Influence of genetic background on human monocyte derived macrophage (MDM) gene expression in response to infection with Mycobacterium tuberculosis H37Rv and treatment with inhalable microparticles containing anti -TB drugs

Abstract

Tuberculosis (TB) is prevalent in most parts of the world, to varying extents (Fig 1.1). However, it is estimated that just about 10% of individuals infected with Mycobacterium tuberculosis (Mtb) and congener pathogens develop active disease (Ducati et al., 2006; Dye et al., 1999, WHO, 2007). Multiple factors are responsible for this phenomenon, such as nutrition, age, living conditions, immune status and genetic predisposition of the host on one hand, and virulence and drug resistance of infectious strains on the other (Lopez, Aguilar et al. 2003; McShane 2003). Recent work in our lab has proposed the use of inhalable microparticles containing a combination of anti-TB drugs for use as adjunct therapy in the treatment of pulmonary TB (Misra, Hickey et al. 2011). Preclinical studies indicate that this drug delivery system possesses high efficacy against experimental TB in mice and guinea pigs (unpublished). High efficacy in animal experiments has been ascribed to the ability of inhaled microparticles to be phagocytosed by alveolar macrophages infected with Mtb. In the macrophage cytosol, microparticles not only deliver a large amount of drug within a small, enclosed space (Sharma, Saxena et al. 2001; Muttil, Kaur et al. 2007; Verma, Kaur et al. 2008) , but also apparently “stimulate the phagocyte” as recommended by Koch ahundred years ago (Young 2001). Such stimulation results in rescue of infected macrophages from alternative activation imposed by the pathogen (Gordon 2003) and elaboration of markers of a classically-activated phenotype. Thus, phagocytosed microparticles induce the production of bactericidal free radicals (Sharma, Muttil et al. 2007), Th1 cytokines (Sharma, Muttil et al. 2007; Yadav, Muttil et al. 2010) from infected macrophages, and drive the cells to apoptosis rather than necrosis (Yadav and Misra 2007). Genome wide-transcription analysis suggests that genes associated with apoptosis, proinflammatory cytokines, and innate defence responses are differentially regulated in macrophages infected in vitro with Mtb as a result of treatment with anti-TB drugs in solution, versus the same drugs in microparticles (unpublished). Since there is a great deal of genetic variation relevant to host defense responses of humans, it is important to investigate whether observations made using a single defined strain of Mtb (H37Rv) infecting macrophages of a single defined genotype (THP-1) would hold in the context of genetic variation observed in a sample of a human population. The present investigations were undertaken to evaluate host response and bacterial survival when primary macrophages derived from human volunteers were infected in vitro with Mtb H37Rv and treated with inhalable microparticles containing isoniazid (INH) and rifabutin (RFB) reported extensively from the lab. TB is a chronic granulomatous disease of humans and animals and has zoonotic importance. It is one of leading cause of death worldwide causing WHO to declare as global health emergency in 1994, the single bacterial disease so far. About 1/3rd of the world population is harboring Mtb in their body and about 10% of infected individuals progress to active TB (Ducati et al., 2006; Dye et al., 1999, WHO, 2007). TB incidence rates are shown in Fig 1. Infections are occurring at the rate of 1 per second (WHO 2006). There were about 8.8 million cases of TB in 2010, with Asia and Africa contributing 59% and 26% of total cases (WHO 2011). India alone accounted for an estimated 26% of all TB cases worldwide, and China and India combined accounted for 38% (WHO 2011). TB is more prevalent in low income countries and is the second largest cause of death worldwide after HIV/AIDS (WHO 2011). Rising numbers of cases in the developed world have been attributed to increase in HIV/AIDS, use of immunosuppressive drugs or substance abuse (WHO 2006). It is estimated that by year 2020, TB will be one of the world top10 global diseases with about 1 billion new cases (Murray & Salomon, 1998; Pasqualoto & Ferreira, 2001). Mtb is usually transmitted by the aerosol route. Coughing by TB patients releases droplets that contain Mtb, and inhalation of such droplets by another person leads to infection (Kaufmann 2001). Once inside the lung, Mtb is phagocytosed by alveolar macrophages, initiating host immune response involving T cell activation, cytokine and other chemokine secretion leading either to elimination of bacteria or progression to disease (Henderson et al., 1997; Roach et al., 2002). On entry into macrophages, bacteria reside in an endocytic vacuole called the phagosme. Mtb and other intracellular bacteria have evolved strategies to prevent phagosome maturation, i.e phagosome- lysosome fusion which would have lead to acidic pH environment, reactive oxygen intermediate release, release of lysosomal content and bactericidal peptides inside the phagosome. Phagosome acidification is hindered due to exclusion of proton ATPases from mycobacterial phagosome. Moreover, mycobacteria inhibit Ca2+ signaling which would have lead to host response against infection. Mtb infection leads to formation of tubercles in the affected part, consisting of defense cells that congregate with the objective of killing these bacilli or restricting the spread of infection. It is the strength of the host cellular response vis-à-vis the virulence of the infecting strain that apparently decides whether infection will be contained or progress to a later stage. The contained infection is known as latent infection and may persist for the whole life without any activation in individuals capable of mounting effective cellular responses. However, at some point of time, if immunity falls because of malnutrition, immunodifficency disease/HIV, ageing or other factors the granuloma become liquefied and bacteria replicate there, followed by spread to nearby areas of the lungs and may disseminate to other parts of the body. Thus the pathogen uses phagocytic cell as its ecological niche, where it lives, replicates and persists (Henderson et al., 1997; Roach et al., 2002). It is well known that host genetics as well as environmental factors have important roles in the outcome of exposure and infection (Casanova & Abel, 2002). It is estimated that 10% of immunocompetent people exposed to Mtb infection develop the disease while the rest 90% never develop do so (Murray et al, 1990). 1.2.1 Mycobacterium tuberculosis complex Six closely related organisms form the Mycobacterium tuberculosis complex implicated in TB: M. tuberculosis (mostly human tuberculosis), M. bovis (mostly bovine and other animals including human), M. microti (mostly in small mammals) M. africanum (mostly in humans and animals in sub Saharan African countries), Bacille Calmette-Guerin (BCG, attenuated strain of M. bovis) and M. canetti, (rarely encountered but can cause human disease) (Greenwood et al., 1997). Mtb is an obligate aerobe, non-motile, non-sporulating, non-capsulating, has straight or slightly curved rods and may occur in clumps or individually (Casanova and Abel 2002). The importance of oxygen for Mtb can be gauged by the predominance of bacteria in oxygen rich tissues like the lung, especially its upper lobe. It has been classified into a distinct group of acid-fast organisms because it does not have characteristics of either Gram‟s positive or negative organisms although it contains peptidoglycan (murein) in the cell wall (Prescott et al., 1996). Mtb falls in the class of slow growers, requiring 3-4 weeks to form colonies with doubling time of about 24 hr. This property is thought to contribute to a less vigorous antimicrobial response by macrophages (McKinney 1998).