Glutathione Primes T Cell Metabolism for Inflammation.

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.

A hyperactive mutant of interferon-regulatory factor 4.

We found that deletion of the final 30 amino acids of transcription factor IRF4's (interferon-regulatory factor) C-terminus creates hyperactive IRF4. When introduced into IRF4-deficient CD4+ or CD8+ T cells, more type 17 differentiation was found compared to WT IRF4. Interestingly, Th9 differentiation and Th2-linked IL-13 production were much less altered.

Subversion of a family of antimicrobial proteins by Salmonella enterica.

Salmonella enterica is a food-borne pathogen able to cause a wide spectrum of diseases ranging from mild gastroenteritis to systemic infections. During almost all stages of the infection process Salmonella is likely to be exposed to a wide variety of host-derived antimicrobial peptides (AMPs). AMPs are important components of the innate immune response which integrate within the bacterial membrane, thus forming pores which lead ultimately to bacterial killing. In contrast to other AMPs Bactericidal/Permeability-increasing Protein (BPI) displayed only weak bacteriostatic or bactericidal effects towards Salmonella enterica sv. Typhimurium (STM) cultures. Surprisingly, we found that sub-antimicrobial concentrations of BPI fold-containing (BPIF) superfamily members mediated adhesion of STM depending on pre-formed type 1 fimbriae. BPIF proteins directly bind to type 1 fimbriae through mannose-containing oligosaccharide modifications. Fimbriae decorated with BPIF proteins exhibit extended binding specificity, allowing for bacterial adhesion on a greater variety of abiotic and biotic surfaces likely promoting host colonization. Further, fimbriae significantly contributed to the resistance against BPI, probably through sequestration of the AMP before membrane interaction. In conclusion, functional subversion of innate immune proteins of the BPIF family through binding to fimbriae promotes Salmonella virulence by survival of host defense and promotion of host colonization.

Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.

The butyrogenic genes from Clostridium difficile DSM 1296(T) have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD(+)-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

A simple and fast system for cloning influenza A virus gene segments into pHW2000- and pCAGGS-based vectors.

The reverse genetics system for influenza A viruses described by Hoffmann et al. (Virology 267(2):310-317, 2000, Proc Natl Acad Sci USA 97(11):6108-6113, 2000, ArchVirol 146(12):2275-2289, 2001) is one of the most commonly used. However, this cloning strategy is rather time-consuming and lacks a selection marker to identify positive clones carrying viral genes. We report here the optimization of the cloning protocol of viral genes into pHW2000 by (i) introducing a selection marker and (ii) simplifying the cloning strategy: now the cloning reaction takes only a few minutes and, in addition, is independent of internal restriction sites for BsmBI/Esp3I, BsaI or AarI. In order to accelerate the whole cloning protocol for the generation of recombinant viruses, we first introduced a lacP/Z-element (lac-promoter/lacZα-fragment) between the two BsmBI sites of pHW2000 to allow selection of positive clones by blue/white screening. Then we optimized the digestion/ligation-protocol: In our system, enzymatic digestion and ligation of PCR products into the vector is performed in a single "one-tube" reaction. Due to this strategy, time and material consumption is reduced by a great amount, as vector and cDNA do not have to be digested and purified prior to the ligation. Therefore, this one-tube reaction yields positive clones with high efficiency and fidelity, again saving time and material, which were formerly required for screening and analyzing clones. Finally, to add more versatility to the system, we also created an entry vector based on TA-cloning. This entry vector provides several advantages: inserted genes can easily be modified, e.g., by site-directed mutagenesis or tag attachment, and then subcloned into pHW2000 or other plasmids containing a similar cloning site (e.g., our modified pCAGGS-Esp-blue) by the same rapid and reliable one-tube reaction protocol described here. In fact, the presented protocol is suitable to be adapted to other reverse genetics systems (e.g., those for members of the order Mononegavirales or the family Bunyaviridae) or cloning of genes in general.

Hypothalamic glycogen synthase kinase 3ß has a central role in the regulation of food intake and glucose metabolism.

GSK3ß (glycogen synthase kinase 3ß) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3ß activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3ß in leptin-deficient Lep(ob/ob) mice and show that intracerebroventricular injection of a GSK3ß inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3ß inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3ß in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3ß signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.

DNA methylation of TH1/TH2 cytokine genes affects sensitization and progress of experimental asthma.

BACKGROUND: Epigenetic changes in DNA methylation have recently been demonstrated to be involved in effector T-cell polarization, resulting in differential secretion of T(H)1 and T(H)2 cytokines. However, the contribution to the development of a chronic inflammatory phenotype remains still unclear. OBJECTIVE: We sought to investigate changes in DNA methylation in marker genes of T-cell subsets during allergen sensitization/challenge and their influence on the development of an allergic airway inflammatory response. METHODS: The relationship between changes in DNA methylation and phenotype development were examined in a well-established model of experimental asthma. DNA methylation was investigated at genomic loci associated with T(H)1 (IFNG promoter) or T(H)2 (conserved noncoding sequence 1 [CNS1]) cytokine production by using bisulfite pyrosequencing. RESULTS: Analysis of CD4(+) T cells revealed a significant increase in DNA methylation at the IFNG promoter after allergen sensitization/challenge, which correlated with decreased IFN-γ cytokine expression, whereas only minor changes were observed at the CNS1 locus. Furthermore, the increase in DNA methylation at the IFNG promoter could be reversed with a DNA methyltransferase (DNMT) inhibitor in vitro and in vivo with beneficial effects on sensitization status and allergic phenotype. The specific importance of the DNA methylation status in CD4(+) T cells could be confirmed by using adoptive transfer experiments. CONCLUSION: We here report the novel finding that epigenetic regulation in T cells contributes to the development of experimental asthma and can be targeted pharmacologically.

Maternal signals for progeny prevention against allergy and asthma.

Allergy and asthma are chronic inflammatory diseases which result from complex gene-environment interactions. Recent evidence indicates the importance of prenatal and postnatal developmental processes in terms of maturation of balanced immune responses. According to the current view, gene-environment interactions during a restricted time frame are responsible for programming of the immune system in favor of allergic immune mechanisms later in life. The interaction between genes and environment is complex and only partially understood; however, heritable epigenetic modifications including chemical additions in and alternative packaging of the DNA have been shown to play a crucial role in this context. Novel data indicate that epigenetic mechanisms contribute to the development of T-helper cell function. Environmental factors, including diesel exhaust particles (DEP), vitamins and tobacco smoke, operate through such mechanisms. Furthermore, the role of environmental microbes provides another and maybe even more important group of exogenous exposures which operates in this critical time frame.

The antimicrobial peptide LL-37 modulates the inflammatory and host defense response of human neutrophils.

The human cathelicidin antimicrobial peptide acts as an effector molecule of the innate immune system with direct antimicrobial and immunomodulatory effects. The aim of this study was to test whether the cathelicidin LL-37 modulates the response of neutrophils to microbial stimulation. Human neutrophils were exposed to LPS, Staphylococcus aureus and Pseudomonas aeruginosa subsequent to incubation with LL-37 and cytokine release was measured by ELISA. The incubation with LL-37 significantly decreased the release of proinflammatory cytokines from stimulated human neutrophils. ROS production of neutrophils was determined by a luminometric and a flow cytometry method. The peptide induced the production of ROS and the engulfment of bacteria into neutrophils. Peritoneal mouse neutrophils isolated from CRAMP-deficient and WT animals were treated with LPS and TNF-alpha in the supernatant was measured by ELISA. Antimicrobial activity of neutrophils was detected by incubating neutrophils isolated from CRAMP-knockout and WT mice with bacteria. Neutrophils from CRAMP-deficient mice released significantly more TNF-alpha after bacterial stimulation and showed decreased antimicrobial activity as compared to cells from WT animals. In conclusion, LL-37 modulates the response of neutrophils to bacterial activation. Cathelicidin controls the release of inflammatory mediators while increasing antimicrobial activity of neutrophils.

NF kappaB activation in embryonic endothelial progenitor cells enhances neovascularization via PSGL-1 mediated recruitment: novel role for LL37.

Embryonal endothelial progenitor cells (eEPCs) are capable of inducing therapeutic angiogenesis in a chronic hind limb model. However, the proportion of eEPCs recruited to the ischemic tissue appears to be a limiting step for the induction of cell-based therapeutic neovascularization. In the present study, we primed eEPCs with the human cathelicidin LL37 (hCAP-18) ex vivo to selectively enhance the eEPC-dependent gain of perfusion in vivo and elucidated the mechanism of action of LL37 on eEPCs. Seven days after femoral artery excision, 5 x 10(6) eEPCs (wt, wild type; p65t, transiently p65 transient; p65s, stable p65-transfected; LL37-eEPCs, LL37 peptide preincubated) were retroinfused into the anterior tibial vein. Recruitment of diI-labeled eEPCs in the ischemic gastrocnemic muscle was investigated 2 days later, whereas collateral growth and perfusion score (obtained by fluorescent microspheres) were assessed at day 7 and day 35 and are given as percentage of day 7 level. Capillary/muscle fiber ratio in the ischemic lower limb was obtained at day 35. Embryonic EPC recruitment in vitro and in vivo was found elevated after LL37 and p65t pretreatment, but not in p65s-eEPCs displaying increased IkappaBalpha or after LL37 in IkappaB-DN overexpressing eEPCs. Using LL37- and p65t-eEPCs, collateral growth (181 +/- 10% and 165 +/- 8%, respectively) surpassed that of wt-eEPCs (135 +/- 7%), increasing perfusion ratio (208 +/- 20% and 210 +/- 17% vs. 142 +/- 12% in wt-eEPCs, respectively), whereas p65s-eEPCs exerted no additive effect (collateral growth 130 +/- 8%; perfusion ratio 155 +/- 15%). Moreover, p65t-eEPC-induced neovascularization was abrogated by blocking antibodies against E-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). We conclude that NF kappaB activation by LL37 or transient p65-transfection increases functionally relevant eEPC recruitment to ischemic muscle tissue via induction of PSGL-1 and E-selectin.

Fetal epigenetic mechanisms and innate immunity in asthma.

Allergy and asthma are chronic inflammatory diseases that result from complex gene-environment interactions. Recent evidence points to the importance of prenatal and postnatal developmental processes in the maturation of balanced immune responses. Novel data indicate that epigenetic mechanisms contribute to the development of T-helper-cell function. Environmental factors, including diesel exhaust particles, vitamins, and tobacco smoke, operate through such mechanisms. Furthermore, the role of environmental microbes provides another-and maybe an even more important-group of exogenous exposures that operate in this critical time frame. A better understanding of fetal immuno-maturation conditions will provide the basis for the development of novel allergo-protective clinical strategies.

Ambient temperature impacts on pH of exhaled breath condensate.

BACKGROUND AND OBJECTIVE: Analysis of exhaled breath condensate (EBC) pH is a non-invasive method to study airway inflammation. Low pH is correlated with inflammatory diseases like asthma and COPD. The aim of this study was to assess the influence of measurement temperature on pH values of EBC. METHODS: EBC was collected using the RTube in 10 healthy non-smoking controls, 10 smokers before and after cigarette smoking, 10 stable COPD patients and 10 patients with exacerbated COPD. pH was determined directly after degassing at temperatures of 23 degrees C and 37 degrees C. RESULTS: When comparing all groups pH was significantly (P = 0.0002) higher (mean +/- SD 7.88 +/- 0.92) at 37 degrees C as compared with 23 degrees C (7.44 +/- 0.90). Specifically, at 23 degrees C pH was significantly lower in the group of exacerbated COPD (6.78 +/- 1.27) and healthy non-smoking controls (8.04 +/- 0.39). In contrast, subgroup analysis of values assessed at 37 degrees C did not display significant differences. CONCLUSIONS: Our data indicate a considerable influence of temperature on pH values in EBC. Thus the temperature at which pH measurements in EBC studies are performed should be declared.

Dietary cellulose induces anti-inflammatory immunity and transcriptional programs via maturation of the intestinal microbiota.

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was Alistipes which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including A. finegoldii showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.

The host defence peptide LL-37/hCAP-18 is a growth factor for lung cancer cells.

Cancer development can be viewed as dysregulated repair. Antimicrobial peptides (AMPs) are effector molecules of the innate immune system with direct antimicrobial activity. Beside this host defence function several AMPs play a role in the regulation of inflammation and tissue repair. The aim of the present study was to investigate whether the human cathelicidin AMP LL-37/hCAP-18 is involved in the biology of lung cancer. Human cancer cell lines were found to express the human cathelicidin LL-37/hCAP-18 mRNA and peptide at different levels. Immunohistochemistry of human lung cancers showed that the peptide is expressed mostly in adenocarcinoma and squamous cell carcinoma. Application of exogenous LL-37 at low concentrations of 5ng/ml to cancer cell lines increased proliferation and growth of anchorage-independent colonies. At the molecular level, LL-37 induced phosphorylation of the epidermal growth factor receptor (EGFR) and activation of downstream MAP kinase signalling pathways. Lung cancer cell lines that stably overexpress the peptide by means of a doxycycline-regulated promoter system also showed a faster growth. When these cell lines were injected subcutaneously into nude mice, cathelicidin overexpression resulted in increased tumourigenicity and the formation of significantly larger tumours. In conclusion, cathelicidin is expressed in human lung cancers. The peptide activates tumour cells resulting in increased cell growth in vitro and in an animal model. The host defence peptide cathelicidin LL-37/hCAP-18 acts as growth factor for human lung cancer.

Reduced viability of neuronal cells after overexpression of protein histidine phosphatase.

Protein histidine phosphatase (PHP) has just recently been discovered in eukaryotes and ATP-citrate lyase (ACL) was shown to be one of its substrates. Since ACL is crucial for cellular energy and fat metabolism we made an attempt to study the influence of PHP on cell viability. Using an adenoviral vector PHP was overexpressed in SN56 cholinergic murine neuroblastoma cells and in primary cultures of hippocampal neurons obtained from embryonic rats (E18). Overexpression of PHP in these cells caused a decrease in ACL activity and consequently impaired viability. To be sure that the reduced cellular viability was achieved by overexpression of PHP we also downregulated ACL in SN56 cells using RNAi-technology. Downregulation of ACL was harmful to the cells similar to what was observed upon overexpression of PHP. Taken together, it is concluded that overexpression of PHP results in increased dephosphorylation with concomitant inactivation of ACL, thus finally leading to cell damage.

Increased vascular smooth muscle contractility in TRPC6-/- mice.

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6(-)(/)(-) smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6(-/-) smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.

Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene.

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

IL-17 and TNF-α Are Key Mediators of Moraxella catarrhalis Triggered Exacerbation of Allergic Airway Inflammation.

Alterations of the airway microbiome are often associated with pulmonary diseases. For example, detection of the bacterial pathogen Moraxella catarrhalis in the upper airways is linked with an increased risk to develop or exacerbate asthma. However, the mechanisms by which M. catarrhalis augments allergic airway inflammation (AAI) remain unclear. We here characterized the cellular and soluble mediators of M. catarrhalis triggered excacerbation of AAI in wt and IL-17 deficient as well as in animals treated with TNF-α and IL-6 neutralizing antibodies. We compared the type of inflammatory response in M. catarrhalis infected, house dust mite (HDM)-allergic and animals infected with M. catarrhalis at different time points of HDM sensitization. We found that airway infection of mice with M. catarrhalis triggers a strong inflammatory response with massive neutrophilic infiltrates, high amounts of IL-6 and TNF-α and moderate levels of CD4+ T-cell-derived IFN-γ and IL-17. If bacterial infection occurred during HDM allergen sensitization, the allergic airway response was exacerbated, particularly by the expansion of Th17 cells and increased TNF-α levels. Neutralization of IL-17 or TNF-α but not IL-6 resulted in accelerated clearance of M. catarrhalis and effectively prevented infection-induced exacerbation of AAI. Taken together, our data demonstrate an essential role for TNF-α and IL-17 in infection-triggered exacerbation of AAI.

Steerable Induction of the Thymosin ß4/MRTF-A Pathway via AAV-Based Overexpression Induces Therapeutic Neovascularization.

Viral vectors have been frequently used in a variety of preclinical animal models to deliver genetic constructs into tissues. Among the vectors used, adeno-associated viral vectors (AAVs) may be targeted to specific tissues, depending on the serotype used. Moreover, they show robust expression for prolonged periods of time and have a low immunogenic potential. Furthermore, AAVs, unlike other vector systems, only display a low rate of genomic integration. However, to ensure efficient transgene production, expression is typically driven by constitutively active promoters, such as the cytomegalovirus (CMV) promoter. Tetracyclin responsive promoters represent a promising alternative to unregulated promoters. The present study compares AAVs encoding either constitutively active CMV or tet-off promoter regions in the preclinical models of hindlimb and chronic myocardial ischemia. Therapeutically, mediators regulating vessel maturation, specifically thymosin beta 4 (Tß4) and the downstream signaling molecule myocardin-related transcription factor A (MRTF-A) as well as the endothelial activator angiopoietin-2 (Ang2) were overexpressed via AAVs using both promotors. In the model of rabbit hindlimb ischemia, temporary (tet-off) expression of Tß4 improved capillary density, collateralization, and perfusion in the ischemic hindlimb, with no detectable difference to constitutive Tß4 overexpression. Similarly, constitutive overexpression of MRTF-A alone was able to improve capillarization, collateralization and perfusion. Temporary expression of Ang2 for 7 days further increased capillary density and pericyte coverage compared with MRTF-A alone, without further improving collateralization or perfusion. In the pig model of chronic myocardial ischemia constitutive expression of Tß4 for 4 weeks induced capillary and collateral growth similarly to a pulsed expression (2 day expression per week for 3 weeks). Taken together these findings demonstrate for two models of preclinical interventions that temporary gene expression may lead to similar results as constitutive expression, highlighting the potential of controlled temporary gene expression for induction of vascular growth as a therapeutic approach.