Wonder P. Drake

Molecular Analysis of Sarcoidosis Tissues for Mycobacterium Species DNA

We performed polymerase chain reaction analysis, for Mycobacterium species 16S rRNA, rpoB, and IS6110 sequences, on 25 tissue specimens from patients with sarcoidosis and on 25 control tissue specimens consisting of mediastinal or cervical lymph nodes and lung biopsies. Mycobacterium species 16S rRNA sequences were amplified from 12 (48%) rpoB sequences from 6 (24%) of the sarcoidosis specimens. In total, 16S rRNA or rpoB sequences were amplified from 15 sarcoidosis specimens (60%) but were not detected in any of the control tissues (p=0.00002, Chi square). In three specimens, the sequences resembled Mycobacterium species other than M. tuberculosis. All specimens with sequences consistent with M. tuberculosis were negative for IS6110. We provide evidence that one of a variety of Mycobacterium species, especially organisms resembling M. tuberculosis, is found in most patients with sarcoidosis.

Cellular Recognition of Mycobacterium tuberculosis ESAT-6 and KatG Peptides in Systemic Sarcoidosis

ABSTRACT Sarcoidosis is an enigmatic disease with a pathology similar to that of tuberculosis. We detected Th-1 immune responses to Mycobacterium tuberculosis ESAT-6 and KatG peptides from peripheral blood mononuclear cells from 15/26 sarcoidosis, 1/24 purified-protein-derivative-negative (PPD−) (P < 0.0001, Fishers exact test), and 7/8 PPD-positive (PPD+) subjects (P = 0.21). This finding provides immunologic links between mycobacteria and systemic sarcoidosis.

Mycobacterial antigens may be important in sarcoidosis pathogenesis.

Purpose of review To describe the most recent epidemiologic, molecular and immunologic literature related to the role of infectious antigens in sarcoidosis pathogenesis, with a focus upon Mycobacterium and Proprionibacterium species. Recent findings Recent studies of successful molecular analysis for and humoral immunity to mycobacterial antigens from sarcoidosis patients have renewed interest in a potential role of mycobacteria in sarcoidosis. One study provided molecular and immunologic evidence for mycobacteria among sarcoidosis subjects from the United States. These studies, while preliminary, provide the groundwork for more in-depth studies of the potential role of mycobacteria in sarcoidosis pathogenesis. Proprionibacteria have also been proposed as a cause of sarcoidosis; a study of the detection of Proprionibacterium species nucleic acids throughout the lung of sarcoidosis and control subjects, however, suggests that these organisms are less likely to be causal. Summary While the studies to date do not fulfill Kochs postulates, they do add further support to the hypothesis that infectious antigens, particularly those from mycobacteria, may have a causal role in some sarcoidosis cases. In future studies that purport to show an association of microbial antigen(s) with sarcoidosis, investigation of genetic risk factors contributing to risk will be important, in order to explain why some patients are found to have an association with microbial antigens and others are not.

Cellular Responses to Mycobacterial Antigens Are Present in Bronchoalveolar Lavage Fluid Used in the Diagnosis of Sarcoidosis

ABSTRACT 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.

Mycobacterium tuberculosis Antigen 85A Induces Th-1 Immune Responses in Systemic Sarcoidosis

Sarcoidosis is a granulomatous disease of unknown etiology, characterized by a Th-1 immunophenotype. Although humoral immune responses by sarcoidosis subjects to mycobacterial proteins have been detected, mycobacterial antigens capable of inducing cellular immune responses in sarcoidosis subjects have not been reported. We used the enzyme-linked immunospot assay to assess for recognition of the Mycobacterium tuberculosis mycolyl transferase, Antigen 85A, by peripheral blood mononuclear cells from 25 sarcoidosis subjects, 22 PPD− (purified protein derivative) healthy volunteers, and 16 PPD+ healthy subjects. Reactivity to Ag85A whole protein was observed in 15 of 25 sarcoidosis subjects compared to 2 of 22 PPD− subjects (p=0.0006, Fisher’s exact test) and to 14 of 16 PPD+ subjects (p=0.084, Fisher’s exact test). Monoclonal antibody against HLA-DR inhibited recognition. In addition to immune recognition of Ag85A whole protein, peptide-mapping studies identified four immunogenic Ag85A peptides, which induced Th-1 immune responses in individual sarcoidosis subjects, suggesting that multiple epitopes from a mycobacterial protein may have a role in sarcoidosis immunopathogenesis.

Dual Analysis for Mycobacteria and Propionibacteria in Sarcoidosis BAL

PurposeSarcoidosis 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).MethodsBAL 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.ResultsCD4+ 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.ConclusionsMALDI-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.

Oral antimycobacterial therapy in chronic cutaneous sarcoidosis: a randomized, single-masked, placebo-controlled study.

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.

Multiple mycobacterial antigens are targets of the adaptive immune response in pulmonary sarcoidosis

IntroductionSarcoidosis 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.MethodsFifty-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.ResultsAlveolar 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.ConclusionsTogether 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.

Blockade of the programmed death-1 pathway restores sarcoidosis CD4(+) T-cell proliferative capacity.

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.

Reversal of Global CD4+ Subset Dysfunction Is Associated with Spontaneous Clinical Resolution of Pulmonary Sarcoidosis

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.