A novel cystatin derived from Trichinella spiralis suppresses macrophage-mediated inflammatory responses

Abstract

Trichinella spiralis can modulate host immune responses to retain a suitable environment for its long-term survival. Incidentally, the parasite elicits regulatory effects through immunomodulatory molecule release, which can suppress host inflammation and may be used for the treatment of unrelated inflammatory diseases in someday. Here we identified and characterized a novel T. spiralis cystatin (TsCstN), which inhibits inflammation mediated by LPS-treated macrophages.Proteins contained in the excretory-secretory (ES) product of muscle-stage T. spiralis (ES-L1) were fractionated, and each was treated with mouse bone marrow-derived macrophages (mBMDMs) before LPS stimulation. The fractions that exhibited high immunomodulatory property by decreasing pro-inflammatory cytokines or increasing anti-inflammatory cytokines were identified by mass spectrometry. Incidentally, the conserved hypothetical protein (Tsp_04814) was selected for further characterization as it presented the most significant MS score. An annotation of Tsp_04814 using protein structural homology comparison suggested that it has high structural similarity to human cystatin E/M (TM score 0.690). The recombinant T. spiralis novel cystatin (rTsCstN) was expressed in Escherichia coli at a molecular weight of approximately 13 kDa. Mouse anti-rTsCstN polyclonal antibody (pAb) could detect native TsCstN in crude worm antigens (CWA) and ES-L1 and be predominantly localized in the stichosome and subcuticular cells. rTsCstN inhibited cysteine proteases in vitro, especially cathepsin L, at an optimal pH of 6. Besides, rTsCstN could be internalized into mBMDMs, which were mostly distributed in the cytoplasm and lysosome both before and after LPS stimulation. To evaluate the rTsCstN immunomodulatory properties on mBMDMs, rTsCstN was incubated with mBMDM before LPS stimulation; this demonstrated that rTsCstN suppressed pro-inflammatory cytokine production and MHC class II expression.T. spiralis L1-derived TsCstN was characterized as a novel cysteine PLOS NEGLECTED TROPICAL DISEASES

Trichinella spiralis:inflammation modulator

“…Breg cells participate in the suppression of experimental autoimmune encephalomyelitis (Wolf et al ., 1996). ES molecules that are secreted from T. spiralis induce Breg cells (Kobpornchai et al ., 2020); however, the molecules that are involved in this induction remain unclear.…”

“…As new identified molecular from T. spiralis , T. spiralis calreticulin (Ts-CRT) can bind to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages (Zhao et al ., 2017). Trichinella spiralis novel cystatin (TsCstN) elicits an anti-inflammatory property by suppressing proinflammatory cytokines and interfering with the antigen presentation process through depletion of MHC class II expression (Kobpornchai et al ., 2020). As an immune-modulator, T. spiralis glutathione-S-transferase (TsGST) participates in regulating maturation and function of DCs and induces the Th2-type immune response of its host (Jin et al ., 2019; Yang et al ., 2020a).…”

“…Trichinella spiralis ES products that enable cross-talk between the parasite and the host have attracted a lot of attention (Wang et al ., 2017). Obtained results indicated that this protein complex mixture from ES products and its particular components (either in native or recombinant form) could largely reproduce the effects of infection on the host immune system (by shifting the immune response from pro- to anti-inflammatory type) (Aranzamendi et al ., 2013a; Yang et al ., 2014; Kobpornchai et al ., 2020). Therefore, almost any research performed in the field seeks to explore the molecular mechanisms and to identify molecules important for creation of the anti-inflammatory milieu achieved by this parasite.…”

The Two Faces of Nematode Infection: Virulence and Immunomodulatory Molecules From Nematode Parasites of Mammals, Insects and Plants

“…Specifically, Bm cystatin led to increased Tregs in the colon and alternative activation of peritoneal macrophages. Recently, cystatin from the ES products of the zoonotic nematode T. spiralis , rTsCstN, was discovered as structurally homologous to human cystatin ( Kobpornchai et al, 2020 ). Functionally, rTsCstN suppressed inflammatory cytokine production by LPS-treated mouse bone marrow derived macrophages.…”

Parasite Cystatin: Immunomodulatory Molecule with Therapeutic Activity against Immune Mediated Disorders

“…Downmodulation of HLA-DR and CD86 expression by the parasite cystatins suggest an alternative receptor-mediated effect of cystatin on APC. Recombinant tick cystatin and Trichinella spiralis cystatin also show similar modulatory effects on APC hampering antigen presentation and downregulation of surface expression of CD80 and CD86 [ 65 , 66 , 67 ].…”