Plasma CXCL10 Research Articles

Increased peripheral levels of TARC/CCL17 in first episode psychosis patients

BackgroundEvidence for a link between the pathophysiology of schizophrenia and the immune system is mounting. Altered levels of chemokines in plasma have previously been reported in patients with schizophrenia under antipsychotic medication. Here we aimed to study both peripheral and central chemokine levels in drug-naïve or short-time medicated first episode psychosis (FEP) patients. MethodWe analyzed nine chemokines in plasma and CSF from 41 FEP patients and 22 healthy controls using electrochemiluminescence assay. ResultsIn plasma four chemokines; TARC/CCL17, eotaxin/CCL11, MDC/CCL22, IP-10/CXCL10 and in CSF one chemokine; IP-10/CXCL10 showed reliable detection in >50% of the cases. FEP patients displayed increased levels of TARC/CCL17 in plasma compared to healthy controls, 89.6 (IQR 66.2–125.8) pg/mL compared to 48.6 (IQR 28.0–71.7) pg/mL (p = 0.001). The difference was not attributed to confounding factors. Plasma TARC/CCL17 was not associated with PANSS, CGI or GAF scores, neither with cognitive functions. The chemokines eotaxin/CCL11, MDC/CCL22, IP-10/CXCL10 in plasma and IP-10/CXCL10 in CSF did not differ between FEP patients and controls. ConclusionIn line with a previous study showing that chronic patients with schizophrenia display increased plasma TARC/CCL17 levels, we here found an elevation in FEP patients suggesting a role of TARC/CCL17 in early stages of schizophrenia. The exact mechanism of this involvement is still unknown and future longitudinal studies as well as studies of central and peripheral chemokine levels would be of great interest.

Comprehensive Analysis of Chemokines in Host Organs and Their Corresponding Receptors on Donor T-Cells in Xenogeneic Gvhd Model

Comprehensive Analysis of Chemokines in Host Organs and Their Corresponding Receptors on Donor T-Cells in Xenogeneic Gvhd Model

Effects of Horticultural Therapy on Asian Older Adults: A Randomized Controlled Trial.

The effect of horticultural therapy (HT) on immune and endocrine biomarkers remains largely unknown. We designed a waitlist-control randomized controlled trial to investigate the effectiveness of HT in improving mental well-being and modulating biomarker levels. A total of 59 older adults was recruited, with 29 randomly assigned to the HT intervention and 30 to the waitlist control group. The participants attended weekly intervention sessions for the first 3 months and monthly sessions for the subsequent 3 months. Biological and psychosocial data were collected. Biomarkers included IL-1β, IL-6, sgp-130, CXCL12/SDF-1α, CCL-5/RANTES, BDNF (brain-derived neurotrophic factor), hs-CRP, cortisol and DHEA (dehydroepiandrosterone). Psychosocial measures examined cognitive functions, depression, anxiety, psychological well-being, social connectedness and satisfaction with life. A significant reduction in plasma IL-6 level (p = 0.02) was observed in the HT intervention group. For the waitlist control group, significant reductions in plasma CXCL12 (SDF-1α) (p = 0.003), CXCL5 (RANTES) (p = 0.05) and BDNF (p = 0.003) were observed. A significant improvement in social connectedness was also observed in the HT group (p = 0.01). Conclusion: HT, in reducing plasma IL-6, may prevent inflammatory disorders and through maintaining plasma CXCL12 (SDF-1α), may maintain hematopoietic support to the brain. HT may be applied in communal gardening to enhance the well-being of older adults.

Plasma levels of the proangiogenic protein CXCL16 remains elevated for 1\xa0month after minimally invasive colorectal cancer resection

BackgroundInflammation-induced endothelial precursor cell recruitment and angiogenesis are thought to be associated with CXCL16-CXCR6 pair activity. This study’s main purpose was to determine plasma CXCL16 levels after minimally invasive colorectal resection (MICR) for colorectal cancer (CRC); an adjunct study assessed wound fluid (WF) and plasma CXCL16 levels in a separate group of CRC patients.MethodsCRC patients who had MICR and for whom plasma was available in a tissue bank were eligible. Plasma samples were collected preoperatively from all patients. Samples were also collected on postoperative days (POD) 1 and 3 and at various late postoperative time points (POD 7–34). In a separate study, blood and intra-abdominal wound fluid (WF) samples were collected from CRC MICR patients (pts). Samples were stored at − 80 °C. CXCL16 levels were determined via ELISA. The Wilcoxon signed-rank and Mann and Whitney tests were used for analysis.ResultsMain study: 86 CRC pts. were included. The mean preoperative plasma CXCL16 level was 2.36 ± 0.57 ng/ml. Elevated mean plasma levels (p < 0.0001 × first 4 time points) were noted on POD 1 (2.82 ± 0.81, n = 86), POD 3 (3.12 ± 0.77, n = 82), POD 7–13 (3.28 ± 0.88, n = 64), POD 14–20 (3.03 ± 0.62, n = 24), POD 21–27 (3.06 ± 0.67, n = 20, p = 0.0003), and POD 28–34 (3.17 ± 0.43, n = 11, p = 0.001) vs. preop levels. WF study: In the adjunct study, plasma and WF CXCL16 levels were determined for 23 CRC MICR pts. WF levels at all time points were significantly elevated over plasma levels.ConclusionPlasma CXCL16 levels were elevated for 4 weeks after minimally invasive colorectal resection for cancer. Also, WF CXCL16 levels were 3–10 times greater than the corresponding plasma concentrations. The source of the late plasma elevations may be the healing wound. Increased plasma CXCL16 levels may promote tumor angiogenesis in the first month after MICR.

Circulating inflammatory monocytes contribute to impaired influenza vaccine responses in HIV-infected participants.

Antibody responses are often impaired in old age and in HIV-positive (HIV+) infection despite virologic control with antiretroviral therapy but innate immunologic determinants are not well understood. Monocytes and natural killer cells were examined for relationships to age, HIV infection and influenza vaccine responses. Virologically suppressed HIV+ (n = 139) and HIV-negative (HIV-) (n = 137) participants classified by age as young (18-39 years), middle-aged (40-59 years) and old (≥60 years) were evaluated preinfluenza and postinfluenza vaccination. Prevaccination frequencies of inflammatory monocytes were highest in old HIV+ and HIV-, with old HIV+ exhibiting higher frequency of integrin CD11b on inflammatory monocytes that was correlated with age, expression of C-C chemokine receptor-2 (CCR2) and plasma soluble tumor necrosis factor receptor-1 (sTNFR1), with inverse correlation with postvaccination influenza H1N1 antibody titers. Higher frequencies of CD11b+ inflammatory monocytes (CD11b(hi), >48.4%) compared with low frequencies of CD11b+ inflammatory monocytes (<15.8%) was associated with higher prevaccination frequencies of total and inflammatory monocytes and higher CCR2 MFI, higher plasma sTNFR1 and CXCL-10 with higher lipopolysaccharide stimulated expression of TNFα and IL-6, concomitant with lower postvaccination influenza antibody titers. In HIV+ CD11b(hi) expressers, the depletion of inflammatory monocytes from peripheral blood mononuclear cells resulted in enhanced antigen-specific CD4+ T-cell proliferation. Immature CD56(hi) natural killer cells were lower in young HIV+ compared with young HIV- participants. Perturbations of innate immunity and inflammation signified by high CD11b on inflammatory monocytes are exacerbated with aging in HIV+ and negatively impact immune function involved in Ab response to influenza vaccination.

Abstract P5-11-03: Tumor CXCL16/CXCR6 expression and soluble CXCL16 in HER2+ breast cancer (BC)

Abstract Background: CXCL16 is a pro-inflammatory chemokine associated with chemotaxis of CXCR6-expressing lymphocytes (T cells, NKT cells, NK cells) and the promotion of breast cancer cell proliferation, migration, and invasion in vitro. CXCL16 exists in a transmembrane or a cleaved, soluble form. There is limited clinically relevant data available on the CXCL16/CXCR6 signaling axis in HER2+ BC. This preliminary study examines tumor CXCL16 and CXCR6 mRNA expression and patient outcome in publicly available datasets and examines soluble CXCL16 in the plasma of 32 HER2+ BC patients enrolled in ICORG 10-05 (neo-adjuvant chemotherapy (docetaxel/carboplatin) +/- trastuzumab, lapatinib or trastuzumab/lapatinib). Methods: CXCL16 and CXCR6 mRNA expression was interrogated in publicly available datasets using BreastMark, a web-based tool for preliminary assessment of putative biomarkers in breast cancer.A median cut-off was used for all analyses. Pre-treatment and post-treatment (2 weeks pre-surgery) blood samples were collected from patients enrolled in ICORG 10-05. Plasma CXCL16 levels were determined by Luminex xMAP assay. Pre- and post-treatment levels of CXCL16 were compared directly or based on response (pathological complete response, CR, n=14 or non-pathological complete response, nCR, n=18). Stromal lymphocyte (SL) and tumor-infiltrating lymphocyte (TIL) levels were determined from Haematoxylin and Eosin-, AE1/AE3- and CD45FFPE- stained formalin-fixed paraffin embedded tissue. Pre-treatment lymphocyte levels were correlated with pre- and post-treatment levels of CXCL16 (Pearson's product-moment correlation). Results: In BC as a whole, analysis of publicly available datasets reveals tumor CXCL16 expression is not associated with outcome (n=1238, HR=0.9953, p=0.9516) but high tumor CXCR6 expression is (n=2652, HR=1.127, p=0.0476). Within a HER2+ cohort, inverse correlative analysis suggests high CXCR6/low CXCL16 tumor expression is significantly associated with a worse outcome (n=61, HR=2.731, p=0.01). For ICORG 10-05 patients, circulating CXCL16 plasma levels were significantly higher post-treatment (p&amp;lt;0.0001). The magnitude of this increase was significantly greater in CR than nCR patients (p&amp;lt;0.009). Post-treatment circulating CXCL16 levels negatively correlate with pre-treatment total (SL+TIL) lymphocyte counts (correlation coefficient -0.50 (CI -0.75- -0.13), p=0.01) in ICORG 10-05 patients. SL and total lymphocyte (SL+TIL) counts were higher in CR (n=13) vs. nCR (n=13) patients but the difference was not significant (SL, p=0.22; SL+TIL, p=0.29). Conclusions: Our preliminary results suggest tumor levels of CXCL16/CXCR6 are associated with patient outcome and circulating levels of soluble CXCL16 are altered by treatment and correlate with tumor immune infiltrate in HER2+ BC patients. Further examination of tumor CXCL16/CXCR6 expression and circulating CXCL16 as potential biomarkers of response is warranted in a larger cohort of HER2+ BC patients. Citation Format: Collins DM, Madden SF, Eustace AJ, Toomey S, Kay EW, Fay J, O'Donovan N, Gallagher WM, Hennessy B, Crown J. Tumor CXCL16/CXCR6 expression and soluble CXCL16 in HER2+ breast cancer (BC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-11-03.

Plasma CXCL1 levels and TRAF3IP2 variants in patients with myocardial infarction.

IL-17A plays an important role in inflammatory responses in myocardial infarction (MI). IL-17A signals through its receptor, for which, Act1 (TRAF3IP2) functions as a key upstream adaptor in the pathway. To compare frequencies of functional polymorphisms of TRAF3IP2 (rs13210247, rs33980500) between patients with MI and healthy controls. The selected SNPs were studied in 201 Iranian MI patients and 201 healthy blood donors from Fars Province by PCR-RFLP in association with clinicopathologic criteria of patients. CXCL1 plasma levels in 126 MI patients and 50 normal subjects were measured by ELISA. A significant increase in the mutant (T) allele of TRAF3IP2 rs33980500 in patients with diastolic dysfunction of the heart (P=.01) was observed. The highest correlation, however, was observed between the TRAF3IP2 rs33980500 TT genotype and T allele with left main coronary artery stenosis (P=.01, P<.001; OR=31.03). T allele of TRAF3IP2 rs33980500 was also associated with female gender, family history of cardiovascular disease, and mechanical complications of heart (P=.04, P=.02, and P=.01, respectively). Moreover, TRAF3IP2 rs13210247 (G) correlated with mechanical complications of the heart (P=.01). A significant increase in the plasma levels of CXCL1 chemokine in patients (P=.0006) associated with TT genotype of TRAF3IP2 (rs33980500) was observed (P=.04). The gene variants of Act1 adaptor are associated with correlates of poor outcome in patients with MI and plasma CXCL1 levels.

Multipl Sklerozda CXCL12'nin (SDF-1 alfa) Prediktif Rolü

Objective: Multiple sclerosis (MS) is an inflammatory, autoimmune, demyelinating disease characterized by multifocal inflammation, progressive myelin loss and consequent axonal demyelination. Pathogenic mechanisms of MS are oligodendrocyte damage as a result of leukocyte chemotaxis to central nervous system and production of inflammatory mediators, demyelination and neuronal injury. CXCL12 (SDF-1 alpha) is a basic chemokine which is chemoattractant for resting and activated T cells. We aimed to evaluate whether CXCL12 levels may be a predictive marker of neuroinflammation in MS patients. Material and Methods: We designed a prospective case control study of fifty-four MS patients and twenty-eight healthy controls were enrolled to the study. Plasma CXCL12 levels of the groups were measured in blood samples. The groups were compared in terms of plasma CXCL12 levels. Lymphocyte count, which is a marker of inflammation, was also compared. In the study group subsequent analysis was demonstrated the association between clinical parameters and plasma CXCL12 levels. Results: CXCL12 levels were statistically higher than the control group. Mean CXCL12 level of MS patients was 2026,5±398,7 pg/ml and the mean CXCL12 level of the controls was 1840,6±256,0 pg/ml. Lymphocyte count of the patients was also statistically higher in the study group. According to the receiver operator characteristic (ROC) curve analysis mean CXCL12 levels were discriminative factors in patients in the study group. There was no statistically significant association between plasma CXCL12 levels and clinical parameters. Conclusion: We conclude that elevations in CXCL12 levels might be a promising marker pointing out the pathogenic role of inflammation in multiple sclerosis.

High CXCL10/IP-10 levels are a hallmark in the clinical evolution of the HIV infection

The aim of this study was to investigate the modulation of plasma CXCL10, CCL20, CCL22, CCL2, CCL17 and CCL24 levels in HIV-positive patients grouped according to extreme phenotypes of progression to AIDS, and at different stages of HIV infection. HIV-positive individuals with extreme phenotypes of AIDS progression (n=58) at different clinical stages (chronic individuals, both pre-HAART and under-HAART) and HIV-negative controls (n=20) were evaluated. Additionally, HIV-positive individuals that initiated HAART with >350CD4+T-cells/mm3 were compared with those who initiated treatment with <350CD4+T-cells/mm3. Plasma levels of six chemokines were quantified by a Luminex assay. Higher CXCL10 levels were observed in individuals immediately before their CD4+T-cell levels were indicative for HAART (pre-HAART), independently of their progressor status, i.e. slow (SPs) or rapid progressors (RPs). SPs pre-HAART showed higher CXCL10 levels compared to elite controllers and RPs under HAART (pc=0.009 and pc=0.007, respectively). CXCL10 levels were higher in SPs HAART CD4<350 (initiated HAART with <350 CD4+T-cells) when compared with SPs HAART CD4>350 (initiated HAART with >350 CD4+T-cells) (1096 vs. 360.33pg/mL, p=0.0101). Normalisation of CXCL10 levels seems to depend on the CD4+T-cell nadir at HAART initiation. CCL20 levels were higher in chronic SPs, SPs pre-HAART, SPs HAART and RPs HAART compared with the HIV-negative controls, indicating persistent CCL20 expression. In conclusion, our results indicate that CXCL10 levels are a hallmark in the clinical evolution of HIV infection. However, our results must be verified in a study evaluating a larger number of AIDS progressors.

Plasma CXCL16 Level is Associated With Cardiovascular Disease in Iranian Hemodialysis Patients

Plasma CXCL16 Level is Associated With Cardiovascular Disease in Iranian Hemodialysis Patients

Inflammatory chemokines and their receptors in human visceral leishmaniasis: Gene expression profile in peripheral blood, splenic cellular sources and their impact on trafficking of inflammatory cells.

Chemokines play an important role in determining cellular composition at inflammatory sites, and as such, influence disease outcome. In this study, we investigated the expression profile and splenic cellular source of various inflammatory chemokines and their receptors in human visceral leishmaniasis (VL). The expression of chemokines or their receptors was measured at the gene and protein level by employing real time qPCR and a cytometric bead array assay, respectively. In addition, the cellular source of chemokines and their receptors in the spleen was identified employing gene expression analyses in sequentially selected cell subsets. We identified elevated expression of CXCL10, CXCL9, CXCL8, and decreased CCL2 from VL patients. Further, we found reduced expression of the chemokine receptors CXCR1, CXCR2, CXCR3 and CCR2, but increased expression of CCR7 on VL PBMC, compared to endemic healthy controls. Additionally, splenic monocytes were found to be the major source of CXCL10, CXCL9 and CCR2, whereas T cells were the main source of CXCR3 and CCR7. We also report a strong association between plasma IFN-γ and CXCL-10, CXCL-9 levels. Enhanced parasite burden positively correlates with increased expression of CXCL10, CXCL9, IFN-γ and IL-10. Overall our result indicates that VL patients have an elevated inflammatory chemokine milieu which correlated with disease severity. However, expression of their chemokine receptors was significantly impaired, which may have contributed to reduced frequencies of blood monocytes and neutrophils in peripheral blood. In contrast, enhanced expression of CCR7 was associated with increased numbers of activated T cells in circulation. These findings highlight the importance of chemokines for recruitment of various cell populations in VL, and the knowledge gained may help in global understandings of the complex interaction between chemokines and pathological processes, and therefore will contribute towards the design of novel chemokine based immunological therapies against VL.

CXCL10 GENE PROMOTER POLYMORPHISM – 1447A>G IS ASSOCIATED WITH MALARIA IN GHANAIAN CHILDREN

BackgroundRecent studies indicate that interferon gamma inducible chemokine, CXCL10, is a strong predictor of both human and experimental cerebral malaria. We hypothesised malaria infection is associated with variation in CXCL10...

Herpes Simplex Virus Suppressive Therapy in Herpes Simplex Virus-2/Human Immunodeficiency Virus-1 Coinfected Women Is Associated With Reduced Systemic CXCL10 But Not Genital Cytokines.

Herpes simplex virus type-2 (HSV-2) may heighten immune activation and increase human immunodeficiency virus 1 (HIV-1) replication, resulting in greater infectivity and faster HIV-1 disease progression. An 18-week randomized, placebo-controlled crossover trial of 500 mg valacyclovir twice daily in 20 antiretroviral-naive women coinfected with HSV-2 and HIV-1 was conducted and HSV-2 suppression was found to significantly reduce both HSV-2 and HIV-1 viral loads both systemically and the endocervical compartment. To determine the effect of HSV-2 suppression on systemic and genital mucosal inflammation, plasma specimens, and endocervical swabs were collected weekly from volunteers in the trial and cryopreserved. Plasma was assessed for concentrations of 31 cytokines and chemokines; endocervical fluid was eluted from swabs and assayed for 14 cytokines and chemokines. Valacyclovir significantly reduced plasma CXCL10 but did not significantly alter other cytokine concentrations in either compartment. These data suggest genital tract inflammation in women persists despite HSV-2 suppression, supporting the lack of effect on transmission seen in large scale efficacy trials. Alternative therapies are needed to reduce persistent mucosal inflammation that may enhance transmission of HSV-2 and HIV-1.

Intracoronary nitrite suppresses the inflammatory response following primary percutaneous coronary intervention

ObjectiveRecent work suggests that intracoronary nitrite reduces myocardial infarct size following primary percutaneous coronary intervention (PPCI) for acute myocardial infarction (AMI), although the exact mechanisms are unclear. We explored the...

Viral and Host Responses After Stopping Long-term Nucleos(t)ide Analogue Therapy in HBeAg-Negative Chronic Hepatitis B.

This prospective study investigated viral and host markers after stopping long-term therapy with nucleos(t)ide analogues in noncirrhotic patients with hepatitis B e antigen-negative chronic hepatitis B. After stopping therapy, 13 of 15 patients experienced a virological relapse. Rebound of hepatitis B virus DNA and hepatitis B core-related antigen was associated with induction of plasma tumor necrosis factor, interleukin (IL) 10 , IL-12p70, CXCL10 and subsequent decline in hepatitis B surface antigen (HBsAg), with 20% HBsAg loss after long-term follow-up. The peak levels of hepatitis B virus DNA and hepatitis B core-related antigen after cessation of therapy were positively correlated with the level of HBsAg decline at week 48. Thus, stopping or interrupting NA treatment should be further investigated as a strategy to accelerate HBsAg loss.

Elevated Basal Pre-infection CXCL10 in Plasma and in the Small Intestine after Infection Are Associated with More Rapid HIV/SIV Disease Onset.

Elevated blood CXCL10/IP-10 levels during primary HIV-1 infection (PHI) were described as an independent marker of rapid disease onset, more robust than peak viremia or CD4 cell nadir. IP-10 enhances the recruitment of CXCR3+ cells, which include major HIV-target cells, raising the question if it promotes the establishment of viral reservoirs. We analyzed data from four cohorts of HIV+ patients, allowing us to study IP-10 levels before infection (Amsterdam cohort), as well as during controlled and uncontrolled viremia (ANRS cohorts). We also addressed IP-10 expression levels with regards to lymphoid tissues (LT) and blood viral reservoirs in patients and non-human primates. Pre-existing elevated IP-10 levels but not sCD63 associated with rapid CD4 T-cell loss upon HIV-1 infection. During PHI, IP-10 levels and to a lesser level IL-18 correlated with cell-associated HIV DNA, while 26 other inflammatory soluble markers did not. IP-10 levels tended to differ between HIV controllers with detectable and undetectable viremia. IP-10 was increased in SIV-exposed aviremic macaques with detectable SIV DNA in tissues. IP-10 mRNA was produced at higher levels in the small intestine than in colon or rectum. Jejunal IP-10+ cells corresponded to numerous small and round CD68neg cells as well as to macrophages. Blood IP-10 response negatively correlated with RORC (Th17 marker) gene expression in the small intestine. CXCR3 expression was higher on memory CD4+ T cells than any other immune cells. CD4 T cells from chronically infected animals expressed extremely high levels of intra-cellular CXCR3 suggesting internalization after ligand recognition. Elevated systemic IP-10 levels before infection associated with rapid disease progression. Systemic IP-10 during PHI correlated with HIV DNA. IP-10 production was regionalized in the intestine during early SIV infection and CD68+ and CD68neg haematopoietic cells in the small intestine appeared to be the major source of IP-10.

Plasma CXCL10, sCD163 and sCD14 Levels Have Distinct Associations with Antiretroviral Treatment and Cardiovascular Disease Risk Factors.

We investigate the associations of three established plasma biomarkers in the context of HIV and treatment-related variables including a comprehensive cardiovascular disease risk assessment, within a large ambulatory HIV cohort. Patients were recruited in 2010 to form the Royal Perth Hospital HIV/CVD risk cohort. Plasma sCD14, sCD163 and CXCL10 levels were measured in 475 consecutive patients with documented CVD risk (age, ethnicity, gender, smoking, blood pressure, BMI, fasting metabolic profile) and HIV treatment history including immunological/virological outcomes. The biomarkers assessed showed distinct associations with virological response: CXCL10 strongly correlated with HIV-1 RNA (p<0.001), sCD163 was significantly reduced among ‘aviraemic’ patients only (p = 0.02), while sCD14 was unaffected by virological status under 10,000 copies/mL (p>0.2). Associations between higher sCD163 and protease inhibitor therapy (p = 0.05) and lower sCD14 with integrase inhibitor therapy (p = 0.02) were observed. Levels of sCD163 were also associated with CVD risk factors (age, ethnicity, HDL, BMI), with a favourable influence of Framingham score <10% (p = 0.04). Soluble CD14 levels were higher among smokers (p = 0.002), with no effect of other CVD risk factors, except age (p = 0.045). Our findings confirm CXCL10, sCD163 and sCD14 have distinct associations with different aspects of HIV infection and treatment. Levels of CXCL10 correlated with routinely monitored variables, sCD163 levels reflect a deeper level of virological suppression and influence of CVD risk factors, while sCD14 levels were not associated with routinely monitored variables, with evidence of specific effects of smoking and integrase inhibitor therapy warranting further investigation.

Circulating T follicular helper cells with increased function during chronic graft-versus-host disease

AbstractChronic graft-versus-host disease (cGVHD) remains a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Previous studies have established that both donor B and T cells contribute to immune pathology in cGVHD but the mechanisms responsible for coordinated B- and T-cell responses directed against recipient antigens have not been understood. T follicular helper cells (TFH) play an important role in the regulation of B-cell immunity. We performed extensive phenotypic and functional analysis of circulating TFH (cTFH) and B cells in 66 patients after HSCT. Patients with active cGVHD had a significantly lower frequency of cTFH compared with patients without cGVHD. This was associated with higher CXCL13 plasma levels suggesting increased homing of TFH to secondary lymphoid organs. In patients with active cGVHD, cTFH phenotype was skewed toward a highly activated profile with predominance of T helper 2 (Th2)/Th17 subsets. Activated cTFH in patients with cGVHD demonstrated increased functional ability to promote B-cell immunoglobulin secretion and maturation. Moreover, the activation signature of cTFH was highly correlated with increased B-cell activation and plasmablast maturation in patients after transplant. These studies provide new insights into the immune pathogenesis of human cGVHD and identify TFH as a key coordinating element supporting B-cell involvement in this disease.

Bacterial Dose-Dependent Role of G Protein-Coupled Receptor Kinase 5 in Escherichia coli-Induced Pneumonia.

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose.

Crucial Factors of the Inflammatory Microenvironment (IL-1 beta/TNF-alpha/TIMP-1) Promote Maintenance of the Malignant Hemopoietic Clone of Myelofibrosis By Stimulating the in Vitro Survival/Proliferation/Migration of Circulating CD34+ stem/Progenitor Cells

Introduction. Myelofibrosis (MF), an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC) with a dysregulation in JAK/STAT signalling (mutations in JAK2, MPL and Calreticulin (CALR) genes), is characterized by a state of chronic inflammation. It is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Only recently, it has been hypothesized that the sustained inflammatory microenvironment of MF can alter crucial biological processes, leading to genomic instability and cancer progression. Here we tested the in vitro functional effects of pivotal players of the inflammatory microenvironment (the extracellular ATP nucleotide and selected cytokines, such as Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)) on the HSPCs from MF patients.Methods: Circulating CD34+/CD34+ CD38- cells from MF patients (JAK2V617F (17 cases) and CALR (9 cases) mutations) or cord blood (CB; 8 samples) were phenotypically and functionally characterized after in vitro incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), phenotype (evaluation of CD63 (TIMP-1 receptor), CXCR4 and CD38 expression), cell cycle and clonogenic capacity. Migration was assessed first towards a CXCL12 gradient in the presence or absence of the pro-inflammatory factors. In parallel experiments, CD34+ cells from MF patients were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence or absence of the pro-inflammatory cytokines and then evaluated for their ability to migrate towards a CXCL12 gradient. Plasma TIMP-1, TNF-α, IL-1β and CXCL12 were measured by ELISA assay.Results: The plasma levels of TIMP-1, TNF-α, IL-1β, CXCL12 and the number of circulating CD34+, CD34+ CD38-, CD34+ CD63+, CD34+ CD184+ cells were increased in MF patients. According to mutational status, the CD34+ CD63+ cells were higher in the CALR+ patients.The survival of MF CD34+ cells was strongly stimulated by in vitro incubation with TNF-α or IL-1β as compared with the CB-derived CD34+ cells or untreated cells. By multiple cytokine combinations, IL-1β/TIMP-1, IL-1β /ATP or IL-1β /TNF-α treatments significantly promote the survival of MF CD34+ cells as compared with the normal counterparts or the untreated cells. Various combinations with IL-1β were also effective in stimulating survival of CD34+CD38- cells.IL-1β/TIMP-1 and IL-1β/TNF-α/TIMP-1, but not factors alone, significantly increased the CFU-C growth of MF patients as compared with the CB-derived counterparts and the untreated cells. Moreover, comparing CALR+ vs JAK2V617F+ patients, the colony formation of JAK2V617F+ patients was mainly promoted by the IL-1β/TNF-α treatment.Along with clonogenic capacity stimulation, exposure of CD34+ cells from MF patients to IL-1β/TNF-α/TIMP-1 significantly increases the S-phase cells, suggesting that these pro-inflammatory factors stimulated cell-cycle progression in dormant CD34+ MF cells.Migration of CD34+ cells from MF was significantly increased in CXCL12 treated cells. In addition, exposure of MF CD34+ cells to IL-1β/TNF-α, IL-1β/TIMP-1 or IL-1β/TNF-α/TIMP-1 significantly promotes cell migration in comparison with the CB-derived counterparts or SDF-1 alone. MF migrated cells in the presence of IL-1β/TNF-α significantly upregulate CD63 expression. Intriguingly, colony formation of MF migrated CD34+ cells in the presence of IL-1β/TNF-α or IL-1β/TNF-α/TIMP-1 was potently increased. Finally, co-culture systems with normal MSCs in the presence of pro-inflammatory factors revealed that MF CD34+ cells display increased migration ability toward CXCL12 gradient.Conclusions: Altogether our findings suggest that in MF the inflammatory niche plays a key role in the maintenance of the malignant clone. Thus, the interplay between the pro-inflammatory cytokines promote and select the HSPCs with higher proliferative activity, clonogenic potential and migration capability. Targeting these microenvironmental interactions may be a clinically relevant approach.D.F. and D.S. equally contributed DisclosuresMartinelli:Pfizer: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy; AMGEN: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy.