Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance*

Background: Dysregulated Toll-like receptor (TLR) signaling is a hallmark of endotoxin-tolerized macrophages in immunosuppression stages of sepsis but Pellino-1 involvement is unknown. Results: Endotoxin tolerization suppresses LPS-induced Pellino-1, Pellino-1 potentiates TLR2/4-driven NF-κB, cytokines and K63-linked IRAK1, TBK1, TAK1, and TRAF6 polyubiquitination. Conclusion: Pellino-1 potentiates TLR2/4 signaling and is decreased by endotoxin tolerization. Significance: Uncovering mechanisms of endotoxin tolerance is critical to understand sepsis-associated immunosuppression. Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.

Pellino was first identified in Drosophila as the protein interacting with Pelle, an ortholog of IRAK4, to activate production of antimicrobial peptides and to protect against infection with Gram-positive bacteria (13,14). Mammalian Pellino-1, Pellino-2, and Pellino-3 share a common structural organization, expressing N-terminal forkhead-associated domains that promote Pellino interactions with phospho-Thr residues of their substrates, and C-terminal RING-like domains responsible for E3 Ub ligase activity (15). Pellino proteins interact with intermediates shared by all Pellinos (e.g. IRAK kinases) but can also target substrates specific individual Pellinos (e.g. SMAD6 for Pellino-1) and regulate IL-1R, TLR, and nucleotide binding and oligomerization domain-like receptor signaling (15). Despite common domain organization and a set of common interacting partners, Pellinos exert non-redundant receptor-, cell-, and species-specific effects whose mechanisms are poorly understood, may be mediated in E3 Ub ligase-dependent-or independent manner (15), and require further studies.
Septic patients surviving the initial cytokine storm become immunocompromised and unable to mount an effective response against secondary infections that often results in fatal outcomes (16,17). Monocytes from immunocompromised septic patients show altered TLR4 responses to lipopolysaccharide (LPS) (18), which is reminiscent of TLR4 responses observed in endotoxin-tolerant cells (19). Endotoxin tolerance is described as re-programming of TLR4 responses to LPS challenge after prior exposure to endotoxin, and is manifested by suppressed expression of proinflammatory cytokines, without inhibition of anti-inflammatory cytokines, antimicrobial effectors, or phagocytosis (16,20,21). It acts as a double-edged sword: on one hand, endotoxin tolerance limits excessive cytokine production during systemic inflammatory response syndrome, sparing organs and tissues from damage, whereas on the other, it is responsible for the failure of the immunocompromised host to counteract secondary infections (21). We previously identified impaired activation of MyD88-and TRIF-dependent signaling pathways, with deregulated IRAK4, IRAK1, and TBK1, as key hallmarks of endotoxin tolerance (22)(23)(24). In view of the importance of Pellino-1 in regulating IL1R/TLR signaling (15,25), we sought to determine the significance of Pellino-1 in endotoxin tolerance and to elucidate its role in TLR2 and TLR4 signaling. To the best of our knowledge, we reported for the first time that induction of endotoxin tolerance in mouse and human macrophages impairs LPS indelibility of Pellino-1 mRNA and protein expression. Using overexpression and gene silencing, Pellino-1 was found to act as a positive regulator of TLR2-and TLR4mediated signaling within MyD88-IRAK1-TAK1-TRAF6 and TRIF-TBK1 axes, and to regulate expression of MyD88-and TRIF-dependent cytokines. Mechanistically, we report that Pellino-1 plays a critical role to promote K63-linked polyubiquitination of IRAK1, TBK1, and TAK-1.

Experimental Procedures
Reagents and Cell Culture-Highly purified, protein-free Escherichia coli K235 LPS was kindly provided by Dr. Stefanie Vogel (University of Maryland School of Medicine, Baltimore, MD), and rabbit antibody (Ab) against human Pellino-1 (26) was a gift from Dr. Peter Cheung (Nanyang Technological University, Singapore).
Mice and Macrophage Isolation-C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Induction of in vivo endotoxin tolerance was carried out as described (23). In brief, peritoneal exudate cells were isolated from mice 4 days after intraperitoneal injection with sterile 3% thioglycollate (REMEL Inc., Lenexa, KS), and 24 h before isolation of cells, mice were administered saline (control) or LPS (20 g/mouse, in vivo induction of endotoxin tolerance). Peritoneal macrophages were obtained by peritoneal lavage and subsequent adherence to plastic as described (23). Cells were plated into 6-well plates (4 ϫ 10 6 cells per well) and cultured in cRPMI 1640. All animal procedures were carried out with institutional IACUC approval.
Lentiviral Transduction of THP1 Cells-GIPZ Lentiviral scrambled or Pellino-1 shRNA were obtained from GE Health Care/Dharmacon, and packaging vectors pSPAX2 (plasmid number 12260) and pMD2.G (plasmid number 12259) were from Addgene. Lentiviral transduction of THP1 cells was performed as published previously (34). In brief, to generate lentiviral particles, HEK293T cells were plated in 6-well plates (2 ϫ 10 6 cells/well), cultured overnight, and transfected with GIPZ shRNA (1 g/well), pSPAX2 (0.75 g/well), and pMD2G (0.25 g/well) using Lipofectamine 2000, according to the manufacturer's instruction. After 48 h, medium containing viral particles was collected and fresh cDMEM was added to the wells, cells were cultured for additional 48 h, and viral particle-containing medium was collected. After filtration through 0.45-m filters, medium was centrifuged (16,000 ϫ g, 2 h), the pellet was resuspended in 0.25 ml of cRPMI with Polybrene (2 g/ml), and the mixture was added to wells of 24-well plates containing THP1 cells. Plates were centrifuged for 45 min at 1200 ϫ g, medium was aspirated and fresh cRPMI was added (0.3 ml/well) to cells. After culturing for 24 h, plates were centrifuged for 10 min at 1200 ϫ g, cells were resuspended in cRPMI containing 5 g/ml of puromycin, and stable bulk transfectants were selected for at least 2 weeks.
Nucleofection-pSuper expression vectors encoding scrambled or Pellino-1 shRNA variants were introduced into THP1 cells by nucleofection, using the Nucleofector I device (Lonza) and the cell line nucleofection kit V (Lonza), as recommended by the manufacturer. After recovery for 24 h, cells were treated for 3 h with medium or LPS (100 ng/ml), RNA was isolated, reverse transcribed, and analyzed by real-time PCR using genespecific primers.
NF-B Reporter Assays-Reporter assays were performed as reported previously (22,24,28,29,32). In brief, cells were transfected with plasmids, using Lipofectamine 2000, recovered for 48 h, and treated for 5 h with medium or stimuli. Cells were lysed in a passive lysis buffer (Promega), and firefly luciferase versus Renilla luciferase activities were measured using dual luciferase reporter assay system (Promega) on a Berthold LB 9507 luminometer (Berthold Technologies).

Pellino-1 Potentiates TLR2/4 Signaling and Is Decreased in Endotoxin Tolerance
Phagocytosis Assay and Flow Cytometry-THP-1 cells were plated in 24-well plates (0.5 ϫ 10 6 cells per well), incubated with 20 ng/ml of PMA for 72 h, washed, and resuspended with cRPMI with or without pHrodo Red-conjugated heat-killed E. coli or S. aureus bioparticles (Life Technologies) at a bacteria per cell ratio of 50:1. After incubation for the various times on ice or at 37°C, cells were detached, washed in icecold PBS containing 3% FBS, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies), fixed for 15 min in 3.7% formaldehyde, and analyzed by FACS on a LSRII Flow Cytometer (BD Biosciences) to measure pHRodo fluorescence. The data were analyzed using the FlowJo software (Tree Star).
Statistical Analysis-Statistical analysis was performed using the GraphPad Prism 5 program for Windows (GraphPad Software Inc.). Statistical differences were evaluated using Student's t test with the confidence interval set at 95% level. The results were expressed as mean Ϯ S.D. values.

Induction of Endotoxin Tolerance Impairs LPS Inducibility of
Pellino-1-In the first series of experiments, we examined the impact of in vitro induction of endotoxin tolerance on basaland LPS-induced expression of Pellino-1 in human primary monocytes and in macrophage-like cell lines, THP-1 and MonoMac-6 cells. THP-1 cells were differentiated for 72 h with 20 ng/ml of PMA to attain macrophage-like characteristics, e.g. plastic adherence, CD14 expression, and phagocytosis (30). In line with previous studies by us and others (22-24, 32, 36 -38), prior exposure to endotoxin significantly decreased induction of TNF-␣ gene expression to subsequent LPS challenge, documenting the induction of endotoxin tolerance in all three cell types (Fig. 1, A-C, left column). LPS stimulation of mediumpretreated monocytes, THP1, and MonoMac-6 cells resulted in robust induction of Pellino-1 mRNA, which was the strongest at 3 h, declined by 8 h, and further decreased by 16 h post-LPS challenge (Fig. 1, A-C, right columns). Notably, at all times, endotoxin-tolerized cells exhibited 65-95% inhibition of LPSmediated induction of Pellino-1 mRNA compared with control medium-pretreated cells (Fig. 1, A-C, right column).
Next, we employed an in vivo model of endotoxin tolerance (23,36), whereby C57BL/6J mice were injected intraperitoneally with LPS (20 g/mouse, endotoxin tolerization in vivo) or PBS (controls), and thioglycollate-elicited peritoneal macrophages were exposed to medium or LPS in vitro, followed by qPCR analyses of cytokine and Pellino-1 gene expression. Macrophages obtained from PBS-injected mice showed 4 -8fold increases in IL-6 mRNA levels following LPS stimulation for 1 and 3 h, respectively, whereas macrophages from LPStreated animals had only 2-fold maximal induction of IL-6 mRNA (Fig. 1D, left), demonstrating endotoxin-tolerant phenotype. LPS caused only 1.4 -2.4-fold increases in the induction of Pellino-1 mRNA in macrophages from mice previously administered with LPS, in contrast to robust LPS induction observed in macrophages from control, PBS-injected mice (Fig.  1D, right).
It was important to confirm inhibited LPS inducibility of Pellino-1 in endotoxin-tolerized macrophages at the protein level.
Because NF-B is critically involved in activating cytokine gene expression (39), we next studied whether Pellino-1 regulates TLR2-and TLR4-inducible expression of IL-8 mRNA. Pellino-1 Significantly Up-regulates TLR-mediated Induction of MyD88-and TRIF-dependent Cytokine Genes in Response to LPS, Pam3Cys, and Heat-killed E. coli or S. aureus but Only Weakly Affects Phagocytosis of Heat-killed Bacteria-Next, we sought to extend our findings on a positive regulatory role of Pellino-1 in TLR2 and TLR4 signaling obtained in HEK293 transfectants to cells with macrophage-like phenotype. We were also interested to define how Pellino-1 affects MyD88and TRIF-dependent signaling pathways. To this end, we employed nucleofection-or lentiviral-based introduction of Pellino-1 or control scrambled shRNA species into THP-1 cells, cells were differentiated with PMA to attain macrophage phenotype, and their responses to TLR2 and TLR4 agonists, as well as to heat-killed E. coli and S. aureus were analyzed. Nucleofec- (C) cells were exposed for 24 h with medium or 100 ng/ml of LPS. Thereafter, cells were washed three times and treated with medium or 100 ng/ml of LPS for the indicated times. D, C57BL/6J mice were intraperitoneally administered thyoglycollate, and 72 h later, were intraperitoneally injected with saline or LPS (20 g/mouse). Peritoneal exudate cells were isolated, and plastic-adherent macrophages were treated for 1 and 3 h with medium or 100 ng/ml of LPS. RNA was isolated, reverse- tion of Pellino-1 shRNA into THP-1 cells decreased Pellino-1 mRNA levels by 52% compared with scrambled shRNA-transfected cells, whereas not affecting Pellino-3 gene expression (Fig. 5A, left column), demonstrating specific ablation of Pellino-1. THP1 cells nucleofected with scrambled shRNA responded to LPS by robust induction of TNF-␣ and IL-6 mRNA, increasing their expression by 36-and 92-fold, respectively, whereas induction of TRIF-dependent IFN-␤ and CCL5 was manifested to a lesser extent (4.2-and 2.5-fold increase, Fig.  5A). Pellino-1 knock-down achieved by nucleofection of Pellino-1 shRNA significantly suppressed LPS inducibility of genes encoding TNF-␣ (54% inhibition), IL-6 (89% inhibition), IFN-␤ (57% inhibition), and CCL5 (33% inhibition) (Fig. 5).
To confirm and extend these results, we obtained THP-1 cell lines stably expressing scrambled or Pellino-1 shRNA after lentivirus transduction and selection in puromycin, and analyzed their responses to TLR2 (Pam3Cys) and TLR4 (LPS) agonists as well as to heat-killed bacteria utilizing TLR2 (S. aureus) and TLR4 (E. coli). Incorporation of Pellino-1 shRNA by lentivirus transduction led to an even higher decrease (66% inhibition) in Pellino-1 mRNA levels compared with scrambled shRNA-expressing cells, whereas not affecting Pellino-3 mRNA expression (Fig. 5B, left column). Pellino-1 ablation decreased LPSinducible TNF-␣ and IFN-␤ gene expression by 60 and 50%, respectively (Fig. 5B, top, middle, and right panels), and reduced Pam3Cys-mediated expression of TNF-␣ and IL-6 mRNA by 57 and 36% (Fig. 5B, bottom middle and right panels). Heat-killed E. coli caused 29-and 10-fold increases in TNF-␣ and pro-IL-1␤ gene expression in THP-1 cells transduced with scrambled shRNA, whereas expression of these cytokine genes in Pellino-1 shRNA-expressing cells was reduced by 38 and 50%, respectively (Fig. 6A, top panel). Similarly, whereas scrambled shRNA-THP1 cells responded to stimulation with heat-killed S. aureus by 14.9-and 4-fold induction of TNF-␣ and pro-IL-1␤ mRNA, Pellino-1 knockdown led to 50 and 60% reduction in their inducibility (Fig. 6A, bottom panel). , and MonoMac-6 (C) cells were pretreated for 20 h with medium or 100 ng/ml of LPS, washed, and restimulated with or without 100 ng/ml of LPS for the indicated times. Cell lysates were examined by Western blot analyses with Abs against Pellino-1, p-p65, IB-␣, phospho-and total p38, and tubulin, as shown. Panels on the right depict quantification of Western blot results using the NIH ImageJ software package. Shown are the data of a representative (n ϭ 4) experiments. JULY 31, 2015 • VOLUME 290 • NUMBER 31

JOURNAL OF BIOLOGICAL CHEMISTRY 19223
Because Pellino proteins regulate TLR signaling (33, 40 -44) and TLR signaling modulates phagocytosis (45-47), we studied the impact of Pellino-1 deficiency on phagocytosis of heat-killed E. coli and S. aureus by PMA-differentiated THP-1 cells. We used pHrodo Red-conjugated heat-killed bacteria and phagocytosis was judged by fluorescence of pH-sensitive  pHrodo Red upon its localization in the phagolysosome compartment undergoing acidification, whereas under basal conditions, this fluorophore does not emit fluorescence (48). As con-trols, we measured autofluorescence of cells incubated in the absence of bacteria and in bacteria-cell cultures incubated on ice (to inhibit phagocytosis). Incubation of THP1 cells express- ing scrambled shRNA with pHrodo Red-conjugated E. coli and S. aureus for 2 h at 37°C markedly increased fluorescence compared with values observed in identical cultures incubated on ice (Fig. 6, B, top, and C). THP1 cells expressing Pellino-1 shRNA and incubated with heat-killed pHrodo Red-conjugated E. coli and S. aureus at 37°C exhibited 19 -25% statistically significant increases in mean fluorescence channel (MFI) values compared with scrambled shRNA-transduced THP-1 cells (Fig.  6, B, bottom, and C). Collectively, these results indicate that Pellino-1 positively regulates expression of MyD88-and TRIFdependent cytokine genes but only weakly affects phagocytosis of heat-killed E. coli and S. aureus.
Pellino-1 Modulates NF-B Reporter Activation Driven by Overexpression of MyD88, IRAK1, TRAF6, TAK1, or by Transfection of TRIF and TBK1 but Does Not Affect p65-driven Responses-To position Pellino-1 within the MyD88-and TRIF-dependent pathways, we initiated activation of a transfected NF-B luciferase reporter in HEK293T cells by overexpression of a series of adapters or kinases, and studied the impact of co-expressed Pellino-1 on these processes. Overexpressed signaling intermediates initiate signaling downstream in a ligand-independent manner, because of their ability to dimerize, bypassing the requirement for agonist-inducible dimerization, and this is a commonly used approach to discern the location of a molecule in question within signaling axes (22,24,32,33,44,49). Fig. 7A demonstrates that ectopic expression of Pellino-1 led to a synergistic up-regulation of MyD88-and TRIF-driven NF-B reporter activation by 2.8 -4.5-and 2.2-3.4-fold (top two panels on the left), and increased NF-B transactivation driven by IRAK1, TAK1, and TBK1 by 3.7-, 3.1-, and 1.5-fold, respectively (bottom panels). Pellino-1 transfection also increased the ability of overexpressed TRAF6 to mediate activation of the pELAM-Luc NF-B luciferase reporter, but did not affect p65-driven NF-B transactivation (Fig. 7A, top two panels on the right). As evidenced by co-immunoprecipitation, expressed Pellino-1 associated with transfected and, to a lesser extent, endogenous IRAK1 and TBK1 (Fig. 7B, top panel). Transfected IRAK1 and TBK1 exhibited patterns of post-translational modifications and phosphorylation (Fig. 7B, bottom  panel), reflecting constitutive activation of overexpressed kinases, in line with reported data (32,33,50,51). Overexpression of Pellino-1 led to increased abundance of modified species of IRAK1 and the phosphorylated form of TBK1 (Fig. 7B, bottom panel). Next, we studied the impact of Pellino-1 overexpression on basal and LPS-mediated phosphorylation of IRAK1 (an MyD88-dependent outcome), TBK1 and IRF3 (TRIF-dependent outcomes). Transfection of Pellino-1 constitutively induced phosphorylation of IRAK1, TBK1, and IRF3, and potentiated LPS-driven phosphorylation of these intermediates (Fig. 7C). These data indicate that Pellino-1 potentiates NF-B activation driven by overexpression of MyD88, TRIF, IRAK1, TBK1, TAK1, and TRAF6 and increases basal and LPS-inducible phosphorylation of IRAK1 and TBK1, although not affecting p65-induced NF-B.

Pellino-1 Potentiates TLR2/4 Signaling and Is Decreased in Endotoxin Tolerance
observed in unstimulated cells (Fig. 8, A-D). Transfected Pellino-1 induced attachment of K63-only HA-Ub to all studied intermediates, in line with constitutive activation of overexpressed Pellino-1 (Figs. 2-7) (33), and markedly enhanced LPSinducible K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and TRAF6 (Fig. 8, A-D). Similarly designed experiments employing transfection of K48-only HA-Ub demon-strated only weak K48-linked polyubiquitination of IRAK1 and TBK1, which was not up-regulated by LPS, and showed the failure of transfected Pellino-1 to affect this signaling outcome (data not shown). Collectively, these results revealed the ability of transfected Pellino-1 to mediate basal and potentiate LPSmediated K63-linked ubiquitination of IRAK1, TBK1, TAK1, and TRAF6. A, HEK293T cells were plated in 24-well plates and co-transfected with or without FLAG-Pellino-1 (0.8 g/well) in the presence of pELAM-Luc (0.4 g/well) and pTK-Renilla Luc (20 ng/well) along with the indicated amounts of pcDNA3-AU1-MyD88 or pcDNA3-TRIF, or with expression vectors encoding IRAK1, TBK1, TRAF6, TAK1, or p65 (10 ng/well each). After recovery for 48 h, cell lysates were assayed for firefly versus Renilla luciferase activities, and data are presented as fold-induction compared with values in cells transfected with pcDNA3, pELAM-Luc, and pTK-Renilla Luc taken as 1. B, HEK293T cells were plated in 6-well plates and transfected with pcDNA3, pRK5-IRAK1, or pSuper-TBK1 (1 g/well) along with pcDNA3 or FLAG-Pellino-1 (1 g/well). After recovery for 48 h, cell lysates were prepared and immunoprecipitated (IP) with anti-Pellino-1 Ab followed by immunoblotting (IB) with anti-IRAK1 (left) or anti-TBK1 (right) Abs (top panels); or examined by Western blot analyzes with Abs for the indicated proteins (bottom panels). C, 293/TLR4 cells were plated in 6-well plates and transfected with pcDNA3-CD14, pEFBOS-MD2 (0.5 g/well each), along with pcDNA3 or FLAG-Pellino-1 (1 g/well each). After recovery for 48 h, cells were treated with medium or 100 ng/ml of LPS for the indicated times. Cell lysates were immunoprecipitated with anti-IRAK1 Ab followed by Western blot analyses with anti-p-IRAK1 or anti-IRAK1 Abs (top two panels) or analyzed by immunoblotting with Abs against the indicated proteins. The data of a representative (n ϭ 3) experiment are shown.

Discussion
Previous studies by us and others demonstrated inhibited activation of the MyD88-and TRIF-dependent pathways in endotoxin-tolerized human monocytes and mouse macrophages, including suppressed induction of MyD88-dependent (pro-IL-1␤, TNF-␣, IL-6, and IL-12 p40) and TRIF-dependent (IFN-␤) cytokine genes (22-24, 32, 36, 52-55). We previously reported that endotoxin tolerance impairs LPS-inducible activation of IRAK4, IRAK1, TBK1, and TAK1 (23,24,54,55), and, in the present study, sought to analyze molecular mechanisms of this phenomenon. Because Pellino-1 has been reported to interact with IRAK4 and IRAK1 kinases (33,56), and is capable of forming a signalosome with TBK1, TAK1, and TRAF6 (26,33,57), we wanted to analyze the role of Pellino-1 in endotoxin tolerance and TLR2/4 signaling. To the best of our knowledge, this paper demonstrates for the first time that in vivo induction of endotoxin tolerance significantly blunted LPS-inducible Pellino-1 gene expression in mouse peritoneal macrophages. Similarly, in vitro endotoxin tolerization of human primary monocytes and macrophage-like MonoMac-6 and THP-1 cells abrogated the capacity of LPS to up-regulate Pellino-1 mRNA and protein expression. The induction of Pellino-1 in endotoxin-tolerized cells was markedly inhibited at all times (by 75-96%). Notably, reduction in the Pellino-1 expression upon Pellino-1 knockdown in THP-1 cells is manifested to a lower extent compared with residual Pellino-1 present in LPS-tolerant monocytes and macrophages, yet it was sufficient to markedly attenuate TLR2/4 signaling. Thus, decreased Pellino-1 levels and, possibly, its deficient activation, dependent on phosphorylation of several Ser and Thr residues, ubiquitination and sumoylation (15), could underlie compromised TLR-inducible activation of NF-B and inflammatory cytokine genes in endotoxin-tolerized cells. Interestingly, Pellino-1 has been reported to interact with positive (IRAK1, IRAK4, TBK1, TRAF6, TAK1, and deformed epidermal autoregulatory factor-1) and negative (SMAD6 and SMAD7) TLR regulators (reviewed in Ref. 15). Whether Pellino-1 can also interact with IRAK-M, SHIP1, or A20, known negative regulators of TLR signaling (reviewed in Ref. 4), is yet to be determined. It is tempting to speculate that altered expression and, possibly, activity of Pellino-1, imposes changes in other regulators of TLR signaling, initiating complex regulatory networks resulting in altered post-translational modifications of TLR intermediates, transcriptional changes, and re-programming of TLR signaling in endotoxin-tolerized cells.
Although the molecular mechanisms behind reduced LPS inducibility of Pellino-1 in endotoxin tolerance is unknown, it is possible that decreased activation of TBK1 and IRF3 in LPStolerant cells is responsible (24), because the TBK-IRF3 axes induces Pellino-1 expression and TBK1 up-regulates its activity (26,57). Interestingly, Pellino-1 interacts with inhibitory Smad6 and Smad7 involved in the TGF-␤ pathway, leading to reduced interactions of IRAK1 with IL1R/TLR, impaired NF-B activation, and cytokine expression (58,59). Such a mechanism could also play a role during endotoxin tolerance, given up-regulation of TGF-␤ expression upon endotoxin tolerization (60). Studies are in progress to dissect the molecu-lar mechanisms responsible for decreased LPS inducibility of Pellino-1 in endotoxin-tolerized cells.
Controversial results have been published regarding the role of Pellino-1 in MyD88-and TRIF-driven cascades in macrophages and other cell types (reviewed in Ref. 15). To establish the role of Pellino-1 in TLR2 and TLR4 signaling, we employed Pellino-1 overexpression and shRNA-mediated ablation strategies. Pellino-1 overexpression constitutively induced NF-B and synergistically up-regulated Pam3Cys-or LPS-inducible NF-B activation and expression of IL-8 mRNA in HEK293 cells expressing TLR2 or TLR4/MD2, whereas Pellino-1 knockdown decreased these responses. We confirmed these results in macrophage-like THP-1 cells, where Pellino-1 ablation resulted in decreased expression of MyD88-(TNF-␣, IL-6) and TRIF-dependent (IFN-␤, CCL5) cytokine genes in response to TLR2 (Pam3Cys) and TLR4 (LPS) agonists and heat-killed S. aureus and E. coli. The involvement of Pellino-1 in MyD88and TRIF-dependent signaling was also supported by our results showing that ectopic overexpression of Pellino-1 synergistically increased NF-B reporter activation driven by transfected MyD88 or IRAK1, on one hand, and by TRIF or TBK1, on the other. Furthermore, co-immunoprecipitation experiments revealed that Pellino-1 interacts with transfected IRAK1 and TBK1 upon their co-expression, and potentiates post-translational modifications of IRAK1 and phosphorylation of TBK1. Subsequent analyses of downstream components of the MyD88 and TRIF pathways showed that Pellino-1 up-regulated NF-B reporter activation driven by overexpression of TAK1 and TRAF6, whereas Pellino-1 did not affect p65-induced NF-B.
Consistent with the ability of Pellino-1 to interact with multiple TLR intermediates, i.e. IRAK1, TBK1, RIP-1, and TRAF6 (26,33,56,57), Pellino-1 could promote the assembly of a multiprotein complex of these intermediates and activate signaling capacities of each in an E3 Ub ligase-dependent and/or independent manner (reviewed in Ref. 15). Conversely, Pellino-1 could primarily target and activate upstream kinases IRAK1 and TBK1. In this scenario, even if signaling is initiated at the level of downstream molecules (e.g. TAK1 and TRAF6) rendered constitutively active by transfection, Pellino-1-activated upstream kinases could potentiate such responses. For instance, this could occur by promoting TRAF6 and TAK1 recruitment to K63-polyubiquitinated IRAK1 (7). Further studies are required to uncover which mechanism is utilized by Pellino-1.
Although in vitro Pellino-1 mediates both K48-linked, "degradative" and K63-linked, "signaling-promoting" polyubiquitination of its targets, such as IRAK1 (61), in vivo studies demonstrated primarily Pellino-1-mediated K63-linked polyubiquitination (33, 50) that promotes protein-protein interactions and enhances downstream signaling (15). This paper shows that co-expression of Pellino-1 exclusively up-regulates K63-linked polyubiquitination of IRAK1, TAK1, and TBK1 in 293/TLR4/MD2 cells stimulated with LPS, whereas it failed to affect weak K48-linked polyubiquitination of these molecules (data not shown), confirming previously published findings (33,56,62). Of importance, we demonstrate for the first time that Pellino-1 also enhances LPS-driven K63linked polyubiquitination of TBK1, which might play a role in promoting the ability of TBK1 to form downstream signalosome complexes and activate its kinase activity. It would be interesting to delineate whether Pellino-1 alone acts as a positive regulator of IRAK1-and TBK1-driven pathways or it can co-opt other members of the Pellino family implicated in positive regulation of IL-1R/TLR pathways, e.g. Pellino-2 (63), and other E3 Ub ligases fine-tuning TBK1 signaling, e.g. Nrdp1 (64).
Modulation of phagocytosis by TLR signaling has been a subject of controversy (33, 40 -44, 65). Because Pellino-1 positively controls TLR2/4-mediated signaling outcomes, we examined whether it regulates phagocytosis. Our data show only a weak impact of Pellino-1 ablation on phagocytosis of heat-killed, pHrodo Red-conjugated S. aureus and E. coli by THP-1 cells, as judged by phagolysosome acidification-induced fluorescence of bacteria-conjugated, pH-sensitive fluorophore. Thus, despite that Pellino-1 acted as a positive regulator of TLR-inducible K63-linked polyubiquitination of IRAK1, TBK1, and activator of MyD88/IRAK1-and TRIF/TBK1-driven cytokine genes, it only weakly affected phagocytosis of heat-killed bacteria. These results may suggest that Pellino-1 is unlikely to affect host innate immune responses during infections by regulating microbial uptake or phagocytosis, but rather acts via potentiation of production of MyD88-and TRIF-dependent inflammatory cytokines. Notably, Pellino-1-deficient and sufficient human pulmonary bronchial epithelial cells had comparable replication of rhinovirus upon in vitro infection, whereas significant reduction of CXCL8 expression was noted in Pellino-1 Ϫ/Ϫ cells (40). Further studies are required to determine a currently unknown impact of Pellino-1 deficiency on microbial loads during infection in vivo and phagocytosis of live bacteria.
In summary, we report herein for the first time that induction of endotoxin tolerance in vivo and in vitro down-regulated LPS inducibility of Pellino-1. Pellino-1 regulated both MyD88-and TRIF-dependent signaling pathways via association with IRAK1 and TBK1 and by positively regulating their LPS-mediated K63-linked polyubiquitination, leading to increased expression of inflammatory cytokines. We also report that Pellino-1 increased NF-B activation driven by the MyD88-IRAK1-TRAF6-TAK1 and TRIF-TBK1 signaling axes and upregulated K63-linked polyubiquitination of TAK1 and TRAF6, but did not affect p65-induced NF-B. In contrast to its prominent regulation of TLR2/4-driven cytokines, Pellino-1 only weakly affected phagocytosis of heat-killed S. aureus and E. coli. Given that Pellino-1 deficiency ameliorates poly(I:C)-and LPSinduced septic shock (25), it would be important to determine consequences of Pellino-1 knock-out on susceptibility to microbial infections in vivo, infection morbidity and mortality, and to assess responses of macrophages and dendritic cells. It is tempting to speculate that small molecule-or peptide-based pharmacological inhibition of Pellino-1 signaling at early stages of sepsis or in patients with autoimmune and inflammatory diseases could provide beneficial therapeutic outcomes. In this respect, it is noteworthy that Pellino-1-interacting SMAD6 blocking peptides were shown to inhibit manifestations of lethal inflammatory responses in mice in sepsis models of cecal ligation and puncture and endotoxemia (61).