Cardif, also known as IPS-1, VISA, and MAVS, is an intracellular adaptor protein that functions downstream of the retinoic acid–inducible gene I family of pattern recognition receptors. Cardif is required for the production of type I IFNs and other inflammatory cytokines after retinoic acid–inducible gene I–like receptors recognize intracellular antigenic RNA. Studies have recently shown that Cardif may have other roles in the immune system in addition to its role in viral immunity. In this study, we find that the absence of Cardif alters normal NK cell development and maturation. Cardif−/− mice have a 35% loss of mature CD27CD11b+ NK cells in the periphery. In addition, Cardif−/− NK cells have altered surface marker expression, lower cytotoxicity, decreased intracellular STAT1 levels, increased apoptosis, and decreased proliferation compared with wild-type NK cells. Mixed chimeric mice revealed that the defective maturation and increased apoptotic rate of peripheral Cardif−/− NK cells is cell intrinsic. However, Cardif−/− mice showed enhanced control of mouse CMV (a DNA β-herpesvirus) by NK cells, commensurate with increased activation and IFN-γ production by these immature NK cell subsets. These results indicate that the skewed differentiation and altered STAT expression of Cardif−/− NK cells can result in their hyperresponsiveness in some settings and support recent findings that Cardif-dependent signaling can regulate aspects of immune cell development and/or function distinct from its well-characterized role in mediating cell-intrinsic defense to RNA viruses.

Pattern recognition receptors recognize pathogen-associated molecular patterns (1, 2). Retinoic acid–inducible gene-I (RIG-I)–like receptors are a subset of pattern recognition receptors that recognize intracellular viral nucleic acids and induce the production of type-I IFNs and NF-κB–regulated genes (3). Two members of the RIG-I–like receptor family, RIG-I and MDA5, have caspase recruitment and activation domains (CARDs) that allow for downstream signaling after activation. The adaptor protein that interacts with RIG-I and MDA5 and allows downstream signaling was discovered by four different groups and is thus known by four names: Cardif (CARD adaptor inducing IFN-β), MAVS (mitochondrial antiviral signaling), IPS-1 (IFN-β promoter stimulator-1), and VISA (virus-induced signaling adaptor) (47). We refer to this protein as Cardif.

RIG-I and MDA5 initiate signaling through CARD–CARD interactions with Cardif, which is a ubiquitously expressed protein that is located on the outer mitochondrial membrane of both immune and nonimmune cells (3, 8). The mitochondrial localization of Cardif is essential to its signaling function. Once Cardif has been engaged by RIG-I or MDA5, it aggregates with other Cardif molecules. This aggregation is essential to propagation of downstream signals (9). Cardif interacts with cytoplasmic adaptor molecules TRAF3, TRAF2, and TRAF6 to activate transcription factors NF-κB, IRF3, and IRF7 to induce expression of type I IFN genes and IFN-induced genes (3, 10).

Cardif is vital for signaling in response to viral pathogenic nucleic acids sensed by MDA5 and RIG-I. However, there have been reports that suggest that both RIG-I and Cardif may play roles in immune regulation that are separate from their roles in viral defense (1115). Wang et al. (12) report that RIG-I−/− mice develop colitis and are more susceptible to dextran sulfate–induced colitis. Xu et al. (13) report that Cardif−/− B cells have a cell-intrinsic defect in CD23 and TLR7 expression. In addition, Cardif−/− mice develop more severe disease in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (16). Cardif is also required for the optimal activation of the NLRP3 inflammasome (14). RIG-I activity under the control of IRF1 has been implicated in the progression of atherosclerosis. Wang et al. (15) propose that 25-hydroxycholesterol induces IL-8 production in macrophages by inducing IRF1 and subsequent RIG-I expression and activation. These reports, as well as others, suggest that Cardif is active even in the absence of pathogenic viral RNA.

NK cells are innate cytotoxic lymphocytes that target virally infected, stressed, or cancerous cells (17). NK cells primarily develop in the bone marrow, although some peripheral organs such as the liver can house and develop NK cells (1820). Mature NK (mNK) cells are the primary NK cells found in peripheral organs such as the spleen, liver, and lymph nodes where they undergo additional maturation (21). CD49b acquisition is the earliest stage of NK maturity. The acquisition of CD11b, CD43, and KLRG1 occur after CD49b, and identify more advanced stages of NK maturation. Surface markers CD27 and CD11b can be used to further delineate stages of maturation within immature NK (iNK) and CD49b+ NK (mNK) cells (2224). Maturation using these markers is divided into four stages that progress in the following order: CD27CD11b (stage 1) → CD27+CD11b (stage 2) → CD27+CD11b+ (stage 3) → CD27CD11b+ (stage 4). Very few CD27CD11b NK cells are found in the peripheral organs, and the majority of CD27CD11b NK cells do not express CD49b. CD27+CD11b NK cells are highly proliferative and have the ability to degranulate but are not as cytotoxic as CD27+CD11b+ or CD27CD11b+ NK cells. CD27+CD11b+ NK cells are highly responsive to cytokine and dendritic cell (DC) stimulation in vitro compared with CD27CD11b+ NK cells (22). CD27CD11b+ NK cells are terminally differentiated, long-lived, and express the inhibitory NK receptor KLRG1 on their surface (25). CD27+CD11b+ NK cells are most prevalent in bone marrow and lymph nodes, whereas CD27CD11b+ NK cells are the predominant stage found in spleen, liver, blood, and lung.

NK cell numbers, maturation, and function are diminished in the absence of type I IFN signaling. IFNAR−/− mice that lack the ability to respond to type I IFN have fewer NK cells and are unable to control the growth of tumor cells that are normally susceptible to NK killing (2629). IFNAR-deficient NK cells are also unable to kill some NK target cells in vitro. It has been suggested that consistent and low levels of type I IFN are required to maintain NK cell numbers and functionality in vivo as well (30). Constitutive type I IFN signaling maintains STAT1 levels in NK cells. High STAT1 levels are required to maintain NK cytotoxicity, whereas STAT4 leads to IFN-γ production (26, 31, 32). It has been suggested that many of the effects of type I IFN deficiency in NK cells can be attributed to the reduction of intracellular STAT1 levels.

Considering the earlier studies and observations, we sought to understand how the absence of Cardif affects the development, maturation, and function of NK cells. In this study, we found that Cardif expression is required for maintaining optimal NK cell numbers in the periphery and for full NK cell maturation. As such, we found a marked reduction in the number of CD49b+ and CD27CD11b+ NK cells with a concomitant increase in CD27+CD11b and CD27+CD11b+ NK cells in the periphery of mice lacking Cardif. These NK cells had decreased cytotoxicity, although they produced comparable amounts of IFN-γ after stimulation in vitro. Mixed bone marrow chimeras revealed that the maturation of peripheral Cardif−/− NK cells is cell intrinsic. Similar to NK cells from IFNAR−/− mice, Cardif−/− NK cells showed decreased proliferation and decreased STAT1 activation. Intriguingly, despite their altered differentiation and effector function in these settings, in response to mouse CMV (MCMV) infection, NK cells from Cardif−/− mice showed heightened IFN-γ production and enhanced control of this dsDNA β-herpesvirus. Together, our results indicate that Cardif imposes intrinsic regulation of NK cell subset maintenance and function in diverse inflammatory settings.

Cardif−/− mice were a kind gift from the Shresta laboratory at La Jolla Institute for Allergy and Immunology. Cardif−/− mice were generated as described by Michallet et al. (33) and are on a C57BL/6 background. C57BL/6 mice purchased from The Jackson Laboratory (000664) or wild-type (WT) littermates were used as controls in experiments. Male and female mice were used at 6–12 wk of age. All experiments adhered to the guidelines outlined by the La Jolla Institute for Allergy and Immunology Animal Care and Use Committee, according to criteria outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Mice were euthanized by CO2 inhalation.

Spleen and liver were harvested and pushed through a 40-μm strainer. Liver was perfused with PBS before collection and placed in RPMI 1640 medium supplemented with 10 μm HEPES and 10% FBS. In addition, lymphocytes were separated from hepatocytes via density centrifugation. RBCs were lysed with RBC lysis buffer according to the manufacturer’s protocol (Biolegend).

Cells were resuspended in FACS buffer (1% BSA and 0.1% sodium azide in PBS), and 1–4 × 106 cells were incubated in 100 μl with anti-CD16/CD32 Ab (2.4G2) for 30 min on ice to block FCγRII/III binding. Samples were then incubated with a mixture of fluorochrome-conjugated Abs (Biolegend, eBioscience, BD Biosciences) for 30 min on ice in the dark. LIVE/DEAD Fixable Dead Cell Stain (Invitrogen) was used to determine cell viability. Intracellular staining of Abs was performed after cells were fixed and permeabilized using 0.55% paraformaldehyde and Permeabilization Buffer (BD Biosciences). Samples were analyzed for cellular fluorescence on an LSR II (BD Biosciences), and data were analyzed with FlowJo software (Tree Star). A complete flow-cytometry gating strategy for peripheral NK cells is shown in Supplemental Fig. 1.

Spleen was harvested and pushed through a 40-μm strainer. RBCs were lysed with RBC lysis buffer according to the manufacturer’s protocol (Biolegend). Splenocytes were cultured in 96-well plates for 4–5 h in the presence of either PMA (50 ng/ml) and ionomycin (1 μg/ml) or IL-12 (20 ng/ml) and IL-18 (20 ng/ml). When measuring degranulation, FITC-conjugated CD107a Ab (0.05 μg/ml) was added to the stimulation media. After stimulation, splenocytes were washed with PBS and then prepared for flow-cytometry analysis as described earlier.

NK cells were enriched using EasySep Mouse NK Cell Enrichment kit (Stem Cell Technologies). Spleens from three or more mice were pooled together. Purity of NK cells was ∼80–85% as reported by supplier. Murine cytokines were used in the following concentrations: IL-15, 50 ng/ml (Peprotech); IL-18, 50 ng/ml (Peprotech); and IFN-β, 10 U/ml (Millipore). There were triplicates of each condition. Cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4 h. After 4 h, cells were harvested and processed for FACS analysis.

For type I IFN rescue experiments, NK cells were cultured in the presence of IL-15 only or IL-15 with IFN-β for 5 d at a concentration of 5e6 cells/ml in a 96-well round-bottom plate. IL-15 was vital for the survival of NK cells in culture. After 5 d of cytokine treatment, NK cells were stimulated with PMA and ionomycin at the aforementioned concentrations and analyzed by flow cytometry.

Enriched NK cells were cultured at various ratios together with calcein-labeled YAC-1 cells for 4 h. YAC-1 cells were labeled with 0.5 μM calcein. Specific lysis was determined by measuring percent specific release of calcein using the following formula: Percent specific release = (experimental release − spontaneous release)/(maximum release − spontaneous release) × 100.

Bone marrow transplantation studies were performed as previously described (34). CD54.1/0.2 mice were irradiated with two doses of 550 rad each, 4 h apart. Bone marrow cells were isolated from CD45.1 and Cardif−/− mice and processed under sterile conditions. A single-cell suspension in PBS was obtained with a 1:1 ratio of CD45.1 and Cardif−/− bone marrow cells. Approximately 5e6 cells were retro-orbitally injected into recipient CD54.1/0.2 mice in a volume of 200 μl. Mice received autoclaved water treated with antibiotics (trimethoprim-sulfamethoxazole) 1 wk before and continued until 1 wk after injection. Mice were euthanized for experiments 8–10 wk after injection.

Annexin V (Invitrogen) and propidium iodide (Invitrogen) were used to identify apoptotic and dead cells, respectively. Cells were stained according to the protocol provided by the manufacturer. FITC-conjugated caspase-3 (BD Biosciences) was also used to identify apoptotic cells. Cells were stained for intracellular caspase-3 according to the manufacturer’s protocol. For proliferation, Ki-67 (eBioscience) was used to identify proliferating cells ex vivo. Splenocytes were stained for intracellular Ki-67 using the Foxp3/Transcription factor staining buffer set (eBioscience), and cells were stained according to the protocol provided by the manufacturer.

NK cells were isolated using EasySep Mouse CD49b positive selection kit (STEMCELL Technologies). Spleens from seven or more mice were pooled together. Purity of NK cells was ∼90% as reported by supplier. Isolated NK cells were then divided and either left untreated or treated with 100 U/ml IFN-β (Millipore) for 15 min at 37°C. Cells were then lysed for protein collection using radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, 1 mM NaF, 0.5% NP-40, 0.1% Brij35, 0.1% deoxycholic acid). Total was quantified using BCA Protein Assay Reagent (Thermo Scientific). Afterward, 30 μg of each protein sample was loaded into SDS-PAGE. The following Abs were used at specified concentrations for immunoblots: STAT1 (1:1000; no. 9172; Cell Signaling), pSTAT1 (Tyr701; 1:1000; no. 9171; Cell Signaling), STAT4 (1:1000; no. 2653; Cell Signaling), p-STAT4 (1:1000; no. 5267; Cell Signaling), and β-Actin (1:2000; no. 9774; Cell Signaling). Western blots were quantified using ImageJ software (National Institutes of Health).

MCMV WT and ∆m157 salivary gland stocks were derived from the bacterial artificial chromosome–cloned MCMV K181 strain (kind gift from Alec Redwood, Murdoch University) after in vivo passage of MEF-derived virus twice in 3-wk-old BALB/c mice for 12 d, as previously described (35). Cardif−/− and WT mice were infected i.p. with 1 × 105 PFU, and viral replication in organs was measured by plaque assay in NIH 3T3 cells as described previously (36). NK cells were depleted by injecting 200 μg anti-NK1.1 Ab (clone PK136) in 200 μl PBS 24 h preinfection.

All data are presented with mean ± SEM as determined by Prism software (GraphPad). Unpaired t tests were used to compare WT and Cardif−/− samples. The p values ≤0.05 were considered significant for all experiments.

Cardif is reportedly active in the absence of a viral infection and is a potent inducer of type I IFN (3, 13, 16). Studies have shown that type I IFN regulates NK cell maturation and function (27, 30). With this information, we hypothesized that Cardif may influence NK cell maturation and function. To test this hypothesis, we characterized NK cells in the bone marrow and in the periphery of WT and Cardif−/− mice. We found that, compared with WT mice, Cardif−/− mice have fewer numbers of CD19CD3NK1.1+ NK cells in spleen, but not in bone marrow (Fig. 1A).

FIGURE 1.

Cardif is required for optimal NK cell numbers in the periphery and for the terminal differentiation of NK cells. (A) The absolute number of NK1.1+CD3CD19 cells was determined in the spleen. (B) Representative plots (left and middle panels) and bar graphs (right panels) show the percentage of CD49b NK cells (NK1.1+CD3CD19) in the spleen (upper panels) and liver (lower panels). (C) Representative plots and bar graphs plots (left and middle panels) and bar graphs (right panels) show the distribution of mNK (NK1.1+CD3CD19 CD49b+) cells based on CD27 and CD11b expression in the spleen (upper panels) and liver (lower panels). Stages 1–4 are as follows: stage 1(CD27CD11b), stage 2 (CD27+CD11b), stage 3 (CD27+CD11b), and stage 4 (CD27CD11b+). Experiments were repeated at least three times. n = 3–5 mice/group/experiment. Data are means ± SEM. **p < 0.01, ***p < 0.001.

FIGURE 1.

Cardif is required for optimal NK cell numbers in the periphery and for the terminal differentiation of NK cells. (A) The absolute number of NK1.1+CD3CD19 cells was determined in the spleen. (B) Representative plots (left and middle panels) and bar graphs (right panels) show the percentage of CD49b NK cells (NK1.1+CD3CD19) in the spleen (upper panels) and liver (lower panels). (C) Representative plots and bar graphs plots (left and middle panels) and bar graphs (right panels) show the distribution of mNK (NK1.1+CD3CD19 CD49b+) cells based on CD27 and CD11b expression in the spleen (upper panels) and liver (lower panels). Stages 1–4 are as follows: stage 1(CD27CD11b), stage 2 (CD27+CD11b), stage 3 (CD27+CD11b), and stage 4 (CD27CD11b+). Experiments were repeated at least three times. n = 3–5 mice/group/experiment. Data are means ± SEM. **p < 0.01, ***p < 0.001.

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The acquisition of CD49b marks the developmental step from iNK to mNK cells (21, 37). We found that the frequency of CD49b (immature) NK cells in Cardif−/− spleen and liver is significantly higher than in WT NK cells (Fig. 1B). This 2-fold increase of CD49b NK cells in the spleens and livers of Cardif−/− mice suggests a delay in mNK development and maintenance in the periphery.

In addition to a reduction in mNK cells (NK1.1+CD3CD19 CD49b+), we observed a significant reduction in terminally differentiated CD27CD11b+ NK cells in the spleen and liver of Cardif−/− mice (Fig. 1C). This reduction of CD27CD11b+ NK cells is accompanied by a concomitant increase in frequencies of CD27+CD11b and CD27+CD11b+ NK cells. Cardif−/− mice have a 35–40% loss of terminally differentiated mature CD27CD11b+ NK cells with a concomitant increase in less mature (CD27+CD11b and CD27+CD11b+) NK cells in the spleen. In addition, there is a >50% decrease in terminally differentiated CD27CD11b+ NK cells and a 50% increase in CD27+CD11b+ NK cells in liver of Cardif−/− mice (Fig. 1C). NK cells in the blood and lymph nodes of Cardif−/− mice displayed similar alterations in NK maturation (data not shown). However, we found no differences in NK maturation in the bone marrow (Supplemental Fig. 2). Taken together, these data suggest that Cardif is important for NK maturation but does not impair NK development in bone marrow.

We next wanted to examine homeostatic NK function in the absence of Cardif signaling. Ly49 receptor expression on NK cells is required for NK cell “licensing,” the process that allows NK cells to become active cytolytic cells after their encounter with self MHC-1–expressing cells (3840). The expression of the inhibitory receptors such as Ly49C/I and KLRG1 on NK cells is associated with NK cell maturity (23, 25). We investigated the expression of KLRG1 and select Ly49 receptors in Cardif−/− mice. We found that significantly fewer Cardif−/− mNK (NK1.1+CD3CD19CD49b+) cells express KLRG1 (Fig. 2A, 2B) (21). Moreover, there is a decrease in the surface expression of KLRG1 on Cardif−/− mNK cells (Fig. 2B). We found that WT and Cardif−/− mNK cells express similar amounts of the activating Ly49H receptor and the inhibitory Ly49G2 receptor (Fig. 2A, 2B). However, the number of Ly49C/I+ mNK cells is markedly decreased in Cardif−/− mice. Likewise, the surface expression of Ly49C/I on Cardif−/− mNK cells is significantly lower than WT mNK cells. The decrease in the frequency and cellular expression of KLRG1 and Ly49C/I, which are associated with NK cell activation and maturation, suggest that Cardif is involved in late mNK cell maturation (18, 41).

FIGURE 2.

Cardif−/− NK cells are less mature. (A) Representative histograms show the percentages of KLRG1+ cells, and cells expressing select activating and inhibitory Ly49 receptors in WT and Cardif−/− mNK populations (NK1.1+CD3CD19 CD49b+). (B) Bar graphs depict the percentage (left panel) and amount of expression (right panel) of NK maturation markers and Ly49 receptors. Amount of expression was determined by flow-cytometric analysis of mean fluorescence intensity (MFI). Experiments were repeated twice. n = 3 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01.

FIGURE 2.

Cardif−/− NK cells are less mature. (A) Representative histograms show the percentages of KLRG1+ cells, and cells expressing select activating and inhibitory Ly49 receptors in WT and Cardif−/− mNK populations (NK1.1+CD3CD19 CD49b+). (B) Bar graphs depict the percentage (left panel) and amount of expression (right panel) of NK maturation markers and Ly49 receptors. Amount of expression was determined by flow-cytometric analysis of mean fluorescence intensity (MFI). Experiments were repeated twice. n = 3 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01.

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To identify mechanistically why there are fewer mNK cells in Cardif−/− mice, we investigated the survival and proliferation of NK cells in Cardif−/− mice. We found that more Cardif−/− NK cells were caspase-3+, suggesting that they were more apoptotic (Fig. 3A). We confirmed the increase in apoptosis using Annexin V, because we found more Annexin V+ NK cells in Cardif−/− mice (Fig. 3A). The greatest increase in apoptotic mNK cells was observed in terminally differentiated CD27CD11b+ NK cells, where 2-fold more CD27CD11b+ Cardif−/− NK cells were apoptotic as noted by caspase-3 expression (Fig. 3B). This increase of caspase-3+ CD27CD11b+ NK cells in Cardif−/− mice accounts for the increase in frequencies of total caspase-3+ NK cells. These data suggest that Cardif−/− NK cells, particularly terminally differentiated CD27CD11b+ NK cells, are more apoptotic in the absence of tonic Cardif signaling. We used Ki-67 to identify proliferating NK cells in Cardif−/− and WT mice. We found an ∼15% decrease in proliferation of total splenic Cardif−/− NK cells compared with WT NK cells (Fig. 3C). However, upon further analysis, we found that CD27+CD11b and CD27+CD11b+ Cardif−/− NK cells have an ∼25 and ∼30% reduction in proliferation, respectively. These two stages of NK cells are known to mature into CD27CD11b+ NK cells (22, 23). Thus, in the absence of Cardif, early-stage NK cells proliferate less whereas terminally differentiated CD27CD11b+ NK cells are prone to apoptosis. These data support our findings that Cardif−/− mice have fewer total NK cells and increased numbers of early-stage NK cells.

FIGURE 3.

Cardif−/− NK cells are more apoptotic and less proliferative. (A) Bar graphs show the percentage of Caspase-3+ (upper panel) and Annexin V+ (lower panel) NK cells (NK1.1+CD3CD19) from WT and Cardif−/− mice. (B) Representative plots and bar graph of the percentage of WT and Cardif−/− caspase-3+ CD27CD11b+ NK cells. (C) Bar graphs depict the percentage of Ki-67+ total NK cells (left panel), as well as stage 1–4 NK cells (middle and right panels). Experiments were repeated twice. n = 4–5 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Cardif−/− NK cells are more apoptotic and less proliferative. (A) Bar graphs show the percentage of Caspase-3+ (upper panel) and Annexin V+ (lower panel) NK cells (NK1.1+CD3CD19) from WT and Cardif−/− mice. (B) Representative plots and bar graph of the percentage of WT and Cardif−/− caspase-3+ CD27CD11b+ NK cells. (C) Bar graphs depict the percentage of Ki-67+ total NK cells (left panel), as well as stage 1–4 NK cells (middle and right panels). Experiments were repeated twice. n = 4–5 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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Because we found that NK cells in Cardif−/− mice are less mature and fewer are Ly49C/I+, we hypothesized that Cardif-deficient NK cells may not function as well as WT NK cells. Fernandez et al. (42) have previously shown that unlicensed Ly49C/I NK cells are less cytotoxic than Ly49C/I+ NK cells. We stimulated WT and NK Cardif−/− NK cells with either IL-12 and IL-18 or PMA and ionomycin ex vivo. We found that granzyme B and CD107a, a marker of degranulation, were significantly reduced in Cardif−/− NK cells compared with WT NK cells, suggesting that Cardif−/− NK cells are likely impaired in their ability to kill target cells (Fig. 4A). The production of IFN-γ, however, was comparable between NK cells from both WT and Cardif−/− mice. Next, we tested the ability of Cardif−/− NK cells to directly kill target cells in vitro. YAC-1 cells served as target cells in the assay. Cardif−/− NK cells had ∼20–25% less cytotoxic activity than WT NK cells when cultured with target B cells (Fig. 4B). These data indicate that Cardif plays an important role in manipulating the functions of NK cells.

FIGURE 4.

Cardif−/− NK cells are less cytotoxic than WT mNK cells. (A) Representative plots and bar graphs show the percentage of CD107a+ (upper panels), granzyme B+ (middle panels), and IFN-γ+ (lower panels) mNK (NK1.1+CD3CD19 CD49b+) cells in WT and Cardif−/− samples. (B) Representative line graph depicting the cytotoxic activity of WT and Cardif−/− NK cells against target YAC-1 cells at various ratios. Ratios are NK/target cells. Experiments were repeated at least twice. n = 3–5 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01.

FIGURE 4.

Cardif−/− NK cells are less cytotoxic than WT mNK cells. (A) Representative plots and bar graphs show the percentage of CD107a+ (upper panels), granzyme B+ (middle panels), and IFN-γ+ (lower panels) mNK (NK1.1+CD3CD19 CD49b+) cells in WT and Cardif−/− samples. (B) Representative line graph depicting the cytotoxic activity of WT and Cardif−/− NK cells against target YAC-1 cells at various ratios. Ratios are NK/target cells. Experiments were repeated at least twice. n = 3–5 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01.

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Reports have shown that the maintenance of STAT1 levels in NK cells is important for NK cell cytotoxicity upon activation, whereas the levels of STAT4 are important for IFN-γ production (31). In earlier experiments, we found that Cardif−/− NK cells are less cytotoxic than WT NK cells, whereas IFN-γ production was the same (Fig. 4A, 4B). With these observations, we decided to measure STAT1 and STAT4 phosphorylation in NK cells and found that there were dramatic reductions in both the phosphorylation and the levels of STAT1 protein in Cardif−/− NK cells (Fig. 5A). STAT4 protein and phosphorylation seemed similar between Cardif−/− and WT NK cells, but we found that the p-STAT4/STAT4 ratio was higher in Cardif−/− NK cells (Fig. 5B). Based on a known role for STAT1 in regulating NK cytolytic functions, these data indicate that the reduced cytolytic function of Cardif−/− NK cells is most likely due to the low intracellular amount of STAT1 and phosphorylated. The effects of an increase in the p-STAT4/STAT4 ratio in Cardif−/− NK cells was most likely masked because of the already high amounts of STAT4 present in WT NK cells.

FIGURE 5.

Cardif−/− mNK cells have decreased levels of STAT1 and p-STAT1. (A) Western blot shows the amount of total STAT1 (top panel), p-STAT1 (second panel), STAT4 (fourth panel), and p-STAT4 (bottom panel) protein in WT (left) and Cardif−/− (right) mNK cells. Cells were treated with PBS or 100 U/ml IFN-β. Afterward, 30 μg protein from cell lysates was loaded per well. β-Actin was used to normalize protein data. (B) pSTAT1/STAT1 and p-STAT4/STAT4 ratios derived Western blot quantification. Experiments were repeated twice. n = 4 mice/group/experiment. Data are means ± SEM.

FIGURE 5.

Cardif−/− mNK cells have decreased levels of STAT1 and p-STAT1. (A) Western blot shows the amount of total STAT1 (top panel), p-STAT1 (second panel), STAT4 (fourth panel), and p-STAT4 (bottom panel) protein in WT (left) and Cardif−/− (right) mNK cells. Cells were treated with PBS or 100 U/ml IFN-β. Afterward, 30 μg protein from cell lysates was loaded per well. β-Actin was used to normalize protein data. (B) pSTAT1/STAT1 and p-STAT4/STAT4 ratios derived Western blot quantification. Experiments were repeated twice. n = 4 mice/group/experiment. Data are means ± SEM.

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To determine whether the impact of Cardif on NK cell maturation is cell intrinsic, we used a mixed chimera approach. WT CD45.1+ bone marrow and CD45.2+Cardif−/− bone marrow were mixed 1:1 and transplanted into irradiated CD45.1+/CD45.2+ recipient mice (Fig. 6A). After 10 wk of reconstitution, we assessed the phenotype of NK cells in various organs. We found that CD45.1+ and Cardif−/− cells contributed equally to the total number of CD45+ cells in the reconstituted bone marrow (Fig. 6B). However, there were slight but consistently lower percentages of Cardif−/− origin NK (CD45.1CD45.2+NK1.1+CD3CD19) cells in the bone marrow of chimeric mice compared with WT CD45.1 origin NK cells (data not shown). We found that this NK cell–specific phenotype was more pronounced in the periphery (Fig. 6C). We found that Cardif−/− NK cells reconstituted only ∼40%, whereas CD45.1+ NK cells reconstituted ∼60% of total NK cells in the spleen and liver (Fig. 6D). We also observed a slight decrease of the CD27CD11b+ subset within Cardif−/− NK cells in the spleen and liver, similar to what we observed in the global knockout mice (Figs. 1C, 6E). The decrease in Cardif−/− CD27CD11b+ NK cells was accompanied by an increase in CD27+CD11b Cardif−/− NK cells in both the spleen and the liver of the mixed bone marrow chimeric mice (Fig. 6E).

FIGURE 6.

Cardif−/− NK cells show maturation defect in 1:1 bone marrow chimeric mice. (A) Schematic diagram of method used to create 1:1 mixed bone marrow chimeric mice. Cardif−/− (CD45.2) and CD45.1 bone marrow were i.v. injected into CD45.1/0.2 recipient mice in a 1:1 ratio. Mice were harvested 10 wk after injection. (B) Representative plot depicting equal amounts of WT and Cardif−/− CD45+ cells in the bone marrow. (C) Representative plots that show the ratios of CD45.1 and Cardif−/− cells in the liver in specific populations: live cells → CD3CD19 → NK1.1+CD3CD19 → NK1.1+CD3CD19 → NK1.1+CD3CD19CD27CD11b+. (D) Representative bar graphs show the contribution of cells from CD45.1 and Cardif−/− origins to total NK1.1+CD3CD19 cells in spleen and liver. Bar graphs (right panels) depict the distribution of WT (CD45.1) and Cardif−/− NK cells based on the four maturation stages, denoted by CD27 and CD11b in the spleen and liver of 1:1 mixed bone marrow chimeric mice. (E) Bar graphs depict the production of CD107a (left panel) and granzyme B (right panel) by NK cells. (F) Bar graphs depict the percentage of Caspase-3+ NK cells. Representative plots and bar graph depict the percentage of Caspase-3+ CD27CD11b+ NK cells (middle and right panels). (G) Bar graphs depict the percentage of Ki-67+ NK cells. Experiments were performed twice (A–E) or once (F and G). n = 6 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

Cardif−/− NK cells show maturation defect in 1:1 bone marrow chimeric mice. (A) Schematic diagram of method used to create 1:1 mixed bone marrow chimeric mice. Cardif−/− (CD45.2) and CD45.1 bone marrow were i.v. injected into CD45.1/0.2 recipient mice in a 1:1 ratio. Mice were harvested 10 wk after injection. (B) Representative plot depicting equal amounts of WT and Cardif−/− CD45+ cells in the bone marrow. (C) Representative plots that show the ratios of CD45.1 and Cardif−/− cells in the liver in specific populations: live cells → CD3CD19 → NK1.1+CD3CD19 → NK1.1+CD3CD19 → NK1.1+CD3CD19CD27CD11b+. (D) Representative bar graphs show the contribution of cells from CD45.1 and Cardif−/− origins to total NK1.1+CD3CD19 cells in spleen and liver. Bar graphs (right panels) depict the distribution of WT (CD45.1) and Cardif−/− NK cells based on the four maturation stages, denoted by CD27 and CD11b in the spleen and liver of 1:1 mixed bone marrow chimeric mice. (E) Bar graphs depict the production of CD107a (left panel) and granzyme B (right panel) by NK cells. (F) Bar graphs depict the percentage of Caspase-3+ NK cells. Representative plots and bar graph depict the percentage of Caspase-3+ CD27CD11b+ NK cells (middle and right panels). (G) Bar graphs depict the percentage of Ki-67+ NK cells. Experiments were performed twice (A–E) or once (F and G). n = 6 mice/group/experiment. Data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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We also investigated the function of Cardif−/− NK cells in the chimeric mice. We found that although Cardif−/− NK cells and WT NK cells displayed a similar amount of degranulation, granzyme B production was lower in Cardif−/− NK cells (Fig. 6F). In addition, we found that more Cardif−/− NK cells are caspase-3+ compared with WT NK cells in chimeric mice (Fig. 6G). Similar to global knockout mice, CD27CD11b+ Cardif−/− NK cells in chimeric mice have the greatest increase in apoptosis (Fig. 6G). We examined proliferation of WT and Cardif−/− NK cells in chimeric mice using Ki-67 and found that both WT and Cardif−/− NK cells proliferated at the same rate (Fig. 6H).

Signaling via Cardif leads to the production of type I IFN. We, along with other groups, have found that similar to Cardif−/− mice, IFNAR1−/− mice have fewer terminally differentiated NK cells and abrogated functionality in the absence of type I IFN signaling (27, 28, 43, 44) (Supplemental Fig. 3). Gough et al. (30) have theorized that low homeostatic levels of type I IFN are required in the absence of infection to sustain levels of STAT proteins in the cytoplasm of various immune cells and consequently maintain normal cell function upon activation. Thus, we hypothesized that IFN-β signaling was likely impaired in response to Cardif deletion because Cardif−/− NK cells have low intracellular levels of STAT1 (Fig. 3). In an attempt to rescue NK maturation, we treated WT and Cardif−/− NK cells with IFN-β in vitro. Similar to Brady et al. (45), we noticed drastically reduced levels of CD11b expression on NK cells in culture. This made it difficult to confidently identify the maturation stages of NK cells. We did, however, measure total expression of CD11b and KLRG1 in the cultured NK cells. Both CD11b and KLRG1 expression significantly increased with IFN-β treatment in WT and Cardif−/− NK cells (Supplemental Fig. 3). Still, CD11b and KLRG1 levels were not restored to those of WT with IFN-β stimulation.

NK cells are a key component of the host innate response required to control MCMV (46), which is a commonly used viral model for assessing NK effector function. MCMV replication in Cardif−/− mice was controlled ∼10-fold better than that seen in WT mice at day 4 postinfection (Fig. 7A). However, infection with an MCMV mutant lacking the viral m157 protein (detected by Ly49h-expressing NK cells) resulted in no difference in viral control between Cardif−/− and WT mice (Fig. 7A) (36, 4749). In addition, depletion of NK cells before MCMV infection also normalized viral replication to WT levels in Cardif−/− mice (Supplemental Fig. 4). These data indicate the better control of MCMV seen in Cardif−/− mice is due to enhanced NK-mediated effector function(s). We have previously shown that a proportion of splenic NK cells express IFN-γ at ∼12 h post MCMV infection in response to the first wave of type I IFN produced, and this is a key time point regulating antiviral control in subsequent days (36, 50). Maslowski et al. (51) have also described an early production of IFN-γ by NK cells that is STAT4- and type I IFN–dependent during LCMV infection. Consequently, we specifically chose to measure NK IFN-γ production at 12 h after MCMV infection because type I IFN produced by infected splenic stromal cells is the primary cytokine driving this at this early time point, as opposed to plasmacytoid DC–produced IL-12, which is the primary inducer of NK IFN-γ at 36 h (36). We hypothesized that the STAT1/STAT4 ratio present in Cardif−/− NK cells might result in enhanced IFN-γ production by NK cells at 12 h after MCMV infection, potentially accounting for the observed increase in antiviral defense. Indeed, at 12 h postinfection, ∼4-fold higher numbers of NK cells in Cardif−/− mice produced IFN-γ than was seen in WT mice (Fig. 7B). All Cardif−/− NK cell subsets produced more IFN-γ+ when compared with their WT counterparts; however, the highest percentage of IFN-γ+cells was seen in the less mature CD27+CD11b+ subset as compared with the more mature CD27CD11b+ NK cells. In addition to the CD27+CD11b+ NK subset containing the highest percentage of IFN-γ+ cells in MCMV-infected WT and Cardif−/− spleens, Cardif−/− spleens also contained nearly twice the normal proportion of these cells (Fig. 7B). These data support a model where increased numbers of CD27+CD11b+ NK cells in Cardif−/− mice, as well as enhanced STAT1/STAT4 ratios, are largely responsible for the increased IFN-γ production observed in their NK cell compartment in response to initial MCMV infection, and likely underlie the enhanced control of MCMV infection in spleens of Cardif-deficient mice.

FIGURE 7.

Cardif−/− mice show heightened NK activation and enhanced control of MCMV infection. (A) WT and Δm157 MCMV replication levels in spleens of day 4 infected WT and Cardif−/− mice. (B) Representative plots of IFN-γ production by NK cells at 12 h in MCMV-infected mice (upper panels), and bar graphs quantifying this in multiple mice and in NK cell subsets (lower panels). Bar graph portraying the percentage of IFN-γ+ cells within NK cell subsets in the spleens of mice 12 h postinfection (lower right panel). n ≥ 3 mice/group. Experiments were performed twice with at least three mice/group. (A) Combined data from two separate experiments, with (B) representative of a single experiment. Data are means ± SEM. *p < 0.05, ***p < 0.001.

FIGURE 7.

Cardif−/− mice show heightened NK activation and enhanced control of MCMV infection. (A) WT and Δm157 MCMV replication levels in spleens of day 4 infected WT and Cardif−/− mice. (B) Representative plots of IFN-γ production by NK cells at 12 h in MCMV-infected mice (upper panels), and bar graphs quantifying this in multiple mice and in NK cell subsets (lower panels). Bar graph portraying the percentage of IFN-γ+ cells within NK cell subsets in the spleens of mice 12 h postinfection (lower right panel). n ≥ 3 mice/group. Experiments were performed twice with at least three mice/group. (A) Combined data from two separate experiments, with (B) representative of a single experiment. Data are means ± SEM. *p < 0.05, ***p < 0.001.

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We have discovered a novel intrinsic role for Cardif in the maturation and function of NK cells. It is widely accepted that Cardif is vital for antiviral signaling in response to RIG-I and MDA5 activation; however, several studies indicate that Cardif and other components in this pathway have relatively unexplored nonviral functions (1316). Cardif has been shown to reduce the severity of experimental autoimmune encephalomyelitis in mice, activate the NLRP3 inflammasome, and regulate TLR7 expression in B cells. However, currently, no reports describe the requirement for, or involvement of, Cardif in NK cell development. In this report, we describe a novel and cell-intrinsic role for Cardif in the homeostatic maturation and function of NK cells.

We discovered that Cardif mice have fewer splenic NK cells when compared with WT mice, and Cardif−/− NK cells are ∼40–45% more likely to lack CD49b, a marker of mNK cells. Splenic and liver Cardif−/− NK cells populations also have ∼30 and ∼60% fewer terminally differentiated NK cells, respectively. Markers associated with NK maturation, Ly49C/I and KLRG1, are also lower in Cardif−/− NK cells. Functionally, we found that Cardif−/− NK cells had lower cytotoxic activity, because Cardif−/− NK cells did not degranulate nor produce as much granzyme B, nor kill target cells and WT NK cells. However, we observed no changes in IFN-γ production in Cardif-deficient NK cells. We did not measure production of other cytokines by Cardif NK cells in this study. Because STAT1 has been shown to regulate the cytotoxicity functions of NK cells, whereas STAT4 governs IFN-γ production by NK cells, we measured total STAT protein and protein phosphorylation. STAT1 protein levels and phosphorylation were considerably reduced in Cardif−/− NK cells. In contrast, p-STAT4/STAT4 protein levels were higher in Cardif−/− NK cells. These data suggest that the diminished cytotoxicity of Cardif−/− NK cells is due to the reduced intracellular levels of STAT1. Although there is an increase of p-STAT4/STAT4 in Cardif−/− NK cells, this increase may not have translated into increased levels of IFN-γ in our ex vivo stimulation assays because the already high overall STAT4 levels in WT NK cells is saturating. However, the altered STAT1/STAT4 ratio could have more dramatic consequences in the context of NK effector function in vivo, as appears to be the case from our studies with MCMV.

Cardif likely plays a role in stabilizing NK cells and preventing their apoptosis. A recent report indicated that Cardif associates with caspase-8 to induce apoptosis via caspase-3 in response to viral infection (52). Our data show that Cardif deficiency increases apoptosis in NK cells, particularly in terminally differentiated CD27CD11b+ NK cells. Moreover, our data suggest that the presence of Cardif decreases NK cell proliferation, predominantly in CD27+CD11b and CD27+CD11b+ NK cells. Cardif’s apparent role in apoptosis and proliferation in NK cells explains much of the immature phenotype seen in NK cells from Cardif−/− mice (Fig. 1). The lack of proliferation in CD27+CD11b and CD27+CD11b+ NK cells suggests a lack of differentiation into terminally differentiated stage 4 NK cells. Together our data suggest that Cardif−/− mice have fewer total NK cells, with a lower percentage of terminally differentiated CD27CD11b+ NK cells, and this is likely caused by an increase in CD27CD11b+ NK cell death and a decrease in proliferation of early-stage CD27+CD11b and CD27+CD11b+ NK cells. NF-κB is a major downstream target of Cardif signaling along with type I IFN production (5, 6). NF-κB is known to be involved in cell survival in multiple cell types, including B and T cells (53). Yet, little is known about NF-κB activity in NK cells. A few reports suggest that changes in NF-κB signaling regulate NK cell proliferation, Ly49 expression, and increased apoptosis (5355). NF-κB signaling is tightly regulated in NK cells and changes in NF-κB activity are likely to perturb NK cell survival, and we surmise that the absence of a Cardif–NF-κB signaling axis may contribute to the differences observed in NK cell survival.

Our mixed bone marrow chimera experiments revealed that Cardif is intrinsically required for optimal terminal differentiation of NK cells. Moreover, Cardif is intrinsically required for the fitness and survival of NK cells in peripheral organs, because Cardif−/− NK cell numbers were lower than WT in the periphery of the mixed chimeric mice, and CD27CD11b+ terminally differentiated NK cells were nearly twice as likely to be derived from WT mice in this mixed chimera scenario. We also found that increased apoptosis and decreased granzyme B levels in Cardif−/− NK cells are due to cell-intrinsic deficiencies. The decreased proliferation and increased degranulation that was observed in Cardif−/− mice, however, is not cell intrinsic.

We initially thought that the cell-extrinsic factor acting upon Cardif−/− NK cells was IFN-β; however, we found that treatment with IFN-β in vitro, although somewhat effective, is not able to completely rescue the maturation phenotype that is present in Cardif−/− NK cells. Type I IFN is known to indirectly affect the differentiation of NK cells by upregulating the amount of IL-15 and IL-15Rα present on DCs (56, 57). IL-15 transpresentation by DCs has been shown to have a dose-dependent effect on the development, maturation, and proliferation of NK and NKT cells (5860). IL-15 transpresentation is required for the expression of NK1.1 on NKT cells in the thymus. We have found that fewer NKT cells in the thymus express NK1.1 in young Cardif−/− mice, which leads us to believe that there may be a perturbation of “basal” type I IFN production in Cardif−/− mice that leads to lower amounts of IL-15 transpresentation (data not shown). We also found that Cardif−/− DCs express lower amounts of IL-15/IL-15Rα complexes when stimulated with TLR9 agonists (data not shown). The involvement of IL-15 transpresentation in the NK phenotype of Cardif−/− mice is also supported by results that the proliferation and cytotoxicity of Cardif−/− NK cells is normal in WT hosts, two effector functions that are mediated by IL-15 transpresentation. Thus, Cardif appears to regulate the maturation and function of NK cells by both direct and indirect mechanisms. Further studies to delineate the role of type I IFNs in the terminal differentiation of NK cells in the absence of Cardif and other RIG-I molecules will be of interest, particularly in the context of viral immunity and autoimmune diseases.

MCMV infection was used as another model to test the function of Cardif−/− NK cells, because these cells are key for innate defense to this virus. Somewhat surprisingly, we found that Cardif−/− mice controlled MCMV replication better than WT mice at times of peak acute infection, and this enhanced control in the absence of Cardif was shown to be NK dependent by both depleting NK cells and using an MCMV∆m157 mutant virus (4749). This amplified innate antiviral defense was consistent with an increased percentage of IFN-γ+ NK cells in the spleen seen at 12 h of infection, a time point when innate defense to MCMV is “kick-started” and the baseline for MCMV replication is being established (61). This initial IFN-γ production by NK cells occurs in response to type I IFN produced by MCMV-infected marginal zone stromal cells in the spleen, a cell type for which MCMV shows specific tropism because of its differentiation by constitutive lymphotoxin-αβ signaling (36, 50). Data from Mack et al. (32) also show that very early after LCMV infection, a short burst of IFN-γ is produced by NK cells via the type I IFN pathway, independent of IL-12. In turn, Döring et al. (62) have shown that Cardif−/− plasmacytoid DCs, and macrophages produce similar levels of IFN-α in response to MCMV, cells that produce the second wave of innate cytokines in the spleen in response to MCMV generated from the infected marginal zone stroma (61). Together, this suggests that instead of type I IFN being altered in MCMV-infected Cardif−/− mice, which is perhaps not surprising because MCMV is a dsDNA virus, that Cardif−/− NK cells are more highly subject to activation by these key innate cytokines. Hayakawa et al. (22) found that CD27+CD11b+ NK cells produced more IFN-γ than CD27CD11b+ NK cells in response to cytokines and DC cross talk. We also found higher IFN-γ production by CD27+CD11b+ NK cells in response to MCMV, accounting for a substantial overall increase in NK cell IFN-γ seen in Cardif−/− mice. This suggests that the CD27CD11b+ NK subset may preferentially contribute to MCMV control in some contexts, although this needs to be tested more directly. In addition, the prevalence of more unlicensed, Ly49C/I NK cells in Cardif−/− mice may also contribute to the observed enhanced control of MCMV, because these cells are activated to a higher extent in the response to this virus because of fewer inhibitory mechanisms (63, 64). In contrast, these unlicensed Ly49C/I NK cells are not as functional as licensed NK cells in contexts other than viral infection, such as tumor immunosurveillance, contributing to the reduced cytotoxicity of Cardif−/− NK cells seen in vitro (39). Also, lower STAT1/STAT4 ratios might prolong IFN-γ production by Cardif−/− NK, because it could take longer to induce high enough STAT1 levels in response to type I IFN to downregulate STAT4.

Increased Cardif NK cell activation in the context of MCMV infection may be indicative of a potentially broader role of Cardif in regulating autoimmunity. Indeed, Cardif has recently been associated with incidence of systemic lupus erythematosus (SLE). Pothlichet et al. (65) described a loss-of-function mitochondrial antiviral signaling (Cardif) variant in humans that is associated with SLE in African Americans. Patients with this variant were characterized by low type I IFN levels and a lack of autoantibodies specific for RNA-binding protein. Similarly, Molineros et al. (66) described a risk allele of IFIH1 (MDA5) in African Americans that is associated with an increased risk for SLE with downregulation of type I IFN signaling. Interestingly, SLE patients have reduced absolute numbers and a reduction in the cytolytic activity of NK cells, similar to the phenotype observed in Cardif−/− mice (6771). Furthermore, just as in Cardif−/− mice, the NK cells of SLE patients have an increase in the number of iNK cells and a concomitant decrease of mature cytotoxic NK cells. Future studies should investigate Cardif signaling in NK cells in the context of SLE to further delineate the effect of Cardif on NK cells and SLE pathogenesis.

Our data suggest that Cardif negatively regulates NK cell maturation and function in the absence of viral infection but may result in their hyperreactivity in the context of pathogen infection. Several other publications have suggested a similar “dual role” for Cardif; however, clear mechanisms have not been established (7, 15, 16, 65). With regard to the role of Cardif in regulating immune cell homeostasis, tonic signaling through the Rig-I/MDA5–Cardif axis triggered by endogenous agonists may play a role. There have been reports of endogenous agonists of Rig-I such as the small self-RNAs produced by RNase-L, IRE1α, and endogenous retroviral elements in the human genome (7277). Recently, Dupuis-Maurin et al. (78) discovered that overexpression of the transcription factor specificity protein 1 activates the Rig-I–Cardif pathway in the absence of a pathogen by stimulating the 2′-5′-oligoadenylate synthetase 1–RNase-L pathway to produce small self-RNAs. Tonic Rig-I/MDA5–Cardif signaling activity stimulated by specificity protein 1 or other transcription factors may lead to low-level production of IFN-β and NF-κB activation. It is also possible that Cardif is involved in other signaling cascades rather than the RIG-I/MDA5–Cardif axis, because Subramanian et al. (14) recently reported the activity of Cardif in the NLRP3 inflammasome.

We thank Dr. Alec Redwood (Murdoch University) for providing the MCMV strain. We thank Amy Blatchley and Deborah Yoakum at La Jolla Institute for Allergy and Immunology for assistance with mice and the Imaging Facility at La Jolla Institute for Allergy and Immunology for excellent technical assistance with flow cytometry.

This work was supported by the National Institutes of Health (Grant F31 HL110668 to L.D.H., Grant R01 HL097368 to C.C.H., Grant F32HL117533 to H.N.N., and Grant R01 AI101423 to C.A.B.), American Heart Association Grant 13POST16990029 (to R.T.), and the American Diabetes Association (Award 1-13-MUI-02 to L.D.H. and C.C.H.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

CARD

caspase recruitment and activation domain

Cardif

CARD adaptor inducing IFN-β

DC

dendritic cell

iNK

immature NK

mNK

mature NK

MCMV

mouse CMV

RIG-I

retinoic acid–inducible gene I

SLE

systemic lupus erythematosus

WT

wild-type.

1
Takeuchi
O.
,
Akira
S.
.
2010
.
Pattern recognition receptors and inflammation.
Cell
140
:
805
820
.
2
Ireton
R. C.
,
Gale
M.
 Jr.
2011
.
RIG-I like receptors in antiviral immunity and therapeutic applications.
Viruses
3
:
906
919
.
3
Eisenächer
K.
,
Krug
A.
.
2012
.
Regulation of RLR-mediated innate immune signaling--it is all about keeping the balance.
Eur. J. Cell Biol.
91
:
36
47
.
4
Meylan
E.
,
Curran
J.
,
Hofmann
K.
,
Moradpour
D.
,
Binder
M.
,
Bartenschlager
R.
,
Tschopp
J.
.
2005
.
Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.
Nature
437
:
1167
1172
.
5
Seth
R. B.
,
Sun
L.
,
Ea
C.-K.
,
Chen
Z. J.
.
2005
.
Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.
Cell
122
:
669
682
.
6
Kawai
T.
,
Takahashi
K.
,
Sato
S.
,
Coban
C.
,
Kumar
H.
,
Kato
H.
,
Ishii
K. J.
,
Takeuchi
O.
,
Akira
S.
.
2005
.
IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.
Nat. Immunol.
6
:
981
988
.
7
Xu
L.-G.
,
Wang
Y.-Y.
,
Han
K.-J.
,
Li
L.-Y.
,
Zhai
Z.
,
Shu
H.-B.
.
2005
.
VISA is an adapter protein required for virus-triggered IFN-beta signaling.
Mol. Cell
19
:
727
740
.
8
Kawai
T.
,
Akira
S.
.
2006
.
TLR signaling.
Cell Death Differ.
13
:
816
825
.
9
Takamatsu
S.
,
Onoguchi
K.
,
Onomoto
K.
,
Narita
R.
,
Takahasi
K.
,
Ishidate
F.
,
Fujiwara
T. K.
,
Yoneyama
M.
,
Kato
H.
,
Fujita
T.
.
2013
.
Functional characterization of domains of IPS-1 using an inducible oligomerization system.
PLoS One
8
:
e53578
.
10
Loo
Y.-M.
,
Gale
M.
 Jr.
2011
.
Immune signaling by RIG-I-like receptors.
Immunity
34
:
680
692
.
11
Li
X. D.
,
Chiu
Y. H.
,
Ismail
A. S.
,
Behrendt
C. L.
,
Wight-Carter
M.
,
Hooper
L. V.
,
Chen
Z. J.
.
2011
.
Mitochondrial antiviral signaling protein (MAVS) monitors commensal bacteria and induces an immune response that prevents experimental colitis.
Proc. Natl. Acad. Sci. USA
108
:
17390
17395
.
12
Wang
Y.
,
Zhang
H.-X.
,
Sun
Y.-P.
,
Liu
Z.-X.
,
Liu
X.-S.
,
Wang
L.
,
Lu
S.-Y.
,
Kong
H.
,
Liu
Q.-L.
,
Li
X.-H.
, et al
.
2007
.
Rig-I-/- mice develop colitis associated with downregulation of G alpha i2.
Cell Res.
17
:
858
868
.
13
Xu
L.-G.
,
Jin
L.
,
Zhang
B.-C.
,
Akerlund
L. J.
,
Shu
H.-B.
,
Cambier
J. C.
.
2012
.
VISA is required for B cell expression of TLR7.
J. Immunol.
188
:
248
258
.
14
Subramanian
N.
,
Natarajan
K.
,
Clatworthy
M. R.
,
Wang
Z.
,
Germain
R. N.
.
2013
.
The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation.
Cell
153
:
348
361
.
15
Wang
F.
,
Xia
W.
,
Liu
F.
,
Li
J.
,
Wang
G.
,
Gu
J.
.
2012
.
Interferon regulator factor 1/retinoic inducible gene I (IRF1/RIG-I) axis mediates 25-hydroxycholesterol-induced interleukin-8 production in atherosclerosis.
Cardiovasc. Res.
93
:
190
199
.
16
Dann
A.
,
Poeck
H.
,
Croxford
A. L.
,
Gaupp
S.
,
Kierdorf
K.
,
Knust
M.
,
Pfeifer
D.
,
Maihoefer
C.
,
Endres
S.
,
Kalinke
U.
, et al
.
2012
.
Cytosolic RIG-I-like helicases act as negative regulators of sterile inflammation in the CNS.
Nat. Neurosci.
15
:
98
106
.
17
Vivier, E., E. Tomasello, M. Baratin, T. Walzer, and S. Ugolini. 2008. Functions of natural killer cells. Nat. Immunol. 9: 503–510
.
18
Gordon
S. M.
,
Chaix
J.
,
Rupp
L. J.
,
Wu
J.
,
Madera
S.
,
Sun
J. C.
,
Lindsten
T.
,
Reiner
S. L.
.
2012
.
The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation.
Immunity
36
:
55
67
.
19
Daussy
C.
,
Faure
F.
,
Mayol
K.
,
Viel
S.
,
Gasteiger
G.
,
Charrier
E.
,
Bienvenu
J.
,
Henry
T.
,
Debien
E.
,
Hasan
U. A.
, et al
.
2014
.
T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow.
J. Exp. Med.
211
:
563
577
.
20
Fathman
J. W.
,
Bhattacharya
D.
,
Inlay
M. A.
,
Seita
J.
,
Karsunky
H.
,
Weissman
I. L.
.
2011
.
Identification of the earliest natural killer cell-committed progenitor in murine bone marrow.
Blood
118
:
5439
5447
.
21
De Colvenaer
V.
,
Taveirne
S.
,
Delforche
M.
,
De Smedt
M.
,
Vandekerckhove
B.
,
Taghon
T.
,
Boon
L.
,
Plum
J.
,
Leclercq
G.
.
2011
.
CD27-deficient mice show normal NK-cell differentiation but impaired function upon stimulation.
Immunol. Cell Biol.
89
:
803
811
.
22
Hayakawa
Y.
,
Smyth
M. J.
.
2006
.
CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity.
J. Immunol.
176
:
1517
1524
.
23
Chiossone
L.
,
Chaix
J.
,
Fuseri
N.
,
Roth
C.
,
Vivier
E.
,
Walzer
T.
.
2009
.
Maturation of mouse NK cells is a 4-stage developmental program.
Blood
113
:
5488
5496
.
24
Fu
B.
,
Wang
F.
,
Sun
R.
,
Ling
B.
,
Tian
Z.
,
Wei
H.
.
2011
.
CD11b and CD27 reflect distinct population and functional specialization in human natural killer cells.
Immunology
133
:
350
359
.
25
Huntington
N. D.
,
Tabarias
H.
,
Fairfax
K.
,
Brady
J.
,
Hayakawa
Y.
,
Degli-Esposti
M. A.
,
Smyth
M. J.
,
Tarlinton
D. M.
,
Nutt
S. L.
.
2007
.
NK cell maturation and peripheral homeostasis is associated with KLRG1 up-regulation.
J. Immunol.
178
:
4764
4770
.
26
Nguyen
K. B.
,
Salazar-Mather
T. P.
,
Dalod
M. Y.
,
Van Deusen
J. B.
,
Wei
X. Q.
,
Liew
F. Y.
,
Caligiuri
M. A.
,
Durbin
J. E.
,
Biron
C. A.
.
2002
.
Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection.
J. Immunol.
169
:
4279
4287
.
27
Swann
J. B.
,
Hayakawa
Y.
,
Zerafa
N.
,
Sheehan
K. C. F.
,
Scott
B.
,
Schreiber
R. D.
,
Hertzog
P.
,
Smyth
M. J.
.
2007
.
Type I IFN contributes to NK cell homeostasis, activation, and antitumor function.
J. Immunol.
178
:
7540
7549
.
28
Mizutani
T.
,
Neugebauer
N.
,
Putz
E. M.
,
Moritz
N.
,
Simma
O.
,
Zebedin-Brandl
E.
,
Gotthardt
D.
,
Warsch
W.
,
Eckelhart
E.
,
Kantner
H. P.
, et al
.
2012
.
Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance.
OncoImmunology
1
:
1027
1037
.
29
Lee
C. K.
,
Rao
D. T.
,
Gertner
R.
,
Gimeno
R.
,
Frey
A. B.
,
Levy
D. E.
.
2000
.
Distinct requirements for IFNs and STAT1 in NK cell function.
J. Immunol.
165
:
3571
3577
.
30
Gough
D. J.
,
Messina
N. L.
,
Clarke
C. J. P.
,
Johnstone
R. W.
,
Levy
D. E.
.
2012
.
Constitutive type I interferon modulates homeostatic balance through tonic signaling.
Immunity
36
:
166
174
.
31
Miyagi
T.
,
Gil
M. P.
,
Wang
X.
,
Louten
J.
,
Chu
W.-M.
,
Biron
C. A.
.
2007
.
High basal STAT4 balanced by STAT1 induction to control type 1 interferon effects in natural killer cells.
J. Exp. Med.
204
:
2383
2396
.
32
Mack
E. A.
,
Kallal
L. E.
,
Demers
D. A.
,
Biron
C. A.
.
2011
.
Type 1 interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection.
MBio
2
:
e00169
e11
.
33
Michallet
M.-C.
,
Meylan
E.
,
Ermolaeva
M. A.
,
Vazquez
J.
,
Rebsamen
M.
,
Curran
J.
,
Poeck
H.
,
Bscheider
M.
,
Hartmann
G.
,
König
M.
, et al
.
2008
.
TRADD protein is an essential component of the RIG-like helicase antiviral pathway.
Immunity
28
:
651
661
.
34
Hanna
R. N.
,
Carlin
L. M.
,
Hubbeling
H. G.
,
Nackiewicz
D.
,
Green
A. M.
,
Punt
J. A.
,
Geissmann
F.
,
Hedrick
C. C.
.
2011
.
The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes.
Nat. Immunol.
12
:
778
785
.
35
McWhorter
A. R.
,
Smith
L. M.
,
Masters
L. L.
,
Chan
B.
,
Shellam
G. R.
,
Redwood
A. J.
.
2013
.
Natural killer cell dependent within-host competition arises during multiple MCMV infection: consequences for viral transmission and evolution.
PLoS Pathog.
9
:
e1003111
.
36
Verma
S.
,
Wang
Q.
,
Chodaczek
G.
,
Benedict
C. A.
.
2013
.
Lymphoid-tissue stromal cells coordinate innate defense to cytomegalovirus.
J. Virol.
87
:
6201
6210
.
37
Seillet
C.
,
Huntington
N. D.
,
Gangatirkar
P.
,
Axelsson
E.
,
Minnich
M.
,
Brady
H. J. M.
,
Busslinger
M.
,
Smyth
M. J.
,
Belz
G. T.
,
Carotta
S.
.
2014
.
Differential requirement for Nfil3 during NK cell development.
J. Immunol.
192
:
2667
2676
.
38
Sun
J. C.
2010
.
Re-educating natural killer cells.
J. Exp. Med.
207
:
2049
2052
.
39
Kim
S.
,
Poursine-Laurent
J.
,
Truscott
S. M.
,
Lybarger
L.
,
Song
Y.-J.
,
Yang
L.
,
French
A. R.
,
Sunwoo
J. B.
,
Lemieux
S.
,
Hansen
T. H.
,
Yokoyama
W. M.
.
2005
.
Licensing of natural killer cells by host major histocompatibility complex class I molecules.
Nature
436
:
709
713
.
40
Yokoyama
W. M.
,
Kim
S.
.
2006
.
Licensing of natural killer cells by self-major histocompatibility complex class I.
Immunol. Rev.
214
:
143
154
.
41
Robbins
S. H.
,
Tessmer
M. S.
,
Van Kaer
L.
,
Brossay
L.
.
2005
.
Direct effects of T-bet and MHC class I expression, but not STAT1, on peripheral NK cell maturation.
Eur. J. Immunol.
35
:
757
765
.
42
Fernandez
N. C.
,
Treiner
E.
,
Vance
R. E.
,
Jamieson
A. M.
,
Lemieux
S.
,
Raulet
D. H.
.
2005
.
A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules.
Blood
105
:
4416
4423
.
43
Beuneu
H.
,
Deguine
J.
,
Bouvier
I.
,
Di Santo
J. P.
,
Albert
M. L.
,
Bousso
P.
.
2011
.
Cutting edge: a dual role for type I IFNs during polyinosinic-polycytidylic acid-induced NK cell activation.
J. Immunol.
187
:
2084
2088
.
44
Marçais
A.
,
Viel
S.
,
Grau
M.
,
Henry
T.
,
Marvel
J.
,
Walzer
T.
.
2013
.
Regulation of mouse NK cell development and function by cytokines.
Front. Immunol.
4
:
450
.
45
Brady
J.
,
Carotta
S.
,
Thong
R. P. L.
,
Chan
C. J.
,
Hayakawa
Y.
,
Smyth
M. J.
,
Nutt
S. L.
.
2010
.
The interactions of multiple cytokines control NK cell maturation.
J. Immunol.
185
:
6679
6688
.
46
Biron
C. A.
,
Nguyen
K. B.
,
Pien
G. C.
,
Cousens
L. P.
,
Salazar-Mather
T. P.
.
1999
.
Natural killer cells in antiviral defense: function and regulation by innate cytokines.
Annu. Rev. Immunol.
17
:
189
220
.
47
Bubić
I.
,
Wagner
M.
,
Krmpotić
A.
,
Saulig
T.
,
Kim
S.
,
Yokoyama
W. M.
,
Jonjić
S.
,
Koszinowski
U. H.
.
2004
.
Gain of virulence caused by loss of a gene in murine cytomegalovirus.
J. Virol.
78
:
7536
7544
.
48
Orr
M. T.
,
Sun
J. C.
,
Hesslein
D. G. T.
,
Arase
H.
,
Phillips
J. H.
,
Takai
T.
,
Lanier
L. L.
.
2009
.
Ly49H signaling through DAP10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection.
J. Exp. Med.
206
:
807
817
.
49
Brown
M. G.
,
Dokun
A. O.
,
Heusel
J. W.
,
Smith
H. R.
,
Beckman
D. L.
,
Blattenberger
E. A.
,
Dubbelde
C. E.
,
Stone
L. R.
,
Scalzo
A. A.
,
Yokoyama
W. M.
.
2001
.
Vital involvement of a natural killer cell activation receptor in resistance to viral infection.
Science
292
:
934
937
.
50
Schneider
K.
,
Loewendorf
A.
,
De Trez
C.
,
Fulton
J.
,
Rhode
A.
,
Shumway
H.
,
Ha
S.
,
Patterson
G.
,
Pfeffer
K.
,
Nedospasov
S. A.
, et al
.
2008
.
Lymphotoxin-mediated crosstalk between B cells and splenic stroma promotes the initial type I interferon response to cytomegalovirus.
Cell Host Microbe
3
:
67
76
.
51
Maslowski
K. M.
,
Vieira
A. T.
,
Ng
A.
,
Kranich
J.
,
Sierro
F.
,
Yu
D.
,
Schilter
H. C.
,
Rolph
M. S.
,
Mackay
F.
,
Artis
D.
, et al
.
2009
.
Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43.
Nature
461
:
1282
1286
.
52
El Maadidi
S.
,
Faletti
L.
,
Berg
B.
,
Wenzl
C.
,
Wieland
K.
,
Chen
Z. J.
,
Maurer
U.
,
Borner
C.
.
2014
.
A novel mitochondrial MAVS/Caspase-8 platform links RNA virus-induced innate antiviral signaling to Bax/Bak-independent apoptosis.
J. Immunol.
192
:
1171
1183
.
53
Samson
S. I.
,
Mémet
S.
,
Vosshenrich
C. A.
,
Colucci
F.
,
Richard
O.
,
Ndiaye
D.
,
Israël
A.
,
Di Santo
J. P.
.
2004
.
Combined deficiency in IkappaBalpha and IkappaBepsilon reveals a critical window of NF-kappaB activity in natural killer cell differentiation.
Blood
103
:
4573
4580
.
54
Pascal
V.
,
Nathan
N. R.
,
Claudio
E.
,
Siebenlist
U.
,
Anderson
S. K.
.
2007
.
NF-kappa B p50/p65 affects the frequency of Ly49 gene expression by NK cells.
J. Immunol.
179
:
1751
1759
.
55
Tato
C. M.
,
Mason
N.
,
Artis
D.
,
Shapira
S.
,
Caamano
J. C.
,
Bream
J. H.
,
Liou
H.-C.
,
Hunter
C. A.
.
2006
.
Opposing roles of NF-kappaB family members in the regulation of NK cell proliferation and production of IFN-gamma.
Int. Immunol.
18
:
505
513
.
56
Lucas
M.
,
Schachterle
W.
,
Oberle
K.
,
Aichele
P.
,
Diefenbach
A.
.
2007
.
Dendritic cells prime natural killer cells by trans-presenting interleukin 15.
Immunity
26
:
503
517
.
57
Baranek
T.
,
Manh
T.-P. V.
,
Alexandre
Y.
,
Maqbool
M. A.
,
Cabeza
J. Z.
,
Tomasello
E.
,
Crozat
K.
,
Bessou
G.
,
Zucchini
N.
,
Robbins
S. H.
, et al
.
2012
.
Differential responses of immune cells to type I interferon contribute to host resistance to viral infection. [Published erratum appears in 2013 Cell Host Microbe 13: 372.]
Cell Host Microbe
12
:
571
584
.
58
Lee
G. A.
,
Liou
Y.-H.
,
Wang
S.-W.
,
Ko
K.-L.
,
Jiang
S.-T.
,
Liao
N.-S.
.
2011
.
Different NK cell developmental events require different levels of IL-15 trans-presentation.
J. Immunol.
187
:
1212
1221
.
59
Castillo
E. F.
,
Schluns
K. S.
.
2012
.
Regulating the immune system via IL-15 transpresentation.
Cytokine
59
:
479
490
.
60
Gordy
L. E.
,
Bezbradica
J. S.
,
Flyak
A. I.
,
Spencer
C. T.
,
Dunkle
A.
,
Sun
J.
,
Stanic
A. K.
,
Boothby
M. R.
,
He
Y.-W.
,
Zhao
Z.
, et al
.
2011
.
IL-15 regulates homeostasis and terminal maturation of NKT cells.
J. Immunol.
187
:
6335
6345
.
61
Verma
S.
,
Benedict
C. A.
.
2011
.
Sources and signals regulating type I interferon production: lessons learned from cytomegalovirus.
J. Interferon Cytokine Res.
31
:
211
218
.
62
Döring
M.
,
Lessin
I.
,
Frenz
T.
,
Spanier
J.
,
Kessler
A.
,
Tegtmeyer
P.
,
Dağ
F.
,
Thiel
N.
,
Trilling
M.
,
Lienenklaus
S.
, et al
.
2014
.
M27 expressed by cytomegalovirus counteracts effective type I interferon induction of myeloid cells but not of plasmacytoid dendritic cells.
J. Virol.
88
:
13638
13650
.
63
Orr
M. T.
,
Murphy
W. J.
,
Lanier
L. L.
.
2010
.
‘Unlicensed’ natural killer cells dominate the response to cytomegalovirus infection.
Nat. Immunol.
11
:
321
327
.
64
Orr, M. T., and L. L. Lanier. 2011. Natural killer cell licensing during viral infection. In Crossroads between Innate and Adaptive Immunity III. Advances in Experimental Medicine and Biology, Vol. 780. B. Pulendran, P. D. Katsikis, and S. P. Schoenberger, eds. New York: Springer, p. 37–44
.
65
Pothlichet
J.
,
Niewold
T. B.
,
Vitour
D.
,
Solhonne
B.
,
Crow
M. K.
,
Si-Tahar
M.
.
2011
.
A loss-of-function variant of the antiviral molecule MAVS is associated with a subset of systemic lupus patients.
EMBO Mol. Med.
3
:
142
152
.
66
Molineros
J. E.
,
Maiti
A. K.
,
Sun
C.
,
Looger
L. L.
,
Han
S.
,
Kim-Howard
X.
,
Glenn
S.
,
Adler
A.
,
Kelly
J. A.
,
Niewold
T. B.
, et al
BIOLUPUS Network
.
2013
.
Admixture mapping in lupus identifies multiple functional variants within IFIH1 associated with apoptosis, inflammation, and autoantibody production.
PLoS Genet.
9
:
e1003222
.
67
Poggi
A.
,
Zocchi
M. R.
.
2014
.
NK cell autoreactivity and autoimmune diseases.
Front. Immunol.
5
:
27
.
68
Park
Y.-W.
,
Kee
S.-J.
,
Cho
Y.-N.
,
Lee
E.-H.
,
Lee
H.-Y.
,
Kim
E.-M.
,
Shin
M.-H.
,
Park
J.-J.
,
Kim
T.-J.
,
Lee
S.-S.
, et al
.
2009
.
Impaired differentiation and cytotoxicity of natural killer cells in systemic lupus erythematosus.
Arthritis Rheum.
60
:
1753
1763
.
69
Henriques
A.
,
Teixeira
L.
,
Inês
L.
,
Carvalheiro
T.
,
Gonçalves
A.
,
Martinho
A.
,
Pais
M. L.
,
da Silva
J. A. P.
,
Paiva
A.
.
2013
.
NK cells dysfunction in systemic lupus erythematosus: relation to disease activity.
Clin. Rheumatol.
32
:
805
813
.
70
Hervier
B.
,
Beziat
V.
,
Haroche
J.
,
Mathian
A.
,
Lebon
P.
,
Ghillani-Dalbin
P.
,
Musset
L.
,
Debré
P.
,
Amoura
Z.
,
Vieillard
V.
.
2011
.
Phenotype and function of natural killer cells in systemic lupus erythematosus: excess interferon-γ production in patients with active disease.
Arthritis Rheum.
63
:
1698
1706
.
71
Schepis
D.
,
Gunnarsson
I.
,
Eloranta
M.-L.
,
Lampa
J.
,
Jacobson
S. H.
,
Kärre
K.
,
Berg
L.
.
2009
.
Increased proportion of CD56bright natural killer cells in active and inactive systemic lupus erythematosus.
Immunology
126
:
140
146
.
72
Malathi
K.
,
Dong
B.
,
Gale
M.
 Jr.
,
Silverman
R. H.
.
2007
.
Small self-RNA generated by RNase L amplifies antiviral innate immunity.
Nature
448
:
816
819
.
73
Ezelle, H. J., and B. A. Hassel. 2012. Pathologic effects of RNase-L dysregulation in immunity and proliferative control. Front. Biosci. (Schol Ed). 4: 767–786
.
74
Cho
J. A.
,
Lee
A. H.
,
Platzer
B.
,
Cross
B. C.
,
Gardner
B. M.
,
De Luca
H.
,
Luong
P.
,
Harding
H. P.
,
Glimcher
L. H.
,
Walter
P.
, et al
.
2013
.
The unfolded protein response element IRE1α senses bacterial proteins invading the ER to activate RIG-I and innate immune signaling.
Cell Host Microbe
13
:
558
569
.
75
Malathi
K.
,
Saito
T.
,
Crochet
N.
,
Barton
D. J.
,
Gale
M.
 Jr.
,
Silverman
R. H.
.
2010
.
RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP.
RNA
16
:
2108
2119
.
76
Zeng
M.
,
Hu
Z.
,
Shi
X.
,
Li
X.
,
Zhan
X.
,
Li
X. D.
,
Wang
J.
,
Choi
J. H.
,
Wang
K. W.
,
Purrington
T.
, et al
.
2014
.
MAVS, cGAS, and endogenous retroviruses in T-independent B cell responses.
Science
346
:
1486
1492
.
77
Grasset
E. K.
,
Cerutti
A.
.
2014
.
Immunology. Retroviral help for B cells.
Science
346
:
1454
1455
.
78
Dupuis-Maurin
V.
,
Brinza
L.
,
Baguet
J.
,
Plantamura
E.
,
Schicklin
S.
,
Chambion
S.
,
Macari
C.
,
Tomkowiak
M.
,
Deniaud
E.
,
Leverrier
Y.
, et al
.
2015
.
Overexpression of the transcription factor Sp1 activates the OAS-RNAse L-RIG-I pathway.
PLoS One
10
:
e0118551
.

The authors have no financial conflicts of interest.

Supplementary data