Luiz E. Bermudez

Recombinant Granulocyte–Macrophage Colony‐Stimulating Factor Activates Human Macrophages to Inhibit Growth or Kill Mycobacterium avium Complex

Organisms belonging to the Mycobacterium avium complex (MAC) are associated with life‐threatening bacteremia in patients with the acquired immunodeficiency syndrome (AIDS). As these organisms survive within macrophages, we examined the ability of recombinant human granulocyte–monocyte colony‐stimulating factor (GM‐CSF) to activate human monocyte‐derived macrophages to inhibit the intracellular growth or kill the most mouse‐virulent MAC strain In our collection that belongs to serotype 1. While unstimulated cells did not inhibit intracellular growth of MAC, macrophages activated by GM‐CSF (10–104 U/ml) inhibited or killed up to 58 ± 5% of the initial inoculum. This activation was dose‐dependent, with maximal change occurring with a dose of 100 U/ml after 72 hr exposure. Inhibition or killing was demonstrated if GM‐CSF was given both before or after establishment of infection. The combination of GM‐CSF (102 U/ml) plus TNF (102 U/ml) augmented macrophage killing (range, 31 ± 4%) compared with GM‐CSF (102 U/ml) alone, but the combination of recombinant human interferon‐gamma (IFNγ) plus GM‐CSF resulted in a significant decrease in intracellular inhibition of growth or killing (13.3 ± 2%) compared with 57.7 ± 5% obtained with GM‐CSF alone. These results indicate that: 1) GM‐CSF can activate macrophages to inhibit intracellular growth or kill MAC; 2) killing may be augmented by TNF; and 3) IFNγ may impair GM‐CSF‐dependent macrophage activation.

Killing of Mycobacterium avium and Mycobacterium tuberculosis by a Mycobacteriophage Delivered by a Nonvirulent Mycobacterium: A Model for Phage Therapy of Intracellular Bacterial Pathogens

Mycobacterium avium causes disseminated infection in patients with acquired immune deficiency syndrome. Mycobacterium tuberculosis is a pathogen associated with the deaths of millions of people worldwide annually. Effective therapeutic regimens exist that are limited by the emergence of drug resistance and the inability of antibiotics to kill dormant organisms. The present study describes a system using Mycobacterium smegmatis, an avirulent mycobacterium, to deliver the lytic phage TM4 where both M. avium and M. tuberculosis reside within macrophages. These results showed that treatment of M. avium-infected, as well as M. tuberculosis-infected, RAW 264.7 macrophages, with M. smegmatis transiently infected with TM4, resulted in a significant time- and titer-dependent reduction in the number of viable intracellular bacilli. In addition, the M. smegmatis vacuole harboring TM4 fuses with the M. avium vacuole in macrophages. These results suggest a potentially novel concept to kill intracellular pathogenic bacteria and warrant future development.

Treatment with recombinant granulocyte colony‐stimulating factor (FilgrastinTM) stimulates neutrophils and tissue macrophages and induces an effective non‐specific response against Mycobacterium avium in mice

A role of neutrophils in the host response against Mycobacterium avium (MAC) has recently been suggested. To investigate this matter further, we determined the effect of granulocyte colony‐stimulating factor (G‐CSF) on the outcome of MAC infection in mice. C57BL/6 bg+/bg− black mice were intravenously infected with 1×107 MAC and then divided into four experimental groups to receive G‐CSF as follows: (i) 10u2003μg/kg/day; (ii) 50u2003μg/kg/day; (iii) 100u2003μg/kg/day; (iv) placebo control. Mice were killed at 2 and 4 weeks of treatment to determine the bacterial load of liver and spleen. Treatment with G‐CSF at both 10 and 50u2003μg/kg/day doses significantly decreased the number of viable bacteria in liver and spleen after 2 weeks (≈70·5% and 69·0%, respectively), and after 4 weeks (≈53% and 52%, respectively, P<0·05 compared with placebo control). Treatment with 100u2003μg/kg/day did not result in decrease of bacterial colony‐forming units in the liver and spleen after 4 weeks. Administration of G‐CSF induced interleukin‐10 (IL‐10) and IL‐12 production by splenocytes. To examine if the protective effect of G‐CSF was accompanied by the activation of phagocytic cells, blood neutrophils and splenic macrophages were purified from mice receiving G‐CSF and their ability to kill MAC was examined ex vivo. Neutrophils and macrophages from G‐CSF‐treated mice were able to inhibit the growth of or to kill MAC ex vivo, while phagocytic cells from untreated control mice had no anti‐MAC effect. These results suggest that activation of neutrophils appears to induce an effective non‐specific host defence against MAC, and further studies should aim for better understanding of the mechanisms of protection.

Stimulation with cytokines enhances penetration of azithromycin into human macrophages.

An effective intracellular concentration of an antimicrobial agent is essential for therapy of infections caused by organisms of the Mycobacterium avium complex. We previously reported on the effect of the combination of azithromycin and tumor necrosis factor (TNF) against M. avium infection in macrophages. We now report that stimulation of macrophages either with recombinant human gamma interferon (IFN-gamma, 10(2) U/ml) or with recombinant human TNF-alpha (10(2) U/ml) resulted in an increase in the intracellular concentration of azithromycin by approximately 200% within 3 h, compared with the concentration in unstimulated macrophages. Infection of macrophages with M. avium complex led to a decrease in the uptake of [14C]azithromycin by infected cells, compared with that by uninfected controls. Stimulation of infected macrophages with recombinant IFN-gamma or TNF-alpha overcame the inhibitory effect associated with infection. These results suggest that the increased bactericidal activity of the TNF-alpha-azithromycin or IFN-gamma-azithromycin combination against M. avium is related to enhanced uptake of the antibiotic by the stimulated phagocyte.

Mycobacterium avium enters intestinal epithelial cells through the apical membrane, but not by the basolateral surface, activates small GTPase Rho and, once within epithelial cells, expresses an invasive phenotype

Mycobacterium avium is a common pathogen in AIDS patients that is primarily (but not exclusively) acquired through the gastrointestinal tract, leading to the development of bacteraemia and disseminated disease. To cause infection through the gut, binding and invasion of the intestinal epithelial barrier are required. To characterize this process further, we determined the cell surface(s) (basolateral vs. apical membrane) that M. avium interacts with in intestinal mucosal cells in vitro. The level of binding and invasion of both HT‐29 and Caco‐2 intestinal cell monolayers by M. avium were similar when the assay was performed with control medium in the presence of Ca2+ (when only the apical surface was exposed), with Ca2+‐depleted medium or with Ca2+‐depleted mediumu2003+u20031u2003mM EGTA (exposure of both apical and basolateral membranes), suggesting that the bacterium enters the apical surface of the epithelial lining. These observations were confirmed by assays in a transwell system and by using fluorescent microscopy. Real‐time video microscopy showed that M. avium entry was not associated with membrane ruffling and the use of pharmacological inhibitors of the small GTPases demonstrated that M. avium invasion is dependent on the activation of the small GTPases Rho, but not on Rac or Cdc42. Passage of M. avium through HT‐29 cells led to a phenotypic change (intracellular growth; IG) that was associated with a significantly greater (between five‐ and ninefold) ability to bind to and invade new monolayers of epithelial cells or macrophages when compared with the invasion by M. avium grown on agar (extracellular growth; EG). IG phenotype invasion of HT‐29 cells also takes place only by the apical surface. M. avium enters intestinal epithelial cells by the apical surface and, once within the cells, changes phenotype, becoming more invasive towards both macrophages and other epithelial cells.

Apoptosis of Mycobacterium avium-infected macrophages is mediated by both tumour necrosis factor (TNF) and Fas, and involves the activation of caspases

Mycobacterium avium causes disseminated infection in AIDS patients and several forms of infection in immunocompetent hosts. Recent studies have shown that M. avium infection of macrophages inu2003vitro leads to apoptosis of significant numbers of infected cells. Several strains of M. avium used to infect human macrophages for 5 days (multiplicity of infection of 10) triggered 28–46% higher levels of apoptosis than observed with uninfected macrophages at the same time points. Mycobacterium avium strains unable to replicate intracellularly (rep−) resulted in a 15% rate of apoptosis, while M. smegmatis‐infected monolayers showed the same percentage of apoptotic cells as the uninfected macrophage control. The presence of anti‐TNF‐α antibody reduced apoptosis to 17% and the presence of anti‐Fas antibody reduced apoptosis to 10%. When both antibodies were used together, the apoptosis level was 5% above the control. Treatment with TGF‐β also reduced the number of apoptotic cells in infected monolayers. If intracellular growth was inhibited, apoptosis of macrophages decreased significantly. It was also shown that apoptosis was associated with IL‐1β‐converting enzyme (ICE) activation and was significantly reduced by a caspase inhibitor. Gaining understanding of the mechanisms of M. avium‐associated apoptosis of macrophages will provide important insight into M. avium pathogenesis.

Recombinant cytokines for controlling mycobacterial infections.

Knowing how mycobacteria exploit host cytokines to survive and which cytokines have important roles in host defense against mycobacteria should allow the use of these molecules in the treatment of mycobacterial infections. Both interleukin 2 and interferon gamma have been used to treat patients with leprosy, and granulocyte-macrophage colony-stimulating factor is presently being administered to AIDS patients infected with Mycobacterium avium.

Pathogenesis of nontuberculous mycobacteria infections

M avium is a microorganism well adapted to living in the environment and in different hosts. During the past 15 years, a substantial amount of information has been accumulated about the mechanisms used by M avium to cross the hosts mucosal barrier, replicate inside cells, circumvent the hosts immune response, and persist inside the host. It turns out that M avium is a fascinating pathogen after all. The increasing knowledge about M avium pathogenesis may one day provide means for a more effective prophylaxis as well as for treatment of the infection.

Mefloquine, Moxifloxacin, and Ethambutol Are a Triple-Drug Alternative to Macrolide-Containing Regimens for Treatment of Mycobacterium avium Disease

Macrolides are the core of effective drug regimens for the treatment of Mycobacterium avium complex (MAC) disease. Mefloquine (MFQ), moxifloxacin (MXF), and ethambutol (EMB), in combination, were evaluated against both clarithromycin-resistant (CLR-R) and CLR-susceptible (CLR-S) MAC; MFQ (40 mg/kg), MXF (100 mg/kg), or EMB (100 mg/kg/day) was given to mice for 4 weeks. MFQ was bactericidal, whereas MXF and EMB were bacteriostatic against both MAC 101 CLR-S and CLR-R. The combination of MFQ and EMB reduced (P<.05, for comparison with controls), and the combination of MFQ and MXF significantly reduced, the load of CLR-R in both the liver and the spleen. Treatment with all 3 drugs was associated with approximately 1-log reduction of CLR-R after 1 week, 2.1-log reduction of CLR-R after 4 weeks, and 2.17-log reduction in MAC/mL blood. Treatment of MAC 101 CLR-S strain had comparable results.

Homologated aza analogs of arabinose as antimycobacterial agents

A series of hydrolytically-stable aza analogs of arabinofuranose was prepared and evaluated against Mycobacterium tuberculosis and M. avium. The compounds were designed to mimic the putative arabinose donor involved in biogenesis of the essential cell wall polysaccharide, arabinogalactan. Though most compounds displayed little activity in cell culture, one compound showed significant activity in infected macrophage models.