**1. Introduction**

Adult-onset Still's disease (AOSD) is an inflammatory disease usually affecting young adults [1]. AOSD is associated with a very heterogeneous clinical picture, a triad of high fever, arthritis, and evanescent pink salmon skin rash are commonly observed [2]. Furthermore, a multiorgan involvement of the disease is recognised, including liver involvement, splenomegaly, and poly-serositis [2]. A typical hyperferritinemia is observed in these patients, associated with increases of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) [1]. Additionally, patients with AOSD experience life-threatening complications, which may rapidly evolve into multiple-organ failure and death [3]. These patients would frequently develop macrophage activation syndrome (MAS), a secondary form of hemophagocytic lymphohistiocytosis (HLH) [4,5]. The latter is characterised by continuous high fever, extreme hyperferritinemia, pancytopenia, and histopathological evidence of hemophagocytosis by activated macrophages, typically in bone marrow [5,6].

Although it is typical, this histological finding is not mandatory for HLH diagnosis since it cannot be recognized at the beginning of the disease in bone marrow biopsies [4]. Another important characterisation of HLH is the organomegaly, splenomegaly, and hepatomegaly frequently recognized in these patients [4]. In addition, it was proposed that AOSD and MAS may be considered part of the same disease spectrum, sharing clinical and pathogenic features, and in which AOSD may represent a milder form [7]. Furthermore,

these diseases have been recently included in the so-called hyperferritinemic syndrome, which, together with catastrophic anti-phospholipid syndrome and septic shock, share similar clinical and laboratory features, including very high levels of ferritin [8].

As far as the pathogenesis is concerned, AOSD is considered at the crossroads between auto-inflammatory and autoimmune diseases [9]. Both the innate and adaptative arms of the immune system are called upon in the pathogenic mechanisms underlying this disease [10]. The pathogenic mechanisms of MAS have not been fully clarified yet, but recently a multi-layer pathogenic model was proposed [6]. Both genetic predisposition and several triggers may contribute to the development of a cytolytic dysfunction, prolonging the survival of target cells and enhancing antigen presentation to overproduce proinflammatory cytokines, leading to full-blown MAS syndrome [5,6,11]. In this context, the role of interferons (IFNs) was pointed out mainly for inducing cytokine storm syndrome and MAS occurrence during AOSD [5,6,11]. On these bases, in this work we reviewed the role of IFNs on AOSD and MAS, focusing on their pathogenic role in promoting the hyperinflammatory response and as new possible therapeutic targets.

### **2. Interferons**

In 1957, a molecule was first described with the ability to "interfere" with viral replication and protect cells from infection, which was called an IFN [12]. Since then, a growing body of evidence has shown that multiple IFNs exist which mediate a variety of biological functions from defence against viral infections to antitumor and immunomodulatory effects [13]. IFNs are classified into three main groups according to chromosomal location, their aminoacidic sequence, and specific receptors: i. type I IFNs (-α, -β, -δ, -ε, -ζ, -κ, -τ, and -ω); ii. type II IFN (-γ); iii. type III IFNs (-λ1, -λ2, -λ3). Type I IFNs and IFN-γ are physiologically expressed and are increased by stress and infections [13]. IFNs are critical effectors of both innate and adaptive immune responses, associated with the development of immune cell populations and their activation in response to pathogens, cancers, and other conditions [14]. In addition, the elevated production of IFNs is recognised during both autoimmune and autoinflammatory diseases [15]. This increases the expression of target genes and the canonical interferon-stimulated genes (ISGs) in affected tissues and in circulating blood cells, thus defining the "IFN signature" [14]. The latter is reported to be a typical characteristic of some diseases [16].

#### **3. IFN I**

#### *3.1. Generalities*

IFN-α and IFN-β are the most studied and characterised members of this class of IFNs [16]. IFN-α is encoded by more than 20 different genes. Among these, 13 lead to a functional protein in humans and 14 in mice, whereas IFN-β is encoded by a single gene in both humans and mice [16,17]. Although IFN-α and -β may regulate an overlapping set of genes, these two cytokines differ slightly in their downstream effects and in their expression pattern [18]. Other type I subtypes (IFN- δ, -ε, -ζ, -κ, -τ, and -ω) are lessoften studied [16]. Type I IFNs act on most cell types and induce an antiviral state by increasing the major histocompatibility complex expression and inducing the production of chemokines and cytokines [19,20]. Furthermore, type I IFNs boost the innate arm of the immune system by stimulating the maturation of dendritic cells and the function of natural killer cells [16]. These IFNs also enhance the adaptive response of the immune system by promoting the activation of T and B cells [14]. As a major component of the innate immune system protecting against viruses, the expression of IFN-α and IFN-β is induced by viral infection [19,20]. Type I IFNs bind to the ubiquitously expressed type I IFN receptor (IFNAR) in an autocrine and paracrine manner, modulating the expression of numerous IFN-stimulated genes (ISG) which are involved in the antiviral and anti-inflammatory responses and the pro-apoptotic and anti-proliferative activities [18].

### *3.2. Pathogenic Implications in AOSD and MAS*

Multiple lines of evidence indicate that type I IFNs also exert anti-inflammatory functions [21–23]. These anti-inflammatory phenomena were proposed because IFN-α may reduce both interleukin (IL)-1α and IL-1β production by two main pathways [23,24]. By acting on the signal transducer and activator of transcription 1 (STAT1), type I IFNs may repress the activity of the Nucleotide Binding Domain (NBD), Leucine-Rich Repeat (LRR) containing (NLR) protein 1 (NLRP1) and NLRP3 inflammasomes, thereby suppressing caspase-1-dependent IL-1β maturation [23]. These molecules could also induce the expression of IL-10 in a STAT1-dependent manner, which in turn may reduce the abundance of the pro-IL-1α and pro-IL-1β signals via STAT3 [23]. Such inflammasome inhibition by type I IFNs may also suggest a mechanism for the observed IFN-dependent suppression of IL-18 maturation, since it would also depend on inflammasome activity [23]. Because of these anti-inflammatory functions, an impaired response of type I IFNs may be implicated in the generation of the hyperinflammatory processes [18]. Patients with more severe COVID-19, during the ongoing catastrophic pandemic by SARS-CoV-2, may provide a virally induced representative model of cytokine storm syndrome, thus suggesting similarities with the underlying pathogenic mechanisms of AOSD and MAS [25,26]. Interestingly, severe coronavirus disease 2019 (COVID-19) may display many common aspects with other disorders included in hyperferritinaemic syndrome, including continuous fever and high levels of ferritin [27]. In the context of COVID-19, Hadjadj et al. observed a distinct phenotype in severe and critical patients, associated with a highly impaired type I IFN response, associated with decreased production and reduced activity [28]. In addition, the presence of neutralizing autoantibodies against type I IFNs was supposed in the inhibition of the type I IFN response [29]. These autoantibodies against type I IFNs seemed to be clinically silent until the infection, suggesting that the small quantities of such molecules could be implicated in the onset of cytokine storm syndrome [29].

Taking these observations together, the impairment of the functions of type I IFNs or their delayed response may be implicated in the development of a cytokine storm syndrome. These pathogenic alterations could be also associated with the development of MAS during AOSD, thus providing food for thought for further mechanistic studies. In fact, limited data are available about the role of IFN I in the pathogenesis of AOSD and MAS, so far. In this setting, sera levels of both IFN-α and IFN-β were studied by enzyme-linked immunosorbent assay (ELISA) in 39 AOSD patients, both during a flare of the disease and when following therapies [30]. Levels of IFN-α were detected in only one of the AOSD patients. Instead, levels of IFN-β were found in both patients with an active flare of the disease and those following therapies, without any statistically significant difference [30]. Notably, the type I IFN response on the HLH experimental model was studied in a murine model with a specific deletion of IFNAR (IFNAR-KO) [31]. HLH was induced by stimulation with an IL-10 receptor-blocking antibody and a Toll-like receptor 9 (TLR9) agonist. When IL-10 signalling was maintained, the administration of the TLR9 agonist resulted in a milder HLH in wild-type (WT) mice, with less severe hepatitis and lack of hemophagocytosis. However, thrombocytopenia and IFN-γ were similar between the IFNAR-KO and the WT mice. Despite IFN-γ levels being comparable to those of the WT mice, the IFNAR-KO mice did not develop anaemia, suggesting that type I IFNs could be involved in leading to this feature during HLH [31]. In the same model, the simultaneous administration of both an IL-10 receptor-blocking antibody and a TLR9 agonist led to fulminant HLH. The IFNAR-KO mice had less weight loss than their WT counterparts but were comparable for thrombocytopenia, hepatitis, and splenic hemophagocytosis. Furthermore, the IFNAR-KO mice treated for fulminant HLH conditions experienced the same degree of anaemia when compared to WT mice. Taking together these findings, a complex interaction between type I and type II IFNs in the pathogenesis of TLR9-mediated HLH could be suggested [31].
