Type I interferons (IFN) are a family of cytokines which are expressed early upon infection in order to guarantee the survival of the host until the adaptive immunity is activated. In order to elicit their pro-inflammatory function, type I IFN are induced e.g. upon sensing of pathogen-associated molecular patterns (PAMPs). PAMPs are recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLR) and the retinoic acid-inducible gene (RIG)-I-like receptors (RLR). Whereas most TLR signal via the adaptor protein myeloid differentiation primary response 88 (MyD88), signaling of RLR is dependent on the adaptor protein mitochondrial antiviral signaling protein (MAVS) (1). Type I IFN can be subdivided into 13 isoforms of IFN-α, one IFN-β, as well as IFN-ϵ, IFN-τ, IFN-κ, IFN-ω, IFN-δ, IFN-ζ, and IFN-v (2). All type I IFN bind to a common type I IFN receptor (IFNAR) in order to regulate the expression of hundreds of IFN-stimulated genes (ISG) (2, 3). It has been shown by several studies in the past that a positive feedback loop via the IFNAR is necessary for the formation of robust type I IFN responses. Small amounts of early type I IFN, mainly IFN-α₄ and IFN-β, bind to the IFNAR in order to induce the production of high amounts of type I IFN (4–6). Nevertheless, depending on dose and route of infection, the IFNAR feedback loop is not strictly necessary for robust type I IFN expression upon infection with a variety of RNA-encoded viruses (7).

Even though type I IFN are generally considered as pro-inflammatory cytokines, they can also exert anti-inflammatory functions. Impaired induction of type I IFN in a model of TLR9 ligand-induced liver injury resulted in increased inflammation and liver damage. Here, the protective role of type I IFN was mediated via the attenuation of IL-1β expression and induction of the IL-1 receptor antagonist (RA) (8). Moreover, IL-1 family member-mediated immune pathology was shown for alcohol-induced hepatitis, fatty liver disease, as well as in a mouse model investigating lipopolysaccharide (LPS)/d-galactosamine (D-GalN)-induced liver injury (9, 10). In addition, in a previous study we showed that the artificial double-stranded RNA polyinosinic:polycytidylic acid (poly(I:C)) induced severe liver injury in IFNAR-deficient mice (IFNAR^-/-). Deficient type I IFN signaling was associated with increased levels of IL-1β, which in turn was causative for the induction of severe liver damage as shown e.g. by high activity of serum alanine aminotransferase (ALT) (11, 12). As many viruses produce double-stranded RNA during their life cycle, poly(I:C) resembles a good model for a viral infection. To further explore the anti-inflammatory role of type I IFN upon a ‘real’ acute viral infection, we aimed to investigate the regulation of IL-1 family members in virus-induced hepatitis.

Rift Valley fever is an emerging zoonotic disease endemic in sub-Saharan African countries and the Arabian Peninsula. The disease is caused by the Rift Valley fever virus (RVFV), which is an enveloped virus with a segmented negative-sensed single-stranded RNA genome. It belongs to the Bunyaviridae family, genus Phlebovirus, and is primarily transmitted via Aedes mcintoshi mosquitos (13–18). RVFV outbreaks in the human population vary in extent, intensity, and location. Thus far, the largest outbreak was documented in Egypt 1977 with 10,000-20,000 cases and a mortality rate of up to 20% (16). RVFV predominantly infects domestic ruminant animals including sheep, cattle, goats, and camels (16). In humans, infection with RVFV usually causes a self-limiting mild disease with influenza-like symptoms. Nevertheless, a small percentage of patients develop complications with clinical symptoms ranging from hemorrhagic fever to acute hepatitis, severe encephalitis, or thrombosis (13, 17, 19). The liver is considered the major target organ for RVFV and viral replication was shown to induce apoptosis in hepatocytes accompanied by necrosis (13, 15, 18). In line with that, RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis (15, 16, 20).

RVFV was described to mainly infect epithelial cells (such as hepatocytes), mesenchymal cells, neural cells, hematopoietic cells such as mononuclear phagocytes, and cells morphologically consistent with dendritic cells (DC) (15). Other studies identified macrophages to be the primarily virus shedding cells while DC and granulocytes are additional target cells for RVFV replication (17).

The genome of RVFV consists of three segments: large (L), medium (M), and small (S) (16, 18). RVFV clone (cl)13 is an avirulent virus variant, which harbors a large deletion in the S segment. It is naturally attenuated and was obtained by plaque purification from a field isolate obtained from a nonfatal human case during an outbreak in 1974 (19). RVFV cl13 has no pathogenicity for mice or hamsters and the animals survive infections with up to 10⁶ plaque-forming units (pfu) without developing any signs of disease. Nevertheless, type I IFN are critical for the survival of mice upon RVFV cl13 infection as IFNAR^-/- mice show up to 100-fold higher titers when compared to their wildtype (WT) counterparts and finally succumb to infection (19).

In order to investigate if type I IFN also exert anti-inflammatory effects upon an acute viral infection, we used RVFV cl13 and analyzed the induction of IL-1 family members. Interestingly, data revealed a critical role for the novel IL-1 family member IL-36γ which was known thus far to play a pro-inflammatory role in some human disorders such as psoriasis, inflammatory bowel disease, pulmonary disease, or rheumatoid arthritis (21–24). IL-36 cytokines are new members of the IL-1 family which comprise IL-36α, IL-36β, and IL-36γ (25–27). All bind to a heterodimeric receptor composed of the IL-36 receptor (IL-36R) and IL-1 receptor accessory protein (IL-1RAcP). IL-36 receptor antagonist (IL-36RA) and IL-38 have been shown to negatively regulate the IL-36 signaling pathway via competitive binding to IL-36R, suppressing agonist recognition and IL-1RAcP recruitment (21, 28–30). IL-36R is expressed in skin (especially on keratinocytes), epithelial cells within the lung, the gastrointestinal tract, and other tissues. Furthermore, several types of immune cells were shown to express IL-36R and respond to stimulation. In addition, human and mouse DC express IL-36R and binding of IL-36 promotes DC maturation and improves antigen presentation via upregulation of HLA-DR, CD83, and CD86 (21, 29, 31–33). IL-36 stimulates the production of various cytokines, chemokines, adhesion molecules, and pro-inflammatory mediators. IL-36 cytokines can be induced upon stimulation with different agents including cytokines, TLR ligands, bacterial or viral infections, or other pathological conditions (30, 34, 35).

In this study, we provide for the first time a mechanism for the type I IFN-mediated regulation of IL-36γ induction in the context of an acute viral infection.

It was shown before that members of the IL-1 family, especially IL-1β, play an important role in liver injury (11, 43). The current study demonstrates a pathological role for IL-36γ upon an acute viral infection. The pro-inflammatory cytokine IL-36γ was known before to be critically involved in several inflammatory disorders such as psoriasis (29, 44, 45), inflammatory bowel disease (23, 32, 44), rheumatoid arthritis (21), or systemic lupus erythematosus (SLE) (24). Accumulating evidences suggest that IL-36γ also plays a role during infectious diseases (24). Nevertheless, it is not fully understood yet if it promotes infection, drives immune-pathology, or rather plays a protective role.

A study by Wang et al. using a mouse model for influenza A virus (IAV) infection revealed that IL-36 contributes to lung damage and mortality by promoting inflammation. Mice deficient for the IL-36R were protected from IAV-induced lung injury and mortality. Furthermore, IL-36R^-/- mice showed reduced lymphocyte activation, accumulation of myeloid cells, reduced permeability of the alveolar epithelial barrier, and less production of pro-inflammatory cytokines and chemokines (24, 46). In line with that, patients with IAV-induced acute respiratory syndrome (ARDS) show higher concentrations of IL-36γ in the plasma when compared to healthy individuals (47). In addition, IL-36γ and IL-36α are significantly upregulated in patients with pulmonary tuberculosis, bacterial pneumonia, or chronic hepatitis B virus (HBV) infection (47, 48).

Others reported a protective role for IL-36. Upon infection of mice, pretreatment with IL-36γ increased the resistance against Herpes simplex virus (HSV)-2 infection and disease (21). Mice lacking IL-36γ showed increased morbidity and mortality upon IAV infection which was associated with increased virus titers and higher levels of inflammatory cytokines (46). Furthermore, IL-36γ^-/- mice showed higher bacterial load in the lung, systemic dissemination, and higher mortality upon infection with Staphylococcus pneumoniae when compared to WT mice (45). Blocking IL-36γ receptor binding and using recombinant IL-36γ (see Figure 3 ), we found IL-36γ to be causative for the liver damage induced upon infection with RVFV cl13 in absence of intact type I IFN signaling. Interestingly, neither IL-36α, IL-36β, nor IL-36RA were elevated in organs of RVFV cl13-infected IFNAR^-/- mice when compared to untreated IFNAR^-/- or WT mice (see Supplementary Figure 2 ).

IL-36γ was shown before to play a role in drug-induced liver injury. In a model of acetaminophen (APAP)-induced hepatitis, Scheiermann et al. showed elevated levels of IL-36γ within the liver of IFNAR^-/- mice. However, application of IL-36RA increased the late phase of liver injury indicating a potential role for IL-36γ as a cytokine contributing to the decision between tissue damage and liver regeneration (40). Moreover, treatment with IL-36RA significantly reduced the production of pro-inflammatory cytokines in BALB/c mice in a model of Concanavalin (Con)A-induced liver injury (49). ConA-treated IL-36R^-/- animals exhibited exaggerated T cell responses as shown by increased infiltration of effector T cells into the liver accompanied by the production of pro-inflammatory cytokines (29).

Interestingly, in our study, IL-36γ-mediated liver injury upon RVFV infection was completely independent from viral loads in various organs tested. In particular, while viral titers in MAVS^-/-IFNAR^-/- animals are comparable to those in IFNAR^-/- mice ( Figures 1 , 6 ), ALT activity in these double-deficient mice is comparable to WT animals ( Figure 6 ). This suggests an immunopathology caused by IL-36γ expression in an IFNAR-deficient situation, whereas the hepatocellular tropism of RVFV and thus a virus-induced hepatitis seem to play a minor role.

Upon drug-induced liver injury, hepatocytes were shown to be the major source of IL-36 promoting the inflammatory response (50). In line with this, IL-6, TNF-α, and IFN-γ were slightly elevated in RVFV cl13-infected IFNAR^-/- mice when compared to RVFV cl13-infected WT mice (for all p-value = 0.0571, Mann-Whitney-Test). In addition, a minor expression of IL-12p70 and IL-18 was observed in RVFV cl13-infected IFNAR^-/- mice while other cytokines such as IL2, IL-4 or IL-5 did not differ between RVFV cl13-infected IFNAR^-/- and WT mice ( Supplementary Figure 3 ). Our study revealed that type I IFN sensing by macrophages as well as DC is critical for the prevention of IL-36γ production and thus the IL-36γ-induced liver injury upon RVFV cl13 infection (see Figure 5 ). The protective role of myeloid cells was also shown before for poly(I:C)-induced liver damage. Here, myeloid-derived suppressor cells infiltrated the liver in a type I IFN-dependent manner in order to produce IL-1RA and therefore prevent IL-1β-mediated liver injury (11). Interestingly, in the current study, no enhanced IL-36RA expression was detected in qRT-PCR analyses of organs of WT mice (see Supplementary Figure 2 ) indicating that other factors are involved in order to regulate the expression of IL-36γ. Within the poly(I:C)-induced model of liver damage as well as in our current study, myeloid cells within the peritoneum are of particular importance. Along this line, deficient type I IFN signaling was associated with decreased liver recruitment of DC in a model of TLR9 ligand-induced liver damage (8). In addition, a study by Pinto et al. demonstrated that deletion of the IFNAR on subsets of myeloid cells such as macrophages and DC, resulted in uncontrolled replication of West Nile virus (WNV), production of pro-inflammatory cytokines, organ damage, and death (51). Interestingly, particularly cells of the macrophage lineage show increased susceptibility to RVFV cl13 infection in the absence of type I IFN. During initial stages of infection of IFNAR^-/- mice, RVFV cl13 replicates within macrophages and DC (17). Ermler et al. demonstrated that murine conventional DC and macrophages express type I IFN in response to RVFV cl13 (14, 18).

In addition, RVFV cl13-infected human monocyte-derived macrophages were shown to secrete TNF-α and type I IFN (18). This together with our data implies that production as well as sensing of type I IFN by myeloid cells such as macrophages and DC, is critical in order to prevent IL-36γ production and thus the IL-36γ-induced liver damage upon RVFV cl13 infection. Besides immune cells, human lung fibroblasts and human bronchial epithelial cells were shown to produce IL-6 and CXCL8 upon treatment with IL-36 (30). Thus, other cells than immune cells such as hepatocytes might contribute to a production of pro-inflammatory cytokines and dysregulated immune reaction upon RVFV cl13 infection.

It was shown in several studies that myeloid cells sense PAMPs rather via RLRs than TLRs. For example, Dutta et al. revealed that MAVS signaling in myeloid cells was critical for the resistance to Ebola virus infection in mice (52). In line with this, a variety of RNA-encoded viruses were shown to induce IFNAR-independent type I IFN responses in a MAVS-dependent manner (7). Here, we show that IL-36γ production and accordingly liver damage were dependent on MAVS in RVFV cl13-infected IFNAR^-/- mice while MyD88-adapted TLR are of no importance for IL-36γ production (see Figure 6 ). Along this line, others showed that upon infection with WNV no cytokine production was observed in MAVS^-/-IFNAR^-/- mice (51). Of note, production of pro-inflammatory cytokines by DC or fibroblasts upon IL-36γ stimulation was shown to be dependent on MyD88 indicating that induction and sensing of IL-36γ can be mediated by different pathways (23, 45).

The interaction between type I IFN and IL-36 was previously observed also in psoriasis patients. Here, the expression of IL-36 strongly correlated with type I IFN overexpression (53). In addition, IL-36 serum levels in patients correlate with SLE disease activity, a disorder characterized by an enhanced type I IFN signature (53). In epithelial cells, IL-36 was shown to increase antiviral immunity by enhancing the expression of type I IFN stimulated genes (ISG) via the IFNAR (54). This mechanism may have evolved in order to control viruses that developed immune evasion strategies by blocking the production of type I IFN.

In conclusion, our study demonstrated that pro-inflammatory IL-36γ is causative for the observed liver injury in IFNAR^-/- mice upon an acute viral infection. The expression of IL-36γ is regulated by type I IFN, which need to be sensed by myeloid cells in order to prevent liver damage.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by Regierungspräsidium Darmstadt, license number F107-1027. The study was conducted in accordance with the local legislation and institutional requirements.

All authors contributed either to research design (MA, ZW, and GK) and/or the acquisition (MA, MN, EK, MD, SK, IB, SO-I), data analysis (MA, ZW, MN, EK, MD, SK, IB, SO-I), or interpretation of data (all authors). MA and ZW drafted the manuscript, which was critically revised by all other authors. All authors contributed to the article and approved the submitted version.