Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, mechanisms these cells use to evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages, without changing total cholesterol levels. Resistance to CDC pore formation requires production of the oxysterol 25-hydroxycholesterol, inhibition of cholesterol synthesis, and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking IFN’s ability to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host-defense strategy.

C holesterol is the most abundant lipid in the plasma membrane and is an important determinant of the membrane's biochemical and biophysical properties [1][2][3][4] . Cellular cholesterol homeostasis is maintained through a combination of synthesis, import, storage and efflux pathways. While each pathway may be engaged independently, the pathways are often co-regulated to ensure that sufficient cholesterol is available for a cell and to avoid deleterious accumulation. Recent studies indicate that sterol metabolism pathways of immune cells, in particular macrophages, can be rapidly reprogrammed to support their effector functions [5][6][7][8] . Toll-like receptors (TLRs) and IFNs have been shown to mediate reprogramming of cholesterol homeostasis by rapidly decreasing cholesterol synthetic flux 9,10 , increasing cholesterol ester storage in lipid droplets 11,12 and stimulating the production of cholesterol derivatives such as 25HC 5,13,14 . This reprogramming of sterol homeostasis facilitates host defense responses through a variety of mechanisms, including blockade of viral entry, regulation of innate immune receptor signaling, immune cell skewing and increasing macrophage phagocytic capacity. Thus, coordinated regulation of sterol metabolic pathways in immune cells is now viewed as a fundamental component of the host defense response.
Given the essential requirement for cholesterol in animal membranes, it is not surprising that microorganisms and viruses have evolved strategies that take advantage of cholesterol availability in membranes to facilitate pathogenesis [15][16][17][18] . CDCs are a group of secreted toxins produced mainly by Gram-positive bacteria that depend on membrane cholesterol for their pathogenic function [19][20][21] . CDC binding to cholesterol in the plasma membrane facilitate CDC oligomerization and pore formation, resulting in loss of membrane integrity, reduced efficacy of antimicrobial function and cell death 22,23 . Cells have repair mechanisms by which they minimize the deleterious effects of CDC toxins. Damaged plasma membranes can be internalized and resealed 24,25 , or they may bleb and be subsequently shed as microvesicles 26 . However, it appears that phagocytes attempting membrane repair may become hyporesponsive to proinflammatory stimuli through shedding of TLRs and cytokine receptors 23 . Thus, host membrane repair mechanisms, which preserve phagocyte survival, also reduce their effectiveness for antimicrobial host defense. Whether phagocytes or other immune cells have evolved specific strategies that facilitate evasion from these pore-forming toxins remains less well understood.
Here we report that activation of pattern recognition receptor (PRR) signaling on macrophages influences susceptibility to CDC-induced loss of membrane integrity and function. We show that IFN signals convey resistance to CDC-mediated membrane damage on phagocytes by reprogramming their cellular cholesterol metabolism, whereas myeloid differentiation primary-response protein 88 (Myd88)-dependent TLR signals do not induce protection. Mechanistic studies revealed that IFNs mediate resistance to CDCs by decreasing their binding to the plasma membrane. The decrease in CDC binding requires coordinated inhibition of new cholesterol synthesis and an increase in the esterification of a small Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins to PFO and SLO (Fig. 1f,g and Extended Data Fig. 1h,i), indicating that IFN signals can induce a 'resistant' phenotype regardless of the PRR engaged. We also found that IFN stimulation (IFN-γ or IFN-β; 4 h) of purified neutrophils from mouse bone marrow resulted in resistance to CDC-mediated loss of membrane integrity (Fig. 1h), demonstrating the ability of IFN to induce protection in other phagocytic, innate immune cells. Importantly, IFN stimulation largely preserved the ability of macrophages to phagocytose extracellular bacteria (that is, Staphylococcus aureus) when cells were challenged with PFO (Fig. 1i). Together, these data demonstrate that autocrine or paracrine IFN signals induce resistance to CDC-mediated pore formation, resulting in preservation of macrophage membrane integrity and phagocytic function.
IFNs decrease binding of CDCs to the plasma membrane. To understand whether the observed changes in CDC sensitivity could be due to altered binding of CDCs to the plasma membrane, we performed studies on IFN-stimulated macrophages with fluorescently labeled ALO domain 4 (ALO-D4), a modified CDC toxin that has markedly reduced pore-forming capacity but retains near-normal binding characteristics to the plasma membrane [33][34][35][36] . We found that the binding of ALO-D4 to IFN-β-or IFN-γ-stimulated macrophages was substantially decreased when compared to control macrophages (Fig. 2a). High-throughput imaging analysis of macrophage cultures (>3,000 macrophages for each condition) stimulated with IFN-β, IFN-γ, STING or TLR3 ligand showed a marked reduction in the average intensity of ALO-D4 binding ( Fig. 2b-e). By contrast, ALO-D4 binding was maintained or modestly increased in TLR1/TLR2-stimulated macrophages (Fig. 2f,g), consistent with our data indicating that TLR1/TLR2-stimulated macrophages maintained sensitivity to CDC-induced loss of membrane integrity. A similar decrease in ALO-D4 binding in response to either IFN-α or IFN-β was observed in macrophages derived from human peripheral blood mononuclear cells (hPBMCs; Fig. 2h,i). Likewise, stimulation (6 h) of purified neutrophils from mouse bone marrow with IFNs also decreased ALO-D4 binding (Extended Data Fig. 2a,b). Time-course studies showed that a decrease in ALO-D4 binding could be observed within 2 h of IFN stimulation of BMDMs (Fig. 2j,k). As expected, the decrease in ALO-D4 signal induced by IFN-β (or PRR-induced type I IFNs) was abrogated in type I IFN receptor (IFNAR)-deficient macrophages (Extended Data Fig. 2c,d). However, overnight IFN-γ treatment of IFNAR-deficient macrophages decreased ALO-D4 binding (Fig. 2l,m), demonstrating that resistance induced by type I and type II IFN cytokines are not interdependent. In combination, these data indicate that protection from CDCs is induced quickly in response to IFNs and is mediated by reduced binding of CDCs to the plasma membrane.
IFNs decrease a small pool of membrane cholesterol targeted by CDCs. Next, we asked whether IFN signals altered the binding of CDCs to the plasma membrane by decreasing macrophage cholesterol content. To address this, we performed gas chromatographymass spectrometry (GC-MS) to quantify total cellular cholesterol levels in IFN-stimulated BMDMs. We observed that IFNs modestly increased total cholesterol in macrophages (Fig. 3a). Likewise, we observed that STING or TLR3 activation, which induces robust type I IFN responses, also increased total cholesterol (Extended Data Fig. 3a). Thus, the protection mediated by IFNs could not be attributed to a global decrease in cholesterol content. We also performed GC-MS on purified plasma membranes from quiescent and IFN-γ-stimulated macrophages; however, no difference in cholesterol pool size was observed ( Fig. 3b and Extended Data Fig. 3b). We also stained BMDMs with filipin III, a bacterial product that binds to unesterified cholesterol in membranes and forms a fluorescent complex 37 . Consistent with our mass spectrometry studies, no change in filipin III staining was observed in response to IFN stimulation (Extended Data Fig. 3c). We also investigated whether IFN stimulation disrupted sphingomyelin-associated cholesterol in the plasma membrane. Ostreolysin A (OlyA) is a protein that specifically binds to cholesterol when associated with sphingomyelin 38 . To assess whether IFNs altered this pool of cholesterol in the plasma membrane of macrophages, we applied nanoscale secondary ion mass spectrometry (NanoSIMS) 39,40 using stable-isotope-labeled ALO-D4 and OlyA proteins. Consistent with the fluorescent ALO-D4 imaging studies, we observed a decrease in 15 N-labeled ALO-D4 binding in response to IFN-β stimulation (Fig. 3c). In contrast, [ 15 N]OlyA binding remained unchanged (Fig. 3c). High-throughput imaging studies with fluorescence-labeled OlyA showed a similar binding pattern for unstimulated and IFN-treated macrophage cultures (Extended Data Fig. 3d,e). Cholera toxin B (CTB) staining also showed no change, indicating that IFN signaling does not disrupt GM1 ganglioside-containing microdomains of the plasma membrane (Extended Data Fig. 3f). Together, these data support the conclusion that substantial amounts of cholesterol remain in the plasma membrane in association with sphingolipids after IFN stimulation 38 . Consistent with this concept, brief treatment of IFN-stimulated macrophages with sphingomyelinase 39 restored ALO-D4 binding (Fig. 3d,e). Thus, we conclude that CDCs are targeting a small fraction of non-sphingomyelin-associated cholesterol within the plasma membrane and that IFN signaling alters the availability of this pool of cholesterol to reduce CDC binding to phagocytes.
Cholesterol synthesis is linked to CDC susceptibility. We and others have previously reported that IFN signaling can reduce de novo cholesterol synthesis in macrophages 7,9,10 . Thus, we considered the possibility that changes in CDC sensitivity were linked to the cholesterol biosynthetic capacity of activated macrophages. To address this, we performed studies of [ 13 C] glucose tracer enrichment on PRR-and IFN-stimulated macrophages. IFN-β and IFN-γ stimulation markedly decreased macrophage cholesterol synthesis (Fig.  4a). Likewise, activation of STING or TLR3 also decreased cholesterol biosynthesis (Extended Data Fig. 4a,b). In contrast, activation of TLR1/TLR2, TLR7 and TLR9 increased the amounts of synthesized cholesterol accumulated (Extended Data Fig. 4b). These data largely mirror the differential sensitivity to CDCs that we observed in PRR-and IFN-stimulated macrophages, leading us to hypothesize that cholesterol biosynthetic flux determines susceptibility to CDC-induced plasma membrane damage. Consistent with this, pharmacologic inhibition of cholesterol synthesis using simvastatin reduced ALO-D4 binding to otherwise unstimulated macrophages and protected against CDC-mediated membrane damage (Fig. 4b,c and Extended Data Fig. 4c). In addition, we asked whether genetic attenuation of cholesterol synthesis would intrinsically convey resistance to CDC recognition. Loss of the cleavage-activating protein SCAP, which is required to chaperone the sterol regulatory element binding protein (SREBP), has been shown to attenuate cholesterol synthesis in macrophages 10 . We observed that quiescent SCAP-deficient macrophages exhibited decreased ALO-D4 binding, and, accordingly, these cells were protected from the PFO challenge ( Fig. 4d,e). The oxysterol 25HC is an endogenous inhibitor of cholesterol biosynthesis through its ability to induce degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis, and inhibition of the SREBP transcriptional axis 41,42 . Thus, we asked whether treating macrophage cultures with 25HC would also alter ALO-D4 binding. To this end, wild-type quiescent macrophages were treated with 25HC (3 μM) for 4 h. We found that treatment with 25HC  markedly reduced ALO-D4 binding ( Fig. 4f,g). Together, these data support the concept that the pool of newly synthesized cholesterol, rather than the total cholesterol available in the plasma membrane, is linked to the sensitivity or resistance of macrophages to CDC-mediated toxicity.
Production of 25HC is required for resistance to CDCs. IFNs have a well-defined role in inducing the conversion of cholesterol into 25HC via the enzyme CH25H in macrophages 5,13,14 . Thus, we next asked whether the endogenous production of 25HC is required for IFN-mediated protection against CDCs. To address this, control and CH25H-deficient macrophages were stimulated with IFNs and ALO-D4 binding was assessed. We observed that neither IFN-β nor IFN-γ treatment of CH25H-deficient macrophage cultures reduced ALO-D4 binding (Fig. 5a,b). Correspondingly, we found that stimulating CH25H-deficient macrophages with IFN-β or IFN-γ was unable to induce protection against either PFO or SLO challenge (Fig. 5c,d and Extended Data Fig. 5a,b). We also observed that, in contrast to control macrophages, IFN treatment of CH25H-deficient macrophages reduced their ability to maintain efficient phagocytosis of bacteria when challenged with PFO (Fig. 5e). Importantly, provisioning of 25HC to cultures of CH25H-deficient or control macrophages protected them from CDC-induced pore formation (Fig. 5f) and largely restored their ability to phagocytose bacteria (Fig. 5e). Thus, endogenous production of 25HC appears to be critical for macrophage protection to CDCs. Next, we sought to better define the mechanism by which IFN signaling and the production of 25HC mediate protection against CDCs. Our studies to this point correlated a decrease in cholesterol biosynthesis with protection against challenge by CDCs, leading us to ask whether the loss of CH25H altered IFN-mediated reduction in the cholesterol synthesis. [ 13 C] glucose tracer enrichment studies showed that loss of CH25H in macrophages resulted in basally higher production of cholesterol as compared to control macrophages but did not alter total cholesterol content (Fig. 5g). IFN-β stimulation was able to reduce cholesterol synthesis in both control and CH25H-deficient macrophages, but the reduction was attenuated in the absence of CH25H, resulting in a synthetic profile that more closely resembled that observed in unstimulated control macrophages (Fig. 5g). Treatment of CH25H-deficient macrophages with simvastatin was sufficient to reduce ALO-D4 binding, supporting the concept that the ability of 25HC to regulate cholesterol synthetic flux is an important mechanism by which IFNs mediate protection from CDCs (Fig. 5h,i).
Cholesterol esterification contributes to CDC resistance of macrophages. We observed that IFNs induce a number of genes involved in cholesterol esterification and efflux (Fig. 6a), prompting us to ask whether these processes were also necessary for IFN-mediated protection from CDCs. As expected, IFN treatment of macrophages increased cholesterol ester formation 43,44 (Fig. 6b). Unexpectedly, we found that cholesterol in culture medium was not required for accumulation of esterified cholesterol in response to IFNs (Extended Data Fig. 6a), indicating that the source of cholesterol for esterification can be derived from host cell membranes rather than from the extracellular milieu. Pharmacologic inhibition of the cholesterol esterification enzymes acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) partially attenuated the protection from PFO-mediated pore formation conveyed by IFN signaling (Fig. 6c and Extended Data Fig. 6b). 25HC has also been shown to mediate cholesterol ester formation 45,46 ; thus, we asked whether CH25H deficiency influenced cholesterol ester production in response to IFN signals. We found that quiescent CH25H-deficient macrophages had modestly increased levels of cholesterol esters and were able to increase this pool to the levels seen in control macrophages in response to IFN stimulation (Extended Data Fig. 6c). Consistent with this, we observed that treatment of CH25H-deficient macrophages with an ACAT inhibitor further increased ALO-D4 binding and increased their susceptibility to CDC-mediated membrane damage ( Fig. 6d and Extended Data Fig. 6d). These data indicate that synthesis of cholesterol esters in macrophages is required, in part, for resistance to CDCs. To test whether cholesterol efflux was required for IFN-mediated protection from CDCs, we activated macrophages deficient in the key cholesterol efflux transporters ATP-binding cassette sub-family A member 1 (ABCA1) or ATP-binding cassette subfamily G member 1 (ABCG1) with IFN 11,12 . We found that IFN treatment (24 h) reduced ALO-D4 binding in both ABCA1-and ABCG1-deficient macrophages to a level similar to that of control macrophages (Extended Data Fig. 6e). No difference in susceptibility to CDC-mediated pore formation was observed between quiescent control and ABCA1-deficient macrophages (Fig. 6e). Furthermore, IFN treatment conveyed resistance to PFO challenge for ABCA1-deficient macrophages, indicating that efflux was unlikely to be an important component of the IFN-mediated protection under these conditions. However, we did find that phar-macological enforcement of cholesterol efflux by treating with liver X receptors (LXR) agonist GW3965 (ref. 47 ) for 24 h decreased ALO-D4 binding and provided robust protection against CDCs in wild-type macrophages (Fig. 6f,g). We also observed that LXR activation with GW3965 decreased ALO-D4 binding and conveyed protection for CH25H-deficient macrophages when challenged with PFO (Fig. 6f,g), indicating that 25HC mediates protection by regulating synthesis of cholesterol rather than by blocking CDCs at the membrane or inducing esterification. Taken together, these data support a model where IFNs reduce cholesterol synthesis and increase esterification of free cholesterol to reduce the cholesterol pool size in the plasma membrane targeted by CDCs.

25HC mediates protection to CDC-induced tissue damage.
Finally, we asked whether reprogramming of cholesterol homeostasis would influence CDC susceptibility in vivo. To this end, SLO was injected into the skin of control and CH25H-deficient mice. Injection sites were visually monitored for up to 48 h for the appearance of erythema and ulceration. The SLO challenge resulted in modest erythema within 24 h and small ulcers at 48 h ( Fig. 7a and Extended Data Fig. 7a). In contrast, CH25H-deficient mice developed severe erythema within 24 h, which progressed to ulcerative lesions that had upwards of a six-fold larger area than those of their wild-type counterparts (Fig. 7a and Extended Data Fig. 7a). Histologic examination confirmed greater epidermal ulceration area, along with a greater depth of tissue necrosis into the deep dermis and, in some cases, the adipose tissue, in CH25H-deficient mice (Fig. 7b). We also asked whether injecting 25HC into the dermis would protect mice from CDC-induced tissue damage. For these studies, we used the ALO challenge, because we found that ALO reliably induces larger skin lesions in control mice compared to SLO. We found that preinjection of 25HC into the dermis of mice for 6 h markedly reduced ALO-induced tissue damage ( Fig. 7c and Extended Data Fig.  7b). Histologic examination revealed that, in general, 25HC pretreatment completely prevented tissue damage, or, in cases with damage, 25HC pretreatment reduced both ulcer area and depth (Fig. 7d). Thus, we conclude that generation of 25HC and the con-sequent ability to reprogram cholesterol metabolism protect mice from CDC-mediated tissue damage.

Discussion
Immune cells have presumably evolved strategies to evade the deleterious effects of virulence factors to efficiently combat pathogens and protect the host from damage. In this study, we delineate a mechanism by which IFN signals alter the abundance of a small pool of cholesterol in the plasma membrane to protect macrophages and neutrophils from pathogen-produced toxins. It is likely that IFN-mediated reprogramming of cholesterol homeostasis will extend into other cell types, such as endothelium, adipocytes and epithelium, given the extent of tissue damage observed in the skin of CH25H-deficient mice in response to CDC challenge. Mechanistically, we find that inhibition of new cholesterol synthesis through the actions of CH25H is required to induce this protection mechanism. We also find that the IFN-mediated conversion of cholesterol into cholesterol esters is required for maximal protection. In combination, IFN regulation of these distinct but interrelated cholesterol metabolic pathways ensures that the cholesterol pool targeted by CDCs remains small, and consequently macrophages and neutrophils retain their function. These data also imply that pharmacologic manipulation of the cholesterol homeostatic machinery in the skin might decrease tissue damage associated with the deleterious effects of Gram-positive infections observed in necrotizing fasciitis or similar soft tissue infections. However, it will be necessary to determine whether manipulating cholesterol homeostasis in infected tissues interferes with other host defense pathways and clearance of microorganisms. One unexpected finding of our studies is that the IFN-induced changes in plasma membrane cholesterol are restricted to a very small fraction of the total membrane cholesterol. The exact pool of cholesterol in the plasma membrane targeted by CDCs remains poorly characterized 19,20 , and our data suggest that a minor fraction of cholesterol in the plasma membrane is required for CDC binding and pore formation. The majority of cholesterol is not evenly distributed across the plasma membrane 2,48 , but rather is tightly associated with sphingomyelins or other phospholipids in microdomains 38,49 . Recent studies provide evidence for distinct pools of cholesterol within the plasma membrane. These studies describe a small pool of cholesterol, termed 'accessible' cholesterol, that is in rapid equilibrium with the endoplasmic reticulum cholesterol pool and, consequently, the cholesterol biosynthetic machinery 48 . We suspect that this is the cholesterol pool that is targeted by CDCs and rapidly reduced upon IFN signaling. Our data indicate that this IFN-dependent reduction of the accessible cholesterol pool occurs through the combined actions of the ACAT enzymes and inhibition of cholesterol biosynthesis. We cannot rule out the possibility that IFN signals also move cholesterol laterally or vertically from the accessible pool into other domains in the plasma membrane. It will be of considerable interest to determine the spatial distribution of the accessible cholesterol pool within the plasma membrane and how this specific cholesterol pool relates to canonical domains in the plasma membrane as described previously. We also believe that other cholesterol metabolic pathways will be involved in this protective mechanism. We found that IFNs induce changes in ALO-D4 binding within 2 h; thus, it seems likely that direct signaling events downstream of the IFN receptors initiate this process, and it will be of interest to define the cholesterol transport machinery involved in the rapid removal of this small cholesterol pool from the plasma membrane. The observation that both type I and type II IFNs regulate a broad cassette of genes involved in cellular cholesterol homeostasis (for example, efflux, esterification, synthesis and intracellular trafficking) leads us to surmise that considerable pressure exists to sequester or limit accessible or free cholesterol from pathogens, and it will be of interest to determine the pathophysiologic context where this reshaping of membrane cholesterol homeostasis contributes to host defense. These observations also imply that other mechanisms that can influence the pool size of accessible cholesterol, such as metabolic dysfunction, could influence the susceptibility of cells to these pore-forming toxins. Consistent with this notion, it has been shown that diabetes and obesity are independent risk factors for necrotizing fasciitis 50,51 , and we posit that restoring cellular cholesterol homeostasis with drugs that target cholesterol metabolic machinery might serve to decrease the severity of disease in these patients. In support of this idea, we find that enforced cholesterol efflux through the activation of LXR can induce protection independently of IFN signaling. Likewise, injecting 25HC into the skin protected the dermis from ALO-induced damage, providing initial proof of concept that metabolic manipulation could be an adjunctive approach to conventional antimicrobials.
In conclusion, these studies provide evidence that rapid remodeling of the lipid content of membranes is used as a host defense strategy by macrophages and neutrophils. Given the breadth of microbial and viral proteins that target host lipids, we propose that signal-specific reprogramming of the lipidome of cells will be a fundamentally important strategy for host cells to evade different pathogens.

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Methods
Mouse strains. Wild-type and gene-targeted mice were purchased from The Jackson Laboratory: WT C57BL/6J (JAX 000664), B6.129S6-Ch25h tm1Rus /J (Ch25h −/− , JAX 016263) and B6(Cg)-Ifnar1 tm1.2Ees /J (Ifnar1 −/− , JAX 028288). SCAP-deficient macrophages were generated from a Lzy2-Cre × Scap fl/fl cross as previously described 10 . ABCA1-and ABCG1-deficient macrophages were generated from Lzy2-Cre × Abca1 fl/fl or Lyz2-Cre × Abcg1 fl/fl animals as previously described 50,51 . All mice used for BMDMs were males aged 8-16 weeks. For in vivo CDC injections, mice were female and aged 8-12 weeks. All mice were maintained in pathogen-free facilities of the University of California, Los Angeles. All experiments on mice and tissues collected from mice were performed in strict accordance with the University of California policy on the humane and ethical treatment of animals.
PBMC-derived macrophages. Human monocyte-derived monocytes were isolated from leukopacks using standard Ficoll isolation procedures and plastic adherence. Isolation of monocytes was conducted by the UCLA Center for AIDS Research Virology Core laboratory. Monocytes were differentiated into macrophages with 50 ng ml -1 human GM-CSF (Peprotech, 300-03) in IMDM medium (HyClone) with 10% FBS and 1% (vol/vol) penicillin-streptomycin for 7 d before experimental use. Cholesterol ester analysis. Macrophages were cultured in six-well dishes (Fisher, 08-772-1B) and stimulated with TLR ligands as described above. At 48 h after stimulation, cells were imaged for cell counting as previously described 10  Plasma membrane purification. BMDMs were seeded at 8 × 10 6 cells per plate on 10-cm tissue-culture-treated dishes. Cells were left for 2 d before stimulation. Cells were then treated with IFNs for 24 h. Plasma membrane purification was carried out by cell surface biotinylation followed by streptavidin affinity chromatography as previously described 36,46 .
Filipin III staining. BMDMs were seeded at 1.5 × 10 5 cells per well on 24-well plates. Cells were left for 1-3 d before stimulation. Cells were pretreated with TLR ligands for 24 h. Filipin III was purchased from Sigma-Aldrich (F4767) and resuspended in ethanol to 2 mg ml -1 . BMDMs were washed once with PBS and fixed with 3% paraformaldehyde for 15 min at 22 °C. Cells were then washed twice and stained with 100 μg ml -1 filipin III for 30 min at 22 °C in darkness. After staining, cells were washed once with PBS and visualized using a Zeiss Axio Observer Z1.
CTB staining. BMDMs were seeded at 1.5 × 10 5 cells per well on 24-well plates and left for 1-3 d before stimulation. Cells were pretreated with IFNs for 24 h. BMDMs were lifted off the plates by scraping with the plunger of a 1-ml syringe and transferred to a 96-well round-bottom plate. CTB was purchased from Thermo Fisher Scientific (C34777). Cells were stained with 1 μg ml -1 CTB for 10 min on ice. Cells were then resuspended in FACS buffer and analyzed with a flow cytometer (Attune NxT). Data were analyzed by FlowJo V10.  Data are representatives of three independent experiments, and are shown as mean ± s.e.m. (n = 3). Statistical significance was determined using an RM one-way ANOVA with Dunnett's correction (a-g) or a two-way ANOVA with Dunnett's correction (h, i). ***P<0.001.