Tuberculosis transcriptomics: host protection and immune evasion mechanisms

Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions

The gut microbiome in tuberculosis susceptibility and treatment response: guilty or not guilty?

CITATION:Osagie A. Eribo, Nelita du Plessis, Mumin Ozturk, Reto Guler, Gerhard Walzl & Novel N. Chegou Cellular and Molecular Life Sciences volume 77, pages 1497–1509 (2020)Although tuberculosis (TB) is a curable disease, it remains the foremost cause of death from a single pathogen. Globally, approximately 1.6 million people died of TB in 2017. Many predisposing factors related to host immunity, genetics and the environment have been linked to TB. However, recent evidence suggests a relationship between dysbiosis in the gut microbiome and TB disease development. The underlying mechanism(s) whereby dysbiosis in the gut microbiota may impact the different stages in TB disease progression, are, however, not fully explained. In the wake of recently emerging literature, the gut microbiome could represent a potential modifiable host factor to improve TB immunity and treatment response. Herein, we summarize early data detailing (1) possible association between gut microbiome dysbiosis and TB (2) the potential for the use of microbiota biosignatures to discriminate active TB disease from healthy individuals (3) the adverse effect of protracted anti-TB antibiotics treatment on gut microbiota balance, and possible link to increased susceptibility to Mycobacterium tuberculosis re-infection or TB recrudescence following successful cure. We also discuss immune pathways whereby the gut microbiome could impact TB disease and serve as target for clinical manipulation

Corneal properties in children with congenital isolated growth hormone deficiency

<b>AIM:</b>To compare the corneal parameters of children with congenital isolated growth hormone deficiency and healthy subjects.<b>METHODS:</b> In this cross-sectional, prospective study, 50 cases with growth hormone (GH) deficiency treated with recombinant GH and 71 healthy children underwent a complete ophthalmic examination. The corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) were measured with the Ocular Response Analyzer (ORA). Central corneal thickness (CCT) was measured by a ultrasonic pachymeter.<b>RESULTS:</b> The mean age was 13.0±3.0 years in the GH deficiency group consisting of 21 females and 29 males and 13.4±2.4 years in the healthy children group consisting of 41 females and 30 males. There was no statistically significant difference between the groups for gender or age (Chi-square test, <i>P</i>=0.09; independent <i>t</i>-test, <i>P</i>=0.28, respectively). The mean duration of recombinant GH therapy was 3.8±2.4y in the study group. The mean CH, CRF, IOPg and IOPcc values were 11.0±2.0, 10.9±1.9, 15.1±3.3, and 15.1±3.2 mm Hg respectively in the study group. The same values were 10.7±1.7, 10.5±1.7, 15.2±3.3, and 15.3±3.4 mm Hg respectively in the control group. The mean CCT values were 555.7±40.6, 545.1±32.5 µm in the study and control groups respectively. There was no statistically significant difference between the two groups for CH, CRF, IOPg, IOPcc measurements or CCT values (independent <i>t</i>-test, <i>P</i>=0.315, 0.286, 0.145, 0.747, 0.13 respectively).<b>CONCLUSION:</b> Our study suggests that GH deficiency does not have an effect on the corneal parameters and CCT values. This observation could be because of the duration between the beginning of disease and the diagnosis and beginning of GH therapy

Omega-3 Fatty Acid and Iron Supplementation Alone, but Not in Combination, Lower Inflammation and Anemia of Infection in Mycobacterium tuberculosis-Infected Mice

Progressive inflammation and anemia are common in tuberculosis (TB) and linked to poor clinical outcomes. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have inflammation-resolving properties, whereas iron supplementation in TB may have limited efficacy and enhance bacterial growth. We investigated effects of iron and EPA/DHA supplementation, alone and in combination, on inflammation, anemia, iron status markers and clinical outcomes in Mycobacterium tuberculosis-infected C3HeB/FeJ mice. One week post-infection, mice received the AIN-93 diet without (control) or with supplemental iron (Fe), EPA/DHA, or Fe+EPA/DHA for 3 weeks. Mice supplemented with Fe or EPA/DHA had lower soluble transferrin receptor, ferritin and hepcidin than controls, but these effects were attenuated in Fe+EPA/DHA mice. EPA/DHA increased inflammation-resolving lipid mediators and lowered lung IL-1α, IFN-γ, plasma IL-1β, and TNF-α. Fe lowered lung IL-1α, IL-1β, plasma IL-1β, TNF-α, and IL-6. However, the cytokine-lowering effects in the lungs were attenuated with Fe+EPA/DHA. Mice supplemented with EPA/DHA had lower lung bacterial loads than controls, but this effect was attenuated in Fe+EPA/DHA mice. Thus, individually, post-infection EPA/DHA and iron supplementation lowered systemic and lung inflammation and mitigated anemia of infection in TB, but not when combined. EPA/DHA also enhanced bactericidal effects and could support inflammation resolution and management of anemia

Adjunct n-3 Long-Chain Polyunsaturated Fatty Acid Treatment in Tuberculosis Reduces Inflammation and Improves Anemia of Infection More in C3HeB/FeJ Mice With Low n-3 Fatty Acid Status Than Sufficient n-3 Fatty Acid Status

Populations at risk for tuberculosis (TB) may have a low n-3 polyunsaturated fatty acid (PUFA) status. Our research previously showed that post-infection supplementation of n-3 long-chain PUFA (LCPUFA) in TB without TB medication was beneficial in n-3 PUFA sufficient but not in low-status C3HeB/FeJ mice. In this study, we investigated the effect of n-3 LCPUFA adjunct to TB medication in TB mice with a low compared to a sufficient n-3 PUFA status. Mice were conditioned on an n-3 PUFA-deficient (n- 3FAD) or n-3 PUFA-sufficient (n-3FAS) diet for 6 weeks before TB infection. Postinfection at 2 weeks, both groups were switched to an n-3 LCPUFA [eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA)] supplemented diet and euthanized at 4- and 14- days post-treatment. Iron and anemia status, bacterial loads, lung pathology, lung cytokines/chemokines, and lung lipid mediators were measured. Following 14 days of treatment, hemoglobin (Hb) was higher in the n-3FAD than the untreated n-3FAS group (p = 0.022), whereas the n-3FAS (drug) treated control and n-3FAS groups were not. Proinflammatory lung cytokines; interleukin-6 (IL-6) (p = 0.011), IL-1a (p = 0.039), MCP1 (p = 0.003), MIP1- a (p = 0.043), and RANTES (p = 0.034); were lower, and the antiinflammatory cytokine IL-4 (p=0.002) and growth factor GMCSF (p=0.007) were higher in the n-3FAD compared with the n-3FAS mice after 14 days. These results suggest that n-3 LCPUFA therapy in TB-infected mice, in combination with TB medication, may improve anemia of infection more in low n-3 fatty acid status than sufficient status mice. Furthermore, the low n-3 fatty acid status TB mice supplemented with n-3 LCPUFA showed comparatively lower cytokine-mediated inflammation despite presenting with lower pro-resolving lipid mediators

Omega-3 long-chain polyunsaturated fatty acids promote antibacterial and inflammation-resolving effects in Mycobacterium tuberculosis-infected C3HeB/FeJ mice, dependent on fatty acid status

AbstractNon-resolving inflammation is characteristic of tuberculosis (TB). Given their inflammation-resolving properties,n-3 long-chain PUFA (n-3 LCPUFA) may support TB treatment. This research aimed to investigate the effects ofn-3 LCPUFA on clinical and inflammatory outcomes ofMycobacterium tuberculosis-infected C3HeB/FeJ mice with either normal or lown-3 PUFA status before infection. Using a two-by-two design, uninfected mice were conditioned on either ann-3 PUFA-sufficient (n-3FAS) or -deficient (n-3FAD) diet for 6 weeks. One week post-infection, mice were randomised to eithern-3 LCPUFA supplemented (n-3FAS/n-3+ andn-3FAD/n-3+) or continued onn-3FAS orn-3FAD diets for 3 weeks. Mice were euthanised and fatty acid status, lung bacterial load and pathology, cytokine, lipid mediator and immune cell phenotype analysed.n-3 LCPUFA supplementation inn-3FAS mice lowered lung bacterial loads (P= 0·003), T cells (P= 0·019), CD4+T cells (P= 0·014) and interferon (IFN)-γ(P< 0·001) and promoted a pro-resolving lung lipid mediator profile. Compared withn-3FAS mice, then-3FAD group had lower bacterial loads (P= 0·037), significantly higher immune cell recruitment and a more pro-inflammatory lipid mediator profile, however, significantly lower lung IFN-γ, IL-1α, IL-1βand IL-17, and supplementation in then-3FAD group provided no beneficial effect on lung bacterial load or inflammation. Our study provides the first evidence thatn-3 LCPUFA supplementation has antibacterial and inflammation-resolving benefits in TB when provided 1 week after infection in the context of a sufficientn-3 PUFA status, whilst a lown-3 PUFA status may promote better bacterial control and lower lung inflammation not benefiting fromn-3 LCPUFA supplementation

Differential Targeting of c-Maf, Bach-1, and Elmo-1 by microRNA-143 and microRNA-365 Promotes the Intracellular Growth of Mycobacterium tuberculosis in Alternatively IL-4/IL-13 Activated Macrophages

Mycobacterium tuberculosis (Mtb) can subvert the host defense by skewing macrophage activation toward a less microbicidal alternative activated state to avoid classical effector killing functions. Investigating the molecular basis of this evasion mechanism could uncover potential candidates for host directed therapy against tuberculosis (TB). A limited number of miRNAs have recently been shown to regulate host-mycobacterial interactions. Here, we performed time course kinetics experiments on bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (MDMs) alternatively activated with IL-4, IL-13, or a combination of IL-4/IL-13, followed by infection with Mtb clinical Beijing strain HN878. MiR-143 and miR-365 were highly induced in Mtb-infected M(IL-4/IL-13) BMDMs and MDMs. Knockdown of miR-143 and miR-365 using antagomiRs decreased the intracellular growth of Mtb HN878, reduced the production of IL-6 and CCL5 and promoted the apoptotic death of Mtb HN878-infected M(IL-4/IL-13) BMDMs. Computational target prediction identified c-Maf, Bach-1 and Elmo-1 as potential targets for both miR-143 and miR-365. Functional validation using luciferase assay, RNA-pulldown assay and Western blotting revealed that c-Maf and Bach-1 are directly targeted by miR-143 while c-Maf, Bach-1, and Elmo-1 are direct targets of miR-365. Knockdown of c-Maf using GapmeRs promoted intracellular Mtb growth when compared to control treated M(IL-4/IL-13) macrophages. Meanwhile, the blocking of Bach-1 had no effect and blocking Elmo-1 resulted in decreased Mtb growth. Combination treatment of M(IL-4/IL-13) macrophages with miR-143 mimics or miR-365 mimics and c-Maf, Bach-1, or Elmo-1 gene-specific GapmeRs restored Mtb growth in miR-143 mimic-treated groups and enhanced Mtb growth in miR-365 mimics-treated groups, thus suggesting the Mtb growth-promoting activities of miR-143 and miR-365 are mediated at least partially through interaction with c-Maf, Bach-1, and Elmo-1. We further show that knockdown of miR-143 and miR-365 in M(IL-4/IL-13) BMDMs decreased the expression of HO-1 and IL-10 which are known targets of Bach-1 and c-Maf, respectively, with Mtb growth-promoting activities in macrophages. Altogether, our work reports a host detrimental role of miR-143 and miR-365 during Mtb infection and highlights for the first time the role and miRNA-mediated regulation of c-Maf, Bach-1, and Elmo-1 in Mtb-infected M(IL-4/IL-13) macrophages

Evaluation of Minor Groove Binders (MGBs) as novel anti-mycobacterial agents, and the effect of using non-ionic surfactant vesicles as a delivery system to improve their efficacy

Objectives: The slow development of major advances in drug discovery for the treatment of Mycobacterium tuberculosis (Mtb) infection have led to a compelling need for evaluation of more effective drug therapies against tuberculosis. New classes of drugs are constantly being evaluated for anti-mycobacterial activity with currently a very limited number of new drugs approved for TB treatment. Minor Groove Binders (MGBs) have previously revealed promising anti-microbial activity against various infectious agents; however have not yet been screened against Mtb. Methods: Mycobactericidal activity of MGB compounds against Mtb was determined using H37Rv-GFP microplate assay. MGB hits were screened for their intracellular mycobactericidal efficacy against clinical Beijing Mtb strain HN878 in bone marrow-derived macrophages using standard colony-forming unit counting. Cell viability was assessed by CellTiter-Blue assays. Selected MGB were encapsulated into non-ionic surfactant vesicles (NIVs) for drug delivery system evaluation. Results: H37Rv-GFP screening yielded a hitlist of 7 compounds at an MIC99 between 0.39 and 1.56 μM. MGB-362 and MGB-364 displayed intracellular mycobactericidal activity against Mtb HN878 at MIC50 of 4.09 μM and 4.19 μM respectively, whilst being non-toxic. Subsequent encapsulation into NIVs demonstrated a 1.6 and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared to MGB alone formulation Conclusions: MGBs anti-mycobacterial activities together with non-toxic properties indicate that MGB compounds constitute an important new class of drug/chemical entity, which holds promise in future anti-TB therapy. Furthermore, NIVs ability to better deliver entrapped MGB compounds to an intracellular Mtb infection has provided merit for further preclinical evaluation

Differential Targeting of c-Maf, Bach-1, and Elmo-1 by microRNA-143 and microRNA-365 Promotes the Intracellular Growth of Mycobacterium tuberculosis in Alternatively IL-4/IL-13 Activated Macrophages.

Mycobacterium tuberculosis (Mtb) can subvert the host defense by skewing macrophage activation toward a less microbicidal alternative activated state to avoid classical effector killing functions. Investigating the molecular basis of this evasion mechanism could uncover potential candidates for host directed therapy against tuberculosis (TB). A limited number of miRNAs have recently been shown to regulate host-mycobacterial interactions. Here, we performed time course kinetics experiments on bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (MDMs) alternatively activated with IL-4, IL-13, or a combination of IL-4/IL-13, followed by infection with Mtb clinical Beijing strain HN878. MiR-143 and miR-365 were highly induced in Mtb-infected M(IL-4/IL-13) BMDMs and MDMs. Knockdown of miR-143 and miR-365 using antagomiRs decreased the intracellular growth of Mtb HN878, reduced the production of IL-6 and CCL5 and promoted the apoptotic death of Mtb HN878-infected M(IL-4/IL-13) BMDMs. Computational target prediction identified c-Maf, Bach-1 and Elmo-1 as potential targets for both miR-143 and miR-365. Functional validation using luciferase assay, RNA-pulldown assay and Western blotting revealed that c-Maf and Bach-1 are directly targeted by miR-143 while c-Maf, Bach-1, and Elmo-1 are direct targets of miR-365. Knockdown of c-Maf using GapmeRs promoted intracellular Mtb growth when compared to control treated M(IL-4/IL-13) macrophages. Meanwhile, the blocking of Bach-1 had no effect and blocking Elmo-1 resulted in decreased Mtb growth. Combination treatment of M(IL-4/IL-13) macrophages with miR-143 mimics or miR-365 mimics and c-Maf, Bach-1, or Elmo-1 gene-specific GapmeRs restored Mtb growth in miR-143 mimic-treated groups and enhanced Mtb growth in miR-365 mimics-treated groups, thus suggesting the Mtb growth-promoting activities of miR-143 and miR-365 are mediated at least partially through interaction with c-Maf, Bach-1, and Elmo-1. We further show that knockdown of miR-143 and miR-365 in M(IL-4/IL-13) BMDMs decreased the expression of HO-1 and IL-10 which are known targets of Bach-1 and c-Maf, respectively, with Mtb growth-promoting activities in macrophages. Altogether, our work reports a host detrimental role of miR-143 and miR-365 during Mtb infection and highlights for the first time the role and miRNA-mediated regulation of c-Maf, Bach-1, and Elmo-1 in Mtb-infected M(IL-4/IL-13) macrophages

Targeting Molecular Inflammatory Pathways in Granuloma as Host-Directed Therapies for Tuberculosis

Globally, more than 10 million people developed active tuberculosis (TB), with 1.4 million deaths in 2020. In addition, the emergence of drug-resistant strains in many regions of the world threatens national TB control programs. This requires an understanding of host-pathogen interactions and finding novel treatments including host-directed therapies (HDTs) is of utter importance to tackle the TB epidemic. Mycobacterium tuberculosis (Mtb), the causative agent for TB, mainly infects the lungs causing inflammatory processes leading to immune activation and the development and formation of granulomas. During TB disease progression, the mononuclear inflammatory cell infiltrates which form the central structure of granulomas undergo cellular changes to form epithelioid cells, multinucleated giant cells and foamy macrophages. Granulomas further contain neutrophils, NK cells, dendritic cells and an outer layer composed of T and B lymphocytes and fibroblasts. This complex granulomatous host response can be modulated by Mtb to induce pathological changes damaging host lung tissues ultimately benefiting the persistence and survival of Mtb within host macrophages. The development of cavities is likely to enhance inter-host transmission and caseum could facilitate the dissemination of Mtb to other organs inducing disease progression. This review explores host targets and molecular pathways in the inflammatory granuloma host immune response that may be beneficial as target candidates for HDTs against TB