Iain Welsby

PARP12, an Interferon-stimulated Gene Involved in the Control of Protein Translation and Inflammation

Background: The individual role of members of the poly(ADP-ribose) polymerase family is unclear. Results: PARP12 displays a dual subcellular localization and effector function, controlling both protein translation and NF-κB signaling. Conclusion: PARP12 mediates two important effector mechanisms linked to the establishment of an anti-viral state. Significance: ADP-ribosylation may play an important role in the innate control of microbial infections. Transcriptome analyses have recently identified PARP12, a member of a large family of ADP-ribosyl transferases, as an interferon-induced gene (ISG), whose function remains incompletely characterized. We demonstrate herein that PARP12 is a genuine ISG, whose expressed protein displays at least two distinct subcellular locations and related functions. Upon ectopic expression or exposure to oxidative stress, PARP12 is recruited to stress-granules (SGs), known sites of mRNA translational arrest. Accordingly, PARP12 was found to block mRNA translation, possibly upon association to the translational machinery. Both the N-terminal domain (containing an RNA-binding domain characterized by the presence of five CCCH-type Zn-fingers) and integrity of the catalytic domain are required for this suppressive function. In contrast, stimulation with LPS leads to the localization of PARP12 to p62/SQSTM1 (an adaptor protein involved in innate signaling and autophagy) containing structures, unrelated to SGs. Deletion of the N-terminal domain promotes the association of the protein to p62/SQSTM1, suggesting that the RNA-binding domain is responsible for the subcellular localization of PARP12. Association to p62/SQSTM1 was found to correlate with increased NF-κB signaling, suggesting a role for PARP12 in inflammation. Collectively, these observations suggest that PARP12 can alternate between two distinct subcellular compartments associated to two distinct cellular functions. The present work therefore identifies PARP12 as an ISG with a potential role in cellular defenses against viral infections.

Complex roles of members of the ADP-ribosyl transferase super family in immune defences: Looking beyond PARP1.

ADP ribosylation has been recently recognised as an important posttranslational modification regulating numerous cellular processes. This enzymatic activity is shared by two major families of enzymes, the extracellular ADP-ribosyl-transferases, or ecto-ARTS and the poly-ADP-ribosyltranferases, whose denomination derives from the capacity of its founding member, PARP1, to synthesise large linear or branched polymers of ADP-ribose on target proteins. This latter post-translational modification has recently attracted much interest based on its role in the cellular response to genotoxic and oxidative stress. Accordingly, a series of PARP-specific pharmacological inhibitors have demonstrated cell survival and anti-inflammatory properties in vivo, promoting a renewed interest in the potential immunoregulatory role of this gene family. More recently, the role of ADP-ribosylation in regulating several aspects of intracellular signalling and gene transcription has been uncovered, in particular within cells of the immune system, revealing the potential immunomodulatory role of several members of this family in addition to PARP1. We review herein the experimental evidence illustrating the complex role played by this gene family in regulating multiple aspects of the immune response, including cell survival, cytokine gene transcription and antiviral innate defences. In particular, the unexpected potential anti-inflammatory role of members of this family (including in particular PARP5a, 5b and PARP14) will be briefly discussed, raising some concern on the use of pan-specific PARP inhibitors to treat chronic inflammatory diseases.

Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes

Significance Interferon regulatory factor (IRF) 3 is one of the key transcription factors implicated in innate antiviral responses. In recent years its role in shaping adaptive immune responses through activation of a specific transcriptional program in antigen-presenting cells has been appreciated. In this work we show that within CD8 T cells, IRF3 interacts with the transcriptional network that controls their polarization, thereby limiting the capacity of these cells to produce IL-17. These findings shed light on the functions of IRF3 in adaptive immune responses. IFN regulatory factor (IRF) 3 plays a key role in innate responses against viruses. Herein we assessed its contribution to T-cell activation. We observed that poly(I:C)-induced IRF3 activation in CD8 T cells represses IL-17 expression in a type I IFN-independent fashion. Even in the absence of poly(I:C), polyclonally activated naïve IRF3−/− CD8 T cells expressed high levels of IL-17 and IL-23R in comparison with wild-type cells. Furthermore, IRF3−/− OT1 cells adoptively transferred into wild-type hosts also produced higher IL-17 levels upon immunization than their wild-type counterparts. This phenotype could be reversed by ectopic expression of IRF3, confirming that this effect is intrinsic to T cells. We show that IRF3 directly interacts with RORγt in the cytoplasm through its IRF interaction domain and limits its ability to bind and transactivate the IL-17 promoter. These observations uncover an unexpected role of IRF3 in the control of CD8 T-cell polarization.

Central Role of CD169 + Lymph Node Resident Macrophages in the Adjuvanticity of the QS-21 Component of AS01

Saponins represent a promising class of vaccine adjuvant. Together with the TLR4-ligand MPL, QS-21 is part of the Adjuvant System AS01, a key component of the malaria and zoster candidate vaccines that display demonstrated clinical efficacy. However, the mechanism of action of QS-21 in this liposomal formulation is poorly understood. Upon intra-muscular immunisation, we observed that QS-21 rapidly accumulated in CD169+ resident macrophages of the draining lymph node where it elicited a local innate immune response. Depletion of these cells abrogated QS-21-mediated innate cell recruitment to the lymph node, dendritic cell (DC) phenotypic maturation as well as the adjuvant effect on T-cell and antibody responses to co-administered antigens. DCs rather than lymph node-resident macrophages were directly involved in T-cell priming by QS-21, as revealed by the decrease in antigen-specific T-cell response in Batf3−/− mice. Further analysis showed that the adjuvant effect of QS-21 depended on the integration of Caspase-1 and MyD88 pathways, at least in part through the local release of HMGB1. Taken together, this work unravels the key role of lymph node sentinel macrophage in controlling the adjuvant effect of a molecule proven to improve vaccine response in humans.

Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

Combining immunostimulants in adjuvants can improve the quality of the immune response to vaccines. Here, we report a unique mechanism of molecular and cellular synergy between a TLR4 ligand, 3-O-desacyl-4’-monophosphoryl lipid A (MPL), and a saponin, QS-21, the constituents of the Adjuvant System AS01. AS01 is part of the malaria and herpes zoster vaccine candidates that have demonstrated efficacy in phase III studies. Hours after injection of AS01-adjuvanted vaccine, resident cells, such as NK cells and CD8+ T cells, release IFNγ in the lymph node draining the injection site. This effect results from MPL and QS-21 synergy and is controlled by macrophages, IL-12 and IL-18. Depletion strategies showed that this early IFNγ production was essential for the activation of dendritic cells and the development of Th1 immunity by AS01-adjuvanted vaccine. A similar activation was observed in the lymph node of AS01-injected macaques as well as in the blood of individuals receiving the malaria RTS,S vaccine. This mechanism, previously described for infections, illustrates how adjuvants trigger naturally occurring pathways to improve the efficacy of vaccines.Adjuvants: Vaccine components working in synergy to improve beneficial effects of vaccinationA mechanism is revealed by which vaccine components co-operate to stimulate the immune system and improve vaccine efficacy. Some vaccines are formulated with adjuvants—compounds that induce a greater immune response to the vaccine and help to elicit greater protection against future infections. Arnaud Didierlaurent and his team of researchers at GSK Vaccines, Belgium, demonstrate that the two immunostimulants in the adjuvant AS01, used in several recently developed vaccines, works in tandem to trigger the activation of important immune system moderators. The synergistic effect of the immunostimulants modulate specific immune cells at the site of the vaccination to better prepare the body against future infection. Studies such as this allow us to better understand how vaccines work and lay the foundation for more informed research into future vaccine development.

Lysosome-Dependent Activation of Human Dendritic Cells by the Vaccine Adjuvant QS-21

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation.

Regulation of Interleukin-23 Expression in Health and Disease

Interleukin (IL)-23 plays a central role in the orchestration of inflammatory responses. Produced by dendritic cells and macrophages, this cytokine promotes the protection of the host against mucosal pathogens through the induction of IL-17 and related cytokines by lymphoid cells. Preclinical disease models and association studies in humans have also clearly demonstrated the implication of IL-23 signalling pathway in inflammatory diseases. Indeed, this cytokine is now considered as a major therapeutic target in immune-based pathologies such as psoriasis, ankylosing spondylitis or Crohns disease. Furthermore, in the context of inflammation-related cancer, IL-23 is thought to contribute to tumorigenesis and progression to metastatic disease. Herein, we review our current understanding of IL-23 regulation at the transcriptional and post-transcriptional levels. We discuss the relevance of these findings in the context of infection, chronic inflammation and cancer.

Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-β.

Lactobacillus acidophilus induces a potent interferon‐β (IFN‐β) response in dendritic cells (DCs) by a Toll‐like receptor 2 (TLR2) ‐dependent mechanism, in turn leading to strong interleukin‐12 (IL‐12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus‐induced IFN‐β and IL‐12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN‐β and IL‐12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) ‐dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk‐independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN‐β and IL‐12 was strongly inhibited. As L. acidophilus‐induced IFN‐β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN‐β induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN‐β and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN‐β and IL‐12 production is inhibited by TLR stimulation from the plasma membrane.

Activation of the endoplasmic reticulum stress sensor IRE1α by the vaccine adjuvant AS03 contributes to its immunostimulatory properties

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants.Adjuvants: ER stress is critical for adjuvant efficacyAdjuvants are the ‘secret sauce’ of vaccines—by stimulating the innate arm of the immune system they potentiate activation of adaptive immunity; however, their mode of action is incompletely understood. A team led by Stanislas Goriely at the Université Libre de Bruxelles studied the functional basis of a major class of clinically-applied adjuvant—AS03—an oil-in-water emulsion adjuvant used in human influenza vaccines. Using an in vivo mouse model and cell lines they observe that AS03 triggered downregulation of genes controlling lipid metabolism. At the same time there was an increase in an ER-stress signature likely as a result of accumulation of intracellular lipid content in myeloid cells such as macrophages. ER stress was critical for the production of inflammatory cytokines by macrophages and the subsequent generation of robust and specific antibody responses following vaccination. Selective enhancement of ER stress might therefore lead to superior vaccines while minimizing side effects.

Author Correction: Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity

In the original version of the article, Fig. 2b was mislabelled. The second row was incorrectly labelled as “MiMPL > MiPBS and MiQS ≈ MPBS” this has now been corrected to “MiQS-21 > MiPBS and MiMPL ≈ MiPBS”. The third row was incorrectly labelled as “MiQS21 > MiPBS and MiMPL ≈ MiPBS” this has now been corrected to “MiMPL > MiPBS and MiQS-21 ≈ MiPBS”. These errors have now been corrected in the PDF and HTML versions of this article.