Caroline Lassnig

Partial impairment of cytokine responses in Tyk2-deficient mice.

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 IFNalpha/beta and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNgamma 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.

Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection.

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.

Type I Interferons Promote Fatal Immunopathology by Regulating Inflammatory Monocytes and Neutrophils during Candida Infections

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.

Inhibition of mTOR blocks the anti-inflammatory effects of glucocorticoids in myeloid immune cells

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.

UME6 is a crucial downstream target of other transcriptional regulators of true hyphal development in Candida albicans.

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.

Stable Production of Human Insulin-like Growth Factor 1 (IGF-1) in the Milk of Hemi- and Homozygous Transgenic Rabbits Over Several Generations

One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 ± 12μg/ml (ranging from 223 ± 61 to 484 ± 39 μg/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 ± 41 μg/ml (ranging from 360 ± 15 to 678 ± 80 μg/ml). Homozygous huIGF-1 females clearly showed a significantly increased production performance of the recombinant protein.

TYK2 kinase activity is required for functional type I interferon responses in vivo.

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 (Tyk2K923E) 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 TYK2K923E in vitro. Tyk2K923E 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.

Intestinal Epithelial Cell Tyrosine Kinase 2 Transduces IL-22 Signals To Protect from Acute Colitis

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.

p38α Senses Environmental Stress To Control Innate Immune Responses via Mechanistic Target of Rapamycin

The MAPK 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 IL-10 but inhibited IL-12 expression via mTOR and blocked the differentiation of proinflammatory CD4+ Th1 cells. Cellular stress induced p38α-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, p38α and PI3K concurrently modulated mTOR to balance IL-12 and IL-10 expression. Our data link p38α to mTOR signaling in myeloid immune cells that is decisive for tuning the immune response in dependence on the environmental milieu.

Diazepam leads to enhanced severity of orthopoxvirus infection and immune suppression.

Benzodiazepines are drugs widely used as tranquilizers and in various other indications. We treated Balb/c mice with diazepam and infected them with cowpox (CPXV) and vaccinia virus (VACV). Disease index, weight loss and the antibody response were determined. Additionally the influence of different benzodiazepines on the mitogen response of human peripheral blood lymphocytes and spleen cells was tested. Diazepam led to earlier disease onset, prolonged duration of symptoms, higher weight loss and overall disease index in VACV infected mice. CPXV infected mice developed poxviral skin lesions only after drug administration and a significant decrease in the specific antibody response was also observed. Diazepam and alprazolam also inhibited the proliferative response of human lymphocytes/spleen cells in vitro but did not show noteworthy apoptotic effects. It is surprising that even a single dose of diazepam has a profound influence on the immune system, sufficient to facilitate symptomatic infectious disease. These data provide first evidence that commonly used drugs like Valium may augment severity of rare poxvirus infections such as CPXV or monkeypox. As VACV is still used as life vaccine against smallpox there is also a risk of enhanced side effects or possible interference with the success of vaccination.