*4.4. Restoration of Microbial Dysbiosis in CST-KO Mice after Fecal Microbial Transplant (FMT) from WT Donor Mice*

FMT is now established as an effective therapeutic modality in the treatment of the following diseases: (i) antibiotic-refractory recurrent *Clostridium difficile* colitis with a success rate of up to 95% [102–105], (ii) constipation, (iii) irritable bowel syndrome, and (iv) inflammatory bowel disease [106–108]. Therefore, attempts were made recently to assess whether gut microbial population in mice can be reversed by reciprocal FMT. WT mice that received FMT from the CST-KO mice (WTFMT-CST-KO) encompassed a reduction of Clostridia and *Akkermansia* [109], which are linked to metabolic disorders and insulin resistance [110,111] and a marked increase in the Proteobacteria population, which are associated with active inflammatory bowel disease (IBD) states [112,113]. Of note, CST-KO mice are insulin-resistant on a normal chow diet [97]. In contrast, insulin-resistant CST-KO mice that received FMT from the WT mice (CST-KOFMT-WT) showed an increase in richness, a notable reduction of *Staphylococcus*, and an increase in the butyrate-producing *Intestinimonas* [109] (Figure 6). Butyrate, taken up directly by colonocytes, serves not only as a direct source of energy that contributes directly to a healthy gut, but also acts as a signaling molecule that affects many factors such as satiety, secretion of hormones, and glucose metabolism [114–116]. Furthermore, reduced levels of butyrate are strongly associated with IBD and metabolic disorders [117,118]. Butyrate has also been shown to restore gut barrier integrity [119], modulates regulatory T cell function [120–122], and regulates certain serine proteases [123,124].

#### **5. Catestatin and Innate Immunity**

The first indication for the role of CST in innate immunity came from a study in rats where intravenous administration of CST was shown to reduce pressor responses by electrical stimulation [125]. The hypotensive effect of CST was revealed to be mediated at least in part by profuse histamine release (by ~21-fold) and action at the H1 receptor [125]. The in vivo studies were later confirmed in peritoneal and pleural mast cells where CST caused dose-dependent release of histamine utilizing signaling pathways established for wasp venom peptide mastoparan and other amphiphilic cationic neuropeptides (the peptidergic pathway) [126]. This pathway is in sharp contrast to the nicotinic-cholinergic pathway used by CST to induce catecholamine secretion from chromaffin cells [5]. Subsequent studies uncover the following: (i) release of immunoreactive CST-containing peptides from

human stimulated polymorphonuclear neutrophils [21]; (ii) detection of CST in mouse peritoneal macrophages by Western blots [98]; (iii) detection of CST in human monocytes and monocyte-derived macrophages by Western blots [127]; (iv) blockade of lipopolysaccharide (LPS)-induced increase in expression of tumor necrosis factor alpha [127]; (v) decreased expression of proinflammatory cytokines by CST in plasma and heart [98]; (vi) inhibition of infiltration of macrophages in obese liver [97]; (vii) degranulation of primary mast cells from human peripheral blood [128]; and (viii) low plasma CST in fatal COVID-19 patients [129]. These findings implicate CST as an immunomodulatory peptide. Since receptor-ligand interactions are an essential driver of host-immune response [130], it is important to examine if CST can bind with a receptor on immune cells and regulate their polarization and function in host defense.

#### **6. Evolutionary Conservation and Selection Pressure on CST in Mammals**
