MCP-1 mediates ischemia-reperfusion-induced cardiomyocyte apoptosis via MCPIP1 and CaSR.

Monocyte chemotactic protein-1 (MCP-1) plays a crucial role in ischemia-reperfusion (I/R) injury; however, the detailed mechanism of MCP-1 in I/R injury-induced cardiomyocyte apoptosis remains unclear. In this study, we explored the cascade downstream of I/R-induced MCP-1 that modulates cell apoptosis and determined whether Ca2+-sensing receptors (CaSRs) are involved in the process. Protein levels were detected in a cardiac muscle cell line (HL-1) and primary cultured neonatal mouse ventricular cardiomyocytes using Western blotting and immunocytochemistry. Released MCP-1 was detected using ELISA. Both Hoechst staining and flow cytometry methods were used to measure cell apoptosis. Specific pharmacological inhibitors of CC chemokine receptor 2 (RS-102895) and CaSR (NPS-2143) as well as a CaSR activator (evocalcet) were applied to confirm the roles of these factors in I/R-induced cell apoptosis. I/R inhibited cell viability and upregulated cell apoptosis. Moreover, I/R induced the release of MCP-1 from both HL-1 cells and primary cardiomyocytes. Further research confirmed that CaSR acted as an upstream effector of monocyte chemotactic protein-1-induced protein-1 (MCPIP1) and coordinately regulated cell apoptosis, which was verified by addition of an inhibitor or activator of CaSR. Moreover, MCPIP1 induced cell apoptosis through endoplasmic reticulum (ER) stress but not autophagy induced by I/R. Based on these findings, I/R-induced MCP-1 release regulates cardiomyocyte apoptosis via the MCPIP1 and CaSR pathways, suggesting a new therapeutic strategy for I/R injury.NEW & NOTEWORTHY Ischemia-reperfusion (I/R)-induced monocyte chemotactic protein-1 release regulates cardiomyocyte apoptosis via the monocyte chemotactic protein-1-induced protein-1 (MCPIP1) and Ca2+-sensing receptor pathway. The functional changes mediated by MCPIP1 involve the activation of endoplasmic reticulum stress, but not the autophagy pathway, after I/R injury.

Association between dyslipidemia and CCL2 in patients undergoing hemodialysis.

INTRODUCTION: Hemodialysis (HD) is associated with high risk for cardiovascular diseases including acute myocardial infarction, stroke and congestive heart failure. C-C Motif Chemokine Ligand 2 (CCL2), also known monocyte chemotactic protein-1 (MCP-1) can be produced by a variety of cells, reaching increased levels in dyslipidemic chronic kidney disease (CKD) patients undergoing HD treatment. The main of this study was to evaluate the association between of CCL2 plasma levels and dyslipidemia in CKD patients undergoing HD. METHODS: A cross-sectional study enrolled 160 Brazilian HD patients. CCL2 plasma levels were measured by capture ELISA. The association between CCL2 levels and dyslipidemia was investigated using linear regression, adjusted for classic and non-classical CVD risk factors. RESULTS: A significant association was observed between CCL2 levels and dyslipidemia (P = 0.029), even after adjustment for possible confounding variables, such as age, gender, body mass index, diabetes mellitus, HD time, urea pre-hemodialysis and interdialytic weight gain (P = 0.045). CONCLUSION: Our findings show that CCL2 levels are associated with dyslipidemia, which suggests a role of this cytokine in the pathogenesis of cardiovascular disease in HD patients. A better understanding of this pathogenesis could contribute to the discovery of new therapeutic targets that would reduce cardiovascular complications in these patients.

IL-8 and MCP-1/CCL2 regulate proteolytic activity in triple negative inflammatory breast cancer a mechanism that might be modulated by Src and Erk1/2.

Inflammatory breast cancer (IBC) is a highly metastatic and lethal breast cancer. As many as 25-30% of IBCs are triple negative (TN) and associated with low survival rates and poor prognosis. We found that the microenvironment of IBC is characterized by high infiltration of tumor associated macrophages (TAMs) and by over-expression of the cysteine protease cathepsin B (CTSB). TAMs in IBC secrete high levels of the cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared to non-IBC patients. Herein, we tested the roles of IL-8 and MCP-1/CCL2 in modulating proteolytic activity and invasiveness of TN-non-IBC as compared to TN-IBC and addressed the underlying molecular mechanism(s) for both cytokines. Quantitative real time PCR results showed that IL-8 and MCP-1/CCL2 were significantly overexpressed in tissues of TN-IBCs. IL-8 and MCP-1/CCL2 induced CTSB expression and activity of the p-Src and p-Erk1/2 signaling pathways relevant for invasion and metastasis in TN-non-IBC, HCC70 cells and TN-IBC, SUM149 cells. Dasatinib, an inhibitor of p-Src, and U0126, an inhibitor of p-Erk1/2, down-regulated invasion and expression of CTSB by HCC70 and SUM149 cells, a mechanism that is reversed by IL-8 and MCP-1/CCL2. Our study shows that targeting the cytokines IL-8 and MCP-1/CCL2 and associated signaling molecules may represent a promising therapeutic strategy in TN-IBC patients.

Empagliflozin Inhibits Basal and IL-1ß-Mediated MCP-1/CCL2 and Endothelin-1 Expression in Human Proximal Tubular Cells.

SGLT2 inhibitors (SGLT2i) slow the progression of chronic kidney disease; however, evidence for the underlying molecular mechanisms is scarce. We investigated SGLT2i-mediated effects on differential gene expression in two independent human proximal tubular cell (HPTC) lines (HK-2 and RPTEC/TERT1) at the mRNA and protein levels under normoglycemic conditions, utilizing IL-1ß as a pro-inflammatory mediator. Microarray hybridization identified 259 genes that were uniformly upregulated by IL-1ß (10 mg/mL) and downregulated by empagliflozin (Empa) (500 nM) after 24 h of stimulation in two independent HPTC lines (n = 2, each). The functional annotation of these genes identified eight pathway clusters. Among 12 genes annotated to the highest ranked cluster (enrichment score, 3.51), monocyte chemoattractant protein-1/CC-chemokine ligand 2 (MCP-1/CCL2) and endothelin-1 (ET-1) were selected for verification at mRNA and protein levels based on their established involvement in the early pathogenesis of chronic kidney disease: IL-1ß upregulated basal MCP-1/CCL2 (15- and 19-fold) and ET-1 (3- and 8-fold) mRNA expression, while Empa downregulated basal MCP-1/CCL2 (0.6- and 0.5-fold) and ET-1 (0.3- and 0.2-fold) mRNA expression as early as 1 h after stimulation and for at least 24 h in HK-2 and RPTEC/TERT1 cells, respectively. The co-administration of Empa inhibited IL-1ß-mediated MCP-1/CCL2 (0.2-fold, each) and ET-1 (0.2-fold, each) mRNA expression as early as 1 h after ligand stimulation and for at least 24 h in both HPTC lines, respectively. This inhibitory effect of Empa on basal and IL-1ß-mediated MCP-1/CCL2 and ET-1 mRNA expression was corroborated at the protein level. Our study presents novel evidence for the interference of SGLT2 inhibition with tubular inflammatory response mechanisms under normoglycemic conditions that might account for SGLT2i-mediated nephroprotection.

Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1: a novel mechanism for regulation of MCP-1 production and function.

The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.

Decreased Motor Neuron Support by SMA Astrocytes due to Diminished MCP1 Secretion.

Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by severe, often fatal muscle weakness due to loss of motor neurons. SMA patients have deletions and other mutations of the survival of motor neuron 1 (SMN1) gene, resulting in decreased SMN protein. Astrocytes are the primary support cells of the CNS and are responsible for glutamate clearance, metabolic support, response to injury, and regulation of signal transmission. Astrocytes have been implicated in SMA as in in other neurodegenerative disorders. Astrocyte-specific rescue of SMN protein levels has been shown to mitigate disease manifestations in mice. However, the mechanism by which SMN deficiency in astrocytes may contribute to SMA is unclear and what aspect of astrocyte activity is lacking is unknown. Therefore, it is worthwhile to identify defects in SMN-deficient astrocytes that compromise normal function. We show here that SMA astrocyte cultures derived from mouse spinal cord of both sexes are deficient in supporting both WT and SMN-deficient motor neurons derived from male, female, and mixed-sex sources and that this deficiency may be mitigated with secreted factors. In particular, SMN-deficient astrocytes have decreased levels of monocyte chemoactive protein 1 (MCP1) secretion compared with controls and MCP1 restoration stimulates outgrowth of neurites from cultured motor neurons. Correction of MCP1 deficiency may thus be a new therapeutic approach to SMA.SIGNIFICANCE STATEMENT Spinal muscular atrophy (SMA) is caused by the loss of motor neurons, but astrocyte dysfunction also contributes to the disease in mouse models. Monocyte chemoactive protein 1 (MCP1) has been shown to be neuroprotective and is released by astrocytes. Here, we report that MCP1 levels are decreased in SMA mice and that replacement of deficient MCP1 increases differentiation and neurite length of WT and SMN-deficient motor-neuron-like cells in cell culture. This study reveals a novel aspect of astrocyte dysfunction in SMA and indicates a possible approach for improving motor neuron growth and survival in this disease.

Nucleotide receptors control IL-8/CXCL8 and MCP-1/CCL2 secretions as well as proliferation in human glioma cells.

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Monocyte chemoattractant protein 1 (MCP-1/CCL2) contributes to thymus atrophy in acute myeloid leukemia.

Recent studies on acute myelogenous leukemia (AML) patients have revealed the existence of T-cell immunodeficiencies, characterized by peripheral T lymphocytes that are unable to interact with blasts, reduced thymic emigrants and oligoclonal restricted repertoires. These observations suggest that there is a profound thymic dysregulation, which is difficult to study in AML patients. Using the C1498 AML mouse model, we demonstrated that leukemia development was associated with thymus atrophy, which was defined by abnormal organ weight and reduced cellularity. In addition, we observed a dramatic loss of peripheral CD4(+) and CD8(+) T-cell numbers with increased frequencies of CD4(+) FoxP3(+) regulatory and activated/memory T cells. Investigating the mechanisms leading to this atrophy, we observed a significant accumulation of the monocyte chemoattractant protein 1 (MCP-1/CCL2) in thymi of leukemic mice. Treatment of AML-bearing animals with a blocking anti-CCL2 antibody revealed a lower tumor burden, augmented antileukemic T-cell responses, and improved survival rate compared to nontreated mice. These results were not observed when neutralization of CCL2 was performed in thymectomized mice. Altogether, we show that the CCL2 protein participates in thymic atrophy in AML mice, and this could have important implications for future immunotherapeutic strategies.

Chemokine MCP1/CCL2 and RANTES/CCL5 gene polymorphisms influence Henoch-Schönlein purpura susceptibility and severity.

BACKGROUND/PURPOSE: Henoch-Schönlein purpura (HSP) is the most common small vessel vasculitis in children. It is considered to be an IgA-containing immune complex-mediated disease. Chemokines are small secreted proteins that attract leukocytes during inflammation. Our aim was to determine the serum levels of chemokines and investigate the association of chemokine gene polymorphisms with childhood HSP. METHODS: Serum levels of chemokines (interleukin-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, MIG/CXCL9, and IP-10/CXCL10) were determined using cytometric beads arrays. We investigated the association of three single-nucleotide polymorphisms (SNPs) MCP1/CCL2 -2518C/T, RANTES/CCL5 -403C/T, and RANTES/CCL5 -28C/G with HSP in 85 HSP patients and 136 healthy controls. RESULTS: Five serum chemokine levels were significantly elevated in patients with the acute stage of HSP compared to the normal controls (p < 0.05). MCP1/CCL2 -2518 TT genotype and T allele were associated with the risk for HSP with OR (95% CI) 3.32 (1.45-7.59) and 1.78 (1.20-2.64), respectively. The RANTES/CCL5 -28 GG genotype was associated with a significantly lower percentage of corticosteroid usage and lower corticosteroid accumulative dose in HSP patients. RANTES/CCL5 -403 TC and TT genotype were significantly associated with renal manifestations with an OR (95% CI) of 4.33 (1.44-12.99), adjusted for sex and age and the other two SNP genotypes. CONCLUSION: Our results support the fact that chemokines play important roles in the pathogenesis of HSP. MCP1/CCL2 gene polymorphisms were associated with susceptibility for HSP. RANTES/CCL5 gene polymorphisms may be related to disease severity and HSP nephritis.

Neurosteroid [3α,5α]3-hydroxypregnan-20-one inhibition of chemokine monocyte chemoattractant protein-1 in alcohol-preferring rat brain neurons, microglia, and astroglia.

BACKGROUND: Neuroimmune dysfunction in alcohol use disorder (AUD) is associated with activation of myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptors (TLR) resulting in overexpression of the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). MCP-1 overexpression in the brain is linked to anxiety, higher alcohol intake, neuronal death, and activation of microglia observed in AUD. The neurosteroid [3α,5α][3-hydroxypregnan-20-one (3α,5α-THP) has been reported as an inhibitor of MyD88-dependent TLR activation and MCP-1 overexpression in mouse and human macrophages and the brain of alcohol-preferring (P) rats. METHODS: We investigated how 3α,5α-THP regulates MCP-1 expression at the cellular level in P rat nucleus accumbens (NAc) and central amygdala (CeA). We focused on neurons, microglia, and astrocytes, examining the individual voxel density of MCP-1, neuronal marker NeuN, microglial marker IBA1, astrocytic marker GFAP, and their shared voxel density, defined as intersection. Ethanol-naïve male and female P rats were perfused 1 h after IP injections of 15 mg/kg of 3α,5α-THP, or vehicle. The NAc and CeA were imaged using confocal microscopy following double-immunofluorescence staining for MCP-1 with NeuN, IBA1, and GFAP, respectively. RESULTS: MCP-1 intersected with NeuN predominantly and IBA1/GFAP negligibly. 3α,5α-THP reduced MCP-1 expression in NeuN-labeled cells by 38.27 ± 28.09% in male and 56.11 ± 21.46% in female NAc, also 37.99 ± 19.53% in male and 54.96 ± 30.58% in female CeA. In females, 3α,5α-THP reduced the MCP-1 within IBA1 and GFAP-labeled voxels in the NAc and CeA. Conversely, in males, 3α,5α-THP did not significantly alter the MCP-1 within IBA1 in NAc or with GFAP in the CeA. Furthermore, 3α,5α-THP decreased levels of IBA1 in both regions and sexes with no impact on GFAP or NeuN levels. Secondary analysis performed on data normalized to % control values indicated that no significant sex differences were present. CONCLUSIONS: These data suggest that 3α,5α-THP inhibits neuronal MCP-1 expression and decreases the proliferation of microglia in P rats. These results increase our understanding of potential mechanisms for 3α,5α-THP modulation of ethanol consumption.

Cytokines are systemic effectors of lymphatic function in acute inflammation.

The response of the lymphatic system to inflammatory insult and infection is not completely understood. Using a near-infrared fluorescence (NIRF) imaging system to noninvasively document propulsive function, we noted the short-term cessation of murine lymphatic propulsion as early as 4h following LPS injection. Notably, the effects were systemic, displaying bilateral lymphatic pumping cessation after a unilateral insult. Furthermore, IL-1ß, TNF-α, and IL-6, cytokines that were found to be elevated in serum during lymphatic pumping cessation, were shown separately to acutely and systemically decrease lymphatic pulsing frequency and velocity following intradermal administration. Surprisingly, marked lymphatic vessel dilation and leakiness were noted in limbs contralateral to IL-1ß intradermal administration, but not in ipsilateral limbs. The effects of IL-1ß on lymphatic pumping were abated by pre-treatment with an inhibitor of inducible nitric oxide synthase, L-NIL (N-iminoethyl-L-lysine). The results suggest that lymphatic propulsion is systemically impaired within 4h of acute inflammatory insult, and that some cytokines are major effectors of lymphatic pumping cessation through nitric oxide-mediated mechanisms. These findings may help in understanding the actions of cytokines as mediators of lymphatic function in inflammatory and infectious states.

Inflammatory markers in cerebrospinal fluid of paediatric spinal muscular atrophy patients receiving nusinersen treatment.

Spinal muscular atrophy (SMA) is a progressive motor neuron disease with onset during infancy or early childhood. Recent therapeutic advances targeting the genetic defect that underlies SMA improved survival in patients with infantile onset SMA (type 1) and improved motor function in SMA type 1-3. The most commonly used therapy for SMA, the antisense oligonucleotide nusinersen, is delivered by repeated intrathecal injections. The long-term safety effects of this procedure, however, have not yet been investigated in detail. We here present case reports of three children with SMA in which routine laboratory investigation revealed increased leukocyte counts in cerebrospinal fluid (CSF) collected during the course of nusinersen treatment. To further characterize this observation, we used a multiplex method to analyse a broad spectrum of inflammatory markers in the CSF of these patients. We found that interleukin-10 (IL10) was consistently elevated in CSF with increased leukocyte counts, but other inflammatory markers were not. Based on this analysis we selected 7 markers for further analysis in a cohort of 38 children with SMA and determined their expression during the course of nusinersen therapy. No consistent association was found between levels of inflammatory markers and the duration of nusinersen therapy in individual patients. However, monocyte chemoactive protein 1 (MCP1/CCL2) -a neuroprotective protein secreted by astrocytes and previously associated with SMA- levels increased over the course of nusinersen treatment, indicating a possible neuroprotective mechanism associated with nusinersen therapy. In summary, our findings confirm that repeated intrathecal injections are safe and do not trigger unwanted immune responses.

Evaluating causality of cellular senescence in non-alcoholic fatty liver disease.

Cellular senescence is a state of irreversible cell cycle arrest that has important physiological functions. However, cellular senescence is also a hallmark of ageing and has been associated with several pathological conditions. A wide range of factors including genotoxic stress, mitogens and inflammatory cytokines can induce senescence. Phenotypically, senescent cells are characterised by short telomeres, an enlarged nuclear area and damaged genomic and mitochondrial DNA. Secretion of proinflammatory proteins, also known as the senescence-associated secretory phenotype, is a characteristic of senescent cells that is thought to be the main contributor to their disease-inducing properties. In the past decade, the role of cellular senescence in the development of non-alcoholic fatty liver disease (NAFLD) and its progression towards non-alcoholic steatohepatitis (NASH) has garnered significant interest. Until recently, it was suggested that hepatocyte cellular senescence is a mere consequence of the metabolic dysregulation and inflammatory phenomena in fatty liver disease. However, recent work in rodents has suggested that senescence may be a causal factor in NAFLD development. Although causality is yet to be established in humans, current evidence suggests that targeting senescent cells has therapeutic potential for NAFLD. We aim to provide insights into the quality of the evidence supporting a causal role of cellular senescence in the development of NAFLD in rodents and humans. We will elaborate on key cellular and molecular features of senescence and discuss the efficacy and safety of novel senolytic drugs for the treatment or prevention of NAFLD.

Data showing differential expression of Monocyte chemoattractant protein-1 in response to symptomatic and asymptomatic T. vaginalis infection.

Trichomoniasis is caused by Trichomonas vaginalis (a protozoan parasite). About 80% of the infected cases remain asymptomatic [1]. The differential response of showing symptoms or no symptoms is not yet explored. However, some studies gave us some insights on the pathogenesis of trichomonas and also about host defense mechanism. Host secretes pro-inflammatory cytokines and chemokines to evade infection. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a strong chemoattractant of monocytes, NK-cells and T-lymphocytes. Many reports have shown high MCP-1 levels during trichomonas infection [2], [3], [4], [5] in human prostate stromal myofibroblast cells (WPMY-1), HeLa cells, vaginal epithelial cells (VECs) but levels in response to symptomatic and asymptomatic isolates is not yet reported. In this article, we have reported MCP-1 levels in the vaginal washes and serum samples of BALB/c mouse infected with symptomatic and asymptomatic T. vaginalis isolates for different time points. We found higher levels of MCP-1 in vaginal washes of symptomatic group on 2nd day post infection (dpi) than control uninfected group. While on 4th dpi and 14th dpi, higher levels of MCP-1 in vaginal washes was observed in asymptomatic group as compared to control group. However, significant level of MCP-1 was observed in asymptomatic group on 14th dpi as compared to symptomatic group in vaginal washes. We have also observed significantly higher levels of MCP-1 in the serum samples of symptomatic group on 2nd, 4th and 14th dpi as compared to control group. A higher level of MCP-1 was found at all the time points in serum samples of asymptomatic group as compared to control group. Interestingly, a significant higher level of MCP-1 was found in the serum samples of BALB/c mice in asymptomatic as compared to symptomatic group. The MCP-1 levels in both vaginal washes and serum were significantly higher in asymptomatic group at later time points.

Peripheral versus central mechanisms of the cannabinoid type 2 receptor agonist AM1710 in a mouse model of neuropathic pain.

The CB2 R agonist AM1710, examined in animal models of peripheral neuropathy, is effective in controlling aberrant light touch sensitivity, referred to as mechanical allodynia. However, nonspecific binding of AM1710 to CB1 R, either peripherally or centrally, could be partially responsible for the analgesic effects of AM1710. Thus, we sought to determine in mice whether spinal (intrathecal; i.t.) or peripheral AM1710 administration could lead to anti-allodynia by reducing the protein expression of spinal and dorsal root ganglia (DRG) proinflammatory cytokines and elevating the anti-inflammatory cytokine interleukin-10 (IL-10) in the absence of CB1 R. Macrophage cell cultures were examined to characterize AM1710-mediated suppression of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Either i.p. or i.t. AM1710 reversed CCI-induced mechanical allodynia to sham levels in CB1 R (-/-), (+/-), (+/+) mice. CCI-induced neuropathy decreased IL-10 immunoreactivity (IR) in the dorsal root ganglia (DRG) and the dorsal horn of the spinal cord, with i.t. AM1710 restoring basal IL-10 IR. CCI-induced elevations in proinflammatory cytokine IR were decreased within the spinal cord only after i.t. AM1710 in all mouse genotypes. Meanwhile, within DRG tissue from neuropathic mice, proinflammatory cytokines were decreased following either i.p. or i.t. AM1710. Analysis of cultured supernatants revealed AM1710 decreased TNF-alpha protein. We conclude that CB1 R is dispensable for either peripheral or central anti-allodynic actions of AM1710 in neuropathic mice. Cannabinoid CB2 R agonists produce heightened spinal IL-10 which may be clinically relevant to successfully treat neuropathic pain.

Association of Plasma Biomarkers for Angiogenesis and Proteinopathy in Indian Amyotrophic Lateral Sclerosis Patients.

Background Amyotrophic lateral sclerosis (ALS) is a rare motor neuron disease with progressive degeneration of motor neurons. Various molecules have been explored to provide the early diagnostic/prognostic tool for ALS without getting much success in the field and miscellaneous reports studied in various population. Objective The study was aimed to see the differential expression of proteins involved in angiogenesis (angiogenin [ANG], vascular endothelial growth factor [VEGF], vascular endothelial growth factor receptor 2 [VEGFR2], etc), proteinopathy (transactive response DNA binding protein-43 [TDP-43] and optineurin [OPTN]), and neuroinflammation (monocyte chemoattractant protein-1[MCP-1]) based on the characteristics of ALS pathology. Though, suitable panel based on protein expression profile can be designed to robust the ALS identification by enhancing the prognostic and diagnostic efficacy for ALS. Methods A total of 89 ALS patients and 98 nonneurological controls were analyzed for the protein expression. Expression of angiogenic (VEGF, VEGFR2, and ANG), neuroinflammation (MCP-1), and proteinopathy (TDP-43 and OPTN) markers were estimated in plasma of the participants. Proteins were normalized with respective value of total protein before employing statistical analysis. Results Analysis has exhibited significantly reduced expression of angiogenic, proteinopathy, and neuroinflammation biomarkers in ALS patients in comparison to controls. Spearman's correlation analysis has showed the positive correlation to each protein. Conclusion Altered expression of these proteins is indicating the prominent function in ALS pathology which may be interdependent and may have a synergistic role. Hence, a panel of expression can be proposed to diagnose ALS patient which may also suggest the modulation of therapeutic strategy according to expression profile of patient.

Ratio of IL-8 in CSF versus Serum Is Elevated in Patients with Unruptured Brain Aneurysm.

Only scarce data pertaining to interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human aneurysm can be found in the current literature. Therefore, the aim of this study was the evaluation of cerebrospinal fluid (CSF) and serum IL-8 and MCP-1 concentration in unruptured intracranial aneurysm (UIA) patients (n = 25) compared to the control group (n = 20). IL-8 and MCP-1 concentrations were measured with ELISA method. We demonstrated that CSF IL-8 concentration of UIA patients is significantly higher (p < 0.001) than that presented in the serum, which can indicate its local synthesis within central nervous system. CSF IL-8 concentration was also significantly related to aneurysm size, which may reflect the participation of IL-8 in the formation and development of brain aneurysms. IL-8 Quotient (CSF IL-8 divided by serum IL-8) in UIA patients was statistically higher compared to control individuals (p = 0.045). However, the diagnostic utility analysis did not equivocally indicate the diagnostic usefulness of the IL-8 Quotient evaluation in brain aneurysm patients. Nevertheless, this aspect requires further study.

MCP-1/CCL2 Mediated by Autocrine Loop of PDGF-BB Promotes Invasion of Lung Cancer Cell by Recruitment of Macrophages Via CCL2-CCR2 Axis.

Platelet-derived growth factor-BB (PDGF-BB) is recognized as a potential player in a paracrine manner in tumor stroma development. PDGF-BB has an autocrine growth function in lung cancer cells; however, the mechanism in nonsmall-cell lung cancer is not fully understood. In this study, we report that PDGF-BB increased monocyte chemoattractant protein-1 (MCP-1)-dependent macrophage recruitment and that expression of metastatic genes increased in A549 cells cocultured with RAW 264.7 macrophages. Similar to exogenous PDGF-BB, PDGF-BB might have a self-stimulation in invasion of cancer cells during reciprocal activation of cancer cells and tumor-associated macrophages through secretion of soluble proteins. Also, we found that PDGF-BB upregulates both mRNA and protein level of MCP-1 expression in human A549 cells through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt cell signaling pathways, binding NFκB to MCP-1 promoter site and PDGF-Rß as critical receptor. These results suggested that MCP-1/chemokine (C-C motif) ligand 2 (CCL2) expression mediated by the autocrine loop of PDGF-BB enhanced recruitment of macrophages through CCL2-CCR2 axis, which could ultimately increase expression of metastatic genes in lung cancer cells, finally promoting invasive potential of cancer cells.

Tear and serum interleukin-8 and serum CX3CL1, CCL2 and CCL5 in sulfur mustard eye-exposed patients.

BACKGROUND: The serum and tear levels of four inflammatory chemokines were evaluated in sulfur mustard (SM)-exposed with serious ocular problems. MATERIALS AND METHODS: In this study, 128 SM-exposed patients and 31 healthy control participants participated. Tear and serum levels of chemokines were assessed by ELISA method. RESULTS: There was no significant difference in the serum level of IL-8/CXCL8, CX3CL1/fractalkine, CCL2/MCP-1, and CCL5/RANTES between all SM-exposed subjects and control groups. The tear level of IL-8 in the SM-exposed group was lower than the control group, but the difference was not significant. In the SM-exposed group with the abnormalities in tear breakup time (TBUT) test, fundus and pannus formation were significantly higher than SM-exposed patients without these problems. CX3CL1 levels have significantly increased in SM-exposed group with blepharitis, pterygium, and conjunctival pigmentation as compared with the control group. Besides, significantly higher levels of CX3CL1 were observed in SM-exposed group with or without bulbar conjunctival hyperemia and abnormal vessels a well as with fundus abnormality compared to the control group. Only, SM-exposed group with subconjunctival fibrosis had significantly lower levels of CCL5 than SM-exposed group without this problem. CONCLUSION: The higher level of CX3CL1 and consistent levels of IL-8/CXCL8, MCP-1/CCL2, and RANTES/CCL5 in SM-exposed individuals may indicate an anti-inflammatory response against the destructive effects of SM gas. High tear level of IL-8/CXCL8 reflects the severity of ocular surface abnormalities, yet significantly low tear level found in mild SM-exposed subgroup compared with the control group. The lower levels of CX3CL1 and RANTES/CCL5 may represent the different pathophysiology which requires further studies.

Silencing TNFα with lipidoid nanoparticles downregulates both TNFα and MCP-1 in an in vitro co-culture model of diabetic foot ulcers.

Diabetes is one of the most formidable diseases facing the world today, with the number of patients growing every year. Poor glycemic control yields a host of complications, such as impaired wound healing. This often results in the formation of diabetic foot ulcers, which carry a poor prognosis because they are notoriously difficult to treat. Current therapies do not address the increased number of infiltrating macrophages to the wound bed that overproduce tumor necrosis factor α (TNFα), which increases fibroblast apoptosis and collagen dismantling and decreases angiogenesis. In this study, we investigated the potential of RNA interference therapy to reduce the inappropriately high levels of TNFα in the wound bed. Although TNFα is a challenging gene silencing target, our lipidoid nanoparticles potently silence TNFα mRNA and protein expression at siRNA doses of 5-100nM without inducing vehicle-related gene silencing or cell death. We also describe the creation of an in vitro macrophage-fibroblast co-culture model, which reflects the TNFα and monocyte chemotactant protein-1 (MCP-1/CCL2) cross-talk that exists in diabetic wounds. Because TNFα induces fibroblasts to produce MCP-1, we show that silencing TNFα results in a downregulation of MCP-1, which should inhibit the recruitment of additional macrophages to the wound. In co-culture experiments, a single lipidoid nanoparticle dose of 100nM siTNFα downregulated TNFα and MCP-1 by 64% and 32%, respectively. These data underscore the potential of lipidoid nanoparticle RNAi treatment to inhibit a positive feedback cycle that fuels the pathogenesis of diabetic foot ulcers. STATEMENT OF SIGNIFICANCE: Diabetic foot ulcers are a rapidly growing issue worldwide, with current ulcer treatments not as effective as desired. RNA interference therapy represents a largely untapped possible solution to impaired wound healing. We show that siRNA-loaded lipidoid nanoparticles silence the overexpression of tumor necrosis factor α (TNFα) in inflammatory macrophages which leads to a subsequent downregulation of fibroblast-produced macrophage chemotactant protein-1 (MCP-1). Both TNFα and MCP-1 are critical components of the inflammatory feedback loop that exists in chronic wounds. In contrast to the majority of wound drug delivery studies, our study utilizes macrophage/fibroblast co-culture experiments to recapitulate a multicellular wound environment in which cytokine signaling influences inflammation. Results underscore the therapeutic potential of siRNA nanoparticles directed against TNFα in inhibiting two key inflammatory targets in chronic wounds.