**4. Discussion**

Given the documented role of ERAP2 in antigen presentation [20] and viral infections [21], we examined the genetic control of ERAP2 transcripts in the human antimicrobial response. In particular, based on the results recently reported by Ye and co-workers [19], we investigated if the expression of the recently characterized ERAP2/Iso3 is flu-specific or if it can be triggered by other microbial stimuli. Our results sugges<sup>t</sup> that: (1) ERAP2/Iso3 mRNA expression is not restricted to flu-infection but it can be prompted by other pathogens including HIV, SARS-CoV-2, CMV, Bacteria (LPS); (2) ERAP2/Iso3 mRNA is translated into a protein following microbial induction; (3) ERAP2/Iso3 mRNA expression is sensitive to viral concentration.

Remarkably, Ye et al. did not detect ERAP2/Iso3 expression in IFNB1 stimulated cells leading to the conclusion that the transcription of novel ERAP2 isoforms is likely initiated by viral sensing pathways upstream of type 1 interferon signaling [19]. Conversely, in our cell culture condition, IFNα-stimulation was able to induce ERAP2/Iso3 expression in a consistent way, suggesting the participation of type 1 interferon cascade to the induction of ERAP2/Iso3 transcription, as already documented for the wild type forms of ERAP1 and 2 [26]. The different cell kinds and IFN subtypes adopted in the two experimental settings could at least partially justify the discrepancies reported in the two studies, but further analyses are needed to clarify the pathways and molecules induced by microbial exposure, directly responsible for ERAP2/Iso3 synthesis. For example, in our study IL-1β stimulus was not able to trigger the expression of any ERAP variants, further strengthening the assumption of a specific response of ERAP gene transcription controlled by pathogen exposure. Furthermore, as IFNα is also strongly associated to various type of I IFN conditions such as Sjogren's disease [27], systemic lupus erythematosus [28] and Scleroderma [29] it would be valuable to verify if ERAP2/Iso3 expression varies in patients affected by these pathologies and/or following administration of IFNα therapy.

The discovery of these new ERAP2 isoforms is of grea<sup>t</sup> importance and gives a plausible explanation to the maintenance of one of the major gene expression quantitative trait loci (eQTL) and alternate isoform usage in most tissues and cell types [19,30]. Indeed, until last year, the preservation of HapB at intermediate frequency in human population was almost unexplained, as its transcript was believed to be addressed to NMD [9,10]. The identification of this previously uncharacterized short isoform, ERAP2/Iso3, transcribed from HapB, results in the partial rescue of ERAP2 expression, suggesting its involvement in the anti-microbial response.

The mechanism of action and functional impact of this new genetic variant to host defense; however, is still indefinite. The lack of the aminopeptidase domain proves that it does not directly participate in the shaping of antigenic peptides to be presented to CD8+ T cells. However, in 2005 Saveanu et al. conducted an immunoblot analysis detecting the presence of ERAP1-ERAP2 heterodimers and possibly homodimers of each enzyme [4]. This crystallographic dimer is described as mediated by domains I and II of the enzyme that is missing from ERAP2/Iso3 [31]. However, this observation has been reiterated by Ye et al. who hypothesized that ERAP2 isoforms could have a dominant-negative effect on either ERAP1 and possibly ERAP2 wt, through the formation of hetero- or homodimeric complexes [15]. Despite still speculative the fact that ERAP2/Iso3 may exert such an effect cannot be ruled out: this, in turn, could lead to an altered peptide processing, which could confer an advantage/disadvantage against infections by presenting a more/less immunogenic antigen repertoire. In relation to this aspect, previous studies have demonstrated that the functional skills of ERAP monomers, homo, or heterodimers may significantly differ in terms of both substrate specificity and trimming efficiency [32]. In particular, ERAP1/2 dimerization creates complexes with superior peptide-trimming efficacy and a higher affinity towards ERAP1 preferential substrates. This is allowed by the adoption of a modified physical conformation by ERAP1, caused by its interaction with ERAP2, which mainly works as an enhancer of ERAP1 role upon dimer assembling [33]. Further studies are needed to verify if also ERAP2/Iso3 physical interaction with the wt ERAP variants prompts an allosteric effect able to modify basic enzymatic parameters and to improve their substrate-binding affinity. However, the observation that ERAP2/Iso3 mRNA is translated into a protein allows to speculate on the generation of a new ERAP

member which further contributes to enrich the non-redundant, ye<sup>t</sup> a complete and potent system of aminopeptidases, warranting an e fficient trimming of various kinds of precursors.

The results obtained in this study definitely establish a link between invading pathogens and ERAP2/Iso3 expression which further strengthens the significance of the results reported by Ye and collaborators. Supporting this hypothesis, we observed that ERAP2/Iso3 expression progressively increased in response to growing doses of viral input in both SARS-CoV-2 and HIV-1 in vitro infection assay, as if the production of this genetic variant, as well as one of the other elements within the ERAP family, were directly dependent on the viral dose of exposure. Additionally, the observation that the increased expression of ERAP2/Iso3 in response to pathogen exposure is accompanied by the modulation of many other determinants (chemokines, cytokines, pathogen recognition receptor, inflammasome, interferon-stimulated genes, adhesion molecules, activation/inhibition markers, antigen presentation elements) orchestrating the anti-microbial immune response, further supports its direct intervention in this defensive pathway. Notably, as its expression is increased by a wide range of microbial stimuli including viral antigens, inactivated virus as well as bacterial by-products, it is possible to assume that ERAP2/Iso3 expression is not pathogen-specific, but it's secondary to the activation of an antimicrobial cascade commonly shared by di fferent pathogens. Meanwhile, we cannot exclude that ERAPs responses observed following microbial stimulations and in vitro viral infections result from an erroneous transcription and translation due to pathogen-induced cellular stress. Indeed, as ERAP1 and ERAP2 expression has been demonstrated to be prompted by IFNγ stimulation, ERAPs production could be secondary to the innate immune response of the cells to infections, rather than to an immune response or an immune evasion mechanism. Further analyses will be necessary to verify this hypothesis.

The involvement of ERAPs in modulating viral infections is widely recognized as recently reviewed in [34]. Several studies, indeed, have demonstrated the intervention of ERAP genetic variants in the life cycle of HCV, flu, CMV, HPV, HIV, and other pathogens at di fferent levels. In particular, studies performed by our research group have established an association between ERAP2/Iso1 and HIV-infection in terms of both susceptibility [17,35] and progression [36]. However, to our knowledge ERAP expression and/or genetic variants have been correlated to the recent coronavirus disease 2019 (COVID-19), provoked by SARS-CoV-2, only by two recent studies [34,37,38]. In the first one by Stamatakis et al. ERAP2 trimming ability has been investigated in SARS-CoV-2 infection together with ERAP1 and IRAP, and it has been proved as the most stable of the enzymes generating optimal length antigenic peptides for HLA binding [38]. In the second one by Lu et al. by examining 193 deaths from 1,412 confirmed infections in a group of 5,871 UK Biobank participants tested for the virus, rs150892504 variant in ERAP2 gene came up as potentially being implicated in risk from SARS-CoV-2 infection. Although rs150892504 variant is not in linkage disequilibrium with rs2248374, this finding suggests the involvement of ERAP2 in the modulation of SARS-CoV-2 infection. Such an assumption is further supported by other intriguing observations. This virus enters the cells by spike protein binding to ACE2 (angiotensin-converting enzyme 2), which is responsible for the conversion of angiotensin I to angiotensin I-9 and of angiotensin II to angiotensin I-7, an e ffective vasodilator, thus working as a negative regulator of the renin-angiotensin system (RAS) Our results, for the first time demonstrate that SARS-CoV-2 exposure triggers the expression of ERAP1, ERAP2/Iso1 and also the recently detected ERAP2/Iso3 in a dose-dependent mode, suggesting its participation in the control of the anti-SARS-CoV-2 response. This observation is far more important, considering that besides their involvement in the antigen presentation pathway, ERAPs display several key anti-SARS-CoV-2 functions. Indeed, they intervene in the RAS, where ERAP1 e fficiently cleaves angiotensin II to angiotensin III and IV, and ERAP2 cuts angiotensin III to angiotensin IV, thus influencing both ACE2 virus receptor bio-availability and blood pressure levels [39]. Furthermore, ERAPs modulate the proteolytic cleavage of IL-6 receptor (IL-6R α) [40] a function which can improve the clinical conditions in COVID-19 patients, as recently documented following its pharmacological inhibition by Tocilizumab [41]. Last but not least, Ranjit and colleagues recently demonstrated that sex-specific di fferences in ERAP1 modulation influence blood pressure and RAS responses [42], and a male

bias in mortality has emerged in the COVID-19 pandemic since the very beginning. As ERAPs genetic variants have been demonstrated to orchestrate and condition the result of infection of coronavirus in other animal species [43,44] detailed studies investigating SARS-CoV-2-host interplay is absolutely mandatory.

Another query which needs to be addressed in the near future concerns the cellular localization of ERAP2/Iso3 isoform. In particular, it would be interesting to verify if, as with the wt ERAP variants [17], even ERAP2/Iso3 may be secreted into the extracellular milieu following inflammatory stimulation, with which substrates it may interact, which functions may eventually exert in this environment and if its administration can interfere with viral replication.

Considering the key role played by ERAPs in antigen processing and presentation, it is plausible that these aminopeptidases may be potential targets and controllers of the pathogenicity of infectious diseases, shaping the susceptibility and response to microbial infections. The recent acquisition of ERAP intervention even in the modulation of innate immunity further reinforces this assumption.

Given the growing number of viral epidemics, the identification of molecular mechanisms driven by factors such as ERAPs that can interfere, control or modulate viral replication is unequivocally needed as they could be widely exploited for the inception of future, still unknown viral infections.

**Author Contributions:** Each author has approved the submitted version and agrees to be personally accountable for the author's own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work. Conceptualization, M.B. and I.S.; Methodology, C.V. (Claudia Vanetti), S.G. and C.V. (Chiara Vicentini); Formal Analysis, I.S., M.G.; Statistical Analyses, E.T.; Investigation, S.G. and C.V. (Chiara Vicentini); Data Curation, C.V. (Claudia Vanetti), M.G.; Writing—Original Draft Preparation, I.S.; Writing—Review & Editing, M.B.; Supervision, M.B. and M.C.; Funding Acquisition, M.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by Falk Renewables, (REC18GZUCC N. 27767).

**Conflicts of Interest:** The authors declare no conflict of interest.
