Mustard seed major allergen Sin a1 activates intestinal epithelial cells and also dendritic cells that drive type 2 immune responses.

Mustard seeds belong to the food category of mandatory labelling due to the severe reactions they can trigger in allergic patients. However, the mechanisms underlying allergic sensitization to mustard seeds are poorly understood. The aim of this work is to study type 2 immune activation induced by the mustard seed major allergen Sin a1 via the intestinal mucosa, employing an in vitro model mimicking allergen exposure via the intestinal epithelial cells (IECs). Sin a1 was isolated from the total protein extract and exposed to IEC, monocyte derived dendritic cells (DCs) or IEC/DC co-cultures. A system of consecutive co-cultures was employed to study the generic capacity of Sin a1 to induce type 2 activation leading to sensitization: IEC/DC, DC/T-cell, T/B-cell and stem cell derived mast cells (MCs) derived from healthy donors. Immune profiles were determined by ELISA and flow cytometry. Sin a1 activated IEC and induced type-2 cytokine secretion in IEC/DC co-culture or DC alone (IL-15, IL-25 and TSLP), and primed DC induced type 2 T-cell skewing. IgG secretion in the T-cell/B-cell phase was enhanced in the presence of Sin a1 in the first stages of the co-culture. Anti-IgE did not induce degranulation but promoted IL-13 and IL-4 release by MC primed with the supernatant from B-cells co-cultured with Sin a1-IEC/DC or -DC primed T-cells. Sin a1 enhanced the release of type-2 inflammatory mediators by epithelial and dendritic cells; the latter instructed generic type-2 responses in T-cells that resulted in B-cell activation, and finally MC activation upon anti-IgE exposure. This indicates that via activation of IEC and/or DC, mustard seed allergen Sin a1 is capable of driving type 2 immunity which may lead to allergic sensitization.

Butyrate Protects Barrier Integrity and Suppresses Immune Activation in a Caco-2/PBMC Co-Culture Model While HDAC Inhibition Mimics Butyrate in Restoring Cytokine-Induced Barrier Disruption.

Low-grade inflammation and barrier disruption are increasingly acknowledged for their association with non-communicable diseases (NCDs). Short chain fatty acids (SCFAs), especially butyrate, could be a potential treatment because of their combined anti-inflammatory and barrier- protective capacities, but more insight into their mechanism of action is needed. In the present study, non-activated, lipopolysaccharide-activated and αCD3/CD28-activated peripheral blood mononuclear cells (PBMCs) with and without intestinal epithelial cells (IEC) Caco-2 were used to study the effect of butyrate on barrier function, cytokine release and immune cell phenotype. A Caco-2 model was used to compare the capacities of butyrate, propionate and acetate and study their mechanism of action, while investigating the contribution of lipoxygenase (LOX), cyclooxygenase (COX) and histone deacetylase (HDAC) inhibition. Butyrate protected against inflammatory-induced barrier disruption while modulating inflammatory cytokine release by activated PBMCs (interleukin-1 beta↑, tumor necrosis factor alpha↓, interleukin-17a↓, interferon gamma↓, interleukin-10↓) and immune cell phenotype (regulatory T-cells↓, T helper 17 cells↓, T helper 1 cells↓) in the PBMC/Caco-2 co-culture model. Similar suppression of immune activation was shown in absence of IEC. Butyrate, propionate and acetate reduced inflammatory cytokine-induced IEC activation and, in particular, butyrate was capable of fully protecting against cytokine-induced epithelial permeability for a prolonged period. Different HDAC inhibitors could mimic this barrier-protective effect, showing HDAC might be involved in the mechanism of action of butyrate, whereas LOX and COX did not show involvement. These results show the importance of sufficient butyrate levels to maintain intestinal homeostasis.

Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition.

Non-communicable diseases are increasing and have an underlying low-grade inflammation in common, which may affect gut health. To maintain intestinal homeostasis, unwanted epithelial activation needs to be avoided. This study compared the efficacy of butyrate, propionate and acetate to suppress IFN-γ+/-TNF-α induced intestinal epithelial activation in association with their HDAC inhibitory capacity, while studying the canonical and non-canonical STAT1 pathway. HT-29 were activated with IFN-γ+/-TNF-α and treated with short chain fatty acids (SCFAs) or histone deacetylase (HDAC) inhibitors. CXCL10 release and protein and mRNA expression of proteins involved in the STAT1 pathway were determined. All SCFAs dose-dependently inhibited CXCL10 release of the cells after activation with IFN-γ or IFN-γ+TNF-α. Butyrate was the most effective, completely preventing CXCL10 induction. Butyrate did not affect phosphorylated STAT1, nor phosphorylated NFκB p65, but inhibited IRF9 and phosphorylated JAK2 protein expression in activated cells. Additionally, butyrate inhibited CXCL10, SOCS1, JAK2 and IRF9 mRNA in activated cells. The effect of butyrate was mimicked by class I HDAC inhibitors and a general HDAC inhibitor Trichostatin A. Butyrate is the most potent inhibitor of CXCL10 release compared to other SCFAs and acts via HDAC inhibition. This causes downregulation of CXCL10, JAK2 and IRF9 genes, resulting in a decreased IRF9 protein expression which inhibits the non-canonical pathway and CXCL10 transcription.