Suggestive linkage of the parathyroid receptor type 1 to osteoporosis

We have investigated the role of 23 candidate genes in the control of bone mineral density (BMD) by linkage studies in families of probands with osteoporosis (lumbar spine [LS] or femoral neck [FN] BMD T score < -2.5) and low BMD relative to an age- and gender-matched cohort (Z score < -2.0). One hundred and fifteen probands (35 male, 80 female) and 499 of their first- or second-degree relatives (223 males and 276 females) were recruited for the study. BMD was measured at the LS and FN using dual-energy X-ray absorptiometry and expressed as age- and gender-matched Z scores corrected for body mass index. The candidate genes studied were the androgen receptor, type I collagen A1 (COLIA1), COLIA2, COLIIA1, vitamin D receptor (VDR), colony-stimulating factor 1, calcium-sensing receptor, epidermal growth factor (EGF), estrogen receptor 1 (ESR1), fibrillin type 1, insulin-like growth factor 1, interleukin-1 alpha (IL-1α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), osteopontin, parathyroid hormone (PTH), PTH-related peptide, PTH receptor type 1 (PTHR1), transforming growth factor-beta 1, and tumor necrosis factors alpha and beta. Sixty-four microsatellites lying close to or within these genes were investigated for linkage with BMD. Using the program MapMaker/Sibs there was suggestive evidence of linkage between BMD and PTHR1 (maximum LOD score obtained [MLS] 2.7-3.5). Moderate evidence of linkage was also observed with EGF (MLS 1.8), COLIA1 (MLS 1.7), COLIIA1/VDR (MLS 1.7), ESR1 (MLS 1.4), IL-1α (MLS 1.4), IL-4 (MLS 1.2), and IL-6 (MLS 1.2). Variance components analysis using the program ACT, correcting for proband-wise ascertainment, also showed evidence of linkage (p ≤0.05) at markers close to or within the candidate genes IL- 1α, PTHR1, IL-6, and COLIIA1/VDR. Further studies will be required to confirm these findings, to refine the location of gene responsible for the observed linkage, and to screen the candidate genes targeted at these loci for mutations.

Uropathogenic Escherichia Coli engages CD14-dependent signaling to enable bladder-macrophage-dependent control of acute urinary tract infection

Background CD14, a coreceptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to gram-negative bacteria. Despite the central role of CD14 in the inflammatory response to lipopolysaccharide and other microbial products and in the dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown. Methods We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14−/− mice and RNA sequencing to define the CD14-dependent transcriptional signature and the role of CD14 in host defense against UTI in the bladder. Results UPEC induced the upregulation of Cd14 and the monocyte/macrophage-related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT mice, compared with Cd14−/− mice. Exacerbation of infection in Cd14−/− mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and interleukin 17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens. Conclusions This study identifies new host protective and signaling roles for CD14 in the bladder during UPEC UTI.

Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to Uropathogenic Escherichia coli

Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

Infection-induced IL-10 and Jak-Stat: A review of the molecular circuitry controlling immune hyperactivity in response to pathogenic microbes

Generation of effective immune responses against pathogenic microbes depends on a fine balance between pro- and anti-inflammatory responses. Interleukin-10 (IL-10) is essential in regulating this balance and has garnered renewed interest recently as a modulator of the response to infection at the JAK-STAT signaling axis of host responses. Here, we examine how IL-10 functions as the “master regulator” of immune responses through JAK-STAT, and provide a perspective from recent insights on bacterial, protozoan, and viral infection model systems. Pattern recognition and subsequent molecular events that drive activation of IL-10-associated JAK-STAT circuitry are reviewed and the implications for microbial pathogenesis are discussed.

The level of FoxO1 and IL-15 in skeletal muscle, serum and synovial fluid in people with knee osteoarthritis: a case control study

Objectives Impaired muscle function is common in knee osteoarthritis (OA). Numerous biochemical molecules have been implicated in the development of OA; however, these have only been identified in the joint and serum. This study compared the expression of interleukin (IL-15) and Forkhead box protein-O1 (FoxO1) in muscle of patients with knee OA asymptomatic individuals, and examined whether IL-15 was also present in the joint and serum. Method Muscle and blood samples were collected from 19 patients with diagnosed knee OA and 10 age-matched asymptomatic individuals. Synovial fluid and muscle biopsies were collected from the OA group during knee replacement surgery. IL-15 and FoxO1were measured in the skeletal muscle. IL-15 abundance was also analysed in the serum of both groups and synovial fluid from the OA group. Knee extensor strength was measured and correlated with IL-15 and FoxO1 in the muscle. Results FoxO1 protein expression was higher (p=0.04), whereas IL-15 expression was lower (p=0.02) in the muscle of the OA group. Strength was also lower in the OA group, and was inversely correlated with FoxO1 expression. No correlation was found between IL-15 in the joint, muscle or serum. Conclusion Skeletal muscle, particularly the quadriceps, is affected in people with knee OA where elevated FoxO1 protein expression was associated with reduced muscle strength. While IL-15 protein expression in the muscle was lower in the knee OA group, no correlation was found between the expression of IL-15 protein in the muscle, joint and serum, which suggests that inflammation is regulated differently within these tissues.

Proinflammatory cytokines regulate cementogenic differentiation of periodontal ligament cells by Wnt/Ca2+ signaling pathway

Periodontal inflammation can inhibit cell differentiation of periodontal ligament cells (PDLCs), resulting in decreased bone/cementum regeneration ability. The Wnt signaling pathway, including canonical Wnt/β-catenin signaling and noncanonical Wnt/Ca2+ signaling, plays essential roles in cell proliferation and differentiation during tooth development. However, little is still known whether noncanonical Wnt/Ca2+ signaling cascade could regulate cementogenic/osteogenic differentiation capability of PDLCs within an inflammatory environment. Therefore, in this study, human PDLCs (hPDLCs) and their cementogenic differentiation potential were investigated in the presence of cytokines. The data demonstrated that both cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) inhibited cell proliferation, relative alkaline phosphatase activity, bone/cementum-related gene/protein expression, and canonical Wnt pathway-related gene/protein expression in hPDLCs. Interestingly, both cytokines upregulated the noncanonical Wnt/Ca2+ signaling-related gene and protein expression in hPDLCs. When the Wnt/Ca2+ pathway was blocked by Ca2+/calmodulin-dependent protein kinase II inhibitor KN93, even in the presence of IL-6 and TNF-α, cementogenesis could be stimulated in hPDLCs. Our data indicate that the Wnt/Ca2+ pathway plays an inhibitory role on PDLC cementogenic differentiation in inflammatory microenvironments. Therefore, targeting the Wnt/Ca2+ pathway may provide a novel therapeutic approach to improve periodontal regeneration for periodontal diseases.

Molecular dynamics simulations of CXCL-8 and its interactions with a receptor peptide, heparin fragments, and sulfated linked cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 310-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment−CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8−receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

A combination of local inflammation and central memory T cells potentiates immunotherapy in the skin

Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro - derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8+ T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation. Copyright © 2012 by The American Association of Immunologists, Inc.

Distinct subpopulations of gamma delta T cells are present in normal and tumor-bearing human liver

Gamma delta T cells are thought to mediate immune responses at epithelial surfaces. We have quantified and characterized hepatic and peripheral blood gamma delta T cells from 11 normal and 13 unresolved tumor-bearing human liver specimens. gamma delta T cells are enriched in normal liver (6.6% of T cells) relative to matched blood (0.9%; P = 0.008). The majority express CD4(-)CD8(-) phenotypes and many express CD56 and/or CD161. In vitro, hepatic gamma delta T cells can be induced to kill tumor cell lines and release interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-4. Analysis of V gamma and V delta chain usage indicated that V delta 3(+) cells are expanded in normal livers (21.2% of gamma delta T cells) compared to blood (0.5%; P = 0.001). Tumor-bearing livers had significant expansions and depletions of gamma delta T cell subsets but normal cytolytic activity. This study identifies novel populations of liver T cells that may play a role in immunity against tumors.

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells

A major group of murine NK T (NKT) cells express an invariant Vα14Jα18 TCR α-chain specific for glycolipid Ags presented by CD1d. Murine Vα14Jα18+ account for 30–50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Vα24Vβ11+ NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3+ cells) and blood (0.02%). In contrast to those in blood, most hepatic Vα24+ NKT cells express the Vβ11 chain. They include CD4+, CD8+, and CD4−CD8− cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Vα24+ T cells are potent producers of IFN-γ and TNF-α, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, α-galactosylceramide. Vα24+Vβ11+ cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-γ in response to α-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.

Disease-associated polymorphisms in ERAP1 do not alter endoplasmic reticulum stress in patients with ankylosing spondylitis

The mechanism by which human leukocyte antigen B27 (HLA-B27) contributes to ankylosing spondylitis (AS) remains unclear. Genetic studies demonstrate that association with and interaction between polymorphisms of endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 influence the risk of AS. It has been hypothesised that ERAP1-mediated HLA-B27 misfolding increases endoplasmic reticulum (ER) stress, driving an interleukin (IL) 23-dependent, pro-inflammatory immune response. We tested the hypothesis that AS-risk ERAP1 variants increase ER-stress and concomitant pro-inflammatory cytokine production in HLA-B27 + but not HLA-B27-AS patients or controls. Forty-nine AS cases and 22 healthy controls were grouped according to HLA-B27 status and AS-associated ERAP1 rs30187 genotypes: HLA-B27 + ERAP1 risk, HLA-B27 + ERAP1 protective, HLA-B27-ERAP1 risk and HLA-B27-ERAP1 protective. Expression levels of ER-stress markers GRP78 (8 kDa glucose-regulated protein), CHOP (C/EBP-homologous protein) and inflammatory cytokines were determined in peripheral blood mononuclear cell and ileal biopsies. We found no differences in ER-stress gene expression between HLA-B27 + and HLA-B27-cases or healthy controls, or between cases or controls stratified by carriage of ERAP1 risk or protective alleles in the presence or absence of HLA-B27. No differences were observed between expression of IL17A or TNF (tumour necrosis factor) in HLA-B27 + ERAP1 risk, HLA-B27 + ERAP1 protective and HLA-B27-ERAP1 protective cases. These data demonstrate that aberrant ERAP1 activity and HLA-B27 carriage does not alter ER-stress levels in AS, suggesting that ERAP1 and HLA-B27 may influence disease susceptibility through other mechanisms. © 2015 Macmillan Publishers Limited.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers

Introduction Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. Methods Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. Results There was no significant difference in HLAclass I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. Discussion Large differences were not seen in HLAB27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms.

The role of 25-hydroxyvitamin D deficiency in promoting insulin resistance and inflammation in patients with chronic kidney disease: A randomised controlled trial

BACKGROUND Approximately 50% of patients with stage 3 Chronic Kidney Disease are 25-hydroxyvitamin D insufficient, and this prevalence increases with falling glomerular filtration rate. Vitamin D is now recognised as having pleiotropic roles beyond bone and mineral homeostasis, with the vitamin D receptor and metabolising machinery identified in multiple tissues. Worryingly, recent observational data has highlighted an association between hypovitaminosis D and increased cardiovascular mortality, possibly mediated via vitamin D effects on insulin resistance and inflammation. The main hypothesis of this study is that oral Vitamin D supplementation will ameliorate insulin resistance in patients with Chronic Kidney Disease stage 3 when compared to placebo. Secondary hypotheses will test whether this is associated with decreased inflammation and bone/adipocyte-endocrine dysregulation. METHODS/DESIGN This study is a single-centre, double-blinded, randomised, placebo-controlled trial. Inclusion criteria include; estimated glomerular filtration rate 30-59 ml/min/1.73 m(2); aged >or=18 on entry to study; and serum 25-hydroxyvitamin D levels <75 nmol/L. Patients will be randomised 1:1 to receive either oral cholecalciferol 2000IU/day or placebo for 6 months. The primary outcome will be an improvement in insulin sensitivity, measured by hyperinsulinaemic euglycaemic clamp. Secondary outcome measures will include serum parathyroid hormone, cytokines (Interleukin-1beta, Interleukin-6, Tumour Necrosis Factor alpha), adiponectin (total and High Molecular Weight), osteocalcin (carboxylated and under-carboxylated), peripheral blood mononuclear cell Nuclear Factor Kappa-B p65 binding activity, brachial artery reactivity, aortic pulse wave velocity and waveform analysis, and indirect calorimetry. All outcome measures will be performed at baseline and end of study. DISCUSSION To date, no randomised controlled trial has been performed in pre-dialysis CKD patients to study the correlation between vitamin D status with supplementation, insulin resistance and markers of adverse cardiovascular risk. We remain hopeful that cholecalciferol may be a safe intervention, with health benefits beyond those related to bone-mineral homeostasis. TRIAL REGISTRATION Australian and New Zealand Clinical Trials Registry ACTRN12609000246280.