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Background: The current gold standard in HIV treatment fails to provide a definitive cure. Infected patients must adhere to a lifelong therapy burden, which on its own can impose side [...]

Background: Persistent latent reservoirs of intact HIV-1 proviruses, capable of rebounding despite suppressive antiretroviral therapy (ART), hinder efforts towards an HIV-1 cure. Hence, assays specifically quantifying intact proviruses are crucial to assess the impact of curative interventions. Two recent assays have been utilized in clinical trials: intact proviral DNA assay (IPDA) and quadruplex quantitative PCR (Q4PCR). While IPDA is more sensitive due to amplifying short fragments, it may overestimate intact fractions by relying only on quantification of 2 proviral regions. Q4PCR samples 4 proviral regions, yet is sequencing-based, favoring amplification of shorter, hence non-intact, proviral sequences., Methods: Leveraging digital PCR (dPCR) advancements, we developed the "Rainbow" 5-plex proviral HIV-1 DNA assay. This first-in-its-kind assay was evaluated using standard materials and samples from 83 people living with HIV-1, enabling simultaneous quantification of both total and intact HIV-1 DNA levels. HIV proviral unique molecular identifier (UMI)-mediated long-read sequencing (HIV-PULSE) was used to validate the specificity of the Rainbow HIV-1 DNA assay., Results: The Rainbow assay proved equally sensitive but more specific than IPDA and is not subjected to bias against full-length proviruses, enabling high-throughput quantification of total and intact reservoir size. The near full-length sequences allowed validation of the Rainbow specificity and the design of personalized Rainbow primer/probe sets, which enabled the detection of intact HIV-1 DNA., Conclusions: This innovation offers potential for targeted evaluation and monitoring of potential rebound-competent reservoirs, contributing to HIV-1 management and cure strategies. ClinicalTrials.gov Registration Numbers: NCT04553081, NCT04305665., (© Association for Diagnostics & Laboratory Medicine 2025.)

To prevent infection by respiratory viruses and consequently limit virus circulation, vaccines need to promote mucosal immunity. The extent to which the currently used messenger RNA (mRNA)-based COVID-19 vaccines induce mucosal immunity remains poorly characterized. We evaluated mucosal neutralizing antibody responses in a cohort of 183 individuals. Participants were sampled at several time points after primary adenovirus vector-based or mRNA-based COVID-19 vaccination and after mRNA-based booster vaccinations. Our findings revealed that repeated vaccination with mRNA boosters promoted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in nasal secretions. Nasal and serum neutralizing antibody titers of both IgG and IgA isotypes correlated to one another. We investigated the source of these mucosal antibodies in a mouse model wherein mice received repeated mRNA vaccines for SARS-CoV-2. These experiments indicated that neutralizing antibody-producing cells reside in the spleen and bone marrow, whereas no proof of tissue homing to the respiratory mucosa was observed, despite the detection of mucosal antibodies. Serum transfer experiments confirmed that circulating antibodies were able to migrate to the respiratory mucosa. Collectively, these results demonstrate that, especially upon repeated vaccination, the currently used COVID-19 mRNA vaccines can elicit mucosal neutralizing antibodies and that vaccination might also stimulate mucosal immunity induced by previous SARS-CoV-2 infection. Moreover, migration of circulating antibodies to the respiratory mucosa might be a main mechanism. These findings advance our understanding of mRNA vaccine-induced immunity and have implications for the design of vaccine strategies to combat respiratory infections.

Background: The events during acute HIV infection (AHI) set the stage for the subsequent course of the disease. Early initiation of antiretroviral therapy (ART) has been associated with favorable immunovirological outcomes, yet the precise impact of ART timing during AHI remains unclear, particularly on lymphoid tissues., Materials and Methods: The ACS cohort is a prospective cohort study in Belgium, collecting longitudinal clinical data and human bodily material (HBM) from people diagnosed and treated during AHI. The aim of the cohort is to study the impact of ART initiation during AHI on HIV reservoir and immune dysfunction in peripheral blood and anatomical sanctuary sites, as well as its effect on the gut microbiome. The cohort consists of two HBM sampling trajectories: one limited (blood, stool and leukapheresis) and a more extensive one (blood, stool, leukapheresis, colonoscopy, inguinal lymph node excision and lumbar puncture). Here we describe the baseline characteristics, immunovirological outcomes, safety and tolerability of HBM sampling., Results: Between March 2016 and April 2024, 47 participants were enrolled, predominantly men who have sex with men (MSM), with a median age of 36 years [IQR 30-43.5]. Almost 90 % of participants initiated ART within 72 h after study inclusion, irrespective of HBM sampling trajectory. The timing of ART initiation according to the Fiebig stage did not significantly impact immune recovery (CD4/CD8 ratio ≥1) or the time to viral suppression. Approximately 40 % of participants opted for the extensive HBM sampling trajectory during AHI. However, the participation rate for the extensive trajectory decreased by nearly half at the longitudinal follow-up timepoint. In general, study-related procedures were safe and well-tolerated, with limited procedure-related adverse events (AEs). Inguinal lymph node excision was associated with the highest AE rate, in line with previous reports., Conclusions: Our findings reaffirm the beneficial effect of ART initiation during AHI on long term immunovirological outcomes, regardless of Fiebig stage at treatment initiation. Additionally, we demonstrate that the collection of HBM during and longitudinally after AHI is safe and feasible, without compromising time to ART initiation. Cohorts that integrate comprehensive clinical data with high-quality HBM samples are essential to longitudinally study the impact of early ART on reservoir dynamics and immune responses across various anatomical sites after AHI., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Linos Vandekerckhove reports financial support and travel were provided by ViiV Healthcare. Jozefien De Clercq reports travel was provided by ViiV Healthcare. Sarah Gerlo reports travel was provided by ViiV Healthcare. Linos Vandekerckhove reports financial support was provided by Collen-Francqui Fund. Linos Vandekerckhove reports financial support provided by Research Foundation Flanders. Linos Vandekerckhove reports financial support provided by the Belgium Research on AIDS and HIV Consortium (BREACH). Linos Vandekerckhove reports a relationship with Gilead Sciences Inc that includes: consulting or advisory. Linos Vandekerckhove, co-author serves as an Editorial Board Member for Journal of Virus Eradication. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Background: Reducing the number of active compounds for lifelong HIV treatment is of interest, especially to reduce potential long-term side effects. So far, available data assessing viral control, support the robustness and safety of 2DR (2-drug regimen) ART compared to 3DR. However, further in-depth investigations of the viral reservoirs are mandatory to guarantee long-term safety of these regimens regarding stable intact HIV-1 DNA copies, HIV-1 RNA transcripts and sustained immunological control., Methods: The Rumba study is the first prospective randomized controlled trial evaluating the impact of switch from 3DR to 2DR on the viral reservoir. Participants on any stable 2nd generation INSTI-based 3DR regimen with HIV-1 RNA<50 copies/ml plasma for at least 3 months were randomized to switch to dolutegravir/lamivudine (DTG/3TC, N=89) or to switch or stay on bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF, N=45). After 48 weeks, virological, immunological and metabolic parameters were evaluated., Results: We did not observe a significant difference in change over time in the mean number of intact HIV-1 DNA copies/million CD4+ T cells with DTG/3TC compared to B/F/TAF. There was no evidence in this study that switching to DTG/3TC increased the active reservoir by HIV-1 transcription. No significant changes in pro-inflammatory cytokines or major immune cell subsets were observed. Changes in exhaustion and activation of specific cellular subsets were small and bidirectional. Metabolic outcomes are similar between the treatment regimens., Conclusions: This study confirms the safety of DTG/3TC compared to B/F/TAF through viral control after in-depth investigations of the intact HIV-1 reservoir, HIV-1 transcription and inflammatory markers., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non-overlapping epitopes., Methods: Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI)-based prediction to select non-competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs., Findings: Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI-based prediction employed with the intention to ensure non-overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor-binding epitope in a remarkably similar manner., Interpretation: Our findings indicate that, even in the Alphafold era, AI-based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection., Funding: Full list of funders is provided at the end of the manuscript., Competing Interests: Declaration of interests Ghent University has filed for patent protection on the antibody sequences described herein, and D.D.A., M.W., R.W., W.W., S.G. and L.V. are named as co-inventors on this patent (European Patent Application: 21186206.5). A.P. is employee of the MAbSilico, H.R. holds a patent regarding neutralizing VHH antibodies binding the Spike RBD (PCT/EP2021/052885) and has filed a priority application for neutralizing VHH antibodies binding Spike S2 (EP 23160838.1). X.S. is a recipient of FWO research project COVID-19 (G0G4920N) and FWO-FNRS project VIREOS (EOS ID: 30981113) grants., (Copyright © 2023. Published by Elsevier B.V.)

Background: Despite the beneficial effects of antiretroviral therapy (ART) initiation during acute HIV infection (AHI), residual immune activation remains a hallmark of treated HIV infection., Methods: Plasma concentrations of 40 mediators were measured longitudinally in 39 early treated participants of a Belgian AHI cohort (HIV+) and in 21 HIV-negative controls (HIV-). We investigated the association of the inflammatory profile with clinical presentation, plasma viral load, immunological parameters, and in-depth characterization of the HIV reservoir., Results: While levels of most soluble mediators normalized with suppressive ART, we demonstrated the persistence of a pro-inflammatory signature in early treated HIV+ participants in comparison to HIV- controls. Examination of these mediators demonstrated a correlation with their levels during AHI, which seemed to be viremia-driven, and suggested involvement of an activated myeloid compartment, IFN-γ-signaling, and inflammasome-related pathways. Interestingly, some of these pro-inflammatory mediators correlated with a larger reservoir size and slower reservoir decay. In contrast, we also identified soluble mediators which were associated with favorable effects on immunovirological outcomes and reservoir, both during and after AHI., Conclusion: These data highlight how the persistent pro-inflammatory profile observed in early ART treated individuals is shaped during AHI and is intertwined with viral dynamics., Competing Interests: LV has received consulting fees and travel grants from Gilead Sciences and ViiV Healthcare, paid to his institution. JDC and SG have received travel grants from ViiV Healthcare. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 De Clercq, De Scheerder, Mortier, Verhofstede, Vandecasteele, Allard, Necsoi, De Wit, Gerlo and Vandekerckhove.)

IL-1R integrates signals from IL-1α and IL-1β, and it is widely expressed across tissues and immune cell types. While the expression pattern and function of IL-1R within the innate immune system is well studied, its role in adaptive immunity, particularly within the CD8 T cell compartment, remains underexplored. Here, we show that CD8 T cells dynamically upregulate IL-1R1 levels during priming by APCs, which correlates with their proliferation status and the acquisition of an effector phenotype. Notably, this IL-1 sensitivity persists in memory CD8 T cells of both mice and humans, influencing effector cytokine production upon TCR reactivation. Furthermore, our study highlights that antiviral effector and tissue-resident CD8 T cell responses against influenza A virus infection become impaired in the absence of IL-1 signaling. Altogether, these data support the exploitation of IL-1 activity in the context of T cell vaccination strategies and warrant consideration of the impact of clinical IL-1 inhibition on the rollout of T cell immunity.

Introduction: To date, with no prophylactic human immunodeficiency virus (HIV) vaccine available, HIV incidence rates remain undefeated. Despite full virological suppression, HIV+ individuals exhibit a higher rate of cardiovascular disorders and cancers what is attributed to the residual, persistent levels of immune activation., Methods: We have established the Virological and Immunological Monitoring (VIM) platform and forty VIM samples that included treated immunological responders (IRs) or nonresponders (INRs), viremic untreated subjects and uninfected controls, were phenotyped by flow cytometry and plasma was used to quantify proinflammatory eicosanoids and the specialized proresolving mediators by liquid chromatography tandem mass spectrometry., Results: While HIV infection profoundly altered lipid mediator (LM) profile, differences were also seen in patients on viral suppressive therapy. IRs exhibited higher levels of proresolving mediators as compared to INRs and notable differences in plasma LM were also seen in early and late treated individuals., Conclusions: This study demonstrated distortions in proinflammatory/proresolution processes in infected patients including those with controlled viremia., (© 2021 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Despite the high efficacy of the BNT162b2 vaccine in the general population, data on its immunogenicity among frail elderly individuals are limited. Recently, levels of anti-SARS-CoV-2 spike IgG antibodies and serum neutralization titers were confirmed as good immune markers of protection against the virus, with evidence showing a reverse correlation between these two parameters and susceptibility to infection. Here we analyzed sera from 138 nursing home residents (median age of 88.9 years) and 312 nursing home staff (median age of 50.7 years) to determine the humoral response to two doses of the BNT162b2 vaccine, and found markedly decreased serum anti-spike antibody levels and neutralization titers in the nursing home resident (NHR) group, with over 11% non-responders compared to only 1.3% among the controls. Moreover, three months post-vaccination, a significant decrease in antibody titers was observed in COVID-19-naive nursing home residents. Subsequent flow cytometry and interferon gamma secretion analyses indicated that antibody non-responders among NHRs also failed to mount cellular responses. The presented data emphasize that additional measures are needed in the population of frail elderly individuals. Given the high proportion of non-responders among NHRs, continued monitoring should be considered in this group.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not always confined to the respiratory system, as it impacts people on a broad clinical spectrum from asymptomatic to severe systemic manifestations resulting in death. Further, accumulation of intra-host single nucleotide variants during prolonged SARS-CoV-2 infection may lead to emergence of variants of concern (VOCs). Still, information on virus infectivity and intra-host evolution across organs is sparse. We report a detailed virological analysis of thirteen postmortem coronavirus disease 2019 (COVID-19) cases that provides proof of viremia and presence of replication-competent SARS-CoV-2 in extrapulmonary organs of immunocompromised patients, including heart, kidney, liver, and spleen (NCT04366882). In parallel, we identify organ-specific SARS-CoV-2 genome diversity and mutations of concern N501Y, T1027I, and Y453F, while the patient had died long before reported emergence of VOCs. These mutations appear in multiple organs and replicate in Vero E6 cells, highlighting their infectivity. Finally, we show two stages of fatal disease evolution based on disease duration and viral loads in lungs and plasma. Our results provide insights about the pathogenesis and intra-host evolution of SARS-CoV-2 and show that COVID-19 treatment and hygiene measures need to be tailored to specific needs of immunocompromised patients, even when respiratory symptoms cease., (© 2021. The Author(s).)

Background: Clinical success of therapeutic cancer vaccines depends on the ability to mount strong and durable antitumor T cell responses. To achieve this, potent cellular adjuvants are highly needed. Interleukin-1β (IL-1β) acts on CD8 + T cells and promotes their expansion and effector differentiation, but toxicity and undesired tumor-promoting side effects hamper efficient clinical application of this cytokine., Methods: This 'cytokine problem' can be solved by use of AcTakines ( Ac tivity-on- Ta rget cyto kines ), which represent fusions between low-activity cytokine mutants and cell type-specific single-domain antibodies. AcTakines deliver cytokine activity to a priori selected cell types and as such evade toxicity and unwanted off-target side effects. Here, we employ subcutaneous melanoma and lung carcinoma models to evaluate the antitumor effects of AcTakines., Results: In this work, we use an IL-1β-based AcTakine to drive proliferation and effector functionality of antitumor CD8 + T cells without inducing measurable toxicity. AcTakine treatment enhances diversity of the T cell receptor repertoire and empowers adoptive T cell transfer. Combination treatment with a neovasculature-targeted tumor necrosis factor (TNF) AcTakine mediates full tumor eradication and establishes immunological memory that protects against secondary tumor challenge. Interferon-γ was found to empower this AcTakine synergy by sensitizing the tumor microenvironment to TNF., Conclusions: Our data illustrate that anticancer cellular immunity can be safely promoted with an IL-1β-based AcTakine, which synergizes with other immunotherapies for efficient tumor destruction., Competing Interests: Competing interests: Financial interests: NK and JT are affiliated and hold equity interests in Orionis Biosciences. The following patent applications are related to the work presented in this paper: WO/2015/007542: Targeted modified IL-1 family members. Applicants: VIB VZW, Universiteit Gent, Centre National de la Recherche Scientifique, Université Montpellier, Centre Hospitalier Regional Universitaire de Montpellier. Inventors: JT, SG, FP and GU This patent application describes IL-1β mutants with reduced bioactivity that can be activated by targeting. WO/2017/134306: CD8 binding agents. Applicants: Orionis Biosciences, VIB VZW, Universiteit Gent. Inventors: JT, AC, NK and SG. This patent application describes sdAbs that bind CD8 and that can be used to target mutant IL-1β to CTLs. WO/2015/007903: Targeted modified TNF family members. Applicants: VIB VZW, Universiteit Gent, Centre National de la Recherche Scientifique, Université Montpellier, Centre Hospitalier Regional Universitaire de Montpellier. This patent application describes TNF mutants with reduced bioactivity that can be activated by targeting. Inventors: JT, Jennyfer Bultinck, FP and GU. The authors have no other, nonfinancial, competing interests to declare., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

The three-signal paradigm tries to capture how the innate immune system instructs adaptive immune responses in three well-defined actions: (1) presentation of antigenic peptides in the context of MHC molecules, which allows for a specific T cell response; (2) T cell co-stimulation, which breaks T cell tolerance; and (3) secretion of polarizing cytokines in the priming environment, thereby specializing T cell immunity. The three-signal model provides an empirical framework for innate instruction of adaptive immunity, but mainly discusses STAT-dependent cytokines in T cell activation and differentiation, while the multi-faceted roles of type I IFNs and IL-1 cytokine superfamily members are often neglected. IL-1α and IL-1β are pro-inflammatory cytokines, produced following damage to the host (release of DAMPs) or upon innate recognition of PAMPs. IL-1 activity on both DCs and T cells can further shape the adaptive immune response with variable outcomes. IL-1 signaling in DCs promotes their ability to induce T cell activation, but also direct action of IL-1 on both CD4 + and CD8 + T cells, either alone or in synergy with prototypical polarizing cytokines, influences T cell differentiation under different conditions. The activities of IL-1 form a direct bridge between innate and adaptive immunity and could therefore be clinically translatable in the context of prophylactic and therapeutic strategies to empower the formation of T cell immunity. Understanding the modalities of IL-1 activity during T cell activation thus could hold major implications for rational development of the next generation of vaccine adjuvants., Competing Interests: JT is affiliated with Orionis Biosciences BV as scientific advisor and holds equity interests in Orionis Biosciences BV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Van Den Eeckhout, Tavernier and Gerlo.)

Since SARS-CoV-2 appeared in the human population, the scientific community has scrambled to gather as much information as possible to find good strategies for the containment and treatment of this pandemic virus. Here, we performed a systematic review of the current (pre)published SARS-CoV-2 literature with a focus on the evidence concerning SARS-CoV-2 distribution in human tissues and viral shedding in body fluids. In addition, this evidence is aligned with published ACE2 entry-receptor (single cell) expression data across the human body to construct a viral distribution and ACE2 receptor body map. We highlight the broad organotropism of SARS-CoV-2, as many studies identified viral components (RNA, proteins) in multiple organs, including the pharynx, trachea, lungs, blood, heart, vessels, intestines, brain, male genitals and kidneys. This also implicates the presence of viral components in various body fluids such as mucus, saliva, urine, cerebrospinal fluid, semen and breast milk. The main SARS-CoV-2 entry receptor, ACE2, is expressed at different levels in multiple tissues throughout the human body, but its expression levels do not always correspond with SARS-CoV-2 detection, indicating that there is a complex interplay between virus and host. Together, these data shed new light on the current view of SARS-CoV-2 pathogenesis and lay the foundation for better diagnosis and treatment of COVID-19 patients., Competing Interests: The authors have declared that no competing interests exist.

Annual administration and reformulation of influenza vaccines is required for protection against seasonal infections. However, the induction of strong and long-lasting T cells is critical to reach broad and potentially lifelong antiviral immunity. The NLRP3 inflammasome and its product interleukin-1β (IL-1β) are pivotal mediators of cellular immune responses to influenza, yet, overactivation of these systems leads to side effects, which hamper clinical applications. Here, we present a bypass around these toxicities by targeting the activity of IL-1β to CD8 + T cells. Using this approach, we demonstrate safe inclusion of IL-1β as an adjuvant in vaccination strategies, leading to full protection of mice against a high influenza virus challenge dose by raising potent T cell responses. In conclusion, this paper proposes a class of IL-1β-based vaccine adjuvants and also provides further insight in the mechanics of cellular immune responses driven by IL-1β., Competing Interests: Competing interestsFinancial interests: N.K. and J.T. are affiliated with Orionis Biosciences Inc. as scientific advisor and/or employee and hold equity interests in Orionis Biosciences Inc. N.K., J.T., S.G., F.P., and G.U. are inventors on the following patent applications related to the work presented in this paper: WO/2015/007542: Targeted modified IL-1 family members. Applicants: VIB VZW, Universiteit Gent, Centre National de la Recherche Scientifique, Université Montpellier, Centre Hospitalier Regional Universitaire de Montpellier. Inventors: J.T., S.G., F.P., and G.U. This patent application describes IL-1β mutants with reduced bioactivity that can be activated by targeting as demonstrated in this paper for IL-1β Q148G. WO/2017/134306: CD8 binding agents. Applicants: Orionis Biosciences Inc., VIB VZW, Universiteit Gent. Inventors: J.T., Anje Cauwels, N.K., and S.G. This patent application describes sdAbs that bind CD8 and that can be used to target mutant IL-1β to CTLs as demonstrated in this paper. The authors have no other, nonfinancial, competing interests to declare., (© The Author(s) 2020.)

Background: Many natalizumab treated patients experience end of dose interval (EDI) symptoms towards the end of the administration cycle. Natalizumab has previously shown to influence cytokine profiles in relapsing remitting MS patients. We hypothesize that EDI symptoms might be explained by variability in serum cytokine levels during natalizumab treatment., Methods: 42 relapsing remitting MS patients were included. Participants were evaluated before natalizumab administration (day 0) and 7 days afterwards (day 7). At both time points fatigue, depressed mood and cognition were evaluated using the fatigue severity scale (FSS), the visual analogue scale for fatigue (VAS-F), the symbol digit modality test (SDMT) and the inventory for depressive symptomatology (IDS-SR). Serum samples were tested for concentrations of IL-6, IFN-γ and TNF-α at both timepoints. On day 7 an additional EDI questionnaire was completed. Data were analyzed with SPSS by means of non-parametric tests., Results: EDI symptoms were reported by 59.5%. Although fatigue was most frequently reported, fatigue scales did not significantly change from day 0 to 7 in (fatigued) EDI patients. Mood and cognition significantly ameliorated in both EDI and non-EDI patients. Cytokines remained stable at day 0 vs 7 except for a significant increase in IFN-γ. On day 0, IFN-γ concentration was positively correlated with a depressed mood in the whole cohort, and with mood and fatigue in the EDI group. Depressed mood positively whilst cognition negatively correlated with IFN-γ concentration on day 0 in the EDI subgroup reporting fatigue. No significant correlations between IL-6 nor TNF-α and symptom scores could be found., Conclusion: In our study EDI symptoms could not be objectified since EDI and non-EDI groups did not differ in terms of change in mood, cognition and fatigue between day 0 and 7 suggesting that symptom recrudescence could be a subjective experience. Although our results need to be interpreted cautiously, we found no clear correlation between studied serum cytokines concentrations and the occurrence of EDI symptoms., Competing Interests: Declaration of Competing Interest This study was sponsored by Biogen as an investigator-initiated trial. The authors maintained full editorial control of all content. Dekeyser Cathérine: has received travel compensation from Biogen; De Pue Annelien: has received travel compensation from Biogen; Sieben Anne: nothing to disclose; Algoed Luc: has received travel compensation from Biogen; Vanhijfte Liesbeth: nothing to disclose; Sarah Gerlo: nothing to disclose; Laureys Guy: has received travel compensations, consultancy and speakers fees from Biogen, (Copyright © 2020 Elsevier B.V. All rights reserved.)

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A + dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker. Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463-74. ©2017 AACR ., (©2017 American Association for Cancer Research.)

KISS (KInase Substrate Sensor) is a recently developed two-hybrid technology that allows in situ analysis of protein-protein interactions in intact mammalian cells. In this method, which is derived from MAPPIT (mammalian protein-protein interaction trap), the bait protein is coupled to the kinase domain of TYK2, while the prey protein is fused to a fragment of the gp130 cytokine receptor chain. Bait and prey interaction leads to phosphorylation of the gp130 anchor by TYK2, followed by recruitment and activation of STAT3, resulting in transcription of a STAT3-dependent reporter system. This approach enables the identification of interactions between proteins, including transmembrane and cytosolic proteins, and their modulation in response to physiological or pharmacological challenges. Here, we describe a detailed step-by-step protocol for the detection of an interaction between two proteins of interest using KISS.

Despite approval for the treatment of various malignancies, clinical application of cytokines such as type I interferon (IFN) is severely impeded by their systemic toxicity. AcTakines (Activity-on-Target cytokines) are optimized immunocytokines that, when injected in mice, only reveal their activity upon cell-specific impact. We here show that type I IFN-derived AcTaferon targeted to the tumor displays strong antitumor activity without any associated toxicity, in contrast with wild type IFN. Treatment with CD20-targeted AcTaferon of CD20 + lymphoma tumors or melanoma tumors engineered to be CD20 + , drastically reduced tumor growth. This antitumor effect was completely lost in IFNAR- or Batf3-deficient mice, and depended on IFN signaling in conventional dendritic cells. Also the presence of, but not the IFN signaling in, CD8 + T lymphocytes was critical for proficient antitumor effects. When combined with immunogenic chemotherapy, low-dose TNF, or immune checkpoint blockade strategies such as anti-PDL1, anti-CTLA4 or anti-LAG3, complete tumor regressions and subsequent immunity (memory) were observed, still without any concomitant morbidity, again in sharp contrast with wild type IFN. Interestingly, the combination therapy of tumor-targeted AcTaferon with checkpoint inhibiting antibodies indicated its ability to convert nonresponding tumors into responders. Collectively, our findings demonstrate that AcTaferon targeted to tumor-specific surface markers may provide a safe and generic addition to cancer (immuno)therapies.

Loss of ER Ca 2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca 2+ . Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca 2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca 2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca 2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

There is growing in vivo evidence that the dipeptide carnosine has protective effects in metabolic diseases. A critical unanswered question is whether its site of action is tissues or plasma. This was investigated using oral carnosine versus β-alanine supplementation in a high-fat diet rat model. Thirty-six male Sprague-Dawley rats received a control diet (CON), a high-fat diet (HF; 60% of energy from fat), the HF diet with 1.8% carnosine (HFcar), or the HF diet with 1% β-alanine (HFba), as β-alanine can increase muscle carnosine without increasing plasma carnosine. Insulin sensitivity, inflammatory signaling, and lipoxidative stress were determined in skeletal muscle and blood. In a pilot study, urine was collected. The 3 HF groups were significantly heavier than the CON group. Muscle carnosine concentrations increased equally in the HFcar and HFba groups, while elevated plasma carnosine levels and carnosine-4-hydroxy-2-nonenal adducts were detected only in the HFcar group. Elevated plasma and urine N(ε)-(carboxymethyl)lysine in HF rats was reduced by ∼50% in the HFcar group but not in the HFba group. Likewise, inducible nitric oxide synthase mRNA was decreased by 47% (p < 0.05) in the HFcar group, but not in the HFba group, compared with HF rats. We conclude that plasma carnosine, but not muscle carnosine, is involved in preventing early-stage lipoxidation in the circulation and inflammatory signaling in the muscle of rats.

β2-Adrenergic receptors (β2-ARs) transduce the effects of (nor)epinephrine on a variety of cell types and act as key mediators of the body's reaction to stress. β2-ARs are also expressed on immune cells and there is ample evidence for their role in immunomodulation. A key regulator of the immune response and a target for regulation by stress-induced signals is the transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB shapes the course of both innate and adaptive immune responses and plays an important role in susceptibility to disease. In this review, we summarise the literature that has been accumulated in the past 20years on adrenergic modulation of NF-κB function. We here focus on the molecular basis of the reported interactions and show that both physiological and pharmacological triggers of β2-ARs intersect with the NF-κB signalling cascade at different levels. Importantly, the action of β2-AR-derived signals on NF-κB activity appears to be highly cell type specific and gene selective, providing opportunities for the development of selective NF-κB modulators., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type 1 diabetes is an autoimmune disease with a strong inflammatory component. The cytokines interleukin-1β and interferon-γ contribute to beta cell apoptosis in type 1 diabetes. These cytokines induce endoplasmic reticulum stress and the unfolded protein response (UPR), contributing to the loss of beta cells. IRE1α, one of the UPR mediators, triggers insulin degradation and inflammation in beta cells and is critical for the transition from "physiological" to "pathological" UPR. The mechanisms regulating inositol-requiring protein 1α (IRE1α) activation and its signaling for beta cell "adaptation," "stress response," or "apoptosis" remain to be clarified. To address these questions, we combined mammalian protein-protein interaction trap-based IRE1α interactome and functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines to identify novel cytokine-induced regulators of IRE1α. Based on this approach, we identified N-Myc interactor (NMI) as an IRE1α-interacting/modulator protein in rodent and human pancreatic beta cells. An increased expression of NMI was detected in islets from nonobese diabetic mice with insulitis and in rodent or human beta cells exposed in vitro to the pro-inflammatory cytokines interleukin-1β and interferon-γ. Detailed mechanistic studies demonstrated that NMI negatively modulates IRE1α-dependent activation of JNK and apoptosis in rodent and human pancreatic beta cells. In conclusion, by using a combined omics approach, we identified NMI induction as a novel negative feedback mechanism that decreases IRE1α-dependent activation of JNK and apoptosis in cytokine-exposed beta cells

With more than 150,000 species, parasitoids are a large group of hymenopteran insects that inject venom into and then lay their eggs in or on other insects, eventually killing the hosts. Their venoms have evolved into different mechanisms for manipulating host immunity, physiology and behavior in such a way that enhance development of the parasitoid young. The venom from the ectoparasitoid Nasonia vitripennis inhibits the immune system in its host organism in order to protect their offspring from elimination. Since the major innate immune pathways in insects, the Toll and Imd pathways, are homologous to the NF-κB pathway in mammals, we were interested in whether a similar immune suppression seen in insects could be elicited in a mammalian cell system. A well characterized NF-κB reporter gene assay in fibrosarcoma cells showed a dose-dependent inhibition of NF-κB signaling caused by the venom. In line with this NF-κB inhibitory action, N. vitripennis venom dampened the expression of IL-6, a prototypical proinflammatory cytokine, from LPS-treated macrophages. The venom also inhibited the expression of two NF-κB target genes, IκBα and A20, that act in a negative feedback loop to prevent excessive NF-κB activity. Surprisingly, we did not detect any effect of the venom on the early events in the canonical NF-κB activation pathway, leading to NF-κB nuclear translocation, which was unaltered in venom-treated cells. The MAP kinases ERK, p38 and JNK are other crucial regulators of immune responses. We observed that venom treatment did not affect p38 and ERK activation, but induced a prolonged JNK activation. In summary, our data indicate that venom from N. vitripennis inhibits NF-κB signaling in mammalian cells. We identify venom-induced up regulation of the glucocorticoid receptor-regulated GILZ as a most likely molecular mediator for this inhibition.

The proinflammatory cytokine Tumour Necrosis Factor (TNF)-α is implicated in a variety of skeletal muscle pathologies. Here, we have investigated how in vitro cotreatment of skeletal muscle C2C12 cells with β-agonists modulates the TNF-α-induced inflammatory program. We observed that C2C12 myotubes express functional TNF receptor 1 (TNF-R1) and β2-adrenoreceptors (β2-ARs). TNF-α activated the canonical Nuclear Factor-κB (NF-κB) pathway and Mitogen-Activated Protein Kinases (MAPKs), culminating in potent induction of NF-κB-dependent proinflammatory genes. Cotreatment with the β-agonist isoproterenol potentiated the expression of inflammatory mediators, including Interleukin-6 (IL-6) and several chemokines. The enhanced production of chemotactic factors upon TNF-α/isoproterenol cotreatment was also suggested by the results from migrational analysis. Whereas we could not explain our observations by cytoplasmic crosstalk, we found that TNF-R1-and β2-AR-induced signalling cascades cooperate in the nucleus. Using the IL-6 promoter as a model, we demonstrated that TNF-α/isoproterenol cotreatment provoked phosphorylation of histone H3 at serine 10, concomitant with enhanced promoter accessibility and recruitment of the NF-κB p65 subunit, cAMP-response element-binding protein (CREB), CREB-binding protein (CBP) and RNA polymerase II. In summary, we show that β-agonists potentiate TNF-α action, via nuclear crosstalk, that promotes chromatin relaxation at selected gene promoters. Our data warrant further study into the mode of action of β-agonists and urge for caution in their use as therapeutic agents for muscular disorders.

Background: The NF-κB signaling pathway orchestrates many of the intricate aspects of neuroinflammation. Astrocytic β₂-adrenergic receptors have emerged as potential regulators in central nervous system inflammation and are potential targets for pharmacological modulation. The aim of this study was to elucidate the crosstalk between astrocytic β₂-adrenergic receptors and the TNF-α induced inflammatory gene program., Methods: Proinflammatory conditions were generated by the administration of TNF-α. Genes that are susceptible to astrocytic crosstalk between β₂-adrenergic receptors (stimulated by clenbuterol) and TNF-α were identified by qPCR-macroarray-based gene expression analysis in a human 1321 N1 astrocytoma cell line. Transcriptional patterns of the identified genes in vitro were validated by RT-PCR on the 1321 N1 cell line as well as on primary rat astrocytes. In vivo expression patterns were examined by intracerebroventricular administration of clenbuterol and/or TNF-α in rats. To examine the impact on the inflammatory cell content of the brain we performed extensive FACS analysis of rat brain immune cells after intracerebroventricular clenbuterol and/or TNF-α administration., Results: Parallel transcriptional patterns in vivo and in vitro confirmed the relevance of astrocytic β₂-adrenergic receptors as modulators of brain inflammatory responses. Importantly, we observed pronounced effects of β2-adrenergic receptor agonists and TNF-α on IL-6, CXCL2, CXCL3, VCAM1, and ICAM1 expression, suggesting a role in inflammatory brain cell homeostasis. Extensive FACS-analysis of inflammatory cell content in the brain demonstrated that clenbuterol/TNF-α co-administration skewed the T cell population towards a double negative phenotype and induced a shift in the myeloid brain cell population towards a neutrophilic predominance., Conclusions: Our results show that astrocytic β₂-adrenergic receptors are potent regulators of astrocytic TNF-α-activated genes in vitro and in vivo, and ultimately modulate the molecular network involved in the homeostasis of inflammatory cells in the central nervous system. Astrocytic β₂-adrenergic receptors and their downstream signaling pathway may serve as potential targets to modulate neuroinflammatory responses.

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal(-/-) mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.

The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4-induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.

Background: Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively., Results: Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively., Conclusions: Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.

It has been known for several decades that cyclic AMP (cAMP), a prototypical second messenger, transducing the action of a variety of G-protein-coupled receptor ligands, has potent immunosuppressive and anti-inflammatory actions. These actions have been attributed in part to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB plays a crucial role in switching on the gene expression of a plethora of inflammatory and immune mediators, and as such is one of the master regulators of the immune response and a key target for anti-inflammatory drug design. A number of fundamental molecular mechanisms, contributing to the overall inhibitory actions of cAMP on NF-κB function, are well established. Paradoxically, recent reports indicate that cAMP, via its main effector, the protein kinase A (PKA), also promotes NF-κB activity. Indeed, cAMP actions appear to be highly cell type- and context-dependent. Importantly, several novel players in the cAMP/NF-κB connection, which selectively direct cAMP action, have been recently identified. These findings not only open up exciting new research avenues but also reveal novel opportunities for the design of more selective, NF-κB-targeting, anti-inflammatory drugs.

Almost a quarter of a century ago, interleukin-6 (IL-6) was discovered as an inflammatory cytokine involved in B cell differentiation. Today, IL-6 is recognized to be a highly versatile cytokine, with pleiotropic actions not only in immune cells, but also in other cell types, such as cells of the central nervous system (CNS). The first evidence implicating IL-6 in brain-related processes originated from its dysregulated expression in several neurological disorders such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. In addition, IL-6 was shown to be involved in multiple physiological CNS processes such as neuron homeostasis, astrogliogenesis and neuronal differentiation. The molecular mechanisms underlying IL-6 functions in the brain have only recently started to emerge. In this review, an overview of the latest discoveries concerning the actions of IL-6 in the nervous system is provided. The central position of IL-6 in the neuroinflammatory reaction pattern, and more specifically, the role of IL-6 in specific neurodegenerative processes, which accompany Alzheimer's disease, multiple sclerosis and excitotoxicity, are discussed. It is evident that IL-6 has a dichotomic action in the CNS, displaying neurotrophic properties on the one hand, and detrimental actions on the other. This is in agreement with its central role in neuroinflammation, which evolved as a beneficial process, aimed at maintaining tissue homeostasis, but which can become malignant when exaggerated. In this perspective, it is not surprising that 'well-meant' actions of IL-6 are often causing harm instead of leading to recovery., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Uncontrolled expression of IL-6 in the central nervous system is associated with neurodegenerative pathology and glioma development. Astrocytes are the predominant source of IL-6 in the central nervous system, and they are characteristically susceptible to synergistic IL-6 expression. Combined β-adrenergic and TNF-receptor triggering induces synergistic IL-6 expression in 1321N1 cells via a transcriptional enhancer mechanism. Here, we have investigated the molecular basis of the very potent "super"-synergistic IL-6 expression that is apparent after combined treatment of astrocytes with a β-adrenergic agonist, isoproterenol, and the inflammatory cytokines TNF-α and IL-1β. We found that IL-1β treatment strengthens the IL-6 synergy by inducing a distinct stabilization of IL-6 mRNA. Surprisingly, the mRNA-stabilizing effect seems to be dependent on protein kinase C (PKC), but not on the prototypical mRNA-stabilizing kinase p38. Moreover, although the mRNA-binding protein HuR basally stabilizes IL-6 mRNA, the mRNA-stabilizing effect of IL-1β is independent of HuR. Our data using pharmacological inhibitors suggest PKC is an important modulator of IL-6 expression in the central nervous system and this might have therapeutic implications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cyclic adenosine monophosphate (cAMP) is a well-known second messenger that operates through different signaling molecules, including protein kinase A (PKA) and guanine exchange proteins directly activated by cAMP (EPAC). Cell-permeable cAMP analogs such as 8-(4-chloro-phenyl-thio)-cAMP (8-pCPT-cAMP) modulate cytokine secretion by different leukocyte subsets, including T cells and monocytes. Since cAMP-modulating drugs such as phosphodiesterase inhibitors are being tested in inflammatory disorders such as asthma and chronic obstructive lung disease, it is important to obtain more insight into the regulation of cytokine production by cAMP. To address the signaling molecules involved in cAMP-mediated modulation of cytokine production, we used cAMP derivatives such as N(6)-benzoyladenosine-cAMP (6-Bnz-cAMP) and 8-pCPT-2-O-methyl cAMP (8-pCPT-2'-O-Me-cAMP), which selectively activate either PKA or EPAC, respectively. We show that in T cells, 6-Bnz-cAMP exerts similar globally inhibiting effects on cytokine secretion as 8-pCPT-cAMP, indicating that these effects are mediated by PKA. On the contrary, 8-pCPT-2'-O-Me-cAMP specifically inhibits the production of interleukin-10 (IL-10) in lipopolysaccharide-activated T-cell-depleted peripheral blood mononuclear cells, whereas the production of IL-1β, tumor necrosis factor α, and IL-12 is not or hardly affected. Inhibition by 8-pCPT-2'-O-Me-cAMP of IL-10 production was confirmed using purified monocytes. Further, in B cells 6-Bnz-cAMP, but not 8-pCPT-2'-O-Me-cAMP, stimulated IL-10 production. In conclusion, cAMP stimulates IL-10 production via PKA in activated B cells, but inhibits IL-10 production in activated monocytes through EPAC. We speculate that selective effects of PKA and EPAC on cytokine production in leukocyte subsets open up therapeutic possibilities using selective activators or inhibitors of EPAC or PKA.

Evidence accumulates for a key role of the beta(2)-adrenergic receptors in the many homeostatic and neuroprotective functions of astrocytes, including glycogen metabolism, regulation of immune responses, release of neurotrophic factors, and the astrogliosis that occurs in response to neuronal injury. A dysregulation of the astrocytic beta(2)-adrenergic-pathway is suspected to contribute to the physiopathology of a number of prevalent and devastating neurological conditions such as multiple sclerosis, Alzheimer's disease, human immunodeficiency virus encephalitis, stroke and hepatic encephalopathy. In this review we focus on the physiological functions of astrocytic beta(2)-adrenergic receptors, and their possible impact in disease states.

Background: Multidrug resistance (MDR) is a major obstacle in cancer treatment and is often the result of overexpression of the drug efflux protein, P-glycoprotein (P-gp), as a consequence of hyperactivation of NFkappaB, AP1 and Nrf2 transcription factors. In addition to effluxing chemotherapeutic drugs, P-gp also plays a specific role in blocking caspase-dependent apoptotic pathways. One feature that cytotoxic treatments of cancer have in common is activation of the transcription factor NFkappaB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFkappaB inhibitors may promote apoptosis in cancer cells and could be used to overcome resistance to chemotherapeutic agents., Results: Although the natural withanolide withaferin A and polyphenol quercetin, show comparable inhibition of NFkappaB target genes (involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance) in doxorubicin-sensitive K562 and -resistant K562/Adr cells, only withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation and apoptosis is delayed only. Interestingly, although withaferin A and quercetin treatments both decrease intracellular protein levels of Bcl2, Bim and P-Bad, only withaferin A decreases protein levels of cytoskeletal tubulin, concomitantly with potent PARP cleavage, caspase 3 activation and apoptosis, at least in part via a direct thiol oxidation mechanism., Conclusions: This demonstrates that different classes of natural NFkappaB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis.

Astrocytes are critical players in the innate immune response of the central nervous system. Upon encountering proinflammatory stimuli, astrocytes produce a plethora of inflammatory mediators. Here, we have investigated how beta(2)-adrenergic receptor activation modulates proinflammatory gene expression in astrocytes. We have observed that treatment of human 1321N1 astrocytes with the beta-adrenergic agonist isoproterenol synergistically enhanced TNF-alpha-induced expression of the cytokine IL-6. The effect of isoproterenol was cAMP-dependent and mediated by the beta(2)-adrenergic subtype. Using pharmacological inhibitors and siRNA we showed that protein kinase A (PKA) is an indispensable mediator of the synergy. Simultaneous induction with isoproterenol and TNF-alpha was moreover associated with combined recruitment of CREB and p65 to the native IL-6 promoter. The role of CREB and NFkappaB in promoting the synergy was corroborated using IL-6 promoter point mutants, as well as via siRNA-mediated silencing of CREB and NFkappaB. Interestingly, whereas CREB and NFkappaB usually compete for the limiting cofactor CREB binding protein (CBP), we detected enhanced recruitment of CBP at the IL-6 promoter in our system. The transcriptional synergy seems to be a gene specific process, occurring at the IL-6 and COX-2 gene, but not at other typical NFkappaB-dependent genes such as IL-8, ICAM-1 or VCAM-1. As astrocytic IL-6 overexpression has been associated with neuroinflammatory and neurodegenerative processes, our findings might have important physiological consequences., (2010 Elsevier Inc. All rights reserved.)

The transcription factor nuclear factor kappaB (NF-kappaB) is one of the central mediators of inflammatory gene expression. Several posttranslational modifications of NF-kappaB, regulating its transactivation ability, have been described. Especially phosphorylation of the NF-kappaB subunit p65 has been investigated in depth and several commercial phosphospecific antibodies, targeting selected p65 residues, are available. One of the p65 residues, that is subject to phosphorylation by protein kinase A (PKA) as well as by mitogen-stimulated kinase-1 (MSK-1), is the serine at position 276. Here, we have performed a detailed analysis of the performance of the most commonly used commercial anti-P-p65 Ser276 antibodies. Our findings indicate that at least three widely used anti-P-p65 Ser276 antibodies do not detect p65 in vivo via Western Blot, but instead crossreact with PKA-regulated proteins. As PKA is one of the main kinases responsible for phosphorylation of p65 at Ser276, this observation warrants cautious interpretation of data generated using the tested antibodies.

In the combat against inflammation, glucocorticoids (GCs) are a widespread therapeutic. These ligands of the glucocorticoid receptor (GR) inhibit the transactivation of various transcription factors, including nuclear factor-kappaB (NF-kappaB), and alter the composition of the pro-inflammatory enhanceosome, culminating in the repression of pro-inflammatory gene expression. However, pharmacological usage of GCs in long-term treatment is burdened with a detrimental side-effect profile. Recently, we discovered that GCs can lower NF-kappaB transactivation and pro-inflammatory gene expression by abolishing the recruitment of mitogen- and stress-activated protein kinase 1 (MSK1) (EC 2.7.11.1) to pro-inflammatory gene promoters and displacing a significant fraction of MSK1 to the cytoplasm. In our current investigation in L929sA fibroblasts, upon combining GCs and MSK1 inhibitors, we discovered a dose-dependent additive repression of pro-inflammatory gene expression, most likely due to diverse and multilayered repression mechanisms employed by GCs and MSK1 inhibitors. Therefore, the combined application of GCs and MSK1 inhibitors enabled a similar level of repression of pro-inflammatory gene expression, using actually a lower concentration of GCs and MSK1 inhibitors combined than would be necessary when using these inhibitors separately. Although H89 can inhibit both MSK1 and PKA, TNF does not activate PKA (EC 2.7.11.11) and as such PKA inhibition does not mediate H89-instigated repression of TNF-stimulated gene expression. Furthermore, the additional repressive effects of liganded GR and inhibition of MSK1, are not mediated via GR transactivation mechanisms. In conclusion, these results could entail a new therapeutic strategy using lower drug concentrations, potentially leading to a more beneficial side-effect profile.