Expression Of Chemotactic Factors Research Articles

Disrupted Tuzzerella abundance and impaired l-glutamine levels induce Treg accumulation in ovarian endometriosis: a comprehensive multi-omics analysis

IntroductionThe microbial community plays a crucial role in the pathological microenvironment. However, the structure of the microbial community within endometriotic lesions and its impact on the microenvironment is still limited.MethodsAll 55 tissue samples, including ovarian ectopic (OEMs) and normal (NE) endometrium, were subjected to 16S rRNA sequencing, metabolomic and proteomic analysis.ResultsWe found the abundance of Tuzzerella is significantly lower in OEMs compared to NE tissue (p < 0.01). We selected samples from these two groups that exhibited the most pronounced difference in Tuzzerella abundance for further metabolomic and proteomic analysis. Our findings indicated that endometriotic lesions were associated with a decrease in l-Glutamine levels. However, proteomic analysis revealed a significant upregulation of proteins related to the complement pathway, including C3, C7, C1S, CLU, and A2M. Subsequent metabolic and protein correlation predictions demonstrated a negative regulation between l-Glutamine and C7. In vitro experiments further confirmed that high concentrations of Glutamine significantly inhibit C7 protein expression. Additionally, immune cell infiltration analysis, multiplex immunofluorescence, and multifactorial testing demonstrated a positive correlation between C7 expression and the infiltration of regulatory T cells (Tregs) in ectopic lesions, while l-Glutamine was found to negatively regulate the expression of chemotactic factors for Tregs.ConclusionIn this study, we found a clear multi-omics pathway alteration, “Tuzzerella (microbe)—l-Glutamine (metabolite)—C7 (protein),” which affects the infiltration of Tregs in endometriotic lesions. Our findings provide insights into endometriosis classification and personalized treatment strategies based on microbial structures.Graphical abstract

Amino Acids Fueling Fibroblast-Like Synoviocyte Activation and Arthritis By Regulating Chemokine Expression and Leukocyte Migration.

We analyzed the impact of amino acid (AA) availability on the inflammatory response in arthritis. We stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) with tumor necrosis factor (TNF) in the presence or absence of proteinogenic AAs and measured their response by QuantSeq 3' messenger RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Signal transduction events were determined by Western blot. We performed K/BxN serum transfer arthritis in mice receiving a normal and a low-protein diet and analyzed arthritis clinically and histologically. Deprivation of AAs decreased the expression of a specific subset of genes, including the chemokines CXCL10, CCL2, and CCL5 in TNF-stimulated FLSs. Mechanistically, the presence of AAs was required for the TNF-induced activation of an interferon regulatory factor 1 (IRF1)-STAT1 signaling circuit that drives the expression of chemotactic factors. The expression of IRF1 and the IRF1-dependent gene set in FLSs was highly correlated with the presence of inflammatory cells in human RA, emphasizing the important role of this AA-dependent pathway in inflammatory cell recruitment to the synovial tissue. Finally, we show that mice receiving a low-protein diet expressed less IRF1 in the inflamed synovium and consequently developed reduced clinical and histologic signs of arthritis. AA deprivation reduces the severity of arthritis by suppressing the expression of IRF1-STAT1-driven chemokines, which are crucial for leukocyte recruitment to the arthritic joint. Overall, our study provides novel insights into critical determinants of inflammatory arthritis and may pave the way for dietary intervention trials in RA.

Expression of immune regulatory factors, chemokines and growth factorsin differentiated gastric cancer cells treated with an anticancer bioactive peptide combined with oxaliplatin.

Gastric cancer is one of the most common malignant tumors of the digestive system. An anticancer bioactive peptide (ACBP) was previously shown to have an important role in inhibiting the differentiation of the MKN-45, N87 and GES-1 cell lines. However, to date, research on the effects of inflammatory factors in MKN-45, N87 and GES-1 cell lines after treatment with ACBP combined with oxaliplatin (OXA) has not been performed. To investigate the expression of immune regulatory factors, tumor growth factors and chemotactic factors in differentiated gastric cancer cells treated with ACBP combined with OXA, the expression of cytokines, including interleukin (IL)-1β, IL-1 receptor antagonist, IL-2, IL-4, IL-6-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, IFN-γ-induced protein-10, macrophage inflammatory protein (MIP)-1α, platelet-derived growth factor (PDGF)-BB, MIP-1β, regulated upon activation, normal T cell expressed and presumably secreted, TNF-α and VEGF, was assessed with cell experiments using the Bio-Plex ProT Human Cytokine 27-plex Assay. The results indicated that immune regulatory factor, tumor growth factor and chemotactic factor expression levels were different after treatment with ACBP alone or ACBP combined with OXA. IFN-γ, IL-1β, IL-17, IL-9, IL-10, IL-15, bFGF, GM-CSF and PDGF-BB expression was decreased in MKN-45 and N87 cells after ACBP treatment (P<0.01) and ACBP+OXA treatment (P<0.01) compared with the control cells, which indicated that ACBP inhibited tumor growth by regulating these cytokines, and the combination treatment inhibited tumor growth by regulating these cytokines. MIP-1β, MCP-1 and IL-13 expression was decreased in MKN-45 and N87 cells after the combination treatment compared with ACBP treatment alone, which indicated that ACBP combined with OXA was able to inhibit tumor growth by regulating these cytokines, while the mechanism of action of the ACBP and OXA is actually different, e.g. for OXA, this would be to cause DNA damage response. Therefore, the ACBP and OXA combination treatment may be closely associated with tumor progression and metastasis with immunological competence by MCP-1, MIP-1β and IL-13 expression.

Helicobacter pylori regulates TIFA turnover in gastric epithelial cells.

The human pathogen Helicobacter pylori induces a strong inflammatory response in gastric mucosa manifested by the recruitment of neutrophils and macrophages to the places of infection, and by changes in epithelial integrity and function. At the molecular level, this innate immune response is essentially dependent on the activation of NF-κB transcription factors regulating the expression of chemotactic factors, e.g., IL-8. Recently, it has been demonstrated that the NF-κB signaling pathway is triggered by the bacterial heptose metabolites, which activate the host ALPK1-TIFA axis. TIFA has been suggested to promote oligomerization and activity of the E3 ubiquitin ligase TRAF6, which further stimulates TAK1-IKK signaling. Here, we demonstrate that ALPK1-dependent TIFA activation in H. pylori-infected gastric epithelial cells is followed in time by a decline in TIFA levels, and that this process is impeded by inhibitors of the proteasomal and lysosomal degradation. According to our data, TRAF2, TRAF6, TAK1 or NEMO are not required for TIFA degradation. Additionally, H. pylori promotes the interaction of TIFA with free polyubiquitin as well as with optineurin, TAX1BP1 and LAMP1, which are known protein adaptors involved in intracellular trafficking to lysosomes.

Analysis of the chemotactic factors for tumor-infiltrating fibrocytes and their prognostic significances in lung cancer.

Fibrocytes, which are bone marrow-derived collagen-producing cells, have been reported to be involved in pathogenesis of pulmonary fibrosis. Our previous study reported that tumor-infiltrating fibrocytes play a role in tumor progression and drug resistance in lung cancer. The present study therefore examined chemotactic factors for fibrocytes in tissues of non-small cell lung cancer (NSCLC) and their prognostic significance. Surgically resected tumor tissues were examined for the expression of chemotactic factors, including C-X-C motif chemokine 12 (CXCL12), CCL2, platelet-derived growth factor (PDGF)-AA and PDGF-BB, as well as tumor-infiltrating fibrocytes by immunostaining. The chemotactic ability of fibrocytes in response to each factor was evaluated using a migration assay by counting the migrated cells microscopically, and expression of receptors for chemotactic factors were analyzed by flow cytometry. The expression of CXCL12, but not CCL2, PDGF-AA, or PDGF-BB, was associated with the number of tumor-infiltrating fibrocytes in lung adenocarcinoma (LUAD), but not lung squamous cell carcinoma (LUSQ). In addition, patients with an increased expression of CXCL12 in LUAD but not LUSQ showed a significantly poorer prognosis compared with those with a decreased expression. However, the expression of CCL2, PDGF-AA and PDGF-BB was not correlated with the prognosis of patients with NSCLC. The number of fibrocytes was associated with a poor prognosis in LUAD. Fibrocytes derived from the peripheral blood of healthy subjects as well as patients with lung cancer expressed higher levels of CXCR4 compared with CCR2, PDGF and receptor-α and receptor-β. Overall, these results suggested that targeting tumor-infiltrating fibrocytes via the CXCL12/CXCR4 axis may be a useful strategy for controlling the progression of NSCLC, particularly LUAD.

3D bioprinting of proangiogenic constructs with induced immunomodulatory microenvironments through a dual cross-linking procedure using laponite incorporated bioink

Vascularization is vital for the survival and functionality of complex tissue-engineered organs, and immune microenvironment is pivotal for effective vascularization. 3D bioprinting is a powerful technique for manufacturing engineered tissues. However, the reconstruction of functionalized vascular scaffolds with immunomodulatory properties through 3D bioprinting has rarely been reported. In this study, we fabricated scaffolds with immunomodulatory properties by incorporating INF-γ loaded laponite into the mixtures of gelatin methacrylate (GelMA)/alginate/4-arm poly(ethylene glycol) acrylate (PEG) (GAP) through coaxial bioprinting method with a sequential cross-linking mechanism that allows for stable production of 3D microfibrous scaffolds. Laponite addition optimized the hydrogel's physical and chemical performance, improved the rheological properties and printing feasibility while enhancing mechanical stress, making the direct fabrication of scaffolds with increased porosity and decreased filament diameter possible. Furthermore, new scaffolds facilitated the expression of chemotactic factors and accelerated EPC migration toward the microfiber peripheries to form a layer of confluent endothelium. Meanwhile, the scaffolds were capable of releasing IFN-γ in the early stage to stimulate macrophage M1 polarization, followed by induction of M2 polarization via the release of Si4+, Mg2+ as the degradation of laponite occurred, which successfully improved the sprouting and mature of newly formed vasculature, as well as vascularized bone regeneration. Our results suggested that a combination of GAP-IFN-γ@Lap bioink with a dual-step cross-linking procedure could regulate the local immune microenvironment, aiding the formation of a confluent endothelium, promoting angiogenesis and tissue regeneration, which potentially provides an efficient and simple strategy for developing complex vascularized tissues.

Maintaining a protective state for human periodontal tissue.

Periodontitis, caused by infection with periodontal pathogens, is primarily characterized by inflammatory bone resorption and destruction of connective tissue. Simply describing periodontitis as a specific bacterial infection cannot completely explain the various periodontal tissue destruction patterns observed. Periodontal tissue damage is thought to be caused by various factors. In recent years, research goals for periodontal pathogens have shifted from searching for specific pathogens to investigating mechanisms that damage periodontal tissues. Bacteria interact directly with the host in several ways, influencing expression and activity of molecules that evade host defenses, and destroying local tissues and inhibiting their repair. The host's innate and acquired immune systems are important defense mechanisms that protect periodontal tissues from attack and invasion of periodontal pathogens, thus preventing infection. Innate and acquired immunity have evolved to confront the microbial challenge, forming a seamless defense network in periodontal tissues. In the innate immune response, host cells quickly detect, via specialized receptors, macromolecules and nucleic acids present on bacterial cell walls, and this triggers a protective, inflammatory response. The work of this subsystem of host immunity is performed mainly by phagocytes, beta-defensin, and the complement system. In addition, the first line of defense in oral innate immunity is the junctional epithelium, which acts as a physical barrier to the entry of oral bacteria and other nonself substances. In the presence of a normal flora, junctional epithelial cells differentiate actively and proliferate apically, with concomitant increase in chemotactic factor expression recruiting neutrophils. These immune cells play an important role in maintaining homeostasis and the protective state in periodontal tissue because they eliminate unwanted bacteria over time. Previous studies indicate a mechanism for attracting immune cells to periodontal tissue with the purpose of maintaining a protective state; although this mechanism can function without bacteria, it is enhanced by the normal flora. A better understanding of the relationship between the protective state and its disruption in periodontal disease could lead to the development of new treatment strategies for periodontal disease.

P057 IL-17 regulates expression of chemotactic chemokines in human colonic subepithelial myofibroblasts

Abstract Background Crohn’s disease (CD) and ulcerative colitis (UC), the two main entities of inflammatory bowel disease (IBD), are characterised by chronic and relapsing/remitting inflammation of the gastrointestinal tract, and occasionally ultimately result in debilitating intestinal fibrosis. Apart from their key role in fibrosis, there is evidence that subepithelial myofibroblasts (SEMFs) participate in the IBD inflammatory cascade, as they express various pro-inflammatory cytokine receptors. We examined the effect of pro-inflammatory IL-17—the hallmark cytokine of T-lymphocytes differentiated to Th17—on the expression of lymphocyte-chemotactic chemokines in SEMFs. Methods SEMFs were isolated from endoscopically obtained colonic biopsies from healthy controls, set to culture and stimulated with 100 ng/ml IL-17 for 6 h. Total RNA was extracted and mRNA expression of CCL5, CXCL1 and CXCL11 was assessed with reverse transcription quantitative (RT-q) PCR. Changes in cytokine mRNA are provided as medians (IQR). Results Untreated SEMFs had a basal expression of chemokines of the CCL and CXCL family groups. So far, our study has shown that the IL-17 stimulation leads to a statistically significant upregulation of CCL and CXCL chemokines in SEMFs (p &amp;lt; 0.001). In detail, CCL5 was upregulated 5.7-fold (4.9–7.5), CXCL1 72.9-fold (63.1–90.7) and CXCL11 25.4-fold (17.3–37.3). Conclusion IL-17 induced the expression of chemotactic factors in SEMFs. Our results further support a potential role of SEMFs in the shaping of intestinal mucosal immunity by serving as immunological intermediates that respond to cytokines of adaptive immunity and amplify the recruitment of immune cells via chemokine production.

Role of Endothelial Cells in Renal Fibrosis.

Renal fibrosis has been regarded as the common pathway of end-stage renal failure. Understanding the fundamental mechanism that leads to renal fibrosis is essential for developing better therapeutic options for chronic kidney diseases. So far, the main abstractions are on the injury of tubular epithelial cells, activation of interstitial cells, expression of chemotactic factor and adhesion molecule, infiltration of inflammatory cells and homeostasis of ECM. However, emerging studies revealed that endothelial cells (ECs) might happen to endothelial-to-mesenchymal transition (EndMT) dependent and/or independent endothelial dysfunction, which were supposed to accelerate renal fibrosis and are identified as new mechanisms for the proliferation of myofibroblasts as well. In this chapter, we are about to interpret the role of ECs in renal fibrosis and analyze the related molecules and pathways of both EndMT and EndMT independent endothelial dysfunction.

Abstract C112: Cabozantinib eradicates advanced murine prostate cancer by activating anti-tumor innate immunity

Abstract Several kinase inhibitors targeting aberrant signaling pathways in tumor cells have been deployed in cancer therapy. However, their impact on the tumor immune microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed striking responses in early phase clinical trials, particularly in cancer patients with bone metastases. Here we show that cabozantinib rapidly eradicates invasive, poorly-differentiated PTEN/p53 deficient murine prostate cancer. This was associated with increased neutrophil chemotactic factor expression, including CXCL12 and HMGB1 production by tumor cells, and robust infiltration of neutrophils into the tumor. Critically, cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte depletion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor, plerixafor. Collectively, these results demonstrate that cabozantinib triggers neutrophil-mediated anti-tumor innate immune response that results in tumor clearance. (Manuscript submitted to Science) Citation Format: Akash Patnaik, Kenneth Swanson, Sabina Signoretti, Huihui Ye, Eva Csizmadia, Jesse Novak, Marina Gehring, Katja Helenius, Athalia Pyzer, Laleh Montaser, Lily Wang, Olivier Elemento, Elena Levantini, John Clohessy, John Asara, Kathleen Kelly, Pier Paolo Pandolfi, Jacalyn Rosenblatt, David Avigan, Steven Balk, Lewis Cantley. Cabozantinib eradicates advanced murine prostate cancer by activating anti-tumor innate immunity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C112.

Stromal Cells Derived from Visceral and Obese Adipose Tissue Promote Growth of Ovarian Cancers.

Obesity, and in particular visceral obesity, has been associated with an increased risk of developing cancers as well as higher rates of mortality following diagnosis. The impact of obesity on adipose-derived stromal cells (ASC), which contribute to the formation of tumor stroma, is unknown. Here we hypothesized that visceral source and diet-induced obesity (DIO) changes the ASC phenotype, contributing to the tumor promoting effects of obesity. We found that ASC isolated from subcutaneous (SC-ASC) and visceral (V-ASC) white adipose tissue(WAT) of lean(Le) and obese(Ob) mice exhibited similar mesenchymal cell surface markers expression, and had comparable effects on ovarian cancer cell proliferation and migration. Obese and visceral derived ASC proliferated slower and exhibited impaired differentiation into adipocytes and osteocytes in vitro as compared to ASC derived from subcutaneous WAT of lean mice. Intraperitoneal co-injection of ovarian cancer cells with obese or visceral derived ASC, but not lean SC-ASC, increased growth of intraperitoneal ID8 tumors as compared to controls. Obese and V-ASC increased stromal infiltration of inflammatory cells, including CD3+ T cells and F4/80+ macrophages. Obese and visceral derived ASC, but not lean SC-ASC, increased expression of chemotactic factors IL-6, MIP-2, and MCP-1 when cultured with tumor cells. Overall, these results demonstrate that obese and V-ASC have a unique phenotype, with more limited proliferation and differentiation capacity but enhanced expression of chemotactic factors in response to malignant cells which support infiltration of inflammatory cells and support tumor growth and dissemination.

Abstract 315: Inflamed HDL Loses Its Anti-inflammatory Properties on Adipocytes: Role of Interaction of Serum Amyloid A with the Extracellular Matrix

Obesity induces adipocyte hypertrophy and accumulation of macrophages in adipose tissue, which associates with insulin resistance and cardiovascular disease risk. HDL and apoA-I have anti-inflammatory properties in addition to facilitating reverse cholesterol transport. We recently showed that HDL from healthy humans and lean mice inhibits palmitate-induced monocyte chemotactic factor expression by decreasing lipid raft (LR) content in adipocytes. We also have shown that adipocytes produced extracellular matrix (ECM) in vivo and in vitro when they became hypertrophic. Moreover, inflamed HDL loses its anti-inflammatory effect and ability to stimulate cholesterol efflux from adipocytes. From gain of function and loss of function experiments, it appears that serum amyloid A (SAA) is partially responsible for these effects. However, the mechanism by which SAA impairs HDL’s anti-inflammatory function is unknown. To generate HDL under conditions of sterile inflammation, HDL was purified from plasma of AgNO 3 -injected C57BL/6 mice. Also, to generate SAA-enriched HDL, HDL was purified after HDL from healthy humans was exposed to 293 HEK cells with lentiviral overexpression of SAA2. Control, inflamed and SAA-enriched HDL were labelled using DiI dye and the ECM of adipocytes was stained by Alexa 488 conjugated wheat germ agglutinin. After exposure of control HDL to adipocytes, there was no co-localization of DiI and Alexa 488 staining. However, we found that inflamed and SAA-enriched HDL were co-localized with the ECM of adipocytes, suggesting that inflamed and SAA-enriched HDL are being trapped at the ECM and therefore are blocked from accessing the plasma membrane. Enzymatic digestion of the ECM restored inflamed and SAA-enriched HDL’s abilities to facilitate cholesterol efflux from adipocytes and to inhibit palmitate-induced chemotactic factor expression. Taken together, our results suggest that the presence of SAA on HDL makes the lipoprotein lose its anti-inflammatory effect on adipocytes by blocking accessibility of HDL to the plasma membrane. The interaction between SAA in HDL and the ECM of adipocytes should be considered as a new therapeutic target for improvement of HDL’s anti-inflammatory properties.

Abstract 114: Serum Amyloid A Plays an Important Role in Making HDL Lose Its Anti-inflammatory Effect on Adipocytes

Adipose tissue inflammation associates with insulin resistance and increased cardiovascular disease risk. HDL has both anti-inflammatory and atheroprotective properties. We recently showed that HDL from healthy humans and lean mice inhibits palmitate-induced monocyte chemotactic factor expression by decreasing lipid raft (LR) content in adipocytes. However, little is known regarding whether inflammation would cause HDL to lose these anti-inflammatory effects on adipocytes. To generate HDL under conditions of sterile inflammation, HDL was purified from plasma of AgNO 3 -injected C57BL/6 mice. In 3T3L-1 adipocytes, “inflamed HDL” did not disrupt the formation of LR or inhibit monocyte chemotactic factor expression. Moreover, inflamed HDL lost its ability to facilitate cholesterol efflux in adipocytes. To determine causal factors of HDL that might be responsible for this loss of anti-inflammatory effect on adipocytes, we investigated role of serum amyloid A (SAA), levels of which are increased up to 1000 fold in HDL by acute inflammation. In a loss of function study, inflamed HDL from AgNO 3 -injectd SAA1/2 double knockout (DKO) mice restored part of its anti-inflammatory and cholesterol efflux capacity. Conversely, in a gain of function study, increasing the SAA content in uninflamed HDL by exposure to 293 HEK cells with lentiviral overexpression of SAA2 resulted in a partial loss of its anti-inflammatory effect and cholesterol efflux capacity. To determine whether HDL from moderately inflamed humans had similar effects on adipocyte inflammation, HDL isolated from patients with systemic lupus erythematosus (n=18) also demonstrated impaired anti-inflammatory effects in adipocytes. The extent of impairment associated positively with plasma SAA levels ( r =0.80; p &lt;0.001), and negatively with cholesterol efflux capacity of the HDL ( r =-0.74; p &lt;0.001). These findings indicate that inflamed HDL loses its anti-inflammatory effect and cholesterol efflux capacity on adipocytes. The presence of SAA in HDL may contribute to the generation of dysfunctional HDL and should be considered as a causal factor, not simply as a biomarker of dysfunctional HDL.

Acute resistance exercise increases the expression of chemotactic factors within skeletal muscle.

Intense resistance exercise causes mechanical loading of skeletal muscle, followed by muscle adaptation. Chemotactic factors likely play an important role in these processes. We investigated the time course of changes in the expression and tissue localization of several key chemotactic factors in skeletal muscle during the early phase of recovery following resistance exercise. Muscle biopsy samples were obtained from vastus lateralis of eight untrained men (22 ± 0.5 years) before and 2, 4 and 24 h after three sets of leg press, squat and leg extension at 80 % 1-RM. Monocyte chemotactic protein-1 (95×), interleukin-8 (2,300×), IL-6 (317×), urokinase-type plasminogen activator (15×), vascular endothelial growth factor (2×) and fractalkine (2.5×) mRNA was significantly elevated 2 h post-exercise. Interleukin-8 (38×) and interleukin-6 (58×) protein was also significantly elevated 2 h post-exercise, while monocyte chemotactic protein-1 protein was significantly elevated at 2 h (22×) and 4 h (21×) post-exercise. Monocyte chemotactic protein-1 and interleukin-8 were expressed by cells residing in the interstitial space between muscle fibers and, in some cases, were co-localized with CD68 + macrophages, PAX7 + satellite cells and blood vessels. However, the patterns of staining were inconclusive and not consistent. In conclusion, resistance exercise stimulated a marked increase in the mRNA and protein expression of various chemotactic factors in skeletal muscle. Myofibers were not the dominant source of these factors. These findings suggest that chemotactic factors regulate remodeling/adaptation of skeletal muscle during the early phase of recovery following resistance exercise.

CD56brightPerforinlow Noncytotoxic Human NK Cells Are Abundant in Both Healthy and Neoplastic Solid Tissues and Recirculate to Secondary Lymphoid Organs via Afferent Lymph

As limited information is available regarding the distribution and trafficking of NK cells among solid organs, we have analyzed a wide array of tissues derived from different human compartments. NK cells were widely distributed in most solid tissues, although their amount varied significantly depending on the tissue/organ analyzed. Interestingly, the distribution appeared to be subset specific, as some tissues were preferentially populated by CD56(bright)perforin(low) NK cells, with others by the CD56(dim)perforin(high) cytotoxic counterpart. Nevertheless, most tissues were highly enriched in CD56(bright)perforin(low) cells, and the distribution of NK subsets appeared in accordance with tissue gene expression of chemotactic factors, for which receptors are differently represented in the two subsets. Remarkably, chemokine expression pattern of tissues was modified after neoplastic transformation. As a result, although the total amount of NK cells infiltrating the tissues did not significantly change upon malignant transformation, the relative proportion of NK subsets infiltrating the tissues was different, with a trend toward a tumor-infiltrating NK population enriched in noncytotoxic cells. Besides solid tissues, CD56(bright)perforin(low) NK cells were also detected in seroma fluids, which represents an accrual of human afferent lymph, indicating that they may leave peripheral solid tissues and recirculate to secondary lymphoid organs via lymphatic vessels. Our results provide a comprehensive mapping of NK cells in human tissues, demonstrating that discrete NK subsets populate and recirculate through most human tissues and that organ-specific chemokine expression patterns might affect their distribution. In this context, chemokine switch upon neoplastic transformation might represent a novel mechanism of tumor immune escape.

Extracellular ATP mediates the late phase of neutrophil recruitment to the lung in murine models of acute lung injury

Acute lung injury (ALI) is a severe inflammatory condition whose pathogenesis is irrevocably linked to neutrophil emigration to the lung. Activation and recruitment of neutrophils to the lung is mostly attributable to local production of the chemokines. However, much of our understanding of neutrophil recruitment to the lung is based on studies focusing on early time points after initiation of injury. In this study, we sought to evaluate the extended temporal relationship between neutrophil chemotactic factor expression and influx of neutrophils into the lung after intratracheal administration of either LPS or bleomycin. In both models, results demonstrated two phases of neutrophil chemotactic factor expression; first, an early phase characterized by high levels of CXCL1/keratinocyte-derived chemokine, CXCL2/monocyte-inhibitory protein-2, and CXCL5/LPS-induced chemokine expression, and second, a late phase distinguished by increases in extracellular ATP. Furthermore, we show that strategies aimed at either enhancing ATP catabolism (ip ecto-5'-nucleotidase administration) or inhibiting glycolytic ATP production (ip 2-deoxy-d-glucose treatment) reduce extracellular ATP accumulation, limit vascular leakage, and effectively block the late, but not the early, stages of neutrophil recruitment to the lung after LPS instillation. In conclusion, this study illustrates that neutrophil recruitment to the lung is mediated by the time-dependent expression of chemotactic factors and suggests that novel strategies, which reduce extracellular ATP accumulation, may attenuate late neutrophil recruitment and limit lung injury during ALI.

Inhibitory effects of dexamethasone against polyinosinic:polycytidylic acid-induced chemotactic factor expression in bronchial epithelial cells and the underlying mechanism

Objective To investigate the effect of dexamethasone on polyinosinic: polycytidylic acid(PIC)-induced chemotactic factor expression in human bronchial epithelial(16hBE) cells and the underlying mechanism.Methods 16hBE cells were treated with different concentrations of PIC(0.001,0.01,0.1,and 1 μg/ml) and dexamethasone(0.1,1,and 10 μmol/L).IL 8 and IP-10 mRNA levels were detected by RT-PCR 6 h after stimulation.IL-8 and IP-10 protein levels in the culture supernatant were detected by ELISA 24 h after stimulation.NF-κB p65 subunit expression was detected by immunohistochemical staining.Results PIC concentration-dependently(0.001,0.01,and 0.1 μg/ml) increased the expression of IL-8 and IP-10 mRNA and protein compared with the control group,with significant differences found when PIC at 0.01 μg/ml and 0.1 μg/ml(P0.05,P0.01).When the concentration of PIC was 1 μg/ml,the expressions of IL-8 and IP-10 were decreased at both mRNA and protein levels.Pretreatment with dexamethasone(1 μmol/L and 10 μmol/L) for 0.5 h significantly inhibited the IL-8 and IP-10 expression at both mRNA and protein levels(P0.05,P0.01).Dexamethasone pretreatment(1 μmol/L) significantly inhibited PIC-induced NF-κB p65 subunit expression(P0.01).Conclusion Glucocorticoids can suppress PIC-induced IL-8 and IP-10 expression in human bronchial epithelial cells,probably through activation of NF-κB pathway.

Apolipoprotein AI and High-Density Lipoprotein Have Anti-Inflammatory Effects on Adipocytes via Cholesterol Transporters

Rationale: Macrophage accumulation in adipose tissue associates with insulin resistance and increased cardiovascular disease risk. We previously have shown that generation of reactive oxygen species and monocyte chemotactic factors after exposure of adipocytes to saturated fatty acids, such as palmitate, occurs via translocation of NADPH oxidase 4 into lipid rafts (LRs). The anti-inflammatory effects of apolipoprotein AI (apoAI) and high-density lipoprotein (HDL) on macrophages and endothelial cells seem to occur via cholesterol depletion of LRs. However, little is known concerning anti-inflammatory effects of HDL and apoAI on adipocytes. Objective: To determine whether apoAI and HDL inhibit inflammation in adipocytes and adipose tissue, and whether this is dependent on LRs. Methods and Results: In 3T3L-1 adipocytes, apoAI, HDL, and methyl-β-cyclodextrin inhibited chemotactic factor expression. ApoAI and HDL also disrupted LRs, reduced plasma membrane cholesterol content, inhibited NADPH oxidase 4 translocation into LRs, and reduced palmitate-induced reactive oxygen species generation and monocyte chemotactic factor expression. Silencing ATP-binding cassette A-1 abrogated the effect of apoAI, but not HDL, whereas silencing ATP-binding cassette G-1 or scavenger receptor B-1 abrogated the effect of HDL but not apoAI. In vivo, apoAI transgenic mice fed a high-fat, high-sucrose, cholesterol-containing diet showed reduced chemotactic factor and proinflammatory cytokine expression and reduced macrophage accumulation in adipose tissue. Conclusions: ApoAI and HDL have anti-inflammatory effects in adipocytes and adipose tissue similar to their effects in other cell types. These effects are consistent with disruption and removal of cholesterol from LRs, which are regulated by cholesterol transporters, such as ATP-binding cassette A-1, ATP-binding cassette G-1, and scavenger receptor B-1.

High-Density Lipoprotein Extinguishes Fire in Fat

Obesity is associated with a state of chronic, low-grade inflammation that reflects the accumulation of macrophages and other leukocytes in adipose tissue.1–3 In obesity cross-talk between adipocytes and macrophages amplifies the production of inflammatory cytokines and chemokines by macrophages and adipocytes,4 leading to local and systemic insulin resistance.5 Macrophage inflammation is also prominent in atherosclerosis, and cholesterol efflux pathways mediated by high-density lipoprotein (HDL), apolipoprotein AI (apoAI), and ATP-binding cassette transporters (ABC) act to suppress inflammation in macrophage foam cells.6 However, a possible role of cholesterol efflux pathways in the inflammation of obesity has not been previously addressed. In this issue of Circulation Research , Umemoto et al7 provide evidence that HDL and apoAI promote cholesterol efflux from adipocytes and reverse the expression of the proinflammatory chemotactic factors induced by saturated fatty acids in adipocytes, suggesting a novel mechanism for anti-inflammatory properties of HDL and the possibility that increasing HDL-mediated cholesterol efflux has beneficial effects on insulin resistance. Article, see p 1345 In previous work the authors of this study have demonstrated that saturated fatty acids such as palmitate stimulate monocyte chemotactic factor expression and production from 3T3-L1 adipocytes.8 This effect involves increased NADPH oxidase 4 (NOX4) activity, recruitment of NOX4 to lipid rafts, and enhanced reactive oxygen species (ROS) production.9 ROS generation has been implicated as an important contributor to insulin resistance in obesity and diabetes mellitus.10 HDL promotes cholesterol efflux and reduces cholesterol content in lipid rafts, altering the activity of signaling molecules in lipid rafts.11 Therefore, Umemoto et al7 investigated whether HDL-mediated cholesterol efflux from adipocytes affected chemotactic factor expression and whether this involved modulating NOX4 recruitment to lipid rafts and its activity. …

ROCK inhibitor fasudil attenuated high glucose-induced MCP-1 and VCAM-1 expression and monocyte-endothelial cell adhesion

BackgroundPrevious studies suggested that the RhoA/ROCK pathway may contribute to vascular complications in diabetes. The present study was designed to investigate whether ROCK inhibitor fasudil could prevent high glucose-induced monocyte-endothelial cells adhesion, and whether this was related to fasudil effects on vascular endothelial cell expression of chemotactic factors, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1).MethodsHUVECs were stimulated with high glucose (HG) or HG + fasudil in different concentration or different time. Monocyte-endothelial cell adhesion was determined using fluorescence-labeled monocytes. The mRNA and protein expression of VCAM-1 and MCP-1 were measured using real-time PCR and western blot. The protein levels of RhoA, ROCKI and p-MYPT were determined using western blot analysis. ELISA was employed to measure the expression of soluble VCAM-1 and MCP-1 in cell supernatants and human serum samples.ResultsFasudil significantly suppressed HG-induced adhesion of THP-1 to HUVECs. Fasudil reduced Rho/ROCK activity (as indicated by lower p-MYPT/MYPT ratio), and prevented HG induced increases in VCAM-1 and MCP-1 mRNA and protein levels. Fasudil also decreased MCP-1 concentration in HUVEC supernatants, but increased sVCAM-1 shedding into the media. In human diabetic subjects, 2 weeks of fasudil treatment significantly decreased serum MCP-1 level from 27.9 ± 10.6 pg/ml to 13.8 ± 7.0 pg/ml (P < 0.05), while sVCAM-1 increased from 23.2 ± 7.5 ng/ml to 39.7 ± 5.6 ng/ml after fasudil treatment (P < 0.05).ConclusionsTreatment with the Rho/ROCK pathway inhibitor fasudil attenuated HG-induced monocyte-endothelial cell adhesion, possibly by reducing endothelial expression of VCAM-1 and MCP-1. These results suggest inhibition of Rho/ROCK signaling may have therapeutic potential in preventing diabetes associated vascular inflammation and atherogenesis.