**5. Impact of Macrophages in the Development of Radiation Fibrosis**

Recent insights regarding the functional role of inflammatory cells suggest that inflammation could play a role beyond the classical "acute" phase. During the radiation wound repair process, recruitment of inflammatory cells occurs at the site of injury, which can contribute to late inflammatory tissue damage through a continuous mechanismbetween inflammation, hypoxia and fibrosis [46]. During normal healing, sequential activation

of the classical, proinflammatory, M1 and alternately activated macrophages, M2a, M2b and M2c, is known to occur, which facilitates the transitions between the inflammatory, proliferative and remodeling phases of the repair process [47]. Thus, macrophage dysfunction or deficient generation can lead to the uncontrolled production of inflammatory mediators and growth factors. This can modify their communications with other cells (epithelial and/or endothelial cells, fibroblasts, progenitors and stem cells) and contribute to a state of persistent injury, which could, in turn, lead to the onset and maintenance of a pathological fibrotic process [48,49]. Macrophages are heterogeneous cells with various phenotypes and functions in part regulated by their micro-environment. Macrophages commonly exist in two (basically defined) distinct subsets, M1 and M2 macrophages, which have different functions and transcriptional profiles (schematically: M1 macrophages are associated with the production of proinflammatory cytokines, while M2 phenotype reprogramming enhances the production of anti-inflammatory cytokines) [50–52]. The representation of macrophage subtypes and their different actions are illustrated in Figure 2. The macrophage reprogramming processes, and steps have been reviewed elsewhere. Briefly, those include 1/an amplified macrophage response, which is a direct amplification following exposure to a reprogramming factor (e.g.,INFγ) but also a cross-amplification consecutive to another factor, such as lipopolysaccharide; 2/a reciprocal suppression of the alternate phenotype (M1/M2); 3/a cascade activation of the reprogramming mechanisms; 4/a feedback phenomenon [53].There are schematically two typical macrophage reprogramming signaling pathways: on the one hand, activation pathways such as JNK, Notch, TLR/NF-κB (p65/p50), PI3K/Akt2, JAK/STAT1, and HIF1αfavor the M1 phenotype; while on the other hand pathways such as PI3K/Akt1, JAK/STAT3/6, TGF-β/SMAD, TLR/NFκB (p50/p50) and HIF2α are mainly involved in M2 phenotype programming [54]. After a fractionatedirradiation, an abnormal wound healing response occurs, which is characterized by the accumulation of M2 macrophages that promote fibrosis through the production of TGF-β1 [48,55]. Temporal and spatial coordination of myofibroblast activities with inflammatory macrophages is crucial for the controlled healing process and restored homeostasis in injured tissue, such as after irradiation. In this context, the M1 phenotype has also been associated with an antifibrotic effect by releasing MMPs (MMP-9, MMP-12 and MMP-13 that degrade ECM). However, if the injury process persists, fibrosis progresses with the proliferation of myofibroblasts and ECM deposition (such as collagen and fibronectin) in and around inflamed or damaged tissue [56,57]. In this context, M1 macrophages represent the starting point of the profibrotic process. Indeed, M1 macrophages release proinflammatory cytokines and chemokines that indirectly promote the proliferation of myofibroblasts. In this deregulated tissue microenvironment. There is a modulating of the macrophage phenotype, in which M2 macrophages phenotype can be generated by apoptotic bodies accumulated, ECM quality modified and Th2 cytokines stimulation [58]. M2 initially involves anti-inflammatory cells, with the release of IL10, arginase, TGFβ and HO-1 [59]. When the tissue microenvironment homeostasis is deregulated, M2 activation leads to fibrocytes recruitment and proliferation, myofibroblastactivation, and fibroblasts proliferation. In addition to collagen production, pro-fibro-fibrotic genes are transcripted, leading to the secretion of a large number of profibrotic factors such as TGF-B1, PDGF, IL6, IL-13 IL-7 and galactin-3and to an increase in tissue inhibitors of metalloproteinases (TIMPs) expression [55,57,60,61]. Recent data highlighted that the exacerbation of radiationinduced pulmonary fibrosis might depend on the mesenchymal transition of epithelial cells, promoted by the TGF-β-secreting M2 macrophages [62]. Therefore, any change in the M1/M2 balance will have a central role in terms of fibrosis control or worsening. In addition, exposure to irradiation may also activate macrophages indirectly. An abnormal wound healing response occurs, which is characterized by the accumulation of M2 macrophages, which promote fibrosis through the production of TGF-β1 [63]. Non-coding RNA appears to be involved in the initiation and progression of radiation-induced lung fibrosis by modulating the M2-mediated signaling pathway [64]. In animal models of lung fibrosis, it was reported a high expression of let-7i and low expression of miR-21 at 3 weeks

post-irradiation. At a later stage (after 26 weeks), let-7i expression decreased, and miR-21 was upregulated. In addition, it has been described that let-7i targets TGFBR1, inhibiting TGF-β signaling, while miR-21 degrades the TGF-β inhibitor SMAD7. In support of this, the miR-21 expression is upregulated in several models of fibrosis [65,66]. Significant functional and temporal differences have been shown among several distinct miRNAs that are found both in the inflammatory phase (immediately post-irradiation) and in the later fibrotic stages [58]. Mukherji et al. proposed that macrophage activation could be a secondary effect of radiation exposure, which may result from cellular damage signals and clearance of radiation-induced apoptotic cells, rather than a direct effect of irradiation [67]. In Oncoimmunology, Meziani and colleagues et al. provide a thorough discussion of the immune system/macrophage responses to radiotherapy and their involvement in the development of radiation injury. Macrophages are described as a promising therapeutic target for the prevention or the treatment of radiation-induced toxicities [68]. This immunomodulatory approach aims not only to increase the antitumor efficacy of radiotherapy but also to limit its side effects. To limit the initiation of fibrosis in healthy irradiated tissue, the migration of type 2 macrophages or the reprogramming of M1/M2 must be controlled [68]. However, the dynamics of myeloid cells in the bladder after pelvic radiotherapy have not yet been elucidated and must be investigated.

**Figure 2.** Role of macrophage polarization in inflammation, phenotype markers and signaling molecules involved in M1/M2, M2a, M2b, M2c, M2d. Importance of M1/M2 balance in tissue repair. The M1 CD68 +/CD80 + macrophages which are pro-inflammatory and the M2 CD68 +/CD206 + macrophages which are anti-inflammatory. Red arrows: elevation. Blue arrow: decrease **Figure 2.** Role of macrophage polarization in inflammation, phenotype markers and signaling molecules involved in M1/M2, M2a, M2b, M2c, M2d. Importance of M1/M2 balance in tissue repair. The M1 CD68 +/CD80 + macrophages which are pro-inflammatory and the M2 CD68 +/CD206 + macrophages which are anti-inflammatory. Red arrows: elevation. Blue arrow: decrease

#### **6. Preclinical Studies of Radiation Cystitis and Cell Therapy: A New Therapeutic Avenue**  *6.1. Preclinical Studies of Radiation-Induced Cystitis*  Animal models of radiation cystitis are preferably performed in rodents. As illus-**6. Preclinical Studies of Radiation Cystitis and Cell Therapy: A New Therapeutic Avenue**
