An important characteristic of adaptive immunity is the capacity of antigen-specific lymphocytes to undergo rapid and extensive proliferation in response to antigenic challenge. Thus, understanding the signaling pathways that impact proliferation is critical to understanding how immune responses are generated and controlled. Previous work from a number of laboratories has outlined the importance of glycolytic metabolism in T cell responses (MacDonald and Cerottini, 1979
; Rathmell et al., 2000
; Vander Heiden et al., 2001
). The influence of lipid metabolism on lymphocyte function, however, is poorly understood. We have shown here that intracellular sterol metabolism has a previously unrecognized regulatory role in the control of acquired immune responses. Cholesterol is an essential component of membranes and therefore the requirement for adequate cholesterol in cell reproduction is obvious. However, our data reveal that the intracellular availability of sterols is dynamically regulated during T cell activation, and that this is linked to transcriptional responses mediated by SREBP and LXR, as well as to cell cycle control. Moreover, we show that the ability of LXR to alter cellular sterol metabolism through regulation of the ABCG1 sterol transporter impacts both lymphocyte proliferation and antigen-stimulated immune responses.
The initial clue to a potential role for LXR signaling in innate immune cells came from analysis of LXR null mice, which exhibit age-dependent splenomegaly and lymphadenopathy. This phenotype reflects expansion in both the T and B cell compartments and is linked to the previously unrecognized ability of LXRβ to regulate mitogen- and antigen-driven lymphocyte proliferation. Analysis of purified primary lymphoid cultures established that activation of LXRβ by physiologic or pharmacologic ligands diminishes the proliferative capacity of B and T cells. Conversely, genetic loss of LXRβ rescues cells from the inhibitory effect of LXR agonist and potentiates mitogen-and antigen-driven expansion. We also demonstrated that the ability of LXR to impact cell proliferation has a functional consequence for lymphoid homeostasis and antigen-driven immune responses in vivo. Lymphocytes lacking LXRβ expression show an exaggerated response in both homeostatic and vaccine-driven proliferation models. Thus, endogenous LXR signaling is a physiologically important determinant of immune responses and pharmacological LXR activation has the potential for immune modulation.
Given the ability of LXR to inhibit NF-κB signaling in macrophages, we initially suspected that LXR might interfere with TCR signaling and proximal pathways involved in lymphocyte activation. However, this appears not to be the case, since markers of activation and IL-2 production were not affected by LXR ligands. We also excluded the possibility that LXR activation was causing apoptosis directly, as is known to occur with GR agonists. Rather, cell cycle analysis indicated that LXR signaling was regulating the G1 to S transition. Collectively, these observations suggested that LXR was controlling the expression of one or more genes whose action was inhibitory to cell cycle progression. To identify such genes we profiled gene expression during lymphocyte activation. We found that a battery of genes linked to cholesterol homeostasis is dynamically regulated during lymphocyte activation, and that exogenous LXR ligand alters this metabolic program. In particular, the genes encoding the sterol transporters ABCA1 and ABCG1–both LXR targets–are rapidly downregulated upon T cell receptor crosslinking. The addition of LXR agonist strongly stimulates their expression. These findings led to the hypothesis that ABCG1 action alters sterol metabolism in lymphocytes in a manner that is inhibitory to proliferation. Definitive support for the requirement of ABCG1 in this model is provided by the demonstration that the ability of LXR to inhibit proliferation is markedly reduced in lymphocytes from Abcg1 null mice. A definitive link to sterol metabolism is established by the observation that the inhibitory effect of LXR is completely blocked if lymphocytes are provided with an excess of mevalonate, the precursor for cholesterol and oxysterols.
Our results reveal the existence of an endogenous sterol signaling pathway that regulates lymphocyte proliferation through coordinate regulation of SREBP and LXR activity. The changes in SREBP and LXR target gene expression we observe during T cell activation are indicative of alterations in endogenous sterol regulators of both transcription factors. Brown and Goldstein have delineated an elegant mechanism for the regulation of cholesterol synthesis in which SREBP is held as an inactive precursor in the ER by the sterol-sensing protein SCAP (Goldstein et al., 2006
). A logical conclusion of our data is that sterol content in the ER (cholesterol and/or oxysterols) must be decreasing rapidly upon lymphocyte activation. Furthermore, the reduction in LXR target gene expression during activation indicates that the nuclear availability of sterol LXR activators is also reduced. Indeed, basal expression of LXR target genes in lymphocytes is dependent on the production of endogenous LXR agonists by the mevalonate pathway.
Unexpectedly, the mechanism by which LXR activity is regulated during T cell activation does not involve alteration in ligand production, rather it is due to induction of enzymatic ligand metabolism. The enzyme SULT2B1 transfers sulfate groups to oxysterols, inactivates them as LXR ligands, and facilitates their export from the cell. We have shown that expression of SULT2B1 is dramatically increased in T cells by proliferative stimuli. This induction would be predicted to deplete the cell of LXR ligand and suppress expression of LXR target genes (Chen et al., 2007
; Fuda et al., 2007
). In fact, expression of SULT2B1 in resting lymphocytes using an adenoviral vector recapitulates the effects of proliferative stimuli on both the LXR and SREBP gene expression programs. Thus, upregulation of SULT2B1 during cell proliferation provides an elegant mechanism to affect changes in cellular cholesterol metabolism required to support new membrane synthesis and cell division. Whether additional mechanisms also contribute to regulation of LXR activity during cell proliferation remains to be addressed.
The ability of ABCG1 expression to block proliferation implicates a sterol substrate of this transporter in a metabolic checkpoint that regulates cell-cycle progression. Early studies on cholesterol synthesis in lymphocytes found that manipulation of the mevalonate pathway by the addition of sterol metabolites, such as 25-hydroxycholesterol, resulted in a G1 arrest (Chakrabarti and Engleman, 1991
). Similarly, HMG-CoA inhibitors block the proliferation of cells in multiple systems. A complicating factor in these studies is that suppression of the mevalonate pathway also perturbs synthesis of non-sterol mevalonate derivatives such as geranylgeraniol and farnesol. However, attempts to uncouple the cholesterol synthetic pathway from non-steroidal protein modifications either using low dose statins or inhibitors of downstream enzymes have revealed an absolute requirement for cholesterol in cell cycle progression and mitosis (Martinez-Botas et al., 2001
; Martinez-Botas et al., 1999
). We have observed a similar arrest in the cell cycle; however, our gene profiling studies show that we are not blocking the SREBP-2 pathway. Rather, we are likely enforcing sterol efflux or redistribution, resulting in a localized depletion of sterols.
ABCG1 is known to play an important role in cholesterol and oxysterol efflux (Kennedy et al., 2005
; Terasaka et al., 2007
; Wang et al., 2004
). Recent studies have also reported that ABCG1 is found in intra-cellular compartments, such as the ER and vesicles, and that ABCG1 expression stimulates SREBP-2 activity through the redistribution of sterols out of the ER (Tarr and Edwards, 2007
). We hypothesize that adequate levels of one or more sterols in a particular cellular compartment, likely the ER, are read by the cell cycle machinery as an indication of appropriate metabolic conditions for cell division. Downregulation of ABCG1 during activation may be necessary to maintain compartmentalization of these sterols. Forced induction of ABCG1 by LXR activation reduces the availability of this signaling sterol. In the absence of LXR, increased sterol levels act as a stimulus to proliferation. At present, we favor the hypothesis that cholesterol itself is the sterol being sensed by the cell cycle machinery, because the ability of LXR agonists to block proliferation indicates that the signaling sterol is not an LXR agonist.
In summary, this work outlines a previously unrecognized role for LXRβ and sterol signaling in the regulation of lymphocyte function. Although our focus in this report has been on lymphocytes, the ability of the SULT2B1-LXR-ABCG1 axis to couple cellular cholesterol metabolism and proliferation is likely to be applicable to many cell types, particularly those undergoing rapid cell division. Finally, given that LXR responds to endogenous lipids whose availability may be altered in disease, our results raise the possibility that LXR signaling may impact acquired immune responses in human metabolic diseases such as dyslipidemia and atherosclerosis.