COORDINADOR ADMINISTRATIVA

The gut is the largest reservoir of macrophages in the body [30]. Gut macrophages are mainly populated in the lamina propria and other parts of the gut such as mucosa, submucosa and muscularis mucosa [146]. Gut resident macrophages exhibit granular cytoplasm and active phagocytic behavior [146] and are identified by different cell

surface markers such as CD11b, CD11c, F4/80, Fc-gamma receptor 1 (FcγRI; CD64),

macrosialin (CD68), CD115, MHCII, CD206, hemoglobin scavenger receptor

(CD163), Siglec F and αEβ7 receptor (CD103) [147]. In fact, distinguishing gut

macrophages from other cells has to be performed by a combination of these markers,

as CD11b+ and F4/80+ eosinophils [146] or CD11b+, CD11c+ and MHCII+ DCs [148]

have been previously reported. Recently, CX3CR1+ or the fractalkine receptor has

been shown to be associated with gut macrophages rather than gut DCs [148, 149].

On these grounds, gut macrophages in a steady state can be characterized by CD11b+,

CD11c+, F4/80+, CX3CR1+, MHCII+, Siglec F-, and CD103-.

Studies have demonstrated that gut resident macrophages mainly originate from bone

marrow-derived Ly6Chigh monocytes [150, 151]. It was further shown that these

recruited Ly6Chigh monocytes differentiate to F4/80+ CD64+ CX3CR1high Ly6C-

resident macrophages at least in part in a CCR2/CCL2-dependent manner [147].

Other chemokines such as TGF-β and IL-8, which are constantly produced by

mucosal IECs and mast cells, have also been shown to be involved in the recruitment of blood monocytes [152, 153].

1.3.2.1

Gut Macrophage Roles

Gut macrophages are key players in maintaining gut immunity and homeostasis. In normal conditions, gut macrophages are tolerant to microbial products [147]. However, they retain their housekeeping functions, such as clearance of apoptotic

cells [154], as well as tissue remodeling [155]. Additionally, gut macrophages are highly phagocytic and bactericidal [156]. They lack respiratory burst activity [157] and do not produce NO [158] or inflammatory cytokines such as IL-1 and IL-6 [147, 156]. They, however, produce high levels of the anti-inflammatory cytokine IL-10 [147]. IL-10 plays a pivotal role in gut homeostasis. It induces production of the scavenger receptors such as CD206 and CD163. Additionally, IL-10 suppresses activation of gut macrophages to inflammatory stimuli [156]. Gut microbiota plays an important role in the production of IL-10 by gut macrophages. In fact, gut macrophages from germ free mice produce less IL-10 [159, 160]. Gut macrophages,

however, produce low levels of TNF-α. Such effects have also been shown to be

beneficial in regulating enterocyte growth, altering the permeability of the IEC barrier and inducing of matrix metalloproteinases and other tissue remodeling enzymes [161, 162]. Furthermore, gut macrophages produce prostaglandin E2 that stimulates proliferation of epithelial progenitors [163]. Overall, in the gut it appears that macrophages display a partial activation state with inflammatory anergy [156].

Gut macrophages also serve as APCs as they express high levels of MHCII [149]. It

has been speculated that gut macrophages can differentiate naïve CD4+ T cells to

forkhead box P3 (FoxP3+) T regulatory cells in the gut [164]. Such effects of gut

macrophages are still debatable, as gut macrophages cannot migrate to MLNs in a steady state [148]. In addition, it has been shown that gut macrophages do not

efficiently activate naïve CD4+ _{T cells in vitro [148]. Thus, other mediators may also}

be involved indirectly in this process. For instance, IL-10 is a substantial cytokine produced by gut macrophages and has been shown to regulate T regulatory cells that have migrated to the gut after initial priming in the MLNs [165, 166]. Gut macrophages may also facilitate the maintenance of other T cell populations. Indeed,

microbiota-dependent IL-1β production in gut macrophages have been shown to

contribute to the development of IL-17 producing T (Th17) cells in non-inflamed conditions [167].

profound presence due to heavily localized bacteria. Any disturbance to this balance may result in gut disorders, such as IBD [168]. IBD mainly comprises Crohn’s disease (CD) and ulcerative colitis (UC). CD is characterized by transmural inflammation in any part of the GI tract, mostly in the ileocecal region and terminal ileum, bowel obstruction, the presence of granulomas and the development of skip lesions. In contrast, UC inflammation is limited to the mucosa of the colon and rectum, and is characterized by bloody stool and severe diarrhea [169, 170]. Both CD and UC are caused by abnormal immune activation in the intestine [171]. It is postulated that the loss of immune tolerance to endogenous flora is responsible for initiating abnormal immune responses in genetically defective hosts [169]. In addition, the microbiome has also been found as a source of inflammation in IBD, as transferring the microbiome from IBD established T-bet-deficient mice (lacking Th1 immune response) to a wild-type mice caused intestinal inflammation [172]. Other theories such as genetic background and environmental factors have also been suggested to play a role in IBD [169, 173-176]. To date no cure has been found for IBD and most patients receive only antibiotics or anti-inflammatory drugs with negative consequences to reduce the symptoms and complications of the disease, which leads to less hospitalization and surgery. Therefore, new therapeutic treatments are needed to overcome these diseases [170, 177].

Macrophages are an abundant immune cells in the gut and are of major importance in the regulation of the immunological and inflammatory responses [30]. Dysregulation of cytokine production in macrophages has also been reported to associate with IBD. Macrophages from CD patients secrete high levels of pro-inflammatory IL-23 but low levels of anti-inflammatory IL-10. However, macrophages from UC patients mainly secrete high levels of pro-inflammatory IL-12 [178]. Macrophage polarization has also been shown to affect outcome of colitis. M1 macrophages contribute to pathogenesis of colitis; where M2 macrophages reduced the severity of the disease [134, 179, 180]. G-CSF was also produced less in IBD patients [181]. Less efficient bacterial clearance resulting from decreased ROS production [182] and phagocytosis [183] in macrophages have also been linked to IBD. Taken together, these findings indicate that an imbalance in any of the physiological functions of gut macrophages,

including inadequate response to GI tract flora, inappropriate clearing of microbes and ineffective switch from pro-inflammatory response to anti-inflammatory response, may tip the balance between GI bacteria and host’s immune system toward gut disorders such as IBD [171]. Indeed, recent genome-wide association studies revealed several single-nucleotide polymorphisms that contribute to the development of IBD: genes for microbial sensing (NOD2, IRF5, NFKB1, RELA, REL, RIPK2, CARD9 and PTPN22), clearance (ATG16L1, IRGM and NCF4) and integrating antimicrobial adaptive immune responses (IL-23R, IL-10, IL-12, IL-18RAP/IL-1R1, IFNGR/IFNAR1, JAK2, STAT3 and TYK2) [171, 184]. These analyses again suggest that defects in macrophage and DC function may contribute to the pathogenesis of IBD.

1.3.2.3

Mouse models of IBD

Animal models have been extensively used to increase our understanding of IBD. Mice can be used as an IBD model due to their inexpensive cost and small size compared to their counterparts such as monkeys. However, no ideal model that mimics all aspects of human IBD currently exists. To date, there are different models of IBD that can be grouped in three major categories based on the nature of inflammation and method of generation; the erosive self-limiting models of acute colitis, spontaneous models induced by targeted gene deletion and models induced by disruption of T-cell homeostasis [185]. As each animal model serves for different purposes, finding an appropriate model to study role of gut macrophages in colitis is an asset.

The erosive, self-limiting model of colitis can be used to resemble acute colitis with similar histopathological characteristics of UC such as neutrophil infiltration, mucosal erosions and ulcerations. This model can be produced by administration of dextran sulfate sodium (DSS) [186, 187]. DSS is a water-soluble heparin-like sulfated polysaccharide that is widely used to investigate the pathogenesis of UC [188]. Supplementing the drinking water of mice with DSS induces colonic mucosal inflammation with ulceration, body weight loss, bloody stools and diarrhea for several

damage through killing IECs and does not induce T cell responses, at least in the acute model [190, 191]. To investigate chronic inflammation, DSS can be administrated in low dose for several cycles [192]. Indeed, DSS-treatment of C.B17 SCID (sever combined immunodeficiency) mice resulted in colitis in these mice [189], indicating that innate immune cells such as macrophages and DCs are responsible for pathogenesis in this model.

In contrast to the DSS model, the trinitrobenzene sulfonic acid (TNBS) model induces colonic inflammation when administrated intrarectally in only susceptible strains of mice (but not C57BL/6j mice) and depends on the gut microbiota [186, 187, 193,

194]. This model closely resembles CD and has been shown to induce a CD4+ T cell-

mediated-Th1 response [193, 194]. In addition, depletion of phagocytes in a TNBS model also showed to be effective in reducing colitis conditions in this model, confirming the role of macrophages and DCs in pathogenesis of IBD [195].

Spontaneous models induced by targeted gene deletion such as IL-10-deficient mice

(IL-10-/-), resembling CD, have also been used [196]. The IL-10-/- mice produce Th1-

mediated immune responses, mucosal hyperplasia, a transmural mononuclear cell infiltration, occasional crypt abscesses and focal erosions [185]. This model has been

used for studies of various immunologic agents (anti-TNF-α and anti-IFN-γ

antibodies), antibiotics and probiotics. Similar to TNBS model, elimination of

phagocytes in IL-10-/- _{mice also ameliorates the disease conditions in these mice,}

again indicating the importance of innate immune cells in this model [197].

Models induced by disruption of T-cell homeostasis have also been shown to be

effective in resembling of IBD. This model is produced by adoptive transfer of CD4+

CD45RBhigh T cells (naive T-cells) from healthy wild-type mice into syngeneic

recipients that lack T and B cells (such as SCID or RAG-deficient mice). This model produces transmural inflammation, epithelial cell hyperplasia, polymorphonuclear neutrophil and mononuclear leukocyte infiltration, crypt abscesses and epithelial cell erosions [185].