Tali Lang

The negative regulation of Toll-like receptor and associated pathways

Toll‐like receptors (TLRs) are essential mediators of both innate and adaptive immunity by recognizing and eliciting responses upon invasion of pathogens. The response of TLRs must be stringently regulated as exaggerated expression of signalling components as well as pro‐inflammatory cytokines can have devastating effects on the host, resulting in chronic inflammatory diseases, autoimmune disorders and aid in the pathogenesis of TLR‐associated human diseases. Therefore, it is essential that negative regulators act at multiple levels within TLR signalling cascades, as well as through eliciting negative‐feedback mechanisms in order to synchronize the positive activation and negative regulation of signal transduction to avert potentially harmful immunological consequences. This review explores the various mechanisms employed by negative regulators to ensure the appropriate modulation of both immune and inflammatory responses.

The Hepatitis B e antigen (HBeAg) targets and suppresses activation of the Toll-like receptor signaling pathway

BACKGROUND & AIMS Viruses target innate immune pathways to evade host antiviral responses. Recent studies demonstrate a relationship between hepatitis B disease states and the hosts innate immune response, although the mechanism of immunomodulation is unknown. In humans, the innate immune system recognizes pathogens via pattern recognition receptors such as the Toll-like receptors (TLR), initiating anti-inflammatory responses. TLR expression and pro-inflammatory cytokine production is reduced in hepatitis B e antigen (HBeAg)-positive patients following TLR stimulation. The aim of this study was to investigate interactions between TLR signaling pathways and the mature HBeAg protein localized in the cytosol. METHODS The ability of HBeAg to inhibit TLR signaling and association with TLR adapters was evaluated by immunoprecipitation, immunostaining, and reporter studies. RESULTS Our findings show that HBeAg co-localizes with Toll/IL-1 receptor (TIR)-containing proteins TRAM, Mal, and TLR2 at the sub-cellular level, which was not observed for Hepatitis B core antigen. Co-immunoprecipitation analysis demonstrated HBeAg interacted with TIR proteins Mal and TRAM, while a mutated HBeAg ablated interaction between Mal and MyD88. Importantly, HBeAg also disrupted homotypic TIR:TIR interaction critical for TLR-mediated signaling. Finally, HBeAg suppressed TIR-mediated activation of the inflammatory transcription factors, NF-κB and Interferon-β promoter activity. CONCLUSIONS Our study provides the first molecular mechanism describing HBeAg immunomodulation of innate immune signal transduction pathways via interaction and targeting of TLR-mediated signaling pathways. These finding suggest the mechanism as to how HBeAg evades innate immune responses contributing to the pathogenesis of chronic hepatitis B infection and the establishment of viral persistence.

Autophagy and inflammasomes

HighlightsLoss of autophagy increases inflammasome activation.Autophagy removes inflammasome‐activating stimuli from the cytosol.Autophagy degrades inflammasome components.Autophagy sequesters IL‐1&bgr;.Further understanding of these processes could lead to new therapeutic targets. Abstract Autophagy is a ubiquitous cellular mechanism for the targeted lysosomal degradation of various cytosolic constituents, from proteins to organelles. As an essential homeostatic mechanism, autophagy is upregulated in response to numerous environmental and pharmacological stimuli, including starvation, where it facilitates the recycling of essential amino acids. In addition, autophagy plays specific roles within the immune system; it serves as a source of peptides for antigen presentation, a mechanism for the engulfment and degradation of intracellular pathogens and as a key regulator of inflammatory cytokines. In particular, autophagy has been shown to play a number of roles in regulating inflammasome activation, from the removal of inflammasome‐activating endogenous signals, to the sequestration and degradation of inflammasome components. Autophagy also plays a role in determining the fate of IL‐1&bgr;, which is concentrated in autophagosomes. This review discusses a growing body of literature that suggests autophagy is a critical regulator of inflammasome activation and the subsequent release of IL‐1 family cytokines.

MIF: Implications in the Pathoetiology of Systemic Lupus Erythematosus

Macrophage migration Inhibitory factor (MIF) was one of the earliest pro-inflammatory cytokines to be identified. Increasing interest in this cytokine in recent decades has followed the cloning of human MIF and the generation of Mif−/− mice. Deepening understanding of signaling pathways utilized by MIF and putative receptor mechanisms have followed. MIF is distinct from all other cytokines by virtue of its unique induction by and counter regulation of glucocorticoids (GCs). MIF is further differentiated from other cytokines by its structural homology to specific tautomerase and isomerase enzymes and correlative in vitro enzymatic functions. The role of MIF in immune and inflammatory states, including a range of human autoimmune diseases, is now well established, as are the relationships between MIF polymorphisms and a number of inflammatory diseases. Here, we review the known pleiotropic activities of MIF, in addition to novel functions of MIF in processes including autophagy and autophagic cell death. In addition, recent developments in the understanding of the role of MIF in systemic lupus erythematosus (SLE) are reviewed. Finally, we discuss the potential application of anti-MIF strategies to treat human diseases such as SLE, which will require a comprehensive understanding of the unique and complex activities of this ubiquitously expressed cytokine.

Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages

ABSTRACT MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]) is a pro-inflammatory cytokine expressed in multiple cells types, including macrophages. MIF plays a pathogenic role in a number of inflammatory diseases and has been linked to tumor progression in some cancers. Previous work has demonstrated that loss of autophagy in macrophages enhances secretion of IL1 family cytokines. Here, we demonstrate that loss of autophagy, by pharmacological inhibition or siRNA silencing of Atg5, enhances MIF secretion by monocytes and macrophages. We further demonstrate that this is dependent on mitochondrial reactive oxygen species (ROS). Induction of autophagy with MTOR inhibitors had no effect on MIF secretion, but amino acid starvation increased secretion. This was unaffected by Atg5 siRNA but was again dependent on mitochondrial ROS. Our data demonstrate that autophagic regulation of mitochondrial ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases and cancers.

Structural and Functional Integrity of Spermatozoa Is Compromised as a Consequence of Acute Uropathogenic E. coli-Associated Epididymitis

ABSTRACT Uropathogenic Escherichia coli (UPEC)-associated epididymitis is commonly diagnosed in outpatient settings. Although the infection can be successfully cleared using antimicrobial medications, 40% of patients unexplainably show persistent impaired semen parameters even after treatment. Our aim was to investigate whether pathogenic UPEC and its associated virulence factor hemolysin (hlyA) perturb the structural and functional integrity of both the epididymis and sperm, actions that may be responsible for the observed impairment and possibly a reduction of fertilization capabilities. Semen collected from patients diagnosed with E. coli-only related epididymitis showed that sperm counts were low 14 days postantimicrobial treatment regardless of hlyA status. At Day 84 following treatment, hlyA production correlated with approximately 4-fold lower sperm concentrations than in men with hlyA-negative strains. In vivo experiments with the hlyA-producing UPEC CFT073 strain in a murine epididymitis model showed that just 3 days postinfection, structural damage to the epididymis (epithelial damage, leukocyte infiltration, and edema formation) was present. This was more severe in UPEC CFT073 compared to nonpathogenic E. coli (NPEC 470) infection. Moreover, pathogenic UPEC strains prematurely activated the acrosome in vivo and in vitro. Raman microspectroscopy revealed that UPEC CFT073 undermined sperm integrity by inducing nuclear DNA damage. Consistent with these observations, the in vitro fertilization capability of hlyA-treated mouse sperm was completely abolished, although sperm were motile. These findings provide new insights into understanding the possible processes underlying clinical manifestations of acute epididymitis.

Acute epididymitis induces alterations in sperm protein composition

OBJECTIVE To use a proteomic approach to evaluate possible postinflammatory alterations in the protein composition of motile sperm in patients 3 months after acute epididymitis. DESIGN Prospective case-control study. SETTING University medical school research laboratory. PATIENT(S) Eight patients 3 months after acute unilateral epididymitis and 10 healthy controls. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Proteome analysis of sperm samples collected by swim-up from control and acute epididymitis patients analyzed by two-dimensional gel electrophoresis and subsequent protein identification by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry; immunofluorescence staining for mitochondrial ATP synthase subunit β (ATP5B), α-tubulin (TUBA1A), and tubulin-β2c (TUBB4B) for validation purposes. RESULT(S) Proteome analysis identified 35 proteins in sperm from epididymitis patients that were down-regulated, irrespective of subcellular localization and biologic function. Furthermore, immunofluorescence microscopy confirmed ATP5B, TUBA1A, and TUBB4B were less abundantly expressed in epididymitis samples compared with controls. CONCLUSION(S) Despite normal semen parameters observed by conventional semen analysis in patients after epididymitis, significant changes to sperm protein composition were observed. These changes may be implicated as additional factors contributing to subfertility/infertility in men after episodes of epididymitis.

Uropathogenic Escherichia coli Modulates Innate Immunity To Suppress Th1-Mediated Inflammatory Responses during Infectious Epididymitis

ABSTRACT Infectious epididymitis in men, a frequent entity in urological outpatient settings, is commonly caused by bacteria originating from the anal region ascending the genitourinary tract. One of the most prevalent pathogens associated with epididymitis is Escherichia coli. In our previous study, we showed that semen quality is compromised in men following epididymitis associated with specific E. coli pathovars. Thus, our aim was to investigate possible differences in immune responses elicited during epididymitis following infection with the uropathogenic E. coli (UPEC) strain CFT073 and the nonpathogenic enteric E. coli (NPEC) strain 470. Employing an in vivo experimental epididymitis model, C57BL/6 mice were infected with UPEC CFT073, NPEC 470, or phosphate-buffered saline (PBS) as a sham control for up to 7 days. After infection with NPEC 470, the expression of proinflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha in the epididymis was significantly increased. Conversely, UPEC CFT073-challenged mice displayed inflammatory gene expression at levels comparable to sham PBS-treated animals. Moreover, by day 7 only NPEC-infected animals showed activation of adaptive immunity evident by a substantial influx of CD3+ and F4/80+ cells in the epididymal interstitium. This correlated with enhanced production of Th1-associated cytokines IL-2 and gamma interferon (IFN-γ). Furthermore, splenocytes isolated from UPEC-infected mice exhibited diminished T-cell responses with significantly reduced secretion of IL-2 and IFN-γ in contrast to NPEC-infected animals. Overall, these findings provide new insights into understanding pathogen-specific modulation of host immunity during acute phases of epididymitis, which may influence severity of disease and clinical outcomes.

Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1β, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.MIF is a cytokine best known for its modulatory effect on expression of proinflammatory cytokines. Here the authors show that MIF facilitates the NLRP3–vimentin interaction, resulting in Nlrp3 inflammasome activation.

Toll-IL1 receptor-mediated innate immune responses vary across HBV genotype and predict treatment response to pegylated-IFN in HBeAg-positive CHB patients

Patients with hepatitis B e antigen (HBeAg)‐positive chronic hepatitis B (CHB) have suppressed TLR2 expression, function and cytokine production. The aim of this study was to explore the importance of hepatitis B virus (HBV) genotype in innate immune responses and investigate whether Toll‐like receptor (TLR) expression/function has potential roles as predictive biomarkers of successful therapy with pegylated interferon (Peg‐IFN) therapy of HBeAg seroconversion in HBeAg‐positive patients. We showed that as early as 4 weeks after initiation of Peg‐IFN, future HBeAg seroconverters had significantly elevated levels of TLR2 expression on monocytes. TLR2‐associated IL‐6 production at baseline and week 4 of therapy and TLR4 IL‐6 production at week 4 were also markedly elevated in HBeAg seroconverters. HBV genotype also influenced treatment response, with genotypes A and B more likely to seroconvert than D. We were able to demonstrate that these differences were due in part to the interaction of the specific HBeAg proteins with TLR pathway adaptor molecules, and these interactions were genotype dependent. HBeAg‐mediated modulation of TLR signalling was also observed in Huh7 cells, following stimulation with Pam3Cys. Importantly, the addition of IFN‐α to TLR2‐stimulated cells cotransfected with an HBeAg expression plasmid reversed HBeAg‐mediated suppression of hepatocytes. These findings demonstrate that patients with an activated inflammatory response are much more likely to respond to IFN therapy, with TLR responses showing promise as potential biomarkers of HBeAg seroconversion in this setting. Furthermore, our findings suggest there is differential genotype‐specific HBeAg suppression of innate signalling pathways which may account for some of the clinical differences observed across the CHB spectrum.