A novel calreticulin of Psoroptes ovis regulated keratinocyte function resulting in host skin barrier dysfunction: implications for involvement in the pathogenesis of psoroptic mange

A novel calreticulin of Psoroptes ovis regulated keratinocyte function resulting in host skin barrier dysfunction: implications for involvement in the pathogenesis of psoroptic mange | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel calreticulin of Psoroptes ovis regulated keratinocyte function resulting in host skin barrier dysfunction: implications for involvement in the pathogenesis of psoroptic mange Yane Li, Guiying Hao, Je Fan, Fangyan Wu, Xiangyue Yao, Youping Liang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6077171/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 May, 2025 Read the published version in Parasites & Vectors → Version 1 posted 5 You are reading this latest preprint version Abstract Background Psoroptes ovis , the causative agent of psoroptic mange, affects a wide range of domestic and wild animals, causing substantial economic losses and threatening wildlife survival. However, the underlying pathogenesis of this ectoparasitic disease remains poorly understood. Methods In this study, we comprehensively characterized the sequence conservation and excretory-secretory properties of P. ovis calreticulin (PsoCRT) using sequence alignment, immunoblotting, and immunofluorescence assays. To investigate the functional impact of recombinant PsoCRT (rPsoCRT), we conducted in vitro studies assessing its effects on keratinocyte proliferation, migration, differentiation, and the expression of immune regulatory factors. Additionally, we employed rabbit ear intradermal injections of rPsoCRT to histologically observe tissue changes and confirm alterations in the expression profiles of immune regulatory factors. Results PsoCRT was expressed across all developmental stages of P. ovis , with peak expression observed in adult males. Notably, PsoCRT was excreted and secreted into the host epidermis, primarily localizing within the stratum granulosum and spinosum. Intriguingly, sera from rabbits infested with P. ovis did not recognize PsoCRT. In vitro studies revealed that rPsoCRT significantly inhibited keratinocyte proliferation and migration, promoted differentiation, and upregulated the expression of IL-1β, IL-6, IL-36, CCL27, and VEGF in vitro , without altering the levels of IFN-γ or TNF-α. In vivo , rabbit ear intradermal injections of rPsoCRT induced epidermal cell differentiation, immune cell infiltration, and an upregulation of IL-6, CCL27, and VEGF expressions. Conclusions PsoCRT disrupted the physical and immune barriers of keratinocytes, leading to skin dysfunction and facilitating a microenvironment conducive to P. ovis parasitization, thereby highlighting its important role in the pathogenesis of psoroptic mange. Psoroptes ovis Calreticulin Tissue localization Keratinocytes Immune regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Psoroptes ovis , the causative agent of psoroptic mange, is a prevalent ectoparasite affecting both domestic and wild animals worldwide. Its clinical manifestations, including emaciation, scabbing, and skin thickening, ultimately result in significant economic losses and severe welfare concerns in the livestock industry [ 1 – 3 ]. Despite this substantial impact on both economic and animal welfare, our understanding of the pathogenesis of psoroptic mange remains limited. As a non-burrowing mite, P. ovis resides on the host’s skin surface [ 4 ], utilizing its mouthparts to abrade the epidermal layer without penetrating it [ 5 , 6 ]. Consequently, keratinocytes, the predominant cell type in the epidermis, become the primary targets of interaction between the mite and its host [ 5 , 7 ]. Keratinocytes, as the fundamental units of the skin's epidermal barrier, play a crucial role in maintaining the structural and functional integrity of the skin. Through their unique ability to synthesize and secrete a variety of bioactive molecules, they finely regulate the skin’s immune response, hydration levels, and permeability, thereby reinforcing the skin’s protective barrier against external threats [ 8 , 9 ]. Importantly, keratinocytes, as the frontline cells directly interacting with P. ovis , are exposed to numerous antigenic molecules secreted and excreted by the mite during its parasitic infestation [ 6 , 10 , 11 ]. These antigenic molecules are known to disrupt keratinocytes function [ 7 ]; however, a substantial portion of the specific protein components that mediate the pathogenic processes of P. ovis remain unidentified and uncharacterized to date. This gap in our knowledge hinders the understanding of the host-pathogen interplay and the development of effective therapeutic strategies. Calreticulin (CRT) is a highly conserved endoplasmic reticulum Ca 2+ binding protein and lectin-like chaperone [ 12 ], ubiquitously across diverse parasites, including protozoa, ectoparasites and helminths [ 13 ]. In parasites, CRT establishes complex interactions with various host target cells, thereby delicately orchestrating the progression of parasite diseases [ 14 ]. For instance, Trichinella spiralis , and Schistosoma japonicum have been shown to secrete or express CRT on their surfaces, which modulates cellular immunity, trigger immune shift, facilitates immune evasion, and participates in other critical biological processes [ 15 , 16 ]. Through these intricate mechanisms, CRT precisely regulates the initiation of host immune response [ 13 , 17 ], ultimately aiding in the establishment and maintenance of parasitism within the host. In a recent study, we identified a novel CRT protein, termed PsoCRT (Genbank accession number: PQ498351), within the excretory-secretory proteins of P. ovis [ 18 ]. However, the functional characterization of PsoCRT remains elusive. To address this knowledge gap, we conducted a comprehensive analysis of PsoCRT gene transcription expression and its tissue localization in rabbit skin lesions. Furthermore, we evaluated the regulatory effects of rPsoCRT on keratinocytes both in vitro and in vivo using rabbit models. Finally, we discussed the potential role of PsoCRT in the pathogenesis of psoroptic mange. Methods Animal, cell, and parasite sources Healthy New Zealand rabbits aged 3 months (n = 9) were purchased from a rabbit farm in Sichuan province that had no history of P. ovis infestation in Sichuan province. Additionally, two rabbits infested with P. ovis (n = 2) were provided by the Department of Parasitology, Sichuan Agricultural University (Sichuan, China). Two healthy female rats aged 6 weeks were obtained from Chengdu Dashuo Laboratory Animal Co., Ltd. Keratinocytes were procured from Zhejiang Meisen Cellular Technology Co. (CTCC) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM) complete medium, supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin. The cells were then incubated at 37℃ for 24 h in a humidified atmosphere with 5% CO 2 . Psoroptes mites were collected from infested rabbits to maintain mite colonies at the Department of Parasitology, Sichuan Agricultural University. Mites at each lifecycle stage (larva, nymph, male, and female) were individually harvested, and a mixed population comprising mites from all lifecycle stages was also collected, following a previously established method [ 19 ]. Prediction of CD4 T cell epitopes, homology analysis, and construction of evolutionary tree CD4 + T cell epitopes were predicted by the Immune Epitope Database (IEDB) Analysis Resource ( http://tools.immuneepitope.org/CD4episcore/ ). Multiple sequence alignment of PsoCRT and its orthologs was performed using DNAMAN version 9.0 (Lynnon Biosoft, Quebec, Canada). A phylogenetic tree was inferred using the neighbor-joining (NJ) method, based on the poisson correction method, with MEGA version 7.0 (bootstrap = 1000). Preparation of rPsoCRT, excretory-secretory (E/S) proteins, and whole-body proteins from P. ovis mites The BL21 (DE3) strain of Escherichia coli harboring the pET32a-PsoCRT plasmid, preserved in the Department of Parasitology, Sichuan Agricultural University, was resuscitated and cultured in Luria-Bertani (LB) medium at 37 ℃ for expansion. Induction was carried out with 1 mmol/L IPTG for 24 h, followed by cells harvesting via centrifugation. The cells were subsequently lysed using ultrasonication to obtain the supernatant, which underwent purification through Ni 2+ -affinity chromatography to yield the purified rPsoCRT. The rPsoCRT was further treated using an Endotoxin Removal Kit (Smart-Life Sciences Biotechnology Co., Ltd., Changzhou, China) to eliminate endotoxin contamination. Mixed-stage mites of P. ovis were washed three times with sterile water, subsequently sterilized in 70% ethanol, and then centrifuged at 500 × g for 30 s to obtain clean mite bodies. Whole-body proteins were extracted from 20 mg of these clean mites using the ExKine™ Pro Animal Cell/Tissue Total Protein Extraction Kit (Abbkine, Wuhan, China). E/S proteins of P. ovis were extracted following the methodology described by Warkin CA et al. [ 7 ]. Briefly, after obtaining the clean mites through the aforementioned washing process, they were placed in Petri dishes and incubated overnight at 28°C with 75% relative humidity (± 15%) to facilitate their recovery and the secretion of E/S products. Following the removal of excess mites, eggs, and debris, the mite secretions were collected from the culture dishes using pre-cooled phosphate-buffered saline (PBS) solution. Subsequently, the secretions were precipitated and concentrated using the TCA-acetone method to obtain the E/S proteins. Polyclonal antibody production and western blotting analysis Polyclonal antibody production and western blotting analysis To prepare polyclonal antibody against rPsoCRT, rats (n = 2) were immunized following the method described by Manunathachar [ 20 ]. Briefly, each rat was initially subcutaneously injected with 0.3 mg purified rPsoCRT mixed an equal volume of Freund's complete adjuvant (Sigma, St. Louis, USA). For the second and third injections at 7-day interval, Freund's incomplete adjuvant (Sigma) was used instead. Sera samples were collected before immunization and 3 days after the third immunization. The anti-preimmune IgG and anti-rPsoCRT IgG were purified from the collected sera using Protein G affinity chromatography (GenScript, Nanjing, China), following the manufacturer’s instructions. Whole-body extracts, E/S proteins of P. ovis , and the purified rPsoCRT were separated by 10% SDS-PAGE. The protein bands were then transferred onto a PVDF membrane using Trans-Blot SD Semi-Dry Transer Cell (Bio-rad, California, USA). The membrane was blocked with a 5% (v/v) solution of skimmed milk in PBS and subsequently incubated overnight at 4℃ with either rabbit P. ovis -positive or -negative sera, or rat anti-preimmune IgG, or rat anti-rPsoCRT IgG (all diluted 1:200 in PBS). Following this, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/rat antibody (diluted 1:2000) (Absin, Shanghai, China). After washing five times with Tween 20 in Tris buffer saline (TBST), the membranes were visualized using the Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, Beijing, China). Transcriptional analysis of calreticulin at different life stages of P. ovis For transcriptional profiling of calreticulin across various life stages of P. ovis , total RNA was extracted from mites at each life-cycle stage using the Trizol UP kit (TransGen Biotech, Beijing, China). Subsequently, the RNA was reverse transcribed into cDNA with the RT Easy™ II kit with gDNase Eraser (Foregene, Chengdu, China). Real-time flrorescence quantitative PCR (qRT-PCR) was performed in a 20 µL reaction mixture containng 10 µL of 2 × Real PCR Easy TM Mix-SYBR (Foregene), each reaction contained 0.8 µL of each primer (10µM), 1.5 µL of total cDNA, and 6.9 µL of RNase-free ddH 2 O. The β-actin gene was employed as an internal reference for normalization of calreticulin gene expression levels, with the nymphal stage serving as the baseline control. Primer sequences were listed in Table 1. Each sample was analyzed in triplicate to ensure reproducibility. Relative gene expression levels were calculated using the 2 −ΔΔCt method [ 21 ]. Immunolocalization of CRT in lesional skin of rabbit infested with P. ovis Skin samples were collected via punch biopsy from the lesional ear skin of rabbits naturally infested with P. ovis (n = 2) and from corresponding skin healthy locations on rabbit (n = 2). These samples were processed into paraffin sections for subsequent hematoxylin and eosin (HE) staining and immunofluorescence analysis. For immunofluorescence staining, the sections were dewaxed, rehydrated, and blocked, followed by overnight incubation at 4°C with either rat anti-preimmune IgG or anti-rPsoCRT IgG (diluted 1:100 in PBS). After thorough washing, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-rat IgG (diluted 1:200 in PBS; Abclonal, Wuhan, China), counterstained with 4', 6-diamidino-2-phenylindole (DAPI), and visualized using an immunofluorescence microscope (BX53, Olympus, Japan). CCK-8 assay Cell viability was assessed by a Cell Counting Kit-8 assay (Oriscience, Chengdu, China) according to the manufacturer’s instructions. HaCaT cells (1 × 10 6 cells/well) were seeded into 96-well plates with different concentrations of rPsoCRT (5, 10, 20, 40, and 80 µg/mL) and incubated for different times (12, 24, and 36 h). Simultaneously, PBS (0.01 mol/L, pH 7.4) and pET-32a protein were set as control groups, respectively. Each treatment was conducted in triplicate. Following the incubation period, 10 µL of CCK-8 solution was added to each well, and the plates were incubated for an additional 2 h. The absorbance at 450 nm was measured using a Benchmark plus microplate reader (Bio-Rad, Hercules, CA). The percentage of viable cells was calculated using the following formula: cell survival rate (%) = [(mean OD 450 value in test wells - mean OD 450 value in blank wells) / (mean OD 450 value in control wells - mean OD 450 value in blank wells)] × 100%. Scratch assay To assess cell migration in HaCaT cells, 1×10 6 cells/well were plated in a 12-well plate and incubated until reaching 80% confluence at 37℃ in a 5% CO 2 incubator (Thermo, Waltham, USA). Following three washes with PBS, a uniform scratch was created across the cell monolayer using a sterilized 200 µL pipette tip. Detached cells were carefully removed, and the remaining cells were co-incubate with the optimal concentration of rPsoCRT determined in the CCK-8 assay for an additional 24 h. Simultaneously, two control groups were set up: one with PBS (0.01 mol/L, pH 7.4) and the other with pET-32a protein at the same concentration as rPsoCRT. Each treatment was performed in triplicate. Cell migration was evaluated by capturing images of three randomly selected, non-overlapping fields using an inverted microscope (ICX41, Ningbo Sunny Instruments Co., Ltd., China) and quantifying the migration area using ImageJ 1.53e software. The cell migration rate (%) was calculated as follows: (initial scratched area- scratched area after incubation) / initial scratched area × 100%. qRT-PCR analysis of immunoregulatory factors and differentiation proteins in HaCaT cells following rPsoCRT treatment HaCaT cells (1 × 10 6 cells/well) were plated in 12-well plates and treated with rPsoCRT (5 µg/ml) or controls (PBS, pET-32a at 5 µg/ml) once they reached 70% confluence. The cells were then incubated for 24 h at 37°C in a 5% CO 2 atmosphere. All treatments were performed in replicates. Total RNA was extracted from HaCaT cells using a Tissue/Cell RNA Extraction Kit (Foregene) and subsequently reverse transcribed into cDNA using a Reverse Transcription Kit (Foregene). qRT-PCR reactions were performed using Real Time PCR Easy TM -SYBR Green I (Foregene) to quantify the relative transcription levels of involucrin (IVL), filaggrin (FLG), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-36 (IL-36), C-C motif chemokine ligand 27 (CCL27), and vascular endothelial growth factor (VEGF). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene, with the PBS-treated group serving as the control for normalization. Each qRT-PCR reaction mixture contained 0.8 µL of each primer, 10 µL of 2 × Real PCR Easy TM Mix-SYBR (Foregene), 1.5 µL of cDNA, and ddH 2 O up to a total volume of 20 µL. The relative expression levels of the targets genes were calculated using the 2 −ΔΔCt method [ 21 ]. The primer sequences are listed in Table 1. Histopathological analysis of rabbit ear skin following rPsoCRT injection using HE staining Nine three-month-old healthy New Zealand White rabbits were used in this study, with three animals assigned to each experimental group. One day prior to the experiment, the dorsal surface of their ears was shaved and cleaned. A single injection site on the dorsal surface of each ear was selected for intradermal injections of 100 µg of rPsoCRT, 100 µL of PBS (as a negative control), or 100 µg of pET-32a empty vector (as an additional control). Twenty-four hours post-injection, the rabbits were euthanized, and skin tissues encompassing the injection sites were carefully excised and fixed in 4% paraformaldehyde for subsequent paraffin embedding. The embedded tissues were then sectioned and stained with hematoxylin and eosin (HE). The stained sections were scanned using an Olympus VS120 S6 pathology scanner (OLYMPUS, Tokyo, Japan). Six random fields of view from each injection site were examined under a microscope, and immunocompetent cells were quantified. qRT-PCR detection of immunoregulatory factors and differentiation proteins in epidermis of rabbit skin tissues following rPsoCRT injection Epidermal tissues were isolated from rabbit skin collected at the aforementioned residual injection sites. The tissues were carefully dissected to remove excess subcutaneous fat and muscular tissues, followed by thorough rinsing with D-Hanks buffer. Subsequently, the skin samples were subjected to overnight digestion at 4°C in 0.4% Dispase II solution, with the epidermis facing downwards. The following day, the epidermal tissue layer was gently separated in a sterile culture dish. Total RNA was extracted from the isolated epidermal tissue using the Tissue/Cell RNA Extraction Kit (Foregene), and reverse transcribed into cDNA with the Reverse Transcription Kit (Foregene), adhering to the protocols previously described in this study. qRT-PCR analysis was then conducted using Real Time PCR EasyTM-SYBR Green I (Foregene), with β-actin serving as the internal control and the PBS injected group as the reference. The mRNA transcription levels of FLG, LOR, IL-1β, IL-6, CCL27, and VEGFwere quantified. The reaction mixture consisted of 0.8 µL of each primer, 10 µL of SYBR qRT-PCR™ Master Mix, 1.5 µL of cDNA template, and ddH 2 O to a final volume of 20 µL. Relative gene expression was calculated using the 2 −ΔΔCt method [ 21 ]. The primer sequences are detailed in Table 1. Statistical analysis Statistical differences between groups were evaluated using one-way analysis of variance (ANOVA). Data are presented as the mean ± standard error (SE). Significance levels was set at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. All experiments were conducted with a minimum of three biological replicates. Data processing and graphical representation were performed using GraphPad Prism 8.0 software. Ethical Approval All animal experimental protocols were reviewed and approved by the Experimental Animal Ethics and Welfare Committee of Sichuan Agricultural University. The animal experiments were conducted in strict accordance with the experimental operating procedures of Sichuan Agricultural University [Permit No. SYSK (Sichuan 2019-187)]. Results Prediction of PsoCRT CD4 + T cell epitope ， homology analysis and evolutionary tree construction Prediction of CD4 + T-cell epitopes within the PsoCRT amino acid sequence revealed the presence of multiple potential epitopes (Table 2), implying broad immune cell recognition of this protein. Homology analysis, conducted through multiple sequence alignment, revealed a high degree of sequence conservation between PsoCRT and calreticulin sequences of other mite species, including Euroglyphus maynei , Dermatophagoides pteronyssinus , Dermatophagoides farinae , and Sarcoptes scabiei , with identity ranges of 86.94-91.45% (Fig. 1A). The NJ tree based on the amino acid alignment of these calreticulins indicated that P. ovis has a closer evolutionary relationship to E. maynei , D. pteronyssinus , and D. farinae than to its hosts (sheep and rabbit) (Fig. 1B). Western blotting analysis Western blotting analysis revealed the presence of PsoCRT native protein in both whole-body proteins and the E/S proteins of P. ovis (Fig. 2A and 2B). This finding indicates that CRT functions as both a structural and a secretory protein in P. ovis . Surprisingly, PsoCRT was not recognized by the sera from rabbits infested with P. ovis (Fig. 2C). Differential transcription levels of Pso CRT mRNA across developmental stages of P. ovis qRT-PCR results revealed that PsoCRT mRNA is transcribed throughout the entire lifecycle of P. ovis . Notably, the highest transcription levels were observed in male mites, closedly followed by larvae mites, and then by adult females. The lowest transcript abundance for PsoCRT was detected in the nymph stage, which was approximately sevenfold lower compared to that in adult male mites ( P < 0.0001) (Fig. 3). Native Pso CRT localization in specific epidermal layers of skin lesion in rabbits infested with P. ovis In the ear skin lesions of rabbits infested with P. ovis , native PsoCRT was predominantly localized to the stratum spinosum and stratum granulosum of the epidermis, with no detectable green fluorescence in healthy skin of rabbits. Control immune-histochemical staining using pre-immune rat serum IgG yielded no fluorescent signals in either the lesional or healthy skin tissues (Fig. 4). rPsoCRT impacted HaCaT cell proliferation at high-concentration and cell migration at low-concentration Using a CCK-8 assay, we found that rPsoCRT concentrations above 10 μg/mL significantly inhibited HaCaT cell proliferation in a dose-dependent manner at 24 h (Fig. 5A, P 0.05). Time-course analysis revealed that 5 μg/mL rPsoCRT inhibited proliferation at 36 h ( P 0.05) (Fig. 5A). Thus, for subsequent experiments, we used 5 μg/mL rPsoCRT for 24 h to avoid proliferation interference. In a scratch assay, 5 μg/mL rPsoCRT significantly inhibited HaCaT cell migration ( P 0.05, Fig. 5B). rPsoCRT affected immunoregulatory and differentiation gene expression in the HaCaT cells qRT-PCR analysis was conducted to assess the effect of rPsoCRT on the transcriptional profiles of immunoregulatory factors and differentiation marker genes in HaCaT cells. Our results revealed no significant alterations in the expression of all target genes between the PBS group and the pET32a group ( P > 0.05) (Fig. 6). Notably, rPsoCRT treatment led to a significant upregulation of IL-1β, IL-6, IL-36, CCL27, and VEGF ( P < 0.0001) transcripts in HaCaT cells compared to both the PBS and pET32a groups. Among these, CCL27 and VEGF exhibited the most pronounced increase, with transcript levels approximately 5-fold higher than those in the controls, followed by IL-6, which was elevated by approximately 3-fold. Conversely, TNF-α and IFN-γ transcript levels did not significant differ across the PBS, pET32a, and rPsoCRT-treated groups ( P > 0.05) (Fig. 6). Additionally, rPsoCRT treatment resulted in a significant increase in the mRNA levels of the differentiation markers FLG and IVL in HaCaT cells ( P < 0.001) (Fig. 6). rPsoCRT induced histological changes including hyperkeratosis and immune cell infiltration in rabbit ear epidermis After 24 h of intradermal injection of rPsoCRT into rabbit ears, skin tissue sections from the injection site were stained with HE for observation. No significant differences were observed between the PBS and pET32a groups ( P > 0.05, Fig. 7). However, in contrast to both control groups, the epidermal tissue of rabbit ears in the rPsoCRT-treated group exhibited hyperkeratosis, along with significant immune cell infiltration in the deeper layers of the skin ( P < 0.0001, Fig. 7). rPsoCRT affected immunoregulatory and differentiation gene expression in the rabbit ear skin epidermis Our in vitro cellular experiments have previously demonstrated that rPsoCRT upregulates the transcription of immunomodulatory factors and differentiation markers in keratinocytes. To further extend these observations, we conducted an investigation to determine whether rPsoCRT similarly influences the transcription levels of these factors within the epidermal layer of rabbit ear skin. Using qPCR, we found that the administration of rPsoCRT led to statistically significant increases in the transcription levels of IL-6 ( P < 0.001), CCL27 ( P < 0.05), VEGF ( P < 0.05), and FLG ( P < 0.0001) at the injection sites compared to the control groups. Conversely, no significant alterations were observed in the transcription levels of IL-1β and LOR (Fig. 8). Discussion Calreticulin (CRT) is a multifunctional, conserved protein with a complex structure and diverse functional domains [13]. It plays roles in antigen presentation, immune regulation, and other functions in parasites [22-24]. However, the functional characteristics of CRT protein in P. ovis remain unexplored. Here, we functionally characterized a novel CRT in P. ovis , termed PsoCRT, and investigated its regulatory role in keratinocytes. We found that PsoCRT, secreted by P. ovis into the lesion area, modulated keratinocytes functions to disrupt skin’s physical and immune barriers, thereby promoting psoroptic mange development. Studies have demonstrated that CRT was expressed in Entamoeba histolytica and Caenorhabditis elegans [25,26], with evidence indicating that the absence of CRT results in impaired growth and development in these organisms [26]. Similarly, P. ovis expresses CRT throughout its entire life cycle (Fig. 3), suggesting a potential role for CRT in the growth and development of this parasite, as seen in other organisms. In C. elegans , the lack of CRT has been shown to result in decreased mating behavior and defective sperm development, thereby hindering male growth and reproduction [26]. Notably, our observations revealed that CRT transcripts are most abundant in males P. ovis , hinting at a possible association with male reproduction and development in this species as well. Additionally, we found that PsoCRT was excreted and secreted by P. ovis directly into the skin lesion areas on the host’s body surface. Strikingly, rPsoCRT showed no detectable antibody reactivity in sera from P. ovis- infested rabbits (Fig. 2), a result consistently replicated in three independent experiments. This immunological feature aligns with documented properties of CRT in Sarcoptes scabiei , which evaded antibody recognition in infested host sera [27]. Previous study has hypothesized that S. scabiei CRT protein may function as a non-antigenic molecule, with its inability to elicit antibody response potentially attributable to epitope masking through post-translational modifications. This adaptation collectively represent an immune evasion strategy that enables the parasite to circumvent host immune defenses. Specifically, PsoCRT’s failure to induce humoral immunity-through epitope concealment-prevents its detection by the host’s adaptive immune response, thereby facilitating immune evasion. Keratinocytes are crucial in maintaining skin homeostasis. They meticulously regulate their proliferation, differentiation, and migration within the epidermis, thereby sustaining the integrity and regenerative potential of the skin barrier, as well as maintaining the dynamic balance of the epidermal architecture [28,29]. However, exposure to exogenous adverse factors or stimulation by external substances can disrupt this delicate balance, leading to functional abnormalities, impaired skin barrier function, and ultimately triggering various skin diseases such as psoriasis, eczema, and atopic dermatitis [30,31]. In our present study, we have uncovered that during parasitism, P. ovis secretes and excretes native CRT protein into the epidermal spinous and granular layers of skin lesions (Fig. 4), which are primarily composed of keratinocytes. This suggests that P. ovis can directly target keratinocytes with its CRT protein during its life cycle. We hypothesize that CRT, upon interacting with keratinocytes, may modulate their functions. Indeed, using an in vitro model with HaCaT cells, we found that rPsoCRT significantly inhibited the proliferation and migration of keratinocytes (Fig. 5), implying that P. ovis might inhibit these process in the host epidermis by secreting PsoCRT, thereby hindering skin wound healing and ultimately leading to defects in the host skin's physical barrier. To validate this hypothesis, we conducted in vivo experiments conducted on New Zealand White rabbits and found that injection of rPsoCRT resulted in abnormal expression of FLG, a differentiation proteins crucial for the skin physical barrier (Fig. 8), further causing dysfunction of the rabbits’ skin physical barriers. A defective skin physical barrier facilitates the infiltration of mite allergen molecules, exacerbating the inflammatory response [32,33]. This, in turn, leads to increased inflammatory exudate production, providing more food sources for the mites [34,35], resulting in an increase in mite populations and further progression of the psoroptic mange. Additionally, our study revealed that rPsoCRT significantly promoted the transcriptional expression of key differentiation-related markers in keratinocytes, with notable upregulation of IVL and FLG (Fig. 6). In vitro experiments also demonstrated that rPsoCRT resulted in significantly elevated expression of FLG in the epidermis of rabbits, furtherly leading to hyperkeratinization at the injection sites of rabbits. Notably, severe hyperkeratosis was observed in the cases of natural Psoroptes infestation [36]. These findings indicate that CRT secreted by P. ovis contributes to the pathogenesis of psoroptic mange. Keratinocytes, as integral components of the skin’s immune barrier, secrete immune-regulatory factors that collaborate with immune cells and bioactive molecules to defend against pathogens and maintain skin health [37]. In the context of parasitic infestations, CRT of parasites has been implicated in modulating host immune and is involved in the parasitic disease initiation and progression, including the establishment of parasite infestation, the immune evasion strategies employed by parasites, and their ability to infiltrate tissue [16,17,38]. In P. ovis , we revealed that rPsoCRT significantly enhances the transcription of IL-1β, IL-6, IL-36, CCL27, and VEGF in keratinocytes (Fig. 6). This upregulation facilitates the recruitment and infiltration of immune cells to the infection site [39,40]. The elevated expression of CCL27, which binds to CC chemokine receptor 10 (CCR10), might promote the migration of immune cells to specific skin regions [41], while increased VEGF expression might enhance vascular permeability [42], enabling immune cells extravasation from blood vessels into localized skin areas. This process, in turn, disrupts the delicate balance of local immune homeostasis [43]. I n vivo experiments corroborate these hypotheses, with rabbit ear skin injected with rPsoCRT exhibiting high expression of IL-6, CCL27, and VEGF in the epidermis, accompanied by immune cell extensive infiltration in the dermis (Fig. 7, 8). These findings demonstrate that PsoCRT might employ a dual mechanism to disrupt epidermal homeostasis, representing an evolved parasitic strategy that both suppress keratinocyte proliferation/migration and induces inflammatory responses in HaCaT cells, thereby simultaneously compromising both physical and immune barriers. This coordinated targeting of distinct host defense systems creates a permissive microenvironment for P. ovis colonization, where impaired re-epithelialization prevents wound closure concurrent with excessive inflammation generating nutrient-rich exudates that sustain mite populations–collectively facilitating parasite establishment and driving chronic psoroptic mange progression. Conclusion PsoCRT, secreted by P. ovis , disrupted keratinocyte physical and immune barriers, resulting in impaired skin dysfunction and creating a conducive microenvironment for P. ovis parasitization, thereby playing an important role in psoroptic mange pathogenesis . Abbreviations P. ovis : Psoroptes ovis ; Pso CRT: calreticulin of P. ovis ; r Pso CRT: recombinant Pso CRT; DMEM: Dulbecco’s Modified Eagle Medium; FBS: fetal bovine serum; LB: Luria-Bertani medium; NJ: neighbor-joining; E/S proteins: excretory-secretory proteins; IPTG: isopropyl-β-D-thiogalactoside; PCR: Polymerase chain reaction; PBS: phosphate-buffered saline solution; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HRP: horseradish peroxidase; TBST: Tween 20 in Tris buffer saline; qRT-PCR: Real-time flrorescence quantitative PCR; HE: hematoxylin and eosin staining; FITC: fluorescein isothiocyanate; DAPI: 4', 6-diamidino-2-phenylindole; CCK-8: Cell Counting Kit-8 assay; HaCaT: Human Keratinocytes Cells; IVL: involucrin ; FLG: filaggrin; TNF-α: tumor necrosis factor-α; IFN-γ: interferon-γ; IL-1β: interleukin-1 beta; IL-6: interleukin-6; IL-36: interleukin-36; CCL27: C-C motif chemokine ligand 27; VEGF: vascular endothelial growth factor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; SD: standard deviation; LOR: loricrin; CCR10: CC chemokine receptor 10. Declarations The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We thank Li Tang and Cuirui Huang (Sichuan Agricultural University) for their help of experimental guidance and suggestions. Availability of data and materials The nucleotide sequence of calreticulin gene from P. ovis var. cuniculi in this article is available in the GenBank database under the accession no. PQ498351. The other data supporting our findings and conclusions are available in the article. Authors’ contributions YEL, GYH and XBG contributed the central idea, analyzed most of the data, and wrote the initial draft of the paper. XBG and GYH revised this paper. The remaining authors contributed to refining the ideas, carrying out additional analyses and finalizing this paper. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This work was supported by a grant from Double-Support Plan of Disciplinary Construction in Sichuan Agricultural University (Grant No. 2421993328). The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. References Van Den Broek AHM, Huntley JF, Halliwell REW, Machell J, Taylor M, Miller HRP, et al. Cutaneous hypersensitivity reactions to Psoroptes ovis and Der p 1 in sheep previously infested with P. ovis -the sheep scab mite. Vet. Immunol. Immunopathol, 2003,91(2): 105-117. He ML, Xu J, He R, Shen NX, Gu XB, Peng XR, et al. Preliminary analysis of Psoroptes ovis transcriptome in different developmental stages. Parasites Vectors, 2016,9(1): 570. Stoeckli MR, McNeilly TN, Frew D,Marr EJ, Nisbet AJ, Van Den Broek, et al. The effect of Psoroptes ovis infestation on ovine epidermal barrier function. Vet. Res, 2013,44(1): 11. 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Keratinocyte: a trigger or an executor of psoriasis? J Leukocyte Biol, 2020,108(2): 485-491. Lewis C J, Mardaryev A N, Poterlowicz K, Sharova T Y, Aziz A, Sharpe D T, et al. Bone morphogenetic protein signaling suppresses wound-induced skin repair by inhibiting keratinocyte proliferation and migration. J Invest Dermatol, 2014,134(3): 827-837. Den Broek V, Huntley, Machell, Bates, Groves. Cutaneous and systemic responses during primary and challenge infestations of sheep with the sheep scab mite, Psoroptes ovis . Parasite Immunol, 2000,22(8): 407-414. Bienvenu A-L, Gonzalez-Rey E, Picot S. Apoptosis induced by parasitic diseases. Parasit Vectors, 2010,3: 1-9. Deloach JR, Wright FC. Ingestion of rabbit erythrocytes containing 51Cr-labeled hemoglobin by Psoroptes spp. (Acari: Psoroptidae) that originated on cattle, mountain sheep, or rabbits. J Med Entomol, 1981,18 4: 345-348. Burgess STG, Greer A, Frew D, Wells Beth, Marr Edward J, Nisbet Alasdair J, et al. Transcriptomic analysis of circulating leukocytes reveals novel aspects of the host systemic inflammatory response to sheep scab mites. PLoS ONE, 2012,7(8):e42778. Brown SJ, Irvine AD. Atopic eczema and the filaggrin story. Semin Cutan Med Surg, 2008,27(2): 128-137. Chieosilapatham P, Kiatsurayanon C, Umehara Y, Trujillo-Paez J V, Peng G, Yue H, et al. Keratinocytes: innate immune cells in atopic dermatitis. Clin Exp Immunol, 2021,204(3): 296-309. Shao S, Hao C, Zhan B, Zhuang QH, Zhao LM, Chen Y, et al. Trichinella spiralis calreticulin S-domain binds to human complement c1q to interfere with c1q-mediated immune functions. Front Immunol, 2020,11:572326. Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: at the crossroads of cell signalling and inflammatory disease. BBA-Mol Cell Res, 2014,1843(11): 2563-2582. Conti P, Pregliasco FE, Bellomo RG, Gallenga C E, Caraffa A, Kritas S K, et al. 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J Immunol, 2012,188(1): 417-425. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.pdf Table2.pdf Cite Share Download PDF Status: Published Journal Publication published 30 May, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Accepted 13 Apr, 2025 Editor assigned by journal 13 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Submission checks completed at journal 01 Apr, 2025 First submitted to journal 30 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6077171","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436772447,"identity":"929fbbfe-7416-4db5-a4d0-bd52fc848b9c","order_by":0,"name":"Yane Li","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yane","middleName":"","lastName":"Li","suffix":""},{"id":436772448,"identity":"5f820c85-fed4-47fb-9f88-558e3115e67c","order_by":1,"name":"Guiying Hao","email":"","orcid":"","institution":"Xichang College","correspondingAuthor":false,"prefix":"","firstName":"Guiying","middleName":"","lastName":"Hao","suffix":""},{"id":436772449,"identity":"e531ca2d-49c8-4dc9-afb7-9c5f0e33d9d9","order_by":2,"name":"Je Fan","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Je","middleName":"","lastName":"Fan","suffix":""},{"id":436772450,"identity":"3508c0b7-27cc-47c7-8a6a-6ce63bfe1fd6","order_by":3,"name":"Fangyan Wu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fangyan","middleName":"","lastName":"Wu","suffix":""},{"id":436772451,"identity":"e706ebfa-c0d9-4434-a0f6-b8a9b4e4b749","order_by":4,"name":"Xiangyue Yao","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiangyue","middleName":"","lastName":"Yao","suffix":""},{"id":436772452,"identity":"bd653c1e-6c99-48dc-a172-d3557463d6c4","order_by":5,"name":"Youping Liang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Youping","middleName":"","lastName":"Liang","suffix":""},{"id":436772453,"identity":"a5c5eac5-8209-4e91-8e17-f7fef8a58595","order_by":6,"name":"Jing Xu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Xu","suffix":""},{"id":436772454,"identity":"8397b9bc-608b-44e5-b5a3-1d149b98d574","order_by":7,"name":"Ran He","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"He","suffix":""},{"id":436772455,"identity":"732b8ce6-2263-4998-8c51-e4dc29dc3be9","order_by":8,"name":"Hui Wang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":436772456,"identity":"eba889bf-3a38-4b8b-92bd-20b681157018","order_by":9,"name":"Yue Xie","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Xie","suffix":""},{"id":436772460,"identity":"9974de0c-cde2-4462-924e-b5ca010cdd4b","order_by":10,"name":"Xiaobin Gu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYJCCA0DEw8DMfPDBh4p/cmzszQeI1MLOlmw448wBYz6eYwlEWsTPYybN23YgcZ5EjgJetfIROYYHflTckTFn5jE24GG7k97GkMPA8KNiG04thjfSEg72nHnGY9nMVvhAgudZbhvD2QOMPWdu49YyI/nAAd62wzwGh5k3GxhIMOe2MfYlMDO24dOS2HDwL1gLg5lEggFzOhszjwFeLfISyQcOQ2xhMZM4kHA4gY2NgBYDnmcJh2WAfjE4DAzkhgNphm08bCDf4bGlPcf445uKO/YG5w8ffPz3n428/PzHBx/8qMBjywFsolgF4bY04JMdBaNgFIyCUQACAGcTX3b/WIwjAAAAAElFTkSuQmCC","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xiaobin","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2025-02-21 07:38:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6077171/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6077171/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06800-4","type":"published","date":"2025-05-30T15:57:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79760070,"identity":"63d9c2b1-aaab-467c-815c-07432ec22a9d","added_by":"auto","created_at":"2025-04-02 11:03:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1042214,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment comparison and phylogenetic analysis of calreticulin amino acid sequences from diverse species. Construction of multiple sequence comparison (A) and neighbor-joining tree (NJ) (B) based on CRT amino acid sequences. (A) The deduced CRT amino acid sequences were compared with homologous sequences of other mite and host CRT proteins, including \u003cem\u003eEuroglyphus maynei\u003c/em\u003e（GenBank：OTF69430.1）, \u003cem\u003eDermatophagoides pteronyssinus \u003c/em\u003e（GenBank：XP_027196726.1）, \u003cem\u003eDermatophagoides farinae\u003c/em\u003e（GenBank：XP_046918009.1）:, \u003cem\u003eSarcoptes scabiei\u003c/em\u003e（GenBank：KAF7491713.1）, \u003cem\u003eOryctolagus cuniculus\u003c/em\u003e (rabbit) （GenBank：NP_001075704.1）and \u003cem\u003eOvis aries\u003c/em\u003e(sheep)（GenBank：XP_004008533.1）. (B) Construction of NJ tree based on PsoCRT and its homologous amino acid sequence.\u003c/p\u003e","description":"","filename":"Binder11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/3cab9121486c4bca190f3019.jpg"},{"id":79759567,"identity":"84cdf787-1006-49c7-9063-e16425503507","added_by":"auto","created_at":"2025-04-02 10:55:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":346825,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting analysis of rPsoCRT. (A) Detection of PsoCRT in whole-body protein extracts of \u003cem\u003eP. ovis\u003c/em\u003e using rat anti-rPsoCRT-IgG serum (Lane 1), with a control lane showing negative serum from pre-immunized rats (Lane 2). (B) Demonstration of PsoCRT in the E/S proteins of \u003cem\u003eP. ovis\u003c/em\u003e using the same antibody (Lane 3), alongside a corresponding control lane with serum from healthy rats (Lane 4). (C) Evaluation of the serological recognition of rPsoCRT by serum from rabbits naturally infested with \u003cem\u003eP. ovis\u003c/em\u003e(Lane 5), compared to a control lane with serum from healthy rabbits (Lane 6). Molecular weight standards (Lane M) are included in each panel for size reference.\u003c/p\u003e","description":"","filename":"Binder12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/8f820b8e46b131a9f1d1dd33.jpg"},{"id":79759575,"identity":"52d6892c-f672-4a14-ac8a-d87219414770","added_by":"auto","created_at":"2025-04-02 10:55:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":333512,"visible":true,"origin":"","legend":"\u003cp\u003eRelative differential expression of PsoCRT genes across various developmental stages of\u003cem\u003e P.ovis\u003c/em\u003e. Realtive PsoCRT gene expression in larvae (L), nymphs (N), adult females (A-F), and adult males (A-M) of \u003cem\u003eP. ovis\u003c/em\u003e is shown, normalized to β-actin. Data are expressed as mean±standard error (SE). Statistical significance is indicated as follows: ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Binder13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/7142a7dbd53f1c14e74591ea.jpg"},{"id":79760918,"identity":"82bcab86-deeb-4eb0-a594-706ca550267f","added_by":"auto","created_at":"2025-04-02 11:11:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1186979,"visible":true,"origin":"","legend":"\u003cp\u003eImmunolocalization of native PsoCRT in healthy (A) and \u003cem\u003eP. ovis\u003c/em\u003e-infested (B) rabbit ear skin. Left column (HE staining): Skin sections were stained with HE for structural analysis. Middle Column (Control IgG): Sections were incubated with pre-immunization rat IgG\u003cem\u003e \u003c/em\u003eas negative control. Right column (anti-rPsoCRT-IgG): Sections were incubated with anti-rPsoCRT-IgG for specific detection of native PsoCRT. scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"Binder14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/259dfb16c2dd10db88f66f69.jpg"},{"id":79759568,"identity":"579a4994-1dd9-49bf-aa21-f592e9994fb0","added_by":"auto","created_at":"2025-04-02 10:55:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":386655,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of rPsoCRT on the proliferation and migration of HaCaT cells. (A) Cell proliferation viability was assessed using the CCK-8 assay. Left panel: Effects of various concentrations of rPsoCRT on HaCaT cells after 24 h. Right panel: Impact of 5 μg/mL rPsoCRT on HaCaT cell proliferation viability over different time periods (12, 24, 36 h). (B) Cell migration was evaluated using a scratch assay. Representative images were captured using an inverted microscope. Data are presented as mean±SE. Statistical significance is indicated as follows: *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; no annotation represents no significant difference.\u003c/p\u003e","description":"","filename":"Binder15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/257758dcb8cb8f510794ecbd.jpg"},{"id":79759570,"identity":"b3d0f36f-1a22-485d-9ffa-2fe1ecf0081c","added_by":"auto","created_at":"2025-04-02 10:55:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":306940,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of rPsoCRT on the transcription levels of immune-regulatory genes and differentiation markers in HaCaT cells. Cells were stimulated with 5 μg/mL rPsoCRT for 24 h prior to collection, and the changes in transcription levels of the aforementioned genes were detected using qRT-PCR. Data are presented as mean ± SE. Statistical significance is indicated as follows: ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, no annotation represents no significant difference.\u003c/p\u003e","description":"","filename":"Binder16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/38530da0ce7f55ea5b8f8a7e.jpg"},{"id":79760073,"identity":"88f832f3-1cff-4f5a-934f-842833808597","added_by":"auto","created_at":"2025-04-02 11:03:15","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1039150,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological observation and immune cell infiltration in rabbit ear skin (HE staining). (A) superficial skin layer; B. deep skin layer; C. localized deep skin tissue magnification. Scale bar = 100 μm/Scale bar = 20 μm. Data are expressed as mean±standard error (SE). Statistical significance is indicated as follows: **** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001, no annotation represents no significant difference, black arrows: immune cells.\u003c/p\u003e","description":"","filename":"Binder17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/c729b7b315ad00800e425e0f.jpg"},{"id":79760076,"identity":"0cd835a3-c085-49b2-af4d-672ca18ac384","added_by":"auto","created_at":"2025-04-02 11:03:15","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":387931,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of rPsoCRT on the transcript levels of immunizing factors and differentiation proteins in the healthy rabbit epidermal layer. Data are expressed as mean ± SE. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, and no annotation represents no significant difference.\u003c/p\u003e","description":"","filename":"Binder18.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/623132d54c7b9e692c9e5455.jpg"},{"id":83783054,"identity":"f4027b53-2f50-48c0-adbb-51c507d69f51","added_by":"auto","created_at":"2025-06-02 16:10:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6308357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/7004935d-c80b-4937-865d-edc5fa4e5204.pdf"},{"id":79759576,"identity":"ca576650-bed1-4f70-ba6a-d0c2e5008f4c","added_by":"auto","created_at":"2025-04-02 10:55:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":83277,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/8f9c48aae3c17232162981bd.pdf"},{"id":79760071,"identity":"f488a055-54d5-4468-952e-b2e146cc739c","added_by":"auto","created_at":"2025-04-02 11:03:15","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":78405,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6077171/v1/d7b881bb692fe5b108dbfdc7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel calreticulin of Psoroptes ovis regulated keratinocyte function resulting in host skin barrier dysfunction: implications for involvement in the pathogenesis of psoroptic mange","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003ePsoroptes ovis\u003c/em\u003e, the causative agent of psoroptic mange, is a prevalent ectoparasite affecting both domestic and wild animals worldwide. Its clinical manifestations, including emaciation, scabbing, and skin thickening, ultimately result in significant economic losses and severe welfare concerns in the livestock industry [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite this substantial impact on both economic and animal welfare, our understanding of the pathogenesis of psoroptic mange remains limited. As a non-burrowing mite, \u003cem\u003eP. ovis\u003c/em\u003e resides on the host\u0026rsquo;s skin surface [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], utilizing its mouthparts to abrade the epidermal layer without penetrating it [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, keratinocytes, the predominant cell type in the epidermis, become the primary targets of interaction between the mite and its host [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Keratinocytes, as the fundamental units of the skin's epidermal barrier, play a crucial role in maintaining the structural and functional integrity of the skin. Through their unique ability to synthesize and secrete a variety of bioactive molecules, they finely regulate the skin\u0026rsquo;s immune response, hydration levels, and permeability, thereby reinforcing the skin\u0026rsquo;s protective barrier against external threats [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Importantly, keratinocytes, as the frontline cells directly interacting with \u003cem\u003eP. ovis\u003c/em\u003e, are exposed to numerous antigenic molecules secreted and excreted by the mite during its parasitic infestation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These antigenic molecules are known to disrupt keratinocytes function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; however, a substantial portion of the specific protein components that mediate the pathogenic processes of \u003cem\u003eP. ovis\u003c/em\u003e remain unidentified and uncharacterized to date. This gap in our knowledge hinders the understanding of the host-pathogen interplay and the development of effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eCalreticulin (CRT) is a highly conserved endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e binding protein and lectin-like chaperone [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], ubiquitously across diverse parasites, including protozoa, ectoparasites and helminths [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In parasites, CRT establishes complex interactions with various host target cells, thereby delicately orchestrating the progression of parasite diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For instance, \u003cem\u003eTrichinella spiralis\u003c/em\u003e, and \u003cem\u003eSchistosoma japonicum\u003c/em\u003e have been shown to secrete or express CRT on their surfaces, which modulates cellular immunity, trigger immune shift, facilitates immune evasion, and participates in other critical biological processes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Through these intricate mechanisms, CRT precisely regulates the initiation of host immune response [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], ultimately aiding in the establishment and maintenance of parasitism within the host.\u003c/p\u003e \u003cp\u003eIn a recent study, we identified a novel CRT protein, termed PsoCRT (Genbank accession number: PQ498351), within the excretory-secretory proteins of \u003cem\u003eP. ovis\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the functional characterization of PsoCRT remains elusive. To address this knowledge gap, we conducted a comprehensive analysis of PsoCRT gene transcription expression and its tissue localization in rabbit skin lesions. Furthermore, we evaluated the regulatory effects of rPsoCRT on keratinocytes both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e using rabbit models. Finally, we discussed the potential role of PsoCRT in the pathogenesis of psoroptic mange.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal, cell, and parasite sources\u003c/h2\u003e \u003cp\u003eHealthy New Zealand rabbits aged 3 months (n\u0026thinsp;=\u0026thinsp;9) were purchased from a rabbit farm in Sichuan province that had no history of \u003cem\u003eP. ovis\u003c/em\u003e infestation in Sichuan province. Additionally, two rabbits infested with \u003cem\u003eP. ovis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) were provided by the Department of Parasitology, Sichuan Agricultural University (Sichuan, China). Two healthy female rats aged 6 weeks were obtained from Chengdu Dashuo Laboratory Animal Co., Ltd. Keratinocytes were procured from Zhejiang Meisen Cellular Technology Co. (CTCC) and were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) complete medium, supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin. The cells were then incubated at 37℃ for 24 h in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePsoroptes\u003c/em\u003e mites were collected from infested rabbits to maintain mite colonies at the Department of Parasitology, Sichuan Agricultural University. Mites at each lifecycle stage (larva, nymph, male, and female) were individually harvested, and a mixed population comprising mites from all lifecycle stages was also collected, following a previously established method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePrediction of CD4 T cell epitopes, homology analysis, and construction of evolutionary tree\u003c/h3\u003e\n\u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cell epitopes were predicted by the Immune Epitope Database (IEDB) Analysis Resource (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tools.immuneepitope.org/CD4episcore/\u003c/span\u003e\u003cspan address=\"http://tools.immuneepitope.org/CD4episcore/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignment of PsoCRT and its orthologs was performed using DNAMAN version 9.0 (Lynnon Biosoft, Quebec, Canada). A phylogenetic tree was inferred using the neighbor-joining (NJ) method, based on the poisson correction method, with MEGA version 7.0 (bootstrap\u0026thinsp;=\u0026thinsp;1000).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of rPsoCRT, excretory-secretory (E/S) proteins, and whole-body proteins from\u003c/b\u003e \u003cb\u003eP. ovis\u003c/b\u003e \u003cb\u003emites\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe BL21 (DE3) strain of Escherichia coli harboring the pET32a-PsoCRT plasmid, preserved in the Department of Parasitology, Sichuan Agricultural University, was resuscitated and cultured in Luria-Bertani (LB) medium at 37 ℃ for expansion. Induction was carried out with 1 mmol/L IPTG for 24 h, followed by cells harvesting via centrifugation. The cells were subsequently lysed using ultrasonication to obtain the supernatant, which underwent purification through Ni\u003csup\u003e2+\u003c/sup\u003e-affinity chromatography to yield the purified rPsoCRT. The rPsoCRT was further treated using an Endotoxin Removal Kit (Smart-Life Sciences Biotechnology Co., Ltd., Changzhou, China) to eliminate endotoxin contamination.\u003c/p\u003e \u003cp\u003eMixed-stage mites of \u003cem\u003eP. ovis\u003c/em\u003e were washed three times with sterile water, subsequently sterilized in 70% ethanol, and then centrifuged at 500 \u0026times; g for 30 s to obtain clean mite bodies. Whole-body proteins were extracted from 20 mg of these clean mites using the ExKine\u0026trade; Pro Animal Cell/Tissue Total Protein Extraction Kit (Abbkine, Wuhan, China). E/S proteins of \u003cem\u003eP. ovis\u003c/em\u003e were extracted following the methodology described by Warkin CA et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, after obtaining the clean mites through the aforementioned washing process, they were placed in Petri dishes and incubated overnight at 28\u0026deg;C with 75% relative humidity (\u0026plusmn;\u0026thinsp;15%) to facilitate their recovery and the secretion of E/S products. Following the removal of excess mites, eggs, and debris, the mite secretions were collected from the culture dishes using pre-cooled phosphate-buffered saline (PBS) solution. Subsequently, the secretions were precipitated and concentrated using the TCA-acetone method to obtain the E/S proteins.\u003c/p\u003e\n\u003ch3\u003ePolyclonal antibody production and western blotting analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003ePolyclonal antibody production and western blotting analysis\u003c/div\u003e \u003cp\u003eTo prepare polyclonal antibody against rPsoCRT, rats (n\u0026thinsp;=\u0026thinsp;2) were immunized following the method described by Manunathachar [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, each rat was initially subcutaneously injected with 0.3 mg purified rPsoCRT mixed an equal volume of Freund's complete adjuvant (Sigma, St. Louis, USA). For the second and third injections at 7-day interval, Freund's incomplete adjuvant (Sigma) was used instead. Sera samples were collected before immunization and 3 days after the third immunization. The anti-preimmune IgG and anti-rPsoCRT IgG were purified from the collected sera using Protein G affinity chromatography (GenScript, Nanjing, China), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eWhole-body extracts, E/S proteins of \u003cem\u003eP. ovis\u003c/em\u003e, and the purified rPsoCRT were separated by 10% SDS-PAGE. The protein bands were then transferred onto a PVDF membrane using Trans-Blot SD Semi-Dry Transer Cell (Bio-rad, California, USA). The membrane was blocked with a 5% (v/v) solution of skimmed milk in PBS and subsequently incubated overnight at 4℃ with either rabbit \u003cem\u003eP. ovis\u003c/em\u003e-positive or -negative sera, or rat anti-preimmune IgG, or rat anti-rPsoCRT IgG (all diluted 1:200 in PBS). Following this, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/rat antibody (diluted 1:2000) (Absin, Shanghai, China). After washing five times with Tween 20 in Tris buffer saline (TBST), the membranes were visualized using the Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, Beijing, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptional analysis of calreticulin at different life stages of\u003c/b\u003e \u003cb\u003eP. ovis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor transcriptional profiling of calreticulin across various life stages of \u003cem\u003eP. ovis\u003c/em\u003e, total RNA was extracted from mites at each life-cycle stage using the Trizol UP kit (TransGen Biotech, Beijing, China). Subsequently, the RNA was reverse transcribed into cDNA with the RT Easy\u0026trade; II kit with gDNase Eraser (Foregene, Chengdu, China). Real-time flrorescence quantitative PCR (qRT-PCR) was performed in a 20 \u0026micro;L reaction mixture containng 10 \u0026micro;L of 2 \u0026times; Real PCR Easy \u003csup\u003eTM\u003c/sup\u003e Mix-SYBR (Foregene), each reaction contained 0.8 \u0026micro;L of each primer (10\u0026micro;M), 1.5 \u0026micro;L of total cDNA, and 6.9 \u0026micro;L of RNase-free ddH\u003csub\u003e2\u003c/sub\u003eO. The β-actin gene was employed as an internal reference for normalization of calreticulin gene expression levels, with the nymphal stage serving as the baseline control. Primer sequences were listed in Table\u0026nbsp;1. Each sample was analyzed in triplicate to ensure reproducibility. Relative gene expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunolocalization of CRT in lesional skin of rabbit infested with\u003c/b\u003e \u003cb\u003eP. ovis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSkin samples were collected via punch biopsy from the lesional ear skin of rabbits naturally infested with \u003cem\u003eP. ovis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) and from corresponding skin healthy locations on rabbit (n\u0026thinsp;=\u0026thinsp;2). These samples were processed into paraffin sections for subsequent hematoxylin and eosin (HE) staining and immunofluorescence analysis. For immunofluorescence staining, the sections were dewaxed, rehydrated, and blocked, followed by overnight incubation at 4\u0026deg;C with either rat anti-preimmune IgG or anti-rPsoCRT IgG (diluted 1:100 in PBS). After thorough washing, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-rat IgG (diluted 1:200 in PBS; Abclonal, Wuhan, China), counterstained with 4', 6-diamidino-2-phenylindole (DAPI), and visualized using an immunofluorescence microscope (BX53, Olympus, Japan).\u003c/p\u003e\n\u003ch3\u003eCCK-8 assay\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed by a Cell Counting Kit-8 assay (Oriscience, Chengdu, China) according to the manufacturer\u0026rsquo;s instructions. HaCaT cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were seeded into 96-well plates with different concentrations of rPsoCRT (5, 10, 20, 40, and 80 \u0026micro;g/mL) and incubated for different times (12, 24, and 36 h). Simultaneously, PBS (0.01 mol/L, pH 7.4) and pET-32a protein were set as control groups, respectively. Each treatment was conducted in triplicate. Following the incubation period, 10 \u0026micro;L of CCK-8 solution was added to each well, and the plates were incubated for an additional 2 h. The absorbance at 450 nm was measured using a Benchmark plus microplate reader (Bio-Rad, Hercules, CA). The percentage of viable cells was calculated using the following formula: cell survival rate (%) = [(mean OD\u003csub\u003e450\u003c/sub\u003e value in test wells - mean OD\u003csub\u003e450\u003c/sub\u003e value in blank wells) / (mean OD\u003csub\u003e450\u003c/sub\u003e value in control wells - mean OD\u003csub\u003e450\u003c/sub\u003e value in blank wells)] \u0026times; 100%.\u003c/p\u003e\n\u003ch3\u003eScratch assay\u003c/h3\u003e\n\u003cp\u003eTo assess cell migration in HaCaT cells, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well were plated in a 12-well plate and incubated until reaching 80% confluence at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator (Thermo, Waltham, USA). Following three washes with PBS, a uniform scratch was created across the cell monolayer using a sterilized 200 \u0026micro;L pipette tip. Detached cells were carefully removed, and the remaining cells were co-incubate with the optimal concentration of rPsoCRT determined in the CCK-8 assay for an additional 24 h. Simultaneously, two control groups were set up: one with PBS (0.01 mol/L, pH 7.4) and the other with pET-32a protein at the same concentration as rPsoCRT. Each treatment was performed in triplicate. Cell migration was evaluated by capturing images of three randomly selected, non-overlapping fields using an inverted microscope (ICX41, Ningbo Sunny Instruments Co., Ltd., China) and quantifying the migration area using ImageJ 1.53e software. The cell migration rate (%) was calculated as follows: (initial scratched area- scratched area after incubation) / initial scratched area \u0026times; 100%.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR analysis of immunoregulatory factors and differentiation proteins in HaCaT cells following rPsoCRT treatment\u003c/h2\u003e \u003cp\u003eHaCaT cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were plated in 12-well plates and treated with rPsoCRT (5 \u0026micro;g/ml) or controls (PBS, pET-32a at 5 \u0026micro;g/ml) once they reached 70% confluence. The cells were then incubated for 24 h at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. All treatments were performed in replicates. Total RNA was extracted from HaCaT cells using a Tissue/Cell RNA Extraction Kit (Foregene) and subsequently reverse transcribed into cDNA using a Reverse Transcription Kit (Foregene). qRT-PCR reactions were performed using Real Time PCR Easy\u003csup\u003eTM\u003c/sup\u003e-SYBR Green I (Foregene) to quantify the relative transcription levels of involucrin (IVL), filaggrin (FLG), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-36 (IL-36), C-C motif chemokine ligand 27 (CCL27), and vascular endothelial growth factor (VEGF). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene, with the PBS-treated group serving as the control for normalization. Each qRT-PCR reaction mixture contained 0.8 \u0026micro;L of each primer, 10 \u0026micro;L of 2 \u0026times; Real PCR Easy \u003csup\u003eTM\u003c/sup\u003e Mix-SYBR (Foregene), 1.5 \u0026micro;L of cDNA, and ddH\u003csub\u003e2\u003c/sub\u003eO up to a total volume of 20 \u0026micro;L. The relative expression levels of the targets genes were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The primer sequences are listed in Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological analysis of rabbit ear skin following rPsoCRT injection using HE staining\u003c/h3\u003e\n\u003cp\u003eNine three-month-old healthy New Zealand White rabbits were used in this study, with three animals assigned to each experimental group. One day prior to the experiment, the dorsal surface of their ears was shaved and cleaned. A single injection site on the dorsal surface of each ear was selected for intradermal injections of 100 \u0026micro;g of rPsoCRT, 100 \u0026micro;L of PBS (as a negative control), or 100 \u0026micro;g of pET-32a empty vector (as an additional control). Twenty-four hours post-injection, the rabbits were euthanized, and skin tissues encompassing the injection sites were carefully excised and fixed in 4% paraformaldehyde for subsequent paraffin embedding. The embedded tissues were then sectioned and stained with hematoxylin and eosin (HE). The stained sections were scanned using an Olympus VS120 S6 pathology scanner (OLYMPUS, Tokyo, Japan). Six random fields of view from each injection site were examined under a microscope, and immunocompetent cells were quantified.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR detection of immunoregulatory factors and differentiation proteins in epidermis of rabbit skin tissues following rPsoCRT injection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEpidermal tissues were isolated from rabbit skin collected at the aforementioned residual injection sites. The tissues were carefully dissected to remove excess subcutaneous fat and muscular tissues, followed by thorough rinsing with D-Hanks buffer. Subsequently, the skin samples were subjected to overnight digestion at 4\u0026deg;C in 0.4% Dispase II solution, with the epidermis facing downwards. The following day, the epidermal tissue layer was gently separated in a sterile culture dish.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from the isolated epidermal tissue using the Tissue/Cell RNA Extraction Kit (Foregene), and reverse transcribed into cDNA with the Reverse Transcription Kit (Foregene), adhering to the protocols previously described in this study. qRT-PCR analysis was then conducted using Real Time PCR EasyTM-SYBR Green I (Foregene), with β-actin serving as the internal control and the PBS injected group as the reference. The mRNA transcription levels of FLG, LOR, IL-1β, IL-6, CCL27, and VEGFwere quantified. The reaction mixture consisted of 0.8 \u0026micro;L of each primer, 10 \u0026micro;L of SYBR qRT-PCR\u0026trade; Master Mix, 1.5 \u0026micro;L of cDNA template, and ddH\u003csub\u003e2\u003c/sub\u003eO to a final volume of 20 \u0026micro;L. Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The primer sequences are detailed in Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical differences between groups were evaluated using one-way analysis of variance (ANOVA). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Significance levels was set at *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. All experiments were conducted with a minimum of three biological replicates. Data processing and graphical representation were performed using GraphPad Prism 8.0 software.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experimental protocols were reviewed and approved by the Experimental Animal Ethics and Welfare Committee of Sichuan Agricultural University. The animal experiments were conducted in strict accordance with the experimental operating procedures of Sichuan Agricultural University [Permit No. SYSK (Sichuan 2019-187)].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePrediction of PsoCRT CD4\u003csup\u003e+\u003c/sup\u003e T cell epitope\u003c/strong\u003e\u003cstrong\u003e，\u003c/strong\u003e\u003cstrong\u003ehomology analysis and evolutionary tree construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrediction of CD4\u003csup\u003e+\u003c/sup\u003e T-cell epitopes within the PsoCRT amino acid sequence revealed the presence of multiple potential epitopes (Table 2), implying broad immune cell recognition of this protein. Homology analysis, conducted through multiple sequence alignment, revealed a high degree of sequence conservation between PsoCRT and calreticulin sequences of other mite species, including \u003cem\u003eEuroglyphus maynei\u003c/em\u003e, \u003cem\u003eDermatophagoides pteronyssinus\u003c/em\u003e, \u003cem\u003eDermatophagoides farinae\u003c/em\u003e, and \u003cem\u003eSarcoptes scabiei\u003c/em\u003e, with identity ranges of 86.94-91.45% (Fig. 1A). The NJ tree based on the amino acid alignment of these calreticulins indicated that \u003cem\u003eP. ovis\u003c/em\u003e has a closer evolutionary relationship to \u003cem\u003eE. maynei\u003c/em\u003e, \u003cem\u003eD. pteronyssinus\u003c/em\u003e, and \u003cem\u003eD. farinae\u003c/em\u003e than to its hosts (sheep and rabbit) (Fig. 1B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blotting analysis revealed the presence of PsoCRT native protein in both whole-body proteins and the E/S proteins of \u003cem\u003eP. ovis\u003c/em\u003e (Fig. 2A and 2B). This finding indicates that CRT functions as both a structural and a secretory protein in \u003cem\u003eP. ovis\u003c/em\u003e. Surprisingly, PsoCRT was not recognized by the sera from rabbits infested with \u003cem\u003eP. ovis\u003c/em\u003e (Fig. 2C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential transcription levels of Pso\u003c/strong\u003e\u003cstrong\u003eCRT mRNA across developmental stages of \u003cem\u003eP. ovis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eqRT-PCR results revealed that PsoCRT mRNA is transcribed throughout the entire lifecycle of \u003cem\u003eP. ovis\u003c/em\u003e. Notably, the highest transcription levels were observed in male mites, closedly followed by larvae mites, and then by adult females. The lowest transcript abundance for PsoCRT was detected in the nymph stage, which was approximately sevenfold lower compared to that in adult male mites (\u003cem\u003eP \u0026lt;\u0026nbsp;\u003c/em\u003e0.0001) (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNative\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePso\u003c/strong\u003e\u003cstrong\u003eCRT\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;localization in specific epidermal layers of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eskin lesion in rabbits infested with \u003cem\u003eP. ovis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the ear skin lesions of rabbits infested with \u003cem\u003eP. ovis\u003c/em\u003e, native PsoCRT was predominantly localized to the stratum spinosum and stratum granulosum of the epidermis, with no detectable green fluorescence in healthy skin of rabbits. Control immune-histochemical staining using pre-immune rat serum IgG yielded no fluorescent signals in either the lesional or healthy skin tissues (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003erPsoCRT impacted HaCaT cell proliferation at high-concentration and cell migration at low-concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing a CCK-8 assay, we found that rPsoCRT concentrations above 10 \u0026mu;g/mL significantly inhibited HaCaT cell proliferation in a dose-dependent manner at 24 h (Fig. 5A, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001), while 5 \u0026mu;g/mL had no significant effect (Fig. 5A, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Time-course analysis revealed that 5 \u0026mu;g/mL rPsoCRT inhibited proliferation at 36 h (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001), but not at 12 or 24 h (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05) (Fig. 5A). Thus, for subsequent experiments, we used 5 \u0026mu;g/mL rPsoCRT for 24 h to avoid proliferation interference. In a scratch assay, 5 \u0026mu;g/mL rPsoCRT significantly inhibited HaCaT cell migration (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001), with no significant difference observed between the PBS group and the pET-32a vector group (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05, Fig. 5B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003erPsoCRT affected immunoregulatory and differentiation gene expression in the HaCaT cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eqRT-PCR analysis was conducted to assess the effect of rPsoCRT on the transcriptional profiles of immunoregulatory factors and differentiation marker genes in HaCaT cells. Our results revealed no significant alterations in the expression of all target genes between the PBS group and the pET32a group (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) (Fig. 6). Notably, rPsoCRT\u003cem\u003e\u0026nbsp;\u003c/em\u003etreatment led to a significant upregulation of IL-1\u0026beta;, IL-6, IL-36, CCL27, and VEGF (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001) transcripts in HaCaT cells compared to both the PBS and pET32a groups. Among these, CCL27 and VEGF exhibited the most pronounced increase, with transcript levels approximately 5-fold higher than those in the controls, followed by IL-6, which was elevated by approximately 3-fold. Conversely, TNF-\u0026alpha; and IFN-\u0026gamma; transcript levels did not significant differ across the PBS, pET32a, and rPsoCRT-treated groups (\u003cem\u003eP \u0026gt;\u0026nbsp;\u003c/em\u003e0.05) (Fig. 6). Additionally, rPsoCRT treatment resulted in a significant increase in the mRNA levels of the differentiation markers FLG and IVL in HaCaT cells (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) (Fig. 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003erPsoCRT induced histological changes including hyperkeratosis and immune cell infiltration in rabbit ear epidermis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 24 h of intradermal injection of rPsoCRT into rabbit ears, skin tissue sections from the injection site were stained with HE for observation. No significant differences were observed between the PBS and pET32a groups (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, Fig. 7). However, in contrast to both control groups, the epidermal tissue of rabbit ears in the rPsoCRT-treated group exhibited hyperkeratosis, along with significant immune cell infiltration in the deeper layers of the skin (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001, Fig. 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003erPsoCRT affected immunoregulatory and differentiation gene expression in the rabbit ear skin epidermis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e cellular experiments have previously demonstrated that rPsoCRT upregulates the transcription of immunomodulatory factors and differentiation markers in keratinocytes. To further extend these observations, we conducted an investigation to determine whether rPsoCRT similarly influences the transcription levels of these factors within the epidermal layer of rabbit ear skin. Using qPCR, we found that the administration of rPsoCRT led to statistically significant increases in the transcription levels of IL-6 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001), CCL27 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), VEGF (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and FLG (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001) at the injection sites compared to the control groups. Conversely, no significant alterations were observed in the transcription levels of IL-1\u0026beta; and LOR (Fig. 8).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCalreticulin (CRT) is a multifunctional, conserved protein with a complex structure and diverse functional domains\u0026nbsp;[13]. It plays roles\u0026nbsp;in antigen presentation, immune regulation, and other functions in parasites\u0026nbsp;[22-24]. However, the functional characteristics of CRT protein in \u003cem\u003eP. ovis\u003c/em\u003e remain unexplored. Here, we functionally characterized a novel CRT in \u003cem\u003eP. ovis\u003c/em\u003e, termed PsoCRT, and investigated its regulatory role in keratinocytes. We found that PsoCRT, secreted by \u003cem\u003eP. ovis\u003c/em\u003e into the lesion area, modulated keratinocytes functions to disrupt skin\u0026rsquo;s physical and immune barriers, thereby promoting psoroptic mange development.\u003c/p\u003e\n\u003cp\u003eStudies have demonstrated that CRT was expressed in \u003cem\u003eEntamoeba histolytica\u003c/em\u003e and \u003cem\u003eCaenorhabditis elegans\u0026nbsp;\u003c/em\u003e[25,26], with evidence indicating that the absence of CRT results in impaired growth and development in these organisms [26]. Similarly, \u003cem\u003eP. ovis\u0026nbsp;\u003c/em\u003eexpresses CRT throughout its entire life cycle (Fig. 3), suggesting a potential role for CRT in the growth and development of this parasite, as seen in other organisms. In \u003cem\u003eC. elegans\u003c/em\u003e, the lack of CRT has been shown to result in decreased mating behavior and defective sperm development, thereby hindering male growth and reproduction [26]. Notably, our observations revealed that CRT transcripts are most abundant in males \u003cem\u003eP. ovis\u003c/em\u003e, hinting at a possible association with male reproduction and development in this species as well. Additionally, we found that PsoCRT was excreted and secreted by \u003cem\u003eP. ovis\u003c/em\u003e directly into the skin lesion areas on the host\u0026rsquo;s body surface. Strikingly, rPsoCRT showed no detectable antibody reactivity in sera from \u003cem\u003eP. ovis-\u003c/em\u003einfested\u003cem\u003e\u0026nbsp;\u003c/em\u003erabbits (Fig. 2), a result consistently replicated in three independent experiments.\u0026nbsp;This immunological feature aligns with documented properties of CRT\u0026nbsp;in \u003cem\u003eSarcoptes scabiei\u003c/em\u003e, which evaded antibody recognition in infested host sera\u0026nbsp;[27]. Previous study has hypothesized that \u003cem\u003eS. scabiei\u0026nbsp;\u003c/em\u003eCRT protein may function as a non-antigenic molecule, with its inability to elicit antibody response potentially attributable to\u0026nbsp;epitope masking through post-translational modifications.\u0026nbsp;This adaptation collectively represent an immune evasion strategy that enables the parasite to circumvent host immune defenses. Specifically,\u0026nbsp;PsoCRT\u0026rsquo;s failure to induce humoral immunity-through epitope concealment-prevents its detection by the host\u0026rsquo;s adaptive immune response, thereby facilitating immune evasion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKeratinocytes are crucial in maintaining skin homeostasis. They meticulously regulate their proliferation, differentiation, and migration within the epidermis, thereby sustaining the integrity and regenerative potential of the skin barrier, as well as maintaining the dynamic balance of the epidermal architecture\u0026nbsp;[28,29]. However, exposure to exogenous adverse factors or stimulation by external substances can disrupt this delicate balance, leading to functional abnormalities, impaired skin barrier function, and ultimately triggering various skin diseases such as psoriasis, eczema, and atopic dermatitis\u0026nbsp;[30,31]. In our present study, we have uncovered that during parasitism, \u003cem\u003eP. ovis\u003c/em\u003e secretes and excretes native CRT protein into the epidermal spinous and granular layers of skin lesions (Fig. 4), which are primarily composed of keratinocytes. This suggests that \u003cem\u003eP. ovis\u003c/em\u003e can directly target keratinocytes with its CRT protein during its life cycle. We hypothesize that CRT, upon interacting with keratinocytes, may modulate their functions. Indeed, using an \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003emodel with HaCaT cells, we found that rPsoCRT significantly inhibited the proliferation and migration of keratinocytes (Fig. 5), implying that \u003cem\u003eP. ovis\u003c/em\u003e might inhibit these process in the host epidermis by secreting PsoCRT, thereby hindering skin wound healing and ultimately leading to defects in the host skin\u0026apos;s physical barrier. To validate this hypothesis, we conducted \u003cem\u003ein vivo\u003c/em\u003e experiments conducted on New Zealand White rabbits and found that injection of rPsoCRT resulted in abnormal expression of FLG, a differentiation proteins crucial for the skin physical barrier (Fig. 8), further causing dysfunction of the rabbits\u0026rsquo; skin physical barriers.\u0026nbsp;A defective skin physical barrier facilitates the infiltration of mite allergen molecules, exacerbating the inflammatory response\u0026nbsp;[32,33]. This,\u0026nbsp;in turn, leads to increased inflammatory exudate production, providing more food sources for the mites\u0026nbsp;[34,35],\u0026nbsp;resulting in an increase in mite populations and further progression of the psoroptic mange.\u0026nbsp;Additionally, our study revealed that rPsoCRT significantly promoted the transcriptional expression of key differentiation-related markers in keratinocytes, with notable upregulation of IVL and FLG (Fig. 6). \u003cem\u003eIn vitro\u003c/em\u003e experiments also demonstrated that rPsoCRT resulted in significantly elevated expression of FLG in the epidermis of rabbits, furtherly leading to hyperkeratinization at the injection sites of rabbits. Notably, severe hyperkeratosis was observed in the cases of natural \u003cem\u003ePsoroptes\u003c/em\u003e infestation\u0026nbsp;[36]. These findings indicate that CRT secreted by \u003cem\u003eP. ovis\u003c/em\u003e contributes to the pathogenesis of psoroptic mange.\u003c/p\u003e\n\u003cp\u003eKeratinocytes, as integral components of the skin\u0026rsquo;s immune barrier, secrete immune-regulatory factors that collaborate with immune cells and bioactive molecules to defend against pathogens and maintain skin health [37]. In the context of parasitic infestations, CRT of parasites has been implicated in modulating host immune and is involved in the parasitic disease initiation and progression, including the establishment of parasite infestation, the immune evasion strategies employed by parasites, and their ability to infiltrate tissue [16,17,38]. In \u003cem\u003eP. ovis\u003c/em\u003e, we revealed that rPsoCRT significantly enhances the transcription of IL-1\u0026beta;, IL-6, IL-36, CCL27, and VEGF in keratinocytes (Fig. 6).\u0026nbsp;This upregulation facilitates the recruitment and infiltration of immune cells to the infection site\u0026nbsp;[39,40].\u0026nbsp;The\u0026nbsp;elevated expression of CCL27, which binds to CC chemokine receptor 10 (CCR10), might promote the migration of immune cells to specific skin regions\u0026nbsp;[41], while increased VEGF expression might enhance vascular permeability\u0026nbsp;[42], enabling immune cells extravasation from blood vessels into localized skin areas. This process, in turn, disrupts the delicate balance of local immune homeostasis\u0026nbsp;[43].\u0026nbsp;\u003cem\u003eI\u003c/em\u003e\u003cem\u003en vivo\u0026nbsp;\u003c/em\u003eexperiments corroborate these hypotheses, with rabbit ear skin injected with rPsoCRT exhibiting high expression of IL-6, CCL27, and VEGF in the epidermis, accompanied by immune cell extensive infiltration in the dermis (Fig. 7, 8). These findings demonstrate that PsoCRT might employ a dual mechanism to disrupt epidermal homeostasis, representing an evolved parasitic strategy that both suppress keratinocyte proliferation/migration and induces inflammatory responses in HaCaT cells, thereby simultaneously compromising both physical and immune barriers. This coordinated targeting of distinct host defense systems creates a permissive microenvironment for \u003cem\u003eP. ovis\u003c/em\u003e colonization, where impaired re-epithelialization prevents wound closure concurrent with excessive inflammation generating nutrient-rich exudates that sustain mite populations\u0026ndash;collectively facilitating parasite establishment and driving chronic psoroptic mange progression.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePsoCRT, secreted by \u003cem\u003eP. ovis\u003c/em\u003e, disrupted keratinocyte physical and immune barriers, resulting in impaired skin dysfunction and creating a conducive microenvironment for \u003cem\u003eP. ovis\u003c/em\u003e parasitization, thereby playing an important\u0026nbsp;role in psoroptic mange pathogenesis\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eP. ovis\u003c/em\u003e: \u003cem\u003ePsoroptes ovis\u003c/em\u003e;\u0026nbsp;\u003cem\u003ePso\u003c/em\u003eCRT: calreticulin of \u003cem\u003eP. ovis\u003c/em\u003e;\u003cem\u003e\u0026nbsp;\u003c/em\u003er\u003cem\u003ePso\u003c/em\u003eCRT: recombinant \u003cem\u003ePso\u003c/em\u003eCRT; DMEM: Dulbecco\u0026rsquo;s Modified Eagle Medium; FBS: fetal bovine serum; LB: Luria-Bertani medium; NJ: neighbor-joining; E/S proteins: excretory-secretory proteins; IPTG: isopropyl-\u0026beta;-D-thiogalactoside; PCR: Polymerase chain reaction; PBS: phosphate-buffered saline solution; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HRP: horseradish peroxidase; TBST: Tween 20 in Tris buffer saline; qRT-PCR: Real-time flrorescence quantitative PCR; HE: hematoxylin and eosin staining; FITC: fluorescein isothiocyanate; DAPI: 4\u0026apos;, 6-diamidino-2-phenylindole; CCK-8: Cell Counting Kit-8 assay; HaCaT: Human Keratinocytes Cells; IVL: involucrin ; FLG: filaggrin; TNF-\u0026alpha;: tumor necrosis factor-\u0026alpha;; IFN-\u0026gamma;: interferon-\u0026gamma;; IL-1\u0026beta;: interleukin-1 beta; IL-6: interleukin-6; IL-36: interleukin-36; CCL27: C-C motif chemokine ligand 27; VEGF: vascular endothelial growth factor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; SD: standard deviation; LOR: loricrin; CCR10: CC chemokine receptor 10.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Li Tang and Cuirui Huang (Sichuan Agricultural University)\u0026nbsp;for their help of experimental guidance and suggestions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequence of calreticulin gene from \u003cem\u003eP. ovis\u0026nbsp;\u003c/em\u003evar.\u003cem\u003e\u0026nbsp;cuniculi\u0026nbsp;\u003c/em\u003ein this article is available in the GenBank database under the accession no. PQ498351. The other data supporting our findings and conclusions are available in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYEL, GYH and XBG contributed the central idea, analyzed most of the data, and wrote the initial draft of the paper. XBG and GYH revised this paper. The remaining authors contributed to refining the ideas, carrying out additional analyses and finalizing this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from Double-Support Plan of Disciplinary Construction in Sichuan Agricultural University (Grant No. 2421993328). The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVan Den Broek AHM, Huntley JF, Halliwell REW, Machell J, Taylor M, Miller HRP, et al. Cutaneous hypersensitivity reactions to \u003cem\u003ePsoroptes ovis\u003c/em\u003e and \u003cem\u003eDer\u003c/em\u003e p 1 in sheep previously infested with \u003cem\u003eP. ovis\u003c/em\u003e-the sheep scab mite. Vet. Immunol. Immunopathol, 2003,91(2): 105-117.\u003c/li\u003e\n\u003cli\u003eHe ML, Xu J, He R, Shen NX, Gu XB, Peng XR, et al. Preliminary analysis of \u003cem\u003ePsoroptes ovis\u003c/em\u003e transcriptome in different developmental stages. Parasites Vectors, 2016,9(1): 570.\u003c/li\u003e\n\u003cli\u003eStoeckli MR, McNeilly TN, Frew D,Marr EJ, Nisbet AJ, Van Den Broek, et al. The effect of \u003cem\u003ePsoroptes ovis\u003c/em\u003e infestation on ovine epidermal barrier function. Vet. Res, 2013,44(1): 11.\u003c/li\u003e\n\u003cli\u003eSanders A, Froggatt P, Wall R, Smith, KE. Life‐cycle stage morphology of psoroptes mange mites. Med. Vet. Entomol, 2000,14(2): 131-141.\u003c/li\u003e\n\u003cli\u003eMathieson BRF, Lehane MJ. 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Mediat Inflamm, 2017,2017: 1-12.\u003c/li\u003e\n\u003cli\u003eSuter MM, Schulze K, Bergman W, Welle M, Roosje P, M\u0026uuml;ller EJ, et al. The keratinocyte in epidermal renewal and defence. Vet Dermatol, 2009,20(5-6): 515-532.\u003c/li\u003e\n\u003cli\u003eHamilton KA, Nisbet AJ, Lehane MJ, Taylor MA, Billingsley PF. A physiological and biochemical model for digestion in the ectoparasitic mite, \u003cem\u003ePsoroptes ovis\u003c/em\u003e (Acari: Psoroptidae). Int J Parasitol, 2003,33(8): 773-785.\u003c/li\u003e\n\u003cli\u003eVan Den Broek AH, Huntley JF. Sheep scab: the disease, pathogenesis and control. J Comp Pathol, 2003,128(2-3): 79-91.\u003c/li\u003e\n\u003cli\u003eMichalak M. Calreticulin: Endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e gatekeeper. J Cell Mol Med, 2024,28(5): e17839.\u003c/li\u003e\n\u003cli\u003eEsperante D, Flisser A, Mendlovic F. The many faces of parasite calreticulin. 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J Immunol, 2012,188(1): 417-425.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Psoroptes ovis, Calreticulin, Tissue localization, Keratinocytes, Immune regulation","lastPublishedDoi":"10.21203/rs.3.rs-6077171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6077171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePsoroptes ovis\u003c/em\u003e, the causative agent of psoroptic mange, affects a wide range of domestic and wild animals, causing substantial economic losses and threatening wildlife survival. However, the underlying pathogenesis of this ectoparasitic disease remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, we comprehensively characterized the sequence conservation and excretory-secretory properties of \u003cem\u003eP. ovis\u003c/em\u003e calreticulin (PsoCRT) using sequence alignment, immunoblotting, and immunofluorescence assays. To investigate the functional impact of recombinant PsoCRT (rPsoCRT), we conducted in \u003cem\u003evitro\u003c/em\u003e studies assessing its effects on keratinocyte proliferation, migration, differentiation, and the expression of immune regulatory factors. Additionally, we employed rabbit ear intradermal injections of rPsoCRT to histologically observe tissue changes and confirm alterations in the expression profiles of immune regulatory factors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePsoCRT was expressed across all developmental stages of \u003cem\u003eP. ovis\u003c/em\u003e, with peak expression observed in adult males. Notably, PsoCRT was excreted and secreted into the host epidermis, primarily localizing within the stratum granulosum and spinosum. Intriguingly, sera from rabbits infested with \u003cem\u003eP. ovis\u003c/em\u003e did not recognize PsoCRT. \u003cem\u003eIn vitro\u003c/em\u003e studies revealed that rPsoCRT significantly inhibited keratinocyte proliferation and migration, promoted differentiation, and upregulated the expression of IL-1β, IL-6, IL-36, CCL27, and VEGF \u003cem\u003ein vitro\u003c/em\u003e, without altering the levels of IFN-γ or TNF-α. \u003cem\u003eIn vivo\u003c/em\u003e, rabbit ear intradermal injections of rPsoCRT induced epidermal cell differentiation, immune cell infiltration, and an upregulation of IL-6, CCL27, and VEGF expressions.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePsoCRT disrupted the physical and immune barriers of keratinocytes, leading to skin dysfunction and facilitating a microenvironment conducive to \u003cem\u003eP. ovis\u003c/em\u003e parasitization, thereby highlighting its important role in the pathogenesis of psoroptic mange.\u003c/p\u003e","manuscriptTitle":"A novel calreticulin of Psoroptes ovis regulated keratinocyte function resulting in host skin barrier dysfunction: implications for involvement in the pathogenesis of psoroptic mange","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 10:55:10","doi":"10.21203/rs.3.rs-6077171/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-14T02:09:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-13T12:16:28+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T08:55:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-01T07:49:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-03-31T02:59:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"390e3866-3344-4de6-abf6-e908634a6e71","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:05:41+00:00","versionOfRecord":{"articleIdentity":"rs-6077171","link":"https://doi.org/10.1186/s13071-025-06800-4","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-05-30 15:57:30","publishedOnDateReadable":"May 30th, 2025"},"versionCreatedAt":"2025-04-02 10:55:10","video":"","vorDoi":"10.1186/s13071-025-06800-4","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06800-4","workflowStages":[]},"version":"v1","identity":"rs-6077171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6077171","identity":"rs-6077171","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}