Coexistent helminth infections are known to modulate T cell and cytokine responses in latent infection with Mycobacterium tuberculosis However, their role in modulating chemokine responses in latent tuberculosis (LTB) has not been explored. Because chemokines play a vital role in the protective immune responses in LTB, we postulated that coexistent helminth infection could modulate chemokine production in helminth-LTB coinfection. To test this, we measured the levels of a panel of CC and CXC chemokines at baseline and following mycobacterial Ag or mitogen stimulation in individuals with LTB with (Strongyloides stercoralis⁺LTB⁺) or without S. stercoralis (S. stercoralis^-LTB⁺) infection and in individuals without both infections, healthy controls (HC). At baseline (in the absence of a stimulus), S. stercoralis⁺LTB⁺ individuals exhibited significantly diminished production of CCL1, CCL2, CCL4, CCL11, CXCL9, CXCL10, and CXCL11 in comparison with S. stercoralis^-LTB⁺ and/or HC individuals. Upon mycobacterial Ag stimulation, S. stercoralis⁺LTB⁺ individuals exhibited significantly diminished production of CCL1, CCL2, CCL4, CCL11, CXCL2, CXCL9, and CXCL10 in comparison with S. stercoralis^-LTB⁺ and/or HC individuals. No differences were observed upon mitogen stimulation. Finally, after anthelmintic treatment, the baseline levels of CCL1, CCL2, CCL4, CCL11, and CXCL11 and mycobacterial Ag-stimulated levels of CCL1, CCL2, CCL11, CXCL2, and CXCL10 were significantly increased in S. stercoralis⁺LTB⁺ individuals. Thus, our data demonstrate that S. stercoralis⁺LTB⁺ individuals are associated with a compromised ability to express both CC and CXC chemokines and that this defect is at least partially reversible upon treatment. Hence, coexistent helminth infection induces downmodulation of chemokine responses in LTB individuals with likely potential effects on tuberculosis pathogenesis.

Breast cancer is commonest cancer in women worldwide. Elucidation of underlying biology and molecular pathways is necessary for improving therapeutic options and clinical outcomes. Molecular alterations in breast cancer are complex and involve cross-talk between multiple signaling pathways. The aim of this study is to extract a unique mRNA fingerprint of breast cancer in Lebanese women using microarray technologies. Gene-expression profiles of 94 fresh breast tissue samples (84 cancerous/10 non-tumor adjacent samples) were analyzed using GeneChip Human Genome U133 Plus 2.0 arrays. Quantitative real-time PCR was employed to validate candidate genes. Differentially expressed genes between breast cancer and non-tumor tissues were screened. Significant differences in gene expression were established for COL11A1/COL10A1/MMP1/COL6A6/DLK1/S100P/CXCL11/SOX11/LEP/ADIPOQ/OXTR/FOSL1/ACSBG1 and C21orf37. Pathways/diseases representing these genes were retrieved and linked using PANTHER®/Pathway Studio®. Many of the deregulated genes are associated with extracellular matrix, inflammation, angiogenesis, metastasis, differentiation, cell proliferation and tumorigenesis. Characteristics of breast cancers in Lebanese were compared to those of women from Western populations to explain why breast cancer is more aggressive and presents a decade earlier in Lebanese victims. Delineating molecular mechanisms of breast cancer in Lebanese women led to key genes which could serve as potential biomarkers and/or novel drug targets for breast cancer.

The mechanisms underlying the exaggerated inflammatory response in Behçet's syndrome (BS) remain poorly understood. We investigated the response of CD14(+) blood monocytes to interferon (IFN)-γ, focusing on the chemokine CXCL10. Chemokine synthesis and release were analysed at a protein and mRNA level following stimulation with IFN-γ. Findings in BS patients were compared with 25 healthy controls (HC), 15 rheumatoid arthritis (RA) and 15 systemic lupus erythematosus (SLE) disease control patients. BS monocytes produced significantly more CXCL10 protein than HC monocytes from 2 h following IFN-γ stimulation, despite equivalent quantities of mRNA, suggesting more efficient translation. This was significantly more pronounced in BS with high disease activity and in those with ocular and neurological clinical manifestations. The imbalance between CXCL10 protein and mRNA expression was not observed in either RA or SLE patients, and was not seen with other chemokines studied (CXCL9, CXCL11 and CCL2). Furthermore, BS monocytes treated with an alternative stimulant (LPS) did not show abnormal tumour necrosis factor (TNF)-α release. Sucrose density gradients to segregate monocyte CXCL10 mRNA into free RNA or polysome-associated RNA showed equal proportions in BS and HC samples, suggesting that the difference between BS and HC may be due to reduced negative control of CXCL10 translation in BS at a post-initiation level. We conclude that BS monocytes have dysfunctional post-transcriptional regulation of CXCL10 mRNA, resulting in over-expression of CXCL10 protein upon IFN-γ stimulation. As CXCL10 is a chemokine that recruits mononuclear cells, this abnormality may contribute to the exaggerated inflammatory responses that characterizes BS.

OBJECTIVE

Increased interferon gamma (IFN-γ) expression in dry eye causes ocular surface epithelial disease termed keratoconjunctivitis sicca (KCS). The purpose of this study was to investigated the effects of the LFA-1 antagonist, lifitegrast, in a mouse desiccating stress (DS) dry eye model that develops KCS similar to Sjögren syndrome.

METHODS

Mice were treated with vehicle or lifitegrast twice daily for 5 days and expression of Th1 family genes (IFN-γ, CXCL9, and CXCL11) was evaluated by real-time polymerase chain reaction. Cornea barrier function was assessed by Oregon Green dextran staining and goblet cell number and area were measured.

RESULTS

Compared to the vehicle-treated group, the lifitegrast-treated group had significantly lower expression of Th1 family genes, less corneal barrier disruption, and greater conjunctival goblet cell density/area.

CONCLUSIONS

These findings indicate that lifitegrast inhibits DS-induced IFN-γ expression and KCS. This suggests that ICAM-LFA-1 signaling is involved with generation of Th1 inflammation in KCS.

BACKGROUND

The role of monokine induced by interferon-γ (IFN-γ, MIG/CXCL9), IFN-γ-inducible protein (IP-10/CXCL10), and IFN-inducible T cell α chemoattractant (I-TAC/CXCL11) in allergic inflammation has not been explored in detail in vivo. The aim of the study was to examine the changes in concentrations of MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in nasal lavages collected from healthy and allergic subjects during nasal allergen challenge.

METHODS

Subjects allergic to grass pollen and healthy controls were included. Nasal allergen challenge preceded by placebo administration was performed outside the pollen season. Nasal lavages were collected before and 30 min after application of the placebo and 30 min after allergen administration. Concentrations of chemokines were determined using ELISA.

RESULTS

We observed significantly higher concentrations of IP-10/CXCL10 in allergic patients compared to the healthy subjects before (354.49 ±329.24 vs. 164.62 ±175.94 pg/ml; p = 0.036), 30 min after placebo (420.3 ±421.28 vs. 246.88 ±353.24 pg/ml; p = 0.021) and 30 min after allergen administration (403.28 ±359.29 vs. 162.68 ±148.69 pg/ml; p = 0.025). IP-10/CXCL10 levels did not change 30 min after allergen provocation. In contrast, MIG/CXCL9 levels were similar in both groups before and after placebo. However, a significant rise in MIG/CXCL9 concentration was noted in allergic patients 30 min after the allergen (138.88 ±109.59 vs. 395.8 ±301.2 pg/ml; p = 0.00026). I-TAC/CXCL11 concentrations increased after placebo as well as the allergen in both groups.

CONCLUSIONS

IP-10/CXCL10 concentrations are elevated in nasal lavages from allergic patients and this chemokine may play a role in chronic allergic inflammation. MIG/CXCL9 levels increase rapidly after allergen application, which may suggest its role in the early allergic response. Results on I-TAC/CXCL11 concentrations remain inconclusive.

Background: Mood disorders (major depressive disorder, MDD, and bipolar disorder, BD) are considered leading causes of life-long disability worldwide, where high rates of no response to treatment or relapse and delays in receiving a proper diagnosis (~60% of depressed BD patients are initially misdiagnosed as MDD) contribute to a growing personal and socio-economic burden. The immune system may represent a new target to develop novel diagnostic and therapeutic procedures but reliable biomarkers still need to be found.

Methods: In our study we predicted the differential diagnosis of mood disorders by considering the plasma levels of 54 cytokines, chemokines and growth factors of 81 BD and 127 MDD depressed patients. Clinical diagnoses were predicted also against 32 healthy controls. Elastic net models, including 5000 non-parametric bootstrapping procedure and inner and outer 10-fold nested cross-validation were performed in order to identify the signatures for the disorders.

Results: Results showed that the immune-inflammatory signature classifies the two disorders with a high accuracy (AUC = 97%), specifically 92% and 86% respectively for MDD and BD. MDD diagnosis was predicted by high levels of markers related to both pro-inflammatory (i.e. IL-1β, IL-6, IL-7, IL-16) and regulatory responses (IL-2, IL-4, and IL-10), whereas BD by high levels of inflammatory markers (CCL3, CCL4, CCL5, CCL11, CCL25, CCL27, CXCL11, IL-9 and TNF-α).

Conclusions: Our findings provide novel tools for early diagnosis of BD, strengthening the impact of biomarkers research into clinical practice, and new insights for the development of innovative therapeutic strategies for depressive disorders.

Keywords: Bipolar disorder; Cytokines; Depression; Inflammation; Machine learning; Mood disorders.

Industrial-scale expansion of mesenchymal stromal cells (MSCs) is often used in clinical trials, and the effect of replicative senescence on MSC functionality is of mechanistic interest. Senescent MSCs exhibit cell-cycle arrest, cellular hypertrophy, and express the senescent marker β-galactosidase. Although both fit and senescent MSCs display intact lung-homing properties in vivo, senescent MSCs acquire a significant defect in inhibiting T-cell proliferation and cytokine secretion in vitro. IFNγ does not upregulate HLA-DR on senescent MSCs, whereas its silencing did not reverse fit MSCs' immunosuppressive properties. Secretome analysis of MSC and activated peripheral blood mononuclear cell coculture demonstrate that senescent MSCs are significantly defective in up (vascular endothelial growth factor [VEGF], granulocyte colony-stimulating factor [GCSF], CXCL10, CCL2) or down (IL-1ra, IFNγ, IL-2r, CCL4, tumor necrosis factor-α, IL-5) regulating cytokines/chemokines. Unlike indoleamine 2,3 dioxygenase (IDO), silencing of CXCL9, CXCL10, CXCL11, GCSF, CCL2, and exogenous addition of VEGF, fibroblast growth factor-basic do not modulate MSCs' immunosuppressive properties. Kynurenine levels were downregulated in senescent MSC cocultures compared with fit MSC counterparts, and exogenous addition of kynurenine inhibits T-cell proliferation in the presence of senescent MSCs. IFNγ prelicensing activated several immunomodulatory genes including IDO in fit and senescent MSCs at comparable levels and significantly enhanced senescent MSCs' immunosuppressive effect on T-cell proliferation. Our results define immune functional defects acquired by senescent MSCs, which are reversible by IFNγ prelicensing.

The successful generation of T cell-mediated immunity for the treatment of cancer has been a major focal point of research. One of the critical strategies of cancer immunotherapy is to efficiently activate antigen-specific CD8 T cells in the immunosuppressive tumor environment. Here, we used transgenic OT-I/CD45.2/Rag^-/- mice as a source of effector CD8 T cells to determine whether irradiation combined with adoptive T cell transfer therapy could improve T cell proliferation and effector function in murine tumor models. Local irradiation combined with adoptive T cell therapy showed a synergistic effect on tumor growth inhibition in mice. Mechanistically, irradiation increased the release of tumor-associated antigens, which facilitated cross-presentation of tumor-associated antigens by dendritic cells and the priming of antigen-specific T lymphocytes. Additionally, irradiation enhanced the homing of the antigen-specific T cells to tumor tissues via the increased release of CCL5, CXCL9, and CXCL11 from tumor cells. Moreover, irradiation enhanced the proliferation and effector function of both adoptively transferred T cells and endogenous antigen-specific T cells. Our findings provide evidence to support that local irradiation enhanced the therapeutic efficacy of adoptive T cell therapy for cancer, indicating that the combination of radiotherapy and adoptive T cell therapy may be a promising strategy for tumor treatment.

Sepsis is one of the most prevalent diseases in the world. The development of cardiac dysfunction in sepsis results in an increase of mortality. It is known that Bruton's tyrosine kinase (BTK) plays a role in toll-like receptor signaling and NLRP3 inflammasome activation, two key components in the pathophysiology of sepsis and sepsis-associated cardiac dysfunction. In this study we investigated whether pharmacological inhibition of BTK (ibrutinib 30 mg/kg and acalabrutinib 3 mg/kg) attenuates sepsis associated cardiac dysfunction in mice. 10-week old male C57BL/6 mice underwent CLP or sham surgery. One hour after surgery mice received either vehicle (5% DMSO + 30% cyclodextrin i.v.), ibrutinib (30 mg/kg i.v.), or acalabrutinib (3 mg/kg i.v.). Mice also received antibiotics and an analgesic at 6 and 18 h. After 24 h, cardiac function was assessed by echocardiography in vivo. Cardiac tissue underwent western blot analysis to determine the activation of BTK, NLRP3 inflammasome and NF-κB pathway. Serum analysis of 33 cytokines was conducted by a multiplex assay. When compared to sham-operated animals, mice subjected to CLP demonstrated a significant reduction in ejection fraction (EF), fractional shortening (FS), and fractional area change (FAC). The cardiac tissue from CLP mice showed significant increases of BTK, NF-κB, and NLRP3 inflammasome activation. CLP animals resulted in a significant increase of serum cytokines and chemokines (TNF-α, IL-6, IFN-γ, KC, eotaxin-1, eotaxin-2, IL-10, IL-4, CXCL10, and CXCL11). Delayed administration of ibrutinib and acalabrutinib attenuated the decline of EF, FS, and FAC caused by CLP and also reduced the activation of BTK, NF-κB, and NLRP3 inflammasome. Both ibrutinib and acalabrutinib significantly suppressed the release of cytokines and chemokines. Our study revealed that delayed intravenous administration of ibrutinib or acalabrutinib attenuated the cardiac dysfunction associated with sepsis by inhibiting BTK, reducing NF-κB activation and the activation of the inflammasome. Cytokines associated with sepsis were significantly reduced by both BTK inhibitors. Acalabrutinib is found to be more potent than ibrutinib and could potentially prove to be a novel therapeutic in sepsis. Thus, the FDA-approved BTK inhibitors ibrutinib and acalabrutinib may be repurposed for the use in sepsis.

Recruitment of myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment (TME) contributes to cancer immune evasion. MDSCs express the chemokine receptor CXCR2, and inhibiting CXCR2 suppresses the recruitment of MDSCs into the tumor and the pre-metastatic niche. Here we compared the growth and metastasis of melanoma and breast cancer xenografts in mice exhibiting or not exhibiting targeted deletion of Cxcr2 in myeloid cells (CXCR2myeΔ/Δ vs. CXCR2 myeWT). Detailed analysis of leukocyte populations in peripheral blood and in tumors from CXCR2myeΔ/Δ mice revealed that loss of CXCR2 signaling in myeloid cells resulted in reduced intratumoral MDSCs and increased intratumoral CXCL11. The increase in intratumoral CXCL11 was derived in part from tumor-infiltrating B1b cells. The reduction in intratumoral MDSCs coupled with an increase in intratumoral B1b cells expressing CXCL11 resulted in enhanced infiltration and activation of effector CD8+ T cells in the TME of CXCR2myeΔ/Δ mice, accompanied by inhibition of tumor growth in CXCR2myeΔ/Δ mice compared to CXCR2myeWT littermates. Treatment of tumor-bearing mice with a CXCR2 antagonist (SX-682) also inhibited tumor growth, reduced intratumoral MDSCs, and increased intratumoral B1b cells expressing CXCL11, leading to an increase in activated CD8+ T cells in the tumor. Depletion of B220+ cells or depletion of CD8+ T cells reversed the tumor inhibitory properties in CXCR2myeΔ/Δ mice. These data revealed a mechanism by which loss of CXCR2 signaling in myeloid cells modulates antitumor immunity through decreasing MDSCs and enriching CXCL11-producing B1b cells in the TME, which in turn increases CD8+ T-cell recruitment and activation in tumors.

BACKGROUND

Nur77, an orphan member of the nuclear receptor superfamily, has been implicated in the regulation of inflammation. However, the in vivo function of Nur77 remains largely unexplored. In the current study, we investigated the role of Nur77 in inflammation and immunity in mice.

RESULTS

We found that elderly 8-month-old Nur77-deficient mice (Nur77(-/-)) developed systemic inflammation. Compared to wild-type (WT) mice (Nur77(+/+)), Nur77(-/-) mice showed splenomegaly, severe infiltration of inflammatory cells in several organs including liver, lung, spleen and kidney, increased hyperplasia of fibrous tissue in the lung and enlargement of kidney glomeruli. Additionally, Nur77(-/-) mice had increased production of pro-inflammatory cytokines and immunoglobulin, and elicited pro-inflammatory M1-like polarization in macrophages as revealed by increased expression of CXCL11 and INDO, and decreased expression of MRC1.

CONCLUSIONS

These in vivo observations provide evidence for a pivotal role for Nur77 in the regulation of systemic inflammation and emphasize the pathogenic significance of Nur77 in vivo.

BACKGROUND

Lymphocyte migration from the blood into the CNS is mediated by chemokines and chemokine receptors. Chemokines CXCL10 and CXCL11 are important for the recruitment of CXCR3-expressing Th1 lymphocytes to the site of inflammation.

OBJECTIVE

To determine the concentrations of CXCL10 and CXCL11 in the CSF and plasma of children with enteroviral aseptic meningitis (EV AM) and controls and the contribution of these chemokines to the chemokine concentration gradient between the periphery and the CNS.

METHODS

The study included 26 pediatric patients with EV AM and 16 controls in whom CNS infection is excluded by negative CSF examination. Chemokines were quantified by using enzyme immunoassay. Etiological diagnosis of EV AM was based on the detection of enteroviral RNA in the CSF using real-time PCR.

RESULTS

CXCL10 (median 12 725 pg/ml) and CXCL11 (median 187 pg/ml) concentrations in CSF of patients with meningitis were significantly higher compared to plasma (median 173 pg/ml and median 110 pg/ml; p < 0.001, p = 0.026 respectively). CXCL10 concentrations in the CSF (median 198 pg/ml) and plasma of controls (median 124 pg/ml) were not significantly different (p = 0.642). CXCL11 concentrations in the CSF of controls (median 89 pg/ml) were significantly lower compared with plasma (median 139 pg/ml, p = 0.004). Chemokine concentration gradient was not influenced by pleocytosis, nor dependent on cytologic CSF formula or the presence of proteinorrachia.

CONCLUSIONS

CXCL10 and CXCL11 concentration gradient between the CSF and plasma in children with EV AM suggests an important role of these chemokines in the T-cells recruitment into the CNS and local immunoreaction.

TNFα has been identified as playing an important role in pathologic complications associated with diabetic retinopathy and retinal inflammation, such as retinal leukostasis. However, the transcriptional effects of TNFα on retinal microvascular endothelial cells and the different signaling pathways involved are not yet fully understood. In the present study, RNA-seq was used to profile the transcriptome of human retinal microvascular endothelial cells (HRMEC) treated for 4 hours with TNFα in the presence or absence of the NFAT-specific inhibitor INCA-6, in order to gain insight into the specific effects of TNFα on RMEC and identify any involvement of NFAT signaling. Differential expression analysis revealed that TNFα treatment significantly upregulated the expression of 579 genes when compared to vehicle-treated controls, and subsequent pathway analysis revealed a TNFα-induced enrichment of transcripts associated with cytokine-cytokine receptor interactions, cell adhesion molecules, and leukocyte transendothelial migration. Differential expression analysis comparing TNFα-treated cells to those co-treated with INCA-6 revealed 10 genes whose expression was significantly reduced by the NFAT inhibitor, including those encoding the proteins VCAM1 and CX3CL1 and cytokines CXCL10 and CXCL11. This study identifies the transcriptional effects of TNFα on HRMEC, highlighting its involvement in multiple pathways that contribute to retinal leukostasis, and identifying a previously unknown role for NFAT-signaling downstream of TNFα.

OBJECTIVE

To characterize the IFNbeta1a-regulated gene expression on leukocytes of Multiple Sclerosis (MS) patients using microarrays with whole human genome representation.

METHODS

Genes differentially expressed by interferon-beta were identified by a microarray in vitro study performed in leukocytes obtained from 5 MS relapsing-remitting patients.

RESULTS

Following the culture of peripheral blood mononuclear cells from MS relapsing-remitting patients for 24 hs with IFNbeta1a, the expression of 868 genes was modified: 545 increased (including CXCL11, CCL8, INDO, IFI27, CFB, CXCL10 and IFIT1) and 323 diminished (including RBP7, SEPT5, RNF8, ADORA2B and FOS).

CONCLUSIONS

Since many of them were previously recognized as involved in MS pathogenesis, the IFNbeta1a mechanism of action could imply a compensatory regulation of systems deregulated in MS.

OBJECTIVE

Atherosclerosis is a chronic disease that involves inflammation, in which cytokines, including interferon-gamma (IFNgamma), participate. Endothelial cells (ECs) exposed to IFNgamma increase the expression of CXC chemokines. ECs subjected to laminar flow (LF) are atheroprotective, despite an unclear mechanism. This study was conducted to analyze whether ECs under LF were protected from IFNgamma-induced responses.

METHODS

IFNgamma-treated human umbilical cord ECs were subjected to LF in a well-defined flow chamber system. IFNgamma-induced STAT1 activation and downstream target genes were examined.

RESULTS

ECs exposed to IFNgamma triggered STAT1 activation via the phosphorylation of Tyr701 and Ser727 in STAT1. ECs exposed to LF alone did not activate STAT1. LF exposure of IFNgamma-treated ECs significantly attenuated IFNgamma-induced Tyr701 phosphorylation in a shear-force- and time-dependent manner, whereas Ser727 phosphorylation was unaffected. Consistently, LF inhibited IFNgamma-induced STAT1 binding to DNA. ECs treated with IFNgamma induced the expression of three T-cell-specific CXC chemokines (CXCL9, CXCL10 and CXCL11) as well as CIITA, a transcriptional regulator of major histocompatibility complex class II (MHCII). Consistently, LF exposure of IFNgamma-treated ECs reduced the expression of CXC chemokines and CIITA.

CONCLUSIONS

LF attenuates IFNgamma-induced responses via the suppression of STAT1 activation. Inhibition by LF of the interferon-induced ECs' response may explain some aspects of LF's atheroprotective effects on the endothelium.

BACKGROUND

Basal cell carcinoma (BCC) is the most common malignancy in humans worldwide. Studies suggest that BCCs exhibit immunoprotection, similar to other keratinocyte carcinomas, although the mechanisms of defence have not been defined.

OBJECTIVE

To examine if indoleamine 2,3-dioxygenase (IDO), an immune privilege-associated enzyme, would be expressed in BCC, regulated in part by CXCR3.

METHODS

We analysed the expression and function of IDO in human BCC (hBCC) tissues using nonlesional skin epithelial (NL) tissues as a control.

RESULTS

Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) revealed significant upregulation of IDO1 and IDO2 (12·5- and 19·14-fold change, respectively) in nodular hBCCs as compared with NL tissues. Immunohistochemistry showed that IDO colocalized with keratin 17, a BCC keratinocyte marker, in hBCC tissues. Western blot identified a full-length IDO (42 kDa) product and a splice variant (∼30 kDa) in BCC tissues. Kynurenine assays and qPCR were conducted to determine IDO enzymatic activity in hBCCs in vitro with CXCL11 supplementation, which has previously been shown to be required for the tumour cell growth. Addition of CXCL11 upregulated IDO2 and increased l-kynurenine concentration in a dose-dependent manner in hBCCs while normal primary keratinocytes exhibited no response.

CONCLUSIONS

The expression of IDO at both mRNA and protein levels in hBCC tissues, the upregulation of IDO2 and the IDO-mediated l-kynurenine production in hBCCs with CXCL11 treatment suggest that functional IDO is synthesized by hBCC tumours and may be used as a method of immunoprotection during tumorigenesis. Also, IDO enzymatic activity may be modulated by CXCR3/CXCL11 signalling in BCCs.

Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.

Noninvasive methods to diagnose and differentiate acute cellular rejection from acute tubular necrosis or acute calcineurin inhibitor toxicity are still missing. Because T lymphocytes play a decisive role in early states of rejection, we investigated the suitability and feasibility of antibody-mediated contrast-enhanced ultrasound by using microbubbles targeted to CD3(+) , CD4(+) , or CD8(+) T cells in different models of renal disease. In an established rat renal transplantation model, CD3-mediated ultrasound allows the detection of acute rejection as early as on postoperative day 2. Ultrasound signal intensities increased with the severity of inflammation. Further, an early response to therapy could be monitored by using contrast-enhanced sonography. Notably, acute tubular necrosis occurring after ischemia-reperfusion injury as well as acute calcineurin inhibitor toxicity could easily be differentiated. Finally, the quantified ultrasound signal correlated significantly with the number of infiltrating T cells obtained by histology and with CD3 mRNA levels, as well as with chemokine CXCL9, CXCL11, and CCL19 mRNA but not with KIM-1 mRNA expression, thereby representing the severity of graft inflammation but not the degree of kidney injury. In summary, we demonstrate that antibody-mediated contrast-enhanced ultrasound targeting T lymphocytes could be a promising tool for an easy and reproducible assessment of acute rejection after renal transplantation.

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1β and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.

To investigate the possible involvement of interferon-inducible T cell alpha chemoattractant (I-TAC or CXCL11) in the salivary gland lesions of Sjögren's syndrome (SS), I-TAC mRNA expression was assayed in cultured salivary epithelial cells by reverse transcriptase-polymerase chain reaction (RT-PCR). This transcript was not expressed by SS salivary epithelial cells in the absence of IFNgamma, but was expressed after cells were stimulated with IFNgamma. We found that IFNgamma was the only cytokine that induced I-TAC mRNA expression. I-TAC proteins were shown by ELISA to be secreted into the culture supernatants of SS salivary epithelial cells following IFNgamma stimulation. Moreover, immunohistochemical assays of minor salivary glands (MSG) showed that I-TAC was predominantly located in the ductal epithelium adjacent to lymphoid infiltrates in the SS salivary gland, indicating that ductal epithelial cells produce I-TAC proteins in response to stimulation with IFNgamma secreted by lymphocytes that infiltrate into SS salivary glands. In contrast, I-TAC proteins were virtually absent from normal salivary glands. These findings suggest that I-TAC is involved in the development of salivary gland lesions observed in SS.

BACKGROUND

Quality of life (QoL) in patients with chronic disease is impaired and cannot be solely explained by disease severity. We explored whether genetic variability and activity contributes to QoL in patients with Marfan syndrome (MFS), a genetic connective tissue disorder.

RESULTS

In 121 MFS patients, patient characteristics (i.e. demographics and MFS-related symptoms) were assessed. Patients completed the SF-36 to measure QoL. In addition, transcriptome wide gene expression and 484 Single Nucleotide Polymorphysms (SNPs) in cytokine genes were available. QoL was first analyzed and associated with patient characteristics. Patients' physical QoL was impaired and weakly related with age and scoliosis, whereas mental quality of life (MCS) was normal. To explain a largely lacking correlation between disease severity and QoL, we related genome wide gene expression to QoL. Patients with lower MCS scores had high expression levels of CXCL9 and CXCL11 cytokine-related genes (p=0.001; p=0.002); similarly, patients with low vitality scores had high expression levels of CXCL9, CXCL11 and IFNA6 cytokine-related genes (p=0.02; p=0.02; p=0.04), independent of patient characteristics. Subsequently, we associated cytokine related SNPs to mental QoL (MCS and vitality). SNP-cluster in the IL4R gene showed a weak association with MCS and vitality (strongest association p=0.0017). Although overall mental QoL was normal, >10% of patients had low scores for MCS and vitality. Post-hoc analysis of systemic inflammatory mediators showed that patients with lowest MCS and vitality scores had high levels of CCL11 cytokine (p=0.03; p=0.04).

CONCLUSIONS

Variation in the cytokine genetic pathway and its activation is related to mental QoL. These findings might allow us to identify and, ultimately, treat patients susceptible to poor QoL.

OBJECTIVE

To study the involvement of the chemokine receptor CXCR3 and its ligands (CXCL9/Mig, CXCL10/IP-10, CXCL11/ITAC) in optic neuritis (ON).

METHODS

30 patients with ON and 10 non-inflammatory neurological disease controls were included. The patients underwent a phlebotomy, lumbar puncture, and MRI scan. CXCR3 expression was studied on blood and cerebrospinal fluid (CSF) T cells by flow cytometry. CXCL9, CXCL10, and CXCL11 were measured in plasma and CSF by ELISA.

RESULTS

CSF concentrations of CXCL10, but not of CXCL9 and CXCL11, were significantly higher in ON patients than in controls. CSF concentrations of CXCL10 correlated with the CSF leucocyte count in ON patients, and CXCR3 expressing cells were significantly enriched in the CSF.

CONCLUSIONS

These data show that the CSF concentration of the CXCR3 ligand CXCL10 is selectively increased in CSF from ON patients, and CXCR3 positive cells are recruited to the subarachnoid space.