Luis F. Barrera

IFNγ Response to Mycobacterium tuberculosis, Risk of Infection and Disease in Household Contacts of Tuberculosis Patients in Colombia

Objectives Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development. Methods A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination. Results Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007). Discussion CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.

Inhibition of Virulent Mycobacterium tuberculosis by Bcgr and Bcgs Macrophages Correlates with Nitric Oxide Production

The Nramp1 gene controls macrophage resistance or susceptibility to several intracellular microorganisms; however, there is conflicting evidence regarding its role during infection with virulent Mycobacterium tuberculosis. Nitric oxide (NO) is a potent antimycobacterial agent produced by macrophages, which is also regulated by Nramp1. The in vitro ability of B10R (resistant) and B10S (susceptible) murine macrophages to inhibit M. tuberculosis H37Rv and to produce NO in response to infection and interferon-gamma (IFN-gamma) was compared. Infected B10R macrophages inhibited [3H]uracil incorporation by M. tuberculosis and produced higher amounts of NO than did B10S macrophages. IFN-gamma increased the inhibitory activity of both cells. Inhibition of M. tuberculosis by IFN-gamma-activated B10R macrophages was reversed by N(G)-monomethyl-L-arginine (N(G)MMA). L-arginine restored NO production and increased the antimycobacterial activity by IFN-gamma-stimulated N(G)MMA-treated macrophages. The Bcg/Nramp1 gene may regulate macrophage resistance or susceptibility to virulent M. tuberculosis by a differential capability of these cells to produce NO.

Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death

Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-kappaB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.

T cell responses to DosR and Rpf proteins in actively and latently infected individuals from Colombia

Mycobacterium tuberculosis DosR regulon-encoded proteins elicit strong immune T-cell responses in individuals with latent tuberculosis (LTBI). Also, resuscitation (Rpf) proteins can induce such responses. However, variations in the immunogenicity of the DosR and Rpf proteins have been observed in European and African populations, and no data are published from other geographic areas. In Colombian LTBI and patients with recently diagnosed PTB, we therefore studied the immune response to DosR, Rpf, stress, and nominal antigens from Mtb, in 7-day stimulated cultures. Three DosR (Rv1737c, Rv2029c, Rv2628c) and 2 Rpf (Rv0867 and Rv2389c) antigens were recognized most prominently on the basis of the net IFNγ production (DosR) or the percentage of responding individuals (Rpf). Results show that the selected DosR antigens induced a higher proportion of CD4-T cells producing IFNγ from LTBI, compared to pulmonary TB patients (PTB), while there were no differences in the proportion of CD8-T cells. An increased frequency of CD4, but not CD8 T-cells with a CD45RO(+)CD27(+) phenotype was observed in LTBI in response to Rv2029c, Rv0867c, and Rv2389c, compared to PTB. The levels of cytokines and chemokines in the supernatants of stimulated cells, showed that the DosR and Rpf antigens induced higher levels of IFNγ in cultures from LTBI compared to PTB, although the induced pattern of cytokines and chemokines was also antigen dependent. In summary, our results are consistent with the significant immunogenicity of Mtb DosR and Rpf antigens in LTBI individuals, and confirm and extend previously reported data from other TB affected human populations.

Evaluation of Toll-like receptor and adaptor molecule polymorphisms for susceptibility to tuberculosis in a Colombian population.

Immunological studies have supported the idea that innate immunity is critical for the control of Mycobacterium tuberculosis (Mtb) infection in humans. Despite the overwhelming evidence showing the critical role of Toll‐like receptors (TLRs) in the in vitro recognition of Mtb, the in vivo significance of individual TLRs has been more difficult to demonstrate consistently. We were interested in examining the role of genes of TLRs and molecules involved in their signalling cascades, and a case–control study was designed to test the association of polymorphisms of these innate immune genes with pulmonary tuberculosis (TB) in a Colombian population. In this study, we did not find an association with TLR2, TLR4, TLR9, MyD88 or MAL/TIRAP polymorphic variants. These findings suggest that those genes are not involved as risk factors for pulmonary TB in our population.

Tuberculin skin test reactivity is dependent on host genetic background in Colombian tuberculosis household contacts.

The tuberculin skin test (TST) measures the intensity of antimycobacterial acquired immunity and is used to diagnose latent infection with Mycobacterium tuberculosis. We report evidence for a codominant gene explaining ∼65% of the TST variability. Disregarding the host genetic background may lead to misclassifications of TST-based diagnosis of latent M. tuberculosis infection.

Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia.

ABSTRACT Multifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with the Mycobacterium tuberculosis RD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to the M. tuberculosis latency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in which M. tuberculosis is endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen of M. tuberculosis induced higher frequencies of CD4+ or CD8+ mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4+ and/or CD8+ T cells with a CD45RO+ CD27+ phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection from M. tuberculosis reactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states of M. tuberculosis infections.

Metabolic adaptation of two in silico mutants of Mycobacterium tuberculosis during infection

BackgroundUp to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb’s vulnerability and metabolic adaptation have been carried out.ResultsA genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports.ConclusionsThis study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Transcriptome profiling of the cysticercus stage of the laboratory model Taenia crassiceps, strain ORF

Neurocysticercosis (NC) is a serious public health problem mainly in developing countries. NC caused by the cysticercus stage from cestode Taenia solium is considered by the WHO and ITFDE as a potentially eradicable disease. Definitive diagnosis of NC is challenging because of the unspecific clinical manifestations such as the non-definitive evidence presented by neuroimaging (in most cases) and the lack of definitive serological test. Taenia crassiceps (ORF strain) is a cestode closely related to T. solium and it has frequently been used as a source of antigens for immunodiagnostics. A murine model to study host immune response to infection has also been established by using T. crassiceps. Despite the extensive use of T. crassiceps for research, molecular information for this cestode is scarce in public databases. With the aim of providing more extensive information on T. crassiceps biology, an RNA-seq experiment and subsequent bioinformatic transcriptome processing of this cestode parasite mRNA in its cysticercus stage were carried out. A total of 227,082 read/ESTs were sequenced using the 454-GS FLX Titanium technology and assembled into 10,787 contigs. This transcriptome dataset represents new and valuable molecular information of the cestode T. crassiceps (ORF). This information will substantially improve public information and will help to achieve a better understanding of the biology of T. crassiceps and to identify target proteins for serodiagnosis and vaccination.

Alveolar macrophages from tuberculosis patients display an altered inflammatory gene expression profile

Alveolar macrophages (AMs) are major targets of Mycobacterium tuberculosis (Mtb) infection, critical during the progression of active tuberculosis (TB). The complex immunopathology of TB generates diverse microenvironments in the lung, which shape immune responses by AMs. In the current study, we perform whole genome microarray transcriptional profiling on RNA isolated from AMs from TB patients (AMsTB) compared to AMs from control subjects (AMsCT) using bronchoalveolar lavage (BAL). Our hypothesis was that systemic effects on the local lung microenvironment during TB affect the transcriptional response of AMsTB. We found a unique gene expression profile of 51 genes, including up-regulated CHIT1, CHI3L1, CCL5, CCL22, CCL8, CXCL9, MMP9, MMP7 and MMP12, associated with a robust pro-inflammatory response, cell recruitment and tissue damage, and genes of the cyclin family (CCND1, CCND2, and CCNA1) associated with cell proliferation. These expression profiles may account for the inflammatory condition in the lungs of TB patients. CXCL5, IL1B, CAMP, and TGFB1 were down-regulated, suggesting an altered control of Mtb infection. Also, MARCO and COLEC12, affecting phagocytosis, and CES1, associated with an increase in free cholesterol, were down-regulated. The observed changes in mRNA expression profiles may partially account for the inability of AMsTB to effectively control Mtb infection, suggesting that a balanced control of pro- and anti-inflammatory immune responses is crucial for infection control.