Publications

INTRODUCTION: Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to Mycobacterium tuberculosis antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to M. tuberculosis antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis.

METHODS: A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established in vitro human granuloma model wherein peripheral blood mononuclear cells were exposed to M. tuberculosis antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation via phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin).

RESULTS: In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction.

CONCLUSIONS: Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.

γ-Tilmanocept (99m Tc-tilmanocept) is a receptor-directed, radiolabeled tracer that is FDA-approved for guiding sentinel lymph node biopsy. Tilmanocept binds the C-type lectin mannose receptor (MR, CD206) on macrophages. In this study, nonradioactive, fluorescently-labeled Cy3-tilmanocept was used to detect CD206+ mononuclear cells in the cartilage of mice with antibody-induced arthritis and in the synovial fluid and tissue of human subjects with rheumatoid arthritis (RA) for comparison with osteoarthritis (OA), and healthy volunteer (HV) controls. Murine arthritis was induced by injection of monoclonal anti-cartilage antibody followed by injection of Escherichia coli lipopolysaccharide. Post-arthritis development (7-11 days), the mice were injected intravenously with Cy3-tilmanocept followed by in vivo and ex vivo epifluorescence imaging. Two-photon imaging, immunofluorescence, and immunohistochemistry were used to identify articular and synovial macrophages (CD206, F4/80, and Cy3-tilmanocept binding) in murine tissues. Cy3-tilmanocept epifluorescence was present in arthritic knees and elbows of murine tissues; no radiographic changes were noted in the skeletons. However, inflammatory arthritic changes were apparent by histopathology and immunohistochemistry (F4/80), immunofluorescence (CD206) and Cy3-tilmanocept binding. In human RA synovial fluid, Cy3-tilmanocept staining correlated with CD206+ /CD16+ cells; negligible labeling was observed in OA samples. Cy3-tilmanocept colocalized with CD206 and staining was significantly higher in RA synovial tissue compared to OA or HV. Our results demonstrate that imaging with Cy3-tilmanocept can detect in vivo inflammatory, CD206+ macrophages in an early arthritis animal model and in human RA patients. These data establish a novel tool for preclinical research of early arthritis and have implications for early RA detection and monitoring of therapeutic efficacy in humans.

Inflammation plays a crucial role in the control of Mycobacterium tuberculosis infection. In this study, we demonstrate that an inflammatory pulmonary environment at the time of infection mediated by lipopolysaccharide treatment in mice confers enhanced protection against M. tuberculosis for up to 6 months postinfection. This early and transient inflammatory environment was associated with a neutrophil and CD11b+ cell influx and increased inflammatory cytokines. In vitro infection demonstrated that neutrophils from lipopolysaccharide-treated mice exhibited increased association with M. tuberculosis and had a greater innate capacity for killing M. tuberculosis. Finally, partial depletion of neutrophils in lipopolysaccharide-treated mice showed an increase in M. tuberculosis burden, suggesting neutrophils played a part in the protection observed in lipopolysaccharide-treated mice. These results indicate a positive role for an inflammatory environment in the initial stages of M. tuberculosis infection and suggest that acute inflammation at the time of M. tuberculosis infection can positively alter disease outcome. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, is estimated to infect one-fourth of the world's population and is one of the leading causes of death due to an infectious disease worldwide. The high-level variability in tuberculosis disease responses in the human populace may be linked to immune processes related to inflammation. In many cases, inflammation appears to exasperate tuberculosis responses; however, some evidence suggests inflammatory processes improve control of M. tuberculosis infection. Here, we show an acute inflammatory stimulus in mice provides protection against M. tuberculosis for up to 6 months, suggesting acute inflammation can positively affect M. tuberculosis infection outcome.

BACKGROUND: Interferon gamma release assays (IGRAs) are used to detect latent Mycobacterium tuberculosis (M.tb) infection (LTBI) in adults, but their performance in older people is not well-established. We evaluated IGRAs for LTBI detection in older Hispanic recent TB contacts (ReC) or community controls (CoC).

METHODS: Cross-sectional assessment of LTBI with T-SPOT.TB and/or QuantiFERON-Gold in-tube or -Plus assay in older (≥60 years) and adult (18-50 years) Hispanic people.

RESULTS: We enrolled 193 CoC (119 adults, 74 older persons) and 459 ReC (361 adults, 98 older persons). LTBI positivity increased with age in CoC (19%-59%, P<0.001), but was similar in ReC (59%-69%, P=0.329). Older people had lower concordance between IGRAs (kappa 0.465 vs 0.688 in adults) and more inconclusive results (indeterminate/borderline 11.6% vs 5.8% in adults, P=0.012). With simultaneous IGRAs, inconclusive results were resolved as positive or negative with the other IGRA. The magnitude of response to M.tb peptides in IGRAs was similar among age groups, but responsiveness to mitogens was lower in older people.

CONCLUSIONS: IGRAs are suitable for LTBI detection in older people. Discordant and inconclusive findings are more prevalent in older people, but results are resolved when IGRA is repeated with a different IGRA test.

Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell population comprised of immature myeloid cells and myeloid progenitors with very potent immunosuppressive potential. MDSCs are reported to be abundant in the lungs of active tuberculosis (TB) patients. We sought to perform an in-depth study of MDSCs during latent TB infection (LTBI) and active TB (ATB) using the nonhuman primate (NHP) model of pulmonary TB. We found a higher proportion of granulocytic, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the lungs of ATB animals compared to those with LTBI or naive control animals. Active disease in the lung, but not LTBI, was furthermore associated with higher proliferation, expansion, and immunosuppressive capabilities of PMN-MDSCs, as shown by enhanced expression of Ki67, indoleamine 2,3-dioxygenase (IDO1), interleukin-10 (IL-10), matrix metallopeptidase 9 (MMP-9), inducible nitric oxide synthase (iNOS), and programmed death-ligand 1 (PD-L1). These immunosuppressive PMN-MDSCs specifically localized to the lymphocytic cuff at the periphery of the granulomas in animals with ATB. Conversely, these cells were scarcely distributed in interstitial lung tissue and the inner core of granulomas. This spatial regulation suggests an important immunomodulatory role of PMN-MDSCs by restricting T cell access to the TB granuloma core and can potentially explain dysfunctional anti-TB responses in active granuloma. Our results raise the possibility that the presence of MDSCs can serve as a biomarker for ATB, while their disappearance can indicate successful therapy. Furthermore, MDSCs may serve as a potential target cell for adjunctive TB therapy. IMPORTANCE Myeloid cells are immunocytes of innate origin that orchestrate the first response toward pathogens via immune surveillance (uptake and killing), antigen presentation, and initiation of adaptive immunity by T cell stimulation. However, MDSCs are a subset of innate immunocytes that deviate to an immunoregulatory phenotype. MDSCs possess strong immunosuppressive capabilities that are induced in autoimmune, malignant neoplastic, and chronic inflammatory diseases. Induction of MDSCs has been found in peripheral blood, bronchoalveolar lavage (BAL) fluid, and pleural effusions of active TB patients, but their precise localization in lung tissue and in TB granulomas remains unclear due to challenges associated with sampling lungs and granulomas from active TB patients. Nonhuman primates (NHPs) are an important animal model with TB granulomas that closely mimic those found in humans and can therefore be used for studies that are otherwise challenging with patient material. Herein, we study MDSC localization in the lungs of NHPs exhibiting latent and active TB. Our findings reveal that MDSCs localize and exert their immunosuppressive roles at the periphery rather than in the core of TB granulomas.

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis ( M . tb ), resulted in almost 1.4 million deaths in 2019 and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M . tb comes in close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M . tb upon contact, defining subsequent M . tb -host cell interactions and infection outcomes in vitro and in vivo . We also demonstrated that ALF from 60+ year old elders (E-ALF) vs . healthy 18- to 45-year-old adults (A-ALF) is dysfunctional with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay demonstrates that M . tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M . tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M . tb exposure to E-ALF shows lesser transcriptional response, with most of the M . tb genes unchanged or downregulated. Overall, this study indicates that M . tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status determined by factors such as age might play an important role in determining infection outcome.

Universal approaches to the prevention and treatment of human diseases fail to take into account profound immune diversity resulting from genetic variations across populations. Personalized or precision medicine takes into account individual lifestyle, environment, and biology (genetics and immune status) and is being adopted in several disease intervention strategies such as cancer and heart disease. However, its application in infectious diseases, particularly global diseases such as tuberculosis (TB), is far more complex and in a state of infancy. Here, we discuss the impact of human genetic variations on immune responses and how they relate to failures seen in current TB diagnostic, therapy, and vaccine approaches across populations. We offer our perspective on the challenges and potential for more refined approaches going forward.

Oxidized cholesterols have emerged as important signaling molecules of immune function, but little is known about the role of these oxysterols during mycobacterial infections. We found that expression of the oxysterol-receptor GPR183 was reduced in blood from patients with tuberculosis (TB) and type 2 diabetes (T2D) compared to TB patients without T2D and was associated with TB disease severity on chest x-ray. GPR183 activation by 7α,25-dihydroxycholesterol (7α,25-OHC) reduced growth of Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis BCG in primary human monocytes, an effect abrogated by the GPR183 antagonist GSK682753. Growth inhibition was associated with reduced IFN-β and IL-10 expression and enhanced autophagy. Mice lacking GPR183 had significantly increased lung Mtb burden and dysregulated IFNs during early infection. Together, our data demonstrate that GPR183 is an important regulator of intracellular mycobacterial growth and interferons during mycobacterial infection.