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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.

Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1β isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α (Stat1α/α) or the STAT1β (Stat1β/β ) isoform. NK cells from Stat1α/α mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from Stat1β/β mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 (Stat1-/- ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in Stat1β/β mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of Stat1β/β mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.

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.

Indian hemp is used since thousands of years as herbal drug. We found that a single dose of cannabis resin was equally active as Δ9-tetrahydrocannabinol (THC) enhancing severity and duration of symptoms in vaccinia virus infected mice. Cowpox virus did not cause symptomatic disease, but some reduction of specific antibody production was observed in drug treated animals. In vitro cannabis was superior to THC alone at inhibiting mitogen stimulated proliferation of human and mouse spleen cells and peripheral blood mononuclear cells. Also resin sub-fractions other than THC, cannabidiol and cannabinol, recovered also from cigarette smoke, were found inhibitory, suggesting additional involvement of constituents other than psychoactive THC. The immunoregulatory effects must be differentiated from apoptotic effects on spleen cells and lymphocytic mouse cell lines, which were observed with resin and THC but not with cannabidiol or cannabinol. A significant contribution of cytotoxic effects seems unlikely as drug treated lymphocytes were still capable of producing cytokines after T-cell receptor-specific stimulation. Considering a recent case of unusually severe cowpox virus infection in a young drug taker these data confirm a risk of “soft drugs” for acquiring poxvirus infection or enhancing side effects of the smallpox vaccine and perhaps also other live vaccines.

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.

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.

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.

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.

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.

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.