Correction: Dissecting the Autocrine and Paracrine Roles of the CCR2-CCL2 Axis in Tumor Survival and Angiogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0028305.].

CCR5+ Myeloid-Derived Suppressor Cells Are Enriched and Activated in Melanoma Lesions.

Accumulation of myeloid-derived suppressor cells (MDSC) in melanoma microenvironment is supported by chemokine receptor/chemokine signaling. Although different chemokines were suggested to be involved in this process, the role of CCR5 and its ligands is not established. Using a Ret transgenic mouse melanoma model, we found an accumulation of CCR5+ MDSCs in melanoma lesions associated with both increased concentrations of CCR5 ligands and tumor progression. Tumor-infiltrating CCR5+ MDSCs displayed higher immunosuppressive activity than their CCR5- counterparts. Upregulation of CCR5 expression on CD11b+Gr1+ myeloid cells was induced in vitro by CCR5 ligands and other inflammatory factors. In melanoma patients, CCR5+ MDSCs were enriched at the tumor site and correlated with enhanced production of CCR5 ligands. Moreover, they exhibited a stronger immunosuppressive pattern compared with CCR5- MDSCs. Blocking CCR5/CCR5 ligand interactions increased survival of tumor-bearing mice and was associated with reduced migration and immunosuppressive potential of MDSCs in tumor lesions. Our findings define a critical role for CCR5 in recruitment and activation of MDSCs, suggesting a novel strategy for melanoma treatment.Significance: These findings validate the importance of the CCR5/CCR5 ligand axis not only for MDSC recruitment but also for further activation of their immunosuppressive functions in the tumor microenvironment, with potentially broad therapeutic implications, given existing clinically available inhibitors of this axis. Cancer Res; 78(1); 157-67. ©2017 AACR.

CCR5 Directs the Mobilization of CD11b+Gr1+Ly6Clow Polymorphonuclear Myeloid Cells from the Bone Marrow to the Blood to Support Tumor Development.

Cells of hematopoietic origin can be subdivided into cells of the lymphoid lineage and those of the myeloid lineage, among which are myeloid-derived suppressor cells (MDSCs). The MDSCs can be further divided into CD11b+Ly6G-Ly6Chi monocytic (Mo) MDSCs and CD11b+Ly6G+Ly6Clow polymorphonuclear (PMN) MDSCs. Both subtypes support tumor growth and suppress anti-tumor immunity. Their accumulation at the tumor site includes mobilization from the bone marrow to the blood followed by colonization at the tumor site. The present study examines the mechanism by which PMN-MDSCs are mobilized from the BM to the blood to later accumulate at the tumor site. We show that the chemokine receptor CCR5 is a key driver of this event. We also show that, beyond chemoattraction, the interaction between CCR5 and its ligands promotes the proliferation of CCR5+ PMN-MDSCs at the BM and, later, potentiates their immune-suppressive activities at the tumor site in part by inducing arginase-1.

CCR8+FOXp3+ Treg cells as master drivers of immune regulation.

The current study identifies CCR8+ regulatory T cells (Treg cells) as drivers of immunosuppression. We show that in human peripheral blood cells, more than 30% of Treg up-regulate CCR8 following activation in the presence of CCL1. This interaction induces STAT3-dependent up-regulation of FOXp3, CD39, IL-10, and granzyme B, resulting in enhanced suppressive activity of these cells. Of the four human CCR8 ligands, CCL1 is unique in potentiating Treg cells. The relevance of these observations has been extended using an experimental model of multiple sclerosis [experimental autoimmune encephalomyelitis, (EAE)] and a stabilized version of mouse CCL1 (CCL1-Ig). First, we identified a self-feeding mechanism by which CCL1 produced by Treg cells at an autoimmune site up-regulates the expression of its own receptor, CCR8, on these cells. Administration of CCL1-Ig during EAE enhanced the in vivo proliferation of these CCR8+ regulatory cells while inducing the expression of CD39, granzyme B, and IL-10, resulting in the efficacious suppression of ongoing EAE. The critical role of the CCL1-CCR8 axis in Treg cells was further dissected through adoptive transfer studies using CCR8-/- mice. Collectively, we demonstrate the pivotal role of CCR8+ Treg cells in restraining immunity and highlight the potential clinical implications of this discovery.

Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis.

OBJECTIVE AND DESIGN: Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease. MATERIALS: The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo. METHODS: Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo. RESULTS: RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE. CONCLUSIONS: RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

Biased signaling pathways via CXCR3 control the development and function of CD4+ T cell subsets.

Structurally related chemotactic cytokines (chemokines) regulate cell trafficking through interactions with 7-transmembrane domain, G protein-coupled receptors. Biased signaling or functional selectivity is a concept that describes a situation where a 7-transmembrane domain receptor preferentially activates one of several available cellular signaling pathways. It can be divided into 3 distinct cases: ligand bias, receptor bias, and tissue or cell bias. Many studies, including those coming from our lab, have shown that only a limited number of chemokines are key drivers of inflammation. We have referred to them as "driver chemokines." They include the CXCR3 ligands CXCL9 and CXCL10, the CCR2 ligand CCL2, all 3 CCR5 ligands, and the CCR9 ligand CCL25. As for CXCR3, despite the proinflammatory nature of CXCL10 and CXCL9, transgenic mice lacking CXCR3 display an aggravated manifestation of different autoimmune disease, including Type I diabetes and experimental autoimmune encephalomyelitis. Recently, we showed that whereas CXCL9 and CXCL10 induce effector Th1/Th17 cells to promote inflammation, CXCL11, with a relatively higher binding affinity to CXCR3, drives the development of the forkhead box P3-negative IL-10(high) T regulatory 1 cell subset and hence, dampens inflammation. We also showed that CXCL9/CXCL10 activates a different signaling cascade than CXCL11, despite binding to the same receptor, CXCR3, which results in these diverse biologic activities. This provides new evidence for the role of biased signaling in regulating biologic activities, in which CXCL11 induces ligand bias at CXCR3 and receptor-biased signaling via atypical chemokine receptor 3.

The Role of Chemokines in Shaping the Balance Between CD4(+) T Cell Subsets and Its Therapeutic Implications in Autoimmune and Cancer Diseases.

Chemokines are the key activators of adhesion molecule and also drivers of leukocyte migration to inflammatory sites and are therefore mostly considered as proinflammatory mediators. Many studies, including ours, imply that targeting the function of several key chemokines, but not many others, could effectively suppress inflammatory responses and inflammatory autoimmunity. Along with this, a single chemokine named CXCL10 could be used to induce antitumor immunity, and thereby suppress myeloma. Our working hypothesis is that some chemokines differ from others as aside from being chemoattractants for leukocytes and effective activators of adhesion receptors that possess additional biological properties making them "driver chemokines." We came up with this notion when studying the interlay between CXCR4 and CXCL12 and between CXCR3 and its three ligands: CXCL9, CXCL10, and CXCL11. The current mini-review focuses on these ligands and their biological properties. First, we elaborate the role of cytokines in directing the polarization of effector and regulatory T cell subset and the plasticity of this process. Then, we extend this notion to chemokines while focusing on CXCL 12 and the CXCR3 ligands. Finally, we elaborate the potential clinical implications of these studies for therapy of autoimmunity, graft-versus-host disease, and cancer.

Duodenal Expression of 25 Hydroxyvitamin D3-1α-hydroxylase Is Higher in Adolescents Than in Children and Adults.

CONTEXT: Puberty is associated with increased dietary calcium absorption. However, little is known about the metabolic adaptations that enhance calcium absorption during puberty. OBJECTIVES: To investigate duodenal 25-hydroxy vitamin D-1α-hydroxylase (CYP 27B1) mRNA expression and duodenal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) production in children, adolescents, and adults. DESIGN AND METHODS: CYP27B1a nd IGF1 mRNA expression and 1,25(OH)2D3 production were determined in duodenal biopsies. CYP27B1 expression was also determined after IGF1R inhibitor treatment of human and mice duodenal explants. mRNA expression was determined by RT-PCR, and CYP27B1 activity was determined by incubating duodenal explants with 25(OH)D3 and measuring 1,25(OH)2D3 production by radioimmunoassay. RESULTS: CYP27B1 mRNA expression was 13.7 and 10.4 times higher in biopsies from adolescents compared to adults and children, respectively. IGF1 mRNA expression was 30% and 45% higher in explants from adolescents and children, respectively, compared to adults. Inhibition of IGF1 receptor activity decreased CYP27B1 expression in explants from both mice (85%) and humans (24%). 1,25(OH)2D3 production reached a maximum velocity of 768 ± 268 pmol/l/mg protein at 748.8 nmol/l of 25(OH)D3 in children and adolescents, whereas the maximum velocity was 86.4 ± 43.2 pmol/l/mg protein in adults. The substrate concentration at which the enzyme shows half of its maximum activity was similar in all groups, ranging between 624 and 837 nmol/L of 25(OH)D3. CONCLUSIONS: Increased CYP27B1 expression and local duodenal 1,25(OH)2D3 production during puberty may be a metabolic adaptation that promotes dietary calcium absorption. IGF1, a major factor in skeletal growth, is also involved in the modulation of CYP27B1 expression in the gut and may increase calcium supply for the growing bone.

The role of chemokines in adjusting the balance between CD4+ effector T cell subsets and FOXp3-negative regulatory T cells.

Chemokines are small (~8-14 kDa) structurally-related chemotactic cytokines that regulate cell trafficking through interactions with specific seven-trans membrane, G protein-coupled receptors (GPCRs). One of the important features of G protein-coupled receptors (GPCRs) is their ability to transmit diverse signaling cascades upon binding different ligands. The current review focuses on the interplay between three ligands: CXCL9, CXCL10 and CXCL11 binding the same receptor (CXCR3) on CD4+ T cells, yet direct different signaling cascades to shape T cell mediated immunity. The review brings about a new concept regarding the biological activities of chemokines in shaping CD4+ T cell immunity, and also a new approach for applying chemokine based therapy of autoimmune diseases.

CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis.

A single G protein-coupled receptor (GPCR) can activate multiple signaling cascades based on the binding of different ligands. The biological relevance of this feature in immune regulation has not been evaluated. The chemokine-binding GPCR CXCR3 is preferentially expressed on CD4+ T cells, and canonically binds 3 structurally related chemokines: CXCL9, CXCL10, and CXCL11. Here we have shown that CXCL10/CXCR3 interactions drive effector Th1 polarization via STAT1, STAT4, and STAT5 phosphorylation, while CXCL11/CXCR3 binding induces an immunotolerizing state that is characterized by IL-10(hi) (Tr1) and IL-4(hi) (Th2) cells, mediated via p70 kinase/mTOR in STAT3- and STAT6-dependent pathways. CXCL11 binds CXCR3 with a higher affinity than CXCL10, suggesting that CXCL11 has the potential to restrain inflammatory autoimmunity. We generated a CXCL11-Ig fusion molecule and evaluated its use in the EAE model of inflammatory autoimmune disease. Administration of CXCL11-Ig during the first episode of relapsing EAE in SJL/J mice not only led to rapid remission, but also prevented subsequent relapse. Using GFP-expressing effector CD4+ T cells, we observed that successful therapy was associated with reduced accumulation of these cells at the autoimmune site. Finally, we showed that very low doses of CXCL11 rapidly suppress signs of EAE in C57BL/6 mice lacking functional CXCL11.

The role of vitamin D receptor in innate and adaptive immunity: a study in hereditary vitamin D-resistant rickets patients.

CONTEXT: Vitamin D has regulatory effects on innate and adaptive immunity. Curiously, hereditary vitamin D-resistant rickets (HVDRR) patients show no increased incidence of infectious or autoimmune diseases. OBJECTIVES: The aim of the study was to investigate the role of vitamin D and the vitamin D receptor (VDR) in innate and adaptive immune responses in monocytes and lymphocytes from HVDRR patients. DESIGN AND METHODS: Fifteen HVDRR patients and 17 controls participated in the investigation. Activated monocytes (lipopolysaccharides) and lymphocytes (anti-CD3, CD28, and α-GalCer) were incubated with and without 25(OH)D3 (100 nM). The mRNA expressions of CYP27B1 and VDR; vitamin D response (TLR2); vitamin D response elements binding protein (hnRNP); antimicrobial peptides cathelicidin and ß-defensin; the transcription factor enhancer binding proteins C/EBPα, C/EBPß, and C/EBPε and enzymes involved in NO generation, Nos2, and Arginase1 were analyzed by RT-PCR. TNF-α, interferon-γ, IL-4, IL-10, and IL-17 concentrations in lymphocyte cultures media were measured by ELISA. RESULTS: Cathelicidin expression was lower in HVDRR monocytes than in control monocytes. 25(OH)D3 increased significantly the expression of cathelicidin in control monocytes (2.3-fold) but only slightly in HVDRR monocytes. 25(OH)D3 increased the expression of VDR (2-fold), C/EBPε (2-fold), C/EBPß (1.7-fold), and hnRNP and suppressed TLR2 only in control monocytes. Unexpectedly, 25(OH)D3 increased the expression of CYP27b1, C/EBPα, Nos2, and Arginase1 in HVDRR monocytes. TNFα and IL-17 concentrations were significantly higher in HVDRR lymphocyte cultures than in controls. 25(OH)D3 suppressed IL-17 only in control lymphocyte. 25(OH)D3 increased IL-4, IL-10, and interferon-γ concentrations in control lymphocyte media but not in HVDRR. CONCLUSIONS: Our results demonstrate impairments in various components of innate immunity in HVDTRR patients' monocytes and a proinflammatory cytokine profile in their lymphocytes. The underlying VDR-independent compensatory mechanisms that protect HVDRR patients from infections and autoimmune diseases remain undetermined.

Dissecting the autocrine and paracrine roles of the CCR2-CCL2 axis in tumor survival and angiogenesis.

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX3CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2-/-, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX3CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX3CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx3cr1(gfp)), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX3CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.

Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.

Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/ß, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/ß, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity.

A fusion protein encoding the second extracellular domain of CCR5 arrests chemokine-induced cosignaling and effectively suppresses ongoing experimental autoimmune encephalomyelitis.

CCR5 is a key CCR that is highly expressed on CD4(+) T cells. It binds three different ligands: CCL3 (MIP-alpha), CCL4 (MIP-beta), and CCL5 (RANTES). Recent studies suggested that the interaction between CCR5 and its ligands is essential not only for attracting these CCR5(+) T cells but also substantial for transuding cosignals for their activation. The current study explores, for the first time, the in vivo consequences of CCR5 as a costimulatory molecule. First, we show redundancy between CCR5 ligands not only in chemoattractive properties but also in their ability to induced cosignals via CCR5. This has motivated us to generate a soluble receptor-based fusion protein that would selectively bind and neutralize all three CCR5 ligands. We show in this study that a 30-aa-based CCR5-Ig fusion protein encoding the second extracellular domain of receptor selectively binds and neutralizes all three CCR5 ligands and, when administered during ongoing experimental autoimmune encephalomyelitis, rapidly suppressed the disease while arresting Ag-specific effector T cell functions. Finally, our results clearly show that although CCR5 ligands induced cosignaling for IL-2 production is directed by CCR5, other proinflammatory properties of these ligands, such as TNF-alpha, IL-17, and IFN-gamma production, are CCR5 independent and therefore likely to be mediated by the other receptors for these ligands. These findings imply that implementing a CCR5-Ig-based therapy would be advantageous over blockade of this receptor or of the use of mAbs for targeting a single CCR5 ligand.

Antigen-specific CD25- Foxp3- IFN-gamma(high) CD4+ T cells restrain the development of experimental allergic encephalomyelitis by suppressing Th17.

The current study identifies within the Th1 subtype two distinct CD4(+) populations: those capable of transferring inflammatory autoimmunity and others that regulate its development by suppressing Th17 in an interferon (IFN)-gamma-dependent manner. These CD4(+)IFN-gamma(high)IL-4(low)IL-10(low)TGF-beta(low)FOXp3(-) cells in fact function as antigen-specific regulatory cells that restrain the development of autoimmunity by increasing the threshold of Th17 activation. We show that development of autoimmune conditions within the central nervous system is dependent on the Fas ligand-mediated apoptosis of these regulatory cells at early stages of disease. We also show that not only is the function of these cells IFN-gamma dependent but also that stable over expression of IFN-gamma in encephalitogenic CD4(+) T cells redirects their biological function to become antigen-specific regulatory cells. This may also explain, in part, the pleiotropic role of IFN-gamma in the regulation of autoimmunity, as previously observed by others.