The Role of M. leprae Hsp65 Protein and Peptides in the Pathogenesis of Uveitis

Abstract

Experimental autoimmune uveitis (EAU) is a well established model for immune-mediated organ-specific disease. Our group has recently shown that the M. leprae Hsp65 aggravated the uveitis in mice; in the present study, we evaluated the action of M. leprae K(409)A mutant protein and the synthetic peptides Leader pep and K(409)A pep (covering amino acids residues 352-371 of WT and K(409)A proteins of M. leprae Hsp65, resp.) on the pathogenesis of EAU. Mice received the 161-180 IRBP peptide and B. pertussis toxin followed by the intraperitoneal inoculation of K(409)A protein or the Leader pep or K(409)A pep. The Leader pep aggravated the disease, but mice receiving the K(409)A pep did not develop the disease and presented an increase in IL-10 levels by spleen cells and a decrease in the percentage of CD4+ IFN-γ+ T cells. Moreover, animals receiving the Leader pep presented the highest scores of the disease associated with increase percentage of CD4+ IFN-γ+ T cells. These results would contribute to understanding of the pathogenesis of EAU and support the concept that immune responses to Hsp are of potential importance in exacerbating, perpetuating, or even controlling organ-restricted autoimmune diseases, and it is discussed the irreversibility of autoimmune syndromes.

Figure 1

Administration of K⁴⁰⁹A protein and Leader pep increased EAU scores, and injection with K⁴⁰⁹A pep decreased EAU scores. B10.RIII mice were immunized with 40 μg 161–180 IRBP peptide on day 0 and followed by a single injection with 2.5 μg of K⁴⁰⁹A, Leader pep, or K⁴⁰⁹A pep (i.p.). Eyes were collected for histopathology 21 days after immunization. EAU scores were assigned by histopathologic examination of the eyes on a scale from 0 to 4 according to the extent of inflammation and tissue damage. **P < 0.01: control versus K⁴⁰⁹A pep; K⁴⁰⁹A versus K⁴⁰⁹A pep; Leader pep versus K⁴⁰⁹A pep. Disease incidence: control group: 75% of animals presented grade 1.5; K⁴⁰⁹A: 75% of animals expressed grade 3; Leader pep: 75% of animals presented grade 3; K⁴⁰⁹A pep: 75% of animals expressed no disease.

Figure 2

Histopathologic features representative of the EAU scores. Mice control shows ocular lesions characterized by cells infiltrating the vitreous and vasculitis (a) and (b); in mice injected with K⁴⁰⁹A, it is possible to observe inflammatory cells in the vitreous and retinal folds (c) and (d); mice injected with Leader pep show cells in the vitreous and retinal folds (e) and (f); mice injected with K⁴⁰⁹A pep exhibit a normal retinal architecture corresponding to nonimmunized naïve mice (g) and (h). Representative photographs (hematoxylin-eosin staining). Original magnifications: 100× (left); 400× (right).

Figure 3

Expansion of CD4+IFN-γ+ T cells in lymph nodes (a) and spleen (b) after K⁴⁰⁹A, Leader pep, and K⁴⁰⁹A pep inoculation. Cells from draining lymph nodes and spleen collected at day 21 after immunization were labeled with anti-CD4 monoclonal antibody, fixed and permeabilized, stained intracellularly, and analyzed by flow cytometry evaluating the CD4, IFN- expression. Results are expressed in mean and SD. ***P < 0.001: K⁴⁰⁹A versus K⁴⁰⁹A pep (A); **P < 0.01: K⁴⁰⁹A versus K⁴⁰⁹A pep (B); ***P < 0.001: Leader pep versus K⁴⁰⁹A pep (B).

Figure 4

Analyses of IL-10 and IL-17 levels. Spleen cells from K⁴⁰⁹A, Leader pep, and K⁴⁰⁹A pep inoculated, or control mice were harvested at day 21 and stimulated in vitro with 30 mg/mL IRBP. After 72 hours, IL-10 (a) and IL-17 (b) levels were determined by ELISA. Results are expressed as mean ± SD. **P < 0.01: control versus K⁴⁰⁹A (A); *P < 0.05: control versus Leader pep (A); **P < 0.01: control versus K⁴⁰⁹A pep (A); ***P < 0.001: K⁴⁰⁹A versus K⁴⁰⁹A pep (A); ***P < 0.001: Leader pep versus K⁴⁰⁹A pep (A).

Figure 5

Autoimmune disease aggravation: Y: the individual state of an irreversible immune responsiveness; a: disease degree; ▲_i: entropy production, where i represents the number of autoimmune episodes being a time-dependent discrete variable.