NOD2 is dispensable for ATG16L1 deficiency-mediated resistance to urinary tract infection

NOD2 (nucleotide-binding oligomerization domain containing 2) functions as a pathogen sensor and is involved in development of Crohn disease, a form of inflammatory bowel disease. NOD2 functions in concert with the autophagy protein ATG16L1, which is also implicated in Crohn disease. Recently, we identified a novel protective role of ATG16L1 deficiency in uropathogenic Escherichia coli-induced urinary tract infections (UTIs), which are common infectious diseases in humans. Given the known roles of NOD2 in recruiting ATG16L1 to the bacterial entry site, autophagy induction, and Crohn disease, we hypothesized that NOD2 may also play an important role in UTI pathogenesis. Instead, we found evidence that NOD2 is dispensable in the pathogenesis of UTIs in mice and humans. First, loss of Nod2 did not affect the clearance of bacteriuria and the recruitment of innate immune cells to the bladder. Second, we showed that, although nod2^?/? mice display increased kidney abscesses in the upper urinary tract, there were no increased bacterial loads or persistence in this niche. Third, although a previous study indicates that loss of Nod2 reverses the protection from intestinal infection afforded by loss of ATG16L1 in mice, we found NOD2 deficiency did not reverse the ATG16L1-deficiency-induced protection from UTI. Finally, a population-based study of a cohort of 1819 patients did not reveal any association of NOD2 polymorphisms with UTI incidence. Together, our data indicated that NOD2 is dispensable for UTI pathogenesis in both mice and humans and does not contribute to ATG16L1-deficiency-induced resistance to UTI in mice.     

ATG16L1 and pathogenesis of urinary tract infections

Autophagy is generally considered to be antipathogenic. The autophagy gene ATG16L1 has a commonly occurring mutation associated with Crohn disease (CD) and intestinal cell abnormalities. Mice hypomorphic for ATG16L1 (ATG16L1^HM) recreate specific features of CD. Our recent study shows that the same ATG16L1^HM mice that are susceptible to intestinal inflammatory disease are protected from urinary tract infections (UTI), a common and important human disease primarily caused by uropathogenic E. coli (UPEC). UPEC colonize the bladder and exhibit both luminal and intra-epithelial stages. The host responds by recruiting innate immune cells and shedding infected epithelial cells to clear infection. Despite these countermeasures, UPEC can persist within the bladder epithelium as membrane-enclosed quiescent intracellular reservoirs (QIRs) that can seed recurrent UTI. The mechanisms of persistence remain unknown. In this study, we show that ATG16L1 deficiency protects the host against acute UTI and UPEC latency. ATG16L1^HM mice clear urinary bacterial loads more rapidly and thoroughly due to ATG16L1-deficient innate immune components. Furthermore, ATG16L1^HM mice exhibit superficial urothelial cell-autonomous architectural aberrations that also result in significantly reduced QIR numbers. Our findings reveal a host-protective effect of ATG16L1 deficiency in vivo against a common pathogen.     

Evidence of expression variation and allelic imbalance in Crohn's disease susceptibility genes NOD2 and ATG16L1 in human dendritic cells

Human dendritic cells (DCs) play an important role in induction and progression of Crohn's disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~ 40% and ~ 70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.     

ATG16L1 and pathogenesis of urinary tract infections

Autophagy is generally considered to be antipathogenic. The autophagy gene ATG16L1 has a commonly occurring mutation associated with Crohn disease (CD) and intestinal cell abnormalities. Mice hypomorphic for ATG16L1 (ATG16L1^HM) recreate specific features of CD. Our recent study shows that the same ATG16L1^HM mice that are susceptible to intestinal inflammatory disease are protected from urinary tract infections (UTI), a common and important human disease primarily caused by uropathogenic E. coli (UPEC). UPEC colonize the bladder and exhibit both luminal and intra-epithelial stages. The host responds by recruiting innate immune cells and shedding infected epithelial cells to clear infection. Despite these countermeasures, UPEC can persist within the bladder epithelium as membrane-enclosed quiescent intracellular reservoirs (QIRs) that can seed recurrent UTI. The mechanisms of persistence remain unknown. In this study, we show that ATG16L1 deficiency protects the host against acute UTI and UPEC latency. ATG16L1^HM mice clear urinary bacterial loads more rapidly and thoroughly due to ATG16L1-deficient innate immune components. Furthermore, ATG16L1^HM mice exhibit superficial urothelial cell-autonomous architectural aberrations that also result in significantly reduced QIR numbers. Our findings reveal a host-protective effect of ATG16L1 deficiency in vivo against a common pathogen.     

The complex interplay of NOD-like receptors and the autophagy machinery in the pathophysiology of Crohn disease

Several coding variants of NOD2 and ATG16L1 are associated with increased risk of Crohn disease (CD). NOD2, a cytosolic receptor of the innate immune system activates pro-inflammatory signalling cascades upon recognition of bacterial muramyl dipeptide, but seems also to be involved in antiviral and anti-parasitic defence programs. The CD associated variant L1007fsinsC leads to impaired pro-inflammatory signalling and diminished bacterial clearance. ATG16L1 is a protein essential for autophagosome formation at the phagophore assembly site. The CD associated T300A variant is located in the c-terminal WD40 domain, whose function is still unknown. Basal autophagy is not affected by the T300A variant, but antibacterial autophagy (xenophagy) is impaired, a finding that relates ATG16L1 as well as NOD2 to pathogen defence. Notably, combination of disease-associated alleles of ATG16L1 and NOD2/CARD15 leads to synergistically increased susceptibility for CD, indicating a possible crosstalk between NOD2- and ATG16L1-mediated processes in the pathogenesis of CD. This review surveys current research results and discusses the functional models of potential interplay between NLR-pathways and xenophagy. Interaction between pathways is discussed in the context of reactive oxygen species (ROS), membrane co-localisation, antigen processing and implications of disturbed Paneth cell vesicle export. These effects on pathogen response might imbalance the intestinal barrier epithelia towards chronic inflammation and promote development of Crohn disease. Further elucidation of NOD2/ATG16L1 interplay in xenophagy is relevant for understanding the aetiology of chronic intestinal inflammation and host-microbe interaction in general and could lead to principal new insights to xenophagy induction.     

ATG16L1 polymorphisms are associated with NOD2-induced hyperinflammation

In recent years considerable advances in understanding the pathogenesis of Crohn disease have been achieved, with the identification of susceptibility variants of genes that are part of the autophagy machinery, i.e., ATG16L1 and IRGM. Subsequent functional studies have been conducted to unravel the underlying mechanism of this genetic association. For the ATG16L1 Thr300Ala polymorphism (c.898A > G, rs2241880), it was demonstrated that the risk variant is associated with a reduced capacity of innate immune cells to induce autophagy upon triggering with specific microbial structures such as peptidoglycans, that are specifically recognized by the intracellular pattern-recognition receptor nucleotide oligomerization domain-2 (NOD2). Due to the impaired autophagy activation, autophagosome formation and the subsequent antigen presentation through the major histocompatibility complex are diminished, leading to decreased immune activation. However, these findings arguing for defective host defense mechanisms in individuals bearing the ATG16L1 300Ala variant, and subsequent bacterial persistence in the gut mucosa, provide no conclusive explanation for the excessive inflammation observed in Crohn disease.     

A role for vimentin in Crohn disease

Crohn disease (CD), one of the major chronic inflammatory bowel diseases, occurs anywhere in the gastrointestinal tract with discontinuous transmural inflammation. A number of studies have now demonstrated that genetic predisposition, environmental influences and a dysregulated immune response to the intestinal microflora are involved. Major CD susceptibility pathways uncovered through genome-wide association studies strongly implicate the innate immune response (NOD2), in addition to the more specific acquired T cell response (IL23R, ICOSLG) and autophagy (ATG16L1, IRGM). Examination of the disease-associated microbiome, although complex, has identified several potentially contributory microorganisms, most notably adherent-invasive E.coli strains (AIEC), which have been isolated by independent investigators in both adult and pediatric CD patients. Here we discuss our recent finding that the type-III intermediate filament (IF) protein VIM/vimentin is a novel NOD2 interacting protein that regulates NOD2 activities including inflammatory NFKB1 signaling, autophagy and bacterial handling.     

ATG16L1: A multifunctional susceptibility factor in Crohn disease

Genetic variations in the autophagic pathway influence genetic predispositions to Crohn disease. Autophagy, the major lysosomal pathway for degrading and recycling cytoplasmic material, constitutes an important homeostatic cellular process. Of interest, single-nucleotide polymorphisms in ATG16L1 (autophagy-related 16-like 1 [S. cerevisiae]), a key component in the autophagic response to invading pathogens, have been associated with an increased risk of developing Crohn disease. The most common and well-studied genetic variant of ATG16L1 (rs2241880; leading to a T300A conversion) exhibits a strong association with risk for developing Crohn disease. The rs2241880 variant plays a crucial role in pathogen clearance, resulting in imbalanced cytokine production, and is linked to other biological processes, such as the endoplasmic reticulum stress/unfolded protein response. In this review, we focus on the importance of ATG16L1 and its genetic variant (T300A) within the elementary biological processes linked to Crohn disease.     

Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.     

ATG16L1 polymorphisms are associated with NOD2-induced hyperinflammation

In recent years considerable advances in understanding the pathogenesis of Crohn disease have been achieved, with the identification of susceptibility variants of genes that are part of the autophagy machinery, i.e., ATG16L1 and IRGM. Subsequent functional studies have been conducted to unravel the underlying mechanism of this genetic association. For the ATG16L1 Thr300Ala polymorphism (c.898A > G, rs2241880), it was demonstrated that the risk variant is associated with a reduced capacity of innate immune cells to induce autophagy upon triggering with specific microbial structures such as peptidoglycans, that are specifically recognized by the intracellular pattern-recognition receptor nucleotide oligomerization domain-2 (NOD2). Due to the impaired autophagy activation, autophagosome formation and the subsequent antigen presentation through the major histocompatibility complex are diminished, leading to decreased immune activation. However, these findings arguing for defective host defense mechanisms in individuals bearing the ATG16L1 300Ala variant, and subsequent bacterial persistence in the gut mucosa, provide no conclusive explanation for the excessive inflammation observed in Crohn disease.     

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.     

A review of major Crohn’s disease susceptibility genes and their role in disease pathogenesis

Crohn’s disease (CD) is a chronic inflammatory bowel disease whose relevance is increasing in industrialized society. Recent genome wide association studies revealed over seventy one loci associated with disease penetrance. Several variants that increase disease risk encode for altered proteins that diminish bacterial host defense. NOD2 alters intracellular bacterial sensing while ATG16L1 is thought to diminish bacterial clearance by impairing autophagy. Additionally, changes in the IBD5 locus are thought to diminish barrier function. Alternatively, recent data indicate a gain of function genetic variant of IL23R is protective amongst European CD patients. These recent genetic discoveries contradict historical theories that Crohn’s disease results from overactive auto-aggressive responses. Rather, new genetic data suggest disease-associated variants encode for dysfunctional proteins that diminish essential innate immune responses against commensal organisms. This review provides an overview of these critical discoveries and places them in their biological context.     

Contribution of NOD2 to lung inflammation during Staphylococcus aureus-induced pneumonia

Staphylococcus aureus is the most commonly found Gram-positive bacterium in patients admitted in intensive-care units, causing septicaemia or pneumonia. In this work, we investigated the role of NOD2 in S. aureus-induced pneumonia. We found that the absence of NOD2 affected weight loss and recovery speed. Nod2?/? mice showed a reduced lung inflammation in comparison to wild-type animals, with lower presence of cytokines in broncho-alveolar lavage fluids and reduced recruitment of neutrophils. Furthermore, histological analysis of the lungs revealed less severe lesions in Nod2?/? mice at day 2 and day 7 post-infection. In conclusion, we demonstrated that NOD2 is not a crucial receptor to fight S. aureus-induced pneumonia, but that it contributes to the inflammatory response in the lungs. Interestingly, the absence of NOD2 led to a lesser inflammation and was finally beneficial for the animal recovery.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA (+) but not VacA (-) strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.     

A CARD9 Polymorphism Is Associated with Decreased Likelihood of Persistent Conjugated Hyperbilirubinemia in Intestinal Failure

Recently, genetic associations have been described in intestinal transplants. Namely, Crohn''s disease susceptibility gene NOD2 polymorphisms have been reported to be more prevalent in patients with graft failure following intestinal transplantation (IT). Therefore, we sought to determine if polymorphisms in the NOD2 signaling cascade, including NOD2, CARD9, RAC1 and ATG16L1 are associated with intestinal failure (IF) or its complications. We carried out a cross-sectional study of 59 children with IF and 500 healthy Caucasian controls. Using the Taqman platform we determined the prevalence of NOD2 as well as ATG16L1, RAC1 and CARD9 SNPs. NOD2 pathway polymorphisms were evaluated in relation to outcomes of episodes of sepsis, ICU admissions, hyperbilirubinemia and need for IT. We found that the minor allele of a CARD9 SNP was associated with protection from developing IF when compared to healthy controls and was also associated with decreased odds of sustained conjugated hyperbilirubinemia. Therefore, IF patients with CARD9 polymorphism are less likely to develop progressive liver disease and suggests that host innate immunity may play a role in IF associated liver disease.     

A role for vimentin in Crohn disease

Crohn disease (CD), one of the major chronic inflammatory bowel diseases, occurs anywhere in the gastrointestinal tract with discontinuous transmural inflammation. A number of studies have now demonstrated that genetic predisposition, environmental influences and a dysregulated immune response to the intestinal microflora are involved. Major CD susceptibility pathways uncovered through genome-wide association studies strongly implicate the innate immune response (NOD2), in addition to the more specific acquired T cell response (IL23R, ICOSLG) and autophagy (ATG16L1, IRGM). Examination of the disease-associated microbiome, although complex, has identified several potentially contributory microorganisms, most notably adherent-invasive E.coli strains (AIEC), which have been isolated by independent investigators in both adult and pediatric CD patients. Here we discuss our recent finding that the type-III intermediate filament (IF) protein VIM/vimentin is a novel NOD2 interacting protein that regulates NOD2 activities including inflammatory NFKB1 signaling, autophagy and bacterial handling.     

Autophagy suppresses interleukin-1β (IL-1β) signaling by activation of p62 degradation via lysosomal and proteasomal pathways

ATG16L1 is an essential component of the autophagasome. The T300A allele of ATG16L1 is associated with the increased susceptibility to Crohn disease. In this study, we identified a novel function of ATG16L1, which suppresses signaling of the pro-inflammatory cytokine IL-1β. Deletion of ATG16L1 in mouse embryonic fibroblasts significantly amplifies IL-1β signal transduction cascades. This amplification is due to elevated p62 levels in ATG16L1-deficient cells. We found that ATG16L1 regulates p62 levels via both autolysosomal and proteasomal pathways. For proteasomal degradation, we found that Cullin-3 (Cul-3) is a E3 ubiquitin ligase of p62 and that ATG16L1 is essential for neddylation of Cul-3, a step required for Cul-3 activation. Taken together our data indicate that loss-of-function of ATG16L1 results in a hyper-responsiveness to the IL-1β signaling because of the increased p62 level.     

Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD) 2 in Host Defense during Pneumococcal Pneumonia

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD) 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2 ^-/-) and wild-type (Wt) alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2 ^-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39), an isogenic capsule locus deletion mutant (D39Δcps) or serotype 3 S. pneumoniae (6303) via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2 ^-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2 ^-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2 ^-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2 ^-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.     

NOD2-mediated autophagy and Crohn disease

Autophagy is important in immune cells as a means of disposing of pathogens and in connecting with the antigen presentation machinery to facilitate immune priming and initiation of a correctly targeted adaptive immune response. While Toll-like receptors (TLRs) are known to regulate autophagy in this context, the extent to which other pattern recognition receptors (PRRs) are involved has been unclear. NOD2 is an intracellular PRR of the Nod-like receptor (NLR) family that is notable in that variants in the ligand recognition domain are associated with Crohn disease (CD). Our recent study shows NOD2 activates autophagy in a manner requiring ATG16L1, another CD susceptibility gene. NOD2 autophagy induction is required for bacterial handling and MHC class II antigen presentation in human dendritic cells (DCs). CD patients DCs expressing CD risk variant NOD2 or ATG16L1 display reduced autophagy induction after NOD2 triggering resulting in reduced bacterial killing and defective antigen presentation. Aberrant bacterial handling and immune priming could act as a trigger for inflammation in CD.     

Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p

Multiple genetic studies have implicated the autophagy-related gene, ATG16L1, in the pathogenesis of Crohn disease (CD). While CD-related research on ATG16L1 has focused on the functional significance of ATG16L1 genetic variations, the mechanisms underlying the regulation of ATG16L1 expression are unclear. Our laboratory has described that microRNAs (miRNAs), key regulators of gene expression, are dysregulated in CD. Here, we report miRNA-mediated regulation of ATG16L1 in colonic epithelial cells as well as Jurkat T cells. Dual luciferase reporter assays following the transfection of vectors containing the ATG16L1 3′-untranslated region (3′UTR) or truncated 3′UTR fragments suggest that the first half of ATG16L1 3′UTR in the 5′ end is more functional for miRNA targeting. Of 5 tested miRNAs with putative binding sites within the region, MIR142-3p, upon transient overexpression in the cells, resulted in decreased ATG16L1 mRNA and protein levels. Further observation demonstrated that the luciferase reporter vector with a mutant MIR142-3p binding sequence in the 3′UTR was unresponsive to the inhibitory effect of MIR142-3p, suggesting ATG16L1 is a gene target of MIR142-3p. Moreover, the regulation of ATG16L1 expression by a MIR142-3p mimic blunted starvation- and L18-MDP-induced autophagic activity in HCT116 cells. Additionally, we found that a MIR142-3p inhibitor enhanced starvation-induced autophagy in Jurkat T cells. Our study reveals MIR142-3p as a new autophagy-regulating small molecule by targeting ATG16L1, implying a role of this miRNA in intestinal inflammation and CD.