The role of inflammation and matrix proteins in airway remodelling

Asthma is a chronic inflammatory disorder of the airways. Remodelling in asthma is defined as the structural changes seen in the airways of asthmatics in comparison to healthy controls. Progressive loss of lung function also seen in asthma might be caused by remodelling. The research aims of this thesis were to investigate inflammation and remodelling in the airways of different types of asthmatics and smokers. The association between inflammation and remodelling was also examined in a mouse model of allergic airway inflammation. Healthy smokers showed increased numbers of macrophages in the BAL with no changes in the inflammatory cells in biopsies. Macrophages seemed to be quite quiescent, since mRNA expression for a wide variety of inflammatory mediators, especially chemokines CCL3, CCL4, CCL5 and CCL20, secreted by macrophages was significantly lower than in healthy non-smokers. Attenuated macrophage activity in the airway lumen may render smokers more susceptible to airway infections and have an impact on the development of other airway pathology. Patients with diisocyanate-induced asthma (DIA) on inhaled corticosteroids (ICS) who still had non-specific bronchial hyperreactivity (NSBHR) at the end of the follow-up showed increased expression of TNF-α, IL-6 and IL-15 mRNA in BAL cells compared to those without NSBHR. In addition to being markers for poor prognosis and possible slight glucocorticoid resistance, these cytokines might aid in guiding the treatment of DIA. The increase in the thickness of tenascin-C layer in the bronchial basement membrane (BM) was much less than usually seen in other types of asthma, which might not make tenascin-C a good marker for DIA. OVA-induced tenascin-C expression in the lung was attenuated in STAT4-/- mice with impaired Th1-type immunity compared to WT mice. Interestingly, STAT6-/- mice with impaired Th2-type immunity showed tenascin-C expression levels similar to those of WT mice. The clearest difference between these two knockout strains in response to OVA was that STAT4-/- mice exhibited no upregulation of IFN-γ and TNF-α mRNA expression. Thus, tenascin-C expression was unexpectedly more related to Th1 type reactions. In vitro studies confirmed the results. Human fibroblasts stimulated by TNF-α and IFN-γ showed increased expression of tenascin-C. Patients with newly diagnosed asthma showed increased expression of laminin α2 in the bronchial BM in comparison to patients with asthma symptoms only and healthy controls. Both patients with asthma and those with only asthma symptoms showed increased expression of the laminin β2 chain in comparison to controls. Thus, laminin α2 expression differentiated patients with clinical asthma from patients with symptoms only. Furthermore, the expression of laminin α2 and β2 was associated with NSBHR, linking very specific remodelling events to clinical findings.

Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4(+) T cell activation

The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H2O2 . In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca2+ concentrations Ca2+](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNF alpha and IFN gamma by CD4+ T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca2+ ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling. (C) 2010 Elsevier B.V. All rights reserved.

NK-and T-cell function in chronic myeloid leukemia patients treated with interferon-? monotherapy

Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.

Intracellular Pathogen Sensor NOD2 Programs Macrophages to Trigger Notch1 Activation

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-alpha or IFN-gamma-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.

Inhibition of hepatitis C virus replication by herbal extract: Phyllanthus amarus as potent natural source

Hepatitis C virus infection is a major health problem worldwide. Developing effective antiviral therapy for HCV is the need of the hour. The viral enzymes NS3 protease and NS5B RNA dependent RNA polymerase are essential enzymes for polyprotein processing and viral RNA replication and thus can be potential targets for screening anti-HCV compounds. A large number of phytochemicals are present in plants, which are found to be promising antiviral agents. In this study, we have screened inhibitory effect of different plant extracts against the NS3 and NS5B enzymes of hepatitis C virus. Methanolic extracts were prepared from various plant materials and their inhibitory effects on the viral enzymes were determined by in vitro enzyme assays. Effect on viral RNA replication was investigated by using TaqMan Real time RT-PCR. Interestingly, Phyllanthus amarus root (PAR) extract showed significant inhibition of HCV-NS3 protease enzyme; whereas P. amarus leaf (PAL) extract showed considerable inhibition of NS5B in the in vitro assays. Further, the PAR and PAL extracts significantly inhibited replication of HCV monocistronic replicon RNA and HCV H77S viral RNA in HCV cell culture system. However, both PAR and PAL extracts did not show cytotoxicity in Huh7 cells in the MTT assay. Furthermore, addition of PAR together with IFN-alpha showed additive effect in the inhibition of HCV RNA replication. Results suggest the possible molecular basis of the inhibitory activity of PA extract against HCV which would help in optimization and subsequent development of specific antiviral agent using P. amarus as potent natural source. (C) 2011 Elsevier B.V. All rights reserved.

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFN gamma and TNF alpha levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

ATP release and autocrine signaling through P2X4 receptors regulate gamma delta T cell activation

Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional alpha beta T cells release ATP upon TCR cross-linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the ``unconventional'' gamma delta T lymphocytes. We observed that gamma delta T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or IPP. Pretreatment of gamma delta T cells with (10)panx-1, CBX, or Bf A reversed the stimulation-induced increase in extracellular ATP concentration, indicating that panx-1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with (10)panx-1 inhibited Ca2+ signaling in response to TCR stimulation. qPCR revealed that gamma delta T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP-ATP inhibited transcriptional up-regulation of TNF-alpha and IFN-gamma in gamma delta T cells stimulated with anti-CD3/CD28-coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human gamma delta T cells. J. Leukoc. Biol. 92: 787-794; 2012.

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/ mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2(-/-) mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnf alpha and Ifn gamma in sera are not significantly affected, Nos2(-/-) mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.

Curcumin reduces the antimicrobial activity of ciprofloxacin against Salmonella Typhimurium and Salmonella Typhi

Typhoidal and non-typhoidal infection by Salmonella is a serious threat to human health. Ciprofloxacin is the last drug of choice to clear the infection. Ciprofloxacin, a gyrase inhibitor, kills bacteria by inducing chromosome fragmentation, SOS response and reactive oxygen species (ROS) in the bacterial cell. Curcumin, an active ingredient from turmeric, is a major dietary molecule among Asians and possesses medicinal properties. Our research aimed at investigating whether curcumin modulates the action of ciprofloxacin. We investigated the role of curcumin in interfering with the antibacterial action of ciprofloxacin in vitro and in vivo. RTPCR, DNA fragmentation and confocal microscopy were used to investigate the modulation of ciprofloxacin-induced SOS response, DNA damage and subsequent filamentation by curcumin. Chemiluminescence and nitroblue tetrazolium reduction assays were performed to assess the interference of curcumin with ciprofloxacin-induced ROS. DNA binding and cleavage assays were done to understand the rescue of ciprofloxacin-mediated gyrase inhibition by curcumin. Curcumin interferes with the action of ciprofloxacin thereby increasing the proliferation of Salmonella Typhi and Salmonella Typhimurium in macrophages. In a murine model of typhoid fever, mice fed with curcumin had an increased bacterial burden in the reticuloendothelial system and succumbed to death faster. This was brought about by the inhibition of ciprofloxacin-mediated downstream signalling by curcumin. The antioxidant property of curcumin is crucial in protecting Salmonella against the oxidative burst induced by ciprofloxacin or interferon (IFN), a pro-inflammatory cytokine. However, curcumin is unable to rescue ciprofloxacin-induced gyrase inhibition. Curcumins ability to hinder the bactericidal action of ciprofloxacin and IFN might significantly augment Salmonella pathogenesis.

Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells Evidence of Anergy

Objective: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a(+)CD207(+) cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1 beta, IL-6, IL-10, IL-12p70, interferon (IFN) alpha, interferon gamma, and tumor necrosis factor (TNF) alpha by Luminex multiplex bead array. Human papillomavirus was genotyped. Results: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs. expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1 beta, and tumor necrosis factor alpha to TLR8 ligand and interferon alpha in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Interferon-gamma- and glucocorticoid-mediated pathways synergize to enhance death of CD4(+) CD8(+) thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S.typhimurium). Significant death of CD4+CD8+ thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4+CD8+ thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon- (IFN-), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifn/ mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN- and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifn/ mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifn/ mice. Importantly, the number of CD4+CD8+ thymocytes is significantly higher in Ifn/ mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN--mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S.typhimurium infection. The implications of this study for host responses during infection are discussed.

Regulation of Chemokines, CCL3 and CCL4, by interferon gamma and Nitric oxide synthase 2 in mouse macrophages and during Salmonella enterica Serovar Typhimurium infection

Background. Interferon gamma (IFN-gamma) increases the expression of multiple genes and responses; however, the mechanisms by which IFN-gamma downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-gamma and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella typhimurium infection of mice was investigated. Methods. Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2(-/-)mice were used to identify the contribution of signaling molecules during IFN-gamma-mediated in vitro regulation of CCL3, CCL4, and CXCL10. In addition, infection of bone marrow-derived macrophages (BMDMs) and mice (C57BL/6, Ifn-gamma(-/), and Nos2(-/-)) with S. typhimurium were used to gain an understanding of the in vivo regulation of these chemokines. Results. IFN-gamma repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)-NOS2-p38 mitogen activated protein kinase (p38MAPK)-activating transcription factor 3 (ATF3) dependent pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-gamma and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-gamma and NOS2 enhanced serum amounts of tumor necrosis factor alpha and CXCL10 but repressed CCL3 and CCL4. Conclusions. This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection.

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2 A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1 alpha, IL-1 beta, G-CSF, GM-CSF, MIP-1 alpha, IFN-gamma, IL-17, and TNF-alpha. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality (C) 2013 Elsevier Inc. All rights reserved.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress Major histocompatibility complex II expression during Mycobacterium bovis Bacillus Calmette-Guerin infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guerin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-gamma-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.