Expression Of MIP-1α Research Articles

IL-17 induces MIP-1α expression in primary mouse astrocytes via TRPC channel.

Our previous study demonstrated IL-17-mediated induction of MIP-1α through its binding to the cognate IL-17RA and MIP-1α was involved in astrocyte activation. Transient receptor potential canonical (TRPC) channel was involved in astrocyte activation, however, whether TRPC channel regulates MIP-1α expression in the context of multiple sclerosis (MS) remains largely unknown. In this study we identify the essential role of TRPC channel in IL-17-mediated MIP-1α expression and astrocyte activation. Moreover, treatment of astrocytes with IL-17 activated MAPKs and PI3K/Akt signaling pathways with downstream NF-κB pathways. Interestingly, the TRPC blocker-SKF96365 (10μM) and Norgestimate (10μM) significantly inhibited the increased expression of MIP-1α via suppression of IL-17-mediated ERK, p38 and JNK MAPKs and PI3K/Akt pathway activation, thereby underscoring the role of TRPC channel in this process. Together these data underpin the role of TRPC channel as a novel target that regulates MIP-1α expression and cell activation-mediated by IL-17 with implications for therapeutic intervention for reversal of neuroinflammation inflicted by IL-17. Understanding the regulation of MIP-1α expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with MS.

Statins inhibited the MIP-1α expression via inhibition of Ras/ERK and Ras/Akt pathways in myeloma cells.

Macrophage inflammatory protein-1alpha (MIP-1α) is detected at high concentrations in patients with multiple myeloma. It is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, inhibiting MIP-1α expression may be useful in developing therapeutic treatments for multiple myeloma-induced osteolysis. In this study, we investigated the potential of statins to inhibit mRNA expression and secretion of MIP-1α in mouse myeloma cells (MOPC-31C). We found that statins inhibited the lipopolysaccharide (LPS)-induced MIP-1α mRNA expression and protein secretion in MOPC-31C cells. This inhibition was reversed when farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), intermediates of the mevalonate pathway, were combined with statins. Furthermore, statins reduced the GTP form of Ras, a phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our results indicate that statins inhibit biosynthesis of FPP and GGPP and thereby down regulate signal transduction of Ras/ERK and Ras/Akt pathways. The net effect suppresses LPS-induced MIP-1α mRNA expression and protein secretion in MOPC-31C cells. Thus, statins hold great promise for developing effective therapies against myeloma-induced osteolysis.

Evaluation of Pro-Inflammatory Cytokines Expression in Mouse Splenocytes After Co-Incubation with the Biofield Energy Treated Formulation: Impact of the Trivedi Effect<sup>®</sup>

With the increasing popularity of herbomineral preparations in healthcare, a new proprietary herbomineral formulation was formulated with ashwagandha root extract and minerals viz. zinc, magnesium, and selenium. The aim of the study was to evaluate the immunomodulatory potential of Biofield Energy Healing (The Trivedi Effect®) on the herbomineral test formulation using mice splenocytes. The test formulation was divided into two parts. One part was the control without the Biofield Treatment. The other part was labelled the Biofield Treated sample, which received the Biofield Energy Healing Treatment remotely from twenty renowned Biofield Energy Healers. The splenocyte cells were exposed with the test formulation at ranges of 0.00001053 to 10.53 µg/mL for cell viability by MTT assay, with cell viability ranging from 77.50% to 176.52%. TNF-α was significantly inhibited by 15.88%, 15.28%, 12.30%, 12.60%, and 22.72% at 0.00001053, 0.001053, 0.1053, 1.053, and 10.53 µg/mL, respectively in the Biofield Treated test formulation compared to the vehicle control (VC). TNF-α was significantly reduced by 2.33% and 8.35% at 1.053 and 10.53 µg/mL, respectively compared to the untreated test formulation. IL-1β was significantly reduced by 30.81%, 27.36%, 23.92%, 18.40%, 11.27%, and 21.16% at 0.00001053, 0.0001053, 0.001053, 0.01053, 0.1053, and 1.053 µg/mL, respectively in the Biofield Treated test formulation compared to the VC. IL-1β was significantly reduced by 48.63% (p≤0.001) and 15.28% at 0.00001053 and 0.0001053 µg/mL, respectively in the Biofield Treated test formulation compared to the untreated test formulation. MIP-1α expression was inhibited by the Biofield Treated test formulation and showed immunosuppressive activity at 0.01053, 0.1053, 1.053, and 10.53 µg/mL by 22.33%, 16.25%, 15.58%, and 21.83%, respectively compared to the VC. The Biofield Treated test formulation significantly reduced the MIP-1α expression by 13.27% and 15.67% (p<0.05) at 0.01053 and 10.53 µg/mL, respectively compared to the untreated test formulation. The results showed the expression of IFN-γ was significantly reduced by 33.45%, 25.38%, 37.15%, 27.74%, 32.44%, 23.03%, and 44.21% at 0.00001053, 0.0001053, 0.001053, 0.01053, 0.1053, 1.053, and 10.53 µg/mL, respectively in the Biofield Treated test formulation compared to the VC. Further, the IFN-γ level was significantly decreased by 19.02% at 10.53 µg/mL in the Biofield Treated test formulation compared to the untreated test formulation. Overall, the results demonstrate that The Trivedi Effect® Biofield Energy Healing (TEBEH) significantly enhanced the anti-inflammatory and immunomodulatory properties of the treated formulation, and may also be useful in organ transplants, anti-aging, and stress management by improving overall health and quality of life.

High Glucose Increases the Expression of Inflammatory Cytokine Genes in Macrophages Through H3K9 Methyltransferase Mechanism.

Recent studies suggest that histone modification is one of the mechanisms regulating inflammatory cytokine gene expression in hyperglycemic conditions. However, it remains unknown how histone methylation is initiated and involved in changes of inflammatory cytokine gene expression under high glucose (HG) conditions. Our aim was to investigate whether H3K9 methylation was involved in HG-induced expression of inflammatory cytokines in macrophages. Expression profile of cytokine genes under hyperglycemia in THP-1-derived macrophages was determined by human cytokine antibody array. Based on the results from the human cytokine antibody array analyses, the H3K9me3 levels of 4 inflammatory cytokine genes, including interleukin-6 (IL-6), IL-12p40, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β under HG were determined by ChIP assays. Furthermore, the expression of these 4 inflammatory cytokine genes under either HG or chaetocin (an inhibitor of SUV39H1 methyltransferase) exposure or overexpression of SUV39H1 (a H3K9me3-specific methyltransferase) was analyzed by quantitative polymerase chain reaction. Macrophages cultured in HG conditions showed increased gene expression and decreased H3K9me3 levels of inflammatory cytokine genes compared with macrophages incubated in normal glucose (NG) culture. Inhibition of SUV39H1 with chaetocin in NG-treated macrophages also increased the expression of IL-6, IL-12p40, MIP-1α, and MIP-1β. Furthermore, inhibition of SUV39H1 with chaetocin in HG-treated macrophages further increased the expression of these inflammatory cytokines. Contrarily, NG-treated macrophages transfected with SUV39H1 plasmids show decreased expression of inflammatory cytokines. Furthermore, overexpression of SUV39H1 in HG-treated macrophages alleviated the expression of inflammatory cytokines under HG conditions. Finally, HG also increases the expression of inflammation cytokines in mouse bone marrow-derived macrophages. Our data demonstrated that HG increases the expression of inflammatory cytokines in macrophages through decreased H3K9me3 levels, which was partly mediated by SUV39H1. Dysregulation of epigenetic histone modification may be one of the underlying mechanisms for HG-induced inflammatory cytokine expression in macrophages.

Donor Pretreatment With IL-1 Receptor Antagonist Attenuates Inflammation and Improves Functional Potency in Islets From Brain-Dead Nonhuman Primates.

Most pancreas and islet grafts are recovered from brain-dead (BD) donors. In this study we characterized the early inflammatory response induced by brain death in pancreata and islets from nonhuman primate donors and evaluated the effect of targeted anti-inflammatory intervention in the protection of pancreatic islets prior to transplantation. BD donors were monitored for 6 h and assigned to three experimental groups: group 1: BD-untreated donors (BD-UT) (n = 7), group 2: BD + donor pretreatment with IL-1ra (n = 6), and group 3: non-BD animals serving as controls (n = 7). We observed an IL-1ra-dependent reduction in the mobilization and activation of neutrophils from bone marrow and a significantly reduced accumulation of CD68(+) leukocytes in the pancreas and islets after brain death induction. Donor treatment with IL-1ra significantly decreased chemokine mRNA expression (MCP-1, IL-8, and MIP-1a) and attenuated the activation of circulating neutrophils and intraislet macrophages as demonstrated by a reduction in intracellular IL-1β, IL-6, MCP-1, and MIP-1α expression. As a result, IL-1ra dramatically improved viability, mitochondrial membrane polarity, and islet engraftment in mice transplanted using a minimal islet mass. These results suggest that early immunomodulation targeting inflammation in the BD donor may represent an effective therapeutic strategy to improve islet quality and function prior to transplantation.

EGCG induces G-CSF expression and neutrophilia in experimental sepsis.

A major green tea component, epigallocatechin-3-gallate (EGCG), has been proven protective against lethal sepsis in experimental setting, but its protective mechanisms remain incompletely understood. Here, we provide evidence to support EGCG's capacities in stimulating G-CSF production and neutrophilia in vivo. In an animal model of sepsis, EGCG significantly elevated peritoneal levels of G-CSF and several chemokines (e.g., MCP-1/CCL2 and MIP-1γ/CCL9), and consequently increased peritoneal neutrophil numbers (neutrophilia) at a late stage. In vitro, EGCG divergently affected HMGB1-mediated production of several chemokines: reducing CXCL15 and RANTES/CCL5, but elevating G-CSF and MIP-1α/CCL3 production by peritoneal macrophages. Similarly, it significantly induced the expression and secretion of G-CSF and MIP-1α/CCL3 in human peripheral blood mononuclear cells. Based on our preliminary data, it may be important to search for anti-inflammatory and G-CSF-stimulating agents for the clinical management of inflammatory diseases.

Adenosine Modulates NR4A Orphan Nuclear Receptors To Attenuate Hyperinflammatory Responses in Monocytic Cells.

Adenosine receptor-mediated regulation of monocyte/macrophage inflammatory responses is critical in the maintenance of tissue homeostasis. In this study, we reveal that adenosine potently modulates the expression of NR4A1, 2, and 3 orphan nuclear receptors in myeloid cells, and this modulation is primarily through the adenosine A2a receptor subtype. We demonstrate that A2a receptor activation of NR4A1-3 receptor synthesis is further enhanced in TLR4-stimulated monocytes. After TLR4 stimulation, NR4A receptor-depleted monocyte/macrophage cells display significantly altered expression of cell-surface markers and produce increased inflammatory cytokine and chemokine secretion rendering the cells an enhanced proinflammatory phenotype. Exposure of TLR4 or TNF-α-stimulated monocytes to adenosine analogs directs changes in the expression of MIP-3α and IL-23p19, with NR4A2 depletion leading to significantly enhanced expression of these factors. Furthermore, we establish that nuclear levels of NF-κB/p65 are increased in TLR/adenosine-stimulated NR4A2-depleted cells. We show that, after TLR/adenosine receptor stimulation, NR4A2 depletion promotes significant binding of NF-κB/p65 to a κB consensus binding motif within the MIP-3α proximal promoter leading to increased protein secretion, confirming a pivotal role for NF-κB activity in controlling cellular responses and gene expression outcomes in response to these mediators. Thus, these data demonstrate that during an inflammatory response, adenosine modulation of NR4A receptor activity acts to limit NF-κB-mediated effects and that loss of NR4A2 expression leads to enhanced NF-κB activity and hyperinflammatory responses in myeloid cells.

Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages.

Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn’s disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.

Recombinant tissue plasminogen activator promotes, and progesterone attenuates, microglia/macrophage M1 polarization and recruitment of microglia after MCAO stroke in rats.

Tissue plasminogen activator (tPA) is one of the few approved treatments for stroke, but its effects on the phenotype of microglia/macrophages are poorly understood. One of its side effects is an increase in the inflammatory response leading to neuronal cell damage and death in the ischemic cascade after stroke. Injury-induced activated microglia/macrophages can have dual functions as pro-inflammatory (M1) and anti-inflammatory (M2) factors in brain injury and repair. Recent studies show that progesterone (PROG) is a potent anti-inflammatory agent which affects microglia/macrophage expression after brain injury. We examined the interaction of tPA-induced expression of microglia/macrophage phenotypes and PROG's anti-inflammatory effects. tPA treatment increased the recruitment of microglia/macrophages, the polarity of M1 reactions, the expression of MIP-1α in neurons and capillaries, and the expression of MMP-3 compared to vehicle, and PROG modulated these effects. PROG treatment attenuates tPA-induced inflammatory alterations in brain capillaries and microglia/macrophages both in vivo and in vitro and thus may be a useful adjunct therapy when tPA is given for stroke.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans induces the expression of chemokines MCP-1, MIP-1α, and IP-10 via similar but distinct signaling pathways in murine macrophages

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium frequently isolated from lesions of patients with localized aggressive periodontitis. Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, stimulates innate immune cells via Toll-like receptor 4 (TLR4) to initiate inflammatory responses. In this study, we purified LPS from A. actinomycetemcomitans (AaLPS) and investigated its ability to induce the expression of chemokines, which play an important role in recruitment of leukocytes to the infection site. AaLPS induced the expression of chemokines, MCP-1, MIP-1α, and IP-10 in murine macrophages, leading to the infiltration of peripheral blood mononuclear cells in a transwell system. Although TLR4 was essential for the induction of all these chemokines by AaLPS, MCP-1 and MIP-1α expressions were MyD88-dependent, but IP-10 expression was MyD88-independent, as determined using macrophages from mice deficient in TLR4 or MyD88. Furthermore, the activation of ERK and JNK were necessary for the expression of MCP-1 and MIP-1α, whereas p38 MAP kinase and JNK activations were required for IP-10 expression. In addition, IFN-β/STAT1 signaling was exclusively involved in IP-10 expression but not in MCP-1 or MIP-1α expression. AaLPS also activated the transcription factors, NF-κB, AP-1, NF-IL6, and ISRE, all of which are involved in chemokine gene expression. These results suggest that AaLPS induces the expression of chemokines MCP-1, MIP-1α, and IP-10 through TLR4 in murine macrophages. Further, the induction of MCP-1 and MIP-1α requires MyD88, ERK, and JNK, whereas the induction of IP-10 requires JNK, p38 MAP kinase, and IFN-β/STAT1.

Apelin activates the expression of inflammatory cytokines in microglial BV2 cells via PI-3K/Akt and MEK/Erk pathways.

This paper aims to observe the changes of the inflammatory cytokines in microglial BV2 cells stimulated by apelin, and investigate the mechanism of inflammatory cytokines secretion after apelin stimulation. Immunofluorescence and quantitative real-time PCR were performed to observe expression of TNF-α, IL-1β, IL-10, MIP-1α, and MCP-1 in BV2 cells. Western blot was used to investigate the expression of phosphorylation PI-3K/Akt and phosphorylation Erk signaling pathways in BV2 cells after stimulation by apelin. Furthermore, PI-3K/Akt inhibitor (LY294402) and Erk inhibitor (U0126) were used as antagonists to detect the secretion mechanisms of cytokines in BV2 cells stimulated by apelin. Exogenous recombinant apelin activated the expression of TNF-α, IL-1β, MCP-1 and MIP-1α in BV2 cells by the detection of fluorescence expression and mRNA. Apelin also unregulated the protein expression of p-PI-3K/Akt and p-Erk in BV2 cells induced by apelin. LY294002 and U0126 inhibited activation of p-PI-3K/Akt and p-Erk expression by Western blot and attenuated the expression of inflammation factors in BV2 cells by fluorescence staining. This study demonstrates that apelin is a potential activator of inflammation factors through the PI3K/Akt and Erk signaling pathway and is potential therapeutically relevant to inflammatory responses of microglia cells.

The Influence of Immunization Route, Tissue Microenvironment, and Cytokine Cell Milieu on HIV-Specific CD8+ T Cells Measured Using Fluidigm Dynamic Arrays.

Thirty different genes including cytokines, chemokines, granzymes, perforin and specifically integrins were evaluated in Peyer's patch-KdGag197–205-specific CD8+ T cells (pools of 100 cells) using Fluidigm 48.48 Dynamic arrays following three different prime-boost immunization strategies. Data revealed that the route of prime or the booster immunization differentially influenced the integrin expression profile on gut KdGag197–205-specific CD8+ T cells. Specifically, elevated numbers of integrin αE and αD expressing gut KdGag197–205-specific CD8+ T cells were detected following mucosal but not systemic priming. Also, αE/β7 and αD/β2 heterodimerization were more noticeable in an intranasal (i.n.)/i.n. vaccination setting compared to i.n./intramuscular (i.m) or i.m./i.m. vaccinations. Moreover, in all vaccine groups tested α4 appeared to heterodimerize more closely with β7 then β1. Also MIP-1β, RANTES, CCR5, perforin and integrin α4 bio-markers were significantly elevated in i.n./i.m. and i.m./i.m. immunization groups compared to purely mucosal i.n./i.n. delivery. Furthermore, when wild type (WT) BALB/c and IL-13 knockout (KO) mice were immunized using i.n./i.m. strategy, MIP-1α, MIP-1β, RANTES, integrins α4, β1 and β7 mRNA expression levels were found to be significantly different, in mucosal verses systemic KdGag197–205-specific CD8+ T cells. Interestingly, the numbers of gut KdGag197–205-specific CD8+ T cells expressing gut-homing markers α4β7 and CCR9 protein were also significantly elevated in IL-13 KO compared to WT control. Collectively, our findings further corroborate that the route of vaccine delivery, tissue microenvironment and IL-13 depleted cytokine milieu can significantly alter the antigen-specific CD8+ T cell gene expression profiles and in turn modulate their functional avidities as well as homing capabilities.

IFNγ-mediated exacerbation of Saccharopolyspora rectivirgula-induced proinflammatory responses is dependent on interferon regulatory factor 1 (INM3P.403)

Abstract Saccharopolyspora rectivirgula (SR) is a thermophilic actinomycetes that causes hypersensitivity pneumonitis (HP). Interferon γ (IFNγ) produced after exposure to SR has been demonstrated to be necessary for development of HP. However, the molecular mechanism by which IFNγ enhances SR-mediated inflammatory responses is not completely understood. We investigated how IFNγ alters SR-induced expression of cytokines/chemokines. IFNγ pretreatment substantially enhanced expression of TNFα, IL-6, IL-23, IFNβ, MIP-1α, and MIP-2, without affecting IL-10 expression, in response to SR. Expression of interferon regulatory factor 1 (IRF1) was rapidly increased in cells pre-exposed to IFNγ, and inhibition of IRF1 synthesis abolished IFNγ-enhanced gene expression in response to SR. In contrast, suppression of IRF1 expression did not alter SR-mediated expression of IL-10. SR stimulation led to the binding of IRF1 to the IL-6 promoter region, but not to the IL-10 promoter region in IFNγ-pretreated cells. This indicates that IRF1 expressed in cells pre-exposed to IFNγ is activated by subsequent SR stimulation and contributes to the synergistic expression of certain proinflammatory genes. Our results imply that IFNγ produced after initial exposure to SR may help direct the proinflammatory responses to subsequent SR exposure toward to pathogenic Th1/Th17 development by selectively amplifying expression of certain cytokines/chemokines without affecting expression of regulatory cytokine IL-10.

Macrophage inflammatory protein-1α promotes the growth of acute myeloid leukemia cells

BACKGROWND: Macrophage inflammatory protein-1α (MIP-l α/CCL3) belongs to the C-C chemokine family (CCL3), which can be secreted by macrophages, other types of hematopoietic cells and bone marrow stromal cells. Higher levels of MIP-1α were found to be associated with several kinds of hematologic malignancies, including multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Moreover, MIP-1α has been reported to be an adverse prognostic factor for CLL. However, the impact of MIP-1α on acute myeloid leukemia (AML) has been poorly investigated. To investigate the influence of MIP-1α on proliferction of AML cells. Using MLL-AF9 induced AML mouse model, the expression of MIP-1α was measured by real time quantitative RT-PCR. AML cell proliferation was examined by cell counting and colony forming assay (CFC). The influence of blocking the MIP-1α action on the growth and pathogenic ability of AML cells was explored by using the small molecule antagonist for interfering interaction of MIP-1α with its receptor CCR1. The MIP-1α could promote the proliferation and colony formation of AML cells, the blocking MIP-1a could inhibit the growth of AML cells and delay onset of AML. The MIP-1a promotes the occurence and progression of AML, therefore blocking the MIP-1α signal pathway may be served as a strategy to inhibit the growth of AML cells, and MIP-1α can be a potential target for treatment of AML.

Size effects of single-walled carbon nanotubes on in vivo and in vitro pulmonary toxicity

To elucidate the effect of size on the pulmonary toxicity of single-wall carbon nanotubes (SWCNTs), we prepared two types of dispersed SWCNTs, namely relatively thin bundles with short linear shapes (CNT-1) and thick bundles with long linear shapes (CNT-2), and conducted rat intratracheal instillation tests and in vitro cell-based assays using NR8383 rat alveolar macrophages. Total protein levels, MIP-1α expression, cell counts in BALF, and histopathological examinations revealed that CNT-1 caused pulmonary inflammation and slower recovery and that CNT-2 elicited acute lung inflammation shortly after their instillation. Comprehensive gene expression analysis confirmed that CNT-1-induced genes were strongly associated with inflammatory responses, cell proliferation, and immune system processes at 7 or 30 d post-instillation. Numerous genes were significantly upregulated or downregulated by CNT-2 at 1 d post-instillation. In vitro assays demonstrated that CNT-1 and CNT-2 SWCNTs were phagocytized by NR8383 cells. CNT-2 treatment induced cell growth inhibition, reactive oxygen species production, MIP-1α expression, and several genes involved in response to stimulus, whereas CNT-1 treatment did not exert a significant impact in these regards. These results suggest that SWCNTs formed as relatively thin bundles with short linear shapes elicited delayed pulmonary inflammation with slower recovery. In contrast, SWCNTs with a relatively thick bundle and long linear shapes sensitively induced cellular responses in alveolar macrophages and elicited acute lung inflammation shortly after inhalation. We conclude that the pulmonary toxicity of SWCNTs is closely associated with the size of the bundles. These physical parameters are useful for risk assessment and management of SWCNTs.

The effect of Indomethacin and Betamethasone on the cytokine response of human neonatal mononuclear cells to gram-positive bacteria

Intrauterine infections with gram-positive bacteria pose a serious threat to neonates since they can result in neonatal sepsis, induce a fetal inflammatory response and also cause preterm birth. Despite intensive care, prematurity remains a leading cause of neonatal death, and is often accompanied by a number of morbidities. In order to prevent premature birth, tocolytic agents like Indomethacin are administered. Betamethasone is used to promote lung maturation and prevent respiratory distress syndrome. A combination of both drugs is assumed to prevent premature delivery while simultaneously facilitating lung maturation. This study investigates the effect of Betamethasone, Indomethacin and a combination of both on the cytokine production of neonatal cord blood mononuclear cells (CBMC) after stimulation with lysates of the gram-positive pathogens Streptococcus agalactiae and Enterococcus faecalis. The aim of the study is to determine the impact of these drugs on the function of the neonatal immune system which should aid clinicians in choosing the optimal therapy in case of preterm birth associated with intrauterine infection. Betamethasone reduced the production of the pro-inflammatory cytokines IL-6, IL-12p40, MIP-1α and TNF and increased the expression of the anti-inflammatory cytokine IL-10, depending on the pathogen used for stimulation. In contrast to Betamethasone, Indomethacin almost exclusively increased IL-10 production. The combination of both drugs decreased the expression of IL-6, IL-12p40, MIP-1α and TNF while increasing IL-10 production, depending on the concentration of Indomethacin and the pathogen used for stimulation. Based on our results, the combination therapy with Indomethacin and Betamethasone has a similar effect on cytokine production as Betamethasone alone, which is generally administered in case of impending preterm birth. However, the combination therapy has the advantage of promoting lung maturation while simultaneously blocking preterm labor effectively.

Effects of anodic titanium dioxide nanotubes of different diameters on macrophage secretion and expression of cytokines and chemokines.

To investigate effects of TiO2 nanotubes of different diameters on J744A.1 macrophage behaviour, secretion and expression of pro-inflammatory cytokines and chemokines. Macrophage-like J744A.1 cells were cultured on three types of Ti surface: mechanically polished titanium plus 30 and 80 nm TiO2 nanotube surfaces, for 4, 24 and 48 h. Macrophage adhesion and proliferation were assessed using CCK-8 assay. Levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokines (MCP-1 and MIP-1α) secreted into the supernatant were measured using the Cytometric Bead Arrays kit. TNF-α, MCP-1 and MIP-1α gene expression were quantitatively analysed by real-time PCR. These show that TiO2 nanotube surfaces, especially of 80 nm TiO2 nanotube, benefited macrophage adhesion and proliferation, and reduced protein secretion and mRNA expression of TNF-α, MCP-1 and MIP-1α. IL-1β and IL-6 were undetectable on all the surfaces at all times. TiO2 nanotube surfaces, especially of 80 nm TiO2 nanotube, reduced inflammatory response in vitro, which might be part of a basis for rapid osseointegration in implants with TiO2 nanotube surfaces in animal studies.

Chemokines CCL14 and CCL3 Facilitate Monocytes/Macrophage Infiltration in Multiple Myeloma Bone Marrow

Drug resistance is the most common reason for treatment failure in multiple myeloma (MM), a plasma cell cancer of the bone marrow (BM). Our previous studies have shown that macrophage (MΦ) infiltration is increased in MM BM, and these MM-associated MΦs induce MM drug resistance by protecting MM cells from chemotherapeutic-induced cell death. A recently published clinical study also suggests that the number of BM MΦs negatively correlates with MM drug response and patient survival. Why MM BM harbors more MΦ is not well understood. In general, MΦs are derived from circulating monocytes, and monocytes are recruited to tissues by chemokines and differentiate into MΦs. In this study, we examined monocyte/MΦ chemokine expression in MM BM and determined that CCL14 and CCL3 were functional chemokines that regulated monocyte/MΦ infiltration in MM BM.To identify chemokines that regulate monocyte/MΦ infiltration in the MM tumor bed, we examined a panel of chemokines expressed in MM patient BM cells. Specifically, total BM cells (both CD138+ and CD138- cells) from MM patients were assayed by qPCR for target gene expression analysis. MM BM cells highly expressed CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and CCL14 (HCC-1), but not CCL8 (MCP-2), CCL7 (MCP-3) or CCL13 (MCP-4). Next, we compared expression of those chemokines in MM BM vs. healthy BM aspirates by ELISA. MIP-1 α and HCC-1 were highly expressed in MM BM aspirates (n=11), but not in BM from healthy donors (n=7; P<0.05). Immunohistochemistry staining also confirmed that MM BM (n=5 patients) highly expressed MIP-1 α and HCC-1. Based on our findings, we hypothesized that elevated MΦ infiltration in MM BM might be due to MM BM overexpression of MIP-1α and HCC-1. To test this hypothesis, we first examined MIP-1α and HCC-1 function in monocyte migration. In vitro chemotactic assay showed that adding MIP-1α or HCC-1 neutralizing antibody inhibited monocyte migration to cocultured BM stromal cells (BMSCs) and MM cells (P<0.05). The antibodies also inhibited monocyte migration to ex vivo cultured BM cells from MM patients (P<0.05). In a murine MM mouse model, C57BL/KawRij mice inoculated with 5TGM1, a murine MM cell line, developed tumors in hind leg bones. Both flow cytometry analysis and immunohistochemistry staining suggested that the tumor-bearing mice had an increased number of MΦs in the BM, compared with healthy mice (P<0.05). Because HCC-1 does not have murine homology but it has the highest amino acid sequence similarity to MIP-1α, we treated the mice with MIP-1α neutralization antibody. Intra-peritoneal injection of mouse MIP-1α antibody decreased the MΦ number in BM (P<0.05). Such in vivo findings strongly suggest that MIP-1α regulates MΦ infiltration in MM BM. We also examined the source cells that contributed to high MIP-1 α and HCC-1 expression in MM BM. Primary MM cells (CD138+) and non-malignant BM cells (CD138-) were isolated from patient (n=5) BM aspirates. qPCR analysis showed that both CD138+ and CD138- cells had high MIP-1α and HCC-1 expression. Further, BMSC/MM coculture stimulated MIP-1α overexpression in BMSCs. Finally, we examined the association between MIP-1α or HCC-1 expression and the number of BM MΦ in MM patients. The chemokine expression in BM aspirates was determined by ELISA, and the number of MΦ was measured by flow cytometry analysis for CD14+/CD68+ cells. We found that MIP-1α expression was positively associated with the number of BM MΦs (P<0.05).To summarize, our findings suggest that CCL14 and CCL3 facilitate monocyte/MΦ infiltration in MM BM. Inhibiting these 2 chemokines may decrease the number of MM-associated MΦs, therefore increasing MM cell vulnerability to chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks

Background: The toxicity of multi-walled carbon nanotubes (MWCNT) may be related to the immune system. The objective of this study was to obtain information for immunotoxic mechanisms of MWCNT in situ.Methods: Using whole-body inhalation, male and female rats were exposed to 0, 0.2, 1 or 5 mg MWCNT/m3 for 13 weeks. Thereafter, spleens were recovered from the rats. Real-time PCR was done to assess expression of TNFα, IL-1β, IL-6, IL-10, MCP-1 and MIP-1α mRNA in the splenic macrophages; splenic T-lymphocytes were examined for IL-2 and TGF-β1 mRNA expression.Results: The relative expression of IL-1β mRNA in the cells from female rats exposed to 5 mg MWCNT/m3 was significantly higher than that in control cells. For IL-6 and IL-10, cells from rats in the 0.2 and 5 mg MWCNT/m3 had significantly higher mRNA expressions than did cells from controls. Expression of IL-1β, IL-6 and TNFα genes in cells from males in all exposure groups were higher than in control cells. Expression of MIP-1α in the cells from female 5-mg group was significantly higher than that in cells in the control. Only IL-2 was expression reduced, i.e. cells from male and female rats in all MWCNT groups had significantly lower mRNA expressions than control cells.Conclusions: Systemic inflammation would likely occur in rats (or other hosts) exposed to MWCNT via inhalation due to increases in the expression of inflammatory cytokines in splenic macrophages. Moreover, decreases in IL-2 expression in T-lymphocytes may be critical to the potential reductions in anti-tumor responses in MWCNT-exposed hosts.

Variability of cytokine gene expression in intestinal tissue and the impact of normalization with the use of reference genes

ObjectiveTo determine the intra- and inter-subject variability of mucosal cytokine gene expression in rectal biopsies from healthy volunteers and to screen cytokine and chemokine mRNA as potential biomarkers of mucosal inflammation. Design and methodsRectal biopsies were collected from 8 participants (3 biopsies per participant) and 1 additional participant (10 biopsies). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to quantify IL-1β, IL-6, IL-12p40, IL-8, IFN-γ, MIP-1α, MIP-1β, RANTES, and TNF-α gene expression in the rectal tissue. The intra-assay, inter-biopsy and inter-subject variance was measured in the eight participants. Bootstrap re-sampling of the biopsy measurements was performed to determine the accuracy of gene expression data obtained for 10 biopsies obtained from one participant. Cytokines were both non-normalized and normalized using four reference genes (GAPDH, β-actin, β2 microglobulin, and CD45). ResultsCytokine measurement accuracy was increased with the number of biopsy samples, per person; four biopsies were typically needed to produce a mean result within a 95% confidence interval of the subject’s cytokine level approximately 80% of the time. Intra-assay precision (% geometric standard deviation) ranged between 8.2 and 96.9 with high variance between patients and even between different biopsies from the same patient. Variability was not greatly reduced with the use of reference genes to normalize data. ConclusionsThe number of biopsy samples required to provide an accurate result varied by target although 4 biopsy samples per subject and timepoint, provided for >77% accuracy across all targets tested. Biopsies within the same subjects and between subjects had similar levels of variance while variance within a biopsy (intra-assay) was generally lower. Normalization of inflammatory cytokines against reference genes failed to consistently reduce variance. The accuracy and reliability of mRNA expression of inflammatory cytokines will set a ceiling on the ability of these measures to predict mucosal inflammation. Techniques to reduce variability should be developed within a larger cohort of individuals before normative reference values can be validated.