cible potentielle d'un vaccin contre l'invasion par T. spiralis, et que le galactomannane pourrait être un nouvel agent thérapeutique adjuvant et un adjuvant vaccinal potentiel contre l'infection à T. spiralis.