Fulvic Acid Attenuates Atopic Dermatitis by Downregulating CCL17/22.

The main pathogenic factor in atopic dermatitis (AD) is Th2 inflammation, and levels of serum CCL17 and CCL22 are related to severity in AD patients. Fulvic acid (FA) is a kind of natural humic acid with anti-inflammatory, antibacterial, and immunomodulatory effects. Our experiments demonstrated the therapeutic effect of FA on AD mice and revealed some potential mechanisms. FA was shown to reduce TARC/CCL17 and MDC/CCL22 expression in HaCaT cells stimulated by TNF-α and IFN-γ. The inhibitors showed that FA inhibits CCL17 and CCL22 production by deactivating the p38 MAPK and JNK pathways. After 2,4-dinitrochlorobenzene (DNCB) induction in mice with atopic dermatitis, FA effectively reduced the symptoms and serum levels of CCL17 and CCL22. In conclusion, topical FA attenuated AD via downregulation of CCL17 and CCL22, via inhibition of P38 MAPK and JNK phosphorylation, and FA is a potential therapeutic agent for AD.

CCL17 Aggravates Myocardial Injury by Suppressing Recruitment of Regulatory T Cells.

BACKGROUND: Recent studies have established that CCR2 (C-C chemokine receptor type 2) marks proinflammatory subsets of monocytes, macrophages, and dendritic cells that contribute to adverse left ventricle (LV) remodeling and heart failure progression. Elucidation of the effector mechanisms that mediate adverse effects of CCR2+ monocytes, macrophages, and dendritic cells will yield important insights into therapeutic strategies to suppress myocardial inflammation. METHODS: We used mouse models of reperfused myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate CCL17 (C-C chemokine ligand 17). We used Ccl17 knockout mice, flow cytometry, RNA sequencing, biochemical assays, cell trafficking studies, and in vivo cell depletion to identify the cell types that generate CCL17, define signaling pathways that controlled its expression, delineate the functional importance of CCL17 in adverse LV remodeling and heart failure progression, and determine the mechanistic basis by which CCL17 exerts its effects. RESULTS: We demonstrated that CCL17 is expressed in CCR2+ macrophages and cluster of differentiation 11b+ conventional dendritic cells after myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation. We clarified the transcriptional signature of CCL17+ macrophages and dendritic cells and identified granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling as a key regulator of CCL17 expression through cooperative activation of STAT5 (signal transducer and activator of transcription 5) and canonical NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling. Ccl17 deletion resulted in reduced LV remodeling, decreased myocardial fibrosis and cardiomyocyte hypertrophy, and improved LV systolic function after myocardial infarction and angiotensin II and phenylephrine infusion. We observed increased abundance of regulatory T cells (Tregs) in the myocardium of injured Ccl17 knockout mice. CCL17 inhibited Treg recruitment through biased activation of CCR4. CCL17 activated Gq signaling and CCL22 (C-C chemokine ligand 22) activated both Gq and ARRB (ß-arrestin) signaling downstream of CCR4. CCL17 competitively inhibited CCL22 stimulated ARRB signaling and Treg migration. We provide evidence that Tregs mediated the protective effects of Ccl17 deletion on myocardial inflammation and adverse LV remodeling. CONCLUSIONS: These findings identify CCL17 as a proinflammatory mediator of CCR2+ macrophages and dendritic cells and suggest that inhibition of CCL17 may serve as an effective strategy to promote Treg recruitment and suppress myocardial inflammation.

Recombinant CCL17 Enhances Hematoma Resolution and Activation of CCR4/ERK/Nrf2/CD163 Signaling Pathway After Intracerebral Hemorrhage in Mice.

Hematoma is a crucial factor leading to poor prognosis after intracerebral hemorrhage (ICH). Promoting microglial phagocytosis to enhance hematoma resolution may be an important therapeutic target for recovery after ICH. C-C chemokine receptor 4 (CCR4) is important for regulating immune balance in the central nervous system. However, whether CCR4 activation can attenuate hematoma after ICH remains unknown. We aimed to evaluate whether CCL17 (a specific ligand of CCR4) treatment can promote hematoma resolution through CCR4/ERK/Nrf2/CD163 pathway after ICH. A total of 261 adult male CD1 mice were used. Mice were subjected to intrastriatal injection of autologous blood to induce ICH and randomly assigned to receive recombinant CCL17 (rCCL17) or vehicle which was administered intranasally at 1 h after ICH. To elucidate the underlying mechanism, C021, a selective inhibitor of CCR4 and ML385 and a selective inhibitor of Nrf2 were administered 1 h prior to ICH induction. Clustered regularly interspaced short palindromic repeats (CRISPR) knockout for CD163 was administered by intracerebroventricular injection at 48 h before ICH. Brain edema, short- and long-term neurobehavior evaluation, hematoma volume, hemoglobin content, western blot, and immunofluorescence staining were performed. Endogenous CCL17, CCR4, and CD163 expression increased and peaked at 72 h after ICH. CCR4 was expressed by microglia. CCR4 activation with rCCL17 significantly improved neurobehavioral scores and reduced hematoma volume and brain edema compared with vehicle. Moreover, rCCL17 treatment significantly promoted phosphorylation of ERK1/2, increased the expression Nrf2, and upregulated CD163 expression after ICH. The protective effects of rCCL17 were abolished by administration of C021, ML385, and CD163 CRISPR knockout. This study demonstrated that CCR4 activation with rCCL17 promoted hematoma resolution by increasing CD163 expression and CCR4/ERK/Nrf2 pathway activation after ICH, thereby reducing brain edema and improving neurological function. Overall, our study suggests that CCR4 activation may be a potential therapeutic strategy to attenuate hematoma in early brain injury after ICH.

Role of Chemokine Receptor CCR4 and Regulatory T Cells in Wound Healing of Diabetic Mice.

Wound healing is a well-coordinated process that involves inflammatory mediators and cellular responses; however, if any disturbances are present during this process, tissue repair is impaired. Chronic wounds are one of the serious long-term complications associated with diabetes mellitus. The chemokine receptor CCR4 and its respective ligands, CCL17 and CCL22, are involved in regulatory T cell recruitment and activation in inflamed skin; however, the role of regulatory T cells in wounds is still not clear. Our aim was to investigate the role of CCR4 and regulatory T cells in cutaneous wound healing in diabetic mice. Alloxan-induced diabetic wild- type mice (diabetic) developed wounds that were difficult to heal, differently from CCR4-/- diabetic mice (CCR4-/- diabetic), and also from anti-CCL17/22 or anti-CD25-injected diabetic mice that presented with accelerated wound healing and fewer regulatory T cells in the wound bed. Consequently, CCR4-/- diabetic mice also presented with alteration on T cells population in the wound and draining lymph nodes; on day 14, these mice also displayed an increase of collagen fiber deposition. Still, cytokine levels were decreased in the wounds of CCR4-/- diabetic mice on day 2. Our data suggest that the receptor CCR4 and regulatory T cells negatively affect wound healing in diabetic mice.

A CCR4 antagonist reverses the tumor-promoting microenvironment of renal cancer.

Elevated expression of the chemokine receptor CCR4 in tumors is associated with poor prognosis in several cancers. Here, we have determined that CCR4 was highly expressed in human renal cell carcinoma (RCC) biopsies and observed abnormal levels of CCR4 ligands in RCC patient plasma. An antagonistic anti-CCR4 antibody had antitumor activity in the RENCA mouse model of RCC. CCR4 inhibition did not reduce the proportion of infiltrating leukocytes in the tumor microenvironment but altered the phenotype of myeloid cells, increased NK cell and Th1 cytokine levels, and reduced immature myeloid cell infiltrate and blood chemokine levels. In spite of prominent changes in the myeloid compartment, the anti-CCR4 antibody did not affect RENCA tumors in T cell-deficient mice, and treatment with an anti-class II MHC antibody abrogated its antitumor activity. We concluded that the effects of the anti-CCR4 antibody required the adaptive immune system and CD4+ T cells. Moreover, CCL17-induced IFN-γ production was reduced when Th1-polarized normal CD4+ T cells were exposed to the CCR4 ligand, evidencing the involvement of CCR4 in Th1/Th2 regulation. The anti-CCR4 antibody, alone or in combination with other immune modulators, is a potential treatment approach to human solid cancers with high levels of CCR4-expressing tumor-infiltrating leukocytes and abnormal plasma CCR4 ligand levels.

Furin-dependent CCL17-fused recombinant toxin controls HTLV-1 infection by targeting and eliminating infected CCR4-expressing cells in vitro and in vivo.

BACKGROUND: Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) infection. However, there are no therapies to prevent ATL development in high-risk asymptomatic carriers. To develop a therapy targeting HTLV-1-infected cells that are known to express CCR4 frequently, we tested whether truncated Pseudomonas exotoxin (PE38) fused to a CCR4 ligand, CCL17/thymus and activation-regulated chemokine (TARC), selectively eliminates such cells. RESULTS: Our data show that TARC-PE38 efficiently killed HTLV-1-infected cell lines. It also shrank HTLV-1-associated solid tumors in an infected-cell-engrafted mouse model. In HTLV-1-positive humanized mice, TARC-PE38 markedly inhibited the proliferation of HTLV-1-infected human CD4(+)CD25(+) or CD4(+)CD25(+)CCR4(+) cells and reduced the proviral loads (PVLs) in peripheral blood mononuclear cells (PBMCs). Importantly, TARC-PE38 significantly reduced the PVLs in PBMCs obtained from asymptomatic carriers. We show that the cytotoxicity of TARC-PE38 is mediated by the expression of the proprotein convertase, furin. The expression of furin was enhanced in HTLV-1-infected cells and correlated positively with PVLs in HTLV-1-infected individuals, suggesting that infected cells are more susceptible to TARC-PE38 than normal cells. CONCLUSIONS: TARC-PE38 robustly controls HTLV-1 infection by eliminating infected cells in both a CCR4- and furin-dependent manner, indicating the excellent therapeutic potential of TARC-PE38.

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists.

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to ß-arrestin and stimulated GTPγS binding however CCL17 did not couple to ß-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.

Targeted reduction of CCR4⁺ cells is sufficient to suppress allergic airway inflammation.

BACKGROUND: Bronchial asthma is characterized by allergic airway inflammation involving C-C chemokine receptor type 4 (CCR4)-positive Th2 cells. As such, we hypothesize that the disease can be alleviated by targeted-elimination of CCR4⁺ cells. Thymus and activation-regulated chemokine (TARC)-PE38, a TARC fused the exotoxin fragment PE38 from Pseudomonas aeruginosa, has been shown to efficiently kill CCR4⁺ cells by delivering the exotoxin fragment PE38 into CCR4⁺ cells. To test our hypothesis, we examined whether TARC-PE38 could suppress allergic airway inflammation in a mouse model of house dust mite (HDM)-induced allergic airway inflammation. METHODS: We evaluated the effect of TARC-PE38 on the major characteristics of HDM-induced allergic airway inflammation. Airway hyperresponsiveness, lung histopathology, lung Th1/Th2 cell populations, and concentrations of Th1/Th2 cytokines in the lungs were assessed in HDM-sensitized and challenged mice in the presence and absence of TARC-PE38. RESULTS: TARC-PE38 efficiently suppressed allergic airway inflammation by significantly reducing airway hyperresponsiveness, the overall area of inflammation, and goblet cell hyperplasia. In HDM-sensitized and challenged mice, TARC-PE38 specifically reduced the numbers of CCR4⁺ cells. This reduction was associated with a significant decrease in the production of Th2 cytokines in the airway,and a decrease in the number of leukocytes, including macrophages, eosinophils and lymphocytes, within the subepithelial area of the lungs and airway lumen. TARC-PE38 had noeffect on Th1 cells. CONCLUSION: Our data suggest that the elimination of CCR4⁺ cells via TARC-PE38 treatment is sufficient to control allergic airway inflammation and airway hyperresponsiveness.

[Expression and function of chemokine TARC/CCR4 at fetal-maternal interface in first trimester].

OBJECTIVE: To investigate the expression and function of thymus and activation regulated chemokine (TARC) and its special receptor CCR4 at placenta villous in the first trimester placenta villous. METHODS: Placenta villous was collected from healthy women undergoing artificial abortion at 6 to 8 weeks of gestation. mRNA levels of TARC, CCR4 were analyzed using semi-quantitative reverse transcription (RT)-PCR methods. Immunohistochemistry assay was used to assess the protein localization and expression of TARC, CCR4. Additionally, extravillous cytotrophoblasts were isolated and cultured. Expression of TARC and CCR4 was measured by immunofluorescence assay. Invasion of cell line HTR8/SVneo was analyzed by transwell assay at concentration of 10, 25, 50 and 100 ng/ml of TARC matched with RPMI 1640 fetal bovine serum free culture medium as control group. In the mean time, blocking experiment was also added to detect TARC regulating cell invasion, which were classified into four groups: control, 100 ng/ml rhTARC, 20 µg/ml anti-TARC+100 ng/ml rhTARC, 100 ng/ml rhTARC+20 µg/ml IgG. The influence of 100 ng/ml TARC on expression level of integrin-α5 and integrin-ß1 were measured by using western-blot assay. RESULTS: (1) In vivo assay:expression of TARC and CCR4 mRNA were detectable in first trimester placenta villous, TARC protein was localized in cytotrophoblasts, syncytiotrophoblasts and cell column especially on the distal portion, while CCR4 protein was localized on invading interstitial cytotrophobalsts. (2) In vitro assay: a. TARC, CCR4 was also expressed in primary isolated extravillous cytotrophoblasts by immunofluorescence assay; b. Matrigel invasion assay demonstrated that TARC had specific dose dependent stimulatory effects on the cells invading through the matrigel precoated filter, the number of cells migration into the lower chamber were:142±31 at 10 ng/ml group, 161±46 at 25 ng/ml group, 201±30 at 50 ng/ml group, 312±48 at 100 ng/ml group, 117±33 at control group, the significant response observed from 25 ng/ml (P<0.05) and reached a peak effect at 100 ng/ml (P<0.01); c. Blocking experiment demonstrated that when trophoblast invasion was monitored in response to TARC neutralizing antibody (15 µg/ml) together with rhTARC 100 ng/ml. The stimulatory activity of rhTARC was completely overcome, with the cells invasion into the lower chambers were 100 ng/ml rhTARC, 20 µg/ml anti-TARC+100 ng/ml rhTARC, 100 ng/ml rhTARC+20 µg/ml IgG, control: 313±47, 113±41, 287±75 and 128±23, respectively; d. Western-blot assay demonstrated that if cells were treated with 100 ng/ml rhTARC, the expression of integrin-α5 were significantly increased (P<0.01), integrin-ß1 level also increased when compared with control (P<0.05). CONCLUSION: TARC was expressed specifically at human fetal-maternal interface. Trophoblast invasion and migration mainly was regulated by up-regulation integrin-α5 and integrin-ß1, which plays an role in trophoblasts differentiation and placentation.

In situ prior proliferation of CD4+ CCR6+ regulatory T cells facilitated by TGF-ß secreting DCs is crucial for their enrichment and suppression in tumor immunity.

BACKGROUND: CD4(+)CD25(+) regulatory T cells (Tregs), a heterogeneous population, were enrichment in tumor mass and played an important role in modulating anti-tumor immunity. Recently, we reported a Treg subset, CCR6(+) Tregs but not CCR6(-)Tregs, were enriched in tumor mass and closely related to poor prognosis of breast cancer patients. However, the underlying mechanism remains elusive. Here, we carefully evaluate the enrichment of CCR6(+)Tregs in tumor mass during progression of breast cancer and explore its possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The frequency of CCR6(+)Tregs in tumor infiltrating lymphocytes (TILs ) was analyzed at early stage and at late stage of tumor in a murine breast cancer model by FACS respectively. The expansion of CCR6(+)Tregs and their CCR6(-) counterpart in tumor mass were determined by BrdU incorporation assay. The effect and its possible mechanism of tumor-resident antigen presenting cells (APCs) on the proliferation of CCR6(+)Tregs also were evaluated. The role of local expansion of CCR6(+)Tregs in their enrichment and suppression in vivo also was evaluated in adoptive cell transfer assay. We found that the prior enrichment of CCR6(+)Tregs but not CCR6(-)Tregs in tumor mass during progression of murine breast cancer, which was dependent on the dominant proliferation of CCR6(+) Tregs in situ. Further study demonstrated that tumor-resident DCs triggered the proliferation of CCR6(+)Treg cells in TGF-ß dependent manner. Adoptive transfer of CCR6(+)Tregs was found to potently inhibit the function of CD8(+)T cells in vivo, which was dependent on their proliferation and subsequently enrichment in tumor mass. CONCLUSIONS/SIGNIFICANCE: Our finding suggested that CCR6(+) Tregs, a distinct subset of Tregs, exert its predominant suppressive role in tumor immunity through prior in situ expansion, which might ultimately provide helpful thoughts for the designing of Treg-based immunotherapy for tumor in the future.

In vivo imaging of cutaneous T-cell lymphoma migration to the skin.

Cutaneous T-cell lymphoma (CTCL) is characterized by the accumulation of malignant CD4(+) T cells in the skin. Although the expression of adhesion molecules and chemokine receptors on CTCL cells has been studied extensively on ex vivo isolated cells, very little is known about the dynamics and mechanisms of CTCL trafficking in vivo. However, detailed knowledge of the molecular cues mediating CTCL migration may be used to interfere with their homing to the skin. We made use of real-time intravital epifluorescence video and two-photon microscopy to visualize malignant T cells from Sezary syndrome (SS), a leukemic variant of CTCL, in dermal microvessels in mouse ear skin. We found that SS cells rolled along dermal venules in a P-selectin- and E-selectin-dependent manner at ratios similar to CD4(+) memory T cells from normal donors. We furthermore show that the chemokine CCL17/TARC, but not CCL27/CTACK, was sufficient to induce the arrest of SS cells in the microvasculature. However, a combination of both chemokines was required to induce extravasation of SS cells. Together, our experiments delineate the molecular adhesion cascade operant in SS cell homing to the skin in vivo.

A systemic granulomatous response to Schistosoma mansoni eggs alters responsiveness of bone-marrow-derived macrophages to Toll-like receptor agonists.

Macrophages play a pivotal role in innate and acquired immune responses to Schistosoma mansoni. Classical (M1) or alternative (M2) activation states of these cells further delineate their roles in tissue damage through innate immunity or fibrotic remodeling, respectively. In the present study, we addressed the following question: Does systemic Th2-type cytokine polarization evoked by S. mansoni affect macrophage differentiation and activation? To this end, we analyzed bone marrow-derived macrophages from mice with S. mansoni egg-induced pulmonary granulomas and unchallenged (or naïve) mice to determine their activation state and their response to specific TLR agonists, including S. mansoni egg antigens. Unlike naïve macrophages, macrophages from Th2-polarized mice constitutively expressed significantly higher "found in inflammatory zone-1" (FIZZ1) and ST2 (M2 markers) and significantly lower NO synthase 2, CCL3, MIP-2, TNF-alpha, and IL-12 (M1 markers). Also, compared with naïve macrophages, Th2-polarized macrophages exhibited enhanced responses to the presence of specific TLR agonists, which consistently induced significantly higher levels of gene and protein levels for M2 and M1 markers in these cells. Together, these data show that signals received by bone marrow precursors during S. mansoni egg-induced granuloma responses dynamically alter the development of macrophages and enhance the TLR responsiveness of these cells, which may ultimately have a significant effect on the pulmonary granulomatous response.