ORIGINAL ARTICLE
Lung granulomas from Mycobacterium tuberculosis/HIV-1 co-infected patients display decreased in situ TNF production

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Abstract

Tuberculosis/HIV-1 co-infection is responsible for thousands of deaths each year, and previous studies have reported that co-infected individuals display major morphological alterations in tissue granulomas. The purpose of this study was to evaluate immunohistopathological characteristics in lung tissues from pulmonary TB/HIV-1-co-infected individuals. Following autopsy, tuberculosis-positive HIV-1-negative cases displayed granulomas with normal architecture, mainly composed of a mononuclear infiltrate with typical epithelioid, as well as giant cells, and exhibiting caseous necrosis. In contrast, lesions from the TB/HIV-1-co-infected group showed extensive necrosis, poorly formed granulomas, and a marked presence of polymorphonuclear cells. More importantly, TNF staining was greatly reduced in the TB/HIV-1-co-infected individuals. Our data suggest that HIV-1 infection alters the organization of pulmonary granulomas by modulating TNF and, possibly, cell trafficking, leading to an impaired anti-tuberculosis response.

Introduction

Mycobaterium tuberculosis infection is endemic throughout the world and causes many deaths. The World Health Organization (WHO) estimates that the occurrence of tuberculosis (TB) exceeds 14 million cases per year, with a mortality of 2 million people per year [33]. The most common form is pulmonary TB, which presents a high prevalence and incidence in the developing world due to poor sanitary conditions and malnutrition. In addition, HIV-1 infection is an important factor that increases the incidence and severity of tuberculosis. While AIDS clearly leads to heightened susceptibility to mycobacterial infection, evidence also suggests that M. tuberculosis may accelerate the progression of HIV disease through a process of immune activation of viral expression [5], [9], [14], [32]. However, the interaction between HIV-1 and M. tuberculosis in different tissues, particularly in lungs, is not fully understood.

The hallmark of M. tuberculosis-infected tissues is the granuloma, a cellular accumulation around the bacilli that is comprised mainly of macrophages, epithelioid cells, multinucleated giant cells, and surrounded by a rim of T lymphocytes [23], [25]. The main functions of granulomas are thought to be the containment of the infection preventing bacterial spread to surrounding tissues and organs, and to concentrate the immune response to a limited infectious area [23]. A number of reports have shown that a well-structured granuloma is critical to control M. tuberculosis infection, and a role for cytokines, such as TNF to restrict mycobacteria growth, has been demonstrated in both human and experimental tuberculosis [4], [7], [13]. For example, it has been shown that in the absence of TNF, the initial recruitment of monocytes into the site of infection is impaired with influx of inflammatory cells (e.g. neutrophils) and development of necrotic lesions [4], [12], thus suggesting that TNF is involved in granuloma development. In contrast, it has been recently reported by Iliopoulus et al. that three patients, receiving anti-TNF therapy, did develop normal granulomas but failed to control disease [16]. Whether TNF plays a direct role in mycobacterial-induced granuloma in humans remains to be determined.

HIV-1 has been shown to influence the clinical course of M. tuberculosis infection [6]. These pathogens are co-endemic in many areas of the world, and patients simultaneously infected with these two agents exhibit greater morbidity and mortality than individuals harboring either pathogen alone [5], [9]. However, immuno-pathological features for the breakdown of host resistance in dually infected individuals are unclear. During TB, the production of pro-inflammatory cytokines, such as TNF, may be important to control disease, and a change in this profile in dually infected patients has been reported [8], [15], [31]. In contrast, M. tuberculosis can induce HIV-1 expression in both TNF-dependent and -independent pathways in vitro, as well as experimental models [2], [3], [26], [31], suggesting that while important in restricting bacterial proliferation, TNF could stimulate HIV-1 expression in infected tissues. Elevated HIV-1 replication may lead to decreased CD4+T cell counts and could affect granuloma formation and IFN-γ synthesis [18]. These findings indicate that complex in situ responses to both pathogens take place in co-infected individuals. Despite its importance, in situ granuloma responses that are present in the lungs in TB/HIV-1-co-infected individuals are not well characterized.

In the present study, we analyzed lung samples obtained from autopsies of TB/HIV-1-co-infected and TB-positive but HIV-1-negative individuals to investigate the local immune response and histopathological patterns of M. tuberculosis-induced granulomas in dually infected patients. We found that, in addition to major alterations in the TB granuloma aspects, TB/HIV-1 co-infected individuals displayed an impaired TNF production in the granuloma. This result suggests that HIV-1 infection modulates immunological responses in the lung granulomas, which may influence bacterial growth restriction, as well as virus replication in situ.

Section snippets

Necropsy study

We performed a retrospective necropsy study of adults who had died from pulmonary tuberculosis between June 1995 and June 2001 at the Prof. Edgar Santos University Hospital, Bahia, Brazil. To prevent autolysis and tissue disintegration, the time between death and autopsy was approximately 4 h (minimum legal interval between death and autopsy required by Brazilian legislation), and the samples were immediately fixed in 10% formalin. Cases had a mean age of 34 years (range: 18–40) with a 1.5:1

Morphological features of TB/HIV-1-positive lung granulomas

To investigate whether HIV-1 co-infection influences granuloma composition in TB patients, we compared several morpho-immunological aspects of TB/HIV-negative and TB/HIV-1-positive lung samples. The number of granulomas per slide varied from 4 to 10, and all lesions displayed a concentric aspect as observed in mature granulomas. As expected, lung collected at autopsy from the TB/HIV-1-negative group displayed well-organized typical granulomas with three defined zones: (1) a central area of

Discussion

The present study investigates the possible effect of HIV on morpho-immunological features of M. tuberculosis-infected lungs. Our results showed that lungs from TB/HIV-1-co-infected cases display a major change in the cellular composition of the tuberculous granuloma associated with differences in acid-fast bacilli burden. Indeed, the morphology of these lesions is completely distinct from the typical granuloma observed in tissues from pulmonary TB (HIV-negative) cases. These findings suggest

Acknowledgment

This work was supported by Conselho Nacional de Pesquisa (CNPq). We are grateful to Dr. Tania Correia for her excellent technical assistance, Dr. Cristiane Cunha Frota for providing us with the IS6110 primers, and Dr. Charles A. Scanga (NIH) and Renata Leal (UFBA) for their helpful discussions during the preparation of this manuscript. MB-N is a Senior CNPq fellow.

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