in maintaining gut barrier function and providing important immunomodulatory and antioxidant effects (**Figure 2**). The gut is an important secondary organ and also a site of starting severe systemic complications during AP. Intestinal barrier dysfunction is associated with translocation of bacteria and their inflammatory and toxic products, responsible for infection of the necrotic pancreas and systemic inflammatory responses. Therefore, maintaining the integrity of the gut barrier in the small intestine is one of the main goals in early-phase treatment of severe AP (23). Optimal nutritional support in AP has been under debate for decades. Bowl at rest (*nothing by mouth*) strategy has been implemented conventionally to treat AP (24, 25). However, dietary restrictions exacerbate patient's malnutrition due to imbalance between reduced food intake and higher nutritional requirements, leading to further catabolism, bacterial translocation (26), and ultimate mortality (27). Evidence of clinical trials has demonstrated parenteral nutrition (PN) in preventing pancreatic stimulation and many benefits of enteral nutrition (EN). However, in daily practice, it remains challenging to predict whether EN will be tolerated in patients with AP (8).

Strategic approaches to include nutritional supplements have also been attempted to provide additional immune regulatory

and antioxidative effects. Probiotics and prebiotics have been shown to stabilize the disturbed intestinal barrier homeostasis and be beneficial in reducing the infection rate in primary clinical trials (28–31). Due to the immunosuppressive and inflammatory nature of the disease, immunonutrients like glutamine and omega-3 fatty acids (ω-3 FAs) have been added to parenteral or enteral formulas to modulate immune functions, suppress the hyper inflammatory responses, and reestablish tissue and organ homeostasis in clinical practice (21, 32, 33). Supplements with antioxidative properties like glutamine and vitamin C have also been suggested to provide additional beneficial effects (34).

The review aims to provide a comprehensive chronological review on latest clinical trials on EN versus PN strategies and nutritional supplements including probiotics (prebiotics and synbiotics), glutamine, ω-3 FAs, and vitamins, hoping to provide the basis for future development of nutritional strategies in clinical AP.

## EN VERSUS PN

Traditionally, AP patients were maintained on *nil per os* or *nothing per mouth* treatment until resolution of pain or normalization of pancreatic enzymes to allow the pancreas to rest (35). Currently, it is widely accepted that early EN may be critical to improve AP-associated malnutrition and the overall outcomes, as bowel rest is associated with intestinal mucosal atrophy and increased infectious complications (9). Gut barrier dysfunction is found in approximately 60% of patients with AP (8, 36). Importantly, EN exerts immunomodulatory effects to preserve gut mucosa integrity, stimulate intestinal motility, and reduce bacterial overgrowth (8, 37). A randomized clinical study demonstrated that immediate oral feeding in patients with mild AP was feasible and safe and accelerated recovery without adverse gastrointestinal events (38). Another randomized controlled trial supported early-stage introduction of initial oral nutrition with either a clear liquid diet or a low-fat solid diet for patients who developed mild AP (39). In these patients, if oral intake is not tolerated, enteral feeding is recommended (9). In patients with severe AP or predicted severe AP, EN with oral or tube feeding thought to preserve the gut barrier function to prevent bacterial translocation is preferred over PN. A multicenter randomized study in the *New England Journal of Medicine* demonstrated that early tube feeding and oral diet after 72 h are equivalent in reducing infection rates or death in AP patients at high risk for complications (40). A Cochrane meta-analysis of eight randomized controlled studies found that EN reduced mortality, systemic infections, and multiorgan failure among patients with AP as compared to PN (41). Another metaanalysis of 381 patients confirmed the benefit of EN versus PN support in patients with severe AP with lower mortality, fewer infectious complications, decreased organ failure and surgical intervention rate (42). Over the optimal route of EN, several trials have suggested the nasogastric route as an alternative to nasoduodenal or nasojejunal routes (43). Multiple randomized controlled trials involving 157 patients with predicted severe AP demonstrated that nasogastric feeding was safe and well tolerated compared with nasojejunal feeding (44). Given its demonstrated beneficial outcomes, it remains challenging to predict whether EN will be tolerated in patients with AP (8). However, as shown by multiple randomized trials that have associated total PN (TPN) with risks of infection and other complications (35), PN should still be minimized unless the enteral route is not available, not tolerated, or not meeting caloric requirements.

## NUTRITIONAL SUPPLEMENTS

## Probiotics, Prebiotics, and Synbiotics

Changes in intestinal motility and microbiome, immune response, and mucosal barrier function during AP lead to bacterial translocation and subsequent pancreatic necrosis infection, which is one of the principal causes of complications and death in severe AP patients (45). Potential roles of probiotics have been proposed for immunomodulatory and health-promoting benefits to restore the gut integrity, modulate immune responses against invading pathogens, and prevent proliferation of harmful bacteria beyond those of basic nutrition, which have been evaluated in a number of clinical trials (**Table 2**).

An early indication of beneficial effects of synbiotics on severe AP-associated endotoxemia came from a randomized, doubleblind clinical trial with 45 patients receiving either live or heatinactivated *Lactobacillus plantarum 299* with oat fiber supplement as early EN. The results suggested that supplementary combined pre- and probiotics was effective in reducing infected pancreatic necrosis and surgical interventions (46, 47). The findings were subsequently supported and extended by a larger study with 62 patients on the Synbiotic 2000 formulated early EN with four different types of prebiotics (inulin, beta-glucan, resistant starch, and pectin) and probiotics (four different *Lactobacilli* preparations). Patients receiving synbiotic therapy had reduced total incidence of SIRS and lower rates of organ failure, supporting that early EN with synbiotics may prevent organ dysfunctions in the late phase of severe AP (48). The effects of *L. plantarum* only enteral feeding were evaluated in 76 patients with AP. Overall, the patients with ecoimmunonutrition showed attenuated disease severity, improved intestinal permeability, and better clinical outcomes (49). Prebiotic fiber alone supplementation with EN assessed in a randomized, double-blind study with 30 consecutive severe AP patients was found to shorten hospital stay, duration of nutrition therapy, and reduce the acute phase response and overall complications compared to standard EN therapy (50). Probiotic prophylaxis in severe AP has been contraindicated. The Dutch Acute Pancreatitis Study Group reported in PROPATRIA, a multicenter, randomized, double-blind, placebo-controlled trial with in a total of 200 patients with predicted severe AP that multispecies probiotic (Ecologic 641: six probiotic strains) prophylaxis did not reduce the risk of infectious complications and was associated with an increased risk of mortality (55, 56), although overall this combination of probiotic strains reduced bacterial translocation (52). Following studies involving multispecies probiotic supplementation with EN early abandoned after the publication of PROPATRIA study seemed to support the results that no significant trend was identified for an effect of probiotics on gut permeability or endotoxemia in AP (53, 57), although a positive effect was observed with reduced endotoxin levels (57).

#### TABLE 2 | Characteristics of clinical trials on probiotic treatment in AP.

*AP, acute pancreatitis; EN, enteral nutrition; MODS, multiple organ dysfunction syndrome; PN, parenteral nutrition; SIRS, systemic inflammatory response syndrome; TPN, total parenteral nutrition.*

Recently, a local study of 70 patients with severe AP comparing PN, EN and EN with addition of the probiotic *Bifidobacterium* found that early EN with *Bifidobacterium* resulted in lower levels of pro-inflammatory cytokines, improved gastrointestinal function, reduced complications, and shorter hospital stay in patients with severe AP (54). These data suggest the potential of single specific probiotic strains supplemented, which however should be further evaluated by validated clinical trials before their beneficial effects could be confirmed.

## Glutamine

Glutamine is an important constituent of intra and extracellular amino acid pool, with immune modulatory and antioxidant effects, and its depletion has been demonstrated in critical illness (58). Glutamine improves immune cell functions and contributes to antioxidative defenses. It can also support the intestinal integrity and decrease bacterial translocation; hence reduce systemic inflammatory responses and sepsis, which are important in critical illnesses such as AP (33).

An early randomized, controlled study with 28 AP patients received either a standard TPN or an isonitrogen, isocaloric TPN containing 0.3 g/kg l-alanine-l-glutamine demonstrated that glutamine supplementation with TPN was associated with a significant increase of cholinesterase, albumin, and lymphocyte count in AP as well a decrease of C-reactive protein compared to standard TPN. AP patients receiving glutamine was associated with a reduced length of TPN and a trend of reduced length of hospital stay, suggesting that glutamine substitution in TPN is beneficial in patients with AP (59). The effects of glutamine enriched (0.3 g/kg/day) TPN when further evaluated in 40 patients with AP. Beneficial effects of glutamine supplementation to TPN were found on acute pancreatic responses with serum lipase, amylase activities, and C-reactive protein levels decreased and the prevention of complications in patients with AP (59). Later, the effect of parenteral glutamine on recovery from severe AP was more thoroughly investigated in a randomized trial with 44 patients. l-alanyl-l-glutamine-supplemented PN increased serum IL-10 levels, improved nitrogen balance, and decreased infectious morbidity in patients with severe AP (60). Enterally, supplementation of glutamine and arginine in patients diagnosed of AP and predicted to develop a severe course was found to improve gut barrier function by reducing the gut permeability and decreasing plasma endotoxin level in the early stage of severe AP (61). Other than glutamine supplemented with TPN and EN, intravenously administered glutamine with early nasojejunal nutrition was also evaluated. In a randomized study, 45 patients with severe AP received glutamine or normal amino acid solution together with nasojejunal nutrition. The results demonstrated that the glutamine-receiving group showed signs of improvement in all end-point measurements including the rate of pancreas-specific infectious complications, organ failure, length of hospital stay, and mortality rate; and statistical significant difference was noted only in the length of hospital stay (62). Furthermore, a randomized trial compared early versus late intravenous infusion of alanylglutamine dipeptide in 76 patients with severe AP and demonstrated that early-stage intervention achieved a better clinical outcome: shortened duration of hospitalization, reduced rate of infection, organ dysfunction, need for surgery, and mortality, compared to the late treatment (63). More recently, glutamine supplemented in combination with normal saline and hydroxyethyl starch in resuscitation fluids were more efficient in relieving inflammation and sustaining the intestinal barrier in patients with severe AP (64). Two recent meta-analysis studies of randomized controlled trials demonstrated that glutamine supplementation resulted in significantly reduced mortality and complications (65, 66). Further analysis suggested a clear advantage for glutamine supplementation in patients who received TPN. In contrast, patients with AP who received EN did not require glutamine supplementation (65). Finally, oral glutamine supplementation did not seem to confer any significant effect on gut permeability and endotoxemia in severe AP (67). Characteristics of clinical studies on glutamine supplementation included in this review have been summarized in **Table 3**. Together, while glutamine supplementation with TPN shows promising clinical outcomes, enteral glutamine supplementation needs to be investigated in future.

## Omega-3 Fatty Acids

Dietary polyunsaturated fatty acids have known immunomodulatory and other beneficial health-promoting effects. A prospective cohort study on the association of fish consumption and non-gallstone-related AP has suggested that total fish (fatty fish and lean fish combined) consumption may be associated with decreased risk of non-gallstone-related AP (68). A randomized prospective clinical trial assessing enteral formula enriched with ω-3 FAs in the treatment of AP suggested that EN supplemented with ω-3 FAs seemed to have clinical benefits based upon the shortened time of jejunal feeding and hospital stay (69). Subsequently, independent studies evaluated the effects of PN with ω-3 FA supplementation on severe AP. Wang et al. compared in a randomized, double-blind trial a total of 40 severe AP patients receiving PN with the same basal nutrients but different lipid compositions: soybean oil-/fish oil-based fat solutions. The study showed that patients with ω-3 FAs-supplemented PN had increased eicosapentaenoic acid concentrations and decreased pro-inflammatory cytokines, together with improved respiratory function and shortened continuous renal replacement therapy time, suggesting attenuated systemic responses to pancreatic and organ injury (70). A parallel study by the same group enrolling 56 patients who received isocaloric and isonitrogenous PN with fats of all ω-6 FAs or 4:1 ω-6:ω-3 FAs demonstrated that ω-3 FAs-supplemented PN elevated the IL-10 level and human

*AD, the interval between admittance to ICU and start of enteral or parenteral nutrition; AP, acute pancreatitis; APACHE, acute physiology and chronic health evaluation; Cont., control; DOS, duration of hospital stay; EN, enteral nutrition; Interv., intervention; PN, parenteral nutrition.*

leukocyte antigen-DR expression in severe AP patients (71). In accordance, during the initial stage of severe AP, parenteral supplementation with ω-3 fish oil emulsion was found to suppress SIRS, modulate the balance of pro-/anti-inflammatory cytokines and thus improve AP-associated severe conditions (72). Clinical studies on ω-3 FA supplementation have been summarized in **Table 4**. Although polyunsaturated FAs remain potential beneficial supplements with EN/PN, further larger trials are needed for formulations and confirmatory beneficial clinical effects.

## Vitamins

Oxidative stress is involved in the onset of AP and also in the development of the systemic inflammatory responses, being glutathione depletion, xanthine oxidase activation, and thiol oxidation in proteins critical features of the disease in the pancreas. Vitamins as important immunonutrients and antioxidants have been inversely associated with AP (73). Plasma concentrations of vitamin A and vitamin C were found significantly lower in AP patients than in healthy controls (*P* < 0.05) (74). Recently, vitamin D, mainly from the milk products, has been inversely associated with gallstone-related AP (73). Vitamin supplementation assessed in combination with other antioxidants or in vitamin-only therapy has been evaluated earlier and yielded mixed outcomes. A multicenter randomized, double-blind, placebo clinical trial by Siriwardena et al. concluded that use of intravenous combination antioxidant therapy containing vitamin C (*N*-acetylcysteine, selenium, vitamin C) was not justified to continue in clinical severe AP (75). Subsequently, another group comparing vitamin C, *N*-acetylcysteine, antoxyl forte antioxidant combination with standard medical treatment in early AP patients suggested that antioxidant supplementation could decrease the length of hospital stay and complications in patients with early AP, but this hypothesis needed to be supported by a larger clinical trial (76). With respect of vitamin-only antioxidant therapies, a study involving 84 AP patients and 40 healthy subjects in China on high-dose vitamin C has demonstrated that it has therapeutic

*AD, the interval between admittance to ICU and start of enteral or parenteral nutrition; AP, acute pancreatitis; APACHE, acute physiology and chronic health evaluation; Cont., control; DOS, duration of hospital stay; EN, enteral nutrition; Interv., intervention; PN, parenteral nutrition;* ω*-3 FAs, omega-3 fatty acids.*

TABLE 5 | Characteristics of clinical trials on vitamins as the nutritional supplements in AP.

*AP, acute pancreatitis; APACHE, acute physiology and chronic health evaluation; Cont., control; DOS, duration of hospital stay; EN, enteral nutrition; Interv., intervention; PN, parenteral nutrition.*

efficacy on the disease and proposed the potential mechanisms to be promoting anti-oxidizing capability in patients, blocking lipid peroxidation and improving cellular immune function (77). In contrast, multiple vitamins-based antioxidant therapy (vitamin A, vitamin C, and vitamin E) in a single-center randomized study involving 39 patients has not been proven beneficial in patients with established severe AP (78). Collectively, data so far on vitamin therapy in AP (**Table 5**) have been mixed and should be carefully evaluated for dosing and timing of intervention for potential promising outcomes in clinical use.

## CONCLUSION AND FUTURE PERSPECTIVES

In most patients, an oral soft or solid diet can be beneficial if tolerated. When oral feeding is not tolerated for a few days, enteral feeding through a nasogastric or nasojejunal feeding tube should be attempted within the first 72 h of administration. PN should be minimized for its risks of infection and other complications. Only if enteral route is not available or tolerated, PN may be considered. Overall, nutritional support plays a critical role in clinical management of severe AP, although the optimal timing remains unclear. Predicting the nutritional tolerance of patients with AP remains challenging as the current evaluation system needs to be improved. Various nutritional supplement(s) together with PN or EN with currently mixed clinical outcomes is a subject of interest for future evaluation and may lead to promising outcomes. In addition, given its heterogeneous etiological factors and varying clinical manifestations, precision medicine, although not much applied in the condition, remains as a temping approach to

## REFERENCES

optimize clinical outcomes on classified individuals based on susceptibility to the condition and its systemic complications.

## AUTHOR CONTRIBUTIONS

JS designed the subject content of the review article. L-LP, JL, MS, and JS conducted initial search of literature, drafted the manuscript, and prepared the figures and tables. MB gave the constructive comments and critically reviewed the manuscript. JS had primary responsibility for final content. All authors read and approved the final manuscript.

## ACKNOWLEDGMENT

The authors would like to thank Prof. Yi Miao from Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, for critical reading of the manuscript and useful suggestions.

## FUNDING

The work was supported by funds from the National Natural Science Foundation of China (Grant nos: 91642114, 31570915, 81573420, and 31400779), Key Program of Fundamental Research Funds for the Central Universities (Grant no: JUSRP51613A), free exploration funding from State Key Laboratory of Food Science and Technology (SKLF-ZZB-201702), "Zhuo Xue" Talent Plan of Fudan University, and Royal Society of New Zealand's "Catalyst: Leaders New Zealand-China Scientist Exchange Programme."

rate in patients with severe acute pancreatitis. *JPEN J Parenter Enteral Nutr* (2008) 32:403–11. doi:10.1177/0148607108319797

and infectious risk: a randomized controlled trial. *Inflammation* (2009) 32:304–9. doi:10.1007/s10753-009-9136-0

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Pan, Li, Shamoon, Bhatia and Sun. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Inulin-Type Fructans Modulates Pancreatic–Gut Innate Immune Responses and Gut Barrier Integrity during Experimental Acute Pancreatitis in a Chain Length-Dependent Manner

*Yue He1,2†, Chengfei Wu1,2†, Jiahong Li 1,2, Hongli Li1,2, Zhenghua Sun1,2, Hao Zhang1,2, Paul de Vos <sup>3</sup> , Li-Long Pan4 \* and Jia Sun1,2,4\**

#### *Edited by:*

*Willem Van Eden, Utrecht University, Netherlands*

> *Reviewed by: Brandt D. Pence, University of Memphis,*

*United States Mourad Aribi, University of Tlemcen, Algeria*

#### *\*Correspondence:*

*Li-Long Pan<EMAIL_ADDRESS>Jia Sun<EMAIL_ADDRESS>† Co-first authors.*

#### *Specialty section:*

*This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology*

*Received: 20 June 2017 Accepted: 12 September 2017 Published: 26 September 2017*

#### *Citation:*

*He Y, Wu C, Li J, Li H, Sun Z, Zhang H, de Vos P, Pan L-L and Sun J (2017) Inulin-Type Fructans Modulates Pancreatic–Gut Innate Immune Responses and Gut Barrier Integrity during Experimental Acute Pancreatitis in a Chain Length-Dependent Manner. Front. Immunol. 8:1209. doi: 10.3389/fimmu.2017.01209*

*1State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China, 2School of Food Science and Technology, Jiangnan University, Wuxi, China, 3Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands, 4School of Medicine, Jiangnan University, Wuxi, China*

Acute pancreatitis (AP) is a common abdominal inflammatory disorder and one of the leading causes of hospital admission for gastrointestinal disorders. No specific pharmacological or nutritional therapy is available but highly needed. Inulin-type fructans (ITFs) are capable of modifying gut immune and barrier homeostasis in a chemistry-dependent manner and hence potentially applicable for managing AP, but their efficacy in AP has not been demonstrated yet. The current study aimed to examine and compare modulatory effects of ITFs with different degrees of fermentability on pancreatic–gut immunity and barrier function during experimentally induced AP in mice. BALB/c mice were fed short (I)- or long (IV)-chain ITFs supplemented diets for up to 3 days before AP induction by caerulein. Attenuating effects on AP development were stronger with ITF IV than with ITF I. We found that long-chain ITF IV attenuated the severity of AP, as evidenced by reduced serum amylase levels, lipase levels, pancreatic myeloperoxidase activity, pancreatic edema, and histological examination demonstrating reduced pancreatic damage. Shortchain ITF I demonstrated only partial protective effects. Both ITF IV and ITF I modulated AP-associated systemic cytokine levels. ITF IV but not ITF I restored AP-associated intestinal barrier dysfunction by upregulating colonic tight junction modulatory proteins, antimicrobial peptides, and improved general colonic histology. Additionally, differential modulatory effects of ITF IV and ITF I were observed on pancreatic and gut immunity: ITF IV supplementation prevented innate immune cell infiltration in the pancreas and colon and tissue cytokine production. Similar effects were only observed in the gut with ITF I and not in the pancreas. Lastly, ITF IV but not ITF I downregulated AP-triggered upregulation of IL-1 receptor-associated kinase 4 (IRAK-4) and phosphor-c-Jun N-terminal kinase (p-JNK), and a net decrease of phosphor-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 (p-NF-κB p65) nuclear translocation and activation in the pancreas. Our findings demonstrate a clear chain length-dependent effect of inulin on AP. The attenuating effects are caused by modulating effects of long-chain inulin on the pancreatic–gut immunity via the pancreatic IRAK-4/p-JNK/p-NF-κBp65 signaling pathway and on prevention of disruption of the gut barrier.

Keywords: dietary fibers, inflammation, pancreatic–intestinal immunity, signaling kinases, tight junction proteins, antimicrobial peptides

## INTRODUCTION

Acute pancreatitis (AP) is a sudden inflammation of the pancreas caused by inappropriate activation of local digestive enzymes. During the past decades, it has become the leading cause of hospital admission for gastrointestinal disorders in many countries and the worldwide incidence is increasing (1). Although the mild form of AP is self-limited, nearly 20–25% of patients with AP develop severe symptoms with systemic inflammatory responses. The mortality rate in these patients is as high as 30% (2). Up to now, a targeted pharmacological or nutraceutical therapy specific for AP management is lacking (3, 4). Novel insight however in function–effector relationship of dietary components and their anti-inflammatory effects has generated optimism about the possibility to create nutraceutical strategies to control local inflammation and prevent systemic complications of AP (2).

Pathophysiological studies of AP have revealed that AP is caused by unregulated intra-acinar activation of trypsin and other digestive enzymes, leading to autodigestion of the pancreas and local inflammation. The main triggering factors include pancreatic hyperstimulation, gallstones, and alcohol abuse (5). The induced local inflammation is accompanied by activation of endothelial cells and transendothelial migration of leukocytes, neutrophils, and macrophages, leading to release of harmful enzymes and cytokines that amplify the local inflammatory responses. As a consequence, pancreatic complications such as acinar cell necrosis, pseudocyst formation, and abscess development might occur in most severe cases, leading to transmission of inflammation to other remote organs, which is ultimately responsible for AP-associated mortality.

Innate immune activation and acinar cell inflammatory signaling play a pivotal role in the pathogenesis of AP (5–8). Local inflammation is initiated by acinar cell damage and local production of pro-inflammatory cytokines/chemokines by these cells, followed by infiltration of neutrophils and macrophages (9). Neutrophils, macrophages, and dendritic cells with distinct cell-surface and intracellular markers have been associated with the development of AP and severity of the inflammatory conditions (10–13). The balance between pro-inflammatory and anti-inflammatory mediators produced from acini and infiltrated immune cells determines the outcome of the AP. Tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-10 are important cytokines in AP (11, 14–16). TNF-α is released by local Ly6Chi monocytes/macrophages in the pancreas and enhances the severity of the experimental disease (11, 17, 18). Regulatory cytokines, such as IL-10, limit the local and systemic consequences of experimental pancreatitis (18). Expression of these cytokines is under the control of the transcription factor NF-κB. NF-κB activation in pancreatic acinar cells is responsible for the expression of a large number of inflammation-related genes (19). NF-κB activation is regulated by its upstream signaling kinases, such as MAP kinases (MAPKs), which may be activated by receptor-associated kinases, such as IL-1 receptor-associated kinase 4 (IRAK-4) and transforming growth factor-β activated kinase (TAK) (20, 21). Consequently, modulation of immune cell activation, inflammatory cytokine production, and pancreatic inflammatory signaling molecules may be rational approaches to alleviate AP symptoms.

Accumulating evidence is available demonstrating that severe AP is associated with changes in the microcirculation, gut permeability/motility, bacterial translocation, and activation of the gut-associated lymphoid tissue. Preventing AP-associated intestinal barrier disruption or inflammation might therefore be a key target for effective therapy (22). A possible nutraceutical approach to prevent intestinal barrier disruption is by intervening with anti-inflammatory food components such as specific dietary fibers. Dietary fibers may be fermented by the gut microbiota to produce health-promoting short-chain fatty acids (SCFAs) and modify intestinal barrier function (23). Therefore, consumption of dietary fibers represents a promising strategy to modulate the progression of AP. We have recently demonstrated that lowmethoxyl lemon pectin (LMP) attenuated inflammatory responses and improved intestinal barrier integrity in experimental AP (2). Another family of nutritional molecules with supporting effect on barrier function and anti-inflammatory effect that might be instrumental for management of AP is inulin-type fructans (ITFs) (23–27). ITFs are a family of dietary fibers belonging to beta(2→1) linear fructan-type carbohydrate subgroup and have an impact on gastrointestinal functions, which is largely related to their biochemical and physiological attributes. In the gut, ITFs are rapidly fermented to produce SCFAs that exert some of the local and systemic effects of ITFs. In addition, ITFs have demonstrated clear chemistry or degree of polymerization (DP) dependent effects (28). Previous studies have demonstrated that DP or chain length of ITFs that affects the magnitude of receptor activation and/or their site of fermentation in the gut determines their prebiotic and immune modulatory effects on immune cells, gut barrier function, and microbiota composition (23–27, 29). ITFs have been implicated in a variety of inflammatory and

**Abbreviations:** AP, acute pancreatitis; AMPs, antimicrobial peptides; ANOVA, analysis of variance; CRAMP, cathelicidin-related antimicrobial peptide; DEFB1, β-defensin-1; DP, degree of polymerization; ERK, extracellular signal-regulated kinase; H&E, hematoxylin and eosin; IL, interleukin; IRAK, IL-1 receptor associated kinase; ITF, inulin-type fructan; JNK, c-Jun N-terminal kinase; LMP, low-methoxyl pectins; MAPKs, mitogen-activated protein kinases; MPO, myeloperoxidase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; RT-qPCR, real-time quantitative polymerase chain reaction; SCFAs, short-chain fatty acids; TAK, TGF-β activated kinase; TGF-β, transforming growth factor-β; TJ, tight junction; TNF-α, tumor necrosis factor-α; ZO-1, zonula occludens-1.

immunological dysfunctions. However, their potential effects have not been studied in the management of AP.

In this study, we examined and compared the effects of longchain ITF IV and the more readily fermentable short-chain ITF I on their protective effects during experimentally induced AP. In a caerulein-induced AP mice model, we studied after ITF treatment severity of pancreatitis, the frequencies of infiltrating neutrophils, macrophages, and dendritic cells in the pancreas and colon as well as production of the cytokines TNF-α, IL-1β, and IL-10. Additionally, we studied colon integrity by determining expression of gut–epithelial tight junction (TJ) modulatory proteins and barrier reinforcing immunomodulatory antimicrobial peptides (AMPs). Finally, we investigated the signaling pathways and kinases modulated by ITFs to gain insight in the mechanisms by which ITFs modulate AP.

## MATERIALS AND METHODS

## Fibers and Structural Characterization

The applied ITF I (frutalose OFP, 2 < DP < 25) and ITF IV (FrutafitTEX, 10 < DP < 60) were extracted from chicory roots (Sensus B.V., Roosendaal, The Netherlands). Their specific chain length profiles (range and distribution) were characterized by high-performance anion exchange chromatography as previously described (23, 24).

## Animals

All animal-related experimental protocols were approved by the Institutional Animal Ethics Committee of Jiangnan University in compliance with the recommendations of national and international guidelines for the Care and Use of Laboratory Animals, and were performed in accordance with the guidelines therein. Eightweek-old female BALB/c mice, 20 ± 2 g (Su Pu Si Biotechnology, Suzhou, Jiangsu, China) reared on *ad-libitum* access to standard laboratory chow and water were used in this study. The animals were maintained at *Animal Housing Unit* of the University under controlled temperature (23–25°C), pathogen-free conditions, and at a 12:12 h light:dark cycle.

## Experimental Design and AP Induction

One-week time was allowed for the acclimatization of animals before starting the experiment(s). Mice were randomly divided into four groups (*n* = 4–6) according to different diets and fed for 72 h (**Table 1**).

After 12 h of fasting, AP was induced in mice by eight repeated intraperitoneal (i.p.) injections of caerulein (50 µg/kg/h)

*AP, acute pancreatitis; ITF IV, inulin-type fructan IV (long-chain inulins); ITF I, inulin-type fructan I (short-chain inulins).*

(Sigma-Aldrich, St. Louis, MO, USA). The littermates in control group were injected (i.p.) with the same volume of normal saline and served as controls.

## Tissues Sampling

Mice were euthanized and sacrificed with a lethal dose of pentobarbitone sodium (100 mg/kg) 1 h after the last caerulein injection. For serum analysis, blood samples were centrifuged at 3,000 × *g* for 15 min, after which serum was collected and stored at –80°C. Tissues, including pancreas and colon, were excised, fixed in 4% paraformaldehyde or snap freeze in liquid nitrogen and stored at –80°C for later analysis.

## Serum Amylase and Lipase Measurements

Serum amylase activities were measured by a serum assay kit (Jian Cheng Bioengineering Institute, Nanjing, China) (30). Lipase activities were measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA).

 Pancreatic water contents were evaluated by the ratio of initial weight (wet weight) of the pancreas to its weight after drying at 80°C for 48 h (dry weight). MPO activity measurements were determined by an MPO assay kit (Jian Cheng Bioengineering Institute, Nanjing, China).

## Histological Evaluation

Fresh pancreatic and colonic tissues were fixed in 4% paraformaldehyde overnight, washed with ddH2O, and rehydrated with ethanol and embedded in paraffin. The Skiving machine Slicer PM2245 (Leica, Wetzlar, Germany) diced 5-µm sections were stained with hematoxylin and eosin (H&E). For pancreatic injury evaluation, a DM2000 light microscope (Leica, Wetzlar, Germany) was used at ×40 magnification. The examination was carried out based on both infiltrating inflammatory cells and other morphological changes in tissues which are considered to be markers of inflammation/tissue damage.

## ELISA Analysis

The levels of cytokines (TNF-α, IL-1β, and IL-10) in serum were measured by ELISA kits (R&D Systems, Minneapolis, MN, USA) following the standard procedure of the manufacturer. To measure the cytokines level of pancreatic and colonic tissues (TNF-α, IL-1β, and IL-10), the tissues were homogenized in a saline solution (1:19, w/v) using a Polytron homogenizer (Scientz-48, Ningbo, Zhejiang, China) at 55 Hz for 1 min. Samples were centrifuged at 4°C, 10,000 × *g* for 10 min. Finally, the supernatant was collected for ELISA analysis.

## RNA Isolation and Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis

Transcription of mRNA of occludin, zonula occludens protein-1 (ZO-1), β-defensin-1 (DEFB1), and cathelicidin-related antimicrobial peptide (CRAMP) was analyzed by RT-qPCR. Total RNA was isolated from pancreas and colonic tissues using TRIzol (Life Technologies, MA, USA) and was subjected to reverse transcription using Prime-Script RT reagent kit (TaKaRa Bio, Japan) following the manufacturer's instructions. SYBR® Green RT-qPCR was performed using real-time PCR system (BIO RAD CFX Connect, CA, USA). The relative mRNA levels were normalized to mRNA levels of β-actin (housekeeping control) and calculations for fold change of each mRNA were made on comparative cycle threshold method (2−ΔΔCt). The primers used in this study are provided in **Table 2**.

## Western Blot Analysis

Pancreatic and colonic tissues were homogenized in ice-cold lysis buffer RIPA (containing cocktail protease inhibitors; Beyotime, Shanghai, China). Samples were centrifuged at 4°C, 10,000 × *g* for 10 min and equal amounts of protein (30 µg), as determined using standard bicinchoninic acid assay (BCA) method by BCA Protein Assay Kit (Beyotime, Shanghai, China). Samples were electrophoresed on blots 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride membranes. Membranes were blocked with blocking buffer for 1.5 h at room temperature, washed with TBS-Tween 20 (TBST), and finally incubated overnight at 4°C with anti-extracellular signal-regulated kinase (ERK)/phospho-ERK, JNK/phospho-JNK, p38/phospho-p38, IRAK-4, phospho-p65, and GAPDH (housekeeping) antibodies. Incubation with fluorescently labeled secondary horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000) was performed for 2 h at room temperature and immunoreactivity was analyzed by Western Lightening Plus enhanced chemiluminescence (PerkinElmer, MA, USA) according to the manufacturer's instructions.

## Flow Cytometry Analysis

Freshly harvested pancreatic and colonic tissues were digested in 1.0 mg/mL collagenase-P (Boehringer, Mannheim, Germany) solution at 37°C for 15 min and filtered through 75 µm filters with hank's solution (Beyotime, Shanghai, China). Single-cell suspensions were incubated for 30 min at 4°C in hank's solution with the following mAbs: APC Rat Anti-Mouse CD11b, BV421 Rat Anti-Mouse F4/80, Alexa Fluor 700 Rat Anti-Mouse Ly-6G, PE Hamster Anti-Mouse CD11c, FITC Rat Anti-Mouse MHCII, and PerCP-Cy5.5 Rat Anti-Mouse CD8a (BD Pharmingen, CA, USA). Gating method of fluorescence-activated cell sorting was programmed as CD11b<sup>+</sup> Ly-6G<sup>+</sup> (for neutrophils), CD11b<sup>+</sup> F4/80<sup>+</sup> (for macrophages), CD11c<sup>+</sup> MHCII<sup>+</sup> (for conventional dendritic

TABLE 2 | List of primers used for RT-qPCR.

*DEFB1,* β*-defensin-1; CRAMP, cathelicidin-related antimicrobial peptide; RT-qPCR, real-time quantitative polymerase chain reaction.*

cells, cDCs), and CD8a<sup>+</sup>CD11c<sup>+</sup> MHCII<sup>+</sup> (for plasmacytoid dendritic cells, pDCs). Flow cytometer was performed on Attune NxT (Thermo Fisher Scientific, MA, USA). Data were analyzed using ACEA NovoExpress software (Novo Express International, Inc., South San Francisco, CA, USA).

## Statistical Analysis

Data are expressed as means ± SEM. The parametric distribution of the results was confirmed using Kolmogorov–Smirnov test. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's *post hoc* test using GraphPad Prism (version 5; GraphPad Software Inc., San Francisco, CA, USA). The values of *P* < 0.05 were considered to indicate a statistically significant difference.

## RESULTS

## ITF IV Supplementation of the Diet Alleviates Severity of Caerulein-Induced AP

Modulatory effects of ITFs were examined in caerulein hyperstimulated mice by supplementing the diets with either ITF I or ITF IV for 3 days before AP induction. While dietary supplementation with 5% ITF I or ITF IV alone without AP induction did not show any effect on pancreatic markers (Figure S1 in Supplementary Material), protective effects of ITF supplementation on AP were observed and the effects were highly ITF chain length dependent. ITF IV had more attenuating effects than ITF I. ITF IV but not ITF I attenuated caerulein-induced increases in serum amylase, serum lipase, pancreatic MPO activities, and edema (**Figures 1A–D**). However, both ITF I and ITF IV modulated serum cytokines. Both types of ITFs reduced serum proinflammatory IL-1β levels and increased the regulatory cytokine IL-10 (**Figures 1E,F**). Histological examination of pancreatic sections confirmed an overall better attenuating AP effect of ITF IV as evidenced by generally improved cellular morphology, restored interlobular space expansion, reduced inflammatory infiltrates, and acini necrosis (**Figure 1G**). Taken together, these data indicate that ITF-IV-supplemented diets alleviate the severity of AP in mice, while ITF I diet has a more minor beneficial effect.

## ITF IV but Not ITF I Supplementation Strengthening Intestinal Barrier Function by Upregulating TJ Proteins and AMPs

Compromised intestinal barrier integrity and intestinal injury, marked by dysregulated expression of TJ modulatory proteins and barrier reinforcing AMPs, develop as AP progresses (22, 31). Therefore, maintaining intestinal barrier function might be key in the ITF-induced attenuating effects on AP. To determine such an effect of ITFs, we measured in ITF-treated animals, the mRNA expression of major structural proteins such as TJ, occludin and ZO-1, as well as AMPs DEFB1 and CRAMP. All these molecules are involved in the physical and chemical barriers of the mucosa (32, 33). The RT-qPCR analysis

IL, interleukin; ITF, inulin-type fructans; MPO, myeloperoxidase.

showed that ITF IV but not ITF I supplementation prevented caerulein-induced downregulation of occludin and ZO-1 as well as the downregulation of DEFB1 and CRAMP (**Figures 2A–D**). Furthermore, histological examination of colon sections revealed that intestinal mucosal epithelia widened and villous apical epithelium peeled off in mice with AP as compared with control mice. This colonic damage was less severe in mice fed on ITF IV supplemented diet (**Figure 2E**). Together, these findings suggest that ITF IV but not ITF I supplementation prevents AP-associated gut integrity damage and injury.

## ITF IV Supplementation Reducing Innate Immune Cell Infiltration into the Pancreas and Pancreatic Cytokine Production during AP

Both infiltration of inflammatory immune cells and an imbalance between pro-inflammatory and anti-inflammatory cytokines are key immunological pathophysiological events in AP development (34–36). For this reason, we determined the frequencies of neutrophils (CD11b<sup>+</sup>Ly-6G<sup>+</sup>), macrophages (CD11b<sup>+</sup>F4/80<sup>+</sup>), cDCs (CD11c<sup>+</sup>MHCII<sup>+</sup>), and pDCs (CD8a<sup>+</sup> CD11c<sup>+</sup>MHCII<sup>+</sup>) that infiltrated into the pancreas of AP mice fed with ITFs. ITF IV supplementation profoundly inhibited the infiltration of neutrophils and macrophages (Figure S2 in Supplementary Material). The percentage of CD8a<sup>+</sup> DCs or pDCs but not cDCs was lowered with AP but normal in mice fed with ITF IV. ITF I did not have such an effect (Figure S2 in Supplementary Material). The percentages of neutrophil and macrophage in the pancreas robustly increased with AP induction and were significantly less in mice fed with ITF IV (**Figures 3A–C**) but this was still increased in mice fed with ITF I. Additionally, pancreatic cytokine production remained enhanced in ITFI but not in animals fed with ITF IV (**Figure 3D**). Collectively, these data demonstrate the immune attenuating effects of ITF IV.

## Both ITF IV and ITF I Supplementation Modulating AP-Induced Innate Immune Cell Infiltration in the Gut and Cytokine Production

Immune compromised gut environment as a result of disrupted barrier function and intestinal injury is associated with major inflammatory cell infiltration and imbalanced inflammatory cytokine production. Therefore, we also investigated infiltration of innate immune cells in the gut during AP. AP induction resulted in robust increases in neutrophils and macrophages in the colon. Both ITF I and ITF IV supplementation reduced the number of infiltrating neutrophils and macrophages (**Figures 4A–C**). The percentage of CD8a<sup>+</sup> DCs or pDCs in the colon lowered with AP, but was prevented by ITF IV and not by ITF I (Figure S3 in Supplementary Material). Furthermore, consistent with reduced inflammatory cell infiltration in the gut, both ITFs prevented AP-induced increases in pro-inflammatory TNF-α and IL-1β levels, and decreases in IL-10 level in the colon as compared with untreated AP control (**Figure 4D**). Taken together, these data suggest positive immunomodulatory effects of both ITFs on AP-associated gut inflammation.

## ITF IV but Not ITF I Supplementation Modulating Pancreatic IRAK-4/JNK/NF-**κ**B Activation in AP

Lastly, we investigated the signaling molecular pathways underlying differential effects by ITF I and ITF IV supplementation. Activation of NF-κB and its upstream signaling kinases (IRAK, TAK1, and MAPKs) were determined by Western blot. It was found that ITF IV significantly modulated AP-induced IRAK-4, JNK, and NF-κB activation, while p-TAK, p-ERK, and p-p38 remained unaffected by ITF (**Figures 5A–D**). In comparison, ITF I attenuated IRAK-4 and p-JNK activation associated with AP, but did not affect NF-κB nuclear translocation and activation (**Figures 5A–D**).

## DISCUSSION

The current study examines and compares effects of different types of ITFs in experimental AP and associated intestinal immune and barrier dysregulation. The effects were ITF type dependent and more pronounced with ITF IV than with ITF I. We observed that dietary long-chain inulin (ITF IV) supplementation mitigated the severity of AP: it suppressed characteristic inflammatory cell infiltration, modulated inflammatory cytokine production in the pancreatic–gut region, and prevented intestinal barrier function integrity. Its attenuating effect was associated with suppression of AP-induced IRAK-4/p-JNK/NF-κB pathway activation. ITF I only demonstrated significant modulatory effects in colonic immune responses and serum inflammatory markers, suggesting that short-chain inulin effects are limited to the gut (**Figure 6**).

Inulin-type fructans have been tested in a variety of inflammatory and immunological disease models and it has been shown that administration of ITF can exert immunomodulatory actions and alleviate acute inflammation (23, 28, 37). To the best of our knowledge, this is the first study in which efficacy of ITF is shown in AP. It has been earlier suggested, however, that dietary fibers are inversely related with pancreatic enzyme activity and can inhibit pancreatic digestive enzyme activities *in vivo* (38). As premature activation of pancreatic zymogens is a triggering event of human AP and inhibited by ITF, this study supports the potential application of ITF in preventing AP.

A particular intriguing observation of this study was that long-chain inulins have a better local pancreatic and gut protective effect in AP than short-chain inulins. Previously, differential prebiotic and immunomodulatory effects have been demonstrated by ITFs of varying chain length. Prolonged fermentation of long-chain inulin (DP > 10) compared with short-chain inulin (DP < 10) ensures more endurable and profound prebiotic and immunomodulatory effects, locally in the colon and systemically in remote organs. Another ITF chain lengthdependent mechanism which might explain the difference is the magnitude by which ITFs can activate toll-like receptor 2, which is higher with long-chain than with short-chain ITFs (24). In addition, the preferential sites of fermentation for these two ITFs are different: colon for long-chain and small intestine for short-chain inulins. This may imply a prolonged and different action of ITF IV as compared with ITF I and might explain the observation that ITF IV but not ITF I increased the colonic expression of the TJ proteins ZO-1 and occluding as well as AMP DEFB1 and CRAMP. Differential regulation of TJ proteins has been observed after dietary interventions earlier (39).

Innate immune activations and pancreatic acinar cell inflammatory signaling are characteristic for AP (7). Moreover, AP induction by caerulein hyperstimulation caused neutrophil and

macrophage infiltration. This infiltration is prevented by dietary ITF IV supplementation. Increased pancreatic Ly6Chi monocytes/ macrophages have been found to be positively correlated with the severity of AP and are dependent on TNF-α production by these cells (11). IL-10 production by M2 macrophages has been shown to impair neutrophil recruitment in inflammatory and infectious conditions (40, 41). Along with reduced infiltration of neutrophils and macrophages in ITF IV-fed mice with AP, the production of TNF-α and IL-1β was found to be suppressed and IL-10 levels increased in pancreatic and colonic tissues. While modulatory effects of ITF IV were observed on pancreatic, colonic, and systemic cytokine levels, those of ITF I was only significant in

FIGURE 5 | Effects of ITFs on AP-mediated activation of MAPK and NF-κB pathways in the pancreas. Mice were fed with 5% ITF I or ITF IV supplemented diets for 72 h before AP induction by caerulein. The activation/expression of pancreatic IRAK4 (A), p-TAK1 (B), p-ERK, p-JNK, p-p38 (C), and NF-κB p-p65 (D) were examined by Western blot. Data shown are means ± SEM*. \*\*P* < 0.01 and *\*\*\*P* < 0.001. AP, acute pancreatitis; ERK, extracellular signal-regulated kinase; IRAK, IL-1 receptor associated kinase; ITF, inulin-type fructans; JNK, c-Jun N-terminal kinase; MAPKs, mitogen-activated protein kinases; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TAK, TGF-β activated kinase.

fructans.

colonic and systemic cytokines. Balanced pro-inflammatory and anti-inflammatory cytokine production prevented an augmented inflammatory response from a positive inflammatory feedback circuit (42).

Pancreatic acinar inflammation with activation of MAPKs and of the transcription factor NF-κB is a key pathological event during the development of AP (43–46). AP induction or supraphysiological concentrations of caerulein are associated with MAPK activation in pancreatic acini (47). Overexpression of p65 protein trans-activated NF-κB aggravates acute pancreatic inflammatory responses and MAPKs regulate NF-κB activation by mediating phosphorylation of its inhibitory-κB IκB protein, to allow NF-κB translocation to the nucleus followed by upregulation of inflammatory genes (19, 47, 48). Here, we found that MAPKs JNK but not ERK1/2 or p38 activation was inhibited by ITF IV, concomitant with an attenuated NF-κB p65 nuclear translocation and activation. It might be speculated that different upstream MAPKs are regulated by dietary fibers. Our data suggest that ITF IV interferes with the development of AP, by reducing pancreatic activation of IRAK-4-JNK-NF-κB p65 signaling pathway and inhibiting the release of inflammatory mediators. It has been demonstrated that IRAK-4 has been demonstrated essential in many innate immune responses and can activate JNK pathway and downstream NF-κB (20, 21, 49). NF-κB translocation in acinar cells increases the severity of pancreatitis in mice, and leads to transcription of cytokines and other inflammatory mediators (19). Our study shows that ITF IV downregulated IRAK-4/p-JNK/NF-κB p65 in the pancreas during AP.

As AP progresses, the inflammatory cytokines produced in the pancreas, including TNF-α and IL-1β, reach the gut by the microcirculation. These cytokines will recruit more leukocytes and induce more inflammatory mediators that ultimately will contribute to intestinal barrier dysfunction and mucosal injury (22, 50, 51). Gut barrier dysfunction and worsened gut permeability in AP are accompanied by reduced expression of TJ reinforcing proteins and AMPs. Lack of physical and chemical barriers of the gut makes the host susceptible to bacterial translocation that contributes to a second golf of inflammatory event in the pancreas, causing excessive inflammation and development of multi-organ dysfunctions. As shown here, dietary ITF IV intervention, by limiting pancreatic–gut inflammation, restoring gut barrier function and integrity to prevent secondary excessive inflammatory responses, represents a promising approach to prevent the progress of AP. Although we have shown the modulatory effects of ITF IV on pancreatic–intestinal immunity and gut barrier function, the detailed mechanisms remain to be fully understood. Future investigation could identify specific receptor involvement and/or examine how their fermentation product SCFAs are regulated to exert protective effects during the condition. Moreover, the translational importance of these safe, value-added prebiotics for clinical AP merits further exploration.

Collectively, our data suggest that dietary ITF IV but not ITF I intake prevents the development of AP by three-graded actions: first, by preventing pancreatic inflammation and damage; second, by preventing AP-associated intestinal barrier dysfunction and intestinal inflammation; and last, by precluding progression of the gut and pancreatic inflammation into a systemic inflammatory response by the two aforementioned mechanisms. The current study provides evidence that nutritional application of long-chain inulins in clinical AP might be efficacious.

## ETHICS STATEMENT

All animal-related experimental protocols were approved by the Institutional Animal Ethics Committee of Jiangnan University in compliance with the recommendations of national and international guidelines for the Care and Use of

## REFERENCES

Laboratory Animals, and were performed in accordance with the guidelines therein.

## AUTHOR CONTRIBUTIONS

YH, CW, JL, and HL performed experiments and analyzed data. P-LL, ZS, HZ, and PDV provided intellectual inputs, contributed to the data acquisition, and critically reviewed the manuscript. JS and P-LL designed and interpreted experiments. JS, P-LL, YH, and PDV wrote the paper.

## FUNDING

The work was supported by funds from the National Natural Science Foundation of China (Grant nos: 91642114, 31570915, 81573420, and 31400779, National Youth 1,000 Talents Plan), Jiangsu Province Recruitment Plan for High-level, Innovative and Entrepreneurial Talents, Key Program of Fundamental Research Funds for the Central Universities (Grant no: JUSRP51613A), free exploration funding from State Key Laboratory of Food Science and Technology (SKLF-ZZB-201702), "Zhuo Xue" Talent Plan of Fudan University, and China Postdoctoral Science Foundation (Grant no: 2016M601719).

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2017.01209/ full#supplementary-material.

FIGURE S1 | Effects of dietary ITF supplementation on markers of pancreatic damage. Mice were fed with 5% ITF I or 5% ITF IV supplemented diets for 72 h. Serum amylase (A), pancreatic MPO activity (B), and pancreatic edema (C) were then determined as described in Section "Materials and Methods," respectively. Data shown are means ± SEM*.* ITF, inulin-type fructans; MPO, myeloperoxidase.

FIGURE S2 | Effects of ITF IV on CD8a+ DCs in the pancreas during AP: representative flow cytometry dot plots of CD11c+MHCII+ DCs (A), histogram (B), and percentage (C) of CD8a+ DCs on DC subsets in the pancreas were shown. Data shown are means ± SEM from four independent experiments with three to four pooled mice per group in each experiment. *\*\*P* < 0.01. AP, acute pancreatitis; DCs, dendritic cells; ITF, inulin-type fructans.

FIGURE S3 | Effects of ITF IV on CD8a+ DCs in the colon during AP: representative flow cytometer dot plots of CD11c+MHCII+ DCs (A), histogram (B), and percentage (C) of CD8a+ DCs on DC subsets in the colon were shown. Data shown are means ± SEM from four independent experiments with three to four pooled mice per group in each experiment. *\*P* < 0.05 and *\*\*P* < 0.01. AP, acute pancreatitis; DCs, dendritic cells; ITF, inulin-type fructans.

and definitions by international consensus. *Gut* (2013) 62:102–11. doi:10.1136/ gutjnl-2012-302779

secretion in the rat cecum. *J Agric Food Chem* (2011) 59:5771–8. doi:10.1021/ jf200859z

51. Machado MC, da Silva FP, Cunha DG, Barbeiro DF, Coelho AMM, Souza HP. Tu1915 intestinal barrier dysfunction in ageing animals with acute pancreatitis: increased intestinal inflammation? *Gastroenterology* (2015) 148:S–1190. doi:10.1016/S0016-5085(15)34066-X

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 He, Wu, Li, Li, Sun, Zhang, de Vos, Pan and Sun. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Heitor A. Paula Neto1 \*, Priscila Ausina2 , Lilian S. Gomez2 , João G. B. Leandro2 , Patricia Zancan3 and Mauro Sola-Penna2*

*<sup>1</sup> Laboratório de Alvos Moleculares, Faculdade de Farmácia, Departamento de Biotecnologia Farmacêutica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2 Laboratório de Enzimologia e Controle do Metabolismo, Faculdade de Farmácia, Departamento de Biotecnologia Farmacêutica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, <sup>3</sup> Laboratório de Oncobiologia Molecular, Faculdade de Farmácia, Departamento de Biotecnologia Farmacêutica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil*

### *Edited by:*

*Jia Sun, Jiangnan University, China*

#### *Reviewed by: Pinyi Lu,*

*Biotherapeutics, Inc., United States Xu Zhang, Scintillon Institute, United States*

*\*Correspondence: Heitor A. Paula Neto<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology*

*Received: 29 June 2017 Accepted: 20 October 2017 Published: 06 November 2017*

#### *Citation:*

*Paula Neto HA, Ausina P, Gomez LS, Leandro JGB, Zancan P and Sola-Penna M (2017) Effects of Food Additives on Immune Cells As Contributors to Body Weight Gain and Immune-Mediated Metabolic Dysregulation. Front. Immunol. 8:1478. doi: 10.3389/fimmu.2017.01478*

Food additives are compounds used in order to improve food palatability, texture, and shelf life. Despite a significant effort to assure safety of use, toxicological analysis of these substances, generally, rely on their direct toxicity to target organs (liver and kidney) or their genotoxic effects. Much less attention is paid to the effects of these compounds on cells of the immune system. This is of relevance given that metabolic dysregulation and obesity have a strong immune-mediated component. Obese individuals present a state of chronic low-grade inflammation that contributes to the establishment of insulin resistance and other metabolic abnormalities known as the metabolic syndrome. Obesity and metabolic syndrome are currently recognized as worldwide epidemics that pose a profound socioeconomic impact and represent a concern to public health. Cells of the immune system contribute to both the maintenance of "lean homeostasis" and the metabolic dysregulation observed in obese individuals. Although much attention has been drawn in the past decades to obesity and metabolic syndrome as a result of ingesting highly processed food containing large amounts of fat and simple sugars, mounting evidence suggest that food additives may also be important contributors to metabolic derangement. Herein, we review pieces of evidence from the literature showing that food additives have relevant effects on cells of the immune system that could contribute to immune-mediated metabolic dysregulation. Considering their potential to predispose individuals to develop obesity and metabolic syndrome, their use should be taken with caution or maybe revisited.

Keywords: food additives, type 2 diabetes, metabolic syndrome, metainflammation, microbiota, citrate, immunometabolism

## INTRODUCTION

## Obesity and Metabolic Syndrome

Obesity has reached worldwide epidemic proportions in the last decades. This is accompanied by an increased prevalence of comorbidities, such as type II diabetes, cardiovascular diseases, cancer, and other chronic diseases. In fact, 7 out of 10 leading causes of death in the U.S. are chronic disease states of which many have associations with obesity (1–3). Altogether, obesity and its comorbidities impose high social and economic costs to individuals and society alike. Therefore, understanding the causes of obesity and designing preventive strategies are fundamental demands.

The factors that contribute to obesity are many (2), but it is generally accepted that obesity results from an association of genetic predisposition (which is currently unmodifiable) with a dysregulated energy balance. The World Health Organization (WHO) defines obesity as a body mass index (BMI, weight in kilograms divided by the square of height in meters) greater or equal to 30 kg/m2 (3). Based on this definition, obesity can be considered a disorder of disproportionate mass of an individual. In that sense, it is reasonable to think of obesity as a result of positive energy balance, that is, increased calorie intake (especially in the form of dense calorie processed food) and decreased calorie expenditure (sedentary lifestyle).

Based on the positive energy balance view, excessive calorie intake and sedentary habits are considered the main modifiable factors contributing to the obesity epidemic. Special attention has been drawn to the often consumed highly processed and energy-dense foods. In fact, many obesity models in experimental animals rely on feeding mice and rats a high-fat or a high-fat, high-sugar diet. Therefore, the sugar and lipid content of foods and drinks are always taken into account when referring to weight gain and obesity.

## Metainflammation and Insulin Resistance

The concept of obesity as excessive weight due to increased fat deposition is simplistic. Over the last two decades, cumulating pieces of evidence support the concept that fat tissue hyperplasia and hypertrophy is accompanied by profound alterations in adipose tissue homeostasis. The breakthrough description of elevated tumor necrosis factor α (TNFα) levels in adipose tissue of obese individuals and the contribution of TNFα to the establishment of insulin resistance (4) paved the way for the concept of an inflammatory component of obesity. This inflammatory component is referred to as "metainflammation," a pathological condition of chronic, low-grade inflammation observed in obese subjects (5, 6).

Later studies have shown macrophage infiltration in obese fat tissue and their contribution to pro-inflammatory cytokine production (including TNFα) and insulin resistance. More recently, studies have shown the participation of many immune cell types in adipose tissue homeostasis, dysregulation, and an important contribution of macrophage polarization. In lean individuals, adipose tissue has a prevalence of alternatively activated (or M2) macrophages. These M2 macrophages produce anti-inflammatory cytokines, such as IL-10, and express arginase-1, which metabolizes arginine to polyamines and away from free radical nitric oxide (NO). Various immune cell types maintain M2 polarization in lean adipose tissue; T cells (Tregs) that contribute to leanness (7) and may be a relevant source of IL-10, a cytokine that polarizes macrophages to a M2 phenotype in adipose tissue (8), and eosinophils, which are the main source of the M2-polarizing cytokine IL-4 in adipose tissues (9). These cytokines and M2 macrophages sustain adipose tissue homeostasis and contribute to insulin sensitivity.

In obese adipose tissue, on the other hand, pro-inflammatory mediators such as TNFα and the CC chemokine ligand C–C motif ligand 2 recruit blood monocytes, where these cells become polarized toward the "classical" pro-inflammatory M1 state (10, 11). Interestingly an important contributor to M1 polarization is the activation of TLR4 by free fatty acids (12), which are found in large amounts due to increased lipolysis in obese adipose tissue. In contrast to their M2 counterparts, M1 macrophages produce pro-inflammatory mediators, such as TNF-α, IL-1β, and leukotriene B4, and express the inducible isoform of NO synthase (iNOS), leading to the production of large amounts of NO from arginine. Altogether, these mediators contribute to adipose tissue insulin resistance and, consequently, a sustained state of lipolysis which contributes to the perpetuation of the local low-grade inflammatory state. Moreover, eosinophil and Treg numbers were shown to lower in obese adipose tissue further favoring M1 polarization.

A macrophage M1–M2 imbalance can also be seen in other target organs, such as the liver (13). In lean individuals, liverresident macrophages, known as Kupffer cells (KCs) show a M2-like phenotype, sustained by their PPARδ expression (14). Mediators derived from obese adipose tissue, especially TNF-α and FFAs are able to polarize KCs to a M1-like state. This is further complicated by the fact that M1-KCs produce mediators that recruit M1-prone monocytes from the circulation to populate the liver (15). This process is directly related to the establishment of liver insulin resistance and the progress of liver steatosis to cirrhosis and liver failure. In fact, depletion of liver macrophages prevents steatohepatitis and insulin resistance in mice (16, 17). Besides endogenous mediators, KCs can be activated by microbial products derived from the gut (18). The liver is anatomically positioned so that it is the first organ to contact gut-derived products. Actually, this is one proposed mechanism by which disturbances in gut barrier—either directly or through an imbalance of the gut microbiome—can result in liver inflammation and metabolic derangements (18).

In summary, the current view is that the modern lifestyle, characterized by overeating associated with low physical activity, leads to increased weight and fat mass gain through a shifted energy balance. This disturbs the homeostasis of target organs, such as the liver and adipose tissue, likely through mechanisms involving the pro-inflammatory activation of immune cells, leading to peripheral insulin resistance. In the longer term, these alterations will culminate in metabolic syndrome, characterized by abdominal fat, hypertension, dyslipidemia, insulin resistance, and steatohepatitis.

## Food Additives As Neglected Players in Obesity and Metabolic Syndrome

In modern societies, the easy access to food, together with the demand for highly palatable and ready-to-serve products lead to the generalized consumption of industrialized processed foods. Processed food lacking in vitamins, fibers, and minerals, and dense in calories in the form of fat and simple sugar are, thus, considered the main villains of modern diets (19). The contribution of excessive calorie intake to the alarming increase in obesity witnessed in the last three decades is undeniable.

However, processed food is composed of not only sugar and fat but also a series of other products that are added in order to increase palatability, modify texture, and prolong shelf life. These products are collectively called food additives. A few of these additives even have well-known beneficial health effects as exemplified by probiotics and prebiotics. These additives are used to directly or indirectly influence gut microbiota and have well-documented benefits for host health and well-being (20). All prospected food additives, however, need to pass a rigorous scrutiny for their potential toxicity and undesired side effects. These toxicity tests are carried out following a series of guidelines, including outcome parameters that must be observed. These parameters comprise analysis of clinical manifestations, biochemical and hematological alterations, and postmortem analysis (21, 22). Based on these results, food additives are allowed to be used in varying amounts and many of them are considered innocuous and safe. However, studies sometimes present evidence that questions food additive safety and asks for a reassessment of their use. In this regard, we present evidence from the current literature that food additives—even some that are generally considered safe—may have relevant effects on immune cells and, thus, could also contribute to the burden of obesity and its related comorbidities. We have selected a few examples to illustrate and base a discussion on effects that food additives may have on cells of the immune system, which may potentially contribute to a series of pathologic and metabolic conditions. These examples are summarized in **Table 1** and discussed in detail in the subsequent sections.

## WHEN "NATURAL" MAY NOT BE HEALTHY: THE CASE OF CITRATE

Citrate is a very common food and drink additive widely used by the food industry as a chemical acidifier, flavoring agent and a preservative (36, 37). Since it is found in large amounts in many fruits and vegetables, especially citric fruits, it is generally considered natural and healthy. The Food and Drug Administration (FDA), for example, does not pose a limit for citric acid addition to foods or drinks.

Apart from being a food additive, citrate is also a metabolite involved in carbohydrate and lipid metabolisms. In situations of anabolism, as after meals, citrate is formed in the mitochondria and exported to the cytosol. Cytosolic citrate is metabolized to acetyl-CoA and oxaloacetate by the enzyme ATP:citrate lyase (ACLY) (38). Cytosolic acetyl-CoA then serves as the main building block for fatty acids and cholesterol synthesis. Despite the well-described fate of endogenous citrate, the fate of ingested citrate remains largely unexplored. Citrate enters the cells through the SLC13A5 citrate transporter encoded by the mINDY (mammalian I'm Not Dead Yet) gene (39). In humans, this gene is primarily expressed in the liver, presenting very low but detectable expression in the brain, testes, and ovary (40). Moreover, expression of this citrate transporter is upregulated in the liver of animals submitted to a high-fat, high-sucrose diet (40). Since ACLY is also highly expressed in the liver (41), it is reasonable to assume that food-derived citrate may contribute to postprandial lipid and cholesterol synthesis. Although expression of SLC13A5 in inflammatory cells such as monocytes, has not been reported, it has been reported that exogenous citrate modulates liposaccharide-induced monocyte inflammatory responses in cell culture (29), raising the question of the role of exogenous citrate in inflammation.

We recently addressed this issue by supplementing citrate in the drinking water of mice fed a standard diet (28). Contrary to our expectations, we did not find any increase in weight gain, adipose tissue mass, ectopic lipid deposition, or plasmatic lipid levels. This suggests that citrate supplementation did not contribute significantly to *de novo* lipid synthesis, at least in the mediumterm duration (75 days) of our experiment. We currently do not know how citrate supplementation may affect lipid metabolism in the long term. On the other hand, we found a significant effect on glucose homeostasis. Mice receiving citrate plus sucrose showed higher fasting glycemia and diminished glucose tolerance. This

Table 1 | Common food additives and their proposed effects on cells of the immune system and metabolic parameters.

*Food additives, their described effects, and their maximum daily intake recommendations established by Food and Drug Administration.*

was accompanied by increased levels of inflammatory cytokines in adipose tissue (28).

The mechanisms by which exogenous citrate may contribute to adipose tissue inflammation and establishment of insulin resistance remains to be determined. In this regard, recent data suggest that citrate may be pro- or anti-inflammatory. For example, citrate administration was shown to protect mice from cerebral and hepatic oxidative damage induced by low-dose LPS (42). Parameters of tissue damage, such as DNA fragmentation and hepatic enzymes in plasma, were significantly diminished in citrate-treated mice. However, this was only observed at lower citrate doses. At the highest dose tested citrate showed no effect or even a potentiating effect. *In vitro*, exogenous citrate was recently shown to potentiate LPS-induced activation of THP-1 monocyte cell line (29). This was dependent on calcium availability, since higher citrate concentrations showed an opposite inhibitory effect that was rescued by calcium supplementation. Potentiation of LPS effects by citrate could be blocked by TCA, an ACLY competitive inhibitor, suggesting the participation of cytosolic citrate metabolism in this effect. Indeed, cytosolic citrate accumulation is a metabolic characteristic of inflammatory macrophages (43) and some effector functions of macrophages depend on citrate metabolism (44). It was recently demonstrated that ACLY is important for macrophage synthesis of lipid mediators and free radical production. Interfering with ACLY expression or activity significantly inhibited prostaglandin synthesis and NO and reactive oxygen species production by LPS- or cytokine-activated macrophage cell lines (45). Moreover, mitochondrial citrate carrier and ACLY expression is upregulated by LPS in a NF-κB-dependent fashion, increasing citrate influx to the cytosol (46).

Furthermore, citrate may also have epigenetic effects on macrophages. ACLY plays a role in epigenetic regulation in diverse mammalian cell types. Histone acetylation is responsive to ACLYdependent glucose availability (41, 47) and ACLY was shown to localize in the nucleus, where it can contribute to the epigenetic program of adipocyte differentiation as a source of acetyl groups for histone acetylation (48). In kidney mesangial cells, ACLY can also promote histone hyperacetylation and upregulation of fibrogenic genes, contributing to high glucose-triggered diabetic renal fibrosis (49). More specifically in macrophages, a recent report showed that epigenetic modifications underlying IL-4 mediated M2 polarization were dependent on ACLY activation triggered by Akt–mTOR pathway (50). Furthermore, citrate carrier acetylation and inhibition strongly decreases LPS-induced inflammatory response (46).

Finally, the contribution of citrate to obesity and its related comorbidities can be inferred indirectly by pharmacological data. Bempedoic acid, a lipid-regulating compound, was recently described as a potent ACLY inhibitor (51, 52). In mouse model of metabolic dysregulation, bempedoic acid treatment significantly reduced adiposity, plasma lipid levels, and attenuated inflammation onset and atherosclerotic lesion development (53). A phase-II clinical trial showed that bempedoic acid safely lowers LDL-c levels and could be used alone or in association with others therapies (51). Moreover, treatment with hydroxycitrate, which functions as ACLY inhibitor, attenuates weight gain, lipid deposition, and adipose tissue inflammation in spontaneous genetically obese rats (54).

These data support the hypothesis that citrate intake from foods and drinks may promote inflammation. Our results show that citrate contributes to adipose tissue inflammation and glucose intolerance even when mice are fed a regular diet. Although citrate effects may vary depending on experimental settings, i.e., may protect from LPS effects (42) or potentiate LPS effects (29), the abovementioned pieces of evidence suggest that it is important to reassess the concept that citrate is inert. Further studies are needed to address the metabolic effects of citrate supplementation in the long-term as well as in association with high-calorie diets.

## SALT: A VIEW BEYOND HYPERTENSION

Salt, especially in the form of sodium chloride, is largely used worldwide to increase food flavor and palatability. High sodium intake correlates with the development of a series of diseases, being hypertension the most popularly known (55). Industrialized and processed foods usually contain high amounts of salt that can be up to 500 times more than a similar home-cooked meal (56). Moreover, associated with their high-sodium amounts, industrialized foods present low or undetectable levels of other minerals, especially those related to beneficial effects, such as magnesium, selenium, zinc, and others (57). The low consumption of these minerals is strongly associated with the prevalence of obesity, type 2 diabetes (T2D) (58), and inflammatory bowel disease (59, 60). The mechanisms underlying the detrimental effects of high salt consumption are still a matter of debate. However, recent data provide pieces of evidence that high salt may have a relevant impact on cells of the immune system.

Two independent studies have shown that a high-salt diet induces T cell polarization toward the pathogenic Th17 phenotype, predisposing mice to develop autoimmune disease (32, 33). This was shown to be dependent on the activation of the serine/ threonine kinase serum glucocorticoid kinase (SGK)-1, p38- MAPK, and nuclear factor of activated T cells (NFAT) 5. Concerted activation of these factors leads to increased IL-23R expression and stabilization of the Th17 phenotype. Since Th17 cells play a pathogenic role in obesity and metabolic syndrome (61, 62), it is reasonable to assume that a similar mechanism may predispose individuals consuming large amounts of salt to develop metabolic derangements.

Similar to the findings in Th17 cells, macrophages also had their effector phenotype modulated by extracellular osmolarity (*in vitro*) and high salt consumption (*in vivo*). Through the same p38–NFAT5 pathway triggered in T cells, increased sodium concentrations were able to boost NO production and the leishmanicidal activity of macrophages (31). This suggests that high salt concentrations shift macrophage activation toward the M1 phenotype. In fact, high salt blunted M2 polarization induced by IL-4 and IL-13 and impaired their effector functions (63). This is interesting when considering, for example, the effects of citrate on macrophage activation. High sodium and citrate are commonly found in processed food and both seem to favor M1 polarization of macrophages. This could have profound influence on the development of insulin resistance and adipose tissue inflammation, especially if a synergic effect could be demonstrated in future studies. Moreover, a recent report described that macrophages are able to sense hyperosmotic stress and activate both the NLRP3 and NLRC4 inflammasomes (64). This was dependent on mitochondrial ROS production and lead to IL-1β secretion that contributed to a biased T cell polarization toward the Th17 phenotype. Given the well-documented participation of NLRP3 inflammasome and IL-1β in insulin resistance (65, 66), this could be a reasonable mechanism for the association between high salt consumption and the metabolic syndrome.

Finally, increased salt concentrations, either *in vitro* or *in vivo*, was shown to impair Treg differentiation and function (67). This effect was also dependent on SGK-1, resulted in loss of Treg suppressive function and increased interferon (IFN)-γ production, thus contributing to the aggravation of both graft-versus-host disease and experimental colitis. Taken together, these studies suggest that the consumption of a high-salt diet would strongly favor a pro-inflammatory immune response with heightened Th17 and M1 activation and decreased Treg and M2 functions. Given the well-documented pathological role of both Th17 T cells and M1 macrophages in obesity, it is reasonable to speculate that high salt consumption would favor obesity and diabetes by an immune-mediated mechanism involving a strong bias toward pro-inflammatory phenotype of both macrophages and T cells. In fact, high salt consumption has been associated with features of the metabolic syndrome, such as obesity, hypertension, and T2D (68). However, the impact of this salt-triggered pro-inflammatory bias to obesity and T2D remains to be determined. Moreover, the effect of chronic exposure to high salt diet has never been tested, neither in obesity nor in autoimmune models. Another open question is the outcome of high salt consumption in association with other additives with potential pro-inflammatory effects, such as citrate (above) or carrageenan and emulsifiers (discussed below). Nevertheless, the straight interrelationship among salt consumption, weight gain, and immune-associated metabolic dysregulation cannot be dismissed.

## THE GUT MICROBIOME: A SYMBIOTIC RELATIOSHIP WITH THE IMMUNE SYSTEM AFFECTING WEIGHT GAIN AND TYPE 2 DIABETES

The gut microbiota comprises a diverse community of microbes that inhabits the intestinal tract. It is now well demonstrated that the gut microbiota provides key signals for the full maturation of the immune system and also has important metabolic benefits for the host. On the other hand, disturbances of the microbiota– host relationship are associated with immune-mediated and metabolic-associated chronic diseases, such as diabetes and the metabolic syndrome (27). Mucus at the intestinal surface prevents direct contact between epithelial cells that line the intestine and the gut microbiota (69). Diverse agents (nutritional, chemical, or even an imbalance among the different bacteria species) that interfere with this multilayered mucus barrier might have the potential to promote or to control diseases associated with gut inflammation.

Regardless of the mucus barrier, gut microbiota interacts with immune cells, providing signals that are fundamental to the normal development of the host immune functions (70–72). Development of Th17 cells in the gut, for example, strictly depends on the presence of segmented filamentous bacteria (73, 74). By contrast, Clostridium and *Bacteroides fragilis* favor Tregs development (75, 76). In fact, any dysbiosis affecting the ileum microbiota deeply affect intestinal innate immunity and CD4 T cell homeostasis—particularly, a decrease in Th17 cells. These changes are the initial steps for the onset of metabolic diseases. Certainly, they are sufficient to induce glucose intolerance and insulin resistance.

In the gut, CD4 T cells contribute to immunity by differentiating into various subsets, notably inflammatory and regulatory cells (77, 78). Th17 cells are the most abundant CD4 T cells in mucosal tissues (79). They secrete two isoforms of IL-17 (IL-17A and IL-17F) and/or IL-22, which confer protection against fungi and pathogenic bacteria. Th17 cell differentiation is mediated by the transcription factor retinoid-related orphan receptor gamma t (80), induced by the cytokines TGFβ1, IL-6, IL-1β, and IL-21, and maintained by IL-23 (81).

It is remarkabe that 90% of the human gut microbiome is made up of organisms from two phyla, Firmicutes and Bacteroidetes. Importantly, an imbalance between these phyla has been directly linked to obesity, T2D, and inflammation (82, 83). Most of the studies report the augmentation in Firmicutes abundance and reduction in Bacteroidetes in obese versus lean individuals (84–87).

Corroborating these previous studies, elegant results from Cani et al. (88) and Turnbaugh et al. (89) demonstrated an association between the development of obesity and T2D, and an increase in the Firmicutes to Bacteroidetes ratio in the gut. This imbalance results in increased energy harvesting from food, as well as an increase in the transcription of genes controlling both lipogenesis in the liver and adipose tissue development (86, 89). Changes in the gut microbiota may also directly influence cells of both the innate (macrophages, dendritic cells, and innate lymphoid cells) and adaptive immune systems, thus contributing to development and maintenance of a state of chronic low-grade inflammation in obese individuals (75, 90).

Hydrolyzed carbohydrates and simple sugars can be fermented by gut microorganisms to short-chain fatty acids (SCFA), such as acetate, propionate, and butyrate. This sugar metabolism can result in an added 10% daily dietary energy to the host (91–93). Consumption of Western diets—low or normal in fiber contents and rich in fat and digestible sugars—can alter gut microbiota composition, impacting the production of SCFA and other gutderived metabolites. Dysbiosis induced by fat- and sugar-rich diets is also associated with a thinner and less protective mucus layer, which results in increased gut permeability, chronic lowgrade inflammation, and metabolic disorders (88, 94). Bacteriaderived SCFA have been shown to have anti-inflammatory properties attenuating the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IFN-γ. Thus, consumption of Western diets that results in decreased abundance of SCFAproducing bacteria could facilitate inflammatory responses in the gut (95).

## FOOD ADDITIVES THAT ALTER GUT MICROBIOME: EMULSIFIERS AND SWEETENERS

Recent studies have demonstrated that the consumption of artificial sweeteners and dietary emulsifiers can alter the gut microbiota, resulting in intestinal disturbance and inflammation, favoring the development of the metabolic syndrome (27, 96).

Sweeteners such as high-fructose corn syrup and low-calorie or calorie-free sugar substitutes (sugar alcohols and artificial sweeteners) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. A series of compounds (sucralose, aspartame, saccharin, steviol, Luo han guo extract, among others) are approved to be used as sweeteners in food, and are generally recognized as safe by the FDA. Cyclamates are banned in the United States but are still allowed in many other countries (97). Moreover, most of the commercial formulations of saccharin, sucralose, or aspartame comprise ~5% sweetener and ~95% glucose, functioning as a sugar source. Despite being considered safe and even beneficial owing to their low caloric content, pieces of evidence in various species, including humans, show that artificial sweeteners can be metabolically active (56, 98–104).

Pieces of evidence from both mice and human studies suggest that the consumption of artificial sweeteners is associated with increased glucose intolerance. This effect seems to result from alterations in the composition and function of the gut microbiota. In fact, several bacterial taxa that changed in response to sweetener consumption were previously associated with T2D development in humans (105, 106). For instance, it is reported that sweetener consumption increases the Bacteroides:Clostridiales ratio by increasing the former and decreasing the later. Indeed, the exposure of rats to Splenda (an artificial sweetener containing 1% sucralose) was associated with both increased weight gain and significant alterations in gut microbiota composition (23). In addition, the exposure to saccharin, sucralose, and especially aspartame-promoted alterations of the gut microbiota leading to an impaired glucose tolerance (24, 26). In fact, individuals consuming aspartame presented elevated fasting glucose and impaired insulin sensitivity, which were associated with gut dysbiosis (25).

Most artificial sweeteners are not chemically modified in the human gastrointestinal tract (107, 108) and, thus, directly encounter the intestinal microbiota. However, aspartame is a target for intestinal esterases and peptidases, which transforms aspartame into amino acids and methanol before reaching the colon (109). These products of aspartame metabolism should not have a significant influence on gut microbiota. Thus, the mechanisms of aspartame-induced dysbiosis remain unclear.

Currently, sucralose is widely used due to its taste (extremely similar to natural sugar) and providing no calories to the human body. Moreover, sucralose does not present the common and undesirable bitter aftertaste present in most of the artificial sweeteners. Therefore, sucralose has been considered a safe food additive mainly for the lack of any toxic effect and noeffect on metabolism (110). However, in a recent study, it was shown that sucralose consumption deeply impacts the gut microbiome composition increasing the release of pro-inflammatory mediators in mice. This effect was accompanied by an increase of inflammatory markers in the liver, such as MMP-2 and iNOS (26).

Saccharin is another sweetener strongly associated with the onset of metabolic diseases. Saccharin decreases the release of glucagon-like peptide-1 (GLP-1), an incretin hormone implicated in the regulation of various physiological processes, such as food intake, glycemic control, and cardiovascular protection (111, 112). Long-term cohort studies of artificial sweetener consumption reported increased risks of adverse outcomes, such as diabetes, cardiovascular diseases, and stroke, a pattern consistent with persistently reduced GLP-1 levels. On the other hand, Daly et al. (113) showed some prebiotic-like effects of saccharin/neohesperidin dihydrochalcone (SUCRAM®). Saccharin significantly increased gut abundance of Lactobacillus in piglets. Lactobacilli are the predominant lactic acid bacteria in the pig intestine and have well-described protective activity in the gastrointestinal tract, influencing gut immune system maturation and regulating intestinal inflammatory responses (113).

Emulsifiers are detergent-like molecules used by food industry as stabilizers for processed foods. Two commonly used emulsifiers are carboxymethylcellulose (CMC) and polysorbate-80 (P80). Toxicological and carcinogenic potential of P80 have been established and P80 is approved by the FDA for use in concentrations of up to 1%. CMC has not been extensively studied but it is used in various processed food at up to 2%. A recent study observed that, in mice, relatively low concentrations of these emulsifiers induced low-grade inflammation, obesity, and metabolic syndrome (27). These effects were associated with emulsifier-induced dysbiosis, bacterial translocation through the mucosal barrier, and increased pro-inflammatory potential. In fact, Roberts et al. (114) have shown that both emulsifiers can increase bacterial translocation across cultured epithelial layer (114). Moreover, bacterial translocation was associated with mucus loss and increased bacterial adherence after chronic exposure to dietary emulsifier (27).

Modest increases in body weight and fasting glycemia were observed in animals given doses as low as 0.1% CMC. Higher doses of 0.5% resulted in low-grade inflammation and increased adiposity. For P80, pieces of evidence of low-grade inflammation and increased adiposity were found in animals consuming amounts of as little as 0.1%. Higher concentrations of 0.5% resulted in a mild increase in fasting glycemia (27). These emulsifier-induced metabolic derangements were long-lasting since they could be observed even 6 weeks after emulsifier consumption withdraw (27).

Emulsifier-induced changes in gut microbiota play a role in driving inflammation and metabolic changes promoted by these food additives. They altered fecal levels of SCFA, including decreased levels of butyrate and bile acids (71, 115). In fact, emulsifiers induce gut barrier alterations and low-grade inflammation. They also result in significantly increased food intake (at least twice as high as the control mice), which drives the development of obesity by increasing fat mass and promoting metabolic alterations. Besides, excessive fat mass accumulation is associated with low-grade inflammation, which plays a crucial role in the onset of obesity and related metabolic disorders. Cani and Everard (116) reported that as the gut microbiota composition and the fecal SCFA profile are modified in emulsifier treated mice, such changes may also impact food intake. Growing evidence suggests that microbial products, for example, butyrate and propionate, may directly bind to specific G-protein-coupled receptors such as GPR41/43 (117), enriched in enteroendocrine L-cells. The stimulation of these receptors triggers the release of enteroendocrine peptides, such as GLP-1 and peptide YY that reduce food intake *via* a gut/brain axis.

In conclusion, emulsifiers and sweeteners, which are freely used by people controlling caloric intake as well as by obese and patients with diabetes, present a verified effect on gut microbiota. This effect results in dysbiosis that normally is associated with the development of insulin resistance and to weight gain. Therefore, these additives should not be considered as innocuous and must have their use controlled and labeled with regard to possible undesirable effects.

## ASSOCIATION OF FOOD ADDITIVES: CARRAGEENAN

Carrageenan is a hydrocolloid extracted from seaweeds and widely used by food industry as thickener and stabilizer. Due to its high molecular weight, it is assumed that carrageenan is not absorbed in the intestine and, thus, can be safely consumed (118). In fact, orally administered carrageenan can be recovered in feces in substantial amounts (119). Recovered carrageenan behaves similarly to the original in chromatographic assays, suggesting that it is stable at low pH and resistant to bacterial degradation, not being substantially altered or metabolized in the gastrointestinal tract (119).

However, recent studies demonstrate that oral carrageenan administration can lead to insulin resistance. This suggests that either there is a mechanism of immune cell activation in the gut or that the absorption of even trace amounts of carrageenan is sufficient to trigger pro-inflammatory responses systemically.

Carrageenan has been widely used as an inducer of inflammation and inflammatory pain (120). Its pro-inflammatory potential results from TLR4 activation, an innate immune receptor also involved in obesity and insulin resistance (12). This renders carrageenan as a potential to trigger for cellular processes that would contribute to the establishment of metabolic syndrome. For example, intratumoral carrageenan administration was recently reported to inhibit tumor progression by shifting macrophages toward a M1 phenotype (121).

In that sense, recent studies report the effect of administering carrageenan in water on metabolic parameters in mice. It was first shown that carrageenan consumption—in doses equivalent to those in foodstuff—leads to glucose intolerance *via* increased IRS-1 serine phosphorylation and interference with insulintriggered PI3K–Akt pathway (34). This was also observed *in vitro*, using the human hepatocyte cell line HepG2, suggesting that a similar mechanism may be operating in human obesity. Further studies by the same group demonstrated that interference with insulin signaling by carrageenan was triggered *via* a signaling pathway involving TLR4, Bcl10, and mitochondrial ROS, which was further sustained by GRB10 (122). GRB10 is an adaptor protein that associates with and inhibits IRS-1 and was upregulated by carrageenan exposure both *in vitro* and *in vivo* (122).

Interestingly, carrageenan consumption exacerbated glucose intolerance and dyslipidemia induced by high-fat diet feeding (35). Of note, carrageenan did not show any effect on lipid profile of mice fed a normal diet, but induced a substantial increase in non-HDL cholesterol levels. This suggests that carrageenan consumption, especially in association with fat rich food (as seen in industrialized foods), may predispose individuals to develop atherosclerosis and increase cardiovascular risk.

The safety of carrageenan consumption is still a matter of debate. Arguments in favor of its safety rely on toxicological studies showing no significant intestinal absorption after oral administration. In these studies, there was no evidence of carrageenan traces in the liver or cells of the gastrointestinal tract of non-human primates and rats (123, 124), although the analytical method applied were not very sensitive. By using a more sensitive method, administering radiolabeled carrageenan, it was observed the presence of absorbed carrageenan in cecal lymph node, Peyer's patches, and the intestinal wall, mainly associated with macrophages (125). Other authors showed the presence of macrophage-borne carrageenan in intestinal lamina propria and the liver, suggesting that sampling of intestinal content by mucosal macrophages may provide a route for carrageenan entry (126, 127).

Despite the lack of consensus on whether carrageenan is absorbed or not, its pro-inflammatory activity is undeniable. Active absorption or passive entry of even trace amounts of carrageenan could have significant effects on metabolic homeostasis, especially with chronic exposure. Given that a high-fat diet or food additives such as emulsifiers were shown to interfere with intestinal mucosal permeability, it is important to reconsider the toxicological properties of carrageenan. For example, the addition of carrageenan (as a thickener) to foods rich in fat could facilitate the extravasation of carrageenan due to high-fat-induced intestinal permeability. Likewise, association of carrageenan with emulsifiers (as seen in many processed foods) would facilitate carrageenan absorption after disruption of the epithelial intestinal barrier. Therefore, in light of these new data showing that the intestinal barrier homeostasis may be disrupted by dietary components, it becomes necessary to reassess the toxicological properties of this widely used food additive.

## CONCLUSION

Modern societies have witnessed an alarming increase in cases of obesity, T2D, and other comorbidities associated with poor eating habits. In addition, the incorporation of processed and industrialized food into everyday eating correlates with this phenomenon. In an attempt to tackle that threat, much attention has been paid to the amount of calories present in food. Specifically, the amount of fat and simple sugar is considered the main villains of modern processed food. Although the detrimental effects of fat and sugar are undeniable, industrialized food is composed of many other substances of which the metabolic effects are usually ignored. Emulsifiers, thickeners, artificial sweeteners, and preservatives may have direct and indirect effects on cells of the immune system, contributing to metabolic dysregulation. Moreover, safety evaluation and estimates of food additives toxicity are usually based on tests of single compounds, thus underestimating the effects of associating two or more additives. Therefore, policies that aim at curbing the evolution of obesity must take into account not only calorie content of food but also all the other components and their effects. Whenever possible, people should avoid readyto-serve, additive rich, processed food and prefer healthier food choices or home-cooked meals.

## AUTHOR CONTRIBUTIONS

HN conceived the text and wrote the manuscript. PA, LG, JL, PZ, and MS-P wrote the manuscript.

## REFERENCES

## ACKNOWLEDGMENTS

Authors thank Dr. Patricia L. Mitchell (Univesité Laval, Québec, QC, Canada) for critical reading and English editing of the manuscript.

## FUNDING

The group research is supported by grants from CAPES, CNPq grant numbers 405214/2015-4 and 447065/2014-9 (for P.Z.) and FAPERJ grant numbers 26/110.164/2014 (for P.Z.) and 26/111.239/2014 (for H.A.P.N.). MS-P receives a CNE fellowship from FAPERJ and a research fellowship from CNPq. PZ receives a JCNE fellowship from FAPERJ. LG is supported by a PhD fellowship from CNPq, PA is supported by a PhD fellowship from CAPES, and JL received a PhD fellowship from FAPERJ and is currently receiving a PhD fellowship from CAPES.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Paula Neto, Ausina, Gomez, Leandro, Zancan and Sola-Penna. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Sarah Leão Fiorini Aguiar1 , Mariana Camila Gonçalves Miranda1 , Mauro Andrade Freitas Guimarães1 , Helton Costa Santiago1 , Camila Pereira Queiroz1 , Pricila da Silva Cunha1 , Denise Carmona Cara2 , Giselle Foureaux2 , Anderson José Ferreira2 , Valbert Nascimento Cardoso3 , Patrícia Aparecida Barros1 , Tatiani Uceli Maioli <sup>4</sup> and Ana Maria Caetano Faria1 \**

*1Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 3Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 4Departamento de Nutrição, Escola de Enfermagem, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil*

## *Edited by:*

*Paul De Vos, University Medical Center Groningen, Netherlands*

#### *Reviewed by:*

*Abdelhadi Saoudi, Institut National de la Santé et de la Recherche Médicale, France Guillermo Docena, Universidad Nacional de La Plata, Argentina*

> *\*Correspondence: Ana Maria Caetano Faria<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology*

*Received: 26 June 2017 Accepted: 19 December 2017 Published: 15 January 2018*

#### *Citation:*

*Aguiar SLF, Miranda MCG, Guimarães MAF, Santiago HC, Queiroz CP, Cunha PS, Cara DC, Foureaux G, Ferreira AJ, Cardoso VN, Barros PA, Maioli TU and Faria AMC (2018) High-Salt Diet Induces IL-17-Dependent Gut Inflammation and Exacerbates Colitis in Mice. Front. Immunol. 8:1969. doi: 10.3389/fimmu.2017.01969*

Excess intake of sodium is often associated with high risk for cardiovascular disease. More recently, some studies on the effects of high-salt diets (HSDs) have also demonstrated that they are able to activate Th17 cells and increase severity of autoimmune diseases. The purpose of the present study was to evaluate the effects of a diet supplemented with NaCl in the colonic mucosa at steady state and during inflammation. We showed that consumption of HSD by mice triggered a gut inflammatory reaction associated with IL-23 production, recruitment of neutrophils, and increased frequency of the IL-17 producing type 3 innate lymphoid cells (ILC3) in the colon. Moreover, gut inflammation was not observed in IL-17–/– mice but it was present, although at lower grade, in RAG−/<sup>−</sup> mice suggesting that the inflammatory effects of HSD was dependent on IL-17 but only partially on Th17 cells. Expression of SGK1, a kinase involved in sodium homeostasis, increased 90 min after ingestion of 50% NaCl solution and decreased 3 weeks after HSD consumption. Colitis induced by oral administration of either dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid was exacerbated by HSD consumption and this effect was associated with increased frequencies of RORγt <sup>+</sup> CD4+ T cells and neutrophils in the colon. Therefore, our results demonstrated that consumption of HSD *per se* triggered a histologically detectable inflammation in the colon and also exacerbated chemically induced models of colitis in mice by a mechanism dependent on IL-17 production most likely by both ILC3 and Th17 cells.

Keywords: sodium chloride, gut inflammation, IL-17, Th17 cells, ILC3

## INTRODUCTION

Sodium is an indispensable nutrient for proper cell functions in living animals when consumed in appropriate amounts (1). It is essential for maintenance of osmotic pressure, normal pH, distribution of body fluids, and for most of metabolic processes. The influx of sodium ions across plasma membrane is required for action potential involved in nerve impulses and muscle contraction. Sodium chloride is readily available in processed food and ingested in large amounts as part of Western diets (2). Excess intake of sodium is often associated with high risk for cardiovascular diseases (2–4).

It has been reported recently that immune cells play an important role in mediating the detrimental effects associated with consumption of high concentration of sodium. Elevated concentration of sodium chloride was found to induce proinflammatory IL-17-producing helper T (Th17) cells *via* p38/ MAPK/nuclear factor of activated T cells 5 pathway and the serum/glucocorticoid-regulated kinase 1/forkhead box protein 1 (SGK1) pathway both *in vitro* and *in vivo* (5, 6). These studies have demonstrated that high-salt diets (HSDs) exacerbate autoimmune encephalomyelitis in mice in a SGK1-dependent fashion (5, 6). In addition, hypertension and renal injury induced by HSD in salt-sensitive rats are accompanied by increased infiltration of T cells in kidneys (7, 8).

Intestinal mucosa is the major surface of contact with the external environment and the first tissue to interact with all types of food components including salt. The gastrointestinal tract is in constant interaction with the microbiota and the antigens from diet. Under normal conditions, exposure to these antigenic proteins would induce oral tolerance. However, any failure in intestinal homeostasis may result in inflammatory reactions toward microbiota and food antigens (9). These reactions are especially present during inflammatory bowel diseases (IBDs) such as Crohn's disease (CD) and ulcerative colitis (UC). It has been proposed that IBD results from the imbalance between regulatory T cells (Treg) and inflammatory Th1, Th2, and Th17 cells (10). Several studies have demonstrated an increased expression of Th17-related cytokines in both UC and CD, although it is not clear whether these cytokines are only produced by T cells (11).

Dietary composition has been long seen as a potential risk factor for the increased incidence of autoimmune diseases and IBD. A specific factor that has changed in the Western diet over time is the increasing amount of sodium-containing salt intake. Some recent studies have demonstrated that diets containing high amounts of sodium chloride can also induce an increase in Th17 cells and aggravation of experimental models of colitis by the activation of SGK1 kinase (12–14). These studies highlighted the importance of SGK1/Th17 axis but did not explore appropriately the possibility that IL-17 could be produced by other sources such as ILC3. It is clear that several cell types other than lymphocytes are important in intestinal homeostasis and also in the onset of inflammatory diseases (15).

Innate lymphoid cells (ILCs) represent a heterogeneous group of hematopoietic cells of the innate immune system that have a common lymphoid progenitor. While ILCs lack rearranged antigen-specific receptors, they express many of the transcription factors and effector molecules expressed by CD4<sup>+</sup> T helper (Th) cell populations, suggesting that ILCs may be an evolutionary precursor of cells of the adaptive immune system. They also have homologous functions with the Th cells. ILC1 express the transcription factor t-bet and is homologous to Th1 cells; ILC2 expresses GATA 3 and are homologous to Th-2 cells and ILC3 express ROR-γt and are homologous to Th-17 cells (16, 17). Considering that Th17 cells have been demonstrated to be pivot cells in pathogenicity of HSD in encephalomyelitis (5, 6) as well as colitis (12–14) models, that ILC3 are very abundant in the gut mucosa and that they are increased in the colon of individuals during the course of IBD (17, 18), it is likely that the effect of HSDs in colitis development is dependent on IL-17 production by both Th17 cells and ILC3.

This study aims to investigate the direct effect of a sodium chloride-rich diet in the gut mucosa as well as in the development of chemically induced colitis. Our working hypothesis is that sodium chloride may have an inflammatory effect *per se* and that both T cells and ILCs may play a role in this effect. In addition, we sought to investigate the synergistic effect of HSD in the inflammatory events taking place during experimental colitis induced by administration of 1% dextran sodium sulfate (DSS) in C57BL/6 mice and in the colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in BALB/c mice.

## MATERIALS AND METHODS

## Animals

Female BALB/c and C57BL/6 mice were purchased from CeBio (Centro de Bioterismo da Universidade Federal de Minas Gerais). Eight-week-old animals were used. IL-17<sup>−</sup>/<sup>−</sup> and RAG<sup>−</sup>/<sup>−</sup> mice were kindly provided by Dr. João Santana from Universidade de São Paulo, Ribeirão Preto, Brazil, and by Dr. Ricardo Gazzinelli, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, and maintained in our animal facility located at Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais. Animals were bred and housed in microisolators. All procedures were approved by the local ethical committee for animal research (CEUA-UFMG, Brazil, protocol 50/2014). Experiments were performed in accordance with guidelines and regulation established by Conselho Nacional de Controle de Experimentação Animal (CONCEA), Brazil.

## High-Salt Diet

Mice received either AIN93G diet as a control diet (15) or modified AIN93G supplemented with 4% NaCl (HSD) throughout the experiments.

## Hemodynamic Measurements Using the Tail-Cuff Method

Mean arterial pressure (MAP) was evaluated by a volume pressure recording sensor and an occlusion tail-cuff, which measures mice's tail blood pressure noninvasively (Kent Scientific Corporation, Torington, CT, United States) (16). Mice were acclimated to restraint and tail-cuff inflation for 2 days before the beginning of experiments. The restraint platform was maintained at 31–34°C. In each session, mice were placed in an acrylic retainer, and the tail was inserted into a compression cuff that measured the blood pressure eight times. Following the measurement cycle, the average of these values was considered for each mouse. All parameters were evaluated weekly, 1 day before the beginning of dietary consumption as well as 7, 14, and 21 days thereafter.

## Intestinal Permeability Test

Intestinal permeability was determined by measuring radioactivity diffusion in the blood after oral administration of diethylenetriamine pentaacetic acid (DTPA) labeled with 99m-technetium (99mTc). After 21 days of diet consumption, animals received 0.1 mL of a DTPA solution labeled with 18.5 mebequerel of 99mTc by gavage. Four hours later, mice were anesthetized; their blood was collected, weighed, and placed in appropriate tubes for radioactivity determination. Blood radioactivity levels were determined using an automated gamma counter (Perkin Elmer Wallac Wizard 1470–020 Gamma Counter; PerkinElmer Inc., Waltham, MA, USA). Results obtained were compared with the standard dose and calculated as a percentage of the dose per gram of blood using the following equation:

% dose/g blood

> , = ( ) cpm ing of blood/cpm dose of standard ×100

where cpm represents the counts of radioactivity per minute.

## Flow Cytometry Analyses

Following euthanasia, colon was harvested, and mesenteric lymph nodes were removed. Intestines were then cut open longitudinally and drawn through a pair of curved forceps while applying gentle pressure to remove intestinal contents. Tissues were cut into 2–4 cm fragments, washed twice to remove feces in calcium- and magnesium-free HBSS containing 2% FCS (at 4°C). Tissues were placed in 50 ml tubes and washed three times in HBSS containing 2% FCS at 4°C. Tissues were transferred to 25 cm3 tissue culture flasks and incubated at 37°C in HBSS containing 10% FCS, 0.2 mmol/l EDTA, 1 mmol/l DTT, 100 U/ml penicillin, and 100 µg/ml streptomycin. After 20 min, flasks were shaken vigorously for 30 s, and supernatant containing IEL was separated from the tissue fragments using a stainless steel sieve. To isolate lamina propria lymphocytes, the remaining tissue was washed three times with RPMI, and intestinal pieces were subsequently incubated for 30 min at 37°C in RPMI supplemented with 100 U/mL liberase (Roche). Cells were separated from tissue debris by purification through a 70 µm nylon filter. This step was repeated with a 40 µm nylon filter. Cell suspensions were adjusted to 106 cells/ml.

Flow cytometry analysis for neutrophils was performed using the following antibodies: CD11c-APC, CD103-Pacific Blue, MHC (IA IE)-PE, Ly6C-Percp, Ly6G-FITC, CD45-APC-Cy7, and F4/80-PE-Cy7. For T lymphocyte analysis, the following antibodies were used: CD4-FITC, ROR-γt-PE, IL-23R-PercepCy5.5, CD45-APC-Cy7. For ILC analysis, the following antibodies were used: Pacific Blue: CD11b, CD11c, or FceR1a; BV421: CD16 or CD19; PERCP-CY5.5: CD8; PE/CY7: CD4; BV570: CD45; BV605: CD117; APC: CD127; F700: CD3; PE-CF594: ROR-γt; PE: t-bet; AF488: GATA 3. All antibodies were purchased from Biolegend (San Diego, CA, USA). Strategies used to analyze these cell populations are shown in Figures S1 and S2 in Supplementary Material. For surface antigen detection, cells were labeled with monoclonal antibodies for 30 min at 4°C. For intracellular labeling, a fixing/permeabilization kit (e-Bioscience, San Diego, CA, USA) was used after this step. Samples were then incubated for 30 min with a solution containing the appropriate antibodies. After washing with PBS containing 0.5% FBS, samples were fixed with 3% paraformaldehyde for 30 min, washed and stored in PBS at 4°C. Cells were acquired using a FACSCanto II cytometer (Becton Dickinson, East Rutherford, NJ, USA) and data was analyzed by FlowJo software (Tree Star, Ashland, OR, USA).

## Intestinal Tissue Preparation for Cytokine Measurement

Colon samples were weighted and homogenized in PBS containing 0.05% Tween-20, 0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KIU Aprotinin A using a tissue homogenizer (100 mg tissue/ml buffer). Suspensions were centrifuged at 12.000g for 20 min at 4°C and the supernatants were transferred to microtubes. Supernatants were collected for cytokine assay levels of IFN-γ and IL-23 by capture ELISA. Briefly, plates were coated with purified monoclonal antibodies reactive with the cytokines IFN-γ and IL-23 (BD-Pharmingen, Franklin, NJ, USA) overnight at 4°C. In the following day, wells were washed, supernatants and standards were added, and plates were incubated overnight at 4°C. In the third day, biotinylated monoclonal antibodies against cytokines were added and plates were incubated for 2 h at room temperature. Color reaction was developed at room temperature with 100 μL/ well orthophenylenediamine (1 mg/mL), 0.04% H2O2 substrate in sodium citrate buffer. Reaction was interrupted by the addition of 20 μL/well of 2 N H2SO4. Absorbance was measured at 492 nm by ELISA reader (Bio-Rad, Philadelphia, PA, USA).

## RNA Isolation, Reverse Transcription, and Quantitative Real-time PCR (qPCR)

Total RNA extraction from frozen colon of C57BL/6 mice that received either standard diet (control group) or HSD group for 3 weeks, 5 days, 3 days, 120 h, 48 h, 36 h, or 24 h and colon of mice that received a 50% NaCl solution by oral gavage and were euthanized after 90 min thereafter, was performed using TRIzol® reagent (Invitrogen, USA), followed by treatment with RQ1 RNase-Free DNase (Promega, USA), and cDNA synthesis using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, UK). qPCR assays were performed to evaluate the mRNA levels of *Sgk1* (serum/glucocorticoid-regulated kinase 1) gene. *Gapdh* (glyceraldehyde-3-phosphate dehydrogenase) gene was used for the normalization of the data. Primers were designed using Primer3 program, version 0.4.0 (http://frodo. wi.mit.edu/) and analyses of parameters such as GC content and formation of homo-dimer, hetero-dimer, and hairpins structures were performed using OligoAnalyzer 3.1 software (https://www. idtdna.com/calc/analyzer). The specificity of the primers was confirmed using Primer-BLAST program (http://www.ncbi.nlm. nih.gov/tools/primer-blast/), to ensure that the primers amplified only the coding region (CDS) of interest. The sequences of the forward and reverse primers used for amplification of *Sgk1* (GenBank: NM\_001161847.2) and *Gapdh* (GenBank: GU214026.1) were 5′-CAAATCAACCTGGGTCCGTC-3′ (Sgk1-F), 5′-TCCAAAACTGCCCTTTCCG-3′ (Sgk1-R); 5′-CATCTTCCAGGAGCGAGACC-3′ (Gapdh-F), and 5′-GAAGGGGCGGAGATGATGAC-3′ (Gapdh-R). Each qPCR reaction contained 5 µL KAPA SYBR® FAST qPCR Master Mix 2× (Kapa Biosystems, USA); 0.2 µL of ROX High 50×; 200 ng of cDNA; each forward and reverse primer at the optimized concentrations (0.4 µM (F)/0.4 μM (R) for Sgk1 and 0.3 µM (F)/0.3 μM (R) for Gapdh) and water up to a final volume of 10 µL. The reaction profile was an initial step of denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 3 s and combined annealing and extension at 60°C for 30 s. A "no template control" was made in all the qPCR reactions for each pair of primers containing all the reagents except cDNA. Reaction specificity was confirmed with melting curves analysis and agarose gel electrophoresis experiments (*Sgk1* amplicon size = 87 bp; *Gapdh* amplicon size = 150 bp). Standard curves were generated with series of log dilutions of cDNA to calculate the amplification efficiency (*Sgk1*: Eff = 98.98%, *R*<sup>2</sup>= 0.99; *Gapdh*: Eff = 96.28%, *R*<sup>2</sup> = 0.99). The qPCR reactions were performed using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, UK), and the data were processed by SDS Software, version 2.4 (Applied Biosystems, UK). The calculation of gene expression was performed using the 2−ΔCt method, where ΔCt= Ct value of target gene − Ct value of reference gene.

## Colitis Induction

Dextran sodium sulfate-induced colitis was triggered by administration of 1% dextran sulfate sodium in the drinking water to C57BL/6 mice for 7 days (19). TNBS-induced colitis was triggered by administration of 2.5 mg TNBS in 35% alcohol intrarectally to BALB/c mice 7 days before euthanasia (19).

## Clinical Score and Histological Assessment of Colitis

Colons were excised and colonic inflammation assessed using a previously defined scoring system (20), which includes macroscopic features such as the presence or absence of adhesions, strictures, and diarrhea (diarrhea was defined as loose, watery stool). Samples of colon were fixed in formalin and processed for microscopic analysis. Hematoxylin-eosin-stained sections were blindly scored based on a semiquantitative scoring system that includes the main alterations observed: goblet cell depletion, muscular layer (erosion/ulceration) destruction, cellular infiltration, and edema; each parameter could receive 0–3 in the score index.

Colitis clinical severity (Disease Activity Index) were evaluated by a scoring system described previously (20) where the following features were graded: extent of destruction of normal mucosal architecture (0: normal; 1, 2, and 3: mild, moderate, and extensive damage, respectively), presence and degree of cellular infiltration (0: normal; 1, 2, and 3: mild, moderate, and transmural infiltration, respectively), extent of muscle thickening (0: normal; 1, 2, and 3: mild, moderate, and extensive thickening, respectively), presence or absence of crypt abscesses (0: absent; 1: present) and the presence or absence of goblet cell depletion (0: absent; 1: present). Scores for each feature were summed up to a maximum possible score of 11.

## Measurement of the Activity of Myeloperoxidase (MPO)

Samples were first homogenized in buffer solution (NaCl 0.1 M, Na3PO4 0.02 M, and Na2EDTA 0.015 M) and then centrifuged. Supernatant was discarded and the pellet resuspended and centrifuged. Supernatant was discarded and the pellet resuspended in another buffer (Na3PO4 and 0.5% w/v HETAB) solution and then stored at room temperature in a ratio of 1.9 ml/100 mg. Samples were homogenized and half the volume was withdrawn for NAG enzyme activity measurement. The other half of the homogenate was used for the determination of MPO activity. From this step on, the samples received different treatments.

Evaluation of NAG activity was performed separately. Salina/ Triton (Saline 0.9% and Triton X-100 0.1%) was added to the homogenate, samples were homogenized, and then centrifuged at 4°C for 10 min at 3000 RPM. Hundred microliters of the supernatant were collected and diluted in citrate phosphate buffer (0.1 M citric acid and 0.1 M Na2HPO4) for NAG measurement. In the microplate, 100 µL of each diluted sample was pipetted, and 100 µL of the substrate *p*-nitrophenyl-*N*-acetyl-β-d-glucosaminide diluted in phosphate citrate buffer was added to each well. Samples were incubated. Then, 100 µL of 0.2 M glycine buffer was added to the samples to stop the reaction. The absorbance was red at 400 nm. The mean of the values was obtained from each duplicate and was used to determine the activity of the enzyme.

## Statistical Analysis

All results were expressed as the mean ± SD of the mean. Significance of differences among groups was determined by either Student's *t*-test or analysis of variance (ANOVA). Most of the data represent results from two or three independent experiments with five mice per group as indicated in the legend of figures. Means were considered statistically different when *p* < 0.05.

## RESULTS

## High-Salt Diet Induced Augmented Arterial Pressure in Mice

To confirm that 4% sodium chloride diet was able to increase the blood pressure, as expected for a HSD, we performed a plethysmography (a technique used to measure changes in body blood volume) in mice. After 3 weeks of HSD consumption, we observed an increase in mean arterial pressure (MAP) in mice fed HSD when compared to mice fed a standard diet (**Figure 1A**).

## High-Salt Diet Induced Histological Signs of Colonic Inflammation and Increase in Intestinal Permeability

Clinical signs of colitis such as weight loss, diarrhea, rectal bleeding, and reduced colon's length were not observed in mice that received HSD when compared to mice fed standard diet (**Figure 1B**). Intestinal permeability was also measured as a more sensitive method to evaluate intestinal damage. **Figure 1C** shows that mice fed HSD had an increased intestinal permeability than control mice. Histological sections where obtained to access microscopic signs of inflammation. HSD-fed mice presented depletion of goblet cells, cellular infiltration in the colonic mucosa, erosion, ulceration, and destruction in *muscularis mucosa* layer, as well as edema in submucosal layer (**Figure 1D**). Histological scores were calculated and it showed inflammatory alterations in the HSD-fed mice (**Figure 1E**).

## High-Salt Diet Increased the Production of IL-23 and the Frequency of IL-23R**<sup>+</sup>** CD4**+** T Cells

Next, we investigated local factors that could lead to intestinal inflammation in HSD-fed mice. Colon extracts were examined for their content of IL-23, a cytokine involved in Th17 and type 3 innate lymphoid cell (ILC3) differentiation. IFN-γ, a typical Th1 cytokine, was also measured. HSD-fed mice presented increased levels of IL-23, but normal levels of IFN-γ in their colons when compared with the control group (**Figures 2A,B**, respectively). An increased frequency of CD4<sup>+</sup> T cells expressing IL-23R<sup>+</sup> in the colonic tissues (**Figure 2C**) was also observed. Th17 are known to secrete IL-17 and this inflammatory cytokine is involved in recruitment of neutrophils, ultimately leading to mucosal inflammation (21). To verify whether increased levels of IL-23 could be related to neutrophil infiltration in the colon, the frequency of these cells was analyzed by flow cytometry. **Figure 2D** shows an increased frequency of neutrophils in the colon of mice fed HSD for 3 weeks. This result was confirmed by the histological analysis of the colonic mucosa showing a significant infiltration of neutrophils in the colons of HSD-fed mice (**Figure 2E**).

## High-Salt Diet Led to Increase in the Frequency of ILC 3 in the Colon

Type 3 innate lymphoid cells (ILC3) are important cells for gut homeostasis but are also involved in the development of intestinal inflammatory diseases (16–18). Considering that IL-23 cytokine, that is elevated in our model, is closely associated with ILC3 differentiation, the frequency of colonic ILC populations where analyzed. ILC1 population was decreased in the colon after 3 weeks of HSD consumption (**Figure 3A**). ILC2 frequency also decreased after 2 and 3 weeks of HSD consumption (**Figure 3B**). However, ILC3 frequency was increased in the colon of mice fed HSD for 2 weeks (**Figure 3C**).

## High-Salt Diet Lead to Gut Inflammation in RAG**−**/**−** Mice but Not in IL-17**−**/**−** Mice

Since we found that consumption of HSD was associated with IL-23 production and increased frequencies of ILC3

(important cells for IL-17 production), we examined next the role of IL-17 as a trigger of inflammation. **Figures 4A,B** show the histological section from colon of wild-type (WT) mice fed either control or HSD, respectively. To evaluate the role of IL-17 in the inflammatory process driven by HSD, we analyzed the histological scores of IL-17<sup>−</sup>/<sup>−</sup> mice that received HSD diet for 3 weeks and no inflammatory infiltration in their colons was found (**Figure 4D**) suggesting the effects of HSD consumption were dependent on IL-17.