The central finding of this study is that KLF2 inhibits HIF-1α dependent myeloid cell activation. Specifically, we find that: (1) KLF2 expression is regulated by hypoxia and bacterial products in a manner that is anti-parallel to that of HIF-1α, (2) this anti-parallel pattern of expression is recapitulated in human subjects with sepsis, (3) KLF2 transcriptionally inhibits the NFκB-HIF-1 axis and attendant myeloid cell functions, (4) myeloid deficiency of KLF2 confers a pro-inflammatory milieu that offers protection in the context of polymicrobial infection but is deleterious in the setting of LPS-induced endotoxic shock, and (5) the phenotype of Lyz2creKlf2fl/fl mice is rescued by inhibition or ablation of HIF-1α. Collectively, these observations identify KLF2 expression and function as a critical component of the innate immune response to bacterial infection and endotoxic shock.
Our studies provide important insights regarding the role of KLF2 as a transcriptional regulator of both myeloid cell quiescence and activation. On the basis of these observations, we propose the model outlined in the Graphical Abstract, available online. Circulating myeloid cells, which are in the quiescent state, express robust levels of KLF2 and low expression of HIF-1α. As a consequence, circulating concentration of pro-inflammatory cytokines are held in check at vanishingly low. The importance of this basal KLF2 expression is underscored by the fact that its deficiency leads to spontaneous activation of myeloid cells – which is manifest as elevated serum concentreation of several pro-inflammatory cytokines in Lyz2creKlf2fl/fl mice. Our studies suggest that this modest increase in basal cytokines is secondary to derepression of HIF-1α. In support, a modest but appreciable increase in basal HIF-1α mRNA and protein are observed in KLF2-deficient macrophages. Furthermore, the increase in basal pro-inflammatory cytokines is completely abolished by compound deficiency of KLF2 and HIF-1α.
Our studies also indicate that release from KLF2-mediated repression is critical for optimal myeloid cell activation. Upon egress into tissue, myeloid cells encounter hypoxic tissues as well as foreign pathogens. Importantly, we find that both hypoxia and bacterial products reduce KLF2 expression while robustly activating HIF-1α expression and/or activity. These findings may be clinically relevant as a reduction in KLF2 mRNA and enhanced HIF-1α mRNA expression were seen in circulating human myeloid cells from patients with sepsis. This reduction in KLF2 expression appears to be required for optimal HIF-1α activation as sustained expression of KLF2 strongly attenuates the NFκB mediated induction of HIF-1α mRNA and protein. The coordinated reduction in KLF2 and induction of HIF-1α allows for optimal bactericidal activity. Consistent with this idea, KLF2 deficient myeloid cells are “primed” for activation in the basal state and exhibit robust bacterial killing in antibiotic protection assays and confer improved survival following CLP with reduced bacterial burden in blood and peripheral tissues. The enhanced expression of HIF-1α observed in KLF2-null cells is clearly an important mediator of the observed phenotype as myeloid cells deficient in both KLF2 and HIF-1α lose bactericidal activity and succumb more readily following CLP challenge. Our observations from the LPS- induced endotoxic shock experiments represent an extension of this line of reasoning. In the event that initial host defense efforts fail, bacterial products can leach out into the circulation and induce an overwhelming inflammatory response. In this case, it is anticipated that KLF2 expression will fall both in circulating and non-circulating myeloid cells leading to an exaggerated induction of HIF-1α and an inflammatory storm. Consistent with this model, Lyz2creKlf2fl/fl
mice exhibit a profound intolerance to LPS-induced sepsis. Further, exceptionally high concentration of cytokines (especially IL-1β, MCP-1, IL-17 and TNF-α) was observed after LPS challenge, rendering animals unable to sustain themselves against elevated concentration of bacterial endotoxins. Again, the importance of HIF-1α in this setting is underscored by the fact that Lyz2creKlf2fl/flSetd2fl/fl
mice exhibit enhanced survival after LPS challenge. The latter finding is also consistent with the observations of Pessyoneux and colleagues who demonstrated that myeloid HIF-1α deficiency renders rodents resistant to LPS-induced sepsis (Peyssonnaux et al., 2007
). Collectively these studies identify a KLF2-HIF-1 axis as critical in regulating the balance between myeloid quiescence and activation.
Our mechanistic insights suggest that in the context of inflammatory stimuli such as LPS, KLF2’s ability to inhibit HIF-1α expression occurs primarily through the inhibition of NFκB-dependent induction of HIF-1α mRNA expression. Our data coupled with previous observations (Das et al., 2006
; SenBanerjee et al., 2004
) suggest that KLF2 does not affect the NFκB pathway at the cytosolic signaling or the recruitment of NFκB to the endogenous HIF-1α promoter. Intriguingly, the main effect lies at the level of co-activator recruitment. Sustained expression of KLF2 inhibits while deficiency augments p300 and PCAF recruitment to the HIF-1α promoter. Previous studies by our group and others reveal that KLF2 can interact directly with both p300 and PCAF and thus, in the setting of sustained KLF2 expression, this likely constitutes the molecular basis for preventing p300-PCAF recruitment to NFκB (Ahmad and Lingrel, 2005
; SenBanerjee et al., 2004
). This type of control at the target gene transcription is atypical and provides a particularly elegant and important “molecular brake” that titrates the inflammatory transcriptional response. As KLF2 expression fall, unbound p300-PCAF can more freely interact with NFκB and induce target genes. We note, however, that in the setting of an infected tissue, HIF-1α expression are induced not only through de novo
transcription but also secondary to protein stabilization. In this regard, a recent report by Kawanami and colleagues showed that in hypoxic endothelial cells KLF2 can also reduce HIF-1α protein stability by disrupting interaction with its chaperone Hsp90 (Kawanami et al., 2009
). Whether a similar mechanism is operative in hypoxic myeloid cells is an important area for future investigation. Collectively, our observations along with previous work, suggest that KLF2 negatively regulates HIF-1α at both transcriptional and post-transcriptional levels.
We note that although the survival phenotype observed in myeloid KLF2 deficient animals supports a dominant role for HIF-1α, there is evidence that additional mechanisms may also be operative. For example, because myeloid deficiency of HIF-1α, KLF2 and HIF-1α mice display similar rates of survival in experimental models of sepsis one may also expect minimal differences in gene expression or functional responses in cell and/or animals bearing these two genotypes. Whereas this appears to be the case for many key myeloid genes functions, several other parameters demonstrate differential responses between the Lyz2creSetd2fl/fl
genotypes. Additionally, although much of our study highlights the induction of pro-inflammatory targets following KLF2 depletion, our microarrays also show reduced expression of numerous factors with potent anti-inflammatory properties. These factors have diverse cellular functions and include enzymes (e.g. arginase-1 and TIMP3), growth factors (e.g. TGFβ superfamily members), secreted molecules (e.g. Retnla and Cyr61), and transcription factors (e.g. PPARδ) (Barish et al., 2008
; Gill et al., 2010
; Nair et al., 2009
). We note that because our microarray study was conducted at a single time point, one may be underestimating the full spectrum of KLF2-regulated targets. Indeed, kinetic microarray and/or RNA-seq approaches (Wang et al., 2009
) will be helpful towards gaining additional insights regarding KLF2 action in myeloid biology. The importance of these additional regulatory pathways will require additional investigation and serve as the focus of future studies.
Finally, the observations presented in this study coupled with previous studies of KLF2 biology bear important implications for the clinical syndrome of sepsis. We note that the multi-organ failure that typifies end-stage sepsis occurs not only through exuberant myeloid cell activation but also secondary to widespread endothelial damage (Aird, 2003
). Characteristically, endothelial injury in sepsis leads to diffuse vascular dysfunction manifest as enhanced permeability, intravascular coagulation, and loss of vascular tone (Schouten et al., 2008
). A robust literature indicates that endothelial KLF2 is an essential regulator of endothelial homeostasis (Atkins and Jain, 2007
) and confers an anti-inflammatory, anti-thrombotic, and anti-adhesive phenotype to the vessel wall (Dekker et al., 2006
; Lin et al., 2006
; Lin et al., 2005
). Indeed, KLF2+/−
mice exhibit a pro-inflammatory (Atkins et al., 2008
) and pro-permeable vasculature (Lin et al., 2010
). Collectively, these observations along with findings presented in the current study, suggest that a reduction in endothelial and myeloid KLF2 expression may be functionally important in regulating the organism’s response to sepsis. As such, manipulation of KLF2 expression may offer new opportunities for therapeutic gain. Indeed, agents known to induce KLF2 expression such as statins have been shown to ameliorate experimental sepsis and are being considered for clinical application(Liappis et al., 2001