Tuberculosis and diabetes: from bench to bedside and back.

People living with diabetes (DM) are at increased risk to become infected with Mycobacterium tuberculosis, to progress from latent tuberculous infection to active tuberculosis (TB) disease, and to suffer adverse TB treatment outcomes. In some low- and middle-income countries, DM prevalence among newly diagnosed TB patients exceeds 40%. Despite the global significance of DM as an acquired TB risk factor, the biochemical and cellular mechanisms of susceptibility are incompletely understood. This review summarizes the landscape of basic research using animal models of the TB-DM interaction, and the extent to which findings in animal studies reflect or may explain the clinical features of TB-DM comorbidity in humans. We conclude that immunopathy results in damage to major organs as a complication of DM, likely operating through biochemical pathways such as those responsible for diabetic nephropathy, neuropathy, retinopathy, cardiovascular disease, and delayed wound healing. Insights gained from animal models can inform optimal management of TB-DM comorbidity and will be essential for pre-clinical development of therapeutic countermeasures.

Convergence of non-communicable diseases and tuberculosis: a two-way street?

The intersection of tuberculosis (TB) with non-communicable diseases (NCDs), including diabetes mellitus (DM), chronic lung disease (CLD), and cardiovascular disease (CVD), has emerged as a critical clinical and public health challenge. Rapidly expanding NCD epidemics threaten TB control in low- and middle-income countries, where the prevention and treatment of TB disease remain a great burden. However, to date, the notion that TB may adversely impact NCD risk and severity has not been well explored. This review summarizes biomedical hypotheses, findings from animal models, and emerging epidemiologic data related to the progression of DM, CLD and CVD during and after active TB disease. We conclude that there is sufficient empirical evidence to justify a greater research emphasis on the syndemic interaction between TB and NCD.

TNF-dependent BALB/c murine macrophage apoptosis following Mycobacterium tuberculosis infection inhibits bacillary growth in an IFN-gamma independent manner.

SETTING: In vitro model of murine macrophage M. tuberculosis infection. OBJECTIVE: To evaluate the association and cytokine control of host cell apoptosis and bacillary killing in M. tuberculosis -infected murine peritoneal macrophage (PM). DESIGN: Murine PM from different strains of mice were infected with H37Ra. Bacillary growth and macrophage apoptosis were evaluated under different cytokine conditions. RESULTS: Like human alveolar macrophages, PM from BALB/c mice were found to undergo apoptosis after infection with M. tuberculosis in a TNF-dependent manner. Neutralizing TNF with anti-TNF antibody inhibited PM apoptosis following infection, and resulted in increased bacillary growth. Pre-treatment of PM with interferon (IFN-gamma) resulted in significant killing of the infecting bacilli, which was not dependent on TNF or apoptosis of the cells. In contrast to BALB/c mice, PM from C3H/HeJ mice did not undergo apoptosis following infection and did not undergo TNF- and apoptosis-dependent inhibition of bacillary growth. CONCLUSION: These findings suggest that TNF contributes to macrophage inhibition of M. tuberculosis growth by a mechanism that is dependent on apoptosis and independent of IFN-gamma activity. This protective phenotype was not seen in all strains of mice and merits investigation as a marker of mycobacterial host susceptibility.

Prospects for IL-16 in the treatment of AIDS.

IL-16 is a multi-functional cytokine that uses CD4 as a receptor to signal diverse biological activities by target cells including T-lymphocytes, monocytes and eosinophils. IL-16 has been shown to repress HIV-1 infection in lymphocytes and monocytic cells and it is active against both laboratory and naturally acquired virus isolates. In lymphocytes, the repressive effect of IL-16 occurs at the level of virus transcription, while it appears to inhibit viral entry in monocytic cells. Clinical studies comparing serum IL-16 levels with the state of HIV-1 disease suggest that this cytokine is a functionally significant endogenous antiviral factor. The antiviral activity of IL-16 may be of therapeutic benefit in HIV/AIDS but its greatest potential is for immune reconstitution. Stimulation of CD4+ T-cells with IL-16 primes cells to respond to IL-2, by upregulating the expression of IL-2 receptor p75 (CD25). Co-treatment of peripheral blood mononuclear cells (PBMC) with IL-16 plus IL-2 (or IL-15) in vitro selectively expands the population of CD4+ T-cells. Clinical trials of recombinant IL-2 have already shown promise in HIV/AIDS. In combination with IL-16, the beneficial effects of IL-2 may be augmented and specifically targeted to CD4+ T-cells. Thus, IL-16 shows considerable promise as an agent for the biological therapy of HIV/AIDS.

Macrophage stimulation with Murabutide, an HIV-suppressive muramyl peptide derivative, selectively activates extracellular signal-regulated kinases 1 and 2, C/EBPbeta and STAT1: role of CD14 and Toll-like receptors 2 and 4.

The smallest unit of bacterial peptidoglycans known to be endowed with biological activities is muramyl dipeptide (MDP). A clinically acceptable synthetic derivative of MDP, namely murabutide (MB), has been found to present interesting pharmacological properties and to suppress HIV-1 replication in monocyte-derived macrophages (MDM). We have addressed the signaling events activated in MDM following stimulation with either MB or the potent immunostimulant LPS. We also examined whether signaling by muramyl peptides involves the use of cell surface receptors, including CD14 and Toll-like receptor 2 (TLR2) or TLR4 that are known to be signal-transducing receptors for other bacterial cell wall components. We demonstrate that, unlike LPS, the safe immunomodulator MB selectively activates extracellular signal-regulated kinases (Erk) 1/2, in the absence of detectable Jun N-terminal kinase (JNK) or p38 mitogen-activated kinase activation. Furthermore, STAT1 activation but weak or no activation of STAT3 or STAT5 respectively, could be detected in MB-stimulated MDM. Using MonoMac6 cells, we observed high C/EBPbeta and AP-1 but weaker and transient NF-kappaB activation by MB.Moreover, the truncated form of C/EBPbeta, known to repress HIV-1 transcription, was detected in extracts from MB-treated THP-1 cells. Surprisingly, neither MB nor MDP were able to transduce signals via CD14 and TLR2 or 4. These findings present major differences in the early cell activation process between LPS and muramyl peptides, and strongly argue for the implication of co-receptors other than TLR2 and TLR4 in mediating the signaling events induced by defined subunits of bacterial peptidoglycans.

Nuclear translocation of the N-terminal prodomain of interleukin-16.

Interleukin-16 (IL-16) is a pleiotropic cytokine that functions as a chemoattractant factor, a modulator of T cell activation, and an inhibitor of human immunodeficiency virus (HIV) replication. These diverse functions are exclusively attributed to the secreted C-terminal peptide of 121 amino acids (mature IL-16), which is cleaved from the precursor protein (pro-IL-16) by caspase-3. Human pro-IL-16 is comprised of 631 amino acids with three PDZ domains, one of which is present in secreted mature IL-16. No cellular localization or biologic functions have been ascribed to the unusually large and highly conserved N-terminal prodomain formed as a result of proteolytic release of the third PDZ domain of pro-IL-16. Here we show that the N-terminal prodomain of pro-IL-16 translocates into the nucleus following cleavage of the C-terminal segment. The nuclear localization signal of pro-IL-16 consists of a classical bipartite nuclear targeting motif. We also show that the nuclear targeting of the IL-16 prodomain induces a G(0)/G(1) arrest in the cell cycle. Taken together, the high degree of conservation of the prodomain among species, the presence of two PDZ motifs, and the nuclear localization and subsequent inhibitory effect on cell cycle progression suggest that pro-IL-16 is cleaved into two functional proteins, a C-terminal-secreted cytokine and an N-terminal product, which affects the cell cycle.

Human immunodeficiency virus type 1 infection of alveolar macrophages impairs their innate fungicidal activity.

Impaired adaptive immunity is the hallmark of AIDS, but the effects of human immunodeficiency virus type 1 (HIV-1) infection on innate immunity are less clear. Cryptococcus neoformans (CN) is a common AIDS-related fungal pathogen acquired by inhalation. Alveolar macrophages (AM) comprise the initial host defense in cryptococcosis and they may arrest infection before dissemination occurs. We hypothesized that HIV-1 infection of AM impairs their anti-cryptococcal activity. This was tested by infection of normal AM with the M-tropic strain HIV-1(Bal). Two weeks postinfection we measured fungistatic activity against CN by colony counting, binding, and internalization of CN by confocal microscopy and AM cell viability by Alamar Blue assay. Uninfected AM from most donors demonstrated innate fungicidal activity against CN. In HIV-1-infected AM, there was a significant reduction, and in most cases loss, of fungicidal activity compared with the uninfected AM. The reduced antifungal activity was not due to any cytotoxic effect of HIV-1, and HIV-1 infection did not impair binding or internalization of yeast by AM. Thus, the innate fungicidal activity of primary human AM is impaired after HIV-1 infection in vitro by a mechanism involving a defect of intracellular antimicrobial processing.

Interleukin-16.

Interleukin 16 (IL-16) was initially described in 1982 as the first T cell chemoattractant. Through interaction with CD4, IL-16 has now been characterized as a chemoattractant for a variety of CD4+ immune cells. Recent in vivo studies have more fully characterized IL-16 as an immunomodulatory cytokine that contributes to the regulatory process of CD4+ cell recruitment and activation at sites of inflammation in association with asthma and several autoimmune diseases. Since its cloning in 1994, IL-16 structure and function have been studied extensively. This review addresses the current data regarding IL-16 protein and gene structure; the expanding list of cells capable of generating IL-16; the direct interaction of IL-16 with its receptor, CD4; and the functional bioactivities of IL-16 as they relate to inflammation and HIV-1 infection. In addition, potential therapeutic modalities for IL-16 relating to inflammation and immune reconstitution in HIV-1 infection are also discussed.

Interleukin 16: implications for CD4 functions and HIV-1 progression.

In this article, David Center and colleagues clarify the controversies that have emerged over the unique structure of interleukin 16 and its anti-HIV-1 activity. Interleukin 16 is a ligand for CD4, and this implies CD4 acts as a sentinel receptor that can switch CD4+ T cells between immune and inflammatory functions.

Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages.

Human alveolar macrophages (AMphi) undergo apoptosis following infection with Mycobacterium tuberculosis in vitro. Apoptosis of cells infected with intracellular pathogens may benefit the host by eliminating a supportive environment for bacterial growth. The present study compared AMphi apoptosis following infection by M. tuberculosis complex strains of differing virulence and by Mycobacterium kansasii. Avirulent or attenuated bacilli (M. tuberculosis H37Ra, Mycobacterium bovis bacillus Calmette-Guérin, and M. kansasii) induced significantly more AMphi apoptosis than virulent strains (M. tuberculosis H37Rv, Erdman, M. tuberculosis clinical isolate BMC 96.1, and M. bovis wild type). Increased apoptosis was not due to greater intracellular bacterial replication because virulent strains grew more rapidly in AMphi than attenuated strains despite causing less apoptosis. These findings suggest the existence of mycobacterial virulence determinants that modulate the apoptotic response of AMphi to intracellular infection and support the hypothesis that macrophage apoptosis contributes to innate host defense in tuberculosis.

Enhanced in vivo human immunodeficiency virus-1 replication in the lungs of human immunodeficiency virus-infected persons with Pneumocystis carinii pneumonia.

The relationship of serum human immunodeficiency virus-1 (HIV-1) RNA levels to HIV-1 RNA levels in other compartments, such as the lungs, is not well characterized. The purpose of this study was to determine the viral burden of HIV-1 in the lungs by comparing HIV-1 RNA in cell-free bronchoalveolar lavage fluid (BALF) with that in serum. Specimens were examined from 77 HIV-seropositive adults (CD4(+) cell counts: 0 to 700 cells/mm(3); 48% receiving prescribed antiretroviral agents), comprising 43 asymptomatic individuals who were compared with 34 persons with active lung disease caused by Pneumocystis carinii (n = 26), bacteria (n = 3), Mycobacterium avium complex (n = 2), Nocardia sp. (n = 1), Aspergillus sp. (n = 1), or pulmonary Kaposi's sarcoma (n = 1). For serum HIV-1 RNA, the proportion of subjects with detectable levels and the mean values were similar for asymptomatic individuals and persons with active lung disease (85% versus 86%, respectively) (6.64 x 10(4) versus 1. 81 x 10(5) HIV-1 RNA copies/ml; p = 0.13). In contrast, HIV-1 RNA in BALF was more often detected (16% versus 62%; p = 0.001), and mean values were higher (1.04 x 10(5) versus 3.31 x 10(6) HIV-1 RNA copies/ml; p = 0.032), in subjects with active lung disease than in asymptomatic subjects, independent of early or advanced clinical stages of HIV-related disease. For both study groups, HIV-1 RNA levels in BALF exceeded those in serum in 56% of cases by up to 66-fold, and did not correlate with local levels of tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, or interleukin-16. HIV-1 proviral DNA in cells from BALF was detected in up to 86% of subjects, more frequently in persons with advanced HIV disease (p = 0.0496), and often involved > 10% of BALF cells, but did not correlate with HIV-1 RNA detected in BALF. These data provide evidence for active HIV-1 replication in the lungs. HIV-1 replication is compartmentalized relative to serum, may be restricted, is independent of HIV-1 proviral DNA and clinical stage of HIV, and may be influenced by pulmonary disease such as P. carinii pneumonia or by other local or lung-specific factors. The lungs represent a large reservoir for HIV-1, and may present a source of persistent HIV-1 replication even during periods of apparent clinical latency of HIV-1 infection.

Macrophage apoptosis in mycobacterial infections.

Mycobacterial diseases are a major public health concern. In the case of tuberculosis, the problem has been acerbated due to the emergence of drug-resistant strains of Mycobacterium tuberculosis, and Mycobacterium avium is the major opportunistic pathogen in HIV-1 infection in the United States. M. tuberculosis and M. avium replicate in human macrophages and induce apoptosis. Incubation of freshly added uninfected autologous macrophages with apoptotic M. avium-infected macrophages results in 90% inhibition of bacterial growth. Apoptosis also prevents the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies. Consistent with the model that host cell apoptosis is a defense mechanism against mycobacteria is the finding that the virulent M. tuberculosis strain H37Rv induces substantially less macrophage apoptosis than the attenuated strain H37Ra. Evasion of apoptosis by this pathogen is achieved by enhanced release of sTNFR2 by H37Rv-infected macrophages and subsequent formation of inactive TNF-alpha-TNFR2 complexes. These observations contribute to the hypothesis that apoptosis of the host macrophage is an important defense mechanism in mycobacterial infections, which prevents the spread of the infection.

Identification of domains in IL-16 critical for biological activity.

IL-16 is a proinflammatory cytokine implicated in the pathogenesis of asthma and other conditions characterized by recruitment of CD4+ T cells to sites of disease. It is postulated that CD4 is an IL-16 receptor, although other receptors or coreceptors may exist. Among several known functions, IL-16 is a chemoattractant factor for CD4+ T cells and it inhibits MLR. We previously reported that an oligopeptide corresponding to the 16 C-terminal residues of human IL-16 inhibits chemoattractant activity. To identify functional domains with greater precision, shorter oligonucleotides containing native or mutated C-terminal IL-16 sequences were tested for IL-16 inhibition. Within the 16 C-terminal residues, the minimal peptide RRKS (corresponding to Arg106 to Ser109) was shown to mediate inhibition of IL-16 chemoattractant activity. Inhibition was lost when either arginine was substituted with alanine. Point mutations in IL-16 revealed that Arg107 is critical for chemoattractant activity, but MLR inhibition was unaffected by mutation of Arg107 or even deletion of the C-terminal tail through Arg106. Deletion of 12 or 22 N-terminal residues of IL-16 had no impact on chemoattractant activity, but MLR inhibition was reduced. Deletion of 16 C-terminal plus 12 N-terminal residues abolished both chemoattractant and MLR-inhibitory activity of IL-16. These data indicate that receptor interactions with IL-16 that activate T cell migration are not identical with those required for MLR inhibition, and suggest that both N-terminal and C-terminal domains in IL-16 participate in receptor binding or activation.

Identification of a CD4 domain required for interleukin-16 binding and lymphocyte activation.

Interleukin-16 (IL-16) activates CD4(+) cells, possibly by direct interaction with CD4. IL-16 structure and function are highly conserved across species, suggesting similar conservation of a putative IL-16 binding site on CD4. Comparison of the human CD4 amino acid sequence with that of several different species revealed that immunoglobulin-like domain 4 is the most conserved extracellular region. Potential interaction of this domain with IL-16 was studied by testing murine D4 sequence-based oligopeptides for inhibition of IL-16 chemoattractant activity and inhibition of IL-16 binding to CD4 in vitro. Three contiguous 12-residue D4 region peptides (designated A, B, and C) blocked IL-16 chemoattractant activity, with peptide B the most potent. Peptides A and B were synergistic for inhibition, but peptide C was not. Peptides A and B also blocked IL-16 binding to CD4 in vitro, whereas peptide C did not. CD4, in addition to its known function as a receptor for major histocompatibility complex class II, contains a binding site for IL-16 in the D4 domain. The D4 residues required for IL-16 binding overlap those previously shown to participate in CD4-CD4 dimerization following class II major histocompatibility complex binding, providing a mechanistic explanation for the known function of IL-16 to inhibit the mixed lymphocyte reaction.

The role of macrophage cell death in tuberculosis.

Studies of host responses to infection have traditionally focused on the direct antimicrobial activity of effector molecules (antibodies, complement, defensins, reactive oxygen and nitrogen intermediates) and immunocytes (macrophages, lymphocytes, and neutrophils among others). The discovery of the systems for programmed cell death of eukaryotic cells has revealed a unique role for this process in the complex interplay between microorganisms and their cellular targets or responding immunocytes. In particular, cells of the monocyte/macrophage lineage have been demonstrated to undergo apoptosis following intracellular infection with certain pathogens that are otherwise capable of surviving within the hostile environment of the phagosome or which can escape the phagosome. Mycobacterium tuberculosis is a prototypical 'intracellular parasite' of macrophages, and the direct induction of macrophage apoptosis by this organism has recently been reported from several laboratories. This paper reviews the current understanding of the mechanism and regulation of macrophage apoptosis in response to M. tuberculosis and examines the role this process plays in protective immunity and microbial virulence.

Processing and release of IL-16 from CD4+ but not CD8+ T cells is activation dependent.

IL-16 is synthesized as a precursor molecule of 68 kDa (pro-IL-16) that is processed by caspase-3, a member of the IL-1 converting enzyme (ICE) family. This cleavage results in a 13-kDa carboxy terminal peptide, which constitutes the bioactive secreted form of IL-16. We have previously reported constitutive IL-16 mRNA expression and pro-IL-16 protein in CD4+ and CD8+ T cells. Although bioactive IL-16 protein is present in unstimulated CD8+ T cells, there is no bioactive IL-16 present in CD4+ T cells. Along these lines, unstimulated CD8+ T cells contain active caspase-3. In the current studies we investigated the regulation of IL-16 protein and mRNA expression in CD4+ T cells and determined the kinetics of secretion following stimulation of the TCR. CD4+ T cells release IL-16 protein following antigenic stimulation, and this release is accelerated in time by costimulation via CD28. However, CD3/CD28 costimulation did not alter IL-16 mRNA appearance or stability in either CD4+ or CD8+ T cells. The secretion of bioactive IL-16 from CD4+ T cells correlated with the appearance of cleavage of pro-caspase-3 into its 20-kDa active form. Thus, resting CD8+ T cells contain active caspase-3 that is capable of cleaving pro-IL-16, whereas CD4+ T cells require activation for the appearance of active caspase-3. The mechanism of release or secretion of bioactive IL-16 is currently unknown, but does not correlate with cellular apoptosis.

Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNF-alpha.

Infection by Mycobacterium tuberculosis (MTB) induces human alveolar macrophage (AMphi) apoptosis by a TNF-alpha-dependent mechanism. The apoptotic response is postulated to be a defense mechanism, limiting the growth of this intracellular pathogen. Consistent with that model, recent studies showed that the virulent MTB strain H37Rv induces substantially less AMphi apoptosis than the attenuated strain H37Ra. We now report that AMphi infection with either H37Rv or H37Ra induces comparable levels of TNF-alpha measured by ELISA but that TNF-alpha bioactivity is reduced in supernatants of H37Rv-infected AMphi. Differential release of soluble TNFR2 (sTNFR2), with formation of inactive TNF-alpha-TNFR2 complexes accounted for the difference in TNF-alpha bioactivity in these cultures. Release of sTNFR2 by H37Rv-infected AMphi was IL-10 dependent since it was inhibited by neutralizing anti-IL-10 Ab. Thus, the effect of TNF-alpha produced by AMphi following infection can be modulated by virulent MTB, using IL-10 as an upstream mediator.

Tissue and T cell distribution of precursor and mature IL-16.

IL-16 is a novel cytokine, which is chemoattractant for CD4+ T cells, macrophages, and eosinophils. Recently, it was reported that IL-16 is synthesized as an approximately 80-kDa precursor molecule, pro-IL-16. Since little is known about the processing and tissue distribution of IL-16 and pro-IL-16, we investigated the distribution of IL-16 mRNA and protein in human lymphoid tissue. Northern blotting identified IL-16 mRNA predominantly in normal lymphoid organs, including PBMC, spleen, and thymus. Immunohistochemistry of human lymph node localized IL-16 protein to lymphocyte cytoplasm within T cell zones and occasionally in lymphocytes in B cell zones. Flow cytometric detection of intracellular IL-16 showed that >70% of CD4+ and CD8+ T cells constitutively expressed IL-16 protein. Western blot analysis of PBMC revealed nearly all of this protein to be approximately 80-kDa pro-IL-16 in unstimulated PBMC, and upon cell activation, the amino terminus of pro-IL-16 is processed into multiple fragments. These results show that pro-IL-16 is widely and constitutively expressed and suggest that the amino terminus of the protein can be processed upon cell activation.