The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

The NLRP3 inflammasome is important for inducing IL-1β and IL-18 inflammatory responses. Here the authors show, by generating and characterizing Peli2 deficient mice and immune cells, that an E3 ubiquitin ligase Pellino2 promotes inflammasome priming by inducing NLRP3 ubiquitination and by targeting IRAK1

Secretory Leucoprotease Inhibitor (SLPI) Promotes Survival during Acute Pseudomonas aeruginosa Infection by Suppression of Inflammation Rather Than Microbial Killing

Secretory leucoprotease inhibitor (SLPI) has multifaceted functions, including inhibition of protease activity, antimicrobial functions, and anti-inflammatory properties. In this study, we show that SLPI plays a role in controlling pulmonary Pseudomonas aeruginosa infection. Mice lacking SLPI were highly susceptible to P. aeruginosa infection, however there was no difference in bacterial burden. Utilising a model of P. aeruginosa LPS-induced lung inflammation, human recombinant SLPI (hrSLPI) administered intraperitoneally suppressed the recruitment of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and resulted in reduced BALF and serum levels of inflammatory cytokines and chemokines. This anti-inflammatory effect of hrSLPI was similarly demonstrated in a systemic inflammation model induced by intraperitoneal injection of LPS from various bacteria or lipoteichoic acid, highlighting the broad anti-inflammatory properties of hrSLPI. Moreover, in bone-marrow-derived macrophages, hrSLPI reduced LPS-induced phosphorylation of p-IkB-α, p-IKK-α/β, p-P38, demonstrating that the anti-inflammatory effect of hrSLPI was due to the inhibition of the NFκB and MAPK pathways. In conclusion, administration of hrSLPI attenuates excessive inflammatory responses and is therefore, a promising strategy to target inflammatory diseases such as acute respiratory distress syndrome or sepsis and could potentially be used to augment antibiotic treatment

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts

ECSIT is a critical limiting factor for cardiac function

Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein with roles in early development, activation of the transcription factor NF-κB, and production of mitochondrial reactive oxygen species (mROS) that facilitates clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here that human ECSIT (hECSIT) is highly labile. To explore whether the instability of hECSIT affects functions previously ascribed to its murine counterpart, we created a potentially novel transgenic mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanized mouse has low levels of hECSIT protein, in keeping with its intrinsic instability. Whereas low-level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early development and activation of the NF-κB pathway, macrophages from humanized mice showed impaired clearance of Salmonella that was associated with reduced production of mROS. Notably, severe cardiac hypertrophy was manifested in aging humanized mice, leading to premature death. The cellular and molecular basis of this phenotype was delineated by showing that low levels of human ECSIT protein led to a marked reduction in assembly and activity of mitochondrial complex I with impaired oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanized hECSIT mice also showed reduced mitochondrial fusion and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte-intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We also show that cardiac fibrosis and damage in humans correlated with low expression of human ECSIT. In summary, our findings identify a role for ECSIT in cardioprotection, while generating a valuable experimental model to study mitochondrial dysfunction and cardiac pathophysiology

Primer sequences for cloning EBV putative DUBs.

*<p>F: forward, R: reverse.</p

Schematic model of BPLF1-mediated TLR evasion during EBV infection.

<p>During EBV infection, EBV components are sensed by host TLRs (a). These receptors activate signaling pathways through adaptor molecules MyD88 and TRIF, culminating in activation of transcription factor NF-κB (b). NF-κB induces production of proinflammatory cytokines (c) and obstructs viral replication. TLR signaling pathways are extensively regulated by ubiquitination, a process governed by cellular ubiquitin ligases and deubiquitinases (e.g. A20, d). The EBV-encoded DUB BPLF1 counteracts TLR-mediated NF-κB activation and can interfere with K63- and K48-linked ubiquitination of signaling intermediates, for example on TRAF6, NEMO, and IκBα (e). BPLF1 is expressed as a full-length protein during the late phase of productive EBV infection, is incorporated into the tegument of viral particles (f), and can subsequently be released into newly infected cells (g). It is as yet unclear where BPLF1 is processed to yield a shorter active fragment of ca. 280 aa (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003960#ppat-1003960-g001" target="_blank">Fig. 1</a>). Thus, (processed) BPLF1 could exert its immunomodulatory functions towards TLR signaling not only in EBV-producing B cells (h), but also during primary infection (g).</p

BPLF1 is expressed during the late phase of lytic EBV infection and is incorporated into viral particles.

<p>EBV⁺ AKBM cells were treated with anti-human IgG (αIgG) to induce productive infection. (<b>a</b>) At the indicated times post-induction, percentages of AKBM cells undergoing productive infection were determined by flow cytometric analysis of ratCD2-GFP reporter expression. (<b>b</b>) EBV protein expression in post-nuclear cell lysates was determined by immunoblotting with Abs specific for BZLF1 (immediate early, IE), BGLF5 (early, E), and gp42 (late, L). β-actin served as loading control. (<b>c</b>) Immunoblot of post-nuclear lysates of EBV B95.8 particles (lane 1), EBV⁻ AK31 cells (lanes 2–4), and EBV⁺ AKBM cells (lanes 5–7). EBV⁺ AKBM cells and EBV⁻ control AK31 cells were treated with αIgG Abs for 24 hours, resulting in productive infection in 26% of AKBM cells; AK31 cells expressed ratCD2-GFP constitutively in ∼40% of the population (data not shown). Phosphonoacetic acid (PAA, 300 µg/mL) treatment starting 1 hour prior to anti-IgG treatment was used to inhibit late protein expression; β-actin served as loading control.</p

EBV BPLF1 is a DUB active during productive infection.

<p>EBV⁺ AKBM BL cells were treated with anti-human IgG (αIgG) to induce viral replication. (<b>a</b>) At the indicated times post induction, percentages of productively EBV-infected AKBM cells were determined by flow cytometric analysis of induced ratCD2-GFP reporter expression. (<b>b</b>) In post-nuclear AKBM cell lysates, active DUBs were labeled with a fluorescent Ub-VME probe, resolved by SDS-PAGE, and visualized by in-gel fluorescence imaging. The arrow indicates a band appearing in AKBM cells after 9 hours of lytic cycle induction (lanes 4 and 5). (<b>c</b>) DUB profiles of EBV⁻ AK31-rCD2-GFP cells (AK31, lanes 1 and 2), EBV⁺ AKBM cells (lanes 3 and 4), and transfected 293T cells (lanes 5–9). In parallel with the EBV⁺ AKBM cells, EBV⁻ control AK31 cells were treated with αIgG for 24 hours; this resulted in productive infection in 56% of AKBM cells; AK31 cells included a population of 45% that expressed ratCD2-GFP from a constitutive promoter irrespective of αIgG treatment (data not shown). For comparison, 293T cells were transfected with constructs encoding three (putative) EBV DUBs: BPFL1, BXLF1, BSLF1, or the 325 aa N-terminal part of BPLF1. The asterisk marks a smaller fragment observed upon transfection of 293T cells with full-length or the N-terminal part of BPLF1 (lanes 6 and 9). Left and right panels are parts of one gel displayed at different exposures. (<b>d</b>) Sixteen hours post-transfection, percentages of 293T cells expressing BPLF1, BXLF1, BSLF1 or the N-terminal fragment of BPLF1 were determined by intracellular FACS staining for the Flag-tag. (<b>e</b>) Immunoblot of part of the gel in c probed with an anti-Flag Ab to detect transfected BXLF1 and BSLF1 (sequential staining following 1F2, see in f). (<b>f</b>) Immunoblot of the gel in c probed with the BPLF1-specific mouse monoclonal Ab 1F2. Both panels are part of one gel presented at different exposures.</p

BPLF1 interferes with TLR-mediated NF-κB activation.

<p>293T cells were transiently transfected with a firefly luciferase reporter construct responsive to either NF-κB activation or IFNβ promoter activity, an HSV TK-promoter driven Renilla luciferase plasmid (for normalization), and plasmids encoding HA-BPLF1, Flag-BXLF1, Flag-BSLF1 or cellular control DUB A20. (<b>a</b>) An NF-κB responsive firefly luciferase reporter and 4 or 8 ng of vectors encoding the EBV or control proteins were cotransfected in 293T cells and the TLR signaling cascade was initiated by expressing adaptor protein MyD88. Sixteen hours post-transfection, cells were lysed and luciferase activity was determined. In addition, protein expression was analyzed in post-nuclear cell lysates by immunoblot using anti-HA and anti-Flag Abs; β-actin served as loading control. (<b>b</b>) 293-TLR2/CD14 cells were cotransfected with an NF-κB responsive reporter and 32 ng of plasmids encoding the indicated proteins. Starting at 16 hours post-transfection, cells were stimulated with 10 ng/mL MALP-2 or 6.5×10⁶ EBV particles/mL for 7 hours, after which they were lysed and luciferase activity was measured. (<b>c</b>) Similarly, 293-TLR3 cells were cotransfected with 16 ng of the indicated genes together with an NF-κB responsive reporter (left panel) or a reporter under control of the IFNβ promoter (right panel). Starting 16 hours post-transfection, cells were stimulated for 7 hours with 10 µg/mL poly(I∶C), after which they were lysed and luciferase activity was determined. Data are presented as percentages firefly luciferase activity relative to stimulated control samples and normalized for transfection efficiency using <i>Renilla</i> luciferase values, mean ± SD. (<b>d</b>) 293-TLR2/CD14 cells were transfected with empty control vector or plasmids encoding EBV BPLF1 or cellular A20. Starting at 24 hours post-transfection, cells were stimulated with 10 µg/mL MALP2 or 6.5×10⁶ EBV particles/mL for 8 hours. IL-8 secretion in the culture supernatants was determined by ELISA.</p

BPLF1 interferes with TLR signal transduction at multiple levels.

<p>(<b>a</b>) 293-TLR2/CD14 cells and control 293-CD14 cells were transiently transfected with plasmids encoding Flag-tagged BPLF1, catalytically inactive mutant BPLF1_C61A, or cellular control DUB A20. At 40 hours post-transfection, cells were treated with 10 ng/mL MALP-2 for indicated time periods and IκBα levels in post-nuclear cell extracts were determined by immunoblotting with an anti-IκBα Ab. Expression of transfected Flag-tagged proteins was assessed by immunoblotting with an anti-Flag Ab; TfR served as loading control. (<b>b</b>) Schematic representation of the signaling cascade that induces NF-κB following TLR activation. Ub in green is K63-linked, Ub in purple is K48-linked. (<b>c</b>) 293T cells were cotransfected with an NF-κB responsive firefly luciferase reporter, HSV TK promoter-driven Renilla luciferase for normalization, and plasmids expressing BPLF1, BPLF_C61A, or controls IκBα and A20 (13.5 ng). TLR signaling was induced by expression of activator proteins MyD88, IRAK1, TRAF6, or IKKα. After 16 hours of transfection, luciferase activity was measured. Results are depicted as percentage firefly luciferase activity normalized using <i>Renilla</i> luciferase values relative to stimulated control sample, mean ± SD.</p