Matías González-Quiroz

Alice Blondel

Alfredo Sagredo

Claudio Hetz

Eric Chevet

Remy Pedeux
email: remy.pedeux@univ-rennes1.fr

When Endoplasmic Reticulum Proteostasis Meets the DNA-Damage Response

Keywords:

Sustaining both proteome and genome integrity requires the integration of a wide range of mechanisms and signaling pathways. The latter comprise, in particular, the unfolded protein response (UPR) and the DNA damage response (DDR).

These adaptive mechanisms take place respectively in the endoplasmic reticulum and in the nucleus. Alterations in UPR and DDR are associated with aging and with the occurrence of pathologies such as degenerative diseases, metabolic and inflammatory disorders, and cancer. Here we discuss the emerging signaling crosstalks between the UPR stress sensors and the DDR and their implication in cancer biology.

Introduction

Maintenance of protein homeostasis (proteostasis) is mediated by a network of interconnected quality-control processes, ensuring a functional proteome [1]. Deregulation of endoplasmic reticulum (ER) proteostasis is a common feature of several metabolic, degenerative, immunological, or neoplastic diseases [2,3]. ER proteostasis surveillance is mediated by the unfolded protein response (UPR), a signal transduction pathway that senses protein biogenesis defects in the ER [2]. Likewise, alteration of genome integrity (GI) and the mechanisms involved in GI maintenance, prevent inherent and sporadic genetic diseases. An evolutionarily conserved mechanism, the DNA damage response (DDR) ensures GI through the recognition of DNA lesions, followed by the initiation of a signaling cascade resulting in DNA repair [4]. Recently, failure in maintaining GI was associated with ER proteostasis alteration [5 10]. In addition, some studies now support a fundamental biological function for UPR sensors in the maintenance of GI and DNA-damage gene expression [11 14].

In this review we describe the UPR and DDR sensors, their mechanisms of action, their impact on global proteostasis and on the activation of the DDR. We discuss emerging connections between the UPR and the DDR and we focus on cancer, given the relevance of both pathways as hallmarks of this disease.

ER Proteostasis and the Unfolded Protein Response

The ER is the gateway to the secretory pathway through which ~30% of the cellular proteins transit. Proteins acquire proper folding and conformation in the ER, thus making this compartment a key contributor to cellular proteostasis [1]. ER proteostasis disruption can be due to i) malfunctions of ER proteostasis control mechanisms, ii) the accumulation of improperly folded proteins, or iii) the imbalance between protein folding capacity and demand, and yields a condition called ER stress.

To cope with ER stress, the UPR, a homeostatic signaling pathway that aims at restoring ER proteostasis, is triggered to increase ER protein folding and clearance capacity or to promote cell death programs if the stress cannot be resolved (see box 1) [15,16]. Under basal conditions (non stressed), it is believed that ER stress sensors are maintained inactive through the binding to the ER luminal chaperone binding immunoglobulin protein (BiP), whereas the accumulation of misfolded proteins triggers BiP release from the stress sensors thereby allowing their activation (Figure 1) [17]. The execution of the UPR results in i) the reduction of misfolded proteins in the ER resulting from the transient attenuation of mRNA translation; (ii) the improvement of the ER folding capacity by increasing the expression of ER-resident chaperones proteins; (iii) the enhancement of ER protein clearance by increasing its degradation capacity (e.g., through the ER-associated degradation (ERAD) [18]); (iv) the enhancement of the export capacity (e.g. through the upregulation of the expression of several genes whose products are involved in ER cargo exit) (Figure 1) [19].

The UPR is transduced by three ER-resident proteins, I E1 , PERK and A F6 , whose primary function is to activate the signalling pathways whose aim is to restore ER proteostasis [2]. However, when ER stress cannot be resolved, these PERK is a ubiquitously expressed type I transmembrane serine/threonine kinase. Under ER stress, PERK oligomerizes and trans-autophosphorylates to acquire full kinase catalytic activity and to phosphorylate the eukaryotic translation initiator factor-2 (eIF2 ), thereby attenuating the general protein synthesis [2,30].

This limits the entry of newly synthesized proteins in the ER while allowing the selective translation of a growing set of specific mRNA such as that coding for the activating transcription factor 4 (ATF4), a transcription factor which promotes the antioxidant response, amino acid metabolism, ER folding capacity, upregulation of macro-autophagy and therefore has an important pro-survival role (Figure 1) [2].

Additionally, ATF4 expression engages the apoptotic program through the expression of CHOP protein (also known as GADD153), a transcription factor that upregulates the pro-apoptotic members of the BCL-2 family and GADD34 (see box 1) [2,31].

ATF6 is a single-pass type II transmembrane protein located in the ER under resting conditions. ATF6 bears a bZIP transcription factor on its cytosolic domain that is released upon ER stress [2,32]. The accumulation of improperly folded proteins in the ER causes ATF6 to be exported to the Golgi apparatus and processed by the S1P and S2P proteases [16]. This process mainly leads to the release of the cytosolic fragment domain of ATF6 [16]. In the nucleus, ATF6 cytosolic domain, simultaneously with XBP1s, upregulates the expression of CHOP and other genes involved in the regulation of ER size, protein-folding capacity, and the ERAD (Figure 1) [33,34].

Remarkably, reprogramming of UPR signaling has been linked with the acquisition of several distinctive hallmarks of cancer [35]. Tumour cells are exposed to several cell-extrinsic and -intrinsic perturbations that promote the selective pressure to engage the UPR signalling [19,35]. In general, IRE1 and PERK signaling contribute to cancer progression by promoting tumour growth and cell survival in different type of tumours [27,36 39]. However, there are only few studies that link ATF6 activity and cancer. The expression of ATF6 is elevated in colorectal cancer but not in normal mucosa [40] and its expression correlates with a poor prognosis [41]. In human epidermoid carcinoma cells, ATF6 signaling increases Rheb expression, which in turn activates mTOR signaling (Figure 2) [42].

In addition, protein disulfide isomerase 5 (PDIA5)-dependent activation of ATF6 was described to be instrumental in the acquisition of Imatinib resistance in chronic myeloid leukemia (Figure 2) [43]. Although the role of UPR signaling in the tumour biology is supported by strong evidence [35,39,44], the specific molecular relationship with the genomic instability has not been studied in depth.

Genome Integrity and the DNA Damage Response

The preservation of genomic integrity represents a challenge because DNA is constantly exposed to endogenous and exogenous sources of damage. To

UPR and DDR signaling in genomic integrity

Maintaining GI is critical to prevent diseases such as cancer, but once cancer occurs it is important to promote the survival of cancer cells. Genotoxic stress is key in most of the cancer treatments, because the collapse of DDR activation and DNA repair mechanisms trigger cell death [52,53]. Interestingly, in patients with a poor prognosis there is a significant correlation between higher BiP expression and chemoresistance [19,39]. Moreover, in a panel of cancer cell lines, genotoxic drugs promote changes in ER structure in a process mediated by the transcriptional activation of p53 (one of the main tumour suppressor and a key player in DDR (see box 1)), which generates the expression of receptor expression-enhancing protein 1 and 2 (REEP1/2) and p53-induced gene 8 (PIG8), three ER-shaping proteins [54].

To date few evidences, connect the UPR and SSB repair. Among them, HMGB1, a protein mainly involved in MMR and BER was shown to be associated with the UPR after ER stress in Huntington s disease. Nonetheless, this link was revealed using a bioinformatic analysis and thus should be carefully considered and confirmed experimentally [55]. In addition, the key BER protein, APEX1, was shown to be induced at the transcriptional level by ER stress in human hepatoma cancer cells [56]. Lastly, ChIP-seq experiments revealed that XBP1s binds to the promoter region of several BER, MMR and NER genes [12,57]. In summary, the links between UPR and SSB are mainly due to transcriptional control of DDR genes (BRCA1, FEN1, H2afx, XRCC1, XRCC4, Parp1, Mre11a and Rad51) by UPR sensors or ER stress stimuli (Figure 2). Aside from this compilation of evidences, most of the links are found to be established upon DSB repair, highlighting the important role of this mechanism in the DDR and UPR proteostasis.

Recently, in human osteosarcoma cells, ATM was shown to differentially regulate proteostasis under DNA damage conditions and oxidative stress.

Oxidative stress is a natural biological process to which all cells are subjected, and that is recurrent among different pathologies such as degenerative, metabolic, immunological diseases, or cancer [58]. In C. elegans, it has been observed that the collapse of proteostasis, associated with the accumulation and aggregation of misfolded proteins, is directly associated with oxidative stress characteristic of aging [59]. Indeed, the expression of mutated forms of ATM resistant to oxidative stress has a slight effect on the DDR, but favors the clearance of toxic protein Under ER stress, PERK activity induces the expression of p47, a truncated p53 isoform, which in turn triggers the upregulation of 14-3-3 proteins that target the phosphatase CDC25. This prevents the activation of the cyclin B/CDK1 complex and promotes G2/M arrest, facilitating the ER stress resolution by acting in conjunction with PERK to repress protein synthesis and the ER protein load [81,82]. Interestingly, PERK signaling promotes radio-resistance in human breast and lung cancer cells by increasing the DSB repair signaling [83,84] Nevertheless, the signaling crosstalk between ATF6 and the DDR and its regulation is not fully characterized, and experimental results are needed to broaden the contribution of this UPR branch to the control, regulation and interaction with the machinery in charge of the genome stability.

Most of the reports available to date focus on the role of IRE1 and PERK as regulators of the transcription of genes coding for DNA damage proteins, which in turn modulate processes such as cell cycle progression and apoptosis engagement impacting the cell fate. The resulting information is pointing towards a direct molecular interconnection between ER proteostasis and DNA damage surveillance as a new and exciting research field where these two homeostatic signaling exert reciprocal and bilateral regulations.

Concluding Remarks

Recently, several reports have pointed towards interactions between the UPR and the DDR. This suggests a relationship between the ER stress signaling and DNA damage and repair pathways, but the significance of these observations on disease onset is unknown and important questions remain unknown (see Outstanding Questions). Furthermore, new insights about the role of UPR sensors in the maintenance of GI, opens up new perspectives regarding therapeutic targets. Recently, a library of chemotherapeutics compound (> 80 compounds) was shown to induce immunogenic cell death through PERK and IRE1 activation.

Future research would be necessary to evaluate more UPR-activation markers in cancer cells [88] after exposure to radiotherapy or chemotherapeutic drugs such as etoposide, doxorubicin, oxaliplatin, paclitaxel or temozolomide; considering the extensive use of these drugs as standard of care in tumour. Moreover, the administration of these drugs is associated with the activation of IRE1 in some models [11,27,72,89,90]. Finally, it is necessary to explore new models to study UPR and the chemotherapeutic response in cancer. Currently, solid tumours represent a suitable model to study the effect of UPR activation in cancer. These types of models have often been useful to reach relevant conclusions about the UPR in cancer, but the inability to correlate with the interaction with extrinsic factors such as metabolic stress, hypoxia, or drug availability will always be a limitation. Furthermore, it would be interesting to study UPR and cancer in suspended or anchor-free cell models. In these models, the current work correlates UPR and its effects on the immune response [91 93], leaving aside the role of chemotherapy or radiotherapy. Thus, the use of anchor-free cancer cells and the generation of UPR sensors-knockout cells eliminates extrinsic factors. However, limitations in anchor-free cell models are related to the expression of surface antigens or the damage-associated molecular patterns (DAMPs) that could be regulated by the UPR under DNA-damage conditions.

The identification of a fundamental biological function for mRNA decay in the maintenance of genome integrity represents a unique example for selective and specific activation of RIDD activity with clear physiological implications.

Remarkably, IRE1 is frequently affected by loss-of-function mutations in various type of cancer [39], contrasting with the notion that cancer cells require IRE1 to survive in hypoxic conditions [35,92]. We speculate that the genetic alterations of IRE1 observed in cancer may synergize with oncogenes to promote genomic instability. Overall, a direct inter-connection is emerging between the pathways that ensure the integrity of the proteome and the genome. It is necessary to explore in depth how unfolded protein response regulates the gene expression, ribosome profile and proteins expression in a context of genotoxic stress, using different multi-omics strategies [94,95], in order to evaluate the global modulation of the DNA damage response. As evidenced in this review, the crosstalk between UPR and DDR is of great interest in the context of UPR biology, especially but not exclusively in cancer biology and treatment. Also, PERK and IRE1 have been identified as components of the ER/MAMs [112,113], suggesting novel interactions between the UPR-sensors and p53.

Finally, cancer cells are exposed to several factors that alter proteostasis. To cope with this, tumour cells engage the UPR to manage these disturbances [35]. As p53 mutations are the most recurrent alterations in cancer, leading to the resistance to stressors as DNA damage, the selective inhibition of pro-survival UPR represent a promising intervention on p53-deficient tumours, engaging apoptosis by the induction of unresolved ER stress.

Glossary:

Proteostasis: Is a network of interconnected quality-control processes in the cell that maintains a functional proteome. Chaperones, foldases, oxidoreductases and glycosylating enzymes ensure that secretory proteins are properly folded, modified and assembled into multi-protein complexes in the ER before they transit further downstream in the secretory pathway.

Unfolded

Gamma-H2AX ( H2AX):

Upon DSB induction, the histone variant H2AX is phosphorylated on serine 139 by ATM, ATR or DNA-PK, generating phosphorylated H2AX, or so-called H2AX. H2AX induction is one of the earliest events detected in cells and human biopsies following exposure to DNA damaging agents. H2AX is a key marker of double-strand DNA damages, allowing the activation and relocalization of repair proteins to DSB sites as well as the signal amplification.

Oxidative stress: Is an imbalance between the production of reactive oxygen species (free radicals) and antioxidant defenses. Amino acids such as proline, arginine, lysine and threonine are particularly vulnerable to oxidative damage, both as free molecules or within proteins. Moreover, oxidative damage can also affect the integrity and stability of DNA and RNA.

  UPR sensors activate death signalling pathways (see Box 1) [1]. IRE1 is a type I transmembrane protein, that exhibits kinase and endoribonuclease (RNase) activities in its cytosolic domain. Under ER stress, IRE1 oligomerizes, then transautophosphorylates which triggers a conformational change that activates the RNase domain. IRE1 RNase activation, together with the tRNA ligase RtcB, induces the non-conventional splicing of X-box binding protein 1 (XBP1) mRNA, [2,20 22]. The spliced XBP1 mRNA encodes the transcription factor XBP1s which promotes the transcription of a number of genes whose products are involved in the ER proteostasis such as foldases, oxidoreductases and ERAD components (Figure 1) [2,16]. Alternatively, IRE1 RNase degrades multiple mRNAs and miRNAs in a sequence-specific process called regulated IRE1 -dependent decay (RIDD) of RNA [23]. Although RIDD activity has been proposed to be necessary for the maintenance of ER homeostasis [24,25] and the pathogenesis of diabetes [26], cancer [27] and inflammatory conditions [28,29] most of the available evidences are difficult to interpret due to the concomitant activation of Xbp1 mRNA splicing and RIDD activity.

  ensure the genome protection, cells have evolved mechanisms for the detection and repair of DNA lesions called the DDR. The DDR comprises different pathways that can be triggered either by single-strand breaks (SSB) (e.g., mismatch mediated repair (MMR), nucleotide excision repair (NER) or base excision repair (BER)) or by double-strand breaks (DSB) (e.g., non-homologous end joining (NHEJ) and homologous recombination (HR)) [45]. DSB are one of the most harmful injuries to the genome [4]. Failure in DSB repair contributes to the genomic instability that drives cancer development [4,46]. The response to DNA double-strand breaks is controlled by three kinases that are members of the phosphoinositide 3-kinase (PI3K)-related kinases family: ATM, ATR, and DNA-PKcs [4]. Those kinases coordinate the phosphorylation of numerous proteins, ultimately regulating a broad spectrum of cellular processes such as DNA replication and repair, cell-cycle progression regulation, and apoptosis or senescence initiation (Figure 1) [47]. Depending on the mechanisms inducing DSB and the cellular context, different kinase pathways are favored such as ATM-Chk2 signaling along with the MRE11-RAD50-NBS1 (MRN) complex [4]. ATR-Chk1 is recruited and activated to DSB by single-stranded DNA coated with replication protein A (RPA) together with its partner ATR-interacting protein (ATRIP) [4]. The MRN complex is recruited to DNA DSB immediately after its occurrence, recruiting ATM to the chromatin and stimulating its kinase activity [4]. ATM activates a widespread DSB-signaling cascade that begins with H2AX phosphorylation ( H2AX). H2AX is a marker of double-strand DNA damage, its phosphorylation is one of the first response after DSB, and the intensity of the response is proportional to the number and size of DSB foci [48]. Chk2 is activated by ATM and redistributed throughout the nucleus where it functions along with Chk1 in the cell cycle checkpoint signaling network and DNA repair [49]. Chk1 activation after DNA damage is a key function of ATR since activated Chk1 is essential for S and G2/M phase cell cycle regulation [4]. The activation of Chk1 and Chk2 induces the phosphorylation of the transcription factor p53 and the subsequent transcription of p53 target genes [50,51]. These cellular mechanisms are critical in the maintenance of genome integrity and prevention of diseases.

  aggregates[6]. The loss of ATM function under oxidative damage causes wide cellular stress as ATM functions are not limited to its participation in DNA-repair or a specific cellular compartment[6,60]. It is possible to speculate that in physiological conditions, ATM plays a role as an oxidative stress sensor, additionally sensing alterations in other cell compartments, including the ER[10,61]. Accordingly, in S. cerevisiae, the ATM/ATR ortholog, Mec1, is a key component of the signaling network promoting survival in response to proteotoxic stress[62]. Mec1 regulates the expression of genes linked to proteostasis, and its inactivation leads to widespread protein aggregation and cell death[62].Interestingly, protein aggregation is resolved by the activation of autophagy, which facilitates aggregate clearance[62]. Similarly, in mouse fibroblasts, inhibition of chaperone-mediated autophagy leads to hyperphosphorylation and destabilization of the MRN complex and regulated degradation of Chk1 protein[63]. This suggests that autophagy may contribute to GI by ensuring nuclear proteostasis (Figure1). It has been demonstrated that alterations in the functionality of DDR proteins, e.g. ATM, correlate with several pathologies other than cancer, such as neurodegenerative syndromes[64,65] or systemic autoimmune diseases [66 68].The UPR sensors in the DNA damage responseSeveral studies have reported functional links between UPR and DDR signaling. Below, we detail the most recent studies in the field, mainly associated with the role of the IRE1 and PERK sensors.IRE1 signalingIn S. cerevisiae, exogenous expression of mammalian XBP1 was found to play a role in NHEJ DSB repair pathway through the regulation of H4 acetylation[69].Initially, the regulatory network governed by XBP1s was studied in mouse cells from plasma, pancreatic , and skeletal myotubes cells, revealing that XBP1s regulates the transcription of a cluster of DNA-repair genes under ER stress[57].Similar experiments were performed in human hepatic cells confirming that XBP1s directly controls the transcription of multiple DDR genes and the levels of H2AX (Figure2)[12]. Moreover, silencing of XBP1s causes an increase in the formation of H2AX foci as well as a reduction in the expression of MRN complex proteins and in ATM phosphorylation (Figure2) suggesting an increase in DNA damage coupled with a reduction in damage recognition and processing[70]. XBP1s is not only involved in the regulation of DDR genes but is also directly linked to genotoxic stress response (Figure2). In human oropharyngeal carcinoma cells, UV-radiation increases the phosphorylation of IRE1 and the expression of XBP1s, thereby triggering an increase in interleukin-6 expression [70]. In addition, in human colorectal cancer cells, exposure to genotoxic drugs, such as doxorubicin and 5fluorouracil, was found to reduce IRE1 expression and XBP1 mRNA splicing in a p53-dependent manner [71]. However, those results should be taken with caution as the genotoxic stress-induced phenotypes are diverse, and the responses depend on the cell type and agent used. XBP1s regulates the expression of Cul5-ASB11, a ubiquitin ligase targeting BIK, a pro-apoptotic protein [71]. A decreased XBP1s expression reduces Cul5-ASB11 level and increases the expression of BIK protein under DNA damage. This leads to increased apoptosis, whereas apoptosis is prevented under ER stress [71]. Consequently, ubiquitination and degradation of BIK regulates cell fate in the opposite way, depending on the stress conditions (Figure 2) [71]. It is important to note that in human multiple myeloma cell lines, doxorubicin has been proposed as a pharmacological inhibitor of IRE1 , since it reduces XBP1 mRNA splicing and RIDD activity which in turns leads to a decrease in cell survival [72,73]. This could extend the hypothesis that some genotoxic drugs could also act as pharmacological inhibitors of IRE1 in cells such as multiple myeloma [72], colorectal cancer [71] and triple-negative breast cancer [74]. The activity of UPR stress sensors can be regulated by their binding to cofactors (activators and inhibitors), in addition to post-translational modifications [16]. The concept of the UPRosome emerged to visualize UPR stress sensors as platforms onto which different components assemble to generate composite signals, but also to crosstalk with other signaling pathways to regulate various cellular processes [75]. An example of these multiple interactions is provided by fortilin, a pro-survival molecule that through p53, inhibits ER stress-induced cell death [76]. Fortilin directly interacts with IRE1 , inhibiting its kinase and endoribonuclease domains [76]. Moreover, fortilin silencing increases the expression of XBP1s, which is associated with increased DNA fragmentation and apoptosis in vivo. This suggests that the expression of XBP1s increases the signaling of DNA damage, modulating the expression of DNA-repair genes (Figure 2) [57,76]. Recently, a novel IRE1 function associated to the decay of mRNA coding proteins involved in DDR has been identified. In mouse embryonic fibroblasts, genotoxic drugs exclusively engaged the RIDD activity in the absence of XBP1 mRNA splicing [11]. In this model, IRE1 deficiency impairs the ability to repair the genome under DNA damage, disrupting the cell cycle control and the phosphorylation of checkpoint kinases and the histone H2AX [11]. At the molecular level, DNA damage triggers the activation of the c-Abl tyrosine kinase, which operates as a scaffold protein to stabilize IRE1 oligomers and to favor RIDD activity [11]. The role IRE1 activity in controlling the DDR through RIDD was also validated in fly and mouse models, highlighting a relevant role of this UPR signaling branch in sustaining cell survival and DNA repair under genotoxic stress [11]. In addition, RIDD activity may play protective roles in glioblastoma, and in an in vitro assay some DNA-damage proteins could be cleaved by endonucleases domains of IRE1 [27]. These studies highlight the new findings around the RNase activity of IRE1 and its role in pathological conditions, where XBP1s promotes cell death while RIDD enables cell survival [27]. PERK signaling A direct molecular relationship between PERK and GI is not well documented. However, the activation of PERK-p-eIF2 -ATF4 signaling by singlestrand breaks (SSB) was recently described to support cell survival under nutrientrestricted conditions [77]. As the PERK-NRF2 branch contributes to the transcriptional regulation of several genes that mediate the antioxidant response, its alteration has been associated with an increase of ROS after ER stress and the accumulation of oxidative DNA lesions [78]. The downregulation of PERK in human breast cancer cells correlates with an increase in the global phosphorylation of ATM as well as the phosphorylation of its downstream effector Chk2, leading to an increase in H2AX (Figure 2) [79]. It is interesting to highlight that, as discussed in the previous section, XBP1s downregulation causes an increase in the H2AX foci but triggers a reduction in ATM phosphorylation [70]. The loss of PERK triggers a significant attenuation of tumour cell proliferation by increased oxidative DNA damage, leading to G2/M cell cycle checkpoint activation [79]. Under ER stress, PERK activation has been shown to operate as a negative regulator of DNA replication in the absence of DNA damage markers by the phosphorylation of the adaptor protein Claspin and the activation of Chk1 [14]. The suppression of the general protein translation by eIF2 phosphorylation reduces cyclin D1 synthesis, generating an impaired activity of cyclin D1-CDK4 complex followed by an inhibition of CDK2, hence ensuring cell cycle arrest at G1 phase (Figure 2) [80].

  and chemoresistance in human colon cancer cells by the PERK/NRF2/MRP1 axis [85]. These data open the possibility that PERK inhibitors could be potentially used as a chemo-sensitization treatment [85]. ATF6 Signaling In a human breast cancer cell model the expression of mutant p53 was shown to enhance the pro-survival activity of ATF6 and to inhibit both IRE1 and PERK branches, damping the activation of CHOP and c-Jun N-terminal kinases (JNK) [13]. This selective activation is necessary for the invasion, migration and cell survival [13]. Furthermore, p53-mutants exhibit increased ATF6 activity (Figure 2) [13]. In addition, ATF6 expression and the engagement of senescence have been described in cells subjected to oncogene activation or to UV-irradiation [86]. Finally, ATF6 expression was shown to contribute to radio-resistance in glioblastoma cells through the upregulation of BiP expression (Figure 2) [87].

BOX1A

  deadly relationship: Crosstalk between UPR-DDR and their involvement in apoptosis In response to unresolved DNA-damage and ER stress the apoptotic program is mainly orchestrated by p53 and CHOP. Both transcription factors are upregulated as a mechanism to monitor the integrity/stability of the genome and proteome. CHOP is upregulated by the UPR arms [96] and it is directly related to the ER stress-induced apoptosis. However, CHOP-deficient cells still undergo apoptosis, suggesting unknown pro-death signaling. CHOP promotes apoptosis through the repression of anti-apoptotic and induction of pro-apoptotic genes, such as, BAD, BIM, NOXA, PUMA and DR5 [97,98]. p53 expression is induced by several stress signals such as DNA damage and oncogene activation [99]. Moreover, p53 has several non-transcriptional functions [100,101]. Senescence, cell-cycle arrest, and apoptosis are the most prominent outcomes of p53 [102]. Principally, p53 engages the apoptosis through the transcriptional regulation of the pro-apoptotic proteins PUMA, BIM, NOXA, and extrinsic apoptotic pathway components [103]. Interestingly, the crosstalk between these two transcription factors has been described. CHOP drives the MDM2 expression, promoting p53 degradation [104]. It has been linked to the function and localization of p53 as a component of the ER stress-induced apoptotic pathway. ER stress promotes p53 expression through NF-B [105] and CHOP co-operates with FOXO3a to regulate the expression of PUMA and BIM under ER stress [44,106]. Moreover, p53 is an important mediator of ER stress dependent apoptosis through the upregulation of PUMA [107]. PERK activation modifies the translation of the p53 mRNA from the full-length to the p53 N40 (p53/47) isoform and actively suppresses the p21 expression during ER stress, promoting G2 cell-cycle [108]. During chronic ER stress, p53 induces BIK expression while at the same time suppressing BiP translation, leading to the dissociation of the BIK/BiP complex and the apoptosis activation [109]. P53 is located at ER/mitochondria-associated membranes (MAMs) contact sites modulating the Ca 2+ transfer to the mitochondria [110]. Moreover, p53 regulates autophagy, by the proper localization of PML protein at ER/MAMs [111].

  Protein Response (UPR): Is a signal transduction pathway that senses the fidelity of protein folding in the ER lumen. The UPR transmits information about protein folding status to the nucleus and cytosol to adjust the protein folding capacity of the cell. The UPR is transduced by three principal ER-resident proteins: inositol-requiring protein 1 (I E1 ), PKR-like ER kinase (PERK), and activating transcription factor 6 (A F6 ). Genomic Instability (GI): Includes all processes that maintain the integrity of DNA such as sensing, signaling and repair of DNA damage, processing of DNA damage in the context of chromatin and chromosomes, cell cycle checkpoint control and apoptosis control. Effective maintenance of genome integrity is essential for healthy organisms, aging, and prevention of diseases. DNA damage response (DDR): Cellular response involving DNA damage recognition, followed by the initiation of a cellular signaling cascade that promotes DNA repair, which can modulate cell-cycle progression, chromatin structure and transcription, both at sites of DNA damage and globally. DDR after DNA-double strand breaks is controlled by three related kinases: Ataxia-telangiectasia mutated (ATM), ATM and Rad3-Related (ATR) and DNA-dependent protein kinase (DNA-PKcs). Binding immunoglobulin protein (BiP): Is a key ER chaperone and master regulator of ER functions under ER stress. Detection of misfolded protein species by the three UPR sensors is partly dependent on BiP. ER-associated degradation (ERAD): Is the principal quality-control mechanism responsible for targeting misfolded ER proteins for cytosolic degradation. ERAD targets are destroyed by the cytoplasmic ubiquitin proteasome system. Many ER chaperones participate into the ERAD complex, including BiP, EDEM1, OS9, and XTP3B. The UPR sensor IRE1 and SEL1L-HRD1 complexes are the two most conserved branches of ER quality-control mechanisms. Cell-extrinsic factor: Any factor that is independent of the genetic background or alteration of DNA, such as hypoxia, glucose deprivation, or inadequate amino acid supplies. Cell-intrinsic factor: Any factor that is dependent on the genetic background or DNA, such as oncogenic activation, alteration in chromosome number or hyperploid. DNA double-strand breaks (DSB): Different classes of DNA damage such as ultraviolet (UV) light, irradiation, DNA-damage drugs or oxidative stress that leads to DNA rupture in both strands. If DNA is not repaired correctly, DSB can cause deletions, translocations, and fusions of the DNA.

Figure 1 .

 1 Figure 1. Unfolded Protein Response and DNA Damage Sensors. All three ER stress sensors (PERK, IRE1 , ATF6) are localized at the ER membrane and under ER stress they activate signaling events that increase protein-folding capacity and reduce protein load on the ER. In response to DNA damage, ATM is activated and recruited to DSBs by the MRE11-RAD50-NBS1 complex and ATR is recruited to RPA-coated ssDNA by its binding partner ATRIP. DNA-PKcs, meanwhile, is recruited and activated by Ku-bound DSB ends. The UPR transcription factors and DNA damage proteins determine cell fate by the regulation of distinct subsets of target genes spanning from the recovery of ER homeostasis to DNA damage response. The green boxes illustrate the common target and functions induced by the ER stress and genotoxic stress. The blue boxes illustrate UPR functions induced by the ER stress response. The grey box illustrates the DDR proteins involved in the UPR. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License.

Figure 2 :

 2 Figure 2: Unfolded Protein Response sensors involved in the DNA Damage Response. IRE1 is maintained in a repressed state through an association with BiP. Upon ER or genotoxic stress, misfolded proteins dock to BiP, thus triggering the dissociation from IRE1 . This triggers IRE1 dimerization and autotransphosphorylation inducing its activation. IRE1 may also bind misfolded proteins to oligomerize. IRE1 allows the splicing of XBP1 mRNA and the degradation of RNAs (RIDD). XBP1s transcriptional factor governs notably the expression of genes involved in DDR and ubiquitin ligases. RIDD activity governs the expression of mRNA, which impact on DDR proteins. IRE1 activity can be modulated by fortilin, c-Abl, p53, doxorubicin or 5-fluorouracil. Knockdown of XBP1 reduces ATM phosphorylation, MRN complex expression and increases H2AX. The mechanism of PERK repression and activation is the same as for IRE1 . Activated PERK phosphorylates eIF2 , which in turn shuts down global translation and concomitantly increases the expression of the transcription factor ATF4.The

Acknowledgments

We apologize to authors whose works we were unable to cite because of space limitations. We thank reviewers for very constructive criticism. We thank Javier Diaz and Dr. Jaime Meléndez for their revisions and commentaries of the manuscript. This work was directly funded by CONICYT fellowship (PCHA/Doctorado Nacional/2016-21160232) (M.G-Q). Doctoral fellowship ARED/INSERM R gion Bretagne (A.B). FONDECYT 3190738 and FONDAP-GERO-15150012 (A.S). FONDAP program 15150012, Millennium Institute P09-015-F, FONDEF ID16I10223 and D11E1007, and FONDECYT-T1180186 and Ecos-Conicyt no C17S02 (C.H.). Institut National du Cancer (INCa PLBIO), ANR under the frame of ERANET (ERAAT) and EU H2020 MSCA ITN-675448 (TRAINERS) and MSCA RISE-734749 (INSPIRED) (E.C). Fondation pour la Recherche Médicale (FMR, DEQ20180339169), Ligue contre le cancer Grand Ouest and Institut National de la Santé et de la Recherche Médicale (INSERM) (R.P).

Highlights

-Alteration in the genome integrity has been associated with disruption of the endoplasmic reticulum (ER) proteostasis -The unfolded protein response (UPR) and the DNA damage response (DDR) play important roles in the development and progression of several diseases, including cancer.

-UPR sensors IRE1α, PERK and ATF6α play a role in response to genotoxic and ER stress in cells by interacting with DNA damage proteins functions (e.g. ATM, ATR, p53, p21, Chk1 or Chk2).

-The crosstalk between UPR and DDR may contribute to cancer progression. What are the most relevant cancer models that can be used to study UPR and its effect on DNA damage response?


Julie Carcreff

François Cheviré

Elodie Galdo

Ronan Lebullenger

Antoine Gautier

Jean-Luc Adam

Laurent Brilland

Gilles Renversez

Johann Troles

Keywords: chalcogenide glasses, microstructured optical fibers, 3D printing

teaching and research institutions in France or abroad, or from public or private research centers.

I Introduction

In recent years, a growing interest has settled for optical materials and fibers for the mid infrared (mid-IR) region. This interest originates from societal needs for health and environment for instance, and also from demand for defense applications. Indeed, the mid-IR spectral region contains the atmospheric transparent windows (3-5 µm) and (8-12 µm) where thermal imaging (military and civilian) can take place. Furthermore, the infrared window is well-suited for sensing (bio)-molecules, whose fingerprints are located at wavelengths between 2 and 15 µm. The development of mid-IR transparent materials and optical fibers is essential. Chalcogenide glasses are good candidates for the realization of new and innovative mid-IR systems. In this context, chalcogenide fibers have attracted great interest in the field of versatile mid-IR fiber transmission, supercontinuum generation , and sensing [START_REF] Tao | Infrared fibers[END_REF].

An original way to obtain fibers is to design microstructured optical fibers (MOFs). Such fibers present unique optical properties thanks to the high degree of freedom in the design of their geometrical structure. The elaboration of chalcogenide MOFs permits to combine the mid-IR transmission of chalcogenide glasses to the light propagation properties of MOFs. Different methods for the realization of chalcogenide preforms and microstructured fibers have been reported, such as the stack and draw method [START_REF] Brilland | Interfaces impact on the transmission of chalcogenide photonic crystal fibres[END_REF], drilling method [START_REF] El-Amraoui | Microstructured chalcogenide optical fibers from As2S3 glass: towards new IR broadband sources[END_REF], extrusion processes [START_REF] Wu | Mid-infrared supercontinuum generation in a suspended-core telluriumbased chalcogenide fiber[END_REF] and molding method [START_REF] Toupin | Small core Ge-As-Se microstructured optical fiber with single-mode propagation and low optical losses[END_REF].

We have investigated an alternative way for fabricating complex microstructured preform by using an original 3D printing process. It has been shown recently that bulk As2S3 can be obtained by additive manufacturing process [START_REF] Baudet | 3D-printing of arsenic sulfide chalcogenide glasses[END_REF]. In the present study, we have 3D-printed a holey chalcogenide preform, and shown that this preform can be drawn into a chalcogenide optical fiber that can transmit light. The attenuation of the printed fiber will be compared to the optical transmission of chalcogenide fiber drawn from a classical melt-quenched preform.

3D-printing of chalcogenide preforms: a novel process for the elaboration of chalcogenide microstructured optical fibers II Elaboration of chalcogenide preform and fiber by 3D printing 1. 3D manufacturing of the preform A new method has been investigated for producing preform for mid-IR fibers: the additive manufacturing. This method can be implemented with Te20As30Se50 glass (TAS), which is very stable against crystallization. Likewise, its low glass transition temperature (Tg) facilitates its shaping at low temperature and makes it a good candidate to explore 3D printing of chalcogenide glasses

The thermal properties of TAS, i.e. Tg around 140°C and an extrusion temperature. around 270-300°C are close to the ones of commercial printable polycarbonate (Tg around 140-150°C and extrusion temperature. around 260-310°C). In order to manufacture TAS glass preforms, a customizable commercial RepRap-style 3D printer (Anet A8) has been modified accordingly with a copper hot end (nozzle diameter = 0.4mm) that can reach printing temperature up to 400°C and a sodalime glass bed that can be heated slightly above the TAS Tg for adhesion purpose. 400-mm long TAS rods (1.75 and 3 mm in diameter), produced by the fiber-drawing method, were utilized to supply the feeding mechanism especially customized for brittle materials. As a proof of concept of the printability of chalcogenide glass preforms, a basic tube of glass has been elaborated, at first. The material is deposited uniformly layer by layer in a continuous circular motion at constant linear speed and extrusion rate under ambient conditions. The resulting piece has an external diameter of 8.2 mm and an internal diameter of 3 mm for a height of 15mm. The tube surface is bright, suggesting that neither crystallization, nor defects are discernible to the naked eye. Only the stripes due to the different layers of the additive process are clearly visible, as can be seen in Figure 1.

Drawing and optical characterizations of the "printed" fiber

The printed preform has been drawn into a hollow-core fiber by using a drawing tower especially built for soft glasses such as chalcogenide glasses. Few meters long samples with an inner diameter around 70 µm and an outer diameter around 280 µm (Fig2a) have been produced. The attenuation curve of the "printed" fibers has been recorded and compared to the attenuation losses measured in the TAS glass cane used for printing the preform (fig 2b). The measured optical losses for the "printed" fiber are much higher than the initial optical losses, by nearly a factor of 4 in the 7-8 µm region. The additional losses are mainly due to scattering effects induced by the presence of bubbles (fig 2) and also water and oxygen pollution during the 3D printing that takes place in air. One can note that the initial glass used for this first attempt was not a high purity chalcogenide glass. Those results open a new way for the elaboration of chalcogenide MOFs, especially for the preparation of hollow core chalcogenide MOFs.

Fig 1 .

 1 Fig 1. Photos of the chalcogenide glass hollow preform obtained by 3D printing.

Fig 2 :

 2 Fig 2 : 3D-printed chalcogenide hollow fiber : (a) fiber cross section, (b) : Optical loss of the chalcogenide (black) and the printed hollow fiber (red).


Małgorzata Kloc
email: mkloc@houstonmethodist.org

Rafik M Ghobrial

Sławomir Lewicki

Jacek Z Kubiak
email: jacek.kubiak@univ-rennes1.fr

Malgorzata Kloc

Macrophages in Diabetes Mellitus (DM) and COVID-19: do they trigger DM?

Keywords: Diabetes mellitus, macrophages, SARS-CoV-2, COVID-19 Pancreatic macrophages

Diabetes mellitus (DM) augments the risk of hospitalization and mortality resulting from viral, bacterial, or fungal pathogen infection. This has been also true for the past SARS and MERS, and current SARS-CoV-2 coronavirus epidemics. Clinical data indicate that SARS-CoV-2 infection triggers a severe course of COVID-19 more frequently in diabetic than nondiabetic patients. Here we overview the cellular and molecular mechanisms associated with this phenomenon. We focus on alterations in the immune cells, especially monocytes and macrophages, involved in innate immune response and inflammatory processes, which differ in type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). We also describe the DM-related changes in the monocyte/macrophages functions, how they could lead to the severe outcome of SARS-CoV-2 infection, and importantly, if and how they could initiate DM in DM-susceptible patients.

organ responsible for the homeostasis of glucose in the bloodstream, is not producing enough insulin. The pancreas is a heterocrine gland that has food digestive (exocrine) and hormonal (endocrine) function. In its exocrine capacity, the pancreas produces hormones insulin, which lowers glucose level, and glucagon, which raises glucose level, and somatostatin, which inhibits the secretion of insulin and glucagon. There are five types of cells in the pancreatic islets: β-cells secreting insulin; alpha cells secreting glucagon, delta cells secreting somatostatin, epsilon cells secreting ghrelin (a so-called "hunger hormone" that stimulates food intake, fat deposition and growth hormone release), and pancreatic polypeptide (PP) secreting cells, which regulate endocrine and exocrine secretory functions of the pancreas. In addition, the pancreatic islets contain a population of resident islet macrophages. The islet macrophages are self-renewed and are rarely, if not at all, replenished by the bone marrow/blood-derived monocytes [START_REF] Carrero | Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice[END_REF]. Islet macrophages are situated in the vicinity of the blood vessels, and they communicate with the cellular and acellular A c c e p t e d M a n u s c r i p t components of the blood through the filopodia that extend to the lumen of the vessels. They also interact with the β-cells, capturing insulin and presenting it to the autoreactive CD + 4 T cells, which are critical for the immune response. Islet macrophages express both stimulatory and suppressive ligands and receptors, chemokines, and cytokines including IL-1β, TNFα, and a high level of major histocompatibility complex MHC-II, and as such are the key regulator of the activation and recruitment of the lymphocytes, and additional monocyte/macrophages into the islets during the infection [START_REF] Calderon | Cellular and molecular events in the localization of diabetogenic T cells to islets of Langerhans[END_REF][START_REF] Calderon | The pancreas anatomy conditions the origin and properties of resident macrophages[END_REF][START_REF] Carrero | Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice[END_REF][START_REF] Ferris | The islet-resident macrophage is in an inflammatory state and senses microbial products in blood[END_REF][START_REF] Vomund | Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells[END_REF]. For example, [START_REF] Carrero | Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice[END_REF] showed that in the autoimmune diabetes nude mouse model, the deepletion of the islet macrophages using monoclonal antibody against the macrophage colony-stimulating factor1 (CSF-1) receptor decreses a recruitment of T cells into the islets.

Pancreatic macrophages in Diabetes mellitus (DM)

Diabetes mellitus (DM) results from the loss of functional β-cell mass in the pancreas. There are two main forms of DM: type 1 diabetes mellitus (T1DM) or Insulin-Dependent Diabetes Mellitus (IDDM; which accounts for 5-10% of all diabetic patients) and type 2 diabetes mellitus (T2DM) also known as Noninsulin-Dependent Diabetes Mellitus (NIDDM; which accounts for 90-95% of all diabetic patients). Preclinical and clinical studies indicate that the increased numbers of innate immune cells, and produced by them inflammatory factors have causative and detrimental effects on the islets and β-cells in diabetes [START_REF] Böni-Schnetzler | Islet inflammation in type 2 diabetes[END_REF]. Molecular mechanisms leading to β-cell failures and cell death by apoptosis in T1DM and T2DM are distinct. While T1DM is immune-mediated, T2DM is metabolic-, mostly lipid-, mediated, and associated with obesity. These two mechanisms differently affect not only β-cells, but also the accompanying immune cells including macrophages, which are involved in the inflammatory processes associated with DM [START_REF] Eizirik | Pancreatic β-cells in type 1 and type 2 diabetes mellitus: different pathways to failure[END_REF][START_REF] Ying | The role of macrophages in obesity-associated islet inflammation and β-cell abnormalities[END_REF]. The β-cell apoptosis is driven by the increase in mitogen-activated protein kinases triggering ER stress and followed by the release of apoptotic signals from the mitochondria both in T1DM and T2DM. However, two different gene cascades seem to be involved in each case: inositol-requiring enzyme 1 (IRE1)-driven genes in T1DM and the protein kinase RNAlike endoplasmic reticulum kinase PERK/ Eukaryotic Initiation Factor 2 alpha (eIF2α)dependent pathway in T2DM [START_REF] Cnop | Mechanisms of pancreatic β-cell death in type 1 and type 2 diabetes: many differences, few similarities[END_REF][START_REF] Eizirik | Pancreatic β-cells in type 1 and type 2 diabetes mellitus: different pathways to failure[END_REF]. Both T1DM and T2DM also affect the function of resident and recruited monocytes/macrophages that are essential for the innate immune response, cytokine secretion, phagocytosis, tissue homeostasis, and A c c e p t e d M a n u s c r i p t remodeling [START_REF] Murray | Protective and pathogenic functions of macrophage subsets[END_REF]. DM, mostly via high glucose levels, modifies monocyte/macrophage metabolism causing failures in the innate immune and inflammatory processes (Ayala et al. 2019;[START_REF] Casqueiro | Infections in patients with diabetes mellitus: a review pathogenesis[END_REF][START_REF] Esper | The effect of diabetes mellitus on organ dysfunction with sepsis: an epidemiological study[END_REF]. This is due in part to the changes in macrophage epigenetic profile, which modify their predisposed inflammatory state [START_REF] Van Diepen | Diabetes propels the risk for cardiovascular disease: sweet monocytes becoming aggressive?[END_REF]. Some of the DM-induced changes in macrophage functions are common to T1DM and T2DM, and some are distinct. For instance, in both DM variants, the proinflammatory cytokine or macrophage migration inhibitory factor Mif, is highly expressed and its level is elevated in the serum. The studies in the mouse model showed that Mif plays a crucial role in macrophages and dendritic cells activation (another type of innate immune system cells), lowers the levels of inflammatory cytokines, hyperglycemia, specific pancreatic islet antigen-(PIAg-) IgG, and reduces macrophages infiltration into the pancreatic islets [START_REF] Sánchez-Zamora | Altered Macrophage and Dendritic Cell Response in Mif-/-Mice Reveals a Role of A c c e p t e d M a n u s c r i p t Mif for Inflammatory-Th1 Response in Type 1 Diabetes[END_REF]).

T1DM causes specific signal transduction dysregulations in the macrophages. The comparison of bone marrow-derived macrophages (BMDM) and peritoneal macrophages in non-diabetic and diabetic C57BL/6 male mice, the animal model for T1DM, has shown, recently, the dysregulation of both Pl3K/AKT, ERK 1/2 and SAPK/JNK protein kinases pathways and the secretion of cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 (Galvão Tessaro e tal. 2020). Thus, in the T1DM, the macrophagedependent innate immune response is significantly altered. In T2DM, both macrophages and NK cells show important modifications in the abilities to chemotaxis, and phagocytosis [START_REF] Lecube | Phagocytic activity is impaired in type 2 diabetes mellitus and increases after metabolic improvement[END_REF], the functions playing a pivotal role in the initial stages of the fight against infection. Decrease of these functions condemns T1DM and T2DM patients to delayed and weaker immune defense already in the initial stages of infection, including SARS-CoV-2, which is today of the particular interest. However, macrophages can not only protect but also aggravate the DM by augmenting pancreatic islets inflammation, and affecting β cell proliferation [START_REF]Expansion of islet-resident macrophages leads to inflammation affecting beta cell proliferation and function in obesity[END_REF]. Studies in diabetes 1 mouse model showed that diabetes promotes an inflammatory monocyte/macrophage phenotype [START_REF] Kanter | Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase[END_REF].

These data indicate that although the macrophages can have both protective and destructive roles depending on the pathologic context [START_REF] Ying | The role of macrophages in obesity-associated islet inflammation and β-cell abnormalities[END_REF], their function in DM is skewed toward the inflammatory (and eventually damaging) response.

A c c e p t e d M a n u s c r i p t

Increased ACE2 expression in DM and the SARS-CoV-2 infection

Although the general understanding is that the frequency of microbial infections in diabetic patients is the same as in the general population, some studies are indicating that diabetic patients have a higher risk of infections [START_REF] Casqueiro | Infections in patients with diabetes mellitus: a review pathogenesis[END_REF][START_REF] Carey | Risk of Infection in Type 1 and Type 2 Diabetes Compared With the General Population: A Matched Cohort Study[END_REF][START_REF] Critchley | Glycemic Control and Risk of Infections Among People With Type 1 or Type 2 Diabetes in a Large Primary Care Cohort Study[END_REF]. Most importantly everybody agrees that infected diabetic patients have higher morbidity and mortality. This is also true for coronaviruses infections, such as SARS, MERS, and the current SARS-CoV-2responsible for the current COVID-19 pandemic. The one of the resaons of high morbidity of COVID-19 is directly related to the overstimulation of lung macrophages and overproduction of inflammatory cytokines, a so called cytokine storm that results in the caute respiratory distress syndrome (ARDS) [START_REF] Kloc | How nicotine can inhibit cytokine storm in the lungs and prevent or lessen the severity of COVID-19 infection?[END_REF]. Higher probability for diabetic patients to develop higher SARS-CoV-2 viral load and more severe symptoms are related to the increased expression of Angiotensin-converting-enzyme 2 or ACE2 -the receptor for coronaviruses entry to human cells [START_REF] Roca-Ho | Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse[END_REF][START_REF]Are patients with hypertension and diabetes mellitus at increased risk for COVID-19 infection[END_REF][START_REF] Richardson | Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area[END_REF][START_REF] Erener | Diabetes, Infection Risk And Covid-19[END_REF]. ACE2 is expressed in the lung, heart, adipose tissue, kidney, pancreas (especially in β-cells), the luminal surface of the small intestine, and blood vessels [START_REF] Hamming | Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus: a first step in understanding SARS pathogenesis[END_REF][START_REF] Monteil | Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-817 Grade Soluble Human ACE2[END_REF]Ziegler et al. 2020).

The ACE2 is also expressed in the macrophages and monocytes [START_REF] Chen | The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly decimates human spleens and lymph nodes[END_REF][START_REF] Rutkowska-Zapała | Human monocyte subsets exhibit divergent angiotensin I-converting activity[END_REF], and its expression is stimulated by the inflammatory signals, such as type I interferon, produced by macrophages (Ziegler et al. (2020). Moreover, the medications with which diabetic patients are treated, e.g. glucagon-like peptide I (GLP-1) agonists, or hypertension medications, and statins, increase the ACE2 expression even more [START_REF] Drucker | Coronavirus Infections and Type 2 Diabetes-Shared Pathways with Therapeutic Implications[END_REF]. Thus, diabetic patients become not only the privileged target for SARS-CoV-2 infection, but they may also develop a higher viral load because of the overexpression of ACE2 viral receptors in many cell types, nurturing SARS-CoV-2 replication.

How can COVID-19 elicit DM?

The possibility that COVID-19 may elicit DM in susceptible patients has been evoked recently [START_REF] Rubino | New-Onset Diabetes in Covid-19[END_REF][START_REF] Caruso | Type 1 diabetes triggered by covid-19 pandemic: A potential outbreak?[END_REF]. It seems to be confirmed by recent case reports [START_REF] Marchand | Type 1 diabetes onset triggered by COVID-19[END_REF]. Such a hypothesis has been already proposed following the SARS epidemic in 2002 [START_REF] Yang | Binding of SARS coronavirus to its receptor damages islets and causes acute diabetes[END_REF]). As we wrote above, SARS-CoV-2 infects many types of cells expressing ACE2 receptors, including the macrophages and β-cell in the pancreas. We postulate that there are several plausible mechanisms responsible for the A c c e p t e d M a n u s c r i p t triggering of DM in COVID-19. One possibility is that, similar to the effect of SARS-CoV-2 in the lungs, the infection of pancreatic macrophages causes inflammatory cytokine and chemokines storm in the pancreas. This, in turn, recruits additional immune cells, including proinflammatory monocyte/macrophage, causing additional damage to the pancreatic islets and β-cells. The second possibility is that because macrophages and monocytes are motile, after being infected with SARS-CoV-2, they disseminate virus to the and within the pancreas (Klepper and Branch, 2015). Another possibility is that the SARS-CoV-2 infection of the βcells, directly damages them, causing apoptosis, and reducing β-cell mass in the pancreas.

Although, this scenario seemed the least likely because the direct damage to the pancreatic islets should result in higher than the reported incidence of COVID-19-induced diabetes, however, recent data presented in Nature News indicate that indead, the SARS-CoV-2 may cause direct damage to the insulin producing β-cells [START_REF] Mallapaty | Mounting clues suggest the coronavirus might trigger diabetes Nature News[END_REF]

  . Although, most probably, the combination of these different mechanisms is responsible for the development of DM in COVID-19 patients further preclinical and clinical studies, and analyses are necessary to give definite answers. Ziegler CGK et al. (2020). SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues. Cell 181(5): 1016-1035.e19. doi: 10.1016/j.cell.2020.04.035 A c c e p t e d M a n u s c r i p t

Acknowledgements

While writing this article JZK was supported by the grant "Kościuszko" # 5508/2017/DA from the Polish Ministry of National Defense.

Conflict of Interest

The authors declare that they have no conflict of interest.

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