Free ISG15 as a dimer generates IL-1β-producing CD8α+ dendritic cells at the site of infection

ISG15 is strongly induced after type I IFN stimulation producing a protein comprised of two ubiquitin-like domains. Intracellularly, ISG15 can be covalently linked and modify the function of target proteins (ISGylation). In addition, free unconjugated ISG15 can be released from cells. We found that ISG15 is released in the serum of Toxoplasma gondii infected mice early after infection in a type-I IFN independent manner. Once in the extracellular space, free ISG15 forms dimers and enhances the release of key cytokines involved in the immune response to the parasite: IL-12, IFN-γ, and IL-1β. Its action is dependent on an actively invading and replicating live parasite. ISG15 induces an increase of IL-1β later during infection by leading to increased IL-1β producing CD8α+ dendritic cells at the site of infection. Here, we define for the first time the molecular determinants of active free ISG15 and link ISG15 to IL-1β production by CD8α+ dendritic cells. Thus we define ISG15 as a novel secreted modulator of the cytokine response during Toxoplasma infection.

ISG15 is a 15 kDa member of the family of interferon stimulated genes (ISGs) that contains two ubiquitin-like domains connected by a proline peptide linker.
Similar to ubiquitin, ISG15 can be conjugated to intracellular host and viral proteinsemploying a cascade of E1 (UBE1L), E2 (UbcH8), and E3 conjugating enzymes. This ISGylation can modify protein function (LIT) and extensive studies have elucidated the role conjugated ISG15, mostly in the context of viral infection [1][2][3] . Recently the function of intracellular conjugated ISG15 has been extended to bacterial infections when it was found that Listeria infection induces ISGylation in non-phagocytic cells 4 . Interestingly, this ISG15 induction was type I IFN independent and lead to the release of IL-8 and IL-6 restricting bacterial replication 4 . Free ISG15 can also be released from the cell to the extracellular space and is detectable in the serum 5 This extracellular free ISG15 it has long been appreciated to play an important role. In vitro studies suggested a role for free ISG15 as a cytokines able to induce IFN-γ secretion from NK cells 3 and CD3 + lymphocytes in vitro 6,7 . Additionally, ISG15 was isolated from red blood cells of mice infected with Plasmodium yoelii and shown to act as a chemotractant for neutrophils 8 .
However the in vivo function of free ISG15 remains ill-defined, and only two studies addressed the role offree ISG15 in an in vivo setting 9,10 . In a neonatal model of infection with Chikungunya virus, free ISG15 functions as an immunomodulator of proinflammatory cytokines, providing the first evidence that free ISG15 contributes to the host response during infection in the whole organism 9 . Most strikingly, it was recently demonstrated that ISG15 is a potent IFN-γ-inducing "cytokine" playing an essential role in anti-mycobacterial immunity 10 . Free extracellular ISG15 is effective alone or in synergy with IL-12 to induce IFN-γ secretion from granulocytes and NK cells in response to mycobacterial infection 10 . The ISG15-IFN-γ circuit may therefore be an "innate" complement to the more "adaptive" IL-12-IFN-γ circuit 10 .
Toxoplasma gondii is an obligate intracellular parasite that can infect virtually any nucleated cell. The active acute infection is believed to be mostly controlled by IFNγ, however, the parasite is never eliminated from an infected host and establishes a chronic infection at immune privileged sites, such as the brain and the heart 11 . In immunocompromised adults, this intracellular parasite dormant state in the brain reactivates and leads to the development of toxoplasmosis. The subsequent uncontrolled parasite replication causes life-threatening brain damage that is characterized by brain abscesses and necrotic areas. Two defence molecules, IL-12 and IFN-γ, orchestrate protective immunity in infected hosts 11 . The role of the IL-12-IFN-γ circuit during Toxoplasma gondii infection has been extensively characterised 11,12 .
Toxoplasma gondii infection triggers the release of a broad spectrum of molecules that can control the immune response to the parasite (LIT). In particular recent work has drawn much attention to the role of the inflammasome and IL-1β in the control of Toxoplasma infection [1][2][3][13][14][15] . Host protective immunity against Toxoplasma is highly dependent on the inflammasome sensors NLRP1 and NLRP3 4,15 . Moreover, the activation of the inflammasome is also dependent on the Toxoplasma strain dependent, with type II parasite being able to induce NLRPs activation and IL-1β release 4,14,15 .
Given the strong dependence of a Toxoplasma infection on IL-12 and IFN-γ, coupled with the parasite's newly discovered ability to induce the inflammasome, we employed Toxoplasma infection to delineate the in vivo activity of free ISG15 in relation to IL-1β production. Here we shown that free extracellular ISG15 is produced during live Toxoplasma type II infection. Free extracellular ISG15, but not intracellular conjugated ISG15, enhances the generation of IL-12, IFN-γ and IL-1β during infection with live Toxoplasma gondii parasites. As a prerequisite for function, ISG15 had to form dimers.
ISG15 production during Toxoplasma infection resulted in recruitment of IL-1β-producing CD8α + dendritic cells (DCs) to the site of infection. These data demonstrate a novel role of ISG15 as an immunomodulatory molecule of IL-1β production within the context of the whole organism and extend the antipathogenic repertoire of ISG15 from viral and bacterial to protozoan infections.

Toxoplasma gondii infection induces to the type I IFN-independent production and release of free ISG15 into the serum
To analyse if ISG15 is released during Toxoplasma gondii infection, we infected mice with Toxoplasma type II and monitored ISG15 levels in the serum during the acute phase of infection. We first determined the serum ISG15 levels by ELISA ( Fig 1A). As early as day 2 post-infection (p.i.) the amount of ISG15 released was higher than in uninfected control mice ( Fig.   1A) and its levels continue to rise until day 4 p.i. (Fig. 1A). To ascertain specificity of the commercial ELISA, we demonstrated that no signal is observed in the serum of Toxoplasma-infected ISG15 -/mice (Fig. S1A).
Likewise, recombinant ISG15 protein was detected in a dose-dependent manner (Fig. S1B). ISG15 may exist in the extracellular spaces as a monomeric or dimeric protein 16 . To analyse the quaternary structure of free ISG15 during Toxoplasma infection, we analysed the serum of infected mice by SDS-PAGE and immunoblotting under non-reducing conditions. We confirmed the release of ISG15 early upon infection and noticed the presence of a 30 kDa band that may represents dimeric ISG15 (Fig. 1B top). The protein band intensity from four experiments was quantified by ImageJ software and the immunoblot for the IgG heavy chain was used to normalize the levels of ISG15 in the serum (Fig. 1B bottom).
The release of ISG15 upon Toxoplasma infection might be an active process requiring with the presence of a live, replicative parasite, or be 7 caused by specific recognition patterns of parasite or its products. To investigate whether ISG15 release depends on active host cell invasion by the parasite, we infected mice with either live parasites, γ-irradiated parasites that are able to invade but not replicate inside a cell, or heat killed parasites that are phagocytosed by cells. Only infection with live, actively replicating parasite leads to high ISG15 release in the serum was observed at day 4 p.i. (Fig. 1C), suggesting that ISG15 release during the early phase of infection is dependent on active invasion and replication of the parasite.
ISG15 belongs to the group of type I IFN inducible genes and is highly induced after type I IFN-generating viral infections 5,17 2,3,18 . However, it has recently been shown that ISG15 can also be produced during bacterial Listeriosis in a type I IFN-independent manner 6,7,19 . The role of type I IFN during protozoan parasite infection is controversial and not yet fully understood 20 . Recently a transcriptomic survey of the host response to different Toxoplasma gondii strains revealed that a subset of atypical strains induce a type I IFN response in macrophages and fibroblasts 8,21 . Other studies showed that Toxoplasma classical strains have the capacity to trigger a type I IFN response, but have evolved strategies to limit the induction of type I IFN and the ability of type I IFN to activate STAT1-dependent  1D). We concluded that early after infection Toxoplasma induces the release of ISG15 in a type-I IFN independent way. To further corroborate this finding we also analysed the expression of a panel of classical IFN target genes in the spleen of infected mice at day 4 p.i. (Fig. S1C). None of these genes were increased in expression upon Toxoplasma infection. To dissect the role of free ISG15 during Toxoplasma infection, we infected mice with Toxoplasma type II and treated them with 1 µg of recombinant murine ISG15 at day 0, 1 and 2 p.i. by intra-peritoneal injection. As controls we employed both untreated Toxoplasma infected mice and naïve mice (Fig.   S2A). We determined the levels of key cytokines released early after infection.
Interestingly, shortly upon infection, ISG15-treated mice showed a modest but significant increase in IFN-γ, IL-12 and IL-1β, at different time points compared to infected but untreated mice ( Fig. 2A). In particular, IL-1β secretion was consistently increased at all time points in ISG15-treated infected mice. Importantly, no increase in these cytokine levels were seen upon ISG15 treatment of uninfected mice, demonstrating that ISG15 alone is not sufficient to increase cytokine secretion, but only does so in the context of an infection (Fig. S2A). Additionally, this result ensures that the preparation of recombinant ISG15 is endotoxin free.
As outlined in the introduction, only two studies have addressed the role of free extracellular ISG15 as a 'cytokine-like' molecule in an in vivo infection model 9,10 . To asses whether the impact on cytokine secretion during Toxoplasma infection is attributable to the free or conjugated form of ISG15, we infected ISG15 -/mice, that completely lack ISG15 11,24 , or UbE1L -/mice, which are devoid of intracellular conjugation of ISG15 through ISG15 E1 enzyme deficiency 11,25 . Upon infection levels of released cytokines were only diminished in ISG15 -/but not UbE1L -/mice (Fig. 2B), strongly suggesting ISGylation is not critical for this process. However we did not observe a difference in survival or parasite load in the ISG15 -/mice as compared to control mice ( Fig. S2B and C). We therefore conclude that, albeit not necessary for survival, the free extracellular form of ISG15 modulates cytokine release during Toxoplasma infection.
To unequivocally assess if the cytokine modulatory function of ISG15 requires a process of active invasion from the parasite, we infected mice with live-, γ-irradiated-, or heat killed Toxoplasma. In each case we either only infected the mice or in addition treated the animals with recombinant ISG15.
Recombinant ISG15 injection only increased cytokine release when mice were infected with live, actively replicating parasites (Fig. 3A), suggesting that ISG15-dependent modulation of cytokine levels during the early phase of infection is strictly dependent on active invasion and replication of the parasite.

Free ISG15 needs to dimerise to enhances the release of IL-12, IFN-γ and IL-1β during Toxoplasma infection
As we had noted the appearance of an anti-ISG15 immunoreactive 30 kDa band that may represent dimeric ISG15, we speculated that a dimeric form of ISG15 there is causative for the cytokine induction (Fig. 1B). As previously described, the disulphide bonds between cysteines in the hinge region might be responsible for ISG15 dimer formation 26

Dimeric free ISG15 increases IL-1β-production by CD8α + DCs at the site of infection
As many cell types have been identified as IL-1β-producers during infections 27,28 , we investigated which cells might release IL-1β in an ISG15 dependent manner during Toxoplasma infection. C57BL/6 mice were infected and left untreated or treated them with recombinant ISG15. At day 4 p.i., representing the peak of ISG15 release, we isolated cells from spleen, lymph nodes and peritoneal exudate and analysed the identity of cells of the innate cell compartment: neutrophils, macrophages, inflammatory monocytes and dendritic cells. In the spleen and the lymph nodes we did not find any notable difference in any cell population in PBS versus ISG15-treated mice (Fig. S3).
However, at the site of infection, within the peritoneal exudate, we found an increased number and frequency of DCs in ISG15-treated infected mice (Fig.   S4A). DCs play a critical role in the immune response to Toxoplasma infection 11,12,29 . Among the different subsets of DCs that are involved in the immune response to Toxoplasma infection, CD8α + DCs play an important role and are involved in the presentation of Toxoplasma-specific epitopes to CD8 + T cells 30 . We therefore analysed the DC compartment of the peritoneal exudate of Toxoplasma-infected ISG15 -/and C57BL6 mice upon infection, the latter left untreated or treated with recombinant ISG15, or with ISG15-C76S/C144S. At day 4 p.i., we observed a higher number and frequency of both CD103 + and CD8α + conventional DCs in infected mice treated with ISG15, whereas a significantly lower number and frequency of these cells was observed in the ISG15 -/infected mice; finally mice treated with ISG15-C76S/C144S resemble the untreated mice, confirming that ISG15 has to form dimers to exert its immunomodulatory functions ( Fig. 4A and S4B).
To determine whether the CD8α + DCs present at the site of Toxoplasma infection are responsible for the increased IL-1β release in ISG15-treated mice, we performed an intracellular staining for IL-1β on these cells at day 4 p.i.. Mice infected and treated with ISG15 had an increased frequency and number of IL-1β-producing CD8α + DCs (Fig. 4B). As control for the specificity of the IL-1β staining, we used both the fluourescence minus one (FMO) setting and an isotype-matched control antibody (Fig. 4B). These results suggest that early during Toxoplasma gondii infection ISG15 release contributes to IL-1β production by CD8α + DCs present at the site of the infection.

Discussion
We demonstrate that free extracellular ISG15 is involved in the regulation of the immune response to a protozoan infection with Toxoplasma gondii in vivo.
We show that this free form of ISG15 is released in the serum early upon In the course of these experiments M. tuberculosis infection in mice was used to demonstrate that ISG15 plays a role as an IFN-γ-inducing secreted molecule essential for antimycobacterial immunity 5,10 . Following these in vivo studies, we found that free ISG15 has immunomodulatory functions during Toxopasma gondii infection, extending its role from viral and bacterial to protozoan infections.
ISG15 can be detected either intracellulary or extracellulary [5][6][7] . During the intracellular ISGylation process, ISG15 dimer formation, through conserved cysteine residues, reduces the amount of ISG15 that can be coupled with the target proteins, a process that can be prevented by nitrosylation of those cysteine residues on the ISG15 molecule 4,26 . Destinct from the ISGylation process, it is unclear whether the quaternary structure of extracellular ISG15 is essential for its function. Here, we show that the formation of ISG15 dimers trough conserved cysteines, is important for the cytokine-like activity of free ISG15 during Toxoplasma gondii infection. Thus, dimerisation of ISG15 is either required for de novo recognition by its putative receptor on target cells or for the downstream signalling response that ensues upon stimulation of its receptor. We unequivocally demonstrated that free ISG15 possesses an immunomodulatory capacity during a protozoan infection, and the protein has to be present as dimers in the extracellular space to exert this function. It will be interesting to determine whether this observation is restricted to the infection with Toxoplasma or whether this is a general feature of free ISG15 function in vivo.
Several cell types were suspected to represent targets for the immunomodulatory function of free ISG15. Among those are neutrophils 8 , NK cells 3,7,9 , and cells of the adaptive immune system, such as T cells 7,10 . We found that ISG15 levels correlate with an increased number and frequency of CD103 + and CD8α + conventional dendritic cells at the site of infection. These two DC subsets are not identical but share a number of phenotypic characteristics, most likely derived from an immediate precursor that develops into either CD8α + DCs or CD103 + DCs, depending on the tissue it seeds 11,31 .
In the context of Toxoplasma infection different studies have shown that CD8α + DC are a critical source of IL-12 during the acute phase of infection and relocate to the T cell area of the spleen to promote IFN-γ production by T cells 14,15 32,33 . Even thought CD8α + DC are mainly defined as lymphoid resident-DCs, there are reports of migratory DCs expressing the CD8 marker presents in lower frequency in different compartments 19,34 . CD8α + have been shown to participate in antigen presentation and T-cell priming for several intracellular pathogens including Listeria monocytogenes 11,12,35 and Salmonella typhimurium [13][14][15]36 , supporting a general role for CD8α + DCs in the priming of immunity to intracellular pathogens. We newly show that those cells can release IL-1β to counteract the parasite. Moreover, based on our results, it is tempting to speculate that CD8α + DCs express the ISG15 receptor and, in response to increasing ISG15 levels during Toxoplasma infection, migrate to the site of infection. Accordingly, while ISG15 alone is not sufficient to lower the parasite load, it can increase IL-1β levels.
In our study we found that free dimeric ISG15 triggers an increase in For in vivo experiments infected mice were treated with ISG15 (1µg/mouse) or treated with either PBS, buffer from the gel filtration or nothing with no difference observed.

Reagents and Antibodies
Anti-ISG15 rabbit sera, gently gift of Peter Knobeloch, was used as primary antibodies for immunoblot. Secondary antibodies for immunoblotting were

Statistical analysis
All statistical significance analyses were performed using Prism software  C57BL6 mice were infected with 25,000 type II tachyzoites i.p. and serum samples were collected at different time points p.i.. A) Serum ISG15 levels during Toxoplasma infection measured by ELISA. 6 mice/group. One of three independent experiments. B) ISG15 immunoblot on serum samples. On the top, one representative blot of ISG15 and total IgG heavy chain used as loading control. On the bottom is plotted the quantification of four independent experiments. C) C57BL6 mice were infected with live or heat killed or γ-irradiated tachyzoites i.p., and serum ISG15 levels was assessed at day 4 p.i.. 3 mice/group, one of two independent experiments. D) qPCR for ISG15 and IFNα genes on RNA extracted from peritoneal exudate cells from uninfected and at day 2, 4, 7 and 9 p.i., 6 mice per group. One of two independent experiments. Statistics for all were analysed by two way ANOVA statistic with Tukey's multiple comparisons test, *p<0.05; ***p<0.0005; ****p<0.00005.          CD8a DCs Figure S4: C57BL6 mice were either infected with 25,000 type II tachyzoite i.p. and treated with recombinant ISG15 or infected only. Mice were sacrified 4 days post infection and cells were purified from peritoneal exudate. A) Gating strategy (top) and quantification (bottom) for the staining used to analyse the innate cell populations, 3 mice per group. One of three independent experiments. B) Gating strategy for the staining used to analyse CD8α+ and CD103 DCs, 3 mice per group. One of three independent experiments. Statistics were analysed by two way ANOVA statistic with Tukey's multiple comparisons test, ***p<0.0005.