Outbreak MDR Strains of M. tuberculosis (Mtb) Induce Differential TNF-alpha Secretion by Macrophages Altering CD54 Expression on Bronchial Epithelial Cells

KVIATCOVSKY DENISE; BALBOA LUCIANA; SCHIERLOH PABLO; LOPEZ BEATRIZ; SASIAIN MARÍA DEL CARMEM; DE LA BARRERA SILVIA

Medellín

Congreso; XI Congress InmunoColombia 2015; 2015

Asociación latinoamericana de Inmunología

Mycobacterium tuberculosis, which causes tuberculosis (TB), is one of the most successful human pathogens and its evolution is not well known. It is probably that over the last century human intervention, particularly massive vaccination with BCG and drug treatment, has exerted a selective pressure from which have survived the best adapted genotypes. The influence of genetic variation of Mtb on tuberculous infection is a recent research area that, as in other infections, demonstrates that they have an essential role in the onset of TB. The modern Mtb evolves through deletion, duplication and recombination events and single nucleotide polymorphism (SNP) that gave rise to the emergence of separate strain lines with different pathogenic characteristics. The understanding of the impact of the genetic diversity of Mtb on the development of TB and host immune response were initiated from works describing strains that produced TB outbreaks worldwide. It is postulated that Mtb prevalent genotypes have developed mechanisms that have allowed them to evade the immune response and adapt to its host. Such mechanisms involve receptors which would recognize antigens in the clinical isolates and not in the reference strain H37Rv, triggering an immune response that would differ from H37Rv.Mtb mainly infects the lung by interacting with airway epithelium and resident macrophages (Mac). The immune response in the lung is finely balanced since the immune system has the ability to mount a protective immune response against certain microorganisms and at the same time is able to establish tolerance mechanisms against commensal microorganisms as well as self-antigens. Lung epithelial cells act as a first line defense against a wide variety of pathogens including mycobacterias. Furthermore the lung epithelium not only acts as a physical barrier but play an important role as a modulator of the immune response by secreting a variety of cytokines (CKs), chemokines (QKS), defensins, etc. The adhesion of pathogens to epithelial cells of the respiratory tract is a fundamental requisite for colonization and subsequent infection. In this context, the respiratory epithelium play a major role through identifying microorganisms by specialized receptors, leading to the initiation of an inflammatory response which will impact on the immune response developed by the host. There is evidence that the bronchial epithelial cells have an important role in the induction, amplification and regulation of the inflammatory response. Bronchial airway epithelial cells are able to recognize lot of pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) that allow a quick response to a wide variety of pathogens. Besides, infected alveolar macrophages produce cytokines such as IL-1β and TNF-α that also induce cytokines production by lung epithelial cells, thus enhancing the inflammatory response.In 2006, the Laboratory of Immunology of Respiratory Diseases (IMEX-CONICET) initiated studies about the impact of the genetic variability of Mtb clinical isolates prevalent in Argentina on the innate and adaptive immune response in humans. For this purpose, sensitive to drugs and drug multiresistant Mtb strains from Haarlem and LAM families have been used. Human intervention, especially the inadequate treatment of TB and/or lack of commitment of the patient to treatment, have favored the multiplication of drug resistant Mtb strains. Epidemiological, bacteriological and genotyping studies have allowed the isolation and identification of outbreak strains Muñiz (M), Ra and H37Rv reference strain, which have spread to the community. We have previously demonstrated that certain soluble factors released by Mac stimulated with MDR strains, M and Ra differentially modulate CD54 expression in the human bronchial epithelial cell line Calu-6. The aim of this work was to identify those soluble factors involved in CD54 regulation in airway epithelial cells. To do so, monocytes derived Mac from healthy donors were stimulated with M, Ra or H37Rv strains for 6h at Mtb:Mac 1:2 or 1:5 ratios and TNF-alpha levels were determined by FACS and ELISA. In addition, Calu-6 cells were co-cultured with Mac or stimulated Mac-derived supernatants (Mac-SN) and CD54 expression was evaluated. To confirm the role of CKs on CD54 modulation, Calu-6 cells were treated with Mac-SN in the presence of a synthetic soluble TNF-alpha receptor (Etanercept) and/or anti-IL-1beta monoclonal antibody.Results: 1) M-stimulated Mac produced lower levels of released (n=7) or intracellular (n=9) TNF-alpha than H37Rv-stimulated Mac (p