Joanne Turner

Immune system changes with healthy aging lead to chronic systemic inflammation termed inflammaging, contributing to age-related pathologies. Alveolar macrophages (AMs) maintain lung homeostasis and health. The impact of inflammaging on AM populations requires further definition. Herein, we examined the effect of healthy old age on the phenotype and ontogeny of AMs from mice, non-human primates and humans. We identify three AM subpopulations in old age, including a CD11c ⁺ CD11b ⁺ population that increases more than 10-fold across mammalian species, leading to significant changes associated with heightened inflammation and immune dysregulation. RNA-seq analysis identifies distinct transcriptional AM subpopulation profiles. Experiments in mice reveal the importance of the alveolar environment in shaping AM phenotype and recruitment in healthy old age. The old age alveolar environment promotes phenotypic reprogramming of tissue-resident AMs (TRAMs) toward a CD11c ⁺ CD11b ⁺ state in addition to enhancing CCR2-dependent recruitment of monocyte-derived AMs, which are relatively short-lived and exhibit high turnover due to Fas-mediated apoptosis. These studies provide important insight regarding the impact of the alveolar environment in healthy old age on AM phenotype and function.

Immune system changes with age lead to chronic systemic inflammation termed "inflammaging", contributing to age-related pathologies. Alveolar macrophages (AMs) maintain lung homeostasis and health. The impact of inflammaging on AM populations requires further definition. Herein, we examined the effect of age on the phenotype and ontogeny of AMs from mice, non-human primates and humans. We identify three AM subpopulations in old age, two of which increase more than 10-fold, leading to significant functional consequences associated with heightened inflammation and immune dysregulation. RNA-seq analysis identifies unique transcriptional AM subpopulation profiles. Adoptive transfer experiments reveal the importance of the alveolar environment in AM recruitment and phenotypic change in old age. Monocyte-derived AM recruitment in old age requires CCR2 and leads to relatively short-lived AMs with high turnover due to Fas-mediated apoptosis. These studies provide new insight on the impact of the alveolar environment in healthy old age on AM phenotype and function.

We previously reported that live, but not dead, virulent Mycobacterium tuberculosis ( Mtb ) H37Rv bacilli induce cell death in human lung fibroblast cell lines, MRC-5, MRC-9, and TIG-1. Here, using two distinct Mtb strains from two different lineages (HN878 lineage 2 and H37Rv lineage 4), we confirmed cell death at day 2 after infection with a device that measures cell growth/cytotoxicity in real time (Maestro-Z [AXION]). Mtb bacilli uptake by the fibroblast was confirmed with a transmission electron microscope on day 2. Expressions of inflammatory cytokines and interleukin (IL)−1β, IL-6, and IL-8 were observed when exposed to live, but not dead bacteria. The cell death of fibroblasts induced by both Mtb strains tested was prevented by caspase-1/4 and NLRP3 inflammasome inhibitors, but not by caspase-3 and caspase-9 inhibitors. Therefore, we classified the fibroblast cell death by Mtb infection as pyroptosis. To investigate the biological and pathological relevance of fibroblast cell death by Mtb infection, we performed dual RNA-Seq analysis on Mtb within fibroblasts and Mtb -infected fibroblasts at day 2. In Mtb bacilli tcrR , secE2 , ahpD , and mazF8 genes were highly induced during infection. These genes play roles in survival in a hypoxic environment, production of a calcium-binding protein-inducing cytokine, and regulation of transcription in a toxin-antitoxin system. The gene expressions of IL-1β, IL-6, and IL-8, caspase-4, and NLRP3, but not of caspase-3 and caspase-9, were augmented in Mtb bacilli-infected fibroblasts. Taken together, our study suggests that Mtb bacilli attempt to survive in lung fibroblasts and that pyroptosis of the host fibroblasts activates the immune system against the infection. IMPORTANCE: The role of “non-classical immune cells,” that is, fibroblasts, epithelial cells, adipocytes, etc., except for the “classical immune cells,” that is, macrophages and lymphoid cells, is not well known in the infection of Mtb bacilli. We have previously found that live, but not dead, Mtb bacilli induce cell death in human lung fibroblasts, except in human macrophages and monocytes. The present study reveals that fibroblasts ingest Mtb bacilli the same as macrophages and that in vivo Mtb bacilli within fibroblasts attempt to survive in the host cells, and pyroptosis, including the production of inflammatory cytokines, is induced in the Mtb-infected fibroblasts. Our results suggest that pyroptosis of the host fibroblasts activates the immune system against the infection.

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 (TB), caused by the bacterium Mycobacterium tuberculosis ( M.tb ), remains a significant health concern worldwide, especially in populations with weakened or compromised immune systems, such as the elderly. Proper adaptive immune function, particularly a CD4 ⁺ T cell response, is central to host immunity against M.tb . Chronic infections, such as M.tb , as well as aging promote T cell exhaustion and senescence, which can impair immune control and promote progression to TB disease. Mitochondrial dysfunction contributes to T cell dysfunction, both in aging and chronic infections and diseases. Mitochondrial perturbations can disrupt cellular metabolism, enhance oxidative stress, and impair T-cell signaling and effector functions. This study examined the impact of mitochondrial transplantation (mito-transfer) on CD4 ⁺ T cell differentiation and function using aged mouse models and human CD4 ⁺ T cells from elderly individuals. Our study revealed that mito-transfer in naïve CD4 ⁺ T cells promoted the generation of protective effector and memory CD4 ⁺ T cells during M.tb infection in mice. Further, mito-transfer enhanced the function of elderly human T cells by increasing their mitochondrial mass and modulating cytokine production, which in turn reduced exhaustion and senescence cell markers. Our results suggest that mito-transfer could be a novel strategy to reestablish aged CD4 ⁺ T cell function, potentially improving immune responses in the elderly and chronic TB patients, with a broader implication for other diseases where mitochondrial dysfunction is linked to T cell exhaustion and senescence.

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.

Mechanisms to shorten the duration of tuberculosis (TB) treatment include new drug formulations or schedules and the development of host-directed therapies (HDTs) that better enable the host immune system to eliminate Mycobacterium tuberculosis. Previous studies have shown that pyrazinamide, a first-line antibiotic, can also modulate immune function, making it an attractive target for combinatorial HDT/antibiotic therapy, with the goal to accelerate clearance of M. tuberculosis. In this study, we assessed the value of anti–IL-10R1 as an HDT along with pyrazinamide and show that short-term anti–IL-10R1 blockade during pyrazinamide treatment enhanced the antimycobacterial efficacy of pyrazinamide, resulting in faster clearance of M. tuberculosis in mice. Furthermore, 45 d of pyrazinamide treatment in a functionally IL-10–deficient environment resulted in sterilizing clearance of M. tuberculosis. Our data suggest that short-term IL-10 blockade with standard TB drugs has the potential to improve clinical outcome by reducing the treatment duration.

Inflammation plays a significant role in lung infection including that caused by Mycobacterium tuberculosis (M.tb), where both adaptive and innate lymphocytes can affect infection control. How inflammation affects infection is understood in a broad sense, including inflammaging (chronic inflammation) seen in the elderly, but the explicit role that inflammation can play in regulation of lymphocyte function is not known. To fill this knowledge gap, we used an acute lipopolysaccharide (LPS) treatment in young mice and studied lymphocyte responses, focusing on CD8 T cell subsets. LPS treatment decreased the total numbers of T cells in the lungs of LPS mice, while also increasing the number of activated T cells. We demonstrate that lung CD8 T cells from LPS mice became capable of an antigen independent innate-like IFN-γ secretion, dependent on IL-12p70 stimulation, paralleling innate-like IFN-γ secretion of lung CD8 T cells from old mice. Overall, this study provides information on how acute inflammation can affect lymphocytes, particularly CD8 T cells, which could potentially affect immune control of various disease states.

The elderly are understudied despite their high risk of tuberculosis (TB). We sought to identify factors underlying the lack of an association between TB and type 2 diabetes (T2D) in the elderly, but not adults. We conducted a case–control study in elderly (≥65 years old; ELD) vs. younger adults (young/middle-aged adults (18–44/45–64 years old; YA|MAA) stratified by TB and T2D, using a research study population (n = 1160) and TB surveillance data (n = 8783). In the research study population the adjusted odds ratio (AOR) of TB in T2D was highest in young adults (AOR 6.48) but waned with age becoming non-significant in the elderly. Findings were validated using TB surveillance data. T2D in the elderly (vs. T2D in younger individuals) was characterized by better glucose control (e.g., lower hyperglycemia or HbA1c), lower insulin resistance, more sulphonylureas use, and features of less inflammation (e.g., lower obesity, neutrophils, platelets, anti-inflammatory use). We posit that differences underlying glucose dysregulation and inflammation in elderly vs. younger adults with T2D, contribute to their differential association with TB. Studies in the elderly provide valuable insights into TB-T2D pathogenesis, e.g., here we identified insulin resistance as a novel candidate mechanism by which T2D may increase active TB risk.