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This systematic review aimed to: (a) generate a descriptive synthesis of preclinical studies assessing the therapeutic potential of regulatory T lymphocytes (Tregs) to arrest periodontitis, (b) evaluate the methodological heterogeneity of the reviewed animal studies and (c) assess the risk of bias (RoB) of the included studies. The electronic search for animal studies included the MEDLINE, EMBASE, Web of Science and LILACS databases. In addition, a manual search assessed the high-ranked scientific journals in "periodontics/immunology" and the references listed in the included studies. There were no language, year or publication status restrictions. Two independent reviewers selected and extracted the data, and Cohen's Kappa coefficient was calculated to determine the inter-examiner agreement. The Systematic Review Center for Laboratory Animal Experimentation's (SYRCLE) tool was used to assess the RoB. A total of 21 of the 425 studies obtained from the database search were included. Treg function was mainly described in Porphyromonas gingivalis-induced periodontitis (57.1%) in mice (76.2%), where Treg suppression was strongly related to disease progression and Treg induction was strongly related to immuno-inflammatory response reduction. Of those 21 studies, eight included eight animal experiments using three distinct therapeutic approaches, including: P. gingivalis-driven immunization (n = 3), retinoic acid inoculation (n = 2) and anti-inflammatory molecules in polymeric carriers (n = 3), which could modulate the Treg activity through cytokine production (interleukin-10 and transforming growth factor-beta1), CC-chemokine- and CC-chemokine receptor-mediated chemoattraction (CCL22 and CCR4) or Th17-associated receptor activator of nuclear factor kappaB ligand (RANKL) downregulation. However, the studies with animal experiments did not specify the randomization sequences and housing conditions that were used, and therefore, 42.11% of the entries were rated as unclear RoB. Distinct therapeutic strategies involving Tregs could potentially suppress the immuno-inflammatory response and restore alveolar bone homeostasis during periodontitis. Nevertheless, important methodological variability, poor reporting of treatment effect estimates and unclear RoB suggest using caution when assessing the results of these studies.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

By assessing the changes in concentration of soluble receptor activator of nuclear factor κ B ligand (RANKL) and osteoprotegrin (OPG) after initiation of combination antiretroviral therapy (cART) in treatment-naive HIV-infected patients we aimed to evaluate whether the initial accelerated bone loss could be mediated by increased soluble RANKL (sRANKL) levels associated with CD4+ T cell recovery. We used multiplex immunoassays to determine sRANKL and OPG concentrations in plasma from 48 HIV patients at baseline and 12, 24, 48 and 96 weeks after cART initiation. Soluble RANKL changed significantly over time (overall p = 0.02) with 25% decrease (95% CI: -42 to −5) at week 24 compared to baseline and stabilized at a lower level thereafter. We found no correlation between CD4+ T cell count increment and changes in sRANKL or between percentage change in BMD and changes in sRANKL. In this study there was no indication that the accelerated bone loss after cART initiation was mediated by early changes in sRANKL due to CD4+ T cell recovery. Future studies should focus on the initial weeks after initiation of cART. Clinical-Trial.gov . id NCT00135460 , August 25, 2005. The study was approved by the Danish Data Protection Agency, Danish Medicines Agency and Regional Ethics Committee.

SummaryThe “blood vulnerability”, resulting from the complex balance between serum molecules and inflammatory cell atherosclerotic activities, is a major determinant in the evaluation of the “global patient cardiovascular vulnerability”. In the present study, we focused on the role of the soluble receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL, a potential marker of coronary calcification and vulnerability) in the release of neutrophilic proteases. Then, the association between these mediators and the degree of coronary calcification (assessed by coronary calcium score [CCS]) was investigated in 20 subjects (aged ≥65 years) asymptomatic for cardiovascular disease. Results showed that RANKL dose-dependently induced matrix metalloprotease (MMP)-8 and MMP-9 release from human primary neutrophils cultured in Teflon dishes (suspension condition, mimicking cells circulating in the blood stream). Conversely, when adherent to polystyrene, neutrophils became unresponsive to RANKL. RANKL did not influence the release of other neutrophilic products in suspension and adherence cultures as well as neutrophil migration. RANKL-induced release of MMPs was dependent on the activation of defined intracellular signalling pathways (PI3K/Akt and ERK1/2). In asymptomatic subjects, serum levels of RANKL, MMP-8 and MMP-9 positively correlated with CCS, reflecting a potential relationship between circulating RANKL and coronary calcification. In conclusion, RANKL increased the release of neutrophilic products potentially related to the “blood” vulnerability via defined intracellular pathways. Serum levels of RANKL might represent a potential biomarker of coronary calcification and related cardiovascular risk.

Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH/3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, numerous mRNAs were enriched without a high ARE score. The enrichment of tetrameric and pentameric sequences suggests a broad AUF1 p42-binding spectrum at short U-rich sequences flanked by A or G. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3’UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure., PLoS ONE, 13 (11), ISSN:1932-6203

Objective. To study the effects of low-dose prednisolone on the osteoclast-regulating proteins osteoprotegerin (OPG) and RANK ligand (RANKL) and on markers of bone resorption, 1CTP generated by MMPs and CTX-1 generated by cathepsin K, in patients with early RA in relation to inflammation and joint destruction. Methods. In 225 patients, who at the start of the first DMARD had been randomized to 7.5 mg prednisolone daily for 2 years, the P-group, or no prednisolone, the NoP-group, OPG and RANKL were analysed at 0-24 months and 1CTP and CTX-1 at 0-12 months. Radiographs of hands and feet were assessed at 0, 1 and 2 years using the modified Sharp-van der Heijde score and radiological progression defined as increase in total Sharp score above 5.8. Data were analysed with a mixed linear model and by the GENMOD procedure. Results. In the P-group, RANKL and the ratio OPG/RANKL were stable between baseline and 24 months, whereas in the NoP-group, RANKL increased and the ratio OPG/RANKL decreased. CTX-1 decreased significantly more in the P-group. 1CTP decreased over time in both groups, but more in the P-group, P

The mouse mammary gland develops postnatally under the control of female reproductive hormones. Estrogens and progesterone trigger morphogenesis by poorly understood mechanisms acting on a subset of mammary epithelial cells (MECs) that express their cognate receptors, estrogen receptor α (ERα) and progesterone receptor (PR). Here, we show that in the adult female, progesterone drives proliferation of MECs in two waves. The first, small wave, encompasses PR(+) cells and requires cyclin D1, the second, large wave, comprises mostly PR(−) cells and relies on the tumor necrosis factor (TNF) family member, receptor activator of NF-κB-ligand (RANKL). RANKL elicits proliferation by a paracrine mechanism. Ablation of RANKL in the mammary epithelium blocks progesterone-induced morphogenesis, and ectopic expression of RANKL in MECs completely rescues the PR −/− phenotype. Systemic administration of RANKL triggers proliferation in the absence of PR signaling, and injection of a RANK signaling inhibitor interferes with progesterone-induced proliferation. Thus, progesterone elicits proliferation by a cell-intrinsic and a, more important, paracrine mechanism.

OBJECTIVE: Interleukin-33 (IL-33; or, IL-1F11) was recently identified as the ligand of the IL-1 family receptor T1/ST2. The aim of this study was to examine IL-33 production in human and mouse joints and to investigate the role of IL-33 and T1/ST2 in experimental arthritis. METHODS: IL-33 expression was examined in human synovial tissue, rheumatoid arthritis (RA) synovial fibroblasts, and arthritic mouse joints. Mice with collagen-induced arthritis (CIA) were treated with blocking anti-ST2 antibody or control antibody beginning at the onset of disease. Arthritis severity was assessed by clinical and histologic scoring. Draining lymph node (LN) cell responses were examined ex vivo, and joint messenger RNA (mRNA) was used for expression profiling. RESULTS: IL-33 was highly expressed in human RA synovium. In cultured synovial fibroblasts, IL-33 expression was strongly induced by IL-1beta and/or tumor necrosis factor alpha. Furthermore, IL-33 mRNA was detected in the joints of mice with CIA and increased during the early phase of the disease. Administration of a blocking anti-ST2 antibody at the onset of disease attenuated the severity of CIA and reduced joint destruction. Anti-ST2 antibody treatment was associated with a marked decrease in interferon-gamma production as well as with a more limited reduction in IL-17 production by ex vivo-stimulated draining LN cells. Finally, RANKL mRNA levels in the joint were reduced by anti-ST2 treatment. CONCLUSION: IL-33 is produced locally in inflamed joints, and neutralization of IL-33 signaling has a therapeutic effect on the course of arthritis. These observations suggest that locally produced IL-33 may contribute to the pathogenesis of joint inflammation and destruction.