Fatty acid amide hydrolase (FAAH) blockade ameliorates experimental colitis by altering microRNA expression and suppressing inflammation.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), which is thought to result from immune-mediated inflammatory disorders, leads to high morbidity and health care cost. Fatty acid amide hydrolase (FAAH) is an enzyme crucially involved in the modulation of intestinal physiology through anandamide (AEA) and other endocannabinoids. Here we examined the effects of an FAAH inhibitor (FAAH-II), on dextran sodium sulphate (DSS)-induced experimental colitis in mice. Treatments with FAAH-II improved overall clinical scores by reversing weight loss and colitis-associated pathogenesis. The frequencies of activated CD4+ T cells in spleens, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and colon lamina propiria (LP) were reduced by FAAH inhibition. Similarly, the frequencies of macrophages, neutrophils, natural killer (NK), and NKT cells in the PPs and LP of mice with colitis declined after FAAH blockade, as did concentrations of systemic and colon inflammatory cytokines. Microarray analysis showed that 26 miRNAs from MLNs and 217 from PPs had a 1.5-fold greater difference in expression after FAAH inhibition. Among them, 8 miRNAs were determined by reverse-transcription polymerase chain reaction (RT-PCR) analysis to have anti-inflammatory properties. Pathway analysis demonstrated that differentially regulated miRNAs target mRNA associated with inflammation. Thus, FAAH-II ameliorates experimental colitis by reducing not only the number of activated T cells but also the frequency of macrophages, neutrophils, and NK/NKT cell, as well as inflammatory miRNAs and cytokine at effector sites in the colon. These studies demonstrate for the first time that FAAH-II inhibitor may suppress colitis through regulation of pro-inflammatory miRNAs expression.

Single Nucleotide Polymorphisms in IL-10, IL-12p40, and IL-13 Genes and Susceptibility to Glioma.

Glioma is one of the most aggressive and most common tumors of the central nervous system (CNS) in humans. The exact causes of glioma are not well known, but evidence suggests the involvement of genetic factors in addition to environmental risk factors. The present study aimed to determine whether polymorphisms in IL-10-1082A/G, IL-12p40 1188C/A, and IL-13+2044G/A (rs20541) are associated with the incidence of glioma in Iraqi patients. Ninety-six patients with different grades of glioma and 40 apparently healthy individuals were recruited. A blood sample and genomic DNA were collected from all subjects. The amplification refractory mutation system and sequence-specific primer polymerase chain reaction (PCR) were used for genotyping of IL-10-1082A/G and IL-12p40 1188C/A, respectively; whereas, the IL-13+2044G/A was detected by DNA sequencing after amplification of the genes by PCR. All SNPs were within Hardy-Weinberg equilibrium and each appeared in three genotypes in patients and controls. In IL-10-1082A/G, these genotypes frequencies were AA (75%), AG (22.93%) and GG (2.07%) in patients as compared to similar frequencies (62.5%), (27.5%) and (10%) respectively, in controls. The variant IL-12p40 1188C/A genotype was AA (72.92%), AC (23.96%), and CC (3.13%%) in patients as compared to 65%, 30%, and 5%, respectively, in controls. The frequencies of IL-13+2044G/A genotypes (GG, GA, and AA) were 89.58%, 9.37%, and 1.04% among patients versus 47.5%, 32.5% and 20%, respectively, among controls. These results suggest a protective role of mutant alleles G and A in IL-10-1082A/G and IL-13+2044G/A against gliomas. Further studies with more rigorous parameter designs will be needed to confirm the current findings.

A genome-wide association scan on the levels of markers of inflammation in Sardinians reveals associations that underpin its complex regulation.

Identifying the genes that influence levels of pro-inflammatory molecules can help to elucidate the mechanisms underlying this process. We first conducted a two-stage genome-wide association scan (GWAS) for the key inflammatory biomarkers Interleukin-6 (IL-6), the general measure of inflammation erythrocyte sedimentation rate (ESR), monocyte chemotactic protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) in a large cohort of individuals from the founder population of Sardinia. By analysing 731,213 autosomal or X chromosome SNPs and an additional ∼1.9 million imputed variants in 4,694 individuals, we identified several SNPs associated with the selected quantitative trait loci (QTLs) and replicated all the top signals in an independent sample of 1,392 individuals from the same population. Next, to increase power to detect and resolve associations, we further genotyped the whole cohort (6,145 individuals) for 293,875 variants included on the ImmunoChip and MetaboChip custom arrays. Overall, our combined approach led to the identification of 9 genome-wide significant novel independent signals-5 of which were identified only with the custom arrays-and provided confirmatory evidence for an additional 7. Novel signals include: for IL-6, in the ABO gene (rs657152, p = 2.13×10(-29)); for ESR, at the HBB (rs4910472, p = 2.31×10(-11)) and UCN119B/SPPL3 (rs11829037, p = 8.91×10(-10)) loci; for MCP-1, near its receptor CCR2 (rs17141006, p = 7.53×10(-13)) and in CADM3 (rs3026968, p = 7.63×10(-13)); for hsCRP, within the CRP gene (rs3093077, p = 5.73×10(-21)), near DARC (rs3845624, p = 1.43×10(-10)), UNC119B/SPPL3 (rs11829037, p = 1.50×10(-14)), and ICOSLG/AIRE (rs113459440, p = 1.54×10(-08)) loci. Confirmatory evidence was found for IL-6 in the IL-6R gene (rs4129267); for ESR at CR1 (rs12567990) and TMEM57 (rs10903129); for MCP-1 at DARC (rs12075); and for hsCRP at CRP (rs1205), HNF1A (rs225918), and APOC-I (rs4420638). Our results improve the current knowledge of genetic variants underlying inflammation and provide novel clues for the understanding of the molecular mechanisms regulating this complex process.

miR-155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses.

Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition that affects millions of people worldwide, results in high morbidity and exorbitant health-care costs. The critical features of both innate and adaptive immunity are to control inflammation and dysfunction in this equilibrium is believed to be the reason for the development of IBD. miR-155, a microRNA, is up-regulated in various inflammatory disease states, including IBD, and is a positive regulator of T-cell responses. To date, no reports have defined a function for miR-155 with regard to cellular responses in IBD. Using an acute experimental colitis model, we found that miR-155(-/-) mice, as compared to wild-type control mice, have decreased clinical scores, a reversal of colitis-associated pathogenesis, and reduced systemic and mucosal inflammatory cytokines. The increased frequency of CD4+ lymphocytes in the spleen and lamina propria with dextran sodium sulphate induction was decreased in miR-155(-/-) mice. Similarly, miR-155 deficiency abrogated the increased numbers of interferon-γ expressing CD4+ T cells typically observed in wild-type mice in this model. The frequency of systemic and mucosal T helper type 17-, CCR9-expressing CD4+ T cells was also reduced in miR-155(-/-) mice compared with control mice. These findings strongly support a role for miR-155 in facilitating pro-inflammatory cellular responses in this model of IBD. Loss of miR-155 also results in decreases in T helper type 1/type 17, CD11b+) and CD11c+ cells, which correlated with reduced clinical scores and severity of disease. miR-155 may serve as a potential therapeutic target for the treatment of IBD.

Distinct energy requirements for human memory CD4 T-cell homeostatic functions.

Differentiation and activation of CD4 memory T cells (T(mem) cells) require energy from different sources, but little is known about energy sources for maintenance and surveillance activities of unactivated T(mem) cells. Mitochondrial fatty acid oxidation (FAO) in human unactivated CD4 T(mem) cells was significantly enhanced by inhibition of glycolysis, with respective means of 1.7- and 4.5-fold for subjects <45 yr and >65 yr, and by stimulation of AMP-activated protein kinase, with respective means of 1.3- and 5.2-fold. However, CCL19 and sphingosine 1-phosphate (S1P), which control homeostatic lymphoid trafficking of unactivated T(mem) cells, altered FAO and glycolysis only minimally or not at all. Inhibition of CD4 T(mem)-cell basal FAO, but not basal glycolysis, significantly suppressed CCL19- and S1P-mediated adherence to collagen by >50 and 20%, respectively, and chemotaxis by >20 and 50%. Apoptosis of unactivated T(mem) cells induced by IL-2 deprivation or CCL19 was increased significantly by >150 and 70%, respectively, with inhibition of FAO and by >110 and 30% with inhibition of glycolysis. Anti-TCR antibody activation of T(mem) cells increased their chemotaxis to CCL5, which was dependent predominantly on glycolysis rather than FAO. The sources supplying energy for diverse functions of unactivated T(mem) cells differ from that required for function after immune activation.

Aging enhances release of exosomal cytokine mRNAs by Aß1-42-stimulated macrophages.

Amyloid-ß1-42 (Aß) peptide effects on human models of central nervous system (CNS)-patrolling macrophages (Ms) and CD4 memory T-cells (CD4-Tms) were investigated to examine immune responses to Aß in Alzheimer's disease. Aß and lipopolysaccharide (LPS) elicited similar M cytokine and exosomal mRNA (ex-mRNA) responses. Aß- and LPS-stimulated Ms from 20 ≥65-yr-old subjects generated significantly more IL-1, TNF-α, and IL-6, but not IL-8 or IL-12, and significantly more ex-mRNAs for IL-6, TNF-α, and IL-12, but not for IL-8 or IL-1, than Ms from 20 matched 21- to 45-yr-old subjects. CD4-Tm generation of IL-2, IL-4, and IFN-γ and, for young subjects, IL-10, but not IL-6, evoked by Aß was significantly lower than with anti-T-cell antigen receptor antibodies (Abs). Abs significantly increased all CD4-Tm ex-mRNAs, but only IL-2 and IL-6 ex-mRNAs were increased by Aß. There were no significant differences between cytokine and ex-mRNA responses of CD4-Tms from the old compared to the young subjects. M-derived serum exosomes from the old subjects had significantly higher IL-6 and IL-12 ex-mRNA levels than those from the young subjects, whereas there were no differences for CD4-Tm-derived serum exosomes. An Aß level relevant to neurodegeneration elicited broad M cytokine and ex-mRNA responses that were significantly greater in the old subjects, but only narrow and age-independent CD4-Tm responses.

Impact of single nucleotide polymorphism in IL-4, IL-4R genes and systemic concentration of IL-4 on the incidence of glioma in Iraqi patients.

Glioma is the most common and believed to be one of the most aggressive tumors of the central nervous system (CNS) in humans. Very little information is available on the etiology and pathogenesis of these tumors to date. A significant gap remains in our current understanding of the molecular pathways involved in the genesis, progression and clinical behavior of these tumors. Recently, several single nucleotide polymorphisms (SNPs) have been identified in cytokine gene sequences, particularly within the promoter region of these genes, and have been shown to be associated with the development of different types of brain tumors. The present study investigates the association of C-33T SNP in the interleukin-4 (IL-4) gene with systemic IL-4 level and the S503P SNP in the IL-4R gene with the incidence of glioma. Blood samples were collected from 100 histologically confirmed adult patients with glioma, and 30 apparently healthy individuals from the same area. DNA was extracted from each blood sample, and the IL-4 and IL-4R genes were amplified using polymerase chain reaction (PCR) with gene-specific primers. Systemic IL-4 concentration was assessed in serum samples from each participant by enzyme-linked immunosorbent assay (ELISA). We observed a negative association between the homozygous genotype (CC) of the SNP C-33T of the IL-4 gene with the incidence of glioma (OR=0.19, 95% CI=0.035-1.02), while the T allele of the SNP demonstrated a significant protective association against glioma. Similarly, the heterozygous (CT) and homozygous mutant (CC) of the SNP S503P of the IL-4R gene demonstrated a significant association with glioma development (OR=0.405, 95% CI=0.17-0.969 and OR=0.147, 95% CI=0.036-0.6 respectively), while the C allele exhibited a highly significant association with protection from glioma formation. These findings suggest that the T allele of the SNP C-33T in the IL-4 gene and the C allele of the SNP S503P in IL-4R may have a protective role against glioma development.

Evidence for miR-181 involvement in neuroinflammatory responses of astrocytes.

Inflammation is a common component of acute injuries of the central nervous system (CNS) such as ischemia, and degenerative disorders such as Alzheimer's disease. Glial cells play important roles in local CNS inflammation, and an understanding of the roles for microRNAs in glial reactivity in injury and disease settings may therefore lead to the development of novel therapeutic interventions. Here, we show that the miR-181 family is developmentally regulated and present in high amounts in astrocytes compared to neurons. Overexpression of miR-181c in cultured astrocytes results in increased cell death when exposed to lipopolysaccharide (LPS). We show that miR-181 expression is altered by exposure to LPS, a model of inflammation, in both wild-type and transgenic mice lacking both receptors for the inflammatory cytokine TNF-α. Knockdown of miR-181 enhanced LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8) and HMGB1, while overexpression of miR-181 resulted in a significant increase in the expression of the anti-inflammatory cytokine IL-10. To assess the effects of miR-181 on the astrocyte transcriptome, we performed gene array and pathway analysis on astrocytes with reduced levels of miR-181b/c. To examine the pool of potential miR-181 targets, we employed a biotin pull-down of miR-181c and gene array analysis. We validated the mRNAs encoding MeCP2 and X-linked inhibitor of apoptosis as targets of miR-181. These findings suggest that miR-181 plays important roles in the molecular responses of astrocytes in inflammatory settings. Further understanding of the role of miR-181 in inflammatory events and CNS injury could lead to novel approaches for the treatment of CNS disorders with an inflammatory component.

Differential G protein subunit expression by prostate cancer cells and their interaction with CXCR5.

BACKGROUND: Prostate cancer (PCa) cell lines and tissues differentially express CXCR5, which positively correlate with PCa progression, and mediate PCa cell migration and invasion following interaction with CXCL13. However, the differential expression of G protein α, ß, and γ subunits by PCa cell lines and the precise combination of these proteins with CXCR5 has not been elucidated. METHODS: We examined differences in G protein expression of normal prostate (RWPE-1) and PCa cell lines (LNCaP, C4-2B, and PC3) by western blot analysis. Further, we immunoprecipitated CXCR5 with different G protein subunits, and CXCR4, following CXCL13 stimulation. To investigate constitutive coupling of CXCR5 with CXCR4 and PAR-1 we performed invasion assay in PCa cells transfected with Gαq/i2 or Gα13 siRNA, following CXCL13 treatment. We also investigated Rac and RhoA activity by G-LISA activation assay in PCa cells following CXCL13/thrombin stimulation. RESULT: Of the 22 G proteins studied, Gαi1-3, Gß1-4, Gγ5, Gγ7, and Gγ10 were expressed by both normal and PCa cell lines. Gαs was moderately expressed in C4-2B and PC3 cell lines, Gαq/11 was only present in RWPE-1 and LNCaP cell lines, while Gα12 and Gα13 were expressed in C4-2B and PC3 cell lines. Gγ9 was expressed only in PCa cell lines. Gα16, Gß5, Gγ1-4, and Gγ13 were not detected in any of the cell lines studied. Surprisingly, CXCR4 co-immunoprecipitated with CXCR5 in PCa cell lines irrespective of CXCL13 treatment. We also identified specific G protein isoforms coupled to CXCR5 in its resting and active states. Gαq/11/Gß3/Gγ9 in LNCaP and Gαi2/Gß3/Gγ9 in C4-2B and PC3 cell lines, were coupled to CXCR5 and disassociated following CXCL13 stimulation. Interestingly, Gα13 co-immunoprecipitated with CXCR5 in CXCL13-treated, but not in untreated PCa cell lines. Inhibition of Gαq/i2 significantly decreased the ability of cells to invade, whereas silencing Gα13 did not affect CXCL13-dependent cell invasion. Finally, CXCL13 treatment significantly increased Rac activity in Gαq/i2 dependent manner, but not RhoA activity, in PCa cell lines. CONCLUSIONS: These findings offer insight into molecular mechanisms of PCa progression and can help to design some therapeutic strategies involving CXCR5 and/or CXCL13 blockade and specific G protein inhibition to abrogate PCa metastasis.

Personality traits and leptin.

OBJECTIVE: Personality traits related to high neuroticism and low conscientiousness are consistently associated with obesity. Hormones implicated in appetite and metabolism, such as leptin, may also be related to personality and may contribute to the association between these traits and obesity. The present research examined the association between leptin and Five Factor Model personality traits. METHODS: A total of 5214 participants (58% women; mean [standard deviation] age = 44.42 [15.93] years; range, 18-94 years) from the SardiNIA project completed the Revised NEO Personality Inventory, a comprehensive measure of personality traits, and their blood samples were assayed for leptin. RESULTS: As expected, lower conscientiousness was associated with higher circulating levels of leptin (r = -0.05, p < .001), even after controlling for body mass index, waist circumference, or inflammatory markers (r = -0.05, p < .001). Neuroticism, in contrast, was unrelated to leptin (r = 0.01, p = .31). CONCLUSIONS: Individuals who are impulsive and lack discipline (low conscientiousness) may develop leptin resistance, which could be one factor that contributes to obesity, whereas the relation between a proneness to anxiety and depression (high neuroticism) and obesity may be mediated through other physiological and/or behavioral pathways.

Distinctive immunoregulatory effects of adenosine on T cells of older humans.

A role for adenosine in immunosenescence was investigated in T cells from older (≥65 yr) and younger (24-45 yr) healthy humans. Adenosine concentrations in cultures of activated T cells were significantly higher (P<0.0001) for older (145±47 nM, mean±sd) than younger (58±5.5 nM) subjects. Expression of the activation coreceptor CD28 was suppressed significantly by 0.1 to 1 µM exogenous adenosine, with greater effects of 1 µM (P<0.01) on T cells of younger (mean suppression of 67 and 65% for CD4 and CD8 T cells, respectively) than older (means of 42 and 46%) subjects. T-cell chemotaxis to CCL21 was suppressed significantly by 0.3 and 1 µM exogenous adenosine, with mean maximum decreases of 39 and 49%, respectively, for younger subjects and 28 and 31% for older subjects. Generation of IL-2 and IFN-γ by T cells of younger and older subjects was suppressed substantially only at adenosine levels of 3 µM or higher. Lower baseline expression of CD28 and chemotaxis to CCL21 and S1P for T cells from older subjects attributable to endogenous adenosine were reversed completely by two different A(2A) adenosine receptor antagonists without affecting T cells of younger subjects. Adenosine is an endogenous T-cell immunosuppressor in older humans, and A(2A) antagonists reverse adenosine-induced T-cell deficiencies of aging.

Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis.

In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the "quiescent" and "proliferative" niches in which hematopoietic stem cells and progenitors reside.

Role of resveratrol-induced CD11b(+) Gr-1(+) myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3(+) T cells and amelioration of chronic colitis in IL-10(-/-) mice.

Resveratrol, a naturally occurring polyphenol has received significant attention as a potent anti-inflammatory agent. Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce proinflammatory cytokines. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population characterized by the co-expression of CD11b(+) and Gr-1(+) and have long been known for their immunosuppressive function. We report that resveratrol effectively attenuated overall clinical scores as well as various pathological markers of colitis in IL-10(-/-) mice by down regulating Th1 responses. Resveratrol lessened the colitis-associated decrease in body weight and increased levels of serum amyloid A (SAA), CXCL10 and colon TNF-α, IL-6, RANTES, IL-12 and IL-1ß concentrations. After resveratrol treatment, the percentage of CXCR3 expressing T cells was decreased in the spleen, mesenteric lymph nodes (MLN), and intestinal lamina propria (LP). However, the percentage and absolute numbers of CD11b(+) and Gr-1(+)cells in the lamina propria (LP) and spleen were increased after resveratrol treatment as compared with the vehicle treatment. Co-culture of resveratrol-induced CD11b(+) Gr-1(+) cells with T cells, attenuated T cell proliferation, and most importantly reduced IFN-γ and GM-CSF production by LP derived T cells from vehicle treated IL-10(-/-) mice with chronic colitis. The current study suggests that administration of resveratrol into IL-10(-/-) mice induces immunosuppressive CD11b(+) Gr-1(+) MDSCs in the colon, which correlates with reversal of established chronic colitis, and down regulation of mucosal and systemic CXCR3(+) expressing effector T cells as well as inflammatory cytokines in the colon. The induction of immunosuppressive CD11b(+) Gr-1(+) cells by resveratrol during colitis is unique, and suggests an as-yet-unidentified mode of anti-inflammatory action of this plant polyphenol.

The GHS-R blocker D-[Lys3] GHRP-6 serves as CCR5 chemokine receptor antagonist.

[D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. This antagonist is one of the most common antagonists utilized in vivo to block GHS-R function and activity. Here, we found that DLS also has the ability to modestly block chemokine function and ligand binding to the chemokine receptor CCR5. The DLS effects on RANTES binding and Erk signaling as well as calcium mobilization appears to be much stronger than its effects on MIP-1α and MIP-1ß. CCR5 have been shown to act as major co-receptor for HIV-1 entry into the CD4 positive host cells. To this end, we also found that DLS blocks M-tropic HIV-1 propagation in activated human PBMCs. These data demonstrate that DLS may not be a highly selective GHS-R1a inhibitor and may also effects on other G-protein coupled receptor (GPCR) family members. Moreover, DLS may have some potential clinical applications in blocking HIV infectivity and CCR5-mediated migration and function in various inflammatory disease states.

Dexamethasone augments CXCR4-mediated signaling in resting human T cells via the activation of the Src kinase Lck.

Dexamethasone (DM) is a synthetic member of the glucocorticoid (GC) class of hormones that possesses anti-inflammatory and immunosuppressant activity and is commonly used to treat chronic inflammatory disorders, severe allergies, and other disease states. Although GCs are known to mediate well-defined transcriptional effects via GC receptors (GCR), there is increasing evidence that GCs also initiate rapid nongenomic signaling events in a variety of cell types. Here, we report that DM induces the phosphorylation of Lck and the activation of other downstream mediators, including p59Fyn, Zap70, Rac1, and Vav in resting but not activated human T cells. DM treatment also augments CXCL12-mediated signaling in resting T cells through its cell surface receptor, CXCR4 resulting in the enhanced actin polymerization, Rac activation, and cell migration on ligand exposure. Lck was found to be a critical intermediate in these DM-induced signaling activities. Moreover, DM-mediated Lck phosphorylation in T cells was dependent on the presence of both the GCR and the CD45 molecule. Overall, these results elucidate additional nongenomic effects of DM and the GCR on resting human T cells, inducing Lck and downstream kinase activation and augmenting chemokine signaling and function.

Reduction of T cell-derived ghrelin enhances proinflammatory cytokine expression: implications for age-associated increases in inflammation.

Ghrelin (Grln) is a peptide hormone that is predominantly produced in the stomach and stimulates appetite and induces growth hormone (GH) release. We have previously reported that ghrelin is also expressed in T cells and exerts prothymic and anti-inflammatory effects. However, the biologic relevance of T cell-derived ghrelin remains to be determined. Here, we report that acylated-bioactive ghrelin is expressed in human T cells and preferentially segregates within the lipid raft domains upon TCR ligation. The RNA interference (RNAi)-mediated down-regulation of ghrelin in primary human T cells activates IkB, and increases Th1 cytokines and IL-17 secretion. Ghrelin expression declines with increasing age in spleen and T cells and exogenous ghrelin administration in old mice reduces proinflammatory cytokines. These findings demonstrate that ghrelin functions in an autocrine and paracrine capacity to regulate proinflammatory cytokine expression in human and murine T cells and may contribute in regulating "inflamm-aging."

Activation of Wnt5A signaling is required for CXC chemokine ligand 12-mediated T-cell migration.

Chemokines mediate the signaling and migration of T cells, but little is known about the transcriptional events involved therein. Microarray analysis of CXC chemokine ligand (CXCL) 12-treated T cells revealed that Wnt ligands are significantly up-regulated during CXCL12 treatment. Real-time polymerase chain reaction and Western blot analysis confirmed that the expression of noncanonical Wnt pathway members (eg, Wnt5A) was specifically up-regulated during CXCL12 stimulation, whereas beta-catenin and canonical Wnt family members were selectively down-regulated. Wnt5A augmented signaling through the CXCL12-CXCR4 axis via the activation of protein kinase C. Moreover, Wnt5A expression was required for CXCL12-mediated T-cell migration, and rWnt5A sensitized human T cells to CXCL12-induced migration. Furthermore, Wnt5A expression was also required for the sustained expression of CXCR4. These results were further supported in vivo using EL4 thymoma metastasis as a model of T-cell migration. Together, these data demonstrate that Wnt5A is a critical mediator of CXCL12-CXCR4 signaling and migration in human and murine T cells.

Human CD4- 8- T cells are a distinctive immunoregulatory subset.

Human CD4(-)8(-) T cells are a minor subset quantitatively but potentially important in immunity because they are predominantly distributed at body surfaces, and their number and activities increase in autoimmune diseases and decrease with aging. Distinguishing characteristics of CD4(-)8(-) T cells are found to include a unique profile of cytokines, including Serpin E1, which is not generated by other T cells, MIF, and TGF-beta. At 2-5% of the total in mixtures with CD4 + CD8 T cells, CD4(-)8(-) T cells enhance the generation of IFN-gamma and IL-17 by up to 12- and 5-fold, respectively, without contributing either cytokine or affecting cytokine production by NK/NKT cells. CD4(-)8(-) T cell-derived MIF is their major enhancer and TGFbeta their principal inhibitor of CD4 and CD8 T cell cytokine production. Decreases in CD4(-)8(-) T cell effects may diminish protective immunity in aging, whereas increases may augment the severity of autoimmune diseases.

Gender specificity of altered human immune cytokine profiles in aging.

Cytokine generation by T cells and monocytes was determined for 50 subjects aged 65 yr or older and concurrently studied young subjects individually matched to each old subject for sex, race, and national origin. Highly significant differences between cytokine levels of old and young subjects all were gender specific. For T cells stimulated with anti-CD3 plus anti-CD28 antibodies, mean ratios of IFN-gamma generation for healthy old to young subjects were 0.22 for men (P<0.001; n=15) and 3.35 for women (P<0.001; n=13), and those of IL-17 were 0.30 for men (P<0.001) and no difference for women. CD8 T cells were the source of high IFN-gamma in healthy old women. For old men with an inflammatory or immune disease (n=10), mean old to young ratios of T-cell-generated IFN-gamma and IL-17 increased with disease severity up to 5.78 and 2.97 (both P<0.01), respectively, without changes for old women with similar diseases (n=12). For differentiated LPS-stimulated monocytes, old to young ratios of TNF-alpha and IL-6 generation were high only in women with immune or inflammatory disease (2.38, P<0.05 and 1.62, P<0.01, respectively), whereas ratios of IFN-gamma-evoked IP-10 chemokine were low in all groups. Alterations in immune cytokine profiles with aging show significant gender specificity.

Heparan sulfate proteoglycan modulation of Wnt5A signal transduction in metastatic melanoma cells.

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.