Eating the enemy in Crohn's disease: An old theory revisited

Abstract

Several old and new observations suggest the existence in Crohn's disease of a phagocytic disorder of macrophages related to impaired bactericidal activity of host cells or to the presence of invasive bacteria that have developed strategies to counteract macrophage killing. It was recently reported that disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease. Secretion of proinflammatory cytokines by CD macrophages was impaired in response to E. coli or specific Toll-like receptor agonists. In addition, major advances in the etiology of Crohn's disease came from the existence of polymorphism in NOD2 and autophagy-related susceptibility genes (ATG16L1 and IRGM) in patients and from the identification of the presence of adherent-invasive E. coli (AIEC) colonizing the CD ileal mucosa and able to resist to macrophage killing. The role of impaired autophagy in Crohn's disease patients has been recently reinforced by the observation that the peptidoglycan receptor NOD2, in addition to sense intracellular bacteria, can induce autophagy by recruiting the critical autophagy protein ATG16L1 to the plasma membrane during bacterial internalization. Defects in autophagy might be the key element of the pathogenic pathway that lead to defective microbial killing, increased exposure to commensal and pathogenic intestinal bacteria and T cell activation. Defects in Paneth cells secreting lysozyme and antimicrobial peptides are observed in patients with ATG16L1 risk allele. Thus, the induction of autophagy or administration of preparations that mirrors the secretion of Paneth cells or both may be regarded as new therapeutic avenues for the treatment of Crohn's disease.

Crohn's disease, Autophagy, Macrophages, Paneth cells, Adherent-invasive E. coli

1 Introduction

Mononuclear phagocytes have three major functions: presentation of antigens, phagocytosis and immunomodulation. They can eliminate cell waste/debris and kill invading pathogens (viruses, bacteria, fungi, protozoa, and helminthes) at sites of infection or tissue damage and in tumor cells. The phagocytic action of monocytes, macrophages and dendritic cells is mediated by the expression of a large number of cell surface proteins, and by communication with other cells via the cytokines and chemokines. Many microbial pattern recognition receptors are expressed in monocytes and macrophages and are essential components of innate immunity. These receptors recognize pathogen-associated molecular patterns and subsequently initiate host cell response to eradicate invading pathogens. The surface mammalian Toll-like receptor family (TLRs) and the intracellular NOD receptors, which activate NF-κB and stimulate proinflammatory cytokine production by monocytes and macrophages are two classes of receptors that play an important role in innate immunity. ¹ , ²

1.1 Phagocytosis

The phagocytic process was first described in 1884, by Metchinikov, who coined the terms “phagocyte” and “phagocytosis”. ³ Phagocytosis consists in the engulfment of an extracellular particle such as a pathogen by the cell membrane to form an internal vesicle called a phagosome; the fusion of lysosomes with the phagosome creates a phagolysosome, in which the content is degraded and can be released extracellularly via exocytosis or intracellularly to undergo further antigen processing. Phagocytosis, with the generation of reactive oxygen species and hypochlorous acid, is still regarded as the critical killing mechanism for most invading pathogens. ⁴ Both neutrophils and mononuclear macrophages are capable of phagocytosis, but each has distinctive functional properties, depending upon their state of differentiation. Mononuclear phagocytes survive longer than neutrophils. This is clinically important because when neutrophil production is transiently interrupted as, for example, in cancer chemotherapy patients are no longer protected against the overwhelming risk of fatal infection. ⁴ Defective phagocytic activity leads to susceptibility to infections and to granuloma formation. ⁵ Granuloma is a circumscribed inflammatory cell infiltrate surrounding a nidus. A granuloma consists of epitheloid macrophages, giant cells, and lymphocytes, which are mostly CD4+. T cells and macrophages produce inflammatory mediators, such as IL-2, IFN- γ and TFN-α which are considered to be pivotal for granuloma formation. ⁶ A recent study reported disordered phagocytic activity in CD patients, in particular a profound defect in the recruitment of neutrophils at the sites of radiolabelled E. coli subcutaneous injection. In addition, the clearance of these organisms was grossly delayed in CD patients compared to controls. ⁷ On the basis of these observations it has been suggested that CD is a primary immunodeficiency of macrophages.

1.2 Autophagy

The phenomenon of cellular autophagy was first observed by Clark, in 1957. ⁸ Autophagy was initially described as a homeostasis process by which eukaryotic cells sequestrated cytoplasm and degraded damaged organelles or protein aggregates via the lysosomal pathway. ⁹ The cellular events during autophagy follow three major distinct stages (i) initiation by the formation of an isolation membrane (phagophore), (ii) elongation and completion of this membrane to give an autophagosome, and (iii) fusion of the double-membraned autophagosome with the lysosome to form an autolysosome ( Fig. 1 ). This last step leads to the formation of an autophagolysosome with the lysis of the inner membrane and intraluminal contents (for review ¹⁰ ). The term autophagy means “to digest oneself”, but it is now clear that this process also plays an important role in the destruction of some intracellular bacteria and for this reason autophagy is also called xenophagy process (for complete review ¹¹ ). Autophagy was strongly induced by several stimuli that occur during infectious diseases including bacterial components, ¹² , ¹³ phagocytic vacuolar membrane remnants from escaped bacteria ¹⁴ and proinflammatory cytokines such as IFN-γ. ¹⁵ Thus, autophagy process is able to target either cytosolic bacteria such as Streptococcus group A ¹⁶ or bacteria enclosed in phagosome such as Mycobacteria . ¹⁷ For example, in murine and human macrophages M. tuberculosis promotes its intramacrophagic survival by interfering with the classical phagosome maturation pathway. Induced autophagy was shown to overcome the trafficking block imposed by M. tuberculosis and subsequently to reduce intracellular bacteria load. ¹⁵ , ¹⁷ , ¹⁸ As a general rule, dysfunction in autophagy leads to persistent infection of several other pathogens such as Salmonella Typhimurium, Streptococcus pyogenes , and M. tuberculosis . ¹⁵ , ¹⁶ , ¹⁹ , ²⁰ Alterations in autophagy could also offer an open gate for low-level invasive bacteria. In addition to playing a role in innate immunity by directly eliminating bacteria, autophagy is also involved in adaptative immunity. It modulates T cell differentiation and promotes antigen presentation by MHC molecules of peptides originating from the digestion of intraluminal contents in autolysosome (for review ¹¹ ).

2 Dyspeptic macrophage, autophagy and Crohn's disease

The concept of phagocyte dysfunction in CD is not new. Thirty years ago an “eating disorder” of macrophages was posited in the pathogenesis of the disease. It was suggested that CD arises from an inability of the macrophage to degrade a variety of normal gut luminal constituents and the term “dyspeptic”macrophage was coined. ²¹ It was postulated that the abnormal macrophage clearance of luminal antigens leads to the retention of microbial constituents in macrophages and subsequently to the development of granuloma, leakage of lysosomal proteinases and persistent stimulus on immune response, in other words a condition of initial immunodeficiency. However, several reports in the early 1980s failed to confirm this hypothesis as the phagocytic activity of monocytes was seemingly not different in UC and CD and bacterial killing appeared to be normal. ²² , ²³ Thus, there was no evidence of a major macrophage defect in IBD, but monocyte activation was largely confirmed. Of note, these old studies were based on the assessment of monocyte phagocytosis and intracellular killing of Staphylococcus aureus , which is not a granuloma-producing organism. Since then, the relationship of phagocyte dysfunction with Crohn's disease is still puzzling.

A genetically determined defect in CD patient macrophages could be linked to polymorphism in autophagy-related genes, which has been observed in CD patients by reliable genome wide association studies. ²⁴ – ²⁸ Defects in autophagy might be the key element in the pathogenic pathway resulting in defective microbial killing, increased persistence of enteroinvasive bacteria and activation of T cells. Indeed, an association between CD and variants in two separate autophagy genes ( ATG16L1 and IRGM ) has been observed. Thr300Ala substitution within the ATG16L1 (autophagy-related 16 like 1) gene is mostly associated with ileal CD. ²⁴ , ²⁶ ATG16L1 is involved in the early stages of autophagy by allowing growth of the isolation membrane and is recruited by NOD-2 receptor following intracellular bacteria sensing. ¹³ Moreover, the CD-risk allele 300A in ATG16L1 induces an autophagy associated defect in Paneth cells that secrete lysozyme and antimicrobial peptides. ²⁹ Paneth cells expressing 300A variant showed defects in the exocytic pathway, which resulted in lysozyme being absent in the protective mucus layer of the ileum. In addition, genes associated with an inflammatory response such as leptin and adiponectin are upregulated in autophagy-deficient Paneth cells. ²⁹ Further weight for the role of autophagy in the regulation of the inflammatory immune response was lent by a study using mice with ATG16L1 hypomorph expression. It was shown that ATG16L1-mediated autophagy controls inflammasome activation and limits production of inflammatory cytokines in response to bacterial components such as LPS and during DSS-induced colitis. ³⁰ In addition to mutations in the ATG16L1 encoding gene, mutations in gene IRGM (immunity-related GTPase family M) have also been reported in CD patients. ²⁷ , ³¹ In IRGM locus, a synonymous mutation C313T is in strong linkage disequilibrium with a 20-kb deletion polymorphism upstream of the IRGM locus close to the promoter. ³¹ , ³² The deletion may be causative by altering the expression of IRGM between the protective and the risk allele. In human macrophages, IRGM plays a role in the control of intracellular pathogen bacteria like M. tuberculosis . ¹⁵ The level of IRGM is critical to control the rate of bacteria-mediated autophagy. Indeed knockdown of IRGM impaired targeting of bacteria by autophagy and induced uncontrolled bacteria overload in infected cells. ¹⁵ , ³³ Autophagy is also involved in the removal of apoptotic cell corps. ³⁴ This process is crucial for the prevention of tissue inflammation. It is believed that defective clearance of apoptotic cells suppresses tolerance to self antigens and leads to autoimmune diseases such as systemic lupus erythematosus ³⁵ .

3 Bacteria resisting macrophage killing in CD

Another possible link between phagocytic dysfunction and bacteria in CD is the ability of some pathogenic bacteria to resist macrophage killing. Macrophages residing in the intestine or attracted to the site of inflammation might normally serve as a first line of defence by nonspecifically eliminating microorganisms that have penetrated from the intestinal lumen. Pathogens that survive within host phagocytes have various mechanisms of survival (i) by avoiding phagocytosis ³⁶ , ³⁷ (ii) by inhibiting fusion of bacteria-containing phagosomes with lysosomes and endosomes ³⁸ (iii) by remodeling their phagosome ³⁹ (iv) by moving out of the phagosome ⁴⁰ , ⁴¹ or (v) by resisting the antimicrobial environment of the mature phagolysosome. ⁴² As examples, to find a successful intracellular replication niche, Mycobacteria and Salmonella enterica serovar Typhimurium within phagocytic cells have developed a strategy of establishing vacuoles specifically retaining or excluding proteins that govern phagosome maturation. ⁴³ – ⁴⁶ The strategy used by a subgroup of vacuolar pathogens such as Brucella abortus , ^47,Coxiella burnetii , ^48,Legionella pneumophila⁴⁹ and Porphyromonas gingivalis⁵⁰ is to infiltrate the autophagic pathway of host cells.

High levels of E. coli colonizing the small intestinal mucosa have been observed in patients with CD. ⁵¹ – ⁵⁵ These E. coli are true pathogens and are termed adherent-invasive E. coli (AIEC) since they are able to survive and to replicate within epithelial cells and macrophages. ⁵⁶ , ⁵⁷ The observation that AIEC are mostly found in biopsies taken from early lesions of CD postoperative recurrence suggests that such bacteria may have a role in the etiopathogenesis of CD. ⁵⁸ CD-associated AIEC strains adhere to the brush border of ileal enterocytes isolated from CD patients. ⁵⁹ The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) abnormally expressed in the ileal mucosa of CD patients acts as a receptor for AIEC, providing a mechanism for their concentration in the ileal mucosa of CD patients. In addition, a recent study using transgenic mice expressing the human CEACAM6 receptor provided evidence that the AIEC binding to CEACAM6 is required for the development of gut inflammation. ⁶⁰

An interesting virulence property of AIEC is their ability to survive within macrophages. ⁵⁷ While most invasive bacteria induce death of infected macrophages, no necrosis and no apoptosis are observed after infection with AIEC. AIEC bacterial replication does not require bacterial escape into the cytoplasmic compartment, in contrast to its behaviour within intestinal epithelial cells. ⁵⁶ Within J774-A1 macrophages, the bacteria induced the formation of a single spacious vacuole by fusion of initial phagosomes ( Fig. 2 ). Such large vacuoles containing numerous bacteria have also been observed with macrophages infected with Salmonella spp. or Yersinia enterocolitica . ³⁸ , ⁶¹ The formation of this specific compartment within AIEC-infected macrophages is probably the key to their ability to resist macrophage killing since spacious-phagosome formation may promote bacterial survival by dilution of toxic lysosomal compounds or attenuation of antimicrobial factors. AIEC-infected macrophages secrete levels of TNF-α much higher than those secreted by LPS-stimulated macrophages. As TNF-α is transcribed and translated de novo after macrophage stimulation, the synthesis of this cytokine demonstrated that macrophages were still active even with numerous intracellular bacteria. Moreover, proinflammatory TNF-α secretion reflects an activation of infected macrophages and thus AIEC-infected macrophages are likely to be continuously activated by the sustained presence of intracellular bacteria.

The behaviour of AIEC with macrophages indicates that these pathogens, which have evolved to resist phagocytosis and to persist within macrophages, may be involved in chronic antigenic stimulation, T cell and macrophage activation, and in the formation of granulomas. Such a hypothesis is also supported by the fact that (i) E. coli antigens are present in CD granulomas ⁶² , ⁶³ (ii) E. coli DNA is detected in 80% of microdissected granulomas of CD patients ⁶⁴ and (iii) diseases with granulomatous responses to E. coli have been reported in animals such as the granulomatous colitis of Boxer dogs or Hjarre's disease in chicken and turkeys. ^65,E. coli strains are isolated from granulomas of 100% Boxer dogs with colitis and share properties with AIEC, and in Hjarre's disease mucoid E. coli have been isolated from tuberculoid lesions of the caecum and liver of chicken and turkeys, and the intramuscular inoculation of pure bacterial cultures or triturated diseased tissues reproduced the disease. ⁶⁵ – ⁶⁷ Another interesting observation reported by Meconi et al. ⁶⁸ is the ability of AIEC LF82 crude protein extracts to mediate an equivalent cell aggregation as that of the live bacteria. In contrast, non pathogenic E. coli K-12 crude protein extracts did not induce greater cell aggregation than the live K-12 bacteria. Thus cellular receptor(s) specialized in the recognition of bacterial antigens present in AIEC but absent in non pathogenic E. coli may be directly involved in this granulomatous response.

4 Behaviour of AIEC in autophagy-deficient host cells

Inside host cells, certain intracellular pathogens control the fate of their membrane-bound compartments, and escape host degradation by interfering with the endocytic pathway or by infiltrating the autophagic route. ⁶⁹ The defective autophagic pathway associated with genetic susceptibility to CD suggests that a primary causal organism would require (i) initial invasive ability and also (ii) an inherent mechanism for chronic intracellular persistence. Thus, the normal enteric flora is not sufficiently invasive or lacks virulence factors that are able to initiate and sustain disease progression. AIEC is a bacterial candidate that exhibits these properties and is detected in CD patients. We recently reported that AIEC bacteria can take advantage of defects in autophagy to replicate within host cells. Infection of autophagy-deficient (Atg5−/−) versus wild-type mouse embryonic fibroblasts indicated that autophagy can restrict the replication of AIEC intracellular bacteria, which are sequestered as single bacteria in multilamellar membrane vacuoles, corresponding to autophagosomes. ³³ Such a property is not found with any other E. coli strains, including non pathogenic, environmental, commensal or pathogenic bacteria such as enterotoxigenic E. coli , enteropathogenic E. coli , enteroinvasive E. coli or diffusely-adhering E. coli . Unlike Shigella spp., an invasive bacterium able to escape autophagy by secreting bacterial effectors by means of the type III secretion system, ⁷⁰ a subpopulation of the AIEC bacteria infiltrate autophagy and their replication can be therefore under host cell control. Functional knockdown of ATG16L1 by siRNA abrogated the autophagy of AIEC bacteria and rescue of ATG16L1 expression using constructs containing protective *300T allele resulted in full efficient autophagy of AIEC bacteria. ¹⁹ , ³³ However, the CD-associated risk *300A allele was unable to mediate fully effective antibacterial autophagy to control intracellular AIEC replication. Likewise, when intracellular IRGM expression was reduced by specific siRNA, a great increase in the number of AIEC intracellular bacteria was observed, indicating a key role of IRGM in controlling intracellular AIEC replication. ³³ Similarly, as shown with dendritic cells (DC) expression of NOD2 CD-associated variant leads to impaired autophagy upon infection or stimulation with muramyl dipeptide, the minimal fragment of peptidoglycan activating NOD2. Interestingly, decreased AIEC killing within DCs in the presence of CD-variant NOD2 has been reported. ⁷¹ The inability of CD-variant NOD2-expressing DCs to traffic bacteria effectively was linked to defects in muramyldipeptide-mediated autophagy. Thus mutations in autophagy-related or in nod2 genes result in similar defects in the autophagy process and in the control of intracellular bacteria replication ( Fig. 3 ).

5 Therapeutic implications

Although CD is a complex genetic disorder in which multiple genetic and non genetic factors contribute to disease susceptibility, impairment of the autophagic machinery as observed in CD patients indicates that agents able to enhance autophagy to promote microbial killing could be therapeutic tools. Such a therapy could even be used to avoid CD patients to be colonized by adherent-invasive E. coli , which are able to replicate within macrophages but which are sensitive to induced autophagy. Autophagy can be achieved by rapamycin, a drug that inhibits the serine/threonine kinase mammalian target of rapamycin (mTOR). An additional important consideration is that rapamycin also has immunosuppressive and antifibrotic effects that may prove beneficial in CD. Two recent case reports showed that rapamycin may be effective in refractory CD. ⁷² , ⁷³ The oral administration of the drug and its safety profile makes the use of rapamycin possible in CD. In addition, lysozyme or preparations that mirror the secretions of Paneth cells could also be of therapeutic interest since impaired function of Paneth cells is observed in CD patients having mutations in NOD2 or autophagy-related genes.

Abbreviations

Abbreviations
• AIEC

  adherent-invasive Escherichia coli

• CD

  Crohn's disease

• CEACAM

  carcinoembryonic antigen-related cell adhesion molecule

• NOD2

  nucleotide-binding oligomerization domain containing 2

• IRGM

  immunity-related GTPase family M

• ATG16L1

  autophagy-related like 1

Figures

Figure 1

Schematic presentation of the formation and maturation of autophagosomes in mammalian cells.

Figure 2

Transmission electron micrograph of AIEC LF82 bacteria-infected J774-A1 macrophages at 48h post-infection. Note the very high number of intracellular AIEC bacteria in a large vacuole.

Figure 3

CD-associated polymorphisms in ATG16L1 or Nod2 lead to impaired autophagy and absence of intracellular bacteria clearance.