Thirty-five years since the discovery of chemotactic cytokines, interleukin-8 and MCAF: A historical overview.

Inflammation is a host defense response to various invading stimuli, but an excessive and persistent inflammatory response can cause tissue injury, which can lead to irreversible organ damage and dysfunction. Excessive inflammatory responses are believed to link to most human diseases. A specific type of leukocyte infiltration into invaded tissues is required for inflammation. Historically, the underlying molecular mechanisms of this process during inflammation were an enigma, compromising research in the fields of inflammation, immunology, and pathology. However, the pioneering discovery of chemotactic cytokines (chemokines), monocyte-derived neutrophil chemotactic factor (MDNCF; interleukin [IL]-8, CXCL8) and monocyte chemotactic and activating factor (MCAF; monocyte chemotactic factor 1 [MCP-1], CCL2) in the late 1980s finally enabled us to address this issue. In this review, we provide a historical overview of chemokine research over the last 35 years.

Analysis of the concentration of CXCL8 chemokine and its CXCR1 and CXCR2 receptors in peritoneal fluid of women with endometriosis.

Endometriosis is an inflammatory estrogen-dependent gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. An important role in the pathogenesis of this disease is played by disorders of the immune system involving chemokines and their receptors, including the CXCL8-CXCR1/ 2 system. AIM: The aim of the study was to assess the concentration of the CXCL8 chemokine and its CXCR1 and CXCR2 receptors in the peritoneal fluid of women with endometriosis. MATERIALS AND METHODS: The study included 32 women aged 21 to 47 years with diagnosed endometriosis and a control group of 8 healthy women aged 21 to 40 years. The material for the research was the peritoneal fluid collected during the laparoscopic procedure. The concentration of chemokines was determined by ELISA tests. RESULTS: The conducted studies showed that the concentration of the CXCL8 chemokine was significantly higher in the peritoneal fluid of the studied women and depended on the clinical advancement of the disease. CONCLUSIONS: Changes in the concentration of the CXCL8 chemokine in the peritoneal fluid of women with endometriosis may indicate impaired immune response and indicate an inflammatory process within the peritoneal cavity. The demonstrated relationship between the concentration of CXCL8 and the stages of clinical advancement indicates a significant role of this chemokine in the development of the disease.

The role of CXCL8 in chronic nonhealing diabetic foot ulcers and phenotypic changes in fibroblasts: a molecular perspective.

INTRODUCTION: A persistent inflammation is perpetuated by infiltrating immune cells and cytokines secreted from these immune cells. Additionally, apoptotic keratinocytes and adipocytes in diabetes causes diabetic foot ulcer (DFU) to arrest in an inflammatory phase without progressing to the resolution phase. This leads to a nonhealing DFU and, despite advanced treatments consisting of wound debridement, off-loading the ulcer of necrotic tissue, wound dressings to keep it moist and control exudate, medication, and preventing infection, DFUs remain a clinical problem. Nonhealing DFUs pose not only an economic burden but also increased morbidity and mortality in the form of psychological stress with and increased chance of amputation, and even death. Thus, investigating the complicated underlying molecular mechanism responsible for nonhealing patterns and designing better therapeutics is warranted. This review article focuses on the role of IL-8-mediated persistent inflammation and phenotypic change of fibroblasts due to this inflammatory cascade. We have discussed various sources of interleukin (IL)-8 secretion and the possible association of IL8-fibroblast plasticity as a cause of nonhealing DFUs. MATERIAL AND METHODS: A literature search on PubMed, Google Scholar, and PMC was done including the terms diabetic foot ulcer, diabetes, diabetic ulcer, chronic inflammation, interleukin 8, diabetic wound, and nonhealing diabetic foot ulcers. The articles in the English language and published in last 10 years were selected. From the pool of these, the articles describing the relationship between IL-8 and nonhealing diabetic foot ulcer and diabetic ulcer were used sorted out and used for this review article following PRISMA guidelines. CONCLUSION: Increased infiltration of inflammatory immune cells, secretion of pro-inflammatory cytokines, altered keratinocyte-fibroblast function, and phenotypic changes of fibroblasts in DFUs seem to be critical to the nonhealing of DFUs. Thus, inhibiting IL-8 secretion and downstream signaling seems to be a goal of potential therapeutics.

Oral Squamous Cell Carcinoma Contributes to Differentiation of Monocyte-Derived Tumor-Associated Macrophages via PAI-1 and IL-8 Production.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation and metastasis, as well as anti-tumor immune suppression. Recent studies have shown that tumors enhance the recruitment and differentiation of TAMs, but the detailed mechanisms have not been clarified. We thus examined the influence of cancer cells on the differentiation of monocytes to TAM subsets, including CD163+, CD204+, and CD206+ cells, in oral squamous cell carcinoma (OSCC) using immunohistochemistry, flow cytometry, and a cytokine array. Furthermore, we investigated the effect of OSCC cells (HSC-2, SQUU-A, and SQUU-B cells) on the differentiation of purified CD14+ cells to TAM subsets. The localization patterns of CD163+, CD204+, and CD206+ in OSCC sections were quite different. The expression of CD206 on CD14+ cells was significantly increased after the co-culture with OSCC cell lines, while the expressions of CD163 and CD204 on CD14+ cells showed no change. High concentrations of plasminogen activator inhibitor-1 (PAI-1) and interleukin-8 (IL-8) were detected in the conditioned medium of OSCC cell lines. PAI-1 and IL-8 stimulated CD14+ cells to express CD206. Moreover, there were positive correlations among the numbers of CD206+, PAI-1+, and IL-8+ cells in OSCC sections. These results suggest that PAI-1 and IL-8 produced by OSCC contribute to the differentiation of monocytes to CD206+ TAMs.

The IL-8-CXCR1/2 axis contributes to diabetic kidney disease.

AIMS/HYPOTHESIS: Inflammation has a major role in diabetic kidney disease. We thus investigated the role of the IL-8-CXCR1/2 axis in favoring kidney damage in diabetes. METHODS: Urinary IL-8 levels were measured in 1247 patients of the Joslin Kidney Study in type 2 diabetes (T2D). The expression of IL-8 and of its membrane receptors CXCR1/CXCR2 was quantified in kidney tissues in patients with T2D and in controls. The effect of CXCR1/2 blockade on diabetic kidney disease was evaluated in db/db mice. RESULTS: IL-8 urinary levels were increased in patients with T2D and diabetic kidney disease, with the highest urinary IL-8 levels found in the patients with the largest decline in glomerular filtration rate, with an increased albumin/creatine ratio and the worst renal outcome. Moreover, glomerular IL-8 renal expression was increased in patients with T2D, as compared to controls. High glucose elicits abundant IL-8 secretion in cultured human immortalized podocytes in vitro. Finally, in diabetic db/db mice and in podocytes in vitro, CXCR1/2 blockade mitigated albuminuria, reduced mesangial expansion, decreased podocyte apoptosis and reduced DNA damage. CONCLUSIONS/INTERPRETATION: The IL-8- CXCR1/2 axis may have a role in diabetic kidney disease by inducing podocyte damage. Indeed, targeting the IL-8-CXCR1/2 axis may reduce the burden of diabetic kidney disease.

VEGF-A, HGF and bFGF are Involved in IL-17A-mediated Migration and Capillary-like Vessel Formation of Vascular Endothelial Cells.

BACKGROUND: Interleukin (IL)-17A possesses biological activities to promote vascular endothelial cell migration and microvessel development. OBJECTIVE: To clarify which angiogenic factors are involved in IL-17A-modified angiogenesis-related functions of vascular endothelial cell migration and microtube development or not. METHODS: The potential contribution of various angiogenic stimulators to in vitro angiogenic activities of IL-17A was assessed with both modified Boyden Chemotaxicell chamber assay and in vitro angiogenesis assay. RESULTS: The addition of a neutralizing antibody (Ab) for hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF)-A to the upper and lower compartments in a modified Boyden Chemotaxicell chamber significantly attenuated human dermal microvascular endothelial cell (HMVEC) migration elicited by IL-17A. Moreover, IL-17A-induced capillary-like microvessel development in human umbilical vein endothelial cell (HUVEC) and human dermal fibroblast (HDF) co-culture system was significantly impaired by a neutralizing Ab against HGF, bFGF, VEGF-A, cysteine-x-cysteine ligand 8 (CXCL8)/IL-8 or cysteine-x-cysteine (CXC) chemokine receptor (CXCR)-2. CONCLUSION: Our findings demonstrate the involvement of HGF, bFGF, VEGF-A and/or CXCL8/IL-8, to various degrees, in migration and microvessel development of vascular endothelial cells mediated by IL-17A.

Upregulation of ACE2 and TMPRSS2 by particulate matter and idiopathic pulmonary fibrosis: a potential role in severe COVID-19.

BACKGROUND: Air pollution exposure and idiopathic pulmonary fibrosis (IPF) cause a poor prognosis after SARS-CoV-2 infection, but the underlying mechanisms are not well explored. Angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are the keys to the entry of SARS-CoV-2. We therefore hypothesized that air pollution exposure and IPF may increase the expression of ACE2 and TMPRSS2 in the lung alveolar region. We measured their expression levels in lung tissues of control non-IPF and IPF patients, and used murine animal models to study the deterioration of IPF caused by particulate matter (PM) and the molecular pathways involved in the expression of ACE2 and TMPRSS2. RESULTS: In non-IPF patients, cells expressing ACE2 and TMPRSS2 were limited to human alveolar cells. ACE2 and TMPRSS2 were largely upregulated in IPF patients, and were co-expressed by fibroblast specific protein 1 (FSP-1) + lung fibroblasts in human pulmonary fibrotic tissue. In animal models, PM exposure increased the severity of bleomycin-induced pulmonary fibrosis. ACE2 and TMPRSS2 were also expressed in FSP-1+ lung fibroblasts in bleomycin-induced pulmonary fibrosis, and when combined with PM exposure, they were further upregulated. The severity of pulmonary fibrosis and the expression of ACE2 and TMPRSS2 caused by PM exposure were blocked by deletion of KC, a murine homologue of IL-8, or treatment with reparixin, an inhibitor of IL-8 receptors CXCR1/2. CONCLUSIONS: These data suggested that risk of SARS-CoV-2 infection and COVID-19 disease severity increased by air pollution exposure and underlying IPF. It can be mediated through upregulating ACE2 and TMPRSS2 in pulmonary fibroblasts, and prevented by blocking the IL-8/CXCR1/2 pathway.

IL-8 induces transdifferentiation of mature hepatocytes toward the cholangiocyte phenotype.

The adult mammalian liver exhibits a remarkable regenerative capacity, with different modes of regeneration according to the type and extent of injury. Hepatocyte-cholangiocyte biphenotypic liver progenitor cell populations appear under conditions of excessive injury. It has been reported that mature hepatocytes can transdifferentiate toward a cholangiocyte phenotype and be a cellular source of progenitor cell populations. Here, we determined that among various plasma cytokines, interleukin (IL)-8 levels were significantly elevated in acute liver failure and severe acute liver injury patients. In vitro assays revealed that administration of IL-8 homologues increases the expression of Sry HMG box protein 9 (SOX9). In liver biopsies of acute liver injury patients, we observed the appearance of SOX9-positive biphenotypic hepatocytes accompanied by elevation of plasma IL-8 levels. Our results suggest that IL-8 regulates the phenotypic conversion of mature hepatocytes toward a cholangiocyte phenotype.

Endothelial cells promote 3D invasion of GBM by IL-8-dependent induction of cancer stem cell properties.

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

Immunomodulatory effects of rhesus monkey bone marrow-derived mesenchymal stem cells in serum-free conditions.

Mesenchymal stem cells (MSCs) have generated tremendous interest for treating various diseases due to their self-renewal and differentiation capacities. Many studies have demonstrated the immunoregulatory capability of MSCs; however, most of these studies were conducted with fetal bovine serum (FBS), which has an uncertain composition. In this study, we established a serum-free, xeno-free, completely chemically defined medium for the proliferation and expansion of rhesus monkey bone marrow (BM)-derived MSCs (rBMSCs) in vitro. The growth kinetics, characteristics, immunophenotype, and immunosuppressive abilities of rBMSCs grown in serum-free media (SFM) were evaluated and compared with those of cells grown in serum-containing media (SCM). Moreover, we employed RNA sequencing to evaluate the expression pattern of genes related to immune responses in both culture conditions. Compared to cells grown in SCM, rBMSCs grown in SFM exhibited better biological characteristics regarding cell proliferation and immunosuppressive abilities. Cells from both media types exhibited similar immunophenotypic expression patterns for CD29, CD34, CD45, HLA-DR, CD73, CD90, and CD105. Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and Gene Set Enrichment Analysis (GSEA) revealed that CXCL8 was downregulated by 4.1 fold in SFM-cultured rBMSCs compared with those in SCM. Furthermore, the mixed lymphocyte culture revealed that the proliferation activity and the expression levels of inflammatory factors of peripheral blood mononuclear cells (PBMCs) were significantly decreased after the addition of the CXCL8 neutralizing antibody, which was related to the elevated immunosuppressive abilities of SFM-suspended rBMSCs. These results suggest a possible cell culture method as well as immunoregulatory mechanisms for clinical cell therapies requiring nonanimal-derived components.

Study of association between interleukin-8 - 845 T/C and + 781 C/T polymorphisms with periodontitis disease among population from Western Iran.

Periodontitis is a chronic inflammatory disease that influences the protective tissues of teeth. IL-8, a member of the chemokine super-family, plays vital roles in the pathogenesis of periodontitis with activation and migration of neutrophils in inflammatory regions. The purpose of present study was to evaluate the association of interleukin-8 - 845 T/C and + 781 C/T polymorphisms with periodontitis in an Iranian population. A total of 65 patients with periodontitis including 18 patients with chronic periodontitis and 47 patients with aggressive periodontitis and 55 controls were enrolled into our study. Interleukin-8 - 845 T/C and + 781 C/T polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. For + 781C/T locus, in the dominant genetic model there was a significant difference between TT vs. CC + CT genotypes that significantly had a protective role against periodontitis disease with a value of 0.38 (95% CI 0.16-0.90, p = 0.02). Also, the analysis of results showed a significant positive association between the distribution of IL-8 - 845 T/C alleles and the risk of periodontitis disease (χ2 = 6.2, p = 0.01) that presence of C allele of IL-8 - 845 increased the risk of periodontitis disease by 9.08-fold [OR 9.08 (95% CI 1.14-72.12, p = 0.03)]. In conclusion, our results demonstrate a positive association between distribution of IL-8 - 845 T/C alleles and risk of periodontitis disease.

MCP-1, KC-like and IL-8 as critical mediators of pathogenesis caused by Babesia canis.

Canine babesiosis caused by the intraerythrocytic protozoan parasite Babesia canis is a tick-borne disease characterized by a host response that involves both cellular and humoral immunity. This study focuses on the secretion of cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Keratinocyte Chemotactic-like (KC-like), Interleukins (IL)-2, IL-7, IL-8, IL-10, IL-15, IL-18 and Monocyte Chemotactic Protein-1 (MCP-1) in babesiosis caused by Babesia canis upon treatment with Imizol®. We assessed time dependent changes in cytokine levels and tested whether these changes correlate with pathogenesis of the disease. Sixteen healthy dogs and 31 dogs infected with Babesia canis, of which 18 showed complications, were treated with Imizol®. One dog died during the study (3.2%). Longitudinal study was perfomed by monitoring dogs at the first day of presentation (day 1) and 6 days later (day 7). Our results show that higher MCP-1 levels on day 1 are positively associated with the occurrence of complications, (complicated vs. uncomplicated; p = 0.00016). A similar pattern was observed for KC-like on day 1 (p = 0.0326) and day 7 (p = 0.044). Moreover, babesiosis caused by B. canis produced a steady increase in IL-8 levels with a moderate to strong negative correlation with erythrocyte counts and hematocrit in uncomplicated diseased dogs only (Spearman's rank correlation coefficient rs = -0.582 and rs = -0.598 respectively). Like for MCP-1, KC-like levels also differed in complicated and uncomplicated diseased dogs on day 1 (p = 0.03236) and day 7 (p = 0.044). Furthermore, KC-like levels were strongly correlated with IL-8 levels (rs = 0.663-0.7) and non-segmented neutrophil counts (rs = 0.572-0.732) in both diseased groups. Analysis of ROC suggests the use of serum levels of MCP-1 and IL-7 as predictors of the occurrence of complications with an AUC of 0.906 and 0.896 respectively and linear combinations of MCP-1, KC-Like, IL-7 and GM-CSF with values up to AUC = 0.983. Cytokine cluster analysis presented in this study can contribute to a better understanding of the pathogenesis of babesiosis and serve as a prognostic tool for the early detection of cases with highest likelihood of developing complications. Overall, our studies show that infection by B. canis elicits a cytokine pattern that is distinct from that observed with B. rossi, and that some of the inflammatory mediators can be useful to predict complications. Our results also suggest targets for the development of novel therapeutic strategies in babesiosis caused by B. canis.

Integrated effect of seasons and lactation stages on the plasma inflammatory cytokines, function and receptor expression of milk neutrophils in Sahiwal (Bos indicus) cows.

Mastitis is a highly prevalent and one of the costliest diseases of dairy cows affecting the mammary gland. Milk neutrophils present in the mammary gland serve as an integral part of the mammary immunity, and their performance is influenced by different environmental conditions and lactation stages. To investigate the combined effects of seasons and lactation stages on the mammary immunity, milk and blood samples were collected from three groups of high producing indigenous Sahiwal cows. Function and receptor expression of milk neutrophils together with cortisol and inflammatory interleukins concentration in blood were studied. The first group of cows started their lactation in winter and completed their lactation in hot-humid season; the second group started their lactation in hot-dry season and completed it in winter. The third group started their lactation in hot-humid and completed by the hot-dry season. Plasma cortisol levels were very high during early lactation in all seasons. An inverse relationship was observed between cortisol levels and glucocorticoid receptor. Elevated phagocytic activity and plasma interleukin-2 levels were seen in winter and during mid lactation of all seasons. A positive correlation was noticed between plasma IL-8, the percentage of milk neutrophils and expression of chemokine receptors (CXCR1 and CXCR2). The highest expression of toll-like receptors (TLR2 and TLR4) and chemokine receptors was in hot-humid season. Reduction in the phagocytic activity of neutrophils, pro-inflammatory cytokines and elevated levels of cortisol in cows which started their lactation and attained peak lactation during hot-humid season indicated more stress in them. Integrated influence of both seasons and lactation stages on the activity of milk neutrophils along with plasma interleukins and cortisol levels may be used to develop suitable managemental strategies to improve mammary health and increase milk production in indigenous dairy breeds experiencing harsh environmental conditions.

The role of interleukin 8 and matrix metalloproteinases 2 and 9 in breast cancer treated with tamoxifen

PURPOSE: To evaluate the role of interleukin 8 (IL8) and matrix metalloproteinase (MMP) 2 and 9 as potential parameters of response to adjuvant tamoxifen and to examine possible associations between biomarkers that might imply possible biological dependence. METHODS: The study included 59 postmenopausal breast cancer patients who received adjuvant tamoxifen. Biomarker levels were determined by ELISA in cytosol tumor extracts. RESULTS: Estrogen receptor (ER) proved as a reliable parameter of response to tamoxifen; patients with ER+ status had significantly longer median relapse-free survival (RFS) compared to those with ER- status (p=0.04). Patients with IL8-status had longer median RFS compared to those with IL8+ status (77 and 53 months, respectively) but without significant difference. Patients with MMP9+ status had longer median RFS compared to those with MMP9-status (92 and 66 months, respectively) but without significant difference. There was no significant difference in RFS between the subgroups formed according to MMP2 median value. A significant positive correlation was found between IL8 and MMP9 levels (p<0.001). Expression of MMP9 was significantly higher in patients with IL8 levels higher than the median (p=0.001). CONCLUSIONS: IL8 showed a tendency to act as an unfavorable parameter while MMP9 showed a tendency to act as a favorable parameter of response to tamoxifen, whereas the role of MMP2 as a potential predictive parameter is more complex. The results indicate that possible existence of positive feedback between IL8 and MMP9 might contribute to progression of breast cancer.

CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment.

The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC-endothelial cell "cuddling," HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.

Insulin-dependent cytoplasmic distribution of Rab4a in mouse adipocytes is inhibited by interleukin-6, -8, and -15.

The purpose of the study was to examine the effect of interleukins, IL-6, IL-8, and IL-15, on insulin-mediated redistribution of Rab4a, an early endosome marker, in mouse 3T3-L1 adipocytes. The interleukins did not affect cell viability; however, cell number was slightly but significantly higher in cultures exposed to IL-8 and IL-15. IL-8 and IL-15 decreased lipid storage in adipocytes, whereas IL-6 had no effect. Rab4A showed cytoplasmic localization, and in control unstimulated adipocytes it was found primarily nearby nucleus, that was supported by cellular fluorescence distribution profile, and by calculated indices, that is, high percentage of near-nuclear area fluorescence and a low mean peripheral cytoplasmic fluorescence/mean near-nuclear fluorescence ratio. Insulin stimulation (100 nmol/l, 30 min) altered the cytoplasmic localization of Rab4a in control adipocytes, which was manifested by its redistribution towards plasma membrane. This effect of insulin was prevented in adipocytes exposed to IL-6, IL-8, or IL-15. We concluded that insulin-dependent Rab4a redistribution, probably reflecting stimulation of vesicle-mediated transport, is inhibited in adipocytes subjected to differentiation in the presence of IL-6, IL-8, or IL-15. Such alterations may be involved in the mechanisms contributing to development of insulin resistance associated with inflammation; however, further studies in this field are required.

Interleukin 8 haplotypes drive divergent responses in uterine endometrial cells and are associated with somatic cell score in Holstein-Friesian cattle.

Interleukin 8 is a proinflammatory chemokine involved in neutrophil recruitment and activation in response to infection and also in the resolution of inflammation. Our previous studies identified a number of genetic polymorphisms in the bovine IL8 promoter region which segregate into two haplotypes, with balanced frequencies in the Holstein-Friesian (HF). We subsequently showed that these haplotypes confer divergent IL8 activity both in vitro in mammary epithelial cells and in vivo in response to LPS. In this study, we hypothesised that the balanced frequency of IL8 haplotype in HF could be explained by divergent selection pressures acting on this locus. To address this hypothesis, an association study was carried out aiming to identify a putative link between the IL8 haplotype and somatic cell score (SCS) in 5746 Holstein-Friesian dairy cows. In addition, the basal and inducible promoter activity of the two IL8 haplotypes was characterised in bovine endometrial epithelial (BEND) cells and in monocyte-derived macrophages. Results showed a significant association between IL8 haplotype 2 (IL8-h2) with increased SCS (P<0.05). Functional analysis showed that the same haplotype was a more potent inducer of IL8 expression in BEND cells in response to LPS and TNFα stimulation. In contrast, co-transfection of the BEND cells with a DNA construct encoding a bovine herpesvirus 4 antigen, induced significantly higher IL8 expression from IL8-h1. The present study sheds light on the molecular mechanisms underlying selection for SCS and provides evidence that the balanced frequencies of the two IL8 haplotypes in HF cattle may occur as a result of opposing directional selection pressures of both bacterial and viral infection.

Downregulation of transgelin blocks interleukin-8 utilization and suppresses vasculogenic mimicry in breast cancer cells.

Vasculogenic mimicry (VM) is a non-classical mechanism recently described in many tumors, whereby cancer cells, rather than endothelial cells, form blood vessels. Transgelin is an actin-binding protein that has been implicated in multiple stages of cancer development. In this study, we investigated the role of transgelin in VM and assessed its effect on the expression of endothelial and angiogenesis-related genes during VM in MDA-MB-231 breast cancer cells. We confirmed the ability of MDA-MB-231 cells to undergo VM through a tube formation assay. Flow cytometry analysis revealed an increase in the expression of the endothelial-related markers VE-cadherin and CD34 in cells that underwent VM, compared with those growing in a monolayer, which was confirmed by immunocytochemistry. We employed siRNA to silence transgelin, and knockdown efficiency was determined by western blot analyses. Downregulation of transgelin suppressed cell proliferation and tube formation, but increased IL-8 levels in Matrigel cultures. RT-PCR analyses revealed that the expression of IL-8, VE-cadherin, and CD34 was unaffected by transgelin knockdown, indicating that increased IL-8 expression was not due to enhanced transcriptional activity. More importantly, the inhibition of IL-8/CXCR2 signaling also resulted in suppression of VM with increased IL-8 levels, confirming that increased IL-8 levels after transgelin knockdown was due to inhibition of IL-8 uptake. Our findings indicate that transgelin regulates VM by enhancing IL uptake. These observations are relevant to the future development of efficient antivascular agents. Impact statement Vasculogenic mimicry (VM) is an angiogenic-independent mechanism of blood vessel formation whereby aggressive tumor cells undergo formation of capillary-like structures. Thus, interventions aimed at angiogenesis might not target the entire tumor vasculature. A more holistic approach is therefore needed in the development of improved antivascular agents. Transgelin, an actin-binding protein, has been associated with multiple stages of cancer development such as proliferation, migration and invasion, but little is known about its role in vasculogenic mimicry. We present here, an additional mechanism by which transgelin promotes malignancy by way of its association with the occurrence of VM. Although transgelin knockdown did not affect the transcript levels of most of the angiogenesis-related genes in this study, it was associated with the inhibition of the uptake of IL-8, accompanied by suppressed VM, indicating that transgelin is required for VM. These observations are relevant to the future development of efficient antivascular agents.

Chemorepellent Semaphorin 3E Negatively Regulates Neutrophil Migration In Vitro and In Vivo.

Neutrophil migration is an essential step in leukocyte trafficking during inflammatory responses. Semaphorins, originally discovered as axon guidance cues in neural development, have been shown to regulate cell migration beyond the nervous system. However, the potential contribution of semaphorins in the regulation of neutrophil migration is not well understood. This study examines the possible role of a secreted chemorepellent, Semaphorin 3E (Sema3E), in neutrophil migration. In this study, we demonstrated that human neutrophils constitutively express Sema3E high-affinity receptor, PlexinD1. Sema3E displayed a potent ability to inhibit CXCL8/IL-8-induced neutrophil migration as determined using a microfluidic device coupled to real-time microscopy and a transwell system in vitro. The antimigratory effect of Sema3E on human neutrophil migration was associated with suppression of CXCL8/IL-8-mediated Ras-related C3 botulinum toxin substrate 1 GTPase activity and actin polymerization. We further addressed the regulatory role of Sema3E in the regulation of neutrophil migration in vivo. Allergen airway exposure induced higher neutrophil recruitment into the lungs of Sema3e-/- mice compared with wild-type controls. Administration of exogenous recombinant Sema3E markedly reduced allergen-induced neutrophil recruitment into the lungs, which was associated with alleviation of allergic airway inflammation and improvement of lung function. Our data suggest that Sema3E could be considered an essential regulatory mediator involved in modulation of neutrophil migration throughout the course of neutrophilic inflammation.