Severe sepsis and septic shock are leading causes of morbidity and mortality worldwide. Infection-associated inflammation promotes the development and progression of adverse outcomes in sepsis. The effects of heterodimeric IL-27 (p28/EBI3) have been implicated in the natural course of sepsis, whereas the molecular mechanisms underlying the regulation of gene expression and release of IL-27 in sepsis are poorly understood. We studied the events regulating the p28 subunit of IL-27 in endotoxic shock and polymicrobial sepsis following cecal ligation and puncture. Neutralizing Abs to IL-27(p28) improved survival rates, restricted cytokine release, and reduced bacterial burden in C57BL/6 mice during sepsis. Genetic disruption of IL-27 signaling enhanced the respiratory burst of macrophages. Experiments using splenectomized mice or treatment with clodronate liposomes suggested that macrophages in the spleen may be a significant source of IL-27(p28) during sepsis. In cultures of TLR4-activated macrophages, the frequency of F4/80(+)CD11b(+)IL-27(p28)(+) cells was reduced by the addition of IL-10. IL-10 antagonized both MyD88-dependent and TRIF-dependent release of IL-27(p28). Genetic deletion of STAT3 in Tie2-Cre/STAT3flox macrophages completely interrupted the inhibition of IL-27(p28) by IL-10 after TLR4 activation. In contrast, IL-10 remained fully active to suppress IL-27(p28) with deletion of SOCS3 in Tie2-Cre/SOCS3flox macrophages. Blockade of IL-10R by Ab or genetic deficiency of IL-10 resulted in 3-5-fold higher concentrations of IL-27(p28) in endotoxic shock and polymicrobial sepsis. Our studies identify IL-10 as a critical suppressing factor for IL-27(p28) production during infection-associated inflammation. These findings may be helpful for a beneficial manipulation of adverse IL-27(p28) release during sepsis.
The members of the signal transducer and activator of transcription (STAT) family of transcription factors modulate the development and function of natural killer (NK) cells. NK cell-mediated tumor surveillance is particularly important in the body's defense against hematological malignancies such as leukemia. STAT3 inhibitors are currently being developed, although their potential effects on NK cells are not clear. We have investigated the function of STAT3 in NK cells with Stat3(?/?)Ncr1-iCreTg mice, whose NK cells lack STAT3. In the absence of STAT3, NK cells develop normally and in normal numbers, but display alterations in the kinetics of interferon-? (IFN-?) production. We report that STAT3 directly binds the IFN-? promoter. In various in vivo models of hematological diseases, loss of STAT3 in NK cells enhances tumor surveillance. The reduced tumor burden is paralleled by increased expression of the activating receptor DNAM-1 and the lytic enzymes perforin and granzyme B. Our findings imply that STAT3 inhibitors will stimulate the cytolytic activity of NK cells against leukemia, thereby providing an additional therapeutic benefit.
Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the Growth hormone receptor gene (Ghr(-/-) , a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2(-/-) ), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr(-/-) ;Mdr2(-/-) mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation and increased collagen deposition relative to Mdr2 (-/-) mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr(-/-) ;Mdr2(-/-) mice had a pronounced down-regulation of hepato-protective genes Hnf6, Egfr and Igf-1, and significantly increased levels of ROS and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr(-/-) ) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis and bile infarcts compared to their wildtype littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr(-/-) ;Mdr2(-/-) mice displayed a significant decrease in tumour incidence compared to Mdr2(-/-) mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. Conclusion: Our findings suggest that GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments. (Hepatology 2014;).
In a murine model of allergic asthma, we found that Tyk-2((-/-)) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4(+) effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13. We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production. This T helper phenotype was accompanied by increased SOCS3 in the lung of Tyk-2 deficient asthmatic mice. Finally, in vivo treatment with rIL-17A inhibited local CD4(+)CD25(+)Foxp3(+) T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung. These results suggest a role of Tyk-2 in different subsets of T helper cells mediated by SOCS3 regulation that is relevant for the treatment of asthma, cancer and autoimmune diseases.
The contribution of the innate immune system to inflammatory bowel disease (IBD) is under intensive investigation. Research in animal models has demonstrated that type I interferons (IFN-Is) protect from IBD. In contrast, studies of patients with IBD have produced conflicting results concerning the therapeutic potential of IFN-Is. Here, we present data suggesting that IFN-Is play dual roles as regulators of intestinal inflammation in dextran sodium sulfate (DSS)-treated C57BL/6 mice. Though IFN-Is reduced acute intestinal damage and the abundance of colitis-associated intestinal bacteria caused by treatment with a high dose of DSS, they also inhibited the resolution of inflammation after DSS treatment. IFN-Is played an anti-inflammatory role by suppressing the release of IL-1? from the colon MHC class II(+) cells. Consistently, IL-1 receptor blockade reduced the severity of inflammation in IFN-I receptor-deficient mice and myeloid cell-restricted ablation of the IFN-I receptor was detrimental. The proinflammatory role of IFN-Is during recovery from DSS treatment was caused by IFN-I-dependent cell apoptosis as well as an increase in chemokine production and infiltrating inflammatory monocytes and neutrophils. Thus, IFN-Is play opposing roles in specific phases of intestinal injury and inflammation, which may be important for guiding treatment strategies in patients.
Evacuation from a smoke filled tunnel requires quick decision-making and swift action from the tunnel occupants. Technical installations such as emergency signage aim to guide tunnel occupants to the closest emergency exits. However, conflicting information may come from the behavior of other tunnel occupants. We examined if and how conflicting social information may affect evacuation in terms of delayed and/or inadequate evacuation decisions and behaviors. To this end, forty participants were repeatedly situated in a virtual reality smoke filled tunnel with an emergency exit visible to one side of the participants. Four social influence conditions were realized. In the control condition participants were alone in the tunnel, while in the other three experimental conditions a virtual agent (VA) was present. In the no-conflict condition, the VA moved to the emergency exit. In the active conflict condition, the VA moved in the opposite direction of the emergency exit. In the passive conflict condition, the VA stayed passive. Participants were less likely to move to the emergency exit in the conflict conditions compared to the no-conflict condition. Pre-movement and movement times in the passive conflict condition were significantly delayed compared to all other conditions. Participants moved the longest distances in the passive conflict condition. These results support the hypothesis that social influence affects evacuation behavior, especially passive behavior of others can thwart an evacuation to safety.
The transcription factor STAT1 is essential for interferon (IFN)-mediated immunity in humans and mice. STAT1 function is tightly regulated, and both loss- and gain-of-function mutations result in severe immune diseases. The two alternatively spliced isoforms, STAT1? and STAT1?, differ with regard to a C-terminal transactivation domain, which is absent in STAT1?. STAT1? is considered to be transcriptionally inactive and to be a competitive inhibitor of STAT1?. To investigate the functions of the STAT1 isoforms in vivo, we generated mice deficient for either STAT1? or STAT1?. As expected, the functions of STAT1? and STAT1? in IFN-?/?- and IFN-?-dependent antiviral activity are largely redundant. In contrast to the current dogma, however, we found that STAT1? is transcriptionally active in response to IFN-?. In the absence of STAT1?, STAT1? shows more prolonged IFN-?-induced phosphorylation and promoter binding. Both isoforms mediate protective, IFN-?-dependent immunity against the bacterium Listeria monocytogenes, although with remarkably different efficiencies. Our data shed new light on the potential contributions of the individual STAT1 isoforms to STAT1-dependent immune responses. Knowledge of STAT1?'s function will help fine-tune diagnostic approaches and help design more specific strategies to interfere with STAT1 activity.
The aim of this study was to test the hypothesis that gene expression and release of IL-27 may be modulated by Tyk2. Macrophages derived from the peritoneum or bone marrow of C57BL/10SnJ (WT) mice produced abundant amounts of IL-27(p28) following TLR4 activation by LPS. In contrast, production of IL-27(p28), but not EBI3, was reduced by ?50% in TLR4-activated macrophages derived from mice with genetic deficiency of Tyk2 compared with WT macrophages. Frequencies of IL-27(p28)+F4/80+CD11b+ cells were lower in TLR4-activated macrophages derived from Tyk2-/- mice. Mechanistically, Tyk2-/- resulted in disruption of a type I IFN-dependent mechanism for production of IL-27(p28), which was induced by type I IFNs, and release of IL-27 was defective in macrophages from IFN-?-/- and IFNAR1-/- mice. In contrast, Tyk2 was not required to mediate the effects of IL-27 on target gene expression in CD4(+) T cells. In vivo, we observed that Tyk2-/- mice have improved survival following endotoxic shock or polymicrobial sepsis induced by CLP. Plasma levels of IL-27(p28) during endotoxic shock or polymicrobial sepsis were markedly reduced in Tyk2-/- mice compared with WT mice. Disruption of IL-27 signaling using IL-27RA-/- mice was protective against sepsis-associated mortality. These data suggest that Tyk2 may mediate adverse outcomes of SIRS by promoting the production of IL-27. In conclusion, this report identifies Tyk2 as a prerequisite factor in the molecular networks that are involved in generation of IL-27.
Collective spin excitations form a fundamental class of excitations in magnetic materials. As their energy reaches down to only a few meV, they are present at all temperatures and substantially influence the properties of magnetic systems. To study the spin excitations in solids from first principles, we have developed a computational scheme based on many-body perturbation theory within the full-potential linearized augmented plane-wave (FLAPW) method. The main quantity of interest is the dynamical transverse spin susceptibility or magnetic response function, from which magnetic excitations, including single-particle spin-flip Stoner excitations and collective spin-wave modes as well as their lifetimes, can be obtained. In order to describe spin waves we include appropriate vertex corrections in the form of a multiple-scattering T matrix, which describes the coupling of electrons and holes with different spins. The electron-hole interaction incorporates the screening of the many-body system within the random-phase approximation. To reduce the numerical cost in evaluating the four-point T matrix, we exploit a transformation to maximally localized Wannier functions that takes advantage of the short spatial range of electronic correlation in the partially filled d or f orbitals of magnetic materials. The theory and the implementation are discussed in detail. In particular, we show how the magnetic response function can be evaluated for arbitrary k points. This enables the calculation of smooth dispersion curves, allowing one to study fine details in the k dependence of the spin-wave spectra. We also demonstrate how spatial and time-reversal symmetry can be exploited to accelerate substantially the computation of the four-point quantities. As an illustration, we present spin-wave spectra and dispersions for the elementary ferromagnet bcc Fe, B2-type tetragonal FeCo, and CrO? calculated with our scheme. The results are in good agreement with available experimental data.
Considerable effort has been expended to identify genes that account for myeloid lineage commitment and development. However, currently available non-invasive mouse models utilize myeloid-specific reporters that are significantly expressed in hematopoietic stem cells as well as lymphoid compartments. Here, we describe a myeloid-specific marker that is not shared by any other lineage. We show that lactotransferrin mRNA is expressed by Gr-1(+)/CD11b(+) cells in the bone marrow, as opposed to hematopoietic stem cells or any peripheral cell population. To follow the progeny of lactotransferrin-expressing bone marrow cells, we generated a mouse model in which a reporter gene is irreversibly activated from the lactotransferrin-promoter. We found that lactotransferrin-reporter labels a majority of neutrophils, monocytes, macrophages and distinct subtypes of dendritic cells, while excluding T, B, natural killer cells, interferon-producing killer dendritic cells, plasmacytoid dendritic cells, erythrocytes and eosinophils. Lactotransferrin-reporter(-) bone marrow cells retain lymphoid, erythroid and long-term repopulating potential, while lactotransferrin-reporter(+) bone marrow cells confer only myeloid, but not lymphoid potential. We conclude that lactotransferrin represents a late stage differentiation marker of neutrophils, macrophages and distinct subtypes of dendritic cells.
Tyrosine kinase 2 (TYK2) has a pivotal role in immunity to infection and tumor surveillance. It is associated with several cytokine receptor chains including type I interferon (IFN) receptor 1 (IFNAR1), interleukin- (IL-) 12 receptor beta 1 (IL-12Rb1) and IL-10R2. We have generated a mouse with a conditional Tyk2 null allele and proved integrity of the conditional Tyk2 locus. TYK2 was successfully removed by the use of ubiquitous and tissue-specific Cre-expressing mouse strains. Myeloid TYK2 was found to critically contribute to the defense against murine cytomegalovirus. Ubiquitous TYK2 ablation severely impaired tumor immunosurveillance, while deletion in myeloid, dendritic or T cells alone showed no effect. The conditional Tyk2 mouse strain will be instrumental to further dissect TYK2 functions in infection, inflammation and cancer.
Although alterations in gut microbiota composition during acute colitis have been repeatedly observed, associated functional changes and the recovery from dysbiosis received little attention. In this study, we investigated structure and function of the gut microbiota during acute inflammation and recovery in a dextran sodium sulfate (DSS)-colitis mouse model using metatranscriptomics, bacterial 16S rRNA gene amplicon sequencing and monitoring of selected host markers. Parallel to an increase of host markers of inflammation during acute colitis, we observed relative abundance shifts and alterations in phylotype composition of the dominant bacterial orders Clostridiales and Bacteroidales, and an increase of the low abundant Enterobacteriales, Deferribacterales, Verrucomicrobiales and Erysipelotrichales. During recovery, the microbiota began to resume, but did not reach its original composition until the end of the experiment. Microbial gene expression was more resilient to disturbance, with pre-perturbation-type transcript profiles appearing quickly after acute colitis. The decrease of Clostridiales during inflammation correlated with a reduction of transcripts related to butyrate formation, suggesting a disturbance in host-microbe signalling and mucosal nutrient provision. The impact of acute inflammation on the Clostridiales was also characterized by a significant downregulation of their flagellin-encoding genes. In contrast, the abundance of members of the Bacteroidales increased along with an increase in transcripts related to mucin degradation. We propose that acute inflammation triggered a selective reaction of the immune system against flagella of commensals and temporarily altered murine microbiota composition and functions relevant for the host. Despite changes in specific interactions, the host-microbiota homeostasis revealed a remarkable ability for recovery.
Extinction is an important mechanism to inhibit initially acquired fear responses. There is growing evidence that the ventromedial prefrontal cortex (vmPFC) inhibits the amygdala and therefore plays an important role in the extinction of delay fear conditioning. To our knowledge, there is no evidence on the role of the prefrontal cortex in the extinction of trace conditioning up to now. Thus, we compared brain structures involved in the extinction of human delay and trace fear conditioning in a between-subjects-design in an fMRI study. Participants were passively guided through a virtual environment during learning and extinction of conditioned fear. Two different lights served as conditioned stimuli (CS); as unconditioned stimulus (US) a mildly painful electric stimulus was delivered. In the delay conditioning group (DCG) the US was administered with offset of one light (CS+), whereas in the trace conditioning group (TCG) the US was presented 4 s after CS+ offset. Both groups showed insular and striatal activation during early extinction, but differed in their prefrontal activation. The vmPFC was mainly activated in the DCG, whereas the TCG showed activation of the dorsolateral prefrontal cortex (dlPFC) during extinction. These results point to different extinction processes in delay and trace conditioning. VmPFC activation during extinction of delay conditioning might reflect the inhibition of the fear response. In contrast, dlPFC activation during extinction of trace conditioning may reflect modulation of working memory processes which are involved in bridging the trace interval and hold information in short term memory.
Signal transducer and activator of transcription (STAT) 1 is a key player in interferon (IFN) signaling, essential in mediating host defense against viruses and other pathogens. STAT1 levels are tightly regulated and loss- or gain-of-function mutations in mice and men lead to severe diseases. We have generated a doxycycline (dox) -inducible, FLAG-tagged Stat1 expression system in mice lacking endogenous STAT1 (i.e. Stat1(ind) mice). We show that STAT1 expression depends on the time and dose of dox treatment in primary cells and a variety of organs isolated from Stat1(ind) mice. In bone marrow-derived macrophages, a fraction of the amount of STAT1 present in WT cells is sufficient for full expression of IFN-induced genes. Dox-induced STAT1 established protection against virus infections in primary cells and mice. The availability of the Stat1(ind) mouse model will enable an examination of the consequences of variable amounts of STAT1. The model will also permit the study of STAT1 dose-dependent and reversible functions as well as of STAT1's contributions to the development, progression and resolution of disease.
Transcriptional activation of the Nos2 gene encoding inducible nitric oxide synthase (iNOS) during infection or inflammation requires coordinate assembly of an initiation complex by transcription factors NF?B and the type I interferon-activated ISGF3. Here we show that infection of macrophages with the intracellular bacterial pathogen Listeria monocytogenes (Lm) causes binding of BET proteins Brd2, Brd3 and, most prominently, Brd4 to the Nos2 promoter and that a profound reduction of Nos2 expression occurred in the presence of the BET inhibitor JQ1. RNA polymerase activity at the Nos2 gene was regulated through Brd-mediated CTD phosphorylation at serine 5 and the rate of transcriptional re-initiation. Underscoring the critical importance of Brd for the regulation of immune responses, application of JQ1 reduced NO production in mice infected with Lm as well as innate resistance to Lm and influenza virus. In a murine model of inflammatory disease JQ1 treatment increased the colitogenic activity of DSS. The data presented in our study suggest that BET protein inhibition in a clinical setting poses the risk of altering the innate immune response to infectious or inflammatory challenge.
Janus kinases (Jak) play essential roles in cytokine and growth factor signaling. Conventional gene targeting of Jak2, creating a null allele, leads to a block in definitive erythropoiesis as a result of failing signal transduction at the homomeric Erythropoietin receptor (EpoR) and at the heteromeric Interferon ? receptor (IFNGR). To investigate the in vivo relevance of the activation loop of Jak2, a Jak2-YY1007/1008FF knock-in mutation was introduced into the germline of mice. The phenotype of the Jak2(FF/FF) mouse line reveals that tyrosine residues 1007/1008 are absolutely essential for kinase function and signal transduction at the homomeric EpoR. Detailed studies using the Jak2 activation loop mutant uncover an essential scaffolding function of Jak2 within the IFNGR receptor complex and reveal that Jak1 can mediate a semi-redundant function for IFNGR signal transduction. These studies are highly important for the molecular understanding of cytokine and growth factor signaling and provide new insights for future strategies in the design of pharmacological blockers of Jak2.
Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a previously unrecognized pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-?/? receptor-dependent manner. This signaling pathway is critical for immune cell-mediated host resistance to liver-stage Plasmodium infection, which we find can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.
Localized plasmon resonance (LSPR) spectroscopy, employing gold nanodisk substrates, is applied for studies of photoswitching in self-assembled monolayers of azobenzene-containing thiols. By choosing customized samples in which the sharp LSPR resonance is well separated from the spectral regime of the molecular absorption bands, the photoisomerization kinetics of the adlayer can be monitored in real time. Quantitative data on the photoinduced trans-cis and cis-trans isomerization processes in inert gas atmosphere were obtained as a function of irradiation intensity and temperature, demonstrating the high sensitivity of this technique to such processes in functional adlayers.
The width and asymmetry of the line shape of the optical transition of a sample of two dimensional (2D) molecular J-aggregates was found to be related to a finite-size effect. The 2D aggregates were domains of the ordered monolayer of the fluorescent dye molecule 3,4,9,10-perylenetetracarboxylic acid dianhydride on a KCl(100) surface. Fluorescence and fluorescence excitation (FLE) spectra were measured as a function of temperature. The system shows a pronounced superradiant emission which yields additional information on the number of coherently coupled molecules participating in the emission. From calculations of the spectra within the tight binding model we find that the finite size of the 2D ordered domains of about N = 7 × 7 molecules, in combination with a Poissonian domain-size distribution, explains the line profile. Line broadening mechanisms due to site disorder or thermal effects--although not excludable straightaway--are not needed to explain the observed FLE line profile. This yields insight into the important, but so far not well understood, relation between the line profile and the size of ordered molecular aggregates.
Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction products has evolved into an alternative strategy to structurally resolve protein complexes. We investigated conformational changes in peroxisome proliferator-activated receptor ? (PPAR?) upon ligand binding. Using E. coli cells with a special tRNA/aminoacyl-tRNA synthetase pair, two PPAR? variants were prepared in which Leu-258 or Phe-273 were site-specifically replaced by the genetically encoded photoreactive amino acid p-benzoylphenylalanine (Bpa). PPAR? variants were subjected to UV-induced cross-linking, both in the absence and in the presence of ligands. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested and peptides were analyzed by mass spectrometry. The inter-residue distances disclosed by the photochemical cross-links served to monitor conformational changes in PPAR? upon agonist and antagonist binding. The data obtained with our strategy emphasize the potential of genetically encoded internal photo-cross-linkers in combination with mass spectrometry as an alternative method to monitor in-solution 3D-protein structures.
Macrophages play a key role in responding to pathogens and initiate an inflammatory response to combat microbe multiplication. Deactivation of macrophages facilitates resolution of the inflammatory response. Deactivated macrophages are characterized by an immunosuppressive phenotype, but the lack of unique markers that can reliably identify these cells explains the poorly defined biological role of this macrophage subset. We identified lipocalin 2 (LCN2) as both a marker of deactivated macrophages and a macrophage deactivator. We show that LCN2 attenuated the early inflammatory response and impaired bacterial clearance, leading to impaired survival of mice suffering from pneumococcal pneumonia. LCN2 induced IL-10 formation by macrophages, skewing macrophage polarization in a STAT3-dependent manner. Pulmonary LCN2 levels were tremendously elevated during bacterial pneumonia in humans, and high LCN2 levels were indicative of a detrimental outcome from pneumonia with Gram-positive bacteria. Our data emphasize the importance of macrophage deactivation for the outcome of pneumococcal infections and highlight the role of LCN2 and IL-10 as determinants of macrophage performance in the respiratory tract.
Plasmonic wave packet propagation is monitored in dielectric-loaded surface plasmon polariton waveguides realized from para-hexaphenylene nanofibers deposited onto a 60 nm thick gold film. Using interferometric time resolved two-photon photoemission electron microscopy we are able to determine phase and group velocity of the surface plasmon polariton (SPP) waveguiding mode (0.967c and 0.85c at ?(Laser) = 812nm) as well as the effective propagation length (39 ?m) along the fiber-gold interface. We furthermore observe that the propagation properties of the SPP waveguiding mode are governed by the cross section of the waveguide.
The combination of chemical cross-linking and high-resolution mass spectrometry is an emerging technique for monitoring conformational changes in proteins induced by drug binding. In this chapter, we describe this approach for gaining insights into the conformational changes of the peroxisome proliferator-activated receptor alpha after binding of low-molecular weight ligands. Our strategy provides a basis to efficiently characterize target protein-drug interactions.
Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the Janus-activated kinase (JAK) tyrosine kinase family, TYK2, and its downstream effector STAT1, in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently showed TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway in T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of interleukin (IL)-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the antiapoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway.
In this study we investigated the role of Brutons tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk (-/-) BMMs showed enhanced TNF-?, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk (-/-) BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk (-/-) BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk (-/-) mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-? and IL-12p70 serum levels in Btk (-/-) mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.
We performed fluorescence (FL) and fluorescence excitation (FLE) spectroscopy on the model molecule perylene-3,4,9,10-tetracarboxyl acid dianhydride (PTCDA) for very low coverages (below 1% of a monolayer) on thin (100) oriented KCl films. Two different states of PTCDA molecules can be distinguished in the spectra: an initial state, which is observed directly after deposition of the molecules onto the cold sample at 20 K, and a final state, which is found after intensive optical excitation or thermal annealing of the sample. The spectrum of the final state is blue-shifted with respect to that of the initial state by 130 ± 15 cm(-1) and exhibits lines with significantly reduced widths. This can be explained by diffusion of molecules from initially populated terrace sites to energetically favoured step edge sites. Polarization dependent spectroscopy reveals the same azimuthal orientation of the molecules on both adsorption sites and leads to a model of the adsorption geometry of PTCDA at the KCl step sites. Our experiment demonstrates how optical spectroscopy can be used to investigate kinetic processes of fluorescent molecules on surfaces.
Small amounts of the model molecule perylene-3,4,9,10-tetracarboxylic acid dianhydride (PTCDA) were vacuum deposited on epitaxial KCl films on Ag(100). The use of a low substrate temperature (20 K) during deposition hampered molecular diffusion resulting in isolated monomers on the surface. Fluorescence and fluorescence excitation spectroscopy performed on these monomers yielded highly resolved spectra with narrow lines corresponding to individual vibronic modes. This high resolution in our spectra is caused by a very small inhomogeneous broadening due to well-defined adsorption sites of the molecule on the substrate. Indeed, by polarization dependent fluorescence spectroscopy we show that the flat-lying molecules exhibit a preferred azimuthal orientation on the surface, the long molecular axis being oriented along the  or the equivalent  direction of the substrate. Furthermore, the high resolution in the spectra allowed a detailed analysis of the vibronic modes. The vibrational modes of the adsorbed molecule are very similar to those of the free PTCDA molecule, but due to the presence of the substrate additional low energy modes which are relevant for the full understanding of the spectra couple to the transition.
The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance.
Gene regulation by cytokine-activated transcription factors of the signal transducer and activator of transcription (STAT) family requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation occurs upon promoter binding by an unknown kinase. Here, we show that the cyclin-dependent kinase 8 (CDK8) module of the Mediator complex phosphorylated regulatory sites within the TADs of STAT1, STAT3, and STAT5, including S727 within the STAT1 TAD in the interferon (IFN) signaling pathway. We also observed a CDK8 requirement for IFN-?-inducible antiviral responses. Microarray analyses revealed that CDK8-mediated STAT1 phosphorylation positively or negatively regulated over 40% of IFN-?-responsive genes, and RNA polymerase II occupancy correlated with gene expression changes. This divergent regulation occurred despite similar CDK8 occupancy at both S727 phosphorylation-dependent and -independent genes. These data identify CDK8 as a key regulator of STAT1 and antiviral responses and suggest a general role for CDK8 in STAT-mediated transcription. As such, CDK8 represents a promising target for therapeutic manipulation of cytokine responses.
Interferons (IFN) are subdivided into type I IFN (IFN-I, here synonymous with IFN-?/?), type II (IFN-?) and type III IFN (IFN-III/IFN-?) that reprogram nuclear gene expression through STATs 1 and 2 by forming STAT1 dimers (mainly IFN-?) or the ISGF3 complex, a STAT1-STAT2-IRF9 heterotrimer (IFN-I and IFN-III). Dominant IFN activities in the immune system are to protect cells from viral replication and to activate macrophages for enhanced effector function. However, the impact of IFN and their STATs on the immune system stretches far beyond these activities and includes the control of inflammation. The goal of this review is to give an overview of the different facets of the inflammatory process that show regulatory input by IFN/STAT.
The MAPK p38? senses environmental stressors and orchestrates inflammatory and immunomodulatory reactions. However, the molecular mechanism how p38? controls immunomodulatory responses in myeloid cells remains elusive. We found that in monocytes and macrophages, p38? activated the mechanistic target of rapamycin (mTOR) pathway in vitro and in vivo. p38? signaling in myeloid immune cells promoted IL-10 but inhibited IL-12 expression via mTOR and blocked the differentiation of proinflammatory CD4(+) Th1 cells. Cellular stress induced p38?-mediated mTOR activation that was independent of PI3K but dependent on the MAPK-activated protein kinase 2 and on the inhibition of tuberous sclerosis 1 and 2, a negative regulatory complex of mTOR signaling. Remarkably, p38? and PI3K concurrently modulated mTOR to balance IL-12 and IL-10 expression. Our data link p38? to mTOR signaling in myeloid immune cells that is decisive for tuning the immune response in dependence on the environmental milieu.
Listeria monocytogenes is a food-borne pathogen which causes mild to life threatening disease in humans. Ingestion of contaminated food delivers the pathogen to the gastrointestinal tract, where it crosses the epithelial barrier and spreads to internal organs. Type I interferons (IFN-I) are produced during infection and decrease host resistance after systemic delivery of L. monocytogenes. Here we show that mice benefit from IFN-I production following infection with L. monocytogenes via the gastrointestinal route. Intragastric infection lead to increased lethality of IFN-I receptor chain 1-deficient (Ifnar1-/-) animals and to higher bacterial numbers in liver and spleen. Compared to infection from the peritoneum, bacteria infecting via the intestinal tract localized more often to periportal and pericentral regions of the liver and less frequently to the margins of liver lobes. Vigorous replication of intestine-borne L. monocytogenes in the livers of Ifnar1-/- mice 48 h post infection was accompanied by the formation of large inflammatory infiltrates in this organ and massive death of surrounding hepatocytes. This was not observed in Ifnar1-/- mice after intraperitoneal infection. The inflammatory response to infection is shaped by alterations in splenic cytokine production, particularly IFN?, which differs after intragastric versus intraperitoneal infection. Taken together, our data suggest that the adverse or beneficial role of a cytokine may vary with the route of infection and that IFN-I are not harmful when infection with L. monocytogenes occurs via the natural route.
Multiparous Stat1-/- mice spontaneously develop mammary tumors with increased incidence: at an average age of 12 months, 55% of the animals suffer from mammary cancer, although the histopathology is heterogeneous. We consistently observed mosaic expression or down-regulation of STAT1 protein in wild-type mammary cancer evolving in the control group. Transplantation experiments show that tumorigenesis in Stat1-/- mice is partially influenced by impaired CTL mediated tumor surveillance. Additionally, STAT1 exerts an intrinsic tumor suppressing role by controlling and blocking proliferation of the mammary epithelium. Loss of STAT1 in epithelial cells enhances cell growth in both transformed and primary cells. The increased proliferative capacity leads to the loss of structured acini formation in 3D-cultures. Analogous effects were observed when Irf1-/- epithelial cells were used. Accordingly, the rate of mammary intraepithelial neoplasias (MINs) is increased in Stat1-/- animals: MINs represent the first step towards mammary tumors. The experiments characterize STAT1/IRF1 as a key growth inhibitory and tumor suppressive signaling pathway that prevents mammary cancer formation by maintaining growth control. Furthermore, they define the loss of STAT1 as a predisposing event via enhanced MIN formation.
Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.
In E?-myc transgenic animals lymphoma formation requires additional genetic alterations, which frequently comprise loss of p53 or overexpression of BCL-2. We describe that the nature of the "second hit" affects the ability of the immune system to contain lymphoma development. Tumors with disrupted p53 signaling killed the host more rapidly than BCL-2 overexpressing ones. Relaxing immunologic control, using Tyk2(-/-) mice or by Ab-mediated depletion of CD8(+) T or natural killer (NK) cells accelerated formation of BCL-2-overexpressing lymphomas but not of those lacking p53. Most strikingly, enforced expression of BCL-2 prolonged disease latency in the absence of p53, whereas blocking p53 function in BCL-2-overexpressing tumors failed to accelerate disease. This shows that blocking apoptosis in p53-deficient cells by enforcing BCL-2 expression can mitigate disease progression increasing the "immunologic visibility." In vitro cytotoxicity assays confirmed that high expression of BCL-2 protein facilitates NK and T cell-mediated killing. Moreover, we found that high BCL-2 expression is accompanied by significantly increased levels of the NKG2D ligand MULT1, which may account for the enhanced killing. Our findings provide first evidence that the nature of the second hit affects tumor immunosurveillance in c-MYC-driven lymphomas and define a potential shortcoming of antitumor therapies targeting BCL-2.
For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.
Tyrosine kinase 2 (Tyk2) is an integral part of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway which relays intracellular signals of various cytokines. Tyk2 crucially contributes to host defense mechanisms against microbial pathogens and to tumor surveillance but also facilitates immune pathologies. Here we investigated the impact of Tyk2 on the macrophage proteome using the synthetic double-stranded RNA analog polyinosinic acid-polycytidylic acid (poly(I:C)) as a mimicry of viral infections. By means of 2D-DIGE in connection with PMF obtained by MALDI-MS and sequence tag determination by MS/MS we unambiguously identified eighteen protein spots corresponding to sixteen distinct proteins that are regulated by poly(I:C) and differentially expressed between wildtype (WT) and Tyk2-deficient macrophages. The majority of these proteins are functionally assigned to cellular immune responses and to metabolism. We show for selected metabolic enzymes, i.e. triosephosphate isomerase (TIM), ATP-citrate synthase (ACLY) and long-chain-fatty-acid-CoA ligase 4 (ACSL4), that Tyk2 affects protein expression transcriptionally and post-transcriptionally. We furthermore confirm the involvement of Tyk2 in the regulation of lipid and carbohydrate metabolism at the level of metabolites. Taken together, our results provide new evidence for important functions of Tyk2 at the molecular interface between innate immunity and cellular metabolism.
T cells and level of the cytokine interferon-? (IFN-?) are increased in adipose tissue in obesity. Hedgehog (Hh) signaling has been shown to potently inhibit white adipocyte differentiation. In light of recent findings in neurons that IFN-? and Hh signaling cross-talk, we examined their potential interaction in the context of adipogenesis.
Antiviral innate immunity relies on the recognition of microbial structures. One such structure is viral RNA that carries a triphosphate group on its 5 terminus (PPP-RNA). By an affinity proteomics approach with PPP-RNA as the bait, we found that the antiviral protein IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) mediated binding of a larger protein complex containing other IFIT family members. IFIT1 bound PPP-RNA with nanomolar affinity and required the arginine at position 187 in a highly charged carboxy-terminal groove of the protein. In the absence of IFIT1, the growth and pathogenicity of viruses containing PPP-RNA was much greater. In contrast, IFIT proteins were dispensable for the clearance of pathogens that did not generate PPP-RNA. On the basis of this specificity and the great abundance of IFIT proteins after infection, we propose that the IFIT complex antagonizes viruses by sequestering specific viral nucleic acids.
The concept of protein cross-linking in combination with mass spectrometry holds great promise to derive structural information on protein conformation and protein-protein interactions. We recently presented a dissociative amine-reactive cross-linker (NHS-BuUrBu-NHS) that is shown herein to be universally applicable to protein structure analysis under matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) conditions, based on the examples of the peptides substance P, luteinizing hormone releasing hormone (LHRH), and the 32-kDa ligand-binding domain of peroxisome proliferator-activated receptor alpha (PPAR?). The characteristic fragment ion patterns and constant neutral losses of the cross-linker greatly simplify the identification of different cross-linked species from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Therefore, this cross-linker holds an enormous potential for deriving structural information of proteins and protein complexes in a highly automated fashion.
A central role for the mammalian target of rapamycin (mTOR) in innate immunity has been recently defined by its ability to limit proinflammatory mediators. Although glucocorticoids (GCs) exert potent anti-inflammatory effects in innate immune cells, it is currently unknown whether the mTOR pathway interferes with GC signaling. Here we show that inhibition of mTOR with rapamycin or Torin1 prevented the anti-inflammatory potency of GC both in human monocytes and myeloid dendritic cells. GCs could not suppress nuclear factor-?B and JNK activation, the expression of proinflammatory cytokines, and the promotion of Th1 responses when mTOR was inhibited. Interestingly, long-term activation of monocytes with lipopolysaccharide enhanced the expression of TSC2, the principle negative regulator of mTOR, whereas dexamethasone blocked TSC2 expression and reestablished mTOR activation. Renal transplant patients receiving rapamycin but not those receiving calcineurin inhibitors displayed a state of innate immune cell hyper-responsiveness despite the concurrent use of GC. Finally, mTOR inhibition was able to override the healing phenotype of dexamethasone in a murine lipopolysaccharide shock model. Collectively, these data identify a novel link between the glucocorticoid receptor and mTOR in innate immune cells, which is of considerable clinical importance in a variety of disorders, including allogeneic transplantation, autoimmune diseases, and cancer.
Interferons (IFNs) are key cytokines in the innate immune response that also bridge the gap to adaptive immunity. Signaling upon stimulation by IFN type I, II and III is mediated by the Jak-Stat pathway. STAT1 is activated by all three IFN receptor complexes and absence of STAT1 from mice increases their susceptibility to pathogens. In addition, depending on the setting, STAT1 can act as tumor suppressor or oncogene. Here we report the generation and detailed functional characterization of a conditional Stat1 knockout mouse. We show the integrity of the conditional Stat1 locus and report successful in vivo deletion by means of a ubiquitous and a tissue-specific Cre recombinase. The conditional Stat1 null allele represents an important tool for identifying novel and cell-autonomous STAT1 functions in infection and cancer.
Human fungal pathogens such as the dimorphic Candida albicans or the yeast-like Candida glabrata can cause systemic candidiasis of high mortality in immunocompromised individuals. Innate immune cells such as dendritic cells and macrophages establish the first line of defense against microbial pathogens and largely determine the outcome of infections. Among other cytokines, they produce type I IFNs (IFNs-I), which are important modulators of the host immune response. Whereas an IFN-I response is a hallmark immune response to bacteria and viruses, a function in fungal pathogenesis has remained unknown. In this study, we demonstrate a novel mechanism mediating a strong IFN-? response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-?/? receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-? release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-? and the ?-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-? release is strongly impaired by Src and Syk family kinase inhibitors and strictly requires completion of phagocytosis as well as phagosomal maturation. Strikingly, TLR7, MyD88, and IRF1 are essential for IFN-? signaling. Furthermore, in a mouse model of disseminated candidiasis we show that IFN-I signaling promotes persistence of C. glabrata in the host. Our data uncover for the first time a pivotal role for endosomal TLR7 signaling in fungal pathogen recognition and highlight the importance of IFNs-I in modulating the host immune response to C. glabrata.
Tyrolean Grey cattle represent a local breed with a population size of ?5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.
In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin ?1 and ?1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal ?1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin ?1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns.
IL-1beta is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1beta is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1beta expression in response to LPS is also regulated at the translational level. LPS-induced IL-1beta protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1beta is found intra- and extracellularly, irrespective of the efficiency of IL-1beta processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1beta mRNA with polysomes. Induction and stability of IL-1beta mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1beta production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1beta synthesis.
Chemical cross-linking combined with a subsequent enzymatic cleavage of the created cross-linked complex and a mass spectrometric analysis of the resulting cross-linked peptide mixture presents an alternative approach to high-resolution analysis, such as NMR spectroscopy or X-ray crystallography, to obtain low-resolution protein structures and to gain insight into protein interfaces. Here, we describe a novel urea-based cross-linker, which allows distinguishing different cross-linking products by collision-induced dissociation (CID) tandem MS experiments based on characteristic product ions and constant neutral losses. The novel cross-linker is part of our ongoing efforts in developing collision-induced dissociative reagents that allow an efficient analysis of cross-linked proteins and protein complexes. Our innovative analytical concept is exemplified for the Munc13-1 peptide and the recombinantly expressed ligand binding domain of the peroxisome proliferator-activated receptor alpha, for which cross-linking reaction mixtures were analyzed both by offline nano-HPLC/MALDI-TOF/TOF mass spectrometry and by online nano-HPLC/nano-ESI-LTQ-orbitrap mass spectrometry. The characteristic fragment ion patterns of the novel cross-linker greatly simplify the identification of different cross-linked species, namely, modified peptides as well as intrapeptide and interpeptide cross-links, from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Our novel urea-based CID cleavable cross-linker is expected to be highly advantageous for analyzing protein 3D structures and protein-protein complexes in an automated manner.
The fragmentation behavior of a novel thiourea-based cross-linker molecule specifically designed for collision-induced dissociation (CID) MS/MS experiments is described. The development of this cross-linker is part of our ongoing efforts to synthesize novel reagents, which create either characteristic fragment ions or indicative constant neutral losses (CNLs) during tandem mass spectrometry allowing a selective and sensitive analysis of cross-linked products. The new derivatizing reagent for chemical cross-linking solely contains a thiourea moiety that is flanked by two amine-reactive N-hydroxy succinimide (NHS) ester moieties for reaction with lysines or free N-termini in proteins. The new reagent offers simple synthetic access and easy structural variation of either length or functionalities at both ends. The thiourea moiety exhibits specifically tailored CID fragmentation capabilities--a characteristic CNL of 85 u--ensuring a reliable detection of derivatized peptides by both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry and as such possesses a versatile applicability for chemical cross-linking studies. A detailed examination of the CID behavior of the presented thiourea-based reagent reveals that slight structural variations of the reagent will be necessary to ensure its comprehensive and efficient application for chemical cross-linking of proteins.
Transcriptional regulation of the Nos2 gene encoding inducible nitric oxide synthase (iNOS) requires type I interferon (IFN-I) signaling and additional signals emanating from pattern recognition receptors. Here we showed sequential and cooperative contributions of the transcription factors ISGF3 (a complex containing STAT1, STAT2, and IRF9 subunits) and NF-kappaB to the transcriptional induction of the Nos2 gene in macrophages infected with the intracellular bacterial pathogen Listeria monocytogenes. NF-kappaB preceded ISGF3 at the Nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor TFIIH with the associated CDK7 kinase for serine 5 phosphorylation of the RNA polymerase II (pol II) carboxyterminal domain (CTD). Subsequent to TFIIH deposition by NF-kappaB, ISGF3 attracted the pol II enzyme and phosphorylation at CTD S5 occurred. Thus, STATs and NF-kappaB cooperate through pol II promoter recruitment and the phosphorylation of its CTD, respectively, as a prerequisite for productive elongation of iNOS mRNA.
Octamer-binding factor 6 (Oct-6, Pou3f1, SCIP, Tst-1) is a transcription factor of the Pit-Oct-Unc (POU) family. POU proteins regulate key developmental processes and have been identified from a diverse range of species. Oct-6 expression is described to be confined to the developing brain, Schwann cells, oligodendrocyte precursors, testes, and skin. Its function is primarily characterised in Schwann cells, where it is required for correctly timed transition to the myelinating state. In the present study, we report that Oct-6 is an interferon (IFN)-inducible protein and show for the first time expression in murine fibroblasts and macrophages.
Tyrosine kinase 2 (Tyk2), a central component of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling, has major effects on innate immunity and inflammation. Mice lacking Tyk2 are resistant to endotoxin shock induced by lipopolysaccharide (LPS), and Tyk2 deficient macrophages fail to efficiently induce interferon alpha/beta after LPS treatment. However, how Tyk2 globally regulates transcription of downstream target genes remains unknown. Here we examine the regulatory role of Tyk2 in basal and inflammatory transcription by comparing gene expression profiles of peritoneal macrophages from Tyk2 mutant and wildtype control mice that were either kept untreated or exposed to LPS for six hours.
Tumor necrosis factor (TNF-alpha) inhibitors, used for the treatment of common inflammatory diseases, currently belong among the most important biotechnologically produced pharmaceuticals. So far four TNF-alpha antagonists have been approved by regulatory authorities for defined subsets of applications. Furthermore, numerous approaches are being taken to develop new protein-based pharmaceuticals and to broaden their application areas in the treatment of TNF-alpha -related diseases. Both the fundamental understanding of disease-related TNF-alpha activity and the subsequent development of corresponding drug candidates demand the availability of large amounts of TNF-alpha as a bioactive protein. We have therefore established a protocol for the rapid high-level synthesis of recombinant human TNF-alpha in Escherichia coli shake-flask cultures and the subsequent purification of the mature protein. Using the advantages of SUMO-fusion technology we were able to produce protein with an authentic N-terminus in high yield. Two immobilized metal ion-affinity chromatography steps with a protease cleavage step in between and subsequent size-exclusion chromatography were utilized to purify the protein. The protein was obtained from the last chromatography step as a trimer, while purity was at least 96% as estimated by SDS-PAGE. The identity of the protein was confirmed by MALDI-TOF mass spectrometry. Recombinant mature TNF-alpha was correctly folded as assessed by CD spectroscopy and its biological activity was confirmed by an L929 cell assay.
Glucocorticoids (GC) are highly potent anti-inflammatory agents frequently administered in clinical medicine. However, even for the most potent GC dexamethasone, only modest effects have been observed in several murine studies. Here we demonstrate that intraperitoneal administration of dexamethasone displays no anti-inflammatory activity in two different mouse models. Low doses of topically applied dexamethasone entirely prevented ear swelling in a contact hypersensitivity model in BALB/c mice, while intraperitoneally injected dexamethasone had no effect on disease progression. Moreover, subcutaneously administered dexamethasone completely inhibited lipopolysaccharide (LPS)-mediated lethality in C57BL/6 mice. In contrast, even ultra-high doses of intraperitoneally injected dexamethasone could not prevent endotoxin-induced death. In conclusion, these results demonstrate that intraperitoneal application of dexamethasone is ineffective in these models of inflammation, which has broad implications for mouse models evaluating the in vivo efficiency of GCs.
Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offsprings oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygotes ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.
The polarization-dependent reflection spectra of fiber Bragg grating (FBG) sensors in polarization-maintaining fibers are influenced by shear strain. This influence can be evaluated from a tensorial coupled-mode theory approach. Yet, this approach requires the numerical integration of the four coupled-mode equations. We present an easy to handle, completely analytical treatment of the polarization-dependent reflection spectra of FBGs. We derive the required equations and compare the results to the numerical integration of the four tensorial coupled mode equations.
Originally identified as antiviral substances produced by infected cells, type I interferons (IFN-I) are now known to have a wide range of additional activities within both the innate and adaptive immune response. Here we review properties of IFN-I contributing to their natural immune adjuvant character, and their important role for the function of complete Freunds adjuvant (CFA) and the TLR9-dependent immune adjuvant IC31. We show data to demonstrate that treatment with IFN-I boosts the ability of vaccine/adjuvant combinations to induce peptide-specific CTL in both young and old mice. We view these findings in the perspective of previous clinical applications of IFN-I for vaccination.
Phosphorylation of transcription factor STAT-1 on Y701 regulates subcellular localization whereas phosphorylation of the transactivating domain at S727 enhances transcriptional activity. In this study, we investigate the impact of STAT-1 and the importance of transactivating domain phosphorylation on the induction of peptide-specific CTL in presence of the TLR9-dependent immune adjuvant IC31. STAT-1 deficiency completely abolished CTL induction upon immunization, which was strongly reduced in animals carrying the mutation of the S727 phospho-acceptor site. A comparable reduction of CTL was found in mice lacking the type I IFN (IFN-I) receptor, whereas IFN-gamma-deficient mice behaved like wild-type controls. This finding suggests that S727-phosphorylated STAT-1 supports IFN-I-dependent induction of CTL. In adoptive transfer experiments, IFN-I- and S727-phosphorylated STAT-1 were critical for the activation and function of dendritic cells. Mice with a T cell-specific IFN-I receptor ablation did not show impaired CTL responses. Unlike the situation observed for CTL development S727-phosphorylated STAT-1 restrained proliferation of naive CD8(+) T cells both in vitro and following transfer into Rag-deficient mice. In summary, our data reveal a dual role of S727-phosphorylated STAT-1 for dendritic cell maturation as a prerequisite for the induction of CTL activity and for T cell autonomous control of activation-induced or homeostatic proliferation.
Self-assembled monolayers of azobenzene-containing thiols on smooth Au(111) surfaces were studied by gap-mode surface-enhanced Raman spectroscopy (gap-mode SERS). By adsorption of colloidal Au nanoparticles on top of the organic adlayer highly reproducible spectra with strongly enhanced intensities are obtained. The observed bands indicate a trans conformation of the azobenzene moieties and are in agreement with structural data for the molecular layer. A characteristic dependency on the terminal and the spacer groups of the molecules is found. Samples prepared during illumination with UV light show pronounced spectral differences that can be attributed to azobenzene in cis conformation.
IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls proinflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is TNF-alpha, a potent proinflammatory mediator that is the target for multiple anti-TNF-alpha clinical strategies in Crohns disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. In this study, we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNF-alpha and IL-1alpha in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3 untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38 MAPK activity. The reduction of p38 MAPK activity releases TTP from the p38 MAPK-mediated inhibition, thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.
We report the combination of chemical cross-linking and high-resolution mass spectrometry for analyzing conformational changes in target proteins that are induced by drug binding. With this approach conformational changes in the peroxisome proliferator-activated receptor alpha (PPARalpha) upon binding of low-molecular weight compounds were readily detected, proving that the strategy provides a basis to efficiently characterize target protein-drug interactions.
Reconstruction of the strain tensor at the position of an embedded fiber Bragg grating sensor has been the goal of recent research. However, ambiguities in the measurand--the polarization resolved reflected intensity spectrum--upon occurrence of shear strain hinder its achievement due to lack of an invertible model. In this work, we derive such a model using coherency matrix properties of unpolarized light. We deduce simplified sensor parameters for the ambiguous shear strain loading case, which possibly lead to a practical inversion of the problem.
True hyphal growth of Candida albicans can be induced by several environmental conditions and contributes significantly to the high virulence of this pathogenic fungus. The transcriptional network that governs hyphal morphogenesis is complex, depends on several regulators and is not completely understood. Recently, CaUME6, a homolog of the Saccharomyces cerevisiae UME6 gene, has been shown to be required for hyphal elongation. In the present study, the C. albicans ume6Delta strain showed a complete defect in hyphae formation under all the growth conditions tested. UME6 was repressed by the Nrg1-Tup1 repressor in yeast-form cells but NRG1 was not repressed by Ume6p under hyphal growth conditions. Wild-type UME6 expression depended on each hyphal regulator tested, and ectopic UME6 expression in efg1Delta, cph1Delta and ras1Delta cells rescued the hyphal defects of these mutants under some hyphal growth conditions. Thus, UME6 is a common downstream target of regulators promoting hyphal development. Ectopic UME6 expression promoted both germ tube formation and hyphal elongation. The expression of all hyphae-specific genes investigated depended on UME6 expression. A model for transcriptional regulation of hyphal development and the role of Ume6p is proposed.
Production of type I interferons (IFN-I, mainly IFNalpha and IFNbeta) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized "interferon-producing cell" (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity. In mice infected with L. monocytogenes, systemic IFN-I was amplified via IFN-beta, the IFN-I receptor (IFNAR), and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characteristic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.
We showed previously that Tyk2(-/-) natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2(-/-) mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2(-/-) OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8(+) cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1(-/-) animals but not on IFNgamma or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1(-/-) and Tyk2(-/-) but not in IFNgamma(-/-) or IL12p35(-/-) mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2(-/-) OT-1 T cells were incapable of controlling EG7-induced tumor growth.
Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFN?-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1(f/f) CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1(-/-) NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1(f/f) animals), maturation was not altered. However, NK cells derived from Ifnar1(f/f) Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1(f/f )Ncr1-iCre compared with Ifnar1(f/f) control mice. Moreover, the ability of Ifnar1(f/f) Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance.
The mammalian Interferon induced transmembrane protein 1 (Ifitm1) gene was originally identified as a member of a gene family highly inducible by type I and type II interferons. Based on expression analyses, it was suggested to be required for normal primordial germ cell migration. The knockdown of Ifitm1 in mouse embryos provided evidence for a role in somitogenesis. We generated the first targeted knockin allele of the Ifitm1 gene to systematically reassess all inferred functions. Sperm motility and the fertility of male and female mutant mice are as in wild type littermates. Embryonic somites and the adult vertebral column appear normal in homozygous Ifitm1 knockout mice, demonstrating that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. Proportions of leucocyte subsets, including granulocytes, monocytes, B-cells, T-cells, NK-cells, and NKT-cells, are unchanged in mutant mice. Based on a normal immune response to Listeria monocytogenes infection, there is no evidence for a dysfunction in downstream IFN? signaling in Ifitm1 mutant mice. Expression from the Ifitm1 locus from E8.5 to E14.5 is highly dynamic. In contrast, in adult mice, Ifitm1 expression is highly restricted and strong in the bronchial epithelium. Intriguingly, IFITM1 is highly overexpressed in tumor epithelia cells of human squamous cell carcinomas and in adenocarcinomas of NSCLC patients. These analyses underline the general importance of targeted in vivo studies for the functional annotation of the mammalian genome. The first comprehensive description of the Ifitm1 expression pattern provides a rational basis for the further examination of Ifitm1 gene functions. Based on our data, the fact that IFITM1 can function as a negative regulator of cell proliferation, and because the gene maps to chromosome band 11p15.5, previously associated with NSCLC, it is likely that IFITM1 in man has a key role in tumor formation.
Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.