Effects of Trichinella spiralis and its serine protease inhibitors on intestinal mucosal barrier function

doi:10.21203/rs.3.rs-4860882/v1

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Effects of Trichinella spiralis and its serine protease inhibitors on intestinal mucosal barrier function

https://doi.org/10.21203/rs.3.rs-4860882/v1

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T. spiralis is a highly pathogenic zoonotic nematode that poses significant public health risks and causes substantial economic losses. Understanding its invasion mechanisms is crucial. This study explored the role of serine protease inhibitors (SPIs) secreted by T. spiralis in disrupting the host intestinal epithelial barrier.

The effects of T. spiralis infection on the jejunal barrier function in mice were investigated. Histopathological analysis showed significant jejunal damage, peaking at day 7 post-infection (dpi). RT-qPCR and Western blotting revealed marked reductions in tight junction proteins (ZO-1, Occludin, Claudin-3), mucins (MUC-1, MUC-2), and anti-inflammatory cytokines (TGF-β, IL-10) from 1 to 15 dpi, along with increased expression of Toll-like receptors (TLR-1, TLR-2, TLR-4) and pro-inflammatory cytokines (TNF-α, IL-1β). Recombinant SPIs (rKaSPI, rAdSPI) were purified and co-cultured with intestinal epithelial cells (IPECs) and used in mouse models. The protein expression changes in IPECs and mice were consistent with those in T. spiralis-infected tissues. Both SPIs downregulated ZO-1, Occludin, Claudin-3, MUC-1, MUC-2, TGF-β, and IL-10, while upregulating TLR-4 and pro-inflammatory cytokines, disrupting the intestinal barrier and exacerbating inflammation. Notably, rAdSPI had a more pronounced effect.

In summary, T. spiralis infection caused significant jejunal damage and disrupted the intestinal barrier. T. spiralis-secreted SPIs, especially AdSPI, played a pivotal role, facilitating invasion by compromising the host’s intestinal barrier and promoting inflammation. This study provides insights into T. spiralis invasion mechanisms and potential targets for trichinellosis prevention and control.

Trichinella spiralis

serine protease inhibitors

intestinal barrier

inflammation

Trichinella spiralis (T. spiralis) is a highly pathogenic zoonotic parasitic nematode, which is a significant foodborne pathogen capable of parasitizing over 150 animal species worldwide [1]. Infection by T. spiralis poses a severe threat to public health and significantly impacts on the livestock industry, particularly swine production, resulting in substantial economic losses [2, 3]. Therefore, researching the mechanisms of interaction between T. spiralis and its host is of great significance. Upon ingestion of muscle tissues containing encapsulated T. spiralis muscle larvae, the released larvae penetrate the intestinal epithelial barrier and invade the intestinal epithelial cells, where they undergo development into adult worms [4, 5]. Consequently, the intestinal epithelium barrier serves as a natural defensive barrier against T. spiralis infection and represents the initial site of mutual interaction between T. spiralis and its hosts [6, 7]. This process determines whether T. spiralis establishes a parasitic relationship with the host. However, the underlying mechanisms of larval invasion remain poorly understood.

Researches indicate that T. spiralis muscle larvae engage in an immunological dialogue with the host by releasing excretory-secretory products (ESPs) to ensure their survival during the intestinal phase of infection [8]. ES antigens secreted by T. spiralis are the initial substances exposed to the host immune system, and participate in interactions with various host cells, including intestinal epithelial cells and immune cells. ES antigens regulate the host’s immune response or influence the gene expression of host cells, thereby creating a suitable environment for their own survival [9, 10]. ES products comprise functional proteins that play distinct roles in inducing host immune responses, including proteases, protease inhibitors, heat shock proteins, phosphatases, endonucleases and so on [11, 12]. Among them, serine protease inhibitors (SPIs) are important components of ES products [13]. SPIs not only inhibit protease activity but also play critical roles in various biological processes such as coagulation, complement activation, and inflammation [14, 15]. SPIs in parasites participate in the survival of the parasite by modulating the host’s immune response [16]. Previous studies have demonstrated that SPI derived from T. spiralis can activate endoplasmic reticulum stress, leading to cell apoptosis, and also activate the NF-κB signaling pathway to promote the occurrence and development of inflammation [17].

Based on the above considerations, the main objective of this study was to analyze whether SPIs secreted from T. spiralis adult worms and larvae could influence the intestinal epithelial barrier function, thereby creating a favorable environment for intestinal colonization. The results of this research were aimed to provide insights into the invasion mechanisms of T. spiralis and contribute to the development of effective strategies for the prevention and control of trichinellosis.

2.1 Animal, cell and parasite

Specific pathogen-free (SPF) female BALB/c and Kunming (KM) mice, aged 6–8 weeks, were purchased from Harbin Medical University. All animal experiments and experimental procedures were conducted in accordance with the “Regulations for the Administration of Laboratory Animals in China” and the animal experimental standards approved by the Animal Management Committee of Northeast Agricultural University. All mice had ad libitum access to food and water and were maintained under SPF conditions with a humidity of 70 ± 10% and a temperature of 20 ± 2℃.

Porcine small intestinal epithelial cells (IPECs) were donated by Harbin Veterinary Research Institute. Cells were cultured in 90% DMEM medium supplemented with 10% fetal bovine serum (FBS) (PAN, Germany) and 1% penicillin/streptomycin (100 U/mL).

T. spiralis (ISS533), originally obtained from swine in Heilongjiang Province, China, propagated and preserved by our laboratory through serial passage in Kunming mice. Infective larvae were isolated from the muscles of infected KM mice using the standard method as described in Reference [18].

2.2 Recombinant protein preparation

KaSPI, T. spiralis adult worm-secreted SPI, has been assigned the GenBank accession number XM_003379851. AdSPI, T. spiralis muscle larvae-secreted SPI, has been assigned the GenBank accession number EU263307.1. In our preliminary laboratory work, the recombinant proteins, pET-30a-TsKaSPI (rKaSPI) and pET-28a-TsAdSPI (rAdSPI) were successfully constructed [19]. The proteins were expressed in E. coli, purified and renatured through a nickel column in accordance with the manufacturer’s protocol (Solarbio, China). The recombinant proteins were confirmed by 12% SDS-PAGE, followed by Coomassie blue staining. The protein concentrations were determined using a bicinchoninic acid (BCA) kit (Meilunbio, China). The contaminated endotoxin was removed by a ToxOut™ High Capacity Endotoxin Removal Kit (GenScriptbio, China).

2.3 Animal grouping and treatment

For T. spiralis infection, each BALB/c mouse was orally infected with 300 larvae of T. spiralis on day 0. Following the life cycle of T. spiralis, six mice were randomly selected on days 1, 3, 5, and 7 post-infection, and small intestinal tissues (duodenum, jejunum, and ileum) were collected and prepared for further analysis. To analyze the effects of recombinant proteins on intestinal epithelial barrier, 24 BALB/c mice were randomly divided into four groups: the blank control group (no treatment, to exclude the potential impact of personal operational errors), the PBS group, the rKaSPI group, and the rAdSPI group. Each mouse in the protein groups received an intraperitoneal injection with 50 µg of the respective protein, and small intestinal tissues and serum were collected on the third day post-injection. Mice in PBS group were intraperitoneally injected with PBS as the negative control.

2.4 Hematoxylin and Eosin Staining and Immunohistochemistry

For Hematoxylin and Eosin (H&E) Staining, the small intestine tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, sectioned at a thickness of 4 µm, stained with H&E, dehydrated again, and mounted for microscopic observation. For immunohistochemical (IHC) staining, 4 µm-thick sections of duodenal tissue underwent antigen retrieval in 0.01 M citrate buffer (pH 6.0) at 121°C for 5 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (H₂O₂) for 15 minutes. Subsequently, the samples were incubated with 1% bovine serum albumin at room temperature for 20 minutes to block non-specific binding. Following this, the sections were incubated overnight at 4°C with the primary antibody (1:100; Wanleibio, China), followed by incubation with HRP-conjugated goat anti-rabbit antibodies (1:500; Abclonal, USA) at 37°C for 30 minutes. Visualization was achieved using 3,3'-diaminobenzidine (DAB; USA, Sigma-Aldrich) staining, followed by counterstaining with Mayer’s hematoxylin solution (Sigma-Aldrich).

2.5 RT-qPCR

Quantitative real-time PCR (RT-qPCR) was performed to detect the relative expression of genes. The total RNA was isolated from Jejunal tissue or IPEC cells with Trizol Reagent (Invitrogen, USA), and then reverse transcribed into the first-strand cDNA using the Prime Script 1st Strand cDNA Synthesis Kit (Takara, Japan). All mRNA primers were designed and synthesized by Sangon Biotech, China (shown in Tables 1 and 2). Then the RT-qPCR was performed according to the instructions of ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) and using Roche Light Cycler 480 system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as a reference gene and the results were calculated using the 2^−∆∆Ct method [20].

Table 1

Primers of the detected genes in Jejunal tissue
Gene name	Primers	Sequence	Access Number
GAPDH	Forward	5’- GTGAAGGTCGGTGTGAACGGATT-3’	NM_001289726.2
GAPDH	Reverse	5’- GGTCTCGCTCCTGGAAGATGGT-3’	NM_001289726.2
IL-1β	Forward	5’- AATCTCGCAGCAGCACATCAAC-3’	XM_006498795.5
IL-1β	Reverse	5’- TGTTCATCTCGGAGCCTGTAGTG-3’	XM_006498795.5
TNF-α	Forward	5’- GCCACCACGCTCTTCTGTCTAC-3’	NM_013693.3
TNF-α	Reverse	5’- GGCTACAGGCTTGTCACTCGAA-3’	NM_013693.3
TGF-β	Forward	5’- CCGCAACAACGCCATCTATGAG-3’	XM_036152883.1
TGF-β	Reverse	5’- ACCAAGGTAACGCCAGGAATTG-3’	XM_036152883.1
IL-10	Forward	5’- GGGTTGCCAAGCCTTATCGGAA-3’	NM_010548.2
IL-10	Reverse	5’- CTGCTCCACTGCCTTGCTCTTA-3’	NM_010548.2
ZO-1	Forward	5’- AAGGCGGATGGTGCTACAAGTG-3’	NM_009386.3
ZO-1	Reverse	5’- GGCTCAGAGGACCGTGTAATGG-3’	NM_009386.3
ZO-2	Forward	5’- GGCGGCTGCTGTATCGGTTA-3’	NM_001198985.2
ZO-2	Reverse	5’- GGCGTGCTTGTCCTGCTCAA-3’	NM_001198985.2
Claudin-3	Forward	5’- CCTTCATCGGCAGCAGCATCAT-3’	NM_009902.4
Claudin-3	Reverse	5’- GCCAGCAGCGAGTCGTACATT-3’	NM_009902.4
Occludin	Forward	5’- TTGGCTACGGAGGTGGCTATGG-3’	NM_008756.2
Occludin	Reverse	5’- AGGAAGCGATGAAGCAGAAGGC-3’	NM_008756.2
TLR1	Forward	5’- ACTATGCTGGTGCTGGCTGTCA-3’	NM_030682.2
TLR1	Reverse	5’- TGTGCCTGGTCTGTGTCCACTG-3’	NM_030682.2
TLR2	Forward	5’- AGACGCTGGAGGTGTTGGATGT-3’	XM_006501460.4
TLR2	Reverse	5’- AAGTGGTTGTCGCCTGCTTCC-3’	XM_006501460.4
TLR4	Forward	5’- GCCATTGCTGCCAACATCATCC-3’	XM_036163964.1
TLR4	Reverse	5’- ACAATTCCACCTGCTGCCTCAG-3’	XM_036163964.1
MUC-1	Forward	5’- CCTGCTGGTGCTGGTCTGTATT-3’	NM_013605.2
MUC-1	Reverse	5’- GTAGCGTCCGTGAGTGTGGTAG-3’	NM_013605.2
MUC-2	Forward	5’- CGACACTCAGCACACCAACCAA-3’	NM_023566.4
MUC-2	Reverse	5’- CAGCGGCACAATCTCCACTACG-3’	NM_023566.4

Table 2

Primers of the detected genes in IPEC-J2 cells
Gene name	Primers	Sequence	Access Number
GAPDH	Forward	5’-ggtgaaggtcggagtgaacg-3’	NM_001206359.1
GAPDH	Reverse	5’-ccgtgggtggaatcatactgg-3’	NM_001206359.1
IL-1β	Forward	5’-aaagataacacgcccaccc-3’	NM_214055.1
IL-1β	Reverse	5’-GGAGTTTCCCAGGAAGACG-3’	NM_214055.1
TNF-α	Forward	5’-gctcttctgcctactgcacttc-3’	NM_214022.1
TNF-α	Reverse	5’-gctgtccctcggctttga-3’	NM_214022.1
TGF-β	Forward	5’-atttacttactgagcatcttggacctta-3’	NM_214015.2
TGF-β	Reverse	5’-gggtgttatcagagtcccttttagc-3’	NM_214015.2
IL-10	Forward	5’-gactcaacgaagaaggcacag-3’	NM_214041.1
IL-10	Reverse	5’-gcaggctggttgggaagt-3’	NM_214041.1
ZO-1	Forward	5’-aggtgctcccatcgt-3’	XM_021098827
ZO-1	Reverse	5’-tttcggtaatactcttcatc-3’	XM_021098827
ZO-2	Forward	5’-ggagcattgacccgacttac-3’	NM_001206404
ZO-2	Reverse	5’-agaccatactcttcattcgcttt-3’	NM_001206404
Claudin-3	Forward	5’-cagagccgttcgcaaccagg-3’	NM_001160075.1
Claudin-3	Reverse	5’-caccacgcagttcatccacagg-3’	NM_001160075.1
Occludin	Forward	5’-cacccagcaacgaca-3’	NM_001163647
Occludin	Reverse	5’-ataacgagcatagacagaat-3’	NM_001163647
TLR1	Forward	5’-ctctgctcaaggacttccgtgta-3’	NM_001031775.1
TLR1	Reverse	5’-agagccagtgccagcccagt-3’	NM_001031775.1
TLR2	Forward	5’-ggtccgatgctggtctttatc-3’	NM_213761.1
TLR2	Reverse	5’-gcaagtcaccctttatgttattca-3’	NM_213761.1
TLR4	Forward	5’-ccagtgctgctttgaatagag-3’	NM_001293316.1
TLR4	Reverse	5’-gaacagaagtgacccggaga-3’	NM_001293316.1
MUC-1	Forward	5’-atgagctgggagcacaggtgg-3’	XM_001926883.5
MUC-1	Reverse	5’-ccaggctcggatggacttcg-3’	XM_001926883.5
MUC-2	Forward	5’-aggacgacaccatctacctcactca-3’	XM_013989745.1
MUC-2	Reverse	5’-gcaaggccagctcgggaat-3’	XM_013989745.1

2.6 Western blotting

Total protein used for Western blotting assays was prepared from Jejunal tissue or IPEC cells, and protein concentration was determined with a BCA assay kit. Equal amount of protein was separated by using 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a 0.45 µm polyvinylidene fluoride (PVDF) filter membrane (Millipore, USA). The PVDF membrane was blocked in 5% non-fat milk for 2 h at room temperature, and then incubated with the primary antibody (Wanleibio, China) overnight at 4 ℃. Subsequently, the PVDF membrane was washed using PBST and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Abclonal, USA) for 2 h at room temperature. Finally, the blot was developed with the ultrasensitive ECL chemiluminescence reagent (Meilunbio, China) and the membrane was exposed. The bands were quantified using a chemiluminescence imaging system (Syngene, USA) and analyzed with Image J software.

2.7 Immunofluorescence

After treatment, IPEC cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes. Following that, the cells were permeabilized using 0.25% TritonX-100 for 10 minutes and blocked with 2% bovine serum albumin for 30 minutes at room temperature. Next, the cells were rinsed with PBS and incubated overnight at 4 ℃ with primary antibodies. Afterward, the wells were washed with PBS and incubated with FITC-labeled secondary antibodies (Bioss, China) for 1 h in the dark at room temperature. The cell nuclei were then stained with 2 µg/mL DAPI for 8 minutes in a dark environment. Finally, the cells were examined under a fluorescence microscope (Syngene, USA).

2.8 ELISA

Mouse or pig double-antibody sandwich ELISA kits (Mlbio, China) were respectively used to measure the cytokine content in mouse serum or cell culture supernatant. Briefly, the microwells were incubated with the experimental or standard samples for 30 min at 37 ℃, followed by the addition of HRP-labeled antibodies. Then, the microwells were thoroughly washed and stained with the substrate TMB for 10 min at 37 ℃. The stop solution was added to terminate the enzyme-substrate reaction and the absorbance at 450 nm was measured using a plate reader (BioTek, USA)

2.9 Statistical analyses

All experimental data were expressed as the mean ± SD by SPSS 22.0 software. Statistical significance was assessed using one-way analysis of variance (ANOVA) and post hoc Duncon’s test. A P-value of < 0.05 was considered statistically significant. The quantification of the protein band intensities was analyzed using Image J software.

3.1 Effects of T. spiralis on jejunal tissue

In previous study, the highest number of worms occurred in the host jejunum following T. spiralis infection. Consequently, the jejunum was selected as the focus of the study. Considering the life cycle of T. spiralis, jejunum samples were collected on days 1 (larvae developing into adults), 3 (adult mating), 7 (numerous newborn larvae), and 15 (newborn larvae migration) post-infection for H&E pathological section analysis. The scoring system, referenced and modified from Juan A. Fernández-Blanco et al. [21], was employed for double-blinded pathological scoring according to the criteria presented in Table 3. The histopathological results indicated that on day 3 post-infection (3 dpi), T. spiralis infection led to significant jejunal damage, characterized by disorganized intestinal cell arrangement, fragmented and shedding villus, extensive inflammatory cell infiltration in the lamina propria and submucosa, and an increased number of goblet cells. On 7 dpi, the damage was more severe, but on 15 dpi, the pathological scores decreased compared to 7 dpi (P < 0.05) (Fig. 1A and D). Additionally, assessments of jejunal villus length and the villus/crypt ratio revealed significant declines in both metrics on 3 dpi, with the most severe damage evident on 7 dpi (Fig. 1B and C). H&E pathological sections demonstrated that T. spiralis infection induced the most pronounced jejunal tissue damage on 7 dpi.

Table 3

Histopathological scoring
Indicators	Score
Indicators	0	1	2	3
Epithelial cell status	No change	Mild	Moderate	Severe
Villus integrity	No change	Mild	Moderate	Severe
Central lacteal expansion	No change	Mild	Moderate	Profound
Leukocyte infiltration	< 10 leukocytes per field	11–15 leukocytes per field	16–20 leukocytes per field	> 20 leukocytes per field
Submucosal oedema	No change	Mild	Moderate	Profound
Mucosal hyperaemia	None	Mild	Moderate	Severe
Number of Goblet cells	< 3 Goblet cells per field	3–5 Goblet cells per field	6–10 Goblet cells per field	> 10 Goblet cells per field
Note: Normal epithelial cells: columnar, closely arranged, monolayer intestinal epithelial cells; Normal villus: intact morphology, no break, no shedding.

3.2 Effects of T. spiralis on host intestinal barrier function

To delve further into the influence of T. spiralis infection on the intestinal mucosal barrier, RT-qPCR and Western blotting were utilized to analyze the expression of key barrier-related factors, including tight junction proteins (TJs), mucins, Toll-like receptors (TLRs), and inflammatory cytokines.

RT-qPCR analysis indicated a marked decline in the tight junction protein ZO-1 within the jejunum at 1 dpi (P < 0.001) compared to the uninfected group (at 0 dpi). Furthermore, Occludin and Claudin-3 expressions underwent significant reductions on 3 dpi (P < 0.001), reaching their lowest levels on 7 dpi. Notably, despite a noteworthy upregulation of ZO-1, Occludin, and Claudin-3 on 15 dpi in comparison to 7 dpi, these levels remained significantly inferior to those on 0 dpi (Fig. 2A), highlighting the persistent effects of T. spiralis infection on the intestinal mucosal barrier. In accordance with the RT-qPCR results, Western blot analysis concurred with the observation of a pronounced decrease in the content of ZO-1, Occludin, and Claudin-3 following T. spiralis infection, with these levels reaching their minimum on 7 dpi (Fig. 2B).

The investigation into the expression changes of jejunal mucins (MUC-1, MUC-2), along with Toll-like receptors (TLR-1, TLR-2, and TLR-4), following T. spiralis infection, utilized similar methods. The results indicate that the expression levels of MUC-1 and MUC-2 underwent a marked reduction as early as day 3 post-infection (P < 0.001), reaching their minimal levels on 7 dpi (Fig. 2C and D). Conversely, the expressions of TLR-1, TLR-2, and TLR-4 significantly augmented on 1 dpi (P < 0.01), peaking at 7 dpi (Fig. 2E and F).

Additionally, the study assessed the effects of T. spiralis infection on the expression of inflammatory cytokines (IL-1β, TNF-α, TGF-β, and IL-10). RT-qPCR and Western blotting results showed that a progressive escalation in the concentrations of TNF-α and IL-1β, commencing at 1 dpi (P < 0.01, P < 0.001, compared to 0 dpi) and culminating at 7 dpi, with a subsequent marked decrease by 15 dpi (compared to 7 dpi). Conversely, the expression of TGF-β and IL-10 exhibited a gradual decline from 1 dpi (P < 0.01, P < 0.001, compared to 0 dpi) to their lowest levels at 7 dpi, followed by a modest recovery at 15 dpi (compared to 7 dpi) (Fig. 2G and H). These results collectively suggested that T. spiralis infection disrupted the barrier function of the jejunal mucosa.

3.3 Isolation and purification of recombinant protein rKaSPI and rAdSPI

SPIs of T. spiralis play a pivotal role within its lifecycle. To investigate the effects of these SPIs on the intestinal mucosal barrier during T. spiralis invasion, the recombinant proteins rKaSPI and rAdSPI previously preserved in our laboratory, were purified. SDS-PAGE gel electrophoresis analysis unveiled that both rKaSPI and rAdSPI displayed clear and precise single protein bands, aligning with the anticipated results. Specifically, the molecular weight of rKaSPI protein was approximated 38 kDa, whereas that of rAdSPI protein was approximated 44 kDa (Fig. 3). The result not only verified the successful expression of both recombinant proteins but also confirmed their accuracy and high purity.

3.4 Effects of recombinant protein rKaSPI and rAdSPI on barrier of IPECs

To examine the impact of rKaSPI and rAdSPI on intestinal epithelial cell barrier function, a study was conducted by co-culturing IPECs with varying concentrations (0, 1, 5, 10, 20, 30 µg/mL) of these two proteins for 24 h. Subsequently, the CCK8 assay was employed to assess their effects on IPEC proliferation. Notably, cell viability remained largely unaffected at concentrations below 5 µg/mL for both proteins. However, a marked decline in cell viability was detected when concentrations exceeded 10 µg/mL (P < 0.001) (Fig. 4), leading to the determination of 10 µg/mL as the optimal working concentration for subsequent experiments involving both rKaSPI and rAdSPI.

To further elucidate their mechanisms, an interaction model between rKaSPI/rAdSPI and IPECs was established, focusing on alterations in tight junction proteins, mucins, Toll-like receptors, and inflammatory cytokines. RT-qPCR and Western blotting analyses revealed that a significant downregulation of ZO-1, Occludin, and Claudin-3 mRNA and protein levels in both IPECs + rKaSPI and IPECs + rAdSPI groups compared to the untreated control (IPECs + PBS). Notably, the inhibitory effect was more pronounced in the IPECs + rAdSPI group (P < 0.001, compared to rKaSPI group), especially regarding Occludin and Claudin-3, as indicated by more significant declines in their mRNA and protein expressions (Fig. 5A and B). This observation was further supported by immunofluorescence assays targeting Claudin-3, which exhibited markedly reduced fluorescence intensity in both treatment groups (Fig. 5C), consistent with the RT-qPCR and Western blotting results.

Furthermore, the expression of mucins MUC-1 and MUC-2 was significantly downregulated (Fig. 5D and E), whereas TLR-4 expression significantly increased in both treated groups (P < 0.001), with no noticeable changes in TLR1 or TLR-2 levels (Fig. 6A and C). Immunofluorescence of MUC-2 further corroborated these findings, demonstrating a substantial decrease in fluorescence intensity post-treatment (Fig. 5F), consistent with the RT-qPCR and Western blotting results.

Given the intricate interplay between inflammatory cytokines and intestinal epithelial barrier function, this study also investigated the expression of TNF-α, IL-1β, TGF-β, and IL-10 in IPECs following treatment with rKaSPI and rAdSPI. Employing RT-qPCR, Western blotting, and sandwich ELISA, we systematically analyzed these cytokines. The results showed that, both rKaSPI and rAdSPI significantly upregulated the mRNA and protein levels of pro-inflammatory cytokines TNF-α and IL-1β (P < 0.001), while downregulating those of anti-inflammatory cytokines TGF-β and IL-10 (P < 0.05, P < 0.001). Remarkably, the upregulation of TNF-α was more pronounced in the IPECs + rAdSPI group compared to the IPECs co-cultured with rKaSPI (Fig. 6B and D). ELISA analysis of cell culture supernatants concurred with RT-qPCR and Western blotting results (Fig. 6E), confirming the regulatory roles of rKaSPI and rAdSPI on inflammatory cytokine expression in IPECs.

3.5 Effects of recombinant protein rKaSPI and rAdSPI on the intestinal barrier of the host

To elucidate the specific effects of recombinant proteins rKaSPI and rAdSPI on the host’s intestinal mucosal barrier function, a mouse trial was designed and executed. Pathological assessment of jejunal tissue sections revealed notable loss of jejunal villus in mice subjected to rKaSPI and rAdSPI injections, compared to the untreated control group. Additionally, marked infiltration of inflammatory cells was observed within the jejunal tissues (Fig. 7A). To quantify the extent of pathological damage, a double-blinded pathological scoring system was applied to jejunal tissues from all groups. The results unequivocally demonstrated a statistically significant elevation in lesion scores in the rKaSPI- and rAdSPI-treated groups than in the control (Fig. 7B).

To delve deeper into the barrier function and its molecular regulatory mechanisms, RT-qPCR and Western blotting were employed to assess the effects of rKaSPI and rAdSPI on the expression levels of ZO-1, Occludin, and Claudin-3 in jejunal tissues. The results showed that, both recombinant proteins significantly downregulated the transcription and translation of ZO-1, Occludin, and Claudin-3 (Fig. 8A and B). Immunohistochemistry analysis further validated the reduction in Claudin-3 expression, which was primarily localized to the lateral membranes of intestinal epithelial cells at the edges of villi and displayed a brownish-yellow staining pattern. A marked decrease was observed in inflammatory infiltrates and adjacent edematous regions in the rKaSPI and rAdSPI groups (Fig. 8E, Left), further supporting the findings from RT-qPCR and Western blotting.

The influence of rKaSPI and rAdSPI on jejunal mucins (MUC-1, MUC-2) and Toll-like receptors (TLR-1, TLR-2, and TLR-4) was also evaluated. RT-qPCR and Western blotting analyses indicated that both proteins effectively decreased MUC-1 and MUC-2 expression (Fig. 8C and D) while significantly upregulating the expression of TLR2 and TLR4 (Fig. 9A and C). Immunohistochemistry of MUC-2 further confirmed its reduced expression in the rKaSPI and rAdSPI groups (Fig. 8E, Right). Notably, rAdSPI exhibited a more pronounced effect on the expression of tight junction proteins (Claudin-3) and mucins (MUC-2) compared to rKaSPI.

To investigate the immunomodulatory roles of rKaSPI and rAdSPI, the expression of TNF-α, IL-1β, TGF-β, and IL-10 in jejunal tissues and serum was systematically analyzed using RT-qPCR, Western blotting, and sandwich ELISA. The two recombinant proteins significantly upregulated TNF-α and IL-1β expression while downregulating TGF-β and IL-10 in jejunal tissues (Fig. 9B and D), corroborated by ELISA results (Figre 9 E). Notably, rAdSPI injection group resulted in significantly lower levels of IL-10 at translational levels in jejunal tissues compared to rKaSPI injection group.

The invasion process of T. spiralis exhibits a highly intricate and multileveled feature, encompassing parasitic life cycles, immune evasion mechanisms, molecular interactions, and genetic regulations. Dissecting the invasion mechanisms of T. spiralis can provide critical insights into the pivotal steps during infection. Throughout the course of evolution, T. spiralis has developed diverse invasion strategies, particularly leveraging its ESPs, which play a pivotal role not only in inter-parasite communication but also in interacting with host cells and evading host immune rejection [22, 23]. The study focuses on a key component of the ESPs of T. spiralis—SPIs—aiming to unravel their potential roles in the parasite’s breach of the host intestinal mucosal barrier and successful establishment of parasitism.

Intestinal epithelial cells (IECs) constitute the first line of physical defense in the gut, safeguarding against luminal antigens, toxins, and harmful substances while actively participating in intestinal mucosal immune responses [24]. They are also the crucial juncture that T. spiralis must traverse during invasion. The study initially probed into the impact of T. spiralis invasion on intestinal mucosal barrier function. By establishing an infection model, the pathological changes in the jejunum of mice infected with T. spiralis were evaluated at various days post-infection (0, 1, 3, 7, 15 dpi). Results indicated that marked jejunal tissue lesions, characterized by extensive villus exfoliation and disruption, accompanied by a substantial infiltration of inflammatory cells, were evident on the 3 dpi. A double-blinded pathological scoring system revealed that lesion scores peaked on the 7 dpi, aligning with the parasitic life cycle within the host, suggesting that this timepoint represents the peak of T. spiralis-induced intestinal tissue damage.

TJs, as crucial intercellular structures, are indispensable for maintaining the functional integrity and stability of the intestinal epithelial barrier [25]. Their functionality relies on intricate and precise interactions among a series of TJ proteins, including members of the claudin, zonula occludens, and TAMP families [26]. Numerous studies have demonstrated that parasites and their products can directly interact with IECs, modulating the expression of TJ proteins, thereby facilitating parasite intracellular parasitism [27, 28]. The study explored the effects of T. spiralis infection on TJ proteins in the host jejunum at different time points. The results revealed that T. spiralis infection significantly downregulated the expression of several key TJ proteins (ZO-1, Occludin, and Claudin-3) in the host jejunum. Specifically, ZO-1 expression decreased significantly early in infection (at dpi 1), whereas Occludin and Claudin-3 levels significantly declined from 3 dpi, reaching their nadir on 7 dpi. This indicates that T. spiralis infection gradually disrupts the intestinal mucosal barrier, with the most severe damage occurring within the first week post-infection.

TLRs, an integral part of the innate immune system, are widely expressed in IECs, immune cells, and other cell types in the gut, crucial for maintaining intestinal barrier function and regulating immune responses [29]. Additionally, mucins exert multifaceted effects on IEC barrier function, including enhancing chemical barriers, maintaining physical barriers, and modulating immune responses, collectively safeguarding intestinal homeostasis and immunity [30]. The study investigated the regulatory effects of T. spiralis infection on two crucial innate immune modalities within the intestinal barrier—TLRs and mucins. Results showed that MUC-1 and MUC-2 expression levels significantly decreased from day 1 post-infection, with this downward trend persisting until day 7, reaching their lowest expression levels. This suggests that T. spiralis infection may impair the mucin barrier of the intestinal mucosa, inhibiting MUC-1 and MUC-2 expression. In contrast, the expression levels of TLR-1, TLR-2, and TLR-4 exhibited an inverse trend compared to mucins. Notably, although the study observed upregulated Toll-like receptor expression, the authors’ previous work found that T. spiralis exosomes possessed the capacity to downregulate Toll-like receptor expression [31]. This intriguing contrast hints at the existence of complex immune regulatory mechanisms during T. spiralis infection, meriting further investigation.

Inflammatory factors impact the integrity and permeability of the intestinal barrier through various mechanisms. For instance, inflammatory factors such as TNF-α and IL-1β can affect TJs in intestinal epithelial cells, thereby increasing their permeability [32]. Conversely, IL-10 protects the intestinal epithelial barrier function from damage by inhibiting the production and activity of inflammatory cytokines [33]. The intensification of inflammation observed in histological sections of the jejunum post-T. spiralis infection was closely related to changes in the expression of inflammatory cytokines. Analysis revealed that TNF-α and IL-1β, important proinflammatory cytokines, exhibited a gradual increase at different days post-infection (1, 3, 7, 15 dpi), peaking on the 7th day. This suggests that as the infection progresses, the host’s inflammatory response gradually intensifies to counter the invasion of T. spiralis. However, on the 15 dpi, the expression levels of TNF-α and IL-1β decreased significantly compared to the 7 dpi, potentially indicating the onset of the resolution phase of the inflammatory response. In contrast to proinflammatory factors, anti-inflammatory cytokines such as TGF-β and IL-10 exhibited a gradual decline in expression during the initial stages of infection, with a recovery observed on the 15th day post-infection. This suggests that as the infection is gradually controlled, the host's anti-inflammatory mechanisms begin to restore and function, balancing and regulating the immune response, promoting tissue repair and restoration.

Serine protease inhibitors are essential components of T. spiralis ESPs, possessing unique biological functions. Previous studies in our laboratory had definitively confirmed that during the invasion of the host by T. spiralis, its secreted serine protease inhibitors could trigger a series of significant phenotypic changes in host cells, such as significantly enhancing endoplasmic reticulum stress [17], and initiating and enhancing autophagy [34]. Subsequently, the study constructed in vitro models of the interaction between two recombinant proteins (rKaSPI and rAdSPI) and IPECs, and further established in vivo models of their effects in mice. By assessing the impact of rKaSPI and rAdSPI on the expression of TJs, mucins, TLRs, and inflammatory cytokines in IPECs and mouse jejunum tissues, the study aimed to thoroughly elucidate the specific mechanisms by which these factors influence intestinal barrier function. Results indicated that both rKaSPI and rAdSPI exhibited regulatory effects on specific gene expression in both cellular and animal models. They collectively downregulated the expression levels of tight junction proteins (e.g., ZO-1, Occludin, Claudin-3), mucins (MUC-1, MUC-2), and anti-inflammatory factors (TGF-β, IL-10), while upregulating the expression of TLR-4 and proinflammatory factors (IL-1β, TNF-α). This pattern of changes was highly consistent with the observed gene expression changes in the jejunum tissues of mice infected with T. spiralis, revealing the crucial roles of rKaSPI and rAdSPI in the invasion process of T. spiralis and their significant impact on intestinal mucosal barrier function. Further observations also noted that while both rKaSPI and rAdSPI promoted TLR-4 expression, T. spiralis infection also involves the regulation of TLR-1 and TLR-2 expression, highlighting the complexity of the parasite’s infection mechanism. When comparing the specific effects of rKaSPI and rAdSPI on gene expression, rAdSPI exhibited a more pronounced regulatory effect, which may be related to the specificity of different life cycle stages of T. spiralis. Considering that AdSPI originates from adult worms parasitizing the small intestine, while KaSPI comes from muscle larvae parasitizing muscle tissue, this difference may explain the stronger efficacy of rAdSPI in influencing small intestine-related gene expression.

In summary, T. spiralis infection in the host led to jejunal tissue lesions, accompanied by significant decreases in the expression of tight junction proteins (e.g., ZO-1, Occludin, Claudin-3) and mucins (MUC-1, MUC-2), along with upregulated Toll-like receptor expression and intensified inflammatory responses. Further in-depth research had revealed that two important proteins secreted by T. spiralis—KaSPI and AdSPI—played pivotal roles in this process. By reducing the levels of tight junction proteins and mucins, they disrupted the tight junction state of the host intestine, subsequently affecting innate immune responses, promoting the expression of Toll-like receptors, and facilitating the release of inflammatory factors (e.g., IL-1β, TNF-α), ultimately weakening the intestinal mucosal barrier function. The study not only clarifies the specific roles of KaSPI and AdSPI in the infection mechanism of T. spiralis but also provides valuable insights into how the parasite successfully invades the host.

Conflict of Interest

The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Author Contributions

YX Lu and RB Wang designed this study. RB Wang, YH Zhang, ZX Li, JB Zhen, LH Lin, F Sun, Yang Han performed the experiments. RB Wang drafted and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (31372427).

Acknowledgements

We thank all the staff members who provided laboratory assistance.

Availability of data and materials

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The experiments were approved by the Animal Ethics Committee of Harbin Medical University and were performed in accordance with animal ethics guidelines and approved protocols (Animal Ethics Committee approval number SYXK [Hei] 2016-007).

Consent for Publication

We, the authors, hereby consent to the publication of our manuscript titled “Effects of Trichinella spiralis and its serine protease inhibitors on intestinal mucosal barrier function” in Veterinary Research. All authors have approved the manuscript and agree to its submission.

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Effects of Trichinella spiralis and its serine protease inhibitors on intestinal mucosal barrier function

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Version 1

Abstract

Figures

1 Introduction

2 Methods

2.1 Animal, cell and parasite

2.2 Recombinant protein preparation

2.3 Animal grouping and treatment

2.4 Hematoxylin and Eosin Staining and Immunohistochemistry

2.5 RT-qPCR

2.6 Western blotting

2.7 Immunofluorescence

2.8 ELISA

2.9 Statistical analyses

3 Results

3.1 Effects of T. spiralis on jejunal tissue

3.2 Effects of T. spiralis on host intestinal barrier function

3.3 Isolation and purification of recombinant protein rKaSPI and rAdSPI

3.4 Effects of recombinant protein rKaSPI and rAdSPI on barrier of IPECs

3.5 Effects of recombinant protein rKaSPI and rAdSPI on the intestinal barrier of the host

4 Discussion

Declarations

References

Status:

Version 1