Macrophage activation screening of ~200 known and predicted kinases revealed the surprising finding that some of the strongest gene ontology enrichment was observed for pathways regulating carbohydrate metabolism. Indeed, AKT1 and STK11, both identified in our kinase screen, while classically thought of as nutrient sensors, also control a fate switch, from cytotoxic effector to memory CD8+
T cells (Finlay and Cantrell, 2011
). More surprising was the robust enrichment of nonprotein kinases potentially capable of contributing directly to metabolic flux through defined pathways. This finding, coupled with our more detailed characterization of CARKL, supports a robust interdependency of metabolism and immune cell function while at the same time highlighting our overall lack of detailed knowledge on the subject.
Indeed, cellular immune activation is a metabolically costly endeavor and cannot operate effectively under energy deficit (Demas et al., 1997
). Among the nonprotein kinase candidates observed to modulate TNFα secretion in our screen, HK, the PFK family, and CARKL are strategically positioned to control glucose usage according to cellular requirements. HK regulates intracellular glucose availability. Fructose-1-6-bisphosphate (F-1-6bp) formation by PFK is an irreversible step in glycolysis that determines glycolytic flux and, not surprisingly, is the target of potent antagonistic regulation by key metabolic hormones, such as glucagon and insulin. Our work adds S7P formation by CARKL as a novel rate-limiting lever for the system, balancing the G3P and S7P intermediates of nonoxidative PPP and glycolysis (Figure S7
). Interestingly, a novel sedhoheptulose-1-7-bisphosphatase, which forms S7P from S1-7bp, was very recently reported as a crucial enzyme in yeast ribosome biogenesis with obvious consequences (Clasquin et al., 2011
One particularly interesting observation was the highly significant drop of NADH levels in pCARKL cells during macrophage activation, which resulted in substantial redox shift (D). PPP activity contributes to reduction of redox couples via NADPH. We therefore expected and observed increased GSH and NADH generation during M1 activation, a phenotype that could be reversed either by administration of DHEA to block the PPP directly or by counterbalancing CARKL loss. M2-like activation, on the other hand, resulted in an upregulation of CARKL which was not followed by increased GSH or NADH formation. These data represent a functional distinction between the two polarization states that is, importantly, CARKL dependent. Further, the pattern of baseline changes observed in the pyridine pool in miCARKL and pCARKL cells (Figure S4
A) provides an interesting potential link to poly ADP-ribose polymerase (PARP) protein family involvement. PARPs are responsible for NAD degradation and important for immune cell function, e.g., activation of NFκB (Oliver et al., 1999; Pollak et al., 2007
). In keeping with this idea, NADP pools were much less affected than NAD in our system, a common feature of PARP-mediated pyridine regulation (Pellny et al., 2009
It has been reported that several master regulators of inflammation are sensitive to ROS and that their function is dependent on cellular redox states (Kazama et al., 2008; Kumar et al., 1992; Toledano and Leonard, 1991; Wang et al., 1999
). For instance, NFκB is sensitive to cellular redox equilibrium (Perl et al., 2002
). This confirms the recent findings of Kawauchi and colleagues reporting the bidirectional influence of NFκB and glucose metabolism in cancer cell physiology (Kawauchi et al., 2008
). Interestingly, although CARKL reduced total active NFκB, relative induction by LPS was only slightly affected. It remains to be established how exactly CARKL and the NFκB system rely on each other. We further report CARKL as negative regulator of LPS-induced SOCS3 expression, without influencing SOCS1 levels, but resulting in enhanced STAT3 phosphorylation. SOCS3 is commonly known as repressor of STAT3. It will be interesting to see whether CARKL directly impacts the SOCS/STAT system (). Specific SOCS isoforms appear critical to eliciting pro- versus anti-inflammatory macrophage states (O'Shea and Murray, 2008; Whyte et al., 2011; Yoshimura et al., 2007
CARKL Regulation of M1/M2 Activation
And what of clinical relevance? Metabolic reconfiguration and mild M1-like polarization are triggered by CARKL loss even in the absence of additional stimulus. While the phenomenon is not fully understood, cystinosis patients, who harbor a 57 kb deletion affecting CARKL
despite showing no strong signs of inflammation, respond positively to continuous anti-inflammatory treatment (Haycock et al., 1982
). Further, our data may provide insight into potential new modes of action for drugs which clearly bridge both immune and metabolic signaling, such as glucocorticoids and rapamycin.
In summary, this study identifies CARKL as a sedoheptulose kinase orchestrating pro- and anti-inflammatory immune responses through metabolic control. Our results highlight the fundamental nature of metabolic reconfiguration in macrophage activation and define unique metabolic demands of distinct activation states.