*2.2. Macrophages*

Intestine contains the largest pool of macrophages in the body [33]. It was long considered that, di fferent from other tissues, embryonic-derived macrophages only populate the colon during neonatal stage. Ly6C (hi) circulating monocytes that recruited and di fferentiated locally into anti-inflammatory macrophages gradually replace embryonic macrophages at the time of weaning. However, a recent study found that there are three subpopulations of macrophage in the mouse gut: Tim-4+CD4+ macrophages are locally maintained, whereas Tim4-CD4+ and Tim4-CD4− macrophages are replenished from blood monocytes [34]. Another study showed that a population of self-maintaining macrophages aroused from embryonic precursors and bone marrow derived monocytes persists in the intestine throughout adulthood. Deficiency of this population leads to vascular leakage, reduced intestinal secretion and motility [35]. In mice, colonic macrophages are identified by the following marker expression profile: CX3CR1int/hi CD64+ CD11b+ CD11clo/int F4/80+ Ly6C-/lo MHCII+ CD172 α+ CD103− SiglecF− CCR7− [36,37]. The lifespan of macrophages is at least 1–2 week [36,38].

## 2.2.1. The Function of Macrophages

Defects in macrophage di fferentiation may contribute to increased susceptibility to IBD [39]. Compared with blood monocytes, human intestinal macrophages display downregulated cytokine production upon bacterial products stimulation but preserve phagocytic and bactericidal activity [40]. Thus, intestinal macrophages (CX3CR1 hi) normally possess an anti-inflammatory phenotype during homeostasis via constitutive production of IL-10 [41], whereas Toll-like receptor-responsive proinflammatory macrophages accumulate in the colon and may contribute to disease severity and progression in IBD [37]. However, colonic anti-inflammatory macrophages are still present and promote tissue repair after injury [42]. Studies in mice lacking macrophages suggested that macrophages are necessary for proper epithelial regeneration after DSS injury [43]. Furthermore, Trem2 expressing macrophages are required for e fficient mucosal regeneration after colonic biopsy injury [44]. In addition, macrophage-secreted WNT ligands enhance intestinal regeneration response against radiation [45]. Transfer of anti-inflammatory macrophages accelerate mucosal repair in 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-treated mice through the activation of the Wnt signaling pathway [46].

## 2.2.2. The Regulation of Macrophages

Macrophage-dependent wound repair in response to DSS-induced colonic injury is markedly diminished in germ-free mice, indicating an essential role of microbiota in macrophage-mediated wound healing [43]. Commensal microbiota-derived local signals in the intestine are essential for recruiting macrophages from circulating monocytes [33]. Breeding of mice in germ-free conditions had a detrimental e ffect on the number of mature macrophages populating the adult colon compared to mice house in conventional conditions.

However, the small intestine macrophages are regulated by dietary amino acids but not microbiota [47]. Mice fed a protein-free diet had significantly lower levels of IL-10-producing macrophages but not IL-10-producing CD4+ T cells in their small intestine, compared with control-diet fed mice [47]. Depletion of commensal bacteria did not a ffect numbers of mature macrophages in the small intestine, spleen, or bone marrow, indicating that the recruitment of macrophages to the small intestine is regulated independently of the microbiota [47]. Depletion of microbiota also has no e ffect on the repair of small intestinal injury [48].

## *2.3. Regulatory T Cells (Treg)*

Treg cells are a subset of CD4+ T cells that can inhibit T helper (Th) cells through the release of anti-inflammatory cytokines, such as IL-10 and TGF-β, or by direct contact with Th cells [49]. Th1 cells

are induced by IL-12 and secrets IFN-γ, whereas Th2 cells are induced by IL-4 and releases IL-5 and IL-13 [50]. Crohn's disease (CD) has been long considered to be driven by a Th1 response, whereas the notion that UC is mediated by Th2 response is still controversial [50]. There are two best-characterized subsets of Treg cells that suppress the immune response: forkhead box P3+ (Foxp3+)-positive Treg cells and Foxp3-negative type 1 Treg (Tr1) cells [51]. Foxp3+ Tregs are mainly derived from the thymus, and some travel to the intestine where they inhibit inappropriate immune reactions. Tregs are significantly reduced in peripheral blood and colonic mucosa of IBD patients [52].

## 2.3.1. The Function of Treg

Foxp3+Tregs promotes the healing of UC through endogenous vascular endothelial growth factor receptor 1 tyrosine kinase (VEGFR1-TK) signaling as mucosal repair of DSS-induced colitis is delayed in VEGFR1-TK knockout mice [53]. For Tr1 cells, in addition to secreting immunosuppressive cytokines IL-10 and TGF-β [54], they secrete IL-22 to regulate repair of the epithelium and protect barrier function of human intestinal epithelial cells [55]. It has been shown recently that patients with refractory in CD were well tolerated with ovalbumin-specific Tr1-based therapy and had a dose-related efficacy [56].

## 2.3.2. The Regulation of Treg

The microbiota affects the frequency and function of mucosal Tregs. The frequency of Tregs increased in the colon and lamina propria of the small intestine after weaning suggesting a role of the microbiota [57]. Post-weaning accumulation of Tregs was impaired in germ-free or antibiotic-treated mice compared with conventionally housed mice. In addition, germ-free mice fed fecal suspensions from conventionally housed mice saw a substantial increase in Treg levels. Indigenous Clostridium species were reported to play a central role in the induction of IL-10 producing Foxp3+ Tregs in the colon and small intestine in mice [57]. Additionally, it appears as though Clostridia bacteria have a direct role in modulating immune cell populations in the gu<sup>t</sup> as many Clostridium-colonized mice were observed to have Tregs negative for Helios—a transcription factor reported to be expressed in thymus-derived "natural" Tregs. Therefore, the absence of Helios suggests that increasing levels of Tregs in the colon may be induced Treg (iTregs). Indeed, the culture of splenic CD4+ cells in the presence of supernatant of intestinal epithelial cells from Clostridium-colonized mice induced the differentiation of FoxP3-expressing cells. Furthermore, this effect was diminished by neutralizing antibody against TGF-β. Interestingly, it appears that iTregs also play a role in maintaining gu<sup>t</sup> homeostasis as demonstrated in a DSS treatment model of colitis. Symptoms of colitis, such as weight loss, rectal bleeding, colon shortening, edema, mucosal erosion, cryp<sup>t</sup> loss, and cellular infiltration, were all reduced in Clostridium-colonized mice treated with DSS compared to controls.

Different from microbiota-induced Treg cells, dietary antigens from solid food induce the main part of the short-lived small intestinal periphery Treg cells [58].

## *2.4. Innate Lymphoid Cells (ILCs)*

ILCs are mainly tissue-resident lymphocytes that lack adaptive antigen receptors expressed on T cells and B cells (Figure 3) [59]. They are generally classified into three subgroups according to their cytokine and transcription factor expression, which parallel with adaptive CD4+ Th cell subsets: group 1 (ILC1), group 2 (ILC2), and group 3 (ILC3) [59–61]. ILC1s are dependent on the T-box transcription factor (T-bet) for their development and function, and they produce IFN-γ and TNF-α [62]. ILC2s are dependent on GATA binding protein 3 (GATA3) and RAR-related orphan receptor alpha (RORα) [63], and produce type 2 cytokines, including IL-4, IL-5, IL-9, and IL-13 [64]. ILC3s are dependent on the transcription factor RAR-related orphan receptor gamma (RORγt) and can produce IL-17 and/or IL-22 [59,65]. ILC1s react to intracellular pathogens, such as viruses and tumors; ILC2s respond to large extracellular parasites and allergens; and ILC3s combat extracellular microbes, such as bacteria and fungi [59]. In addition, a recent report identified a regulatory subpopulation of ILCs (called ILCregs) that exists in the mouse and human gu<sup>t</sup> and Id3 is a fate decision marker for their development [66]. Compared with these ILC subsets, conventional natural killer (NK) cells has a similar developmental process and quick effector functions, thus NK cells are defined as cytotoxic ILCs, which parallel with adaptive CD8+ cytotoxic T lymphocytes [61]. Mature NK cells are dependent on the transcription factor eomesodermin (Eomes), and produce perforins, IFNγ, and granzymes [67]. NK cells control certain viruses such as herpesviruses and cytomegalovirus and tumors [68].

**Figure 3.** ILCs contribute to the process of intestinal wound repair. During wound healing, ILC1 secretes TNF-α and IFN-γ; ILC2 secretes IL-4, IL-5, IL-13, and AREG; ILC3 secretes IL-17 and IL-22; ILCreg secretes IL-10; and cytotoxic ILC (NK cell) secrete perforins, IFN-γ, and granzymes.

## 2.4.1. The Function of ILCs

ILCs maintain tissue homeostasis but also contribute to inflammatory diseases including IBD [69]. ILCs promote the resolution of inflammation and tissue repair [70].

ILC1s have a crucial role in promoting innate immunity to intracellular pathogens, such as T. gondii, by secreting TNF-α and IFN-γ to recruit inflammatory myeloid cells [70]. Intraepithelial ILC1s expand in CD patients and depletion of intraepithelial ILC1s reduced proximal colon inflammation in the anti-CD40-induced colitis model in mice [71].

ILC2s rapidly respond to helminth parasite infection [70]. ILC2s are increased in patients with ulcerative colitis (UC) and play an important role in the tissue reparative response [72]. ILC2s secreted IL-13 binds with its receptor IL-13Rα1 and activates transcription factor Foxp1 to promote β-catenin pathway-dependent intestinal stem cell renewal [73]. In addition, IL-33 can stimulate ILC2s to produce AREG in the colon and promote intestinal epithelial cell regeneration in a model of DSS-induced colitis [74].

ILC3s promotes innate immunity to extracellular bacteria and fungi, such as *Citrobacter rodentium* and *Candida albicans* [70]. ILC3s are decreased in inflamed tissue in both CD and UC patients [72] and are required for tissue repair and regeneration in the inflamed intestine [75]. Adherent CD-associated microbiota induces the CX3CR1+ mononuclear phagocyte-derived TNF-like ligand 1A (TL1A) [76], which stimulates the production of ILC3-derived IL-22 and increases mucosal healing in human IBD [77]. ILC3s are the main source of intestinal IL-22 and the symbiotic commensal microbiota represses this IL-22 production via inducing epithelial expression of IL-25 [75]. In graft versus host disease, radioresistant ILC3s-produced IL-22 protects intestinal stem cells from immune-mediated tissue damage [78]. Mechanistically, IL-22 activates signal transducer and activator of transcription 3 (STAT3) signaling to increase antiapoptotic proliferative response in Lgr5+ stem cells, promoting epithelial regeneration and reducing intestinal pathology and mortality from graft-versus-host disease [79]. Moreover, dietary aryl hydrocarbon receptor aryl hydrocarbon receptor (Ahr) ligands such as glucosinolates promote IL-22 production from ILC3s and protect intestinal stem cells against genotoxic stress [80]. In addition, ILC3s produced IL-22 production also protects damage to the intestine induced by infection and chemotherapy [81,82]. Apart from IL-22, ILC3s secreted IL-17 and IFN-γ is dependent on IL-23 stimulation and is required in *Helicobacter hepaticus*-mediated innate colitis [83].

ILCregs suppress the activation of ILC1s and ILC3s via secretion of IL-10 and promote innate intestinal inflammation resolution induced by several inflammatory stimuli including DSS, anti-CD40 antibody, *Salmonella typhimurium*, and *Citrobacter rodentium* in Rag1 −/− mice [59].

NK cells with cytolytic potential are accumulated in colonic lamina propria of individuals with active IBD [84], and thiopurines can normalize NK cell numbers by inhibition of Rac1 activity to induce apoptosis [85]. Activated NK cells produce proinflammatory cytokines such as IFN-γ and TNF-α to augmen<sup>t</sup> CD4+ T cell proliferation and Th17 differentiation, which contributes to exacerbated inflammatory response [86].

## 2.4.2. The Regulation of ILCs

Commensal microbiota regulates the transcriptional gene expression and epigenetic regulation in ILCs [87]. RNA- and ATAC-seq integration identified that c-MAF and BCL6 regulate the plasticity between ILC1 and ILC3 in the intestine [69]. Moreover, the Ahr signaling is critical in regulating intestinal ILC2-ILC3 balance. This was demonstrated by the fact that Ahr knockout mice have altered gu<sup>t</sup> ILC2 transcription with increased expression of anti-helminth cytokines such as IL-5 and IL-13, whereas Ahr activation increases gu<sup>t</sup> ILC3 to better control *Citrobacter rodentium* infection [88]. Furthermore, ILC1 and ILC3 undergo retinoic-acid dependent upregulation of gu<sup>t</sup> homing receptors CCR9 and α4β7, while ILC2 acquire these receptors during development in the bone marrow [89]. These gu<sup>t</sup> homing receptors are also critical for optimal control of *Citrobacter rodentium* infection. For ILCregs, autocrine TGF-β1 is critical for their expansion during inflammation [66]. NK cells are regulated by various cytokines such as type I IFN, IL-12, IL-18, IL-15, IL-2, and TGF-β1 [90].
