Search

Iron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi. ispartof: Blood vol:141 issue:23 pages:2878-2890 ispartof: location:United States status: published

Environmental microbial triggers shape the development and functionality of the immune system. Alveolar macrophages (AMs), tissue-resident macrophages of the lungs, are in constant and direct contact with inhaled particles and microbes. Such exposures likely impact AM reactivity to subsequent challenges by immunological imprinting mechanisms referred to as trained immunity. Here, we investigated whether a ubiquitous microbial compound has the potential to induce AM training in vivo. We discovered that intranasal exposure to ambient amounts of lipopolysaccharide (LPS) induced a pronounced AM memory response, characterized by enhanced reactivity upon pneumococcal challenge. Exploring the mechanistic basis of AM training, we identified a critical role of type 1 interferon signaling and found that inhibition of fatty acid oxidation and glutaminolysis significantly attenuated the training effect. Notably, adoptive transfer of trained AMs resulted in increased bacterial loads and tissue damage upon subsequent pneumococcal infection. In contrast, intranasal pre-exposure to LPS promoted bacterial clearance, highlighting the complexity of stimulus-induced immune responses, which likely involve multiple cell types and may depend on the local immunological and metabolic environment. Collectively, our findings demonstrate the profound impact of ambient microbial exposure on pulmonary immune memory and reveal tissue-specific features of trained immunity.

Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21{degrees}C represents a chronic cold stress or the 30{degrees}C of the thermoneutral zone constitutes heat stress for the animals. In this study we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20{degrees}C, 25{degrees}C, and 30{degrees}C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that more pups were weaned from B6D2F1 hybrids compared to C57BL/6N mothers and that the number of weaned pups was strongly reduced if mothers of both strains were kept at 30{degrees}C. Furthermore, at 30{degrees}C mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any impact on adrenocortical activity in breeding and control mice. Independent of the ambient temperature however, we found that females raising pups showed elevated levels of fecal corticosterone metabolites (FCMs) compared to controls. Increased levels of stress hormone metabolites were measured specially around birth and during the third week of lactation. Our results provide no evidence for reduced or improved wellbeing of lactating mice at different ambient temperatures, but we found that a 30{degrees}C cage temperature impairs reproductive performance.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALLs) or precursor B-ALLs. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2P760Ltransformed cell models and ex vivo cultured TYK2P760L-mutated patient-derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell- type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection- associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH- promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen.

Tyrosine kinase 2 (TYK2) is a widely expressed receptor-associated kinase that is involved in signaling by a variety of cytokines with important immune regulatory activities. Absence of TYK2 in mice results in impaired NK cell maturation and antitumor activity, although underlying mechanisms are largely unknown. Using conditional ablation of TYK2 in NK cells we show that TYK2 is required for IFN-γ production by NK cells in response to IL-12 and for an efficient immune defense against Listeria monocytogenes. Deletion of TYK2 in NK cells did not impact NK cell maturation and IFN-γ production upon NK cell activating receptor (actR) stimulation. Similarly, NK cell–mediated tumor surveillance was unimpaired upon deletion of TYK2 in NK cells only. In line with the previously reported maturation-associated Ifng promoter demethylation, the less mature phenotype of Tyk2−/− NK cells correlated with an increased CpG methylation at the Ifng locus. Treatment with the DNA hypomethylating agent 5-aza-2-deoxycytidine restored the ability of Tyk2−/− NK cells to produce IFN-γ upon actR but not upon IL-12 stimulation. NK cell maturation was dependent on the presence of TYK2 in dendritic cells and could be rescued in Tyk2-deficient mice by treatment with exogenous IL-15/IL-15Rα complexes. IL-15 treatment also rescued the in vitro cytotoxicity defect and the impaired actR-induced IFN-γ production of Tyk2−/− NK cells. Collectively, our findings provide the first evidence, to our knowledge, for a key role of TYK2 in the host environment in promoting NK cell maturation and antitumor activity.

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-α/β 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.

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.

The mammalian target of rapamycin (mTOR) is a key signaling kinase associated with a variety of cellular functions including the regulation of immunological and inflammatory responses. Classical mTOR inhibitors such as rapamycin or everolimus are commonly used in transplant as well as cancer patients to prevent transplant rejection or cancer progression, respectively. Noninfectious drug-induced pneumonitis is a frequent side effect in mTOR-inhibitor-treated patients. Therefore, we tested the effects of the mTOR inhibitor everolimus and the novel dual PI3K/mTOR inhibitor NVP-BEZ235 in a murine lipopolysaccharide (LPS)-induced acute lung injury model. C57BL/6 mice were treated with either everolimus or NVP-BEZ235 on two consecutive days prior to intratracheal administration of LPS. LPS administration induced a significant increase in total cell, neutrophil and erythrocyte numbers in the bronchoalveolar lavage fluid. Histological examination revealed a serious lung injury as shown by interstitial edema, vascular congestion and mononuclear cell infiltration in these mice after 24 hours. Everolimus as well as NVP-BEZ235 did not noticeable affect overall histopathology of the lungs in the lung injury model. However, NVP-BEZ235 enhanced IL-6 and TNF-α expression after 24 hours. In contrast, everolimus did not affect IL-6 and TNF-α levels. Interestingly, both inhibitors reduced inflammatory cytokines in an LPS/oleic acid-induced lung injury model. In conclusion, the mTOR inhibitors did not worsen the overall histopathological severity, but they exerted distinct effects on proinflammatory cytokine expression in the lung depending on the lung injury model applied.

Humanized mice are crucial tools for studying human pathogens in systemic situations. An animal model of human coronavirus infectious disease has been generated by gene transfer of the human receptor for virus-cell interaction (aminopeptidase N, APN, CD13) into mice. We showed that in vitro and in vivo infections across the species barrier differ in their requirements. Transgenic cells were susceptible to human coronavirus HCoV-229E infection demonstrating the requirement of hAPN for viral cell entry. Transgenic mice, however, could not be infected suggesting additional requirements for in vivo virus susceptibility. Crossing hAPN transgenic mice with interferon unresponsive Stat1(-/- )mice resulted in markedly enhanced virus replication in vitro but did not result in detectable virus replication in vivo. Adaptation of the human virus to murine cells led to successful infection of the humanized transgenic mice. Future genetic engineering approaches are suggested to provide animal models for the better understanding of human infectious diseases.

Human coronavirus (HCoV) 229E is a group 1 coronavirus and is specific to humans. So far, no animal model is available to study the pathogenesis of infection by HCoV-229E. We show here that the expression of aminopeptidase N (APN, also termed CD13), the receptor for HCoV-229E, is required but not sufficient to confer susceptibility in vivo . HCoV-229E infection was facilitated by crossing APN transgenic mice into signal transducers and activators of transcription ( Stat ) 1 null mice and by adaptation of HCoV-229E to grow in primary APN transgenic, Stat1 null fibroblasts. Double transgenic mice allow the study of human coronavirus group 1 infections in an animal model, in particular, viral tropism, replication, recombination, and spread in an immunocompromised situation. Furthermore, these mice provide an important tool for the evaluation of biosafety and efficacy of coronavirus-based vectors.

In the intestinal tract, IL-22 activates STAT3 to promote intestinal epithelial cell (IEC) homeostasis and tissue healing. The mechanism has remained obscure, but we demonstrate that IL-22 acts via tyrosine kinase 2 (Tyk2), a member of the Jak family. Using a mouse model for colitis, we show that Tyk2 deficiency is associated with an altered composition of the gut microbiota and exacerbates inflammatory bowel disease. Colitic Tyk2(-/-) mice have less p-STAT3 in colon tissue and their IECs proliferate less efficiently. Tyk2-deficient primary IECs show reduced p-STAT3 in response to IL-22 stimulation, and expression of IL-22-STAT3 target genes is reduced in IECs from healthy and colitic Tyk2(-/-) mice. Experiments with conditional Tyk2(-/-) mice reveal that IEC-specific depletion of Tyk2 aggravates colitis. Disease symptoms can be alleviated by administering high doses of rIL-22-Fc, indicating that Tyk2 deficiency can be rescued via the IL-22 receptor complex. The pivotal function of Tyk2 in IL-22-dependent colitis was confirmed in Citrobacter rodentium-induced disease. Thus, Tyk2 protects against acute colitis in part by amplifying inflammation-induced epithelial IL-22 signaling to STAT3.

Bacterial pathogens are known for their wide range of strategies to specifically adapt to host environments and infection sites. An in-depth understanding of these adaptation mechanisms is crucial for the development of effective therapeutics and new prevention measures. In this study, we assessed the suitability of Fourier Transform Infrared (FTIR) spectroscopy for monitoring metabolic adaptations of the bacterial pathogen Listeria monocytogenes to specific host genotypes and for exploring the potential of FTIR spectroscopy to gain novel insights into the host-pathogen interaction. Three different mouse genotypes, showing different susceptibility to L. monocytogenes infections, were challenged with L. monocytogenes and re-isolated bacteria were subjected to FTIR spectroscopy. The bacteria from mice with different survival characteristics showed distinct IR spectral patterns, reflecting specific changes in the backbone conformation and the hydrogen-bonding pattern of the protein secondary structure in the bacterial cell. Coupling FTIR spectroscopy with chemometrics allowed us to link bacterial metabolic fingerprints with host infection susceptibility and to decipher longtime memory effects of the host on the bacteria. After prolonged cultivation of host-passaged bacteria under standard laboratory conditions, the host's imprint on bacterial metabolism vanished, which suggests a revertible metabolic adaptation of bacteria to host environment and loss of host environment triggered memory effects over time. In summary, our work demonstrates the potential and power of FTIR spectroscopy to be used as a fast, simple and highly discriminatory tool to investigate the mechanism of bacterial host adaptation on a macromolar and metabolic level.

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, STAT1α and STAT1β, differ with regard to a C-terminal transactivation domain, which is absent in STAT1β. STAT1β is considered to be transcriptionally inactive and to be a competitive inhibitor of STAT1α. To investigate the functions of the STAT1 isoforms in vivo, we generated mice deficient for either STAT1α or STAT1β. As expected, the functions of STAT1α and STAT1β in IFN-α/β- and IFN-λ-dependent antiviral activity are largely redundant. In contrast to the current dogma, however, we found that STAT1β is transcriptionally active in response to IFN-γ. In the absence of STAT1α, STAT1β shows more prolonged IFN-γ-induced phosphorylation and promoter binding. Both isoforms mediate protective, IFN-γ-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 STAT1β's function will help fine-tune diagnostic approaches and help design more specific strategies to interfere with STAT1 activity.

STAT1 is an essential transcription factor regulating interferon (IFN) dependent gene expression and STAT1-deficient humans and mice are highly susceptible to bacterial and viral infections. Alternative splicing of STAT1 results in a full-length STAT1α and a truncated STAT1β isoform, which lacks parts of the C-terminal transactivation domain. We generated STAT1α and STAT1β only expressing mice and could show that STAT1β is not merely a dominant negative factor but is capable of contributing to innate host defence against bacterial infection in vivo , albeit less efficient than STAT1α. Consistently, we found that STAT1α and STAT1β drive distinct transcriptional profiles in response to IFNγ: around half of STAT1-regulated genes were equally well induced by either STAT1α or STAT1β, whereas other target genes showed reduced, delayed or even no induction in the presence of STAT1β only. Phosphorylation and nuclear localisation of STAT1β was prolonged in the absence of STAT1α. This correlated with prolonged binding to target gene promoters but, surprisingly, did not lead to a generally enhanced response as only a small subset of genes showed increased expression at late time points. Phosphorylated STAT1β was not resistant to phosphatases, suggesting that the impaired induction of the negative regulator suppressor of cytokine signalling 1 (SOCS1) by STAT1β causes its more persistent activation. In contrast to IFNγ, IFNα/β- and IFNλ-dependent immune responses were similar in the presence of either one of the STAT1 isoforms. Using site-directed chromatin immunoprecipitation (ChIP) analysis we are currently investigating the molecular mechanisms of the STAT1 isoform specificity in IFNγ-mediated transcriptional activation. Supported by the Austrian Science Fund (FWF, SFB-F28 , DK-W1212 ).

The mitogen-activated protein kinase p38α senses environmental stressors and orchestrates inflammatory and immunomodulatory reactions. However, the molecular mechanism how p38α controls immunomodulatory responses in myeloid cells remains elusive. We found that in monocytes and macrophages, p38α activated the mechanistic target of rapamycin (mTOR) pathway in vitro and in vivo. p38α signaling in myeloid immune cells promoted interleukin (IL)-10 but inhibited IL-12 expression via mTOR and blocked the differentiation of proinflammatory CD4+ T helper 1 cells. Cellular stress induced p38α-mediated mTOR activation that was independent of phosphoinositide 3-kinase (PI3K) but dependent on the kinase MK2 and on the inhibition of TSC1/TSC2 (tuberous sclerosis gene 1 and 2), a negative regulatory complex of mTOR signaling. Remarkably, p38α and PI3K concurrently modulated mTOR to balance IL-12 and IL-10 expression. Our data links p38α to mTOR signaling in myeloid immune cells that is decisive for tuning the immune response in dependence on the environmental milieu.

In this study we investigated the role of Bruton's 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-α, 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-γ 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.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E)) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923E)in vitro. Tyk2(K923E) mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

Invasive fungal infections by Candida albicans (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (Ifnar1−/−), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6Chi monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in Ifnar1−/− mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage., Author Summary Inflammation constitutes a major host response in many microbial infections. Innate immune cells orchestrate the inflammatory response to kill pathogens and clear infections. However, invasive infections by pathogenic microbes including the fungus Candida albicans, can result in an uncontrolled hyper-inflammatory response, leading to severe host damage and sepsis. Type I interferons constitute a hallmark of protective innate immunity in viral and bacterial infections, but at the same time have been notoriously known for their sepsis-promoting effects in numerous experimental inflammation models. Here, we show that type I interferon-signaling mediates the lethal hyper-inflammatory response during systemic mouse infections with C. albicans. Following fungal infections, type I interferons promote the recruitment and activation of inflammatory monocytes and neutrophils to infected organs. The high abundance and activity of inflammatory phagocytes lead to fatal tissue damage. Remarkably, we show that the pharmacological suppression of these inflammatory cells with the drug pioglitazone reduces immunopathology and sepsis-related lethality, suggesting a novel therapeutic option to combat fungal sepsis. In conclusion, our data couple the sepsis-promoting role of type I interferons to the host-destructive activity of inflammatory monocytes and neutrophils. We propose that therapeutic approaches dampening hyper-inflammation might be of general importance in microbial diseases where deleterious immunopathology occurs.

Sepsis is initiated by the presence of bacteria, or their products (e.g. endotoxins), in the bloodstream. It still represents a major problem in health care due to high mortality rates and incomplete understanding of the molecular mechanisms driving the disease. The Janus kinase Tyk2 is part of the signal transduction cascade utilized by different cytokines that are crucial for innate and adaptive immune responses. We have reported previously that absence of Tyk2 in mice leads to an increased resistance against lipopolysaccharide (LPS)-induced endotoxin shock. Within this study, we show that mice expressing kinase-inactive Tyk2 (Tyk2K923E) show an intermediate resistance against LPS between Tyk2 knockout mice (Tyk2−/−) and wildtype (WT) mice. Systemic levels of pro-inflammatory cytokines, such as IL-1 β , IL-17A, IL-6 and IL-18, were decreased in Tyk2−/− and Tyk2K923E compared to WT mice, suggesting that kinase-independent functions of Tyk2 affect either local or late-stage immune responses. IFN α / β and IFN γ production as well as IFN α / β responsiveness were strongly impaired in both Tyk2−/− and Tyk2K923E mice. In line with this, activation of STAT1/2/3/4 in response to IL-12 and IFN β was similarly impaired in Tyk2−/− and Tyk2K923E NK cells and macrophages, respectively. Thus kinase-independent functions of Tyk2 are likely not connected to Tyk2’s function in IL-12 and IFN α / β signalling cascades. In addition to the kinase-independent functions of Tyk2, we provide evidence for a kinase-dependent involvement of Tyk2 in the regulation of the early hypothermic response to LPS. We are currently investigating late-stage immune responses and organ pathology. This project is funded by the Austrian Science Fund (FWF, grants P25642-B22 and SFB-F28 ).

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.

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-κB 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.

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.

Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing.

H.K. is supported by a grant from the affirmative action program for women of the School of Veterinary Medicine Hannover. T.L. is supported by a grant from the German Research council (DFG LE1032/14-1). The authors would like to thank Dr. Martine Yerle and Dr. Denis Milan, INRA, Laboratoire de Genetique Cellulaire, 31326 Castanet-Tolosan, France for providing the IMpRH panel and excellent information on the porcine RH map (http://imprh.toulouse.inra.fr/). We would also like to thank Pieter de Jong and his laboratory for providing the RPCI-44 BAC library.

Signal transducers and activators of transcription (STAT) 1 is a key transcription factor involved in innate and adaptive immune responses by governing the responses to interferons (IFNs) type I, II and III. In order to elucidate the cell type specific contributions of STAT1 to the host response to viral infections our lab generated conditional Stat1 mice. Cytomegalovirus (CMV) can trigger severe disease in immune suppressed individuals, is a frequent cause of congenital birth defects and still represents a major problem in health care. Immunity to acute and persistent CMV infection is intensively studied with respect natural killer (NK) and T cell function. Systemic STAT1 is required for defence against CMV but little is known about the contribution of STAT1 and innate immune cell function. Stat1ΔLysz mice showed highly efficient Stat1 allele depletion efficiencies in the myeloid compartment. Challenges of Stat1ΔLysz with various amounts of MCMV revealed no apparent effect of myeloid STAT1 to survival compared to wildtype mice. However, Stat1ΔLysz mice were deficient to restrict viral load in various organs and showed a high persistence of MCMV in salivary glands. With respect to early infection and viral spread we investigate cytokine expression and response activities, including cell type specific STAT activation. With respect to MCMV persistence we test the requirement of myeloid STAT1 for the activation of CD4+ and CD8+ T cells. This work was funded by the Austrian Science Fund (FWF) IAI DK W1212 and FWF SFB F28.

Partial reverse transcriptase sequencing of 16S rRNA from Mobiluncus curtisii and M. mulieris clearly indicate that the genus Mobiluncus is not a member of the Bacteroidaceae but belongs to the order Actinomycetales. The highest degree of relationship is found with the genus Actinomyces which is supported by the presence of common physiological properties.

To assess the role of the Janus kinase (Jak) family member Tyk2, we have generated Tyk2 -/- mice. In contrast to other Jaks, where inactivation leads to a complete loss of the respective cytokine receptor signal, Tyk2 -/- mice display reduced responses to IFNα/β and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNγ signaling is also impaired in Tyk2 -/- mice. Tyk2 -/- macrophages fail to produce nitric oxide upon lipopolysaccharide induction. Tyk2 -/- mice are unable to clear vaccinia virus and show a reduced T cell response after LCMV challenge. These data imply a selective contribution of Tyk2 to the signals triggered by various biological stimuli and cytokine receptors.