From December 2019 up to now, the novel coronavirus named SARS-CoV-2 has caused a huge impact on health worldwide due to life-threatening COVID-19 disease. Risk factors predisposing to severe COVID-19 are male sex, the presence of comorbidities and an older age, whereas children commonly develop a mild disease after SARS-CoV-2 infection (1). The mechanisms that protect the younger from severe COVID-19 disease are yet to be defined; reasons may be a low expression of the ACE2 receptor, trained immunity, a higher capacity to regenerate cells after viral damage but also a strong and effective innate and/or adaptive immune response to SARS-CoV-2 (1–3). SARS-CoV-2 replication starts in nasopharyngeal mucosal cells and recognition of viral molecular patterns leads to a rapid activation of antiviral and inflammatory responses (4, 5). The antiviral response is largely mediated by the transcription of Interferon (IFN) genes, in particular the type I (IFN-α, -β) and type III (IFN-λ1-3) that are the most important IFNs produced by many cell types. Once the IFNs are secreted from infected cells and bind to specific receptors, their signaling upregulates the expression of numerous IFN-stimulated genes (ISGs). In infected cells, SARS-CoV-2 non-structural proteins are able to inhibit the innate immune receptor signaling and IFN production (6); on the other hand, SARS-CoV-2 is highly sensitive to type I/III IFNs when exogenously administered (7, 8). In patients, the key protective role of the IFN-response was confirmed by genetic studies showing that mutations in genes of the type I IFN pathway are far more frequent in patients with severe COVID-19 than in those with a mild disease (9). Moreover, circulating auto-antibodies (Abs) against type 1 IFNs were detected in around 10% of critically ill COVID-19 patients (10). While a fraction of severely COVID-19 patients is highly suppressed in their IFN production and activity due to genetic defects, the presence of anti-IFN auto-Abs or other causes (9–12), on the other hand, delayed and disproportionate IFN responses are involved in the progression to severe disease (4, 13, 14). Thus, to achieve control of SARS-CoV-2 infection, a fast and effective mucosal innate response appears crucial (15), as seen in adults that developed a mild COVID-19 (14–17). Consistent with this notion, children, which usually develop asymptomatic or very mild COVID-19, present a more robust early innate immune response in comparison with adults (18, 19). In particular, a higher expression of genes associated with IFN signaling was found in SARS-CoV-2 infected children with respect to adults, not stratified by age (18). Loske et al. found that basal levels of several Pattern Recognition Receptors and Interferon-stimulated genes (ISG) were pre-activated in children and that, upon SARS-CoV-2 infection, their expression reached higher levels in children compared to adults (19); in this study, the expression of the IFN coding genes could not be detected. Contrastingly, Gilbert et al. measured the mRNA levels of type I (IFN-α and -β) and type III (IFN-λ1-3) IFNs, reporting that IFN α and -β transcript levels were induced by SARS-CoV-2 infection, reaching more elevated levels in the adult/elderly with respect to the pediatric age in which only IFN- λ1 was comparatively higher (20).

In order to determine which IFNs are activated during an effective anti-SARS-CoV-2 immune response in the upper respiratory tract, we sought to compare this response among different age groups, with respect to negative controls of the same age. Specifically, we measured mRNA expression levels of genes encoding type I (including the less studied IFN-ε and -ω) and type III IFNs, and IFN-stimulated genes in nasopharyngeal cells from subjects of ages ranging from 3 to 90 years, that were evaluated also for symptoms and the need for hospitalization.

Out of 192 individuals (114 males and 78 females) enrolled in this study, 106 tested positive for SARS-CoV-2 RNA and 86 resulted negative to SARS-CoV-2 and to the other respiratory viruses. Levels of expression of genes coding for type I (IFN-α, -β, -ε and -ω), type III (IFNs λ1-3) and for ISG15 and ISG56 were measured in 192 NP samples. Transcript levels of all study genes were significantly higher levels in the SARS-CoV-2 positive- than in the negative- samples (p<0.001 for comparisons of all genes between the two groups; Figure 1 ). Males and females had comparable gene expression levels when considering all samples together or when comparing by sex SARS-CoV-2 negative and positive-samples separately (data not shown). To understand the impact of age on IFN genes expression during SARS-CoV-2 infections, samples were stratified in age groups: children up to 16 years of age (y), young adults (17-40 y), middle-aged adults (41-60 y), older adults (>60 y) and on the base of SARS-CoV-2 negative- or -positive test. Demographic and other data of enrolled subjects stratified by age groups are reported in Table 1 . Median and interquartile range values for each study gene in the 8 groups are shown in Table 2 .

Recent studies proposed that the mild course of SARS-CoV-2 infections in children is associated with their ability to mount an early and effective antiviral response (18, 19, 23, 24). Their common finding was pre-activated levels of innate immunity genes in the nasal epithelium of healthy children compared to adults (18, 19, 23, 24). Our results confirm that IFN basal levels are up-regulated in children comparing with adults of different age groups, provide further insights into this response in the, less-studied, young adults group, and novel findings regarding IFN-ε and IFN-ω expression.

We detected elevated basal expression of type I/III IFN genes in children and observed age-related decrease of their expression. In particular, basal expression of type I IFNs in young adults was intermediate (IFN-α, IFN-ε and IFN-ω) with respect to pediatric and older adults ages, or similar to that in older adults (IFN-β); among type III IFNs, basal expression of IFN-λ1 and -λ2 was similar between children and young adults whereas IFN-λ3 values in the young were intermediate to those found in the other groups. Collectively, these differences in basal transcriptional levels suggest that, in the NP mucosa, IFN types are differently regulated by age.

Upon SARS-CoV-2 infection, children upregulated transcription of IFN-α and ISG56, and moderately also IFN-ω and ISG15; young adults showed moderate transcriptional activation of all study genes with the exception of IFN-λ1, whereas all study genes were significantly upregulated at older ages. Considering that transcript levels of the IFNs coding genes showed more differences with age in healthy controls than in infected individuals, it is plausible to speculate that IFNs are produced during SARS-CoV-2 infection at any age but are more readily available in the respiratory mucosa at younger ages than in the older. This scenario is coherent with the current view that pre-activated levels of the IFN genes are essential to a fast and effective anti-SARS-CoV-2 response as seen in children and in young adults (18, 19, 24) whereas delayed and sustained IFN activation may be detrimental to the respiratory mucosa and associated to the worsening of respiratory symptoms (4). In accordance with this hypothesis, we observed that several IFN genes reached higher levels in children presenting symptoms with respect to the asymptomatic, and in hospitalized patients with respect to not-hospitalized adults.

Coherently with our findings, in the more recent and larger transcriptome study (24) of cells from NP swabs, the most striking difference by age in gene expression signature was observed in the IFN-α signaling that was pre-activated in not-infected children and only slightly induced after SARS-CoV-2 infection; adults (not differentiated by age in that study) showed very low basal levels of IFN-α related genes but these were strongly activated upon infection (24). In contrast with our data, Yoshida et al. (24) found, in adults’ epithelial cells, a higher IFN-α in mild COVID-19 compared to severe disease. In this regard, it should be underlined that while a protective role of IFNs in the first phases of SARS-CoV-2 replication in the nasal mucosa is well documented, the issue of a favorable or detrimental role of sustained IFN production in older hospitalized patients is still debated and conflicting results have been published on comparative levels of IFNs and ISGs induction found in mild vs severe COVID-19 (13, 14, 20, 25).

Remarkably, we detected age-related differences, not previously reported, also in the expression of IFN-ε and IFN-ω.

IFN-ϵ is dissimilar from other type I IFNs mainly due its regulation; its expression is constitutively high mainly in the epithelial cells of the female reproductive tract (26). In addition, in vitro studies demonstrated its ability to restrict HIV replication in epithelial cells, monocyte derived macrophages and T cells (26, 27); in mouse lung, IFN-ϵ is constitutively expressed and able to stimulate an antiviral response upon infection (28) but no such data have been published on IFN-ϵ expression in human respiratory mucosal cells. Thus, we showed for the first time in NP cells, higher basal levels of IFN-ϵ in children compared to adults with no difference according to sex. Interestingly, upon SARS-CoV-2 infection, IFN-ϵ activation was more intense in young adults, reaching levels observed in SARS-CoV-2 positive children If confirmed in a larger number of cases, IFN-ϵ elevated levels at younger ages may suggest its possible role with regards to a successful antiviral response. On the other hand, as seen for the other type I IFNs, more IFN-ϵ was expressed in those who were hospitalized, leading to detrimental effects; interestingly, IFN-ε expression can be upregulated by TNF-α, one of the pro-inflammatory cytokine commonly upregulated in the course of COVID-19 (29).

As regards IFN-ω expression at baseline and upon SARS-CoV-2 infection, differences by age were similar to those seen for IFN-ϵ. Even in the absence of experimental evidences, a potential anti-SARS-CoV-2 activity of IFN-ω was hypothesized (15) based on detection of anti-IFN-ω auto-Abs in a fraction of severe COVID-19 patients together with anti IFN-α2 auto-Abs (10, our submitted data). IFN-ω shares ∼60% amino acid similarity to IFN-α, the type I IFN more phylogenetically close; in humans only one IFN-ω is produced compared to the 13 IFN-α subtypes (30). Intriguingly, IFN-ω is still diversifying in many species, including bats and pigs and protect them from excessive tissue damage following infection by several viruses, including Coronaviridae and Orthomixoviridae that can cause severe diseases in humans (30, 31). Further data are needed to confirm IFN-ω induction in respiratory mucosal cells and its possible role as an anti-SARS-CoV-2 weapon in our innate responses.

As regards type III IFNs, we found that their expression was higher in children at baseline as well as upon infection; their levels were not related to SARS-CoV-2 viral load nor to the occurrence of symptoms in children nor with hospitalization in adults. Specifically, IFN-λ1 relative expression was very low compared to that of IFN-λ2 and -λ3 in all tested samples but the age-related differences in SARS-CoV-2-negative and -positive samples and their relative levels of activation were similar among type III IFNs. In the study by Sposito et al, IFN-λ1 was the more potent ISGs inducer with respect to λ2 and - λ3 and to type I IFNs in mild COVID-19 adults’ patients; IFN-λ1 potency could be a reason to keep its basal expression and its activation low upon infections. In fact, besides being a first line defense against mucosal pathogens, type III IFNs can also exert harmful effects during respiratory viral infections including SARS-CoV-2, in particular by lowering lung epithelial repair (32–34). Indeed, a well-balanced baseline expression of type I/III IFNs’ is involved in the physiological regulation of anti-microbial response in the intestinal, cervical and respiratory mucosa (35–37). A scenario is emerging in which coordinated, quick and effective antiviral actions, relying on pre-activated IFNs’ expression, can halt SARS-CoV-2 replication in the first days post-infection in the nasal mucosa, leading to asymptomatic or mild course whereas a later response, triggering high and persistent expression of antiviral genes, may be less effective and cause respiratory damage (14–19, 24, 38).

As concerns basal ISG expression, ISG56 level was higher in children and young adults than in older adults while ISG15 level was lower only in the elderly. Upon SARS-CoV-2 infection, the two ISGs reached far higher levels in adults, coherently with the overall lower IFN activation at younger ages than in adults. Moreover, ISGs transcripts were related to the amount of SARS-CoV-2 RNA as estimated by the diagnostic Ct value in adult patients. The latter finding agrees with previous studies finding a relationship between ISG combined levels and viral loads (11, 17, 21); our analysis focused only on two of the very numerous ISG stimulated by type I and III IFNs and also by IFN-independent pathways and cannot add much to this issue. Nonetheless, the biological value of testing two ISGs only is strengthened by transcriptomic analysis reporting that ISGs expression was co-regulated in SARS-CoV-2 infected respiratory cells (11, 17, 19).

Our study has several limitations: no information on days since SARS-CoV-2 infection; lack of follow-up samples; the relatively low amount of NP cells obtained by residual diagnostic specimens that limited the number of measurements. Regarding this last point, it may have been informative to measure the expression of Pattern Recognition Receptors genes and those triggered in their signaling cascade, as these genes were shown to be up-regulated in children with respect to adults (19). Another limitation due to low amount of NP specimens is that we could not evaluate cell types composition. Indeed, the relative abundance of resident immune cells in the NP mucosa is higher in the younger compared to adults (39); accordingly, the pre-activated innate immune response in children may be due to differential transcriptional programs in epithelial cells and/or in immune cells. In this study, activation of the IFN-response was measured on mixed NP cells and this method may appear basic and less informative with respect to state of the art single cell RNA sequencing techniques. Nevertheless, Real time PCR assays performed with gene-specific probes are reliable, not-expensive, quantitative tests that allowed to analyze nearly two hundred of residual diagnostic samples from different age groups and add to the existing knowledge.

In conclusion, we could confirm in NP samples including all ages, higher basal levels of type I and III IFNs in children and suggest that the IFN response is partially pre-activated also in young adults.

Moreover, we recognized a possible anti-SARS-CoV-2 role of the less well-characterized IFN-ϵ and IFN-ω, that should be further investigated in vitro and in vivo. On the other hand, IFN-ϵ and IFN-ω seem to be no exception to the rule that IFNs may also exert detrimental roles and tissue damage. These data encourage further investigation on the possible protective effects of several different IFN types against severe COVID-19.

The original contributions presented in the study are included in the article/supplementary files, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Institutional Review board and the Ethics Committee (Policlinico Umberto I Hospital, Sapienza, University of Rome, Rif. 5836, Prot. 0690/2021. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Conceptualization: CS, and AP. Methodology: AP and RN. Formal analysis: GC, LM and LP. Investigation: FF, MF, AV, LS and GO. Data acquisition and analysis: MG, FM, GD’E. Writing, review and editing: AP and CS. Supervision: AP, GA. Funding acquisition, GA, CS. All authors have read and agreed to the published version of the manuscript.

This research was funded by a grant from the Italian Ministry of Health (COVID-2020-12371817) to GA. A grant from Sapienza University (ATENEO H2020, PH120172B4BA8CAF) to GA. A grant from Sapienza University (RM12117A513C1DDD) to CS. Funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of this manuscript; and in the decision to submit the article for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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