ABSTRACT
Investigation of the Th1 immune response in sarcoidosis CD4+ T cells has revealed reduced proliferative capacity and cytokine expression upon TCR stimulation. In other disease models, such cellular dysfunction has been associated with a step-wise, progressive loss of T cell function that results from chronic antigenic stimulation. T cell exhaustion is defined by decreased cytokine production upon TCR activation, decreased proliferation, increased expression of inhibitory cell surface receptors, and increased susceptibility to apoptosis. We characterized sarcoidosis CD4+ T cell immune function in systemic and local environments among subjects undergoing disease progression compared to those experiencing disease resolution. Spontaneous and TCR-stimulated Th1 cytokine expression and proliferation assays were performed in 53 sarcoidosis subjects and 30 healthy controls. PD-1 expression and apoptosis were assessed by flow cytometry. Compared to healthy controls, sarcoidosis CD4+ T cells demonstrated reductions in Th1 cytokine expression, proliferative capacity (p < 0.05), enhanced apoptosis (p < 0.01), and increased PD-1 expression (p < 0.001). BAL-derived CD4+ T cells also demonstrated multiple facets of T cell exhaustion (p < 0.05). Reversal of CD4+ T cell exhaustion was observed in subjects undergoing spontaneous resolution (p < 0.05). Sarcoidosis CD4+ T cells exhibit loss of cellular function during progressive disease that follows the archetype of T cell exhaustion.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Sarcoidosis, Pulmonary/immunology , Th1 Cells/immunology , Adult , Aged , Apoptosis , Cell Proliferation , Cells, Cultured , Clonal Anergy , Cytokines/genetics , Cytokines/metabolism , Disease Progression , Female , Gene Expression Regulation , Humans , Lymphocyte Activation , Male , Middle Aged , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Young AdultABSTRACT
RATIONALE: Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. OBJECTIVES: To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. METHODS: Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. MEASUREMENTS AND MAIN RESULTS: Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. CONCLUSIONS: Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.
Subject(s)
B7-H1 Antigen/metabolism , CD4-Positive T-Lymphocytes/physiology , Programmed Cell Death 1 Receptor/metabolism , Sarcoidosis, Pulmonary/immunology , Adult , Aged , Antibodies , B7-H1 Antigen/immunology , Biomarkers/metabolism , Case-Control Studies , Cell Proliferation , Female , Flow Cytometry , Humans , Immunohistochemistry , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Male , Middle Aged , Programmed Cell Death 1 Receptor/immunology , Remission, Spontaneous , Sarcoidosis, Pulmonary/metabolism , Up-RegulationABSTRACT
Ocular and cutaneous sarcoidosis is a chronic manifestation of sarcoidosis that remains difficult to treat. Recent investigations demonstrating efficacy with antimicrobial therapy in pulmonary and cutaneous sarcoidosis have been reported. Here, we report dual clinical improvement in cutaneous and ocular sarcoidosis following administration of oral antimycobacterial therapy.
ABSTRACT
The chytrid fungus, Batrachochytrium dendrobatidis, causes chytridiomycosis and is a major contributor to global amphibian declines. Although amphibians have robust immune defenses, clearance of this pathogen is impaired. Because inhibition of host immunity is a common survival strategy of pathogenic fungi, we hypothesized that B. dendrobatidis evades clearance by inhibiting immune functions. We found that B. dendrobatidis cells and supernatants impaired lymphocyte proliferation and induced apoptosis; however, fungal recognition and phagocytosis by macrophages and neutrophils was not impaired. Fungal inhibitory factors were resistant to heat, acid, and protease. Their production was absent in zoospores and reduced by nikkomycin Z, suggesting that they may be components of the cell wall. Evasion of host immunity may explain why this pathogen has devastated amphibian populations worldwide.
Subject(s)
Amphibians/immunology , Amphibians/microbiology , Chytridiomycota/pathogenicity , Host-Pathogen Interactions/immunology , Lymphocytes/immunology , Lymphocytes/microbiology , Mycoses/veterinary , Aminoglycosides/pharmacology , Animals , Apoptosis/immunology , Cell Proliferation , Lymphocytes/drug effects , Mycoses/immunology , Spores, Fungal/pathogenicity , Xenopus laevisABSTRACT
IMPORTANCE: Sarcoidosis is a chronic granulomatous disease for which there are limited therapeutic options. This is the first randomized, placebo-controlled study to demonstrate that antimycobacterial therapy reduces lesion diameter and disease severity among patients with chronic cutaneous sarcoidosis. OBJECTIVE: To evaluate the safety and efficacy of once-daily antimycobacterial therapy on the resolution of chronic cutaneous sarcoidosis lesions. DESIGN AND PARTICIPANTS: A randomized, placebo-controlled, single-masked trial on 30 patients with symptomatic chronic cutaneous sarcoidosis lesions deemed to require therapeutic intervention. SETTING: A tertiary referral dermatology center in Nashville, Tennessee. INTERVENTIONS: Participants were randomized to receive either the oral concomitant levofloxacin, ethambutol, azithromycin, and rifampin (CLEAR) regimen or a comparative placebo regimen for 8 weeks with a 180-day follow-up. MAIN OUTCOMES AND MEASURES: Participants were monitored for absolute change in lesion diameter and decrease in granuloma burden, if present, on completion of therapy. OBSERVATIONS: In the intention-to-treat analysis, the CLEAR-treated group had a mean (SD) decrease in lesion diameter of -8.4 (14.0) mm compared with an increase of 0.07 (3.2) mm in the placebo-treated group (P = .05). The CLEAR group had a significant reduction in granuloma burden and experienced a mean (SD) decline of -2.9 (2.5) mm in lesion severity compared with a decline of -0.6 (2.1) mm in the placebo group (P = .02). CONCLUSIONS AND RELEVANCE: Antimycobacterial therapy may result in significant reductions in chronic cutaneous sarcoidosis lesion diameter compared with placebo. These observed reductions, associated with a clinically significant improvement in symptoms, were present at the 180-day follow-up period. Transcriptome analysis of sarcoidosis CD4+ T cells revealed reversal of pathways associated with disease severity and enhanced T-cell function following T-cell receptor stimulation. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01074554.
Subject(s)
Anti-Bacterial Agents/therapeutic use , CD4-Positive T-Lymphocytes/metabolism , Sarcoidosis/drug therapy , Skin Diseases/drug therapy , Administration, Oral , Adult , Aged , Anti-Bacterial Agents/administration & dosage , Azithromycin/administration & dosage , Azithromycin/therapeutic use , Chronic Disease , Drug Therapy, Combination , Ethambutol/administration & dosage , Ethambutol/therapeutic use , Female , Follow-Up Studies , Humans , Levofloxacin , Male , Middle Aged , Ofloxacin/administration & dosage , Ofloxacin/therapeutic use , Rifampin/administration & dosage , Rifampin/therapeutic use , Sarcoidosis/microbiology , Sarcoidosis/pathology , Severity of Illness Index , Single-Blind Method , Skin Diseases/microbiology , Skin Diseases/pathology , Transcriptome , Treatment Outcome , Young AdultABSTRACT
Sarcoidosis pathogenesis is characterized by peripheral anergy and an exaggerated, pulmonary CD4(+) Th1 response. In this study, we demonstrate that CD4(+) anergic responses to polyclonal TCR stimulation are present peripherally and within the lungs of sarcoid patients. Consistent with prior observations, spontaneous release of IL-2 was noted in sarcoidosis bronchoalveolar lavage CD4(+) T cells. However, in contrast to spontaneous hyperactive responses reported previously, the cells displayed anergic responses to polyclonal TCR stimulation. The anergic responses correlated with diminished expression of the Src kinase Lck, protein kinase C-θ, and NF-κB, key mediators of IL-2 transcription. Although T regulatory (Treg) cells were increased in sarcoid patients, Treg depletion from the CD4(+) T cell population of sarcoidosis patients did not rescue IL-2 and IFN-γ production, whereas restoration of the IL-2 signaling cascade, via protein kinase C-θ overexpression, did. Furthermore, sarcoidosis Treg cells displayed poor suppressive capacity indicating that T cell dysfunction was a global CD4(+) manifestation. Analyses of patients with spontaneous clinical resolution revealed that restoration of CD4(+) Th1 and Treg cell function was associated with resolution. Conversely, disease progression exhibited decreased Th1 cytokine secretion and proliferative capacity, and reduced Lck expression. These findings implicate normalized CD4(+) T cell function as a potential therapeutic target for sarcoidosis resolution.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Remission, Spontaneous , Sarcoidosis, Pulmonary/immunology , T-Lymphocyte Subsets/immunology , Adult , Aged , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , CD4-Positive T-Lymphocytes/metabolism , Clonal Anergy/immunology , Female , Humans , Interleukin-2/metabolism , Male , Middle Aged , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Young AdultABSTRACT
RATIONALE: Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells. METHODS: Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation. RESULTS: Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p = 0.03 and p = 0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (p < 0.001 and p = 0.03, respectively). After polyclonal stimulation, Th-17 cells from sarcoidosis patients produced less IFN-γ than healthy controls. CONCLUSIONS: Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis.
Subject(s)
Antigens, Bacterial/immunology , Interferon-gamma/biosynthesis , Sarcoidosis/immunology , Sarcoidosis/metabolism , Th17 Cells/immunology , Adult , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Case-Control Studies , Female , Humans , Immunophenotyping , Interleukin-17/metabolism , Male , Middle Aged , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Th1 Cells/immunology , Th17 Cells/metabolismABSTRACT
BACKGROUND: Necrotising enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Immaturity of gastrointestinal immune regulation may predispose preterm infants to NEC as FOXP3 T regulatory cells (Treg) are critical for intestinal immune homoeostasis. OBJECTIVE: To investigate the hypothesis that abnormal developmental regulation of lamina propria Treg would define premature infants with NEC. DESIGN: Lamina propria mononuclear cell populations from surgically resected ileum from 18 patients with NEC and 30 gestational age-matched non-NEC surgical controls were prospectively isolated. Polychromatic flow cytometry was performed to phenotype and analyse lamina propria T cell populations. The cytokine gene expression profile in NEC tissue was compared with that of non-NEC controls. RESULTS: The total number of Treg, CD4, or CD8 T cells in each ileum section was independent of gestational age, age or postmenstrual age and similar between patients with NEC and controls. In contrast, the ratio of Treg to CD4 T cells or Treg to CD8 T cells was significantly lower in NEC ileum than in infants without NEC (medians 2.9% vs 6.6%, p=0.001 and medians 6.6% vs 25.9%, p<0.001, respectively). For any given number of CD4 or CD8 T cells, Treg were, on average, 60% lower in NEC ileum than in controls. NEC tissue cytokine gene expression profiles were characteristic of inhibited Treg development or function. Treg/CD4 and Treg/CD8 ratios recovered between initial resection for NEC and reanastomosis. CONCLUSION: The proportion of lamina propria Treg is significantly reduced in the ileum of premature infants with NEC and may contribute to the excessive inflammatory state of this disease.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Enterocolitis, Necrotizing/immunology , Forkhead Transcription Factors/metabolism , Infant, Premature, Diseases/immunology , Intestinal Mucosa/immunology , T-Lymphocytes, Regulatory/metabolism , Biomarkers/metabolism , CD4-Positive T-Lymphocytes/metabolism , Case-Control Studies , Female , Flow Cytometry , Gene Expression Profiling , Humans , Infant, Newborn , Infant, Premature , Lymphocyte Count , Male , Prospective StudiesABSTRACT
PURPOSE: Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious antigens continues to strengthen. Recent studies have reported molecular evidence of mycobacteria or propionibacteria. We assessed for immune responses against mycobacterial and propionibacterial antigens in sarcoidosis bronchoalveolar lavage (BAL) using flow cytometry, and localized signals consistent with microbial antigens with sarcoidosis specimens, using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). METHODS: BAL cells from 27 sarcoidosis, 14 PPD- controls, and 9 subjects with nontuberculosis mycobacterial (NTM) infections were analyzed for production of IFN-γ after stimulation with mycobacterial ESAT-6 and Propionibacterium acnes proteins. To complement the immunological data, MALDI-IMS was performed to localize ESAT-6 and Propionibacterium acnes signals within sarcoidosis and control specimens. RESULTS: CD4+ immunologic analysis for mycobacteria was positive in 17/27 sarcoidosis subjects, compared to 2/14 PPD- subjects, and 5/9 NTM subjects (p = 0.008 and p = 0.71 respectively, Fisher's exact test). There was no significant difference for recognition of P. acnes, which occurred only in sarcoidosis subjects that also recognized ESAT-6. Similar results were also observed for the CD8+ immunologic analysis. MALDI-IMS localized signals consistent with ESAT-6 only within sites of granulomatous inflammation, whereas P. acnes signals were distributed throughout the specimen. CONCLUSIONS: MALDI-IMS localizes signals consistent with ESAT-6 to sarcoidosis granulomas, whereas no specific localization of P. acnes signals is detected. Immune responses against both mycobacterial and P. acnes are present within sarcoidosis BAL, but only mycobacterial signals are distinct from disease controls. These immunologic and molecular investigations support further investigation of the microbial community within sarcoidosis granulomas.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Bronchoalveolar Lavage Fluid/immunology , Mycobacterium/immunology , Propionibacterium acnes/immunology , Sarcoidosis/immunology , Adult , Aged , Bronchoalveolar Lavage Fluid/cytology , CD4-Positive T-Lymphocytes/immunology , Enterotoxins/pharmacology , Female , Humans , Interferon-gamma/immunology , Male , Middle Aged , Mycobacterium Infections/immunology , Peptides/immunology , Sarcoidosis/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Young AdultABSTRACT
Sarcoidosis is characterized by noncaseating granulomas containing CD4(+) T cells with a Th1 immunophenotype. Although the causative antigens remain unknown, independent studies noted molecular and immunologic evidence of mycobacterial virulence factors in sarcoidosis specimens. A major limiting factor in discovering new insights into the pathogenesis of sarcoidosis is the lack of an animal model. Using a distinct superoxide dismutase A peptide (sodA) associated with sarcoidosis granulomas, we developed a pulmonary model of sarcoidosis granulomatous inflammation. Mice were sensitized by a subcutaneous injection of sodA, incorporated in incomplete Freund's adjuvant (IFA). Control subjects consisted of mice with no sensitization (ConNS), sensitized with IFA only (ConIFA), or with Schistosoma mansoni eggs. Fourteen days later, sensitized mice were challenged by tail-vein injection of naked beads, covalently coupled to sodA peptides or to schistosome egg antigens (SEA). Histologic analysis revealed hilar lymphadenopathy and noncaseating granulomas in the lungs of sodA-treated or SEA-treated mice. Flow cytometry of bronchoalveolar lavage (BAL) demonstrated CD4(+) T-cell responses against sodA peptide in the sodA-sensitized mice only. Cytometric bead analysis revealed significant differences in IL-2 and IFN-γ secretion in the BAL fluid of sodA-treated mice, compared with mice that received SEA or naked beads (P = 0.008, Wilcoxon rank sum test). ConNS and ConIFA mice demonstrated no significant formation of granuloma, and no Th1 immunophenotype. The use of microbial peptides distinct for sarcoidosis reveals a histologic and immunologic profile in the murine model that correlates well with those profiles noted in human sarcoidosis, providing the framework to investigate the molecular basis for the progression or resolution of sarcoidosis.
Subject(s)
Bacterial Proteins/immunology , Granuloma/etiology , Mycobacterium/enzymology , Mycobacterium/immunology , Sarcoidosis, Pulmonary/etiology , Superoxide Dismutase/immunology , Amino Acid Sequence , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Disease Models, Animal , Female , Granuloma/immunology , Granuloma/pathology , Humans , Mice , Mice, Inbred C57BL , Mycobacterium/genetics , Peptide Fragments/genetics , Peptide Fragments/immunology , Sarcoidosis, Pulmonary/immunology , Sarcoidosis, Pulmonary/pathology , Superoxide Dismutase/genetics , Th1 Cells/immunologyABSTRACT
Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.
Subject(s)
Antigens/immunology , Enzyme-Linked Immunospot Assay/methods , Th1 Cells/immunology , Adaptive Immunity/immunology , Epitopes, T-Lymphocyte/immunology , HumansABSTRACT
INTRODUCTION: Sarcoidosis is a multisystem granulomatous disease for which the association with mycobacteria continues to strengthen. It is hypothesized that a single, poorly degradable antigen is responsible for sarcoidosis pathogenesis. Several reports from independent groups support mycobacterial antigens having a role in sarcoidosis pathogenesis. To identify other microbial targets of the adaptive immune response, we tested the ability of CD4+ and CD8+ T cells to recognize multiple mycobacterial antigens. METHODS: Fifty-four subjects were enrolled in this study: 31 sarcoidosis patients, nine non-tuberculosis mycobacterial (NTM) infection controls, and 14 PPD- controls. Using flow cytometry, we assessed for Th1 immune responses to ESAT-6, katG, Ag85A, sodA, and HSP. RESULTS: Alveolar T-cells from twenty-two of the 31 sarcoidosis patients produced a CD4+ response to at least one of ESAT-6, katG, Ag85A, sodA, or HSP, compared to two of 14 PPD- controls (p = 0.0008) and five of nine NTM controls (p = 0.44), while eighteen of the 31 sarcoidosis subjects tested produced a CD8+ response to at least one of the mycobacterial antigens compared to two of 14 PPD- controls (p = 0.009) and three of nine NTM controls (0.26). Not only did the BAL-derived T cells respond to multiple virulence factors, but also to multiple, distinct epitopes within a given protein. The detection of proliferation upon stimulation with the mycobacterial virulence factors demonstrates that these responses are initiated by antigen specific recognition. CONCLUSIONS: Together these results reveal that antigen-specific CD4+ and CD8+ T cells responses to multiple mycobacterial epitopes are present within sites of active sarcoidosis involvement, and that these antigen-specific responses are present at the time of diagnosis.
Subject(s)
Adaptive Immunity/immunology , Antigens, Bacterial/immunology , Mycobacterium/immunology , Sarcoidosis, Pulmonary/immunology , Th1 Cells/immunology , Adult , Aged , Cells, Cultured , Female , Humans , Male , Middle AgedABSTRACT
Sarcoidosis is a disease of unknown etiology, characterized pathologically by noncaseating granulomas that most commonly involve the lung, skin, lymph nodes, and eyes. Syndromes with similar pathological and immunologic features to sarcoidosis such as chronic beryllium disease, hypersensitivity pneumonitis, and tuberculosis illustrate that granulomatous diseases may or may not have an infectious etiology. Although the etiology of sarcoidosis remains unknown, recent molecular, genetic, and immunologic studies strengthen the association of sarcoidosis with infectious antigens. Currently, the strongest agents considered include PROPIONIBACTERIUM and MYCOBACTERIUM species. Independent studies report the presence of microbial nucleic acids and proteins within sarcoidosis specimens. Th-1 immune responses to mycobacterial proteins have been detected within sarcoidosis diagnostic bronchoalveolar lavage (BAL). These proteins are actively secreted by the mycobacterial SecA 2 secretion system and are important to evade the host immune system. Recent discoveries regarding MHC class II alleles provide additional insight regarding the role of microbial antigens in sarcoidosis pathogenesis. Although further investigation is warranted, the recent progress of independent laboratories, using complementary techniques, strengthens the role of microbial antigens in sarcoidosis pathogenesis. These studies lay a strong foundation toward identifying therapeutic targets.
Subject(s)
Antigens, Bacterial/immunology , Sarcoidosis/immunology , Bronchoalveolar Lavage/methods , Humans , Mycobacterium/immunology , Propionibacterium/immunology , Sarcoidosis/microbiology , Th1 Cells/immunologyABSTRACT
INTRODUCTION: Genetic associations of American sarcoidosis susceptibility implicate MHC class II allele, DRB1*1101. We previously reported immune recognition of Mycobacterium peptides from peripheral cells of 26 sarcoidosis subjects, 24 PPD- healthy volunteers, and eight with latent tuberculosis infection. MATERIALS AND METHODS: In order to further link these genetic and immunologic pillars of sarcoidosis pathogenesis, we performed flow cytometry on these same subjects to identify the cells responsible for immune responses to ESAT-6 and katG peptides, followed by HLA typing to determine allelic associations with recognition. DISCUSSION AND CONCLUSION: Sarcoidosis CD4+ T cells were primarily responsible for the systemic responses. Recognition was inhibited by monoclonal antibody against HLA-DR and HLA-DQ, but not HLA-DP. Immune recognition of ESAT-6 peptide NNALQNLARTISEAG was associated with possession of DRB1*1101. ESAT-6 and katG presented by antigen-presenting cells expressing DRB1*1101-induced Th-1 responses from sarcoidosis T cells, thus providing a mechanistic insight for the association of HLA DRB1*1101 with sarcoidosis, and sarcoidosis T cell interaction with microbial antigens.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , CD4-Positive T-Lymphocytes/metabolism , Catalase/metabolism , HLA-DR Antigens/metabolism , Latent Tuberculosis/genetics , Latent Tuberculosis/immunology , Mycobacterium/immunology , Alleles , Amino Acid Sequence , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Catalase/immunology , Cell Separation , Cells, Cultured , Flow Cytometry , Genetic Association Studies , Genetic Predisposition to Disease , HLA-DR Antigens/genetics , HLA-DR Antigens/immunology , HLA-DRB1 Chains , Histocompatibility Testing , Host-Pathogen Interactions/genetics , Humans , Interferon-gamma/metabolism , Latent Tuberculosis/microbiology , Latent Tuberculosis/physiopathology , Lymphocyte Activation , Molecular Sequence Data , Mycobacterium/pathogenicity , Sarcoidosis, Pulmonary , United StatesABSTRACT
Considerable evidence supports the concept that CD4(+) T cells are important in sarcoidosis pathogenesis, but the antigens responsible for the observed Th1 immunophenotype remain elusive. The epidemiologic association with bioaerosols and the presence of granulomatous inflammation support consideration of mycobacterial antigens. To explore the role of mycobacterial antigens in sarcoidosis immunopathogenesis, we assessed the immune recognition of mycobacterial antigens, the 6-kDa early secreted antigenic protein (ESAT-6) and catalase-peroxidase (KatG), by T cells derived from bronchoalveolar lavage (BAL) fluid obtained during diagnostic bronchoscopy. We report the presence of antigen-specific recognition of ESAT-6 and KatG in T cells from BAL fluid of 32/44 sarcoidosis subjects, compared to 1/27 controls (P < 0.0001). CD4(+) T cells were primarily responsible for immune recognition (32/44 sarcoidosis subjects), although CD8(+) T-cell responses were observed (25/41 sarcoidosis subjects). Recognition was significantly absent from BAL fluid cells of patients with other lung diseases, including infectious granulomatous diseases. Blocking of Toll-like receptor 2 reduced the strength of the observed immune response. The presence of immune responses to mycobacterial antigens in cells from BAL fluid used for sarcoidosis diagnosis suggests a strong association between mycobacteria and sarcoidosis pathogenesis. Inhibition of immune recognition with monoclonal antibody against Toll-like receptor 2 suggests that induction of innate immunity by mycobacteria contributes to the polarized Th1 immune response.
Subject(s)
Antigens, Bacterial/immunology , Bronchoalveolar Lavage Fluid/immunology , Mycobacterium/immunology , Sarcoidosis/diagnosis , Adult , Aged , Aged, 80 and over , Amino Acid Sequence , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Female , Humans , Lymphocyte Activation , Male , Middle Aged , Molecular Sequence Data , Sarcoidosis/immunology , Th1 Cells/immunology , Toll-Like Receptor 2/physiologyABSTRACT
CD4+CD25+ human regulatory T cells (Treg cells), which express the transcription factor FoxP3, suppress T cell activation. In this study, we sought to define cellular and molecular mechanisms of human Treg cell differentiation. A subset of human naive CD4+ T cells that are CD25+ express high levels of FoxP3. We show that upon activation through the TCR, these FoxP3-expressing naive T cells (termed TNreg cells) greatly expand in vitro. Expanded TNreg cells acquire a full Treg phenotype with potent suppressive activity and display low IL-2 production upon TCR stimulation. TNreg cells in which FoxP3 expression was reduced through RNA interference lost their suppressive activity, but retained their low IL-2 secretion in response to TCR stimulation. Furthermore, in support of the notion that TNreg cells represent a separate lineage of naive cells, we found that they were more susceptible to HIV infection as compared with naive CD4+ T cells. Based on these findings, we propose that TNreg cells are precursors for human Treg cells and that these cells require a high level of FoxP3 expression to maintain their suppressive function. Accordingly, modulation of TNreg cell numbers during infections such as HIV may disrupt human Treg cell development, and contribute to chronic immune activation.
Subject(s)
Forkhead Transcription Factors/metabolism , HIV , Immune Tolerance , Lymphocyte Activation , Precursor Cells, T-Lymphoid/immunology , Precursor Cells, T-Lymphoid/virology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/virology , Cells, Cultured , Disease Susceptibility , Female , Forkhead Transcription Factors/genetics , Humans , Immune Tolerance/genetics , Interleukin-2 Receptor alpha Subunit/analysis , Lymphocyte Activation/genetics , Male , Phenotype , RNA Interference , Receptors, Antigen, T-Cell, alpha-beta/analysisABSTRACT
Infection with HIV-1 perturbs homeostasis of human T cell subsets, leading to accelerated immunologic deterioration. While studying changes in CD4(+) memory and naïve T cells during HIV-1 infection, we found that a subset of CD4(+) effector memory T cells that are CCR7(-)CD45RO(-)CD45RA(+) (referred to as TEMRA cells), was significantly increased in some HIV-infected individuals. This T cell subset displayed a differentiated phenotype and skewed Th1-type cytokine production. Despite expressing high levels of CCR5, TEMRA cells were strikingly resistant to infection with CCR5 (R5)-tropic HIV-1, but remained highly susceptible to CXCR4 (X4)-tropic HIV-1. The resistance of TEMRA cells to R5-tropic viruses was determined to be post-entry of the virus and prior to early viral reverse transcription, suggesting a block at the uncoating stage. Remarkably, in a subset of the HIV-infected individuals, the relatively high proportion of TEMRA cells within effector T cells strongly correlated with higher CD4(+) T cell numbers. These data provide compelling evidence for selection of an HIV-1-resistant CD4(+) T cell population during the course of HIV-1 infection. Determining the host factors within TEMRA cells that restrict R5-tropic viruses and endow HIV-1-specific CD4(+) T cells with this ability may result in novel therapeutic strategies against HIV-1 infection.
Subject(s)
HIV Infections/immunology , HIV-1/immunology , Receptors, CCR5/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/virology , AIDS Vaccines/immunology , Apoptosis/immunology , CD3 Complex/metabolism , CD4 Antigens/metabolism , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , Cell Division/immunology , Humans , Immunologic Memory/immunology , Immunophenotyping , Leukocyte Common Antigens/metabolism , Receptors, CCR5/immunology , Receptors, CCR7 , Receptors, Chemokine/metabolism , T-Lymphocyte Subsets/metabolismABSTRACT
Human CD4(+) T cells are major targets for human immunodeficiency virus (HIV) infection. Resting T cells are resistant to HIV infection unless activated through the T-cell receptor (TCR) or by cytokine signals. How T-cell signaling promotes susceptibility of T cells to HIV infection remains poorly understood. Here we demonstrate that the VacA toxin produced by Helicobacter pylori can inhibit HIV infection of primary T cells, stimulated through the TCR or by cytokines alone. This activity of VacA was dependent on its ability to form membrane channels. VacA suppressed HIV infection of T cells at a stage after viral entry, post-reverse transcription and pre-two-long-terminal-repeat circle formation, similar to the cytokine signaling inhibitor rapamycin. Mechanistically, neither VacA nor rapamycin inhibited the activation of cytokine signal transduction components (STAT5, p42/44 mitogen-activated protein kinase, or p38), but both blocked activation of key regulatory proteins required for G(1) cell cycle transition. In contrast to rapamycin, VacA did not suppress phosphorylation of p70 S6 kinase but caused mitochondrial depolarization and ATP depletion within primary T cells. These results suggest that VacA inhibits T-cell activation and HIV infection via a novel mechanism. Identifying the host cell targets of VacA could be useful for elucidating the HIV life cycle within primary T cells.
Subject(s)
Bacterial Proteins/pharmacology , HIV-1/drug effects , T-Lymphocytes/drug effects , Cell Line , HIV Infections/immunology , HIV-1/immunology , HIV-1/physiology , Humans , T-Lymphocytes/virology , Virus Replication/drug effectsABSTRACT
Emerging evidence suggests critical roles for APCs in suppressing immune responses. Here, we show that zymosan, a stimulus for TLR2 and dectin-1, regulates cytokine secretion in DCs and macrophages to induce immunological tolerance. First, zymosan induces DCs to secrete abundant IL-10 but little IL-6 and IL-12(p70). Induction of IL-10 is dependent on TLR2- and dectin-1-mediated activation of ERK MAPK via a mechanism independent of the activation protein 1 (AP-1) transcription factor c-Fos. Such DCs stimulate antigen-specific CD4+ T cells poorly due to IL-10 and the lack of IL-6. Second, zymosan induces F4-80+ macrophages in the splenic red pulp to secrete TGF-beta. Consistent with these effects on APCs, injection of zymosan plus OVA into mice results in OVA-specific T cells that secrete little or no Th1 or Th2 cytokines, but secrete robust levels of IL-10, and are unresponsive to challenge with OVA plus adjuvant. Finally, coinjection of zymosan with OVA plus LPS suppresses the response to OVA via a mechanism dependent on IL-10, TGF-beta, and lack of IL-6. Together, our data demonstrate that zymosan stimulates IL-10+ IL-12(p70)- IL-6low regulatory DCs and TGF-beta+ macrophages to induce immunological tolerance. These data suggest several targets for pharmacological modulation of immune responses in various clinical settings.
Subject(s)
Antigen-Presenting Cells/immunology , Immune Tolerance , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Toll-Like Receptor 2/metabolism , Zymosan/pharmacology , Animals , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fluorescent Antibody Technique , Humans , Inflammation/immunology , Inflammation/metabolism , Interleukin-10/metabolism , Interleukin-12/metabolism , Interleukin-6/metabolism , Lectins, C-Type , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Models, Biological , Proto-Oncogene Proteins c-fos/metabolism , Spleen , Time Factors , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/metabolism , Yeasts/metabolism , Zymosan/administration & dosage , Zymosan/metabolismABSTRACT
Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs.