Dual analysis for mycobacteria and propionibacteria in sarcoidosis BAL

Abstract

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.

Fig. 1

Sarcoidosis BAL T cells demonstrated higher CD4+ T cell specific recognition of mycobacterial antigen compared to P. acnes. Intracellular cytokine staining for IFN-γ was performed on BAL cells from sarcoidosis, PPD-, and NTM subjects, after stimulation with neoantigen (KLH), ESAT-6 peptide, P. acnes supernatant, or SEB (positive control). a Shown are representative flow cytometry dot plots indicating percentage of CD3+CD4+ T cell responses to mycobacterial antigen from a sarcoidosis subject who demonstrated dual recognition of ESAT-6 and P. acnes (Sarcoidosis AA), a subject who recognized ESAT-6 solely (Sarcoidosis BB), a subject who displayed no recognition of either bacteria (Sarcoidosis CC), a NTM control, and a PPD-control. b Percent of CD3+CD4+ T cells that produced IFN-γ in response to KLH, ESAT-6, or P. acnes stimulation with no peptide control subtracted out for all 27 sarcoidosis subjects, 14 PPD- controls, and 9 NTM controls. Differences in the distributions of immune recognition of mycobacterial antigen by BAL CD3+CD4+ T cells were noted between sarcoidosis and disease controls, using the Wilcoxon rank sum test. Medians depicted by lines on figure. A response was considered positive when the frequency of recognition was at least twice background fluorescence and greater than 0.5 %. Stimulation of BAL cells with SEB resulted in a positive IFN-γ response for sarcoidosis subjects and disease controls. c The six sarcoidosis and three NTM subjects who displayed P. acnes recognition also recognized ESAT-6

Fig. 2

Sarcoidosis BAL cells secrete Th1 cytokines and proliferate in response to mycobacterial antigen stimulation. Supernatants were collected from BAL cells 24 h after stimulation with neoantigen (KLH), ESAT-6 peptide, P. acnes supernatant, or SEB (positive control). Data for three sarcoidosis subjects who displayed immune responses (IR) to ESAT-6 and P. acnes, three sarcoidosis subjects who displayed responses to ESAT-6 but not P. acnes, and three PPD-controls who displayed responses to only P. acnes. a IFN-γ. b IL-2. There was a significant difference in extra-cellular Th1 cytokine production to mycobacterial antigens between sarcoidosis and control subjects. c Sarcoidosis BAL cells from subject who displayed responses to both ESAT-6 and P. acnes (Sarcoidosis AB) and subject who displayed responses only to ESAT-6 (Sarcoidosis BD) were CFSE-labeled and activated with neo-antigen (KLH), ESAT-6 peptide, P. acnes supernatant, or SEB. Day 4 post-activation, antigen-specific proliferation of CD4+ cells was assessed by gating on CD3+CD4+ T cells and analyzing the CFSE expression of this subset by flow cytometry. Percent proliferation is indicated by bracket above peaks. Similar results were found in the three other tested subjects

Fig. 3

Sarcoidosis BAL CD8+ T cells display antigen-specific Th1 immune responses to mycobacterial antigen. Intracellular cytokine staining for IFN-γ was performed on BAL cells from sarcoidosis subjects and disease controls, after stimulation with neoantigen (KLH), ESAT-6 peptide, P. acnes supernatant, or SEB. a Shown are representative flow cytometry dot plots indicating percentage of CD3+CD4+ T cell responses to mycobacterial antigen from a sarcoidosis subject who demonstrated dual recognition of ESAT-6 and P. acnes (Sarcoidosis AA), a subject who recognized ESAT-6 solely (Sarcoidosis BB), a subject who displayed no recognition of either bacteria (Sarcoidosis CC), a NTM control, and a PPD- control. b Percent of CD3+CD8+ T cells that produced IFN-γ in response to KLH, ESAT-6, P. acnes supernatant stimulation with no peptide control subtracted out for sarcoidosis subjects and disease controls. Differences in the distributions of immune recognition of mycobacterial antigen by BAL CD3+CD8+ T cells were noted between the sarcoidosis and control subjects using the Wilcoxon rank sum test. Medians depicted by lines on figure. Stimulation of BAL cells with SEB resulted in a positive IFN-γ response for sarcoidosis subjects and disease controls. c The six sarcoidosis and two NTM subjects who displayed P. acnes recognition also recognized ESAT-6. d In the six sarcoidosis subjects who demonstrated dual recognition of the antigens, the trend was the CD8+ T cell response was stronger than the CD4+ T cell response. Differences in the distributions of immune recognition of ESAT-6 and P. acnes by BAL CD3+CD4+ and CD3+CD8+ T cells were measured using the Wilcoxon signed rank test

Fig. 4

Presence of bacteria does not correlate with radiographic stages of sarcoidosis. Tested immune responses to ESAT-6 and P. acnes across radiographic stage. Medians depicted by lines on figure. Differences in the distribution of immune recognition in sarcoidosis patients across radiographic stage I and II were assessed using the Kruskal-Wallis rank sum test. There was no significant difference in the strength of response between stages. a CD4+ T cell immune response. b CD8+ T cell immune response

Fig. 5

MALDI-IMS localizes signals consistent with mycobacterial ESAT-6 and P. acnes within sarcoidosis granulomatous inflammation of a lymph node and a breast biopsy. Distinct protein signals corresponding to ESAT-6 and P. acnes were present within sarcoidosis specimens. (A/B) Signals consistent with ESAT-6 (~11,190 m/z) and P. acnes (~4,560 m/z) were present within sarcoidosis specimens. A representation of the peaks visualized with MALDI analysis of 15 snap-frozen sarcoidosis and four snap-frozen control specimens is depicted; 10 of 15 contained either ESAT-6 and/or P. acnes, compared to 1of 4 control specimen containing signals consistent with P. acnes. No signals consistent with ESAT-6 were noted in the control specimens. c Histologic staining of lymph nodes and the breast biopsy revealed granulomas throughout the lymph node, but only distinct areas of involvement within the breast biopsy (red circle). Localization of signals consistent with ESAT-6 and P. acnes were detected throughout both specimens. ESAT-6 signals were noted within areas of granulomatous involvement; whereas, signals consistent with P. acnes were present irrespective of granuloma involvement in the sarcoidosis specimen