STAT1 and CXCL10 involve in M1 macrophage polarization that may affect osteolysis and bone remodeling in extrapulmonary tuberculosis.

OBJECTIVE: This study was aimed to reveal the molecular mechanism of bone destruction due to macrophage polarization leading to during extrapulmonary tuberculosis (EPTB) infection. METHODS: The dataset GSE83456 was downloaded from the GEO database, and the xCell tool was used to obtain the 64 types of immune cells. The flow cytometry was performed to identified the differences between M1 and M2 macrophages between EPTB and the healthy controls (HCs). The enrichment analyses were performed on the differentially expressed genes (DEGs) and their functionally related modules. The hub genes were screened out, and their relationships with EPTB and the immune cell subtypes were further analyzed. RESULTS: The flow cytometric analysis validated this hypothesis of M1-macrophage polarization correlated with the pathogenesis of EPTB. Of the obtained 103 DEGs, 97 genes were upregulated, and 6 genes were downregulated. The GO and KEGG pathway analyses showed that the DEGs were particularly involved in the immune-related processes. The hub genes (STAT1 and CXCL10) might be involved in M1-macrophage polarization and correlated with the pathogenesis of EPTB. STAT1 and CXCL10 could also behave as biomarkers for EPTB. CONCLUSION: STAT1 and CXCL10 were involved in the M1-macrophage polarization and correlated with the pathogenesis of EPTB. Besides, both of them could also behave as biomarkers for EPTB diagnosis and provide the required clues for targeted therapy in the future.

Targeting interferon-γ in hyperinflammation: opportunities and challenges.

Interferon-γ (IFNγ) is a pleiotropic cytokine with multiple effects on the inflammatory response and on innate and adaptive immunity. Overproduction of IFNγ underlies several, potentially fatal, hyperinflammatory or immune-mediated diseases. Several data from animal models and/or from translational research in patients point to a role of IFNγ in hyperinflammatory diseases, such as primary haemophagocytic lymphohistiocytosis, various forms of secondary haemophagocytic lymphohistiocytosis, including macrophage activation syndrome, and cytokine release syndrome, all of which are often managed by rheumatologists or in consultation with rheumatologists. Given the effects of IFNγ on B cells and T follicular helper cells, a role for IFNγ in systemic lupus erythematosus pathogenesis is emerging. To improve our understanding of the role of IFNγ in human disease, IFNγ-related biomarkers that are relevant for the management of hyperinflammatory diseases are progressively being identified and studied, especially because circulating levels of IFNγ do not always reflect its overproduction in tissue. These biomarkers include STAT1 (specifically the phosphorylated form), neopterin and the chemokine CXCL9. IFNγ-neutralizing agents have shown efficacy in the treatment of primary haemophagocytic lymphohistiocytosis in clinical trials and initial promising results have been obtained in various forms of secondary haemophagocytic lymphohistiocytosis, including macrophage activation syndrome. In clinical practice, there is a growing body of evidence supporting the usefulness of circulating CXCL9 levels as a biomarker reflecting IFNγ production.

Blood Leukocyte Signaling Pathways as Predictors of Severity of Acute Pancreatitis.

OBJECTIVES: Clinical practice lacks biomarkers to predict the severity of acute pancreatitis (AP). We studied if intracellular signaling of circulating leukocytes could predict persistent organ dysfunction (OD) and secondary infections in AP. METHODS: A venous blood sample was taken from 174 patients with AP 72 hours or less from onset of symptoms and 31 healthy controls. Phosphorylation levels (p) of appropriately stimulated signal transducer and activator of transcription 1 (STAT1), STAT6, nuclear factor-κB (NF-κB), Akt, and nonstimulated STAT3 in monocytes, neutrophils, and lymphocytes was measured using phosphospecific flow cytometry. RESULTS: The patients showed higher pSTAT3 and lower pSTAT1, pSTAT6, pNF-κB, and pAkt than healthy controls. pSTAT3 in all leukocyte subtypes studied increased, and pSTAT1 in monocytes and T cells decreased in an AP severity-wise manner. In patients without OD at sampling, high pSTAT3 in monocytes and T lymphocytes were associated with development of persistent OD. In patients with OD, low interleukin-4-stimulated pSTAT6 in monocytes and neutrophils and Escherichia coli-stimulated pNF-κB in neutrophils predicted OD persistence. High pSTAT3 in monocytes, CD8+ T cells, and neutrophils; low pSTAT1 in monocytes and T cells; and low pNF-κB in lymphocytes predicted secondary infections. CONCLUSIONS: Leukocyte STAT3, STAT1, STAT6, and NF-κΒ phosphorylations are potential predictors of AP severity.

JAK-STAT Activity in Peripheral Blood Cells and Kidney Tissue in IgA Nephropathy.

BACKGROUND AND OBJECTIVES: IgA nephropathy is the most common primary glomerular disease in the world. Marked by mesangial inflammation and proliferation, it generally leads to progressive kidney fibrosis. As the Janus kinase signal transducer and activator of transcription pathway has been implicated as an important mediator of diabetic kidney disease and FSGS, detailed investigation of this pathway in IgA nephropathy was undertaken to establish the basis for targeting this pathway across glomerular diseases. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Well characterized patients with IgA nephropathy and controls were studied, allowing us to compare 77 patients with biopsy-proven IgA nephropathy with 45 healthy subjects. STAT phosphorylation was assessed in peripheral blood monocytes (PBMCs) by phosphoflow before and after cytokine stimulation. Kidney Janus kinase signal transducer and activator of transcription activity was studied by immunofluorescence and by transcriptomic studies. An STAT1 activity score was established using downstream transcriptional targets of pSTAT1 and associated with disease and clinical outcomes. RESULTS: We found PBMCs to have upregulated pSTAT production at baseline in patients with IgA nephropathy with a limited reserve to respond to cytokine stimulation compared with controls. Increased staining in glomerular mesangium and endothelium was seen for Jak-2 and pSTAT1 and in the tubulointerstitial for JAK2, pSTAT1, and pSTAT3. Activation of the Janus kinase signal transducer and activator of transcription pathway was further supported by increased pSTAT1 and pSTAT3 scores in glomerular and tubulointerstitial sections of the kidney (glomerular activation Z scores: 7.1 and 4.5, respectively; P values: <0.001 and <0.001, respectively). Clinically, phosphoflow results associated with proteinuria and kidney function, and STAT1 activation associated with proteinuria but was not associated with progression. CONCLUSIONS: Janus kinase signal transducer and activator of transcription signaling was activated in patients with IgA nephropathy compared with controls. There were altered responses in peripheral immune cells and increased message and activated proteins in the kidney. These changes variably related to proteinuria and kidney function.

Association between the methylation of the STAT1 and SOCS3 in peripheral blood and gastric cancer.

BACKGROUND AND AIM: DNA methylation is an important epigenetic modification that can promote the development of various cancers. The STAT1 and SOCS3 have been observed to be hypermethylated in tumor tissues and peripheral blood. This study aimed to explore the relationship between the methylation status of the STAT1 and SOCS3 in peripheral blood and gastric cancer (GC). METHODS: This hospital-based case-control study involved 372 patients with GC and 379 controls. The methylation status of the STAT1 and SOCS3 was semiquantitatively determined using the methylation-sensitive high-resolution melting method. Logistic regression analysis was used to analyze the relationship between the STAT1 and SOCS3 methylation status and GC susceptibility. Moreover, propensity scores were used to control confounding factors. RESULTS: Compared with negative methylation, the positive methylation of SOCS3 significantly increased the risk of GC (ORa  = 1.820, 95% CI: 1.247-2.658, P = 0.002). This trend was also found via stratified analysis, and methylation positivity of the SOCS3 significantly increased the risk of GC in the < 60 years group, in the ≥ 60 years group, and in the positive Helicobacter pylori infection group (ORa  = 1.654, 95% CI: 1.029-2.660, P = 0.038; ORa  = 1.957, 95% CI: 1.136-3.376, P = 0.016; ORa  = 2.084, 95% CI: 1.270-3.422, P = 0.004, respectively). Additionally, no significant association was found between STAT1 methylation and GC risk (ORa  = 0.646, 95% CI: 0.363-1.147, P = 0.135). This study found that the interaction between the methylation status of STAT1 and SOCS3 and environmental factors did not have an impact on GC risk. CONCLUSION: SOCS3 methylation may serve as a new potential biomarker for GC susceptibility.

STAT1 and its related molecules as potential biomarkers in Mycobacterium tuberculosis infection.

Tuberculosis (TB) is a severe infectious disease that seriously endangers human health. The immune defence mechanism of the body against TB is still unclear. The purpose of this study was to find the key molecules involved in the immune defence response during TB infection, and provide reference for the treatment of TB and further understanding of the immune defence mechanism of the body. Data from GSE83456 were downloaded from GEO data sets for analysis, and a total of 192 differentially expressed genes were screened out. Most of these genes are enriched in the interferon signalling pathway and are defence response-related. We also found that STAT1 plays an important role in the immune defence of TB infection and it is one of the key genes related to interferon signalling pathway. STAT1-related molecules including hsa-miR-448, hsa-miR-223-3p, SAMD8_hsa_circRNA 994 and TWF1_hsa_circRNA 9897 were therefore screened out. Furthermore, expression levels of hsa-miR-448 and hsa-miR-223-3p were then verified by qRT-PCR. Results showed that both hsa-miR-448 and hsa-miR-223-3p were down-regulated in plasma from patients with pulmonary TB. Taken together, our data indicate that an mRNA-miRNA-circRNA interaction chain may play an important role in the infection of MTB, and STAT1 and related molecules including hsa-miR-223-3p, has-miR-448, SAMD8_hsa_circRNA994 and TWF1_hsa_circRNA9897 were identified as potential biomarkers in the development of active TB.

Dysregulated expression of STAT1, miR-150, and miR-223 in peripheral blood mononuclear cells of coronary artery disease patients with significant or insignificant stenosis.

Coronary artery disease (CAD) is a multicellular disease characterized by chronic inflammation. Peripheral blood-mononuclear cells (PBMCs), as a critical component of immune system, actively cross-talk with pathophysiological conditions induced by endothelial cell injury, reflecting in perturbed PBMC expression. STAT1 is believed to be relevant to CAD pathogenesis through regulating key inflammatory processes and modulating STAT1 expression play key roles in fine-tuning CAD-related inflammatory processes. This study evaluated PBMC expressions of STAT1, and its regulators (miR-150 and miR-223) in a cohort including 72 patients with CAD with significant ( ≥ 50%) stenosis, 30 patients with insignificant ( < 50%) coronary stenosis (ICAD), and 74 healthy controls, and assessed potential of PBMC expressions to discriminate between patients and controls. We designed quantitative real-time polymerase chain reaction (RT-qPCR) assays and identified stable reference genes for normalizing PBMC quantities of miR-150, miR-223, and STAT1 applying geNorm algorithm to six small RNAs and five mRNAs. There was no significant difference between CAD and ICAD patients regarding STAT1 expression. However, both groups of patients had higher levels of STAT1 than healthy controls. miR-150 and miR-223 were differently expressed across three groups of subjects and were downregulated in patients compared with healthy controls, with the lowest expression levels being observed in patients with ICAD. ROC curves suggested that PBMC expressions may separate between different groups of study subjects. PBMC expressions also discriminated different clinical manifestations of CAD from ICADs or healthy controls. In conclusion, the present study reported PBMC dysregulations of STAT1, miR-150, and miR-223, in patients with significant or insignificant coronary stenosis and suggested that these changes may have diagnostic implications.

JAK-STAT signaling is activated in the kidney and peripheral blood cells of patients with focal segmental glomerulosclerosis.

Focal segmental glomerular sclerosis (FSGS) is a devastating disease with limited treatment options and poor prognosis. Activated JAK-STAT signaling has been implicated in other kidney diseases. Since new technologies allow us to better evaluate changes in systemic and renal JAK-STAT activity as it relates to kidney function, we examined this in 106 patients with biopsy-proven FSGS compared to 47 healthy control individuals. Peripheral immune function was assessed in peripheral blood mononuclear cells by phosphoflow studies before and after cytokine stimulation. Kidney JAK-STAT activity was measured by immunofluorescence and by transcriptomics. A STAT1 activity score was calculated by evaluating message status of downstream targets of pSTAT 1. Peripheral blood mononuclear cells were found to be upregulated in terms of pSTAT production at baseline in FSGS and to have limited reserve to respond to various cytokines. Increased staining for components of the JAK-STAT system in FSGS by microscopy was found. Furthermore, we found transcriptomic evidence for activation of JAK-STAT that increased pSTAT 1 and pSTAT 3 in glomerular and tubulointerstitial sections of the kidney. Some of these changes were associated with the likelihood of remission of proteinuria and progression of disease. JAK-STAT signaling is altered in patients with FSGS as compared to healthy controls with activated peripheral immune cells, increased message in the kidney and increased activated proteins in the kidney. Thus, our findings support immune activation in this disease and point to the JAK-STAT pathway as a potential target for treatment of FSGS.

[Inflammatory and immune biomarkers of radiation response]. / Biomarqueurs inflammatoires et immunologiques de réponse à la radiothérapie.

In radiotherapy, the treatment is adapted to each individual to protect healthy tissues but delivers most of time a standard dose according to the tumor histology and site. The only biomarkers studied to individualize the treatment are the HPV status with radiation dose de-escalation strategies, and tumor hypoxia with dose escalation to hypoxic subvolumes using FMISO- or FAZA-PET imaging. In the last decades, evidence has grown about the contribution of the immune system to radiation tumor response. Many preclinical studies have identified some of the mechanisms involved. In this context, we have realised a systematic review to highlight potential inflammatory and immune biomarkers of radiotherapy response. Some are inside the tumor microenvironment, as lymphocyte infiltration or PD-L1 expression, others are circulating biomarkers, including different types of hematological cells, cytokines and chemokines.

Enhanced perforin expression associated with dasatinib therapy in natural killer cells.

We investigated the effects of dasatinib on natural killer (NK) cell-induced signaling protein and perforin expression as well as plasma cytokine levels by analyzing blood samples from patients with well-controlled chronic myeloid leukemia receiving tyrosine kinase inhibitor (TKI) therapy. Perforin expression and phosphorylation of signal transducer and activator of transcription (STAT) 1, STAT3, Janus kinase (JAK) 1, and JAK2 in NK cells were evaluated by flow cytometry, and the levels of plasma cytokines, including interferon (IFN)-γ and interleukin (IL)-2, were determined by enzyme-linked immunosorbent assays in 40 patients (dasatinib, n = 23; imatinib, n = 11; and nilotinib, n = 6). Perforin levels in NK cells were higher in dasatinib-treated patients before TKI treatment; phospho (p)-STAT1 levels were closely correlated with pJAK1 and perforin levels, and pSTAT3 levels were correlated with pJAK2 and perforin levels. The correlation between pJAK1 and pSTAT1 was apparent in dasatinib-treated patients but not in other TKI-treated patients, and the correlation between pJAK2 and pSTAT3 was apparent in patients treated with other TKIs. Constitutive expression of IFN-γ was higher in patients treated with dasatinib or with other TKIs than in those who were in treatment-free remission (TFR). In contrast, constitutive expression of IL-2 was lower in patients treated with other TKIs than in those treated with dasatinib or in those who were in TFR. These results provided insights into the effects of dasatinib on JAK/STAT signaling in NK cells in vivo and the mechanisms underlying NK cell activation induced by dasatinib therapy.

STAT6 and STAT1 Pathway Activation in Circulating Lymphocytes and Monocytes as Predictor of Treatment Response in Rheumatoid Arthritis.

OBJECTIVE: To find novel predictors of treatment response to disease-modifying antirheumatic drugs (DMARDs), we studied activation of STAT (signal transducers and activators of transcription) 6 and 1 in circulating leukocytes of patients with rheumatoid arthritis (RA). METHODS: 19 patients with untreated recent-onset RA, 16 patients with chronic RA irresponsive to synthetic DMARDs and 37 healthy volunteers provided blood samples for whole blood flow cytometric determination of intracellular STAT6 and STAT1 phosphorylation, expressed as relative fluorescence units, in response to IL-4 and IFN-γ, respectively. Phosphorylation was restudied and treatment response (according to European League Against Rheumatism) determined after 1-year treatment with synthetic DMARDs in recent-onset RA and with biological DMARD in synthetic DMARD-irresponsive RA. Estimation-based exact logistic regression was used to investigate relation of baseline variables to treatment response. 95% confidence intervals of means were estimated by bias-corrected bootstrapping and the significance between baseline and follow-up values was calculated by permutation test. RESULTS: At baseline, levels of phosphorylated STAT6 (pSTAT6) induced by IL-4 in monocytes were higher in those who achieved good treatment response to synthetic DMARDs than in those who did not among patients with untreated RA (OR 2.74, 95% CI 1.05 to 9.47), and IFN-γ -stimulated lymphocyte pSTAT1 levels were higher in those who achieved good treatment response to a biological drug than in those who did not among patients with chronic RA (OR 3.91, 95% CI 1.12 to 20.68). During follow-up, in recent-onset RA patients with good treatment response to synthetic DMARDS, the lymphocyte pSTAT6 levels decreased (p = 0.011), and, consequently, the ratio of pSTAT1/pSTAT6 in lymphocytes increased (p = 0.042). CONCLUSION: Cytokine-stimulated STAT6 and STAT1 phosphorylation in circulating leukocytes was associated with treatment response to DMARDs in this pilot study. The result, if confirmed in larger studies, may aid in developing personalized medicine in RA.

Activated Phosphorylated STAT1 Levels as a Biologically Relevant Immune Signal in Schizophrenia.

OBJECTIVE: Activation of STAT1 is directly downstream of the cytokine receptors that signal from specific proinflammatory cytokines known to be dysregulated in schizophrenia (SZ), such as IFNγ, IL6, IL2 and IL10, as well as hypoxia, viral/bacterial infections and peptide growth factors. If the increased cytokine protein levels repeatedly observed in SZ have biological consequences, then the measurement of pSTAT1 is a logical step forward. METHODS: Peripheral blood mononuclear cells (PBMCs) from controls (n = 13) and subjects with SZ (n = 22) were extracted using the Ficoll method. Participants with SZ were diagnosed using the SCID, clinical symptomatology was measured using the Positive and Negative Syndrome Scale (PANSS), and cognitive functioning was measured using the MATRICS Consensus Cognitive Battery. Levels of activated STAT1 (Y701), i.e. phosphorylated STAT1 (pSTAT1), were measured by a commercially available ELISA in nuclear extracts from PBMCs. RESULTS: There was a significant bimodal distribution in the sample, with an SZ subgroup expressing significantly greater levels of activated pSTAT1 than the remainder of the participants. In this subgroup, levels of pSTAT1 were significantly higher than in the control group, as well as significantly higher than in the remainder of the SZ subjects. Furthermore, this subsample of patients manifested significantly poorer cognitive performance on several measures of the MATRICS. DISCUSSION: pSTAT1 levels may provide a measure of the biological relevance of widely reported elevations in levels of cytokines in SZ over the past several decades. Activation of kinase cascades can be used to partition or disassemble the composite immune signal in patients living with SZ.

[Expression profiles of PI3K, NF-κB, and STAT1 in peripheral blood mononuclear cells in children with bronchial asthma].

OBJECTIVE: To study the expression profiles of PI3K, NF-κB, and STAT1 in peripheral blood mononuclear cells (PBMCs) in children with bronchial asthma, as well as their roles in the pathogenesis of asthma. METHODS: Thirty children with acute exacerbation of bronchial asthma were enrolled as the asthma group, and 20 healthy children were enrolled as the control group. RT-PCR and Western blot were used to measure the mRNA and protein expression levels of PI3K, NF-κB, and STAT1 in PBMCs. A spirometer was used to compare the pulmonary function between the two groups. The correlations between the mRNA expression of PI3K, NF-κB, and STAT1 and pulmonary function in children with bronchial asthma were analyzed. RESULTS: The asthma group had significantly higher mRNA and protein expression levels of PI3K, NF-κB, and STAT1 than the control group (P<0.05). Compared with the control group, the asthma group showed significant reductions in pulmonary function indices such as FEV1%, FEV1/FVC, and PEF% (P<0.05). In children with bronchial asthma, the mRNA expression levels of PI3K, NF-κB, and STAT1 were negatively correlated with FEV1%, FEV1/FVC, and PEF% (P<0.05). CONCLUSIONS: The expression levels of PI3K, NF-κB, and STAT1 increase in children with asthma, and are negatively correlated with pulmonary function indices, suggesting that PI3K, NF-κB and STAT1 are involved in the development and progression of bronchial asthma in children.

Diagnostic value of blood gene expression signatures in active tuberculosis in Thais: a pilot study.

Tuberculosis (TB) is a major global health problem. Routine laboratory tests or newly developed molecular detection are limited to the quality of sputum sample. Here we selected genes specific to TB by a minimum redundancy-maximum relevancy package using publicly available microarray data and determine level of selected genes in blood collected from a Thai TB cohort of 40 active TB patients, 38 healthy controls and 18 previous TB patients using quantitative real-time PCR. FCGR1A, FCGR1B variant 1, FCGR1B variant 2, APOL1, GBP5, PSTPIP2, STAT1, KCNJ15, MAFB and KAZN had significantly higher expression level in active TB individuals as compared with healthy controls and previous TB cases (P<0.01). A mathematical method was applied to calculate TB predictive score, which contains the level of expression of seven genes and this score can identify active TB cases with 82.5% sensitivity and 100% specificity as compared with conventional culture confirmation. In addition, TB predictive scores in active TB patients were reduced to normal after completion of standard short-course therapy, which was mostly in concordant with the disease outcome. These finding suggested that blood gene expression measurement and TB Sick Score could have potential value in terms of diagnosis of TB and anti-TB treatment monitoring.

Simvastatin attenuates rhinovirus-induced interferon and CXCL10 secretion from monocytic cells in vitro.

RV infections frequently trigger exacerbations of respiratory diseases, such as asthma, yet treatment and intervention options remain limited. Statin drugs are the treatment of choice for dyslipidemia and can also modulate immune cell function. To determine whether statin drugs modify antiviral responses of human monocytic cells, we obtained blood monocytes from donors with allergies and/or asthma and treated the cells with sim prior to challenge with RV. RV-induced secretion of CXCL10 was attenuated significantly, irrespective of RV type (RV-16, -14, or -1A), which corresponded with decreases in IFN-α secretion and pSTAT1. Sim pretreatment also reduced RV-induced CXCL10 secretion from human alveolar macrophages. The addition of mev and GGPP-two intermediates of the cholesterol biosynthesis pathway-was able to rescue CXCL10 release fully, demonstrating that effects of sim were related to inhibition of cholesterol biosynthesis and not to an off-target effect. In addition, sim pretreatment attenuated IFN-α-induced pSTAT1 and CXCL10 secretion, providing evidence that sim additionally can affect type I IFNR signaling. SOCS1 and 3 mRNA are both induced with RV stimulation, but sim did not elevate SOCS1 or SOCS3 mRNA expression basally or in the presence of RV. Our findings suggest that sim inhibition of the cholesterol biosynthesis pathway leads to decreased RV-induced chemokine secretion in monocytes and macrophages. These findings suggest that statin drugs have the potential to curb the inflammatory response to RV infection.

Elevated signal transducers and activators of transcription 1 correlates with increased C-C motif chemokine ligand 2 and C-X-C motif chemokine 10 levels in peripheral blood of patients with systemic lupus erythematosus.

INTRODUCTION: The present study examines the levels of recently reported biomarkers, adenosine deaminase acting on RNA (ADAR), C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine 10 (CXCL10), signal transducers and activators of transcription 1 (STAT1), and miR-146a in systemic lupus erythematosus (SLE) patients over multiple visits. METHODS: Peripheral blood leukocytes were collected from 65 healthy donors and 103 SLE patients, 60 of whom had samples from 2 or more visits. Total RNA was isolated and analyzed for the expression of mRNA and microRNA using Taqman real time PCR assays. Relative expression of I-IFN signature genes, chemokines, and miR-146a were determined by the ΔΔCT method. Results were correlated with clinical data and analyzed by Wilcoxon/Kruskal-Wallis test and Fisher's exact test. RESULTS: Levels of ADAR, CCL2, CXCL10, and STAT1 in SLE were significantly elevated compared with the healthy controls (P <0.0001). ADAR, CCL2, and CXCL10 showed significant correlation with IFN score in both healthy donors (P <0.0033) and SLE patients (P <0.0001). In SLE patients, miR-146a level was not significantly different from healthy controls nor correlated to the IFN score. Two STAT1 populations were identified: a low STAT1 and a high STAT1 group. High STAT1 patient visits displayed higher (P ≤0.0020) levels of CCL2 and CXCL10 than the low STAT1 patient visits. STAT1 levels correlated with IFN score in low STAT1 group but not in high STAT1 group. More importantly, high STAT1 levels appeared as an enhancer of CCL2 and CXCL10 as indicated by the significantly stronger correlation of CCL2 and CXCL10 with IFN score in high STAT1 patient visits relative to low STAT1 patient visits. CONCLUSION: Our data indicate a novel role for STAT1 in the pathogenesis of SLE as an expression enhancer of CCL2 and CXCL10 in SLE patients with high levels of STAT1. Future study is needed to examine the exact role of STAT1 in the etiology of SLE.

Reduced levels of CCL2 and CXCL10 in systemic lupus erythematosus patients under treatment with prednisone, mycophenolate mofetil, or hydroxychloroquine, except in a high STAT1 subset.

INTRODUCTION: Our recent data showed that signal transducers and activators of transcription 1 (STAT1), adenosine deaminase acting on RNA (ADAR), C-C motif chemokine ligand 2 (CCL2), and C-X-C motif chemokine 10 (CXCL10) were significantly elevated in a systemic lupus erythematosus (SLE) cohort compared to healthy donors. High and low STAT1 subsets were identified in SLE patient visits. The present study analyzed the correlation of common treatments used in SLE with the levels of these biomarkers. METHODS: Peripheral blood leukocytes were collected from 65 healthy donors and 103 SLE patients, of whom 60 had samples from two or more visits. Total RNA was isolated and analyzed for the expression of mRNA and microRNA using Taqman real-time polymerase chain reaction (PCR) assays. Relative expression of interferon signature genes, CCL2, and CXCL10 were determined by the ΔΔCT method. Results were correlated with therapy using prednisone, mycophenolate mofetil, and hydroxychloroquine and analyzed by Wilcoxon/Kruskal-Wallis test and Fisher's exact test. RESULTS: CCL2 and CXCL10 were significantly higher in untreated patients compared to treated patients, however, in high STAT1 patient visits there is no significant difference between treated and untreated patients' visits. When comparing linear regression fits of interferon (IFN) score with CCL2 and CXCL10, untreated patients and high STAT1 patients displayed significantly higher slopes compared to treated patients. There was no significant difference between the slopes of high STAT1 and untreated patients indicating that CCL2 and CXCL10 were correlated with type-I IFN in high STAT1 patients similar to that in untreated patients. CCL2 and CXCL10 levels in the high STAT1 subset remained high in treated patient visits compared to those of the low STAT1 subset. CONCLUSIONS: Among the biomarkers analyzed, only CCL2 and CXCL10 showed significantly reduced levels in treated compared to untreated SLE patients. STAT1, CCL2, and CXCL10 are potentially useful indicators of therapeutic action in SLE patients. Further work is needed to determine whether high STAT1 levels convey resistance to therapies commonly used to treat SLE and whether STAT1 inhibitors may have therapeutic implication for these patients.

Inflammatory myopathy with abundant macrophages (IMAM): the immunology revisited.

We describe a patient with a clinically atypical presentation of inflammatory myopathy with abundant macrophages (IMAM) but with convincing muscle biopsy features of this subform of inflammatory myopathy. IMAM is characterized mainly by a conspicuous infiltration of muscle and connective tissue by numerous macrophages remote from necrotic and basophilic regenerating muscle fibers. Typically few, mostly CD4(+) T helper (Th) cells are also present. Here, we report a patient with IMAM and demonstrate, that most macrophages express the macrophage mannose receptor 1 (CD206) corresponding to alternatively activated (M2) polarization. Accordingly, signal transducer and activator of transcription 6 (STAT6), involved in Th2-M2 immunity, was expressed at high levels in skeletal muscle. However, TNFα, IFNγ and STAT1, mediators of the T helper 1-classically activated (M1) response were elevated in skeletal muscle and in blood, while expression of CD206 was elevated in skeletal muscle only. Our results argue that IMAM could be a distinct entity between the inflammatory myopathies rather than a subform of dermatomyositis.

Inhibition of CXCL10 release by monomeric C3bi and C4b.

Cellulose acetate (CA) beads are often used for leucocyte apheresis therapy against inflammatory bowel disease. In order to clarify the mechanism of the anti-inflammatory effects of CA, global analysis of the molecules generated in blood by the interaction with CA beads was performed in this study. An activated medium was collected from whole blood that had been preincubated with CA beads, and the effects of the CA-activated medium on leucocyte function were investigated. Fresh blood was stimulated with lipopolysaccharide (LPS) or interferon (IFN)-ß in the presence of the activated medium, and levels of chemokines and cytokines, including CXCL10 (IFN-inducible protein-10), and phosphorylated STAT1 (signal transducer and activator of transcription 1), which is known to be essential for CXCL10 production in leucocytes, were measured. IFN-ß- or LPS-induced CXCL10 production, expression of CXCL10 mRNA and phosphorylation of STAT1 were significantly reduced in the presence of the medium pretreated with CA beads compared with the control without the CA bead treatment. The factors inhibiting CXCL10 production were identified as the C3 and C4 fragments by mass spectrometry. The monomeric C3bi and C4b proteins were abundant in the medium pretreated with CA beads. Furthermore, purified C3bi and C4b were found to inhibit IFN-ß-induced CXCL10 production and STAT1 phosphorylation. Thus, STAT1-mediated CXCL10 production induced by stimulation with LPS or IFN was potently inhibited by monomeric C3bi and C4b generated by the interaction of blood with CA beads. These mechanisms mediated by monomeric C3bi and C4b may be involved in the anti-inflammatory effects of CA.