The role of antidrug antibodies in ustekinumab therapy and the impact of methotrexate.

OBJECTIVE: We investigated the impact of concomitant MTX on ustekinumab (UST) levels and antidrug antibody (ADA) formation in PsA and evaluated consequences in pharmacodynamics and pharmacokinetics. METHODS: We conducted a post-hoc analysis on 112 PsA serum samples of subjects treated with open-label UST and either concomitant MTX (UST/MTX, n = 58) or placebo (UST/pbo, n = 54) obtained in a randomized (1:1), double-blind, multicentre trial. A validated antibody-binding-based multitiered testing was used to detect ADA and ADA with neutralizing capacity (nADA). The impact of MTX on UST immunogenicity was analysed by comparison of UST/pbo with UST/MTX cohorts at different time points. Patient- and disease-related predispositions for ADA formation were investigated with multiple linear regression analysis. Immunogenicity impact on pharmacokinetics, safety and efficacy was determined by cohort comparison between patients with and without ADA formation. RESULTS: Over 52 weeks, 11 UST/pbo- and 19 UST/MTX-treated patients developed ADA (P > 0.05). In the UST/pbo cohort, the visit-dependent UST levels were in the range of 0.047 (0.05) -0.110 (0.07) µg/ml overall, and 0.037 (0.04)-0.091 (0.08) µg/ml in ADA-confirmed subjects. In UST/MTX-treated patients, the UST levels exhibited an intervisit variation in the range of 0.0502 (0.04)-0.106 (0.07) µg/ml overall and 0.029 (0.03)-0.097 (0.07) µg/ml in ADA positive subjects (P > 0.05). At week 52, ADA-confirmed patients did not differ significantly (P > 0.05) in safety or clinical outcomes from ADA-negative patients. CONCLUSION: Concomitant MTX had no significant impact on UST immunogenicity. Furthermore, ADA formation was not associated with impairments in UST safety, efficacy or trough levels. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT03148860.

Comparing the Effects of Rocaglates on Energy Metabolism and Immune Modulation on Cells of the Human Immune System.

A promising new approach to broad spectrum antiviral drugs is the inhibition of the eukaryotic translation initiation factor 4A (elF4A), a DEAD-box RNA helicase that effectively reduces the replication of several pathogenic virus types. Beside the antipathogenic effect, modulation of a host enzyme activity could also have an impact on the immune system. Therefore, we performed a comprehensive study on the influence of elF4A inhibition with natural and synthetic rocaglates on various immune cells. The effect of the rocaglates zotatifin, silvestrol and CR-31-B (-), as well as the nonactive enantiomer CR-31-B (+), on the expression of surface markers, release of cytokines, proliferation, inflammatory mediators and metabolic activity in primary human monocyte-derived macrophages (MdMs), monocyte-derived dendritic cells (MdDCs), T cells and B cells was assessed. The inhibition of elF4A reduced the inflammatory potential and energy metabolism of M1 MdMs, whereas in M2 MdMs, drug-specific and less target-specific effects were observed. Rocaglate treatment also reduced the inflammatory potential of activated MdDCs by altering cytokine release. In T cells, the inhibition of elF4A impaired their activation by reducing the proliferation rate, expression of CD25 and cytokine release. The inhibition of elF4A further reduced B-cell proliferation, plasma cell formation and the release of immune globulins. In conclusion, the inhibition of the elF4A RNA helicase with rocaglates suppressed the function of M1 MdMs, MdDCs, T cells and B cells. This suggests that rocaglates, while inhibiting viral replication, may also suppress bystander tissue injury by the host immune system. Thus, dosing of rocaglates would need to be adjusted to prevent excessive immune suppression without reducing their antiviral activity.

Mechanism of anti-inflammatory effects of rifampicin in an ex vivo culture system of hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease of the hair follicles leading to painful lesions, associated with increased levels of pro-inflammatory cytokines. Numerous guidelines recommend antibiotics like clindamycin and rifampicin in combination, as first-line systemic therapy in moderate-to-severe forms of inflammation. HS has been proposed to be mainly an auto-inflammatory disease associated with but not initially provoked by bacteria. Therefore, it has to be assumed that the pro-inflammatory milieu previously observed in HS skin is not solely dampened by the bacteriostatic inhibition of DNA-dependent RNA polymerase. To further clarify the mechanism of anti-inflammatory effects of rifampicin, ex vivo explants of lesional HS from 8 HS patients were treated with rifampicin, and its effect on cytokine production, immune cells as well as the expression of Toll-like receptor 2 (TLR2) were investigated. Analysis of cell culture medium of rifampicin-treated HS explants revealed an anti-inflammatory effect of rifampicin that significantly inhibiting interleukin (IL)-1ß, IL-6, IL-8, IL-10 and tumour necrosis factor (TNF)-α production. Immunohistochemistry of the rifampicin-treated explants suggested a tendency for it to reduce the expression of TLR2 while not affecting the number of immune cells.

Ibuprofen, Flurbiprofen, Etoricoxib or Paracetamol Do Not Influence ACE2 Expression and Activity In Vitro or in Mice and Do Not Exacerbate In-Vitro SARS-CoV-2 Infection.

SARS-CoV-2 uses the human cell surface protein angiotensin converting enzyme 2 (ACE2) as the receptor by which it gains access into lung and other tissue. Early in the pandemic, there was speculation that a number of commonly used medications-including ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDs)-have the potential to upregulate ACE2, thereby possibly facilitating viral entry and increasing the severity of COVID-19. We investigated the influence of the NSAIDS with a range of cyclooxygenase (COX)1 and COX2 selectivity (ibuprofen, flurbiprofen, etoricoxib) and paracetamol on the level of ACE2 mRNA/protein expression and activity as well as their influence on SARS-CoV-2 infection levels in a Caco-2 cell model. We also analysed the ACE2 mRNA/protein levels and activity in lung, heart and aorta in ibuprofen treated mice. The drugs had no effect on ACE2 mRNA/protein expression and activity in the Caco-2 cell model. There was no up-regulation of ACE2 mRNA/protein expression and activity in lung, heart and aorta tissue in ibuprofen-treated mice in comparison to untreated mice. Viral load was significantly reduced by both flurbiprofen and ibuprofen at high concentrations. Ibuprofen, flurbiprofen, etoricoxib and paracetamol demonstrated no effects on ACE2 expression or activity in vitro or in vivo. Higher concentrations of ibuprofen and flurbiprofen reduced SARS-CoV-2 replication in vitro.

T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model.

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.

Natural antiviral compound silvestrol modulates human monocyte-derived macrophages and dendritic cells.

Outbreaks of infections with viruses like Sars-CoV-2, Ebola virus and Zika virus lead to major global health and economic problems because of limited treatment options. Therefore, new antiviral drug candidates are urgently needed. The promising new antiviral drug candidate silvestrol effectively inhibited replication of Corona-, Ebola-, Zika-, Picorna-, Hepatis E and Chikungunya viruses. Besides a direct impact on pathogens, modulation of the host immune system provides an additional facet to antiviral drug development because suitable immune modulation can boost innate defence mechanisms against the pathogens. In the present study, silvestrol down-regulated several pro- and anti-inflammatory cytokines (IL-6, IL-8, IL-10, CCL2, CCL18) and increased TNF-α during differentiation and activation of M1-macrophages, suggesting that the effects of silvestrol might cancel each other out. However, silvestrol amplified the anti-inflammatory potential of M2-macrophages by increasing expression of anti-inflammatory surface markers CD206, TREM2 and reducing release of pro-inflammatory IL-8 and CCL2. The differentiation of dendritic cells in the presence of silvestrol is characterized by down-regulation of several surface markers and cytokines indicating that differentiation is impaired by silvestrol. In conclusion, silvestrol influences the inflammatory status of immune cells depending on the cell type and activation status.

The Specific IKKε/TBK1 Inhibitor Amlexanox Suppresses Human Melanoma by the Inhibition of Autophagy, NF-κB and MAP Kinase Pathways.

Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types. Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this "difficult-to-treat" cancer type has not been investigated so far. We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients. Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes. The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion. Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells. The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects. In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth. In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

The relevance of ceramides and their synthesizing enzymes for multiple sclerosis.

Ceramide synthases (CerS) synthesize chain length specific ceramides (Cer), which mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that the genetic deletion of CerS2 suppresses EAE pathology by interaction with granulocyte-colony stimulating factor (G-CSF) signaling and CXC motif chemokine receptor 2 (CXCR2) expression, leading to impaired neutrophil migration. In the present study, we investigated the importance of Cers and their synthesizing/metabolizing enzymes in MS. For this purpose, a longitudinal study with 72 MS patients and 25 healthy volunteers was performed. Blood samples were collected from healthy controls and MS patients over 1- or 3-year periods, respectively. Immune cells were counted using flow cytometry, ceramide levels were determined using liquid chromatography-tandem mass spectrometry, and mRNA expression was analyzed using quantitative PCR. In white blood cells, C16-LacCer and C24-Cer were down-regulated in MS patients in comparison with healthy controls. In plasma, C16-Cer, C24:1-Cer, C16-GluCer, and C24:1-GluCer were up-regulated and C16-LacCer was down-regulated in MS patients in comparison with healthy controls. Blood samples from MS patients were characterized by an increased B-cell number. However, there was no correlation between B-cell number and Cer levels. mRNA expression of Cer metabolizing enzymes and G-CSF signaling enzymes was significantly increased in MS patients. Interestingly, G-CSF receptor (G-CSFR) and CXCR2 mRNA expression correlated with CerS2 and UDP-glucose Cer glucosyltransferase (UGCG) mRNA expression. In conclusion, our results indicate that Cer metabolism is linked to G-CSF signaling in MS.

Alpha-methylacyl-CoA racemase deletion has mutually counteracting effects on T-cell responses, associated with unchanged course of EAE.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Altering the metabolism of immune cells is an attractive strategy to modify their activity during autoimmunity in MS. We investigated the effect of modulating fatty acid metabolism in an animal model of MS, EAE. Alpha-methylacyl-CoA racemase (AMACR) converts R-configuration branched fatty acids into the S-configuration, thereby preparing them for ß-oxidation. We observed a significant, disease-dependent elevation of AMACR expression in monocytes and T cells from blood, draining lymph nodes and spleen of EAE mice during the preclinical phase. In vitro analysis revealed that the proliferation of T cells was inhibited in AMACR KO mice, but T-cell polarization was switched toward a pathogenic state involving the production of more IFN-γ and IL-17, but less IL-4. These opposing effects appeared to cancel out each other in vivo, because AMACR KO EAE mice showed a marginal increase in the severity of early clinical symptoms. AMACR was not regulated in the white blood cells of MS patients. Our data show that AMACR is regulated in immune cells during EAE, but it is not a suitable target for the treatment of MS due to its opposing effects.

Machine-Learned Data Structures of Lipid Marker Serum Concentrations in Multiple Sclerosis Patients Differ from Those in Healthy Subjects.

Lipid metabolism has been suggested to be a major pathophysiological mechanism of multiple sclerosis (MS). With the increasing knowledge about lipid signaling, acquired data become increasingly complex making bioinformatics necessary in lipid research. We used unsupervised machine-learning to analyze lipid marker serum concentrations, pursuing the hypothesis that for the most relevant markers the emerging data structures will coincide with the diagnosis of MS. Machine learning was implemented as emergent self-organizing feature maps (ESOM) combined with the U*-matrix visualization technique. The data space consisted of serum concentrations of three main classes of lipid markers comprising eicosanoids (d = 11 markers), ceramides (d = 10), and lyosophosphatidic acids (d = 6). They were analyzed in cohorts of MS patients (n = 102) and healthy subjects (n = 301). Clear data structures in the high-dimensional data space were observed in eicosanoid and ceramides serum concentrations whereas no clear structure could be found in lysophosphatidic acid concentrations. With ceramide concentrations, the structures that had emerged from unsupervised machine-learning almost completely overlapped with the known grouping of MS patients versus healthy subjects. This was only partly provided by eicosanoid serum concentrations. Thus, unsupervised machine-learning identified distinct data structures of bioactive lipid serum concentrations. These structures could be superimposed with the known grouping of MS patients versus healthy subjects, which was almost completely possible with ceramides. Therefore, based on the present analysis, ceramides are first-line candidates for further exploration as drug-gable targets or biomarkers in MS.

Exacerbation of experimental autoimmune encephalomyelitis in ceramide synthase 6 knockout mice is associated with enhanced activation/migration of neutrophils.

Ceramides are mediators of inflammatory processes. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that CerS6 mRNA expression was upregulated 15-fold in peripheral blood leukocytes before the onset of EAE symptoms. In peripheral blood leukocytes from MS patients, a 3.9-fold upregulation was found. Total genetic deletion of CerS6 and the selective deletion of CerS6 in peripheral blood leucocytes exacerbated the progression of clinical symptoms in EAE mice. This was associated with enhanced leukocyte, predominantly neutrophil infiltration and enhanced demyelination in the lumbar spinal cord of EAE mice. Interferon-gamma/tumor necrosis factor alpha (IFN-γ/TNF-α) and granulocyte colony-stimulating factor (G-CSF) both drive EAE development and induce expression of the integrin CD11b and the chemokine receptor C-X-C motif chemokine receptor 2 (CXCR2), and we found they also induce CerS6 expression. In vivo, the genetic deletion of CerS6 enhanced the activation/migration of neutrophils, as reflected by an enhanced upregulation of CD11b and CXCR2. In vitro, the genetic deletion of CerS6 enhanced the activation status of IFN-γ/TNF-α-stimulated neutrophils, as shown by increased expression of nitric oxide and CD11b and an increased adhesion capacity. In G-CSF-stimulated neutrophils, the migration status was enhanced, as reflected by an elevated level of CXCR2 and an increased migration capacity. These data suggest that CerS6/C16-Cer mediates feedback regulation by inhibiting the formation of CD11b and CXCR2, which are induced either by IFN-γ/TNF-α or by G-CSF, respectively. We conclude that CerS6/C16-Cer mediates anti-inflammatory effects during the development of EAE and MS possibly by suppressing the migration and deactivation of neutrophils.

Lack of ceramide synthase 2 suppresses the development of experimental autoimmune encephalomyelitis by impairing the migratory capacity of neutrophils.

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b(+) cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

Ceramide synthase 6 plays a critical role in the development of experimental autoimmune encephalomyelitis.

Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C(16:0)-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C(16:0)-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ-activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ-mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C(16:0)-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C(16:0)-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C(16:0)-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C(16:0)-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.

Fine tuning of the innate and adaptive immune responses by Interleukin-2.

Novel immunotherapies for cancer and other diseases aim to trigger the immune system to produce durable responses, while overcoming the immunosuppression that may contribute to disease severity, and in parallel considering immunosafety aspects. Interleukin-2 (IL-2) was one of the first cytokines that the FDA approved as a cancer-targeting immunotherapy. However, in the past years, IL-2 immunotherapy is not actively offered to patients, due to limited efficacy, when compared to other novel immunotherapies, and the associated severe adverse events. In order to design improved in vitro and in vivo models, able to predict the efficacy and safety of novel IL-2 alternatives, it is important to delineate the mechanistic immunological events triggered by IL-2. Particularly, in this review we will discuss the effects IL-2 has with the bridging cell type of the innate and adaptive immune responses, dendritic cells. The pathways involved in the regulation of IL-2 by dendritic cells and T-cells in cancer and autoimmune disease will also be explored.

IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

TMPRSS2 Impacts Cytokine Expression in Murine Dendritic Cells.

BACKGROUND: The transmembrane protease serine 2 (TMPRSS2) proteolytically activates the envelope proteins of several viruses for viral entry via membrane fusion and is therefore an interesting and promising target for the development of broad-spectrum antivirals. However, the use of a host protein as a target may lead to potential side effects, especially on the immune system. We examined the effect of a genetic deletion of TMPRSS2 on dendritic cells. METHODS: Bone marrow cells from wild-type (WT) and TMPRSS2-deficient mice (TMPRSS2-/-) were differentiated to plasmacytoid dendritic cells (pDCs) and classical DCs (cDCs) and activated with various toll-like receptor (TLR) agonists. We analyzed the released cytokines and the mRNA expression of chemokine receptors, TLR7, TLR9, IRF7 and TCF4 stimulation. RESULTS: In cDCs, the lack of TMPRSS2 led to an increase in IL12 and IFNγ in TLR7/8 agonist resiquimod or TLR 9 agonist ODN 1668-activated cells. Only IL-10 was reduced in TMPRSS2-/- cells in comparison to WT cells activated with ODN 1668. In resiquimod-activated pDCs, the lack of TMPRSS2 led to a decrease in IL-6, IL-10 and INFγ. ODN 1668 activation led to a reduction in IFNα. The effect on receptor expression in pDCs and cDCs was low. CONCLUSION: The effect of TMPRSS2 on pDCS and cDCs depends on the activated TLR, and TMPRSS2 seems to affect cytokine release differently in pDCs and cDCs. In cDCs, TMPRSS2 seems to suppress cytokine release, whereas in pDCS TMPRSS2 possibly mediates cytokine release.

Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin.

Introduction: Hepatotoxicity induced by immunotherapeutics is an appearing cause for immune-mediated drug-induced liver injury. Such immuno-toxic mechanisms are difficult to assess using current preclinical models and the incidence is too low to detect in clinical trials. As hepatotoxicity is a frequent reason for post-authorisation drug withdrawal, there is an urgent need for immuno-inflammatory in vitro models to assess the hepatotoxic potential of immuno-modulatory drug candidates. We developed several immuno-inflammatory hepatotoxicity test systems based on recombinant human interleukin-2 (aldesleukin). Methods: Co-culture models of primary human CD8+ T cells or NK cells with the hepatocyte cell line HepaRG were established and validated with primary human hepatocytes (PHHs). Subsequently, the HepaRG model was refined by increasing complexity by inclusion of monocyte-derived macrophages (MdMs). The main readouts were cytotoxicity, inflammatory mediator release, surface marker expression and specific hepatocyte functions. Results: We identified CD8+ T cells as possible mediators of aldesleukin-mediated hepatotoxicity, with MdMs being implicated in increased aldesleukin-induced inflammatory effects. In co-cultures of CD8+ T cells with MdMs and HepaRG cells, cytotoxicity was induced at intermediate/high aldesleukin concentrations and perforin was upregulated. A pro-inflammatory milieu was created measured by interleukin-6 (IL-6), c-reactive protein (CRP), interferon gamma (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1) increase. NK cells responded to aldesleukin, however, only minor aldesleukin-induced cytotoxic effects were measured in co-cultures. Results obtained with HepaRG cells and with PHHs were comparable, especially regarding cytotoxicity, but high inter-donor variations limited meaningfulness of the PHH model. Discussion: The in vitro test systems developed contribute to the understanding of potential key mechanisms in aldesleukin-mediated hepatotoxicity. In addition, they may aid assessment of immune-mediated hepatotoxicity during the development of novel immunotherapeutics.

Venomics of the milos viper (Macrovipera schweizeri) unveils patterns of venom composition and exochemistry across blunt-nosed viper venoms.

Introduction: Snakebite is a neglected tropical disease and a globally important driver of death and morbidity. Vipers of the genus Macrovipera (Viperidae: Viperinae) are among the snakes of higher medical importance in the Old World. Despite the medical relevance of Macrovipera venoms, the knowledge regarding them is heterogeneously distributed with virtually all works conducted so far focusing on subspecies of Macrovipera lebetinus, while other species within the genus are largely overlooked. Here we present the first proteomic evaluation of the venom from the Greek endemic Milos viper (Macrovipera schweizeri). In line with clinical symptoms typically elicited by Macrovipera envenomations, Milos viper venom primarily comprises coagulotoxic and cytotoxic protein families, such as metalloproteinases (svMP) and serine proteases (svSP). Methods: We conducted comparative bioactivity assays on venoms from M. schweizeri and the M. lebetinus subspecies M. lebetinus cernovi, M. lebetinus obtusa, and M. lebetinus turanica, and showed that they all exhibit similarities in levels of cytotoxicity proteolytic activity, and inhibition of prokaryotic growth. Lastly, we compared Macrovipera venom profiles by 1D-SDS-PAGE and RP-HPLC, as well as our proteomic data with previously published Macrovipera venom proteomes. Results and discussion: The analyzes performed to reveal that a general venom profile seems to be conserved across blunt-nosed vipers, and that, M. schweizeri envenomations, similarly to those caused by other blunt-nosed vipers, are able to cause significant tissue damage. The present work represents an important starting point for the development of comparative studies across the full taxonomic range of the genus Macrovipera and can potentially help optimize the treatment of envenomations caused by M. schweizeri.

The Pharmacological Potential of Novel Melittin Variants from the Honeybee and Solitary Bees against Inflammation and Cancer.

The venom of honeybees is composed of numerous peptides and proteins and has been used for decades as an anti-inflammatory and anti-cancer agent in traditional medicine. However, the bioactivity of specific biomolecular components has been evaluated for the predominant constituent, melittin. So far, only a few melittin-like peptides from solitary bee species have been investigated, and the molecular mechanisms of bee venoms as therapeutic agents remain largely unknown. Here, the preclinical pharmacological activities of known and proteo-transcriptomically discovered new melittin variants from the honeybee and more ancestral variants from phylogenetically older solitary bees were explored in the context of cancer and inflammation. We studied the effects of melittin peptides on cytotoxicity, second messenger release, and inflammatory markers using primary human cells, non-cancer, and cancerous cell lines. Melittin and some of its variants showed cytotoxic effects, induced Ca2+ signaling and inhibited cAMP production, and prevented LPS-induced NO synthesis but did not affect the IP3 signaling and pro-inflammatory activation of endothelial cells. Compared to the originally-described melittin, some phylogenetically more ancestral variants from solitary bees offer potential therapeutic modalities in modulating the in vitro inflammatory processes, and hindering cancer cell viability/proliferation, including aggressive breast cancers, and are worth further investigation.