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. 2013 Sep;218(9):1207–1216. doi: 10.1016/j.imbio.2013.04.009

ISG12 is a critical modulator of innate immune responses in murine models of sepsis☆

P Uhrin 1, T Perkmann 1, B Binder 1, G Schabbauer 1,⁎
PMCID: PMC3748340  PMID: 23747037

Abstract

Sepsis is still a major burden for our society with high incidence of morbidity and mortality each year. Molecular mechanisms underlying the systemic inflammatory response syndrome (SIRS) associated with sepsis are still ill defined and most therapies developed to target the acute inflammatory component of the disease are insufficient.

Recently the role of nuclear receptors (NRs) became a major topic of interest in transcriptional regulation of inflammatory processes. Nuclear receptors, such as the peroxisome proliferators-activated receptors (PPARs), have been demonstrated to exert anti-inflammatory properties by interfering with the NFκB pathway.

We identified the nuclear envelope protein, interferon stimulated gene 12 (ISG12), which directly interacts with NRs. ISG12 is a co-factor stimulating nuclear export of NRs, thereby reducing the anti-inflammatory potential of NRs such as NR4A1. To examine the role of ISG12 in acute inflammatory processes we used recently generated ISG12 deficient mice.

We can clearly demonstrate that lack of ISG12 prolongs survival in experimental sepsis and endotoxemia. Furthermore we can show that several acute inflammatory parameters, such as systemic IL6 cytokine levels, are downregulated in septic ISG12−/− animals. Consistently, similar results were obtained in in vitro experiments in peritoneal macrophages derived from ISG12 deficient mice.

In contrast, mice deficient for the nuclear receptor NR4A1 exhibited an exacerbated innate immune response, and showed a significantly higher mortality after lethal endotoxemic challenge. This dramatic phenotype could be restored in ISG12/NR4A1 double deficient mice.

We conclude from our data in vitro and in vivo that ISG12 is a novel modulator of innate immune responses regulating anti-inflammatory nuclear receptors such as NR4A1.

Keywords: Innate immunity, Sepsis, ISG12

Introduction

Sepsis is the leading cause of death among patients admitted to intensive care units in the United States. Morbidity and mortality are very high, ranging from 30% to 50% (Angus et al. 2001). Unfortunately treatment strategies are limited and a number of promising attempts to implement anti-inflammatory therapeutic approaches failed in clinical trials (Riedemann et al. 2003).

Sepsis is defined by an overwhelming systemic inflammatory response syndrome (SIRS) to pathogens or pathogen associated molecular patterns (PAMPs). Therefore appropriate management of inflammation is critical in septic patients. Although anti-cytokine (TNF-Ab) and anti-PAMP (LPS-Ab) strategies failed, some anti-inflammatory treatment approaches have been applied successfully in acutely ill patients, such as low dose glucocorticoid treatment. The beneficial clinical as well as experimental use of glucocorticoids underscores the relevance of nuclear receptors in acute inflammatory diseases (Annane 2005; Goodwin et al. 2013).

Members of the nuclear receptor family have been implicated in a plethora of physiologic processes such as development, metabolism and inflammation. The most prominent member of the NR-family, in respect of sepsis and sepsis treatment, is the glucocorticoid receptor (GR). Glucocorticoids, among only a few others, are clinically approved drugs in the treatment of septic patients (Annane 2005). Glucocorticoids, such as dexamethasone, have been demonstrated to exert potent anti-inflammatory properties and beneficial effects in sepsis. GRs down-modulate pro-inflammatory processes by specific, trans-repressing mechanisms. The anti-inflammatory action of GR is thought to result from direct interference with NFκB- and AP-1-dependent gene regulation (De et al. 2003).

Furthermore other members of the NR family have also been found to trans-repress pro-inflammatory transcription. LXRs, peroxisome proliferator activated receptors (PPARs) and estrogen receptors, all of which are regulated by small secondary metabolites like lipids and hormones, have been elegantly shown to play important roles in immune regulatory mechanisms (Castrillo and Tontonoz 2004; Devchand et al. 1996; Jiang et al. 1998; McKay and Cidlowski 1999; Ricote et al. 1998). Recently some of those NRs became a major topic of interest in immune biology, since the laboratory of Chris Glass could successfully prove a direct repressing effect of different NRs on NFκB signaling, the major pro-inflammatory pathway governing the expression of secondary inflammatory mediators such as TNFα (Ogawa et al. 2005).

Still the role of several other members of the NR family, such as the orphan nuclear receptors NR4A, in inflammation remains controversial. Evidence for a pro-inflammatory role of NR4As has been published before. It was shown that overexpression of NR4As regulates pro-inflammatory genes in a murine macrophage cell line (Pei et al. 2006). On the other hand we and others could demonstrate that NR4As and NR4A1 (Nurr77, Nak1) in particular exert potent anti-inflammatory and protective functions in vitro and in in vivo models of chronic inflammation such as atherosclerosis and restenosis (Bonta et al. 2006; Gruber et al. 2003; Papac-Milicevic et al. 2012; Hanna et al. 2012). Recently it was shown that NR4A1 blocked vascular remodeling by macrophage inhibition (Bonta et al. 2010). It was shown that NR4A1 is rapidly induced by various inflammatory stimuli, such as LPS (Pei et al. 2005). NR4A1 is involved in negative selection and T-cell apoptosis by the conversion anti- to pro-apoptotic Bcl-2 (Thompson and Winoto 2008). Moreover NR4A1 is involved in myeloid cell differentiation and controls the survival of Ly6C-monocytes (Hanna et al. 2011). Still the role of NR4A1 in acute inflammatory processes such as sepsis appears to be elusive.

Recent findings from our laboratory indicate that NR4As directly interact with the interferon stimulated gene 12 (ISG12). We can demonstrate that NR4A function and cellular distribution is efficiently modulated by ISG12 (Papac-Milicevic et al. 2012). ISG12, IFI27 in mice, belongs to the ISG12 subfamily of small interferon inducible genes that are up-regulated in cells upon stimulation by interferons (Martensen and Justesen 2004; Platanias 2005). ISG12 is a member of a plethora interferon stimulated genes, among which the ubiquitin-like ISG15 has been described to play an important role in innate immunity (Malakhova et al. 2003; Ritchie et al. 2004). To study biological effects of ISG12 in vivo we have recently generated ISG12 gene targeted mice (Papac-Milicevic et al. 2012).

The biological function of the ISG12 subfamily members (6–16, ISG12, ISG12S) is largely unknown. ISG12 was found to be up-regulated in gene expression arrays from patient samples of infectious diseases (Bieche et al. 2005; Schwab et al. 2004; Dooley et al. 2004; Izmailova et al. 2003), inflammatory bowel disease (Dooley et al. 2004) and cancer (Suomela et al. 2004; Bani et al. 2004). The limited amount of available data, points at a distribution of ISG12 at the nuclear envelope (Martensen et al. 2001). Together with findings from our laboratory, we believe that ISG12 that is localized at the nuclear/cytoplasmic border, modulates the subcellular distribution of NRs such as NR4A1.

Since we have found a direct interaction of ISG12 with NR4A1 in different biochemical assays (Papac-Milicevic et al. 2012) and deleterious effects of ISG12 in models of chronic inflammation, we sought to investigate the acute phase immune-modulatory function of ISG12 and NR4A1.

Here we can demonstrate for the first time that NR4A1 has potent immune-protective properties in animal models of sepsis. Furthermore we provide evidence that ISG12 deficiency is beneficial in acute inflammation, which can be explained in part by the increased anti-inflammatory activity of NRs. These findings suggest that ISG12 and NR4A1 are critical players in the regulation of innate immune responses.

Materials and methods

Mice

ISG12 deficient as well as ISG12/NR4A1 double deficient mice were generated at our institute as previously described (Papac-Milicevic et al. 2012). In contrast to the original paper ISG12 deficient mice were backcrossed to a C57BL/6J background for at least 8 generations. Mice deficient for NR4A1 were purchased from Jackson laboratories. This strain is backcrossed to a C57BL/6J background for at least 13 generations. Littermate-controlled experiments were performed using 8–12 week old male mice. All animal studies were approved and comply with institutional guidelines (BMWF-66.009/0103-C/GT/2007).

Isolation and stimulation of primary murine cells

Thioglycollate-elicited peritoneal macrophages were isolated in all cases from gene deficient mice and wildtype littermate controls. In brief, 2 ml of a 4% sterile thioglycollate medium were injected intraperitoneally and macrophages were harvested three days later by peritoneal lavage with 5 ml of Ringer's Solution. Macrophages were counted and seeded at a concentration of 106 cells per ml in RPMI-1640 medium (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin/fungizone (PSF) and 1% l-glutamine. After 2 h, medium was exchanged to remove non-adherent cells; adherent cells were allowed to recover overnight. Macrophages were stimulated with 100 ng/ml pure LPS derived from E. coli O111:B4 (Invivogen, San Diego).

Blood mononuclear cells have been isolated using OptiPrep™, according to the manufacturer's protocols (Sigma, St. Louis, MO).

Western blotting and ELISA

Macrophage cell lysates were separated by SDS–PAGE, blotted to Immobilon PVDF Transfer Membrane (Millipore, Bedford, MA, USA) and probed with primary antibodies against TNFalpha, iNOS and tubulin (Sigma, St. Louis, MO). For detection, a secondary antibody conjugated with horseradish peroxidase (Amersham, Piscataway, NJ) was used. Membranes were developed using the chemi-luminescence reagent assay SuperSignal West Femto and exposed in the FluorChem HD2 Chemiluminescence Imager (Alpha Innotech Corp., San Leandro, CA). Bands were analyzed according to their molecular weight. Tubulin was used for normalization.

For ELISA measurements macrophage supernatant, peritoneal lavage and plasma samples were analyzed for TNFα, IL6, KC/Gro-alpha and MCP-1 using the Duoset ELISA kits (R&D Systems, Minneapolis).

Quantitative real-time RT-PCR

Total RNA was isolated from naïve and stimulated macrophages and PBMCs using TRIzol Reagent (Invitrogen Corp., Carlsbad, CA) according to manufacturer's protocol and reverse transcribed. Semi-quantitative real-time PCR was performed using Fast SYBR Green Mastermix (Applied Biosystems, Foster City, CA) and StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA). Transcription levels of target genes were assayed in duplicates, normalized to pbgd levels and depicted as fold induction of unstimulated control cells.

FACS analysis

Influx of leukocytes upon CLP/sepsis into the peritoneum was analyzed in peritoneal lavage by 2-color flow cytometry using FITC-conjugated anti-LY6G/Gr-1 and Rhodamin-conjugated anti-CD11b/Mac-1 (Ebioscience, Vienna, Austria). Cytometric analyzes were performed using a FACSCalibur flow cytometer and Cell Quest software (BD, Schwechat, Austria).

Endotoxemia

ISG12 or NR4A1 gene deficient mice and respective littermate controls have been treated i.p. with 10–15 mg/kg LPS derived from E. coli O111:B4 (Sigma, St. Louis). Blood was drawn at the indicated time points. Survival of mice was monitored for up to 5 days.

Polymicrobial peritonitis/cecal ligation and puncture (CLP)

ISG12 or NR4A1 gene deficient mice and respective littermate controls have been anesthetized and a 1.5–2 cm midline incision was made through the linea alba. The cecum was located and tightly ligated at its base without causing bowel obstruction. The cecum was punctured once (21 gauge). A small amount of stool was extruded to ensure wound patency. The cecum was then placed in its original position within the abdomen. Immediately after surgery mice will receive a subcutaneous injection of 0.5 ml warm saline, placed under a heating lamp and constantly monitored until they recover. Blood was drawn at the indicated time points. Survival of mice was monitored for up to 5 days.

Lung wet/dry weight

Total lungs have been isolated from healthy or septic animals and dry-blotted immediately. After measurement of wet weight, lungs have been dried overnight at 50 °C and dry weight was measured subsequently. Data is represented as lung wet weight to dry weight ratio.

Statistical analysis

Data were analyzed by GraphPad Prism 5 software using unpaired Student's t-test followed by post hoc tests, when appropriate. Values are expressed as mean ± standard deviation. For survival analysis we performed log rank tests with Kaplan–Meier curves. Criteria for significance for all experiments were p < 0.05.

Results

LPS-mediated upregulation of pro-inflammatory cytokines is positively regulated by ISG12

Previous work has shown that ISG12 expression is regulated by secondary inflammatory mediators, type I interferons (IFNα). In addition to interferons, synthetic polyI:C could be identified as potent inducer of ISG12 (Gjermandsen et al. 2000). The finding that polyI:C drives ISG12 expression is significant for this study, since this agonist utilizes the TLR3 signaling pathway. TLRs are highly activated in acute inflammatory responses to pathogens (Qureshi and Medzhitov 2003).

Initially we wanted to rule out the possibility that other agonists, beside IFNs, activate ISG12. Among others, we used E. coli LPS (100 ng/ml) in vitro on the murine macrophage cell line RAW 264.7 as well as murine peritoneal macrophages (mϕ). Interestingly we found that LPS consistently upregulated isg12 (Fig. 1A and B).

Fig. 1.

Fig. 1

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ISG12 is upregulated by LPS/TLR4 and modulates the LPS-mediated cytokine expression. (A) RAW 264.7 macrophages (B) thioglycollate-elicited peritoneal macrophages derived from ISG12+/+ and −/− mice were treated with E. coli LPS O111:B4 (100 ng/ml) for the indicated time points. isg12 mRNA levels were determined by qPCR. LPS-induced transcription of (C) il6 (D) tnfa and (E) cox2 after 4 h stimulation in peritoneal macrophages was determined by qPCR. (E) LPS-induced expression of TNFα after 16 h stimulation was determined by ELISA. * indicates p < 0.05.

To assess the function of ISG12 in LPS-mediated gene transcription, we compared LPS effects on thioglycollate-elicited peritoneal macrophages derived from either ISG12+/+ or −/− littermate mice, which cell recruitment was not different (Suppl. Fig. S2). We focused on TNFα regulation, since TNFα, among others such as IL-1β and IL-12, plays a key role in the propagation of inflammatory responses, mounting the host defense upon encounter of a pathogen. TNFα is absolutely required in the initial phase of inflammation. However if uncontrolled, it will contribute to an overwhelming inflammatory response, harmful for the host (Hatherill et al. 2000; Hotchkiss and Karl 2003).

In order to clarify the role of ISG12 on LPS-mediated responses in macrophages, we measured tnfα transcription in activated peritoneal mϕ. Fig. 1C shows almost undetectable levels at 0 h. Importantly, tnfα mRNA levels were significantly lower in ISG12 deficient macrophages 4 h post LPS treatment. Interestingly, tnfα levels 2 h after LPS treatment have not been significantly different (data not shown). Similar results could be found for il6 and cox2 on mRNA level (Fig. 1D and E). Among the pro-inflammatory genes we have tested that were not different we found mcp1 (data not shown). Next we assessed the levels of TNFα protein released in the medium upon activation. We found that ISG12+/+ macrophages produced and sequestered significantly more TNFα than ISG12 deficient cells 8 h after LPS treatment (Fig. 1F). This finding was also confirmed by cell-based detection of soluble- and membrane-TNFα (Fig. 1G). Interestingly, when we analyzed the protein expression of another important pro-inflammatory M1 marker gene, iNOS, we did not find significant differences on mRNA and protein levels (Fig. 1G and H).

Thus, it appears that ISG12 critically interferes in the expression of some pro-inflammatory molecules, such as TNFα as well as IL6.

ISG12 modulates innate immune responses in vivo

To further evaluate the immunemodulatory function of ISG12 on inflammatory responses in vivo, we used endotoxemia as a classic murine model of acute inflammation.

Endotoxemia is characterized by the innate response to the pathogen associated molecular pattern (PAMP), lipopolysaccharide (LPS). The encounter of LPS in vivo by immune competent cells such as monocytes and macrophages, leads to a significant immune response, which requires tight control.

The clinical relevance of this model is disputed. However in septic patients, endotoxemia is detected in about 30–40% of all cases associated with gram-negative bacterial infection (Hurley 2000). Furthermore LPS could be detected in septic mice, which underwent cecal ligation and puncture (CLP) treatment as early as 2 h post surgery (Buras et al. 2005).

We assessed the role of ISG12 in a murine model of endotoxemia using the same batch of E. coli LPS B4:O111 at a dose of 15 mg/kg throughout the study. Initially we determined the expression of ISG12 in response to LPS in myeloid cells. We found a marked upregulation of isg12 over time (Fig. 2B). This finding confirms data we obtained in vitro in LPS-treated macrophages. We conclude that presumably LPS and/or secondary inflammatory mediators, such as IFNs upregulate ISG12 in endotoxemic mice.

Fig. 2.

Fig. 2

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ISG12 deficiency protects from lethal endotoxemia. ISG12+/+ and ISG12−/− mice were treated with E. coli LPS O111:B4 at 15 mg/kg. (A) A Kaplan–Meier survival plot is shown for the time-dependent reduction of survival (%) (n = 16; p = 0.01). (B + C) isg12, tnfa and il6 mRNA transcription in myeloid cells derived from endotoxemic mice at the indicated time points (6 h for tnfa and il6) was measured by qPCR. (D) Time course for IL6 cytokine release in endotoxemic mice was determined by ELISA. * indicates p < 0.05.

After endotoxin challenge in vivo, wildtype ISG12 animals showed a modest survival rate with approximately 40% mice surviving the observation period of 4 days. In contrast ISG12 deficient mice were protected in our model, showing significantly reduced mortality (Fig. 2A).

Furthermore we used the cytokine IL6 as an inflammatory marker for the systemic inflammatory response. IL6, among other such as KC/IL8, MIP2 and later c reactive protein (CRP), at least in rats, has been reported to serve as reliable predictor of severity of acute inflammatory processes in animal models of acute inflammation (Buras et al. 2005). We found that levels of IL6 directly correlate with the mortality observed in septic or endotoxemic mice.

The observation that ISG12 deficiency is beneficial was confirmed by analysis of reduced il6 and tnfα mRNA expression in mononuclear cells 6 h post challenge (Fig. 2C) and IL6 cytokine release into the circulation in ISG12 deficient animals 24 h post challenge (Fig. 2D). Circulating TNFα could not be detected at the late time point. Those findings clearly demonstrate that ISG12 critically modulates the innate immune response not only in vitro, but also in vivo.

As stated above, ISG12 was found in a yeast two hybrid screen by our laboratory (Papac-Milicevic et al. 2012). In this assay we used the nuclear receptor NR4A1 as bait. Subsequently we found functional and physical interaction between ISG12 and NR4A1. Thus, we wanted to assess the role of NR4A1 in acute inflammatory responses. Furthermore we wanted to rule out the possibility that immune-modulatory effects of ISG12 are mediated by NR4A1.

NR4A1 is a beneficial nuclear receptor in the acute inflammatory response

It has been reported that the orphan nuclear receptor NR4A1 is regulated on a transcriptional and post-translational level rather than by binding to ligands unlike other nuclear receptors such as GRs and PPARs. NR4A1 is regulated by a plethora of stimuli. Among those we find several inflammatory stimuli (Pei et al. 2005). Furthermore work from Hanna et al. support the notion that NR4A1 is intricately involved in inflammatory processes. They could provide evidence that NR4A1 regulates the polarization toward the IFNγ-driven inflammatory M1 macrophage phenotype in vitro as well as in vivo (Hanna et al. 2012)

Initially we wanted to analyze NR4A1 expression in response to LPS (100 ng/ml) in murine macrophages. In RAW macrophages we found that LPS upregulates nr4a1 immediately, within 3.5 h post LPS treatment (Fig. 3A). This was also proven for thioglycollate-elicited inflammatory macrophages as well as bone marrow derived murine macrophages (data not shown). Next we determined the role of NR4A1 in the LPS-mediated expression of pro-inflammatory TNFα in genetically modified macrophages. Detection of TNFα levels in the supernatants of activated macrophages by ELISA revealed significantly increased TNFα release in NR4A1 deficient cells as compared to wildtype control macrophages (Fig. 3B). This was evident as early 4 h post LPS treatment. Interestingly IL6 production was not significantly affected by NR4A1 deficiency, which was consistent with results obtained in ISG12 deficient macrophages, at least on secreted cytokine levels (data not shown).

Fig. 3.

Fig. 3

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NR4A1 deficiency aggravates the endotoxemic shock increasing mortality. ISG12 deficiency ameliorates the NR4A1 phenotype. (A) nr4a1 mRNA transcription was determined in LPS-induced (100 ng/ml) RAW 264.7 macrophages by qPCR. (B) Time-dependent analysis of TNFα release from LPS-induced (100 ng/ml) NR4A1+/+ and −/− peritoneal macrophages was performed by ELISA. Cytokine release in endotoxemic NR4A1+/+ and −/− mice was determined after 2 h for TNFα (C) and 8 h for IL6 (D) by ELISA. (E) A Kaplan–Meier survival plot is shown for the time-dependent reduction of survival (%) in endotoxemia for NR4A1−/−, ISG12−/−/NR4A1−/− and wildtype mice. * indicates p < 0.05; ** indicates p < 0.05 NR4A1−/− vs. wt; *** indicates p < 0.05 vs. NR4A1−/−.

To further examine the role of NR4A1 in acute inflammation we performed endotoxemia experiments in NR4A1+/+ and −/− mice. Consistent with data obtained in vitro we found significant upregulation of TNFα 2 h post LPS challenge in NR4A−/− mice as compared to wildtype controls (Fig. 3C). Secondly, we found that subsequent cytokine release of the inflammatory cytokine IL6 was elevated in NR4A1−/− mice 8 h post LPS challenge (Fig. 3D).

The upregulation of inflammatory cytokines clearly indicates that the host response in NR4A1 animals is highly dysregulated. Importantly this finding was confirmed by significantly increased mortality (vs. wildtype) in NR4A1 deficient animals (Fig. 3E). All NR4A1 deficient mice included in the study died within the 2d observation period. Wildtype controls on the other hand showed improved survival (approx. 30%) (Fig. 3E).

To test the hypothesis whether ISG12 deficiency could rescue the NR4A1 phenotype we created NR4A1/ISG12 double deficient animals. In the same experimental setting, double deficient endotoxemic mice showed significantly improved survival (vs. ISG12+/+/NR4A1−/−), which was not different from wildtype animals (Fig. 3E). Moreover we performed peritoneal sepsis by cecal ligation and puncture and found accelerated morbidity and increased mortality in NR4A1 deficient mice (Suppl. Fig. S1).

These results indicate that NR4A1 is a beneficial nuclear receptor in acute inflammation downmodulating the exacerbated inflammatory response induced by high dose LPS. ISG12 deficiency could rescue the dramatic phenotype observed in NR4A1−/− mice.

ISG12 deficient mice are protected from polymicrobial sepsis

Next we wanted to assess the function of ISG12 in a clinically relevant model of sepsis, namely cecal ligation and puncture (CLP). CLP is widely accepted by the scientific community, mimicking most of the hallmarks of developing sepsis observed in human patients (Buras et al. 2005). It is interesting to note that in CLP and related animal models of sepsis free circulating LPS can be detected as early as 2 h post laparotomy (Zantl et al. 1998).

CLP causes polymicrobial infection in the peritoneum with varying severity, depending on the puncture size of the cecum. If the innate immune response is not mounted properly and the infection is not readily contained, the bacteria multiply and spread into the circulation (bacteraemia). This process induces a systemic inflammatory response of the host with pathologic consequences such as secondary tissue- or organ- damage. Therefore CLP-induced sepsis is often accompanied by acute lung dysfunction (ALI).

We compared the pathophysiologic effects of CLP in either ISG12+/+ or ISG12−/− mice. To test whether ISG12 is upregulated in our model system at all, we performed density gradient cell isolation and measured isg12 expression analysis in circulating mononuclear cells before and after CLP surgery. Indeed we found a marked upregulation of isg12 mRNA 6 h post challenge (see Fig. 4B).

Fig. 4.

Fig. 4

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Detrimental effects of polymicrobial sepsis are ameliorated in ISG12 deficient mice. (A) A Kaplan–Meier survival plot is shown for the time-dependent reduction of survival (%) in CLP for ISG12−/− and wildtype mice. (B) isg12 mRNA transcription in mononuclear cells derived from septic wildtype mice at the indicated time point was measured by qPCR. (C) Acute lung injury in CLP-induced septic mice was analyzed by evaluating the wet to dry weight ratio (n = 3 sham control; n = 7–9 CLP). Systemic levels of IL6 (D) and KC (E) 24 h post CLP were determined by ELISA (n = 2 sham; n = 15 CLP).

Moreover we monitored CLP-induced septic mice for up to 3 days post surgery. Most of the wildtype mice succumbed between 24 h and 48 h, with an overall mortality of 90%. Surprisingly ISG12 deficient mice were significantly protected from septic challenge. We found that more than 50% of septic ISG12−/− animals survived past the 3 d observation period (Fig. 4A).

These results were also confirmed by the analysis of other pathologic and inflammatory parameters. We compared the severity of lung injury induced by CLP measuring edema formation in whole lungs upon septic challenge. The lungs were dissected 24 h post surgery, right at the start of mortality in our model. Indeed ISG12+/+ mice developed significantly more severe lung injury as compared to ISG12−/− mice (Fig. 4C).

Furthermore we analyzed the systemic inflammatory response in the sepsis model by measurement of serum levels of the cytokine Interleukin 6 (IL6) and the chemokine KC, which is the mouse ortholog for Interleukin 8 (IL8), 24 h post surgery. Similar to endotoxemia, IL6 turned out to be reliable predictor of mortality (Remick et al. 2002). Although the inflammatory status in septic mice is highly variable, ISG12+/+ mice showed significantly higher IL6 levels present in the circulation as compared to littermate ISG12−/− mice (Fig. 4D). This finding indicted to us that the ISG12−/− mice could either contain the infection or successfully suppress the systemic response, since IL6 could not be detected in most of the ISG12 deficient septic animals. At the same time point KC/IL8 levels were slightly, but not significantly, lower in ISG12 deficient animals (Fig. 4E). Furthermore the results are consistent with results obtained in the endotoxemia experiments (Fig. 2C and D).

These findings indicated to us that ISG12 wildtype animals suffered from dysregulated SIRS, which contributed to the observed accelerated death. In contrast ISG12 deficient animals could better cope with the septic challenge. As a consequence the systemic inflammatory burden was successfully reduced.

However we do not know how the initial trigger of sepsis was dealt with in ISG12 wildtype as compared to ISG12 deficient animals. Therefore we evaluated the preceding inflammatory events in the peritoneum, at the site of first encounter with the pathogenic microorganisms.

ISG12 deficiency leads to increased neutrophil granulocyte influx into the peritoneum

CLP rapidly activates cells, which constitutively monitor the peritoneal cavity for pathogens. Important cells in this regard are peritoneal macrophages, as the first line of defense. In mice approximately 40–50% of resident cells are comprised of peritoneal macrophages (unpublished observation). In total 3–5 × 106 cells are found in the peritoneum of untreated mice.

On the basis of our previous findings, we speculated that some of the initial events fighting the infection might be affected by ISG12 upregulation in developing sepsis. Therefore we analyzed the cell infiltration into the peritoneum 24 h post surgery comparing ISG12 wildtype and deficient mice. Initially we did not find differences in inflammatory cell accumulation in the peritoneum in a thioglycollate-based model of sterile inflammation (Suppl. Fig. S2).

Upon septic challenge on the other hand, we found a significant difference in the number of inflammatory cells present in the peritoneum. Much to our surprise, ISG12 deficient mice contained more inflammatory cells in the peritoneal lavage (Fig. 5A). Analysis of the cell type revealed that a major shift from predominant resident peritoneal macrophages to neutrophil granulocytes (Mac-1+, Gr-1+) took place. In this regard we could not detect any differences in the relative cellular composition (approx. 75%) between the genotypes (Fig. 5B).

Fig. 5.

Fig. 5

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Chemokines and peritoneal neutrophil influx are increased in ISG12 deficient septic mice. (A) Peritoneal cells from mice that underwent CLP have been isolated and enumerated 24 h post surgery. (B) FACS analysis from isolated peritoneal cells derived from septic mice has been performed with PE-labeled Mac-1/CD11b and FITC-labeled GR-1/Ly6-G. Percentage of double-positive peritoneal cells is indicated. (C, D) Systemic chemokine levels 8 h post surgery have been measured by ELISA.

When we determined the inflammatory status in the peritoneum no significant changes between the different genotypes could be found for either TNFα, IL6 and MCP-1 or KC. TNFα and IL6 appeared to be lower, but the reduction in ISG12 deficient animals turned out to be not significant (data not shown).

When we determined the time course of systemic chemokine release in septic mice, we found an early peak, preceding the peritoneal neutrophil influx, 6–8 h post surgery (data not shown). Analysis of chemokine release in the circulation revealed that ISG12 deficient mice showed significant increased levels for KC/IL8, which is one of the major chemokines required for neutrophil recruitment 8 h post CLP induction (Fig. 5G). Furthermore we found a trend albeit not significantly different for the monocyte/macrophage chemokine MCP-1/CCL2 (Fig. 5H). Thus we conclude that initially the lack of ISG12 leads to increased production of chemoattractants, which support the influx of neutrophil granulocytes to fight the infection in the peritoneal cavity.

Discussion

To date the molecular mechanisms required to regulate Sepsis/SIRS are poorly characterized. There is no doubt that in developing sepsis the early onset of the inflammatory response is necessary to fight off the infection successfully. It is a well described phenomenon that immune-suppressed patients, like diabetes or cancer patients, have a clear disadvantage surviving septic events (Lederer et al. 1999; Meakins et al. 1977; Oberholzer et al. 2001). Furthermore it is known that immune stimulation with IFNγ restored monocyte/macrophage responsiveness and mortality was reduced (Docke et al. 1997).

Among other recently described immune-suppressing molecules in innate immunity such as SOCS-1 and IRAK-M (Deng et al. 2006; Nakagawa et al. 2002), nuclear receptors have also been implicated in the downmodulation of acute inflammatory processes.

Nuclear receptors, directly act on the transcriptional machinery used by pro-inflammatory pathways. Nuclear receptors compete with pro-inflammatory transcription factors, such as NFκB and AP-1, for readily available transcriptional co-factors, such as CBP, and thereby limit efficiently the trans-activating potential of factors such as NFκB and AP-1. This phenomenon has been shown recently in context of TLR-mediated signaling for the nuclear receptors PPAR and the glucocorticoid receptor (Ogawa et al. 2005).

The role of other nuclear receptors, such as the orphan nuclear receptor family NR4A, in the regulation of acute immune responses is still a controversial issue. Pei et al. could provide evidence for a positive regulatory function of NR4As in the transcriptional regulation of a number of pro-inflammatory genes. This phenomenon was observed upon overexpression of NRs in a murine macrophage cell line (Pei et al. 2006). Even though the results are well documented, artificial overexpression of NR4As does not resemble the physiologic conditions observed in response to an inflammatory challenge in vitro or in vivo. In contrast to Pei and coworkers, our laboratory found a protective role for NR4A1 in a model of restenosis using NR4A1 deficient mice (Papac-Milicevic et al. 2012). These findings are also supported by reports on NR4A1 transgenic as well NR4A1 gene deficient mice in models of atherosclerosis, which prove that NR4A1 expression is beneficial in cardiovascular disease (Bonta et al. 2006; Hanna et al. 2012; Bonta et al. 2010). Moreover recent reports underline the importance of NR4A family members in the modulation of inflammatory responses in innate immune cells (Pei et al. 2005; Pires et al. 2007; Saijo et al. 2009).

Since several preceding publications could show that NR4A1 is rapidly induced in response to PAMPs and secondary inflammatory mediators, as well as its involvement in the M1/M2 macrophage cell fate decision (Pei et al. 2005; Hanna et al. 2012), we speculated that NR4A1 plays a key role in the modulation of the innate immune response in sepsis. Indeed we found that NR4A1 deficiency leads to exacerbated inflammation and increased mortality in murine models of acute inflammation. Nevertheless it is unclear how NR4A1 is regulated on a molecular level. We found in different biochemical screenings that NR4A1 directly interacts with a small nuclear envelope protein ISG12. From our data we conclude that ISG12 modulates the cellular distribution and thereby the functional activity of NR4As. This phenomenon holds true not only for NR4As but also for other NRs (Papac-Milicevic et al. 2012). When we analyzed the expression profile of ISG12, we found that ISG12 is regulated by a variety of inflammatory stimuli, which are also capable of NR4A1 induction in a similar timely manner.

The generation of ISG12 deficient animals gave us the opportunity to analyze the biological properties of ISG12 on a physiologic level. In different animal models of acute inflammation, among which the CLP model is the most clinically relevant, we found that ISG12 deficiency is beneficial and protects mice from endotoxemic as well as septic shock.

Recent publications indicate that the sepsis animal model of cecal ligation puncture can be differentiated into an early acute inflammatory phase and late chronic phase (Xiao et al. 2006). In the first phase management of SIRS and the pathogen elimination characterize the pathogenicity of this peritonitis model. Indeed this is exactly what we observed in CLP experiments in ISG12 and NR4A1 deficient animals. ISG12 deficient animals, which have increased NR activity, show a markedly diminished cytokine expression profile in this critical phase of sepsis. In contrast NR4A1 deficient animals cannot reduce the inflammatory burden and are therefore more likely to succumb to the disease as compared to control or NR4A1/ISG12 double deficient septic animals.

Importantly the ISG12 deficiency rescued the phenotype observed by the lack of NR4A1 in endotoxemia. This result was rather surprising to us, since it indicates that ISG12 not only regulates NR4A1 but also affects the activity of other beneficial molecules as well. Taken together we believe that ISG12 contributes to the regulation of acute inflammatory processes by interacting and regulating transcription factors such as NR4A1. NR4A1 is an important factor dampening the innate immune response. ISG12 on the other hand mitigates the effects mediated by NR4A1 possibly on other transcription factors such as NFκB and AP-1.

Acknowledgment

This article is dedicated to the memory of Prof. Dr. Bernd R. Binder, who supported and inspired all of us with his endless enthusiasm.

This work was funded by FWF projects P19850 and P24802 to GS.

Footnotes

☆

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Fig. S1. NR4A1 deficiency leads to increased mortality in CLP-induced polymicrobial sepsis. (A) A Kaplan–Meier survival plot is shown for the time-dependent reduction of survival (%) in CLP for NR4A1−/− and wildtype mice (wt n = 7; NR4A1−/− n = 10).

Supplementary Fig. S2. Leukocyte recruitment in sterile peritoneal inflammation is not altered by ISG12 deficiency. Total cell numbers from peritoneal lavage of thioglycollate (4%) treated animals (ISG12−/− and wt littermates) are shown (n = 4 each).

mmc1.pdf (9.4KB, pdf)

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