P139 EFFECT OF LYSOSOMAL SHORT CHAIN FATTY ACID DELIVERY ON IMMUNE RESPONSE
Authors: Strass Simon , Heinzel Constanze , Cloos Natascha , Keppler Manuel , Guse Jan , Burnet Michael , Laufer Stefan 
Published: January 2020

Short chain fatty acids (SCFAs) are known as metabolites produced from gut microbiome fermenting dietary fibers and resistant starch. They are regulators of the interplay between the microbiome and its host and may have a role in the pathogenesis of inflammatory bowel disease (IBD). Free SCFAs influence intestinal epithelial and immune cells (e.g. macrophages and primary monocytes) through activation of free fatty acid receptors FFAR2 and FFAR3, inhibition of histone deacetylases (HDAC) and as a source of energy. SCFAs are known to impact cytokine production and differentiation of immune cells (e.g. Tumor necrosis factor alpha (TNFa), Interleukin (IL)-10, IL-6, IL-18, IL-1ß).

We investigated the role of SCFAs in dextran sulfate sodium-induced colitis and their effect on the cytokine production by primary human immune cells. Treatment with SCFAs (acetate, propionate or butyrate) stimulated release of IL-1ß and IL-18 by buffy coat leukocytes or U937 cells without causing increased expression of corresponding genes. This raises the possibility of activation via the NLRP3 (NOD-, LRR- and pyrin domain- containing protein 3) inflammasome. NLRP3 is a multimeric inflammasome complex. Once activated, NLRP3 inflammasome releases caspase 1 leading to formation of mature IL-1ß and IL-18. Recent studies have shown that IL-1ß promotes phagocytosis and clearance of bacteria and aids the gut in eliciting an effective response in early stages of IBD.

We asked whether this effect is mediated via surface or lysosomal (phagosomal) receptors? To answer this question, we prepared a series (SYD010) of novel compounds which are able to accumulate in the phagolysosome of immune cells through their macrolide backbone and deliver SCFAs, bound as esters to the lumen of the activated lysosome. In vitro, together with LPS stimulation, the substances modulated secretion of TNFa, IL-1ß, IL-10 and IL-6 at concentrations about 100x lower than free SCFAs (Figure 1). When tested in a DSS colitis mouse model, the SYD010 series caused a decrease in diarrhea scoring compared to the vehicle-treated control group at a concentration of 0.1 mg/kg (Figure 2) which corresponds to a total dose of ca. 100 nmol/kg (the compounds are systemically distributed). This is lower than the known luminal concentrations of SCFAs which is in the range of 40 mM. Our underlying hypothesis is that lysosomal reception of SCFAs leads to beneficial immune modulation in colitis in so far as stimulation of IL-1ß release promotes bacterial clearance. Furthermore, that concentrative uptake to the phagolysosome leads to enhanced stimulation of these receptors leading to responses at lower ambient concentrations or doses. We are assessing this substance class as potential IBD therapeutics.

Figure 1. Effect of sodium butyrate or CSY4286 (lysosomal butyrate donor) on cytokine production by U937 cells. Supernatants were harvested after 24 h (IL-6) and 48 h (IL-1ß, IL-6) incubation with the test substances and cytokines determined by ELISA. The dotted line represents levels with LPS stimulation alone. SEM was applied for error bars.

Figure 2. Results from DSS-induced (2.5% in drinking water) IBD study in BALB/c mice (8 mice per group). Scoring of body weight and diarrhea score over 8 days (a and b show data from final day of study). SEM was applied for error bars.