Administration of Mycobacterium leprae rHsp65 aggravates experimental autoimmune uveitis in mice

Abstract

The 60 kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Figure 1. Administration of M. leprae rHsp65 increased EAU scores.

[A]: B10.RIII mice were immunized with 161–180 IRBP peptide followed by injection with rHsp65 [i.p.]. Eyes were collected for histopathology 21 days later. EAU scores were assigned on a 0 to 4 scale. [B]: Histopathological findings: a normal retinal architecture corresponding to non immunized naïve mice [normal]; IRBP immunized mice [average score 3; control]; mice immunized with IRBP followed by rHsp65 inoculation [average score 4; rHsp65]. Representative photographs [hematoxilin-eosin staining] of three independent experiments with n = 6–8 mice/group. Magnification: 200x.

Figure 2. rHsp65 inoculation increased IFN-γ and IL-10 levels.

Draining lymph nodes cells from either rHsp65 inoculated or control mice were harvested at day 21 and stimulated in vitro [10⁶ cells/ml] with 30 µg/ml IRBP [A, C, E] and 20 µg/ml M. leprae rHsp65 [B, D, F]. After 48 hours, IFN-γ [A, B], IL-10 [C, D] and IL-6 [E, F] levels were determined by ELISA. Data represent three independent experiments with n = 5 mice/group [*p<0.05 versus control group; t-test analysis].

Figure 3. Expansion of CD4⁺IFN-γ⁺ and CD4⁺IL-17⁺ T cells after rHsp65 inoculation.

Cells from draining lymph nodes collected at day 21 after immunization were labeled with anti-CD4 mAb, fixed and permeabilized, stained intracellularly and analyzed by flow-cytometry evaluating the CD4, IFN-γ and IL-17 expression. Results are expressed in mean ± SD. Data represent three independent experiments with n = 5 mice/group [*p<0.05; **p<0.01 versus control group; t-test analysis].

Figure 4. rHsp65 showed no effect on CD4+Foxp3+ T cells.

Cells from draining lymph nodes were harvested at day 21 after immunization, stained and analyzed by flow-cytometry evaluating the CD4 and Foxp3 expressions. Results are expressed in mean ± SD. Data represent three independent experiments with n = 5 mice/group.

Figure 5. rHsp65 modulated anti-IRBP IgG2a levels.

Serum levels of anti-IRBP IgG1 and IgG2a isotypes [A] and IgG anti-mouse Hsp60 and anti-rHsp65 [B] antibody production in IRBP-immunized mice either or not inoculated with rHsp65. Antibody levels were determined by ELISA at day 21 after immunization. Results are expressed in titers ± SD in each experimental group. Data represent three independent experiments with n = 6–8 mice/group [*p<0.05 versus control group; t-test analysis].