Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.

Connexin26 Modulates Radiation-Induced Skin Damage by Regulating Chemokine CCL27 through MAPK Signaling.

Connexin26 (Cx26) plays an important role in ionizing radiation-induced damage, and CC chemokine ligand 27 (CCL27) regulates the skin immune response. However, the relationship between Cx26 and CCL27 in radiation-induced skin damage is unclear. After X-ray irradiation, clonogenic survival and micronucleus formation were assessed in immortalized human keratinocytes (HaCaT). Proteins in the mitogen activated protein kinase (MAPK) signaling pathway and CCL27-related proteins were detected by immunoblotting. HaCaTCx26-/- cells were constructed to verify the effects of Cx26 on CCL27 secretion. A mouse model was established to examine the expression of CCL27 and skin inflammation in vivo. The degree of skin injury induced by 6 MV of X rays was closely related to CCL27. The phosphorylation of ERK, p38 and NF-κB was significantly increased in irradiated cells. The secretion of CCL27 was significantly decreased in HaCaT wild-type cells relative to HaCaTCx26-/- cells. Whereas cell survival fractions decreased, and the micronuclei formation rate increased as a function of increasing X-ray dose in HaCaT cells, the opposite trend occurred in HaCaTCx26-/- cells. Our findings show that Cx26 likely plays a role in the activation of the MAPK and NF-κB/COX-2 signaling pathways and regulates the secretion of CCL27 in keratinocytes after X-ray radiation-induced skin damage.

ZIM3 activation of CCL25 expression in pulmonary metastatic nodules of osteosarcoma recruits M2 macrophages to promote metastatic growth.

Tumor-associated macrophages (TAMs) play an important role in tumor growth and metastasis. However, the involvement of TAMs infiltration in pulmonary osteosarcoma (OS) metastasis remains poorly understood. Therefore, the effect of OS cells on macrophages migration was investigated by in vivo and in vitro experiments to evaluate the infiltration and mechanism of TAMs in pulmonary OS metastases. The results showed that the zinc finger protein ZIM3 was upregulated in OS cells than in osteoblasts and activated the expression of CCL25, which subsequently promoted the migration of M2 macrophages. CCL25 or ZIM3 silencing in OS cells inhibited the infiltration of M2 macrophages and the formation of pulmonary metastatic nodules in a mouse model of pulmonary OS metastasis and prolonged the survival of the mice. Furthermore, bioinformatics analyses revealed that CCL25 and ZIM3 expressions are negatively correlated with the prognosis of OS patients. In conclusion, this study found that a large number of M2 TAMs were recruited into pulmonary metastatic nodules of OS through the activation of the ZIM3-CCL25 axis in OS cells, thereby facilitating OS metastasis. Therefore, the suppression of ZIM3-CCL25-induced recruitment of M2 TAMs to the metastatic sites might be considered as a therapeutic approach to inhibit the growth of pulmonary OS metastases.

Therapies with CCL25 require controlled release via microparticles to avoid strong inflammatory reactions.

BACKGROUND: Chemokine therapy with C-C motif chemokine ligand 25 (CCL25) is currently under investigation as a promising approach to treat articular cartilage degeneration. We developed a delayed release mechanism based on Poly (lactic-co-glycolic acid) (PLGA) microparticle encapsulation for intraarticular injections to ensure prolonged release of therapeutic dosages. However, CCL25 plays an important role in immune cell regulation and inflammatory processes like T-cell homing and chronic tissue inflammation. Therefore, the potential of CCL25 to activate immune cells must be assessed more thoroughly before further translation into clinical practice. The aim of this study was to evaluate the reaction of different immune cell subsets upon stimulation with different dosages of CCL25 in comparison to CCL25 released from PLGA particles. RESULTS: Immune cell subsets were treated for up to 5 days with CCL25 and subsequently analyzed regarding their cytokine secretion, surface marker expression, polarization, and migratory behavior. The CCL25 receptor C-C chemokine receptor type 9 (CCR9) was expressed to a different extent on all immune cell subsets. Direct stimulation of peripheral blood mononuclear cells (PBMCs) with high dosages of CCL25 resulted in strong increases in the secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-1ß (IL-1ß), tumor-necrosis-factor-α (TNF-α) and interferon-γ (IFN-γ), upregulation of human leukocyte antigen-DR (HLA-DR) on monocytes and CD4+ T-cells, as well as immune cell migration along a CCL25 gradient. Immune cell stimulation with the supernatants from CCL25 loaded PLGA microparticles caused moderate increases in MCP-1, IL-8, and IL-1ß levels, but no changes in surface marker expression or migration. Both CCL25-loaded and unloaded PLGA microparticles induced an increase in IL-8 and MCP-1 release in PBMCs and macrophages, and a slight shift of the surface marker profile towards the direction of M2-macrophage polarization. CONCLUSIONS: While supernatants of CCL25 loaded PLGA microparticles did not provoke strong inflammatory reactions, direct stimulation with CCL25 shows the critical potential to induce global inflammatory activation of human leukocytes at certain concentrations. These findings underline the importance of a safe and reliable release system in a therapeutic setup. Failure of the delivery system could result in strong local and systemic inflammatory reactions that could potentially negate the benefits of chemokine therapy.

Innate Cytokine Induced Early Release of IFNγ and CC Chemokines from Hypoxic Human NK Cells Is Independent of Glucose.

Natural killer (NK) cells are among the first innate immune cells to arrive at sites of tissue inflammation and regulate the immune response to infection and tumors by the release of cytokines including interferon (IFN)γ. In vitro exposure to the innate cytokines interleukin 15 (IL-15) and IL-12/IL-18 enhances NK cell IFNγ production which, beyond 16 h of culture, was shown to depend on metabolic switching to glycolysis. NK effector responses are, however, rapid by comparison. Therefore, we sought to evaluate the importance of glycolysis for shorter-term IFNγ production, considering glucose deprivation and hypoxia as adverse tissue inflammation associated conditions. Treatments with IL-15 for 6 and 16 h were equally effective in priming early IFNγ production in human NK cells in response to secondary IL-12/IL-18 stimulation. Short-term priming was not associated with glycolytic switching but induced the release of IFNγ and, additionally, CCL3, CCL4 and CCL5 from both normoxic and hypoxic NK cells in an equally efficient and, unexpectedly, glucose independent manner. We conclude that release of IFNγ and CC chemokines in the early innate immune response is a metabolically autonomous NK effector program.

Intratumoral delivery of CCL25 enhances immunotherapy against triple-negative breast cancer by recruiting CCR9+ T cells.

CCR9+ T cells have an increased potential to be activated and therefore may mediate strong antitumor responses. Here, we found, however, that CCL25, the only chemokine for CCR9+ cells, is not expressed in human or murine triple-negative breast cancers (TNBCs), raising a hypothesis that intratumoral delivery of CCL25 may enhance antitumor immunotherapy in TNBCs. We first determined whether this approach can enhance CD47-targeted immunotherapy using a tumor acidity-responsive nanoparticle delivery system (NP-siCD47/CCL25) to sequentially release CCL25 protein and CD47 small interfering RNA in tumor. NP-siCD47/CCL25 significantly increased infiltration of CCR9+CD8+ T cells and down-regulated CD47 expression in tumor, resulting in inhibition of tumor growth and metastasis through a T cell-dependent immunity. Furthermore, the antitumor effect of NP-siCD47/CCL25 was synergistically enhanced when used in combination with programmed cell death protein-1/programmed death ligand-1 blockades. This study offers a strategy to enhance immunotherapy by promoting CCR9+CD8+ T cell tumor infiltration.

CCL28-induced RARß expression inhibits oral squamous cell carcinoma bone invasion.

Oral squamous cell carcinoma (OSCC) frequently invades the maxillary or mandibular bone, and this bone invasion is closely associated with poor prognosis and survival. Here, we show that CCL28 functions as a negative regulator of OSCC bone invasion. CCL28 inhibited invasion and epithelial-mesenchymal transition (EMT), and its inhibition of EMT was characterized by induced E-cadherin expression and reduced nuclear localization of ß-catenin in OSCC cells with detectable RUNX3 expression levels. CCL28 signaling via CCR10 increased retinoic acid receptor-ß (RARß) expression by reducing the interaction between RARα and HDAC1. In addition, CCL28 reduced RANKL production in OSCC and osteoblastic cells and blocked RANKL-induced osteoclastogenesis in osteoclast precursors. Intraperitoneally administered CCL28 inhibited tumor growth and osteolysis in mouse calvaria and tibia inoculated with OSCC cells. RARß expression was also increased in tumor tissues. In patients with OSCC, low CCL28, CCR10, and RARß expression levels were highly correlated with bone invasion. Patients with OSCC who had higher expression of CCL28, CCR10, or RARß had significantly better overall survival. These findings suggest that CCL28, CCR10, and RARß are useful markers for the prediction and treatment of OSCC bone invasion. Furthermore, CCL28 upregulation in OSCC cells or CCL28 treatment can be a therapeutic strategy for OSCC bone invasion.

CCL25-Supplemented Hyaluronic Acid Attenuates Cartilage Degeneration in a Guinea Pig Model of Knee Osteoarthritis.

There is evidence that the application of mesenchymal stromal cells (MSCs) counteracts osteoarthritis (OA) progression. However, the prospect of extracting and expanding these cells might be limited. The aim of this study was to investigate whether hyaluronic acid (HA) supplemented with MSC-recruiting chemokine C-C motif ligand 25 (CCL25) can influence the natural course of spontaneous OA in the guinea pig. CCL25 concentration in synovial fluid (SF) was quantified with enzyme-linked immunosorbent assay. Boyden chamber cell migration assay was used to test CCL25-mediated migration of guinea pig MSC. Forty-nine 11-month-old male guinea pigs were divided into seven groups. The main treatments consisted of five intra-articular injections of HA in pure form and in combination with three doses of CCL25 (63, 693, and 6,993 pg) given at a weekly interval. The severity of cartilage damage was assessed by using a modified Mankin score. The measured average physiological concentration of CCL25 in SF of animals is 85 ± 39 pg/ml. MSC showed a 3.2-fold increase in cell migration at 1,000 nM CCL25 in vitro demonstrating the biological migratory activity of CCL25 on these cells. In vivo, treatment with HA alone did not reduce OA progression. Similarly, OA scores were not found significantly reduced after treatment with 63 pg CCL25 + HA. However, when compared to pure HA, treatment with 693 pg CCL25 + HA and 6,993 pg CCL25 + HA significantly reduced the OA score from 10.1 to 7.4 (-28%) and 8.4 (-20%), respectively. These data suggest that intra-articular injections of HA supplemented with CCL25 attenuates OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1723-1729, 2019.

TAFA2 Induces Skeletal (Stromal) Stem Cell Migration Through Activation of Rac1-p38 Signaling.

Understanding the mechanisms regulating recruitment of human skeletal (stromal or mesenchymal) stem cells (hMSC) to sites of tissue injury is a prerequisite for their successful use in cell replacement therapy. Chemokine-like protein TAFA2 is a recently discovered neurokine involved in neuronal cell migration and neurite outgrowth. Here, we demonstrate a possible role for TAFA2 in regulating recruitment of hMSC to bone fracture sites. TAFA2 increased the in vitro trans-well migration and motility of hMSC in a dose-dependent fashion and induced significant morphological changes including formation of lamellipodia as revealed by high-content-image analysis at single-cell level. Mechanistic studies revealed that TAFA2 enhanced hMSC migration through activation of the Rac1-p38 pathway. In addition, TAFA2 enhanced hMSC proliferation, whereas differentiation of hMSC toward osteoblast and adipocyte lineages was not altered. in vivo studies demonstrated transient upregulation of TAFA2 gene expression during the inflammatory phase of fracture healing in a closed femoral fracture model in mice, and a similar pattern was observed in serum levels of TAFA2 in patients after hip fracture. Finally, interleukin-1ß was found as an upstream regulator of TAFA2 expression. Our findings demonstrate that TAFA2 enhances hMSC migration and recruitment and thus is relevant for regenerative medicine applications. Stem Cells 2019;37:407-416.

CCL25 chemokine-guided stem cell attraction: an assessment of possible benefits and risks.

Due to its chemoattraction potential on mesenchymal stromal cells of the CCL25/CCR9 axis, local application of CCL25 to severely damaged tissues may be a promising approach for regenerative therapies. Analysis of the given data revealed that CCL25/CCR9 signaling has a crucial role in regulation of an adult immune homeostasis. CCR9 expression variations resulted in dysfunctional immune response in colitis, rheumatoid arthritis and endometriosis. Regarding oncology, different neoplastic tissues exploit CCL25-dependent CCR9 signaling for either local proliferation or migration processes. The CCR9 pathway likely can trigger crosstalk between the Akt and NOTCH pathway and thus participate in the regulation of the neoplastic behavior. In conclusion, the designated application-tissue requires precise molecular analysis of possible CCR9 expression due to its proto-oncogenic characteristics.

A prognosis and impact factor analysis of DC-CIK cell therapy for patients with hepatocellular carcinoma undergoing postoperative TACE.

Dendritic cell-cytokine-induced killer (DC-CIK) cell therapy has been experimentally implemented for enhancing anti-tumoral immunity in patients with hepatocellular carcinoma (HCC) undergoing postoperative transcatheter arterial chemoembolization (POTACE). We performed a retrospective study to evaluate the clinical efficacies of DC-CIK cell therapy and its correlations with several immune factors of the primary tumors. The overall survival time of HCC patients with HBV infection in the study group (POTACE plus DC-CIK cell therapy) was significantly longer than that of the control group (POTACE alone). The expression level of PD-L1 but not the tumor-infiltrated CD8 and CD4 T cells in the tumor tissues showed significant negative correlations with relapse-free survival (RFS) and overall survival (OS), which was also an independent prognostic factor for the five-years' suvival of patients with HCC receiving POTACE treatment. Furthermore, our study validated that PD-L1 expression was significantly inversely correlated with the survival time of HCC patients receiving POTACE plus DC-CIK cell therapy treatment. More importantly, DC-CIK cell therapy provided the best clinical benefits to HCC patients with the low PD-L1 expression receiving POTACE, which indicate that PD-L1 expression level can serve as a pivotal predictor for the therapeutic efficacy of DC-CIK cell therapy for HCC patients receiving POTACE treatment.

Delayed release of chemokine CCL25 with bioresorbable microparticles for mobilization of human mesenchymal stem cells.

Chemokines are guiding cues for directional trafficking of mesenchymal stem cells (MSC) upon injury and local chemokine delivery at injury sites is an up-to-date strategy to potentiate and prolong recruitment of MSC. In this study we present the chemokine CCL25, also referred to as thymus-expressed chemokine, to mobilize human MSC along positive but not along negative gradients. We hence proceeded to design a biodegradable and injectable release device for CCL25 on the basis of poly(lactic-co-glycolic acid) (PLGA). The conducted studies had the objective to optimize PLGA microparticle fabrication by varying selected formulation parameters, such as polymer type, microparticle size and interior phase composition. We found that microparticles of DV,50∼75 µm and fabricated using end-capped polymers, BSA as carrier protein and vortex mixing to produce the primary emulsion yielded high chemokine loading and delayed CCL25 release. To determine bioactivity, we investigated CCL25 released during the microparticle erosion phase and showed that deacidification of the release medium was required to induce significant MSC mobilization. The designed PLGA microparticles represent an effective and convenient off-the-shelf delivery tool for the delayed release of CCL25. However, continuative in vivo proof-of-concept studies are required to demonstrate enhanced recruitment of MSC and/or therapeutical effects in response to CCL25 release microparticles. STATEMENT OF SIGNIFICANCE: With the discovery of chemokines, particularly CXCL12, as stimulators of stem cell migration, the development of devices that release CXCL12 has proceeded quickly in the last few years. In this manuscript we introduce CCL25 as chemokine to induce mobilization of human MSC. This study proceeds to demonstrate how selection of key formulation parameters of CCL25 loading into PLGA microparticles exerts considerable influence on CCL25 release. This is important for a broad range of efforts in in situ tissue engineering where the candidate chemokine and the delivery device need to be selected carefully. The use of such a cell-free CCL25 release device may provide a new therapeutic option in regenerative medicine.

Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets.

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1.

Thymus-expressed chemokine enhances Porphyromonas gingivalis LPS-induced osteoclast formation via NFATc1 activation.

OBJECTIVE: P. gingivalis is a gram-negative anaerobic bacterium and a major periodontal pathogen. LPS produced by P. gingivalis promotes osteoclast formation. TECK is a CC chemokine whose expression is increased in gingival epithelial cells exposed to P. gingivalis LPS. In this study, we investigated the effect of TECK in osteoclastogenesis induced by P. gingivalis LPS. DESIGNS: Real time reverse transcriptase polymerase chain reaction (RTPCR) analysis and western blotting were performed to confirm TECK in MG63, human osteoblast cell line and primary murine osteoblasts and CCR9 in RAW 264.7 cells and murine bone marrow macrophages (BMMs) as osteoclast precursors. P. gingivalis LPS-treated BMMs and Raw 264.7 cells were cultured with or without TECK or TECK antibody to examine the effect of TECK on osteoclast formation. Cocultures with murine osteoblasts and bone marrow cells were also treated with or without TECK or TECK antibody. Luciferase assay and western blotting were used to determine whether TECK-CCR9 induced osteoclastogenesis was mediated through NFATc1 or NF-kB signaling. RESULTS: TECK was shown to be expressed by osteoblasts, and its receptor, CCR9, by osteoclast precursors. TECK increased P. gingivalis LPS-induced osteoclast numbers in an in vitro osteoclast formation assay using osteoclast precursors. The enhanced osteoclast formation by TECK was mediated by NFATc1, but not by NF-kB signaling. CONCLUSION: TECK may be a novel regulator of osteoclast formation induced by P. gingivalis LPS in periodontitis.

Let-7a mimic attenuates CCL18 induced breast cancer cell metastasis through Lin 28 pathway.

BACKGROUND: MicroRNAs are believed to influence breast cancer cell tumorgenicity by interacting with the production of tumor associated macrophages. At this stage, this hypothesis lacks sufficient empirical evidence. Our study is an investigation of the effects of let-7a on the function of human breast cancer cell lines that had undergone chemokine ligand 18 (CCL18) stimulation. METHODS: Two breast cancer cell lines MDA-MB-231 and MCF-7 were transfected with let-7a mimics with or without CCL18 simulation. The expression level of let-7a was evaluated with qRT-PCR. Our study examined cell proliferation, migration and cell cycles following let-7a treatment. The predicted target of let-7a was identified and confirmed in vitro by a dual luciferase reporter system. The associations between let-7a, CCL18 and target gene expression were evaluated using RT-PCR and the Western blotting method. RESULTS: The downregulated expression level of let-7a was observed in both breast cancer cell lines. When compared to the control and CCL18 stimulation groups, cell proliferation and migration in MDA-MB-231 and MCF-7 cells were significantly inhibited by let-7a. Furthermore, the cell cycle was dramatically blocked at the G2/M phase. The luciferase reporter identified Lin28 as the direct binding target of let-7a in both breast cancer cell lines. CONCLUSION: Upregulation of let-7a carries the potential to reverse CCL18 induced cell proliferation and migration alteration in breast cancer cells by regulating Lin28 expression. Our results provided evidence which suggests the use of let-7a as a therapeutic agent in the treatment of breast cancer.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice.

INTRODUCTION: Biological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn's disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA. METHODS: CCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b+ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted. RESULTS: CCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b+ splenocytes into the synovial tissues. CONCLUSIONS: The interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.

Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages.

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD4(+) CD25(hi) FoxP3(+) T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-ß1 mediates induction of Tregs. TGF-ß1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-ß1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-ß1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC.

A real time chemotaxis assay unveils unique migratory profiles amongst different primary murine macrophages.

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14(+) human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

Identification of the chemokine CCL28 as a growth and survival factor for human hematopoietic stem and progenitor cells.

In an attempt to discover novel growth factors for hematopoietic stem and progenitor cells (HSPCs), we have assessed cytokine responses of cord blood (CB)-derived CD34(+) cells in a high-content growth factor screen. We identify the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor that directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins. CCL28 enhances the functional progenitor cell content of cultured cells by stimulating cell cycling and induces gene expression changes associated with survival. Importantly, addition of CCL28 to cultures of purified putative hematopoietic stem cells (HSCs) significantly increases the ability of the cells to long-term repopulate immunodeficient mice compared with equivalent input numbers of fresh cells. Together, our findings identify CCL28 as a potent growth-promoting factor with the ability to support the in vitro and in vivo functional properties of cultured human hematopoietic cells.