Background

Sarcoidosis is a granulomatous inflammatory disease with limited blood markers to predict outcomes. The interferon-gamma (IFN-γ)-inducible chemotactic cytokines (chemokines), CXCL9 and CXCL10, are both increased in sarcoidosis patients, yet they possess important molecular differences. Our study determined if serum chemokines correlated with different aspects of disease severity.

Methods

We measured CXCL9 and CXCL10 serum levels at initial study visits and longitudinally in sarcoidosis subjects using ELISA. We examined these chemokines' relationships with pulmonary and organ involvement outcomes, their gene expression, peripheral blood immune cell populations, and immunosuppression use.

Results

Higher CXCL10 levels negatively correlated with FVC, TLC, and DLCO at subjects' initial visit and when measured repeatedly over two years. CXCL10 also positively correlated with longitudinal respiratory symptom severity. Additionally, for every log₁₀(CXCL10) increase, the risk of longitudinal pulmonary function decline increased 8.8 times over the 5-year study period (95% CI 1.6-50, p = 0.014, log₁₀(CXCL0) range 0.84-2.7). In contrast, CXCL9 levels positively correlated with systemic organ involvement at initial study visit (1.5 additional organs involved for every log₁₀(CXCL9) increase, 95% CI 1.1-2.0, p = 0.022, log₁₀(CXCL9) range 1.3-3.3). CXCL10, not CXCL9, positively correlated with its own blood gene expression and monocyte level. Immunosuppressive treatment was associated with lower levels of both chemokines.

Conclusions

In sarcoidosis subjects, serum CXCL9 levels correlated with systemic organ involvement and CXCL10 levels strongly correlated with respiratory outcomes, which may ultimately prove helpful in clinical management. These differing associations may be due to differences in cellular regulation and tissue origin.

Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not.

Rationale

Pulmonary sarcoidosis is classically defined by T-helper (Th) cell type 1 inflammation (e.g., IFN-γ production by CD4(+) effector T cells). Recently, IL-17A-secreting cells have been found in lung lavage, invoking Th17 immunity in sarcoidosis. Studies also identified IL-17A-secreting cells that expressed IFN-γ, but their abundance as a percentage of total CD4(+) cells was either low or undetermined.

Objectives

Based on evidence that Th17 cells can be polarized to Th17.1 cells to produce only IFN-γ, our goal was to determine whether Th17.1 cells are a prominent source of IFN-γ in sarcoidosis.

Methods

We developed a single-cell approach to define and isolate major Th-cell subsets using combinations of chemokine receptors and fluorescence-activated cell sorting. We subsequently confirmed the accuracy of subset enrichment by measuring cytokine production.

Measurements and main results

Discrimination between Th17 and Th17.1 cells revealed very high percentages of Th17.1 cells in lung lavage in sarcoidosis compared with controls in two separate cohorts. No differences in Th17 or Th1 lavage cells were found compared with controls. Lung lavage Th17.1-cell percentages were also higher than Th1-cell percentages, and approximately 60% of Th17.1-enriched cells produced only IFN-γ.

Conclusions

Combined use of surface markers and functional assays to study CD4(+) T cells in sarcoidosis revealed a marked expansion of Th17.1 cells that only produce IFN-γ. These results suggest that Th17.1 cells could be misclassified as Th1 cells and may be the predominant producer of IFN-γ in pulmonary sarcoidosis, challenging the Th1 paradigm of pathogenesis.

The combination of expression patterns of AGR2 (anterior gradient 2) and CD10 by prostate cancer provided four phenotypes that correlated with clinical outcome. Based on immunophenotyping, CD10(low)AGR2(high), CD10(high)AGR2(high), CD10(low)AGR2(low), and CD10(high)AGR2(low) were distinguished. AGR2(+) tumors were associated with longer recurrence-free survival and CD10(+) tumors with shorter recurrence-free survival. In high-stage cases, the CD10(low)AGR2(high) phenotype was associated with a ninefold higher recurrence-free survival than the CD10(high)AGR2(low) phenotype. The CD10(high)AGR2(high) and CD10(low)AGR2(low) phenotypes were intermediate. The CD10(high)AGR2(low) phenotype was most frequent in high-grade primary tumors. Conversely, bone and other soft tissue metastases, and derivative xenografts, expressed more AGR2 and less CD10. AGR2 protein was readily detected in tumor metastases. The CD10(high)AGR2(low) phenotype in primary tumors is predictive of poor outcome; however, the CD10(low)AGR2(high) phenotype is more common in metastases. It appears that AGR2 has a protective function in primary tumors but may have a role in the distal spread of tumor cells.