*2.1. Neutrophils*

Neutrophils are the first responding leukocytes to sites of inflammation when the intestinal epithelial barrier is breached and the gu<sup>t</sup> microbiota invade [5]. Mouse neutrophils migrate to wounded tissues begins 4 h and reach peak numbers 18 to 24 h after injury [6]. Neutrophils are short-lived cells with a half-life in the circulation of approximately 1.5 h and 19 h in mice and humans, respectively [7,8]. However, proinflammatory cytokines such as TNF-<sup>α</sup>, IL-1β, and IL-6 increase the lifespan of neutrophils [9], which may contribute to the resolution of inflammation [7].

#### 2.1.1. The Function of Neutrophils

The neutrophils can exert both destructive and protective effects in wound healing (Figure 2) [10]. Excess neutrophils in injured tissues impair healing and correlate with the cryp<sup>t</sup> destruction and ulceration [11,12]. During intestinal inflammation, neutrophils undergo transepithelial migration and secrete a large amount of matrix metalloproteinase-9 (MMP-9) to disrupt epithelial intercellular adhesions, which leads to enhanced epithelial injury [13]. Neutrophil-derived miR-23a–and miR-155-containing microparticles also promote accumulation of double-strand breaks, which leads to impaired colonic healing [14].

**Figure 2.** Neutrophils are a double edge sword in intestinal wound repair. Neutrophils damage intestinal mucosal through secreting MMP-9 and miRNA containing microparticles at acute phase of injury, but they can also promote wound repair through killing bacteria, modulating HIF-1α/ITF signaling and secreting pro-repair cytokines, chemokines, and growth factors.

As neutrophils have a key role in controlling microbial contamination and attracting monocytes and/or macrophages [15], individuals with too few neutrophils display not only higher risk for developing wound infections, but also delayed wound healing [16]. However, blocking neutrophil invasion or neutrophil depletion led to aggravated experimental colitis in animals, indicating a protective role of neutrophils in mucosal repair process [17].

Neutrophils kill bacteria through phagocytosis, neutrophils extracellular traps [18], antimicrobial peptides (including cathelicidins and β-defensins), microbicidal reactive oxygen species, and cytotoxic enzymes such as elastases, myeloperoxidase, and MMPs [19]. Infiltrating neutrophils deplete local oxygen to stabilize the transcription factor hypoxia inducible factor (HIF)-1α in wounded human and murine intestinal mucosa and promote resolution of inflammation. HIF-1α stabilization also protects barrier function through induction of intestinal trefoil factor (ITF) [20,21]. It has been shown that the probiotic *Lactobacillus rhamnosus* GG restored alcohol-reduced ITF in a HIF dependent manner [22].

In addition to eliminating bacteria and adjusting the wound microenvironment through oxygen metabolism, neutrophils promote wound repair by secreting pro-repair cytokines, chemokines, and growth factors. After dextran sodium sulfate (DSS)-induced mucosal injury, neutrophil-derived transforming growth factor-beta (TGF-β) activates MEK1/2 signaling and induces the production of the EGF-like molecule amphiregulin (AREG) in intestinal epithelial cells, which protects intestinal epithelial barrier function and ameliorates DSS-induced colitis [23].

## 2.1.2. The Regulation of Neutrophils

Antibiotic treatment of dams reduced circulating and bone marrow neutrophils via reducing IL-17-producing cells in the intestine and their production of granulocyte colony-stimulating factor (G-CSF) [24]. In contrast to the mucosal protective effects of acute HIF-1α activation described above, we have previously showed that chronic activation of epithelial HIF-2α increased the proinflammatory response [25] and cancer development [26,27]. Among various mechanisms, HIF-2α can directly regulate the expression of neutrophil chemokine CXCL1, which facilitates the recruitment of neutrophils in colitis associated colon tumor [28]. Similarly, during intestinal inflammation, the intestinal epithelial production of neutrophil chemotactic cytokine IL-8 (chemokine C-X-C motif ligand 8, CXCL8) is increased by proinflammatory cytokines IL-1β, TNF-<sup>α</sup>, or interferon-γ (IFN-γ) [29]. A recent report also showed that IFN-γ induced expression of a neutrophil ligand intercellular adhesion molecule-1 (ICAM-1) on the intestinal epithelium apical membrane, which led to enhanced epithelial permeability and facilitated neutrophil transepithelial migration [30]. Interestingly, the enhanced ICAM-1 and neutrophil binding results in decreased neutrophil apoptosis, activation of Akt and β-catenin signaling, increased epithelial cell proliferation, and wound repair [31]. Il-23 signaling is also required for maximal neutrophil recruitment after DSS treatment [32].
