**4. In Vitro, CGA-Derived Peptides Modify the Immunological Activities of Specific Cells Belonging to Defense**

CGA has a prohormone function; numerous cleavage products of this protein display activity in the domain of defense [21,22]. Therefore, we speculated that the CGA-derivedpeptides Chromofungin (CHR, CGA47-66) and Catestatin (CAT, CGA344-364) might impact

the functioning of human cells involved in immunity. We harvested polymorphonuclear cells (PMNs) and monocytes from healthy controls. We exposed them ex vivo to the peptides to assess the consequences as they may occur in vivo during systemic inflammation.

We first performed experiments on PMNs [23]. We demonstrated that, after intracellular penetration, both CHR and CAT provoked a rapid and synergic Ca++- entry with no lytic effect on the cells. This occurred provided free calcium was available in the extracellular space. It was concentration-dependent and in the range of concentrations relevant for clinical effects. We also explored the impact of scrambled isomers and amino-acid substitutions. In doing so, we identified the need for a perfect respect of the chemical primary structure of both CAT and CHR to obtain the expected pharmacodynamical impact, indicating that a precise mechanism of both cell entry and action is required for physiological effects. The Ca++- entry evoked by CHR and CAT is consistent with Ca++ selective store-operated calcium channel activity. Once inside the cells, CAT and CHR interact with calmodulin, thereby allowing the release of lysophospholipids by membranebound iPLA2 and subsequent store-operated calcium channels. As an ultimate result of intracellular Ca++ concentration increase, the PMNs release secretions, among which we isolated factors involved in innate immunity such as lactotransferrin, neutrophil gelatinase, lysozyme, S100 A, and S100B calcium-binding proteins. These data point to a role of CAT and CHR in Ca++ signaling outside the chromaffin cells, with an impact on the activation of PMNs through a mechanism not related to a cellular membrane-bound receptor but in line with a cell-penetrating peptide activity. This action explains how the neuro-hormonal response to stress may trigger a rapid-onset effective enhanced pro-inflammatory PMNsrelated mechanism of defense in vivo in any vascularized tissue where an insult occurs. It is also of note that CAT has been reported to act via the nicotinic acetylcholine receptor (nAChR), a classical surface receptor, which can also participate in anti-inflammatory responses through neural immunity regulation [6].

Our group also explored monocytes. We assessed a possible effect of the CGA47-70 derived peptide (which includes CHR) detected in the plasma of multiple trauma patients who are prone to develop care-related infections [13]. This molecule entered the monocyte progressively over 5 to 15 min with at least two intracytoplasmic localizations, one of which was detected in the perinuclear region. In further cellular investigations, including luciferase assays, we showed that CGA47-66 could inhibit both NF-kappa B and AP-1, which play a role in amplifying and perpetuating the inflammatory processes in vivo. Such activities suggest an anti-inflammatory potential for this peptide with a risk of deleterious imbalance of innate immunity.

#### **5. CGA-Derived Peptides as Actors against Superbugs**

Antibiotic-resistant microbes (bacteria, fungi, and yeasts) are detected increasingly in samples harvested from ICU patients, and they trigger significant morbidity when comorbidities are present. There is, therefore, a need for better tools to cure patients, in addition to the discovery of new anti-microbial drugs.

Based on the observation that bacterial host cells genetically engineered to express CGA for industrial production are dying upon the induction of CGA expression, our group hypothesized that chromogranins impact bacterial survival (see in [22]). Following the HPLC of the protein material secreted by chromaffin cells, it was concluded that several CGA-derived peptides and CGA itself, proenkephalin-A, and free ubiquitin participated partially in the struggle for survival after a stressing challenge. A summary of the antibacterial, antifungal, and anti-malaria properties of some of these chromaffin cell-derived peptides was given in a recent review [22]. Interestingly, while these properties are related to the CGA-derived molecules, they may sometimes be enhanced through synergic associations with either therapeutic albumin or commercially available anti-microbial drugs. Indeed, since AMPs interact with cell membranes, they represent candidates to potentiate anti-microbial drugs, as shown for some molecules marketed for ICU patients [24,25]. Our data also suggest that, sometimes, infectious diseases occur when these AMPs fail to fulfill their missions in oxidative stressing conditions.

Recently, our group also reported that CTS (CGA344-364), but not Cateslytin (CTL, CGA352-366), interacts with circulating albumin, which underlines the important role of the C-terminal part of CTS for the binding process [26]. This interaction improves the anti-microbial activity of this peptide against C. albicans at a concentration of 4 μM, demonstrating a synergistic effect [26].

Finally, human intervention on some of the L-isomers of these molecules may also modify their potency in an attempt to coat medical implants [24,27]. In recent studies, we have tested natural peptides against superbugs carried by ICU patients. It emerged that some natural and some synthetic peptides, though not all, recovered significant bactericidal activity in vitro when associated with or chemically modified. Investigations further showed that AMPs act as either cell-penetrating peptides destabilizing the cell wall of the microbes or as intracellular molecular actors interacting with calmodulin with subsequent limitation of the rate of activity of calmodulin-activated enzymes, some of which play a role in hyphal growth. These data prompted the conclusion that biomaterials intended for human implantation may benefit from the coating by such molecules or from the latter being sprayed (on wounds, for instance). Indeed, contaminations of medical devices and surgical sites continue to contribute to significant hospital morbidity. We have, therefore, successfully moved to functionalize bioprostheses with biomaterials designed to combat biofilm-associated infections. Interestingly, a self-killing approach with the bacterial-controlled release of AMPs has been reported [28]. Lastly, CGA-derived peptides were included in hydrogels to prevent oral cavity infection [29]. CAT was functionalized with polyarginine and hyaluronic acid on a silver platform: this, in addition to CAT's anti-microbial activity, strongly limited the local production of inflammatory cytokines. This is a matter of interest in relation to buccal implantation [30], with potential as regards devices requiring transient implantation in ICU patients.
