Mechanistic computational modeling of monospecific and bispecific antibodies targeting interleukin-6/8 receptors.

The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.

Molecular insights into the microtubules depolymerizing activity of the IL-8 receptor B antagonist SB225002.

OBJECTIVE: We have previously reported the novel off-target microtubules destabilizing activity of SB225002, a compound that was originally designed as a selective and potent IL-8 receptor B antagonist. In the present study we investigated the reversibility of SB225002 antimitotic effect and provided additional mechanistic insights underlying cell death induction in SW480 human colorectal adenocarcinoma cells. MATERIALS AND METHODS: Mitotically arrested cells by SB225002 treatment were isolated by shake-off, and their identity was verified by both flow cytometry and immunoblotting. The reversibility of SB225002 antimitotic effects was investigated by flow cytometry and immunoblotting. Prometaphase arrested cells were imaged via indirect immunofluorescence and confocal microscopy. Activation of CHK1 in mitotically arrested cells was assessed by immunoblotting, and the relationship between CHK1 and mitotic arrest was examined via siRNA-mediated knockdown of CHK1. JNK signaling was evaluated via immunoblotting as well as pharmacological inhibition, followed by flow cytometry. The role of reactive oxygen species (ROS) in cytotoxicity was evaluated by ROS scavenging and flow cytometry. RESULTS: Following SB225002 washout, the mitotic checkpoint was abrogated, and cell cycle perturbations were gradually restored with induction of cell death. Mechanistically, CHK1 checkpoint was activated by SB225002 and occurred downstream of the mitotic checkpoint. In addition, SB225002 activated JNK signaling which contributed to cell death and restrained polyploidy. Furthermore, SB225002 increased intracellular ROS which played a role in mediating SB225002 cytotoxicity. CONCLUSIONS: Findings of the present study warrants further development of SB225002 as a lead compound that uniquely targets microtubules dynamics and IL-8 signaling.

Ladarixin, an inhibitor of the interleukin-8 receptors CXCR1 and CXCR2, in new-onset type 1 diabetes: A multicentre, randomized, double-blind, placebo-controlled trial.

AIM: To evaluate the ability of ladarixin (LDX, 400 mg twice-daily for three cycles of 14 days on/14 days off), an inhibitor of the CXCR1/2 chemokine receptors, to maintain C-peptide production in adult patients with newly diagnosed type 1 diabetes. MATERIALS AND METHODS: A double-blind, randomized (2:1), placebo-controlled study was conducted in 45 males and 31 females (aged 18-46 years) within 100 days of the first insulin administration. The primary endpoint was the area under the curve (AUC) for C-peptide in response to a 2-hour mixed meal tolerance test (AUC[0-120 min] ) at week 13 ± 1. Secondary endpoints included C-peptide AUC(15-120 min) , HbA1c, daily insulin requirement, severe hypoglycaemic events (SHE), the proportion of subjects achieving HbA1c less than 7.0% without SHE and maintaining a residual beta cell function. Follow-up assessments were scheduled at weeks 13 ± 1, 26 ± 2 and 52 ± 2. RESULTS: In total, 26/26 (100%, placebo) and 49/50 (98%, LDX) patients completed week 13. The mean change from baseline to week 13 in C-peptide AUC(0-120 min) was -0.144 ± 0.449 nmol/L with placebo and 0.003 ± .322 nmol/L with LDX. The difference was not significant (0.149 nmol/L, 95% CI -0.04 to 0.33; P = .122). At week 26, the proportion of patients with HbA1c less than 7.0% without SHE was transiently higher in the LDX group (81% vs. 54%, P = .024). Otherwise, no significant secondary endpoint differences were noted. Transient metabolic benefit was seen at week 26 in favour of the LDX group in the prespecified subpopulation with fasting C-peptide less than the median value at screening. CONCLUSIONS: In newly diagnosed patients with type 1 diabetes, short-term LDX treatment had no appreciable effect on preserving residual beta cell function.

CXCR1 correlates to poor outcomes of EGFR-TKI against advanced non-small cell lung cancer by activating chemokine and JAK/STAT pathway.

OBJECTIVE: CXCR1, a member of the seven-transmembrane chemokine receptor family, promotes cell proliferation and metastasis in many tumors. The present study was undertaken to explore the interrelation between CXCR1 expression and the prognosis of advanced non-small cell lung cancer (NSCLC) in addition to the efficacy of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma (LUAD). METHODS: The expression of CXCR1 in NSCLC tissues was assessed by immunohistochemistry. The relationships between CXCR1 expression and clinical-pathological factors were investigated. Concomitantly, the relationship between CXCR1 expression and EGFR-TKI treatment efficacy was investigated. Gene set enrichment analysis (GSEA) was employed for the exploration of pathway enrichment, tumor immune estimation resource (TIMER) and gene expression profiling interactive analysis (GEPIA) for the inspection of the interrelationship between infiltration immune cells and CXCR1. After gain-and loss-of-function of CXCR1 in NSCLC cells, qRT-PCR and Western blot were applied to measure the levels of proteins associated with the chemokine pathway (CCL3 and CXCL2) and the JAK/STAT pathway (IL9R, PIAS4 and STAT5A). RESULTS: CXCR1 significantly correlated with poor prognosis of NSCLC patients. Additionally, CXCR1 limited the clinical efficacy of EGFR-TKIs in advanced LUAD (P = 0.029). In the tumor microenvironment, CXCR1 was positively associated with infiltration levels of immune markers in lung squamous cell carcinoma (LUSC) and LUAD. High expression of CXCR1 was implicated in the NOD-like receptor (NLR), cytokine/cytokine receptor, JAK/STAT and chemokine signaling pathways in LUAD and LUSC. Overexpression of CXCR1 in NSCLC cell lines enhanced expressions of CCL3, CXCL2, IL9R, PIAS4 and STAT5A, while knockdown of CXCR1 repressed expressions of CCL3, CXCL2, IL9R, PIAS4 and STAT5A. CONCLUSION: CXCR1 is correlated with poor prognosis of NSCLC and affects the efficacy of EGFR-TKIs in LUAD.

IL-8 promotes cell migration through regulating EMT by activating the Wnt/ß-catenin pathway in ovarian cancer.

Interleukin-8 (IL-8), as an inflammatory chemokine, has been previously shown to contribute to tumorigenesis in several malignancies including the ovarian cancer. However, little is known about how IL-8 promotes the metastasis and invasion of ovarian cancers cells. In this study, we found that IL-8 and its receptors CXCR1 and CXCR2 were up-regulated in advanced ovarian serous cancer tissues. Furthermore, the level of IL-8 and its receptors CXCR1 and CXCR2 expression were associated with ovarian cancer stage, grade and lymph node metastasis. In vitro, IL-8 promoted ovarian cancer cell migration, initiated the epithelial-mesenchymal transition (EMT) program and activated Wnt/ß-catenin signalling. However, when treated with Reparixin (inhibitor of both IL-8 receptors CXCR1 and CXCR2), effect of both endogenous and exogenous IL-8 was reversed. Together, our results indicated that IL-8 triggered ovarian cancer cells migration partly through Wnt/ß-catenin pathway mediated EMT, and IL-8 may be an important molecule in the invasion and metastasis of ovarian cancer.

Staphylococcus aureus intra-mammary infection affects the expression pattern of IL-R8 in goat.

IL-1R8 is a member of Interleukin-1 receptor family acting as a negative regulator of inflammation reliant on ILRs and TLRs activation. IL-1R8 role has never been evaluated in acute bacterial mastitis. We first investigated IL-1R8 sequence conservation among different species and its pattern of expression in a wide panel of organs from healthy goats. Then, modulation of IL-1R8 during natural and experimental mammary infection was evaluated and compared in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected goats. IL-1R8 has a highly conserved sequence among vertebrates. Goat IL-1R8 was ubiquitously expressed in epithelial and lymphoid tissues with highest levels in pancreas. IL-1R8 was down-regulated in epithelial mammary cells following S. aureus infection. Interestingly it was up-regulated in leukocytes infiltrating the infected mammary tissues suggesting that it could represent a target of S. aureus immune evasion.

Total Synthesis of Bioactive Marine Meroterpenoids: The Cases of Liphagal and Frondosin B.

Liphagal and frondosin B are two marine-derived secondary metabolites sharing a very similar polyfused-benzofuran skeleton. The two tetracyclic meroterpenoids were isolated from marine sponges, both featuring a 6-5-7-6 fused ring system. A preliminary bioactive study shows that (+)-liphagal is a selective kinase (PI3K α) inhibitor, while (+)-frondosin B is shown to inhibit the binding of the cytokine interleukin-8 (IL-8) to its receptor, CX-CLR1/2. The unique structures and interesting biological profiles of these two meroterpenoids have attracted considerable attention from synthetic chemists. Herein we summarize the synthetic efforts with respect to (+)-liphagal and (+)-frondosin B during the past two decades.

Revisiting the role of interleukin-8 in chronic lymphocytic leukemia.

The proliferation and survival of malignant B cells in chronic lymphocytic leukemia (CLL) depend on signals from the microenvironment in lymphoid tissues. Among a plethora of soluble factors, IL-8 has been considered one of the most relevant to support CLL B cell progression in an autocrine fashion, even though the expression of IL-8 receptors, CXCR1 and CXCR2, on leukemic B cells has not been reported. Here we show that circulating CLL B cells neither express CXCR1 or CXCR2 nor they respond to exogenous IL-8 when cultured in vitro alone or in the presence of monocytes/nurse-like cells. By intracellular staining and ELISA we show that highly purified CLL B cells do not produce IL-8 spontaneously or upon activation through the B cell receptor. By contrast, we found that a minor proportion (<0.5%) of contaminating monocytes in enriched suspensions of leukemic cells might be the actual source of IL-8 due to their strong capacity to release this cytokine. Altogether our results indicate that CLL B cells are not able to secrete or respond to IL-8 and highlight the importance of methodological details in in vitro experiments.

Direct effects of interleukin-8 on growth and functional activity of T lymphocytes.

CD3+ T-lymphocytes were isolated from the normal donors by positive magnetic separation. Activation of the T cells with particles conjugated with antibodies to CD3, СD28 and СD2 molecules led to a marked increase in T-cell production of interleukine-8 (IL-8). We present evidence that IL-8 receptor α-chain (CXCR1, CD181) is expressed on the cell surface of 13.3% T cells. Activation of T-lymphocytes resulted in significant enhancement of CD181+ cells both in naive CD4+ T cell and terminally differentiated effector CD4+ T cell compartments with concomitant reduction of CD181+ cells in effector memory CD4+ T cell subset. The level of T cell activation was assessed judging from the surface expression of CD25 (IL-2 receptor α-chain). We demonstrate that IL-8 treatment (0.01-10.0ng/ml concentration range) reduced the activation status of both CD4- and CD4+ effector memory T cells, as well as terminally differentiated effector T cells, without significantly affecting the activation of naive T cells or central memory T cells. In addition, IL-8 up-regulated IL-2 and down-regulated IL-10 production by activated T cells, with no effect on interferon-gamma (IFN-γ) and IL-4 production. Data obtained suggests the importance of IL-8 in the direct regulation of adaptive T cell reactivity.

Expression of CXCR1 (IL-8 receptor A) in splenic, peritoneal macrophages and resident bone marrow cells after acute live or heat killed Staphylococcus aureus stimulation in mice.

Literature reveals that interaction with live Staphylococcus aureus (S. aureus) or heat killed S. aureus (HKSA) promotes secretion of CXCL-8 or interleukin-8 (IL-8) from leukocytes, however, the expressions of CXCR1 in murine splenic (SPM), peritoneal macrophages (PM) and resident fresh bone marrow cells (FBMC) have not been identified. Currently, very few studies are available on the functional characterization of CXCR1 in mouse macrophage subtypes and its modulation in relation to acute S. aureus infection. SPM, PM and FBMCs were infected with viable S. aureus or stimulated with HKSA in presence and absence of anti-CXCR1 antibody in this study. We reported here that CXCR1 was not constitutively expressed by macrophage subtypes and the receptor was induced only after S. aureus stimulation. The CXCR1 band was found specific as we compared with human polymorphonuclear neutrophils (PMNs) as a positive control (data not shown). Although, we did not show that secreted IL-8 from S. aureus-infected macrophages promotes migration of PMNs. Blocking of cell surface CXCR1 decreases the macrophage's ability to clear staphylococcal infection, attenuates proinflammatory cytokine production and the increased catalase and decreased superoxide dismutase (SOD) enzymes of the bacteria might indicate their role in scavenging macrophage derived hydrogen peroxide (H2O2). The decreased levels of cytokines due to CXCR1 blockade before S. aureus infection appear to regulate the killing of bacteria by destroying H2O2 and nitric oxide (NO). Moreover, functional importance of macrophage subpopulation heterogeneity might be important in designing new effective approaches to limit S. aureus infection induced inflammation and cytotoxicity.

Induced Pluripotent Stem Cell-Derived Endothelial Cells Overexpressing Interleukin-8 Receptors A/B and/or C-C Chemokine Receptors 2/5 Inhibit Vascular Injury Response.

Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) C-C chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat-induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS-ECs overexpressing IL8RA/B (RiPS-IL8RA/B-ECs), CCR2/5 (RiPS-CCR2/5-ECs), or both receptors (RiPS-IL8RA/B+CCR2/5-ECs) will inhibit inflammatory responses and neointima formation in balloon-injured rat carotid artery. Twelve-week-old male Sprague-Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS-Null-ECs (ECs transduced with empty virus), (c) RiPS-IL8RA/B-ECs, (d) RiPS-CCR2/5-ECs, or (e) RiPS-IL8RA/B+CCR2/5-ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS-ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injury-induced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS-ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS-ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases. Stem Cells Translational Medicine 2017;6:1168-1177.

Acidic microenvironments induce lymphangiogenesis and IL-8 production via TRPV1 activation in human lymphatic endothelial cells.

Local acidosis is one of the characteristic features of the cancer microenvironment. Many reports indicate that acidosis accelerates the proliferation and invasiveness of cancer cells. However, whether acidic conditions affect lymphatic metastasis is currently unknown. In the present study, we focused on the effects of acidosis on lymphatic endothelial cells (LECs) to assess the relationship between acidic microenvironments and lymph node metastasis. We demonstrated that normal human LECs express various acid receptors by immunohistochemistry and reverse transcriptase-polymerase chain reaction (PCR). Acidic stimulation with low pH medium induced morphological changes in LECs to a spindle shape, and significantly promoted cellular growth and tube formation. Moreover, real-time PCR revealed that acidic conditions increased the mRNA expression of interleukin (IL)-8. Acidic stimulation increased IL-8 production in LECs, whereas a selective transient receptor potential vanilloid subtype 1 (TRPV1) antagonist, 5'-iodoresiniferatoxin, decreased IL-8 production. IL-8 accelerated the proliferation of LECs, and inhibition of IL-8 diminished tube formation and cell migration. In addition, phosphorylation of nuclear factor (NF)-κB was induced by acidic conditions, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that acidic microenvironments in tumors induce lymphangiogenesis via TRPV1 activation in LECs, which in turn may promote lymphatic metastasis.

Synthesis and Characterization of ¹8F-Interleukin-8 Using a Cell-Free Translation System and 4-¹8F-Fluoro-L-Proline.

UNLABELLED: Macromolecules such as proteins are attracting increasing interest for molecular imaging. We previously proposed a novel strategy for preparing macromolecules labeled with a PET radionuclide, (11)C, using a cell-free translation system with (11)C-methionine. However, macromolecules tend to exhibit slower kinetics, thus requiring a longer scanning time. Here, we expand our strategy using (18)F, which has a longer half-life, with the cell-free translation system with 4-(18)F-fluoro-L-proline ((18)F-FPro). We evaluated (18)F-interleukin-8 ((18)F-IL-8) produced by this method in vitro and in vivo to provide a proof of concept of our strategy. METHODS: We tested some fluorinated amino acids to be incorporated into a protein. Trans-(18)F-FPro was radiolabeled from the corresponding precursor. (18)F-IL-8 was produced using the cell-free translation system with trans-(18)F-FPro instead of natural L-proline with incubation at 37°C for 120 min. An in vitro binding assay of (18)F-IL-8 was performed using IL-8 receptor-expressing cells. After intravenous administration of (18)F-IL-8, in vivo PET imaging of IL-8 receptor-expressing xenograft-bearing mice was performed using a small-animal PET system. RESULTS: FPro was identified as an amino acid incorporated into the protein. (18)F-IL-8 was successfully prepared using the cell-free translation system and trans-(18)F-FPro with the radiochemical yield of 1.5% (decay-corrected) based on trans-(18)F-FPro. In vitro binding assays of (18)F-IL-8 demonstrated its binding to IL-8 receptor. In vivo PET imaging demonstrated that (18)F-IL-8 clearly accumulated in IL-8 receptor-expressing xenografts in mice, unlike trans-(18)F-FPro. CONCLUSION: (18)F-IL-8 produced by this method binds to IL-8 receptors in vitro, and (18)F-IL-8 PET clearly visualizes its target receptor-expressing xenograft in vivo. Therefore, this technique might be useful for labeling macromolecules and performing preclinical evaluations of proteins of interest in vitro and in vivo.

Brugia malayi Asparaginyl-tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis.

A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis.

Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat.

Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. METHODS: Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. RESULTS: Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. CONCLUSIONS: We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism.

OBJECTIVE: An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation. METHODS: Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin. RESULTS: Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin. CONCLUSIONS: The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

A surface membrane protein of Entamoeba histolytica functions as a receptor for human chemokine IL-8: its role in the attraction of trophozoites to inflammation sites.

Entamoeba histolytica trophozoites respond to the presence of IL-8, moving by chemotaxis towards the source of the chemokine. IL-8 binds to the trophozoite membrane and triggers a response that activates signaling pathways that in turn regulate actin/myosin cytoskeleton organisation to initiate migration towards the chemokine, suggesting the presence of a receptor for IL-8 in the parasite. Antibodies directed to the human IL-8 receptor (CXCR1) specifically recognised a 29 kDa protein in trophozoite membrane fractions. The same protein was immunoprecipitated by this antibody from total amebic extracts. Peptide analysis of the immunoprecipitated protein revealed a sequence with high homology to a previously identified amebic outer membrane peroxiredoxin and a motif within the third loop of human CXCR1, which is an important site for IL-8 binding and activation of signaling processes. Immunodetection assays demonstrated that the anti-human CXCR1 antibody binds to the 29 kDa protein in a different but close site to where IL-8 binds to the trophozoite surface membrane, suggesting that human and amebic receptors for this chemokine share common epitopes. In the context of the human intestinal environment, a receptor for IL-8 could be a great advantage for E. histolytica trophozoite survival, as they could reach an inflammatory milieu containing abundant nutrients. In addition, it has been suggested that the high content of accessible thiol groups of the protein and its peroxidase activity could provide protection in the oxygen rich milieu of colonic lesions, allowing trophozoite invasion of other tissues and escape from the host immune response.

Loss of RACK1 Promotes Metastasis of Gastric Cancer by Inducing a miR-302c/IL8 Signaling Loop.

Gastric cancer remains the third leading cause of cancer-related mortality worldwide, and invasion and metastasis of gastric cancer represent the major reason for its poor prognosis. In this study, we found that loss of the receptor for activated C-kinase 1 (RACK1) promoted the metastasis of gastric cancer by enhancing the autocrine expression of IL8 in vitro and in vivo. microRNA (miRNA; miR) array identified that RACK1 modulated the expression of a series of miRNAs, including the miR-302 cluster, and RACK1 modulated the IL8 expression and tumor invasion through miRNA-302c. Moreover, upregulation of IL8 in turn decreased the level of miRNA-302c and induced IL8 expression in a feedback manner. Tissue microarray also indicated that RACK1 was correlated with invasion/metastasis phenotype, IL8 expression, as well as 5-year survival in clinical cases of gastric cancer. Together, our results imply that loss of RACK1 in gastric cancer links epigenetics to inflammatory cytokines to promote tumor metastasis.

PYK2 integrates growth factor and cytokine receptors signaling and potentiates breast cancer invasion via a positive feedback loop.

The involvement of ErbB family members in breast cancer progression and metastasis has been demonstrated by many studies. However, the downstream effectors that mediate their migratory and invasive responses have not been fully explored. In this study, we show that the non-receptor tyrosine kinase PYK2 is a key effector of EGFR and HER2 signaling in human breast carcinoma. We found that PYK2 is activated by both EGF and heregulin (HRG) in breast cancer cells, and positively regulates EGF/HRG-induced cell spreading, migration and invasion. PYK2 depletion markedly affects ERK1/2 and STAT3 phosphorylation in response to EGF/HRG as well as to IL8 treatment. Importantly, PYK2 depletion also reduced EGF/HRG-induced MMP9 and IL8 transcription, while IL8 inhibition abrogated EGF-induced MMP9 transcription and attenuated cell invasion. IL8, which is transcriptionally regulated by STAT3 and induces PYK2 activation, prolonged EGF-induced PYK2, STAT3 and ERK1/2 phosphorylation suggesting that IL8 acts through an autocrine loop to reinforce EGF-induced signals. Collectively our studies suggest that PYK2 is a common downstream effector of ErbB and IL8 receptors, and that PYK2 integrates their signaling pathways through a positive feedback loop to potentiate breast cancer invasion. Hence, PYK2 could be a potential therapeutic target for a subset of breast cancer patients.