Glucocorticoid-induced leucine zipper modulates macrophage polarization and apoptotic cell clearance.

Macrophages are professional phagocytes that display remarkable plasticity, with a range of phenotypes that can be broadly characterized by the M1/M2 dichotomy. Glucocorticoid (GC)-induced leucine zipper (GILZ) is a protein known to mediate anti-inflammatory and some pro-resolving actions, including as neutrophil apoptosis. However, the role of GILZ in key macrophage function is not well understood. Here, we investigated the role of GILZ on macrophage reprogramming and efferocytosis. Using murine bone-marrow-derived macrophages (BMDMs), we found that GILZ was expressed in naive BMDMs and exhibited increased expression in M2-like macrophages (IL4-differentiated). M1-like macrophages (IFN/LPS-differentiated) from GILZ-/- mice showed higher expression of the M1 markers CD86, MHC class II, iNOS, IL-6 and TNF-α, associated with increased levels of phosphorylated STAT1 and lower IL-10 levels, compared to M1-differentiated cells from WT mice. There were no changes in the M2 markers CD206 and arginase-1 in macrophages from GILZ-/- mice differentiated with IL-4, compared to cells from WT animals. Treatment of M1-like macrophages with TAT-GILZ, a cell-permeable GILZ fusion protein, decreased the levels of CD86 and MHC class II in M1-like macrophages without modifying CD206 levels in M2-like macrophages. In line with the in vitro data, increased numbers of M1-like macrophages were found into the pleural cavity of GILZ-/- mice after LPS-injection, compared to WT mice. Moreover, efferocytosis was defective in the context of GILZ deficiency, both in vitro and in vivo. Conversely, treatment of LPS-injected mice with TAT-GILZ promoted inflammation resolution, associated with lower numbers of M1-like macrophages and increased efferocytosis. Collectively, these data indicate that GILZ is a regulator of important macrophage functions, contributing to macrophage reprogramming and efferocytosis, both key steps for the resolution of inflammation

Eye Movements Predict Recollective Experience

Previously encountered stimuli can bring to mind a vivid memory of the episodic context in which the stimulus was first experienced ("remembered'' stimuli), or can simply seem familiar ("known'' stimuli). Past studies suggest that more attentional resources are required to encode stimuli that are subsequently remembered than known. However, it is unclear if the attentional resources are distributed differently during encoding and recognition of remembered and known stimuli. Here, we record eye movements while participants encode photos, and later while indicating whether the photos are remembered, known or new. Eye fixations were more clustered during both encoding and recognition of remembered photos relative to known photos. Thus, recognition of photos that bring to mind a vivid memory for the episodic context in which they were experienced is associated with less distributed overt attention during encoding and recognition. The results suggest that remembering is related to encoding of a few distinct details of a photo rather than the photo as a whole. In turn, during recognition remembering may be trigged by enhanced memory for the salient details of the photos

How Emotion Strengthens the Recollective Experience: A Time-Dependent Hippocampal Process

Emotion significantly strengthens the subjective recollective experience even when objective accuracy of the memory is not improved. Here, we examine if this modulation is related to the effect of emotion on hippocampal-dependent memory consolidation. Two critical predictions follow from this hypothesis. First, since consolidation is assumed to take time, the enhancement in the recollective experience for emotional compared to neutral memories should become more apparent following a delay. Second, if the emotion advantage is critically dependent on the hippocampus, then the effects should be reduced in amnesic patients with hippocampal damage. To test these predictions we examined the recollective experience for emotional and neutral photos at two retention intervals (Experiment 1), and in amnesics and controls (Experiment 2). Emotional memories were associated with an enhancement in the recollective experience that was greatest after a delay, whereas familiarity was not influenced by emotion. In amnesics with hippocampal damage the emotion effect on recollective experience was reduced. Surprisingly, however, these patients still showed a general memory advantage for emotional compared to neutral items, but this effect was manifest primarily as a facilitation of familiarity. The results support the consolidation hypothesis of recollective experience, but suggest that the effects of emotion on episodic memory are not exclusively hippocampally mediated. Rather, emotion may enhance recognition by facilitating familiarity when recollection is impaired due to hippocampal damage

The human hepatitis B e antigen targets and suppresses toll-like receptor signalling pathways

HBV infection is characterised by a prolonged ‘immunotolerant hepatitis B e antigen (HBeAg)-positive phase’ where patients are persistently infected with high viral loads, yet they demonstrate no overt inflammation or liver damage. This represents a highly evolved host-parasite relationship. Viruses are known to target the host innate immune system as a means of establishing infection and maintaining chronicity. In humans, the innate immune system recognises pathogens via pattern recognition receptors such as the Toll-like receptors (TLRs), initiating a pro-inflammatory responses. TLRs and associated signalling proteins are characterised by their transmembrane and cytoplasmic Toll-IL1 receptor (TIR) domain. The TIR domain is critical for homotypic TIR:TIR interactions with downstream adaptor proteins leading to activation of downstream transcription factors Nuclear Factor-kappa B (NF-κB) and interferon regulatory factor (IRF)-3. Previous studies have demonstrated that both TLR expression and pro-inflammatory cytokine production is reduced in HBeAg-positive patients compared to HBeAg-negative patients following TLR stimulation. HBeAg is a secreted, non-particulate version of the HBV nucleocapsid protein, the hepatitis B core antigen (HBcAg). It is transcribed, translated and secreted very early in the HBV replication cycle, although importantly, 20-30% of HBeAg is retained in the cytoplasm. No known role is ascribed to this cytosolic HBeAg. The aim of this study was to investigate interactions between TLR signal transduction pathways and the mature HBeAg protein localised in the cytosol. In this thesis I have provided a novel molecular mechanism illustrating how HBeAg inhibits TIR-mediated inflammatory response through direct interaction with a sub-set of TIR-containing proteins. Bio-informatics analysis of HBeAg identified a novel N-terminal 10 amino acid sequence referred to as the Precore Specific Sequence (PSS) distinct from HBcAg, as homologous to the TIR motif found in TLR and associated signal transduction proteins. I have demonstrated by co-localisation and immunoprecipitation that HBeAg, but not HBcAg, is able to interact with a sub-set of membrane proximal TIR-proteins including MyD88 adaptor-like (Mal) and Toll-like receptor adaptor molecule-2 (TRAM). This specificity of this interaction between HBeAg and TIR-containing proteins is demonstrated by the requirement of three critical amino acids “LCL” within the PSS, as mutation of these amino acids ablates all interaction and exerts no inhibitory properties. Critically interactions between HBeAg and TIR-containing proteins were specific as no interaction was observed with MyD88 as illustrated by co-immunoprecipitation or co-localisation studies. Luciferase reporter assays were employed to demonstrate the biological consequences of HBeAg interaction with TIR-containing receptor and adaptor proteins. Co-expression of HBeAg with TLR-2, Mal, TRAM and TIR domain containing adaptor inducing interferon (TRIF) resulted in the significant suppression of both NF-κB and Interferon-β (IFN-β) activation. Consistent with previous findings, NF-κB activation was unaffected by co-expression of HBeAg with MyD88. Importantly, HBcAg which lacks the PSS and the HBeAg mutant had no effect on downstream activation of NF-κB or IFN-β. HBeAg appears to function by sequestering TIR-containing proteins from homotypic TIR:TIR signalling complexes as co-expression. This was illustrated by co-expression of HBeAg inhibiting homotypic TIR:TIR interactions between Mal and MyD88, therefore disrupting downstream signalling. There are several genotypes of HBV found globally and previous studies have reported variation in responsiveness to IFN-β treatment between genotypes. I wished to determine if there were differences in modulation of innate responses amongst genotypes. Interestingly it appears that genotypes A and B suppress predominantly NF-κB mediated responses, whereas genotypes C and D are were found to significantly inhibit both NF-κB and IFN-β mediated responses. Furthermore, TLR-2-induced Interleukin-6 (IL-6) expression is reduced in HBV genotypes C and D compared to genotypes A and B following treatment with IFN-β and TLR-2 agonist Pam3Cys. Critically my findings demonstrate that HBV genotype significantly influences the degree of immuno-modulation on TIR-mediated responses. These findings correlate with previous clinical studies which show individuals with genotypes A and B have higher response rates to IFN treatment facilitating seroconversion compared to genotypes C and D. Critically I have provided a mechanism describing how administration of IFN enhances host immunity in CHB individuals which may explain differences in responsiveness to IFN treatment between HBV genotypes. Overall HBV genotype is an important predictor of response to IFN-based therapy in CHB infection. For best clinical practice and treatment outcome it is strongly recommend that determination of HBV genotype is established before commencing treatment. In conclusion findings presented in this thesis provide a novel molecular mechanism as to how HBeAg interacts with TLR signal transduction pathways to immuno-modulate TIR-mediated innate responses. Importantly the discovery of the PSS, which acts to negatively regulate TLR signalling, may be used in future designs for a novel therapeutic against not only HBV but other TLR-inflammatory diseases circumventing chronicity and poor clinical outcome

The human hepatitis B e antigen targets and suppresses toll-like receptor signalling pathways

HBV infection is characterised by a prolonged ‘immunotolerant hepatitis B e antigen (HBeAg)-positive phase’ where patients are persistently infected with high viral loads, yet they demonstrate no overt inflammation or liver damage. This represents a highly evolved host-parasite relationship. Viruses are known to target the host innate immune system as a means of establishing infection and maintaining chronicity. In humans, the innate immune system recognises pathogens via pattern recognition receptors such as the Toll-like receptors (TLRs), initiating a pro-inflammatory responses. TLRs and associated signalling proteins are characterised by their transmembrane and cytoplasmic Toll-IL1 receptor (TIR) domain. The TIR domain is critical for homotypic TIR:TIR interactions with downstream adaptor proteins leading to activation of downstream transcription factors Nuclear Factor-kappa B (NF-κB) and interferon regulatory factor (IRF)-3. Previous studies have demonstrated that both TLR expression and pro-inflammatory cytokine production is reduced in HBeAg-positive patients compared to HBeAg-negative patients following TLR stimulation. HBeAg is a secreted, non-particulate version of the HBV nucleocapsid protein, the hepatitis B core antigen (HBcAg). It is transcribed, translated and secreted very early in the HBV replication cycle, although importantly, 20-30% of HBeAg is retained in the cytoplasm. No known role is ascribed to this cytosolic HBeAg. The aim of this study was to investigate interactions between TLR signal transduction pathways and the mature HBeAg protein localised in the cytosol. In this thesis I have provided a novel molecular mechanism illustrating how HBeAg inhibits TIR-mediated inflammatory response through direct interaction with a sub-set of TIR-containing proteins. Bio-informatics analysis of HBeAg identified a novel N-terminal 10 amino acid sequence referred to as the Precore Specific Sequence (PSS) distinct from HBcAg, as homologous to the TIR motif found in TLR and associated signal transduction proteins. I have demonstrated by co-localisation and immunoprecipitation that HBeAg, but not HBcAg, is able to interact with a sub-set of membrane proximal TIR-proteins including MyD88 adaptor-like (Mal) and Toll-like receptor adaptor molecule-2 (TRAM). This specificity of this interaction between HBeAg and TIR-containing proteins is demonstrated by the requirement of three critical amino acids “LCL” within the PSS, as mutation of these amino acids ablates all interaction and exerts no inhibitory properties. Critically interactions between HBeAg and TIR-containing proteins were specific as no interaction was observed with MyD88 as illustrated by co-immunoprecipitation or co-localisation studies. Luciferase reporter assays were employed to demonstrate the biological consequences of HBeAg interaction with TIR-containing receptor and adaptor proteins. Co-expression of HBeAg with TLR-2, Mal, TRAM and TIR domain containing adaptor inducing interferon (TRIF) resulted in the significant suppression of both NF-κB and Interferon-β (IFN-β) activation. Consistent with previous findings, NF-κB activation was unaffected by co-expression of HBeAg with MyD88. Importantly, HBcAg which lacks the PSS and the HBeAg mutant had no effect on downstream activation of NF-κB or IFN-β. HBeAg appears to function by sequestering TIR-containing proteins from homotypic TIR:TIR signalling complexes as co-expression. This was illustrated by co-expression of HBeAg inhibiting homotypic TIR:TIR interactions between Mal and MyD88, therefore disrupting downstream signalling. There are several genotypes of HBV found globally and previous studies have reported variation in responsiveness to IFN-β treatment between genotypes. I wished to determine if there were differences in modulation of innate responses amongst genotypes. Interestingly it appears that genotypes A and B suppress predominantly NF-κB mediated responses, whereas genotypes C and D are were found to significantly inhibit both NF-κB and IFN-β mediated responses. Furthermore, TLR-2-induced Interleukin-6 (IL-6) expression is reduced in HBV genotypes C and D compared to genotypes A and B following treatment with IFN-β and TLR-2 agonist Pam3Cys. Critically my findings demonstrate that HBV genotype significantly influences the degree of immuno-modulation on TIR-mediated responses. These findings correlate with previous clinical studies which show individuals with genotypes A and B have higher response rates to IFN treatment facilitating seroconversion compared to genotypes C and D. Critically I have provided a mechanism describing how administration of IFN enhances host immunity in CHB individuals which may explain differences in responsiveness to IFN treatment between HBV genotypes. Overall HBV genotype is an important predictor of response to IFN-based therapy in CHB infection. For best clinical practice and treatment outcome it is strongly recommend that determination of HBV genotype is established before commencing treatment. In conclusion findings presented in this thesis provide a novel molecular mechanism as to how HBeAg interacts with TLR signal transduction pathways to immuno-modulate TIR-mediated innate responses. Importantly the discovery of the PSS, which acts to negatively regulate TLR signalling, may be used in future designs for a novel therapeutic against not only HBV but other TLR-inflammatory diseases circumventing chronicity and poor clinical outcome

Analysis of Serum Interleukin (IL)-1β and IL-18 in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease characterized by biological and clinical heterogeneity. The interleukin (IL)-1 superfamily is a group of innate cytokines that contribute to pathogenesis in many autoimmune diseases. IL-1β and IL-18 are two members that have been shown to play a role in murine lupus-like models, but their role in human SLE remains poorly understood. Here, IL-1β and IL-18 were quantified by enzyme-linked immunosorbent assay in the serum of healthy controls (HCs) and SLE patients from a prospectively followed cohort. Disease activity and organ damage were assessed using SLE disease activity index 2000 (SLEDAI-2K) and SLE damage index scores (SDI), respectively. 184 SLE patients (mean age 44.9 years, 91% female, 56% double-stranded deoxyribonucleic acid positive) were compared to 52 HC. SLE patients had median [IQR] SLEDAI-2K of 4 [2,6], and SDI of 1 [0–2]. Serum IL-18 levels were statistically significantly higher in SLE patients compared to HCs. Univariable linear regression analyses showed that patients with active renal disease or irreversible organ damage had statistically significantly elevated serum IL-18 levels. The association between serum IL-18 and active renal disease was confirmed in multivariable analysis after adjusting for ethnicity and organ damage. High baseline serum IL-18 levels were associated with organ damage at the subsequent visit. Serum IL-1β levels were not significantly elevated in SLE patients when compared to HCs and had no association with overall or organ-specific disease activity or organ damage in cross-sectional and longitudinal analyses. Our data suggest that serum IL-18 and IL-1β have different clinical implications in SLE, with IL-18 being potentially associated with active renal disease