Abstract

   To assess the safety and efficacy of oral immune interventions, it is
   important and required by regulation to assess the impact of those
   interventions not only on the immune system, but also on other organs
   such as the gut as the porte d'entrée. Despite clear indications that
   the immune system interacts with several physiological functions of the
   gut, it is still unknown which pathways and molecules are crucial to
   assessing the impact of nutritional immune interventions on gut
   functioning. Here we used a network-based systems biology approach to
   clarify the molecular relationships between immune system and gut
   functioning and to identify crucial biomarkers to assess effects on gut
   functions upon nutritional immune interventions. First, the different
   gut functionalities were categorized based on literature and EFSA
   guidance documents. Moreover, an overview of the current assays and
   methods to measure gut function was generated. Secondly, gut-function
   related biological processes and adverse events were selected and
   subsequently linked to the physiological functions of the GI tract.
   Thirdly, database terms and annotations from the Gene ontology database
   and the Comparative Toxicogenomics Database (CTD) related to the
   previously selected gut-function related processes were selected. Next,
   database terms and annotations were used to identify the pathways and
   genes involved in those gut functionalities. In parallel, information
   from CTD was used to identify immune disease related genes. The
   resulting lists of both gut and immune function genes showed an overlap
   of 753 genes out of 1,296 gut-function related genes indicating the
   close gut-immune relationship. Using bioinformatics enrichment tools
   DAVID and Panther, the identified gut-immune markers were predicted to
   be involved in motility, barrier function, the digestion and absorption
   of vitamins and fat, regulation of the digestive system and gastric
   acid, and protection from injurious or allergenic material. Concluding,
   here we provide a promising systems biology approach to identify genes
   that help to clarify the relationships between immune system and gut
   functioning, with the aim to identify candidate biomarkers to monitor
   nutritional immune intervention assays for safety and efficacy in the
   general population. This knowledge helps to optimize future study
   designs to predict effects of nutritional immune intervention on gut
   functionalities.

   Keywords: gut assays, biomarkers, safety assessment, efficacy
   assessment, systems biology, immune intervention, network databases

Introduction

   A well balanced immune system is key for overall health and well-being,
   therefore the concept of boosting immunity is gaining in popularity as
   today's complex world presents many potential health challenges. These
   health challenges range from environmental pollution, infections, the
   use of medication, the harmful effects of lifestyle stress and the
   effects of intense physical workouts on the body's natural ability to
   stay healthy. Risk reduction measures or immune health interventions
   may be effective for reducing the loss of health, loss of quality of
   life as well as the costs to society and health care due to immune
   related diseases and disorders.

   Besides beneficial effects of oral immunotherapy and –prophylaxis on
   the functioning of the immune system itself ([41]1–[42]10), (re/un)
   balancing the immune system may also generate dis-immune toxicities.
   These so-called “immune-related adverse events” can come forward in the
   immune system itself [recently addressed in ([43]11)], resulting in,
   for instance, increased incidences or severity of inflammatory
   diseases, but may also affect other immune system-interacting organ
   systems. The gut being the porte d'entrée of oral immunotherapy has
   been selected as one of the key priority organs to include in this
   study. However, despite established association studies that
   immunotherapy can influence gut functioning ([44]12–[45]16), it is
   still largely unknown which immune and gut function-related pathways
   and biomarkers are crucial to monitor in relation to gut functioning
   upon nutritional immune interventions.

   Previously, immune cells and immune cell mediators have been described
   as affecting gut functions in several studies. Yu et al. showed that
   the mucosa of the gastrointestinal tract contains large numbers of
   immunocompetent cells, including mast cells, lymphocytes and
   granulocytes ([46]17). These mast cells play an important role in
   normal physiology functions (regulation of vasodilation, vascular
   homeostasis, innate and adaptive immune responses, angiogenesis, and
   venom detoxification) and pathophysiology (including allergy, asthma,
   anaphylaxis, gastrointestinal disorders, many types of malignancies,
   and cardiovascular diseases) ([47]18). For instance, mast cells are
   known to be involved in gastrointestinal motility, abdominal pain,
   discomfort, and gut barrier function. Mast cells are present in all
   compartments of the gastrointestinal (GI) tract. Upon activation, they
   release an array of inflammatory mediators including histamine,
   5-hydroxytryptamine (5-HT), neutral proteases (tryptases, chymases, and
   carboxypeptidase A), prostaglandins, leukotrienes, platelet activating
   factor (PAF), and several cytokines including tumor necrosis factor-α
   (TNF-α), interleukin (IL)−3,−4,−5,−6 and granulocyte macrophage colony
   stimulating factor (GM-CSF) ([48]17). When the gut is sensitized e.g.,
   in case of irritable bowel syndrome, the infiltration of mast cells and
   the release of mediators are proven to be associated with disturbed
   motility ([49]19). The motility is disturbed by, for example, an
   increase in colonic and intestinal myoelectric spike activity ([50]20),
   the contraction of circular and longitudinal smooth muscle ([51]21),
   and intense duodenal clusters of contraction ([52]22). Additionally,
   mast cells have shown to play a role in chronic pain, mainly at the
   visceral level ([53]23). There is also a positive relationship between
   the intestinal permeability and the number of mast cells with diarrhea
   predominant irritable bowel syndrome ([54]24). The mast cell derived
   tryptase has been identified as a key factor that disrupts the
   intestinal barrier ([55]25). Additionally mast cell mediators, such as
   interferon-γ (IFN-γ), TNF-α, IL-1β, IL-4, IL-13, and prostaglandin E2
   (PGE2), have been shown to have destructive effects on both trans- and
   paracellular permeability ([56]26).

   Aside from mast cells, other immune cells and immune cell mediators can
   also affect gut functions, such as the intestinal barrier function.
   Multiple factors regulate the intestinal barrier, including exogenous
   factors, epithelial apoptosis, cytokines, and immune cells.
   Immune-induced intestinal barrier dysfunction is thought to be critical
   in the predisposition to and exacerbation of numerous autoimmune and
   inflammatory conditions, including inflammatory bowel disease (IBD),
   food allergy, celiac disease and diabetes ([57]27, [58]28). Several
   mucosal immune cells have been implicated in the breakdown of
   intestinal barrier function such as gamma/delta-positive intestinal
   intraepithelial (iIELγδ+) T cells ([59]29) and eosinophils ([60]28).
   Their mediators, such as IFN-γ, TNF-α and some eosinophil granular
   proteins (e.g., major basic protein, eosinophil peroxidase, and
   eosinophilic cationic protein), promote the reorganization of several
   tight junction (TJ) proteins (e.g., zonula occludens-1, junctional
   adhesion molecule A, occludin, claudins-1 and−4) ([61]30) or their
   expression ([62]31), all resulting in a decreased epithelial barrier
   function. In contrast, IL-10 has been shown to positively regulate
   intestinal barrier function ([63]32).

   Food transit is another important feature of gut function. Food transit
   is influenced by the gastrointestinal motility which can be altered by
   inflammation and immune activation. Though the innate immune response
   does not seem to have a major role in muscle function, animal studies
   have shown that a Th1 immune response is associated with
   hypocontractility ([64]33) and a Th2 immune response with
   hypercontractility ([65]34) of inflamed intestinal smooth muscle
   ([66]35). It has been reported that IL-1β plays an important role in
   decreased GI smooth muscle contractility in Th1 cytokines-dominant
   colitis, by downregulating C-kinase-activated protein phosphatase-1
   (PP1) inhibitor, 17 kDa (CPI-17) expression ([67]36–[68]38). The
   inhibitory effect of IL-1β on the GI smooth muscle contraction can also
   be mediated by the upregulation of regulator of G protein signaling 4
   (RGS4) expression by inhibiting NF-κB activation ([69]38, [70]39). Th2
   cytokines may have opposing mechanisms to downregulate RGS4 expression.

   These examples illustrate that the risks and benefits of restoring or
   changing the immune balance by novel treatment strategies of
   immune-related disorders are not limited to immune resistance or the
   inflammatory status as such, but that these immune interventions can
   also impact gut physiology.

   The aim of this study is to illustrate how systems biology can help in
   clarifying the relationships between the immune system and gut
   function, as well as the identification of crucial biomarkers to
   monitor effects on gut functions upon nutritional immune interventions
   in the general population. In this paper we will focus on effects of
   immunonutrition on five major physiological functions of the gut:
   transport/transit of ingested material, extracellular digestion of
   ingested material, intracellular digestion and metabolism, uptake of
   essential nutrients, and protection from injurious or allergenic
   material.

Materials and Methods

Literature Study on the Immune System—Gut Function Relationships

   An inventory of the available literature regarding the relationship
   between the immune system and gut functioning was performed. The
   following biomedical databases were searched: CAB ABSTRACTS, Embase^®,
   MEDLINE^®, Current Contents^® Search, and BIOSIS Previews^®. The
   databases were searched between 15th of February 2017 and 15th of March
   2018.

   The following search string was used: ti,ab ((immune^* OR immuno^* OR
   immuni^*) AND ((intestin^* n/3 function) OR (gut P/0 barrier) OR
   (intestin^* P/0 barrier) OR (gut P/0 integrity) OR (intestin^* P/0
   integrity) OR ((gut OR intestin^*) AND (nutrient^* P/0 absorption)) OR
   (intestin^* p/0 brush p/0 border) OR (gut p/0 brush p/0 border) OR
   ((gut OR intestin^*) AND (peristalsis OR motility Or peristaltic)) OR
   ((gut OR intestin^*) AND (water p/0 absorption)) OR ((gut OR
   intestin^*) AND (transit p/0 time))) NOT (HIV OR “immunodeficiency
   virus” OR AIDS OR “acquired immunodeficiency syndrome” OR “cell line”
   OR dog OR dogs OR canine OR cat OR cats OR feline OR horse OR horses OR
   cattle OR animal OR animals OR veterinary OR broiler OR broilers OR
   chick OR chicks OR chicken. The following criteria were applied to
   obtain the most relevant hits and reduce number of hits: 2007–2018;
   English; Excluding: Conference Abstract, Editorial Material, Editorial,
   Book Chapter, Short Survey, Conference Review, Letter, Chapter, Meeting
   Poster, Note, Erratum, Patent, Book, Corrected and Republished Article,
   Correction, Correspondence, Meeting Summary, Published Erratum, Thesis,
   News.

   During the selection process of relevant manuscripts describing the
   interaction between the immune system and gut function, information
   from manuscripts was collected that describe several key functions and
   clinical endpoints of the gut. In addition, several guidance documents
   were studied to identify those key functions and clinical endpoints of
   the gut that are usually indicated by the regulatory authorities
   ([71]40–[72]42).

Selection of Candidate Biomarkers Linking the Immune System to Gut
Functionality

   First, the different gut functionalities were categorized based on
   literature and EFSA guidance documents, and an overview of the current
   assays and methods to measure gut function was generated. Secondly,
   related adverse events and biological processes were selected based on
   literature and expert knowledge and linked to the physiological
   functions of the GI tract. Thirdly, database terms and annotations from
   Gene Ontology [GO; ([73]www.geneontology.org/) and CTD
   ([74]http://ctdbase.org/); ([75]43)] databases were selected that were
   related to the biological processes as identified previously. Next, key
   pathways involved in those gut functionalities and the involved genes
   were retrieved from the GO and CTD databases: The GO database links
   genes to processes (so called GO terms) related to gut function. The
   CTD database—enabled us to connect genes/proteins to diseases which are
   uniquely identified by MeSH identification numbers (so called MeSH
   IDs). Next, the gut-function related genes were compared with the
   previously identified set of immune health related genes ([76]11).
   Neo4J is a graph-database with query-based calculations (Neo4j, Inc.,
   San Mateo, CA, USA) and is used, together with Venn-diagrams ([77]44)
   to visualize and calculate the unique and overlapping genes/proteins
   among the health endpoints/processes. Using bioinformatics enrichment
   tools DAVID 6.8 [[78]https://david.ncifcrf.gov; bioinformatics
   resources using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   output, updated 2019] and Panther 14.1 [web-based software;
   [79]www.pantherdb.org/pathway, updated 19th December 2018; ([80]45)],
   the identified gut-immune markers were evaluated by predicting the
   effects of these gut-immune genes on gut functions. DAVID was used as
   tool to display the candidate biomarkers on pathway maps from KEGG to
   facilitate the biological interpretation in a network context. To this
   end, the gut-immune genes associated with a specific gut function were
   entered into DAVID after which the top 15 KEGG pathways related to the
   entered genes were retrieved.

   Panther was used as tool to identify GO processes shared between the
   gut and immune system. Hereto, the gut-immune genes associated with a
   specific gut function were entered into Panther after which the top 15
   GO processes related to the entered genes were retrieved. [81]Figure 1
   depicts the workflow and steps for the identification of potential
   candidate markers.

Figure 1.

   [82]Figure 1
   [83]Open in a new tab

   Schematic overview of the work flow to identify gut-function related
   genes possibly affected by immune interventions and to identify
   commonly used assays to measure gut function. The literature search
   resulted in a total of 1,514 manuscripts, of which 1,054 manuscripts
   were considered relevant to gut and immune functions. The information
   from the 1,054 manuscripts was collected resulting in the definition of
   six key gut functions. An overview was made of the commonly used assays
   to study gut function (depicted in [84]Tables 1–[85]4). The adverse
   events related to malfunctioning of the gut and key biological
   processes related to the gut functions were selected from the databases
   Comparative Toxicogenomics Database (CTD; 20 MeSH IDs) and GeneOntology
   (GO; 16 GO processes). Next the genes associated with the 16 GO
   processes and 20 MeSH IDs were retrieved from respectively GO (1,239
   genes) and CTD (741 genes). Thereafter, CTD was used again to retrieve
   3,774 immune disease related genes of the immune health endpoints
   hypersensitivity, autoimmunity, resistance to infection and resistance
   to cancer, as described previously ([86]11). In total 753 genes were
   predicted to be involved in both gut functions and immune functions,
   indicating the strong relationship between the immune system and the
   gut functions. To study which biological processes in the different gut
   functions are predicted to be influenced by the 753 gut-immune related
   genes, a GO enrichment analysis (Panther web-based software;
   [87]www.pantherdb.org/pathway) and an enrichment analysis using DAVID
   (bioinformatics resources using Kyoto Encyclopedia of Genes and Genomes
   (KEGG) pathway output) was performed, resulting in a top 15 selection
   of processes and pathways per gut function (depicted in [88]Tables
   6–[89]9).

Proof of Principle/in silico Test Case

   After selecting a large set of genes involved in the four gut
   functionalities, the next step was to check whether this set of genes
   could be validated by predicting whether an oral immune intervention
   can result in a disturbance of any of the gut functionalities. To this
   end, vitamin D was selected because it is known for its effects on (i)
   the immune response and (ii) adverse/beneficial effects on gut function
   are described, and (iii) top interacting genes are described in the
   CTD. The CTD contains curated data on the top interacting genes
   affected by a chemical/food substance. Using these curated data, the
   top interacting genes were compared with our previous set of
   gut-function related genes, to predict the effects on those
   gut-functionalities after which it was checked whether the predicted
   gut-effects could be confirmed in previously described
   adverse/beneficial effects on gut functionalities.

Results

Literature Study on Immune System—Gut Function Relationships

   The literature study resulted in a total of 1514 articles which were
   reviewed. From these, 30.4% (460) were rejected and 69.6% ([90]1,
   [91]54) were considered relevant. The reasons for rejections were:
   article not in English, species with low physiological similarity
   toward human GUT/immune system (e.g., fish, horses) or not the right
   focus. Of the remaining articles, 54.5% (575) were review articles and
   44.5% (479) were original research articles. During the selection
   process of relevant articles about the interaction between immune
   system and gut function, information describing key functions and
   clinical endpoints of the gut was collected. In addition, several
   guidance documents were studied to identify those key functions and
   clinical endpoints of the gut that are usually specified by the
   regulatory authorities.

Key Gut Functions and Gut Assays to Study the Effects of Oral Immunotherapy

   All collected clinical endpoints and coinciding analyzed parameters
   from literature and guidance documents were structured (see [92]Tables
   1–[93]4). Based on this, we propose that the possible effects of immune
   interventions should be measured on the following four major
   physiological functions of the gut using the corresponding currently
   used assays:

Table 1.

   Methods described in literature to measure transport and transit
   ingested material.
   Gut function Process Method Read out Applicable sites Biological
   samples Example references