Hypoxia is an integral component of the tumor microenvironment. Either as chronic or cycling hypoxia, it exerts a similar effect on cancer processes by activating hypoxia-inducible factor-1 (HIF-1) and nuclear factor (NF-κB), with cycling hypoxia showing a stronger proinflammatory influence. One of the systems affected by hypoxia is the CXC chemokine system. This paper reviews all available information on hypoxia-induced changes in the expression of all CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 (IL-8), CXCL9, CXCL10, CXCL11, CXCL12 (SDF-1), CXCL13, CXCL14, CXCL15, CXCL16, CXCL17) as well as CXC chemokine receptors-CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 and CXCR8. First, we present basic information on the effect of these chemoattractant cytokines on cancer processes. We then discuss the effect of hypoxia-induced changes on CXC chemokine expression on the angiogenesis, lymphangiogenesis and recruitment of various cells to the tumor niche, including myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), regulatory T cells (T_regs) and tumor-infiltrating lymphocytes (TILs). Finally, the review summarizes data on the use of drugs targeting the CXC chemokine system in cancer therapies.

Keywords: CXC chemokine; HIF-1α; IL-8; NF-κB; SDF-1; cancer; cycling hypoxia; hypoxia; hypoxia-inducible factor; tumor.

The mortality rate of patients with critical illness has decreased significantly over the past two decades, but the rate of decline has slowed recently, with organ dysfunction as a major driver of morbidity and mortality. Among patients with the systemic inflammatory response syndrome (SIRS), acute lung injury is a common component with serious morbidity. Previous studies in our laboratory using a murine model of SIRS demonstrated a key role for NADPH oxidase 2 (Nox2)-derived reactive oxygen species in the resolution of inflammation. Nox2-deficient (gp91^phox-/y) mice develop profound lung injury secondary to SIRS and fail to resolve inflammation. Alveolar macrophages from gp91^phox-/y mice express greater levels of chemotactic and pro-inflammatory factors at baseline providing evidence that Nox2 in alveolar macrophages is critical for homeostasis. Based on the lung pathology with increased thrombosis in gp91^phox-/y mice, and the known role of platelets in the inflammatory process, we hypothesized that Nox2 represses platelet activation. In the mouse model, we found that platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) and CXCL7 were increased in the bronchoalveolar fluid of gp91^phox-/y mice at baseline and 24 h post intraperitoneal zymosan-induced SIRS consistent with platelet activation. Activated platelets interact with leukocytes via P-selectin glycoprotein ligand 1 (PSGL-1). Within 2 h of SIRS induction, alveolar neutrophil PSGL-1 expression was higher in gp91^phox-/y mice. Platelet-neutrophil interactions were decreased in the peripheral blood of gp91^phox-/y mice consistent with movement of activated platelets to the lung of mice lacking Nox2. Based on the severe lung pathology and the role of platelets in the formation of neutrophil extracellular traps (NETs), we evaluated NET production. In contrast to previous studies demonstrating Nox2-dependent NET formation, staining of lung sections from mice 24 h post zymosan injection revealed a large number of citrullinated histone 3 (H3CIT) and myeloperoxidase positive cells consistent with NET formation in gp91^phox-/y mice that was virtually absent in WT mice. In addition, H3CIT protein expression and PAD4 activity were higher in the lung of gp91^phox-/y mice post SIRS induction. These results suggest that Nox2 plays a critical role in maintaining homeostasis by regulating platelet activation and NET formation in the lung.

Bone homeostasis comprises the balance between bone-forming osteoblasts and bone-resorbing osteoclasts (OCs), with an acceleration of osteoclastic bone resorption leading to osteoporosis. OCs can be generated from bone marrow cells (BMCs) under the tightly regulated local bone environment. However, it remained difficult to identify the critical cells responsible for providing an osteoclastogenesis niche. In this study, we used a fluorescence-activated cell sorting technique to determine the cell populations important for forming an appropriate microenvironment for osteoclastogenesis and to verify the associated interactions between osteoclast precursor cells and non-OCs. We isolated and removed a small cell population specific for osteoclastogenesis (CXCR4+ CD45- ) from mouse BMCs and cultured the remaining cells with receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage-colony stimulating factor. The resulting cultures showed significantly less large osteoclast formation. Quantitative RT-PCR analysis revealed that these CXCR4+ CD45- cells expressed low levels of RANK and RANKL, but high levels of critical chemokines including stromal cell derived factor 1 (SDF-1), chemokine (C-X-C motif) ligand 7 (CXCL7), and chemokine (C-X3-C motif) ligand 1 (CX3CL1). Furthermore, an SDF-1-specific antibody strongly suppressed OC formation in RAW264.7 cells and antibodies against SDF-1, CXCL7, and CX3CL1 suppressed OC formation in BMCs. These results suggest that isolated CXCR4+ CD45- cells support an appropriate microenvironment for osteoclastogenesis with a direct effect on the cells expressing SDF-1, CXCL7, and CX3CL1 receptors. The regulation of CXCR4+ CD45- cell function might therefore inform therapeutic strategies for diseases involving loss of bone homeostasis. Stem Cells 2016;34:2733-2743.

OBJECTIVE

Chemokines exert different inflammatory responses which can potentially be related to certain fetal chromosomal abnormalities. The aim of the study was to determine the concentration of selected chemokines in plasma and amniotic fluid of women with fetal Down syndrome.

METHODS

Out of 171 amniocentesis, we had 7 patients with confirmed fetal Down syndrome (15th-18th weeks of gestation). For the purpose of our control, we chose 14 women without confirmed chromosomal aberration. To assess the concentration of chemokines in the blood plasma and amniotic fluid, we used a protein macroarray, which allows the simultaneous determination of 40 chemokines per sample.

RESULTS

We showed significant decrease in the concentration of 4 chemokines, HCC-4, IL-28A, IL-31, and MCP-2, and increase in the concentration of CXCL7 (NAP-2) in plasma of women with fetal Down syndrome. Furthermore, we showed decrease in concentration of 3 chemokines, ITAC, MCP-3, MIF, and increase in concentration of 4 chemokines, IP-10, MPIF-1, CXCL7, and 6Ckine, in amniotic fluid of women with fetal Down syndrome.

CONCLUSIONS

On the basis of our findings, our hypothesis is that the chemokines may play role in the pathogenesis of Down syndrome. Defining their potential as biochemical markers of Down syndrome requires further investigation on larger group of patients.

BACKGROUND

Listeria monocytogenes meningitis is the third most common cause of bacterial meningitis and is associated with high rates of mortality and unfavorable outcome.

METHODS

We analyzed 101 cytokines, chemokines and complement factors in CSF of adult patients with Listeria meningitis included in a prospective cohort study and compared these biomarkers between Listeria meningitis patients and negative controls, and between Listeria meningitis patients with a favorable and an unfavorable outcome.

RESULTS

CSF was available from 26 of 62 (42%) Listeria meningitis patients and 19 negative controls. Fifteen (58%) Listeria meningitis patients had an unfavorable outcome. In Listeria meningitis CSF levels of 51 biomarkers were significantly elevated compared to negative controls after Bonferroni correction. The 11 most significantly elevated (P < .01) biomarkers of unfavorable outcome in Listeria meningitis were markers of T-cell activation (sIL-2Rα, sCD40L and IL-1), interferon-related (IFN-α2, IL-18, CX3CL1, CCL20), markers of complement activation (C3a), and endothelial growth factor related (VEGF, CXCL7).

CONCLUSIONS

Our data suggest that T-cell activation, complement activation, interferon- and endothelial growth factor production are important in the immune response to Listeria meningitis, and thereby influence outcome.

CONCLUSIONS

Our study provides target pathways for further studies in the pathophysiology of Listeria meningitis.

Although human stromal vascular fraction (SVF) has been regarded as an attractive stem cell source, its therapeutic mechanism in wound healing has not been fully elucidated.

In this study, we investigated the molecular characteristics and therapeutic property of SVF for wound healing.

Microarray data showed that SVF cells are enriched with a higher level of wound healing or epithelium development-related genes and micro RNA.

Quantitative polymerase chain reaction (PCR) and reverse transcriptase PCR results revealed that the epithelialization growth factor, epidermal growth factor (EGF), chemokines, stromal cell-derived factor (SDF-1 or CXCL12), neutrophil-activating protein-2 (NAP-2 or CXCL7), chemokine receptors (CXCR1, CCR2 and CCR3) and wound healing genes were up-regulated in SVF compared with those in adipose-derived mesenchymal stem cells (ASCs). An in vitro scratch wound closure experiment demonstrated that co-culture with SVF substantially accelerated the wound closure of fibroblasts. Wounds in nude mice were created by skin excisions followed by injections of SVF with Pluronic hydrogel. SVF implantation highly accelerated wound closure and increased cellularity and re-epithelialization. In addition, the transplanted SVF exhibited high engraftment rates in the wound area, suggesting direct benefits for cutaneous closure.

Taken together, these data suggest that SVF possesses high therapeutic capability for wound healing via the secretion of epithelialization and chemotactic growth factors and enhanced engraftment properties.

Background: Microvascular, arterial and venous thrombotic events have been largely described during severe coronavirus disease 19 (COVID-19). However, mechanisms underlying hemostasis dysregulation remain unclear.

Methods: We explored two independent cross-sectional cohorts to identify soluble markers and gene-expression signatures that discriminated COVID-19 severity and outcomes.

Results: We found that elevated soluble (s)P-selectin at admission was associated with disease severity. Elevated sP-selectin was predictive of intubation and death (ROC AUC = 0.67, p = 0.028 and AUC = 0.74, p = 0.0047, respectively). An optimal cutoff value was predictive of intubation with 66% negative predictive value (NPV) and 61% positive predictive value (PPV), and of death with 90% NPV and 55% PPV. An unbiased gene set enrichment analysis revealed that critically ill patients had increased expression of genes related to platelet activation. Hierarchical clustering identified ITG2AB, GP1BB, PPBP and SELPLG to be upregulated in a grade-dependent manner. ROC curve analysis for the prediction of intubation was significant for SELPLG and PPBP (AUC = 0.8, p = 0.046 for both). An optimal cutoff value for PBPP was predictive of intubation with 100% NPV and 45% PPV, and for SELPLG with 100% NPV and 50% PPV.

Conclusion: We provide evidence that platelets contribute to COVID-19 severity. Plasma sP-selectin level was associated with severity and in-hospital mortality. Transcriptional analysis identified PPBP/CXCL7 and SELPLG as biomarkers for intubation. These findings provide additional evidence for platelet activation in driving critical COVID-19. Specific studies evaluating the performance of these biomarkers are required.

Keywords: COVID-19; Platelets; Primary hemostasis; Thrombo-inflammation.

BACKGROUND

It is becoming increasingly evident that platelet chemokines are involved in distinct aspects of atherosclerosis. The aim of this study was to examine the effects of long-term supplementation with purified anthocyanins on platelet chemokines in hypercholesterolemic individuals and to identify correlations of decreased platelet chemokine levels with serum lipid and inflammatory marker levels.

METHODS

A total of 146 hypercholesterolemic individuals were recruited and treated with 320 mg of purified anthocyanins (n = 73) or a placebo (n = 73) daily for 24 weeks in this randomized, double-blind, placebo-controlled trial.

RESULTS

Anthocyanin supplementation for 24 weeks significantly decreased the plasma CXCL7 (-12.32% vs. 4.22%, P = 0.001), CXCL5 (-9.95% vs. 1.93%, P = 0.011), CXCL8 (-6.07% vs. 0.66%, P = 0.004), CXCL12 (-8.11% vs. 5.43%, P = 0.023) and CCL2 levels (-11.63% vs. 12.84%, P = 0.001) compared with the placebo. Interestingly, the decreases in the CXCL7 and CCL2 levels were both positively correlated with the decreases in the serum low-density lipoprotein-cholesterol (LDL-C), high-sensitivity C-reactive protein (hsCRP) and interleukin-1β (IL-1β) levels after anthocyanin supplementation for 24 weeks. The decrease in the CXCL8 level was negatively correlated with the increase in the how-density lipoprotein-cholesterol (HDL-C) level and was positively correlated with the decrease in the soluble P-selectin (sP-selectin) level in the anthocyanin group. In addition, a positive correlation was observed between the decreases in the CXCL12 and tumornecrosis factor-α (TNF-α) levels after anthocyanin supplementation. However, the plasma CXCL4L1, CXCL1, macrophage migration inhibitory factor (MIF) and human plasminogen activator inhibitor 1 (PAI-1) levels did not significantly change following anthocyanin supplementation.

CONCLUSIONS

The present study supports the notion that platelet chemokines are promising targets of anthocyanins in the prevention of atherosclerosis.

BACKGROUND

ChiCTR-TRC-08000240. Registered: 10 December 2008.

Intrauterine growth restriction (IUGR) is a multifactorial condition in that the fetus does not reach its genetically given growth potential. Besides its contribution to perinatal mortality, it is a risk factor for cardiovascular and metabolic diseases later in life. The diagnosis is based on antenatal sonography, which allows differentiating between IUGR and fetuses that are small by constitution (small for gestational age [SGA]). Yet, neither a clinical nor a biochemical tool is available to confirm reliably the diagnosis of IUGR postnatally. Recently, we identified umbilical cord blood proteins of the apolipoprotein family by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with differential signal intensities between the IUGR group and a control group. We hypothesized that identified molecules have the potential to generate a proteome profile specific for IUGR. A total of 114 serum samples (42 from the IUGR group, 12 from the SGA group, and 60 from the control group) of the umbilical vein (99 samples) and umbilical artery (15 samples) were analyzed. Sample quality was estimated by determining the abundance of hemoglobin (hemolysis) and CXC-motif chemokines CXCL4 and CXCL7 (platelet activation). Samples passing the quality criteria were forwarded to multiplex apolipoprotein profiling. Assay performance was tested with the sample sets, resulting in a sensitivity of 0.91 and a specificity of 0.85 in the test set with venous blood samples. Arterial cord blood samples followed the trend (sensitivity, 0.67; specificity, 0.85). SGA samples grouped together with the control samples. We conclude that the proteome profiling signature is confirmatory to clinical surveillance with the potential to identify neonates with IUGR postnatally in low-birth weight babies born at uncertain gestational age when antenatal sonography data have not been recorded.

The relationship has become clear between the expression of chemokine/chemokine receptors on cancer cells and the invasion, metastasis and peritoneal dissemination. Many cancer cells express chemokine receptors which are not expressed on the surface of normal tissues. Recently, it has been reported that overexpression of CXCR4/CXCL12 is related with metastasis to lung, liver, lymph nodes and bone marrow, while the overexpression of CCR7/CCL21 is mainly related with lymph node metastasis. We performed a comparative analysis of differential gene expressions related to chemokines/chemokine receptors, and cytokines in established gastric cancer cell lines by cDNA microarray. Upregulated chemokine genes were CCL21, CCL5, CXCL14, CCL2, CXCL1, CXCL8, CXCL7 and CXCL12, which the downregulated chemokines genes were MIP-1alpha and TECK. The upregulated gene of chemokine receptors was CCR-6. In the cancer microenvironment, cancer cells readily formed edematous and inflammatory conditions, easily metastasizing to other organs with the suppression of dendritic cells. The chemokines/chemokine receptors will hopefully become the new targets for cancer therapies for the regulation of metastasis.

The CXC chemokines are a family of closely related chemoattractant cytokines that bind to, attract, and activate neutrophils to variable degrees. In this study, the relationship between neutrophil-binding affinity and suitability for infection imaging was investigated in a selected group of CXC chemokines. Neutrophil-activating peptide-2 (NAP-2, 70 residues; also called CXCL7) binds with high affinity to the CXCR2 receptor on neutrophils. Recently, C-terminally truncated NAP-2-variants have been described that have enhanced neutrophil-binding affinity and neutrophil-stimulating capacity. Here, NAP-2 and its C-terminal shortened variants NAP-2(1-68), NAP-2(1-66), and NAP-2(1-63) were labeled with (99m)Tc via the hydrazinonicotinamide (HYNIC) chelator and their potential for imaging of infection was investigated in a rabbit model of infection. The CXC chemokine interleukin-8 (IL-8) was used for comparison. In addition, a series of (99m)Tc-labeled CXC chemokines were screened for their potential to image infection, including CTAP-III, GCP-2, ENA-78, PF-4, and IP-10.

METHODS

The receptor-binding affinity of HYNIC-conjugated NAP-2 and its analogs was compared in competitive binding assays on Jurkat cells transfected with the CXCR2 receptor gene. Biodistribution of labeled NAP-2 (analogs) and other CXC chemokines in rabbits with intramuscular Escherichia coli infections was determined both by gamma-camera imaging and by counting dissected tissues at 6 h after injection.

RESULTS

The CXCR2-binding affinity of the HYNIC-conjugated NAP-2 analogs relative to NAP-2 was as follows: NAP-2(1-68), 2.5-fold; NAP-2(1-66), 10-fold; and NAP-2(1-63), 3-fold. In the rabbit model, uptake in the abscess (in percentage injected dose per gram +/- SEM) was 0.084 +/- 0.015 for NAP-2, 0.098 +/- 0.010 for NAP-2(1-68), 0.189 +/- 0.044 for NAP-2(1-66), and 0.114 +/- 0.017 for NAP-2(1-63) at 6 h after injection. In comparison, higher uptake in the abscess was found for labeled IL-8, a modest uptake was found for GCP-2 and ENA-78, and a low uptake was found for CTAP-III, PF-4, and IP-10.

CONCLUSIONS

This study showed a clear relationship between affinity to receptors on neutrophils and suitability for infection imaging. Of the NAP-2 variants, NAP-2(1-66) combined highest affinity to CXCR2 with the best characteristics for imaging. IL-8 binds to both CXCR1 and CXCR2 with high affinity and showed a superior imaging quality. The other CXC chemokines tested bind to neutrophils with lower affinity and were shown to be less suitable for infection imaging in this study.

Ossification of the posterior longitudinal ligament (OPLL), a spinal ligament, reduces the range of motion in limbs. No treatment is currently available for OPLL, which is why therapies are urgently needed. OPLL occurs in obesity, is more common in men, and has an onset after 40 years of age. The mechanisms underlying OPLL remain unclear. In this study, we performed a serum proteomic analysis in both OPLL patients and healthy subjects to identify factors potentially involved in the development of OPLL, and found reduced levels of a protein that might underlie the pathology of OPLL. We isolated the protein, determined its amino acid sequence, and identified it as chemokine (C-X-C motif) ligand 7 (CXCL7). Based on these proteomics findings, we generated a CXCL7 knockout mouse model to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossification in the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7 deficiency in OPLL, we searched for single nucleotide polymorphisms and altered DNA exons, but no abnormalities were found. Although miR-340 levels were found to be high in an miRNA array, they were insufficient to reduce CXCL7 levels. Ubiquitin C-terminal hydrolase1 (UCHL1) was found to be overexpressed in CXCL7-null mice and in the sera of patients with OPLL, and was correlated with OPLL severity. Post-translational modifications of proteins with ubiquitin and ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) enzymes, are thought to control a wide range of cellular processes, and alterations in the ubiquitin-proteasome system have been associated with several degenerative disorders. In addition, the OPLL tissue of CXCL7-null mouse and its primary cells expressed the antibody to ubiquitin (linkage-specific K48). Our data clearly show decreased CXCL7 levels in patients with OPLL, and that OPLL developed in mice lacking CXCL7. Tumor necrosis factor receptor-associated factor (TRAF)6 expression was decreased in CXCL7-null mouse primary cells. Furthermore, K48 polyubiquitination was found in posterior longitudinal ligament ossified tissue and primary cells from CXCL7-null mice. We performed a phosphoproteomics analysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase (ME3K)15, ubiquitin protein ligase E3C (UBE3C) and protein kinase C (PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7 deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitination in the onset of OPLL. In conclusion, CXCL7 levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7 levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL.

Chemokines play a central role in cellular communication in response to bacterial infection. However, the knowledge of the chemokine responses to bacterial infections in dogs remains limited. Uterine bacterial infection (pyometra) is one of the most common bacterial diseases in dogs and causes sepsis in most of the cases. We have shown previously that dogs with pyometra have higher messenger RNA (mRNA) levels of chemokines in uterus. To assess whether the stromal part of the endometrium expresses chemokines in response to bacterial infection, we cultured endometrial stromal cells isolated from healthy dogs and exposed them to either live pathogenic Escherichia coli, isolated from the uterus of a dog with pyometra, or lipopolysaccharide. Changes in the mRNA expression of ELR(+) CXC chemokines, IL-8, CXCL5, CXCL7, and ELR(-) CXC chemokine, CXCL10, were measured after 24 hours using quantitative real-time polymerase chain reaction. Levels of IL-8, CXCL5, and CXCL10 were upregulated in endometrial stromal cells exposed to E coli and lipopolysaccharide, whereas the level of CXCL7 was decreased or unaffected. In addition, levels of IL-8 and CXCL5, but not CXCL7 or CXCL10, were significantly higher in dogs with pyometra than those in healthy dogs. Our findings show that pathogenic uterine-derived E coli induces a CXC chemokine response both in cultured endometrial stromal cells within 24 hours and in pyometra-affected uteri from dogs. Stromal cells could therefore play an important role in early neutrophil and T cell recruitment to the site of inflammation during gram-negative bacterial infection of the uterus. Further studies are needed to clarify the role of chemokines in host response to bacterial infection in dogs and the possibility of using chemokines as diagnostic parameters for bacterial infection in this species.

Metastasis is responsible for the majority of cancer associated fatalities. Tumor cells leaving the primary tumor and entering the blood flow immediately interact with platelets. Activated platelets contribute in different ways to cancer cell survival and proliferation, e.g. in formation of the early metastatic niche by release of different growth factors and chemokines. Here we show that a direct interaction between platelets and MV3 melanoma or MCF7 breast cancer cells induces platelet activation and a VEGF release in citrated plasma that cannot be further elevated by the coagulation cascade and generated thrombin. In contrast, the release of platelet-derived chemokines CXCL5 and CXCL7 depends on both, a thrombin-mediated platelet activation and a direct interaction between tumor cells and platelets. Preincubation of platelets with therapeutic concentrations of unfractionated heparin reduces the tumor cell initiated VEGF release from platelets. In contrast, tumor cell induced CXCL5 and CXCL7 release from platelets was not impacted by heparin pretreatment in citrated plasma. In defibrinated, recalcified plasma, on the contrary, heparin is able to reduce CXCL5 and CXCL7 release from platelets by thrombin inhibition. Our data indicate that different chemokines and growth factors in diverse platelet granules are released in tightly regulated processes by various trigger mechanisms. We show for the first time that heparin is able to reduce the mediator release induced by different tumor cells both in a contact and coagulation dependent manner.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown cause characterized by alveolar epithelial damage, patchy interstitial fibrosis and diffuse microvascular abnormalities. In IPF, alveolar clustering of iron-laden alveolar macrophages-a common sign of microhemorrhage, has been associated with vascular abnormalities and worsening of pulmonary hypertension. As iron-dependent ROS generation has been shown to induce unrestrained macrophage activation in disease models of vascular damage, we explored alveolar macrophage activation phenotype in IPF patients (n = 16) and healthy controls (CTR, n = 7) by RNA sequencing of bronchoalveolar lavage (BAL) cells. The frequencies of macrophages in BAL cells were 86+4% and 83.4+8% in IPF and CTR groups, respectively (p-value = 0.41). In IPF patients, BAL cells showed increased iron-dependent ROS generation (p-value<0.05 vs CTR). Gene expression analysis showed overrepresentation of Gene Ontology processes/functions and KEGG pathways enriched in upregulated M1-type inflammatory (p-value<0.01), M2-type anti-inflammatory/tissue remodeling (p-value<0.0001), and MTPP-type chronic inflammatory/angiogenic (p-value<0.0001) chemokine and cytokine genes. The ex vivo finding was confirmed by the induction of iron-dependent ROS generation and chemokine/cytokine overexpression of Ccl4, Cxcl10 (M1), Il1rn (M2), Cxcl2, and Cxcl7 (MTPP) in MH-S murine immortalized alveolar macrophages exposed to ferric ammonium citrate in culture (p-value<0.05 vs CTR). The data show alveolar macrophage expression of a pro-inflammatory, tissue remodeling and angiogenic complex activation pattern, suggesting that iron accumulation may play a role in macrophage activation.

Background: The presence of neutrophil-rich inflammation in colon tissues of patients with ulcerative colitis (UC) is one of the most important histological characteristics of this disease. However, the expression of CXCL chemokines governing the infiltration of neutrophils in UC has not been well elucidated. Materials and methods: In this experimental study, the UC model was induced in Wistar rats by administration of 2 mL 4% acetic acid into the large colon through the rectum. Animals were anesthetized after 48 hrs; their colon tissue samples were isolated for macroscopic and histopathological examinations. The expression of CXCL family was assessed by reverse transcription polymerase chain reaction (qRT-PCR) technique. Results: Heavy infiltration of neutrophils, coagulation necrosis, and ulcers were observed in H&E staining, which pathologically proved the UC model. qRT-PCR results showed that ELR⁺ CXC chemokines such as CXCL6 and CXCL3 had the highest expression in the UC group, which was 49 and 28 times higher than that of the control group, respectively. In addition, other chemokines of this group including CXCL1, CXCL2, and CXCL7 had a significant increase compared to the control group (P≤0.05). However, ELR^- CXC chemokines such as CXCL4, CXCL13, and CXCL16 showed a smaller upregulation, while CXCL14 chemokine showed a significant decrease compared to the control group (P≤0.05). However, the expression of CXCL9-12 and CXCL17 did not change. Conclusion: The results showed that the ELR⁺ CXC chemokines, especially CXCL6 and CXCL3, many involved in the pathogenesis of UC; therefore, CXCL6 and CXCL3 chemokines can be used as therapeutic targets for UC, although more studies using human samples are required.

BACKGROUND

Cardiac regenerative responses are responsive to paracrine factors. We hypothesize that chronic heart failure (HF) in pediatric patients affects cardiac paracrine signaling relevant to resident c-kit(+)cluster of differentiation (CD)34- cardiac stem cells (CSCs).

METHODS

Discarded atrial septum (huAS) and atrial appendages (huAA) from pediatric patients with HF (huAA-HF; n = 10) or without HF (n = 3) were explanted and suspension explant cultured in media. Conditioned media were screened for 120 human factors using unedited monoclonal antibody-based arrays. Significantly expressed (relative chemiluminescence >30 of 100) factors are reported (secretome). Emigrated cells were immunoselected for c-kit and enumerated as CSCs.

RESULTS

After culture Day 7, CSCs emigrate from huAA but not huAS. The huAA secretome during CSC emigration included hepatocyte growth factor (HGF), epithelial cell-derived neutrophil attractant-78 (ENA-78)/chemokine (C-X-C motif) ligand (CXCL) 5, growth-regulated oncogene-α (GRO-α)/CXCL1, and macrophage migration inhibitory factor (MIF), candidate pro-migratory factors not present in the huAS secretome. Survival/proliferation of emigrated CSCs required coculture with cardiac tissue or tissue-conditioned media. Removal of huAA (Day 14) resulted in the loss of all emigrated CSCs (Day 28) and in decreased expression of 13 factors, including HGF, ENA-78/CXCL5, urokinase-type plasminogen activator receptor (uPAR)/CD87, and neutrophil-activating protein-2 (NAP-2)/CXCL7 candidate pro-survival factors. Secretomes of atrial appendages from HF patients have lower expression of 14 factors, including HGF, ENA-78/CXCL5, GRO-α/CXCL1, MIF, NAP-2/CXCL7, uPAR/CD87, and macrophage inflammatory protein-1α compared with AA from patients without HF.

CONCLUSIONS

Suspension explant culturing models paracrine and innate CSC interactions in the heart. In pediatric patients, heart failure has an enduring effect on the ex vivo cardiac-derived secretome, with lower expression of candidate pro-migratory and pro-survival factors for CSCs.

Many cells, including leucocytes and stromal cells, express CXCL7, a member of the CXC chemokine family, also known as platelet basic protein. CXCL7 is a potent chemoattractant and activator of neutrophil function. Dendritic cells (DCs) play a pivotal role in antigen processing and presentation. Very little information is available on the ability of DCs to recruit neutrophils by producing chemokines. In this work, we have cloned canine CXCL7. Based on the predicted gene sequence and using the 3'RACE technique, the full-length gene was amplified from LPS-treated canine peripheral blood mononuclear cells. The cloned cDNA sequence consisted of 357 nucleotides and encoded a 118 amino acid protein, including a 38 amino acid signal peptide. The use of CXCL7-containing supernatants from CXCL7-transfected BALB/3T3 in the neutrophil migration assay confirmed that canine CXCL7 had chemoattractive activity for neutrophils. We then used canine monocyte-derived DCs to generate CXCL7 for the rest of the experiment. Expression of CXCL7 by DCs treated with LPS, IL-1beta, IL-6, TGF-beta, TNF-alpha, or IFN-gamma was compared using real-time RT-PCR and Western blotting. When treated with IL-1beta, IL-6, TNF-alpha, or TGF-beta, canine DCs expressed significantly higher levels of CXCL7 mRNA and protein than when treated with IFN-gamma or LPS. It is concluded that dog DCs express high levels of the neutrophil chemotactic factor CXCL7 when stimulated by proinflammatory cytokines, including IL-1beta, IL-6, TNF-alpha, or TGF-beta, and may play an important role in modulating inflammatory responses.

Improved diagnostic and treatment methods are needed for chronic graft-versus-host disease (cGVHD), the leading cause of late nonrelapse mortality (NRM) in long-term survivors of allogenic hematopoietic cell transplantation. Validated biomarkers that facilitate disease diagnosis and classification generally are lacking in cGVHD. Here, we conducted whole serum proteomics analysis of a well-established murine multiorgan system cGVHD model. We discovered 4 upregulated proteins during cGVHD that are targetable by genetic ablation or blocking antibodies, including the RAS and JUN kinase activator, CRKL, and CXCL7, CCL8, and CCL9 chemokines. Donor T cells lacking CRK/CRKL prevented the generation of cGVHD, germinal center reactions, and macrophage infiltration seen with wild-type T cells. Whereas antibody blockade of CCL8 or CXCL7 was ineffective in treating cGVHD, CCL9 blockade reversed cGVHD clinical manifestations, histopathological changes, and immunopathological hallmarks. Mechanistically, elevated CCL9 expression was present predominantly in vascular smooth muscle cells and uniquely seen in cGVHD mice. Plasma concentrations of CCL15, the human homolog of mouse CCL9, were elevated in a previously published cohort of 211 cGVHD patients compared with controls and associated with NRM. In a cohort of 792 patients, CCL15 measured at day +100 could not predict cGVHD occurring within the next 3 months with clinically relevant sensitivity/specificity. Our findings demonstrate for the first time the utility of preclinical proteomics screening to identify potential new targets for cGVHD and specifically CCL15 as a diagnosis marker for cGVHD. These data warrant prospective biomarker validation studies.

Hypocalcemia in dairy cows is often associated with inflammation-related disorders such as metritis and mastitis. The protein encoded by the Ca2+ release-activated calcium modulator 1 (ORAI1) gene is a membrane Ca2+ channel subunit that is activated when Ca2+ stores are depleted. Polymorphonuclear neutrophils (PMNL) have a crucial role in the defense against infection through migration, adhesion, chemotaxis, phagocytosis, and reactive oxygen species (ROS) production in response to pathogens. Whether hypocalcemia affects the activity of PMNL and if ORAI1 is involved remains unknown. To address this, PMNL were isolated at 3 d of calving from dairy cows diagnosed as clinically healthy (n = 20, CONTROL) or with plasma concentration of calcium < 2.0 mmol/L as a criterion for diagnosis of subclinical hypocalcemia (n = 20, HYPOCAL). PMNL isolated from both groups of cows were treated with or without the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin, Ca2+ ionophore Ionomycin, and ORAI1 blocker 2APB. The intracellular Ca2+ concentration, ORAI1 abundance, ROS, phagocytosis rate, migration, and adhering capacity of treated PMNL were evaluated. Some of the in vitro assays also included use of small interfering ORAI1 RNA (siORAI1), 100 nM 1,25(OH)2D3, or 100 nM parathyroid hormone (PTH). Intracellular Ca2+ concentration was markedly lower in HYPOCAL. In addition, ORAI1 was detected in PMNL plasma membrane via FACS and was markedly lower in cows with HYPOCAL. Migration, adhesion capacity, and phagocytosis rate of PMNL were lower in response to HYPOCAL. Furthermore, plasma and PMNL concentration of nucleosome assembly protein (NAP2) and pro-platelet basic protein (CXCL7) was markedly lower with HYPOCAL. All these changes were associated with lower ROS production by PMNL. Thapsigargin and ionomycin treatment in vitro increased ORAI1 expression, migration of PMNL, adhering capacity, phagocytosis rate, and ROS production; conversely, those effects were abrogated by siORAI1 and ORAI1 inhibitor 2APB treatment. Also cytosolic Ca2+ concentration and ORAI1 abundance were increased by 1,25(OH)2D3 and PTH supplementation. Overall, the data indicate that failure of PMNL to uptake Ca2+ due to downregulation of ORAI1 during subclinical hypocalcemia is a factor contributing to impaired PMNL function. In addition, plasma PTH or 1,25(OH)2D3 could regulate ORAI1 and also participate in the regulation of PMNL activity.

Although targeted therapy is usually the first-line treatment for advanced renal cell carcinoma (RCC), some patients can experience drug resistance. Cancer stem cells are tumour-initiating cells that play a vital role in drug resistance, metastasis and cancer relapse, while galectins (Gal) participate in tumour progression and drug resistance. However, the exact role of galectins in RCC stemness is yet unknown. In this study, we grew a subpopulation of RCC cells as tumour spheres with higher levels of stemness-related genes, such as Oct4, Sox2 and Nanog. Among the Gal family, Gal-3 in particular was highly expressed in RCC tumour spheres. To further investigate Gal-3's role in the stemness of RCC, lentivirus-mediated knockdown and overexpression of Gal-3 in RCC cells were used to examine both in vitro and in vivo tumorigenicity. We further assessed Gal-3 expression in RCC tissue microarray using immunohistochemistry. Upon suppressing Gal-3 in parental RCC cells, invasion, colony formation, sphere-forming ability, drug resistance and stemness-related gene expression were all significantly decreased. Furthermore, CXCL6, CXCL7 and CXCR2 were down-regulated in Gal-3-knockdown tumour spheres, while CXCR2 overexpression in Gal-3-knockdown RCC restored the ability of sphere formation. Gal-3 overexpression in RCC promoted both in vitro and in vivo tumorigenicity, and its expression was correlated with CXCR2 expression and tumour progression in clinical tissues. RCC patients with higher co-expressions of Gal-3 and CXCR2 demonstrated a worse survival rate. These results indicate that highly expressed Gal-3 may up-regulate CXCR2 to augment RCC stemness. Gal-3 may be a prognostic and innovative target of combined therapy for treating RCC.

BACKGROUND

The cell line HaCaT/SM was derived from the human keratinocyte cell line HaCaT. HaCaT/SM cells display a high resistance against sulfur mustard (SM). Intention of the presented study was to determine the cellular and molecular differences between HaCaT/SM and HaCaT so as to evaluate which changes might be responsible for being resistant against SM.

METHODS

Both cell lines HaCaT and HaCaT/SM were analyzed with respect to their cell growth, nuclei perimeter, clonogenicity and secretion profile. Moreover DNA alkylation pattern under presence of SM was investigated.

RESULTS

In comparison to HaCaT, the HaCaT/SM showed a significant smaller nuclei perimeter. For DNA alkylation a significant difference was observed over time. The clonogenicity of HaCaT/SM was increased to 150%. The secretion profile of these cells demonstrated a strong increase of ANG, PDGF-AA, TIMP1, TIMP2, and a decrease of AREG, CCL5, CXC1, CXC2/3, CXCL6, CXCL7, CXCL8, CXCL10, MIF, Trappin-1.

CONCLUSIONS

The sulfur mustard (SM) resistant cell line HaCaT/SM demonstrates a wide range of significant differences to their origin cell line HaCaT. These differences might be responsible to provide resistance against SM and might also be useful to establish treatment concepts for humans after SM exposure.