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INTRODUCTION: Population-wide interventions offer a pathway to tuberculosis (TB) and leprosy elimination, but 'real-world' implementation in a high-burden setting using a combined approach has not been demonstrated. This implementation study aims to demonstrate the feasibility and evaluate the effect of population-wide screening, treatment and prevention on TB and leprosy incidence rates, as well as TB transmission. METHODS AND ANALYSIS: A non-randomised 'screen-and-treat' intervention conducted in the Pacific atoll of South Tarawa, Kiribati. Households are enumerated and all residents ≥3 years, as well as children <3 years with recent household exposure to TB or leprosy, invited for screening. Participants are screened using tuberculin skin testing, signs and symptoms of TB or leprosy, digital chest X-ray with computer-aided detection and sputum testing (Xpert MTB/RIF Ultra). Those diagnosed with disease are referred to the National TB and Leprosy Programme for management. Participants with TB infection are offered TB preventive treatment and those without TB disease or infection, or leprosy, are offered leprosy prophylaxis. The primary study outcome is the difference in the annual TB case notification rate before and after the intervention; a similar outcome is included for leprosy. The effect on TB transmission will be measured by comparing the estimated annual risk of TB infection in primary school children before and after the intervention, as a co-primary outcome used for power calculations. Comparison of TB and leprosy case notification rates in South Tarawa (the intervention group) and the rest of Kiribati (the control group) before, during and after the intervention is a secondary outcome. ETHICS AND DISSEMINATION: Approval was obtained from the University of Sydney Human Research Ethics Committee (project no. 2021/127) and the Kiribati Ministry of Health and Medical Services (MHMS). Findings will be shared with the MHMS and local communities, published in

Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

Cigarette smoke (CS) exposure is a key risk factor for both active and latent tuberculosis (TB). It is associated with delayed diagnosis, more severe disease progression, unfavourable treatment outcomes and relapse after treatment. Critically, CS exposure is common in heavily populated areas with a high burden of TB, such as China, India and the Russian Federation. It is therefore prudent to evaluate interventions for TB while taking into account the immunological impacts of CS exposure. This review is a mechanistic examination of how CS exposure impairs innate barrier defences, as well as alveolar macrophage, neutrophil, dendritic cell and T-cell functions, in the context of TB infection and disease.

Multiple SARS-CoV-2 vaccine candidates have been approved for use and have had a major impact on the COVID-19 pandemic. There remains, however, a significant need for vaccines that are safe, easily transportable and protective against infection, as well as disease. Mucosal vaccination is favored for its ability to induce immune memory at the site of infection, making it appealing for SARS-CoV-2 vaccine strategies. In this study we performed in-depth analysis of the immune responses in mice to a subunit recombinant spike protein vaccine formulated with the delta-inulin adjuvant Advax when administered intratracheally (IT), versus intramuscular delivery (IM). Both routes produced robust neutralizing antibody titers (NAb) and generated sterilizing immunity against SARS-CoV-2. IT delivery, however, produced significantly higher systemic and lung-local NAb that resisted waning up to six months post vaccination, and only IT delivery generated inducible bronchus-associated lymphoid tissue (iBALT), a site of lymphocyte antigen presentation and proliferation. This was coupled with robust and long-lasting lung tissue-resident memory CD4+ and CD8+ T cells that were not observed in IM-vaccinated mice. This study provides a detailed view of the lung-resident cellular response to IT vaccination against SARS-CoV-2 and demonstrates the importance of delivery site selection in the development of vaccine candidates.

Uropathogenic Escherichia coli (UPEC) causes urinary tract infections that can result in sepsis. The haemostatic system is protective in the pyelonephritis stage of ascending UPEC infection, but the role of the haemostatic system has not been investigated during sepsis. Here we utilize a zebrafish-UPEC systemic infection model to visualize infection-induced coagulation and examine the effects of commonly prescribed anti-haemostatic medications on the infection severity. Treatment of systemically infected zebrafish with warfarin, aspirin, or ticagrelor reduced host survival, while stabilization of clots with aminocaproic acid increased host survival. Anti-haemostatic drug treatment increased UPEC burden. Our findings provide evidence that commonly prescribed anti-haemostatic medications may worsen the outcome of severe UPEC infection.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam2Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4+ T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam2Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses.

Workers in the mining and construction industries are at increased risk of respiratory and other diseases as a result of being exposed to harmful levels of airborne particulate matter (PM) for extended periods of time. While clear links have been established between PM exposure and the development of occupational lung disease, the mechanisms are still poorly understood. A greater understanding of how exposures to different levels and types of PM encountered in mining and construction workplaces affect pathophysiological processes in the airways and lungs and result in different forms of occupational lung disease is urgently required. Such information is needed to inform safe exposure limits and monitoring guidelines for different types of PM and development of biomarkers for earlier disease diagnosis. Suspended particles with a 50% cut-off aerodynamic diameter of 10 µm and 2.5 µm are considered biologically active owing to their ability to bypass the upper respiratory tract's defences and penetrate deep into the lung parenchyma, where they induce potentially irreversible damage, impair lung function and reduce the quality of life. Here we review the current understanding of occupational respiratory diseases, including coal worker pneumoconiosis and silicosis, and how PM exposure may affect pathophysiological responses in the airways and lungs. We also highlight the use of experimental models for better understanding these mechanisms of pathogenesis. We outline the urgency for revised dust control strategies, and the need for evidence-based identification of safe level exposures using clinical and experimental studies to better protect workers' health.

Regulatory T cells (Tregs) play a critical role in immune regulation and peripheral tolerance. While different types of Tregs have been identified in both mice and humans, much of our understanding about how these cells maintain immune homeostasis is derived from animal models. In this study, we examined two distinct human lymphoid organs to understand how repeated exposure to infections at the mucosal surface influences the phenotype and tissue localization of Tregs. We show that while Tregs in both tonsils and spleen express a tissue-resident phenotype, they accumulate in greater numbers in tonsils. Tonsillar-resident Tregs exhibit a highly suppressive phenotype with significantly increased expression of CD39, ICOS and CTLA-4 compared with their counterparts in circulation or in the spleen. Functionally, resident Tregs are able effectively to suppress T cell proliferation. We further demonstrate that tonsillar-resident Tregs share key features of T follicular helper cells. Spatial analysis reveals that the vast majority of resident Tregs are localized at the border of the T-zone and B cell follicle, as well as within the lymphocyte pockets enriched with resident memory T cells. Together our findings suggest that resident Tregs are strategically co-localized to maintain immune homeostasis at sites of recurrent inflammation.

Objectives: Non-sputum-based tests to accurately identify active tuberculosis (TB) disease and monitor response to therapy are urgently needed. This study examined the biomarker capacity of a panel of plasma proteins alone, and in conjunction with a previously identified miRNA signature, to identify active TB disease. Methods: The expression of nine proteins (IP-10, MCP-1, sTNFR1, RANTES, VEGF, IL-6, IL-10, TNF and Eotaxin) was measured in the plasma of 100 control subjects and 100 TB patients, at diagnosis (treatment naïve) and over the course of treatment (1-, 2- and 6-month intervals). The diagnostic performance of the nine proteins alone, and with the miRNA, was assessed. Results: Six proteins were significantly up-regulated in the plasma of TB patients at diagnosis compared to controls. Receiver operator characteristic curve analysis demonstrated that IP-10 with an AUC = 0.874, sensitivity of 75% and specificity of 87% was the best single biomarker candidate to distinguish TB patients from controls. IP-10 and IL-6 levels fell significantly within one month of commencing treatment and may have potential as indicators of a positive response to therapy. The combined protein and miRNA panel gave an AUC of 1.00. A smaller panel of only five analytes (IP-10, miR-29a, miR-146a, miR-99b and miR-221) showed an AUC = 0.995, sensitivity of 96% and specificity of 97%. Conclusions: A novel combination of miRNA and proteins significantly improves the sensitivity and specificity as a biosignature over single biomarker candidates and may be useful for the development of a non-sputum test to aid the diagnosis of active TB disease.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.

Introduction Treatment of latent tuberculosis infection (LTBI) plays a substantial role in the prevention of drug-susceptible tuberculosis (TB). However, clinical trials to evaluate the efficacy of preventive therapy for presumed multidrug-resistant (MDR) LTBI are lacking. This trial aims to evaluate the efficacy of the antibiotic levofloxacin in preventing the development of active TB among latently infected contacts of index patients with MDR-TB. Methods and analysis A double-blind placebo-controlled parallel group randomised controlled trial will be conducted in 10 provinces of Vietnam. Household contacts living with patients with bacteriologically confirmed rifampicin-resistant or MDR-TB will be eligible for recruitment if they have a positive tuberculin skin test or are known to be immunosuppressed, and do not have active TB. Participants will be randomised to receive either levofloxacin or placebo tablets once per day for 6 months. Screening for incident TB will be performed at 6 months intervals. The primary study outcome is the incidence of bacteriologically confirmed TB within 30 months after randomisation. Analysis will be by intention to treat, using Poisson regression. Ethics Ethical approval from the University of Sydney Human Research Ethics Committee was obtained on 29 April 2015 (2014/929), and from the Vietnam Ministry of Health Institutional Review Board on 30 September 2015 (4040/QD-BYT). Dissemination Findings of the study will be published in peer-reviewed publications and conference presentations. Trial registration number ACTRN12616000215426.

COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

T-lymphocytes are critical for protection against respiratory infections, such as Mycobacterium tuberculosis and influenza virus, with chemokine receptors playing an important role in directing these cells to the lungs. CXCR6 is expressed by activated T-lymphocytes and its ligand, CXCL16, is constitutively expressed by the bronchial epithelia, suggesting a role in T-lymphocyte recruitment and retention. However, it is unknown whether CXCR6 is required in responses to pulmonary infection, particularly on CD4+ T-lymphocytes. Analysis of CXCR6-reporter mice revealed that in naïve mice, lung leukocyte expression of CXCR6 was largely restricted to a small population of T-lymphocytes, but this population was highly upregulated after either infection. Nevertheless, pulmonary infection of CXCR6-deficient mice with M. tuberculosis or recombinant influenza A virus expressing P25 peptide (rIAV-P25), an I-Ab-restricted epitope from the immunodominant mycobacterial antigen, Ag85B, demonstrated that the receptor was redundant for recruitment of T-lymphocytes to the lungs. Interestingly, CXCR6-deficiency resulted in reduced bacterial burden in the lungs 6 weeks after M. tuberculosis infection, and reduced weight loss after rIAV-P25 infection compared to wild type controls. This was paradoxically associated with a decrease in Th1-cytokine responses in the lung parenchyma. Adoptive transfer of P25-specific CXCR6-deficient T-lymphocytes into WT mice revealed that this functional change in Th1-cytokine production was not due to a T-lymphocyte intrinsic mechanism. Moreover, there was no reduction in the number or function of CD4+ and CD8+ tissue resident memory cells in the lungs of CXCR6-deficient mice. Although CXCR6 was not required for T-lymphocyte recruitment or retention in the lungs, CXCR6 influenced the kinetics of the inflammatory response so that deficiency led to increased host control of M. tuberculosis and influenza virus.

Tuberculosis is a chronic inflammatory disease caused by persistent infection with Mycobacterium tuberculosis. The rise of antibiotic resistant strains necessitates the design of novel treatments. Recent evidence shows that not only is M. tuberculosis highly resistant to oxidative killing, it also co-opts host oxidant production to induce phagocyte death facilitating bacterial dissemination. We have targeted this redox environment with the cyclic nitroxide derivative 4-methoxy-TEMPO (MetT) in the zebrafish-M. marinum infection model. MetT inhibited the production of mitochondrial ROS and decreased infection-induced cell death to aid containment of infection. We identify a second mechanism of action whereby stress conditions, including hypoxia, found in the infection microenvironment appear to sensitise M. marinum to killing by MetT both in vitro and in vivo. Together, our study demonstrates MetT inhibited the growth and dissemination of M. marinum through host and bacterial targets.

BACKGROUND:Infection-induced thrombocytosis is a clinically important complication of tuberculosis infection. Recent studies have highlighted the utility of aspirin as a host-directed therapy modulating the inflammatory response to infection but have not investigated the possibility that the effect of aspirin is related to an antiplatelet mode of action. METHODS:In this study, we utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin markedly reduced mycobacterial burden. This effect is reproduced by treatment with platelet-specific glycoprotein IIb/IIIa inhibitors demonstrating a detrimental role for infection-induced thrombocyte activation. RESULTS:We find that the reduction in mycobacterial burden is dependent on macrophages and granuloma formation, providing the first in vivo experimental evidence that infection-induced platelet activation compromises protective host immunity to mycobacterial infection. CONCLUSIONS:Our study illuminates platelet activation as an efficacious target of aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.

Host lipid metabolism is an important target for subversion by pathogenic mycobacteria such as Mycobacterium tuberculosis. The appearance of foam cells within the granuloma are well-characterised effects of chronic tuberculosis. The zebrafish-Mycobacterium marinum infection model recapitulates many aspects of human-M. tuberculosis infection and is used as a model to investigate the structural components of the mycobacterial granuloma. Here, we demonstrate that the zebrafish-M. marinum granuloma contains foam cells and that the transdifferentiation of macrophages into foam cells is driven by the mycobacterial ESX1 pathogenicity locus. This report demonstrates conservation of an important aspect of mycobacterial infection across species.

Changes to lipid metabolism are well-characterised consequences of human tuberculosis infection but their functional relevance are not clearly elucidated in these or other host-mycobacterial systems. The zebrafish-Mycobacterium marinum infection model is used extensively to model many aspects of human-M. tuberculosis pathogenesis but has not been widely used to study the role of infection-induced lipid metabolism. We find mammalian mycobacterial infection-induced alterations in host Low Density Lipoprotein metabolism are conserved in the zebrafish model of mycobacterial pathogenesis. Depletion of LDLR, a key lipid metabolism node, decreased M. marinum burden, and corrected infection-induced altered lipid metabolism resulting in decreased LDL and reduced the rate of macrophage transformation into foam cells. Our results demonstrate a conserved role for infection-induced alterations to host lipid metabolism, and specifically the LDL-LDLR axis, across host-mycobacterial species pairings.

© 2018 The British Infection Association Objective: microRNA expression profiles are of interest as a biomarker of tuberculosis (TB). How anti-TB therapy effects miRNA profiles is unknown and was examined. Methods: We identified 87 plasma miRNAs that were significantly modified in an exploratory group of 19 Chinese pulmonary TB (PTB) patients compared to 14 healthy controls. We selected 10 of these miRNAs for analysis in a cohort of 100 PTB patients prior to, and at one, two and six months during treatment. Results: Five miRNAs were differentially expressed in PTB patients compared to controls at diagnosis; miRs −29a and −99b were up-regulated, whilst miRs −21, −146a and −652 were down-regulated. A combination of 5 miRNA distinguished TB from healthy controls with a sensitivity of 94%, a specificity of 88%, and an AUC of 0.976. Within one month of treatment, significant changes in miRs −29a, −99b, −26a and 146a levels occurred in successfully treated patients, although not all miRNAs returned to baseline by treatment completion. Conclusion: A 5-miRNA signature shows potential for development as a novel biomarker for TB disease with potential to predict response to treatment. The failure of all miRNA to return to baseline levels may reflect ongoing remodelling in the lung parenchyma that continues after completion of anti-TB therapy.

© 2017 Elsevier Inc. The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103-, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections.

© 2015 The Authors. Tuberculosis (TB) remains a major public health issue. New tests to aid diagnoses and monitor the response to therapy are urgently required. There is growing interest in the use of microRNA (miRNA) profiles as diagnostic, prognostic or predictive markers in a range of clinical and infectious diseases, including Mycobacterium tuberculosis infection, however, challenges exist to accurately normalise miRNA levels in cohorts. This study examined the appropriateness of 12 miRs and RNU6B to normalise circulating plasma miRNA levels in individuals with active TB from 2 different geographical and ethnic regions. Twelve miRs (let-7, miR-16, miR-22, miR-26, miR-93, miR-103, miR-191, miR-192, miR-221, miR-423, miR-425 and miR-451) and RNU6B were selected based on their reported production by lung cells, expression in blood and previous use as a reference miRNA. Expression levels were analysed in the plasma of newly diagnosed TB patients from Australia and China compared with individuals with latent TB infection and healthy volunteers. Analysis with both geNorm and NormFinder software identified miR-93 as the most suitable reference miR in both cohorts, either when analysed separately or collectively. Interestingly, there were large variations in the expression levels of some miRs, in particular miR-192 and let-7, between the two cohorts, independent of disease status. These data identify miR-93 is a suitable reference miR for normalizing miRNA levels in TB patients, and highlight how environmental, and possibly ethnic, factors influence miRNA expression levels, demonstrating the necessity of assessing the suitability of reference miRs within the study population.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Microparticles (MPs) are small membranous particles (100-1000 nm) released under normal steady-state conditions and are thought to provide a communication network between host cells. Previous studies demonstrated that Mycobacterium tuberculosis (M. tb) infection of macrophages increased the release of MPs, and these MPs induced a proinflammatory response from uninfected macrophages in vitro and in vivo following their transfer into uninfected mice. To determine how M. tb infection modulates the protein composition of the MPs, and if this contributes to their proinflammatory properties, we compared the proteomes of MPs derived from M. tb-infected (TBinf-MP) and uninfected human THP-1 monocytic cells. MP proteins were analyzed by GeLC-MS/MS with spectral counting revealing 68 proteins with statistically significant differential abundances. The 42 proteins increased in abundance in TBinf-MPs included proteins associated with immune function (7), lysosomal/endosomal maturation (4), vesicular formation (12), nucleosome proteins (4), and antigen processing (9). Prominent among these were the type I interferon inducible proteins, ISG15, IFIT1, IFIT2, and IFIT3. Exposure of uninfected THP-1 cells to TBinf-MPs induced increased gene expression of isg15, ifit1, ifit2, and ifit3 and the release of proinflammatory cytokines. These proteins may regulate the proinflammatory potential of the MPs and provide candidate biomarkers for M. tb infection.

Background: Tuberculosis (TB) is an infectious disease that remains a major cause of morbidity and mortality worldwide, yet the reasons why only 10% of people infected with Mycobacterium tuberculosis go on to develop clinical disease are poorly understood. Genetically determined variation in the host immune response is one factor influencing the response to M. tuberculosis. SP110 is an interferon-responsive nuclear body protein with critical roles in cell cycling, apoptosis and immunity to infection. However association studies of the gene with clinical TB in different populations have produced conflicting results. Methods: To examine the importance of the SP110 gene in immunity to TB in the Vietnamese we conducted a case-control genetic association study of 24 SP110 variants, in 663 patients with microbiologically proven TB and 566 unaffected control subjects from three tertiary hospitals in northern Vietnam. Results: Five SNPs within SP110 were associated with all forms of TB, including four SNPs at the C terminus (rs10208770, rs10498244, rs16826860, rs11678451) under a dominant model and one SNP under a recessive model, rs7601176. Two of these SNPs were associated with pulmonary TB (rs10208770 and rs16826860) and one with extra-pulmonary TB (rs10498244). Conclusion: SP110 variants were associated with increased susceptibility to both pulmonary and extra-pulmonary TB in the Vietnamese. Genetic variants in SP110 may influence macrophage signaling responses and apoptosis during M. tuberculosis infection, however further research is required to establish the mechanism by which SP110 influences immunity to tuberculosis infection. © 2014 Fox et al.

BCG vaccine is one of the most commonly-administered vaccines worldwide. Studies suggest the protective efficacy of BCG against TB is better for children than for adults. One potential explanation is that BCG induces a better protective immune response in children. Twenty six children and adults were immunised with BCG. The proportion of Th1-cytokine-producing mycobacterial-specific T cells, and the concentrations of secreted cytokines, were measured before and 10 weeks after BCG immunisation. A significant increase in the proportion of mycobacterial-specific cytokine-producing T cells was observed in both age groups. After BCG immunisation, children and adults had comparable proportions of mycobacterial-specific polyfunctional CD4 T cells when measured relative to the total number of CD4 T cells. However, relative to the subset of Th-1-cytokine-producing CD4 T cells, the proportion of polyfunctional cells was greater in children. Concentrations of secreted cytokines were comparable in children and adults. These findings suggest that the mycobacterial-specific cell-mediated immune response induced by BCG immunisation in children and adults is similar. The implication of a shift to a more polyfunctional immune response within the Th1-cytokine-producing CD4 T cells in children is uncertain as this aspect of the immune response has not been assessed as a potential correlate of protection against TB.

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis. © 2012 Blumenthal et al.