Claudia Manca

A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response

Fifty million new infections with Mycobacterium tuberculosis occur annually, claiming 2–3 million lives from tuberculosis worldwide. Despite the apparent lack of significant genetic heterogeneity between strains of M. tuberculosis, there is mounting evidence that considerable heterogeneity exists in molecules important in disease pathogenesis. These differences may manifest in the ability of some isolates to modify the host cellular immune response, thereby contributing to the observed diversity of clinical outcomes. Here we describe the identification and functional relevance of a highly biologically active lipid species—a polyketide synthase-derived phenolic glycolipid (PGL) produced by a subset of M. tuberculosis isolates belonging to the W-Beijing family that show ‘hyperlethality’ in murine disease models. Disruption of PGL synthesis results in loss of this hypervirulent phenotype without significantly affecting bacterial load during disease. Loss of PGL was found to correlate with an increase in the release of the pro-inflammatory cytokines tumour-necrosis factor-α and interleukins 6 and 12 in vitro. Furthermore, the overproduction of PGL by M. tuberculosis or the addition of purified PGL to monocyte-derived macrophages was found to inhibit the release of these pro-inflammatory mediators in a dose-dependent manner.

Differential Monocyte Activation Underlies Strain-Specific Mycobacterium tuberculosis Pathogenesis

ABSTRACT In vitro infection of monocytes with Mycobacterium tuberculosis HN878 and related W/Beijing isolates preferentially induced interleukin-4 (IL-4) and IL-13, which characterize Th2 polarized immunity. In contrast, CDC1551 induced more IL-12 and other molecules associated with phagocyte activation and Th1 protective immunity. The differential cytokine-chemokine response was mediated by extracted lipids, suggesting that these molecules regulate host responses to infection.

The Phenolic Glycolipid of Mycobacterium tuberculosis Differentially Modulates the Early Host Cytokine Response but Does Not in Itself Confer Hypervirulence

ABSTRACT Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by the clinical M. tuberculosis isolate HN878 has been associated with a less efficient Th1 response and increased virulence in mice and rabbits. It has been suggested that the W-Beijing family is the only group of M. tuberculosis strains with an intact pks1-15 gene, required for the synthesis of PGL-tb and capable of producing PGL-tb. We have found that some strains with an intact pks1-15 do not produce PGL-tb while others may produce a variant of PGL-tb. We examined the early host cytokine response to infection with these strains in vitro to better understand the effect of PGL-tb synthesis on immune responses. In addition, we generated a PGL-tb-producing H37Rv in order to determine the effect of PGL-tb production on the host immune response during infection by a strain normally devoid of PGL-tb synthesis. We observed that PGL-tb production by clinical M. tuberculosis isolates affected cytokine production differently depending on the background of the strain. Importantly, while ectopic PGL-tb production by H37Rv suppressed the induction of several pro- and anti-inflammatory cytokines in vitro in human monocytes, it did not lead to increased virulence in infected mice and rabbits. Collectively, our data indicate that, while PGL-tb may play a role in the immunogenicity and/or virulence of M. tuberculosis, it probably acts in concert with other bacterial factors which seem to be dependent on the background of the strain.

Mycobacterium tuberculosis Inhibits Maturation of Human Monocyte-Derived Dendritic Cells In Vitro

To induce effector immunity, dendritic cells (DCs) must differentiate into fully mature cells. We show that, after human monocyte-derived DCs were infected with virulent Mycobacterium tuberculosis, up-regulation of cellular-surface maturation markers was minimal and reversible. In the presence of a potent stimulus for maturation (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and prostaglandin E2 [PGE2]), M. tuberculosis inhibited phenotypic DC maturation. M. tuberculosis-infected DCs had an impaired ability to induce allogeneic lymphoproliferation and activated autologous memory CD4+ and CD8+ T cells optimally only in the presence of TNF-alpha, IL-1beta, and PGE2. Thus, virulent M. tuberculosis inhibits phenotypic and functional maturation of human monocyte-derived DCs. This mechanism, which has been described elsewhere for various viruses and for the virulent mycobacterium M. leprae, may be a novel mechanism that this pathogen uses to evade the hosts immune response.

Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis

Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens. Since purification of protein from M. tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M. tuberculosis from E. coli cells. Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography. The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E. coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells. The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M. tuberculosis) counterparts. Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.

Effect of standard tuberculosis treatment on plasma cytokine levels in patients with active pulmonary tuberculosis

Background Sputum Mycobacterium tuberculosis (Mtb) culture is commonly used to assess response to antibiotic treatment in individuals with pulmonary tuberculosis (TB). Such techniques are constrained by the slow growth rate of Mtb, and more sensitive methods to monitor Mtb clearance are needed. The goal of this study was to evaluate changes in plasma cytokines in patients undergoing treatment for TB as a means of identifying candidate host markers associated with microbiologic response to therapy. Methods Twenty-four plasma cytokines/chemokines were measured in 42 individuals diagnosed with active pulmonary TB, 52% were HIV co-infected. Individuals, undergoing a 26-week standard TB treatment, were followed longitudinally over 18 months and measurements were associated with HIV status and rates of sputum culture conversion. Results Plasma concentrations of interferon-inducible protein-10 (IP-10) and vascular endothelial growth factor (VEGF) were significantly reduced upon TB treatment, regardless of HIV status. By the end of treatment, IP-10 concentrations were significantly lower in HIV negative individuals when compared to HIV-positive individuals (p = 0.02). Moreover, in HIV negative patients, plasma VEGF concentrations, measured as early as 2-weeks post TB treatment initiation, positively correlated with the time of sputum conversion (p = 0.0017). No significant changes were observed in other studied immune mediators. Conclusions These data suggest that VEGF plasma concentration, measured during early TB treatment, could represent a surrogate marker to monitor sputum culture conversion in HIV uninfected individuals.

Mycobacterium tuberculosis 19-Kilodalton Lipoprotein Inhibits Mycobacterium smegmatis-Induced Cytokine Production by Human Macrophages In Vitro

ABSTRACT Vaccination of mice with Mycobacterium vaccae orM. smegmatis induces some protection against M. tuberculosis challenge. The 19-kDa lipoprotein of M. tuberculosis, expressed in M. vaccae or M. smegmatis (M. smeg19kDa), abrogates this protective immunity. To investigate the mechanism of this suppression of immunity, human monocyte-derived macrophages (MDM) were infected with M. smeg19kDa. Infection resulted in reduced production of tumor necrosis factor alpha (TNF-α) (P < 0.01), interleukin-12 (IL-12) (P < 0.05), IL-6 (P < 0.05), and IL-10 (P < 0.05), compared to infection with M. smegmatis vector (M. smegV). Infection with M. smeg19kDa and with M. smegV had no differential effect on expression of costimulatory molecules on MDM, nor did it affect the proliferation of presensitized T cells cocultured with infected MDM. When MDM were infected withM. smegmatis expressing mutated forms of the 19-kDa lipoprotein, including non-O-glycosylated (M. smeg19NOG), nonsecreted (M. smeg19NS), and nonacylated (M. smeg19NA) variants, the reduced production of TNF-α or IL-12 was not observed. When the purified 19-kDa lipoprotein was added directly to cultures of infected monocytes, there was little effect on either induction of cytokine production or its inhibition. Thus, the immunosuppressive effect is dependent on glycosylated and acylated 19-kDa lipoprotein present in the phagosome containing the mycobacterium. These results suggest that the diminished protection against challenge with M. tuberculosis seen in mice vaccinated with M. smegmatis expressing the 19-kDa lipoprotein is the result of reduced TNF-α and IL-12 production, possibly leading to reduced induction of T-cell activation.

In Vivo Phenotypic Dominance in Mouse Mixed Infections with Mycobacterium tuberculosis Clinical Isolates

Clinical isolates of Mycobacterium tuberculosis demonstrate significant heterogeneity in virulence potential in animal models of infection. Isolate CDC1551, for example, has previously been described in mouse survival studies as being hypovirulent, and isolate HN878 has been described as being hypervirulent. Observed differences in this mouse infection experiment have been proposed to reflect differential engagement of the host immune response. To assess whether this is a local or a systemic effect, C57BL/6 mice were infected simultaneously with mixtures of CDC1551 and HN878 in varying ratios and were monitored for mycobacterial growth kinetics, strain proportions during infection, and mouse survival. Strain mixtures that contained primarily HN878 grew more quickly during the first 5 weeks of infection and were more lethal for mice, and HN878 was enriched during in vivo growth. The absolute number of implanted HN878 bacilli at infection correlated inversely with mouse survival and was independent of concomitant infection with CDC1551. In infections of nonactivated mouse macrophages, HN878 grew more quickly. However, phagocyte preactivation reduced and equalized the growth rate of both strains. These results suggest that HN878 exerts a dominant immunosuppressive effect limited to the granuloma in which it is contained.

Phosphodiesterase 4 inhibition reduces innate immunity and improves isoniazid clearance of Mycobacterium tuberculosis in the lungs of infected mice.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the leading infectious disease causes of morbidity and mortality worldwide. Though current antibiotic regimens can cure the disease, treatment requires at least six months of drug therapy. One reason for the long duration of therapy is that the currently available TB drugs were selected for their ability to kill replicating organisms and are less effective against subpopulations of non-replicating persistent bacilli. Evidence from in vitro models of Mtb growth and mouse infection studies suggests that host immunity may provide some of the environmental cues that drive Mtb towards non-replicating persistence. We hypothesized that selective modulation of the host immune response to modify the environmental pressure on the bacilli may result in better bacterial clearance during TB treatment. For this proof of principal study, we compared bacillary clearance from the lungs of Mtb-infected mice treated with the anti-TB drug isoniazid (INH) in the presence and absence of an immunomodulatory phosphodiesterase 4 inhibitor (PDE4i), CC-3052. The effects of CC-3052 on host global gene expression, induction of cytokines, and T cell activation in the lungs of infected mice were evaluated. We show that CC-3052 modulates the innate immune response without causing generalized immune suppression. Immune modulation combined with INH treatment improved bacillary clearance and resulted in smaller granulomas and less lung pathology, compared to treatment with INH alone. This novel strategy of combining anti-TB drugs with an immune modulating molecule, if applied appropriately to patients, may shorten the duration of TB treatment and improve clinical outcome.

Mycobacterium leprae Actively Modulates the Cytokine Response in Naïve Human Monocytes

ABSTRACT Leprosy is a chronic but treatable infectious disease caused by the intracellular pathogen Mycobacterium leprae. Host immunity to M. leprae determines the diversity of clinical manifestations seen in patients, from tuberculoid leprosy with robust production of Th1-type cytokines to lepromatous disease, characterized by elevated levels of Th2-type cytokines and a suboptimal proinflammatory response. Previous reports have indicated that M. leprae is a poor activator of macrophages and dendritic cells in vitro. To understand whether M. leprae fails to elicit an optimal Th1 immune response or actively interferes with its induction, we have examined the early interactions between M. leprae and monocytes from healthy human donors. We found that, in naïve monocytes, M. leprae induced high levels of the negative regulatory molecules MCP-1 and interleukin-1 (IL-1) receptor antagonist (IL-1Ra), while suppressing IL-6 production through phosphoinositide-3 kinase (PI3K)-dependent mechanisms. In addition, low levels of proinflammatory cytokines were observed in association with reduced activation of nuclear factor-κB (NF-κB) and delayed activation of IL-1β-converting enzyme, ICE (caspase-1), in monocytes stimulated with M. leprae compared with Mycobacterium bovis BCG stimulation. Interestingly, although in itself a weak stimulator of cytokines, M. leprae primed the cells for increased production of tumor necrosis factor alpha and IL-10 in response to a strongly inducing secondary stimulus. Taken together, our results suggest that M. leprae plays an active role to control the release of cytokines from monocytes by providing both positive and negative regulatory signals via multiple signaling pathways involving PI3K, NF-κB, and caspase-1.