Molecular Mechanism responsible for the priming of macrophage activation

Host macrophages can be preprogrammed into opposing primed or tolerant states depending upon the nature and quantities of external stimulants. The paradigm of priming and tolerance has significant implications in the pathogenesis and resolution of both acute and chronic inflammatory diseases. However, the responsible mechanisms are not well understood. Here, we report that super low dose bacterial endotoxin lipopolysaccharide (LPS), as low as 5 pg/ml, primes the expression of proinflammatory mediators in macrophages upon a second high dose LPS challenge (100 ng/ml), although 5 pg/ml LPS itself does not trigger noticeable macrophage activation. Mice primed with super low dose LPS (0.5 μg/kg body weight) in vivo experience significantly elevated mortality following a second hit of high dose LPS as compared with saline-primed control mice. Mechanistically, we demonstrate that LPS primes macrophages by removing transcriptional suppressive RelB through interleukin receptor-associated kinase 1 and Tollip (Toll-interacting protein)-dependent mechanisms. This is in sharp contrast to the well documented RelB stabilization and induction by high dose LPS, potentially through the phosphoinositide 3-kinase (PI3K) pathway. Super low dose and high dose LPS cause opposing modulation of interleukin receptor-associated kinase 1 and PI3K pathways and lead to opposing regulation of RelB. The pathway switching induced by super low versus high dose LPS underscores the importance of competing intracellular circuitry during the establishment of macrophage priming and tolerance.


SUMMARY
Host macrophages can be preprogrammed into opposing primed or tolerant states depending upon the nature and quantities of external stimulants. The paradigm of priming and tolerance has significant implications in the pathogenesis and resolution of both acute and chronic inflammatory diseases. However, the responsible mechanisms are not well understood. Here, we report that super low dose bacterial endotoxin lipopolysaccharide (LPS), as low as 5 pg/mL, primes the expression of proinflammatory mediators in macrophages upon a second high dose LPS challenge (100 ng/mL), although 5 pg/mL LPS itself does not trigger noticeable macrophage activation. Mice primed with super low dose LPS (0.5g/kg body weight) in vivo experience significantly elevated mortality following a second-hit high dose LPS as compared to saline-primed control mice.
Mechanistically, we demonstrate that LPS primes macrophages by removing transcriptional suppressive RelB through interleukin receptor-associated kinase 1 (IRAK-1) and Toll-interacting protein (Tollip) dependent mechanisms. This is in sharp contrast to the well-documented RelB stabilization and induction by high dose LPS, potentially through the phosphoinositide-3-kinase (PI3K) pathway. Super low dose and high dose LPS cause opposing modulation of IRAK-1 and PI3K pathways, and lead to opposing regulation of RelB. The pathway switching induced by super low vs high dose LPS underscores the importance of competing intracellular circuitry during the establishment of macrophage priming and tolerance. ____________________________________

INTRODUCTION
Host innate immune system can not only recognize the structural nature of foreign molecular patterns, but also discern the history and concentration of foreign stimulants. This rudimentary innate immune memory is best reflected in the paradigm of endotoxin priming and tolerance (1). During disseminated endotoxin shock and acute sepsis, bacterial endotoxin (lipopolysaccharide, LPS) ranging from 1 to 300 ng/mL can induce a robust cytokine storm (2,3). Intriguingly, cells pre-exposed to LPS are hypo-responsive to a second LPS challenge in terms of pro-inflammatory gene expression, a phenomenon known as endotoxin tolerance (4). Consequently, a rapid termination of inflammatory responses soon ensues that serves to dampen collateral inflammatory damage.
In contrast, a subclinical super low dose of LPS (<100pg/mL) causes a distinct effect by priming cells for more robust expression of pro-inflammatory mediators in response to a second LPS challenge (5)(6)(7). Recently, super low levels of LPS have been detected in circulation of humans and experimental animals with adverse health conditions including obesity, chronic smoking, infection, and aging (8)(9)(10)(11)(12). Humans with these adverse conditions tend to have elevated mortality associated with septic shock (13,14). Endotoxin priming and exacerbated mortality is also observed in experimental animals (15,16).
The molecular mechanism responsible for the opposing effects of endotoxin priming and tolerance is not well understood. Endotoxin tolerance has drawn most of the research effort in the past. LPS (>1ng/mL) is known to induce an initial wave of robust NFB activation, followed by NFBinduced negative regulators including inhibitor of kappa-B alpha (IB, MAPK phosphatase 1 (MKP-1), interleukin receptor associated kinase M (IRAK-M) and RelB (17)(18)(19). These negative regulators shut down signaling pathways and the expression of pro-inflammatory mediators at multiple levels. For example, IB suppresses p65/RelA by secluding it in the cytoplasm; MKP-1 suppresses the activities of MAP kinases (20); IRAK-M suppresses interleukin receptor associated kinase 1 (IRAK-1) (21); and RelB blocks the transcription of pro-inflammatory mediators by assembling a suppressive complex on their promoters (19). In addition, LPS is known to activate the phosphoinositide-3kinase (PI3K) pathway which dampens the expression of pro-inflammatory mediators through inducing negative regulators including MKP-1, IRAK-M, and suppressing IRAK-1 (22)(23)(24)(25)(26). As a consequence, LPS (>1ng/mL) causes a robust yet transient expression of proinflammatory mediators, followed by a refractory tolerant state.
However, the mechanism of priming effect is less studied and understood.
We first reported that low dose LPS (50-100pg/mL) fails to activate either the robust NFB pathway or the MAP kinase pathway (27). As a consequence, low dose LPS fails to induce robust expression of proinflammatory mediators. On the other hand, we documented that low dose LPS is capable of removing transcriptional suppressors from the promoters of inflammatory genes (27).
The objective of this study is to examine the molecular mechanism responsible for the paradigm of LPS priming and tolerance. By utilizing wild type (WT), IRAK-1 and Tollinteracting protein (Tollip) deficient primary murine bone marrow derived macrophages (BMDM), we identified an opposing effect of super low dose LPS vs high dose LPS on the regulation of PI3K and RelB. While high dose LPS activates PI3K pathway and increases of RelB levels, super low dose LPS suppresses PI3K, and reduces the levels of RelB. The pathway switching triggered by super low dose vs high dose LPS underlies the paradigm of LPS priming and tolerance.

MATERIALS AND METHODS
Reagents-LPS (Escherichia coli 0111:B4) and lipoteichoic acid (LTA) were purchased from Sigma Aldrich. Anti-IB α, ATF2, RelB, p65, IRAK-1, IRAK-M, GAPDH, -Actin antibodies were obtained from Santa Cruz Biotechnology. Anti-mouse IgG and anti-rabbit IgG horseradish peroxidaselinked (HRP) antibodies were purchased from Cell Signaling Technology. The proteasome inhibitor MG132 was from Calbiochem. Mice and cell culture-Wild type (WT) C57BL/6 mice were purchased from the Charles River laboratory. IRAK-1 deficient mice from C57BL/6 background were kindly provided by Dr. James Thomas from the University of Texas Southwestern Medical School. Tollip deficient mice from C57BL/6 background were provided by Dr. Jürg Tschopp from the University of Lausanne at Switzerland. All mice were housed and bred at Derring Hall animal facility in compliance with approved Animal Care and Use Committee protocols at Virginia Polytechnic Institute and State University.
Bone marrow derived macrophages (BMDM) were isolated as we previously described (27). Wild type (WT) Raw264.7 and GP96 knocked-down (GP96KD) Raw264.7 cells defective in cell surface TLR4 were maintained as described previously (28). Analysis of protein and mRNA-Western blots and RNA extractions were performed as previously described (27). The relative levels of different transcripts were calculated using the ΔΔCt method and results were normalized based on the expression of Gapdh. The relative level of mRNA in untreated WT cells was adjusted to 1 and served as the basal reference value. The following primer pairs were used: Western Blots and Analyses of cytokines-Subsequent to indicated treatments and 3 washes with PBS, whole cell lysates were harvested from cells using 1×SDS lysis buffer containing protease inhibitor cocktail (Sigma). Equal amounts of protein extracts were subjected to SDS-PAGE gel (12%), and transferred to PVDF membranes, and visualized with specific Abs using the Amersham Biosciences ECL Plus chemiluminescent detection system(GE Healthcare). Meanwhile, plasma of mice or cell supernatants were collected after treatments by centrifugation and stored at -70 o C before examination. Analyses of cytokines, including IL6 and TNFα were carried out according to manufacturer's procedure (Bio-Rad laboratories, Hercules, California, USA). Briefly, samples and standards were diluted and tested by ELISA which involving several incubation and washes steps. Magnetic Beads coupled with antibodies, biotinylated detector antibodies and phycoerythrin-conjugated streptavidin were added in samples subsequently and incubated with certain time. The test plate was then read on a Bio-Plex system (Bio-Rad laboratories). Raw data were analyzed, and standard curves were generated by reference cytokine concentrations. All samples and standards were tested in duplicates.
Chromatin-immunoprecipitation analysis-Chromatin immunoprecipitation (ChIP) analyses were performed as described previously using Chip-IT Express Kit (Active Motif TM ) (27). The immunoprecipitated DNA fragments were analyzed by PCR using primers spanning the binding sites of RelB on the proximal promoter of mouse IL6. The following are the primer sequences used to amplify the enriched chromatin samples: promoter of mouse IL-6 forward: 5'-TCC CAT CAA GAC ATG CTC AAG TGC-3', reverse: 5'-AGC AGA ATG AGC TAC AGA CAT CCC-3'. Statistical analysis-For all in vitro studies, statistical significances between groups were determined using the Student's t test and indicated by an asterisk in figures; p values < 0.05 were considered statistically significant.
For the in vivo cytokine analyses, the Mann-Whitney U test was performed. The log-rank test was used to assess significant difference in the mortality rate, with p values < 0.05.

Super low dose LPS primes, while high dose LPS tolerizes, the induction of proinflammatory mediators in macrophages
We first determined the expression of interleukin 6 (Il-6) and tumor necrosis factor alpha (Tnf in WT BMDM treated with varying dosages of LPS. As shown in Fig This is consistent with our early report that ~50pg/mL LPS is the threshold concentration required to trigger mild and low grade expression of pro-inflammatory mediators. Indeed, we observed that a super low dose LPS (5 pg/mL) failed to induce noticeable expression of Il-6 and Tnf (Fig.  1A).
We then tested whether super low dose and high dose LPS may cause differential priming and tolerance of macrophages in response to subsequent LPS challenge. WT macrophages were pretreated with 0, 5 pg/mL, or 10ng/mL LPS for 4 hours, followed by washes with PBS and fresh medium. Pretreated cells were re-stimulated with 100ng/mL LPS for 4 hours. We chose the 4 hour-time interval between the two consecutive LPS treatments based on wellestablished observations regarding LPS priming and tolerance (5,7,29). Furthermore, we chose this shorter-time interval rather than the alternative overnight interval to avoid potential complications of cell proliferation, death, and other paracrine effects with an extended LPS treatment. As shown in Fig. 1A, despites its failure to induce any expression of Il-6 and Tnf, 5 pg/mL LPS potently primed the induction of Il-6 and Tnf in macrophages challenged with a second dose LPS. In contrast, high dose LPS (10ng/mL) significantly suppressed the induction of Il-6 and Tnfupon secondary challenge. To further confirm that low dose LPS indicate prime the protein expression of pro-inflammatory mediators, we measured the protein levels of IL-6 in macrophages treated with a high dose LPS alone or primed with a low dose LPS prior to a high dose LPS treatment. As shown in Fig. 1B, low dose LPS significantly primed the production of IL-6 protein in WT macrophages.

Opposing intracellular effects of super low dose and high dose LPS
Our previously study revealed that low dose LPS (50-100pg/mL) fails to induce classical NFB pathway and MAP Kinases (27). Further confirming this finding, we observed that super low dose LPS fails to cause IB degradation, or induction of activating transcription factor 2 (ATF2), a member of the AP-1 family of transcription factors ( Fig. 2A). To further examine the molecular mechanisms responsible for the opposing effects of super low dose vs high dose LPS, we examined the proximal signaling components of the Toll-like receptor 4 (TLR4) pathway. As shown in Fig. 2B, super low dose LPS (5 pg/mL) did not trigger any noticeable IRAK-1 degradation (Fig. 2B). This is in contrast to the effect of high dose LPS (>100 ng/mL) that is known to cause IRAK-1 degradation (30).
High dose LPS is also known to induce IRAK-M, a negative regulator of IRAK-1 and the TLR4 pathway that contributes to LPS tolerance (30).
In contrast, we observed that super low dose LPS failed to induce IRAK-M protein levels in macrophages (Fig. 2B). Instead, a slight yet significant reduction in IRAK-M occurs after 2 h of super low dose LPS treatment. Other negative regulatory pathways induced by high dose LPS include the PI3K pathway, that contributes to the suppression of IRAK-1/TNF receptor associated factor 6 (TRAF6) (22), the induction of negative regulators including IRAK-M (25,31), and the induction of negative transcriptional suppressors of inflammatory genes such as RelB and cAMP response element-binding (CREB) (32,33). Thus, we tested the effects of super low dose LPS on the activation status of PI3K pathway. As shown in Fig.  2C, high dose LPS induced robust phosphorylation of Akt, an indication of PI3K activation. In contrast, super low dose LPS triggered a decrease in the basal levels of phosphorylated Akt.

Differential regulation of RelB by super low vs high dose LPS
RelB has been identified as a crucial modulator of endotoxin tolerance by assembling a suppressive complex on the promoters of pro-inflammatory genes (19). Deletion of RelB leads to excessive pro-inflammatory responses both in vitro and in vivo (34)(35)(36). High dose LPS is welldocumented to induce RelB, through NFB as well as PI3K mediated expression (33,35). However, no report is available regarding the status of RelB in cells challenged with super low dose LPS. We observed that super low (5pg/mL) or low dose (50pg/mL) LPS led to a reduction of RelB protein in macrophages (Fig. 3A). This is in contrast to a robust induction of RelB by high dose LPS (Fig. 3A). It is worth mentioning that high dose LPS led to an initial transient decrease in RelB (at 0.5 hour), followed by a significant increase at the 2 hour time point. The initial decrease of RelB correlates with the transient induction of pro-inflammatory mediators by high dose LPS.
To further confirm the involvement of RelB in differential gene expression induced by super low and high dose LPS, we performed chromatin immunoprecipitation analysis (ChIP) on the promoter of the Il-6 gene. As shown in Fig. 3B, 4 hour priming with 5pg/mL LPS removed RelB from the Il-6 promoter, and prevented RelB association with the Il-6 promoter following additional high dose LPS treatment.

IRAK-1 and Tollip are required for the suppression of RelB
We then studied the upstream molecular mechanisms responsible for the opposing regulation of RelB by super low dose and high dose LPS. Based on previous studies that IRAK-1 and Tollip are preferentially responsible for mediating the proinflammatory responses of low dose LPS (27,37,38), we tested the regulation of RelB in WT, IRAK-1 knockout, and Tollip knockout BMDM. As shown in Fig. 4A, super low dose LPS preferentially reduced the levels of RelB in WT, but not in IRAK-1 or Tollip deficient cells. It is interesting to note that the resting levels of RelB were significantly higher in untreated IRAK-1 deficient and Tollip deficient cells. Given the fact that IRAK-M counteracts the function of IRAK-1, we also tested the levels of RelB in IRAK-M deficient BMDM. We observed that RelB was lower in IRAK-M deficient cells as compared to that in WT cells (Fig. 4B).
In addition to its induction, RelB was recently reported to undergo proteasomemediated degradation (39). To test whether proteasome-mediated degradation may account for LPS-induced decrease in RelB levels, we applied the proteasome inhibitor MG-132. As shown in Fig. 4C, MG-132 effectively stabilized RelB and blocked RelB level decrease initiated by super low dose LPS challenge.
The degradation of RelB was recently shown to be regulated by GSK3 (39). To further determine whether low dose LPS may differentially modulate GSK3 activation in WT, IRAK-1 and Tollip deficient cells, we examined the phosphorylation status of GSK3. GSK3 activity can be differentially regulated by two distinct phosphorylation events at Serine 9 and Tyrosine 216. Ser9 phosphorylation suppresses, while Tyr216 phosphorylation activates GSK3 activity. We observed that 5 pg/mL LPS induced Tyr216 phosphorylation, and suppressed Ser9 phosphorylation of GSK3 (Fig. 5D), an indication of GSK3 activation. The resting levels of p-Y216-GSK3 were lower, and the resting levels of p-S9-GSK3 were higher in IRAK-1 and Tollip deficient cells. Furthermore, low dose LPS failed to induce Tyr216 in either IRAK-1 or Tollip deficient macrophages (Fig. 4D).

IRAK-1 and Tollip are required for the priming of macrophages
Based on our finding, we tested whether IRAK-1 and Tollip may contribute to the priming effects of super low dose LPS in the induction of pro-inflammatory mediators. WT, IRAK-1 deficient and Tollip deficient BMDM were primed with 0 or 5pg/mL LPS for 4 hours. Following washes with PBS and fresh medium, they were treated with 100ng/mL LPS for an additional 4 hours. As shown in Fig. 5, super low dose LPS effectively primed the induction of Il-6, Tnf, Scavenger Receptor A (Sr-a), and kynurenine 3-monooxygenase (Kmo) in WT cells. In sharp contrast, the priming effect of super low dose LPS was ablated in IRAK-1 or Tollip deficient cells.

In vivo priming by super low dose LPS
We then tested the in vivo effect of LPS priming. WT and IRAK1 deficient mice were injected with either PBS or an initial super low dose LPS (0.5g/kg body weight). Four hours later, they were injected with a high dose of LPS (25 mg/kg body weight). The mortality was closely observed every four hours for two days. As shown in Fig.  6A, PBS-primed mice only experienced initial adverse signs including lack of movement, eye discharge, and black, tar-like feces, but eventually survived. In sharp contrast, priming with super low dose LPS caused 70% mortality in WT mice. On the other hand, all IRAK-1 deficient mice survived the priming with either PBS or super low dose LPS, although some displayed the symptoms described above.
We also measured the plasma levels of TNF and IL-6 in PBS and LPS-primed mice. As shown in Fig. 6B, priming by super low dose LPS alone caused only modest expression of IL-6 and TNF. Expectedly, the high dose LPS dramatically increased the plasma levels of IL-6 and TNF in WT mice primed by super low dose LPS, as compared to the PBS control group (a 6.2-fold increase for IL-6 and a 1.8fold increase for TNF). In contrast, the priming effect of super low dose LPS on the expression of IL-6 and TNF was ablated in IRAK-1 deficient mice.

Cross-priming by lipoteichoic acid and oxLDL
To test whether priming can be induced by other TLR agonists in addition to LPS, we tested the effect of selected TLR2 agonist lipoteichoic acid (LTA) and TLR4 agonist oxidized low density lipoprotein (oxLDL). As shown in Figure 7, both low dose LTA (5 ng/mL) and oxLDL can significantly prime macrophages for a more robust expression of Il-6 gene following a second dose LPS.
In terms of the mechanism, we observed that LTA similarly reduces the levels of RelB, as well as the phosphorylation of Akt, and increase the GSK3 phosphorylation. The cross-priming by oxLDL may underlie the pathogenesis of atherosclerosis. A potential paradigm for the dynamic priming and tolerance is proposed in Figure 8.

DISCUSSION
This study reveals potential mechanisms responsible for the paradigm of LPS priming and tolerance (Fig. 7). Our data suggest that super low dose LPS primes the expression of pro-inflammatory mediators by removing the transcriptional suppressive RelB, thus enabling a more robust expression of proinflammatory mediators in cells challenged with an additional dose of LPS. On the other hand, high dose LPS initially reduces, and later induces RelB. This may underlie the transient induction and eventual tolerization/suppression of pro-inflammatory mediators. IRAK-1 and Tollip contribute to RelB degradation induced by super low dose LPS. The differential modulation of RelB by super low and high dose LPS may be mediated through opposing regulation of upstream signaling molecules involving IRAK-1, PI3K.
Super low dose LPS deactivates PI3K responsible for RelB induction. On the other hand, super low dose LPS leaves IRAK-1 intact, and may enable IRAK-1 mediated degradation of RelB. In contrast, high dose LPS activates PI3K and induces RelB.
Our findings reveal anew the paradigm of opposing effects by varying dosages of stimulants. In addition to LPS, other highly important cellular mediators including cytokines, hormones, and adipokines can manifest the similar paradigm. For example, high levels of interleukin-1 (IL-1) (>1 ng/mL) are known to induce robust cytokine storm and cell death (40-42). In contrast, low levels of IL-1 (~10pg/mL) can induce cellular proliferation (42). Likewise, both insulin and leptin are known to trigger either cellular sensitivity or resistance/tolerance (43,44). These phenomena underscore the complex adaptation of cellular systems that ensure proper physiological adjustments to changing environments, with the intended goal of bringing the cellular systems back to homeostatic states. However, unintended side-effects of these adjustments often occur that lead to both chronic and acute human inflammatory diseases. For example, LPS tolerance during the late stage of septic shock is intended to reduce and minimize the harmful pro-inflammatory cytokine storm (45).
Yet this very hyporesponsiveness may subject the host to more severe secondary infection and death (4,46). On the other hand, LPS priming may alert the host to the presence of bacterial infection and facilitate bacterial clearance (5). However, exacerbated responses in low dose LPS-primed individuals during severe bacterial infection and disseminated sepsis may contribute to higher mortality. Maintaining the proper balance between these adjustments is crucial for survival and long-term health.
Our findings provide mechanistic insight regarding the dynamic maintenance of LPS priming and tolerance. Specifically, our data support the theory that competing intracellular players including PI3K and IRAK-1 are differentially modulated by super low and high dose LPS, and that their opposing regulation may bear critical significance in LPS priming and tolerance. The competitive interactions between these molecules have been noticed previously (22,26), although their opposing regulation by low and high dose LPS has not been previously studied. High dose LPS was extensively used in previous mechanistic studies and can degrade IRAK-1, activate PI3K pathway, and induce IRAK-M (30,47,48) (Figure 6). IRAK-M was shown to inhibit IRAK-1 function (21). PI3K pathway was shown to increase IRAK-M and down-regulate both the levels and activities of IRAK-1 (22,23,26,31). Furthermore, PI3K also induces RelB (49). As a consequence, these studies explain the phenomenon of LPS tolerance. Our data indicate that the effect of super low dose LPS on these competing players flips as compared to high dose LPS. We found that super low dose LPS maintains IRAK-1 and deactivates the PI3K pathway. This may enable macrophage priming instead of tolerance. It is worth mentioning that an analogous paradigm has recently been defined during the differentiation of T helper 17 (Th17) and T regulatory (Treg) cells. Transforming growth factor beta (TGF was shown to be involved in the opposing differentiation of both Th17 and Treg cells (50,51). The competing circuit between the transcription factors forkhead box P3 (FoxP3) and retinoic acid-related orphan receptor gamma T (RORT) can be differentially modulated by varying dosages of TGF, and as a consequence, the differentiation of CD4 + T cells can be skewed toward either Th17 or Treg predominance (52). Our current study adds another example of the dynamic and competing circuitry that controls the fate of immune cell function.
Although our findings provide a first step toward the complex regulation of LPS priming and tolerance, the proximal sensing of super low and high dose LPS remains a mystery. Although it is beyond the scope of this study, we hypothesize that differential dimerization of TLR4 with other coreceptors may play a role. It is known that TLR4 can heterodimerize with CD36, SR-A, or MAC-1/CD11b (53,54). MAC-1/TLR4 may preferentially induce PI3K activation and suppression of inflammatory responses (55).
In contrast, CD36/TLR4 was implicated in the propagation of chronic inflammatory processes (53). Future studies are clearly warranted to determine the usage of TLR4 co-receptors by super low dose LPS. Consequently, super low dose LPS and high dose LPS may cause pathway switching at multiple levels, ultimately leading to the opposing regulation of IRAKs and PI3K. Corroborating this, it has been reported that multiple upstream kinases including IRAK-4 and protein kinase C (PKC) can potentially activate IRAK-1. (56,57). We postulate that low versus high dose LPS may differentially activate IRAK-1 upstream kinases that may either cause IRAK-1 degradation and subsequent tolerance, or persistent activation and priming.
Further detailed studies are warranted to test this hypothesis. In addition to IRAKs and PKC, TLR4 signaling is known to activate protein tyrosine kinases such as Btk, Hck, and Pyk2 (58-60). High dose LPS can activate PI3K through MyD88 tyrosine phosphorylation and recruitment of PI3K adaptor p85 (61). On the other hand, a recent report revealed that PI3K can be potently suppressed by PKC (62). It is reasonable to speculate that differential activation of protein tyrosine kinases, PKC, and IRAK-4 may be induced by super low dose vs high dose LPS. As a consequence, opposing regulation of PI3K and IRAK-1 may ensue, skewing their competitive balance towards a priming or tolerance phenotype.
Our data reveal the intriguing regulation of RelB by IRAK-1 and Tollip following super low dose LPS challenge. Although high dose LPS is known to induce RelB, we observed that super low dose LPS fails to induce RelB, potentially due to the lack of NFB and PI3K pathway activation. Instead, super low dose LPS significantly decreased RelB levels, potentially through proteasome-mediated degradation. This unique regulation of RelB is through IRAK-1 and Tollip mediated activation of GSK3. Based on our data, we postulate that RelB may be degraded through two-step modifications that involve phosphorylation and ubiquitination. LPS first induces GSK3 activation through IRAK-1 and Tollip. Activated GSK3 then contributes to RelB phosphorylation and subsequent ubiquitination and degradation. This is consistent with a recent report demonstrating that RelB can be ubiquitinated and degraded through GSK3 dependent process (39). It was shown that GSK3 leads to RelB degradation through RelB phosphorylation (39). Although Tollip has been identified over a decade, its physiological function is poorly understood, perhaps due to the high dosage of TLR stimulants used in past studies. Biochemical analyses indicate that Tollip can bind with phospholipids as well as ubiquitin through its CUE domain (63,64).
Further biochemical studies are needed to clarify whether Tollip may also directly contribute to the ubiquitination process of RelB.
In addition to LPS priming, other agents can potentially cause cross priming. One of the classical examples for cross priming is mediated by interferon gamma (IFN. IFN significantly increases the inflammatory response of macrophages to subsequent LPS challenge (65). Interestingly, the underlying mechanisms also involve suppression of PI3K pathway by IFN (66). We tested other TLR agonists including lipoteichoic acid (LTA, a TLR2 agonist) and oxidized LDL (TLR4 agonist), and observed that low levels of LTA and oxidized LDL can prime macrophages for an elevated pro-inflammatory response to subsequent LPS challenge. Mechanistically, low levels of LTA suppress PI3K pathway and decrease basal levels of phosphorylated Akt. Our data indicate that TLR2 and TLR4 agonists may share similar mechanisms in priming the macrophage activation. Intriguingly, we observed that oxLDL failed to suppress PI3K, failed to reduce RelB levels. Thus, oxLDL may cause crosspriming of macrophage to subsequent LPS challenge through a distinct mechanism. Our data partially explain the detrimental pro-inflammatory effects of oxidized LDL during the pathogenesis of atherosclerosis.
This study reveals potential pathway switching mechanisms responsible for the paradigm of LPS priming and tolerance. These mechanisms may have general implications in diverse cellular processes that govern the fate of immune cell activation. Future studies are needed in order to further define these mechanisms, the critical thresholds for pathway switching, and intervention strategies to aid in the maintenance of a proper homeostatic balance. These studies may hold the keys for effective prevention and treatment of devastating inflammatory diseases.

ACKNOWLEDGMENTS
We would like to thank Alwiya Ahmed for the assistance of BMDM culture.    Western blots analysis of P-Y216-GSK3, P-S9-GSK3, and total GSK3 levels in whole cell lysates of WT, Tollip deficient, and IRAK1 deficient BMDM. WT, Tollip deficient, and IRAK1 deficient BMDM were pre-treated with or without a pretreatment of super low dose LPS (5pg/mL) for 4 h, followed by a high dose LPS (100g/mL) treatment for 4h. Quantitative RT-PCR analysis for fold change of indicated genes were carried out using total harvested RNA samples. The relative transcript levels were standardized against Gapdh levels. Data were representative of at least three independent experiments (mean ± SD, *P < 0.05).   Although super low dose LPS fails to induce any noticeable expression of pro-inflammatory mediators due to the lack of robust NFB pathway, the clearance of RelB on the promoters of inflammatory genes can prime their further induction when cells are further stimulated with a higher dose LPS. In contrast, High dose LPS switches to the robust activation of NFB through IRAK-4/2 that leads to dramatic induction of pro-inflammatory mediators. High dose LPS also activates PI3K pathway and IRAK-M that suppresses IRAK-1, and induces RelB. This leads to subsequent tolerance.
by guest on March 25, 2020