Genome-wide non-CpG methylation of the host genome during M. tuberculosis infection

A mammalian cell utilizes DNA methylation to modulate gene expression in response to environmental changes during development and differentiation. Aberrant DNA methylation changes as a correlate to diseased states like cancer, neurodegenerative conditions and cardiovascular diseases have been documented. Here we show genome-wide DNA methylation changes in macrophages infected with the pathogen M. tuberculosis. Majority of the affected genomic loci were hypermethylated in M. tuberculosis infected THP1 macrophages. Hotspots of differential DNA methylation were enriched in genes involved in immune response and chromatin reorganization. Importantly, DNA methylation changes were observed predominantly for cytosines present in non-CpG dinucleotide context. This observation was consistent with our previous finding that the mycobacterial DNA methyltransferase, Rv2966c, targets non-CpG dinucleotides in the host DNA during M. tuberculosis infection and reiterates the hypothesis that pathogenic bacteria use non-canonical epigenetic strategies during infection

In vivo switch to IL-10–secreting T regulatory cells in high dose allergen exposure

High dose bee venom exposure in beekeepers by natural bee stings represents a model to understand mechanisms of T cell tolerance to allergens in healthy individuals. Continuous exposure of nonallergic beekeepers to high doses of bee venom antigens induces diminished T cell–related cutaneous late-phase swelling to bee stings in parallel with suppressed allergen-specific T cell proliferation and T helper type 1 (Th1) and Th2 cytokine secretion. After multiple bee stings, venom antigen–specific Th1 and Th2 cells show a switch toward interleukin (IL) 10–secreting type 1 T regulatory (Tr1) cells. T cell regulation continues as long as antigen exposure persists and returns to initial levels within 2 to 3 mo after bee stings. Histamine receptor 2 up-regulated on specific Th2 cells displays a dual effect by directly suppressing allergen-stimulated T cells and increasing IL-10 production. In addition, cytotoxic T lymphocyte–associated antigen 4 and programmed death 1 play roles in allergen-specific T cell suppression. In contrast to its role in mucosal allergen tolerance, transforming growth factor β does not seem to be an essential player in skin-related allergen tolerance. Thus, rapid switch and expansion of IL-10–producing Tr1 cells and the use of multiple suppressive factors represent essential mechanisms in immune tolerance to a high dose of allergens in nonallergic individuals

Cross-presentation Is A Source of Tumor Antigens For Multiple Myeloma Immunotherapy

Cross-presentation is an essential bridge between the innate and adaptive arms of the immune system where antigen presenting cells (APCs) prime cytotoxic T cell responses. We have recently identified cross-presentation as a mechanism by which solid tumors present exogenous antigens. We therefore hypothesized that multiple myeloma would be capable of cross-presentation as these cells are derived from B cells, known APCs. We explored the capacity of multiple myeloma to cross-present PR1, a human leukocyte antigen (HLA)-A2 nonameric peptide that is derived from neutrophil elastase (NE) and proteinase 3 (P3), and the ability to treat multiple myeloma using PR1-targeting immunotherapies. Here we demonstrate that multiple myeloma cells lack endogenous NE and P3 expression, possess the ability to take up exogenous NE and P3 and cross-present PR1. This process employs the cytosolic antigen presentation machinery including the proteasome, Golgi, and TAP. Subsequent PR1 cross-presentation renders multiple myeloma cells susceptible to PR1-CTL and anti-PR1/HLA-A2 antibody, both in vitroand in vivo. To our knowledge, this is the first report of multiple myeloma cross-presenting tumor antigens. Collectively, our data demonstrate that PR1 is a novel tumor antigen in multiple myeloma and can be effectively targeted using PR1-targeting immunotherapies. Our study suggests that the multiple myeloma antigen repertoire is much larger than previously appreciated, and that there is a new catalogue of potential immunotherapeutic targets in multiple myeloma that can be derived from exogenous antigens

Development of a Novel, Peptide-based Vaccine for Lyme Disease

Lyme disease (LD) is the most prevalent arthropod borne illness in the US. Currently, there is no vaccine to prevent infection with LD in humans, rather, prevention of this disease relies on avoiding exposure to the tick vector or treating for LD retroactively. Present research towards a new LD vaccine has focused on the idea of using multimeric, chimeric, and multivalent molecules. The antigens targeted in this approach are highly heterologous between strains and species of Borrelia burgdorferi, and as such, this vaccine may require reformulation of antigens to remain relevant. As such, this dissertation explored the idea of using a novel, highly conserved peptide antigen derived from B. burgdorferi to prevent infection with LD. This approach utilized reverse vaccinology, in silico and in vitro analysis of potential protein candidates, and in vivo vaccination studies using selected proteins and peptides to evaluate the feasibility of a novel peptide vaccine with potential to be broadly protective against LD. Using this methodology, a previously uncharacterized vonWillebrand factor A domain containing protein, BB0173, was characterized and found to localized to the inner membrane with the VWFA domain exposed to the periplasmic space. Further, a novel, highly-conserved peptide antigen of B. burgdorferi (PepB) was identified the extracellularly exposed VWFA domain containing protein BB0172 that demonstrated the ability to generate a protective immune response against B. burgdorferi challenge both using the needle and tick based methods of infection

Metabolic Reprogramming of Cystic Fibrosis Macrophages through the Unfolded Protein Response

Cystic fibrosis (CF) is a life-threatening autosomal genetic disease, which affects approximately 48,204 individuals in Europe and 29,887 in the USA. This condition is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In CF, the mutated CFTR, in the case of DF508, causes accumulation of misfolded proteins leading to endoplasmic reticulum (ER) stress, with activation of the IRE1a-XBP1 pathway. This pathway is essential in the regulation of a subset of genes controlling proinflammatory and metabolic responses in immune cells; nevertheless, the metabolic rates of immune cells and the role of this pathway in CF remain elusive. In this study, it was shown that innate immune cells from patients with CF show significantly higher levels of ER stress, particularly in the IRE1a-XBP1 signalling pathway. Interestingly, ER stress was only present in neutrophils, monocytes and macrophages from patients with CF. Overactivation of the IRE1a-XBP1 signalling pathway rewires M1 macrophages from patients with CF, and increases macrophages’ metabolic rates, with high glycolytic rates and mitochondrial function. The increased activity of the IRE1a-XBP1 signalling pathway and the increased metabolic rates were associated with excessive production of TNF and IL-6. Specific inhibition of the RNase domain of the IRE1 arm decreased the excessive glycolytic rates, mitochondrial function and production of inflammatory cytokines. Furthermore, Orkambi, Symkevi and Ivacaftor had an essential impact in changing the metabolic profile of cells with CF mutations. This study shows how innate immune cells from CF patients are affected by ER stress, in particular, M1 macrophages. Moreover, the IRE1a-XBP1 signalling pathway is essential for the increased metabolic rates seen in M1 macrophages with CF mutations. Modulation of ER stress might be an exciting option to recover the metabolic fitness of cells with CF mutation

The Evening Herald (Albuquerque, N.M.), 06-10-1921

https://digitalrepository.unm.edu/abq_eh_news/3291/thumbnail.jp

The Evening Herald (Albuquerque, N.M.), 04-05-1921

https://digitalrepository.unm.edu/abq_eh_news/3226/thumbnail.jp

Functional and structural characterization of Aquifex aeolicus sulfide:quinone oxidoreductase

This work presents the first complete structure of the membrane protein sulfide:quinone oxidoreductase (SQR), obtained by X-ray crystallography. Its description is complemented by the results of biochemical and functional experiments. SQRs are ubiquitous flavoprotein disulfide reductases (FDRs), present in all domains of life, including in humans. Their physiological role extends from sulfide detoxification to sulfide-dependent respiration and photosynthesis (in archaea and bacteria), to heavy metal tolerance (in yeast) and possibly to sulfide signalling (in higher eukaryotes). Until now understanding the function of SQRs was difficult because of the poor level of sequence conservation in this enzyme family, the limited functional characterization available and the absence of any structural data. SQR was identified in the native membranes of the hyperthermophilic bacterium Aquifex aeolicus by peptide mass fingerprinting (PMF) and by a spectrophotometric activity assay. The protein was solubilized in the detergent dodecyl-beta-D-maltoside (DDM) and purified to homogeneity in a functionally active state. It binds one FAD molecule per protein monomer and FAD is its only cofactor. Its structure was determined in the “as-purified”, substrate-bound and inhibitor-bound forms at resolutions of 2.3, 2.0 and 2.9 Å, respectively. It is composed of two Rossmann-fold domains and of one membrane-attachment region. Despite the overall monomeric architecture being similar to that of FDRs, the structure reveals properties that had not been observed in FDRs until now and that have strong implications for the SQR catalytic mechanism. Surprisingly, A. aeolicus SQR is trimeric in the crystal structure and in solution, as determined by density-matched analytical ultracentrifugation, cross-linking and single particle electron microscopy. The trimer creates an appropriate surface for binding lipids and thus ensures that SQR exclusively reduces hydrophobic quinones. SQR inserts to a depth of about 12 Å into the membrane as an integral monotopic membrane protein. The interaction is mediated by an amphipathic helix-turn-helix tripodal motif and two lipid clamps. A channel in the membrane-binding domain extends towards the si-side of FAD and represents the quinone-binding site. The quinone ring is sandwiched between the conserved amino acids Phe 385 and Ile 346 and is possibly protonated upon reduction via Glu 318, Lys 382 and/or neighboring solvent molecules. Sulfide polymerization occurs on the re-side of FAD, where the highly conserved Cys 156 and Cys 347 appear to be covalently bound to the putative product of the reaction, a polysulfur chain which takes the form of an S8 ring in some monomers. Finally, the structure shows that FAD is covalently connected to the protein in an unprecedented way, via a putative disulfide bridge between the 8-methyl group of the isoalloxazine moiety and Cys 124. The high resolution insight into the protein and all unexpected structural observations presented in this work suggest that the catalytic mechanism of SQRs is significantly different from that of FDRs. In agreement with the structural and functional data, two reaction schemes are proposed for A. aeolicus SQR. They both provide a detailed description of how sulfide and quinones reach and bind the active site, how electrons are transferred from sulfide to quinone via FAD and how the elongating polysulfur product is attached to the polypeptide and is finally released. The two hypotheses differ in defining the structure of the covalent protein-FAD intermediate that forms during the reaction cycle and whose identity still remains experimentally undetermined. Remarkably, the structure of the active site and the FAD-binding mode of A. aeolicus SQR are not conserved in another SQR structure which also became available recently, that of the archaeon Acidianus ambivalens. The variability in SQRs suggests that not all of these enzymes follow the same catalytic mechanism, despite having been considered homologous. Consequently, the currently available but contradictory sequence-based classifications of the SQR family were revised. A structure-based alignment calculated on the increasing number of available sequences allowed to define new SQR groups and their characteristic sequence fingerprints in agreement with the reported structural and functional data. In conclusion, the results obtained in this work offer for the first time a detailed look into the intriguing but complicated reactions catalysed by SQRs and provide a stimulus for further genetic, biochemical and structural investigation.Diese Arbeit stellt die erste vollständige Röntgenkristallstruktur des Membranproteins Sulfid:Chinon Oxidoreduktase (SQR) vor. Die Beschreibung der Struktur, wird durch die biochemische und funktionelle Charakterisierung des Enzyms ergänzt. SQRs sind ubiquitäre Flavoprotein-Disulfid-Reduktasen (FDRs), die in allen Domänen des Lebens, darunter auch im Menschen, vertreten sind. Ihre physiologische Funktion reicht von der Sulfidentgiftung bis hin zur sulfidabhängigen Atmung und Photosynthese (in Archaea und Bakterien), zur Schwermetalltoleranz (in Hefen) und vermutlich zur sulfidabhängigen Signalübertragung (in höheren Eukaryoten). Bis heute war es schwierig, die Funktion der SQRs zu verstehen, da diese Familie eine geringe Sequenzidentität aufweist, nur wenig funktionell charakterisiert war und keine strukturellen Daten zur Verfügung standen. Die SQR wurde in den Plasmamembranen des hyperthermophilen Bakteriums Aquifex aeolicus durch einen Peptidmassen Fingerabdruck (PMF) und einen spektrophotometrischen Aktivitätstest identifiziert. Das Protein wurde mit dem Detergenz Dodecyl-β-D-maltosid (DDM) solubilisiert und in aktiver Form bis zur Homogenität gereinigt. Jedes Monomer bindet ein FAD Molekül, welches der einzige Cofaktor ist. Die Struktur der SQR wurde in der „wie-gereinigten“, der Substrat-gebundenen und der Inhibitor-gebundenen Form mit einer Auflösung von 2,3, 2,0 bzw. 2,9 Å bestimmt. Sie besteht aus zwei Rossmann-Domänen und einer Membrankontaktregion. Obwohl die Architektur des Monomers derjenigen von FDRs ähnelt, zeigt die Struktur Merkmale, die bisher noch nicht beobachtet wurden, jedoch groβe Auswirkungen auf den katalytischen Mechanismus der SQRs haben. Die SQR von A. aeolicus liegt überraschenderweise im Kristall als Trimer vor, was durch analytische Ultrazentrifugation (AUC), Crosslinking und Einzelpartikelanalyse auch als native Form in Lösung bestätigt wurde. Das Trimer erzeugt eine passende Oberfläche für die Bindung an die Membran und stellt dadurch sicher, dass die SQR ausschließlich membranassoziierte Chinone reduziert. Die SQR sitzt als integrales monotopisches Membranprotein bis zu einer Tiefe von etwa 12 Å in der Membran. Die Interaktion wird durch zwei amphipatische Helices und zwei spezifische Lipid-Bindestellen vermittelt. Ein Kanal, der von der Membranbindedomäne zur si-Seite des FAD reicht, repräsentiert die Chinon-Bindestelle. Der Chinon-Ring ist zwischen den zwei konservierten Aminosäuren Phe 385 und Ile 346 gebunden und wird nach der Reduktion vermutlich von Glu 318, Lys 382 und/oder den in der Nähe lokalisierten Wassermolekülen protoniert. Die Sulfid-Polymerisierung hingegen geschieht auf der re-Seite des FAD. Hier sind die hoch konservierten Aminosäuren Cys 156 und Cys 347 kovalent an eine Polyschwefelkette gebunden, die in manchen Monomeren die Form eines S8 Rings annimmt und vermutlich das Produkt der Reaktion ist. Schließlich zeigt die Struktur eine ungewöhnliche kovalente Bindung der 8-Methylgruppe von FAD zu Cys 124, welche vermutlich eine Disulfidbrücke darstellt. Der detaillierte Einblick in das Protein und alle unerwarteten strukturellen Beobachtungen, die hier in dieser Arbeit gezeigt werden, deuten auf einen katalytischen Mechanismus der SQRs hin, der sich signifikant zu dem der anderen FDRs unterscheidet. In Übereinstimmung mit den strukturellen und funktionellen Daten werden zwei Reaktionsmechanismen für SQRs vorgeschlagen. Beide erklären wie Sulfid und Chinon in das aktive Zentrum gelangen und binden, wie Elektronen vom Sulfid über FAD zum Chinon übertragen werden und wie das Polyschwefelprodukt, das an die Polypeptidkette gebunden ist, verlängert und schließlich abgegeben wird. Der Unterschied beider Hypothesen liegt in dem kovalenten Protein-FAD Intermediat, das sich während des Reaktionszyklus bildet und dessen Struktur experimentell noch nicht bestimmt wurde. Interessanterweise zeigt die Struktur der SQR von Acidianus ambivalens, welche seit kurzem auch zur Verfügung steht, Unterschiede im aktiven Zentrum und in der FAD-Bindestelle zur SQR von A. aeolicus. Diese Variabilität der SQRs deutet darauf hin, dass nicht alle diese Enzyme dem gleichen katalytischen Mechanismus folgen, obwohl sie bisher als homolog betrachtet wurden. Folglich, wurden die verfügbaren aber widersprüchlichen sequenzbasierten Klassifizierungen der SQR Familie wieder aufgegriffen. Da die entscheidenden Sequenzabschnitte nun bekannt waren, erfolgte die Einteilung der Gruppen durch einen strukturbasierten Abgleich der steigenden Anzahl an verfügbaren Sequenzen und wurde mit den vorhandenen strukturellen und funktionellen Daten in Einklang gebracht. Diese Arbeit liefert erstmals einen tieferen Einblick in den faszinierenden aber komplexen Reaktionen, die SQRs katalysieren, und bietet eine Grundlage für weitere genetische, biochemische und strukturelle Untersuchungen

Environ Health Perspect

The expression of 10 genes implicated in regulation of the inflammatory processes in the lung was studied after exposure of alveolar macrophages (AMs) to silica in vitro or in vivo. Exposure of AMs to silica in vitro up-regulated the messenger RNA (mRNA) levels of three genes [interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2)] without a concomitant increase in the protein levels. AMs isolated after intratracheal instillation of silica up-regulated mRNA levels of four additional genes [granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-1beta, IL-10, and inducible nitric oxide synthase]. IL-6, MCP-1, and MIP-2 protein levels were elevated in bronchoalveolar lavage fluid. Fibroblasts under basal culture conditions express much higher levels of IL-6 and GM-CSF compared with AMs. Coculture of AMs and alveolar type II cells, or coculture of AMs and lung fibroblasts, in contact cultures or Transwell chambers, revealed no synergistic effect. Therefore, such interaction does not explain the effects seen in vivo. Identification of the intercellular communication in vivo is still unresolved. However, fibroblasts appear to be an important source of inflammatory mediators in the lung