Granulomatous Lung Research Articles

Effects of intravenous and oral di(2-ethylhexyl) phthalate (DEHP) and 20% Intralipid vehicle on neonatal rat testis, lung, liver, and kidney

The highest human exposures to the plasticizer di(2-ethylhexyl) phthalate (DEHP) occur through intravenous (iv) exposure from medical procedures. Rodent toxicity studies, mainly using oral exposures, have identified male reproductive toxicity after developmental exposure to DEHP as the primary concern. Other organs are also affected by DEHP and route may influence the degree of target organ involvement. Cammack et al. (2003) reported a critical study focused on testicular toxicity using oral and iv exposures of neonatal Sprague-Dawley rats to 60, 300, or 600 mg/kg body weight/day DEHP in Intralipid vehicle. The present study followed the same dosing paradigm and included assessment of additional organs to evaluate the potential utility of this design for DEHP alternatives. Reduction of testis weight was observed in all DEHP treatment groups and germ cell and Sertoli cell toxicity was observed at the two highest doses with both routes. Lung granulomas occurred in all iv DEHP groups, possibly related to increased fat particle size in DEHP lipid emulsions. Lung alveolar development was inhibited after both oral and iv high dose DEHP. Toxicity of oral Intralipid vehicle was observed in germ and Sertoli cells. The lack of such effects after iv vehicle exposure suggested that this may be a gut-mediated effect.

Local Lung Immune Response to Mycobacterium bovis Challenge after BCG and M. bovis Heat-Inactivated Vaccination in European Badger (Meles meles).

Tuberculosis (TB) vaccination could be used as a key part of integrated strategies for the disease’s control if an effective and safe vaccine under field conditions is obtained. Recent studies in Spain have evaluated the protective efficacy of two oral vaccines against experimental challenge with live intra-bronchial Mycobacterium bovis in captive badgers: the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. With the objective of increasing the knowledge of the cellular development progress of infection and generating further tools to discriminate between mild and severe TB lesions between and within animals, the immunopathology of tuberculous lesions was studied to characterize the local immune response (cell type profile) within lung granulomas from control (non-vaccinated), BCG vaccinated and HIMB-vaccinated experimentally infected badgers with M. bovis. Four immunohistochemical protocols, for the specific detection of macrophages, T lymphocytes, B lymphocytes and plasma cells within TB granulomas in formalin fixed sections of the right middle lung lobe (lobe targeted for the M. bovis delivery), were performed. Immunolabelled sections were scanned and five randomly selected areas were analyzed with digital image analysis software. The results were expressed as the proportion of the positively immunolabelled area within the total area of the selected site. Data was analyzed using the statistical analysis software (SAS). In the three treatment groups, macrophages were the most abundant inflammatory cells within the granulomas, followed by B lymphocytes and plasma cells. T lymphocyes were absent in those granulomas. This would suggest a predominance of a non-specific innate response mediated by phagocytic cells over an adaptative humoral immune response. The proportion of macrophages and plasma cells was higher in BCG and HIMB-vaccinated badgers, respectively, suggesting the establishment of an adaptative humoral response in HIMB-vaccinated badgers. The lower bacterial load at the lung level, as well as the volume of lesions in lungs using magnetic resonance imaging in badgers with the HIMB vaccine in relation with local immune response presented, must be highlighted, since it would be an advantage in favor of its use under field conditions in terms of reducing TB transmission and environmental contamination.

Gut Microbiota Abrogates Anti-α-Gal IgA Response in Lungs and Protects against Experimental Aspergillus Infection in Poultry.

Naturally occurring human antibodies (Abs) of the isotypes IgM and IgG and reactive to the galactose-α-1,3-galactose (α-Gal) epitope are associated with protection against infectious diseases, caused by pathogens expressing the glycan. Gut microbiota bacteria expressing α-Gal regulate the immune response to this glycan in animals lacking endogenous α-Gal. Here, we asked whether the production of anti-α-Gal Abs in response to microbiota stimulation in birds, confers protection against infection by Aspergillus fumigatus, a major fungal pathogen that expresses α-Gal in its surface. We demonstrated that the oral administration of Escherichia coli O86:B7 strain, a bacterium with high α-Gal content, reduces the occurrence of granulomas in lungs and protects turkeys from developing acute aspergillosis. Surprisingly, the protective effect of E. coli O86:B7 was not associated with an increase in circulating anti-α-Gal IgY levels, but with a striking reduction of anti-α-Gal IgA in the lungs of infected turkeys. Subcutaneous immunization against α-Gal did not induce a significant reduction of lung anti-α-Gal IgA and failed to protect against an infectious challenge with A. fumigatus. Oral administration of E. coli O86:B7 was not associated with the upregulation of lung cytokines upon A. fumigatus infection. We concluded that the oral administration of bacteria expressing high levels of α-Gal decreases the levels of lung anti-α-Gal IgA, which are mediators of inflammation and lung damage during acute aspergillosis.

A computational model tracks whole-lung Mycobacterium tuberculosis infection and predicts factors that inhibit dissemination.

Mycobacterium tuberculosis (Mtb), the causative infectious agent of tuberculosis (TB), kills more individuals per year than any other infectious agent. Granulomas, the hallmark of Mtb infection, are complex structures that form in lungs, composed of immune cells surrounding bacteria, infected cells, and a caseous necrotic core. While granulomas serve to physically contain and immunologically restrain bacteria growth, some granulomas are unable to control Mtb growth, leading to bacteria and infected cells leaving the granuloma and disseminating, either resulting in additional granuloma formation (local or non-local) or spread to airways or lymph nodes. Dissemination is associated with development of active TB. It is challenging to experimentally address specific mechanisms driving dissemination from TB lung granulomas. Herein, we develop a novel hybrid multi-scale computational model, MultiGran, that tracks Mtb infection within multiple granulomas in an entire lung. MultiGran follows cells, cytokines, and bacterial populations within each lung granuloma throughout the course of infection and is calibrated to multiple non-human primate (NHP) cellular, granuloma, and whole-lung datasets. We show that MultiGran can recapitulate patterns of in vivo local and non-local dissemination, predict likelihood of dissemination, and predict a crucial role for multifunctional CD8+ T cells and macrophage dynamics for preventing dissemination.

Temporal patterns in single cell immune profile of tuberculosis lung granulomas

Abstract Infection with Mycobacterium tuberculosis results in the formation of tuberculosis lung granulomas, which are complex structures with a network of multiple immune cells closely intertwined and function together. Within a given host, granulomas have unique trajectories and outcomes resulting in bacterial control or persistent bacterial replication and dissemination. The timing of establishment and immune composition of the granuloma in the lung plays a crucial role in the control of bacterial burden. Using high-throughput single-cell mRNA sequencing (SeqWell) on individual TB lung granulomas and computational algorithms, we are generating models to understand infection dynamics that incorporate timing of establishment, immune cell composition, its functional interactions; bacterial burden and trajectories of the granulomas to identify robust drivers of bacterial control. We evaluated original and new lung granulomas identified by PET CT scan from 15 cynomolgus macaques infected with M. tuberculosis (Erdman strain) for 4, 6 or 10 weeks. We identified over 30 functional clusters of cells spanning multiple immune and non-immune cells types including T cells, B cells, plasma cells, dendritic cells, mast cells, macrophages, neutrophils, pneumocytes, fibroblasts and endothelial cells. We observe differential cellular composition associated with temporal patterns (original vs. new) in granulomas that are associated with bacterial burden. By understanding these immune features associated with temporal patterns that limit bacterial replication, we hope to identify novel targets for host-directed immune therapies and prophylactics in M. tuberculosis infection and other granulomatous diseases.

Defining the role of CD8 T cell subsets in tuberculosis in non-human primates

Abstract Tuberculosis (TB) is the number one cause of death by a single infectious agent with no protective vaccine available. Challenges in vaccine development are partially attributed to a deficiency in knowledge regarding specific immune cells’ contribution to protection. CD8 T cells comprise ~30–40% of T cells in the lung granuloma, an organized collection of immune cells that is a hallmark of Mycobacterium tuberculosis (Mtb) infection. However, few studies have explored the diversity, function and contribution to protection of CD8 subsets during TB. We investigated the roles of conventional CD8αβ T cells and unconventional CD8αα T cells during early Mtb infection. Cynomolgus macaques were CD8-depleted using anti-CD8α or anti-CD8β antibodies prior to and during Mtb infection and necropsied at 6 weeks post-infection. CD8α depleted NHPs (CD8αα and CD8αβ depletion) had higher bacterial burdens, worse pathology and overall poorer disease outcome compared to IgG controls, while CD8β depleted (CD8αβ only depletion) displayed an intermediate phenotype. This provides the first evidence for an important early role for non-conventional CD8 T cells in TB. Using spectral flow cytometry we identified multiple CD8 subsets and assessed their functions in granulomas for the first time in NHPs. After CD8α depletion, the repertoire of CD8 subsets dramatically changed in granulomas. This was associated with functional alterations in other immune cell populations which we validated and expanded upon using single-cell RNA sequencing. These studies provide a functional immune landscape of early events in Mtb granulomas as well as the effects of CD8 depletion, shedding light on the protective potential of CD8 T cell subsets during primary Mtb infection.

SIVmac239 infection dysregulates anti-mycobacterial immunity at the granuloma level and contributes to severe tuberculosis in cynomolgus macaques

Abstract Tuberculosis (TB) is a global pandemic caused by Mycobacterium tuberculosis (Mtb) infection that is characterized by formation of lung lesions called granulomas. TB has a synergistic relationship with HIV and TB often occurs in HIV-infected individuals with near normal CD4+ T cell counts. Despite the importance of this co-infection to public health, interactions between HIV and lesion-level immunity remain poorly understood. To address this issue, we used SIVmac239-infected or SIV-negative Mauritian cynomolgus macaques with active TB to identify which cell types are infected in granulomas and how SIV modulates local immunity. Using RNAscope and immunohistochemistry, we found that CD11c+ macrophages were the most-commonly infected cell type in granulomas. To identify how viral infection changes the immune landscape in granulomas, we performed a transcriptional analysis on RNA isolated from lung granulomas. We found that SIV infection downregulated large numbers of macrophage-activating cytokines and receptors, toll-like receptors, and T cell activation markers. In contrast, anti-viral responses including type 1 interferons and receptors expressed by NK cells were upregulated. IL-10 was among the genes that were not differentially regulated. Pathway analysis indicated that many pathways were downregulated, including pathways associated with Th1-, Th2-, and NFkB-mediated responses. These data suggest that SIV infection deactivates protective responses in granulomas but activates antiviral responses that are counterproductive in TB while leaving detrimental IL-10 expression intact. We hypothesize that this disequilibrium leads to an inability to kill bacteria and restrict dissemination that exacerbates TB.

SIV and CD4 depletion distinctly reactivate latent Mycobacterium. tuberculosis infection

Abstract Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB) regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model, we compared reactivation of latent M. tuberculosis (Mtb) infection induced by simian immunodeficiency virus (SIV) or anti-CD4 antibody (αCD4). Reactivation, as defined by the appearance of a new lung lesion by PET CT, occurred in 5 of 7 αCD4 treated animals compared to 4 of 8 SIV infected animals within 2 months of treatment. αCD4 animals had significantly fewer CD4 T cells within granulomas compared to SIV/Mtb infected animals. However, compared to αCD4 groups, SIV infection caused more dissemination of lung granulomas and higher granuloma bacterial burdens. Within the granulomas, SIV replication was associated with greater Mtb growth and reduced Mtb killing. Granulomas from SIV/Mtb animals displayed a distinctly different profile of T cell cytokine and granzyme B expression compared to αCD4 animals and controls. PET CT imaging prior to treatment could predict reactivation in αCD4-treated animals, but not in SIV/Mtb animals. These data suggest that SIV infection disrupts protective immune responses against Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.

FX11 limits Mycobacterium tuberculosis growth and potentiates bactericidal activity of isoniazid through host-directed activity.

ABSTRACTLactate dehydrogenase A (LDHA) mediates interconversion of pyruvate and lactate, and increased lactate turnover is exhibited by malignant and infected immune cells. Hypoxic lung granuloma in Mycobacterium tuberculosis-infected animals present elevated levels of Ldha and lactate. Such alterations in the metabolic milieu could influence the outcome of host-M. tuberculosis interactions. Given the central role of LDHA for tumorigenicity, targeting lactate metabolism is a promising approach for cancer therapy. Here, we sought to determine the importance of LDHA for tuberculosis (TB) disease progression and its potential as a target for host-directed therapy. To this end, we orally administered FX11, a known small-molecule NADH-competitive LDHA inhibitor, to M. tuberculosis-infected C57BL/6J mice and Nos2−/− mice with hypoxic necrotizing lung TB lesions. FX11 did not inhibit M. tuberculosis growth in aerobic/hypoxic liquid culture, but modestly reduced the pulmonary bacterial burden in C57BL/6J mice. Intriguingly, FX11 administration limited M. tuberculosis replication and onset of necrotic lung lesions in Nos2−/− mice. In this model, isoniazid (INH) monotherapy has been known to exhibit biphasic killing kinetics owing to the probable selection of an INH-tolerant bacterial subpopulation. However, adjunct FX11 treatment corrected this adverse effect and resulted in sustained bactericidal activity of INH against M. tuberculosis. As a limitation, LDHA inhibition as an underlying cause of FX11-mediated effect could not be established as the on-target effect of FX11 in vivo was unconfirmed. Nevertheless, this proof-of-concept study encourages further investigation on the underlying mechanisms of LDHA inhibition and its significance in TB pathogenesis.

Non-infectious Complications of Common Variable Immunodeficiency: Updated Clinical Spectrum, Sequelae, and Insights to Pathogenesis.

Non-infectious complications in common variable immunodeficiency (CVID) have emerged as a major clinical challenge. Detailed clinical spectrum, organ-specific pathologies and associated sequelae from 623 CVID patients followed in New York since 1974 were analyzed, and recent insights to pathogenesis were reviewed. Non-infectious manifestations were present in 68.1% of patients, and they do not tend to be present in isolation. They include autoimmunity (33.2%), chronic lung disease (30.3%), lymphoid hyperplasia/splenomegaly (20.9%), liver disease (12.7%), granulomas (9.3%), gastrointestinal disease (7.3%), lymphoma (6.7%), and other malignancies (6.4%). In the lungs, interstitial disease and bronchiectasis were the most common findings, with lymphoma at this site being a rare (n = 6), but serious, manifestation. Bronchiectasis was not a prerequisite for the development of interstitial disease. In the liver, granulomas and nodular regenerative hyperplasia were the most common. Gastrointestinal disease may affect any segment of the intestinal tract, with lymphoid infiltrations and villous blunting being the leading histologic findings. With progression of organ-specific diseases, a wide spectrum of associated sequelae was observed. Lymphoma was more common in females (P = 0.036)—all B cell types except in one subject. Solid organ transplantations (liver, n = 5; lung, n = 4; combined lung and heart, n = 2) and hematopoietic stem cell transplantations (for B cell lymphoma, n = 1) have rarely been performed in this cohort, with mixed outcomes. Recent identification of monogenic defects, in ~10–30% of various CVID cohorts, has highlighted the molecular pathways that can affect both antibody production and broader immune regulation. In addition, cellular defects in both innate and adaptive immune systems are increasingly recognized in this syndrome.

IL-10 Impairs Local Immune Response in Lung Granulomas and Lymph Nodes during Early Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.

Single Center Experience with Hematopoietic Cell Transplantation in Young Children with Chronic Granulomatous Disease

Rationale Chronic granulomatous disease (CGD) is a primary immune deficiency due to defects in the phagocyte oxidative burst leading to life-threatening infections and inflammatory disorders. Due to high mortality and morbidity, hematopoietic cell transplantation (HCT) is now considered a potentially curative option. We evaluated outcomes of HCT at our institution over 13 years. Methods Retrospective chart review of CGD HCTs performed at John Hopkins All Children's Hospital between August 2005-October 2018. Results Eight patients (median age=2.2 years, range=1.1-4.3 years) with CGD underwent HCT: 6 patients had X-linked (CYBB, gp91phox), 2 patients had autosomal recessive (CYBA, p22phox). Pre-HCT infections were controlled including; pneumonia (n=3, 2 Aspergillus, 2 requiring lobectomy) and staphylococcal lymphadenitis/skin abscesses(n=3). Five of eight (62%) had inflammatory disease pre-HCT: lung granulomas (n=2), autoimmune hepatitis (n=3), and colitis (n=2). Myeloablative conditioning was used in 3 and reduced intensity/toxicity (RIC) in 5. Four received matched related donor and 4 received matched unrelated-donor HCTs. Median neutrophil engraftment was 18.5 days (range=10-21 days). Overall survival was 87.5%. One patient died (day +21) from disseminated Trichosporon. All patients achieved primary engraftment; 42% (3/7) maintained full donor chimerism (≥99%) with normalization of DHR, median follow-up 4.3 years (range=4-8.3 years); 42% (3/7) are mixed chimera (58-84% myeloid donor), median follow-up of 0.9 years (range=0.8-1.2 years). One patient who received RIC (Fludarabine/Melphalan/Campath) had secondary graft failure 1 year after HCT, has since developed inflammatory colitis, uveitis, and pneumonitis and is awaiting second HCT. There was no grade III-IV GVHD; 2 had acute grade I GVHD. Post-HCT complications included EBV viremia (without PTLD) in 1, pericarditis in 1, autoimmune cytopenias in 1 and hypothyroidism in 1. Conclusions Patients with CGD are at high risk of early mortality. HCT is a viable treatment option but has considerable risk, often deterring providers and patients from pursuing HCT early. Our institution's experience shows safety and efficacy with early HCT approach.

The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.

Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1β; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1β pathway in granuloma formation and sarcoidosis.19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For in vivo experiments we used the trehalose 6,6'-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1β antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1β in lung granuloma, and increased IL-1β release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p<0.005). NLRP3 knockout mice showed decreased and miR-223 knockout mice increased granuloma formation compared to wild-type mice. Pharmacological interference using NLRP3 pathway inhibitor MCC950 or an anti-IL-1β antibody resulted in reduced granuloma formation (p<0.02).In conclusion, our data provide evidence of upregulated inflammasome and IL-1β pathway activation in sarcoidosis and suggest both as valid therapeutic targets.

Submandibular salivary gland involvement in granulomatosis with polyangiitis

IntroductionGranulomatosis with polyangiitis (GPA) is one of the forms of small vessel vasculitis. It is a rare condition that needs a high degree of suspicion to reach the diagnosis. It is one of the causes of diffuse parenchymal lung disease, with a very wide differential diagnosis. It is commonly misdiagnosed with malignant, granulomatous, and infectious lung diseases.Case presentationWe report a case of a 31-year-old male who presented with productive cough, shortness of breath, hemoptysis, nasal obstruction, and epistaxis together with submandibular salivary gland swelling. Diagnosis of GPA was based on characteristic cavitary lung lesions, nasal and salivary gland involvement, pathological samples that revealed necrotizing granulomatous inflammation, characteristic positive Cytoplasmic- ANCA (C-ANCA), together with exclusion of malignancy and tuberculosis.ConclusionGPA is a rare condition. Salivary gland involvement should raise suspicion about GPA, in addition to other systemic manifestations.

Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis.

Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host-pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell-mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti-M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell-mediated response. Furthermore, adult anti-M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti-M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens.

Adjunctive Host-Directed Therapy With Statins Improves Tuberculosis-Related Outcomes in Mice.

Tuberculosis (TB) treatment is lengthy and complicated and patients often develop chronic lung disease. Recent attention has focused on host-directed therapies aimed at optimizing immune responses to Mycobacterium tuberculosis (Mtb), as adjunctive treatment given with antitubercular drugs. In addition to their cholesterol-lowering properties, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have broad anti-inflammatory and immunomodulatory activities. In the current study, we screened 8 commercially available statins for cytotoxic effect, anti-TB activity, synergy with first-line drugs in macrophages, pharmacokinetics and adjunctive bactericidal activity, and, in 2 different mouse models, as adjunctive therapy to first-line TB drugs. Pravastatin showed the least toxicity in THP-1 and Vero cells. At nontoxic doses, atorvastatin and mevastatin were unable to inhibit Mtb growth in THP-1 cells. Simvastatin, fluvastatin, and pravastatin showed the most favorable therapeutic index and enhanced the antitubercular activity of the first-line drugs isoniazid, rifampin, and pyrazinamide in THP-1 cells. Pravastatin modulated phagosomal maturation characteristics in macrophages, phenocopying macrophage activation, and exhibited potent adjunctive activity in the standard mouse model of TB chemotherapy and in a mouse model of human-like necrotic TB lung granulomas. These data provide compelling evidence for clinical evaluation of pravastatin as adjunctive, host-directed therapy for TB.

In Vivo Activation and Pro-Fibrotic Function of NF-κB in Fibroblastic Cells During Pulmonary Inflammation and Fibrosis Induced by Carbon Nanotubes.

Exposure to insoluble particles in the lung elicits inflammatory responses that eliminate deposited particulates and repair damaged tissue. Overzealous or prolonged responses lead to chronic conditions, such as fibrosis and malignancy, which are frequently progressive and refractory to drug therapy leading to high rates of disability and mortality. The molecular events underlying the progression of lung inflammation to chronic pathology, in particular, the conversion to fibrosis, remain poorly understood. Fibrogenic multi-walled carbon nanotubes (MWCNTs) have been shown to stimulate prominent acute inflammation that evolves into chronic lesions characterized by chronic inflammation, interstitial fibrosis, and granulomas in mouse lungs. In this communication, we examined the in vivo activation of nuclear factor-κB (NF-κB) signaling in fibroblastic cells during the inflammatory and fibrotic progression induced by MWCNTs. Wild-type C57BL/6J male mice were exposed to two fibrogenic MWCNTs (Mitsui XNRI MWNT-7 and long MWCNTs) by pharyngeal aspiration. Both MWCNTs strongly stimulated the nuclear translocation of NF-κB p65 in lung fibroblasts and myofibroblasts during the acute and chronic responses. Phosphorylated NF-κB p65 at serine 276, a marker of NF-κB activation, was markedly induced by MWCNTs in the nucleus of fibroblastic cells. Moreover, two NF-κB-regulated genes encoding pro-fibrotic mediators, tissue inhibitor of metalloproteinase 1 (TIMP1), and osteopontin (OPN), respectively, were significantly induced in lung fibroblasts and myofibroblasts. These results demonstrate that NF-κB is activated to mediate transactivation of pro-fibrotic genes in fibroblastic cells during pulmonary acute and chronic responses to CNTs, providing a mechanistic framework for analyzing gene regulation in pulmonary fibrotic progression through NF-κB signaling.

Neutrophils express pro- and anti-inflammatory cytokines in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques

Neutrophils are implicated in the pathogenesis of tuberculosis (TB), a disease caused by Mycobacterium tuberculosis infection, but the mechanisms by which they promote disease are not fully understood. Neutrophils can express cytokines that influence TB progression, and so we compared neutrophil and T-cell expression of the Th1 cytokines IFNγ and TNF, the Th2 cytokine IL-4, and regulatory cytokine IL-10 in M. tuberculosis-infected macaques to determine if neutrophil cytokine expression contributes to dysregulated immunity in TB. We found that peripheral blood neutrophils produced cytokines after stimulation by mycobacterial antigens and inactive and viable M. tuberculosis. M. tuberculosis antigen-stimulated neutrophils inhibited antigen-specific T-cell IFNγ production. In lung granulomas, neutrophil cytokine expression resembled T-cell cytokine expression, and although there was histologic evidence for neutrophil interaction with T cells, neutrophil cytokine expression was not correlated with T-cell cytokine expression or bacteria load. There was substantial overlap in the spatial arrangement of cytokine-expressing neutrophils and T cells, but IL-10-expressing neutrophils were also abundant in bacteria-rich areas between caseum and epithelioid macrophages. These results suggest that neutrophils contribute to the cytokine milieu in granulomas and may be important immunoregulatory cells in TB granulomas.

Deciphering multicellular microenvironment of tuberculosis lung granulomas associated with bacterial control using high-throughput single-cell mRNA sequencing

Abstract Infection with Mycobacterium tuberculosis results in the formation of tuberculosis (TB) lung granulomas, which are complex structures with a network of multiple immune cells closely intertwined and function together. Within a given host, granulomas have unique trajectories and outcomes. Some granulomas are able to control bacterial replication, while others fail resulting in persistent bacterial replication. The molecular features associated with these outcomes are not well understood. Using high-throughput single-cell mRNA sequencing on individual TB lung granulomas and computational algorithms, we are generating models to understand infection dynamics that incorporate immune cell composition, its function and interaction; bacterial burden and trajectories of the granulomas to identify robust drivers of bacterial control. We evaluated 28 granulomas from 4 cynomolgus macaques, that were infected for 10 weeks with the Erdman strain of M. tuberculosis. We identified over 30 functional clusters of cells spanning multiple immune and non-immune cells types including T cells, B cells, plasma cells, dendritic cells, mast cells, macrophages, neutrophils, type 2 pneumocytes, fibroblasts and endothelial cells. We observe differential cellular composition and host-cell intercellular communication constituting granulomas that are associated with bacterial burden, that may inform systems level properties across granulomas and animals. By understanding features of the cellular microenvironment that limit bacterial replication, we hope to identify novel targets for host-directed immune therapies and prophylactics in M. tuberculosis infection and other granulomatous disease.

Type I Interferon confers susceptibility to Mycobacterium tuberculosis infection within granulomas of SIV co-infected non-human primates

Abstract Mycobacterium tuberculosis (Mtb) co-infection with human immunodeficiency virus (HIV) typically results in poor disease outcome for tuberculosis (TB). While the loss of CD4+ T cells due to HIV is known to cause increased TB disease susceptibility, the mechanisms which govern the loss of disease control in co-infected individuals are not well understood particularly within the lung granulomas that keep TB contained. This study aims to profile the transcriptional patterns of lung granulomas from non-human primates (NHP) co-infected with Mtb and simian immunodeficiency virus (SIV) as a model for TB-SIV co-infection in humans. We compared the transcriptional profiles of granulomas from four Mauritian cynomolgus macaques that were infected with SIV for six months and then co-infected with Mtb for eight weeks against animals that were infected with Mtb for eight weeks. Granulomas from co-infected animals were found to have generally elevated transcripts involved in Type I interferon pathways (IFIT1), anti-bacterial effectors (defensin A, cathepsin G) and evidence of increased neutrophilic inflammation compared to Mtb-only control lung granulomas. Pathways that were downregulated in co-infected animals were related to Type II interferon signaling (IRF8). These results suggest that SIV infection alters the host immune response in ways that may be advantageous to Mtb growth by lowering IFNγ responsiveness and promoting myeloid cell-associated inflammation. We hypothesize this leads to a more permissive and less antibacterial environment that promotes bacterial survival, dissemination, and ultimately leads to increased susceptibility of co-infected individuals towards TB disease.