Differential role of lipoteichoic acids isolated from Staphylococcus aureus and Lactobacillus plantarum on the aggravation and alleviation of atopic dermatitis.

Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, up-regulates inflammatory cytokine production through the toll-like receptor 2 (TLR2) signaling pathway, and also contributes to anti-inflammatory responses against immune cells stimulated by lipopolysaccharides. In the current study, we examined the effects of LTAs isolated from Staphylococcus aureus (aLTA) and Lactobacillus plantarum (pLTA) on the aggravation and alleviation of atopic dermatitis (AD). aLTA strongly induced CCL2 production in THP-1 cells. CCL2 was regulated by the TLR2 pathway including the activation of IRAK2, NF-κB and JNK. CCL2 induced Th2 polarization of CD4+T cells through induction of interleukin (IL)-2, -4, and -5 and inhibition of interferon-gamma (IFN-γ). CCL2 levels and immunoglobulin E (IgE) production were increased in aLTA-injected mice. On the other hand, pLTA moderately affected CCL2 production and it inhibited aLTA-mediated CCL2 production. The serum levels of CCL2 and IgE were inhibited by pLTA pre-injection followed by aLTA reinjection, which resulted in the alleviation of irritant contact dermatitis (ICD) symptoms. Our results suggest that S. aureus infection causes an increase in CCL2 production, and may exacerbate atopic dermatitis (AD)-like symptoms through the excessive IgE production. Alternatively, pLTA alleviated AD-like symptoms by inhibiting aLTA-induced CCL2 and IgE production.

CCL2 is Upregulated by Decreased miR-122 Expression in Iron-Overload-Induced Hepatic Inflammation.

BACKGROUND/AIMS: Iron overload (IO) is accompanied by hepatic inflammation. The chemokine (C-C motif) ligand 2 (CCL2) mediates inflammation, and its overexpression is associated with IO. However, whether IO results in CCL2 overexpression in the liver and the underlying mechanisms are unclear. METHODS: We subjected mice to IO by administering intraperitoneal injections of dextran-iron or by feeding mice a 3% dextran-iron diet to observe the effects of IO on miR-122/CCL2 expression through real-time qPCR and Western blot analysis. We also used indicators, including the expression of the inflammatory cytokine, the inflammation score based on H&E staining and the serum content of ALT and AST to evaluate the effects of IO on hepatic inflammation. Meanwhile, we observed the effects of vitamin E on IO-induced hepatic inflammation. In cells, we used 100 µΜ FeSO4 or 30 µΜ Holo-Tf to produce IO and observed the roles of miR-122 in regulating CCL2 expression by using miR-122 mimics or inhibitors to overexpress or inhibit miR-122. Then, we used a dual-luciferase reporter assay to prove that miR-122 regulates CCL2 expression through direct binding to its complementary sequence in the CCL2 mRNA 3'UTR. RESULTS: IO induces the downregulation of miR-122 and the upregulation of CCL2, as well as inflammatory responses both in vitro and in vivo. Although IO-induced oxidative stress is eliminated by the antioxidant vitamin E, IO-induced hepatic inflammation still exists, which probably can be explained by the fact that vitamin E has no effects on the miR-122/CCL2 pathway. In in vitro experiments, the overexpression and inhibition of miR-122 significantly reduced and increased CCL2 expression, respectively. The dual-luciferase reporter assay indicates that miR-122 binds CCL2 mRNA 3'UTR. CONCLUSION: We propose the roles of miR-122/CCL2 in IO-induced hepatic inflammation. Our studies should provide a new clue for developing clinical strategies for patients with IO.

Adipocyte-Macrophage Cross-Talk in Obesity.

Obesity is characterized by the chronic low-grade activation of the innate immune system. In this respect, macrophage-elicited metabolic inflammation and adipocyte-macrophage interaction has a primary importance in obesity. Large amounts of macrophages are accumulated by different mechanisms in obese adipose tissue. Hypertrophic adipocyte-derived chemotactic monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2) pathway also promotes more macrophage accumulation into the obese adipose tissue. However, increased local extracellular lipid concentrations is a final mechanism for adipose tissue macrophage accumulation. A paracrine loop involving free fatty acids and tumor necrosis factor-alpha (TNF-alpha) between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes in the adipose tissue. Adipocyte-specific caspase-1 and production of interleukin-1beta (IL-1beta) by macrophages; both adipocyte and macrophage induction by toll like receptor-4 (TLR4) through nuclear factor-kappaB (NF-kappaB) activation; free fatty acid-induced and TLR-mediated activation of c-Jun N-terminal kinase (JNK)-related pro-inflammatory pathways in CD11c+ immune cells; are effective in macrophage accumulation and in the development of adipose tissue inflammation. Old adipocytes are removed by macrophages through trogocytosis or sending an "eat me" signal. The obesity-induced changes in adipose tissue macrophage numbers are mainly due to increases in the triple-positive CD11b+ F4/80+ CD11c+ adipose tissue macrophage subpopulation. The ratio of M1-to-M2 macrophages is increased in obesity. Furthermore, hypoxia along with higher concentrations of free fatty acids exacerbates macrophage-mediated inflammation in obesity. The metabolic status of adipocytes is a major determinant of macrophage inflammatory output. Macrophage/adipocyte fatty-acid-binding proteins act at the interface of metabolic and inflammatory pathways. Both macrophages and adipocytes are the sites for active lipid metabolism and signaling.

Estrogen-dependent regulation of human uterine natural killer cells promotes vascular remodelling via secretion of CCL2.

STUDY QUESTION: Does intrauterine biosynthesis of estrogen play an important role in early pregnancy by altering the function of uterine natural killer (uNK) cells? SUMMARY ANSWER: Estrogens directly regulate the function of human uNK cells by increasing uNK cell migration and secretion of uNK cell-derived chemokine (C-C motif) ligand 2 (CCL2) that critically facilitates uNK-mediated angiogenesis. WHAT IS KNOWN ALREADY: uNK cells are a phenotypically distinct population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. Recently we discovered that decidualisation of human endometrial stromal cells results in the generation of an estrogen-rich microenvironment in areas of decidualised endometrium. We hypothesize that intrauterine biosynthesis of estrogens plays an important role in early pregnancy by altering the function of uNK cells. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used primary human uNK cells which were isolated from first trimester human decidua (n = 32). PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary uNK cells were isolated from first trimester human decidua using magnetic cell sorting. The impact of estrogens on uNK cell function was assessed. Isolated uNK cells were treated with estrone (E1, 10(-8) M) or estradiol (E2, 10(-8) M) alone or in combination with the anti-estrogen ICI 182 780 (ICI, 10(-6) M). uNK cell motility was assessed by transwell migration assay and time-lapse microscopy. Expression of chemokine receptors was assessed by quantitative PCR (qPCR) and immunohistochemistry, and angiogenic factors were assessed by qPCR and cytokine array. Concentrations of CCL2 in supernatants were measured by enzyme-linked immunosorbent assay. Angiogenesis was assessed in a human endometrial endothelial cell network formation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with either E1 or E2 increased uNK cell migration (P = 0.0092 and P = 0.0063, respectively) compared with control. Co-administration of the anti-estrogen ICI blocked the effects of E1 and E2 on cell migration. Concentrations of C-X-C chemokine receptor type 4 (CXCR4) mRNA in uNK cells were increased by E2 treatment. The network formation assay revealed that conditioned media from uNK cells treated with E2 significantly increased human endometrial endothelial cell (HEEC) angiogenesis (P = 0.0029 versus control). Analysis of media from uNK cells treated with E2 using an antibody array identified CCL2 as the most abundant cytokine. Validation assays confirmed concentrations of CCL2 mRNA and protein were increased by E2 in uNK cells (P < 0.05 versus controls). Compared with the control, recombinant human CCL2 was found to increase HEEC network formation (P < 0.05) and neutralization of CCL2 in uNK conditioned media significantly decreased E2-dependent uNK-mediated network formation (P = 0.0006). LIMITATIONS, REASONS FOR CAUTION: Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in vivo in humans. Primary human uNK cells were isolated from first trimester decidua at a range of gestations (8-12 weeks), which may be a source of variation. Primary human uNK cells from non-pregnant endometrium were not assessed and therefore the responses of uNK cells to E2 treatment described in this study may be distinct to uNK cells from first trimester decidua. WIDER IMPLICATIONS OF THE FINDINGS: E2 is an essential regulator of reproductive competence. This study demonstrates a critical role for E2 in regulating cellular cross-talk within the endometrium during early pregnancy. We provide the first evidence that E2 directly regulates the function of human uNK cells by altering uNK cell migration and the secretion of uNK-derived angiogenic factors. We describe a novel mechanism of estrogen-dependent secretion of CCL2 which critically mediates uNK-dependent endometrial angiogenesis. Dysregulation of uNK cell function has been implicated in the aetiology of early implantation disorders and disorders of pregnancy. These novel findings provide unique insight into the regulation of uNK cell activity during the establishment of pregnancy in women and highlight key processes which may be targeted in future therapeutic strategies. STUDY FUNDING/COMPETING INTERESTS: Studies undertaken in the authors' laboratory were supported by MRC Programme Grant G1100356/1 to P.T.K.S. The authors have no conflicts of interest to disclose.

Chemokine CCL2 up-regulated in the medullary dorsal horn astrocytes contributes to nocifensive behaviors induced by experimental tooth movement.

To test the hypothesis that the astrocytic chemokine (C-C motif) ligand 2 (CCL2) plays an important role in nocifensive behaviors after experimental tooth movement (ETM), the expression and cellular localization of CCL2 and astrocyte activation in the medullary dorsal horn (MDH) were determined by immunohistochemistry in rats. The dose-dependent effects of intrathecal C-C chemokine receptor type 2 (CCR2) antagonists on these changes in nocifensive behaviors were evaluated. Exogenous CCL2 was added to medullary dorsal horn slices to evaluate its contributory role in the induction of extracellular signal-regulated kinase (ERK) activation ex vivo. We found a significant increase in the expression of CCL2 and glial fibrillary acidic protein (GFAP), corresponding well to the nocifensive behaviors after ETM. In addition, application of recombinant CCL2 led to ERK activation, which could be attenuated effectively by pretreatment with CCL2-neutralizing antibody ex vivo. The magnitude of the nocifensive behavior could be reduced by medullary CCR2 antagonists in a dose-dependent manner. Therefore, the astrocytic CCL2 is actively involved in the development and maintenance of tooth-movement pain and thus may be a potential target for analgesics in orthodontic nocifensive responses control.

Targeting chemoattractant chemokine (C-C motif) ligand 2 derived from astrocytes is a promising therapeutic approach in the treatment of neuromyelitis optica spectrum disorders.

Introduction: Aquaporin-4 immunoglobulin G (AQP4-IgG)-induced astrocytes injury is a key mechanism in the pathogenesis of neuromyelitis spectrum disorder (NMOSD), and although CCL2 is involved, its specific role has not been reported. We aimed to further investigate the role and potential mechanisms of CCL2 in AQP4-IgG-induced astrocyte injury. Methods: First, we evaluated CCL2 levels in paired samples of subject patients by automated microfluidic platform, Ella®. Second, we knock down astrocyte's CCL2 gene in vitro and in vivo to define the function of CCL2 in AQP4-IgG-induced astrocyte injury. Third, astrocyte injury and brain injury in live mice were assessed by immunofluorescence staining and 7.0T MRI, respectively. Western blotting and high-content screening were conducted to clarify the activation of inflammatory signaling pathways, and changes in CCL2 mRNA and cytokine/chemokines were measured by qPCR technique and flow cytometry, respectively. Results: There were greatly higher CSF-CCL2 levels in NMOSD patients than that in other non-inflammatory neurological diseases (OND) groups. Blocking astrocyte CCL2 gene expression can efficiently mitigate AQP4-IgG-induced damage in vitro and in vivo. Interestingly, prevention of CCL2 expression could decrease other inflammatory cytokines released, including IL-6 and IL-1ß. Our data suggest that CCL2 involves in the initiation and plays a pivotal role in AQP4-IgG-damaged astrocytes. Discussion: Our results indicate that CCL2 may serve as a promising candidate target for inflammatory disorder therapy, including NMOSD.

Monocyte chemoattractant protein-1: a proinflammatory cytokine elevated in sarcopenic obesity.

OBJECTIVE: Sarcopenic obesity (SO) is associated with poorer physical outcomes and functional status in the older adult. A proinflammatory milieu associated with central obesity is postulated to enhance muscle catabolism. We set out to examine associations of the chemokine monocyte chemoattractant protein-1 (MCP-1) in groups of older adults, with sarcopenia, obesity, and the SO phenotypes. METHODS: A total of 143 community dwelling, well, older adults were recruited. Cross-sectional clinical data, physical performance, and muscle mass measurements were collected. Obesity and sarcopenia were defined using revised National Cholesterol Education Program (NCEP) obesity guidelines and those of the Asian Working Group for Sarcopenia. Serum levels of MCP-1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In all, 25.2% of subjects were normal, 15.4% sarcopenic, 48.3% obese, and 11.2% were SO. The SO groups had the lowest appendicular lean mass, highest percentage body fat, and lowest performance scores on the Short Physical Performance Battery and grip strength. The MCP-1 levels were significantly different, with the highest levels found in SO participants (P<0.05). CONCLUSION: Significantly raised MCP-1 levels in obese and SO subjects support the theory of chronic inflammation due to excess adiposity. Longitudinal studies will reveal whether SO represents a continuum of obesity causing accelerated sarcopenia and cardiovascular events, or the coexistence of two separate conditions with synergistic effects affecting functional performance.

Contribution of chemokine CCL2/CCR2 signaling in the dorsal root ganglion and spinal cord to the maintenance of neuropathic pain in a rat model of lumbar disc herniation.

UNLABELLED: Lumbar disc herniation (LDH) is a major cause of sciatica, but the underlying mechanisms are not well understood. Chemokine CCL2 has been implicated to play a vital role in the neuroinflammation and central sensitization after spinal nerve ligation. Here we investigated the expression and the role of CCL2 and its receptor CCR2 in LDH-induced pain. Implantation of autologous nucleus pulposus induced persistent pain hypersensitivity, associated with increased mRNA expression of CCL2 and CCR2 in the dorsal root ganglion and spinal cord. Interestingly, CCL2 was increased in neurons and CCR2 was mainly increased in macrophages in the dorsal root ganglion, whereas CCL2 and CCR2 were increased in astrocytes and neurons, respectively, in the spinal cord. Intrathecal injection of CCR2 antagonist RS504393 at 3 days or 10 days significantly attenuated nucleus pulposus-induced mechanical allodynia. The results suggest that CCL2/CCR2 in the dorsal root ganglion and spinal cord is involved in the maintenance of LDH-induced pain. Targeting CCL2/CCR2 signaling may be a potential treatment for chronic radicular neuropathic pain. PERSPECTIVE: These results suggest that CCL2/CCR2 signaling in the dorsal root ganglion and spinal cord is involved in LDH-induced pain via distinct mechanisms. These findings provide evidence of the antinociceptive effect of CCR2 antagonist on radicular neuropathic pain.

Gene expression in subcutaneous adipose tissue differs in women with polycystic ovary syndrome and controls matched pair-wise for age, body weight, and body mass index.

Adipose tissue dysfunction may be a central factor in the pathogenesis of insulin resistance in women with polycystic ovary syndrome (PCOS). Gene expression in subcutaneous adipose tissue in PCOS and its relation to metabolic and endocrine features of the syndrome have been fragmentarily investigated. The aim was to assess in subcutaneous adipose tissue the expression of genes potentially associated with adipose tissue dysfunction and to explore their relation to features of the syndrome. Twenty-one women with PCOS (body mass index [BMI] 18.2-33.4 kg/m(2)) and 21 controls (BMI 19.2-31.7 kg/m(2)) were matched pair-wise for age, body weight, and BMI. Tissue biopsies were obtained to measure mRNA expression of 44 genes (TaqMan Low Density Array). Differential expression levels were correlated with BMI, glucose infusion rate (GIR), sex hormone binding globulin (SHBG), and sex steroids. In PCOS, expression of adiponectin receptor 2 (ADIPOR2), LPL, and twist-related protein 1 (TWIST1) was decreased, while expression of chemokine (C-C motif) ligand 2 (CCL2) and heme oxygenase (decycling 1) (HMOX1) was increased. TWIST1 and HMOX1, both novel adipokines, correlated with BMI and GIR. After BMI adjustment, LPL and ADIPOR2 expression correlated with plasma estradiol, and CCL2 expression correlated with GIR, in all women. We conclude that adipose tissue mRNA expression differed in PCOS women and controls and that two novel adipokines, TWIST1 and HMOX1, together with adiponectin, LPL, and CCL2, and their downstream pathways merit further investigation.

ß2- and ß3-adrenergic receptors drive COMT-dependent pain by increasing production of nitric oxide and cytokines.

Decreased activity of catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, contributes to pain in humans and animals. Previously, we demonstrated that development of COMT-dependent pain is mediated by both ß2- and ß3-adrenergic receptors (ß2ARs and ß3ARs). Here we investigated molecules downstream of ß2- and ß3ARs driving pain in animals with decreased COMT activity. Based on evidence linking their role in pain and synthesis downstream of ß2- and ß3AR stimulation, we hypothesized that nitric oxide (NO) and proinflammatory cytokines drive COMT-dependent pain. To test this, we measured plasma NO derivatives and cytokines in rats receiving the COMT inhibitor OR486 in the presence or absence of the ß2AR antagonist ICI118,551+ß3AR antagonist SR59320A. We also assessed whether the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) and cytokine-neutralizing antibodies block the development of COMT-dependent pain. Results showed that animals receiving OR486 exhibited higher levels of NO derivatives, tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) in a ß2- and ß3AR-dependent manner. Additionally, inhibition of NO synthases and neutralization of the innate immunity cytokines TNFα, IL-1ß, and IL-6 blocked the development of COMT-dependent pain. Finally, we found that NO influences TNFα, IL-1ß, IL-6, and CCL2 levels, whereas TNFα and IL-6 influence NO levels. Altogether, these results demonstrate that ß2- and ß3ARs contribute to COMT-dependent pain, at least partly, by increasing NO and cytokines. Furthermore, they identify ß2- and ß3ARs, NO, and proinflammatory cytokines as potential therapeutic targets for pain patients with abnormalities in COMT physiology.

Advanced application of porcine intestinal epithelial cells for the selection of immunobiotics modulating toll-like receptor 3-mediated inflammation.

PURPOSE: In this study, we aimed to characterize toll-like receptor (TLR)-3-mediated inflammatory immune response in porcine intestinal epithelial (PIE) cells and in PIE-immune cell co-cultures and, to evaluate if these in vitro systems are useful for selecting immunomodulatory lactic acid bacteria. RESULTS: We demonstrated that these systems are valuable tools for the in vitro study of the inflammatory response triggered by TLR3 in intestinal epithelial cells (IECs) and of the interaction between IECs and immune cells. In addition, we showed that PIE cells could be used for the selection of immunobiotic lactobacilli strains with anti-inflammatory activities. We found that Lactobacillus casei MEP221114 is an immunobiotic candidate for modulation of TLR3-mediated inflammatory responses. CONCLUSION: The present study deepened our understanding of the mechanisms of immunobiotic action by demonstrating that the interaction between some lactobacilli strains and IECs can up-regulate the mRNA expression of TLR negative regulators and that this effect could help to regulate the production of inflammatory mediators during the generation of a TLR3-mediated immune response.