The Effect of NOD2 on the Microbiota in Crohn’s Disease

Abstract

Recent advancements toward the treatment of Crohn’s disease (CD) indicate great promise for long-term remission. CD patients suffer from a complex host of dysregulated interactions between their innate immune system and microbiome. The most predominant link to the onset of CD is a genetic mutation in the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (NOD2). NOD2 responds to the presence of bacteria and stimulates the immune response. Mutations to NOD2 promote low diversity and dysbiosis in the microbiome, leading to impaired mucosal barrier function. Current treatments suppress the immune response rather than enhancing the function of this critical protein. New progress towards stabilizing NOD2 signaling through its interactions with chaperone proteins holds potential in the development of novel CD therapeutics.

Graphical abstract

Introduction

Crohn’s disease (CD) is a debilitating, inflammatory bowel disorder that is proposed to arise from an atypical reaction to commensal bacteria. Traditional treatments for CD target reducing inflammation by suppressing the immune response. In instances of severe intestinal damage, antibiotics are also implemented to allow the intestinal tissue to heal. If medical treatments fail, surgical removal of the effected intestine is required to prevent potentially life-threatening complications [1]. Recent discoveries indicate that by decreasing the biodiversity of the microbiome and weakening the immune response, current therapies may be harmful [2]. A better approach for therapeutics may be to enhance microbial diversity and the immune response to avoid relapse.

Recognizing this issue, attention has turned to probing the interactions between the host and microbiome. Genetic predisposition is highly correlated to the onset of CD. Specifically, mutations to the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (NOD2) are the strongest genetic factor in the advancement of CD and development of an aggressive phenotype [3]. NOD2 variants contribute to various aspects of the pathogenesis. CD patients with NOD2 mutations possess a distinct and compromised microbial composition that allows harmful bacteria to thrive [4]. In order to replenish the microbiome with a stable microbial composition, fecal transplant therapy has been explored with astounding success [5–7]. NOD2 mutations are also linked to low levels of mucosal defensins, resulting in a compromised mucosal barrier [8]. Defensin-based therapeutics could compensate for the decreased expression of the necessary anti-microbial peptides without eliminating commensal bacteria [9]. Recent efforts have demonstrated that the NOD2 mutants are unstable, and by enhancing their half-life through interactions with a chaperone protein, appropriate signaling was restored [10]. Use of a pharmacological chaperone to mimic this heightened function has the potential to directly target all mis-signaling events of NOD2 mutants. This review will highlight recent biochemical and basic science advancements towards new therapeutic targets that are based on enhancing the stability of the critical signaling protein, NOD2.

Role of Host Genetics in Disease Predisposition

The emergence of CD has rapidly increased worldwide, steadily augmenting both its prevalence and incidence. Each year approximately 20 new cases (per 100,000 people) are diagnosed in North America, 12 in Europe, and 5 in Asia and the Middle East. Higher incidences in developed countries may be attributed to better diagnostics that can differentiate CD from other irritable bowel disorders [11]. A growing number of environmental factors that are more frequent in developed countries are also linked to pathogenesis including smoking, diet, stress and appendectomy [12].

In addition to environmental influences, genetic predisposition is predominantly linked with increased CD susceptibility. Recent efforts have identified 140 distinct loci with genome-wide significant evidence for CD association [13]. When evaluating all of the single nucleotide polymorphisms associated with CD, NOD2 mutations stand out as statistically most significant [3]. The prevalent mutations include the point mutations R702W and G908R, as well as a frame shift mutation at residue 1007 [14]. At least one NOD2 mutation is present in 30–40% of CD cases compared with 6–7% present in non-diseased controls [15]. NOD2 CD mutations are the strongest genetic factor in determining the complexity of the disease and need for surgical intervention because they are associated with more severe phenotypes [3,15,16].

NOD2 is an intracellular sensor of bacterial cell wall fragments, present in intestinal epithelial cells. It directly binds to a component of the bacterial peptidoglycan called muramyl dipeptide (MDP) which then elicits an immune response through the NF-κB pathway [17,18]. Several genes that affect NOD2 NF-κB signaling and NOD2-induced IL-8 secretion are present in loci associated with CD risk, increasing the significance of NOD2′s central role in CD [19,20]. NOD2 is the most significant therapeutic target for CD based upon its statistical prevalence, severity of the disease phenotype, and interplay with other CD-associated proteins.

Microbiome Shifts in Crohn’s Disease Patients with NOD2 mutants

The microbiome comprises a vast number of diverse microorganisms and greatly influences the immune response. A hallmark of CD is a shift in an individual’s microbiome composition (Table 1). NOD2 mutations lead to an inability to properly regulate commensal bacteria resulting in decreased bacterial diversity and increased susceptibility to pathogenic bacteria. Individuals with irritable bowel disorders typically have a decrease in the commensal Firmicutes and Bacteroides and an increase in Proteobacteria, which includes common pathogens such as E. coli, Salmonella, Helicobacter, and Vibrio [4,21–29]. These changes are often exacerbated by antibiotic treatment in CD patients [1,4].

Table 1.

Shifts in the microbiota of Crohn’s disease patients compared to healthy individuals

Bacteria	Shift	Significance	Ref
Proteobacteria	+	Include pathogenic strains Salmonella, Vibrio, and E.coli	[4,22,24- 26]
Fusobacteriaceae	+	Pathogenic genus of gram negative bacteria	[4]
B. Vulgatus	+	Has been demonstrated to increase inflammatory cytokines	[21,27]
Neisseriaceae	+	Pathogenic bacteria which can cause gonorrhea and meningitis	[4]
H. Hepaticus	+	Pathogenic bacteria	[23,25]
Bifidobacteriaceae	−	Help regulate pathogens and aid digestion	[4]
Bacteroides	−	Can regulate pathogenic invasion and aid digestion	[4,22,25]
Firmincutes	−	Most common flora in healthy individuals	[4,22,26]
F. Prausnitizii	−	Have been demonstrated to decrease IL-12 and IFN-γ production	[4,28]

There is a symbiotic relationship between NOD2 and the microbiome, with NOD2 maintaining the proper balance of commensal bacteria and commensal bacteria stimulating the appropriate levels of NOD2. Germfree mice show decreased levels of NOD2; normal NOD2 gene expression can be restored through inoculation with commensal bacteria [24]. In contrast, NOD2 mutants promote an increased bacterial load and a decrease in diversity, which is associated with diminished bactericidal activity [25,26]. With many changes in the microbiome associated with CD, it is logical that treatments focus on restoring balance to the microbiome. Current treatments include probiotic supplementation [1] and fecal transplants [5–7] to restore the proper balance of commensal bacteria.

Increasing evidence supports a role of inheritance in the composition of the microbiome [30]. However, the abnormal microbiome of CD patients is distinct from family members [31] where the correct composition is directly altered by an improper immune response. Although typically stable, alterations to the microbiome can rapidly occur in response to environmental changes, making relapses inevitable [32,33]. Therefore the long term impact of fecal transplant therapy is of limited use when CD NOD2 mutants result in a reversion of the microbiome to its diseased state. Many long-term successful fecal transplants have been for invasive pathogens not permanent genetic mutations [34]. Therefore, fecal transplants must be coupled with treatments that continue to maintain a proper immune response.

Role of NOD2 in the Impaired Function of the Mucosal Barrier

The gastric mucosal barrier is an integral part of the body’s natural defense against invading pathogens. Permeability of the mucosal layer indicates that the mucosal immune response has been compromised [35]. The mucosal layer of CD patients is often damaged, allowing for pathogen penetration through the epithelium and leading to severe symptoms [36]. To protect the integrity of the mucosal layer, anti-microbial peptides, such as α- and β-defensins, are released extracellularly from epithelial cells. Defensins are effective against a range of pathogens including Gram-positive and Gram-negative bacteria, mycobacteria, and fungi [37]. Defensin levels are significantly reduced in CD patients, with patients harboring a NOD2 mutation showing a greater decrease [38].

Reduced expression of α-defensins predisposes patients to a complicated phenotype of CD, common to NOD2 mutations [38]. NOD2 can up-regulate expression of α-defensins mainly through induction of the NF-κB pathway, but the mutants have diminished activation [8]. Additionally NOD2 mutants interfere with release of α-defensins because they cannot activate ATG16L, a protein involved in packaging and secretion (Figure 1) [39,40]. NOD2 mutations are also linked to low levels of β-defensins. Activation of NOD2 with MDP induces β-defensins and in contrast, overexpression of NOD2 mutant 1007fs, the most frequent NOD2 variant, results in defective induction [41,42]. Additionally the CD mutants have dysregulated β-defensins induction with Vitamin D, a critical component of NOD2 induced β-defensin expression [43].

Figure 1. NOD2 promotes the expression and secretion of α-defensins in Paneth cells.

A. NOD2 activates the NF-κB pathway upon induction with MDP, resulting in the expression of α-defensins. NOD2 interacts with ATG16L, which leads to the secretion of α-defensins. Commensal bacteria remain distant from Paneth cells. B. CD mutant NOD2 has reduced activation of α-defensin expression. It is unable to recruit ATG16L, decreasing secretion of α-defensins. Harmful bacteria can invade the mucosal layer with decreased protection. C. The structure of NOD2-activating ligand MDP.

Defensin-like drugs have successfully been developed as antibacterial treatments for various applications [37]. Prominent members of the microbiome are resilient to antimicrobial peptides; thus the healthy microbiome is not destroyed by introducing defensin-like drugs [9]. Defensin-like drugs have the benefit of having low molecular weights, resistance to proteolysis and potent anti-microbial activity. However, their production costs, potential toxicity and low stability in vivo make them less attractive as a potential CD therapeutic [37].

Interactions with Chaperone Proteins Stabilize NOD2

In the crowded environment of the cell, efficient folding of newly synthesized proteins can be compromised resulting in misfolding and aggregation. To overcome these challenges and ensure protein homeostasis, a variety of molecular chaperones assist in proper folding. Chaperone proteins improve the stability of proteins by enhancing folding energetics [44]. NOD2 recruits the chaperone proteins heat-shock protein 70 (HSP70) and heat-shock protein 90 to confer stability and dissociation from either result in increased degradation [10,45].

In comparison to the wild-type, the NOD2 CD mutants have displayed significant in vivo instability. Interestingly, by overexpressing HSP70, the NOD2 CD mutants have restored stability and correct inflammatory responses [10]. Interactions between HSP70 and NOD2 are critical for proper function as there are also CD mutations of HSP70 [46]. Modulating the interactions between NOD2 and HSP70 becomes a potential target for the inflammatory response brought about by poor signaling. Variation of HSP70 levels has already proven to be successful in reducing inflammation through the innate immune system following brain injury [47,48].

Commensal bacteria have also been shown to regulate HSP70 levels. Bacteroidetes and Firmicutes secrete n-butyrate, which regulates gene expression in intestinal macrophages [49] and when administered at physiological concentrations increases levels of HSP70, inducing an anti-inflammatory effect [50–52]. Thus, it appears that commensal bacteria are potentially utilizing butyrate as a means to induce HSP70 expression to stabilize NOD2 in order to regulate the microbiome and prevent the spread of pathogenic bacteria.

Enhancing the function of NOD2 was previously targeted through small molecules that are derivatives of its ligand, MDP (Figure 1C) [53,54]. The success of these derivatives could be further enhanced by small molecules that mimic HSP70 stabilization (Table 2). Pharmacological chaperones are small molecule mimics of chaperones proteins that bind to a protein, directly affecting its stability. They have successfully been applied to various proteins that lead to a disease state due to instability (e.g. G protein–coupled receptors, neurotransmitter receptors, and glycosidases [55,56]). We propose that the next trend in CD therapeutics will be the implemention of pharmacological chaperones to stabilize the NOD2 mutants.

Table 2. Proposed mechanism of correcting Crohn’s mutant function.

WT NOD2 has appropriate NF-κB signaling upon induction with MDP and enhanced stability. These effects are amplified when HSP70 is overexpressed. The NOD2 CD mutants would similarly benefit to have enhanced stability and NF-κB signaling with overexpressed HSP70 [10].

NOD2 Isoform	Bacterial Cell Wall Ligand	HSP70	Effects
Wild-type	+	−	Increased stability and NF-κB activation
Wild-type	−	+	Increased stability
Wild-type	+	+	Increased stability and enhanced NF-κB activation
CD mutant	+	−	Increased stability and decreased NF-κB activation
CD mutant	−	+	Increased stability
CD mutant	+	+	Increased stability and enhanced NF-κB activation

Conclusions and Outlook

The CD mutants of NOD2 have more widespread influence on the development of CD than any other factor. The NOD2 mutants contribute to pathogenesis by promoting microbial dysbiosis and reducing defensin levels. Potential therapeutics such as fecal transplants could modify the microbiome to a healthy composition. However, the rebounding effect of the highly dynamic microbiome necessitates a solution that addresses the causes of microbiome imbalance. It is evident that a biochemical approach is necessary to correct the response of the permanent genetic mutations that influence CD. Defensin-like drugs could compensate for the negative effects of aberrated signaling but would only mask the underlying issues of the improper microbiome. A superior approach is to enhance the stability and correct the mis-signaling of CD NOD2 mutants through pharmacological chaperones. Manipulating the stability of this central protein would broadly target each of the mechanisms in which it contributes to the pathogenesis of CD.

Highlights.

• NOD2 mutations are strongly linked to Crohn’s disease and its most severe phenotype

• Host microbiome of NOD2 variant patients promotes harmful bacteria and dysbiosis

• Decreased mucosal defensin expression and secretion correlated to NOD2 mutations

• Instability and mis-signaling of NOD2 mutants can be corrected through chaperones