Extending the IMQ Model: Deep Characterization of the Human TLR7 Response for Early Drug Development.

Imiquimod (IMQ; brand name Aldara®) is a registered topical agent that has been proven to induce local inflammation via the Toll-like receptor (TLR)7 pathway. The purpose of this study was to characterize TLR7-mediated inflammation following 7 days (168 h) of topical IMQ exposure in healthy volunteers, and to compare the effects of short exposure (48 h-72 h) with prolonged exposure (120 h-168 h). IMQ (100mg) was applied under occlusion to 5 different tape-stripped treatment sites on the back of 10 healthy participants for a maximum of 7 consecutive days. Erythema and skin perfusion were measured daily up to 168h. Biopsies for immunohistochemical staining and RNA sequencing were collected at 0h, 48h, 72h, 120h and 168h post IMQ application. IMQ triggered an inflammatory response starting at 48h after application, including erythema and perfusion of the skin. At the transcriptomic level, IMQ induced TLR7 signalling, IRF involvement and activation of TNF signalling via NF-κB. Furthermore, an enhanced inflammatory response at the cellular level was observed after prolonged IMQ exposure, with cellular infiltration of dendritic cells, macrophages and T cells which was also corroborated by transcriptomic profiles. No difference was found in the erythema and perfusion response after 168h of IMQ exposure compared to 72h. Prolonged IMQ exposure revealed enhanced cellular responses and additional pathways with modulated activity compared to short exposure and can therefore be of interest as a model for investigational compounds targeting innate and adaptive immune responses.

A SUCTION BLISTER MODEL TO CHARACTERIZE EPIDERMAL WOUND HEALING AND EVALUATE THE EFFICACY OF THE TOPICAL WOUND HEALING AGENT INM-755 IN HEALTHY VOLUNTEERS.

Non-healing wounds represent a substantial medical burden with few effective treatments available. To address this challenge, we developed a novel epidermal wound healing model using suction blisters in healthy volunteers. This model allowed for the comprehensive assessment of wound healing dynamics and the evaluation of INM-755, a topical cream containing cannabinol, as a potential therapeutic agent. Two clinical studies were conducted: an observational study and an interventional study. In both studies, healthy volunteers underwent a suction blister procedure on their lower back, creating open epidermal wounds. Wound healing parameters were assessed using advanced imaging systems. Skin barrier function and perfusion were evaluated through trans epidermal water loss (TEWL) and dynamic optical coherence tomography (D-OCT), respectively. The observational study demonstrated the successful and reproducible Induction of blisters and the removal of epidermal sheet, enabling quantifiable measurements of wound healing parameters over time. Re-epithelialization was observed, revealing recovery of skin barrier function and perfusion. In the interventional study, differences of treatments over time were quantified using the above-described techniques. Despite differences from disease-specific blistering, our developed model provides a valuable platform for studying wound healing mechanisms and assessing novel therapeutic interventions. The sensitivity to treatment effects demonstrated in our study underscores the potential utility of this model in early-phase clinical drug development programs targeting wound healing disorders.

Immunomodulating effects of the single bacterial strain therapy EDP1815 on innate and adaptive immune challenge responses - a randomized, placebo-controlled clinical trial.

The gut microbiome can modulate systemic inflammation and is therefore target for immunomodulation. Immunomodulating effects of EDP1815, a bacterial commensal strain of Prevotella histicola, were studied in healthy participants. Effects on adaptive immunity were evaluated by a neo-antigen challenge with keyhole limpet haemocyanin (KLH), while effects on innate immunity were evaluated by topical toll-like receptor 7 (TLR7) agonist imiquimod. Capsules with two enteric coating levels (EC1, EC2) were compared. Thirty-six healthy participants were included and received a daily dose of 8 × 1010 cells EDP1815-EC1, EDP1815-EC2 or placebo (randomization 1:1:1) for 60 days. They received KLH vaccinations at days 8, 24 and 36, with intradermal skin challenge at day 57. KLH challenge outcomes were antibody levels, and skin blood flow and erythema after skin challenge, measured by imaging techniques. Imiquimod administration started at day 57, for 72 h. Outcomes consisted of imaging measurements similar to the KLH challenge, and the influx of inflammatory cells and cytokines in blister fluid. There was no effect of EDP1815 treatment on the KLH challenge, neither on the imaging outcomes of the imiquimod challenge. There was a consistently lower influx of inflammatory cells in the blister fluid of EDP1815-treated participants (neutrophils, p = 0.016; granulocytes, p = 0.024), more pronounced in EC1. There was a lower influx of interleukin [IL]-1ß, IL-6, IL-8, IL-10, interferon [IFN]-γ and tumour necrosis factor in blister fluid of EDP1815-treated participants. EDP1815 had immunomodulatory effects on the innate immune response driven by imiquimod, but no effect on the KLH challenge was observed. Trial registration number: NCT05682222; date: 22 July 2022.

The oral IRAK4 inhibitors zabedosertib and BAY1830839 suppress local and systemic immune responses in a randomized trial in healthy male volunteers.

This study evaluated and characterized the pharmacological activity of the orally administered interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitors BAY1834845 (zabedosertib) and BAY1830839 in healthy male volunteers. Participants received one of either IRAK4 inhibitors or a control treatment (prednisolone 20 mg or placebo) twice daily for 7 days. Localized skin inflammation was induced by topical application of imiquimod (IMQ) cream for 3 days, starting at Day 3 of treatment. The inflammatory response was evaluated by laser speckle contrast imaging (skin perfusion) and multispectral imaging (erythema). At Day 7, participants received 1 ng/kg intravenous lipopolysaccharide (LPS). Circulating inflammatory proteins, leukocyte differentiation, acute phase proteins, and clinical parameters were evaluated before and after the systemic LPS challenge. Treatment with BAY1834845 significantly reduced the mean IMQ-induced skin perfusion response (geometric mean ratio [GMR] vs. placebo: 0.69 for BAY1834845, 0.70 for prednisolone; both p < 0.05). Treatment with BAY1834845 and BAY1830839 significantly reduced IMQ-induced erythema (GMR vs. placebo: 0.75 and 0.83, respectively, both p < 0.05; 0.86 for prednisolone, not significant). Both IRAK4 inhibitors significantly suppressed the serum TNF-α and IL-6 responses (≥80% suppression vs. placebo, p < 0.05) and inhibited C-reactive protein, procalcitonin, and IL-8 responses to intravenous LPS. This study demonstrated the pharmacological effectiveness of BAY1834845 and BAY1830839 in suppressing systemically and locally induced inflammatory responses in the same range as prednisolone, underlining the potential value of these IRAK4 inhibitors as future therapies for dermatological or other immune-mediated inflammatory diseases.

Oral prednisolone suppresses skin inflammation in a healthy volunteer imiquimod challenge model.

Imiquimod (IMQ) is a topical agent that induces local inflammation via the Toll-like receptor 7 pathway. Recently, an IMQ-driven skin inflammation model was developed in healthy volunteers for proof-of-pharmacology trials. The aim of this study was to profile the cellular, biochemical, and clinical effects of the marketed anti-inflammatory compound prednisolone in an IMQ model. This randomized, double-blind, placebo-controlled study was conducted in 24 healthy volunteers. Oral prednisolone (0.25 mg/kg/dose) or placebo (1:1) was administered twice daily for 6 consecutive days. Two days after treatment initiation with prednisolone or placebo, 5 mg imiquimod (IMQ) once daily for two following days was applied under occlusion on the tape-stripped skin of the back for 48 h in healthy volunteers. Non-invasive (imaging and biophysical) and invasive (skin punch biopsies and blister induction) assessments were performed, as well as IMQ ex vivo stimulation of whole blood. Prednisolone reduced blood perfusion and skin erythema following 48 h of IMQ application (95% CI [-26.4%, -4.3%], p = 0.0111 and 95% CI [-7.96, -2.13], p = 0.0016). Oral prednisolone suppressed the IMQ-elevated total cell count (95% CI [-79.7%, -16.3%], p = 0.0165), NK and dendritic cells (95% CI [-68.7%, -5.2%], p = 0.0333, 95% CI [-76.9%, -13.9%], p = 0.0184), and classical monocytes (95% CI [-76.7%, -26.6%], p = 0.0043) in blister fluid. Notably, TNF, IL-6, IL-8, and Mx-A responses in blister exudate were also reduced by prednisolone compared to placebo. Oral prednisolone suppresses IMQ-induced skin inflammation, which underlines the value of this cutaneous challenge model in clinical pharmacology studies of novel anti-inflammatory compounds. In these studies, prednisolone can be used as a benchmark.

Immune Monitoring of Mycophenolate Mofetil Activity in Healthy Volunteers Using Ex Vivo T Cell Function Assays.

Mycophenolate mofetil (MMF) is part of the standard immunosuppressive treatment after transplantation and usually given as "one-dose-fits-all" together with a calcineurin inhibitor (CNI). Although drug concentrations are frequently monitored, there is still a group of patients who experience side effects related to excessive or insufficient immune suppression. We therefore aimed to identify biomarkers that reflect the overall immune status of the patient and might support individualized dosing. We previously studied immune biomarkers for CNIs and aimed to investigate whether these are also suitable to monitor MMF activity. Healthy volunteers received a single dose of MMF or placebo, after which IMPDH enzymatic activity, T cell proliferation, and cytokine production were measured and compared to MPA (MMF's active metabolite) concentration in three different matrices (plasma, peripheral blood mononuclear cells, and T cells). MPA concentrations in T cells exceeded those in PBMCs, but all intracellular concentrations correlated strongly with plasma concentrations. At clinically relevant MPA concentrations, IL-2 and IFN-γ production was mildly suppressed, while MPA T cell proliferation was strongly inhibited. Based on these data, it is expected that monitoring of T cell proliferation in MMF-treated transplantation patients may be a valid strategy to avoid excessive immune suppression.

Correction: Wind et al. Topical Bimiralisib Shows Meaningful Cutaneous Drug Levels in Healthy Volunteers and Mycosis Fungoides Patients but No Clinical Activity in a First-in-Human, Randomized Controlled Trial. Cancers 2022, 14, 1510.

The authors wish to make the following corrections to this paper [...].

Immunosuppression by hydroxychloroquine: mechanistic proof in in vitro experiments but limited systemic activity in a randomized placebo-controlled clinical pharmacology study.

Based on its wide range of immunosuppressive properties, hydroxychloroquine (HCQ) is used for the treatment of several autoimmune diseases. Limited literature is available on the relationship between HCQ concentration and its immunosuppressive effect. To gain insight in this relationship, we performed in vitro experiments in human PBMCs and explored the effect of HCQ on T and B cell proliferation and Toll-like receptor (TLR)3/TLR7/TLR9/RIG-I-induced cytokine production. In a placebo-controlled clinical study, these same endpoints were evaluated in healthy volunteers that were treated with a cumulative dose of 2400 mg HCQ over 5 days. In vitro, HCQ inhibited TLR responses with IC50s > 100 ng/mL and reaching 100% inhibition. In the clinical study, maximal HCQ plasma concentrations ranged from 75 to 200 ng/mL. No ex vivo HCQ effects were found on RIG-I-mediated cytokine release, but there was significant suppression of TLR7 responses and mild suppression of TLR3 and TLR9 responses. Moreover, HCQ treatment did not affect B cell and T cell proliferation. These investigations show that HCQ has clear immunosuppressive effects on human PBMCs, but the effective concentrations exceed the circulating HCQ concentrations under conventional clinical use. Of note, based on HCQ's physicochemical properties, tissue drug concentrations may be higher, potentially resulting in significant local immunosuppression. This trial is registered in the International Clinical Trials Registry Platform (ICTRP) under study number NL8726.

Monitoring of Ex Vivo Cyclosporin a Activity in Healthy Volunteers Using T Cell Function Assays in Relation to Whole Blood and Cellular Pharmacokinetics.

Therapeutic drug monitoring (TDM) of calcineurin inhibitors (i.e., tacrolimus and cyclosporin A) is standard of care after solid organ transplantation. Although the incidence of acute rejection has strongly decreased, there are still many patients who experience severe side effects or rejection after long-term treatment. In this healthy volunteer study we therefore aimed to identify biomarkers to move from a pharmacokinetic-based towards a pharmacodynamic-based monitoring approach for calcineurin inhibitor treatment. Healthy volunteers received a single dose of cyclosporine A (CsA) or placebo, after which whole blood samples were stimulated to measure ex vivo T cell functionality, including proliferation, cytokine production, and activation marker expression. The highest whole blood concentration of CsA was found at 2 h post-dose, which resulted in a strong inhibition of interferon gamma (IFNy) and interleukin-2 (IL-2) production and expression of CD154 and CD71 on T cells. Moreover, the in vitro effect of CsA was studied by incubation of pre-dose whole blood samples with a concentration range of CsA. The average in vitro and ex vivo CsA activity overlapped, making the in vitro dose-effect relationship an interesting method for prediction of post-dose drug effect. The clinical relevance of the results is to be explored in transplantation patients on calcineurin inhibitor treatment.

Topical Bimiralisib Shows Meaningful Cutaneous Drug Levels in Healthy Volunteers and Mycosis Fungoides Patients but No Clinical Activity in a First-in-Human, Randomized Controlled Trial.

Mycosis fungoides (MF) is a subtype of CTCL with a low incidence and high medical need for novel treatments. The objective of this randomized, placebo-controlled, double-blinded, first-in-human study was to evaluate safety, efficacy, cutaneous and systemic pharmacokinetics (PK) of topical bimiralisib in healthy volunteers (HVs) and MF patients. In this trial, a total of 6 HVs and 19 early-stage MF patients were treated with 2.0% bimiralisib gel and/or placebo. Drug efficacy was assessed by the Composite Assessment of Index Lesion Severity (CAILS) score, supported by objective measuring methods to quantify lesion severity. PK blood samples were collected frequently and cutaneous PK was investigated in skin punch biopsies on the last day of treatment. Local distribution of bimiralisib in HVs showed a mean exposure of 2.54 µg/g in the epidermis. A systemic concentration was observed after application of a target dose of 2 mg/cm2 on 400 cm2, with a mean Cavg of 0.96 ng/mL. Systemic exposure of bimiralisib was reached in all treated MF patients, and normalized plasma concentrations showed a 144% increased exposure compared to HVs, with an observed mean Cavg of 4.49 ng/mL and a mean cutaneous concentration of 5.3 µg/g. No difference in CAILS or objective lesion severity quantification upon 42 days of once-daily treatment was observed in the MF patient group. In general, the treatment was well tolerated in terms of local reactions as well as systemic adverse events. In conclusion, we showed that topical bimiralisib treatment leads to (i) meaningful cutaneous drug levels and (ii) well-tolerated systemic drug exposure in MF patients and (iii) a lack of clinical efficacy, in need of further exploration due to numerous unknown factors, before depreciation of topical bimiralisib as a novel therapeutic drug for CTCLs.

Clinical, Cellular, and Molecular Effects of Corticosteroids on the Response to Intradermal Lipopolysaccharide Administration in Healthy Volunteers.

The intradermal lipopolysaccharide (LPS) challenge in healthy volunteers has proven to be a valuable tool to study local inflammation in vivo. In the current study the inhibitory effects of oral and topical corticosteroid treatment on intradermal LPS responses were evaluated to benchmark the challenge for future investigational drugs. Twenty-four healthy male volunteers received a two-and-a-half-day twice daily (b.i.d.) pretreatment with topical clobetasol propionate 0.05% and six healthy volunteers received a two-and-a-half-day b.i.d. pretreatment with oral prednisolone at 0.25 mg/kg body weight per administration. Participants received one injection regimen of either 0, 2, or 4 intradermal LPS injections (5 ng LPS in 50 µL 0.9% sodium chloride solution). The LPS response was evaluated by noninvasive (perfusion, skin temperature, and erythema) and invasive assessments (cellular and cytokine responses) in suction blister exudate. Both corticosteroids significantly suppressed the clinical inflammatory response (erythema P = 0.0001 for clobetasol and P = 0.0016 for prednisolone; heat P = 0.0245 for clobetasol, perfusion P < 0.0001 for clobetasol and P = 0.0036 for prednisolone). Clobetasol also significantly reduced the number of monocytes subsets, dendritic cells, natural killer cells, and T cells in blister exudate. A similar effect was observed for prednisolone. No relevant corticosteroid effects were observed on the cytokine response to LPS. We successfully demonstrated that the anti-inflammatory effects of corticosteroids can be detected using our intradermal LPS challenge model, validating it for evaluation of future investigational drugs, as an initial assessment of the anti-inflammatory effects of such compounds in a minimally invasive manner.

Intradermal lipopolysaccharide challenge as an acute in vivo inflammatory model in healthy volunteers.

AIMS: Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies, systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers, and as such qualify the method as local inflammation model for clinical pharmacology studies. METHODS: Eighteen healthy male volunteers received 2 or 4 intradermal 5 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion, skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate. RESULTS: LPS elicited a visible response and returned to baseline at 48 hours. Erythema, perfusion and temperature were statistically significant (P < .0001) over a 24-hour time course compared to saline. The protein response was dominated by an acute interleukin (IL)-6, IL-8 and tumour necrosis factor response followed by IL-1ß, IL-10 and interferon-γ. The cellular response consisted of an acute neutrophil influx followed by different monocyte subsets and dendritic cells. DISCUSSION: Intradermal LPS administration in humans causes an acute, localized and transient inflammatory reaction that is well-tolerated by healthy volunteers. This may be a valuable inflammation model for evaluating the pharmacological activity of anti-inflammatory investigational compounds in proof of pharmacology studies.

Effect of hydroxychloroquine on the cardiac ventricular repolarization: A randomized clinical trial.

AIMS: Hydroxychloroquine has been suggested as possible treatment for severe acute respiratory syndrome-coronavirus-2. Studies reported an increased risk of QTcF-prolongation after treatment with hydroxychloroquine. The aim of this study was to analyse the concentration-dependent effects of hydroxychloroquine on the ventricular repolarization, including QTcF-duration and T-wave morphology. METHODS: Twenty young (≤30 y) and 20 elderly (65-75 y) healthy male subjects were included. Subjects were randomized to receive either a total dose of 2400 mg hydroxychloroquine over 5 days, or placebo (ratio 1:1). Follow-up duration was 28 days. Electrocardiograms (ECGs) were recorded as triplicate at baseline and 4 postdose single recordings, followed by hydroxychloroquine concentration measurements. ECG intervals (RR, QRS, PR, QTcF, J-Tpc, Tp-Te) and T-wave morphology, measured with the morphology combination score, were analysed with a prespecified linear mixed effects concentration-effect model. RESULTS: There were no significant associations between hydroxychloroquine concentrations and ECG characteristics, including RR-, QRS- and QTcF-interval (P = .09, .34, .25). Mean ΔΔQTcF-interval prolongation did not exceed 5 ms and the upper limit of the 90% confidence interval did not exceed 10 ms at the highest measured concentrations (200 ng/mL). There were no associations between hydroxychloroquine concentration and the T-wave morphology (P = .34 for morphology combination score). There was no significant effect of age group on ECG characteristics. CONCLUSION: In this study, hydroxychloroquine did not affect ventricular repolarization, including the QTcF-interval and T-wave morphology, at plasma concentrations up to 200 ng/mL. Based on this analysis, hydroxychloroquine does not appear to increase the risk of QTcF-induced arrhythmias.

Erratum to "Hydroxychloroquine Effects on TLR Signalling: Underexposed but Unneglectable in COVID-19".

[This corrects the article DOI: 10.1155/2021/6659410.].

Hydroxychloroquine Effects on TLR Signalling: Underexposed but Unneglectable in COVID-19.

The main basis for hydroxychloroquine (HCQ) treatment in COVID-19 is the compound's ability to inhibit viral replication in vitro. HCQ also suppresses immunity, mainly by interference in TLR signalling, but reliable clinical data on the extent and nature of HCQ-induced immunosuppression are lacking. Here, we discuss the mechanistic basis for the use of HCQ against SARS-CoV-2 in a prophylactic setting and in a therapeutic setting, at different stages of the disease. We argue that the clinical effect of prophylactic or therapeutic HCQ treatment in COVID-19 depends on the balance between inhibition of viral replication, immunosuppression, and off-target side effects, and that the outcome is probably dependent on disease stage and disease severity. This is supported by the initial outcomes of the well-designed randomized controlled trials: so far, evidence for a beneficial effect of HCQ treatment for COVID-19 is weak and conflicting.

Targeting of tolerogenic dendritic cells to heat-shock proteins in inflammatory arthritis.

BACKGROUND: Autologous tolerogenic dendritic cells (tolDC) are a promising therapeutic strategy for inflammatory arthritis (IA) as they can regulate autoantigen-specific T cell responses. Here, we investigated two outstanding priorities for clinical development: (i) the suitability of using heat-shock proteins (HSP), abundant in inflamed synovia, as surrogate autoantigens to be presented by tolDC and (ii) identification of functional biomarkers that confirm tolDC regulatory activity. METHODS: Cell proliferation dye-labelled human peripheral blood mononuclear cells of IA (rheumatoid arthritis (RA) and psoriatic arthritis (PsA)) patients or healthy donors were cultured with HSP40-, HSP60- and HSP70-derived peptides or recall antigens (e.g. tuberculin purified protein derivative (PPD)) in the presence or absence of tolDC or control DC for 9 days. Functional characteristics of proliferated antigen-specific T-cells were measured using flow cytometry, gene expression profiling and cytokine secretion immunoassays. Repeated measures analysis of variance (ANOVA) with Bonferroni correction for comparisons between multiple groups and paired Student t test for comparisons between two groups were used to determine significance. RESULTS: All groups showed robust CD4+ T-cell responses towards one or more HSP-derived peptide(s) as assessed by a stimulation index > 2 (healthy donors: 78%, RA: 73%, PsA: 90%) and production of the cytokines IFNγ, IL-17A and GM-CSF. Addition of tolDC but not control DC induced a type 1 regulatory (Tr1) phenotype in the antigen-specific CD4+ T-cell population, as identified by high expression of LAG3, CD49b and secretion of IL-10. Furthermore, tolDC inhibited bystander natural killer (NK) cell activation in a TGFß dependent manner. CONCLUSIONS: HSP-specific CD4+ T-cells are detectable in the majority of RA and PsA patients and can be converted into Tr1 cells by tolDC. HSP-loaded tolDC may therefore be suitable for directing T regulatory responses to antigens in inflamed synovia of IA patients. Tr1 markers LAG3, CD49b and IL-10 are suitable biomarkers for future tolDC clinical trials.

Matured Tolerogenic Dendritic Cells Effectively Inhibit Autoantigen Specific CD4+ T Cells in a Murine Arthritis Model.

Tolerogenic dendritic cells (tolDCs) are a promising treatment modality for diseases caused by a breach in immune tolerance, such as rheumatoid arthritis. Current medication for these diseases is directed toward symptom suppression but no real cure is available yet. TolDC-based therapy aims to restore immune tolerance in an antigen-specific manner. Here we used a mouse model to address two major questions: (i) is a maturation stimulus needed for tolDC function in vitro and in vivo and is maturation required for functioning in experimental arthritis and (ii) can tolDCs modulate CD4+ T cell responses? To answer these questions, we compared matured and immature dexamethasone/vitamin D3-generated tolDCs in vitro. Subsequently, we co-transferred these tolDCs with naïve or effector CD4+ T cells to study the characteristics of transferred T cells after 3 days with flow cytometry and Luminex multiplex assays. In addition, we tested the suppressive capabilities of tolDCs in an experimental arthritis model. We found that tolDCs cannot only modulate naïve CD4+ T cell responses as shown by fewer proliferated and activated CD4+ T cells in vivo, but also effector CD4+ T cells. In addition, Treg (CD4+CD25+FoxP3+) expansions were seen in the proliferating cell population in the presence of tolDCs. Furthermore, we show that administered tolDCs are capable to inhibit arthritis in the proteoglycan-induced arthritis model. However, a maturation stimulus is needed for tolDCs to manifest this tolerizing function in an inflammatory environment. Our data will be instrumental for optimization of future tolDC therapies for autoimmune diseases.

Lipidoid-polymer hybrid nanoparticles loaded with TNF siRNA suppress inflammation after intra-articular administration in a murine experimental arthritis model.

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.

Heat Shock Proteins Can Be Surrogate Autoantigens for Induction of Antigen Specific Therapeutic Tolerance in Rheumatoid Arthritis.

Technologies that enable induction of therapeutic tolerance may revolutionize the treatment of autoimmune diseases by their supposed potential to induce drug-free and lasting disease remission. In combination with diagnostic tests that screen for individuals at risk, these approaches may offer chances to halt disease before serious damage in the tissues can occur. In fact, for healthy individuals at risk, this could lead to a preventive form of vaccination. For therapeutic tolerance to re-instate natural self-tolerance it seems essential to induce tolerance for the critical autoantigens involved in disease. However, for most autoimmune diseases such antigens are poorly defined. This is the case for both disease inciting autoantigens and antigens that become involved through epitope spreading. A possible source of surrogate auto-antigens expressed in tissues during inflammation are heat shock proteins (HSP) or stress proteins. In this mini-review we discuss unique characteristics of HSP which provide them with the capacity to inhibit inflammatory processes. Various studies have shown that epitopes of HSP60 and HSP70 molecules can function as vaccines to downregulate a variety of autoimmune inflammatory diseases. Currently, several research groups are developing cell therapies with the intention to reach therapeutic tolerance. In this review, in which we are proposing to ex vivo load tolerant dendritic cells with a Treg inducing HSP70 derived peptide called B29, we are discussing the chances to develop this as an autologous tolDC therapeutic tolerance therapy for rheumatoid arthritis.

Routing dependent immune responses after experimental R848-adjuvated vaccination.

Most traditional vaccines are administered via the intramuscular route. Other routes of administration however, can induce equal or improved protective memory responses and might provide practical advantages such as needle-free immunization, dose sparing and induction of tissue-specific (mucosal) immunity. Here we explored the differences in immunological outcome after immunization with model antigens via two promising immunization routes (intradermal and intranasal) with or without the experimental adjuvant and TLR7/8-agonist R848. Because the adaptive immune response is largely determined by the local innate cells at the site of immunization, the effect of R848-adjuvation on local cellular recruitment, antigenic uptake by antigen-presenting cells and the initiation of the adaptive response were analyzed for the two routes of administration. We show a general immune-stimulating effect of R848 irrespective of the route of administration. This includes influx of neutrophils, macrophages and dendritic cells to the respective draining lymph nodes and an increase in antigen-positive antigen-presenting cells which leads for both intradermal and intranasal immunization to a mainly TH1 response. Furthermore, both intranasal and intradermal R848-adjuvated immunization induces a local shift in DC subsets; frequencies of CD11b+DC increase whereas CD103+DC decrease in relative abundance in the draining lymph node. In spite of these similarities, the outcome of immune responses differs for the respective immunization routes in both magnitude and cytokine profile. Via the intradermal route, the induced T-cell response is higher compared to that after intranasal immunization, which corresponds with the local higher uptake of antigen by antigen-presenting cells after intradermal immunization. Furthermore, R848-adjuvation enhances ex vivo IL-10 and IL-17 production after intranasal, but not intradermal, T-cell activation. Quite the opposite, intradermal immunization leads to a decrease in IL-10 production by the vaccine induced T-cells. This knowledge may lead to a more rational development of novel adjuvanted vaccines administered via non-traditional routes.