Fiber rich food suppressed airway inflammation, GATA3 + Th2 cells, and FcεRIα+ eosinophils in asthma

Background Allergic Asthma is a disease presenting various endotypes and no current therapies act curative but alleviate disease symptoms. Dietary interventions are gaining increasing importance in regulating immune responses. Furthermore, short chain fatty acids (SFCA), as the main products of dietary fiber’s fermentation by the gut bacteria, ameliorate the pathogenesis and disease burden of different illnesses including asthma. Nevertheless, the connection and crosstalk between the gut and lung is poorly understood. Objective In this work, the role of high fiber diet on the development of allergic asthma at baseline and after exacerbation of disease induced by respiratory viruses was investigated. Methods Hereby, SCFA in serum of asthmatic and non-asthmatic pre-school children before and after airway disease symptoms were analyzed. Moreover, the effect of high fiber diet in vivo in a murine model of house dust mite extract (HDM) induced allergic asthma and in the end in isolated lung and spleen cells infected ex vivo with Rhinovirus was analyzed. Results In this study, a decrease of the SCFA 3-Hydroxybutyric acid in serum of asthmatic children after symptomatic episodes at convalescent visit as compared to asthmatic and control children at baseline visit was observed. In experimental asthma, in mice fed with high fiber diet, a reduced lung GATA3 + Th2 type mediated inflammation, mucus production and collagen deposition and expression of Fc epsilon receptor Ia (FcεRIa) in eosinophils was observed. By contrast, the CD8+ memory effector T cells were induced in the lungs of asthmatic mice fed with high fiber diet. Then, total lung cells from these asthmatic mice fed with either standard food or with fiber rich food were infected with RV ex vivo. Here, RV1b mRNA was found significantly reduced in the lung cells derived from fiber rich food fed mice as compared to those derived from standard food fed asthmatic mice. Looking for the mechanism, an increase in CD8+ T cells in RV infected spleen cells derived from fiber rich fed asthmatic mice, was observed. Conclusion Convalescent preschool asthmatic children after a symptomatic episode have less serum ß-Hydroxybutyric acid as compared to control and asthmatic children at baseline visit. Fiber rich diet associated with anti-inflammatory effects as well as anti-allergic effects by decreasing Type 2 and IgE mediated immune responses and inducing CD8+ memory effector T cells in a murine model of allergic asthma. Finally, ex vivo infection with Rhinovirus (RV) of total lung cells from asthmatic mice fed with fiber rich food led to a decreased RV load as compared to mice fed with standard food. Moreover, spleen cells derived from asthmatic mice fed with fiber rich food induced CD8+ T cells after ex vivo infection with RV. Clinical implications Dietary interventions with increased content in natural fibers like pectins would ameliorate asthma exacerbations. Moreover, respiratory infection in asthma downregulated SCFA in the gut contributing to asthma exacerbations.


Airway hyperresponsiveness
On day 35 invasive measurement of AHR was performed.After deeply euthanizing the mice via intraperitoneal injection with pentobarbital, tracheotomy was performed and a cannula (18G) was inserted into the trachea.The mice were then connected with the flexivent system (EMKA, Paris, France) and AHR was measured by exposing the mice to increasing doses of Methacholine (MKCK9552, Sigma-Aldrich, St. Louis, USA) (0mg/ml (PBS); 12.5mg/ml; 25mg/ml; 50 mg/ml).

Serum preparation from blood
Before organs were removed, blood was collected using a syringe.The blood was transferred to a 1.5ml Eppendorf tube and centrifuged (600xg RT 30min) to receive the serum phase.Serum was removed and transferred to another tube and immediately stored at -80°C.

Total cell isolation of lung
Total lung cell isolation was performed after overnight storage in MACS Tissue storage solution (Miltenyi Biotec, Bergisch Gladbach, Germany).Therefore, the lungs were transferred to a petri dish and minced into small pieces.For digestion of the lung, the organ was incubated in 10ml of Collagenase/DNAse at 37°C for 45 min (Cat: C98991, Sigma-Aldrich, St. Louis, USA; Cat: 10104159001, Roche Diagnostics, Basel, Suisse).Afterwards the digested lungs were push through a cell strainer (40µm) into a 50ml falcon to obtain a single cell suspension.
The cell strainer was rinsed with 5ml RPMI 1640 medium (without supplementation) (AC-LM-0060, Anprotec, Bruckberg, Germany) to wash remaining cells.Next, the isolated cell suspension was centrifuged (1500rpm 4°C 10 min) and the pellet was resuspended in 10ml ACK-Lysis (8.29g NH4Cl, 1g KHCO3 and 0.367g Na2 -EDTA dissolved in 1L distilled water and at the end the pH value was adjusted with NaOH to 7.2 -7.4) for two minutes at room temperature (RT) to lyse red blood cells.After another centrifugation (1500rpm 4°C 5 min), the cell pellet is resuspended in 10ml PBS (RPMI supplemented with 10% FCS, 5% PenStrep, 5% L-Glutamine) and fat was removed by slowly pipetting the suspension.After centrifugation (1500rpm 4°C 5 min), the supernatant was discarded, and the cell pellet resuspended in 5ml medium to count the cells subsequently.Cell counting was performed by using a Neubauer counting chamber.Lymph nodes single cell suspension was isolated immediately after isolating the lymph nodes.Therefore, the lymph nodes were also pushed through a cell strainer (40µm) into a 50ml falcon to obtain a single cell suspension and washed with 5ml RPMI Medium.
After centrifugation (1500rpm 4°C 5 min), the cells were taken up into 5ml RPMI medium again and counted in the same manner as the lung cell suspension.Table E1:Serum B-hydroxybutiric acid (mmol/L) analysis in the cohorts of children with asthma (A) and control children without asthma (C).

Figure S2 :
Figure S2: Regulatory CD4+CD25+Foxp3+ T cells in the lungs by fiber rich food in naïve and HDM treated mice.(A) Gating strategy for lung staining with antibodies against CD3, CD4, CD25 and FoxP3 to distinguish Tregs.(B) Analysis of CD4+ T cells in the lungs.(C) Flow cytometry analysis of Tregs in the lungs of control and asthmatic mice fed with fiber rich diet or standard diet.Representative dot plots are shown.All data is presented as mean ± SEM.Data is analyzed statistically by two-way ANOVA and Sidak's multiple comparisons test.n=4-10 animals per group.

AHR was not altered by fiber rich diet
(A)Invasive lung function measurement with increasing doses of methacholine.All data is shown as mean ± SEM from 6-10 animals per group.Statistical analysis was performed using a two-way ANOVA and Sidak's multiple comparisons test.(B) Gating strategy of Flow cytometry analysis for lung granulocytes.A representative dot plot is shown.(C) Flow cytometry analysis of lung cells.A representative dot plot is shown.All data is presented as mean ± SEM.Data is analyzed statistically by two-way ANOVA and Sidak's multiple comparisons test.n=6-10 animals per group.