Tissue Iron Promotes Wound Repair via M2 Macrophage Polarization and the Chemokine (C-C Motif) Ligands 17 and 22.

Macrophages are important for effective iron recycling and erythropoiesis, but they also play a crucial role in wound healing, orchestrating tissue repair. Recently, we demonstrated a significant accumulation of iron in healing wounds and a requirement of iron for effective repair. Herein, we sought to determine the influence of iron on macrophage function in the context of wound healing. Interestingly, wound macrophages extensively sequestered iron throughout healing, associated with a prohealing M2 phenotype. In delayed healing diabetic mouse wounds, both macrophage polarization and iron sequestration were impaired. In vitro studies revealed that iron promotes differentiation, while skewing macrophages toward a hypersecretory M2-like polarization state. These macrophages produced high levels of chemokine (C-C motif) ligands 17 and 22, promoting wound reepithelialization and extracellular matrix deposition in a human ex vivo wound healing model. Together, these findings reveal a novel, unappreciated role for iron in modulating macrophage behavior to promote subsequent wound repair. These findings support therapeutic evaluation of iron use to promote wound healing in the clinic.

[The role of CD4 ⁺ CD25 ⁺ T regs and CCL17, CCL22 in the pathogenesis of head and neck squamous cell carcinoma].

Objective:To investigate the role of CD4 ⁺ CD25 ⁺ T regs and CCL17 and CCL22 in the pathogenesis of HNSCC.Method:Twenty cases of HNSCC were enrolled. All patients were primary or recurrent after treatment (chemotherapy, surgery). The primary tumor was taken as the experimental group, and the adjacent normal tissues from the primary tumor 1-3 cm were taken as control group. CD4 ⁺ /Foxp3 and CD25⁺/Foxp3 were detected by immunofluorescence, while CCL17 and CCL22 were detected by ELISA. The difference and correlation between the amount of CD4⁺,CD25⁺ and the expression of CCL17, CCL22 were observed and analyzed.Result:The difference of mean optical density between CD4⁺/Foxp3 and CD25⁺/Foxp3 was statistically significant between the experimental group and the control group (P<0.05). The concentration of CCL17 and CCL22 was statistically different between the two groups (P<0.01). There was a positive correlation between CD25⁺and CCL17,CCL22(r=0.595, 0.720,P<0.01).Conclusion:CD4⁺CD25⁺T regs and CCL17,CCL22 played an important role in the pathogenesis of head and neck squamous cell carcinoma,both of which interacted with each other,and promoted the recurrence and metastasis of HNSCC.

Fully human antagonistic antibodies against CCR4 potently inhibit cell signaling and chemotaxis.

BACKGROUND: CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis. METHODOLOGY: Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies. SIGNIFICANCE: For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer.

Long-term IKK2/NF-κB signaling in pancreatic ß-cells induces immune-mediated diabetes.

Type 1 diabetes is a multifactorial inflammatory disease in genetically susceptible individuals characterized by progressive autoimmune destruction of pancreatic ß-cells initiated by yet unknown factors. Although animal models of type 1 diabetes have substantially increased our understanding of disease pathogenesis, heterogeneity seen in human patients cannot be reflected by a single model and calls for additional models covering different aspects of human pathophysiology. Inhibitor of κB kinase (IKK)/nuclear factor-κB (NF-κB) signaling is a master regulator of inflammation; however, its role in diabetes pathogenesis is controversially discussed by studies using different inhibition approaches. To investigate the potential diabetogenic effects of NF-κB in ß-cells, we generated a gain-of-function model allowing conditional IKK2/NF-κB activation in ß-cells. A transgenic mouse model that expresses a constitutively active mutant of human IKK2 dependent on Pdx-1 promoter activity (IKK2-CA(Pdx-1)) spontaneously develops full-blown immune-mediated diabetes with insulitis, hyperglycemia, and hypoinsulinemia. Disease development involves a gene expression program mimicking virus-induced diabetes and allergic inflammatory responses as well as increased major histocompatibility complex class I/II expression by ß-cells that could collectively promote diabetes development. Potential novel diabetes candidate genes were also identified. Interestingly, animals successfully recovered from diabetes upon transgene inactivation. Our data give the first direct evidence that ß-cell-specific IKK2/NF-κB activation is a potential trigger of immune-mediated diabetes. Moreover, IKK2-CA(Pdx-1) mice provide a novel tool for studying critical checkpoints in diabetes pathogenesis and mechanisms governing ß-cell degeneration/regeneration.

Thymus and activation-regulated chemokine (TARC)/CC chemokine ligand (CCL) 17 accelerates wound healing by enhancing fibroblast migration.

BACKGROUND: Some chemokines are known to accelerate wound healing. However, there has been no report on the relationship between Thymus and activation-regulated chemokine (TARC)/CC chemokine ligand (CCL) 17 and wound healing. The purpose of this study was to determine whether CCL17 enhances response to cutaneous injury. METHODS: We made a full-thickness dorsal wound in transgenic (Tg) mice, in which CCL17 was overexpressed and in control mice. Wound size was compared over the course of time. We evaluated the effect of CCL17 on fibroblast migration by a Boyden chamber assay and a scratch wound assay. RESULTS: Wound closure in Tg mice was more accelerated than in control mice. CCL17 enhanced nerve growth factor (NGF) production by 2B4, which is mouse T cell hybridoma. Further, in the wound area of Tg mice, the number of CCR4(+) fibroblasts, CCR4(+) lymphocytes and mast cells was increased compared to control mice, as was the number of NGF(+) lymphocytes around the wound area. In vitro assay, CCL17 was shown to enhance the migration of fibroblasts. CONCLUSION: These results suggest that CCL17 accelerates wound healing, mainly by enhancing fibroblast migration, and possibly by increasing NGF(+) lymphocytes and mast cells, which have independently been reported to enhance wound healing.

CCL22 is involved in the recruitment of CD4+CD25 high T cells into tuberculous pleural effusions.

BACKGROUND AND OBJECTIVE: CD4(+)CD25(high) regulatory T cells are increased in tuberculous pleural effusions (TPE). However, the mechanism by which CD4(+)CD25(high) T cells infiltrate into the pleural cavity is unknown. The aim of this study was to investigate whether the chemokines CCL22 and CCL17 are present in TPE, and the chemoattractant activity of these chemokines for infiltration of CD4(+)CD25(high) T cells into the pleural space. METHODS: The concentrations of CCL22 and CCL17 were measured in pleural effusions from 33 patients with tuberculous pleurisy, 21 patients with pleural bacterial infections and 18 patients with transudative pleural effusions. T lymphocyte subsets in pleural effusions were assessed by flow cytometry. Pleural effusion cells were analysed for the expression of CCL22. The chemoattractant activity of CCL22 for CD4(+)CD25(high) T cells was assessed in vitro. RESULTS: The frequency of CD4(+)CD25(high) T cells was significantly higher in TPE than in blood. High concentrations of CCL22 were detected in tuberculous effusions, but not in bacterial effusions or transudates. Macrophages and T cells in TPE expressed CCL22. Tuberculous pleural fluid was chemotactic for CD4(+)CD25(high) T cells in vitro, and anti-CCL22 antibody partly inhibited this chemotactic activity. CONCLUSIONS: CCL22 appeared to be increased in TPE compared with bacterial pleural effusions or transudates. CCL22 may be responsible for the infiltration of CD4(+)CD25(high) T cells into the pleural space of patients with tuberculous pleurisy.

T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model.

For the adoptive transfer of tumor-directed T lymphocytes to prove effective, there will probably need to be a match between the chemokines the tumor produces and the chemokine receptors the effector T cells express. The Reed-Stemberg cells of Hodgkin lymphoma (HL) predominantly produce thymus- and activation-regulated chemokine/CC chemokine ligand 17 (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), which preferentially attract type 2 T helper (Th2) cells and regulatory T cells (Tregs) that express the TARC/MDC-specific chemokine receptor CCR4, thus generating an immunosuppressed tumor environment. By contrast, effector CD8(+) T cells lack CCR4, are nonresponsive to these chemokines and are rarely detected at the tumor site. We now show that forced expression of CCR4 by effector T cells enhances their migration to HL cells. Furthermore, T lymphocytes expressing both CCR4 and a chimeric antigen receptor directed to the HL associated antigen CD30 sustain their cytotoxic function and cytokine secretion in vitro, and produce enhanced tumor control when infused intravenously in mice engrafted with human HL. This approach may be of value in patients affected by HL.

Role of CCL17 in the generation of cutaneous inflammatory reactions in Hu-PBMC-SCID mice grafted with human skin.

CCL17 may be of interest in skin inflammation, because it mainly attracts T cells expressing the cutaneous homing receptor and binds the chemokine receptor CCR4, preferentially expressed on Th-2 cells. We evaluated the in vivo effect of CCL17 injection in a humanized mouse model. (125)I-CCL17 injection into human skin grafted on severe combined immunodeficient (SCID) mice reconstituted with peripheral blood mononuclear cells resulted in a rapid transportation of CCL17 from the skin to the homolateral lymph nodes, followed 3 hours later by a lymph node infiltration of human memory CD4+ cells and dendritic cells. Intradermal injection of CCL17 resulted 24 hours later in a cutaneous recruitment of human memory CD4+ cells, monocytes, and basophils, but also murine eosinophils. In SCID mice reconstituted with polarized Th-1 or Th-2 cells, intradermal injection of CCL17 resulted in the recruitment of IL-4+ Th-2 cells but not of IFN-gamma+ Th-1 cells, whereas CCL17 was able to recruit both subsets in vitro. These results suggest that, in a humanized in vivo model, CCL17 is sufficient per se to induce a lymph node recruitment of memory CD4+ and dendritic cells and a cutaneous recruitment of Th-2-type cells, stressing it as an important actor in the initiation and development of Th-2-associated skin inflammation.

IL-12 produced by dendritic cells augments CD8+ T cell activation through the production of the chemokines CCL1 and CCL17.

IL-12 family members are an important link between innate and adaptive immunity. IL-12 drives Th1 responses by augmenting IFN-gamma production, which is key for clearance of intracellular pathogens. IL-23 promotes the development of IL-17-producing CD4(+) T cells that participate in the control of extracellular pathogens and the induction of autoimmunity. However, recent studies have shown that these cytokines can modulate lymphocyte migration and cellular interactions. Therefore, we sought to determine the individual roles of IL-12 and IL-23 in naive CD8(+) T cell activation by addressing their ability to influence IFN-gamma production and cellular interaction dynamics during priming by Listeria monocytogenes-infected dendritic cells (DC). We found that IL-12 was the major cytokine influencing the level of IFN-gamma production by CD8(+) T cells while IL-23 had little effect on this response. In addition, we observed that IL-12 promoted longer duration conjugation events between CD8(+) T cells and DC. This enhanced cognate interaction time correlated with increased production of the chemokines CCL1 and CCL17 by WT but not IL-12-deficient DC. Neutralization of both chemokines resulted in reduced interaction time and IFN-gamma production, demonstrating their importance in priming naive CD8(+) T cells. Our study demonstrates a novel mechanism through which IL-12 augments naive CD8(+) T cell activation by facilitating chemokine production, thus promoting more stable cognate interactions during priming.

CCR4 and CCR10 are expressed on epidermal keratinocytes and are involved in cutaneous immune reaction.

CC chemokine ligand (CCL)17 and CCL27 produced by epidermal keratinocytes (KCs) recruit CC chemokine receptor (CCR)4 and CCR10 expressing T cells into the skin, respectively, resulting in enhanced skin inflammation. However, CCR4/CCL17 and CCR10/CCL27 interactions in epidermal KCs have not been investigated. The purpose of this study was to evaluate the role of the CCR4/CCL17 and CCR10/CCL27 loops in cutaneous immune reaction. Normal human KCs (NHKs) and HaCaT KCs expressed both CCR4 and CCR10 at mRNA and protein levels. CCR4 ligand CCL17 but not CCR10 ligand CCL27 induced production of IL-12 p40, granulocyte/monocyte colony-stimulating factor (GM-CSF) and nerve growth factor (NGF) by KCs. Both CCL17 and CCL27 induced migration of KCs in Boyden chamber assay and wound scratch assay. This study revealed that CCR4 and CCR10 are expressed on epidermal KCs and that both are functional in terms of skin cytokine production and/or migration to their ligand CCL17 and CCL27, respectively. Thus this study provided new insight into chemokine/chemokine receptors of KCs.