Linking Lipid Metabolism to the Innate Immune Response in Macrophages through Sterol Regulatory Element Binding Protein -1a

doi:10.1016/j.cmet.2011.04.001

Cell Metab. Author manuscript; available in PMC 2012 May 4.

Published in final edited form as:

Cell Metab. 2011 May 4; 13(5): 540–549.

doi: 10.1016/j.cmet.2011.04.001

PMCID: PMC3090630

NIHMSID: NIHMS289608

Linking Lipid Metabolism to the Innate Immune Response in Macrophages through Sterol Regulatory Element Binding Protein -1a

Seung-Soon Im,^1,² Leyla Yousef,² Christoph Blaschitz,³ Janet Z. Liu,³ Robert A. Edwards,⁴ Stephen G. Young,⁵ Manuela Raffatellu,³ and Timothy F. Osborne^1,^2,⁶

¹Metabolic Signaling and Disease Program, Sanford-Burnham Medical Research Institute, Orlando, Florida

²Department of Molecular Biology and Biochemistry, University of California, Irvine, California

³Department of Microbiology and Molecular Genetics and Institute for Immunology, University of California, Irvine, California

⁴Department of Pathology and Laboratory Medicine and Institute for Immunology, University of California, Irvine California

⁵Department of Medicine and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, California

⁶To whom correspondence should be addressed at: Timothy F. Osborne, Metabolic Signaling and Disease Program, Sanford-Burnham Medical Research Institute, 6400 Sanger Rd Orlando FL 32827, Phone: 407-745-2098; Fax: 407-745-2001; Email: tosborne/at/sanfordburnham.org

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Summary

We show mice with a targeted deficiency in the gene encoding the lipogenic transcription factor SREBP-1a are resistant to endotoxic shock and systemic inflammatory response syndrome induced by cecal ligation and puncture (CLP). When macrophages from the mutant mice were challenged with bacterial lipopolysaccharide they failed to activate lipogenesis as well as two hallmark inflammasome functions, activation of Caspase-1 and secretion of IL-1β. We show that SREBP-1a not only activates genes required for lipogenesis in macrophages but also the gene encoding Nlrp1a, which is a core inflammasome component. Thus, SREBP-1a links lipid metabolism to the innate immune response, which supports our hypothesis that SREBPs evolved to regulate cellular reactions to external challenges that range from nutrient limitation and hypoxia to toxins and pathogens.

Keywords: SREBP-1a deficiency, Macrophage, Inflammasome, Nlrp1/Nalp1, lipogenesis

Individual cells monitor their surrounding environment and react to extracellular challenges that require adaptation or threaten viability. The lipid-rich plasma membrane forms a barrier between a cell and its surroundings and participates in the initial response to changes in nutrient availability and chemical or biological attack. The lipids of the membrane bilayer are key to the dynamics of all cell-environment interactions, and regulation of lipid concentration and composition is essential to optimize protein-lipid microenvironments required for transport, signaling, internalization and shape alterations such as blebbing and invagination. A reliable supply of lipids is essential for each of these functions; thus, the regulation of lipid levels is fundamentally critical for cell-environment interactions.

The sterol regulatory element binding proteins (SREBPs) are a small family of bHLHLZ transcription factors that were first identified in mammalian cells as key regulators of cellular lipid levels (Horton et al., 2002; Osborne and Espenshade, 2009). They are conserved from fission yeast to humans, and there is emerging evidence that in addition to regulating lipid metabolism in response to nutrient levels, SREBPs may play roles in responses to other environmental challenges (Osborne and Espenshade, 2009). These range from oxygen sensing in S. pombe (Hughes et al., 2005), responding to pore-forming bacterial toxins in mammalian fibroblasts (Gurcel et al., 2006) and intestinal adaptations to limit absorption of bitter-tasting toxic dietary components (Jeon et al., 2008).

There are three major mammalian SREBP isoforms: two encoded by the Srebf-1 gene through a combination of alternative promoters and differential splicing and one encoded by the Srebf-2 gene (Osborne and Espenshade, 2009). In general, SREBP mRNAs are ubiquitously expressed but their ratios vary across different tissues. For example, SREBP-1c, an essential regulator of the hepatic insulin response, is 10 times more abundant than SREBP-1a in the liver (Horton et al., 1998; Kim et al., 1998).

In contrast, SREBP-1a is the major isoform in all cultured cell lines examined (Shimomura et al., 1997) and our current studies show it is the most abundant SREBP-1 species in macrophages (Figure 1). Based on this observation and the expanding role of SREBPs in environmental sensing, we hypothesized that SREBP-1a may directly participate in a primary role of macrophages such as pathogen engagement. This is also supported by earlier studies with cultured fibroblast cell lines that indicated SREBPs are activated after exposure to pore-forming bacterial toxins (Gurcel et al., 2006) and during phagocytosis of latex beads (Castoreno et al., 2005).

Figure 1

Tissue patterns for SREBP mRNA expression

Here, we show that mice with a targeted deficiency that specifically inactivates SREBP-1a are resistant to the toxic effects of both lipopolysaccharide (LPS) challenge and sepsis following cecal ligation and puncture (CLP) consistent with a defective innate immune response. Investigating the mechanism in cultured macrophages, we show that SREBP-1a mutant macrophages secrete reduced IL-1β because they have a defect in expression of Nlrp1a which is an NLR family protein that contains both NACHT and leucine-rich repeats (LRRs) (Franchi et al., 2009). Nlrps interact with and coordinate the activation of Caspase-1 as part of an inflammasome (Franchi et al., 2009; Martinon et al., 2007; Schroder and Tschopp) to cleave proinflammatory cytokines IL-1 and IL-18 before their secretion (Martinon et al., 2007; Schroder and Tschopp). We also show SREBP-1a directly activates the Nlrp1a gene and LPS stimulates lipogenesis through an SREBP-1a dependent mechanism. Thus, SREBP-1a couples lipogenesis with the macrophage innate immune response.

Results

SREBP-1a is abundantly expressed in macrophages

We have generated a mouse line with an insertion of a β-geo expression cassette into the intron between the two alternative 5’ exons of the mouse SREBP-1 gene (Figure S1). The insertion significantly reduces normal splicing required from the most 5’ exon containing the longer and more potent SREBP-1a activation domain but has no effect on expression from the other promoter responsible for generating the weaker SREBP-1c isoform (Im et al., 2009). The resulting SREBP-1a deficient mice (SREBP-1aDF) are viable and manifest a mild hepatic defect during fasting (Im et al., 2009).

SREBP-1a is expressed at a far lower level relative to SREBP-1c in liver (Shimomura et al., 1997) so the mild hepatic phenotype was not surprising. To identify a tissue where loss of SREBP-1a might be more consequential, we surveyed multiple tissues to determine where it is the predominant isoform. We analyzed serial dilutions of plasmid DNA in parallel in these studies, making it possible to define isoform-specific expression patterns in a quantitative fashion. Interestingly, the absolute levels of the transcripts, and the ratios of SREBP-1a and 1c, varied dramatically across several adult tissues (Figure 1). In particular, SREBP-1a mRNA was very high, and much more abundant than SREBP-1c, in macrophages cultured from bone marrow (BMDM) or elicitied from the peritoneal cavity after an injection of thioglycolate (PTDM). Bone marrow dendritic cells (BMDC), heart, and small intestine also expressed mostly the 1a message.

SREBP-1aDF mice are resistant to proinflammatory Toxic Shock

Macrophages play a key role in the innate immune response to pathogen challenge so we tested the hypothesis that SREBP-1aDF mice might have an impaired innate immune response using three different in vivo model systems to evaluate responses to pathogen engagement. In the first model, we challenged the mice with injection of the bacterial outer membrane component lipopolysaccharide (LPS). LPS induces a robust inflammatory response in mice, and high doses are lethal (Ianaro et al., 2009). The SREBP-1aDF mice exhibited significant protection from the toxic effects of LPS relative to control mice (Figure 2A; p = 0.03). These results suggest that the SREBP-1aDF mice might have a reduced proinflammatory response so we measured the serum levels of several proinflammatory cytokines. Serum levels of IL-1β were dramatically lower in the SREBP-1aDF mice (Figure 2B) and IL-12 was the only other cytokine that had significantly lower levels relative to WT (Figure S2).

Figure 2

Role of SREBP-1a in Inflammatory signaling in mice

To further explore the in vivo effect of SREBP-1a deficiency on the innate immune system, we assessed the response of SREBP-1aDF mice to induction of polybacterial sepsis following a CLP protocol (Rittirsch et al., 2009). In this experimental model of generalized sepsis (Remick and Ward, 2005), the cecum is punctured allowing intestinal bacteria to enter the peritoneal cavity and mice typically die as a consequence of the resulting systemic inflammatory response syndrome (SIRS). The SREBP-1aDF mice also showed enhanced survival relative to WT mice after CLP (Figure 2C). Interestingly, serum levels of IL-1β were also dramatically reduced in the SREBP-1aDF mice following the CLP (Figure 2D). The reduced levels of proinflammtory IL-1β are consistent with the enhanced survival in both of these hyperinflammatory model systems.

Protection from oral infection by bacterial pathogens such as S. typhimurium require a normal proinflammatory response and we hypothesized that the SREBP-1aDF mice might be more susceptible to bacterial infection by this route. When WT and SREBP-1aDF mice were challenged with an oral dose of S. typhimurium, there was increased inflammatory cell infiltration, especially neutrophils, into the cecum of the SREBP-1aDF mice (Figures 2E–F) despite lower levels of bacterial colonization (Figure 2G). Consistent with the pathological assessment, expression of the neutrophil selective chemokine CXCL1 was also signficantly increased in the SREBP-1aDF cecum (Figure 2H).

SREBP-1aDF macrophages have reduced SREBP-1 protein levels

The in vivo responses to LPS, CLP, and S. typhimurium are consistent with an impaired innate immune response and because SREBP-1a is abundantly expressed in macrophages (Figure 1) we evaluated a potential mechanism for the in vivo responses through studies in cultured macrophages. First, we showed that SREBP-1a mRNA was dramatically reduced in the SREBP-1aDF macrophages without significant effects on expression of the other SREBPs (Figure 3). Immunoblotting with an antibody that reacts with both SREBP-1 isoforms revealed that the level of nuclear SREBP-1 protein was also reduced (Figure 3D). Because the level of 1c mRNA was not reduced, we can conclude that the major SREBP-1 protein in BMDM is SREBP-1a.

Figure 3

SREBP expression levels in BMDM of WT and SREBP-1aDF mice

SREBP-1a activates genes encoding inflammasome subunits in macrophages

To search for a phenotypic difference in SREBP-1aDF macrophages, we first compared global mRNA expression patterns in BMDM isolated from WT and SREBP-1aDF mice. The genes that exhibited statistically significant changes in gene expression of 1.5 fold or higher are highlighted in Figure 4A. The expression of two genes, Nlrp1a and Nlrp1c, were reduced over 95% in the SREBP-1aDF macrophages which was even more dramatic than the effect on expression of SREBP-1 itself (Figure 4B). This finding was confirmed by gene specific qPCR analysis (Figure 4C). We analyzed expression of mRNAs for the related Nlrp1b, Nlrp3, Nlrc4, and ASC as well as IL-1β (Figures 4B and S3) and none were affected by the loss of SREBP-1a.

Figure 4

SREBP-1a regulates Nlrp1a expression level in BMDM

Nlrp1a and 1c encode nucleotide oligomerization domain and leucine rich repeats receptor family members (NOD/LRR) (Franchi et al., 2009; Martinon et al., 2007). These particular NOD/LRRs are predicted to function as scaffold components of an inflammasome, which is a multi-subunit complex(es) that stimulates Caspase-1 to cleave proinflammatory cytokines pro IL-1, pro IL-18, and possibly pro IL-33 (Franchi et al., 2009) to produce the mature inflammatory cytokines. IL-1 and IL-18 are released from macrophages as part of the proinflammatory response to bacterial engagement through Toll-like receptor 4 (TLR4) signaling (Kaisho and Akira, 2000). This is a primary macrophage innate immune response to microbial pathogens and our in vivo results would be consistent with reduced inflammsome activity in the SREBP-1aDF macrophages.

Inflammasome activity is compromised in SREBP-1aDF macrophages

To evaluate whether SREBP-1aDF macrophages have impaired inflammasome function, we performed comparative studies in isolated macrophages (Figure 5). First, we showed that the LPS dependent increase in Caspase-1 activity in WT macrophages was blunted in SREBP-1aDF cells (Figure 5A). To measure IL-1 production from mouse production from mouse maccrophages a pre-challenge with LPS followed by an acute treatment with an additonal exogenous signal such as ATP is typically required (Mariathasan et al., 2004; Sutterwala et al., 2006). Figure 5B shows that the increase in IL-1 release observed in WT was significantly reduced in the SREBP-1aDF macrophages. Caspase-1 mRNA levels as well as IL-1 mRNA induction by LPS were not altered (Figure S4) so the results are consistent with reduced Caspase-1 activation and confirm that the SREBP-1aDF macrophages have impaired inflammasome function.

Figure 5

LPS mediated IL-1β production is decreased in SREBP-1aDF macrophages

IL-1β production in response to S. typhimurium infection was also reduced in the SREBP-1aDF macrophages (Figure 5C) as well which is consistent with a defective inflammasome response. Because serum levels of IL-12 were also lower in the SREBP-1aDF mice treated with LPS (Figure S2), we also measured IL-12 mRNA and production of extracellular IL-12 cytokine. The mRNA levels for both subunits as well as the secreted IL-12 cytokine were also lower in the SREBP-1aDF samples (Figure S4E–G).

IL-1 secretion in response to LPS plus ATP was also reduced in Nlrp3 −/− macrophages and the corresponding mice were protected from lethal challenge by LPS injection (Sutterwala et al., 2006). Both of these responses are similar to the results reported here for the SREBP-1aDF mice. To compare the roles of Nlrp1a and Nlrp3 in LPS dependent IL-1 secretion, we used an siRNA approach to reduce expression of each Nlrp in WT macrophages and the results in Figure 5D demonstrate that both Nlrp1a and Nlrp3 contribute to IL-1 secretion and thee effects may be additive. RNA expression studies confirmed that each Nlrp was specifically targeted by the corresponding siRNA (Figure 5E–F).

The siRNA results also indicate that the reduced inflammasome activity in the SREBP-1aDF macrophages is unlikely to result from the loss of another SREBP-1a dependent process. Additionally, when SREBP-1a expression was restored in the mutant macrophages by electroporation that LPS dependent IL-1 secretion was also restored (Figure 5G). Thus, thee reduced inflammasome activity in the SREBP-1aDF macrophages results from reduced Nlrp1a.

Nlrp1a is a direct SREBP-1a target gene

To determine whether Nlrp1a or Nlrp1c might be gene targets for SREBP-1a, we re-introduced SREBP-1a by adenovirus infection into the SREBP-1aDF macrophages and expression of Nlrp1a, but not Nlrp1c, returned to normal levels (Figure 6A). We also showed that SREBP-1 both activates the promoter through a region containing a canonical SREBP-1 binding site in transfection assays (Figure 6C–E) and binds to the Nlrp1 gene proximal promoter by ChIP analysis, and (Figure 6F–G). Taken together, these results strongly suggest that SREBP-1a directly activates expression of the Nlrp1a gene through a binding site in its proximal promoter.

Figure 6

SREBP-1a activates and binds to Nlrp1a promoter

LPS signaling in macrophages activates lipogenesis through SREBP-1a

A recent report showed that LPS stimulated de novo lipogenesis in macrophages in vivo (Posokhova et al., 2008). SREBP-1c is a key activator of lipogenesis in liver where it is the major SREBP-1 species (Horton et al., 2002), so we tested the idea that SREBP-1a might stimulate lipogenesis in macrophages in response to LPS where it is the more abundant SREBP-1 isoform. In Figure 7A, we show that de novo lipogenesis in BMDM is increased by LPS in WT but not SREBP-1aDF macrophages. LPS also stimulated accumulation of SREBP-1 nuclear protein (Figure 7B) and mRNA levels (Figure 7D) but SREBP-1c mRNA was not affected. These results strongly support that LPS stimulates lipogenesis through SREBP-1a in BMDM.

Figure 7

SREBP-1a activates lipogenesis and is a proinflammatory gene in BMDM

In prior studies, we noted the presence of two binding sites for Sp1 flanking one NF-κB site in the SREBP-1a promoter (Figure 7C) (Zhang et al., 2005). NF-κB is a key downstream transducer of LPS signaling and so we tested the idea that the LPS dependent increase in mRNA might be direcly through NF-κB recruitment to the SREBP-1a promoter. ChIP studies with an antibody to the p62 subunit of NF-κB confirmed this prediction (Figure 7D). NF-κB activates promoters by enhancing Sp1 co-recruitment (Perkins et al., 1993) and because there are two functional Sp1 sites in the SREBP-1a promoter, we also performed ChIP with an antibody to Sp1. Interestingly, Sp1 recruitment was also enhanced by LPS treatment (Figure 7E). These results provide strong evidence that in macrophages LPS stimulates lipogenesis through increasing NF-κB/Sp1 synergistic activation of the SREBP-1a promoter.

Discussion

In the current study, we show that SREBP-1a deficient mice have an impaired innate immune response in vivo and we show SREBP-1a is required to activate Caspase-1 and stimulate both IL-1β production and lipogenesis in response to LPS challenge in isolated macrophages. These results with cultured macrophages provide a mechanism for the impaired innate immune response in the SREBP-1aDF mice.

An intriguing question is why SREBP-1a, a key lipogenic transcription factor, evolved to also directly regulate genes of the innate immune response in macrophages? This is likely because cell proliferation and membrane expansion/rearrangements are both essential in the macrophage response to pathogen challenge and both require new lipid synthesis. Consistent with this idea, LPS was recently shown to increase de novo lipogenesis in macrophages in vivo (Posokhova et al., 2008). Interestingly, our experiments show that LPS stimulates lipogenesis in WT but not SREBP-1aDF macrophages (Figure 7) and we also show that SREBP-1a activates expression of key lipogenic genes in macrophages as well as Nlrp1a in response to macrophage lipid depletion (Figure S5).

Our studies show that SREBP-1a is required for LPS stimulated IL-1β production and this is likely through SREBP-1a activating expression of the gene encoding Nlrp1a, which is a mouse orthologue of human Nlrp1. Human Nlrp1 is part of a protein complex called an inflammasome that is required to activate Caspase-1 to cleave proinflammatory cytokines such as IL-1 during the proinflammatory response (Martinon et al., 2007). Nlrp1a expression, Caspase-1 activity, and IL-1 production were all dramatically reduced in SREBP-1aDF macrophages. Because we observed a similar phenotype when we knocked down Nlrp1a in WT macrophages using siRNA, the effect is unlikely to be a result of another consequence of the SREBP-1a deficiency.

SREBP-1a mRNA and nuclear protein levels were also both increased following LPS treatment (Figure 7). This response is probably through NF-κB activating expression of SREBP-1a after binding to its promoter (Zhang et al., 2005). The increased production of mature SREBP-1 protein is probably driven by increased synthesis of the ER targeted precursor, because in other settings where SREBP gene expression is increased there is also an elevation of nuclear SREBP levels (Repa et al., 2000; Shin and Osborne, 2003).

Nlrp1a mRNA was not further induced by LPS in WT macrophages (Figure S4B), which is likely because the basal SREBP-1a levels in macrophages are sufficient and further induction is both not required and limited by other factors. This is not unexpected as our results also show that known SREBP-1 target genes involved in lipid metabolism are also differentially affected by genetic and metabolic conditions that alter SREBP-1a levels (Figure S5) and knockout and pharmacological studies of nuclear receptors show similar differential effects on subsets of target genes (Wagner et al., 2003).

Earlier studies demonstrated that a knockout of Nlrp3 decreased LPS mediated IL-1 production. However, the response to S. typhimurium challenge was normal (Sutterwala et al., 2006) indicating that an inflammasome composed of other Nlrp family members must be involved. A more recent study indicated that both Nlrp3 and Nlrc4 have redundant roles in host defense against S. typhimurium, suggesting that multiple Nlrs play a cooperative role in host defense against intracellular pathogens (Broz et al., 2010). Because IL-1 production after S. typhimurium challenge was reduced in the SREBP-1aDF macrophages, our results suggest that Nlrp1a may be an additional component of this inflammasome. Using siRNA knockdown of both Nlrp1a and Nlrp3, we also showed that both contribute to LPS dependent IL-1 production (Figure 5).

There was reduced mortality in the SREBP-1aDF mice following both LPS and CLP challenge. These are two situations where a hyper-inflammatory response is lethal. SREBP-1aDF mice also secreted lower levels of IL-1 following LPS injection and CLP treatment. The mutant mice were also more susceptible to infection when challenged with an oral dose of S. typhimurium, consistent with the reduced ability of the SREBP-1aDF mice to effectively mount an anti-inflammatory response to bacterial infection. These results are similar to another report where S. typhimurium infection caused more edema, neutrophil infiltration and epithelial destruction in the cecum of caspase-1 knockout mice (Lara-Tejero et al., 2006). Despite the increased inflammatory response in this study, there was no difference in cecal levels of S. typhimurium in caspase-1−/− versus wild-type mice at 48h post-infection. Similar to this study, we found reduced colonization by S. typhimurium in the cecum of SREBP-1a def mice at 72h post-infection (Fig. 2G).

IL-1 knockout mice are not resistant to a lethal LPS challenge (Fantuzzi et al., 1996), whereas Caspase-1 deficient mice are protected similar to the SREBP-1aDF animals (Li et al., 1995). Thus, the reason for the increased resistance in the SREBP-1aDF mice is likely due to more than just reduced IL-1 production. Indeed, there is evidence that Nlrp1 deficiency results in lower levels of IL-12 production (Witola et al.) and mice deficient in the p40 subunit that is shared between IL-12 and IL-23 have an altered response to CLP (Moreno et al., 2006). Because the SREBP-1aDF mice also have reduced serum levels of IL-12 following LPS challenge, it is possible that at least part of the reason they are more resistant is because of lower levels of IL-12.

All of the in vivo responses suggested there was an altered innate immune response and our more focused studies demonstrate there was an impaired inflammatory response in the SREBP-1aDF macrophages, which provides a mechanism for the in vivo effects. However, because the whole animal setting is much more complicated and the model is a global knockout of SREBP-1a, it is possible that the in vivo response may be more complex and warrants further study.

Mice lacking the inflammasome adaptor ASC are completely resistant to challenge by very high concentrations of LPS (Sutterwala et al., 2006), whereas SREBP-1aDF mice, like the Nlrp3 knockout (Sutterwala et al., 2006), are only partially resistant. This suggests that inflammasomes containing ASC are crucial to the proinflammatory response in vivo and that the functions for Nlrp1a and Nlrp3 might be partially redundant and the results from our siRNA knockdown studies of Nlrp1a and Nlrp3 are consistent with this hypothesis (Figure 5D).

Different NLRs have been proposed to channel Caspase-1 into pathways required for different physiological responses (Schroder and Tschopp, 2010). These include inflammation in response to LPS (Sutterwala et al., 2006), protection from lethal toxin produced by B. anthracis (Boyden and Dietrich, 2006), activation of cell death by S. typhimurium (Mariathasan et al., 2004) and a distinct Caspase-1 dependent pathway for apoptosis in response to bacterial pore forming toxins that also requires SREBPs (Gurcel et al., 2006). This is also consistent with results from other recent studies suggesting there may be multiple inflammasomes that are constituted with different combinations of individual Nlrp species (Broz et al., 2010; Wu et al., 2010).

Production of mature IL-18 is also stimulated by an inflammasome/Caspase-1 dependent mechanism (Gu et al., 1997). However IL-18 levels were not affected in LPS challenged SREBP-1aDF mice (Figure S2). This was not completely unexpected based on other reports where secretion of IL-1 and IL-18 can be uncoupled. In one study, lethal toxin from B. anthracis stimulated macrophage production of both IL-18 and IL-1, whereas LPS stimulation resulted in production of IL-1, but not IL-18 (Cordoba-Rodriguez et al., 2004). Another study also showed that LPS stimulated release of IL-1 from human monocytes but there was no effect on IL-18 (Kankkunen et al., 2009).

In macrophages, the nuclear receptor LXR activates genes involved in cholesterol efflux and reverse cholesterol transport to limit macrophage cholesterol accumulation (Lafitte BA, 2001; Venkateswaran et al., 2000). Additionally, LXR and PPAR-γ signaling in macrophages limits inflammation by inhibiting NF-κB (Ghisletti et al., 2007; Joseph et al., 2003; Welch et al., 2003). These effects are predicted to attenuate the proinflammatory response and would be beneficial in preventing accumulation of lipid in macrophages during critical times such as the early stages of atherosclerotic plaque deposition where inflammatory signaling contributes to the severity of lesion development and progression (Lusis, 2000; Ross, 1999).

Our studies show that SREBP-inflammation cross-talk is important for the initial proinflammatory response, which requires new lipid synthesis for membrane expansion and proliferation. SREBPs pre-date nuclear receptors in evolution and their co-evolution with the proinflammatory response is likely a more primitive response that occurred to couple lipid production to survival pathways in response to pathogen challenge.

Experimental Procedures

Animals

SREBP-1a deficient mice (SREBP-1aDF) were described previously (Im et al., 2009). Mice were maintained in 12-h light/12-h dark cycles with free access to food and water. All procedures were performed in accordance with the Institutional Animal Care and Use Committees at the University of California Irvine and the Sanford-Burnham Medical Research Institute at Lake Nona.

Mouse bone marrow–derived macrophages

Bone marrow–derived macrophages were isolated from either C57BL/6 or SREBP-1aDF essentially as described (Gilchrist et al., 2006). After 7~10 days in culture, the differentiation of bone marrow derived macrophages (BMDM) were confirmed by FACS analysis using anti-CD11b and there was no difference in differentiation between WT and SREBP-1aDF cells. BMDM were then used for experiments as described below and in the figure legends (Joshi et al., 2008).

Cytokine ELISA measurements

BMDMs (2×10⁴/well) were plated in 24 well plates and incubated with 100 ng/ml LPS or 50 ng/ml LPS for various times and then culture supernatants were assayed for IL-1β by ELISA (R&D Systems). Where indicated, 1 mM ATP was added 30 min. prior to sampling the supernatant. For measuring serum cytokine levels in mice, 8–10 week old male mice (WT or SREBP-1aDF) were used. Lipopolysaccharide (LPS; Escherichia coli serotype 055:B5; Sigma) was suspended in endotoxin-free PBS and vigorously mixed for 15 min before use. A single dose of LPS was injected intraperitoneally into mice (10 mg/kg body weight) and blood was collected after the indicated times and cytokine levels were measured in serum using a commercial mouse IL-1β inflammatory cytokine ELISA kit (R&D Systems).

Caspase-1 activity assay

BMDMs were cultured as above and a Caspase-1 enzymatic assay using YVAD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin) as substrate was used (Biovision, Palo Alto, CA). Fluorometric readings were performed over a 30 min period at a wave-length pair of 400/505 nm excitation/emission, using a Spectramax M5 plate reader (Molecular Devices, Sunnyvale, CA). Kinetic analysis (determination of V_max) of AFC fluorescence was used to calculate enzymatic activity, which was normalized by protein content and expressed as fold increase over the basal level (un-stimulated cells).

Adenovirus infection in bone marrow-derived macrophages in vitro

Macrophages were seeded in 100-mm dishes (5×10⁶ cells/dish). Recombinant adenovirus (10 MOI) were added to cultures in DMEM medium including 15% L929 conditioned medium without FBS at 37 °C with 5% CO₂ for 6 h and then medium (plus FBS) was added back. After a further 24 h incubation, cells were harvested for analysis.

Endotoxicosis and Cecal ligation and puncture (CLP)

Male WT or SREBP-1aDF mice (8–10 week old; n=10 per each genotype) were injected with 10 mg/kg of LPS and animals were monitored for 7 days. For induction of polymicrobial sepsis, mice (n=10 per each genotype group) underwent CLP or a sham procedure, as previously described (Delano et al., 2007). Briefly, a laparotomy was performed under general anesthesia, the cecum was isolated, and 0.5 cm of cecum was ligated below the ileocecal valve and a distal region was punctured all the way through one time with a 23-gauge needle. Sham operation was performed by isolating the cecum without ligation or puncture. Blood was collected after 24 h and IL-1β levels were measured by ELISA (SABiosciences) as described above.

S. typhimurium infection of mice and BMDM

BMDM prepared as above were seeded at a concentration of 2×10⁶ cells/ml in 24 well plates. A nalidixic acid resistant derivative of Salmonella typhimurium ATCC14028 was grown overnight static in Luria Bertani (LB) broth. BMDM were infected with S. typhimurium with multiplicities of infection (MOI) of 1 and 10 and were incubated at 37 °C 5% CO₂. To kill extracellular bacteria, the supernatant was replaced with media containing gentamicin at a concentration of 100 µg/ml one hour post-infection, and by media with a lower concentration of gentamicin (25 µg/ml) after an additional hour. Cells were incubated overnight, and supernatant was analyzed for IL-1β by ELISA (eBioscience). The experiment was repeated 5 times with similar results. For the mouse experiments, groups of six SREBP-1aDF or C57/B6 mice were infected with 1×10⁹ CFUs of S. typhimurium 24 hours after treatment with streptomycin as previously described (Raffatellu et al., 2009). At 72 hours after infection, the cecum was collected as described (Raffatellu et al., 2009). S. typhimurium colonization of the cecum was measured by collecting the cecal content which was weighed, and resuspended by vigorous shaking in 1 ml of Phosphate Buffered Saline for 15 min. The colony forming units were enumerated by plating serial dilutions of the resuspended cecal content in LB+Nalidixic acid (50mg/L). Data were normalized by the weight of the cecal content collected from each mouse, which varied between 15–60mg. This experiment was repeated three times with similar results.

Introduction of si-RNA for Nlrp1a and Nlrp3 into bone marrow-derived macrophages

WT BMDMs (2×10⁶ cells), 100 µl of Nucleofector solution, and 100 pmol of smart pool siRNA for Nlrp1a, Nlrp3 or a negative control (Dharmacon) were mixed into a cuvette for the Amaxa Nucleofector and samples were electroporated with program Y-001 designed by the manufacturer. Following electroporation, cells were seeded onto 12-cm dishes. Forty hours after transfection, cells were stimulated with LPS (200 ng/ml) for 6 hrs followed by addition of ATP (1 mM) for 30 min. Cultured media were collected to measure IL-1β by ELSIA and cells were harvested with Trizol (Invitrogen) for isolation of RNA. The smart pool strategy decreases off-target effects and increases specificity as described (http://www.dharmacon.com/uploadedFiles/Home/Resources/Product_Literature/smartpool-technote.pdf)

RNA isolation and quantitative real-time polymerase chain reaction (qPCR)

Total RNA was isolated from macrophages of WT and SREBP-1aDF mice using the Trizol method (Invitrogen). cDNA was synthesized by cDNA superscript kit (Bio-Rad) and used for qPCR with Real-Time PCR Detection (Bio-Rad). mRNA levels were normalized for expression of ribosomal protein L32 mRNA as control and calculated by the comparative threshold cycle method. Plasmids containing partial cDNAs for SREBP-1a, -1c and -2 that encompass the oligonucleotides used for PCR were isolated and the purified DNA was quantified by A260. Serial dilutions of the plasmid DNAs were included in each qPCR analysis for use as a standard curve for quantitation. All qPCR reactions were repeated in triplicate. All primers were designed to amplify RNA across intron junctions and the melting curves in the qPCR reactions all showed a simple melting curve indicative of a single amplified product. qPCR primer sequences were either as described in Table S1 or as published (Im et al., 2009).

Immunoblotting

Nuclear extracts were prepared from BMDM of WT and SREBP-1aDF mice. Immunoblotting was performed following a modified protocol based on a previously described method (Sheng et al., 1995). nuclear proteins were resolved by 8% Tris-HCl SDS/PAGE gel electrophoresis and transferred onto nitrocellulose (Bio-Rad). All Immunoblots were developed using HRP-conjugated secondary antibody with an ECL detection system (GE Healthcare).

Biosynthesis of Fatty acids

BMDM were treated with LPS as described in the text. Cell media was removed form the plates, which were then incubated in fresh medium with sodium [¹⁴C] acetate for 6 hrs. After washing labeled cells, cells were dissolved by 0.1 N NaOH. Prior to saponification, 500 ul of 75% KOH, 10 ul of [³H] cholesterol nonsaponified TLC recovery carrier solution, and 10 ul [³H]oleic acid carrier solution was added to each sample. After extraction with petroleum ether, the fatty acid fraction was dried under nitrogen gas, dissolved in 30 ul of choloroform for fatty acid. Extract samples were spotted onto plastic-backed silica gel TLC plates and stained the TLC plates with iodine vapor. Excise spots were excised and put in scintillation vials including 10 ml scintillation solution to count [¹⁴C] and [³H].

Statistical analysis

Three to five experiments of all studies were performed, using triplicate replications of each sample. The data are represented as mean ± S.D. All data sets were analyzed for statistical significance using a 2-tailed unpaired Student’s t test. All p values below 0.05 were considered significant. Statistical analysis was carried out using Microsoft Excel (Microsoft).

Acknowledgments

We thank Dr. Linda Hammond for contributions to the early portions of this work and Dr. Craig Walsh for helpful discussions and laboratory members for comments on the project. We thank Vanessa Arias and Dr. Andrea Tenner for assistance with macrophage culture, Dr. Dat Tran for assistance with the CLP procedure, Peter Phelan for technical assistance and Dr. Young-Kyo Seo for preparing the SREBP-1 antibody. We also acknowlege the UCI microarray facility for technical assistance and Sang-Hee Yuh for analysis of microarray data. This manuscript was also significantly improved by comments from the anonymous reviewers (especially reviewer 2). This study was supported in part by grants from the NIH to T.O. (HL48044) and M. R. (AI083619, AI083663). M. R. is the recipient of an IDSA ERF/NIFID Astellas Young Investigator Award. JZL is supported by NIH Immunology Research Training Program grant NIH T32 AI60573.

Footnotes

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Author Contributions

SSI, LY, CB and JL performed experiments; RE performed the histopathological analysis; SY provided key reagents; SSI, MR, RE and TO designed experiments and analyzed data; SSI, SY, MR and TO wrote the manuscript.

Competing Interest Statement

The authors declare they have no competing financial interests

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