S. scabiei mites and New Zealand white rabbits were provided by the Department of Parasitology at Sichuan Agricultural University. Mites (adults, nymphs, and larvae) were collected first, then live adult mites (male and female mites) were separated from the sample after morphological identification, and larvae and nymphs were left and collected together. Eggs are laid in the epidermis of the host skin and cannot be collected. Eight female Sprague-Dawley rats (6 weeks old, 150–200 g), 6-week-old female BALB/c mice (6 weeks old, 20–22 g), and 6-week-old male C57BL/6J mice (6 weeks old, 20–22 g) were purchased from SPF Biotechnology Company (Beijing).

Healthy and naturally infected New Zealand rabbit toe skins were fixed overnight with a 4% paraformaldehyde fixative (Biosharp, Guangzhou, China). The paraffin-embedded skin samples were sectioned to 5 μm, dried in an oven at 37°C for 24 h, deparaffinized, immersed in 0.1 M sodium citrate for 15 min for antigen retrieval, and washed three times with phosphate-buffered saline (PBS). The 5% goat serum was used to block at room temperature for 2 h, and then the primary antibody, F4/80 Rabbit pAb (1:200) (ABclonal, Wuhan, China), was incubated overnight at 4°C and washed three times with PBS, followed by the secondary antibody, FITC Goat Anti-Rabbit IgG (H+L) (1:400) (ABclonal, Wuhan, China), incubated for 45 min at room temperature. After washing four times, DAPI (Solarbio, Beijing, China) was used to incubate for 5 min at room temperature. Following mounting, tissue sections were examined and captured on a camera using a digital slice microscope [OLYMPUS, VS120-S6-W (BX61VS), Japan].

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to validate the mRNA expression levels of SsGAL-1, SsGAL-2, SsGAL-3, and SsGAL-5 in different developmental stages of scabies mites, and β-actin was used as a housekeeping gene. The collected mites of different stages were ground in a pre-cooled mortar. Total RNAs of larvae/nymph and adult mites were extracted using the RNAprep Pure Tissue Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. RNA quality and quantity were assessed using the 1.5% agarose gel electrophoresis and the NanoDrop 2000 spectrophotometer. An amount of 1 μg of total RNA was reverse-transcribed to cDNA using the RevertAi™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The qPCR primers (Supplementary Table S1) were designed using the nucleic acid sequences of Galectins in NCBI (SsGAL-1: JXLN01009935.1, SsGAL-2: JXLN01010345.1, SsGAL-3: JXLN01010568.1, SsGAL-5: JXLN01013773.1) as templates, and the qPCR was carried out using the LightCycler480 System (Roche Diagnostics) after the primers were synthesized. The reaction mix (20 μl) includes SYBR Permix Ex TaqII (FOREGENE, Chengdu, China) 10 μl, upstream primer 0.8 μl, downstream primer 0.8 μl, cDNA 2 μl, and ddH₂O 6.4 μl. The cycling conditions were as follows: an initial denaturation at 95 °C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s, and then melting curve analysis at 95°C for 5 s, 60°C for 60 s, and 95°C for 1 s. qPCR was conducted in triplicate, and the relative gene expression levels were calculated using 2^−ΔΔCt method (Livak and Schmittgen, 2001).

Total RNA from S. scabiei mite (larvae+nymphs, and adults) was isolated and reverse-transcribed. Primers were created using the nucleic acid sequence of Galectins from NCBI as a guide (Supplementary Table S2), and PCR amplification was performed using scabies mite cDNA as a template. PCR products were separated and cloned into the PMD19-T vector (TaKaRa, Beijing, China) and subcloned into the pET32a (+) vector (Novagen, USA) using restriction sites (underlined in the above primers); then, the recombinant plasmids were sequenced (Sango, Shanghai, China). The identified recombinant plasmid was transformed into E. coli BL21 (DE3) cells (Invitrogen, USA), and 1 mM isopropyl β-D-1-thiogalactopyranoside was used to induce the expression of cultured cells. The recombinant protein was purified by chromatography with a Ni-NTA His-tag affinity kit (Bio-Rad, USA) according to the manufacturer's instructions. Recombinant protein was analyzed for purity by SDS-PAGE using a 12% gel and subsequently with a bicinchoninic acid protein assay kit (Pierce, USA) to estimate protein concentration. A Western blot was used to identify recombinant Galectins. Protein samples were boiled for 10 min in electrophoresis sample buffer before being separated by 12% SDS-PAGE. An electrophoretic transfer cell (Bio-Rad, USA) was used to transfer the protein onto a PVDF membrane, then the membranes were incubated with 5% skim milk for 2 h after washing three times with TBST, followed by 1:100 diluted serum samples [S. scabiei negative rabbit serum, positive rabbit serum, rat negative serum, and rat anti-Ss-GAL (1,2,3,5) IgG, respectively] at 4°C overnight after washing three times with TBST. Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody or horseradish peroxidase (HRP)-conjugated goat anti-rat IgG antibody (1:3,000; Boster, China) for 2 h. After washing four times with TBST, the Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, China) was used to visualize the protein signal according to the manufacturer's instructions.

Paraffin sections of scabies mite skin and scabies mite bodies were produced in the same way as shown in macrophage localization, and the immunofluorescence procedure was performed as previously described. Eight healthy rats were prepared and randomly divided into four groups of two rats each. To prepare murine anti-rSsGalectins-IgG, the purified rSsGAL-1, rSsGAL-2, rSsGAL-3, and rSsGAL-5 were used as antigens for immunization for each group by subcutaneous injection, respectively. Purified rat anti-rSsGAL-1-IgG, rSsGAL-2-IgG, rSsGAL-3-IgG, rSsGAL-5-IgG, and rat negative serum were used as primary antibodies (diluted in 1:400), and FITC Goat Anti-Rat IgG (H+L) (1:400) (ABclonal, Wuhan, China) was used as secondary antibody to conduct immunofluorescence localization experiments on scabies skin and scabies mites, respectively, to determine the distribution of SsGAL-1, SsGAL-2, SsGAL-3, and SsGAL-5 in scabies mites and secretion in scabies skin. H&E staining was done as a control to make the fluorescent immunohistochemistry results more understandable.

On day 0, a 3 cm by 3 cm portion of the mice's backs had their hair removed. Then, 200 μl of 1% 1-chloro-2,4-dinitrobenzene (DNCB) (olive oil:acetone = 4:1) (Sigma-Aldrich, St. Louis, MO) was evenly applied to the region that has been shaved on days 1–3. An amount of 100 μg of dialyzed and purified rSsGAL-1, rSsGAL-2, rSsGAL-3, and rSsGAL-5 was administered subcutaneously at the same place on days 8 through 14. The control group received the same dose of tacrolimus (MedChemExpress, USA), histamine (VETEC, USA), recombinant pET32a protein, and PBS injections, respectively (10 BALB/c mice for each group), and on days 8, 11, and 14, 4% SDS was applied for 2 h, and then 0.5% DNCB was applied to keep the dermatitis condition. Tacrolimus (Tac) is a macrolide immunosuppressant with anti-inflammatory, antipruritic, and immunomodulatory properties that are extensively used in the treatment of AD skin disorders. Histamine (His) can elicit allergy symptoms such as skin irritation, itching, erythema, and wheal and can be utilized as a positive control in AD models. The tissue samples from the skin lesions were collected on the 15th day, the skin thickness was measured with a micrometer (DeQing, Zhejiang, China), and the samples were kept in a −80°C refrigerator or in a 4% paraformaldehyde fixative solution.

Paraffin sections of mouse AD model skin were prepared as previously described. Stained with hematoxylin and eosin (H&E) to determine the thickening and cellular infiltration of the skin. Furthermore, the immunofluorescence procedure was performed in the same way as shown in macrophage localization. F4/80 Rabbit pAb (1:200) (ABclonal, Wuhan, China) was employed as the primary antibody and FITC Goat Anti-Rabbit IgG (H+L) (1:400) (ABclonal, Wuhan, China) as the secondary antibody to observe macrophage infiltration in experimental mice. A digital section microscope was used to monitor and photograph both H&E and F4/80 practical sections.

The collection of bone marrow cells and differentiation into bone marrow-derived macrophages were performed according to Toda et al. (2021). Cells were resuspended in 400 μl PBS and evenly divided into four tubes. CD11b⁺ (Elabscience, Wuhan, China) and F4/80⁺ (BioLegend, Beijing, China) fluorescent dyes were used for staining at 4°C for 30 min. Before flow cytometry, cells were resuspended in 400 μl PBS. The Kaluza 2.1 program was used to examine the data. More than 85% of the cells bearing CD11b⁺ and F4/80⁺ double-labeled antibodies could be recognized as macrophages.

The working concentration range of recombinant Galectins (0, 0.1, 1, 10, 50, and 100 μg) was established using the CCK-8 (Solarbio, Beijing, China) assay. Based on the results of the CCK-8 cytotoxicity assay, 20 μg rSsGAL-1, rSsGAL-2, rSsGAL-3, and rSsGAL-5 proteins were added to each well and incubated for 24 h, while 20 μg of pET32a protein and 20 μl PBS were added to each well and incubated for 24 h as a blank control group. A volume of 1 ml of BMDM suspension was added to each well of a 6-well plate (the number of cells was approximately 10^*6), and every group received three samples that were identical. Cell supernatant was gathered and kept at −80°C in storage. The cells were washed two times with PBS, and the protein was immediately removed in preparation for further research.

The impact of recombinant galectins on IL-4, IL-12, TGF-β, IL-6, IL-10, IL-1β, inducible nitric oxide synthase (iNOS), and Arg1 sections was examined using real-time PCR analysis (Na, 2019; Feng et al., 2020). The BMDM cells were collected, the total RNA was extracted as directed, and then the RNA obtained was reverse-transcribed into cDNA. The reaction mixture and cycling conditions were maintained as described previously. The qPCR was carried out using the LightCycler480 System (Roche Diagnostics) after primers were synthesized according to the reported primer sequences (Supplementary Table S3). The β-actin gene was simultaneously used as a reference to normalize the data. qPCR was conducted in triplicate, and the relative gene expression levels were calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001).

The above cell supernatant was collected, and then, IL-1β, IL-6, TGF-β, IL-10, iNOS, and Arg1 were detected according to the ELISA kit instructions (4A BIOTECH/CUSABIO, China).

Statistics are expressed as mean ± standard deviation (X ± SD, n = 3) and were determined using one-way analysis of variance (ANOVA) using the GraphPad Prism version 8.0 software (GraphPad Software, USA). Probabilities (p) less than 0.05, 0.01, or 0.001 were considered significant (^*p < 0.05, ^**p < 0.01, or ^***p < 0.001), and a p-value of above 0.05 means no significant difference (p > 0.05). Graphs were created using the GraphPad Prism version 8.0 software.

The results showed that the green fluorescence intensity of scabies mite skin was higher than that of healthy skin, indicating that scabies mite disease can cause an increased infiltration of macrophages in rabbit skin. F4/80 was used as a macrophage surface marker to label macrophages for immunofluorescence staining (Figure 1).

The SsGAL-1, SsGAL-2, SsGAL-3, and SsGAL-5 were expressed in adult mites and in the mixed sample of larvae and nymphs. The expression levels of SsGAL-2 and SsGAL-5 in adult mites were higher than in mixed samples; however, the expression levels of SsGAL-1 and SsGAL-3 in adult mites were slightly lower than those in mixed samples (Figure 2).

The SsGAL-1, SsGAL-2, SsGAL-3, and SsGAL-5 genes were successfully cloned from the cDNA of the mixed sample (larvae, nymph, and adult mites) with specific primers. The size was all around 1,000 bp (1,131, 1,080, 1,008, and 1,020 bp, respectively). The rSsGAL-1 and rSsGAL-5 presented as soluble proteins, while the rSsGAL-2 and rSsGAL-3 presented in inclusion bodies. SDS-PAGE analysis revealed that galectins were purified as clear and single bands at locations 59, 57, 54, and 55 kDa, respectively (the His-tag protein of the expression vector was 18 kDa). Four rSsGalectins were specifically recognized by S. scabiei-positive rabbit serum and anti-Ss-GAL (1,2,3,5) IgG, but no band was observed after incubation with S. scabiei-naive rabbit serum (Figure 3).

No green fluorescence was detected in the scabies skin or scabies mites using negative serum from rats. On the body surface of scabies mites, SsGAL-1 and SsGAL-2 were discovered to include the mouthpiece, limbs, and epidermis, whereas SsGAL-3 and SsGAL-5 were widely distributed within and on the bodies of scabies mites and also included the gut and stomach (Figure 4). Furthermore, green fluorescence was discovered not only on the mites but also on the scabies skin (Figures 5, 6).

On the 5th day, after 3 days of continuous application of 1% DNCB, erythema, edema, and a minor number of scabs occurred on the back skin of mice. With continued application of 0.5% DNCB, the mice exhibited severe scratching, dry skin, scab thickness, erosion, and bleeding. There was no significant difference in clinical symptoms between the rSsGAL-1 and rSsGAL-5 protein groups compared to the AD group after 8–14 days of recombinant protein stimulation. Compared to the AD group, the rSsGAL-2 and rSsGAL-3 protein groups experienced more severe crusting and exudation on the skin surface, similar to histamine group symptoms. The Tacrolimus group had reduced erythema on the skin's surface, no significant scabs, and more hair. The pET32a (+) group exhibited slightly thicker crusts than the AD group, but there was no difference in symptoms between the PBS and AD groups (Figure 7).

It was clear that the mouse AD model was developed successfully because the stratum corneum of the back skin of the mice in the AD group had been damaged, showing hyperkeratosis, parakeratosis, thickness of the spinous layer, and infiltration of inflammatory cells in the dermis. Furthermore, inflammatory cell infiltration in the dorsal skin of mice was higher in the rSsGAL-1, rSsGAL-2, rSsGAL-3, and rSsGAL-5 groups than in the AD group, and the pathological alterations in the skin tissue were more severe. In the rSsGAL-2 group, some necrotic subcutaneous tissues were discovered. The His group had the most severe pathological abnormalities as compared to the positive control group. In contrast, the Tac group had typical stratum corneum structure, thickening of the spinous layer, and spinous layer edema. Inflammatory cells infiltrated more in the pET32a group than in the AD group, but there was no significant difference in the PBS group (Figure 8).

The green fluorescence of the AD group was higher than that of the healthy group. The fluorescence intensity of the rSsGAL-1, rSsGAL-2, rSsGAL-3, and rSsGAL-5 groups rose compared to the AD group, particularly the rSsGAL-3 group, which was similar to the His group. The Tac group's fluorescence intensity was reduced; there was no significant difference between the PBS group, the pET32a group, and the AD group (Figure 9).

Flow cytometry revealed that 99.7% of F4/80⁺ cells were present, 97.2% of CD11b⁺ cells were present, and 97% of F4/80⁺ CD11b⁺ cells were present (Figure 10), showing that this approach successfully converted bone marrow cells into BMDM. The CCK8 test findings indicated that the cell survival rates of rSsGAL-1, rSsGAL-2, rSsGAL-3, rSsGAL-5, and recombinant pET32a proteins were all over 90% at 0.1–100 μg, showing that the above recombinant proteins were effective at 0.1–100 μg. The viability of BMDM has no influence, and further tests can be conducted within this dose range.

The mRNA transcription levels of IL-1β, IL-6, IL-12, iNOS, and Arg1 were higher in the rSsGAL-1, rSsGAL-2, rSsGAL-3, rSsGAL-5, and recombinant pET32a groups than in the control group (p < 0.01). Simultaneously, the levels of IL-4 and IL-10 mRNA transcription were increased (p < 0.05) in the rSsGAL-1 group, while the transcription of IL-10 mRNA was increased (p < 0.05) in the rSsGAL-3 and rSsGAL-5 groups. The PBS group and the blank control group had no significant change in the relative transcription levels of inflammatory-related factors (p > 0.05) (Figure 11).

ELISA detection of inflammatory cytokines in BMDM cell supernatant revealed that rSsGAL-1, rSsGAL-2, rSsGAL-3, and rSsGAL-5 increased IL-10, IL-1β, IL-6, and iNOS (p < 0.01), but Arg1 reduced (p < 0.01). TGF-β increased in the rSsGAL-3 group, but there was no significant difference between the rSsGAL-1, rSsGAL-2, and rSsGAL-5 groups (p > 0.05). Meanwhile, IL-10, IL-1β, and iNOS levels were higher in the rSsGAL-1 and rSsGAL-3 groups than in the rSsGAL-2 and rSsGAL-5 groups. Only Arg1 rose considerably in the PBS group (p > 0.05) (Figure 12).