Tick cysteine protease inhibitors suppress immune responses in mannan-induced psoriasis-like inflammation

Abstract

Protease inhibitors regulate various biological processes and prevent host tissue/organ damage. Specific inhibition/regulation of proteases is clinically valuable for treating several diseases. Psoriasis affects the skin in the limbs and scalp of the body, and the contribution of cysteine and serine proteases to the development of skin inflammation is well documented. Cysteine protease inhibitors from ticks have high specificity, selectivity, and affinity to their target proteases and are efficient immunomodulators. However, their potential therapeutic effect on psoriasis pathogenesis remains to be determined. Therefore, we tested four tick cystatins (Sialostatin L, Sialostatin L2, Iristatin, and Mialostatin) in the recently developed, innate immunity-dependent mannan-induced psoriasis model. We explored the effects of protease inhibitors on clinical symptoms and histological features. In addition, the number and percentage of immune cells (dendritic cells, neutrophils, macrophages, and γδT cells) by flow cytometry, immunofluorescence/immunohistochemistry and, the expression of pro-inflammatory cytokines (TNF-a, IL-6, IL-22, IL-23, and IL-17 family) by qPCR were analyzed using skin, spleen, and lymph node samples. Tick protease inhibitors have significantly decreased psoriasis symptoms and disease manifestations but had differential effects on inflammatory responses and immune cell populations, suggesting different modes of action of these inhibitors on psoriasis-like inflammation. Thus, our study demonstrates, for the first time, the usefulness of tick-derived protease inhibitors for treating skin inflammation in patients.

Keywords: autoimmune disease; immune responses; protease inhibitors; psoriasis; tick.

Figure 1

Tick protease inhibitors decreased psoriasis severity and microscopical disease manifestations. Effect of Sialostatin L, L2 (A) Iristatin or Mialostatin (B) treatment in MISI. (C, D) Statistical results showing the epidermal thickness and histological scores of psoriatic skins from sialostatin L, Sialostatin L2, Iristatin, or Mialostatin-treated mice (n = 12/group). (E) Representative pictures of H&E-stained skin sections (n = 5/group). The scale bar is 200 μm. (F, G) Expression of VEGF and TGF-β in psoriatic skin (n = 5/group). Man, Mannan; α-LA, alpha-lactalbumin, negative control. Results are shown from a representative experiment. All mice developed psoriasis-like inflammation. Statistical analysis was performed using an unpaired t-test; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. #, p < 0.05; ##, p < 0.01; ###, p < 0.001. *** in Panel A was used to show the difference between mannan and Sialostatin L group. ### in Panel A showed significance between mannan and Sialostatin L2 group, and indicates p < 0.001.

Figure 2

Tick protease inhibitors improved mannan-induced skin inflammation by modulating infiltrated immune cells in the skin lesions. (A) Representative pictures of immunofluorescence staining and statistical results show CD11c⁺ cells (green) expression in MISI after Sialostatin L, Sialostatin L2, Iristatin, or Mialostatin treatment (n = 3/group). Nuclei were counterstained with DAPI (blue). (B, C) Immunohistochemistry staining and statistical results show F4/80⁺ and Ly6G⁺ cell expression in the tick protease inhibitors treated mice (n = 3/group). The scale bar is 100 μm. Positive cells in each mm² were calculated under optical and confocal microscopy. Man, mannan; α-LA, alpha-lactalbumin, negative control. Results are from a representative experiment. All mice developed psoriasis-like inflammation. Statistical analysis was performed using an unpaired t-test; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3

Inhibiting innate immune cells from the spleen improves MISI by tick protease inhibitors. (A, B) Statistical results showing the effects of Sialostatin L, Sialostatin L2, Iristatin, and Mialostatin on dendritic cells (CD11b⁺CD11c⁺) at days 4 and 7 (n = 5/group). (C) Gating strategy and representative pictures showing CD11b⁺CD11c⁺ cells in the spleen at day 4 in the tick protease inhibitors treated mice (n = 5/group). (D, E) Effects of Sialostatin L, Sialostatin L2, Iristatin or Mialostatin on macrophages (CD11b⁺F4/80⁺) expression at day 4 and 7 (n = 5/group). (F) Representative plots of spleen cells from mice in MISI at day 7 that were treated with Sialostatin L, Sialostatin L2, Iristatin, or Mialostatin (n = 5/group). (G, H) Effects of Sialostatin L, Sialostatin L2, Iristatin, or Mialostatin on neutrophils (CD11b⁺Ly6G⁺ cells) expression in the spleen cells at day 4 and 7 were analyzed by flow cytometry (n = 5/group). (I) Representative pictures showing macrophage staining (CD11b⁺ cells) within the spleen cells from protease inhibitors treated mice (n = 5/group). Man, mannan; α-LA, alpha-lactalbumin, negative control. Results are shown from a representative experiment. All mice developed psoriasis-like inflammation. Statistical analysis was performed using an unpaired t-test; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4

Effect of tick protease inhibitors on γδ T and Th17 cells at the peak and end of psoriasis. (A, B) Statistical analysis on the effect of Sialostatin L, Sialostatin L2, Iristatin, or Mialostatin on γδ T cells from draining lymph nodes at the peak (day 4) as well as the end (day 7) of psoriasis (n = 5/group). (C) Gating strategy and representative blots showing γδ T cells (CD45⁺γδ T⁺) expression at day 4 in the tick protease inhibitors treated mice (n = 5/group). (D, E) The spleen cells’ frequency is depicted as a fraction of total Th17 cells at days 4 and 7 after Sialostatin L, Sialostatin L2, Iristatin, or Mialostatin treatment in MISI (n = 5/group). (F) Representative pictures show CD4⁺ IL-17A⁺ positive cells from the spleen at day 4 in MISI after tick protease inhibitor treatment (n = 5/group). Man, mannan; α-LA, alpha-lactalbumin, negative control. Results are shown from a representative experiment. All mice developed psoriasis-like inflammation. Statistical analysis was performed using an unpaired t-test; n ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

Tick protease inhibitors decreased mannan-induced skin inflammation by modulating the expression of pro-inflammatory cytokines. Statistical analysis showing the effects of Sialostatin L, Sialostatin L2, Iristatin, or Mialostatin on the mRNA expression of (A) TNF-α, (B) IL-6, (C) IL-22, (D) IL-23 (E) IL-17A, (F) IL-17E, (G) IL-17F, (H) IL-4 and (I) IL-10 in the skin lesions at the peak (day 4) as well as the end (day 7) of mannan induced skin inflammation after Sialostatin L, Sialostatin L2, Iristatin or Mialostatin treatment (n = 5/group). (A–G) PBS group was normalized to 1, and (H, I) mannan group was normalized to 1 for showing the differences between each group. Man, mannan; α-LA, alpha-lactalbumin, negative control. All mice developed psoriasis-like inflammation. Statistical analysis was performed using an unpaired t-test; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.