Cathelicidin Protects the Brain from Mitochondrial DNA Damage in Health, but not Following Septic Shock

Denise Frediani barbeiro Universidade de São Paulo Faculdade de Medicina: Universidade de Sao Paulo Faculdade de Medicina Suely Kubo Ariga Universidade de São Paulo Faculdade de Medicina: Universidade de Sao Paulo Faculdade de Medicina Hermes Vieira Barbeiro Universidade de São Paulo Faculdade de Medicina: Universidade de Sao Paulo Faculdade de Medicina Nadja C Souza-Pinto Universidade de Sao Paulo Campus de Sao Paulo: Universidade de Sao Paulo Fabiano Pinheiro da Silva (  pinheirofabiano@hotmail.com ) Universidade de Sao Paulo https://orcid.org/0000-0003-2673-2202


Introduction
On one hand, we know that DNA is highly susceptible to chemical damage. The DNA replication and repair machinery, moreover, make mistakes. On the other hand, cells possess a sophisticated DNA repair system [1].
Mitochondria are particularly susceptible to DNA damage, since they act as the cellular powerhouses and have to deal with a permanent production of reactive oxygen species (ROS). It is true that an imbalance between ROS generation and cellular system's ability for clearance, promotes damage to lipids, proteins and nucleic acids throughout the cell [2]. Mitochondrial DNA, however, besides its close contact with the respiratory chain, is not protected by histones or a nuclear envelope, becoming an easy target to oxidative lesions. Finally, it is important to cite that besides small, the mitochondrial genome encodes 13 proteins that take part in the oxidative phosphorylation complex [3] and mutations in such genes can also serve to increase ROS cellular levels. Such factors contribute to a high mutagenesis rate [3].
ROS accumulation can lead to DNA base modi cations, deletions, strand breaks and crosslinks.
Oxidative stress and DNA damage have been linked with multiple chronic conditions, such as cancer [4], neurodegenerative processes [5], diabetes [6], cardiovascular diseases [7,8], chronic in ammatory diseases [9] and aging [10]. Here, we hypothesize that DNA damage may also be an important phenomenon to the pathophysiology of sepsis, an acute condition characterized by deregulation of the immune response and intense systemic in ammation. Since the brain is particularly susceptible in sepsis and mitochondrial and immune functions are tightly linked [11,12], we decided to investigate DNA damage in the prefrontal cortex of wild-type and CRAMP-de cient mice, submitted or not to experimental sepsis.
Cathelin-derived antimicrobial peptide (CRAMP) is an antimicrobial peptide that modulates several aspects of the immune response [13]. It is the only cathelicidin in rodents and its counterpart in humans is named LL-37. Cathelicidins are a family of antimicrobial peptides able to directly kill a range of pathogens, including bacteria, protozoa and virus. Despite that, cathelicidins also play a dual role in the immune-in ammatory response through intriguing and poorly understood mechanisms. Indeed, depending on the disease and cellular context, cathelicidins can stimulate or inhibit the immunein ammatory system [14].

Materials And Methods
Cecal ligation and puncture Young (8 weeks-old) and aged (18 months-old) male CRAMP−/− mice on a C57BL/6 genetic background and their matched WT controls were purchased from The Jackson Laboratory (ME, USA).
We induced peritonitis using the model of cecal ligation and puncture (CLP), as previously described [15].
Brie y, animals were anesthetized and the cecum ligated and punctured twice with a 21G needle, allowing fecal material to be released into the peritoneal cavity. Animals were sacri ced 24 hours after the surgery and plasma and tissue samples of the brain (prefrontal cortex) were collected for further analyses.

DNA extraction
The samples were prepared according to the instructions in the Qiagen DNeasy Blood and Tissue kit (#69506 Qiagen). DNA was eluted in 100 µl of elution buffer. The concentration of genomic DNA was determined using Nanoview (GE). Samples were diluted in elution buffer for the PCR assays (6 ng/µL).

Statistical Analysis
Results were analyzed using Kruskal-Wallis test, followed by Mann-Whitney U test with Bonferroni adjustment. Results are shown in boxplots. All analyses were performed using R statistical software (www.r-project.org). A p-value < 0.05 was considered signi cant.

Results
CRAMP protects the brain from mtDNA damage under normal conditions, but not following experimental sepsis.
Both young wild-type mice and young CRAMP-de cient submitted to experimental sepsis showed signi cant mtDNA damage in the brain, when compared to the control groups (p < 0.001 and 0.003, respectively). Secondly, the presence of CRAMP protected the brain of wild-type mice from further mtDNA damage under normal conditions, but not following sepsis (p = 0.011). Aged mice were used as positive controls. As expected, aged mice exhibited more DNA damage in the brain than young mice.

Discussion
It is widely accepted that oxidative stress has a crucial role in sepsis evolution [16,17]. Mitochondrial dysfunction and several ultrastructural changes have been reported in many organs during sepsis [18][19][20][21][22]. It has even been postulated that mitochondrial dysfunction plays a central role in the pathogenesis of the multiple organ dysfunction syndrome (MODS) that frequently follows the course of septic shock and many other in ammatory catastrophes [23,24]. The topic, however, remains controversial. Some authors argue that the studies are still very heterogeneous and inconsistent [25]. DNA damage, for example, as far as we know, had never been investigated in sepsis. Here, we show that mitochondrial DNA damage is aggravated in the brain of wild-type and CRAMP-de cient mice, 24 hours after the induction of experimental sepsis, when compared to the control groups, putting in evidence that sepsis induces signi cant mtDNA damage (Fig. 1). Mitochondrial DNA damage, moreover, is more severe in the brain of healthy CRAMP KO mice, when compared to healthy wild-type mice, showing that CRAMP protects from mitochondrial DNA damage under normal conditions (Fig. 1) We believe that the increase in mtDNA damage in CRAMP-de cient mice, detected only under normal conditions, but not following sepsis, occurred because sepsis induces such a robust in ammatory response that the protective effects of CRAMP became subtle in this situation. Septic encephalopathy patients, thus, may bene t from a targeted therapy directed to restore mitochondrial integrity

Con icts of interest/Competing interests
The authors have no relevant nancial or non-nancial interests to disclose.