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Background: Restoring impaired peripheral immune tolerance is the primary challenge in treating autoimmune diseases. Our previous research demonstrated the effectiveness of small spleen peptides (SSPs), a fraction of low molecular weight proteins, in inhibiting the progression of psoriatic arthritis, even in the presence of high levels of the proinflammatory cytokine TNFα in the bloodstream. When specifically targeting dendritic cells (DCs), SSPs transform them into tolerogenic cells, which efficiently induce the development of regulatory Foxp3 + Treg cells. In this study, we provide further insights into the mechanism of action of SSPs., Results: We found that SSPs stimulate the activation of the mTOR signaling pathway in dendritic cells, albeit in a different manner than the classical immunogenic stimulus LPS. While LPS-induced activation is rapid, strong, and sustained, the activity induced by SSPs is delayed, less intense, yet still significant. These distinct patterns of activation, as measured by phosphorylation of key components of the pathway are also observed in response to other immunogenic and tolerogenic stimuli such as GM-CSF + IL-4 or IL-10 and TGFβ. The disparity in mTOR activation between immunogenic and tolerogenic stimuli is quantitative rather than qualitative. In both cases, mTOR activation primarily occurs through the PI3K/Akt signaling axis and involves ERK and GSK3β kinases, with minimal involvement of AMPK or NF-kB pathways. Furthermore, in the case of SSPs, mTOR activation seems to involve adenosine receptors. Additionally, we observed that DCs treated with SSPs exhibit an energy metabolism with high plasticity, which is typical of tolerogenic cells rather than immunogenic cells., Conclusion: Hence, the decision whether dendritic cells enter an inflammatory or tolerogenic state seems to rely on varying activation thresholds and kinetics of the mTOR signaling pathway., (© 2024. The Author(s).)

Influenza virus is a well-known respiratory pathogen, which still leads to many severe pulmonary infections in the human population every year. Morbidity and mortality rates are further increased if virus infection coincides with co-infections or superinfections caused by bacteria such as Streptococcus pneumoniae ( S. pneumoniae ) and Staphylococcus aureus ( S. aureus ). This enhanced pathogenicity is due to complex interactions between the different pathogens and the host and its immune system and is mainly governed by altered intracellular signaling processes. In this review, we summarize the recent findings regarding the innate and adaptive immune responses during co-infection with influenza virus and S. pneumoniae or S. aureus , describing the signaling pathways involved and how these interactions influence disease outcomes.

Cells respond to extracellular agents by activation of intracellular signaling pathways. Viruses can be regarded as such agents, leading to a firework of signaling inside the cell, primarily induced by pathogen-associated molecular patterns (PAMPs) that provoke safeguard mechanisms to defend from the invader. In the constant arms race between pathogen and cellular defense, viruses not only have evolved mechanisms to suppress or misuse supposedly antiviral signaling processes for their own benefit but also actively induce signaling to promote replication. This creates viral dependencies that may be exploited for novel strategies of antiviral intervention. Here, we will summarize the current knowledge of activation and function of influenza virus-induced signaling pathways with a focus on nuclear factor (NF)-κB signaling, mitogen-activated protein kinase cascades, and the phosphatidylinositol-3-kinase pathway. We will discuss the opportunities and drawbacks of targeting these signaling pathways for antiviral intervention., (Copyright © 2021 Cold Spring Harbor Laboratory Press; all rights reserved.)

Background: The airway epithelium is a major target tissue in respiratory infections, and its antiviral response is mainly orchestrated by the interferon regulatory factor-3 (IRF3), which subsequently induces type I (β) and III (λ) interferon (IFN) signalling. Dual specificity mitogen-activated protein kinase kinase (MEK) pathway contributes to epithelial defence, but its role in the regulation of IFN response in human primary airway epithelial cells (AECs) is not fully understood. Here, we studied the impact of a small-molecule inhibitor (MEKi) on the IFN response following challenge with two major respiratory viruses rhinovirus (RV2) and respiratory syncytial virus (RSVA2) and a TLR3 agonist, poly(I:C)., Methods: The impact of MEKi on viral load and IFN response was evaluated in primary AECs with or without a neutralising antibody against IFN-β. Quantification of viral load was determined by live virus assay and absolute quantification using qRT-PCR. Secretion of cytokines was determined by AlphaLISA/ELISA and expression of interferon-stimulated genes (ISGs) was examined by qRT-PCR and immunoblotting. A poly(I:C) model was also used to further understand the molecular mechanism by which MEK controls IFN response. AlphaLISA, siRNA-interference, immunoblotting, and confocal microscopy was used to investigate the effect of MEKi on IRF3 activation and signalling. The impact of MEKi on ERK and AKT signalling was evaluated by immunoblotting and AlphaLISA., Results: Here, we report that pharmacological inhibition of MEK pathway augments IRF3-driven type I and III IFN response in primary human AECs. MEKi induced activation of PI3K-AKT pathway, which was associated with phosphorylation/inactivation of the translational repressor 4E-BP1 and activation of the protein synthesis regulator p70 S6 kinase, two critical translational effectors. Elevated IFN-β response due to MEKi was also attributed to decreased STAT3 activation, which consequently dampened expression of the transcriptional repressor of IFNB1 gene, PRDI-BF1. Augmented IFN response translated into inhibition of rhinovirus 2 replication in primary AECs but not respiratory syncytial virus A2., Conclusions: Our findings unveil MEK as a key molecular mechanism by which rhinovirus dampens the epithelial cell's antiviral response. Our study provides a better understanding of the role of signalling pathways in shaping the antiviral response and suggests the use of MEK inhibitors in anti-viral therapy against RV.

Influenza is still one of the major plagues worldwide with the threatening potential to cause pandemics. In recent years, increasing levels of resistance to the four FDA approved anti-influenza virus drugs have been described. This situation underlines the urgent need for novel anti-virals in preparation for future influenza epidemics or pandemics. Although the anti-virals currently in use target viral factors such as the neuraminidase or the M2 ion channel, there is an increase in pre-clinical approaches that focus on cellular factors or pathways that directly or indirectly interact with virus replication. This does not only include inhibitors of virus-supportive signaling cascades but also interaction blockers of viral proteins with host cell proteins. This review aims to highlight some of these novel approaches that represent a paradigm change in anti-viral strategies against the influenza virus. Although most of these approaches are still in an early phase of preclinical development they might be very promising particularly with respect to the prevention of viral resistance to potential drugs.

H5N1 influenza virus infections in humans cause a characteristic systemic inflammatory response syndrome; however, the molecular mechanisms are largely unknown. Endothelial cells (ECs) play a pivotal role in hyperdynamic septic diseases. To unravel specific signaling networks activated by H5N1 we used a genome-wide comparative systems biology approach analyzing gene expression in human ECs infected with three different human and avian influenza strains of high and low pathogenicity. Blocking of specific signaling pathways revealed that H5N1 induces an exceptionally NF-κB-dependent gene response in human endothelia. Additionally, the IFN-driven antiviral program in ECs is shown to be dependent on IFN regulatory factor 3 but significantly impaired upon H5N1 infection compared with low pathogenic influenza virus. As additional modulators of this H5N1-specific imbalanced gene response pattern, we identified HMGA1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not antiviral response, whereas NFATC4 was found to regulate transcription of specifically H5N1-induced genes. We describe for the first time, to our knowledge, defined signaling patterns specifically activated by H5N1, which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of an overwhelming proinflammatory and impaired antiviral gene program.

Pathogens such as influenza A viruses (IAV) have to overcome a number of barriers defined and maintained by the host, to successfully establish an infection. One of the initial barriers is collectively characterized as the innate immune system. This is a broad anti-pathogen defense program that ranges from the action of natural killer cells to the induction of an antiviral cytokine response. In this article we will focus on new developments and discoveries concerning the interaction of IAV with the cellular innate immune signaling. We discuss new mechanisms of interference of IAV with the pathogen recognition receptor RIG-I and the type I IFN antagonist NS1 in the background of already known and established concepts. Further we summarize progress related to recently identified IFN induced proteins and the role of RNA interference in the context of IAV infection.

Influenza is still one of the major plagues worldwide with the potential to cause pandemics. The increasing frequency of viral resistance to the four US Food and Drug Administration (FDA)-approved anti-influenza virus drugs underlines the urgent need for novel antivirals to be prepared for future influenza epidemics or pandemics. While the antivirals currently in use exclusively target viral factors, such as neuraminidase or the M2 ion channel, several pre-clinical approaches now focus on cellular factors or pathways that directly or indirectly interact with virus replication. Among these, inhibitors of intracellular signalling cascades that are essential for virus replication have been unravelled as the most promising candidates. This short article aims to highlight two of these novel approaches, namely, inhibition of the classical mitogenic Raf/MEK/ERK kinase cascade and blockade of the pathway that leads to activation of the transcription factor NF-kappaB. It has been shown that inhibition of both virus-induced pathways leads to impaired virus production in vitro and in vivo without side effects or the tendency to induce resistant virus variants. Besides the direct antiviral effect, such inhibitors may also exert additional beneficial effects by blocking the cytokine burst that contributes to the severity of infections by highly pathogenic influenza virus strains. Although these novel strategies are still in an early phase of pre-clinical development they might be very promising, especially with regard to prevention of viral resistance.

Upon influenza A virus infection of cells, a wide variety of antiviral and virus-supportive signalling pathways are induced. Phosphatidylinositol-3-kinase (PI3K) is a recent addition to the growing list of signalling mediators that are activated by these viruses. Several studies have addressed the role of PI3K and the downstream effector protein kinase Akt in influenza A virus-infected cells. PI3K/Akt signalling is activated by diverse mechanisms in a biphasic manner and is required for multiple functions during infection. While the kinase supports activation of the interferon regulatory factor-3 during antiviral interferon induction, it also exhibits virus supportive functions. In fact, PI3K not only regulates a very early step during viral entry but also results in suppression of premature apoptosis at later stages of infection. The latter function is dependent on the expression of the viral non-structural protein-1 (A/NS1). It has been shown that PI3K activation occurs by direct interaction of A/NS1 with the p85 regulatory subunit and interaction sites of A/NS1 and p85 have now been mapped in detail. Here, we summarize the current knowledge on influenza virus-induced PI3K signalling and how this pathway supports viral propagation.

The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNbeta gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNbeta gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNalpha/beta, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5' triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3beta and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.

Replication and transcription of the influenza virus genome takes place exclusively within the nucleus of the infected cells. The viral RNA genome, polymerase subunits, and nucleoprotein form ribonucleoprotein (RNP) complexes. Late in the infectious cycle RNPs have to be exported from the nucleus to be enwrapped into budding progeny virions at the cell membrane. This process requires viral activation of the cellular Raf/MEK/ERK (mitogen-activated protein kinase (MAPK)) signaling cascade that is activated late in the infection cycle. Accordingly, block of the cascade results in retardation of RNP export and reduced titers of progeny virus. In the present study we have analyzed the importance of cell-membrane association of the viral hemagglutinin glycoprotein for viral MAPK activation. We show that hemagglutinin membrane accumulation and its tight association with lipid-raft domains trigger activation of the MAPK cascade via protein kinase Calpha activation and induces RNP export. This may represent an auto-regulative mechanism that coordinates timing of RNP export to a point when all viral components are ready for virus budding.

Small RNA viruses such as influenza viruses extensively manipulate host-cell functions to support their replication. At the same time the infected cell induces an array of defence mechanisms to fight the invader. These processes are mediated by a variety of intracellular signalling cascades. Here we will review the current knowledge of functional kinase signalling and apoptotic events in influenza virus infected cells and how these viruses have learned to misuse these cellular responses for efficient replication.

Influenza virus is a paradigm for a pathogen that frequently crosses the species barrier from animals to humans, causing severe disease in the human population. This ranges from frequent epidemics to occasional pandemic outbreaks with millions of death. All previous pandemics in humans were caused by animal viruses or virus reassortants carrying animal virus genes, underlining that the fight against influenza requires a One Health approach integrating human and veterinary medicine. Furthermore, the fundamental question of what enables a flu pathogen to jump from animals to humans can only be tackled in a transdisciplinary approach between virologists, immunologists and cell biologists. To address this need the German FluResearchNet was established as a first nationwide influenza research network that virtually integrates all national expertise in the field of influenza to unravel viral and host determinants of pathogenicity and species transmission and to explore novel avenues of antiviral intervention. Here we focus on the various novel anti-flu approaches that were developed as part of the FluResearchNet activities. [ABSTRACT FROM AUTHOR]

MK2 and MK3 are downstream targets of p38 and ERK1/2. They control the mRNA stability of several inflammatory cytokines, including TNF-α and IL-10. Whereas MK2 is expressed ubiquitously, the expression of MK3 is restricted to muscle, liver, and heart tissues and T and NK cells. Using Aft-deficient and wild-type (WT) mice, we demonstrated an inhibitory effect of MK3, but not of MK2, on interferon (IFN)-γ expression in T and NK lymphocytes. The results provided evidence that the inhibitory effect of MK3 is based on negative feedback phosphorylation of p38 and ERK1/2, which causes decreased binding of Stat4 to the IFN-γ promoter and reduced expression of IFN-γ mRNA and protein. Consequently, all Mk3-/- mice challenged with the Thl-inducing influenza A virus (IAV) survived the WT LD50 virus dose. The reduced disease severity in the Mk3-/- mice was accompanied by a > 10-fold reduction in viral lung titer and an increase in the number of activated NK cells and enhanced Th1 activation of CD4 T cells. Thus, our data describe the protein kinase MK3 as a novel regulator of the innate and adaptive immune responses. [ABSTRACT FROM AUTHOR]

Infections by influenza A viruses (IAV) are a major health burden to mankind. The current antiviral arsenal against IAV is limited and novel drugs are urgently required. Medicinal plants are known as an abundant source for bioactive compounds, including antiviral agents. The aim of the present study was to characterize the anti-IAV potential of a proanthocyanidin-enriched extract derived from the aerial parts of Rumex acetosa (RA), and to identify active compounds of RA, their mode of action, and structural features conferring anti-IAV activity. In a modified MTT (MTTIAV) assay, RA was shown to inhibit growth of the IAV strain PR8 (H1N1) and a clinical isolate of IAV(H1N1)pdm09 with a half-maximal inhibitory concentration (IC50) of 2.5 µg/mL and 2.2 µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 32 and 36, respectively. At RA concentrations>1 µg/mL plaque formation of IAV(H1N1)pdm09 was abrogated. RA was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. TNF-α and EGF-induced signal transduction in A549 cells was not affected by RA. The dimeric proanthocyanidin epicatechin-3-O-gallate-(4β→8)-epicatechin-3′-O-gallate (procyanidin B2-di-gallate) was identified as the main active principle of RA (IC50 approx. 15 µM, SI≥13). RA and procyanidin B2-di-gallate blocked attachment of IAV and interfered with viral penetration at higher concentrations. Galloylation of the procyanidin core structure was shown to be a prerequisite for anti-IAV activity; o-trihydroxylation in the B-ring increased the anti-IAV activity. In silico docking studies indicated that procyanidin B2-di-gallate is able to interact with the receptor binding site of IAV(H1N1)pdm09 hemagglutinin (HA). In conclusion, the proanthocyanidin-enriched extract RA and its main active constituent procyanidin B2-di-gallate protect cells from IAV infection by inhibiting viral entry into the host cell. RA and procyanidin B2-di-gallate appear to be a promising expansion of the currently available anti-influenza agents. [ABSTRACT FROM AUTHOR]

Influenza A virus remains a major public health concern, both in its annual toll in death and debilitation and its potential to cause devastating pandemics. Like any other virus, influenza A viruses are strongly dependent on cellular factors for replication. One of the hallmark signaling factors activated by viral pathogens is the transcription factor NF-κB. Activation of NF-κB leads to the up-regulation of a variety of antiviral genes. Thus, the factor is commonly regarded as a major regulator of the innate immune defense to infection. However, several recent studies indicate that influenza viruses have acquired the capability to reprogram this antiviral activity and to exploit the factor for efficient replication. These data provide novel insights into the pathophysiological function of NF-κB in the special environment of a virus-infected cell. Furthermore, the unexpected viral dependency on a cellular signaling factor may pave the path for novel antiviral approaches targeting essential cellular components rather than viral factors. [ABSTRACT FROM AUTHOR]

Influenza virus infection results in the activation of a variety of intracellular signaling responses. With regard to the function of these responses, the overall picture that has emerged suggests that most of the signaling events are initiated as a cellular response to defend the invading pathogen. While on the one hand influenza viruses have evolved strategies to keep these responses in a tolerable limit, there is accumulating evidence that the virus has also acquired the capability to exploit the remaining activities to ensure efficient replication. Here we will summarize the current knowledge on influenza virus-induced signaling processes and how these pathogens take advantage of some of these activities within the infected cell to support its propagation. [ABSTRACT FROM AUTHOR]

The anti-viral type I interferon (IFN) response is initiated by the immediate induction of IFNβ, which is mainly controlled by the IFN-regulatory factor-3 (IRF-3). The signaling pathways mediating viral IRF-3 activation are only poorly defined. We show that the Rho GTPase Rac1 is activated upon virus infection and controls IRF-3 phosphorylation and activity. Inhibition of Rac1 leads to reduced IFNβ promoter activity and to enhanced virus production. As a downstream mediator of Rac signaling towards IRF-3, we have identified the kinase p21-activated kinase (PAK1). Furthermore, both Rac1 and PAK1 regulate the recently described IRF-3 activators, IκB kinase-#x03B5; and TANK-binding kinase-1, establishing a first canonical virus-induced IRF-3 activating pathway. [Copyright &y& Elsevier]

Abstract: Despite the essential role of the fibrinogen γ-chain as a blood clotting factor, the fibrinogen γ-chain contains a number of interaction sites to recruit other factors such as leukocytes important for prevention of pathogen entry and propagation of the repair process. Interleukin-6 (IL-6) is known as the major inducer of γ-fibrinogen synthesis in hepatocytes, whereas IL-1β has been shown to act as a potent inhibitor of γ-fibrinogen expression. Studies on the rat fibrinogen γ-chain promoter suggest that nuclear factor (NF)-κB replaces the signal transducer and activator of transcription (STAT) 3 from binding to overlapping NF-κB/STAT3 binding sites within the 5′ regulatory region of the rat γ-chain gene promoter. However, despite its physiological relevance, the underlying mechanism responsible for the inhibitory effect of IL-1β in humans is still not understood and apparently more complex. In contrast to the mechanism described for the rat gene our results indicate that IL-1β suppresses the IL-6-induced activation of the human γ-fibrinogen gene particularly by blocking the late phase STAT3-tyrosine phosphorylation NF-κB-dependently but independent from de novo protein synthesis. Consequently, blocking NF-κB activation restores specifically late phase STAT3 activation as well as the induction of the human γ-fibrinogen gene. In contrast, specifically early STAT3 activation could be restored by a block of the p38 mitogen-activated protein kinase (p38MAPK) pathway. In summary, our results indicate that expression of the γ-fibrinogen gene is mainly controlled by the strength of late phase STAT3 activation, which in turn is negatively regulated by the extent of IL-1β-mediated NF-κB activity. [Copyright &y& Elsevier]