After a saturated fat-rich meal, MLNs are exposed to extremely high concentrations of chylomicrons via the chyle, which might lead to generation of large amounts of pro-inflammatory saturated fatty acids upon TG lipolysis. Our data indicate that MLN and specifically resident macrophages are protected from the pro-inflammatory effect of saturated fatty acids via expression of Angptl4, which is strongly induced by chyle and fatty acids and which via inhibition of LPL prevents lipolysis of chylomicron-TG. In the absence of this protective autocrine mechanism, feeding a diet rich in saturated fat rapidly leads to enhanced lipid uptake into MLN-resident macrophages, triggering foam (Touton) cell formation and a massive inflammatory response characterized by severe mesenteric lymphadenitis. The concomitant induction of numerous cytokines leads to a massive hepatic acute phase response via the connecting portal circulation, which further evolves into a progressive, uncontrolled inflammation that culminates in fibrinopurulent peritonitis, chylous ascites, intestinal fibrosis and cachexia. The data thus show that Angptl4 is for a key player in the protection against the severe pro-inflammatory effects of dietary saturated fat. Based on our data in mice, it can be hypothesized that human subjects homozygous for the E40K mutation in Angptl4, which has reduced ability to inhibit LPL and is associated with lower plasma triglycerides (Romeo et al., 2007
; Shan et al., 2008
; Yin et al., 2009
), may be particularly sensitive to the pro-inflammatory effects of dietary saturated fat.
According to our microarray analysis, LPL was among the most highly expressed genes in mouse peritoneal macrophages. The ability of macrophage LPL to facilitate lipid uptake into macrophages is well recognized (Babaev et al., 1999
; Ostlund-Lindqvist et al., 1983
). The locally released fatty acids may serve as energy source for active macrophages (Yin et al., 1997
), but may also constitute a potential pro-inflammatory stimulus. Consistent with this notion, fatty acids offered to macrophages as VLDL-TG are taken up and engage MAPK-mediated inflammatory pathways along with increased expression of several pro-inflammatory cytokines (Saraswathi and Hasty, 2006
). Our data indicate that exposure of macrophages to elevated yet physiologically relevant concentrations of chylomicrons containing saturated fatty acids unleashes a vast inflammatory response characterized by marked induction of numerous chemokines and other inflammation-related genes, which is entirely dependent on TG-lipolysis. Thus, lipolysis of TG-rich lipoproteins by macrophages is an important process that regulates intracellular fatty acid accumulation and contributes to initiation of pro-inflammatory signaling cascades. We propose that expression of Angptl4 in macrophages and its potent induction by chylomicron-derived fatty acids are part of a feedback mechanism aimed at protecting MLN-resident macrophages against post-prandial lipid overload and associated inflammation.
Ablation of Angptl4 is associated with decreased plasma TG levels caused by increased peripheral LPL activity (Koster et al., 2005
). Recent data indicate that endothelium-bound LPL is stabilized by the protein GPIHBP1, which partially prevents LPL inhibition by Angptl4 (Sonnenburg et al., 2009
). It can be hypothesized that the almost complete blockage of lipid uptake by Angptl4 in macrophages as opposed to its more modest effect in muscle and adipose tissue may be explained by the minimal expression of GPIHBP1 in macrophages (Fig. S7B
) (Sonnenburg et al., 2009
). Future studies will have to address this issue in more detail.
In our study, feeding Angptl4−/− mice a diet rich in polyunsaturated fatty acids did not elicit an inflammatory response, which is consistent with the data in peritoneal macrophages showing a lack of induction of Gdf15 and Cxcl2 by oleic and linoleic acid. In contrast, oleic and linoleic acid were much more potent inducers of Angptl4 expression compared to palmitic acid, suggesting that the Angptl4-mediated feedback inhibition of LPL-dependent fatty acid uptake and consequent suppression of inflammation is only weakly activated by saturated fatty acids.
An important question is how chyle induces inflammation in macrophages. Use of specific chemical inhibitors indicated that the response is not mediated by LPS, is not dependent on Cd36-mediated fatty acid transport, and does not require sphingolipid synthesis. Strikingly, we observed that chyle caused pronounced activation of different branches of the ER stress pathway in macrophages. It has been shown that ER stress can promote inflammation by various mechanisms, including via IRE1a-mediated activation of stress kinases such as the c-Jun N-terminal kinase (Urano et al., 2000
), and via PERK-mediated activation of NF-κB (Jiang et al., 2003
). We found that chyle stimulated IRE1α phosphorylation to promote XBP1 splicing, and activated PERK, eIF2α and their downstream targets. Activation of ER stress in peritoneal macrophages could be reproduced by free palmitic acid but not oleic acid or linoleic acid, suggesting the response to chyle is mediated by saturated fatty acids.
The mechanism by which saturated fatty acids induces ER stress has been the subject of recent investigations. Palmitate but not palmitoleate induced ER stress in pancreatic beta cells (Diakogiannaki et al., 2008
). In liver cells saturated fatty acids induced ER stress independently of ceramide synthesis (Wei et al., 2006
). Stimulation of ER stress by palmitate may occur via increasing the saturated lipid content of the ER membrane phospholipids and triglycerides, leading to compromised ER morphology and integrity and impaired function of protein-folding chaperones (Borradaile et al., 2006
). Data also point to an important role for aP2 (Fabp4) in linking saturated fatty acids to ER stress in macrophages via alterations in lipid composition (Erbay et al., 2009
Several studies have attributed the pro-inflammatory effect of saturated fatty acids to activation of TLR4 (Lee et al., 2001
; Shi et al., 2006
; Suganami et al., 2007
). Recently, interplay between TLR4 (and TLR2) and the ER stress pathway was demonstrated. Specifically, IRE1α was shown to be a positive regulator of the inflammatory response to TLR activation in macrophages, while the PERK pathway was not induced by TLR signaling (Martinon et al., 2010
). These data hint at a possible role for TLR signaling in the response to chyle in macrophages. However, unlike TLR signaling, chyle treatment dramatically induced ER stress as evidenced by the activation of ER stress sensors IRE1α and PERK as well as their downstream targets. Additionally, systematic whole genome analysis of gene regulation by chyle versus the TLR4 agonist LPS revealed some overlap, but chyle clearly did not mimic the effects of LPS, which is illustrated by the differential response of the classic LPS/TLR4-target IL-1β. Although these data do not rule out a role for TLR signaling in mediating the inflammatory effects of chyle, induction of ER stress seems to be a more plausible mechanism.
A previous report briefly eluded to the development of chylous ascites in Angptl4−/− mice after 20 weeks of HFD (Desai et al., 2007
). In the same study it was found that repeated injections of WT mice fed HFD with a monoclonal antibody directed against the N-terminal portion of Angptl4 recapitulated the phenotype of Angptl4−/− mice. Since the antibody is directed against the N-terminal portion of Angptl4 and abolishes its ability to inhibit LPL, these data support the notion that the clinical abnormalities in Angptl4−/− mice fed HFD are related to altered LPL activity, and are independent of the signaling function of the C-terminal fragment of Angptl4.
(Chylous) ascites is a rare phenotype among transgenic mouse models. It has been observed in mice heterozygous for the transcription factor Prox1 as well as in mice lacking Angiopoietin-2. Both proteins are essential for development of the lymphatic vasculature (Gale et al., 2002
; Harvey et al., 2005
). Accordingly, it is tempting to hypothesize a similar role for Angptl4. However, it should be emphasized that the Prox1+/− and Angiopoietin2−/− mice develop chylous ascites shortly after birth, reflecting a severe developmental defect. In contrast, Angptl4−/− mice do not show any changes in lymphatic endothelial integrity and do not exhibit ascites unless challenged with HFD for at least 12 weeks. Rather, the data suggest that the ascites was secondary to progressive inflammation originating in the MLN macrophages, leading to massive lymphadenitis and consequent obstruction in mesenteric lymph flow, which in turn caused dilation of intestinal lymphatic vessels. Furthermore, inflammation of mesenteric lymp nodes and mesenteric fat led to increased local lymphatic and vascular permeability, as shown by chylous ascites and low SAAG, respectively, which is indicative of exudative ascites. The more than two-fold higher protein concentration in ascites fluid compared to chyle supports an important contribution of vascular leakage next to leakage from chyle. Increased circulatory leakage caused fibrinogen extravasation, which after clotting accumulated as fibrin and covered abdominal organs. Chronic inflammation likely gave rise to impaired intestinal barriers function and translocation of enteric bacteria, causing peritonitis which ultimately caused the death of the animals.
In conclusion, we demonstrate that Angptl4 protects against the severe pro-inflammatory effects of dietary saturated fat in MLN by inhibiting macrophage LPL, thereby reducing lipolytic release of fatty acids, macrophage foam cell formation, ER stress, and initiation of a marked inflammatory response. The data provide a clear illustration how the unique anatomy of intestinal lymphatic system, in which immune cells residing in mesenteric lymph nodes are exposed to excessive postprandial TG concentrations, requires the activation of an effective cellular mechanism that serves to protect against elevated lipid uptake and its complications. It can be speculated that the inability to effectively recruit this mechanism may contribute to pro-inflammatory changes related to elevated saturated fat consumption.