The recruitment of leukocytes to infectious foci depends strongly on the local release of chemoattractant mediators. The human CXC chemokine receptor 3 (CXCR3) is an important node in the chemokine signaling network and is expressed by multiple leukocyte lineages, including T cells and macrophages. The ligands of this receptor originate from an ancestral CXCL11 gene in early vertebrates. Here, we used the optically accessible zebrafish embryo model to explore the function of the CXCR3-CXCL11 axis in macrophage recruitment and show that disruption of this axis increases the resistance to mycobacterial infection. In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis to bacterial infections was attenuated, although migration to infection-independent stimuli was unaffected. Additionally, attenuation of macrophage recruitment to infection could be mimicked by treatment with NBI74330, a high-affinity antagonist of CXCR3. We identified two infection-inducible CXCL11-like chemokines as the functional ligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependent chemoattraction when locally administrated in vivo. During infection of zebrafish embryos with Mycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2 deficiency limited the macrophage-mediated dissemination of mycobacteria. Furthermore, the loss of Cxcr3.2 function attenuated the formation of granulomatous lesions, the typical histopathological features of tuberculosis, and led to a reduction in the total bacterial burden. Prevention of mycobacterial dissemination by targeting the CXCR3 pathway, therefore, might represent a host-directed therapeutic strategy for treatment of tuberculosis. The demonstration of a conserved CXCR3-CXCL11 signaling axis in zebrafish extends the translational applicability of this model for studying diseases involving the innate immune system.

BACKGROUND

The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738.

RESULTS

In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment.

CONCLUSIONS

SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

The transcriptome has considerable potential for improving biopsy diagnoses. However, to realize this potential the relationship between the molecular phenotype of disease and histopathology must be established. We assessed 186 consecutive clinically indicated kidney transplant biopsies using microarrays, and built a classifier to distinguish rejection from nonrejection using predictive analysis of microarrays (PAM). Most genes selected by PAM were interferon-gamma-inducible or cytotoxic T-cell associated, for example, CXCL9, CXCL11, GBP1 and INDO. We then compared the PAM diagnoses to those from histopathology, which are based on the Banff diagnostic criteria. Disagreement occurred in approximately 20% of diagnoses, principally because of idiosyncratic limitations in the histopathology scoring system. The problematic diagnosis of 'borderline rejection' was resolved by PAM into two distinct classes, rejection and nonrejection. The diagnostic discrepancies between Banff and PAM in these cases were largely due to the Banff system's requirement for a tubulitis threshold in defining rejection. By examining the discrepancies between gene expression and histopathology, we provide external validation of the main features of the histopathology diagnostic criteria (the Banff consensus system), recommend improvements and outline a pathway for introducing molecular measurements.

We investigated roles for chemoattractants in dissemination of HIV-1 by examining the induction of T cell-active chemokines in HIV-1-infected human monocyte-derived macrophages and dendritic cells. Of the 12 chemokines analyzed, mRNAs for two, CXCL10 and CXCL11, ligands for the chemokine receptor CXCR3, were up-regulated in both cell types upon infection by HIV-1. Induction of these chemokine genes in infected cultures was dependent on both viral entry and reverse transcriptase activity, but not on the HIV-1 envelope glycoprotein. Conditioned medium from infected cells was chemotactic for freshly isolated human CD4+ T cells, and chemotaxis was abolished by pretreatment with an Ab against CXCR3. A lymph node from an HIV-1-infected individual expressed CXCL10 and CXCL11 mRNAs in the paracortex, including venules, as detected by in situ hybridization, whereas neither mRNA was detected after highly active antiretroviral therapy. Because CCR5 on CD4+ T cells is found predominantly on cells that also express CXCR3, these data implicate CXCL10 and CXCL11 in the recruitment of susceptible T cells to HIV-1-infected lymph nodes, macrophages, and dendritic cells. This recruitment might enhance the sequestration of T cells in infected lymphoid organs and the spread of infection between cells, contributing to the immunopathology of AIDS.

The vitamin D receptor (VDR) is a hormone nuclear receptor regulating bone and calcium homeostasis. Studies revealing the expression of VDR on immune cells point toward a role for VDR-dependent signaling pathways in immunity. Here we verified the ability of the natural VDR ligand, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) to interfere in inflammatory and T cell stimulatory capacity of macrophages, in particular within a chronic inflammatory disease features of experimental type 1 diabetes (T1D). We demonstrated that VDR is constitutively expressed in macrophages and both the levels of VDR and its downstream targets, are clearly induced by 1,25(OH)(2)D(3). In control mice, macrophage programming with 1,25(OH)(2)D(3) partially abrogated the activation-provoked expression of IL-12p40, TNFα and iNOS as well as the effector T cell-recruiting chemokines, CXCL9, CXCL10 and CXCL11. Targeting VDR signaling in macrophages counteracted their T-cell stimulatory ability despite essentially unaltered expression of antigen-presenting and costimulatory molecules. Furthermore, even in non-obese diabetic (NOD) mice, where macrophages/monocytes featured a heightened responsiveness toward danger signals and a superior T cell stimulatory capacity, 1,25(OH)(2)D(3) successfully curtailed these basic macrophage-mediated functions. Interestingly, the inhibitory action of the active compound was associated with an IL-10-dependent mechanism since 1,25(OH)(2)D(3)-treatment of IL-10-deficient macrophages failed to reproduce the characteristic repression on inflammatory mediators or T cell proliferation. Combined, these results highlight the possible therapeutic applicability of this natural immunomodulator, due to its ability to counteract macrophage inflammatory and T cell-activating pathways.

Adoptive transfer of CD4+CD25+ regulatory T cells has been shown to have therapeutic effects in experimental graft-vs-host disease (GVHD) models. Chemokines play an important role in the recruitment of alloreactive donor T cells into target organs during GVHD. In this study, we investigated the effectiveness of targeted delivery of CD4+CD25+ regulatory T cells via a transfected chemokine receptor on reduction of organ damage during acute GVHD. High levels of expression of Th1-associated chemokines (CXCL9, CXCL10 and CXCL11) and their receptor CXCR3 were observed in the liver, lung and intestine of GVHD-induced recipient mice. Recipient mice that had undergone transfer of CD4+CD25+Foxp3+ CXCR3-transfected T cells (CXCR3-Treg cells) showed significant amelioration of GVHD changes in the liver, lung and intestine in comparison with recipient mice that had received CD4+CD25+Foxp3+ T cells (Treg cells) or naturally occurring CD4+CD25+ regulatory T cells. This was due to more pronounced migration of CXCR3-Treg cells and their localization for a longer time in Th1-associated chemokine-expressing organs, resulting in stronger suppressive activity. We succeeded in preparing chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression upon accumulation in target organs. This method may provide a new therapeutic approach for organ damage in acute GVHD.

To explore the biological significance of gene expression in the pathogenesis of inflammatory myopathies, we performed microarray experiments followed by real-time PCR and immunohistochemistry on muscle biopsies obtained before and after therapy from patients with dermatomyositis (DM) who improved and patients with inclusion body myositis (sIBM) who did not improve after controlled trials with three monthly intravenous immunoglobulin (IVIg) infusions. The pretreatment biopsies showed high expression of immunoglobulin, adhesion molecules, chemokines and cytokine genes in both sIBM and DM (sIBM>> DM). In the repeated biopsies of DM patients who clinically improved, 2206 genes were downregulated more than 1.5-fold; in contrast, 1700 of the same genes remained unchanged in sIBM patients who did not improve. Genes markedly downregulated in DM, but not sIBM, were interleukin 22, Kallmann syndrome 1 (KAL-1), an adhesion molecule shown for the first time in muscle, ICAM-1, complement C1q, and several structural protein genes. Because mRNA for KAL-1 was selectively upregulated in vitro by transforming growth factor (TGF) beta1, a fibrogenic cytokine immunolocalized in the endomysial connective tissue of pretreatment DM muscles, the downregulation of both TGF-beta and KAL-1 after IVIg only in DM suggests that these molecules have a functional role in connective tissue proliferation and fibrosis. The improved muscles of DM, but not sIBM, showed upregulation of chemokines CXCL9 (Mig) and CXCL11, and several immunoglobulin-related genes, suggesting an effect on muscle remodelling and regeneration. The results suggest that IVIg modulates several immunoregulatory or structural muscle genes, but only a subset of them associated with inflammatory mediators, fibrosis and muscle remodelling are connected with the clinical response. Gene arrays, when combined with clinical assessments, may provide important information in the pathogenesis of inflammatory myopathies.

CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor 3 (CXCR3) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR3 or TLR4 ligands, respectively) synergized with interferon-gamma (IFN-gamma) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-gamma was combined with TLR3 ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-gamma-dependent CXCR3 ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), in combination with IFN-gamma, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG(1)Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-alpha plus IFN-gamma, but not synergy between IFN-gamma and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the physiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gα(i)-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.

The goal to attenuate inflammation without inducing generalized immunosuppression has focused the attention on chemokines, a family of chemotactic peptides that regulate the leukocyte traffick into tissues. However, the development of drugs that block ckemokine activity may be hampered by the observation that some chemokines display pleiotropic biologic functions. For example, the chemokines CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC exhibit the ability to recruit different leukocytes subsets, the capacity to induce the proliferation of vascular pericytes as well as powerful anti-tumor effects, which are mediated by a common receptor, named CXCR3. Because of their pleiotropic biologic effects, these chemokines have been proposed as possible therapeutic targets in cancer, allograft rejection, glomerulonephritis, diabetes, multiple sclerosis, and autoimmune disorders of the thyroid. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, including angiostatic effects, although its specific receptor has remained unknown for a long time. Recently, we provided evidence that the different functions of CXCL9, CXCL10, and CXCL11 on distinct cell types can be at least partly explained by the interaction of these chemokines with two distinct receptors. Indeed, in addition to the classic form of CXCR3 receptor, which we have renamed as CXCR3-A, a novel CXCR3 receptor variant (CXCR3-B) was identified, that not only mediates the angiostatic activity of CXCR3 ligands, but also acts as functional receptor for CXCL4. In this review, we focus on the accumulating evidence demonstrating the pivotal role of CXCR3-binding chemokines in several human diseases. Studies based on CXCR3 targeting have shown its importance in different pathologic conditions and orally active small molecules capable of inhibiting this receptor are now being developed in order to be tested for their activity in humans.

BACKGROUND

Dendritic cells (DCs) are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) induction in order to characterize the usefulness of mature DCs (mDCs) for immune therapy and to identify biomarkers for assessing the quality of mDCs.

METHODS

Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-gamma and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis.

RESULTS

After 24 hours of LPS and IFN-gamma stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs.

CONCLUSIONS

DCs, matured with LPS and IFN-gamma, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.

BACKGROUND

The effectiveness of systemic treatment of psoriasis with fumaric acid esters has been proven, but their mode of action at the cellular and molecular level has not yet been fully elucidated.

OBJECTIVE

To study the effect of dimethylfumarate (DMF) on the production of the chemokines CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11, formerly known as GROalpha, interleukin-8, Mig, IP-10 and IP-9/I-TAC, respectively, in human keratinocytes and peripheral blood mononuclear cells (PBMC).

METHODS

Cultured keratinocytes were stimulated with interferon (IFN) -gamma to produce CXCL9, CXCL10 and CXCL11 and with phorbol myristate acetate to produce CXCL1 and CXCL8 in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). PBMC were stimulated with either IFN-gamma to produce CXCL9 and CXCL10 or lipopolysaccharide to produce CXCL8, in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). RNA preparations from isolated keratinocytes were analysed by Northern blotting; protein production by keratinocytes and PBMC was monitored by an enzyme-linked immunosorbent assay.

RESULTS

Northern blot analysis on isolated keratinocyte RNA preparations showed a dose-dependent inhibition of CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11 transcription by DMF. At 45 micromol L(-1) the inhibition was almost complete. In addition, keratinocytes and PBMC showed in the presence of DMF a dose-dependent inhibition of CXCL8, CXCL9 and CXCL10 protein production.

CONCLUSIONS

These results show the ability of DMF to inhibit the production of chemokines that may be critically involved in the development and perpetuation of psoriatic lesions. This might explain, at least in part, the beneficial effects of treatment with fumaric acid esters in psoriasis patients.

OBJECTIVE

To investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF).

METHODS

The expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. RASF were stimulated with tumour necrosis factor (TNF)-α, interleukin (IL)-1β and toll-like receptor (TLR) ligands (Pam3, pIC and lipopolysaccharide (LPS)) in the presence or absence of the BET inhibitor I-BET151, or siRNA targeting BRD2, BRD3 and BRD4. RASF expression of inflammatory mediators, including MMP1, MMP3, IL-6 and IL-8, was measured by q-PCR, q-PCR array and ELISA. Cellular viability, apoptosis, proliferation and chemoattractive properties of RASF were investigated using MTT, cell apoptosis ELISA, BrdU-based proliferation and transwell migration assays.

RESULTS

BRD2, BRD3 and BRD4 proteins were detected in rheumatoid arthritis (RA) synovial tissue, expressed in both RASF and macrophages. I-BET151 suppressed cytokine and TLR ligand-induced secretion of MMP1, MMP3, IL-6 and IL-8, and mRNA expression of more than 70% of genes induced by TNF-α and IL-1β. Combined silencing of BRD2, BRD3 and BRD4 significantly reduced cytokine and TLR ligand-induced expression of a subset of gene products targeted by I-BET151, including MMP1, CXCL10 and CXCL11. I-BET151 treatment of RASF reduced RASF proliferation, and the chemotactic potential for peripheral blood leucocytes of RASF conditioned medium.

CONCLUSIONS

Inhibition of BET family proteins suppresses the inflammatory, matrix-degrading, proliferative and chemoattractive properties of RASF and suggests a therapeutic potential in the targeting of epigenetic reader proteins in RA.

Th1 and Th2 effector CD4+ T cells orchestrate distinct counterregulatory biological responses. To deliver effective tissue Th1- and Th2-type responses, Th1 and Th2 cell recruitment into tissue must be differentially regulated. We show that tissue-derived STAT1 controls the trafficking of adoptively transferred, Ag-specific, wild-type Th1 cells into the lung. Trafficking of Th1 and Th2 cells is differentially regulated as STAT6, which regulates Th2 cell trafficking, had no effect on the trafficking of Th1 cells and STAT1 deficiency did not alter Th2 cell trafficking. We demonstrate that STAT1 control of Th1 cell trafficking is not mediated through T-bet. STAT1 controls the recruitment of Th1 cells through the induction of CXCL9, CXCL10, CXCL11, and CXCL16, whose expression levels in the lung were markedly decreased in STAT1-/- mice. CXCL10 replacement partially restored Th1 cell trafficking in STAT1-deficient mice in vivo, and deficiency in CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, impaired the trafficking of adoptively transferred Th1 cells in wild-type mice. Our work identifies that STAT1 in peripheral tissue regulates the homing of Ag-specific Th1 cells through the induction of a distinct subset of chemokines and establishes that Th1 and Th2 cell trafficking is differentially controlled in vivo by STAT1 and STAT6, respectively.

OBJECTIVE

Phenotypic differences between vascular smooth muscle cell (VSMC) subtypes lead to diverse pathological processes including atherosclerosis, postangioplasty restenosis and vein graft disease. To better understand the molecular mechanisms underlying functional differences among distinct SMC subtypes, we compared gene expression profiles and functional responses to oxidized low-density lipoprotein (OxLDL) and platelet-derived growth factor (PDGF) between cultured SMCs from human coronary artery (CASM) and saphenous vein (SVSM).

RESULTS

OxLDL and PDGF elicited markedly different functional responses and expression profiles between the 2 SMC subtypes. In CASM, OxLDL inhibited cell proliferation and migration and modified gene expression of chemokines (CXCL10, CXCL11 and CXCL12), proinflammatory cytokines (IL-1, IL-6, and IL-18), insulin-like growth factor binding proteins (IGFBPs), and both endothelial and smooth muscle marker genes. In SVSM, OxLDL promoted proliferation partially via IGF1 signaling, activated NF-kappaB and phosphatidylinositol signaling pathways, and upregulated prostaglandin (PG) receptors and synthases. In untreated cells, alpha-chemokines, proinflammatory cytokines, and genes associated with apoptosis, inflammation, and lipid biosynthesis were higher in CASM, whereas some beta-chemokines, metalloproteinase inhibitors, and IGFBPs were higher in SVSM. Interestingly, the basal expression levels of these genes seemed closely related to their responses to OxLDL and PDGF. In summary, our results suggest dramatic differences in gene expression patterns and functional responses to OxLDL and PDGF between venous and arterial SMCs, with venous SMCs having stronger proliferative/migratory responses to stimuli but also higher expression of atheroprotective genes at baseline.

CONCLUSIONS

These results reveal molecular signatures that define the distinct phenotypes characteristics of coronary artery and saphenous vein SMC subtypes.

The recent recognition that antibody-mediated rejection (ABMR) is the major cause of kidney transplant loss creates strong interest in its pathogenesis. We used microarray analysis of kidney transplant biopsies to identify the changes in pure ABMR. We found that the ABMR transcript changes in the initial Discovery Set were strongly conserved in a subsequent Validation Set. In the Combined Set of 703 biopsies, 2603 transcripts were significantly changed (FDR < 0.05) in ABMR versus all other biopsies. In cultured cells, the transcripts strongly associated with ABMR were expressed in endothelial cells, e.g. cadherins CDH5 and CDH13; IFNG-treated endothelial cells, e.g. phospholipase PLA1A and chemokine CXCL11; or NK cells, e.g. cytotoxicity molecules granulysin (GNLY) and FGFBP2. Other ABMR transcripts were expressed in normal kidney but not cell lines, either increased e.g. Duffy chemokine receptor (DARC) or decreased e.g. sclerostin (SOST). Pathway analysis of ABMR transcripts identified angiogenesis, with roles for angiopoietin and vascular endothelial growth factors; leukocyte-endothelial interactions; and NK signaling, including evidence for CD16a Fc receptor signaling elements shared with T cells. These data support a model of ABMR involving injury-repair in the microcirculation induced by cognate recognition involving antibody and CD16a, triggering IFNG release and antibody-dependent NK cell-mediated cytotoxicity.

B7 family members and their receptors play a central role in the regulation of T-cell responses through T-cell co-stimulation and co-inhibition pathways that constitute attractive targets for the development of immunotherapeutic drugs. In this study, we report that VSIG-3/IGSF11 is a ligand of B7 family member VISTA/PD-1H and inhibits human T-cell functions through a novel VSIG-3/VISTA pathway. An extensive functional ELISA binding screening assay reveals that VSIG-3 binds to the new B7 family member VISTA but does not interact with other known members of the B7 family. Under the same experimental conditions, we did not observe any significant interaction between VSIG-8 and VISTA. In addition, VSIG-3 inhibits human T-cell proliferation in the presence of T-cell receptor signaling. Furthermore, VSIG-3 significantly reduces cytokine and chemokine production by human T cells including IFN-γ, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-1α, and CXCL11/I-TAC. Anti-VISTA neutralization antibodies attenuate the binding of VSIG-3 and VISTA, as well as VSIG-3-induced T-cell inhibition. Hence, we have identified a novel ligand for VISTA that is able to inhibit human T-cell proliferation and cytokine production. This unique VSIG-3/VISTA co-inhibitory pathway may provide new strategies for the treatment of human cancers, autoimmune disorders, infection, and transplant rejection and may aid in the design of better vaccines.

OBJECTIVE

Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS).

METHODS

RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays.

RESULTS

HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11.

CONCLUSIONS

Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.

OBJECTIVE

Interferon-γ (IFNγ) is the pivotal mediator in murine models of primary haemophagocytic lymphohistiocytosis (pHLH). Given the similarities between primary and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), we investigate the involvement of the IFNγ pathway in MAS by evaluating levels of IFNγ and of the induced chemokines, and their relation with laboratory parameters of MAS in systemic juvenile idiopathic arthritis (sJIA) patients with MAS and in a murine MAS model.

METHODS

The Luminex multiplexing assay was used to assess serum levels of interleukin (IL)-1β, IL-6, IFNγ and of the IFNγ-induced chemokines CXCL9, CXCL10 and CXCL11 in patients with sec-HLH (n=11) and in patients with sJIA (n=54), of whom 20 had active MAS at sampling. Expression of IFNγ-induced chemokines was assessed in IL-6 transgenic mice in which MAS is induced by TLR4 stimulation with lipopolysaccharide.

RESULTS

Levels of IFNγ and of IFNγ-induced chemokines were markedly elevated during active MAS and sec-HLH and were significantly higher in patients with MAS compared with active sJIA without MAS. Levels in patients with active sJIA without MAS were comparable to those of patients with clinically inactive sJIA. During MAS, ferritin and alanine transferase levels and neutrophil and platelet counts were significantly correlated with serum levels of IFNγ and CXCL9. In murine MAS, serum levels of ferritin were significantly correlated with mRNA levels of Cxcl9 in liver and spleen.

CONCLUSIONS

The high levels of IFNγ and of IFNγ-induced chemokines and their correlation with the severity of laboratory abnormalities of MAS suggest a pivotal role of IFNγ in MAS.

Immune-mediated cancer regression requires tumor infiltration by antigen-specific effector T cells, but lymphocytes are commonly sparse in melanoma metastases. Activated T cells express CXCR3, whose cognate chemokines are CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. Little is known about expression of these chemokines in lymph node (LN) metastases of melanoma. We evaluated whether metastatic melanoma induces these CXCR3-cognate chemokines in human LN-derived tissues. In addition, as these chemokines can be induced by interferon (IFN), we evaluated whether type I or II IFNs (IFN-α or IFN-γ, respectively) can modulate chemokine expression in an in vitro model of the human tumor microenvironment. Production of CXCL9-11 by melanoma-infiltrated nodes (MIN) was no different than tumor-free nodes; both produced less chemokine than activated LN (sentinel immunized nodes, SIN). These data suggest that melanoma infiltration into LN neither induces nor reduces CXCL9-11. Stimulation with IFN-α or IFN-γ increased production of CXCL10-11 from MIN, but not tumor-free node or SIN. IFN-γ also increased production of CXCL9 in MIN. In IFN-treated SIN, CD14+ cells were the primary source of CXCL9-11, whereas melanoma cells were the source of chemokine in MIN. Melanoma cells in MIN express IFN receptors. Consistent with these observations, multiple human melanoma lines expressed IFN receptors and produced CXCL9-11 in response to IFN treatment. Thus, melanoma infiltration of LN is insufficient to induce the production of CXCL9-11, but melanoma may be a significant source of IFN-induced chemokines. Collectively, these data suggest that IFN-α or IFN-γ may act in the tumor microenvironment to increase the chemotactic gradient for CXCR3+ T cells.

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.

Subsets of NK cells can have distinct functions. Here, we report that >25% of human peripheral blood NK cells express HLA-DR after culture with IL-2. This can be driven by an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLA-DR is upregulated on previously negative cells. HLA-DR-expressing NK cells showed enhanced degranulation to susceptible target cells and expressed chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggesting HLA-DR-expressing NK cells have an important role in an immune response, stimulation of PBMCs with Mycobacterium bovis BCG (BCG) triggered expansion of this subset. Importantly, the magnitude of an individual's NK cell IFN-γ response triggered by BCG was associated with the initial frequency of HLA-DR-expressing NK cells in PBMCs. More directly indicating the importance of HLA-DR-expressing NK cells, enriching the frequency of this subset in PBMCs substantially augmented the IFN-γ response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in circulating blood but readily expanded by IL-2, that can play an important role during immune responses to BCG.

We noted a marked increase in IFNγ mRNA in newborn (NB) murine lungs after exposure to hyperoxia. We sought to evaluate the role of IFNγ in lung injury in newborns. Using a unique triple-transgenic (TTG), IFNγ-overexpressing, lung-targeted, externally regulatable NB murine model, we describe a lung phenotype of impaired alveolarization, resembling human bronchopulmonary dysplasia (BPD). IFNγ-mediated abnormal lung architecture was associated with increased cell death and the upregulation of cell death pathway mediators caspases 3, 6, 8, and 9, and angiopoietin 2. Moreover, an increase was evident in cathepsins B, H, K, L, and S, and in matrix metalloproteinases (MMPs) 2, 9, 12, and 14. The IFNγ-mediated abnormal lung architecture was found to be MMP9-dependent, as indicated by the rescue of the IFNγ-induced pulmonary phenotype and survival during hyperoxia with a concomitant partial deficiency of MMP9. This result was concomitant with a decrease in caspases 3, 6, 8, and 9 and angiopoietin 2, but an increase in the expression of angiopoietin 1. In addition, NB IFNγ TTG mice exhibited significantly decreased survival during hyperoxia, compared with littermate controls. Furthermore, as evidence of clinical relevance, we show increased concentrations of the downstream targets of IFNγ chemokine (C-X-C motif) ligands (CXCL10 and CXCL11) in baboon and human lungs with BPD. IFNγ and its downstream targets may contribute significantly to the final common pathway of hyperoxia-induced injury in the developing lung and in human BPD.

Interferon-gamma-inducible protein-10 (IP-10)/CXCL10 is a CXC chemokine that attracts T lymphocytes and NK cells through activation of CXCR3, the only chemokine receptor identified to date that binds IP-10/CXCL10. We have found that several nonhemopoietic cell types, including epithelial and endothelial cells, have abundant levels of a receptor that binds IP-10/CXCL10 with a Kd of 1-6 nM. Surprisingly, these cells expressed no detectable CXCR3 mRNA. Furthermore, no cell surface expression of CXCR3 was detectable by flow cytometry, and the binding of 125I-labeled IP-10/CXCL10 to these cells was not competed by the other high affinity ligands for CXCR3, monokine induced by IFN-gamma/CXCL9, and I-TAC/CXCL11. Although IP-10/CXCL10 binds to cell surface heparan sulfate glycosaminoglycan (GAG), the receptor expressed by these cells is not GAG, since the affinity of IP-10/CXCL10 for this receptor is much higher than it is for GAG, its binding is not competed by platelet factor 4/CXCL4, and it is present on cells that are genetically incapable of synthesizing GAG. Furthermore, in contrast to IP-10/CXCL10 binding to GAG, IP-10/CXCL10 binding to these cells induces new gene expression and chemotaxis, indicating the ability of this receptor to transduce a signal. These high affinity IP-10/CXCL10-specific receptors on epithelial cells may be involved in cell migration and, perhaps, in the spread of metastatic cells as they exit from the vasculature. (All of the lung cancer cells we examined also expressed CXCR4, which has been shown to play a role in breast cancer metastasis.) CXCR3-negative endothelial cells may also use this receptor to mediate the angiostatic activity of IP-10/CXCL10, which is also expressed by these cells in an autocrine manner.