Blas Peixoto

Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants

The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCGs variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB.

Phosphodiesterase-4 inhibition alters gene expression and improves isoniazid-mediated clearance of Mycobacterium tuberculosis in rabbit lungs.

Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.

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.

Phosphodiesterase-4 Inhibition Combined with Isoniazid Treatment of Rabbits with Pulmonary Tuberculosis Reduces Macrophage Activation and Lung Pathology

Tuberculosis (TB) is responsible for significant morbidity and mortality worldwide. Even after successful microbiological cure of TB, many patients are left with residual pulmonary damage that can lead to chronic respiratory impairment and greater risk of additional TB episodes due to reinfection with Mycobacterium tuberculosis. Elevated levels of the proinflammatory cytokine tumor necrosis factor-α and several other markers of inflammation, together with expression of matrix metalloproteinases, have been associated with increased risk of pulmonary fibrosis, tissue damage, and poor treatment outcomes in TB patients. In this study, we used a rabbit model of pulmonary TB to evaluate the impact of adjunctive immune modulation, using a phosphodiesterase-4 inhibitor that dampens the innate immune response, on the outcome of treatment with the antibiotic isoniazid. Our data show that cotreatment of M. tuberculosis infected rabbits with the phosphodiesterase-4 inhibitor CC-3052 plus isoniazid significantly reduced the extent of immune pathogenesis, compared with antibiotic alone, as determined by histologic analysis of infected tissues and the expression of genes involved in inflammation, fibrosis, and wound healing in the lungs. Combined treatment with an antibiotic and CC-3052 not only lessened disease but also improved bacterial clearance from the lungs. These findings support the potential for adjunctive immune modulation to improve the treatment of pulmonary TB and reduce the risk of chronic respiratory impairment.

Chronic pulmonary cavitary tuberculosis in rabbits: a failed host immune response.

The molecular determinants of the immune response to Mycobacterium tuberculosis HN878 infection in a rabbit model of pulmonary cavitary tuberculosis were studied. Aerosol infection of rabbits resulted in a highly differentially expressed global transcriptome in the lungs at 2 weeks, which dropped at 4 weeks and then gradually increased. While IFNγ was progressively upregulated throughout the infection, several other genes in the IFNγ network were not. T-cell activation network genes were gradually upregulated and maximally induced at 12 weeks. Similarly, the IL4 and B-cell activation networks were progressively upregulated, many reaching high levels between 12 and 16 weeks. Delayed peak expression of genes associated with macrophage activation and Th1 type immunity was noted. Although spleen CD4+ and CD8+ T cells showed maximal tuberculosis antigen-specific activation by 8 weeks, macrophage activation in lungs, lymph nodes and spleen did not peak until 12 weeks. In the lungs, infecting bacilli grew exponentially up to 4 weeks, followed by a steady-state high bacillary load to 12 weeks that moderately increased during cavitation at 16 weeks. Thus, the outcome of HN878 infection of rabbits was determined early during infection by a suboptimal activation of innate immunity and delayed T-cell activation.

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.

Spontaneous Latency in a Rabbit Model of Pulmonary Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune-competent individuals in the absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by a positive immunological response to Mtb antigens, with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered generally by an inability to achieve complete bacillary clearance. Herein, we have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathological characteristics occurs spontaneously. The evidence that Mtb-CDC1551-infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated after immune suppression. These rabbits showed early activation of T cells and macrophages and an early peak in the TNFα level, which decreased in association with clearance of bacilli from the lungs. In the absence of sustained tumor necrosis factor-α production, no necrosis was seen in the evolving lung granulomas. In addition, bacillary control was associated with down-regulation of several metalloprotease genes and an absence of lung fibrosis. This model will be used to characterize molecular markers of protective immunity and reactivation.

Host Targeted Activity of Pyrazinamide in Mycobacterium tuberculosis Infection

Pyrazinamide (PZA) is one of the first line antibiotics used for the treatment of tuberculosis (TB). In the present study, we have used in vitro and in vivo systems to investigate whether PZA, in addition to its known anti-mycobacterial properties, modulate the host immune response during Mycobacterium tuberculosis (Mtb) infection. In vitro we have examined the effect of PZA on cytokine and chemokine release by Mtb-infected or Toll-like receptor (TLR) -stimulated primary human monocytes. In vivo, we have investigated at the transcriptional levels using genome-wide microarray gene expression analysis, whether PZA treatment of Mtb-infected mice alters the host immune response to Mtb infection in the lungs. Here, we report that PZA treatment of Mtb-infected human monocytes and mice significantly reduces the release of pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, TNF-α and MCP-1 at the protein and at the gene transcription levels, respectively. Data from microarray analysis also reveal that PZA treatment of Mtb-infected mice significantly alters the expression level of genes involved in the regulation of the pro-inflammatory mediators, lung inflammatory response and TLR signaling networks. Specifically, genes coding for adenylate cyclase and Peroxisome-Proliferator Activated Receptor (PPAR), molecules known for their anti-inflammatory effect, were found to be up-regulated in the lungs of PZA-treated Mtb-infected mice. Based on the microarray findings, we propose that PZA treatment modulates the host immune response to Mtb infection by reducing pro-inflammatory cytokine production, probably through PPAR- and NF-kB- dependent pathways. In addition, our results suggest that inclusion or exclusion of PZA in the TB treatment regimen could potentially affect the biomarker signature detected in the circulation of TB patients.

Modulation of the Cytokine Response in Human Monocytes by Mycobacterium leprae Phenolic Glycolipid-1

Leprosy is a chronic but treatable infectious disease caused by the intracellular pathogen Mycobacterium leprae. M. leprae cell wall is characterized by a unique phenolic glycolipid-1 (PGL-1) reported to have several immune functions. We have examined the role of PGL-1 in the modulation of monocyte cytokine/chemokine production in naive human monocytes. PGL-1 in its purified form or expressed in a recombinant Mycobacterium bovis Bacillus Colmette-Guérin (BCG) background (rBCG-PGL-1) was tested. We found that PGL-1 selectively modulated the induction of specific monocyte cytokines and chemokines and, when used as prestimulus, exerted priming and/or inhibitory effects on the induction of selected cytokines/chemokines in response to a second stimulus. Taken together, the results of this study support a modulatory role for PGL-1 in the innate immune response to M. leprae. Thus, PGL-1 may play an important role in the development of the anergic clinical forms of disease and in tissue damage seen in lepromatous patients and during the reactional states of leprosy.

Etanercept Exacerbates Inflammation and Pathology in a Rabbit Model of Active Pulmonary Tuberculosis

Treatment of chronic inflammatory diseases with tumor necrosis factor alpha (TNF-α) antagonists has been associated with increased risk of tuberculosis (TB). We examined the usefulness of the rabbit model of active pulmonary TB for studying the impact of the human immune modulatory reagent etanercept on the host immune response. Control of Mycobacterium tuberculosis (Mtb) infection, disease pathology, and the global transcriptional response in Mtb-infected lungs of rabbits were studied. Etanercept treatment exacerbated disease pathology and reduced bacillary control in the lungs, compared with infected untreated animals. Reduced collagen and fibrin deposition in the granulomas was associated with significant downregulation of the collagen metabolism and fibrosis network genes and upregulation of genes in the inflammatory response and cell recruitment networks in the lungs of etanercept treated, compared with untreated rabbits. Our results suggest that targeting the TNF-α signaling pathway disrupts the tissue remodeling process, which is required for the formation and maintenance of well-differentiated granulomas and for control of Mtb growth in the lungs. These results validate the use of the rabbit model for investigating the impact of selected human immune modulatory drugs, such as a TNF-α antagonist, on the host immune response and pathogenesis in TB.