Regulation of intestinal immunity by dietary fatty acids

Fatty acids from various types of food are absorbed through the intestine and provide energy and structural components for mammalian cells. The intestine is in charge of primary digestion then absorption of fatty acids and their biological functions are intensively affected by dietary fat^1,2,3. This includes the alteration of the intestinal immune system, the dysregulation of which is associated with inflammatory diseases including intestinal bowel disease (IBD), tumor, infections and allergies^1,2,3. Systemic change of metabolism occurs upon uptake of a fat-containing meal^4,5,6. Dietary fatty acids present in the food in the form of triacylglycerol acid (TG) are incorporated with cholesterol, hydrophobic vitamins into micelles, which are further emulsified by bile salts and digested by pancreatic lipases into free fatty acids (FFAs) and 2-monoglycerides for them to be absorbed by enterocytes^4,5,6. In enterocytes, fatty acids and monoglycerides are reassembled into TG, which are converted to lipoproteins to form chylomicrons. The chylomicrons are transported through the lymphatic system into the thoracic duct and drained into circulation. During this process, partial FFAs are dissociated from chylomicrons through the interaction with apolipoprotein ApoC-2 present on the arteriolar endothelial cells^4,5,6. The remaining chylomicron remnants are transported to the liver, where the liver TGs, cholesterol and other fats are packed together with ApoB-100 into very low density lipoprotein (VLDL)^4,5,6. VLDLs sequentially become intermediate-density lipoproteins (IDLs) and low-density proteins (LDLs) as TGs and apolipoproteins are gradually removed from the capillaries of muscle and adipose tissues (Fig. 1)^4,5,6. As a note, medium-chain and short-chain fatty acids do not enter the lymphatic system but directly enter the circulation through portal vein^4,5,6. Taken together, fatty acids could be present in the circulation in the form of chylomicrons, LDL and FFA. Over-consumption of dietary fat will increase the serum levels of LDL and FFA. Uptake of lipids is mediated through LDL receptor (LDLR), scavenger receptors such as CD36 and Scavenger Receptor-B1 (SR-B1) expressed on the cell surface⁷. Notably, these receptors are also expressed on immune cells^8,9,10,11. SR-B1 expressed by B cells has been shown to negatively regulate downstream signaling of TLR9 in B cells⁹. LDLR is induced to be expressed by T and B cells upon stimulation¹². CD36 has been shown to mediate uptake of oxidized low-density lipoproteins by CD8⁺ T cells in the tumor microenvironment, leading to lipid peroxidation and cell ferroptosis^10,11. Therefore, the functions of intestinal immune cells could be modulated by directly sensing fatty acids and their cell-permeable metabolites. It remains to be demonstrated whether these receptors work redundantly or cooperatively to regulate fatty acids uptake by immune cells and affect the function of immune cells.

Dietary fatty acids are packaged into micelles and digested by bile acids and lipase into FFAs and 2-monoglycerides, which are resynthesized into TGs in the enterocytes. TGs are converted to chylomicrons, which become chylomicron remnants with dissociation of FFAs. Chylomicron remnants are synthesized into VLDLs in the liver and delivered to the circulation, and gradually become IDLs and LDLs as TGs are removed from the capillaries of muscle and adipose tissues.

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Major types of dietary fatty acids could be categorized into saturated long-chain fatty acids (LCFAs, e.g., C16:0 palmitic acid, C18:0 stearic acid), long-chain monounsaturated fatty acids (LC-MUFAs, e.g., C18:1 oleic acid), long-chain polyunsaturated fatty acids (LC-PUFAs, e.g., C18:2 linoleic acid, and C18:3 α-linolenic acid), medium-chain fatty acids (MCFAs, e.g., C8:0 caprylic acid, C10:0 capric acid, and C12:0 lauric acid)¹³. Short-chain fatty acids (SCFAs) including acetate, propionate and butyrate are not derived from food, but are generated by intestinal microbiota through fermentation of dietary fibers^13,14. From the metabolic perspective, LCFAs but not MCFAs or SCFAs require carnitine shuttle to enter the mitochondria for oxidation^13,15. Furthermore, SCFAs have been shown to bind to and agonize G-protein-coupled receptors (GPCRs)¹⁴. These differential characteristics in metabolic and signaling pathways of fatty acids may lead to distinct effects on intestinal immunity. HFD used in research contains mixed types of fatty acids and is rich in saturated LCFAs. One of the broadly used HFD contains 35% fat by weight (60% by calories), 25% carbohydrate and 26% protein^16,17,18,19. Lard is supplied as a source of fat composed of 38.7% saturated LCFA (mainly palmitic aid, stearic acid), 44.5% MUFA (mainly oleic acid) and 16.9% PUFA (mainly linoleic acid)²⁰. Systemic metabolism of HFD also changes serum levels of cholesterol, fasting glucose and bile acids (BAs)^21,22,23. These components in addition to fatty acids may be sensed by intestinal epithelial cells (IECs) and immune cells directly or indirectly to regulate intestinal immunity.

A central mechanism of how HFD regulates intestinal immunity is through the modulation of the intestinal microbiota²³. It occurs even with a short-term HFD feeding (e.g., less than a week or temporarily during infancy) and could imprint a long term impact on intestinal immunity^19,24. HFD has been found to alter the abundance of bacteria species responsible for induction of specific types of effector cells, which partially explains the HFD-skewed pro-inflammatory profile²⁵. HFD-shaped microbiota also acts through pattern recognition receptors (PRRs) on IECs and antigen presenting cells (such as dendritic cells and macrophages), which may crosstalk with innate lymphoid cells and T cells and expand the inflammation²⁶. The microbiota mediates the generation of secondary BAs, and different BAs act as agonists or antagonists on their receptors expressed by the IECs and further regulate liver BA synthesis²⁷. BAs could in turn alter the microbiota profile by serving as nutrients to specific bacteria species and promoting their growth²⁸. BAs could also directly act on intestinal immune cells to regulate their properties^29,30,31. Therefore, the maintenance of intestinal homeostasis requires the intricate network coordinated by microbiota, IECs and immune cells.

In this review, we outline effects of dietary fatty acids, focusing on the HFD, on intestinal immunity under steady state and pathological conditions. We dissect the mechanisms of how HFD affects intestinal immunity into direct effect of fatty acids on intestinal immune cells and indirect regulation of intestinal immune responses by microbiota and IECs especially under the context of colitis, intestinal infections and tumor. Understanding these mechanisms may facilitate the development of potential strategies to treat intestinal inflammatory diseases.

Maintenance of immune homeostasis by intestinal immune cells

Intestinal immune cells localize in the epithelial layer, lamina propria and gut-associated lymphoid tissues (GALT), where they undergo efficient activation to eliminate pathogens and exhibit immune regulatory roles for timely immune resolution to prevent tissue damage. This includes the innate immune cell branch, including innate lymphoid cells (ILCs), macrophages, dendritic cells (DCs) and granulocytes, and the adaptive immune cell branch consisting of T and B cells³².

Effects of T cells and innate lymphoid cells on intestinal immunity

The lymphoid lineage of intestinal immune cells encompasses the ILCs, T cells and B cells. T cells are one of the most abundantly present types of immune cells in the intestine³³. Due to frequent communication with trillions of microbiota, intestinal T cells are highly activated³⁴. CD4⁺ T helper cells are classified into Th1, Th2, Th17, follicular helper T (Tfh) and regulatory T (Treg) cells based on their transcription factor expression and cytokine production^35,36. Helper-like ILCs, lacking antigen specific receptors, mirror the characters of Th subsets^37,38,39,40. ILC1s and Th1 cells express transcription factor T-bet, produce IFN-γ and facilitate the clearance of intracellular pathogens^41,42,43. ILC2s and Th2 cells, expressing GATA3 and mainly secreting IL-5 and IL-13, are important for the expulsion of parasites and mediating allergic responses^43,44. ILC3s share the expression of RORγt with Th17 cells, and both of them could produce IL-17 and IL-22^43,45. ILC3s contain heterogeneous subsets including CCR6⁺NKp46^–ILC3, CCR6^–NKp46^–ILC3 and NKp46⁺ILC3^46,47,48. The NKp46^–ILC3s are stronger producers for IL-17 and IL-22, whereas the T-bet-expressing NKp46⁺ILC3s also secrete IFN-γ and TNF-α ⁴⁶. Studies indicate that NKp46⁺ILC3s could be converted from CCR6^–NKp46^–ILC3s, and NKp46⁺ILC3s may lose RORγt expression and become ILC1s^38,46,49. This process is driven by pro-inflammatory cytokines such as IL-12, IL-15 and IL-18, and is considered to be implicated in the pathogenesis of Crohn’s disease^38,50,51. NK cells are cytotoxic ILCs. They are similar to the CD8⁺ T cells and possess cytotoxic functions mediated by Perforin and Granzyme B^52,53,54. Notably, intestinal γδT cells are capable of secreting effector cytokines including IFN-γ, IL-17, and IL-22^55,56. Intestinal Tregs are essential for controlling overt inflammation by suppressing the activity of CD4⁺ effector T cells and myeloid cells^57,58,59. Based on the origin and route of induction, intestinal Tregs could be divided into Nrp1⁺RORγt^– thymic-derived Tregs, Nrp1^–RORγt^– food antigen-induced Tregs and Nrp1^–RORγt⁺ microbiota-induced Tregs^60,61,62. In the large intestine, RORγt⁺ Tregs are major source of IL-10, an immunoregulatory cytokine that suppresses pathogenic T cells and supports the function of Tregs themselves^63,64.

Pro-inflammatory cytokines produced by lymphocytes exhibit immuno-defensive function to clear pathogens through recruiting and activating myeloid cells, including macrophages, neutrophils and eosinophils. IFN-γ activates macrophages by enhancing their chemokine secretion and microbial killing activity⁶⁵. IL-5 promotes the differentiation and maturation of eosinophils⁶⁶. IL-22 could act in synergy with IL-17 to stimulate chemokine expression such as CXCL1, which accumulates neutrophils to the inflammatory loci^67,68. Macrophages and neutrophils are able to eliminate pathogens by phagocytosis and oxidative burst. However, this process also causes cell necrosis and tissue damage^69,70. Importantly, IL-17 and IL-22 are critical for maintenance of intestinal homeostasis under steady state. The receptors of IL-17 and IL-22 are expressed on IECs^71,72. IL-17 stimulates the IECs to express tight junction proteins⁷³, and IL-22 has been shown to promote epithelial cell regeneration by increasing the proliferation of intestinal stem cells^71,74. Lacking IL-17 or IL-22 exacerbates experimental colitis^73,75,76.

Role of major histocompatibility complex class II (MHCII)-expressing cells in intestinal immunity

The intestinal macrophages and DCs expressing MHCII present antigens to CD4⁺ Th cells for their activation⁷⁷. Macrophages grouped into M1 and M2 macrophages are correlated with the activation of Th1 and Th2 cells respectively⁷⁸. M1 macrophages are considered to be pro-inflammatory, whereas M2 macrophages have been indicated to play a protective role in IBD⁷⁹. Recently, studies have uncovered complex features of tissue-resident macrophages outside of the M1/M2 macrophage realm^80,81. Functional subsets of intestinal macrophages and DCs have been characterized based on surface markers into MHCII⁺CD11c⁺CD103^–CD11b⁺CX3CR1⁺F4/80⁺CD64⁺ macrophages and MHCII⁺CD11c⁺CX3CR1^int/–F4/80^–CD64^– DCs⁸². The DCs could be further divided into subsets based on the expression of CD11b and CD103 to CD103⁺CD11b^– DCs, CD103⁺CD11b⁺DCs and CD103^–CD11b⁺ DCs⁸². CD11b⁺CX3CR1⁺ macrophages have been shown to play a regulatory role in intestinal inflammation through producing IL-10 and maintaining Tregs⁸³. They also respond to microbiota to produce IL-1β under steady state to support ILC3s to produce GM-CSF, which also supports intestinal Treg maintenance by sustaining TGF-β and retinoic acid (RA)-generating DCs⁸². Notably, among the DC subsets, the CD103⁺CD11b⁺DCs are the most potent generator of RA and contribute to both Tregs and Th17 cells generation^82,84,85.

Aside from the professional antigen presenting cells (DCs, macrophages and B cells) that express MHCII, intestinal MHCII has also been found to be expressed by ILC2s, ILC3s and IECs^86,87,88,89. ILC3-derived MHCII has been shown to present antigens derived from intestinal microbiota to CD4⁺ T cells to suppress their expansion and activation^86,87. On the contrary, ILC2-derived MHCII enhances intestinal Th2 responses and accelerates the elimination of parasites⁸⁸. IEC-derived MHCII communicates with intraepithelial CD4⁺ T cells for them to maintain immunoregulatory functions^17,89. Intestinal B cells and plasma cells are in charge of producing immunoglobulins essential for neutralization of pathogens and prevention of dysbiosis. B cells are particularly accumulated in Peyer’s patches and isolated lymphoid follicles (ILFs) where they interact with T cells and DCs to mediate humoral responses^90,91. Plasma cells are mainly located in the lamina propria⁹⁰. Majority of bacteria-coated immunoglobulins are IgAs⁹². A larger proportion of intestinal IgAs are T cell independent, but both T cell dependent and independent IgA participate in regulating microbiota homeostasis^92,93,94,95. Next, we will summarize how dietary fatty acids, focusing on the HFD, affect the functions of intestinal immune cells to modulate intestinal immunity.

Disrupted T and ILC3 responses by HFD associate with dysbiosis and colitis

Two forms of IBD in clinics are defined as the Crohn’s disease (CD) and ulcerative colitis (UC). Different types of dietary fatty acids exhibit differential effects on IBD. It has been reported that intake of animal protein and n-6 PUFAs with less n-3 PUFAs increases the risk of CD, and increased intake of n-3 PUFAs reduces the risk of UC, highlighting a possible beneficial role of n-3 PUFAs in IBD^96,97. It has been broadly demonstrated with animal experiments that increased uptake of saturated LCFAs (such as HFD feeding) induces obesity accompanied with exacerbated intestinal inflammation. However, a causal link for human obesity and IBD is not explicit⁹⁸. Epidemiology studies have revealed a correlation between obesity and IBD, but this correlation differs in terms of age of patients, forms of IBD and requirement for surgery⁹⁹. Research on pediatric cohorts has revealed that obesity is more likely to occur in UC than CD and is positively correlated with surgery requirements in UC, suggesting obesity a risk factor for UC^100,101,102.

Major changes of T and ILC3 responses by HFD

A concept acknowledged by the field is that HFD feeding or obesity is associated with an intestinal low-grade inflammation (LGI) that contributes to metabolic disorders and the susceptibility to colitis¹⁰³. The LGI is accompanied with disrupted epithelial integrity and dysbiosis, which exacerbates the situation of each other and promotes the progression of LGI.

The LGI is reflected by aberrant T and ILC3 responses, and is present in both the lamina propria and among IECs. It has been reported that feeding of a HFD (60 kcal% fat) for 12–16 weeks leads to increased Th1, IFN-γ-producing CD8⁺ T cells, IL-17-producing γδT cells and decreased Tregs in both the small intestine and colon lamina propria¹⁰³. Ileum upregulation of Th1 cells and downregulation of Tregs have also been reported by a study using 30-day HFD feeding (72 kcal% fat)²⁵. The effect of HFD on Th17 cells has not been consistently observed. The same case exists for IL-6, a master inducer for Th17 differentiation in vitro and a cytokine increased with obesity. Some research has found that both IL-6 and Th17 cells are increased in fat-enriched diet (72 kcal% fat, 10 weeks; 30.9% crude fat, 4 weeks)-fed mice under steady state or in pathological conditions^{104,105,106,107}. This may explain HFD-worsened 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis or autoimmunity experimental encephalomyelitis (EAE), where Th17 cells play pathogenic roles^104,106,107. Intriguingly, other studies show contradictory phenotypes with unchanged or reduced IL-6 and Th17 cells in mice fed with HFD (72 kcal% fat, 30 days; 60 kcal% fat, 6 weeks or 10 weeks)^25,103,108. It has been observed that HFD (60 kcal% fat, 12–14 weeks) suppresses the maintenance of CX3CR1⁺ macrophages and CD11b⁺CD103⁺ DCs, which may contribute to the reduction of Tregs and Th17 cells^83,84,109. Notably, ILC3s with overlapping functions with Th17 cells, have reduced IL-22 production after HFD feeding (60 kcal% fat, 4 weeks or 8 weeks)^16,110. Another study found that mRNA expression of IL-22 was reduced in the colon of HFD-fed mice (60% fat, starting at the age of 4–8 weeks old for at least 8 weeks) upon Citrobacter rodentium infection¹¹¹. Serum IL-22 level was found to be decreased in HFD-fed mice after flagellin injection¹¹¹. Furthermore, the percentage of CD4⁺IL-22⁺ cells, which could be the IL-22-producing Th cells and/or the IL-22-producing CD4⁺ ILC3s⁴³, was decreased in the draining lymph nodes of HFD-fed mice upon ovalbumin/complete Freund’s adjuvant immunization¹¹¹. Interestingly, increased T-bet⁺NKp46⁺ILC3s and ILC1s are found in high-fat and high-sucrose diet (40% fat, 12 weeks) fed mice¹¹⁰, implying the likely conversion of IL-22 competent NKp46^–ILC3 to NKp46⁺ILC3 and ILC1, a process implicated in the development of IBD^50,51. Mucosal-associated invariant T (MAIT) cells, expressing one invariant TCR α-chain and a limited number of β-chains, recognize antigens in the major histocompatibility complex-related molecule 1 (MR1)-restricted manner and play a critical role in mucosal immunity¹¹². It has been shown that ileum MAIT cells are reduced but produce higher IL-17 in HFD-fed mice (60 kcal% fat, 12 weeks)¹¹³. Moreover, MAIT cells exacerbate intestinal inflammation by promoting M1 macrophage differentiation and inhibiting Tregs¹¹³. As discussed above, IL-17 acts as a double-edge sword in intestinal immunity. It is possible that IL-17 originated from different types of cells exhibits distinct roles by communicating with surrounding cells and initiates distinct downstream cascades. The inconsistency in HFD-induced Th17 responses may be due to variations in microbiota, time points of observation and disease situations. These precise mechanisms remain to be further investigated. Intraepithelial lymphocytes (IELs) contain heterogeneous subtypes, including the immunoregulatory T cells that secrete TGF-β and IL-10^{114,115,116,117}. HFD depletes both intraepithelial CD4⁺ and CD4⁺CD8⁺ αβ Τ cells probably by downregulating the gut-homing molecules including CD103 and CCR9 expressed by IELs (60 kcal% fat, 7–46 weeks)¹¹⁸. Switching HFD to normal diet restores the αβ T cells of the IELs and resolves intestinal inflammation¹¹⁸. Taken together, disrupted immune homeostasis reflected by decreased immunoregulatory Tregs, IELs and IL-22⁺ILC3s, in addition to increased pathogenic Th1 and IL-17⁺ MAIT cells, may cooperatively contribute to LGI and susceptibility to colitis (Fig. 2).

HFD induces a pro-inflammatory profile manifested by reduced Tregs and IL-22-producing ILC3s, as well as increased Th1 cells in the intestinal lamina propria. The decrease of Tregs may be attributed to the ablated maintenance of Treg-promoting CX3CR1⁺ macrophages and CD11b⁺CD103⁺ DCs. Moreover, HFD suppresses the SCFAs that have been shown to cell-intrinsically promote intestinal Treg induction. The decrease of Tregs may contribute to uncontrolled pro-inflammatory Th1 responses. In addition, HFD-induced dysbiosis may cause increased Th1 response while inhibiting IL-22 production by ILC3s. Fatty acids could directly act on Th cells. Both LCFAs and MCFAs increase Th1 differentiation, whereas MCFAs suppress Treg differentiation demonstrated by in vitro experiments.

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Direct effects of fatty acids on T cells

One mechanism explaining the immunological perturbation by HFD is through direct action of fatty acids on T cells. Different types of fatty acids have been found to play distinct roles in intestinal immune responses. Long-chain and medium-chain saturated fatty acids could promote the differentiation of Th1 and Th17 cells, and lauric acid as one type of the medium-chain saturated fatty acids also suppresses the differentiation of Tregs in vitro¹⁰⁶. In vivo, feeding mice with lauric acid-enriched diet (30.9% crude fat rich in the lauric acid, 4 weeks) increased small intestinal Th1 and Th17 responses. A study showed that lauric acid-enriched diet resulted in decreased level of SCFAs possibly due to decreased Bacteroidetes facilitating fermentation of fiber-rich nutrition into SCFAs¹⁰⁶. SCFAs have been consistently demonstrated to promote Tregs through directly activating FFAR2 expressed by Tregs and inhibiting JNK/p38 pathway^106,119,120. These findings support a role of lauric acid in suppressing Tregs in vivo, although further data are needed to validate this hypothesis. PUFAs inhibit Th17 cells ex vivo and ameliorate experimental colitis in vivo^121,122. Oleic acid is found to be one of the highest represented LCFA in human serum and promotes the induction and suppressive function of Tregs in vitro¹²³. Mechanistically, it has been indicated that LCFAs support the proliferation and function of Tregs possibly through increasing fatty acid oxidation (FAO) and mitochondria oxidative phosphorylation¹²⁴. But this concept has been challenged by the evidence that the rate-limiting enzyme carnitine palmitoyltransferase 1 (Cpt1a) for LCFA FAO may be dispensable for the induction or maintenance of Tregs both in vitro and in vivo^125,126. It is interesting to observe that some types of fatty acids exhibit consistent functions on T cell responses in vitro and in vivo, suggesting that in vitro experiments can be an efficient system for screening fatty acids candidates to achieve specific regulation of intestinal immunity. However, it remains to be demonstrated if Th cells increase their fatty acids uptake and undergo metabolic change in vivo following dietary fatty acids or HFD feeding.

HFD shapes microbiota to modulate T and ILC3 responses

The microbiota plays a central role in regulating intestinal immunity in the context of HFD feeding. Revisiting different cohorts of studies indicate that HFD reproducibly alters the microbiota pattern by decreasing the ratio of Bacteroidetes over Firmicutes^13,16,127. Many reports also consistently observe increased Proteobacteria and reduced Bifidobacterium^13,16,128. Notably, Lactococcus species have been consistently found to be increased in HFD-fed animals¹²⁷. However, this has been demonstrated to be due to nonviable Lactococcus contamination from the irradiated HFD. Such a bias needs to be considered when performing bacteria sequencing analysis. Furthermore, Lactococcus has been indicated to have proinflammatory properties, which may affect the interpretation of altered intestinal immunity affected by live bacteria upon HFD feeding¹²⁷. Intestinal Th cell and ILC3 responses are closely related to microbiota profile shaped by dietary fatty acids or HFD. Increased intestinal Th1 and Th17 responses in mice fed with lauric acid-enriched diet could be recapitulated in germ free mice fed with normal diet by fecal transplantation¹⁰⁶. This may be attributed to the change of specific bacteria species being able to induce distinct immune responses. For example, Th17-inducing bacteria including Porphyromonadaceae, Segmented filamentous bacteria (SFB), and Bifidobacterium are decreased by lauric acid²⁵. Restoration of Bifidobacterium lactis recovers the lauric acid-suppressed Th17 cells²⁵. Notably, it has been consistently observed that HFD reduces the level of SCFAs (60 kcal% fat, 4 weeks)¹⁶, which plays a regulatory role in intestinal immunity through induction of Tregs and supporting IL-22 production by ILC3s^119,129. One of the explanations for this decreased SCFAs is that the recipe of HFD used by research is lack of soluble fiber, which is critical source for SCFA generation through bacterial fermentation¹⁶. But in a study without specifying the content of fibers, feeding mice with lauric acid could actively downregulate fecal SCFAs level, suggesting the participation of fiber-independent mechanisms such as microbiota¹⁰⁶. Consistently, the addition of soluble fiber could upregulate SCFAs and recover reduced IL-22-producig ILC3s caused by HFD feeding. Nevertheless, the recovery of IL-22 production of ILC3s by fiber supplementation is through microbiota-dependent but SCFA-independent mechanism¹⁶. Although it has been well-acknowledged that the change of microbiota by HFD feeding affects intestinal T and ILC responses, a clear mechanistic link between microbiota and specific immune responses haven’t been established. Antigens, virulence factors, or metabolites derived from different microbiota that contribute to skewed T/ILC3 responses upon HFD feeding remain to be determined.

HFD curtails intestinal IgA production

IgA is the major type of immunoglobulin generated in the intestine and is essential for defense against pathogenic bacteria and prevention of dysbiosis^93,95. Studies indicate that IgA generation is reduced by HFD (60% kcal diet, 12–16 weeks), which possibly contributes to the disorganized microbiota and intestinal inflammation¹⁰⁹. Reduction of intestinal IgA, IgG and IgM upon HFD (34.6% fat, 2 months) feeding has been revealed by proteomics analysis¹³⁰. It has been shown that the colonic, rather than small intestinal or Peyer’s patches, plasma cells have decreased IgA production in HFD-fed mice¹⁰⁹. A potential mechanism is that HFD leads to decreased frequencies of colonic CD11b⁺CD11c⁺CD103⁺ DCs, capable of converting vitamin A to RA to facilitate IgA generation¹⁰⁹. Previous research has also demonstrated that dietary cholesterol could raise the level of 25-hydroxycholesterol, which inhibits Peyer’s patches B cell activation and the generation of IgA through regulating SREBP2 expression in B cells¹³¹. This lack of IgA results in exacerbated Salmonella infection. As HFD increases serum level of cholesterol, there is a chance that metabolites of cholesterol such as 25-hydroxycholesterol is also increased and suppresses IgA production²¹. Interestingly, when supplied at a low ratio in contrast to the HFD recipe, palm oil (enriched in palmitic acid, 4%) but not soybean oil or coconut oil has been found to increase the intestinal IgA. Palm oil also boosts IgA against orally immunized antigens, proposing it being an adjuvant for oral vaccination¹³². This indicates that low dose of LCFA may have a beneficial role in intestinal immunity by promoting IgA production. It will be interesting to investigate how different types and doses of fatty acids affect the generation of T cell dependent or independent IgA production, which may affect intestinal microbiota distribution and intestinal immunity.

HFD disrupts IEC homeostasis and promotes colitis and tumor

IECs contain specialized and heterogeneous populations, including enterocytes, Paneth cells, goblet cells, enterochromaffin cells and tuft cells, in charge of nutrient absorption, mucin secretion, mucosal defense, transmission of lumen antigens and neuroendocrine functions¹³³. We have summarized HFD-perturbed T cell and ILC responses that are closely related to dysbiosis and damaged epithelial integrity, leading to intestinal inflammation. Importantly, IECs could directly sense fatty acids and microbiota, act as an initiator of downstream inflammation cascades, and communicate with macrophages and T cells to affect intestinal immunity under the context of HFD feeding. HFD-disturbed IEC homeostasis exacerbates colitis and even leads to carcinogenesis.

Disruption of the mucin layer by HFD

Damage of the intestinal mucus layer by HFD is closely linked to dysbiosis. Transmembrane or gel-forming mucins secreted by intestinal enterocytes or goblet cells establish a barrier to prevent the intrusion of pathogens¹³⁴. HFD (40% fat, 4 weeks) has been shown to cause decreased mucin production^128,135. This could partly be due to the increased abundance of mucin-degrading bacteria¹²⁸. Notably, one of the specialized mucin-degrading bacteria Akkermansia muciniphila has been observed to be decreased in HFD-fed mice (60% fat, 4 weeks) or obese people¹³⁶. We speculate that while pathological amount of A. muciniphila disturbs intestinal mucus layer and is harmful to intestinal homeostasis, A. muciniphila at the physiological level utilizes the mucin glycan for their maintenance without causing damage to the mucus layer. Rather, A. muciniphila is required for intestinal homeostasis possibly by generating SCFAs and detoxifying hydrogen sulfide (H₂S)¹³⁷. Another report has shown that A. muciniphila-derived extracellular vesicles increase occluding expression by human epithelial cell line¹³⁸, thereby improving the epithelial tight junction function. Restoration of A. Muciniphila significantly improves the epithelial barrier function, and A. muciniphila has been shown to play a protective role in preventing dextran sulfate sodium (DSS)-induced colitis^136,139. Interestingly, it has been shown that peroxisome proliferator-activated receptor-γ (PPARγ) agonist ameliorates whereas PPARγ specific deletion in IECs exacerbates dysbiosis and mucin loss under HFD feeding¹²⁸. Therefore, it is likely that IECs may directly sense fatty acids through PPARγ to interplay with intestinal microbiota and maintain a beneficially mutualistic environment.

Activation of microbiota-IEC-macrophage axis

Disruption of the mucin layer expands the interface for pathogen intrusion. The IECs express a series of PRRs sensing pathogens and MyD88 has been indicated as a common adaptor downstream of a group of Toll-like receptors (TLRs)¹⁴⁰. MyD88 expression by IECs has been shown to play a pathogenic role in HFD-induced obesity and inflammation¹⁴¹. Deletion of MyD88 in IECs restores Tregs and improves dysbiosis. This may further lead to uncontrolled activation of pro-inflammatory T cells and exacerbate dysbiosis in a feed-forward loop. In addition to MyD88-dependent signaling, a study using zebra fish has revealed that acute HFD or high-cholesterol diet (HCD) feeding induces an IL-1β-dependent accumulation of myeloid cells in the intestine¹⁴². Further analysis has revealed that dietary fat facilitates HCD-induced inflammasome activation in IECs, which is responsible for recruitment of CD11b⁺ and CD11c⁺ myeloid cells. This process is dependent on the microbiota suggesting that the activation of inflammasomes may be attributed to upstream activation of PRRs such as the NOD-like receptor (NLR) family members by intruded pathogenic bacteria derivatives due to disrupted epithelial barrier¹⁴³. Similar observations have also been reported in mouse studies. It has been shown that HFD (60% of fat, 24 weeks)-induced CCL2 expression by IECs is required for the recruitment of CCR2⁺ macrophages to the intestine. Inflammasome activation was found in colonic macrophages dependent on the CCL2-CCR2 axis¹⁴⁴. This further led to overt production of IL-1β and IL-18, which may increase the susceptibility to colitis^145,146. The recruited macrophages could also respond to increased IL-6 in the colon and enhance their production of CCL20¹⁴⁷. The latter accumulates CCR6-expressing B cells, αβ T and IL-17-producing γδ T cells to the inflammatory site, which contributes to the pathogenesis of colitis-associated colorectal cancer (CAC)¹⁴⁷. The above observations propose a model that epithelial integrity damaged by HFD promotes aberrant activation of IECs and macrophages to further expand the inflammatory circuit by aggravating dysbiosis and triggering the activities of proinflammatory lymphocytes (Fig. 3).

HFD leads to decreased mucin production by intestinal goblet cells. Dysbiosis caused by HFD may exacerbate the destruction of mucin layer. This leads to intrusion of the intestinal pathogenic bacteria which activates IECs and macrophages through PRRs. HFD enhances CCL2 expression by IECs, accumulating CCR2⁺ macrophages to the intestine. Inflammasome activation of the CCR2⁺ macrophages mediates production of IL-1β and IL-18, which may increase intestinal inflammation. Moreover, increased IL-6 in the colon has been shown to induce CCL20 production by macrophages, which recruits the CCR6-expressing T and B cells to worsen CAC.

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HFD suppresses MHCII expression by IECs

In addition to the bacteria-IEC-macrophage axis, IECs including the Lgr5⁺ intestinal stem cells (ISCs) have been indicated to directly communicate with the T cells (IELs) through MHCII^17,18,89. The MHCII expression by ISCs could be upregulated by activation of TLR2/NOD2 and IFN-γ¹⁸. Moreover, the expression of MHCII on IECs is diurnally regulated by dietary contents and microbiota¹⁷. Both HFD and antibiotics treatment ablate MHCII expression by IECs, and restoration of germ-free mice with microbiota of HFD-fed mice shows deficiency of MHCII expression by IECs¹⁷. Bacterial mono-colonization studies have revealed Akkermansia, Lachnospiraceae and SFB as “inducers” and Lactobacillus murinus as “suppressor” of MHCII expression by IECs¹⁷. The correlated change of microbiota in HFD-fed mice may account for decreased expression of MHCII by IECs. ISCs, localizing at the base of the crypt and giving rise to heterogeneous subtypes of IECs, are considered to be the origin of precancerous dysplasias and are key targets of the HFD-promoted carcinogenesis¹⁴⁸. Genetic deletion of MHCII expression in ISCs specifically impairs the homeostasis of ISC-adjacent Th cells and the immunoregulatory CD4⁺IL-10⁺ IELs, therefore exacerbating Crohn-like enteritis¹⁷. This is likely to be a mechanism for HFD-sensitized colitis accompanied with depletion of IELs. HFD (60 kcal% fat, 9 to 14 months)-suppressed MHCII expression by ISCs also increases their tumor initiating capacity¹⁸. This is correlated with a loss of intraepithelial T cells possibly involved in anti-tumor immunity. These evidences support a notion that disruption of microbiota-MHCII-IEL axis by HFD contributes to the increased incidence of IBD and intestinal tumor (Fig. 4).

Expression of MHCII by IECs could be induced by TLR2 and NOD2 signaling, as well as type 1 cytokine IFN-γ. IEC-derived MHCII sustains IELs including the immunoregulatory CD4⁺IL-10⁺ IELs and other tumor-suppressive CD3⁺ T cells. HFD perturbs MHCII expression by IECs accompanied with decreased IELs. HFD has also been shown to reduce the expression of gut-homing molecules including CD103 and CCR9 by IELs. These mechanisms are considered to contribute to the HFD-increased susceptibility to Crohn-like enteritis, intestinal tumor and colitis.

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Single cell transcriptome sequencing will be helpful to identify phenotypes and functions of specific subsets of IECs, which may account for distinct immune responses affected by HFD feeding. This will facilitate the discovery of potential targets for treatment of IBD and colon cancer linked with western diet consumption or obesity. IECs also express sensors for different BAs altered by HFD to regulate intestinal immunity^149,150. As increased studies have discovered the immunoregulatory role of BAs, we discuss these findings as a separate section below.

Emerging roles of BAs in HFD-caused intestinal pathogenesis

Primary BAs synthesized in the liver are present as chenodeoxycholic acid (CDCA, rapidly converted to muricholic acid (MCA) in rodents) and cholic acid (CA)¹⁵¹. The primary BAs stored in the gall bladder are conjugated with taurine (in mice) or taurine and glycin (in humans)¹⁵¹. Food ingestion stimulates the release of BAs to the duodenum to facilitate the digestion and absorption of fatty acids, cholesterol and fat-soluble vitamins. In the intestine, primary BAs undergo a series of bio-transformation procedures including deconjugation, 7α-dehydroxylation, oxidation and epimerization for generation of secondary BAs^27,151,152. Intestinal microbiota plays a key role in BA metabolism^27,152. Different types of bacteria are enriched in specific types of enzymes that catalyze distinct procedures of BA bio-transformation. As the HFD quickly and profoundly alters the microbiota profile, it also significantly changes the BA composition²². Different BAs act as agonists or antagonists for their receptors, categorized into several family members of proteins including farnesoid X receptor (FXR), the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), the liver X receptor (LXR), and the vitamin D receptor (VDR), GPCRs, muscarinic receptors and sphingosine 1 (SP-1) receptor¹⁵². The above receptors could be expressed on IECs and intestinal immune cells, which serve as a base for the bioactivity of BAs on intestinal immunity.

Both primary and secondary BAs have been reported to be changed by HFD²². Clostridioides difficile infection, often caused by long-term antibiotics administration and one of the most common nosocomial infections, is reported to be accelerated by HFD (60 kcal% fat, 14–16 weeks)¹⁵³. This has been found to be correlated with increased primary conjugated BAs that promote spore germination of C. difficile¹⁵³. The composition of BA is differentially affected by different types of fatty acids. Milk fat (rich in saturated fatty acids, 37 kcal% fat, 5 weeks) but not safflower oil (rich in PUFAs) increases taurocholic acid, which could serve as sulfur source for sulfite-reducing bacteria Bilophila wadsworthia¹⁵⁴. B. wadsworthia has been shown to promote Th1 responses and exacerbate the colitis of Il10^–/– mice¹⁵⁴. Notably, IECs directly sense HFD -induced BAs through receptors such as FXR and TGR-5 to affect their homeostasis. HFD increases the generation of tauro-β-muricholic acid (T-β-MCA) and deoxycholic acid (DCA) acting as FXR antagonist to relieve the proliferation of cancer stem cells (60 kcal% fat, 6 weeks)¹⁴⁹. Interestingly, activation of TGR-5 expressed on ISCs by secondary BAs including the lithocholic acid (LCA) and DCA facilitates the growth of intestinal organoids¹⁵⁰. These observations may be served as supplementary mechanisms for cancer-promoting effect of HFD described in the previous section.

Recently, several kinds of secondary BAs have been found to regulate intestinal Th cells. Through functional screening and validation, 3-oxoLCA has been identified to inhibit intestinal Th17 cells³⁰, whereas isoalloLCA and isoDCA promote intestinal Tregs^30,31. The induction of Tregs by secondary BAs is protective against intestinal inflammation²⁹. Importantly, the generation of RORγt⁺Treg-inducing secondary BAs is dependent on bacterial expression of bile salt hydrolase²⁹. Mechanistically, secondary BAs could signal through VDR expressed by Tregs or by inhibiting FXR activity expressed on DCs to promote intestinal Tregs^29,31. These findings open an exciting research area to establish specific relationships among different types of fatty acids, microbiota and BAs-induced immune responses, which are valuable for potential treatment of IBD by targeting BAs.

Maternal HFD imprints a long-term effect on intestinal immunity in offspring

Recent studies suggest that maternal HFD increases the susceptibility to colitis in offspring. Even when the young descendants are switched back to and maintained on normal diet after weaning, they are still susceptible to colitis when grown to adults^24,105. Infancy is found to be a critical period of time when there is a fluctuation of microbiota profile and stimulated immune responses, discovered as the “weaning reaction”¹⁵⁵. It occurs between 2–4 weeks old in mice and is featured by transient increase in proinflammatory cytokine production accompanied with the generation of RORγt⁺Tregs¹⁵⁵. RA and SCFAs have been found to play a critical role in the generation of RORγt⁺Tregs during weaning reaction, the lack of which predispose the offspring to colitis, allergy and cancer in adult stage^105,155. Maternal HFD (60 kcal% fat, 6 weeks before gestation till the end of lactation) has been found to ablate the weaning reaction by causing overproduction of pro-inflammatory cytokines including TNF-α and IFN-γ, responsible for increased childhood intestinal permeability and adult sensitivity to colitis²⁴. Meanwhile, maternal HFD (60% fat, at least 5 weeks before gestation till the end of lactation) increases in IL-17 expression by ILC3s through a microbiota dependent and adaptive immune system independent manner, resulting in childhood necrotizing enterocolitis¹⁵⁶. Infancy HFD feeding also increases harmful H₂S production by pathogenic bacteria, and pharmacological inhibition of bacterial H₂S production or antibiotics treatment corrects intestinal inflammation and adult susceptibility to colitis²⁴. Importantly, it has been shown that co-house of HFD-nursed (60 kcal% fat, during gestation and lactation) pups with normal diet nursed-pups eliminates potential inflammation¹⁰⁵. Supplementing SCFAs, fermentation product of bacteria, to young mice under maternal HFD ameliorates colitis in a RORγt⁺Treg dependent manner²⁴. Strategies of blocking bacterial H₂S production or antibiotics treatment were performed during maternal HFD feeding. The above evidence strongly indicates a critical role of microbiota, rather than increased calorie intake, in imprinting a susceptibility to colitis in maternal HFD-fed offspring. However, it remains to be determined whether fatty acids or microbiota from the breast milk contributes to the dysbiosis in maternal HFD-fed offspring.

Summary and concluding remarks

HFD-induced obesity has been found to be closely associated with high incidence of intestinal inflammation and tumor. This usually starts with an intestinal LGI featured by pro-inflammatory immune cell activation, damaged epithelial integrity and dysbiosis. We have summarized the direct effects of fatty acids on specific immune cell subsets, which may explain the changed intestinal immune responses by HFD, as well as indirect effects of HFD on intestinal immune subsets by affecting microbiota or intestinal epithelial cells (Table 1). As a central change in HFD feeding, the shift of microbiota profile breaks the epithelial mucin layer and causes pathogen intrusion. This activates IECs or macrophages through PRRs and may further expand the inflammatory circuits by recruitment and activation of inflammatory T cells. HFD also inhibits MHCII expression by IECs, which may contribute to depletion of IELs and increase the occurrence of colitis and tumor. Although mechanisms of how HFD affects intestinal inflammation have been investigated extensively, there are variations in phenotypes and conclusions in terms of the change of specific types of inflammatory responses and altered bacteria species. This may be due to different types of dietary fatty acids, variation in percentages of calories from dietary fatty acids in HFD recipes, as well as different microbiota distribution in research facilities. However, consistency or inconsistency in these findings may be helpful to interpret complex situations in human populations with strong diversities in microbiota and types of dietary fat consumption. It will be interesting to dissect how specific types of fatty acids affect intestinal immunity and delineate their corresponding targets involving bacteria species, bacterial metabolites and their downstream signaling pathways.

HFD-induced obesity is accompanied with metabolic disorders manifested by elevated level of cholesterol, increased blood glucose and insulin resistance. Sensors for cholesterol metabolites¹⁵⁷, glucose transporters^158,159 and insulin receptor (InsR)^160,161 have been reported to be expressed by immune cells and affect the function of immune cells. It will be interesting to examine whether cholesterol and glucose metabolism, as well as insulin signaling, altered by HFD affect the function of intestinal immune cells and regulate intestinal immunity. The understanding of these mechanisms will be helpful for developing potential strategies for the diagnosis, prevention and treatment of intestinal inflammatory diseases.