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 () 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 (). 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 ().
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 ().
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 ().
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.