Macrophages Versus Escherichia coli: A Decisive Fight in
Crohn
’s Disease
Anthony Buisson, MD, PhD,*
,†Marie-Agnès Bringer, PhD,
‡Nicolas Barnich, PhD,* and Emilie Vazeille, PhD*
,†Abstract: The pathophysiology of Crohn’s disease (CD), a chronic inflammatory bowel disease, remains imperfectly elucidated. Consequently, the therapeutic armamentarium remains limited and has not changed the natural history of CD hitherto. Accordingly, physicians need to identify new therapeutic targets to be able to alter the intestinal damage. The most recent hypothesis considered CD as resulting from an abnormal interaction between microbiota and host immune system influenced by genetics and environmental factors. Several experimental and genetic evidence point out intestinal macrophages in CD etiology. An increase of macrophages number and the presence of granulomas are especially observed in the intestinal mucosa of patients with CD. These macrophages could be defective and particularly in responses to infectious agents like CD-associated Escherichia coli. This review focuses on, what is currently known regarding the role of macrophages, macrophages/E. coli interaction, and the impact of CD therapies on macrophages in CD. We also speculate that macrophages modulation could lead to important translational implications in CD with the end goal of promoting gut health.
(Inflamm Bowel Dis 2016;22:2943–2955)
Key Words: E. coli, Macrophages, Crohn’s disease
T
he intestine is the largest reservoir of macrophages in the whole body, and macrophages are the most abundant mono-nuclear phagocytes in the gut lamina propria (LP).1 Gutmacro-phages are strategically located in the subepithelial area of intestinal LP and in the gut-associated lymphoid tissue, particu-larly in Peyer’s patches (PPs) which form the interface between the gut-associated lymphoid tissue and the luminal microenviron-ment. These locations allow intestinal macrophages to be very close to luminal bacteria and antigens.2 Functions of intestinal
macrophages include the maintenance of homeostasis due to the tolerance of the commensal flora, the protection of the mucosa from pathogens, the elimination of cellular debris, and the regu-lation of inflammatory responses against luminal bacteria.
Crohn’s disease (CD) is a chronic disorder that causes inflam-mation of the gastrointestinal tract and affects as many as 2 million patients worldwide.3This disease can be particularly debilitating in
affecting the entire digestive tract and can alter significantly patients’ quality of life. The therapeutic armamentarium remains limited (steroids, immunosuppressants, anti-TNF agents, and anti-integrins therapies) and noncurative in CD, hence the necessity to better under-stand the disease to identify the best targets. Although the complex etiology of CD has not been yet fully elucidated, several experimental and genetic evidence point out defective innate immunity in CD. Thus, several studies have shown that the number of intestinal macrophages is increased in patients with CD4–7 and that CD is characterized by the frequency of aggregates of epithelioid macro-phages referred to as granulomas, the histopathological hallmark of CD.8,9 In addition, macrophages from patients with CD present
disorders of their cytokine secretion profiles and particularly in response to infectious agents.10–13Taken together, these defects could result in impaired detection and clearance of bacteria and unappropri-ated inflammatory response to infection. Gut bacterial dysbiosis asso-ciated with CD should also play a causal role in chronic inflammation. Patients with CD, compared with healthy controls, have fewer bacteria with anti-inflammatory properties such as Faecalibacterium prausnit-zii in the Firmicutes phylum, and more bacteria with proinflammatory properties such as Escherichia coli, in the Proteobacteria phylum, and more particularly adherent-invasive E. coli a.k.a. AIEC.14
Here, we report the synthesis of the current knowledge and recent advances on macrophages in CD pathogenesis. This review consists of three main parts. In thefirst part, we present intestinal macrophages and describe abnormalities related to macrophages that have been observed in CD. The second part is dedicated to interactions between CD-associated E. coli and macrophages. In the last part, we discuss the impact of CD therapy on macrophages and their activities.
Received for publication August 3, 2016; Accepted August 17, 2016.
From the *Clermont Université, UMR 1071 Inserm/Université d’Auvergne,
USC-INRA 2018, Clermont-Ferrand, France;†Department of Hepato-Gastroenterology,
University Hospital Estaing of Clermont-Ferrand, Université d’Auvergne,
Clermont-Ferrand, France; and‡INRA UMR1324, CNRS UMR6265, University of Bourgogne
Franche-Comté, Centre des Sciences du Goût et de l’Alimentation, Dijon, France.
This review is dedicated to the memory of Arlette Darfeuille-Michaud. Address correspondence to: Anthony Buisson, MD, PhD, Department of Hepatogastroenterology, University Hospital Estaing, 1 place Aubrac, 63000 Clermont-Ferrand, France (e-mail: a_buisson@hotmail.fr); or Emilie Vazeille,
PhD, UnitéM2iSH, UMR Inserm/Université d’Auvergne U1071, Université
d’Auvergne, CBRV, 28 17 place Henri Dunant, 63000 Clermont-Ferrand, France
(e-mail: emilie.vazeille@udamail.fr).
Copyright © 2016 Crohn’s & Colitis Foundation of America, Inc.
DOI 10.1097/MIB.0000000000000946 Published online 4 November 2016.
INTESTINAL MACROPHAGES
Intestinal Macrophages and Their Functions
Primitive macrophages rising from yolk sac–blood islands spread into embryos on the establishment of the blood circulatory system and colonize the whole embryo.15In parallel, the
hema-topoiesis occurring in the fetal liver generates monocytes that will gain access to the blood and reach tissues, except the brain, a few days after yolk sac–derived macrophages. Fetal liver–derived monocytes will thereafter proliferate and differentiate into mac-rophages. After birth, these embryonic precursor cells are replaced in the intestine around the time of weaning by cells derived from blood monocytes which express the glycoprotein lymphocyte antigen 6C (Ly6C).16These cells are continuously recruited in the
intestine to renew or enrich the pool of resident macrophages. Factors influencing monocytes recruitment in the intestine include expression of C-C chemokine receptor type 2 (CCR2), cytokines (transforming growth factor-
b
[TGF-beta], interleukin-8 [IL-8]), and the microbiota.17During intestinal macrophages differentiation and through different steps, cells acquire several surface markers such as major histocompatibility complex II (MHC II), glycoprotein F4/80, clusters of differentiation (CD) 64, 11c, 11b, 40, 68, 80, 86, and 14, CCR2, toll-like receptors (TLRs) 2 and 4, and CX3C chemokine receptor 1 (CX3CR1).18A large heterogeneity of resident macrophages exists
in the intestine, and molecular signaling of their differentiation is not yet fully understood.16 This may reflect exposure of monocytes to
local signals such as epithelial or stromal secretions (thymic stromal lymphopoietin, TGF-
b
, prostaglandin E2 [PGE2], semaphorin 7A, and colony stimulating factors 1 and 2), and exposure to specific cellular microenvironment such as abundance of innate and adaptive lymphocytes (type 2 innate lymphoid cell [ILC2], natural killer [NK] and natural killer T [NKT] cells, B cells,ab
and gd T lymphocytes), mast cells, and other myeloid cells. Macrophage differentiation is also under the influence of the microbiota.19,20 Mortha et al20showed that intestinal microbiota modulates the production of the colony stimulating factor 2 and, as a consequence, the number of colonic macrophages.
Alongside dendritic cells, intestinal macrophages ensure the role of guardians of the gut.18They maintain intestinal homeostasis,
epithelial integrity, wound healing, and are key actors in intestinal inflammatory responses.17,21 They do not mediate strong
proin-flammatory responses after bacterial recognition and thereby main-tain symbiotic relationships with the intestinal microbiota.22 On
injury or infection, the intestinal inflammatory response is related to the recruitment of new monocytes from the blood circulation with proinflammatory properties that will differentiate into proin-flammatory macrophages. These cells have a TLR-responsive CX3CR1intLy6Chiprofile and produce several inflammatory
me-diators (inducible nitric oxide synthase, IL-1
b
, tumor necrosis fac-tor [TNF-a], IL-12 and IL-6) that favor the amplification of immune responses and tissue injury.17,23 Intestinal macrophagescontribute especially to the polarization of intestinal T helper cells
(Th1/Th2 and Th17 cells), CD4+T cells subsets, within inflamed
tissues,24 which are implicated in the pathogenesis of chronic
inflammatory bowel diseases.25,26 In a normal situation,
macro-phages contribute to return to the tissue homeostasis by eliminating the causal agent of inflammation and the products resulting from the degradation of damaged tissue. They express arginase-1 (Arg-1), mannose receptor, and release anti-inflammatory cytokines (IL-4, IL-13, IL-10, and TGF-
b
1) to suppress all processes initiated during the inflammatory response and promote tissue repair.27Macrophages in CD Mucosa
Several studies have shown that the number of intestinal macrophages is increased in patients with CD.4–7 More speci fi-cally, macrophage aggregations, focal subepithelial dense accu-mulations, and infiltration throughout the mucosa have been reported in patients with CD.28 In addition, the colonic LP of
patients with CD contains an increased number of macrophages that express cysteine-rich scavenger receptors such as CD68 and CD163.4,5,28These proteins are implicated in
monocytes/macro-phages maturation and play a role in the development of the immune system and the regulation of the immune response. In inflamed mucosa of patients with CD, the presence of CD68hi and CD163hi macrophages is associated with an increase of IL-17 and TNF-a expression.5,29These data suggest a role for CD68hi
and CD163hi macrophages in the amplification and perpetuation
of the ongoing mucosal inflammation in CD and subsequently with CD activity. The presence of a large number of CD14+
macrophages was also observed in patients with CD compared with healthy subjects.6,7 These cells secreted high amounts of
proinflammatory cytokines such as IL-23 and TNF-a and could favor Th17-related inflammatory pathways and innate pathways implicated in CD pathogenesis. The triggering receptor expressed on myeloid cells 1 (TREM1) is also overexpressed by intestinal LP macrophages of patients with CD compared with control sub-jects. TREM1+ macrophages contribute to the intestinal inflam-mation by secreting proinflammatory mediators such as TNF-a, IL-6, IL-8, monocyte chemotactic protein-1, and IL-1
b
.30The hallmark of CD is the presence of epithelioid granulomas,8,9a collection of macrophages and other in
flamma-tory cells which are among the most uniformly observed and specific microscopic features of CD.31However, the significance
of epithelioid granulomas in CD is unclear. Several studies have attempted to identify an etiological agent in granulomas. The occasional finding of microorganisms-specific DNA may repre-sent either a bystander phenomenon or the cause of the disease. It is conceivable that particulate matter from dead organisms or very slow growing organisms can trigger the granulomatous reaction. There are various lines of evidence suggesting that E. coli is involved in the formation of granulomas: (1) E. coli antigens are present in CD granulomas32,33; (2) E. coli DNA is detected
in 80% of microdissected granulomas from patients with CD,34
and (3) E. coli is able to induce the aggregation of infected human macrophages and that these aggregates recruit surrounding lym-phocytes.35A multivariate analysis of the studies focusing on the
presence of epithelioid granulomas in CD revealed that their fre-quency may indicate a more aggressive disease process.8 This
could be, in part, explained by the leakage of activated macro-phages from granulomas. These cells could then secrete proin-flammatory cytokines such as TNF-a, reactive oxygen species such as H2O2, O22, OH, O2, nitrogen derivatives and
proteo-lytic enzymes that damage the neighboring tissue. In addition, granulomas were frequently identified in biopsies with PPs,36
the immune sensors of the intestine. PPs are also composed of activated macrophages and supposed origin of the earliest muco-sal lesions in ileal CD.37,38Thus, the implementation of studies to
investigate the role of intestinal macrophages during CD could bring major elements in understanding CD pathogenesis and in the identification of new therapeutic targets.
CD-Associated Polymorphisms: Impact on
Macrophage Functions
Many genetic variants have been identified as CD suscepti-bility factors.39 The successful genome-wide association studies
have provided a rational framework for new mechanistic insights and directions regarding research in CD. Overall, 30 loci were spe-cifically associated with CD. They are involved in defective intra-cellular bacteria killing and innate immunity (CARD15/nucleotide oligomerization domain 2 [NOD2], immunity-related GTPase fam-ily M [IRGM], IL23R, LRRK2, and ATG16L1) and deregulated adaptive immune responses (IL-23 and Th17 cell pathway: IL23R, IL12B, STAT3, JAK2, and TYK2).40,41
Here, we report CD loci that may affect directly the function of macrophages. Thefirst locus identified as a risk factor for CD was NOD2.42,43 NOD2 is a member of the NOD-like
receptor family, which encompass intracellular sensors of pathogen/microbe-associated molecular patterns playing crucial roles in the innate immunity.44NOD2 recognizes muramyl
dipep-tide, a component of the peptidoglycan present in the bacterial cell wall and has several functions which have been highlighted to provide a potential link to its association with CD: (1) the main-tain of the intestinal epithelial barrier integrity,45–48(2) the regu-lation of immune homeostasis in the gut,49–51 and (3) the regulation of the microbiota.52,53Three mutations in NOD2 have
been documented to be strongly associated with CD onset. They are located within the leucine-rich repeat domain of the NOD2 protein, which corresponds to the microbe-associated molecular pattern recognition region.54These mutations result in a truncated
NOD2 protein (frameshift mutation, L1007fsinsC) or in amino acid substitutions (R702W and G908R).42,43,55 NOD2 mutations
lead to decreased muramyl dipeptide–induced cytokine responses which are critical in the control of commensal microbiota in the lumen of the gut.56NOD2 mutations have also important
func-tions in relation to autophagy, a complex intracellular protein degradation mechanism by which the cell forms double-membrane vacuoles that ultimately fuse with lysosomes whose functions include the elimination of proteins arising from cellular stress responses or ingested pathogenic bacteria, and the pro-cessing of antigens for their presentation in immune
responses.57,58 The most prevalent NOD2 mutation in patients
with CD (L1007fsinsC) result in a frameshift mutation that gen-erates a truncated NOD2 protein and had a profound effect on the autophagy response triggered by intracellular bacteria.59
Besides NOD2, other genes involved in autophagy are associated with CD. Indeed, several genome-wide association studies indicate a highly significant and replicated association between CD and three variants of autophagy genes, i.e., ATG16L1 (Autophagy-related like 1),60,61IRGM,62–64and ULK1 (unc-51
like autophagy activating kinase).65 Other CD genetic
associa-tions were also defined for genes encoding several proteins impli-cated in autophagy regulating pathways such as PTPN2 (protein tyrosine phosphatase nonreceptor type 2),66,67 LRRK2
(leucine-rich repeat kinase 2),41,68CYLD (cylindromatosis),69and XBP-1
(X-box binding protein 1).70Macrophages expressing autophagy
risk variants (ATG16L1, NOD2, and IRGM) harbor defects in autophagy induction, bacterial trafficking, and antigen presentation.71–73 Autophagy-deficient macrophages fail to kill intracellular bacteria and to present antigen by MHC class II, leading to inappropriate activation of the adaptive immune system and to a high production of IL-1
b
and IL-18. This mechanism induces a severe inflammation and participate to the maintenance of chronic inflammatory status.74Phagocytic Capability of CD Macrophages
and Cytokine Secretion Pro
files
Data regarding monocyte (the precursor cell of macro-phages) phagocytosis in CD are conflicting, some report phago-cytic dysfunction,75–77 whereas some do not.78,79 In the same
way, results regarding cytokine secretion profiles of macrophages in CD are also conflicting (Table 1) but most of them highlight large defects of cytokine secretion profiles of macrophages from patients with CD. This could favor chronic inflammatory status and merit further investigations to bring major elements in the understanding of CD pathogenesis and in the identification of new therapeutic targets. For that, it will be important to take into account the complexity of the cytokine response and of the phe-notype of macrophages from patients with CD, but also a wide range of factors which could deeply influence the results such as (1) the stimulating signal considered (e.g., TLRs ligand versus bacteria), (2) the macrophage origin (e.g., monocyte-derived mac-rophages [MDMs] versus intestinal macmac-rophages), (3) the cell preparation, (4) the disease status (e.g., active versus quiescent), (5) treatment of the patients, (6) genetic factors, and others.
E.
COLIAND MACROPHAGES IN CD
Dysbiosis in CD: Abnormal Colonization by
E. Coli
Chronic inflammation in CD results from the activation of mucosal immune system by the gut microbiota. Over the last 15 years, genome-wide association studies identified links between disease and genes involved in detection, microbial signaling, and
TABLE 1. Cytokine Secretion Pro
files of Macrophages in CD Reported
Cytokines
Macrophage origin Increase Decrease Stable
Marks et al79a Quiescent CD Peripheral venous blood CD MDP IL-8 IL-1b TNF-a IL-10 HC IL-8 IL-1b TNF-a IL-10 CD versus HC MDP IL-8 Woundfluid C5a TNF-a LPS Sewell et al11 Quiescent CD Peripheral venous blood
CD versus HC Pam3 TNF-a
Flagellin LPS Smith et al12 Quiescent CD
Peripheral venous blood
CD versus HC Heat-killed E. coli clone NCTC 10418
TNF-a INF-g Campos et al13 CD (undefined status)
Peripheral venous blood
CD E. faecalis IL-12 TNF-a
IL-23 IL-10
E. Coli IL-12 IL-10
TNF-a IL-23
M. avium subsp. paratuberculosis TNF-a IL-12
IL-23 IL-10
M. avium subsp. avium IL-12 TNF-a
IL-23 IL-10
HC E. faecalis TNF-a IL-12
IL-23 IL-10
E. Coli TNF-a IL-12
IL-23 IL-10 M. avium subsp. paratuberculosis TNF-a IL-12
IL-23 IL-10
M. avium subsp. avium TNF-a IL-12 IL-23 IL-10
TABLE 1. (Continued)
Cytokines
Macrophage origin Increase Decrease Stable
CD versus HC Unstimulated IL-12 TNF-a
IL-23 IL-10
E. faecalis IL-23 IL-10 TNF-a IL-12
E. Coli IL-12 TNF-a
IL-23 IL-10 M. avium subsp. paratuberculosis IL-12 TNF-a
IL-23 IL-10 M. avium subsp. avium IL-12 TNF-a
IL-23 IL-10 Vazeille et al10 Quiescent and active CD
Peripheral venous blood
CD AIEC LF82 IL-8
IL-6 TNF-a Nonpathogenic E. coli K-12 IL-8
IL-6 TNF-a
HC AIEC LF82 IL-8
IL-6 TNF-a Nonpathogenic E. coli K-12 IL-8
IL-6 TNF-a
CD versus HC Unstimulated TNF-a
IL-8 IL-6
AIEC LF82 IL-8 TNF-a
IL-6
Nonpathogenic E. coli K-12 IL-8 IL-6 TNF-a Active CD versus
quiescent CD
AIEC LF82 IL-6 IL-8
TNF-a
Nonpathogenic E. coli K-12 IL-8 IL-6 TNF Elliott et al79 CD (undefined status)
Peripheral venous blood
CD versus HC CD-derived E. coli isolates TNF-a IL-10 IL-23 IL-8 E. coli K-12 TNF-a IL-10 IL-8 LPS TNF-a Flanagan et al79b CD (undefined status) Peripheral venous blood
CD versus HC AIEC HM605 TNF-a
IL-8 IL-6
CD, Crohn’s disease; HC, healthy controls; LPS, lipopolysaccharides; MDP, muramyl dipeptide; UC, ulcerative colitis.
clearance of bacteria, shedding in light of the role of gut microbiota in CD pathogenesis.40As both genetic mutation and
inflammation induced remodeling of the gut microbiota,80–82it is
not surprising to observe dysbiosis in patients with CD. Reduced diversity in microbiota of patients with CD has been reported, especially within Firmicutes and Bacteroidetes phyla.83,84At the
species rank, imbalance was also observed with reduction of bac-teria with anti-inflammatory properties such as Faecalibacterium prausnitzii and increased amount of bacteria with proin flamma-tory potential such as E. coli.14,85,86
Particular efforts have been performed to identify specific infectious agents involved in CD pathogenesis over the past 2 decades and still continue. As disorders observed in CD resemble those of intestinal tuberculosis and Johne’s disease, mycobacteria have been suspected to play a role in CD.87,88In addition,
asso-ciations of CD with measles virus or intestinal pathogens such as Listeria or Yersinia were reported but not confirmed.
Besides involvement of pathogens, the hypothesis is that remodeling of the gut microbiota that occurs in patients with CD may favor emergence and/or expansion of pathobionts. Abnormal colonization of ileum and colon mucosa of patients with CD by E. coli strains with adhesive and invasive properties has been convincingly reported by independent groups.86,89–99Association of AIEC with terminal ileum mucosa90,95 should be related, at
least in part, to increased ileal expression of carcinoembryonic antigen–related cell adhesion molecule 6 (CEACAM6), which act as a receptor for AIEC bacteria.90,95,100 Other important
pathogenicity mechanisms of AIEC have been identified and are nicely described in reviews.101–103The next section will focus on CD-associated E. coli and macrophage interactions.
The Presence of
E. Coli in CD Macrophages
Macrophages are, with dendritic cells, key cellular vehicles that enable bacterial trafficking to mesenteric lymph nodes (MLNs). By using culture-dependent techniques, independent groups showed that bacteria are more frequently isolated from MLN in patients with CD than in controls.104,105E. coli has beenimmunodetected in MLN of patients with CD and among bacteria isolated and cultured from MLN. E. coli was the dominant spe-cies.33,105Recently, O’Brien et al106have characterized the
micro-bial communities of resected nodes from patients with CD and controls by high-throughput sequencing analysis. They found that E. coli was more abundant in nodes from patients with CD than from controls and that patients with terminal ileal CD had a greater proportion of E. coli reads in their nodes than patients with colonic or ileocolonic disease.106
Comparison of microbial communities of mucosa and nodes from a same patient showed that they were similar.106
Whether or not, E. coli bacteria recovered in MLN of patients with CD are AIEC remains to be determined. In transgenic mice expressing human CEACAM6 (CEABAC10 mouse model), we recently showed that AIEC bacteria are able to disseminate to MLN.14,107Abilities of AIEC to translocate across M cells lining
PPs and to resist macrophage killing (see Handling of AIEC by
Macrophages) are both required for bacteria to gain access to MLN. These properties might also explain the high proportion of E. coli observed in ileal MLNs of patients with CD.
The presence of E. coli within macrophages underneath ulcers, along fissures, and in the mucosal LP of patients with CD was also reported.33,108Prevalence of LP macrophages that
contain E. coli was compared in healthy controls and patients with CD and ulcerative colitis (UC).108E. coli-containing macrophages
were found in LP in 71% of patients with CD versus 11% of patients with UC and were not found in healthy controls.
Handling of AIEC by Macrophages
Comparison of bacteria uptake in MDMs obtained from healthy donors and patients with CD revealed that higher numbers of AIEC are internalized within MDM irrespectively of their origin compared with nonpathogenic E. coli, suggesting that AIEC bacteria may express specific factors or variants that confer them specific ability or best affinity to interact with macro-phages.10Classically, vacuoles that contain phagocytized material
transit along the endocytic pathway and fuse sequentially with early and late endosome andfinally with lysosome to form deg-radative organelles termed phagolysosomes. Although nonpatho-genic bacteria are rapidly degraded in phagolysosomes, many pathogens have evolved strategies to escape early from the vacu-ole, to block the transit of the vacuvacu-ole, to resist harsh environment of phagolysosomes, or even gain access to intracellular niches that support their intracellular replication.109–111 In 2001, Glasser et al112 showed for thefirst time that AIEC bacteria are able to
survive and replicate within macrophages. In the absence of spe-cific molecular marker of AIEC, this feature is used in combina-tion with the ability of bacteria to adhere to and to invade intestinal epithelial cells, to phenotypically define whether an E. coli strain belongs to the AIEC pathovar.90 Recently, it has
been shown that, in contrast to nonpathogenic bacteria that are efficiently killed by macrophages, AIEC bacteria are able to resist macrophage killing within MDM isolated from healthy controls, and patients with CD and UC.10,108Intramacrophagic replication
of AIEC bacteria was observed in MDM from patients with CD but not in MDM from UC and healthy controls, highlighting inability of CD macrophages to control intracellular AIEC.10In
vitro studies performed in murine and human macrophage cell lines demonstrated that most of the AIEC-containing vacuoles mature without diverting from the classical endocytic pathway and deliver AIEC to lysosomes.113To survive in such harsh
envi-ronment, bacteria should be equipped. Interestingly, AIEC take advantage of the acidic pH of phagolysosomes to replicate.113
Several bacterial factors involved in survival and/or replication of AIEC within macrophages have been identified and are described in the next section.
Part of AIEC bacteria entering macrophages are rapidly, at their site of entry in the cell, targeted by autophagy, which is a lysosomal degradative pathway involved in detection and elimination of intracellular pathogens.72,114In contrast to bacteria
that traffic along the “classical” endocytic pathway, targeting of
AIEC within autophagosomes leads to their rapid degradation.72
Strong association of polymorphisms in several autophagy-related genes including ATG16L1, IRGM, LRRK2, and NOD2 and risk of CD have been identified.42,43,60,63,68 Decrease of ATG16L1
expression with specific siRNA impairs xenophagy against AIEC in the human macrophage cell line THP-1 and favors their intra-cellular persistence.72 Interestingly, pharmacological induction
and physiological induction of autophagy enable to overcome defects in intracellular AIEC clearance.72Independent groups
re-ported that monocytes and MDM from patients with CD who are homozygous for the ATG16L1-T300A risk allele are defective in controlling intracellular AIEC.10,115 In addition, dendritic cells
isolated from donors with CD-associated Nod2 variants and peri-toneal macrophages isolated from Nod2 knockout mice exhibited also impaired autophagy against AIEC.72,116These results suggest
that AIEC persistence in CD macrophages could be favored by genetic polymorphisms in autophagy-related genes. However, it has been also reported that ATG16L1, NOD2, IRGM, and IL-23R genotypes have no influence on the handling of AIEC bacteria by MDM.108,117
Beside genetic conditions, environmental conditions could impact on AIEC–macrophage interactions. Anti–Saccharomyces cerevisiae antibodies (ASCAs) are detectable in the serum of patients with CD.118 Epitope for these antibodies is a mannan (mannose
a1–3 mannose) that is present in the yeast cell wall and trans-membrane glycoproteins of some Mycobacterium species.119–122In vitro studies showed that exposition of human adherent monocytes and MDMs to mannans from microbial origins impaired bactericidal activity of these cells and support intramacrophagic persistence of CD-associated E. coli.122However, the cellular mechanism involved
remains to be identified. To note, other environmental conditions such as iron and production of cellulose by AIEC might also impact on phagocytosis of these bacteria by macrophages and modulate the inflammatory response.123
Factors Involved in AIEC Survival and
Replication Within Macrophages
Ability of AIEC bacteria to survive and replicate within the harsh environment of phagolysosomes may be supported by expression of bacterial factors. Expression of almost all factors identified so far to be essential for the intramacrophagic survival and/or replication of AIEC is induced on stress encountered in the phagolysosomes (e.g., acidic pH or nutritional and oxidative stress). However, it is important to notice that no virulence factor specific of all AIEC strains and involved in bacteria resistance to macrophage killing has been identified to date. Hypothesis is that it is rather expression of a set of factors involved in AIEC resistance to stress that may account to their ability to survive intracellularly rather than a specific virulence factor.
Hfq is a central mediator of sRNA-based gene regulation in bacteria that controls virulence andfitness of several intracellular bacteria.124 Hfq is required for AIEC to survive and replicate
in vitro within the mouse macrophage cell line J774.125Disability
of AIEC Hfq–negative mutant to resist macrophage killing could
be attributed to the important role of Hfq in promoting bacterial adaptation to stress encountered in the intravacuolar milieu such as acidic pH or reactive oxygen and nitrogen species.125
Screening of a transposon mutant library constructed in the paradigm AIEC LF82 strain points atfive bacterial genes (htrA, dsbA, yfgL, slyB, and yraP) involved in the ability of bacteria to resist macrophage killing.126Among these genes, particular
atten-tion has been paid in studying the role of HtrA, a serine protease and chaperone in the periplasmic space, and DsbA, a periplasmic oxidoreductase, in intramacrophagic behavior of AIEC bacteria. In vitro experiments showed that expression of htrA and dsbA genes is both induced by stressful conditions of the intracellular environment and required for intracellular bacteria replication (HtrA) and survival (DsbA).126,127Given that htrA and dsbA are
not virulence factors per se since they are also expressed by commensal and nonpathogenic E. coli, one could speculate that this is rather regulation of their expression that may account for their role in AIEC intracellular survival and replication.
Recently, involvement of the GipA (growth in PPs) factor in the ability of AIEC bacteria to disseminate to MLN and to replicate within macrophages has been shed on light.107Except
for the probiotic E. coli Nissle 1917 strain, none of the sequenced commensal and nonpathogenic E. coli strains harbors the gipA gene. Interestingly, a higher number of E. coli harboring the gipA gene were observed in patients with CD (27.3%) than in controls (17.2%).107 Expression of gipA is induced by reactive oxygen
species, a stress to which bacteria are particularly exposed in the intracellular environment.107 In addition to its role in PPs
colonization by AIEC, GipA is also involved in the ability of AIEC to resist oxidative stress and to replicate within macro-phages, and it has been shown in a mouse model that deletion of gipA impaired translocation of the AIEC to MLN.107Besides,
Cieza et al128have also reported a role for IbeA (invasion of the
brain endothelium protein A) in the ability of AIEC to invade and translocate across M cells and to survive within macrophage. As for GipA, IbeA might favor intramacrophagic persistence of AIEC by supporting resistance to oxidative stress because dele-tion of ibeA in APEC (avian pathogenic E. coli) makes bacteria more sensitive to hydrogen peroxide.129
Other bacterial factors are suspected to support intracel-lular survival/replication of AIEC within macrophages. Com-parison of genomes of AIEC strains with non-AIEC strains and study of their interaction with macrophages identified that growth of AIEC required iron and that the presence of chuA gene, which encodes a heme transporter, correlated with persis-tence in macrophages.130
It cannot also be excluded that, as already described for other pathogens such as Salmonella typhimurium, phenotypic het-erogeneity among intravacuolar AIEC exists with bacteria that replicate and others that switch on vacuolar conditions toward a nonreplicative phenotype of persisters.131Persisters are bacterial
cells that are multidrug tolerant and are suspected to play a role in disease relapse.131Identification of such AIEC bacterial
popula-tions and involved factors need to be investigated.
Macrophages, Cytokines, and AIEC
AIEC induce strong inflammation in mouse models.132–134
Invasive CD-associated E. coli strains, particularly those isolated from inflamed CD tissues, induced secretion of high levels of TNF-a by macrophages.97,112 Secretions of cytokines by MDM
obtained from healthy donors and patients with CD or UC in response to infection with AIEC, non-AIEC or laboratory E. coli strains have been compared in two recent studies.10,108 In the
study by Elliott et al108, decreased secretions of TNF-a and
IL-23 and increased secretion of IL-10 by MDM obtained from patients with CD compared with MDM obtained from healthy controls were observed in response to infection by AIEC, non-AIEC, and laboratory E. coli strains. No modification of IL-8 secretion was observed between MDM obtained from CD and healthy controls infected with different E. coli strains.108 In
another study, Vazeille et al10 reported that AIEC-infected
MDM obtained from patients with CD secrete higher amount of IL-6 than AIEC-infected MDM obtained from patients with UC and healthy controls. Increased secretions of IL-6 and TNF-a were observed in MDM obtained from patients with CD infected with AIEC compared with nonpathogenic E. coli.10 In addition, the
levels of IL-8 secreted by MDM obtained from patients with CD infected with AIEC and nonpathogenic E. coli were decreased compared with infected MDM obtained from patients with UC or healthy controls.10Interestingly, a correlation between the number
of intramacrophagic AIEC and the amount of secreted TNF-a has been reported in the murine macrophage cell line J774-A1 and in MDM from patients with CD .10,135 Conversely, data obtained
with J774-A1 macrophages suggest that replication of AIEC within macrophages could be dependent of TNF-a secretion.135
However, the cellular mechanism driving TNF-a effect on AIEC replication within macrophages has not been yet elucidated.
Autophagy plays a crucial role in the regulation of inflammation.136,137Elliott et al108showed that polymorphisms in
ATG16L1, NOD2, IRGM, and IL-23R genes do not modulate the level of TNF-a secreted by AIEC-infected MDM from patients with CD. However, these results might be re-evaluated in a larger cohort. Defects in autophagy induced by altered expression of ATG16L1 and IRGM due to siRNA treatment in human THP-1 macrophages or by deletion of Nod2 in mice result in exacerbated inflammatory responses in AIEC-infected macrophages.72
Hypoth-esis is that increased secretion of proinflammatory cytokines in macrophages with impaired autophagy is related to uncontrolled intracellular persistence of AIEC. In patients with CD, polymor-phisms in ATG16L1, IRGM, or NOD2 could favor persistence of intracellular such as AIEC by impairing autophagy and as a conse-quence profoundly tip the balance toward inflammation.
EFFECTS OF CD THERAPY ON MACROPHAGES
AND THEIR ACTIVITY
The therapeutic armamentarium remains limited in CD encompassing steroids, immunosuppressant therapies (thiopur-ines, methotrexate), and anti-TNF agents. Recently, a new class of
treatment has been approved for CD targeting the intestinal T-cells homing (anti-integrins therapies).138Here, we review the
effect of the main treatments used in CD on macrophages and their activity. Owing to available data, we focused on steroids, immunosuppressant therapies, and anti-TNF agents.
Effect of Corticosteroids and
Immunosuppressant Therapies on
Macrophages in CD
For many years, corticosteroids have been used effectively in clinical practice to achieve clinical remission in CD.139Despite
their wide use, their exact mechanism of action is still elusive. In fact, although the interaction between corticosteroids and macro-phages is known for many years, data interesting in this relation-ship in CD remain very sparse. Particular effect of corticosteroids is their ability to downregulate the NF-
k
B pathway140–143which is markedly induced in patients with CD and strongly influences the course of mucosal inflammation.144 This effect could be inpart explained by the ability of corticosteroids to induce the expression and the production of the glucocorticoid-induced leu-cine zipper, a protein constitutively produced by macrophages to inhibit the production of RANTES (regulated on activation nor-mal T cell expressed and secreted), macrophage inflammatory protein 1a (MIPa also known as CCL3),145–148 and TLR-2.145
In addition to their effect on the NF-
k
B pathway, corticosteroids could have an impact on macrophage phenotype,149,150TH2-typeimmune responses initiation and amplification,151–153 NLRP3 inflammasome induction and subsequently on the production on IL-6, TNF-a, and IL-1
b
.154All of these processes have been putforward directly or indirectly in CD pathogenesis.108,155,156
Among compounds used in immunosuppressant therapies, azathioprine is mostly known for its mechanism of action related to decreased purine synthesis and subsequent apoptosis of fast proliferating cells such as CD4+T cells.145This cytotoxic effect
was, for a long time, the only proposed mechanism for azathioprine-induced immunosuppression.151However, other
stud-ies have shown that thiopurines could also affect macrophage inflammatory and proliferative processes157,158and functions such
as phagocytosis and chemotactic responses.159,160 Azathioprine
could act on macrophage proliferation, polarization, and activation through inhibition of inducible nitric oxide synthase157,158,161,162
whose activity is increased in CD,163and through downregulation
of CD163 expression, a specific macrophage activation marker whose expression is increased in mucosa of active CD patient.5,164
Besides, 6-mercaptopurine, another immunosuppressant molecule, has been described to robustly suppress mRNA macrophage expression of monocyte chemotactic protein-1,165one of the key
chemokines that regulate migration and infiltration of monocytes/ macrophages and which are upregulated in CD.166,167 Finally,
in vitro data show that methotrexate induces apoptosis in primary murine macrophages at low concentrations in vitro168 and
sup-presses NF-
k
B activity.169Proinflammatory effects of methotrexatehas also been suggested,170,171but the mechanisms involved remain
to be explored.
Anti-TNF, Macrophages, and CD
Effect of Anti-TNF on Macrophages in CD
Although, anti-TNF agents are, to date, the most effective therapeutic class in CD,172–174their mechanisms of action are not fully understood and depend on their nature. Several hypothesis have been advanced to explain their efficacy such as induction of apoptosis of immune cells by reverse signaling through trans-membrane TNF,175 neutralization of soluble and transmembrane
TNF, alteration of cytokine secretion, as detected in serum and in cultures of LP biopsies, with decreased interferon-g produc-tion176,177 and, more recently induction of immunoregulatory
macrophages.178,179
It has been suggested that active CD is associated with increased macrophage activation. In this line, Dige et al180have
assessed macrophage activation before and after starting anti-TNF treatment using the soluble form of hemoglobin–haptoglobin scavenger receptor CD163, a specific biomarker of macrophage activation.164,181They reported that anti-TNF therapy using
in-fliximab and adalimumab rapidly decreased the levels of soluble CD163 in patients with CD supporting the hypothesis that anti-TNF agents may directly target macrophage activation in CD, as suggested by previous in vitro data182,183 and observations in
peripheral blood.182 However, macrophage activation did not
reach the basal level observed in healthy controls.180 It could
mean that anti-TNF therapy was not able to fully annihilate macrophage hyperactivation, even in patients in remission, if the level of soluble CD163 was not correlated to the disease activity. Among the drugs targeting TNF-a, some of them have different functional properties which could explain their different efficacy in inflammatory bowel diseases.178 Data from Vos and
colleagues showed that an anti-TNF needs to bind to membranous TNF-a on activated T cells and possess an Fc region interacting with the Fc receptor on antigen-presenting cells, to inhibit T-cell proliferation in vitro. These interactions induce the differentiation of blood-derived monocytes to macrophages with a regulatory phenotype (CD206+). These cells harbor anti-inflammatory prop-erties, for example, by inhibiting proliferation of activated T cells and by producing anti-inflammatory cytokine, such as IL-10, and might contribute to the resolution of inflammation. Interestingly, these regulatory macrophages are observed in mixed lymphocyte reaction exposed to anti-TNF agents effective in CD (infliximab and adalimumab) but not in mixed lymphocyte reaction exposed to anti-TNF therapy ineffective in CD such as etanercept.178
Ac-cording to the expression of CD14 and human leukocyte antigen-antigen D related (HLA-DR) and the induced anti-inflammatory cytokines, the authors postulated that these macrophages were probably type 2 or M2 macrophages. This is also supported by the wound-healing capacities of these regulatory macrophages.179
Since the publication of the SONIC trial, it is clearly established that infliximab/azathioprine combination therapy is more effective than monotherapy to achieve mucosal healing in CD anti-TNF–naïve and immunosuppressive therapy–naïve
patients.174 Interestingly, Vos et al179 observed no macrophage
induction in patients treated with azathioprine alone, but found an increased expression of regulatory macrophages CD206+ in pa-tients treated with combination therapy compared with infliximab alone. In addition, they showed that macrophages exposed to the infliximab/azathioprine combination therapy display a stronger immunosuppressive phenotype showing enhanced ability to inhibit T-cell proliferation than macrophages exposed to in-fliximab or azathioprine alone.
Effects of Anti-TNF on Bacterial Replication Within
Macrophages in CD
An altered macrophage response to microorganisms is observed in CD. Nazareth and colleagues have investigated the impact of infliximab treatment on circulating monocyte subsets and on macrophage cytokine production in response to bacterial infection. They showed that infliximab induce elevated TNF-a macrophage secretion regardless the type of bacteria used for the infection (E. coli, Mycobacterium avium paratuberculosis [MAP] and M. avium avium).184They concluded that infliximab
treatment increased the production of CD macrophage–induced TNF-a in response to bacteria, which seemed to depend on enrichment of CD16+circulating monocytes.184Thereafter, they
hypothesized that infliximab therapy was associated with the presence of CD macrophages that were more able to induce a dormant MAP phenotype, suggesting that the predominant induction of this form may explain the lack of MAP reactivation during anti-TNF therapy in CD.185
Finally, to date, regarding AIEC bacteria and anti-TNF therapies, the capability of macrophages from patients with CD treated with anti-TNF to control intramacrophagic AIEC infection remains to be explored since few data are available and depend on the model used.10,135
CONCLUSION
Anomalies in macrophages are suspected to account for important immunological defects associated with the development of CD. In addition, strong data suggest that chronic inflammation in CD results from activation of mucosal immune system by unbalanced gut microbiota in CD, and that CD macrophages response to bacteria are disturbed. Interestingly, lines of evidence point out macrophages as a target of some potent molecules in the CD therapeutic armamentarium. Understanding better the features and roles of macrophages in CD etiopathogenesis and how they are regulated through their interaction with the whole microbiota or specific microorganisms should open avenues for the design of new therapeutic tools.
REFERENCES
1. Lee SH, Starkey PM, Gordon S. Quantitative analysis of total macro-phage content in adult mouse tissues. Immunochemical studies with
monoclonal antibody F4/80. J Exp Med. 1985;161:475–489.
2. Jung C, Hugot JP, Barreau F. Peyer’s patches: the immune sensors of the
intestine. Int J Inflam. 2010;2010:823710.
3. Peyrin-Biroulet L, Loftus EV Jr, Colombel JF, et al. The natural history of
adult Crohn’s disease in population-based cohorts. Am J Gastroenterol.
2010;105:289–297.
4. Demetter P, De Vos M, Van Huysse JA, et al. Colon mucosa of patients
both with spondyloarthritis and Crohn’s disease is enriched with
macro-phages expressing the scavenger receptor CD163. Ann Rheum Dis. 2005;
64:321–324.
5. Franze E, Caruso R, Stolfi C, et al. Lesional accumulation of
CD163-expressing cells in the gut of patients with inflammatory bowel disease.
PLoS One. 2013;8:e69839.
6. Thiesen S, Janciauskiene S, Uronen-Hansson H, et al. CD14(hi)HLA-DR (dim) macrophages, with a resemblance to classical blood monocytes,
dom-inate inflamed mucosa in Crohn’s disease. J Leukoc Biol. 2014;95:531–541.
7. Kamada N, Hisamatsu T, Okamoto S, et al. Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via
IL-23/IFN-gamma axis. J Clin Invest. 2008;118:2269–2280.
8. Heresbach D, Alexandre JL, Branger B, et al. Frequency and significance
of granulomas in a cohort of incident cases of Crohn’s disease. Gut.
2005;54:215–222.
9. Molnar T, Tiszlavicz L, Gyulai C, et al. Clinical significance of
granu-loma in Crohn’s disease. World J Gastroenterol. 2005;11:3118–3121.
10. Vazeille E, Buisson A, Bringer MA, et al. Monocyte-derived
macro-phages from Crohn’s disease patients are impaired in the ability to
con-trol intracellular adherent-invasive Escherichia coli and exhibit
disordered cytokine secretion profile. J Crohns Colitis. 2015;9:410–420.
11. Sewell GW, Rahman FZ, Levine AP, et al. Defective tumor necrosis
factor release from Crohn’s disease macrophages in response to Toll-like
receptor activation: relationship to phenotype and genome-wide
associ-ation susceptibility loci. Inflamm Bowel Dis. 2012;18:2120–2127.
12. Smith AM, Sewell GW, Levine AP, et al. Disruption of macrophage
pro-inflammatory cytokine release in Crohn’s disease is associated with
reduced optineurin expression in a subset of patients. Immunology.
2015;144:45–55.
13. Campos N, Magro F, Castro AR, et al. Macrophages from IBD pa-tients exhibit defective tumour necrosis factor-alpha secretion but
otherwise normal or augmented pro-inflammatory responses to
infec-tion. Immunobiology. 2011;216:961–970.
14. Chassaing B, Darfeuille-Michaud A. The commensal microbiota and
enteropathogens in the pathogenesis of inflammatory bowel diseases.
Gastroenterology. 2011;140:1720–1728.
15. Ginhoux F, Jung S. Monocytes and macrophages: developmental
path-ways and tissue homeostasis. Nat Rev Immunol. 2014;14:392–404.
16. Bain CC, Scott CL, Uronen-Hansson H, et al. Resident and
pro-inflammatory macrophages in the colon represent alternative
context-dependent fates of the same Ly6Chi monocyte precursors.
Mucosal Immunol. 2013;6:498–510.
17. Mowat AM, Bain CC. Mucosal macrophages in intestinal homeostasis
and inflammation. J Innate Immun. 2011;3:550–564.
18. Gross M, Salame TM, Jung S. Guardians of the gut - murine intestinal macrophages and dendritic cells. Front Immunol. 2015;6:254. 19. Lavin Y, Winter D, Blecher-Gonen R, et al. Tissue-resident macrophage
enhancer landscapes are shaped by the local microenvironment. Cell. 2014;159:1312–1326.
20. Mortha A, Chudnovskiy A, Hashimoto D, et al. Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeosta-sis. Science. 2014;343:1249288.
21. Kuhl AA, Erben U, Kredel LI, et al. Diversity of intestinal macrophages
in inflammatory bowel diseases. Front Immunol. 2015;6:613.
22. Smythies LE, Sellers M, Clements RH, et al. Human intestinal macro-phages display profound inflammatory anergy despite avid phagocytic
and bacteriocidal activity. J Clin Invest. 2005;115:66–75.
23. Cassetta L, Cassol E, Poli G. Macrophage polarization in health and
disease. ScientificWorldJournal. 2011;11:2391–2402.
24. Arnold CE, Gordon P, Barker RN, et al. The activation status of human
macrophages presenting antigen determines the efficiency of Th17
re-sponses. Immunobiology. 2015;220:10–19.
25. Neurath MF, Finotto S, Glimcher LH. The role of Th1/Th2 polarization
in mucosal immunity. Nat Med. 2002;8:567–573.
26. Weaver CT, Elson CO, Fouser LA, et al. The Th17 pathway and in
flam-matory diseases of the intestines, lungs, and skin. Annu Rev Pathol.
2013;8:477–512.
27. Murray PJ, Wynn TA. Protective and pathogenic functions of
macro-phage subsets. Nat Rev Immunol. 2011;11:723–737.
28. Yao K, Iwashita A, Yao T, et al. Increased numbers of macrophages in
noninflamed gastroduodenal mucosa of patients with Crohn’s disease.
Dig Dis Sci. 1996;41:2260–2267.
29. Fujino S, Andoh A, Bamba S, et al. Increased expression of interleukin
17 in inflammatory bowel disease. Gut. 2003;52:65–70.
30. Schenk M, Bouchon A, Seibold F, et al. TREM-1–expressing intestinal
macrophages crucially amplify chronic inflammation in experimental colitis
and inflammatory bowel diseases. J Clin Invest. 2007;117:3097–3106.
31. Cook MG, Dixon MF. An analysis of the reliability of detection and
diagnostic value of various pathological features in Crohn’s disease and
ulcerative colitis. Gut. 1973;14:255–262.
32. Cartun RW, Van Kruiningen HJ, Pedersen CA, et al. An immunocyto-chemical search for infectious agents in Crohn’s disease. Mod Pathol.
1993;6:212–219.
33. Liu Y, van Kruiningen HJ, West AB, et al. Immunocytochemical evi-dence of Listeria, Escherichia coli, and Streptococcus antigens in
Crohn’s disease. Gastroenterology. 1995;108:1396–1404.
34. Ryan P, Kelly RG, Lee G, et al. Bacterial DNA within granulomas of
patients with Crohn’s disease–detection by laser capture microdissection
and PCR. Am J Gastroenterol. 2004;99:1539–1543.
35. Meconi S, Vercellone A, Levillain F, et al. Adherent-invasive
Escher-ichia coli isolated from Crohn’s disease patients induce granulomas
in vitro. Cell Microbiol. 2007;9:1252–1261.
36. Shikuwa S, Isomoto H, Mizuta Y, et al. Magnifying videoendoscopic findings of Peyer’s patches in the terminal ileum of Crohn’s disease. Gut.
2007;56:894–895.
37. Fujimura Y, Owen RL. M cells as portals of infection: clinical
and pathophysiological aspects. Infect Agents Dis. 1996;5:144–156.
38. Van Kruiningen HJ, Ganley LM, Freda BJ. The role of Peyer’s patches
in the age-related incidence of Crohn’s disease. J Clin Gastroenterol.
1997;25:470–475.
39. Cho JH, Brant SR. Recent insights into the genetics of inflammatory
bowel disease. Gastroenterology. 2011;140:1704–1712.
40. Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature.
2012;491:119–124.
41. Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis
increases to 71 the number of confirmed Crohn’s disease susceptibility
loci. Nat Genet. 2010;42:1118–1125.
42. Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2
leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature. 2001;
411:599–603.
43. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2
associated with susceptibility to Crohn’s disease. Nature. 2001;411:603–606.
44. Strober W, Watanabe T. NOD2, an intracellular innate immune sensor
involved in host defense and Crohn’s disease. Mucosal Immunol. 2011;
4:484–495.
45. Kobayashi KS, Chamaillard M, Ogura Y, et al. Nod2-dependent regula-tion of innate and adaptive immunity in the intestinal tract. Science.
2005;307:731–734.
46. Wehkamp J, Harder J, Weichenthal M, et al. NOD2 (CARD15)
muta-tions in Crohn’s disease are associated with diminished mucosal
alpha-defensin expression. Gut. 2004;53:1658–1664.
47. Geddes K, Rubino SJ, Magalhaes JG, et al. Identification of an innate T
helper type 17 response to intestinal bacterial pathogens. Nat Med. 2011;
17:837–844.
48. Hiemstra IH, Bouma G, Geerts D, et al. Nod2 improves barrier function of intestinal epithelial cells via enhancement of TLR responses. Mol
Immunol. 2012;52:264–272.
49. van Beelen AJ, Zelinkova Z, Taanman-Kueter EW, et al. Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to pro-mote interleukin-17 production in human memory T cells. Immunity.
2007;27:660–669.
50. Brain O, Owens BM, Pichulik T, et al. The intracellular sensor NOD2 induces microRNA-29 expression in human dendritic cells to limit IL-23
release. Immunity. 2013;39:521–536.
51. Jing X, Zulfiqar F, Park SY, et al. Peptidoglycan recognition protein 3
and Nod2 synergistically protect mice from dextran sodium
sulfate-induced colitis. J Immunol. 2014;193:3055–3069.
52. Couturier-Maillard A, Secher T, Rehman A, et al. NOD2-mediated dys-biosis predisposes mice to transmissible colitis and colorectal cancer. J Clin Invest. 2013;123:700–711.
53. Knights D, Silverberg MS, Weersma RK, et al. Complex host genetics
influence the microbiome in inflammatory bowel disease. Genome Med.
2014;6:107.
54. Homer CR, Richmond AL, Rebert NA, et al. ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in
Crohn’s disease pathogenesis. Gastroenterology. 2010;139:1630–1641,
1641 e1631–1632.
55. Begue B, Dumant C, Bambou JC, et al. Microbial induction of CARD15 expression in intestinal epithelial cells via toll-like receptor 5 triggers an antibacterial response loop. J Cell Physiol. 2006;209:241–252.
56. Salem M, Seidelin JB, Eickhardt S, et al. Species-specific engagement of
human nucleotide oligomerization domain 2 (NOD)2 and Toll-like receptor (TLR) signalling upon intracellular bacterial infection: role of
Crohn’s associated NOD2 gene variants. Clin Exp Immunol. 2015;179:
426–434.
57. Mizushima N, Levine B, Cuervo AM, et al. Autophagyfights disease
through cellular self-digestion. Nature. 2008;451:1069–1075.
58. Shibutani ST, Saitoh T, Nowag H, et al. Autophagy and autophagy-related proteins in the immune system. Nat Immunol. 2015;16:
1014–1024.
59. Ramjeet M, Hussey S, Philpott DJ, et al.“Nodophagy”: new crossroads
in Crohn disease pathogenesis. Gut Microbes. 2010;1:307–315.
60. Hampe J, Franke A, Rosenstiel P, et al. A genome-wide association scan
of nonsynonymous SNPs identifies a susceptibility variant for Crohn
disease in ATG16L1. Nat Genet. 2007;39:207–211.
61. Rioux JD, Xavier RJ, Taylor KD, et al. Genome-wide association study
identifies new susceptibility loci for Crohn disease and implicates
autophagy in disease pathogenesis. Nat Genet. 2007;39:596–604.
62. McCarroll SA, Huett A, Kuballa P, et al. Deletion polymorphism upstream of IRGM associated with altered IRGM expression and
Crohn’s disease. Nat Genet. 2008;40:1107–1112.
63. Parkes M, Barrett JC, Prescott NJ, et al. Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to
Crohn’s disease susceptibility. Nat Genet. 2007;39:830–832.
64. Prescott NJ, Dominy KM, Kubo M, et al. Independent and
population-specific association of risk variants at the IRGM locus with Crohn’s
disease. Hum Mol Genet. 2010;19:1828–1839.
65. Henckaerts L, Cleynen I, Brinar M, et al. Genetic variation in the
au-tophagy gene ULK1 and risk of Crohn’s disease. Inflamm Bowel Dis.
2011;17:1392–1397.
66. Glas J, Wagner J, Seiderer J, et al. PTPN2 gene variants are associated
with susceptibility to both Crohn’s disease and ulcerative colitis
sup-porting a common genetic disease background. PLoS One. 2012;7: e33682.
67. Scharl M, Mwinyi J, Fischbeck A, et al. Crohn’s disease-associated
polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy. Inflamm Bowel Dis. 2012;
18:900–912.
68. Barrett JC, Hansoul S, Nicolae DL, et al. Genome-wide association
defines more than 30 distinct susceptibility loci for Crohn’s disease.
Nat Genet. 2008;40:955–962.
69. Cleynen I, Vazeille E, Artieda M, et al. Genetic and microbial factors
modulating the ubiquitin proteasome system in inflammatory bowel
dis-ease. Gut. 2014;63:1265–1274.
70. Hoefkens E, Nys K, John JM, et al. Genetic association and functional role of Crohn disease risk alleles involved in microbial sensing, autophagy, and endoplasmic reticulum (ER) stress. Autophagy. 2013;9:2046–2055.
71. Lapaquette P, Glasser AL, Huett A, et al. Crohn’s disease-associated
adherent-invasive E. coli are selectively favoured by impaired autophagy
to replicate intracellularly. Cell Microbiol. 2010;12:99–113.
72. Lapaquette P, Bringer MA, Darfeuille-Michaud A. Defects in autophagy favour adherent-invasive Escherichia coli persistence within
macro-phages leading to increased pro-inflammatory response. Cell Microbiol.
2012;14:791–807.
73. Singh SB, Davis AS, Taylor GA, et al. Human IRGM induces autophagy
to eliminate intracellular mycobacteria. Science. 2006;313:1438–1441.
74. Nguyen HT, Lapaquette P, Bringer MA, et al. Autophagy and Crohn’s
disease. J Innate Immun. 2013;5:434–443.
75. Caradonna L, Amati L, Lella P, et al. Phagocytosis, killing, lymphocyte-mediated antibacterial activity, serum autoantibodies, and plasma endo-toxins in inflammatory bowel disease. Am J Gastroenterol. 2000;95:
1495–1502.
76. Beeken WL, St Andre-Ukena S, Gundel RM. Comparative studies of
mononuclear phagocyte function in patients with Crohn’s disease and
colon neoplasms. Gut. 1983;24:1034–1040.
77. Okabe N, Ikura S, Uchida Y, et al. Immunological studies on Crohn’s
disease. VIII. Diminished phagocytic activity in peripheral blood
mono-cytes. J Clin Lab Immunol. 1990;32:29–31.
78. Mee AS, Berney J, Jewell DP. Monocytes in inflammatory bowel
dis-ease: absolute monocyte counts. J Clin Pathol. 1980;33:917–920.
79. Elliott TR, Hudspith BN, Rayment NB, et al. Defective macrophage
handling of Escherichia coli in Crohn’s disease. J Gastroenterol
Hep-atol. 2015;30:1265–1274.
79a. Marks DJ, Harbord MW, MacAllister R, et al. Defective acute in
flam-mation in Crohn’s disease: a clinical investigation. Lancet. 2006;25:
367:668–678. Erratum in: Lancet. 2007;28:318.
79b. Flanagan PK, Chiewchengchol D, Wright HL, et al. Killing of Escherichia coli by Crohn’s Disease monocyte-derived macrophages
and its enhancement by hydroxychloroquine and vitamin D. Inflamm
Bowel Dis. 2015;21:1499–1510.
80. Mondot S, Barreau F, Al Nabhani Z, et al. Altered gut microbiota
com-position in immune-impaired Nod2(-/-) mice. Gut. 2012;61:634–635.
81. Elinav E, Strowig T, Kau AL, et al. NLRP6 inflammasome regulates
colonic microbial ecology and risk for colitis. Cell. 2011;145:745–757.
82. Lupp C, Robertson ML, Wickham ME, et al. Host-mediated in
flamma-tion disrupts the intestinal microbiota and promotes the overgrowth of
Enterobacteriaceae. Cell Host Microbe. 2007;2:119–129.
83. Willing BP, Dicksved J, Halfvarson J, et al. A pyrosequencing study
in twins shows that gastrointestinal microbial profiles vary with
inflammatory bowel disease phenotypes. Gastroenterology. 2010;
139:1844–1854. e1841.
84. Frank DN, St Amand AL, Feldman RA, et al. Molecular-phylogenetic
characterization of microbial community imbalances in human in
flam-matory bowel diseases. Proc Natl Acad Sci U S A. 2007;104:
13780–13785.
85. Sokol H, Pigneur B, Watterlot L, et al. Faecalibacterium prausnitzii is an
anti-inflammatory commensal bacterium identified by gut microbiota
analysis of Crohn disease patients. Proc Natl Acad Sci U S A. 2008;
105:16731–16736.
86. Lopez-Siles M, Martinez-Medina M, Busquets D, et al. Mucosa-associ-ated Faecalibacterium prausnitzii and Escherichia coli co-abundance can
distinguish irritable bowel syndrome and inflammatory bowel disease
phenotypes. Int J Med Microbiol. 2014;304:464–475.
87. Liverani E, Scaioli E, Cardamone C, et al. Mycobacterium avium
sub-species paratuberculosis in the etiology of Crohn’s disease, cause or
epiphenomenon?. World J Gastroenterol. 2014;20:13060–13070.
88. Selby W, Pavli P, Crotty B, et al. Two-year combination antibiotic
therapy with clarithromycin, rifabutin, and clofazimine for Crohn’s
dis-ease. Gastroenterology. 2007;132:2313–2319.
89. Darfeuille-Michaud A, Neut C, Barnich N, et al. Presence of adherent
Escherichia coli strains in ileal mucosa of patients with Crohn’s disease.
Gastroenterology. 1998;115:1405–1413.
90. Darfeuille-Michaud A, Boudeau J, Bulois P, et al. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in
Crohn’s disease. Gastroenterology. 2004;127:412–421.
91. Baumgart M, Dogan B, Rishniw M, et al. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli
of novel phylogeny relative to depletion of Clostridiales in Crohn’s
disease involving the ileum. ISME J. 2007;1:403–418.
92. Martin HM, Campbell BJ, Hart CA, et al. Enhanced Escherichia coli
adherence and invasion in Crohn’s disease and colon cancer.
Gastroen-terology. 2004;127:80–93.
93. Fujita H, Eishi Y, Ishige I, et al. Quantitative analysis of bacterial DNA from Mycobacteria spp., Bacteroides vulgatus, and Escherichia coli in
tissue samples from patients with inflammatory bowel diseases.
J Gastroenterol. 2002;37:509–516.
94. Gophna U, Sommerfeld K, Gophna S, et al. Differences between
tissue-associated intestinal microfloras of patients with Crohn’s disease and
ulcerative colitis. J Clin Microbiol. 2006;44:4136–4141.
