Marcel A. Schijf

Regulatory T-cells have a prominent role in the immune modulated vaccine response by specific oligosaccharides

Regulatory T-cells are increasingly important in vaccine strategies. In a Flu-vaccination model the role of CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) and the immune modulation by orally supplied prebiotic oligosaccharides consisting of scGOS/lcFOS/pAOS, were assessed using anti-CD25 (PC61) mediated depletion studies. As expected, in C57BL/6J mice the Flu-vaccination resulted in significantly (p<0.001) increased DTH responses when receiving scGOS/lcFOS/pAOS. In addition, increased T-bet expression of activated CD4(+) T-cells was detected compared to placebo. In vivo depletion of CD25(+) Tregs significantly (p<0.05) increased basal DTH responses, indicating the suppressive function of these CD25(+) Tregs normally present. Surprisingly, in vivo Tregs depletion diminished scGOS/lcFOS/pAOS induced immune modulation completely to control levels (p<0.05). Although no difference in number, percentage or activation of Tregs could be determined after scGOS/lcFOS/pAOS supplementation, changes in Treg function still remains to be investigated. In conclusion, CD25(+) Tregs have an important role in modulated Flu-vaccine responses induced by scGOS/lcFOS/pAOS.

The respiratory local lymph node assay as a tool to study respiratory sensitizers.

The local lymph node assay (LLNA) is used to test the potential of low molecular weight (LMW) compounds to induce sensitization via the skin. In the present study, a respiratory LLNA was developed. Male BALB/c mice were exposed head/nose-only during three consecutive days for 45, 90, 180, or 360 min/day to various LMW allergens. Ear application (skin LLNA) was used as a positive control. Negative controls were exposed to the vehicle. Three days after the last exposure, proliferation was determined in the draining mandibular lymph nodes, and the respiratory tract was examined microscopically. Upon inhalation, the allergens trimellitic anhydride, phthalic anhydride, hexamethylene diisocyanate, toluene diisocyanate, isophorone diisocyanate (IPDI), dinitrochlorobenzene, and oxazolone were positive and showed stimulation indices (SIs) up to 11, whereas trimeric IPDI, formaldehyde, and methyl salicylate were negative (viz. SI < 3). All compounds, except trimeric IPDI, induced histopathological lesions predominantly in the upper respiratory tract. Exposure by inhalation is a realistic approach to test respiratory allergens. However, based on the local toxicity, the dose that can be applied is (generally) much lower than can be achieved by skin application. It is concluded that strong LMW allergens, regardless their immunological nature, besides the skin can also sensitize the body via the respiratory tract. In addition, the contact allergens were as potent as the respiratory allergens, although the potency ranking differed from that in a skin LLNA.

Mycobacterium vaccae administration during allergen sensitization or challenge suppresses asthmatic features

Background and objective  The hygiene hypothesis suggests that a lack of bacterial infections would favour the development of allergic disease. For this reason, bacteria or their components can be used as potential treatment for allergic asthma. We investigated whether heat‐killed Mycobacterium vaccae is either able to suppress the induction of allergic asthma or able to suppress already established allergic asthma.

Contact and respiratory sensitizers can be identified by cytokine profiles following inhalation exposure

There are currently no validated animal models that can identify low molecular weight (LMW) respiratory sensitizers. The Local Lymph Node Assay (LLNA) is a validated animal model developed to detect contact sensitizers using skin exposure, but all LMW respiratory sensitizers tested so far were also positive in this assay. Discrimination between contact and respiratory sensitizers can be achieved by the assessment of cytokine profiles. In a LLNA using the inhalation route, both contact and respiratory sensitizers enhanced proliferation in the draining lymph nodes. The question was if their cytokine profiles were affected by the route of exposure. Male BALB/c mice were exposed head/nose-only during 3 consecutive days to the respiratory sensitizers trimellitic anhydride, phthalic anhydride, toluene diisocyanate, hexamethylene diisocyanate (HDI), and isophorone diisocyanate; the contact sensitizers dinitrochlorobenzene (DNCB), oxazolone (OXA) and formaldehyde (FA), and the irritant methyl salicylate (MS). Three days after the last exposure the draining lymph nodes were excised and cytokine production was measured after ex vivo stimulation with Concanavalin A. Skin application was used as a positive control. After inhalation exposure the respiratory sensitizers induced more interleukin-4 (IL-4) and interleukin (IL-10) compared to the contact sensitizers, whereas the contact sensitizers, except formaldehyde, induced relatively more interferon-gamma (IFN-gamma) production. When IL-4 and IFN-gamma were plotted as a function of the proliferative response, it was shown that IL-4 could be used to identify respiratory sensitizers, except HDI, at concentration levels inducing intermediate stimulation indices. HDI could be distinguished from DNCB and OXA at high SI values. In contrast, contact sensitizers could only be identified when IFN-gamma was measured at high stimulation indices. The skin positive control, tested at high concentrations, showed comparable results for IL-4 and IL-10, whereas IFN-gamma levels could not be used to discriminate between respiratory and contact sensitizers. The contact sensitizer FA and the irritant MS did not induce significant cytokine production after inhalation and skin exposure. In conclusion, the respiratory LLNA is able to identify and distinguish strong contact and respiratory sensitizers when simultaneously proliferation and cytokine production are assessed in the upper respiratory tract draining LNs.

The contact allergen dinitrochlorobenzene (DNCB) and respiratory allergy in the Th2-prone Brown Norway rat

All LMW respiratory allergens known to date can also induce skin allergy in test animals. The question here was if in turn skin allergens can induce allergy in the respiratory tract. Respiratory allergy was tested in Th2-prone Brown Norway (BN) rats by dermal sensitization with the contact allergen dinitrochlorobenzene (DNCB; 1%, day 0; 0.5%, day 7) and a head/nose-only inhalation challenge of 27mg/m3 of DNCB (15 min, day 21), using a protocol that successfully identified chemical respiratory allergens. Skin allergy to DNCB was examined in BN rats and Th1-prone Wistar rats in a local lymph node assay followed by a topical patch challenge of 0.1% DNCB. Sensitization of BN rats via the skin induced DNCB-specific IgG in serum, but not in all animals, and an increased number of CD4+ cells in the lung parenchyma. Subsequent inhalation challenge with DNCB did not provoke apneas or allergic inflammation (signs of respiratory allergy) in the BN rats. However, microarray analysis of mRNA isolated from the lung revealed upregulation of the genes for Ccl2 (MCP-1), Ccl4 (MIP-1beta), Ccl7 and Ccl17. Skin challenge induced considerably less skin irritation and allergic dermatitis in the BN rat than in the Wistar rat. In conclusion, the Th2-prone BN rat appeared less sensitive to DNCB than the Wistar rat; nevertheless, DNCB induced allergic inflammation in the skin of BN rats but even a relatively high challenge concentration did not induce allergy in the respiratory tract, although genes associated with allergy were upregulated in lung tissue.

Local innate and adaptive immune responses regulate inflammatory cell influx into the lungs after vaccination with formalin inactivated RSV

Inactivated respiratory syncytial virus (RSV) vaccines tend to predispose for immune mediated enhanced disease, characterized by Th2 responses and airway hypersensitivity reactions. We show in a C57BL/6 mouse model that the early innate response elicited by the challenge virus (RSV versus influenza virus) influences the outcome of the Th1/Th2 balance in the lung after intramuscular priming with inactivated vaccine. Priming of CD4(+)/IFN-γ(+) T cells by mature dendritic cells administered intravenously and/or priming of a virus specific CD8(+) T cell response ameliorated the Th2-mediated inflammatory response in the lung, suggesting that vaccination procedures are feasible that prevent vaccine induced immune pathology.

Respiratory syncytial virus induced type I IFN production by pDC is regulated by RSV-infected airway epithelial cells, RSV-exposed monocytes and virus specific antibodies.

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14+ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.

Specific Dietary Oligosaccharides Increase Th1 Responses in a Mouse Respiratory Syncytial Virus Infection Model

ABSTRACT Breast feeding reduces the risk of developing severe respiratory syncytial virus (RSV) infections in infants. In addition to maternal antibodies, other immune-modulating factors in human milk contribute to this protection. Specific dietary prebiotic oligosaccharides, similar to oligosaccharides present in human milk, were evaluated in a C57BL/6 mouse RSV infection model. During primary RSV infection, increased numbers of RSV-specific CD4+ T cells producing gamma interferon (IFN-γ) were found in the lungs at days 8 to 10 postinfection in mice receiving diet containing short-chain galactooligosacharides, long-chain fructooligosaccharides, and pectin-derived acidic oligosaccharides (termed scGOS/lcFOS/pAOS). In a Th2-skewed formalin-inactivated (FI)-RSV vaccination model, the prebiotic diet reduced RSV-specific Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13)-producing CD4+ T cells in the lung and the magnitude of airway eosinophilia at day 4 and 6 after infection. This was accompanied by a decreased influx of inflammatory dendritic cells (CD11b+/CD11c+) and increased numbers of IFN-γ-producing CD4+ and CD8+ T cells at day 8 after viral challenge. These findings suggest that specific dietary oligosaccharides can influence trafficking and/or effector functions of innate immune, CD4+, and CD8+ T cell subsets in the lungs of RSV-infected mice. In our models, scGOS/lcFOS/pAOS had no effect on weight but increased viral clearance in FI-RSV-vaccinated mice 8 days after infection. The increased systemic Th1 responses potentiated by scGOS/lcFOS/pAOS might contribute to an accelerated Th1/Th2 shift of the neonatal immune system, which might favor protective immunity against viral infections with a high attack rate in early infancy, such as RSV.

Intranasal Administration of Antibody-Bound Respiratory Syncytial Virus Particles Efficiently Primes Virus-Specific Immune Responses in Mice

ABSTRACT Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to the currently licensed palivizumab. While clearly beneficial during primary infections, preexisting antibodies might affect the onset of adaptive immune responses and the ability to resist subsequent RSV infections. Therefore, we addressed the question of how virus neutralizing antibodies influence the priming of subsequent adaptive immune responses. To test a possible role of the neonatal Fc receptor (FcRn) in this process, we compared the responses in C57BL/6 wild-type (WT) and FcRn−/− mice. We observed substantial virus-specific T-cell priming and B-cell responses in mice primed with RSV IgG immune complexes resulting in predominantly Th1-type CD4+ T-cell and IgG2c antibody responses upon live-virus challenge. RSV-specific CD8+ T cells were primed as well. Activation of these adaptive immune responses was independent of FcRn. Thus, neutralizing antibodies that localize to the airways and prevent infection-related routes of antigen processing can still facilitate antigen presentation of neutralized virus particles and initiate adaptive immune responses against RSV.

Trimellitic anhydride-conjugated serum albumin activates rat alveolar macrophages in vitro

BackgroundOccupational exposure to airborne low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter these inhaled compounds and were previously shown to influence TMA-induced asthma-like symptoms in the Brown Norway rat. TMA is a hapten that will bind to endogenous proteins upon entrance of the body. Therefore, in the present study we determined if TMA and TMA conjugated to serum albumin induced the production of the macrophage mediators nitric oxide (NO), tumour necrosis factor (TNF), and interleukin 6 (IL-6) in vitro using the rat AM cell line NR8383 and primary AMs derived from TMA-sensitized and naïve Brown Norway rats.MethodsCells were incubated with different concentrations of TMA, TMA conjugated to bovine serum albumin (BSA), and BSA as a control for 24 h and the culture supernatant was analyzed for mediator content.ResultsTMA alone was not able to induce the production of mediators by NR8383 cells and primary AMs from sensitized and sham-treated rats. TMA-BSA, on the contrary, dose-dependently stimulated the production of NO, TNF, and IL-6 by NR8383 cells and of NO and TNF, but not IL-6, by primary AMs independent of sensitization.ConclusionResults suggest that although TMA is a highly reactive compound, conjugation to a suitable protein is necessary to induce mediator production by AMs. Furthermore, the observation that effects of TMA-BSA were independent of sensitization suggests involvement of an immunologically non-specific receptor. In the discussion it is argued that a macrophage scavenger receptor is a likely candidate.