Human CCR6+ Th Cells Show Both an Extended Stable Gradient of Th17 Activity and Imprinted Plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.

Human CCR6 + Th cells show both an extended stable gradient of Th17 activity and imprinted plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related (type 17) cells and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo . By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The CCR6 + cells' phenotypes and epigenomes are stable across cell divisions under homeostatic conditions. Nonetheless, activation in polarizing and non-polarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the continuum to yield the unusual plasticity ascribed to type 17 cells.

Chemokine positioning determines mutually exclusive roles for their receptors in extravasation of pathogenic human T cells.

Pro-inflammatory T cells co-express multiple chemokine receptors, but the distinct functions of individual receptors on these cells are largely unknown. Human Th17 cells uniformly express the chemokine receptor CCR6, and we discovered that the subgroup of CD4+CCR6+ cells that co-express CCR2 possess a pathogenic Th17 signature, can produce inflammatory cytokines independent of TCR activation, and are unusually efficient at transendothelial migration (TEM). The ligand for CCR6, CCL20, was capable of binding to activated endothelial cells (ECs) and inducing firm arrest of CCR6+CCR2+ cells under conditions of flow - but CCR6 could not mediate TEM. By contrast, CCL2 and other ligands for CCR2, despite being secreted from both luminal and basal sides of ECs, failed to bind to the EC surfaces - and CCR2 could not mediate arrest. Nonetheless, CCR2 was required for TEM. To understand if CCR2's inability to mediate arrest was due solely to an absence of EC-bound ligands, we generated a CCL2-CXCL9 chimeric chemokine that could bind to the EC surface. Although display of CCL2 on the ECs did indeed lead to CCR2-mediated arrest of CCR6+CCR2+ cells, activating CCR2 with surface-bound CCL2 blocked TEM. We conclude that mediating arrest and TEM are mutually exclusive activities of chemokine receptors and/or their ligands that depend, respectively, on chemokines that bind to the EC luminal surfaces versus non-binding chemokines that form transendothelial gradients under conditions of flow. Our findings provide fundamental insights into mechanisms of lymphocyte extravasation and may lead to novel strategies to block or enhance their migration into tissue.

Bile Acids Improve Psoriasiform Dermatitis through Inhibition of IL-17A Expression and CCL20-CCR6-Mediated Trafficking of T Cells.

Bile acids (BAs), produced in the liver and further transformed in the gut, are cholesterol-derived molecules involved in essential physiological processes. Recent studies suggest that BAs regulate T helper 17 cell function, but the underlying mechanism of this action and their therapeutic value in disease models remains unclear. Using an IL-23 minicircle DNA-based murine model of psoriasiform dermatitis, we showed that oral administration of secondary BAs, including lithocholic acid (LCA), deoxycholic acid, and 3-oxoLCA, significantly improved psoriasiform dermatitis without inducing apparent hepatotoxicity. Of the BAs tested, LCA possessed the greatest potency in treating psoriasiform dermatitis. Intravenous administration of LCA at a much lower dosage (compared with oral treatment) showed a comparable antipsoriatic effect and markedly suppressed the IL-17A response. Ex vivo experiments revealed that LCA reduced IL-17A production in IL-23-stimulated murine T cells in the absence of BA receptors TGR5 or FXR. Strikingly, BAs inhibited CCL20 expression in keratinocytes, which led to reduced migration of CCR6-expressing Jurkat cells cultured in the conditioned medium of stimulated keratinocytes. Thus, BAs improve psoriasiform dermatitis with minimal toxicity via direct inhibition of IL-17A production and blockade of CCL20-mediated trafficking, supporting the potential use of BAs in psoriasis.

A Flow Chamber Assay for Studying MAIT Cell Trafficking.

Human MAIT cells show little expression of the selectin CD62L and the chemokine receptor CCR7, which are important for entering lymph nodes, and high expression of selectin ligands and chemokine receptors that mediate trafficking into inflamed tissue. Extravasation of leukocytes into tissue requires sequential steps including rolling, firm arrest, crawling, and transendothelial migration, and can be modeled using endothelial cell monolayers in flow chambers that approximate the sheer stress found in post-capillary venules. Using MAIT cells purified from elutriated lymphocytes by fluorescence-activated cell sorting, we have used flow chambers to demonstrate roles for individual chemokine receptors in specific steps required for extravasation. These methods provide a general way to study the molecular mechanisms underlying MAIT cell trafficking from blood into tissue.

CXCR3 Identifies Human Naive CD8+ T Cells with Enhanced Effector Differentiation Potential.

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

IL-23- and Imiquimod-Induced Models of Experimental Psoriasis in Mice.

Genome-wide association studies have found that polymorphisms in genes for IL-23 and its receptor are important in psoriasis, and blocking IL-23 is an effective therapy in the disease. The use of Aldara™ , a cream that contains the TLR7 and TLR8 agonist imiquimod (IMQ), was found to exacerbate psoriasis in some patients with pre-existing disease. Intradermal injections of IL-23 and topical application of Aldara/IMQ induce skin inflammation in mice with features similar to psoriasis-including epidermal hyperplasia and accumulation of inflammatory cells in epidermis and dermis-which is mediated by IL-17A, IL-22, and other factors implicated in the human disease. Consequently, these models can be used in preclinical studies to investigate the molecular and cellular pathogenesis of psoriasis, as well as in the evaluation of potential therapies. This article provides detailed methodologies for creating and evaluating the IL-23- and Aldara/IMQ-induced mouse models of psoriasis. The article also provides a protocol for analyzing skin leukocytes by flow cytometry. © 2019 by John Wiley & Sons, Inc.

Correction: Screening of cancer tissue arrays identifies CXCR4 on adrenocortical carcinoma: correlates with expression and quantification on metastases using 64Cu-plerixafor PET.

[This corrects the article DOI: 10.18632/oncotarget.19945.].

C/EBPδ drives interactions between human MAIT cells and endothelial cells that are important for extravasation.

Many mediators and regulators of extravasation by bona fide human memory-phenotype T cells remain undefined. Mucosal-associated invariant T (MAIT) cells are innate-like, antibacterial cells that we found excelled at crossing inflamed endothelium. They displayed abundant selectin ligands, with high expression of FUT7 and ST3GAL4, and expressed CCR6, CCR5, and CCR2, which played non-redundant roles in trafficking on activated endothelial cells. MAIT cells selectively expressed CCAAT/enhancer-binding protein delta (C/EBPδ). Knockdown of C/EBPδ diminished expression of FUT7, ST3GAL4 and CCR6, decreasing MAIT cell rolling and arrest, and consequently the cells' ability to cross an endothelial monolayer in vitro and extravasate in mice. Nonetheless, knockdown of C/EBPδ did not affect CCR2, which was important for the step of transendothelial migration. Thus, MAIT cells demonstrate a program for extravasastion that includes, in part, C/EBPδ and C/EBPδ-regulated genes, and that could be used to enhance, or targeted to inhibit T cell recruitment into inflamed tissue.

The homozygous CX3CR1-M280 mutation impairs human monocyte survival.

Several reports have demonstrated that mouse Cx3cr1 signaling promotes monocyte/macrophage survival. In agreement, we previously found that, in a mouse model of systemic candidiasis, genetic deficiency of Cx3cr1 resulted in increased mortality and impaired tissue fungal clearance associated with decreased macrophage survival. We translated this finding by showing that the dysfunctional CX3CR1 variant CX3CR1-M280 was associated with increased risk and worse outcome of human systemic candidiasis. However, the impact of this mutation on human monocyte/macrophage survival is poorly understood. Herein, we hypothesized that CX3CR1-M280 impairs human monocyte survival. We identified WT (CX3CR1-WT/WT), CX3CR1-WT/M280 heterozygous, and CX3CR1-M280/M280 homozygous healthy donors of European descent, and we show that CX3CL1 rescues serum starvation-induced cell death in CX3CR1-WT/WT and CX3CR1-WT/M280 but not in CX3CR1-M280/M280 monocytes. CX3CL1-induced survival of CX3CR1-WT/WT monocytes is mediated via AKT and ERK activation, which are both impaired in CX3CR1-M280/M280 monocytes, associated with decreased blood monocyte counts in CX3CR1-M280/M280 donors at steady state. Instead, CX3CR1-M280/M280 does not affect monocyte CX3CR1 surface expression or innate immune effector functions. Together, we show that homozygocity of the M280 polymorphism in CX3CR1 is a potentially novel population-based genetic factor that influences human monocyte signaling.

Screening of cancer tissue arrays identifies CXCR4 on adrenocortical carcinoma: correlates with expression and quantification on metastases using 64Cu-plerixafor PET.

Expression of the chemokine receptor CXCR4 by many cancers correlates with aggressive clinical behavior. As part of the initial studies in a project whose goal was to quantify CXCR4 expression on cancers non-invasively, we examined CXCR4 expression in cancer samples by immunohistochemistry using a validated anti-CXCR4 antibody. Among solid tumors, we found expression of CXCR4 on significant percentages of major types of kidney, lung, and pancreatic adenocarcinomas, and, notably, on metastases of clear cell renal cell carcinoma and squamous cell carcinoma of the lung. We found particularly high expression of CXCR4 on adrenocortical cancer (ACC) metastases. Microarrays of ACC metastases revealed correlations between expression of CXCR4 and other chemokine system genes, particularly CXCR7/ACKR3, which encodes an atypical chemokine receptor that shares a ligand, CXCL12, with CXCR4. A first-in-human study using 64Cu-plerixafor for PET in an ACC patient prior to resection of metastases showed heterogeneity among metastatic nodules and good correlations among PET SUVs, CXCR4 staining, and CXCR4 mRNA. Additionally, we were able to show that CXCR4 expression correlated with the rates of growth of the pulmonary lesions in this patient. Further studies are needed to understand better the role of CXCR4 in ACC and whether targeting it may be beneficial. In this regard, non-invasive methods for assessing CXCR4 expression, such as PET using 64Cu-plerixafor, should be important investigative tools.

Monocyte-derived inflammatory Langerhans cells and dermal dendritic cells mediate psoriasis-like inflammation.

Dendritic cells (DCs) have been implicated in the pathogenesis of psoriasis but the roles for specific DC subsets are not well defined. Here we show that DCs are required for psoriasis-like changes in mouse skin induced by the local injection of IL-23. However, Flt3L-dependent DCs and resident Langerhans cells are dispensable for the inflammation. In epidermis and dermis, the critical DCs are TNF-producing and IL-1ß-producing monocyte-derived DCs, including a population of inflammatory Langerhans cells. Depleting Ly6Chi blood monocytes reduces DC accumulation and the skin changes induced either by injecting IL-23 or by application of the TLR7 agonist imiquimod. Moreover, we find that IL-23-induced inflammation requires expression of CCR6 by DCs or their precursors, and that CCR6 mediates monocyte trafficking into inflamed skin. Collectively, our results imply that monocyte-derived cells are critical contributors to psoriasis through production of inflammatory cytokines that augment the activation of skin T cells.

Scan-rescan precision of subchondral bone curvature maps from routine 3D DESS water excitation sequences: Data from the Osteoarthritis Initiative.

BACKGROUND: Subchondral bone (SCB) undergoes changes in the shape of the articulating bone surfaces and is currently recognized as a key target in osteoarthritis (OA) treatment. The aim of this study was to present an automated system that determines the curvature of the SCB regions of the knee and to evaluate its cross-sectional and longitudinal scan-rescan precision METHODS: Six subjects with OA and six control subjects were selected from the Osteoarthritis Initiative (OAI) pilot study database. As per OAI protocol, these subjects underwent 3T MRI at baseline and every twelve months thereafter, including a 3D DESS WE sequence. We analyzed the baseline and twenty-four month images. Each subject was scanned twice at these visits, thus generating scan-rescan information. Images were segmented with an automated multi-atlas framework platform and then 3D renderings of the bone structure were created from the segmentations. Curvature maps were extracted from the 3D renderings and morphed into a reference atlas to determine precision, to generate population statistics, and to visualize cross-sectional and longitudinal curvature changes. RESULTS: The baseline scan-rescan root mean square error values ranged from 0.006mm(-1) to 0.013mm(-1), and from 0.007mm(-1) to 0.018mm(-1) for the SCB of the femur and the tibia, respectively. The standardized response of the mean of the longitudinal changes in curvature in these regions ranged from -0.09 to 0.02 and from -0.016 to 0.015, respectively. CONCLUSION: The fully automated system produces accurate and precise curvature maps of femoral and tibial SCB, and will provide a valuable tool for the analysis of the curvature changes of articulating bone surfaces during the course of knee OA.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific.

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D(3.49)R(3.50)Y(3.51) sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains D(3.49)R(3.50)F(3.51)I(3.52)V(3.53) at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments.

PLZF regulates CCR6 and is critical for the acquisition and maintenance of the Th17 phenotype in human cells.

Th17 cells, which express the chemokine receptor CCR6, are implicated in many immune-mediated disorders, such as psoriasis and multiple sclerosis. We found that expression levels of CCR6 on human effector/memory CD4(+) T cells reflect a continuum of Th17 differentiation. By evaluating the transcriptome in cells with increasing CCR6, we detected progressive upregulation of ZBTB16, which encodes the broad complex, tramtrack, bric-à-brac-zinc finger transcription factor promyelocytic leukemia zinc finger protein (PLZF). Using chromatin immunoprecipitation for modified histones, p300, and PLZF, we identified enhancer-like sites at -9/-10 and -13/-14 kb from the upstream transcription start site of CCR6 that bind PLZF in CCR6(+) cells. For Th cells from adult blood, both in the CCR6(+) memory population and in naive cells activated ex vivo, knockdown of ZBTB16 downregulated CCR6 and other Th17-associated genes. ZBTB16 and RORC (which encodes the "master regulator" RORγt) cross-regulate each other, and PLZF binds at the RORC promoter in CCR6(+) cells. In naive Th cells from cord blood, ZBTB16 expression was confined to CD161(+) cells, which are Th17 cell precursors. ZBTB16 was not expressed in mouse Th17 cells, and Th17 cells could be made from luxoid mice, which harbor an inactivating mutation in Zbtb16. These studies demonstrate a role for PLZF as an activator of transcription important both for Th17 differentiation and the maintenance of the Th17 phenotype in human cells, expand the role of PLZF as a critical regulator in the human adaptive immune system, and identify a novel, essential element in a regulatory network that is of significant therapeutic interest.

Conventional NK cells can produce IL-22 and promote host defense in Klebsiella pneumoniae pneumonia.

It was reported that host defense against pulmonary Klebsiella pneumoniae infection requires IL-22, which was proposed to be of T cell origin. Supporting a role for IL-22, we found that Il22(-/-) mice had decreased survival compared with wild-type mice after intratracheal infection with K. pneumoniae. Surprisingly, however, Rag2(-/-) mice did not differ from wild-type mice in survival or levels of IL-22 in the lungs postinfection with K. pneumoniae. In contrast, K. pneumoniae-infected Rag2(-/-)Il2rg(-/-) mice failed to produce IL-22. These data suggested a possible role for NK cells or other innate lymphoid cells in host defense and production of IL-22. Unlike NK cell-like innate lymphoid cells that produce IL-22 and display a surface phenotype of NK1.1(-)NKp46(+)CCR6(+), lung NK cells showed the conventional phenotype, NK1.1(+)NKp46(+)CCR6(-). Mice depleted of NK cells using anti-asialo GM1 showed decreased survival and higher lung bacterial counts, as well as increased dissemination of K. pneumoniae to blood and liver, compared with control-treated mice. NK cell depletion also led to decreased production of IL-22 in the lung. Within 1 d postinfection, although there was no increase in the number of lung NK cells, a subset of lung NK cells became competent to produce IL-22, and such cells were found in both wild-type and Rag2(-/-) mice. Our data suggest that, during pulmonary infection of mice with K. pneumoniae, conventional NK cells are required for optimal host defense, which includes the production of IL-22.

International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors.

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145-176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

Chemokine receptors in psoriasis.

INTRODUCTION: Psoriasis is a prevalent immune-mediated disease involving primarily the skin. Infiltrating leukocytes play key roles in driving the disease. Along with an array of adhesion proteins, leukocyte trafficking into tissue is controlled by chemoattractants, including chemokines, and their receptors. This review summarizes the data on roles for the chemokine system in psoriasis in order to highlight opportunities for inhibiting this system for therapeutic benefit. AREAS COVERED: The review covers the roles of various leukocyte subsets in psoriasis, focusing on the chemokine receptors that are thought to be responsible for the trafficking of these cells into tissue. The review also discusses in some detail the significance of the IL-23/IL-17/IL-22 cytokine axis in disease. EXPERT OPINION: Many of the new therapies developed for psoriasis are antibodies to neutralize cytokines that have pleiotropic functions in host defense. Inhibiting chemokine receptors can be accomplished using small molecules, and would be expected to block inflammation while resulting in more limited immunosuppression. There has been limited success to date in treating inflammatory disease with chemokine receptor antagonists, which has often been ascribed to the system's redundancy. Other factors may also be important, however, including sub-optimal choices of targets based on incomplete understandings of disease pathogenesis.

CCR6 is required for epidermal trafficking of γδ-T cells in an IL-23-induced model of psoriasiform dermatitis.

A subset of CC chemokine receptor-6(+) (CCR6(+)), γδ-low (GDL) T cells that express Th17 cytokines in mouse skin participates in IL-23-induced psoriasiform dermatitis. We use CCR6-deficient (knockout, KO) and wild-type (WT) mice to analyze skin trafficking patterns of GDL T cells and function-blocking mAbs to determine the role of CCR6 in IL-23-mediated dermatitis. Herein, CCL20 was highly upregulated in IL-23-injected WT mouse ear skin as early as 24 hours after initial treatment, and large numbers of CCR6(+) cells were observed in the epidermis of IL-23-injected WT mice. Anti-CCL20 mAbs reduced psoriasiform dermatitis and blocked recruitment of GDL T cells to the epidermis. In CCR6 KO mice, GDL T cells failed to accumulate in the epidermis after IL-23 treatment, but the total numbers of GDL T cells in the dermis of WT and CCR6 KO mice were equivalent. There was an ∼70% reduction in the proportion of IL-22(+) GDL T cells in the dermis of CCR6 KO mice (vs WT mice), suggesting that effector function and epidermal recruitment of GDL T cells are impaired in CCR6-deficient mice. Thus, these data show that CCR6 regulates epidermal trafficking of γδ-T-cell subsets in the skin and suggest the potential of CCR6 as a therapeutic target for psoriasis.