Amblyostatin-1, the first salivary cystatin with host immunomodulatory and anti-inflammatory properties from the Neotropical tick , vector of Brazilian spotted fever.

INTRODUCTION

The Neotropical tick is the primary vector of , the causative agent of Brazilian spotted fever, a disease associated with high fatality rates. Tick saliva, a complex mixture of bioactive molecules essential for successful blood feeding, facilitates pathogen transmission and modulates host immune responses. A comprehensive evaluation of the salivary gland transcriptome database reveals that protease inhibitors are abundantly expressed molecules in tick saliva during feeding. Thus, this study aims to describe and characterize the most expressed member of the cystatin family identified in salivary transcriptome, named Amblyostatin-1.

METHODS

Bioinformatic tools were employed for analysis of the Amblyostatin-1 sequence and structure. A recombinant version of Amblyostatin-1 was expressed in an system, evaluated against a panel of cysteine proteases in biochemical assays, and used to generate antibodies in immunized mice. The biological activities of Amblyostatin-1 were assessed by its effects on dendritic cell maturation and in a carrageenan-induced inflammation model .

RESULTS

Based on its sequence and predicted three-dimensional structure, Amblyostatin-1 is classified as an I25B cystatin, and its recombinant form selectively inhibits cathepsins L, C, and S at different rates, with a low nanomolar value of 0.697 ± 0.22 nM against cathepsin L. Regarding its biological activities, recombinant Amblyostatin-1 partially affects LPS-induced dendritic cell maturation by downmodulating the costimulatory molecules CD80 and CD86 at higher micromolar concentrations (3 µM) while promoting IL-10 production at nanomolar concentrations (100 nM). The apparent lack of Amblyostatin-1-specific antibody responses in immunized mice suggests an impairment of antigen processing and presentation . Furthermore, in a carrageenan-induced inflammation model, Amblyostatin-1 decreased edema formation and neutrophil infiltration into the skin without affecting other myeloid cells.

DISCUSSION

These findings establish Amblyostatin-1 as a novel salivary cystatin with immunomodulatory and anti-inflammatory properties, highlighting its potential as an immunobiological agent.