*3.2. CgA-Derived Peptides and Immune Cells Activities and Inflammation*

The involvement of CgA and its derived peptides in innate immunity is well known for its antimicrobial activity. In addition, the role of these MF-AMPs is also reported in immune cells, conferring them a complex profile of immunity modulators. This section analyzes this profile studied using in vitro and in vivo models. The role of CgA-derived peptides on immune cells was studied for the first time in 2009; in particular, the effects of Chr and Cts were evaluated on polymorphonuclear neutrophils. After the treatments with the peptides, Chr and Cts were observed inside the cells, demonstrating their ability to penetrate the mammalian membrane and the profile of CPPs [34]. Then, in the presence of extracellular calcium, the two peptides induced a transient calcium influx in the cells, binding Calmodulin-binding factors (W7 and CMZ) and activating iPLA2 [34]. In addition, the pharmacological block of these channels inhibited the calcium flux induced by Chr and Cts [34]. On the other hand, when extracellular calcium is absent, the peptides cannot induce calcium secretion [34]. Notably, the secretion of polymorphonuclear neutrophils treated with the two CgA-derived peptides induced the secretion of several important factors for innate immunity and inflammation, such as Lactotransferrin, Lysozyme, Neutrophil Gelatinase Associated Lipocalin and S100 calcium-binding protein A8/A9 [34]. Several studies reported the role of Vs-1 as an anti-atherogenesis and anti-inflammatory factor suppressing the adhesion of monocytes to endothelial cells by adhesion molecule down-regulation [59,60]. Xiong and coworkers reported the anti-inflammatory role of Vs-II (N-terminal fragment of CgA containing Vs-I; CgA1-113) in an apolipoprotein E-deficient (ApoE−/−) mice model fed with a high-fat diet developing atherosclerosis. In this study, Vs-II treatment reduced the occurrence of atherosclerotic plaque and attenuated lesions [59]. Furthermore, Vs-II significantly reduced the production of pro-inflammatory cytokines in aortic tissue, such as Tumor Necrosis Factor-α (TNF-α), Monocyte Chemoattractant Protein-1 (MCP-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) [59]. The same authors demonstrated, by several in vivo analyses, that these anti-inflammatory properties are based on the ability of Vs-II to reduce leukocytes adhesion on ApoE−/<sup>−</sup> mice arteries but also on the recruitment, transmigration and accumulation of M1 macrophages in the lesions [59]. In the same animal model, Sato et al. showed that Vs-I treatment reduces aortic atherosclerotic lesions development due to reductions in intra-plaque inflammation, macrophage infiltration and aortic smooth muscle cells proliferation and plasma glucose level [60]. From a cellular point of view, Vs-I suppressed the lipopolysaccharide (LPS) induced production of chemokine MCP1 and vascular damage markers, such as VCAM-1 and E-selectin, in human endothelial cells [60]. At the same time, Vs-I was found to reduce M1 pro-inflammatory macrophages differentiation and IL6 release but also oxidized low-density lipoprotein (oxLDL)-induced foam cell formation of macrophages [60]. Of great clinical interest, Vs-I is expressed around Monckeberg's medial calcific sclerosis in human radial arteries [60]. Additionally, the immunomodulatory role of Chr has been reported in a mice model of ulcerative colitis induced by dextran sulfate sodium administration [61,62]. This model decreased Chr expression [62]. Furthermore, Chr treatment, by intracolonic administration, significantly reduced the inflammation and severity of colitis. The anti-inflammatory effects were due to the differentiation of macrophages into M2 anti-inflammatory clones with the consequent reduction of released IL-18 and the increased expression of M2 markers [61,62]. Using the same animal model, Kapoor and colleagues demonstrated that intrarectal Chr treatment reduced colitis severity and inflammation [63]. In parallel, this was associated with a significant decrease in the expression of CD11c, CD40, CD80, CD86 IL6 and IL12p40 in the inflamed colonic mucosa, mesenteric lymph nodes and spleen [63]. In addition, Chr reduces in CD11c positive cells the expression of CD80, CD86 and NF-κB in the spleen and colon, respectively [63]. All these in vivo data demonstrated that Chr has protective properties against intestinal inflammation and exerts the role of immunomodulator for intestinal macrophages and dendritic cells (DCs). These effects were also demonstrated in vitro with macrophages showing that Chr increased the production of anti-inflammatory factors and the M2 differentiation [61]. At the same

time, Chr treatment significantly reduced the expression of M1 macrophage markers and the activation of the NF-κB pathway [62]. Additionally, in this case, in vitro experiments with M1 macrophages demonstrated that this peptide could decrease cellular migration, proinflammatory cytokines production and release, and NF-κB phosphorylation [62]. Furthermore, treatment with Chr or a conditioned medium of Chr-treated macrophages M2 induced epithelial cell proliferation and migration but also decreased oxidative stress and pro-inflammatory cytokine production [61]. In addition, the Chr treatment of naïve bone marrow-derived CD11c positive DCs reduced the LPS-induced expression of CD40, CD80, CD86 IL-6 and IL-12p40 [63]. These results were also confirmed in intestinal tissue isolated from patients with ulcerative colitis, demonstrating that Chr expression was down-regulated in these patients compared to healthy controls [62]. Indeed, the mRNA levels of Chr were positively correlated with the mRNA expression of M2 macrophages activation markers and negatively to the expression of collagen, IL-8 and IL-18, but also with M1 activation markers (TLR-4 expression and NF-kB activation) and consequent pro-inflammatory cytokines production [61,62]. In these patients, the reduction of Chr level is also associated with a negative linear relationship with CD11c and CD86 [63]. Moreover, another study confirmed the anti-inflammatory effects of Chr on monocytes. Treatment with this peptide significantly inhibited the transcription of pro-inflammatory factors, such as NF-kB and AP-1, in these cells [64]. Furthermore, Rabbi and colleagues showed that Cts reduced intestinal inflammation and the onset of colitis lesions by a Stat-3 activation [65]. At the same time, the markers of M1 macrophage activation and the colonic levels of pro-inflammatory cytokines, such as IL-6, IL-1β and TNF-α, were significantly decreased by Cts treatment [66]. However, Cts did not influence M2 macrophage markers [66]. Furthermore, these anti-inflammatory effects were confirmed in cellular experiments using macrophages isolated from the peritoneal cavity and the bone marrow, demonstrating that in vitro treatment with Cts significantly decreased the production of the pro-inflammatory cytokines and phosphorylation of Stat-3 [65]. In addition, peritoneal macrophages isolated from naïve mice and treated with Cts and LPS displayed a reduction in the expression and production of pro-inflammatory cytokines blocking the activation of M1 macrophages [66]. In addition, the genetic deletion of Cts induces hypertensive conditions and left ventricular hypertrophy accompanied by significant macrophage infiltration in cardiac tissue and adrenal gland [67]. In this context, the absence of Cts induced an increased level of pro-inflammatory cytokines TNF-α, C-C motif chemokine ligand (CCL)-2, 3, C-X-C motif chemokine ligand (CXCL)-1 and catecholamines but also an elevated inflammation in heart with the up-regulation of cardiac genes, such as *Tnfa*, *Ifng*, *Emr1*, *Itgam*, *Itgax*, *Nos2a*, *IL12b*, *Ccl2* and *Cxcl1* [67]. It is of great interest that the intraperitoneal administration of Cts reversed this phenotype. In addition, macrophage depletion blocked the onset of hypertension in Cts-knockout (KO) mice [67]. Furthermore, bone-marrow transfer of KO animals in wild-type (WT) counterparts induced hypertension and cardiac inflammation, while opposite conditions showed the opposite phenotype [67]. All these data strongly suggest that the anti-hypertensive effects of Cts are partially mediated by an immunosuppressive action of this peptide on macrophages [67]. The role of Cts as an immunomodulator was also explored in the context of atherosclerosis and vascular injury. In fact, in vitro treatment with Cts on endothelial cells significantly reduces the release of TNF-α and vascular damage markers, such as ICAM-1 and VCAM-1, after LPS exposure [68]. At the same time, Cts treatment suppresses inflammatory responses and oxidizes the low-density lipoprotein-induced foam cell formation of human macrophages [68]. Kojima and colleagues demonstrated that Cts injection to ApoE−/<sup>−</sup> mice significantly reduces macrophage infiltration and the consequent atherosclerotic lesions onset in the aorta but also suppresses aortic smooth muscle cells proliferation and collagen deposition in atheromatous plaques [68]. Furthermore, in vitro experiments with human aortic smooth muscle cells showed that Cts treatment can block collagen-1 and fibronectin expression and migration, proliferation and apoptotic process [68]. Of significant clinical impact, coronary artery disease patients displayed a substantial reduction of plasmatic levels of Cts but an increased expression

in coronary atheromatous plaques [68]. In an acute pulmonary embolism in vivo model and cellular experiments with human pulmonary artery endothelial cells, Cts treatment was found to abolish thrombin-induced inflammation blocking TLR-4 expression and p38 phosphorylation, decreasing the consequent acute pulmonary embolism [69]. In conclusion, Vs-I and Chr than Cts are key attenuators of inflammation in different tissue and pathological conditions by reducing immune cell infiltration and inflammatory activation (Figure 2).

**Figure 2. ChromgraninA** (**CgA**)**-derived Peptides and Inflammation**. Damage or stress stimuli induce the migration, proliferation and activation of macrophages with consequent pro-inflammatory cytokines and chemokines release. This phenomenon generates cell death and inflammation. CgA-derived peptides can reduce M1 polarization and promote M2 anti-inflammatory macrophages differentiation. C-C motif chemokine ligand (CCL); C-X-C motif chemokine ligand (CXCL); interleukin (IL); monocyte chemoattractant protein (MCP); toll-like receptor (TLR); tumor necrosis factor (TNF).
