Jan Louis Ceuppens

Regulatory T cells (Tregs) that express the transcription factor Foxp3 have a critical role in limiting inflammatory processes and tissue damage. Whether Tregs are functional in maintaining epithelial barriers and in control of tight junction expression has not yet been explored. In this study, we investigated the effect of Treg deficiency on the airway epithelial barrier in an experimental murine model in which diphtheria toxin was repeatedly injected in Foxp3-diphtheria toxin receptor (DTR) mice to deplete Tregs. This resulted in spontaneous peribronchial inflammation and led to a systemic and local increase of IL-4, IL-5, CCL3, IFN-γ, and IL-10 and a local (lung) increase of IL-6 and IL-33 and decreased amphiregulin levels. Moreover, Treg depletion increased airway permeability and decreased epithelial tight junction (protein and mRNA) expression. CTLA4-Ig treatment of Treg-depleted mice almost completely prevented barrier dysfunction together with suppression of lung inflammation and cytokine secretion. Treatment with anti–IL-4 partly reversed the effects of Treg depletion on tight junction expression, whereas neutralization of IL-6 of IFN-γ had either no effect or only a limited effect. We conclude that Tregs are essential to protect the epithelial barrier at the level of tight junctions by restricting spontaneous T cell activation and uncontrolled secretion of cytokines, in particular IL-4, in the bronchi.

Introduction: Insight in the pathogenesis of intestinal fibrosis is an unmet medical need in inflammatory bowel diseases. Studies in murine models and human organ fibrosis point to a potential role of innate lymphoid cells (ILC) in chronic intestinal inflammation and fibrosis.Materials and Methods: Dextran sodium sulfate (DSS) in drinking water was used to induce chronic colitis and remodeling in C57Bl/6 wild type (WT), RAG‐deficient, RAG^−/− common γ chain deficient and anti‐CD90.2 monoclonal antibody treated RAG^−/− mice. Inflammation was scored by macroscopic and histological examination and fibrosis was evaluated by hydroxyproline quantification and histology.Results: In RAG^−/− mice (which have a normal ILC population but no adaptive immunity), chronic intestinal inflammation and fibrosis developed similarly as in WT mice, with a relative increase in ILC2 during repeated DSS exposure. Chronic colitis could also be induced in the absence of ILC (RAG^−/−γc^−/− or anti‐CD90.2 treated RAG^−/− mice) with no attenuation of fibrosis. Importantly, clinical recovery based on weight gain after stopping DSS exposure was impaired in ILC‐deficient or ILC‐depleted mice.Conclusion: These data argue against a profibrotic effect of ILC in chronic colitis, but rather suggest that ILC have a protective and recovery‐enhancing effect after repeated intestinal injury.

Allergic asthma is a highly prevalent inflammatory disease of the lower airways, clinically characterized by airway hyperreactivity and deterioration of airway function. Immunomodulatory probiotic bacteria are increasingly being explored to prevent asthma development, alone or in combination with other treatments. In this study, wild-type and recombinant probiotic Lactobacillus rhamnosus GR-1 were tested as preventive treatment of experimental allergic asthma in mice. Recombinant L. rhamnosus GR-1 was designed to produce the major birch pollen allergen Bet v 1, to promote allergen-specific immunomodulation. Administration of wild-type and recombinant L. rhamnosus GR-1 prevented the development of airway hyperreactivity. Recombinant L. rhamnosus GR-1 also prevented elevation of airway total cell counts, lymphocyte counts and lung IL-1β levels, while wild-type L. rhamnosus GR-1 inhibited airway eosinophilia. Of note, a shift in gut microbiome composition was observed after asthma development, which correlated with the severity of airway inflammation and airway hyperreactivity. In the groups that received L. rhamnosus GR-1, this asthma-associated shift in gut microbiome composition was not observed, indicating microbiome-modulating effects of this probiotic. These data demonstrate that L. rhamnosus GR-1 can prevent airway function deterioration in allergic asthma. Bet v 1 expression by L. rhamnosus GR-1 further contributed to lower airway inflammation, although not solely through the expected reduction in T helper 2-associated responses, suggesting involvement of additional mechanisms. The beneficial effects of L. rhamnosus GR-1 correlate with increased gut microbiome resilience, which in turn is linked to protection of airway function, and thus further adds support to the existence of a gut-lung axis.

Background: Increased epithelial permeability has been reported in allergic rhinitis, with histamine and type‐2 inflammation being responsible for tight junction dysfunction. The impact of an epithelial barrier defect on allergic sensitization and mast cell (MC) degranulation remains speculative.Methods: Transepithelial passage of allergens was evaluated on primary human nasal epithelial cell cultures. Active sensitization was attempted by repeated intranasal ovalbumin (OVA) applications in Naïve mice. In a passive sensitization model, mice were injected with IgE to Dermatophagoides pteronyssinus (rDer p)2 and then exposed intranasally to the allergen. Chitosan was used to disrupt nasal epithelial integrity in vitro and in vivo.Results: Chitosan strongly reduced transepithelial electrical resistance and facilitated transepithelial allergen passage in cultured primary nasal epithelial cells. In vivo, intranasal chitosan affected occludin expression and facilitated allergen passage. After epithelial barrier disruption, intranasal OVA application induced higher OVA‐specific IgG1 and total IgE in serum, and increased eosinophilia and interleukin‐5 in bronchoalveolar lavage (BAL) compared to sham‐OVA mice. Chitosan exposure, prior to rDer p2 allergen challenge in passively sensitized mice, resulted in increased β‐hexosaminidase levels in serum and BAL compared to sham‐rDer p2 mice. Intranasal treatment with the synthetic glucocorticoid fluticasone propionate prevented chitosan‐induced barrier dysfunction, allergic sensitization, and MC degranulation.Conclusion: Epithelial barrier dysfunction facilitates transepithelial allergen passage, allergic sensitization, and allergen‐induced MC degranulation even in the absence of inflammatory environment. These results emphasize the crucial role of an intact epithelial barrier in prevention of allergy.

Innate lymphoid cells (ILCs) reside at mucosal barriers where they exhibit high cytokine producing capacity to maintain homeostasis and control infections. Natural cytotoxicity receptor (NCR) positive ILC3s, an important source of intestinal IL-22, have been shown to be decreased in the mucosa of patients with Crohn’s disease (CD) and ulcerative colitis (UC) in favour of pro-inflammatory ILC1s. To study whether current biological anti-TNF, ustekinumab (UST) or vedolizumab (VDZ) therapy can restore the intestinal ILC balance, ILC frequencies were determined in serial blood and biopsies samples. We included 26 CD patients initiating UST, 14 patients initiating VDZ (9 CD, 5 UC), 14 CD patients initiating anti-TNF (8 infliximab, 6 adalimumab) and 10 healthy controls (HC) without endoscopic abnormalities. All cohorts were matched for age, gender, and age at diagnosis. Colonic biopsies and blood were taken at baseline and during endoscopic assessment (Week 8–14 UC, 24 CD). Peripheral blood and lamina propria mononuclear cells were stained for flow cytometric analysis. Pairwise comparisons were performed on ILC numbers determined as frequency of total ILC or total leucocyte population. Intestinal NCR+ ILC3 levels before initiation of biological treatment were significantly decreased in anti-TNF and VDZ cohort (42.0, 37.5 vs. 86.8% of total ILC, both p < 0.001) while ILC1 levels were increased (15.7, 7.7 vs. 2.7, both p < 0.01) when compared with HC. In contrast, ILC subgroup levels were not different in the UST cohort (NCR+ ILC3: 74.8, ILC1:2.4, p = 0.9). In the anti-TNF and VDZ cohort, recovery of NCR+ ILC3s compared with start (p = 0.04, p = 0.03) was observed after first endoscopic evaluation independent of treatment response. Mucosal ILC levels could not be correlated to peripheral ILC levels (r = 0.39, p = 0.27); however, an increase of peripheral NCR+ ILC3s in the total ILC (Figure 1) and leucocyte population could be observed in both the anti-TNF (p = 0.01) and UST (p = 0.001) cohort when compared with the start of therapy. In contrast, no effect of VDZ (p = 0.47) was observed on peripheral ILC levels. Biological therapy can restore the intestinal ILC levels towards homeostatic proportions even in absence of endoscopic response. Anti-TNF and UST treatment increased NCR+ ILC3s levels in the circulation, which are not described in physiological conditions. In contrast, no increased NCR+ ILC3s levels were not observed in VDZ-treated patients. NCR+ ILC3 level will be correlated to cytokine levels in future.

The worldwide prevalence of allergic diseases has drastically increased in the past decades. Recent studies underline the importance of microbial exposure for the development of a balanced immune system. Consequently, probiotic bacteria are emerging as a safe and natural strategy for allergy prevention and treatment. However, clinical probiotic intervention studies have so far yielded conflicting results. There is increasing awareness about the importance of host-associated factors that determine whether an individual will respond to a specific probiotic treatment, and it is therefore crucial to promote a knowledge-based instead of an empirical selection of promising probiotic strains and their administration regimen. In this Review, we summarize the insights from animal model studies of allergic disease, which reveal how host-related factors – such as genetic makeup, sex, age and microbiological status – can impact the outcomes of preventive or curative probiotic treatment. We explore why and how these factors can influence the results of probiotic studies and negatively impact the reproducibility in animal experiments. These same factors might profoundly influence the outcomes of human clinical trials, and can potentially explain the conflicting results from probiotic intervention studies. Therefore, we also link these host-related factors to human probiotic study outcomes in the context of airway allergies.

Background: There is an increasing interest in targeted application of probiotic bacteria for prevention and treatment of airway diseases, including allergies. Here, we investigated the beneficial effects of preventive intranasal treatment with probiotics Lactobacillus rhamnosus GG and L. rhamnosus GR‐1 in a mouse model of allergic asthma.Methods: Lactobacillus rhamnosus was administered intranasally eight times on days 1‐4 and 8‐11 at 5 × 10⁸ CFU/dose, followed by a 2‐week asthma induction protocol with birch pollen extract on alternating days. Effects of preventive treatment were analyzed based on serum antibody levels, bronchoalveolar lavage cell counts, lung histology, lung cytokine levels, and airway hyperreactivity. Colonization and translocation of L. rhamnosus were assessed by bacterial cell counts in nasal mucosa, fecal samples, cervical lymph nodes, and blood. Binding of fluorescent L. rhamnosus to fixed murine nasal mucosal cells and airway macrophages was visualized by fluorescence microscopy.Results: Transient colonization of the murine upper airways by L. rhamnosus GG was demonstrated and was approximately ten times higher compared to L. rhamnosus GR‐1. Marked binding of fluorescent L. rhamnosus GG to murine nasal mucosal cells and airway macrophages was visualized. Preventive treatment with L. rhamnosus GG (but not L. rhamnosus GR‐1) resulted in a significant decrease in bronchoalveolar lavage eosinophil counts, lung interleukin‐13 and interleukin‐5 levels, and airway hyperreactivity. A tendency toward a decrease in serum Bet v 1‐specific immunoglobulin G1 was likewise observed.Conclusion: Intranasally administered L. rhamnosus GG prevents the development of cardinal features of birch pollen‐induced allergic asthma in a strain‐specific manner.

Background: Nasal hyperreactivity (NHR) is an important clinical feature of allergic rhinitis (AR). The efficacy of MP29‐02 (azelastine hydrochloride (AZE) and fluticasone propionate [FP]) nasal spray on local inflammatory mediators and NHR in AR is unknown. We tested if MP29‐02 decreases inflammatory mediators and NHR in AR and if this effect is due to restoration of nasal epithelial barrier function.Methods: A 4‐week double‐blinded placebo‐controlled trial with MP29‐02 treatment was conducted in 28 patients with house dust mite (HDM) AR. The presence of NHR was evaluated by measuring reduction in nasal flow upon cold dry air exposure. The effects of AZE ± FP on barrier integrity and airway inflammation were studied in a murine model of HDM‐induced NHR and on reduced activation of murine sensory neurons and human mast cells.Results: MP29‐02 but not placebo reduced NHR (P < .0001 vs P = .21), levels of substance P (P = .026 vs P = .941), and β‐hexosaminidase (P = .036 vs P = .632) in human nasal secretions. In wild‐type C57BL6 mice, the reduction in β‐hexosaminidase levels (P < .0001) by AZE + FP treatment upon HDM challenge was found in parallel with a decreased transmucosal passage (P = .0012) and completely reversed eosinophilic inflammation (P = .0013). In vitro, repeated applications of AZE + FP desensitized sensory neurons expressing the transient receptor potential channels TRPA1 and TRPV1. AZE + FP reduced MC degranulation to the same extent as AZE alone.Conclusion: MP29‐02 treatment reduces inflammatory mediators and NHR in AR. The effects of AZE + FP on MC degranulation, nasal epithelial barrier integrity, and TRP channels provide novel insights into the pathophysiology of allergic rhinitis.