BACKGROUND: Diagnosis of nontuberculous mycobacteria (NTM) infections remains a challenge. In this study, we describe the evaluation of an immunological NTM-interferon (IFN)-γ release assay (IGRA) that we developed using glycopeptidolipids (GPLs) as NTM-specific antigens.

METHODS: We tested the NTM-IGRA in 99 samples from pediatric patients. Seventy-five were patients with lymphadenitis: 25 were NTM confirmed, 45 were of unknown etiology but compatible with mycobacterial infection and 5 had lymphadenitis caused by an etiologic agent other than NTM. The remaining 24 samples were from control individuals without lymphadenitis (latently infected with M. tuberculosis , uninfected controls and active tuberculosis patients). Peripheral blood mononuclear cells were stimulated overnight with GPLs. Detection of IFN-γ producing cells was evaluated by enzyme-linked immunospot assay.

RESULTS: NTM culture-confirmed lymphadenitis patient samples had a significantly higher response to GPLs than the patients with lymphadenitis of unknown etiology but compatible with mycobacterial infection ( P < 0.001) and lymphadenitis not caused by NTM ( P < 0.01). We analyzed the response against GPLs in samples from unknown etiology lymphadenitis but compatible with mycobacterial infection cases according to the tuberculin skin test (TST) response, and although not statistically significant, those with a TST ≥5 mm had a higher response to GPLs when compared with the TST <5 mm group.

CONCLUSIONS: Stimulation with GPLs yielded promising results in detecting NTM infection in pediatric patients with lymphadenitis. Our results indicate that the test could be useful to guide the diagnosis of pediatric lymphadenitis. This new NTM-IGRA could improve the clinical handling of NTM-infected patients and avoid unnecessary misdiagnosis and treatments.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still an ongoing global health crisis. Clinical data indicate that the case fatality rate (CFR) is age dependent, with a higher CFR percentage in the elderly population. We compared the pathogenesis of SARS-CoV-2 in young and aged K18-hACE2 transgenic mice. We evaluated morbidity, mortality, viral titers, immune responses, and histopathology in SARS-CoV-2-infected young and old K18-hACE2 transgenic mice. Within the limitation of having a low number of mice per group, our results indicate that SARS-CoV-2 infection resulted in slightly higher morbidity, mortality, and viral replication in the lungs of old mice, which was associated with an impaired IgM response and altered cytokine and chemokine profiles. Results of this study increase our understanding of SARS-CoV-2 infectivity and immuno-pathogenesis in the elderly population.

Tuberculosis is the leading cause of death for people living with HIV (PLWH). We hypothesized that altered functions of innate immune components in the human alveolar lining fluid of PLWH (HIV-ALF) drive susceptibility to Mycobacterium tuberculosis (M.tb) infection. Our results indicate a significant increase in oxidation of innate proteins and chemokine levels and significantly lower levels and function of complement components and Th1/Th2/Th17 cytokines in HIV-ALF versus control-ALF (non-HIV-infected people). We further found a deficiency of surfactant protein D (SP-D) and reduced binding of SP-D to M.tb that had been exposed to HIV-ALF. Primary human macrophages infected with M.tb exposed to HIV-ALF were significantly less capable of controlling the infection, which was reversed by SP-D replenishment in HIV-ALF. Thus, based on the limited number of participants in this study, our data suggest that PLWH without antiretroviral therapy (ART) have declining host innate defense function in their lung mucosa, thereby favoring M.tb and potentially other pulmonary infections.

The elderly population is highly susceptible to developing respiratory diseases, including tuberculosis, a devastating disease caused by the airborne pathogen Mycobacterium tuberculosis (M.tb) that kills one person every 18 seconds. Once M.tb reaches the alveolar space, it contacts alveolar lining fluid (ALF), which dictates host-cell interactions. We previously determined that age-associated dysfunction of soluble innate components in human ALF leads to accelerated M.tb growth within human alveolar macrophages. Here we determined the impact of human ALF on M.tb infection of alveolar epithelial type cells (ATs), another critical lung cellular determinant of infection. We observed that elderly ALF (E-ALF)-exposed M.tb had significantly increased intracellular growth with rapid replication in ATs compared to adult ALF (A-ALF)-exposed bacteria, as well as a dampened inflammatory response. A potential mechanism underlying this accelerated growth in ATs was our observation of increased bacterial translocation into the cytosol, a compartment that favors bacterial replication. These findings in the context of our previous studies highlight how the oxidative and dysfunctional status of the elderly lung mucosa determines susceptibility to M.tb infection, including dampening immune responses and favoring bacterial replication within alveolar resident cell populations, including ATs, the most abundant resident cell type within the alveoli.

PURPOSE: Repeated instillations of bacillus Calmette et Guérin (BCG) are the gold standard immunotherapeutic treatment for reducing recurrence for patients with high-grade papillary non-muscle invasive bladder cancer (NMIBC) and for eradicating bladder carcinoma-in situ. Unfortunately, some patients are unable to tolerate BCG due to treatment-associated toxicity and bladder removal is sometimes performed for BCG-intolerance. Prior studies suggest that selectively delipidated BCG (dBCG) improves tolerability of intrapulmonary delivery reducing tissue damage and increasing efficacy in preventing Mycobacterium tuberculosis infection in mice. To address the lack of treatment options for NMIBC with BCG-intolerance, we examined if selective delipidation would compromise BCG's antitumor efficacy and at the same time increase tolerability to the treatment.

MATERIALS AND METHODS: Murine syngeneic MB49 bladder cancer models and in vitro human innate effector cell cytotoxicity assays were used to evaluate efficacy and immune impact of selective delipidation in Tokyo and TICE BCG strains.

RESULTS: Both dBCG-Tokyo and dBCG-TICE effectively treated subcutaneous MB49 tumors in mice and enhanced tumor-infiltrating CD8+ T and natural killer cells, similar to conventional BCG. However, when compared to conventional BCG, only dBCG-Tokyo retained a significant effect on intratumoral tumor-specific CD8+ and γδ T cells by increasing their frequencies in tumor tissue and their production of antitumoral function-related cytokines, i.e., IFN-γ and granzyme B. Further, dBCG-Tokyo but not dBCG-TICE enhanced the function and cytotoxicity of innate effector cells against human bladder cancer T24 in vitro.

CONCLUSIONS: These data support clinical investigation of dBCG-Tokyo as a treatment for patients with BCG-intolerant NMIBC.

UNLABELLED: Upon infection, Mycobacterium tuberculosis ( M.tb ) reaches the alveolar space and comes in close contact with human alveolar lining fluid (ALF) for an uncertain period of time prior to its encounter with alveolar cells. We showed that homeostatic ALF hydrolytic enzymes modify the M.tb cell envelope, driving M.tb -host cell interactions. Still, the contribution of ALF during M.tb infection is poorly understood. Here, we exposed 4 M.tb strains with different levels of virulence, transmissibility, and drug resistance (DR) to physiological concentrations of human ALF for 15-min and 12-h, and performed RNA sequencing. Gene expression analysis showed a temporal and strain-specific adaptation to human ALF. Differential expression (DE) of ALF-exposed vs. unexposed M.tb revealed a total of 397 DE genes associated with lipid metabolism, cell envelope and processes, intermediary metabolism and respiration, and regulatory proteins, among others. Most DE genes were detected at 12-h post-ALF exposure, with DR- M.tb strain W-7642 having the highest number of DE genes. Interestingly, genes from the KstR2 regulon, which controls the degradation of cholesterol C and D rings, were significantly upregulated in all strains post-ALF exposure. These results indicate that M.tb -ALF contact drives initial metabolic and physiologic changes in M.tb , with potential implications in infection outcome.

IMPORTANCE: Tuberculosis, caused by airborne pathogen Mycobacterium tuberculosis ( M.tb ), is one of the leading causes of mortality worldwide. Upon infection, M.tb reaches the alveoli and gets in contact with human alveolar lining fluid (ALF), where ALF hydrolases modify the M.tb cell envelope driving subsequent M.tb -host cell interactions. Still, the contributions of ALF during infection are poorly understood. We exposed 4 M.tb strains to ALF for 15-min and 12-h and performed RNA sequencing, demonstrating a temporal and strain-specific adaptation of M.tb to ALF. Interestingly, genes associated with cholesterol degradation were highly upregulated in all strains. This study shows for the first time that ALF drives global metabolic changes in M.tb during the initial stages of the infection, with potential implications in disease outcome. Biologically relevant networks and common and strain-specific bacterial determinants derived from this study could be further investigated as potential therapeutic candidates.

UNLABELLED: Alveolar macrophages (AMs) are unique lung resident cells that contact airborne pathogens and environmental particulates. The contribution of human AMs (HAM) to pulmonary diseases remains poorly understood due to difficulty in accessing them from human donors and their rapid phenotypic change during in vitro culture. Thus, there remains an unmet need for cost-effective methods for generating and/or differentiating primary cells into a HAM phenotype, particularly important for translational and clinical studies. We developed cell culture conditions that mimic the lung alveolar environment in humans using lung lipids, i.e. , Infasurf (calfactant, natural bovine surfactant) and lung-associated cytokines (GM-CSF, TGF-β, and IL-10) that facilitate the conversion of blood-obtained monocytes to an AM-Like (AML) phenotype and function in tissue culture. Similar to HAM, AML cells are particularly susceptible to both Mycobacterium tuberculosis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. This study reveals the importance of alveolar space components in the development and maintenance of HAM phenotype and function, and provides a readily accessible model to study HAM in infectious and inflammatory disease processes, as well as therapies and vaccines.

IMPORTANCE: Millions die annually from respiratory disorders. Lower respiratory track gas-exchanging alveoli maintain a precarious balance between fighting invaders and minimizing tissue damage. Key players herein are resident AMs. However, there are no easily accessible in vitro models of HAMs, presenting a huge scientific challenge. Here we present a novel model for generating AML cells based on differentiating blood monocytes in a defined lung component cocktail. This model is non-invasive, significantly less costly than performing a bronchoalveolar lavage, yields more AML cells than HAMs per donor and retains their phenotype in culture. We have applied this model to early studies of M. tuberculosis and SARS-CoV-2. This model will significantly advance respiratory biology research.

Mitochondrial dysfunction alters cellular metabolism, increases tissue oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, it is investigated whether the transfer of functional mitochondria into CD4+ T cells that are isolated from old mice (aged CD4+ T cells), can abrogate aging-associated mitochondrial dysfunction, and improve the aged CD4+ T cell functionality. The results show that the delivery of exogenous mitochondria to aged non-activated CD4+ T cells led to significant mitochondrial proteome alterations highlighted by improved aerobic metabolism and decreased cellular mitoROS. Additionally, mito-transferred aged CD4+ T cells showed improvements in activation-induced TCR-signaling kinetics displaying markers of activation (CD25), increased IL-2 production, enhanced proliferation ex vivo. Importantly, immune deficient mouse models (RAG-KO) showed that adoptive transfer of mito-transferred naive aged CD4+ T cells, protected recipient mice from influenza A and Mycobacterium tuberculosis infections. These findings support mitochondria as targets of therapeutic intervention in aging.

In the past few decades, drug-resistant (DR) strains of Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), have become increasingly prevalent and pose a threat to worldwide public health. These strains range from multi (MDR) to extensively (XDR) drug-resistant, making them very difficult to treat. Further, the current and future impact of the Coronavirus Disease 2019 (COVID-19) pandemic on the development of DR-TB is still unknown. Although exhaustive studies have been conducted depicting the uniqueness of the M.tb cell envelope, little is known about how its composition changes in relation to drug resistance acquisition. This knowledge is critical to understanding the capacity of DR-M.tb strains to resist anti-TB drugs, and to inform us on the future design of anti-TB drugs to combat these difficult-to-treat strains. In this review, we discuss the complexities of the M.tb cell envelope along with recent studies investigating how M.tb structurally and biochemically changes in relation to drug resistance. Further, we will describe what is currently known about the influence of M.tb drug resistance on infection outcomes, focusing on its impact on fitness, persister-bacteria, and subclinical TB.

The presence of lipoarabinomannan (LAM) in the Mycobacterium tuberculosis (Mtb) cell envelope was first reported close to 100 years ago. Since then, numerous studies have been dedicated to the isolation, purification, structural definition, and elucidation of the biological properties of Mtb LAM. In this review, we present a brief historical perspective on the discovery of Mtb LAM and the herculean efforts devoted to structurally characterizing the molecule because of its unique structural and biological features. The significance of LAM remains high to this date, mainly due to its distinct immunological properties in conjunction with its role as a biomarker for diagnostic tests due to its identification in urine, and thus can serve as a point-of-care diagnostic test for tuberculosis (TB). In recent decades, LAM has been thoroughly studied and massive amounts of information on this intriguing molecule are now available. In this review, we give the readers a historical perspective and an update on the current knowledge of LAM with information on the inherent carbohydrate composition, which is unique due to the often puzzling sugar residues that are specifically found on LAM. We then guide the readers through the complex and myriad immunological outcomes, which are strictly dependent on LAM's chemical structure. Furthermore, we present issues that remain unresolved and represent the immediate future of LAM research. Addressing the chemistry, functions, and roles of LAM will lead to innovative ways to manipulate the processes that involve this controversial and fascinating biomolecule.