Discovery of Two Novel Immunoepitopes and Development of Peptide-based Sarcoidosis Immunoassay.

RATIONALE: Sarcoidosis is a systemic granulomatous disorder associated with hypergammaglobulinemia and the presence of autoantibodies. The specific antigens initiating granulomatous inflammation in sarcoidosis are unknown and there is no specific test available to diagnose sarcoidosis. To discover novel sarcoidosis antigens, we developed a high-throughput T7 phage display library derived from the sarcoidosis cDNA and identified numerous clones differentiating sarcoidosis from other respiratory diseases. After clone sequencing and homology search, we identified two epitopes (Cofilinµ and Chain A) that specifically bind to serum IgGs of sarcoidosis patients. OBJECTIVES: To develop and validate an epitope-specific IgG-based immunoassay specific for sarcoidosis. METHODS: We chemically synthesized both immunoepitopes (Cofilinµ and Chain A), and generated rabbit polyclonal antibodies against both neoantigens. After extensive standardization, we developed a direct peptide ELISA and measured epitope-specific IgG in sera of 386 subjects including, healthy controls (n=100), three sarcoidosis cohorts (n=186), pulmonary tuberculosis (n=70) and lung cancer (n=30). MEASUREMENTS AND MAIN RESULTS: To develop a model to classify sarcoidosis from other groups, data were analyzed using five-fold cross-validation when adjusting for confounders. The Cofilinµ IgGs model yielded a mean sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of 0.97, 0.9, 0.9 and 0.96, respectively. Those same measures for Chain A IgG antibody were 0.9, 0.83, 0.84 and 0.9 respectively. Combining both biomarkers improved AUC, sensitivity, specificity, PPV and NPV. CONCLUSIONS: These results provide a novel immunoassay for sarcoidosis. The discovery of two neoantigens facilitates the development of biospecific drug discovery and the sarcoidosis-specific model.

MIF modulates p38/ERK phosphorylation via MKP-1 induction in sarcoidosis.

Macrophage migration inhibitory factor (MIF) is a versatile cytokine that influences a variety of cellular processes important for immune regulation and tissue homeostasis. Sarcoidosis is a granulomatous disease characterized by extensive local inflammation and increased T helper cell mediated cytokines. We have shown that MIF has a modulatory role in cytokine networks in sarcoidosis. We investigated the effect of exogenous MIF on sarcoidosis alveolar macrophages (AMs), CD14+ monocytes and peripheral blood mononuclear cells (PBMCs). Our results showed that MIF negatively regulates the increased MAPKs (pp38 and pERK1/2) activation by inducing Mitogen-activated protein kinase phosphatase (MKP)-1. We found that MIF decreased IL-6 and IL-1ß production, increased the percentage of regulatory T-cells (Tregs), and induced IL-1R antagonist (IL-1RA) and IL-10 production. Thus, the results of our study suggest that exogenous MIF modulates MAPK activation by inducing MKP-1and Tregs as well as IL-10 and IL-1RA, and hence plays a modulatory role in immune activation in sarcoidosis.

Modulatory role of macrophage migration inhibitory factor on cytokines and clinical features of sarcoidosis.

Sarcoidosis is a systemic granulomatous disease of unknown etiology with significant heterogeneity in organ manifestations and clinical course. Subjects with sarcoidosis share several features such as, non-necrotizing granuloma, hypergammaglobulinemia, increased local and circulating inflammatory cytokines. Macrophage migration inhibitory factor (MIF) is a pluripotent chemokine modulating cellular function. Study included healthy controls (n = 28) and sarcoidosis patients (n = 65). Sera and BAL of sarcoidosis patients were collected and patients were followed longitudinally for 3 years, and demographics, stages, pulmonary function tests, and organ involvements were recorded. We evaluated MIF in the serum and bronchoalveolar lavage (BAL) fluid of sarcoidosis patients in association with clinical features and cytokines, IL-18, IL-10, IL-6, IFN-γ. We found serum MIF had a positive correlation with IL-10 and IFN-γ and % predicted total lung capacity (%TLC). Serum IL-18 had a significant positive correlation with serum lysozyme, but a negative correlation with %TLC and %DLCO. We identified two groups of sarcoidosis subjects with distinct clinical and cytokine features. A group with prominent extrapulmonary involvement, and low serum MIF, IL-10 and IFN-γ and a group with elevated serum MIF, IL-10 and IFN-γ levels. Our work provides understanding of phenotypic diversity in association with heterogeneity in cytokine landscape in sarcoidosis.

MKP-1 modulates ubiquitination/phosphorylation of TLR signaling.

Ubiquitination and phosphorylation are reversible posttranslational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK MAPKs. We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1-/- showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1, and an increased K63 polyubiquitination on TRAF6. Increased K63 polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, ubiquitin-specific protease-13 (USP13), which cleaves polyubiquitin-chains on client proteins, was substantially enhanced in murine MKP-1-deficient BMDMs. An inhibitor of USP13 decreased the K63 polyubiquitination on TRAF6, TAK1 phosphorylation, IL-1ß, and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

Derangement of Metabolic and Lysosomal Gene Profiles in Response to Dexamethasone Treatment in Sarcoidosis.

Glucocorticoids (GCs) play a central role in modulation of inflammation in various diseases, including respiratory diseases such as sarcoidosis. Surprisingly, the specific anti-inflammatory effects of GCs on different myeloid cells especially in macrophages remain poorly understood. Sarcoidosis is a systemic granulomatous disease of unknown etiology that occurs worldwide and is characterized by granuloma formation in different organs. Alveolar macrophages play a role in sarcoidosis granuloma formation and progressive lung disease. The goal of the present study is to identify the effect of GCs on transcriptomic profiles and the cellular pathways in sarcoidosis alveolar macrophages and their corresponding blood myeloid cells. We determined and compared the whole transcriptional signatures of alveolar macrophages from sarcoidosis patients and blood CD14+ monocytes of the same subjects in response to in vitro treatment with dexamethasone (DEX) via RNA-sequencing. In response to DEX, we identified 2,834 genes that were differentially expressed in AM. Predominant pathways affected were as following: metabolic pathway (FDR = 4.1 × 10-10), lysosome (FDR = 6.3 × 10-9), phagosome (FDR = 3.9 × 10-5). The DEX effect on AMs is associated with metabolic derangements involving glycolysis, oxidative phosphorylation and lipid metabolisms. In contrast, the top impacted pathways in response to DEX treatment in blood CD14+ monocytes were as following; cytokine-cytokine receptor interaction (FDR = 6 × 10-6) and transcriptional misregulation in cancer (FDR = 1 × 10-4). Pathways similarly affected in both cell types were genes involved in lysosomes, cytoskeleton and transcriptional misregulation in cancer. These data suggest that the different effects of DEX on AMs and peripheral blood monocytes are partly dictated by lineage specific transcriptional programs and their physiological functions.

HIF-1α regulates IL-1ß and IL-17 in sarcoidosis.

Sarcoidosis is a complex systemic granulomatous disease of unknown etiology characterized by the presence of activated macrophages and Th1/Th17 effector cells. Data mining of our RNA-Seq analysis of CD14+monocytes showed enrichment for metabolic and hypoxia inducible factor (HIF) pathways in sarcoidosis. Further investigation revealed that sarcoidosis macrophages and monocytes exhibit higher protein levels for HIF-α isoforms, HIF-1ß, and their transcriptional co-activator p300 as well as glucose transporter 1 (Glut1). In situ hybridization of sarcoidosis granulomatous lung tissues showed abundance of HIF-1α in the center of granulomas. The abundance of HIF isoforms was mechanistically linked to elevated IL-1ß and IL-17 since targeted down regulation of HIF-1α via short interfering RNA or a HIF-1α inhibitor decreased their production. Pharmacological intervention using chloroquine, a lysosomal inhibitor, decreased lysosomal associated protein 2 (LAMP2) and HIF-1α levels and modified cytokine production. These data suggest that increased activity of HIF-α isoforms regulate Th1/Th17 mediated inflammation in sarcoidosis.

MEK2 Negatively Regulates Lipopolysaccharide-Mediated IL-1ß Production through HIF-1α Expression.

LPS-activated macrophages require metabolic reprogramming and glucose uptake mediated by hypoxia-inducible factor (HIF)-1 α and glucose transporter 1 (Glut1) expression for proinflammatory cytokine production, especially IL-1ß. This process is tightly regulated through activation of MAPK kinases, including the MEK/ERK pathway as well as several transcription factors including HIF-1α. Although MAPK kinase (MEK) 2 deficiency had no significant effect on NO, TNF-α, or IL-12 production in response to LPS challenge, MEK2-deficient murine bone marrow-derived macrophages (BMDMs) exhibited lower IL-10 production. Importantly, MEK2-deficient BMDMs exhibited a preserved ERK1/2 phosphorylation, higher HIF-1α and Glut1 levels, and substantially increased IL-1ß as well as IL-6 production in response to LPS stimulation. Knockdown of HIF-1α expression via short interference RNA decreased the level of HIF-1α expression in MEK2-deficient BMDMs and decreased IL-1ß production in response to LPS treatment. Furthermore, we performed gain of function experiments by overexpressing MEK2 protein in RAW264.7 cells. LPS stimulation of MEK2 overexpressed in RAW264.7 cells led to a marked decreased IL-1ß production. Finally, we investigated the role of Mek1 and Mek2 double and triple mutation on ERK phosphorylation, HIF-1α expression, and IL-1ß production. We found that MEK2 is the major kinase, which inversely proportionally regulates HIF-1α and IL-1ß expression independent of ERK activation. Our findings demonstrate a novel regulatory function for MEK2 in response to TLR4 activation in IL-1ß production through modulating HIF-1α expression.

K63-Linked Polyubiquitination on TRAF6 Regulates LPS-Mediated MAPK Activation, Cytokine Production, and Bacterial Clearance in Toll-Like Receptor 7/8 Primed Murine Macrophages.

Post viral infection bacterial pneumonia is a major cause of morbidity and mortality associated with both seasonal and pandemic influenza virus illness. Despite much efforts put into the discovery of mechanisms of post viral-bacterial infections and their complications in recent years, the molecular mechanisms underlying the increased susceptibility to bacterial infection remain poorly understood. In this study, we focused on the pathways regulating immune responses in murine macrophages and modeled post viral-bacterial infections through pretreatment of bone marrow-derived macrophages (BMDMs) with a toll-like receptor (TLR) 7/8 ligand (R848) and subsequent challenge with TLR2/4 agonists to mimic bacterial infection. We found R848-primed BMDMs upon subsequent exposure to TLR2/4 ligands respond with enhanced inflammatory cytokine production, especially IL-6 and TNF-α. The enhanced cytokine production in R848-primed BMDMs in response to TLR2/4 was due to increased TGF-ß-activated kinase (TAK) 1 phosphorylation with subsequent activation of ERK and p38 MAPKs. Furthermore, we identified that R848 priming leads to increased K63-linked polyubiquitination on TRAF6. K63-linked polyubiquitination on TRAF6 is a signal leading to enhanced activation of downstream pathways including TAK1. Importantly, R848-primed BMDMs infected with live bacteria exhibited decreased bacterial clearance. Small-molecule enhancer of rapamycin 3, an ubiquitin ligase inhibitor reversed the K63-linked polyubiquitination on TRAF6 in R848-primed BMDMs and subsequently decreased TAK1 and MAPK phosphorylation, and cytokine production as well as reversed the decreased bacterial clearance capacity of BMDMs. Our study may provide a novel molecular target to alleviate post viral-bacterial infections.

RNA-sequencing Identifies Novel Pathways in Sarcoidosis Monocytes.

Sarcoidosis is a complex systemic granulomatous disorder of unknown etiology. Genome-wide association studies have not been able to explain a causative role for nucleotide variation in its pathogenesis. The goal of the present study was to identify the gene expression profile and the cellular pathways altered in sarcoidosis monocytes via RNA-sequencing. Peripheral blood monocytes play a role in sarcoidosis inflammation. Therefore, we determined and compared the transcriptional signature of monocytes from peripheral blood from sarcoidosis patients and healthy controls via RNA-sequencing. We found 2,446 differentially expressed (DE) genes between sarcoidosis and healthy control monocytes. Analysis of these DE genes showed enrichment for ribosome, phagocytosis, lysosome, proteasome, oxidative phosphorylation and metabolic pathways. RNA-sequencing identified upregulation of genes involved in phagocytosis and lysosomal pathway in sarcoidosis monocytes, whereas genes involved in proteasome degradation and ribosomal pathways were downregulated. Further studies are needed to investigate the role of specific genes involved in the identified pathways and their possible interaction leading to sarcoidosis pathology.

Dual Inhibition of Rip2 and IRAK1/4 Regulates IL-1ß and IL-6 in Sarcoidosis Alveolar Macrophages and Peripheral Blood Mononuclear Cells.

Sarcoidosis is a multisystem granulomatous disease of unknown etiology that primarily affects the lungs. Our previous work indicates that activation of p38 plays a pivotal role in sarcoidosis inflammatory response. Therefore, we investigated the upstream kinase responsible for activation of p38 in sarcoidosis alveolar macrophages (AMs) and PBMCs. We identified that sustained p38 phosphorylation in sarcoidosis AMs and PBMCs is associated with active MAPK kinase 4 but not with MAPK kinase 3/6. Additionally, we found that sarcoidosis AMs exhibit a higher expression of IRAK1, IRAK-M, and receptor interacting protein 2 (Rip2). Surprisingly, ex vivo treatment of sarcoidosis AMs or PBMCs with IRAK1/4 inhibitor led to a significant increase in IL-1ß mRNA expression both spontaneously and in response to TLR2 ligand. However, a combination of Rip2 and IRAK-1/4 inhibitors significantly decreased both IL-1ß and IL-6 production in sarcoidosis PBMCs and moderately in AMs. Importantly, a combination of Rip2 and IRAK-1/4 inhibitors led to decreased IFN-γ and IL-6 and decreased percentage of activated CD4(+)CD25(+) cells in PBMCs. These data suggest that in sarcoidosis, both pathways, namely IRAK and Rip2, are deregulated. Targeted modulation of Rip2 and IRAK pathways may prove to be a novel treatment for sarcoidosis.

MEK1 dependent and independent ERK activation regulates IL-10 and IL-12 production in bone marrow derived macrophages.

The mitogen activated protein kinases ERK1/2 play an important role in response to toll like receptor (TLR) activation and cytokine production, including IL-10 and IL-12. Here, we examined the role of MEK1 in ERK1/2 activation in response to TLR4 agonist by using bone marrow-derived macrophages (BMDMs) from wild type (WT) and Mek1(d/d)Sox2(Cre) mice. Our data demonstrates that MEK1 is essential for ERK1/2 activation in response to LPS. Furthermore, stimulation of the TLR4 receptor of BMDMs derived from Mek1(d/d)Sox2(Cre) mice showed enhanced STAT4 phosphorylation and increased IL-12 secretion, but exhibited a significantly lower IL-10 production as compared to WT macrophages. Most interestingly, TLR ligation in the presence of recombinant IL-10 (rIL-10) or retinoic acid (RA) led to ERK1/2 activation independent of MEK1 in BMDMs derived from Mek1(d/d)Sox2(Cre) mice and led to inhibition of STAT4 and decreased IL-12 levels. Collectively, these data suggest that MEK1 is required for TLR4 mediated ERK activation and in turn regulates the production of IL-10 and IL-12. It also indicates that ERK1/2 can be activated independent of MEK1 in the presence of IL-10 and RA and this activation negatively regulates IL-12, but positively regulates IL-10 production. These findings may have significant implications for the development of drugs that modulate MEK1 activity in the treatment of inflammatory, autoimmune and proliferative diseases such as cancer.

Constitutive expression of functionally active protease-activated receptors 1 and 2 in human conjunctival epithelial cells.

Protease-activated receptors (PARs) are G-protein-coupled receptors which initiate inflammatory responses when activated by specific serine proteases. This study was conducted to examine whether human conjunctival epithelial cells (HCECs) express functionally active PAR1 and PAR2 using Chang conjunctival epithelial cells as in vitro model. We performed RT-PCR and immunofluorescence analyses to determine the expression of PAR1 and PAR2, and monitored the production of IL-6 after activating HCECs with PAR1 activating agents (thrombin or TFLLRN) or PAR2 activating agents (tryptase, trypsin, or SLIGKV). The results show that HCECs constitutively express PAR1 and PAR2 mRNA and proteins, and produce significant amounts of IL-6 when incubated with specific PAR-activating enzymes or agonist peptides. Thrombin- and tryptase-induced HCEC activation was blocked by PAR1 and PAR2 neutralizing antibodies, respectively, and by specific enzyme inhibitors. The constitutive expression of PAR1 and PAR2, and their activation by thrombin and tryptase, respectively, may have important implications in ocular inflammation.

Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation.

Inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components are initiated by Toll-like receptor 2 (TLR2) and TLR4, respectively. Therefore, the existence of functionally active TLR2 and TLR4 in human conjunctival epithelial cells (HCEC) are critical for the effective host defense against bacterial infections in the eye. We examined the ability of HCEC to respond to TLR4 ligand, lipopolysaccharide (LPS), or TLR2 ligands, lipoteichoic acid (LTA) and peptidoglycan (PGN) using the Chang conjunctival epithelial cell line and the primary conjunctival epithelial cell line (IOBA-NHC) as in vitro models. Incubation of Chang cells with LPS (1 to 1,000 ng/ml) failed to stimulate IL-6 production where as stimulation with LTA or PGN resulted in marked increases in IL-6 production. Semi-quantitative RT-PCR and immunofluorescence analyses showed that Chang cells express TLR2 and TLR4 mRNA and proteins. However, these cells expressed little or no mRNA encoding MD2, an accessory molecule required for TLR4 signaling. Incubation of Chang epithelial cells with interferon-gamma (IFNgamma), but not TNF-alpha, stimulated MD2 mRNA expression and restored LPS responsiveness. In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production. The lack of LPS response, deficient expression of MD2, and induction of MD2 expression and LPS response after IFNgamma priming, were also evident in IOBA-NHC cells. These results demonstrate that HCEC lack LPS responsiveness due to deficient expression of MD2 and that the response can be restored by IFN-gamma priming or MD2 supplementation.

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components.

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.