Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr^−/− mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.Supplementary key words: Apolipoprotein A-I (apoA-I) mimetic peptides, atherosclerosis, regenerating islet-derived family members 3 alpha (Reg3a), 3 beta (Reg3b), and 3 gamma (Reg3g), lipopolysaccharide (LPS), mucin 2 (Muc2), surfactant A, Interleukins 36 (IL-36), 23 (IL-23), and 22 (IL-22), Paneth cells, goblet cells, Akkermansia muciniphila

The main protein in high density lipoprotein (HDL) is apolipoprotein A-I (apoA-I), which contains 243 amino acids. Peptide mimetics of apoA-I with 10% or less of the number of amino acid residues in apoA-I have been reported by multiple laboratories to bind lipids similar to apoA-I. Most of our work has focused on apoA-I mimetic peptides that are class A amphipathic helical peptides with 18 amino acid residues arranged in a sequence that is not homologous with any portion of the amino acid sequence of apoA-I. However, these mimetic peptides have a tertiary structure that allows them to bind nonoxidized lipids similar to apoA-I. Mimetic peptides with 18 amino acid residues that contained 4–6 phenylalanine residues on the hydrophobic face (named 4F, 5F, and 6F) were found to bind oxidized lipids (particularly oxidized phospholipids) with much higher affinity than apoA-I. Despite their much smaller size, the cost of chemically synthesizing these memetic peptides is prohibitively high for the treatment of chronic diseases (1). Consequently, our laboratory developed a transgenic tomato that expresses the 6F peptide (1). As a control, a transgenic tomato without the 6F peptide was also generated and named EV (1). The content of the potent tomato anti-oxidant, lycopene, was slightly higher in wild-type and in the EV control tomatoes compared with the transgenic tomatoes expressing the 6F peptide. However, adding the transgenic tomatoes expressing the 6F peptide to a Western diet (WD) that was fed to Ldlr^−/− mice inhibited aortic atherosclerosis, but adding the control EV tomatoes did not (1). After feeding WD to which transgenic tomatoes expressing the 6F peptide had been added, intact 6F peptide was found only in the lumen of the small intestine and not in the plasma (1). Despite the absence of 6F peptide in plasma, feeding the transgenic tomatoes expressing the 6F peptide dramatically improved disease processes in animal models in which inflammation plays an important role (1, 2, 3, 4, 5, 6, 7, 8, 9). Zhao et al. (10) (with commentary by Wool, Reardon, and Getz (11)) reported similar findings regarding the efficacy of administering apoA-I mimetics by the oral route despite having undetectable plasma levels of peptide after oral administration. They added HDL-like nanolipid particles containing a peptide mimetic of apoA-I, (which is not structurally related to the 6F peptide) to the drinking water of Ldlr^−/− mice fed a high-fat high-cholesterol diet and observed a dramatic reduction in aortic atherosclerosis. The reduction in aortic atherosclerosis after oral administration was similar to that obtained after injection of the particles (10, 11). Administering the particles by injection resulted in high plasma peptide levels, but when the peptide was given in the drinking water, despite the similar reduction in aortic atherosclerosis, the peptide could not be detected in plasma (10, 11).The apoA-I mimetic peptide 4F is structurally related to 6F but not structurally related to the peptide used by Zhao et al. (10). The 4F peptide differs from 6F by having two fewer phenylalanine residues and is more water soluble than 6F. When 4F was administered to mice by the oral route or by subcutaneous injection, at equal doses there was a similar reduction in plasma serum amyloid A (SAA) levels, and a similar reduction in HDL inflammatory properties, despite peptide plasma levels that were ∼1,000-fold higher after injection compared with after oral administration (12). In these experiments, the amount of peptide found in the feces was similar at each dose of 4F regardless of whether the peptide was administered by the oral route or by subcutaneous injection (12). It was concluded that the intestine was a major site of action for the peptide, regardless of the route of administration (12). Subsequently it was found that, after injection of 4F into the tail vein of mice, the peptide rapidly appeared in the lumen of the duodenum and jejunum at levels greater than that found in the liver and promoted transintestinal cholesterol efflux (13). Together these studies strongly suggested that oral apoA-I mimetic peptides act on the luminal side of enterocytes to reduce systemic inflammation. These studies, however, did not provide mechanistic insight into how the peptides could profoundly decrease systemic inflammation without being absorbed.The mucous layers of the small and large intestines provide a critical interface between enterocytes and the bacteria rich lumen. In the colon, there are two mucous layers; the inner layer is dense and prevents bacteria from directly interacting with the enterocytes (14, 15). In contrast, the mucous layer in the small intestine is much less of a barrier to bacteria (16). Ermund et al. (16) concluded that, in the small intestine, instead of a physical barrier as is the case in the colon, separation of bacteria from enterocytes is more dependent on an array of antibacterial peptides and proteins that are secreted into the mucus to regulate the number of bacteria and their interaction with the enterocytes.The experiments reported here surprisingly demonstrate that WD contains lower levels of oxidized phospholipids compared with the chow diet. However, when WD was fed to Ldlr^−/− mice the content of reactive oxygen species (ROS) and oxidized phospholipids in jejunum mucus increased and the content of antibacterial peptides and proteins in jejunum mucus decreased compared with when the mice were fed the chow diet. As a consequence, dysbiosis, increased gut permeability, and systemic inflammation resulted from feeding WD. All of these changes were ameliorated by adding to WD a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F). The experiments reported here also show that adding oxidized phospholipids ex vivo to jejunum from mice fed a chow diet reproduced the gene expression changes seen in vivo when Ldlr^−/− mice were fed WD, and these changes were prevented when 6F peptide was added ex vivo with the oxidized phospholipids. The data in this article identify an important role for oxidized phospholipids in the mucus of the jejunum in causing dysbiosis and systemic inflammation and provide strong evidence that the mechanism of action of oral apoA-I mimetic peptides in mice fed WD is to protect against the increased levels of oxidized phospholipids that are formed on this diet.