Chemokine Network Research Articles

Using evasins to target the chemokine network in inflammation.

Inflammation, is driven by a network comprising cytokines, chemokines, their target receptors and leukocytes, and is a major pathologic mechanism that adversely affects organ function in diverse human diseases. Despite being supported by substantial target validation, no successful anti-chemokine therapeutic to treat inflammatory disease has yet been developed. This is in part because of the robustness of the chemokine network, which emerges from a large total chemokine load in disease, promiscuous expression of receptors on leukocytes, promiscuous and synergistic interactions between chemokines and receptors, and feedforward loops created by secretion of chemokines by leukocytes themselves. Many parasites, including viruses, helminths and ticks, evade the chemokine network by producing proteins that bind promiscuously to chemokines or their receptors. Evasins - three small glycoproteins identified in the saliva of the brown dog tick - bind multiple chemokines, and are active in several animal models of inflammatory disease. Over 50 evasin homologs have recently been identified from diverse tick species. Characterization of the chemokine binding patterns of evasins show that several have anti-chemokine activities that extend substantially beyond those previously described. These studies indicate that evasins function at the site of the tick bite by reducing total chemokine load. This not only reduces chemokine signaling to receptors, but also interrupts feedforward loops, thus disabling the chemokine network. Taking the lead from nature, a goal for the development of new anti-chemokine therapeutics would be to reduce the total chemokine load in disease. This could be achieved by administering appropriate evasin combinations or by smaller peptides that mimic evasin action.

A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe & Dohme: Research Funding.

Versican (VCAN) Proteolysis Predicts Survival in Multiple Myeloma (MM) after High Dose Therapy and Autologous Hematopoietic Cell Transplantation (HDT/AHCT)

Background: Despite unprecedented response rates associated with "novel agents", HDT/AHCT is a therapeutic mainstay for transplant eligible patients with MM. However, relapse is universal and almost all patients diagnosed with symptomatic myeloma will die of their disease. The immunosuppressive network orchestrated by regulatory myeloid or lymphoid cells and effector dysfunction characterize relapses in MM. We have shown that myeloma-infiltrating myeloid cells produce versican (VCAN), a large matrix proteoglycan that promotes tumor sustaining inflammation and immunosuppression. VCAN proteolysis by a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs(ADAMTS) proteases generates versikine, a bioactive fragment ("matrikine") that regulates Batf3-dendritic cells that control CD8+-attracting chemokine networks. We have shown that VCAN proteolysis predicts post AHCT survival in myeloma in a small series of patients. Here, we explore whether VCAN proteolysis is prognostic of outcomes in an expanded cohort. Methods: Bone marrow core biopsies from patients who underwent HDT/AHCT for MM from 2015-2018 were analyzed at day 90-100 (n=66). ADAMTS protease-mediated VCAN proteolysis was analyzed by immunohistochemistry from the biopsies through detection of neo-epitope DPEAAE as described previously. CD8+ T cells per high power field (HPF) was calculated at 400X magnification (10X ocular with a 40X objective) for available patients (N=35). Patients were divided into low (1+), moderate (2+) and high (3+) groups based on the scoring intensity. Patient characteristics were compared between the three groups using chi-square test or ANOVA as appropriate. Correlation analyses for VCAN proteolysis and CD8+ T cells/HPF were performed using ANOVA and linear regression in patients with available data (n=35). Kaplan Meier estimates were used to generate overall survival (OS) and progression-free survival (PFS). Multivariate analysis using Cox regression model were performed to correlate the association between VCAN proteolysis with PFS and OS using factors that affect outcomes: age, gender, cytogenetics, and treatment regimens. Logistic regression analyses were used to estimate predictors of 2-year OS and PFS. Data were analyzed using SPSS and p<0.05 was considered statistically significant. Results: A total of 66 patients were included and VCAN proteolysis intensity was observed as low (1+) in 32 (48.5%), moderate (2+) in 23 (35%) and high (3+) in 11 (17%) patients respectively. Baseline characteristics between the three groups were similar (Table 1). VCAN proteolysis did not correlate with pre-AHCT response rates but was associated with post AHCT response (>very good partial response, VGPR in low: 87.5%, moderate: 65%, high: 45.5%; p=0.016, Figure 1A). The median follow-up of survivors was 37 months (range 7-116). Higher VCAN proteolysis had worse median PFS (months) [9 (95% CI 0-24) in high vs. 29 (95% CI 11-47) in moderate vs. 53 (95% CI 43-63) in low; p=0.019, Fig 1B) but no difference was observed in OS (Figure 1C). On multivariate analysis, moderate/high VCAN proteolysis had significantly worse PFS compared to low cohort (HR 2.62, 95% CI 1.20-5.74; p=0.016), but not significant for OS (HR 1.82, 95% CI 0.78-4.20; p=0.164). High/moderate VCAN proteolysis was associated with worse 2-year PFS (OR 2.79, 95% CI 1.02-7.64; p=0.046) and 2-year OS (OR 4.26, 95% CI 1.32-13.74; p=0.015) respectively. VCAN proteolysis intensity also correlated with higher CD8+ T cells (median in low: 15.5, moderate: 26, high: 66; regression coefficient 21.9, 95% CI 11.8-32.0, p<0.001). Of 25 patients with VCAN status available at diagnosis, 4 (16%) converted from low to moderate/high status, while 5 (20%) converted from moderate/high to low VCAN status post AHCT respectively. Conclusions: We demonstrate in an expanded cohort that VCAN proteolysis status is associated independently with post AHCT response status and PFS. Increasing VCAN proteolysis signal intensity was associated with progressively worse outcomes consistent with the hypothesis that VCAN proteolysis is a surrogate marker for an immunosuppressive environmental network that promotes MM survival and CD8 T cell dysfunction. VCAN accumulation and turnover maybe predictive of early risk of relapse and can form the basis of future clinical trials aimed at restoring anti-MM immunity guided by VCAN proteolysis status. Disclosures Dhakal: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Hari:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; Cell Vault: Equity Ownership; AbbVie: Consultancy, Honoraria.

How to dissect the plasticity of antigen-specific immune response: a tissue perspective.

Generation of antigen-specific humoral responses following vaccination or infection requires the maturation and function of highly specialized immune cells in secondary lymphoid organs (SLO), such as lymph nodes or tonsils. Factors that orchestrate the dynamics of these cells are still poorly understood. Currently, experimental approaches that enable a detailed description of the function of the immune system in SLO have been mainly developed and optimized in animal models. Conversely, methodological approaches in humans are mainly based on the use of blood-associated material because of the challenging access to tissues. Indeed, only few studies in humans were able to provide a discrete description of the complex network of cytokines, chemokines and lymphocytes acting in tissues after antigenic challenge. Furthermore, even fewer data are currently available on the interaction occurring within the complex micro-architecture of the SLO. This information is crucial in order to design particular vaccination strategies, especially for patients affected by chronic and immune compromising medical conditions who are under-vaccinated or who respond poorly to immunizations. Analysis of immune cells in different human tissues by high-throughput technologies, able to obtain data ranging from gene signature to protein expression and cell phenotypes, is needed to dissect the peculiarity of each immune cell in a definite human tissue. The main aim of this review is to provide an in-depth description of the current available methodologies, proven evidence and future perspectives in the analysis of immune mechanisms following immunization or infections in SLO.

ROLE OF CXCR3 CHEMOKINE RECEPTOR AND ITS LIGANDS IN CERTAIN DISEASES

Chemokines are a special family of cytokines whose main function is to control cell migration; they are key players in the innate and adaptive immune responses. Directed chemotaxis of specific leukocyte subpopulations is necessary not only to maintain homeostasis, but also in development of some immunopathological conditions such as cancer, inflammation, infection, allergies and autoimmune disorders. Chemokines are pleiotropic molecules that are involved in physiological and pathophysiological processes. For example, the CXCR3 chemokine receptor is expressed on various cells: activated T and B lymphocytes, natural killers, eosinophils and neutrophils, dendritic cells, fibroblasts, endothelial and epithelial cells. Hence, CXCR3 and its ligands have a wide range of functional activity. CXCR3 ligands are the IFNγ-induced chemokines: CXCL9, CXCL10, CXCL11, and platelet-derived chemokines: CXCL4, CXCL4L1. All the CXCR3 ligands share common angiostatic properties due to lack of the Glu-Leu-Arg (ELR) motif. IFNγ-induced ligands of the CXCR3 are proinflammatory chemokines, they mainly recruit activated T cells and exert an effect on T cell polarization. Due to wide spectrum of biological activity, the ligands of CXCR3 receptor are involved in pathogenesis of various disorders, such as inflammation, infection, cancer, allergies and autoimmune disorders. In this review, we discuss the role of CXCR3 ligands in immunopathogenesis of various diseases, including the results of our studies in chronic hepatitis C, rheumatoid arthritis and pulmonary tuberculosis. Moreover, we have also discussed the potential laboratory diagnostic applicability of the chemokines in various diseases. This review illustrates a universal role of IFNγ-induced chemokines as mediators of immune responses in various diseases. The studies of CXCR3 ligands, their isoforms and receptors, interactions between themselves and with their receptors can provide a significant contribution to our understanding of the chemokine network. Understanding the system of IFNγ-dependent chemokines may have clinical implications, both for diagnostic tasks, and for therapeutic purposes.

Induction of regional chemokine expression in response to human umbilical cord blood cell infusion in the neonatal mouse ischemia-reperfusion brain injury model.

Regenerative medicine using umbilical cord blood (UCB) cells shows promise for the treatment of cerebral palsy. Although the efficacy of this therapy has been seen in the clinic, the mechanisms by which UCB cells interact and aid in the improvement of symptoms are not clear. We explored the chemokine expression profile in damaged brain tissue in the neonatal mouse ischemia-reperfusion (IR) brain injury model that was infused with human UCB (hUCB) cells. IR brain injury was induced in 9-day-old NOD/SCID mice. hUCB cells were administered 3 weeks post brain injury. Chemokine expression profiles in the brain extract were determined at various time points. Inflammatory chemokines such as CCL1, CCL17, and CXCL12 were transiently upregulated by 24 hours post brain injury. Upregulation of other chemokines, including CCL5, CCL9, and CXCL1 were prolonged up to 3 weeks post brain injury, but most chemokines dissipated over time. There were marked increases in levels of CCL2, CCL12, CCL20, and CX3CL1 in response to hUCB cell treatment, which might be related to the new recruitment and differentiation of neural stem cells, leading to the induction of tissue regeneration. We propose that the chemokine expression profile in the brain shifted from responding to tissue damage to inducing tissue regeneration. hUCB cell administration further enhanced the production of chemokines, and chemokine networks may play an active role in tissue regeneration in neonatal hypoxic-ischemic brain injury.

CCL2 But Not CCR2 Is Required for Spontaneous Articular Cartilage Regeneration Post-Injury.

The role of the inflammatory response in articular cartilage degeneration and/or repair is often debated. Chemokine networks play a critical role in directing the recruitment of immune cells to sites of injury and have been shown to regulate cell behavior. In this study, we investigated the role of the CCL2/CCR2 signaling axis in cartilage regeneration and degeneration. CCL2-/- , CCR2-/- , CCL2-/- CCR2-/- , and control (C57) mice were subjected to full-thickness cartilage defect (FTCD) injuries (n = 9/group) within the femoral groove. Cartilage regeneration at 4 and 12 weeks post-FTCD was assessed using a 14-point histological scoring scale. Mesenchymal stem cells (MSCs) (Sca-1+ , CD140a+ ), macrophages (M1:CD38+ , M2:CD206+ , and M0:F4/80+ ) and proliferating cells (Ki67+ ) were quantified within joints using immunofluorescence. The multi-lineage differentiation capacity of Sca1+ MSCs was determined for all mouse strains. ACL transection (ACL-x) was employed to determine if CCL2-/- CCR2-/- mice were protected against osteoarthritis (OA) (n = 6/group). Absence of CCR2, but not CCL2 nor both (CCL2 and CCR2), enhanced spontaneous articular cartilage regeneration by 4 weeks post-FTCD. Furthermore, increased chondrogenesis was observed in MSCs derived from CCR2-/- mice. CCL2 deficiency promoted MSC homing to the adjacent synovium and FTCD at both 4 and 12 weeks post-injury; with no MSCs present at the surface of the FTCD in the remaining strains. Lower OA scores were observed in CCL2-/- CCR2-/- mice at 12 weeks post-ACL-x compared with C57 mice. Our findings demonstrate an inhibitory role for CCR2 in cartilage regeneration after injury, while CCL2 is required for regeneration, acting through a CCR2 independent mechanism. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2561-2574, 2019.

Multiplex immunohistochemistry assay to evaluate the melanoma tumor microenvironment.

The tumor microenvironment (TME) is composed of a set of cellular compartments comprising vascular, neuroendocrine, stromal, epithelial and immune cells. These compartments constitute a heterogeneous and dynamic set, where intercellular communication is driven by a complex network of cytokines, chemokines, growth factors, and inflammatory and matrix remodeling enzymes. Based on this complexity, an increasing number of assays may be required to identify and locate specific proteins in the tissue section and the standard procedure is to perform one stain at a time on serial sections. Recently, interest in performing multiple assays on formalin-fixed, paraffin embedded (FFPE) specimens has gained ground, and is referred to as multiplexing, i.e., multiple staining of the same section at the same time. Multiple staining is a promising approach that may help to improve understanding of the interactions between the different cellular components of the TME, stratify cancer patients, and help clinicians in their patient management. In this chapter, we detail a simple methodological approach to perform multiple staining on the same section using tissue obtained from patients with melanoma. This procedure evaluates the presence and location of three different proteins, human leukocyte antigen (HLA), forkhead box protein 3 (FoxP3) and Granzyme B (GRZB).

A Comprehensive Profile of Chemokine Gene Expression in the Tissues of the Female Reproductive Tract in Mice

ABSTRACTHomeostatic leukocyte trafficking into and within the female reproductive tract (FRT) contributes to fertility and reproductive health. It is unclear how this process is regulated in the anatomically distinct reproductive tissues, or whether the genes involved are affected by cyclical changes in reproductive hormones. In tissues such as skin and intestine, mouse studies have defined evolutionarily conserved molecular mechanisms for tissue-specific homing, interstitial positioning, and leukocyte egress. Chemokine family members are invariably involved, with the chemokine expression profile of a tissue regulating leukocyte content. Reproductive tissues (ovary, vagina, cervix, uterine horn) of 8 week old virgin female C57BL/6 mice (n = 20) were collected, and expression of mRNA for leukocyte markers and chemokines conducted by qPCR. Lymphocytic and myeloid cell populations within the uterus, cervix, bone marrow and PALN from virgin C57BL/6 mice were determined by flow cytometric analysis. Variation in leukocyte content between reproductive tissues is evident, with the uterus and cervix containing complex mixtures of lymphocytes and myeloid cells. Twenty-six chemokine genes are expressed in the FRT, many by several component tissues, some preferentially by one. Most striking are Xcl1 and Ccl28, which are restricted to the uterus. Ccl20 and genes encoding CXCR2 ligands are primarily transcribed in cervix and vagina. Ovary shows the lowest expression of most chemokine genes, with the notable exception of Ccl21 and Ccl27. We also identify eight chemokines in the vagina whose expression fluctuates substantially across the oestrous cycle. These data reveal complex chemokine networks within the FRT, and provide a framework for future studies of homeostatic leukocyte trafficking into and within these tissues.Abbreviations: BM: bone marrow; DC: dendritic cell; DN: double negative; FRT: female reproductive tract; FSC: forward scatter; NK: natural killer; PALN: para-aortic lymph node; SSC: side scatter; Tregs: regulatory T cells

Inflammatory Breast Cancer Promotes Development of M2 Tumor-Associated Macrophages and Cancer Mesenchymal Cells through a Complex Chemokine Network.

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine-paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer. SIGNIFICANCE: This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3360/F1.large.jpg.

Indispensable Role of CX3CR1+ Dendritic Cells in Regulation of Virus-Induced Neuroinflammation Through Rapid Development of Antiviral Immunity in Peripheral Lymphoid Tissues.

A coordinated host immune response mediated via chemokine network plays a crucial role in boosting defense mechanisms against pathogenic infections. The speed of Ag presentation and delivery by CD11c+ dendritic cells (DCs) to cognate T cells in lymphoid tissues may decide the pathological severity of the infection. Here, we investigated the role of CX3CR1 in the neuroinflammation induced by infection with Japanese encephalitis virus (JEV), a neurotrophic virus. Interestingly, CX3CR1 deficiency strongly enhanced susceptibility to JEV only after peripheral inoculation via footpad. By contrast, both CX3CR1+/+ and CX3CR1−/− mice showed comparable susceptibility to JEV following inoculation via intranasal and intraperitoneal routes. CX3CR1−/− mice exhibited lethal neuroinflammation after JEV inoculation via footpad route, showing high mortality, morbidity, pro-inflammatory cytokine expression, and uncontrolled CNS-infiltration of peripheral leukocytes including Ly-6Chi monocytes and Ly-6Ghi granulocytes. Furthermore, the absence of CX3CR1+CD11c+ DCs appeared to enhance susceptibility of CX3CR1−/− mice to JE after peripheral JEV inoculation. CX3CR1 ablation impaired the migration of CX3CR1+CD11c+ DCs from JEV-inoculated sites to draining lymph nodes (dLNs), resulting in decreased NK cell activation and JEV-specific CD4+/CD8+ T-cell responses. However, CX3CR1-competent mice showed rapid temporal expression of viral Ags in dLNs. Subsequently, JEV was rapidly cleared, with concomitant generation of antiviral NK cell activation and T-cell responses mediated by rapid migration of JEV Ag+CX3CR1+CD11c+ DCs. Using biallelic functional CX3CR1 expression system, the functional expression of CX3CR1 on CD11chi DCs appeared to be essentially required for inducing rapid and effective responses of NK cell activation and Ag-specific CD4+ T cells in dLNs. Strikingly, adoptive transfer of CX3CR1+CD11c+ DCs was found to completely restore the resistance of CX3CR1−/− recipients to JEV, as corroborated by the rapid delivery of JEV Ags in dLNs and attenuation of neuroinflammation in the CNS. Collectively, these results indicate that CX3CR1+CD11c+ DCs play an important role in generating rapid and effective responses of antiviral NK cell activation and Ag-specific T cells after peripheral inoculation with the virus, thereby resulting in conferring resistance to viral infection by reducing the peripheral viral burden.

Residue Mutations in Murine Herpesvirus 68 Immunomodulatory Protein M3 Reveal Specific Modulation of Chemokine Binding.

The M3 protein (M3) encoded by murine gammaherpesvirus 68 (MHV-68) is a unique viral immunomodulator with a high-affinity for a broad spectrum of chemokines, key mediators responsible for the migration of immune cells to sites of inflammation. M3 is currently being studied as a very attractive and desirable tool for blocking the chemokine signaling involved in some inflammatory diseases and cancers. In this study, we elucidated the role of M3 residues E70 and T272 in binding to chemokines by examining the effects of the E70A and T272G mutations on the ability of recombinant M3, prepared in Escherichia coli cells, to bind the human chemokines CCL5 and CXCL8. We found that the E70A mutation enhanced binding of M3 to CCL5 two-fold but had little effect on its binding to CXCL8. In contrast, the T272G mutation was found to be important for the thermal stability of M3 and significantly decreased M3's binding to both CCL5 (by about 4×) and CXCL8 (by about 5×). We also constructed in silico models of the wild-type M3–CCL5 and M3–CCL8 complexes and found substantial differences in their physical and chemical properties. M3 models with single mutation E70A and T272G suggested the role of E70 and T272 in binding M3 protein to chemokines. In sum, we have confirmed that site-directed mutagenesis could be an effective tool for modulating the blockade of particular chemokines by M3, as desired in therapeutic treatments for severe inflammatory illnesses arising from chemokine network dysregulation.

Role of CXCR3 in natural killer cell migration to melanoma cells

Abstract Natural killer (NK) cell recruitment to solid tumors is essential for direct contact between NK and cancer cells and is regulated by chemokine networks. Tumor-derived CXCL10 increases the infiltration of NK cells into solid tumors. In a xenograft mouse model, adoptively transferred human NK cells efficiently infiltrate CXCL10-transduced tumors and enhance regression of tumor growth compared with mock-transduced tumors. Although this study showed that NK cells homed to solid tumor beds in a CXCL10-depedent manner, several questions remain. How do NK cells find cancer cells after extravasation? Which chemokines do B16F10 cells produce? Which chemokine receptors do NK cells use? How do cancer cell-derived chemokines affect NK cell motility? Do these chemokines directly affect NK cell cytotoxicity? To address these issues, we examined the migration dynamics of NK and cancer cells using time-lapse imaging. Although naïve B16F10 cancer cells produce low levels of chemokines, IFN-g-treated B16F10 cells secreted high levels of CXCL10, low levels of CCL5, but did not secrete CCL2, CCL7, or CXCL12. Wild-type NK cells migrated well toward cancer cells and killed them, whereas NK cells deficient in CXCR3 did not. CXCR3-deficient NK cells also showed slower migration speed than did wild-type NK cells. Taken together, our data show that NK cells find cancer cells, at least in part, by sensing CXCL10 produced by cancer cells and suggest that a strategy to increase CXCL10 secretion by cancer cells may improve the efficacy of NK cell–based immunotherapy.

Acquisition of gemcitabine resistance enhances angiogenesis via upregulation of IL‑8 production in pancreatic cancer.

Gemcitabine (Gem) is widely used as chemotherapy for pancreatic cancer (PaCa), but its effect is not fully satisfactory. One of the reasons for this is the acquisition of Gem resistance (Gem‑R). To elucidate the mechanism of Gem‑R, two Gem‑R PaCa cell lines were established from AsPC‑1 and MIA PaCa‑2 cells. It was demonstrated that expression of interleukin‑8 (IL‑8) mRNA was significantly upregulated in Gem‑R PaCa cells by cDNA microarray and RT‑qPCR analyses. Increased IL‑8 secretion by Gem‑R cells was confirmed by cytokine array and enzyme‑linked immunosorbent assay. Moreover, we found that co‑culture with Gem‑R PaCa cells significantly enhanced tube formation of human umbilical vein endothelial cells, and treatment with an anti‑CXCR2 (main receptor for IL‑8) antibody significantly prevented this effect. We previously reported that a chemokine network centered on the IL‑8/CXCR2 axis plays an important role in PaCa angiogenesis, and suppression of this axis has an antitumor effect. Since acquisition of Gem‑R increased IL‑8 production and consequently increased tumor angiogenesis, the IL‑8/CXCR2 axis may be a potential novel therapeutic target for PaCa after acquiring Gem‑R.

Altered T Cell Migratory Capacity in the Progression from Barrett Oesophagus to Oesophageal Adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is an inflammation-driven cancer with poor prognosis and incidence is increasing rapidly. OAC arises from gastro-oesophageal reflux disease (GORD) and reflux-induced Barrett oesophagus (BO). The role of T cells in this disease progression is not yet fully understood. We have previously demonstrated higher proportions of pro-tumour Th2 cells in BO tissue, implicating them in its pathogenesis. While a Th2 immune profile is thought to underlie the metaplastic transformation in BO and promote OAC development, our studies suggest that the abundance of Th2 cells in BO tissue is likely to occur through altered T cell recruitment. This study examined the chemokine networks governing T cell migration to oesophageal tissue during disease progression. Here, we have identified that circulating T cells in OAC patients, exhibit impaired migratory capacity with decreased frequencies of Th1-associated CXCR3+ and Th17-associated CCR6+ cells. Despite the abundance of Th1 chemokines RANTES (CCL5) and MIP-1α (CCL3) in OAC tumour, enrichments of intratumoural T cells expressing corresponding receptors were not observed. These data suggest that T cell infiltration of oesophageal tissue is compromised in OAC and suggest that future therapies targeting T cell trafficking should occur at the pre-neoplastic stage. This is supported by the finding that antagonism of Th2-biased CCR4 significantly reduces T cell migration in BO but not OAC patients. Since we have previously reported a predominant Th2 immune profile in BO, we suggest that chemokine receptor antagonism may be a viable treatment option to alleviate Th2-predominance in BO and interrupt progression to OAC.

Primary Chronic Venous Insufficiency Is Distinguished by Attenuated Circulating Inflammatory Mediators and Healing Networks

Inflammation promotes venous leg ulcers (VLUs) in post-thrombotic syndrome. However, it is not clear how inflammation affects VLU in primary venous reflux. Computational modeling has demonstrated differences in cytokine and chemokine networks in other wound healing paradigms. We hypothesize that serum inflammatory mediators are differentially expressed and disorganized in primary chronic venous insufficiency (CVI), which may be a mechanism for future VLU. Participants were recruited prospectively with Institutional Review Board approval. Blood was obtained during sclerotherapy or endovenous thermal ablation for primary CVI without ulcer (Clinical, Etiology, Anatomy, and Pathophysiology [CEAP] class C2-C4). Control patients without CVI underwent phlebotomy from great saphenous and antecubital veins. Demographics, Venous Clinical Severity Score, and body mass index (BMI) were collected. Twenty-five mediators previously shown to be important in wound healing were measured in serum with Luminex. Values were compared using Mann-Whitney U test. Pearson correlations among mediators (nodes; Fig, ●) that were above a specific threshold prompted connection between nodes (edges; Fig, —). Correlations were mapped as networks (MATLAB; MathWorks, Natick, Mass). “Complexity” was determined from number of connections for each mediator and total number of mediators. A robustness index measuring network strength was calculated by dividing number of connections at a Pearson correlation threshold of 0.95/0.7 Demographics, BMI, Venous Clinical Severity Score, and mean and standard error of the mean mediator values for patients (N = 42) and controls (leg; N = 7) are shown in the Table. Significant differences (P < .05) were demonstrated in 20 of 25 mediators; most reflected lower concentrations in patients. In controls, arm and leg values were nearly identical to one another and across patients. The Fig demonstrates networks of inflammatory mediators for each group, showing lower network complexity and robustness in CVI vs controls. The robustness index for patients, control leg, and control arm was 0.096, 0.169, and 0.241, respectively. CVI associates not only with age and BMI but also with diminished expression of many inflammatory compounds instrumental for wound healing. The relationship among these mediators is similarly weak. In contrast, mediators within competent veins show little variability and a high degree of correlation that is lacking in CVI. Dysregulated inflammatory networks in response to injury from venous hypertension may be a predisposing factor to further venous damage and VLU. Normalizing venous competency may improve baseline cytokine and chemokine interactions and the response to microstresses that can lead to wounds.TableDemographics for inflammatory mediators drawn from leg veins of patients and controlsPatients (N = 42)Controls (N = 7)PMeanSEMMeanSEMGM-CSF19.13.661.60.0<.001IFN-γ235.458.90.0<.001IL-12p7012.92.6410.0<.001IL-17A10.12.227.50.0<.001IL-710.31.530.50.0<.001MIG1227.2195.4313.40.0<.001sIL-2RA696.962.3252.10.0<.001IL-10183.151.50.0.001IL-1b7.51.520.90.0.001IL-437.79107.40.0.001IFN-α251.812.4110.50.0.002IL-1RA117.821.9306.90.0.002MIP-1β33.32.655.60.0.003IL-12p40116.628.7252.10.0.005IP-10685.365.3311.10.0.005IL-1512.92.4240.0.009TNF-α27.64.848.20.0.011IL-551.57.80.2.012IL-24.91.17.60.1.015IL-1332.812.737.20.3.045IL-68.71.520.80.0.059IL-814.23.214.80.4.157Eotaxin100.68.11130.4.265MCP-1343.729.9334.70.3.658MIP-1α5.91.82.60.21RangeRangeAge, years55.731-8834.721-47.008BMI, kg/m23219-482320-26.004VCSS6.52-141.10-2<.001Female71%43%BMI, Body mass index; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; MCP, monocyte chemotactic protein; MIG, monokine induced by γ interferon; MIP, macrophage inflammatory protein; SEM, standard error of the mean; sIL, soluble interleukin; TNF, tumor necrosis factor; VCSS, Venous Clinical Severity Score.Values are reported as picogram/milliliter unless otherwise indicated. Open table in a new tab

Macrophage Migration Inhibitory Factor (MIF)-Based Therapeutic Concepts in Atherosclerosis and Inflammation.

Chemokines orchestrate leukocyte recruitment in atherosclerosis and their blockade is a promising anti-atherosclerotic strategy, but few chemokine-based approaches have advanced into clinical trials, in part owing to the complexity and redundancy of the chemokine network. Macrophage migration inhibitory factor (MIF) is a pivotal mediator of atherosclerotic lesion formation. It has been characterized as an inflammatory cytokine and atypical chemokine that promotes atherogenic leukocyte recruitment and lesional inflammation through interactions with the chemokine receptors CXCR2 and CXCR4, but also exhibits phase-specific CD74-mediated cardioprotective activity. The unique structural properties of MIF and its homologue MIF-2/D-DT offer intriguing therapeutic opportunities including small molecule-, antibody- and peptide-based approaches that may hold promise as inhibitors of atherosclerosis, while sparing tissue-protective classical chemokine pathways. In this review, we summarize the pros and cons of anti-MIF protein strategies and discuss their molecular characteristics and receptor specificities with a focus on cardiovascular disease.

Potential Roles of Innate Immune Chemokine and Cytokine Network on Lipopolysaccharide-Based Therapeutic Approach in Ovarian Cancer.

Ovarian cancer (OC), the deadliest gynecological cancer, results in poor overall survival, urgently requiring a novel therapeutic approach. As cumulative exposures to endotoxins decreased OC risk epidemiologically, we evaluated if LPS, a Toll-like receptor 4 agonist known as active component of endotoxins, could increase survival in the murine peritoneal dissemination model of SKOV-3 OC cells. LPS significantly increased the mean survival time of more than 116 days compared with 63 days in the control. Furthermore, no tumor burden was present in three mice among eight LPS-treated mice. SKOV-3 cells were not responsive to LPS and showed unaltered chemokine signature. Rather than direct effects to OC cells, LPS was found to increase proinflammatory chemokines and cytokines, such as CXCL1, CXCL8, TNF, and IL-1B, in innate immune system. Taken together, LPS is likely to potentiate the cytotoxic-related innate immunogenicity via proinflammatory chemokines and cytokines, which attenuates the peritoneal dissemination of OC.

Chemokines and Chemokine Receptors: Orchestrating Tumor Metastasization.

Metastasis still represents the primary cause of cancer morbidity and mortality worldwide. Chemokine signalling contributes to the overall process of cancer growth and metastasis, and their expression in both primary tumors and metastatic lesions correlate with prognosis. Chemokines promote tumor metastasization by directly supporting cancer cell survival and invasion, angiogenesis, and by indirectly shaping the pre-metastatic niches and antitumor immunity. Here, we will focus on the relevant chemokine/chemokine receptor axes that have been described to drive the metastatic process. We elaborate on their role in the regulation of tumor angiogenesis and immune cell recruitment at both the primary tumor lesions and the pre-metastatic foci. Furthermore, we also discuss the advantages and limits of current pharmacological strategies developed to target chemokine networks for cancer therapy.

Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo.

Cytomegaloviruses (CMVs) encode cellular homologs to evade host immune functions. In this study, we analyzed the roles of GP33, a guinea pig CMV (GPCMV)-encoded G protein-coupled receptor (GPCR) homolog, in cellular signaling, viral growth and pathogenesis. The cDNA structure of GP33 was determined by RACE. The effects of GP33 on some signaling pathways were analyzed in transient transfection assays. The redET two-step recombination system for a BAC containing the GPCMV genome was used to construct a mutant GPCMV containing an early stop codon in the GP33 gene (Δ33) and a rescued GPCMV (r33). We found the following: 1) GP33 activated the CRE- and NFAT-, but not the NFκB-mediated signaling pathway. 2) GP33 was dispensable for infection in tissue cultures and in normal animals. 3) In pregnant animals, viral loads of r33 in the livers, lungs, spleens, and placentas at 6 days post-infection were higher than those of Δ33, although the viruses were cleared by 3 weeks post-infection. 4) The presence of GP33 was associated with frequent lesions, including alveolar hemorrhage in the lungs, and inflammation in the lungs, livers, and spleens of the dams. Our findings suggest that GP33 has critical roles in the pathogenesis of GPCMV during pregnancy. We hypothesize that GP33-mediated signaling activates cytokine secretion from the infected cells, which results in inflammation in some of the maternal organs and the placentas. Alternatively, GP33 may facilitate transient inflammation that is induced by the chemokine network specific to the pregnancy.