Integrated transcriptomic analysis of human tuberculosis granulomas and a biomimetic model identifies therapeutic targets.

Tuberculosis (TB) is a persistent global pandemic, and standard treatment for it has not changed for 30 years. Mycobacterium tuberculosis (Mtb) has undergone prolonged coevolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesized that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a noninfectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared 3 primary human cell culture models at the whole transcriptome level and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified 12 intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.

New Findings of Immunodysregulation, Polyendocrinopathy, and Enteropathy X-linked Syndrome (IPEX); Granulomas in Lung and Duodenum.

Immune dysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome is a rare disorder caused by loss-of-function mutations in the gene forkhead box protein 3 (FOXP3). IPEX patients frequently show chronic diarrhea (enteropathy) associated with villous atrophies in the small intestine. Our case is different from this classical information in the literature, since he presented with neonatal onset inflammatory bowel disease within the first months of life accompanied by deep ulcers throughout colonic mucosa. Moreover, he developed chronic lung disease during follow-up and histopathological examinations showed granulomas in both gastrointestinal tract and lung parenchyma. Genetic analysis revealed the diagnosis of IPEX syndrome with a germline mutation in FOXP3. Thus, our study provides an unusual presentation of IPEX syndrome with colitis and granulomas presence in histopathological examinations.

Current Sarcoidosis Models and the Importance of Focusing on the Granuloma.

The inability to effectively model sarcoidosis in the laboratory or in animals continues to hinder the discovery and translation of new, targeted treatments. The granuloma is the signature pathological hallmark of sarcoidosis, yet there are significant knowledge gaps that exist with regard to how granulomas form. Significant progress toward improved therapeutic and prognostic strategies in sarcoidosis hinges on tractable experimental models that recapitulate the process of granuloma formation in sarcoidosis and allow for mechanistic insights into the molecular events involved. Through its inherent representation of the complex genetics underpinning immune cell dysregulation in sarcoidosis, a recently developed in vitro human granuloma model holds promise in providing detailed mechanistic insight into sarcoidosis-specific disease regulating pathways at play during early stages of granuloma formation. The purpose of this review is to critically evaluate current sarcoidosis models and assess their potential to progress the field toward the goal of improved therapies in this disease. We conclude with the potential integrated use of preclinical models to accelerate progress toward identifying and testing new drugs and drug combinations that can be rapidly brought to clinical trials.

Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes.

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53+/-) mice has not been investigated. We hypothesized that p53+/- mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53+/+) or p53+/- mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53+/+ or p53+/- mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53+/- and p53+/+ mice. A constitutively larger area of CD45R+/CD3+ lymphoid tissue was observed in p53+/- mice compared to p53+/+ mice. Importantly, p53+/- mice had larger granulomas induced by rMWCNTs as compared to p53+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

The transpeptidase PbpA and noncanonical transglycosylase RodA of Mycobacterium tuberculosis play important roles in regulating bacterial cell lengths.

The cell wall of Mycobacterium tuberculosis (Mtb) is a complex structure that protects the pathogen in hostile environments. Peptidoglycan (PG), which helps determine the morphology of the cell envelope, undergoes substantial remodeling under stress. This meshwork of linear chains of sugars, cross-linked through attached peptides, is generated through the sequential action of enzymes termed transglycosylases and transpeptidases. The Mtb genome encodes two classical transglycosylases and four transpeptidases, the functions of which are not fully elucidated. Here, we present work on the yet uncharacterized transpeptidase PbpA and a nonclassical transglycosylase RodA. We elucidate their roles in regulating in vitro growth and in vivo survival of pathogenic mycobacteria. We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth. Biochemical analyses show PbpA to be a classical transpeptidase, whereas RodA is identified to be a member of an emerging class of noncanonical transglycosylases. Phosphorylation of RodA at Thr-463 modulates its biological function. In a guinea pig infection model, RodA and PbpA are found to be required for both bacterial survival and formation of granuloma structures, thus underscoring the importance of these proteins in mediating mycobacterial virulence in the host. Our results emphasize the fact that whereas redundant enzymes probably compensate for the absence of RodA or PbpA during in vitro growth, the two proteins play critical roles for the survival of the pathogen inside its host.

Interleukin-17A and Neutrophils in a Murine Model of Bird-Related Hypersensitivity Pneumonitis.

Hypersensitivity pneumonitis (HP) is an immune mediated lung disease induced by the repeated inhalation of a wide variety of antigens. Bird-related hypersensitivity pneumonitis (BRHP) is one of the most common forms of HP in human and results from the inhalation of avian antigens. The findings of a recent clinical analysis suggest that in addition to Th1 factors, the levels of interleukin(IL)-17 and IL-17-associated transcripts are increased in the setting of HP, and that both IL-17A and neutrophils are crucial for the development of pulmonary inflammation in murine models of HP. Our objectives were to investigate the roles of IL-17A and neutrophils in granuloma-forming inflammation in an acute HP model. We developed a mouse model of acute BRHP using pigeon dropping extract. We evaluated the process of granuloma formation and the roles of both IL-17A and neutrophils in a model. We found that the neutralization of IL-17A by the antibody attenuated granuloma formation and the recruitment of neutrophils, and also decreased the expression level of chemokine(C-X-C motif) ligand 5 (CXCL5) in the acute HP model. We confirmed that most of the neutrophils in the acute HP model exhibited immunoreactivity to the anti-IL-17 antibody. We have identified the central roles of both IL-17A and neutrophils in the pathogenesis of granuloma formation in acute HP. We have also assumed that neutrophils are an important source of IL-17A in an acute HP model, and that the IL-17A-CXCL5 pathway may be responsible for the recruitment of neutrophils.

Lesion-Specific Immune Response in Granulomas of Patients with Pulmonary Tuberculosis: A Pilot Study.

The formation and maintenance of granulomas is central to the host response to Mycobacterium tuberculosis (Mtb) infection. It is widely accepted that the lungs of patients with tuberculosis (TB) usually contain multiple infection foci, and that the granulomas evolve and differentiate independently, resulting in considerable heterogeneity. Although gene expression profiles of human blood cells have been proposed as biomarkers of Mtb infection and/or active disease, the immune profiles of discrete lesion types has not been studied extensively. Using histology, immunopathology and genome-wide transcriptome analysis, we explored the immunological profile of human lung TB granulomas. We show that although the different granulomas share core similarities in their immunological/inflammatory characteristics, they also exhibit significant divergence. Despite similar numbers of CD68+ macrophages in the different lesions, the extent of immune reactivity, as determined by the density of CD3+ T cells in the macrophage rich areas, and the extent of fibrosis, shows considerable variation. Both quantitative and qualitative differences among significantly differentially expressed genes (SDEG) were noted in each of the lesion types studied. Further, network/pathway analysis of SDEG revealed differential regulation of inflammatory response, immune cell trafficking, and cell mediated immune response in the different lesions. Our data highlight the formidable challenges facing ongoing efforts to identify peripheral blood biomarkers due to the diversity of lesion types and complexity of local immune responses in the lung.

Granulomatous lung disease in a patient with a family history of hematological disorders.

BACKGROUND: A 29-year old patient presented with granulomatous lung disease and a family history of myelodysplastic syndrome/acute myeloid leukemia. She appeared to be a carrier of a mutation in the transcription factor GATA2. The case adds to the recent described heterogeneous clinical manifestations and syndromes in which, against a background of hematologic disorders, GATA2 mutations have been demonstrated, such as the Monomac and Emberger syndromes. In patients with a granulomatous disease and a history of (familial) hematologic disorders, the occurence of GATA2 mutations should be considered, as to gain further insight in the occurrence of granulomatous disease in a possible distinct phenotype among GATA2 mutation carriers.

ICAM-1 deficiency exacerbates sarcoid-like granulomatosis induced by Propionibacterium acnes through impaired IL-10 production by regulatory T cells.

Propionibacterium acnes has been implicated as one of the suggested causative antigens for sarcoidosis, a systemic granulomatous disease. By injecting heat-killed P. acnes into the dorsal skin of C57BL/6J mice on days 1, 3, 5, and 14, sarcoid-like granulomatosis was induced in skin and lungs of these mice on day 28. To clarify the role of cell adhesion molecules in cutaneous sarcoidosis, we induced sarcoid-like granulomatosis in mice deficient of intercellular adhesion molecule (ICAM)-1, L-selectin, P-selectin, or E-selectin via repeated P. acnes injection. Histopathologic analysis revealed that granuloma formation was aggravated in the skin and lungs of ICAM-1-deficient mice compared with wild-type mice. Within skin granulomas of ICAM-1-deficient mice, P. acnes immunization up-regulated mRNA expression of tumor necrosis factor-α, although it failed to induce IL-10 mRNA expression in contrast to wild-type mice. Infiltration of regulatory T cells into skin granuloma was similar between wild-type mice and ICAM-1-deficient mice. P. acnes immunization induced IL-10 production by CD4(+)CD25(+)Foxp3(+) regulatory T cells in lymph nodes of wild-type mice in vivo, which was absent in regulatory T cells of ICAM-1-deficient mice. Our results indicate that ICAM-1 is imperative for inducing regulatory T cells to produce IL-10 in vivo, which would prevent granuloma formation.

Nitric oxide inhibits the accumulation of CD4+CD44hiTbet+CD69lo T cells in mycobacterial infection.

Animals lacking the inducible nitric oxide synthase gene (nos2(-/-)) are less susceptible to Mycobacterium avium strain 25291 and lack nitric oxide-mediated immunomodulation of CD4(+) T cells. Here we show that the absence of nos2 results in increased accumulation of neutrophils and both CD4(+) and CD8(+) T cells within the M. avium containing granuloma. Examination of the T-cell phenotype in M. avium infected mice demonstrated that CD4(+)CD44(hi) effector T cells expressing the Th1 transcriptional regulator T-bet (T-bet(+)) were specifically reduced by the presence of nitric oxide. Importantly, the T-bet(+) effector population could be separated into CD69(hi) and CD69(lo) populations, with the CD69(lo) population only able to accumulate during chronic infection within infected nos2(-/-) mice. Transcriptomic comparison between CD4(+)CD44(hi)CD69(hi) and CD4(+)CD44(hi)CD69(lo) populations revealed that CD4(+)CD44(hi)CD69(lo) cells had higher expression of the integrin itgb1/itga4 (VLA-4, CD49d/CD29). Inhibition of Nos2 activity allowed increased accumulation of the CD4(+) CD44(hi)T-bet(+)CD69(lo) population in WT mice as well as increased expression of VLA-4. These data support the hypothesis that effector T cells in mycobacterial granulomata are not a uniform effector population but exist in distinct subsets with differential susceptibility to the regulatory effects of nitric oxide.

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway.

Trehalose 6,6'-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle⁻/⁻ mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.

Lack of cathepsin activities alter or prevent the development of lung granulomas in a mouse model of sarcoidosis.

BACKGROUND: Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis. METHODS: Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor. RESULTS: When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed. CONCLUSIONS: Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.

Necrotizing sarcoid granulomatosis in a family of patients with sarcoidosis reinforces the association between both entities.

Necrotizing sarcoid granulomatosis (NSG) is a rare entity mainly characterized by a prominent granulomatous vasculitis affecting middle-aged or old individuals and with a favorable prognosis. Although many believe it is a variant of sarcoidosis, the proper classification is still a matter of debate as some of its features are found in sarcoidosis but also in Churg-Strauss syndrome, Wegener's disease and hypersensitivity pneumonitis. In this paper, we described for the first time a case of NSG in a family with several cases of sarcoidosis, reinforcing the relationship between NSG and sarcoidosis. Additional interesting findings were the young age of the patient (15 years old), the symptoms limited to the respiratory tract (uncommon when NSG affects youngsters) and the increase in serologic markers of autoimmune disease. Though complete criteria for autoimmune disease were not present, systemic lupus erythematosus and Sjogren's syndrome are possible candidates. As sarcoidosis is described to be associated with several autoimmune diseases, this finding is an additional suggestion of the relationship between both entities.

B cells moderate inflammatory progression and enhance bacterial containment upon pulmonary challenge with Mycobacterium tuberculosis.

Though much is known about the function of T lymphocytes in the adaptive immune response against Mycobacterium tuberculosis, comparably little is understood regarding the corresponding role of B lymphocytes. Indicating B cells as components of lymphoid neogenesis during pulmonary tuberculosis, we have identified ectopic germinal centers (GCs) in the lungs of infected mice. B cells in these pulmonary lymphoid aggregates express peanut agglutinin and GL7, two markers of GC B cells, as well as CXCR5, and migrate in response to the lymphoid-associated chemokine CXCL13 ex vivo. CXCL13 is negatively regulated by the presence of B cells, as its production is elevated in lungs of B cell-deficient (B cell(-/-)) mice. Upon aerosol with 100 CFU of M. tuberculosis Erdman, B cell(-/-) mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils. Infected B cell(-/-) mice show increased production of IL-10 in the lungs, whereas IFN-gamma, TNF-alpha, and IL-10R remain unchanged from wild type. B cell(-/-) mice have enhanced susceptibility to infection when aerogenically challenged with 300 CFU of M. tuberculosis corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell(-/-) mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus. As components of ectopic GCs, moderators of inflammatory progression, and enhancers of local immunity against bacterial challenge, B cells may have a greater role in the host defense against M. tuberculosis than previously thought.

Sarcoidosis--immunopathogenetic concepts.

Sarcoidosis is a chronic granulomatous disorder characterized by an accumulation of activated lymphocytes, predominantly T helper cells, expressing the Th1 phenotype and macrophages at sites of disease activity. Although the cause of sarcoidosis has not been elucidated, several lines of evidence suggest that granuloma formation results from exposure to one or more antigens, eliciting a T lymphocyte response. The induction and evolution of granuloma formation results from a complex interplay between diverse cell types, cytokines, and chemokines. Genetic polymorphisms may influence the clinical expression of the granuloma formation and disease outcome. This article discusses in depth the key cellular elements and signals that generate and orchestrate the sarcoid granulomatous response. The precise factors inciting the sarcoid granulomatous response have not yet been identified, but chronic exposure to microbial agents, their products, or inorganic substances may be important in the pathogenesis.

Genetics of sarcoidosis.

Sarcoidosis likely results from an interplay of environmental and genetic factors. Despite a recent large multicenter study, A Case-Control Etiologic Study of Sarcoidosis (ACCESS), no single causative environmental agent has been identified. Family clustering and differences in racial incidence of sarcoidosis support an inherited susceptibility to sarcoidosis. Siblings of patients with sarcoidosis have about a fivefold increased risk of developing sarcoidosis. Certain human leukocyte antigen (HLA) alleles have been consistently associated with sarcoidosis susceptibility. Furthermore, HLA-DRB1*0301/DQB1*0201 has been associated with good prognosis in Löfgren's syndrome. Many candidate genes studied based on their potential function in immunopathogenesis have been evaluated in case-control studies, but few have been consistently associated with sarcoidosis across populations. Two genome scans have been reported in sarcoidosis. One in African Americans reporting linkage to chromosome 5 and the other in German families reporting linkage to chromosome 6. Follow-up studies on chromosome 6 identified the BTNL2 gene, a B7 family costimulatory molecule to be associated with sarcoidosis. Advances in genotyping and statistical analysis are helping to elucidate the genetics of sarcoidosis.

Epidemiology of sarcoidosis: recent advances and future prospects.

Sarcoidosis is by definition a disease of "unknown causes," but recent epidemiologic advances suggest that the long-standing definition of sarcoidosis may soon need to be amended. The recently completed ACCESS (A Case-Control Etiologic Study of Sarcoidosis) study was not able to definitively identify the "cause" of sarcoidosis, but yielded important findings regarding familial and environmental risks that have advanced our understanding of this disease. The HLA-DRB1 associations reported in ACCESS along with the results of two recently completed genome scans of sarcoidosis in German Caucasians and African-Americans, respectively, have further defined the genetics of sarcoidosis. These studies suggest genetic heterogeneity of sarcoidosis risk between Caucasians and African-Americans and multiple susceptibility genes that interact together and with environmental factors in the disease pathogenesis. Genes that influence sarcoidosis clinical phenotypes may also be largely separate from sarcoidosis susceptibility genes. Although genetic studies of sarcoidosis in African-American populations are confounded by Caucasian admixture, this same admixture may be useful in identifying sarcoidosis genes linked with African ancestry. Case-only methods may be useful in identifying recent acute exposures linked to disease, genetic variants of risk, and gene-environment interactions. In summary, the epidemiology of sarcoidosis has a promising future that should eventually provide the answers to the etiologic origins of this complex disease.

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses.

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.

Underlying chronic granulomatous disease in a patient with bronchocentric granulomatosis.

We present a case of bronchocentric granulomatosis in a woman with no history of asthma who was colonised with Aspergillusfumigatus. A family history of chronic granulomatous disease prompted further testing that demonstrated severely depressed neutrophil oxidant production and gp91(phox) deficiency compatible with the X linked carrier state of chronic granulomatous disease. Only one report of the association of these two rare diseases has previously appeared in the literature. We postulate that an ineffective immune response led to the prolonged colonisation of Afumigatus resulting in a hypersensitivity reaction that was manifest clinically as bronchocentric granulomatosis.