The effects of microbacterial mycolic acids on rodent tuberculosis and adjuvant arthritis

Abstract

Mycolic acids, the characteristic, abundant waxes of the cell wall of Mycobacteria were purified by Counter Current Distribution (CCD) from alkaline methanolytic crude extracts of bacteria, aiming at investigating their role in eliciting immune responses. Crude mycobacterial cell-wall extracts were first made by saponification in potassium hydroxide methanol solution. Purification was then performed with CCD using a bi-phasic tricomponent system, consisting of double distilled deionized water (dddH2O), chloroform and methanol. Emulsions were formed in this system which in turn extended the purification time. The addition of a preliminary funnel extraction step, to reduce the saponified fatty acids in the crude extract, before CCD and the addition of NaCI as an emulsions breaker in the CCD solvent system, produced a high yield of pure mycolic acids. The purity of these mycolic acids were assessed using reversed-phase HPLC-analysis. This method proved not only to be applicable to purify mycolic acids from M. tuberculosis but was also applicable in purifying mycolic acids from other sources, such as M. vaccae. The immunogenic properties of the purified mycolic acids were confirmed in experiments in which they induced the formation of antibodies in Sprague-Dawley rats when immunized in Marcol 52 oil. The antibody response was monitored by ELISA after 3 months of repeated immunization every second week. A dose-related response was observed for the induction of antibodies specific for mycolic acids, immobilized on the ELISA plates. Mycolic acids also appeared to influence adjuvant arthritis. Pure mycolic acids, suspended in mineral oil were administered intradermally into Lewis rats one week before the intradermal administration of an arthritis-inducing dose of lyophilized M. tuberculosis H37Ra. Animals receiving Mycobacteria, but no mycolic acids treatment, developed severe symptoms of arthritis within two weeks after bacterial challenge. No arthritis symptoms were apparent in mycolic acids treated rats. Mycolic acids treatment alone, did not produce arthritis. Mycolic acids pre-treatment of M. tuberculosis H37Rv-infected mice, rendered tuberculosis susceptible Balb/c mice more resistant. This resistance was equivalent to that observed in tuberculosis resistant C57Bl/6 mice. Post-infection treatment of M. tuberculosis H37Rv-infected mice with MA had no effect. Resistance of C57Bl/6 mice is commonly associated with the expression of IL-12 and IFN-ã. The effect of mycolic acids in the spleens of M. tuberculosis-infected Balb/c mice was investigated. It was observed that there was no significant change on the THI and TH2 cytokines. The absence of mycolic acids-induced THI/TH2 cytokine bias implied that protection was not provided by the expression of IL-12 and IFN-ã in the spleen. These results support the hypothesis that mycolic acids are immunogenic in respect of being able to induce specific antibodies, to provide resistance against tuberculosis and to prevent the development of adjuvant arthritis. The mechanism by which mycolic acids perform these tasks is unknown, particularly in these rodent models, which differ from humans, in that they do not have the CD1b that presents mycolic acids in humans. Unravelling this mechanism, can possibly aid the development of a pharmaceutical formulation that introduces MA into the body to enhance resistance to TB and prevent arthritis as an associated side-reaction.