Impact of Moderna mRNA-1273 Booster Vaccine on Fully Vaccinated High-Risk Chronic Dialysis Patients after Loss of Humoral Response.

The long-term effect of protection by two doses of SARS-CoV-2 vaccination in patients receiving chronic intermittent hemodialysis (CIHD) is an urging question. We investigated the humoral and cellular immune response of 42 CIHD patients who had received two doses of SARS-CoV-2 vaccine, and again after a booster vaccine with mRNA-1273 six months later. We measured antibody levels and SARS-CoV-2-specific surrogate neutralizing antibodies (SNA). Functional T cell immune response to vaccination was assessed by quantifying interferon-γ (IFN-γ) and IL-2 secreting T cells specific for SARS-CoV-2 using an ELISpot assay. Our data reveal a moderate immune response after the second dose of vaccination, with significantly decreasing SARS-CoV-2-specific antibody levels and less than half of the study group showed neutralizing antibodies six months afterwards. Booster vaccines increased the humoral response dramatically and led to a response rate of 89.2% for antibody levels and a response rate of 94.6% for SNA. Measurement in a no response/low response (NR/LR) subgroup of our cohort, which differed from the whole group in age and rate of immunosuppressive drugs, indicated failure of a corresponding T cell response after the booster vaccine. We strongly argue in favor of a regular testing of surrogate neutralizing antibodies and consecutive booster vaccinations for CIHD patients to provide a stronger and persistent immunity.

Clinical Significance of Micronutrient Supplementation in Critically Ill COVID-19 Patients with Severe ARDS.

The interplay between inflammation and oxidative stress is a vicious circle, potentially resulting in organ damage. Essential micronutrients such as selenium (Se) and zinc (Zn) support anti-oxidative defense systems and are commonly depleted in severe disease. This single-center retrospective study investigated micronutrient levels under Se and Zn supplementation in critically ill patients with COVID-19 induced acute respiratory distress syndrome (ARDS) and explored potential relationships with immunological and clinical parameters. According to intensive care unit (ICU) standard operating procedures, patients received 1.0 mg of intravenous Se daily on top of artificial nutrition, which contained various amounts of Se and Zn. Micronutrients, inflammatory cytokines, lymphocyte subsets and clinical data were extracted from the patient data management system on admission and after 10 to 14 days of treatment. Forty-six patients were screened for eligibility and 22 patients were included in the study. Twenty-one patients (95%) suffered from severe ARDS and 14 patients (64%) survived to ICU discharge. On admission, the majority of patients had low Se status biomarkers and Zn levels, along with elevated inflammatory parameters. Se supplementation significantly elevated Se (p = 0.027) and selenoprotein P levels (SELENOP; p = 0.016) to normal range. Accordingly, glutathione peroxidase 3 (GPx3) activity increased over time (p = 0.021). Se biomarkers, most notably SELENOP, were inversely correlated with CRP (rs = -0.495), PCT (rs = -0.413), IL-6 (rs = -0.429), IL-1ß (rs = -0.440) and IL-10 (rs = -0.461). Positive associations were found for CD8+ T cells (rs = 0.636), NK cells (rs = 0.772), total IgG (rs = 0.493) and PaO2/FiO2 ratios (rs = 0.504). In addition, survivors tended to have higher Se levels after 10 to 14 days compared to non-survivors (p = 0.075). Sufficient Se and Zn levels may potentially be of clinical significance for an adequate immune response in critically ill patients with severe COVID-19 ARDS.

Effects of Bacillus firmus e volumine ex muris cellulae 6x on cytolytic activity of natural killer cells in vitro

Natural killer (NK) cells are among the first in defense of the innate immune system by eliminating a variety of abnormal or stressed cells such as cancer cells or virus-infected cells. Individuals who exhibit low cytolytic NK cell activity are believed to be at higher risk of viral infection, tumorigenesis, and various other diseases of the immune system. Therefore, restoration of impaired NK cell function might be an essential step in immunostimulatory therapy of immunocompromised patients. Bacillus firmus is a non-pathogenic gram-positive bacterium of the environment, which possesses various immunomodulatory properties in vitro and in vivo. This retrospective study reports on the effect of B. firmus on the activity of NK cells in vitro. Basal cytolytic NK cell activity against tumor cells among peripheral blood mononuclear cells (PBMCs) of routine patients was determined in a standardized NK cell cytotoxicity assay. The impact of cultivation of PBMCs with B. firmus preparation Bacillus firmus e volumine ex muris cellulae (Bacillus firmus (evc)) 6x on tumor cell killing by NK cells was monitored in relation to basal NK cell activity. This study showed that stimulation of PBMCs with Bacillus firmus (evc) 6x in vitro led to a significant increase in NK cell function. Substantial improvement in cytolytic NK cell activity (more than 1.3-fold of basal activity) was much more pronounced for patients with compromised NK cell function. Due to its immunostimulatory mode of action, Bacillus firmus (evc) may be of particular importance in therapy of patients with NK cell deficiency.

Inhibition of antigen-specific immune responses by co-application of an indoleamine 2,3-dioxygenase (IDO)-encoding vector requires antigen transgene expression focused on dendritic cells.

We have previously shown that particle-mediated epidermal delivery (PMED) of plasmids encoding ß-galactosidase (ßGal) under control of the fascin-1 promoter (pFascin-ßGal) yielded selective production of the protein in skin dendritic cells (DCs), and suppressed Th2 responses in a mouse model of type I allergy by inducing Th1/Tc1 cells. However, intranasal challenge of mice immunized with pFascin-ßGal induced airway hyperreactivity (AHR) and neutrophilic inflammation in the lung. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. Here we investigated the consequences of co-application of an IDO-encoding vector on the modulatory effect of DNA vaccination by PMED using pFascin-ßGal in models of eosinophilic allergic and non-eosinophilic intrinsic airway inflammation. IDO-encoding plasmids and pFascin-ßGal or pCMV-ßGal were co-applied to abdominal skin of BALB/c mice without, before or after sensitization with ßGal protein. Immune responses in the lung were analysed after intranasal provocation and airway reactivity was determined by whole body plethysmography. Co-application of pCMV-IDO with pFascin-ßGal, but not pCMV-ßGal inhibited the Th1/Tc1 immune response after PMED. Moreover, AHR in those mice was attenuated following intranasal challenge. Therapeutic vaccination of ßGal-sensitized mice with pFascin-ßGal plus pCMV-IDO slightly suppressed airway inflammation and AHR after provocation with ßGal protein, while prophylactic vaccination was not effective. Altogether, our data suggest that only the combination of DC-restricted antigen and ubiquitous IDO expression attenuated asthma responses in mice, most probably by forming a tryptophan-depleted and kynurenine-enriched micromilieu known to affect neutrophils and T cells.

Role of Protein Kinase C and Nox2-Derived Reactive Oxygen Species Formation in the Activation and Maturation of Dendritic Cells by Phorbol Ester and Lipopolysaccharide.

Aims. Activation/maturation of dendritic cells (DCs) plays a central role in adaptive immune responses by antigen processing and (cross-) activation of T cells. There is ongoing discussion on the role of reactive oxygen species (ROS) in these processes and with the present study we investigated this enigmatic pathway. Methods and Results. DCs were cultured from precursors in the bone marrow of mice (BM-DCs) and analyzed for ROS formation, maturation, and T cell stimulatory capacity upon stimulation with phorbol ester (PDBu) and lipopolysaccharide (LPS). LPS stimulation of BM-DCs caused maturation with moderate intracellular ROS formation, whereas PDBu treatment resulted in maturation with significant ROS formation. The NADPH oxidase inhibitors apocynin/VAS2870 and genetic gp91phox deletion both decreased the ROS signal in PDBu-stimulated BM-DCs without affecting maturation and T cell stimulatory capacity of BM-DCs. In contrast, the protein kinase C inhibitors chelerythrine/Gö6983 decreased PDBu-stimulated ROS formation in BM-DCs as well as maturation. Conclusion. Obviously Nox2-dependent ROS formation in BM-DCs is not always required for their maturation or T cell stimulatory potential. PDBu/LPS-triggered BM-DC maturation rather relies on phosphorylation cascades. Our results question the role of oxidative stress as an essential "danger signal" for BM-DC activation, although we cannot exclude contribution by other ROS sources.

Nutritional control of IL-23/Th17-mediated autoimmune disease through HO-1/STAT3 activation.

The nutritional curcumin (CUR) is beneficial in cell-mediated autoimmune diseases. The molecular mechanisms underlying this food-mediated silencing of inflammatory immune responses are poorly understood. By investigating antigen-specific immune responses we found that dietary CUR impairs the differentiation of Th1/Th17 cells in vivo during encephalomyelitis and instead promoted Th2 cells. In contrast, feeding CUR had no inhibitory effect on ovalbumin-induced airway inflammation. Mechanistically, we found that CUR induces an anti-inflammatory phenotype in dendritic cells (DC) with enhanced STAT3 phosphorylation and suppressed expression of Il12b and Il23a. On the molecular level CUR readily induced NRF2-sensitive heme oxygenase 1 (HO-1) mRNA and protein in LPS-activated DC. HO-1 enhanced STAT3 phosphorylation, which enriched to Il12b and Il23a loci and negatively regulated their transcription. These findings demonstrate the underlying mechanism through which a nutritional can interfere with the immune response. CUR silences IL-23/Th17-mediated pathology by enhancing HO-1/STAT3 interaction in DC.

Gliptin and GLP-1 analog treatment improves survival and vascular inflammation/dysfunction in animals with lipopolysaccharide-induced endotoxemia.

Dipeptidyl peptidase (DPP)-4 inhibitors are used to treat hyperglycemia by increasing the incretin glucagon-like peptide-1 (GLP-1). Previous studies showed anti-inflammatory and antiatherosclerotic effects of DPP-4 inhibitors. Here, we compared the effects of linagliptin versus sitagliptin and liraglutide on survival and vascular function in animal models of endotoxic shock by prophylactic therapy and treatment after lipopolysaccharide (LPS) injection. Gliptins were administered either orally or subcutaneously: linagliptin (5 mg/kg/day), sitagliptin (50 mg/kg/day) or liraglutide (200 µg/kg/day). Endotoxic shock was induced by LPS injection (mice 17.5-20 mg/kg i.p., rats 10 mg/kg/day). Linagliptin and liraglutide treatment or DPP-4 knockout improved the survival of endotoxemic mice, while sitagliptin was ineffective. Linagliptin, liraglutide and sitagliptin ameliorated LPS-induced hypotension and vascular dysfunction in endotoxemic rats, suppressed inflammatory parameters such as whole blood nitrosyl-iron hemoglobin (leukocyte-inducible nitric oxide synthase activity) or aortic mRNA expression of markers of inflammation as well as whole blood and aortic reactive oxygen species formation. Hemostasis (tail bleeding time, activated partial thromboplastin time) was impaired in endotoxemic rats and recovered under cotreatment with linagliptin and liraglutide. Finally, the beneficial effects of linagliptin on vascular function and inflammatory parameters in endotoxemic mice were impaired in AMP-activated kinase (alpha1) knockout mice. The improved survival of endotoxemic animals and other data shown here may warrant further clinical evaluation of these drugs in patients with septic shock beyond the potential improvement of inflammatory complications in diabetic individuals with special emphasis on the role of AMP-activated kinase (alpha1) in the DPP-4/GLP-1 cascade.

Regulation of IgE production and airway reactivity by CD4⁻CD8⁻ regulatory T cells.

The mechanisms of tolerance induction occurring in the course of allergen-specific immunotherapy have not been elucidated in full detail. Our study aimed to characterize high zone tolerance in mouse models of type I allergy and of allergic airway inflammation induced by subcutaneous sensitization of mice with high doses of the model allergen ovalbumin (OVA) without the use of adjuvant. Mice were immunized by subcutaneous injection of high doses (HD) of OVA or, for comparison, low doses (LD) of OVA in saline. HD-mice showed lower specific IgE, but augmented IgG in sera than LD-mice. Pre-treatment of mice with HD-OVA antigen-specifically inhibited IgE production subsequently induced by LD-OVA. OVA-restimulated splenocytes from HD-mice revealed hypoproliferation and impaired production of Th2-associated cytokines. HD-mice exhibited lower airway reactivity, goblet cell hyperplasia and mucus production, as well as IL-5 and IL-13 production in the lungs than LD-mice following local provocation. Recruitment of inflammatory cells into the airways was comparable, while the number of eosinophils in the bronchoalveolar lavage was substantially higher in HD-mice. Adoptive transfer of dnTC from HD-mice into naïve mice, which were subsequently sensitized with LD-OVA, suppressed IgE production in the recipients. The number of dnTC was higher in the spleens of HD-mice than LD-mice. In conclusion, our study demonstrates that subcutaneous sensitization of mice with high doses of allergen in the absence of adjuvant results in attenuated airway reactivity as compared with LD-sensitization and induces CD4(-)CD8(-) dnTC with regulatory function on IgE production.

Dendritic cell-specific biolistic transfection using the fascin gene promoter.

The transcriptional targeting of gene expression to selected cells by cell type-specific promoters displays a fundamental tool in gene therapy. In immunotherapy, dendritic cells (DCs) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection. Here we report on transcriptional targeting of murine skin DCs using plasmids which include the promoter of the gene of the cytoskeletal protein fascin to control antigen production. Fascin, which is mandatory for the formation of dendrites, is synthesized among the hematopoietic cells exclusively by activated DCs. The activity of the promoter of the fascin gene reflects the endogenous production of the protein, being high in mature DCs but almost absent in immature DCs or other cutaneous cells. Here we describe the analysis of transgene-specific immune responses after DC-focused biolistic transfection. In conclusion, the murine fascin promoter can be readily used to target DCs in DNA immunization approaches and thus offers new opportunities for gene therapy.

Efficiency of biolistic DNA vaccination in experimental type I allergy.

Gene gun-mediated delivery of allergen-encoding plasmid DNA has been in focus for many years now as being a needle-free alternative to the protein-based desensitization regimen used in specific immunotherapy. Biolistic immunization with the Helios gene gun has proven to be potent in the induction of antigen-specific CD4(+) and CD8(+) T cells. Here we describe biolistic vaccination in experimental mouse models of IgE-mediated type I allergy as well as allergen-induced airway inflammation.

Glucose-independent improvement of vascular dysfunction in experimental sepsis by dipeptidyl-peptidase 4 inhibition.

AIMS: Dipeptidyl peptidase-4 (DPP-4) inhibitors are a novel class of drugs for the treatment of hyperglycaemia. Preliminary evidence suggests that their antioxidant and anti-inflammatory effects may have beneficial effects on the cardiovascular complications of diabetes. In the present study, we investigate in an experimental sepsis model whether linagliptin exerts pleiotropic vascular effects independent of its glucose-lowering properties. METHODS AND RESULTS: Linagliptin (83 mg/kg chow for 7 days) was administered in a rat model of lipopolysaccharide (LPS) (10 mg/kg, single i.p. dose/24 h)-induced sepsis. Vascular relaxation, reactive oxygen species (ROS) formation, expression of NADPH oxidase subunits and proinflammatory markers, and white blood cell infiltration in the vasculature were determined. Oxidative burst and adhesion of isolated human neutrophils to endothelial cells were measured in the presence of different DPP-4 inhibitors, and their direct vasodilatory effects (isometric tension in isolated aortic rings) were compared. In vivo linagliptin treatment ameliorated LPS-induced endothelial dysfunction and was associated with reduced formation of vascular, cardiac, and blood ROS, aortic expression of inflammatory genes and NADPH oxidase subunits in addition to reduced aortic infiltration with inflammatory cells. Linagliptin was the most potent inhibitor of oxidative burst in isolated activated human neutrophils and it suppressed their adhesion to activated endothelial cells. Of the inhibitors tested, linagliptin and alogliptin had the most pronounced direct vasodilatory potency. CONCLUSION: Linagliptin demonstrated pleiotropic vasodilatory, antioxidant, and anti-inflammatory properties independent of its glucose-lowering properties. These pleiotropic properties are generally not shared by other DPP-4 inhibitors and might translate into cardiovascular benefits in diabetic patients.

Allergen-induced IgE-dependent gut inflammation in a human PBMC-engrafted murine model of allergy.

BACKGROUND: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. OBJECTIVE: In this study we developed a human PBMC-engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. METHODS: Nonobese diabetic (NOD)-scid-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. RESULTS: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergen-specific proliferation and cytokine production of human CD4(+) T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. CONCLUSION: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions.

Mast cells induce migration of dendritic cells in a murine model of acute allergic airway disease.

BACKGROUND: The migration of dendritic cells (DCs) from the lungs to the regional lymph nodes is necessary for the development of allergic airway disease. Following activation, mast cells release a variety of stored or de novo-produced inflammatory mediators, several of them being capable of activating DCs. In this study, the role of mast cells on DC migration from the lungs to the thoracic lymph nodes was investigated in sensitized mice. METHODS: Mast cell-deficient mice (Kit(W-sh/W-sh)) and their wild-type counterparts were sensitized intraperitoneally with ovalbumine (OVA) in saline and challenged by a single intranasal administration of OVA labeled with a fluorescent dye (OVA-Alexa). RESULTS: Following challenge, the relative and absolute amount of OVA- Alexa-positive DCs was clearly increased in sensitized wild-type mice compared to nonsensitized mice. In contrast, sensitized Kit(W-sh/W-sh) showed no increase in OVA-Alexa-positive DCs compared to nonsensitized mast cell-deficient animals. In sensitized Kit(W-sh/W-sh) mice reconstituted with bone marrow-derived mast cells (BMMCs), the number of OVA- Alexa-positive DCs was comparable to that in sensitized wild-type animals. However, transfer of allergen-exposed BMMCs to sensitized mice prior to airway challenge augmented airway inflammation similarly in wild-type and mast cell-deficient mice. In line with this, sensitization with allergen-pulsed DCs induced allergic airway disease independently of mast cells. CONCLUSIONS: This study shows an interaction between mast cells and DCs following allergen challenge in sensitized hosts. However, the function of mast cells can be bypassed in models utilizing activated allergen-exposed DCs to initiate the development of allergic airway disease.

A novel plasmid DNA electroporation method allows transfection of murine DC.

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)-derived peptide induced proliferation of MOG-reactive CD4(+) T cells as potently as did non-transfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.

Uptake and presentation of exogenous antigen and presentation of endogenously produced antigen by skin dendritic cells represent equivalent pathways for the priming of cellular immune responses following biolistic DNA immunization.

Gene gun-mediated biolistic DNA vaccination with beta-galactosidase (betaGal)-encoding plasmid vectors efficiently modulated antigen-induced immune responses in an animal model of type I allergy, including the inhibition of immunoglobulin E (IgE) production. Here we show that CD4(+) as well as CD8(+) T cells from mice biolistically transfected with a plasmid encoding betaGal under the control of the fascin promoter (pFascin-betaGal) are capable of inhibiting betaGal-specific IgE production after adoptive transfer into naïve recipients. Moreover, suppression of IgE production was dependent on interferon (IFN)-gamma. To analyse the modalities of activation of CD4(+) and CD8(+) T cells regarding the localization of antigen synthesis following gene gun-mediated DNA immunization, we used the fascin promoter and the keratin 5 promoter (pK5-betaGal) to direct betaGal production mainly to dendritic cells (DCs) and to keratinocytes, respectively. Gene gun-mediated DNA immunization with each vector induced considerable activation of betaGal-specific CD8(+) cytotoxic T cells. Cytokine production by re-stimulated CD4(+) T cells in draining lymph nodes and immunoglobulin isotype profiles in sera of immunized mice indicated that immunization with pFascin-betaGal induced a T helper type 1 (Th1)-biased immune response, whereas immunization with pK5-betaGal generated a mixed Th1/Th2 immune response. Nevertheless, DNA vaccination with pFascin-betaGal and pK5-betaGal, respectively, efficiently inhibited specific IgE production in the mouse model of type I allergy. In conclusion, our data show that uptake of exogenous antigen produced by keratinocytes and its presentation by untransfected DCs as well as the presentation of antigen synthesized endogenously in DCs represent equivalent pathways for efficient priming of cellular immune responses.

Divergent effects of biolistic gene transfer in a mouse model of allergic airway inflammation.

Particle-mediated epidermal delivery (PMED) of allergen genes efficiently prevents systemic sensitization and suppresses specific immunoglobulin E synthesis. We investigated in a mouse model of allergic airway disease the effect of PMED on the elicitation of local inflammatory reactions in the lung. BALB/c mice were biolistically transfected with plasmids encoding beta-galactosidase (betaGal) as model allergen under control of the DC-targeting fascin promoter and the ubiquitously active cytomegalovirus promoter, respectively. Mice were challenged intranasally with betaGal-protein with or without intermediate sensitization with betaGal adsorbed to aluminiumhydroxide. Subsequently, local cytokine production and recruitment of IFN-gamma-producing CD8(+) effector T cells into the airways were determined, and inflammatory parameters such as cellular infiltration in the bronchoalveolar lavage (BAL) and airway hyperresponsiveness (AHR) were measured. PMED of betaGal-encoding plasmids before sensitization significantly reduced frequencies of eosinophils in the BAL and shifted the local T helper (Th) cell response from a distinct Th2 response toward a Th1-biased response. However, AHR triggered by allergen challenge via the airways was not alleviated in vaccinated mice. Most important, we show that PMED using betaGal-encoding DNA without subsequent sensitization recruited Tc1 cells into the lung and caused a Th1-prone local immune response after subsequent intranasal provocation, accompanied by neutrophilic infiltration into the airways and elicitation of AHR. We conclude that robust Th1/Tc1 immune responses, although highly effective in the counter-regulation of local Th2-mediated pathology, might as well trigger local inflammatory reactions in the lung and provoke the induction of AHR in the mouse model of allergic airway disease.

Formalin-fixed Staphylococcus aureus particles prevent allergic sensitization in a murine model of type I allergy.

BACKGROUND: Bacterial infections are supposed to act counterregulatory to the development of allergen-specific Th2 immune responses. We analyzed whether administration of extracellular Staphylococcus aureus inhibited experimental sensitization against allergens. METHODS: BALB/c mice were immunized with alum-adsorbed ovalbumin (OVA) together with formalin-fixed Staphylococcus particles. OVA-specific antibody production and cytokine synthesis by spleen cells was analyzed. Airway reactivity and cellular infiltration into the airways was assessed after intranasal challenge of mice with OVA. In addition, the capacity of Staphylococcus particles to modulate cytokine production by bone marrow-derived dendritic cells was analyzed in vitro. RESULTS: Simultaneous application of OVA and Staphylococcus particles very efficiently inhibited production of specific IgE and IgG1 as well as secretion of IL-4 and IL-5 by splenocytes, while enhancing IgG2a formation and production of IFN-gamma, indicating a shift from a Th2 response towards a Th1-biased response. This effect was not dependent on the expression of protein A by Staphylococcus. An enhanced frequency or activity of regulatory T cells after administration of Staphylococcus particles was not apparent. Treatment of mice with Staphylococcus particles during the sensitization phase prevented lung inflammation (airway hyperreactivity, eosinophilia) after local challenge with OVA. Culture of bone marrow-derived dendritic cells with Staphylococcus particles induced IL-12p35 and p40 mRNA expression as well as secretion of IL-12p70, and increased production of IL-10 mRNA and protein. CONCLUSIONS: Administration of formalin-fixed Staphylococcus particles induced Th1-biased immune responses and prevented allergic sensitization.

A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid.

The phenotype and function of murine dendritic cells (DCs) are primarily studied using bone-marrow-derived DCs (BM-DCs), but may be hampered by the heterogeneous phenotype of BM-DCs due to their differential state of maturation. Here we characterize a newly established murine DC line (SP37A3) of myeloid origin. During maintainance in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, SP37A3 cells resemble immature DCs characterized by low expression of major histocompatibility complex (MHC) II and costimulatory molecules and low T-cell stimulatory capacity. Upon stimulation, SP37A3 cells acquire a mature phenotype and activate naive T cells as potently as BM-DCs. Similar to BM-DCs, SP37A3 cells activated in the presence of dexamethasone-induced regulatory T cells, which were anergic upon restimulation and suppressed proliferation of naive T cells. This tolerogenic state was reflected by lower expression levels of costimulatory molecules and proinflammatory cytokines compared with mature cells, as well as up-regulated expression of FcgammaRIIB and interleukin-1RA (IL-1RA). SP37A3 cells were responsive to dexamethasone even when applied at later time points during activation, suggesting functional plasticity. Thus, DC line SP37A3 represents a suitable model to study functions of immature and mature as well as tolerogenic myeloid DCs, circumventing restrictions associated with the use of primary DCs and BM-DCs.

Interleukin-10-treated dendritic cells do not inhibit Th2 immune responses in ovalbumin/alum-sensitized mice.

BACKGROUND: It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). METHODS: OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. RESULTS: Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. CONCLUSIONS: Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.

Induction of regulatory T cells by leflunomide in a murine model of contact allergen sensitivity.

Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-specific activation of CD8+ and CD4+ T cells and the production of cytokines resulting in an inflammatory response and tissue damage. We show here that the immunosuppressive compound leflunomide (N-[4-trifluoro-methylphenyl]-5-methylisoxazol-4 carboxamide, HWA 486) (LF) inhibited the contact allergic response induced in mice by epicutaneous application of the haptens dinitrofluorobenzene (DNFB) and oxazolone. The extent of ear swelling remained significantly reduced following repeated challenge with DNFB for up to 18 weeks. LF and DNFB had to be applied simultaneously for inhibition to occur. The loss of CHS responses was shown to be antigen-specific. Adoptive transfer of leukocytes from LF-treated mice into naïve mice resulted in a loss of CHS responsiveness. Transfer of both CD4+ and CD8+ cells was required for maximal loss of CHS responses, with CD8+ cells playing a major role. Significantly enhanced levels of IL-10 mRNA were detected in CD8+ T cells, but not in CD4+ T cells, following LF treatment of mice. LF also suppressed CHS responses in mice previously sensitized and challenged with hapten, when administered together with the hapten. Our data suggest that LF induces a long-lived tolerance in mice by inducing CD8+ and CD4+ regulatory T cells.