Highly infective multidrug-resistant strain interferes with macrophage apoptosis

YOKOBORI N; GEFFNER L; LÓPEZ B; SCHIERLOH P; BALBOA L; ROMERO M; ALEMÁN M; RITACCO V; BARRERA L; DE LA BARRERA S; SASIAIN MC

Bangkok,Tailandia

Congreso; Pathogenesis and Control of Emerging Infections and Drug-Resistant Organisms; 2008

Keystone Symposia TM

In the early 90’s, Argentina was declared by WHO and IUATLD as  a “hot spot” for multidrug-resistant tuberculosis (MDR-TB).  Muñiz (M) and 410 strains are both MDR strains belonging to the Haarlem lineage. M was isolated in a hospital outbreak while 410, despite its identical RFLP genotype (has a single additional band) didn’t spread to the community. Although both were epidemiological, bacteriological and genetically characterized, little is known about their interaction with immune cells. The ability to inhibit macrophage apoptosis by Mycobacterium tuberculosis has been proposed as a strategy to subvert host immunity, but controversy remains. The best characterized cell death programs are the extrinsic or death receptor apoptotic pathway and the intrinsic or mitochondrial pathway both ending in caspase3 activation, but alternative mechanisms have been proposed, such as caspase independent cell death, autophagy and pyroptosis. We have previously reported that heat-killed (hk) M strain induced lower levels of apoptosis and TNF-α than the reference strain H37Rv (Rv) in monocyte derived macrophages (MDM). Moreover, we have determined that while M interferes with Rv-induced apoptosis, 410 does not. The aim of this study was to characterize the pathways that might be involved in the inhibition of Rv-induced apoptosis by the M strain. TNF-α, through its binding to TNFR1, has been proposed as the main mediator of mycobacteria-induced apoptosis. However, exogenously added rTNF-α didn’t increase M-induced MDM apoptosis after a 5 hour stimulation (%Annexin V+: Rv=25.3±5%, M=8.9±1.3%; M+50ng/ml rTNF-α=8.6±2.0%; 1:10 MDM:Mtb ratio). The intrinsic pathway was evaluated in MDM pre-treated with the strains and afterwards apoptosis was induced with staurosporin (ST,0.5µM). We observed that M and 410 strains induced higher resistance to death than Rv (ST =31.0±0.4%, Rv+ST=26.6±4.6%, M+ST=19.1±4.4%, 410+ST=15.5±3.8%; Rv vs M/410: p<0.01; 1:2 MDM:Mtb ratio). In the presence of PDTC (an inhibitor of NFκB activation) M and Rv induced higher MDM apoptosis while 410 did not. Activation of caspase3 was only observed with Rv and in a lesser extent with M.  Mtb owes a great part of its pathogenicity to the complex cell wall it has. Many virulence factors have been described, some of them have antagonistic effects when isolated: e.g. the secretory protein ESAT6 is pro-apoptotic while the cell wall component ManLam is anti-apoptotic. The immunogenic capacity of Mtb will rely on the balance between different signals induced by the whole cell. Heat inactivation of Mtb leads to the loss of activity and structure of labile antigens,  so  next we assed the death inducing capacity of γ-irradiated (γ -i) strains.  All  of the treatments induced  increasing levels of cell death,  but we found a striking difference between hk and  γ-i  Mtb:  while the first group induced  mainly apoptosis,  in the second  prevailed a necrotic phenotype.  This was particularly pronounced for γ-i 410 strain,  even at low doses.  As a control, the vaccine strain Mycobacterium bovis BCG  was used. Our data  shows that although M and 410 are closely related strains, the mechanisms they employ to induce MDM apoptosis are different. In addition, hk-M could be interfering with the Rv-triggered intrinsic pathway. Necrotic cells release intracellular content after plasma membrane rupture and this leads to a inflammatory response. The low infectivity of 410 may rely in part to the high necrosis it induces. Further studies must be performed  to better characterize  this phenomenon.