Metabolic pathways in immune senescence and inflammaging: Novel therapeutic strategy for chronic inflammatory lung diseases. An EAACI position paper from the Task Force for Immunopharmacology.

The accumulation of senescent cells drives inflammaging and increases morbidity of chronic inflammatory lung diseases. Immune responses are built upon dynamic changes in cell metabolism that supply energy and substrates for cell proliferation, differentiation, and activation. Metabolic changes imposed by environmental stress and inflammation on immune cells and tissue microenvironment are thus chiefly involved in the pathophysiology of allergic and other immune-driven diseases. Altered cell metabolism is also a hallmark of cell senescence, a condition characterized by loss of proliferative activity in cells that remain metabolically active. Accelerated senescence can be triggered by acute or chronic stress and inflammatory responses. In contrast, replicative senescence occurs as part of the physiological aging process and has protective roles in cancer surveillance and wound healing. Importantly, cell senescence can also change or hamper response to diverse therapeutic treatments. Understanding the metabolic pathways of senescence in immune and structural cells is therefore critical to detect, prevent, or revert detrimental aspects of senescence-related immunopathology, by developing specific diagnostics and targeted therapies. In this paper, we review the main changes and metabolic alterations occurring in senescent immune cells (macrophages, B cells, T cells). Subsequently, we present the metabolic footprints described in translational studies in patients with chronic asthma and chronic obstructive pulmonary disease (COPD), and review the ongoing preclinical studies and clinical trials of therapeutic approaches aiming at targeting metabolic pathways to antagonize pathological senescence. Because this is a recently emerging field in allergy and clinical immunology, a better understanding of the metabolic profile of the complex landscape of cell senescence is needed. The progress achieved so far is already providing opportunities for new therapies, as well as for strategies aimed at disease prevention and supporting healthy aging.

A multi-center assessment to compare residual allergenicity of partial hydrolyzed whey proteins in a murine model for cow's milk allergy - Comparison to the single parameter guinea pig model.

INTRODUCTION: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas. AIM: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers. METHODS: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study. RESULTS: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers. CONCLUSIONS: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account.

Altered microbial community structure and metabolism in cow's milk allergic mice treated with oral immunotherapy and fructo-oligosaccharides.

Previously, we showed enhanced efficacy of oral immunotherapy (OIT) using fructo-oligosaccharides (FOS, prebiotics) added to the diet of cow's milk allergic mice indicated by a reduction in clinical symptoms and mast cell degranulation. Prebiotics are fermented by gut bacteria, affecting both bacterial composition and availability of metabolites (i.e. short-chain fatty acids (SCFA)). It is thus far unknown which microbial alterations are involved in successful outcomes of OIT with prebiotic supplementation for the treatment of food allergy. To explore potential changes in the microbiota composition and availability of SCFA induced by OIT+FOS. C3H/HeOuJ mice were sensitised and received OIT with or without a FOS supplemented diet. After three weeks, faecal samples were collected to analyse gut microbiota composition using 16S rRNA sequencing. SCFA concentrations were determined in cecum content. FOS supplementation in sensitised mice changed the overall microbial community structure in faecal samples compared to sensitised mice fed the control diet (P=0.03). In contrast, a high level of resemblance in bacterial community structure was observed between the non-sensitised control mice and the OIT+FOS treated mice. OIT mice showed an increased relative abundance of the dysbiosis-associated phylum Proteobacteria compared to the OIT+FOS mice. FOS supplementation increased the relative abundance of genus Allobaculum (Firmicutes), putative butyrate-producing bacteria. OIT+FOS reduced the abundances of the genera's unclassified Rikenellaceae (Bacteroidetes, putative pro-inflammatory bacteria) and unclassified Clostridiales (Firmicutes) compared to sensitised controls and increased the abundance of Lactobacillus (Firmicutes, putative beneficial bacteria) compared to FOS. OIT+FOS mice had increased butyric acid and propionic acid concentrations. OIT+FOS induced a microbial profile closely linked to non-allergic mice and increased concentrations of butyric acid and propionic acid. Future research should confirm whether there is a causal relationship between microbial modulation and the reduction in acute allergic symptoms induced by OIT+FOS.

Video-head impulse test results in patients with Menière's disease related to duration and stage of disease.

BACKGROUND: The video-head impulse test employs the vestibulo-ocular reflex (VOR) to assess vestibular function. To this day, no consensus has been reached among scientists in terms of whether or not vHIT results change in MD patients as the disease progresses. OBJECTIVE: To assess whether the vHIT is more often abnormal in later stages of MD compared to earlier stages. METHODS: We retrospectively analyzed patients with 'definite' MD who had undergone a vHIT and caloric test between 2012 and 2015. Patients were evaluated based on duration of disease in years (≤1, >1≤5, >5≤10, >10) and stage of disease (stage I and II versus III and IV). For the vHIT, an abnormal vestibulo-ocular reflex was defined as a gain cut-off value of≤0.8 and presence of correction saccades including subanalyses using a cut-off value of≤0.9. RESULTS: In 89 definite MD patients (42 (47%) male, mean age 55±5 (SD)), data on both the caloric test and the vHIT were available. The risk of an abnormal vHIT was 25% in patients with a duration of disease over 10 years compared to 22% in the patients with a disease duration of 10 years or less (risk difference 3%, 95% CI:- 28% to 35%), p = 0.82). The risk for an abnormal vHIT in the Stage I and Stage II was 17% compared to 26% in Stage III and IV (risk difference 9%, 95% CI:- 30% to 11%). When using a cut-off value of 0.9 we also did not demonstrate a relationship between the duration of disease and the proportion of abnormal vHIT test results. CONCLUSIONS: There is no relationship between the proportion of abnormal vHIT test results in patients with MD in either duration or stage of disease.

Age of onset of Ménière's disease in the Netherlands: data from a specialised dizziness clinic.

OBJECTIVES: To determine the age of onset of Ménière's disease in patients who visited a specialised dizziness clinic, and to verify whether the trend of a delayed onset age of Ménière's disease as reported for the Japanese population also occurs in the Netherlands. METHOD: A retrospective data analysis was performed of patients diagnosed with 'definite' Ménière's disease who visited our clinic between January 2000 and December 2013. RESULTS: Mean onset age of Ménière's disease among the 296 patients was 53.0 ± 14.1 years; 209 patients (71 per cent) were diagnosed between the fifth and seventh decades of life. No trend towards a later onset of Ménière's disease was found (regression beta co-efficient for year of presentation was 0.03; 95 per cent confidence interval = -0.34-0.61; p = 0.58). CONCLUSION: Ménière's disease has a peak incidence between 40 and 69 years of age. No shift towards a later onset age of Ménière's disease was found.

Determining vestibular hypofunction: start with the video-head impulse test.

Caloric testing is considered the 'reference standard' in determining vestibular hypofunction. Recently, the video-head impulse test (vHIT) was introduced. In the current study we aimed to assess the diagnostic value of the vHIT as compared to caloric testing in determining vestibular function. In a cross-sectional study between May 2012 and May 2013, we prospectively analysed patients with dizziness who had completed caloric testing and the vHIT. For the left and right vestibular system we calculated the mean vHIT gain. We used a gain cut-off value of 0.8 for the vHIT and presence of correction saccades to define an abnormal vestibular-ocular reflex. An asymmetrical ocular response of 22 % or more (Jongkees formula) or an irrigation response with a velocity below 15°/s was considered abnormal. We calculated sensitivity, specificity, positive and negative predictive values with 95 % confidence intervals for the dichotomous vHIT. Among 324 patients [195 females (60 %), aged 53 ± 17 years], 39 (12 %) had an abnormal vHIT gain and 113 (35 %) had an abnormal caloric test. Sensitivity was 31 % (23-40 %), specificity 98 % (95-99 %), positive predictive value was 88 % (74-95 %), and negative predictive value 73 % (67-77 %). In case of vHIT normality, additional caloric testing remains indicated and the vHIT does not replace the caloric test. However, the high positive predictive value of the vHIT indicates that an abnormal vHIT is strongly related to an abnormal caloric test result; therefore, additional caloric testing is not necessary. We conclude that the vHIT is clinically useful as the first test in determining vestibular hypofunction in dizzy patients.

The mycotoxin deoxynivalenol facilitates allergic sensitization to whey in mice.

Intestinal epithelial stress or damage may contribute to allergic sensitization against certain food antigens. Hence, the present study investigated whether impairment of intestinal barrier integrity by the mycotoxin deoxynivalenol (DON) contributes to the development of whey-induced food allergy in a murine model. C3H/HeOuJ mice, orally exposed to DON plus whey once a week for 5 consecutive weeks, showed whey-specific IgG1 and IgE in serum and an acute allergic skin response upon intradermal whey challenge, although early initiating mechanisms of sensitization in the intestine appeared to be different compared with the widely used mucosal adjuvant cholera toxin (CT). Notably, DON exposure modulated tight-junction mRNA and protein levels, and caused an early increase in IL-33, whereas CT exposure affected intestinal γδ T cells. On the other hand, both DON- and CT-sensitized mice induced a time-dependent increase in the soluble IL-33 receptor ST2 (IL-1R1) in serum, and enhanced local innate lymphoid cells type 2 cell numbers. Together, these results demonstrate that DON facilitates allergic sensitization to food proteins and that development of sensitization can be induced by different molecular mechanisms and local immune responses. Our data illustrate the possible contribution of food contaminants in allergic sensitization in humans.

Dietary long chain n-3 polyunsaturated fatty acids prevent allergic sensitization to cow's milk protein in mice.

BACKGROUND: Cow's milk allergy is one of the most common food allergies in children and no treatment is available. Dietary lipid composition may affect the susceptibility to develop allergic disease. OBJECTIVE: Assess whether dietary supplementation with long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) prevents the establishment of food allergy. METHODS: Mice were fed a control or fish oil diet before and during oral sensitization with whey. Acute allergic skin response, serum immunoglobulins as well as dendritic cell (DC) and T cell subsets in mesenteric lymph nodes (MLN), spleen and/or small intestine were assessed. RESULTS: The acute allergic skin response was reduced by more than 50% in sensitized mice fed the fish oil diet compared to the control diet. In addition, anti-whey-IgE and anti-whey-IgG1 levels were decreased in the fish oil group. Serum transfer confirmed that the Th2-type humoral response was suppressed since sera of fish oil fed sensitized mice had a diminished capacity to induce an allergic effector response in naïve recipient mice compared to control sera. Furthermore, the acute skin response was diminished upon passive sensitization in fish oil fed naïve recipient mice. In addition, the percentage of activated Th1 cells was reduced by fish oil in spleen and MLN of sham mice. The percentage of activated Th2 cells was reduced in both sham- and whey-sensitized mice. In contrast, whey-sensitized mice showed an increased percentage of CD11b+CD103+CD8α- DC in MLN in association with enhanced FoxP3+ regulatory T cells (Treg) in spleen and intestine of fish oil fed whey-sensitized mice compared to sham mice. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary n-3 LCPUFA largely prevented allergic sensitization in a murine model for cow's milk allergy by suppressing the humoral response, enhancing local intestinal and systemic Treg and reducing acute allergic symptoms, suggesting future applications for the primary prevention of food allergy.

Interlaboratory evaluation of a cow's milk allergy mouse model to assess the allergenicity of hydrolysed cow's milk based infant formulas.

This study describes two phases of a multi-phase project aiming to validate a mouse model for cow's milk allergy to assess the potential allergenicity of hydrolysed cow's milk based infant formulas (claim support EC-directive 2006/141/E). The transferability and the discriminatory power of this model was evaluated in 4 research centers. Mice were sensitized by oral gavage with whey or extensively hydrolysed whey (eWH) using cholera toxin as an adjuvant. Whey-specific antibodies, mMCP-1 levels, anaphylactic shock symptoms, body temperature and the acute allergic skin response were determined upon whey challenge. In phases I and II, all 4 centers detected elevated levels of whey-specific IgE/IgG1 in whey sensitized animals. Elevated levels of mMCP-1, anaphylactic symptoms, body temperature drop and acute allergic skin response were scored upon whey challenge in 3 out of 4 research centers. In contrast, none of the evaluated parameters were elevated in eWH orally exposed groups. The cow's milk allergy mouse model is capable to distinguish the sensitizing capacity of complete or hydrolysed cow's milk protein. The model uses straightforward parameters relevant to food allergic responses and can be effectively transferred between different laboratories. We propose this mouse model as a new strategy for the screening of new hypoallergenic cow's milk formulas.

CD25+ regulatory T cells transfer n-3 long chain polyunsaturated fatty acids-induced tolerance in mice allergic to cow's milk protein.

BACKGROUND: Recently, we have shown that dietary long-chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) largely prevent allergic sensitization in a murine model for cow's milk allergy. The aim of this study was to assess the contribution of regulatory T cells (Treg) in the prevention of food allergy by n-3 LCPUFA. METHODS: C3H/HeOuJ female donor mice were fed a control or fish oil diet before and during oral sensitization with cow's milk protein whey. Acute allergic skin response (ASR), anaphylaxis, body temperature, serum immunoglobulins, and mouse mast cell protease-1 (mmcp-1) were assessed. Splenocytes of sham- or whey-sensitized donor mice fed either control or fish oil diet were adoptively transferred to naïve recipient mice. Recipient mice received a whole splenocyte suspension, splenocytes ex vivo depleted of CD25+ cells, or MACS-isolated CD4+ CD25+ Treg. Recipient mice were sham- or whey-sensitized and fed control diet. RESULTS: The ASR as well as whey-specific IgE and whey-specific IgG1 levels were reduced in whey-sensitized donor mice fed the fish oil diet as compared to the control diet. Splenocytes of control-diet-fed whey-sensitized donors transferred immunologic memory. By contrast, splenocytes of fish-oil-fed whey-sensitized - but not sham-sensitized - donors transferred tolerance to recipients as shown by a reduction in ASR and serum mmcp-1, and depletion of CD25+ Treg abrogated this. Transfer of CD25+ Treg confirmed the involvement of Treg in the suppression of allergic sensitization. CONCLUSIONS: CD25+ Treg are crucial in whey allergy prevention by n-3 LCPUFA.

Galectin-9 induced by dietary synbiotics is involved in suppression of allergic symptoms in mice and humans.

BACKGROUND: Prebiotic galacto- and fructo-oligosaccharides (scGOS/lcFOS) resembling non-digestible oligosaccharides in human milk reduce the development of atopic disorders. However, the underlying mechanisms are still unclear. Galectins are soluble-type lectins recognizing ß-galactoside containing glycans. Galectin-9 has been shown to regulate mast cell degranulation and T-cell differentiation. In this study, the involvement of galectin-9 as a mechanism by which scGOS/lcFOS in combination with Bifidobacterium breve M-16V protects against acute allergic symptoms was investigated. METHODS: Mice were sensitized orally to whey, while being fed with a diet containing scGOS/lcFOS and Bifidobacterium breve M-16V (GF/Bb) or a control diet. Galectin-9 expression was determined by immunohistochemistry in the intestine and measured in the serum by ELISA. T-cell differentiation was investigated in the mesenteric lymph nodes (MLN) as well as in galectin-9-exposed peripheral blood mononuclear cells (PBMC) cultures. Sera of the mice were evaluated for the capacity to suppress mast cell degranulation using a RBL-2H3 degranulation assay. In addition, in a double-blind, placebo-controlled multicenter trial, galectin-9 levels were measured in the sera of 90 infants with atopic dermatitis who received hydrolyzed formulae with or without GF/Bb. RESULTS: Galectin-9 expression by intestinal epithelial cells and serum galectin-9 levels were increased in mice and humans following dietary intervention with GF/Bb and correlated with reduced acute allergic skin reaction and mast cell degranulation. In addition, GF/Bb enhanced T(h)1- and T(reg)-cell differentiation in MLN and in PBMC cultures exposed to galectin-9. CONCLUSIONS: Dietary supplementation with GF/Bb enhances serum galectin-9 levels, which associates with the prevention of allergic symptoms.

Perinatal programming of murine immune responses by polyunsaturated fatty acids.

Linoleic acid and α-linolenic acid are essential fatty acids (eFAs) and have to be acquired from the diet. eFAs are the precursors for long-chain polyunsaturated fatty acids (lcPUFAs), which are important immune-modulating compounds. lcPUFAs can be converted into eicosanoids and other mediators. They affect membrane structure and fluidity and can alter gene expression. There has been a marked change in dietary fatty acid intake over the last several decades. Since eFAs are acquired from the diet and immune development occurs mainly perinatally, the maternal diet may influence fetal and neonatal eFA levels, and thereby lcPUFA status, and thus immune development and function. To study whether early exposure to eFAs can program immune function, mice were fed diets varying in the ratio of ω-3 to ω-6-eFAs during pregnancy and/or lactation. After weaning, pups received a Western-style diet. At 11 weeks of age, the effects of maternal diet on the offspring's allergic and vaccination responses were examined using the T-helper 2 driven ovalbumin-induced allergy model and the T-helper 1 driven influenza-vaccination model, respectively. Offspring of dams fed a high α-linolenic acid diet during lactation showed an enhanced vaccination response. As diets with either low or high ω-3/ω-6-eFA ratio attenuated the T-helper 2 allergic response, the high α-linolenic acid diet fed during lactation had the most pronounced effect. These results indicate that there is a programming effect of maternal diet on the offspring's immune response and that in mice the window of greatest susceptibility to maternal dietary intervention is the lactation/suckling period.

Depletion of CD4+CD25+ T cells switches the whey-allergic response from immunoglobulin E- to immunoglobulin free light chain-dependent.

BACKGROUND: Symptoms of allergy are largely attributed to an IgE-mediated hypersensitivity response. However, a considerable number of patients also exhibit clinical features of allergy without detectable systemic IgE. Previous work showed that Ig-free light chains (IgLC) may act as an alternate mechanism to induce allergic responses. CD4+CD25+ T cells are crucial in the initiation and regulation of allergic responses and compromised function might affect the response to allergens. OBJECTIVE: To examine the contribution of CD4+CD25+ T cells and IgLC towards the whey-allergic response. METHODS: Mice were sensitized orally with whey using cholera toxin as an adjuvant. CD25+ T cells were depleted in vivo using a CD25 mAb. The acute allergic skin response to whey and ex vivo colon reactivity was measured in the presence or absence of F991, a specific inhibitor of IgLC. Serum whey-specific antibodies and IgLC in serum and mesenteric lymph node (MLN) supernatants were measured. Depletion of CD4+CD25+ T cells was confirmed in the spleen. RESULTS: Anti-CD25 treatment strongly reduced whey-specific antibody levels and resulted in a partial depletion of effector T cells and a major depletion of Foxp3(+) regulatory T cells. Surprisingly, despite the abolished specific IgE response, the acute allergic skin response to whey was not affected. IgLC levels were enhanced in the serum and MLN supernatants of CD25-depleted sensitized mice. F991 inhibited the acute skin response and colon hyperreactivity in anti-CD25-treated mice, indicating that these responses were mainly IgLC dependent. CONCLUSIONS: Depletion of CD4+CD25+ T cells resulted in a switch from an IgE- to an IgLC-dependent acute skin response and functional hyperresponsiveness of the colon. Our data suggest that CD25+ T cells play a crucial role in balancing cow's milk allergy between IgE and IgE-independent responses and both mechanisms might play a role in allergic responses to the same allergen.

Dietary supplementation with specific oligosaccharide mixtures decreases parameters of allergic asthma in mice.

Specific mixtures of prebiotic oligosaccharides showed immune modulatory effects in previous murine vaccination experiments, suggesting a shift towards T-helper 1 (Th1) immunity. These mixtures consisted of short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) in a 9:1 ratio (Immunofortis), with or without pectin-derived acidic oligosaccharides (pAOS). To investigate whether these mixtures could suppress Th2-related responses, they were tested in an ovalbumin (OVA)-induced model for experimental allergic asthma in BALB/c mice. Supplementation with two mixtures of scGOS/lcFOS and scGOS/lcFOS/pAOS at approximately 1% (w/w% net oligosaccharides) in the diet, starting two weeks before OVA sensitization and lasting until the end of the experiment, decreased of several parameters of allergic asthma. The OVA-induced airway inflammation and hyperresponsiveness was significantly suppressed by both mixtures. Moreover, OVA-specific IgE titers were decreased by more than 25%, although this effect was not significant. The effects of the oligosaccharide mixture with pAOS appeared to be more pronounced than the effects of the scGOS/lcFOS mixture without pAOS, but a direct comparison between the mixtures was not made. Overall, the results further support the hypothesis that specific mixtures of oligosaccharides modulate the Th1/Th2 balance by enhancing Th1-related and suppressing Th2-related parameters.

Macrophages induce an allergen-specific and long-term suppression in a mouse asthma model.

Increasing evidence suggests that macrophages (Mphi) play a crucial downregulatory role in the initiation and progression of allergic asthma. Recently, the current authors demonstrated that ovalbumin (OVA)-loaded Mphi (OVA-Mphi) suppress subsequent OVA-induced airway manifestations of asthma and that this effect could be potentiated upon selective activation. In the present study, the authors further delineated the underlying pathway by which Mphi exert this immunosuppressive effect. To examine the migration of OVA-Mphi, cells were labelled with 5'chloromethylfluorescein diacetate (CMFDA) and were administered (i.v.) into OVA-sensitised BALB/c mice. After 20 h, the relevant organs were dissected and analysed using fluorescent microscopy. Allergen-specificity was investigated by treating OVA-sensitised mice with keyhole limpet haemocyanin (KLH)-Mphi activated with immunostimulatory sequence oligodeoxynucleotide (ISS-ODN). By lengthening the period between treatment and challenge to 4 weeks it was examined whether OVA-Mphi exerted an immunosuppressive memory response. Strikingly, CMFDA-labelled Mphi were not trapped in the lungs, but migrated to the spleen. ISS-ODN-stimulated KLH-Mphi failed to suppress OVA-induced airway manifestations of asthma. Moreover, treatment with ISS-ODN-stimulated OVA-Mphi was still effective after lengthening the period between treatment and challenge. These data demonstrate that allergen-loaded macrophages can induce an indirect immunosuppressive response that is allergen-specific and long lasting, which are both hallmarks of a memory lymphocyte response.

CD28/CTLA4 double deficient mice demonstrate crucial role for B7 co-stimulation in the induction of allergic lower airways disease.

BACKGROUND: The existence of a third B7-1/B7-2 receptor was postulated in a recent study using a novel mouse strain lacking both CD28 and CTLA4 (CD28/CTLA4-/-). OBJECTIVE: In the present study, it was investigated if T cell co-stimulation via the putative B7-1/B7-2 receptor plays a role in the induction of Th2-mediated asthma manifestations in mice. METHODS: BALB/c wild-type, CD28/CTLA4-/- and B7-1/B7-2-/- mice were sensitized and aerosol challenged with ovalbumin (OVA). RESULTS: At 24 h after the last aerosol, wild-type mice showed airway hyper-responsiveness in vivo and up-regulated levels of serum OVA-specific IgE compared with the situation shortly before OVA challenge. In addition, eosinophil numbers and IL-5 levels in the broncho-alveolar lavage fluid and Th2 cytokine production by lung cells upon OVA re-stimulation in vitro were observed. In agreement with an earlier study, we failed to induce any of the asthma manifestations in B7-1/B7-2-/- mice. Importantly, also CD28/CTLA4-/- mice showed no asthma manifestations upon OVA sensitization and challenge. CONCLUSION: These data clearly demonstrate that T cell co-stimulation via the putative B7-1/B7-2 receptor appears to have no role in the induction of Th2-mediated asthma manifestations in this murine model and, conversely, that CD28 signalling is crucial.

CTLA4-IgG reverses asthma manifestations in a mild but not in a more "severe" ongoing murine model.

We investigated whether CTLA4-Ig can reverse established asthma manifestations in a novel murine model of ongoing disease. In BALB/c mice, sensitized to ovalbumin (OVA) without adjuvant, airway inflammation was induced by a first series of OVA aerosol challenges. Murine CTLA4-IgG was then administered, followed by a second series of OVA inhalations. In control-treated mice, two series of OVA challenges induced upregulation of OVA-specific IgE in serum, eosinophils in the bronchoalveolar lavage fluid (BALF), and IL-5 production by lung lymphocytes upon OVA restimulation in vitro, compared with saline-challenged mice. CTLA4-IgG significantly inhibited all of these parameters in OVA-challenged mice. Importantly, mCTLA4-IgG performed better than the gold-standard dexamethasone because this corticosteroid did not inhibit the upregulation of OVA-specific IgE in serum. In a more "severe" ongoing model, induced by sensitization to OVA emulsified in aluminum hydroxide, resulting in airway hyperresponsiveness to methacholine and stronger inflammatory responses, mCTLA4-IgG was less effective in that only the number of eosinophils in the BALF was reduced (P = 0.053), whereas dexamethasone inhibited both BALF eosinophilia and cytokine production by lung lymphocytes. Thus, CTLA4-Ig might be an effective alternative therapy in established allergic asthma, especially in situations of mild disease.

Allergen immunotherapy inhibits airway eosinophilia and hyperresponsiveness associated with decreased IL-4 production by lymphocytes in a murine model of allergic asthma.

In the present study, we investigated whether allergen immunotherapy is effective in a murine model with immunologic and pathophysiologic features reminiscent of allergic asthma. Ovalbumin-sensitized mice received increasing (1 microgram to 1 mg) subcutaneous doses of ovalbumin twice a week for 8 wk according to a semirush immunotherapy protocol as used in allergic patients. During immunotherapy, an initial rise in serum levels of ovalbumin-specific antibodies (immunoglobulin [Ig]G1, IgE, IgG2a) occurred, after which IgE levels decreased sharply concomitant with an increase in IgG2a levels. The increase in IgG2a levels, with the decline in IgE levels, suggests that during immunotherapy interferon-gamma production is increased or interleukin (IL)-4 production is decreased. After immunotherapy, inhalation challenge of the mice with ovalbumin revealed almost complete inhibition (98%, P < 0.01) of eosinophil infiltration into bronchoalveolar lavage and airway hyperresponsiveness (100% at 320 microgram/kg methacholine, P < 0.05) compared with sham-treated animals. In addition, IL-4 production of thoracic lymph node cells stimulated with ovalbumin in vitro was largely reduced (60%, P < 0.05) after immunotherapy. Thus, effective immunotherapy in this animal model appears to be due to modulation of antigen-specific T cells. Similar effects on airway symptoms and IL-4 production can be obtained within 1 wk by three injections of the highest dose of ovalbumin (1 mg). This animal model will be used as a preclinical model to improve allergen immunotherapy and to gain more insight into the mechanisms involved.

Involvement of IL-16 in the induction of airway hyper-responsiveness and up-regulation of IgE in a murine model of allergic asthma.

Experiments were designed to investigate the role of IL-16 in a mouse model of allergic asthma. OVA-sensitized mice were repeatedly exposed to OVA or saline aerosols. Bronchoalveolar lavage fluid (BALF) was collected after the last aerosol, and the presence of IL-16 was evaluated using a migration assay with human lymphocytes. Migration of lymphocytes was significantly increased in the presence of cell-free BALF from OVA-challenged mice compared with BALF from saline-challenged controls. This response was significantly inhibited after addition of antibodies to IL-16, demonstrating the presence of IL-16 in BALF of OVA-challenged animals. Immunohistochemistry was performed and revealed IL-16 immunoreactivity particularly in airway epithelial cells but also in cellular infiltrates in OVA-challenged mice. IL-16 immunoreactivity was absent in nonsensitized animals; however, some reactivity was detected in epithelial cells of sensitized but saline-challenged mice, suggesting that sensitization induced IL-16 expression in airway epithelium. Treatment of mice with antibodies to IL-16 during the challenge period significantly suppressed up-regulation of OVA-specific IgE in OVA-challenged animals. Furthermore, antibodies to IL-16 significantly inhibited the development of airway hyper-responsiveness after repeated OVA inhalations, whereas the number of eosinophils in bronchoalveolar lavage or airway tissue was not affected. In conclusion, IL-16 immunoreactivity is present in the airways after sensitization. After repeated OVA inhalation, IL-16 immunoreactivity is markedly increased and IL-16 is detectable in BALF. Furthermore, IL-16 plays an important role in airway hyper-responsiveness and up-regulation of IgE but is not important for eosinophil accumulation in a mouse model of allergic asthma.

Development of airway hyperresponsiveness is dependent on interferon-gamma and independent of eosinophil infiltration.

In this study the role of interleukin (IL)4, IL5, interferon (IFN) gamma, and tumor necrosis factor (TNF) alpha in the development of airway hyperresponsiveness and inflammatory cell infiltration was investigated using a murine model for allergic asthma. Mice were sensitized with ovalbumin and subsequently challenged repeatedly with ovalbumin aerosols. During the challenge period, mice were treated with monoclonal antibodies directed against IL4, IL5, IFN gamma, or TNF alpha. Control antibody-treated mice showed airway hyperresponsiveness to methacholine and the presence of eosinophils in bronchoalveolar lavage (BAL). Treatment with antibodies to IFN gamma completely abolished development of airway hyperresponsiveness in ovalbumin-challenged animals. After treatment with antibodies to TNF alpha, airway hyperresponsiveness in the ovalbumin-challenged animals was partially but not significantly inhibited. Antibodies to IL4 or IL5 did not inhibit airway hyperresponsiveness. The presence of eosinophils in BAL of ovalbumin-challenged mice was completely inhibited after treatment with antibodies to IL5. Treatment with antibodies to IL4, IFN gamma, or TNF alpha had no effect on eosinophilia. Because IFN gamma and IL5 have either an effect on the induction of airway hyperresponsiveness or on the development of eosinophil infiltration, our results suggest that the two phenomena are differentially regulated.