Mechanisms of NLRP3 activation and pathology during neurodegeneration.

Inflammasomes are multiprotein complexes that are mainly present in resident and infiltrating immune cells in the central nervous system. Inflammasomes function as intracellular sensors of immunometabolic stress, infection and changes in the local microenvironment. Inflammasome assembly in response to these 'danger signals', triggers recruitment and cluster-dependent activation of caspase-1 and the subsequent proteolytic activation of inflammatory cytokines such as interleukin-1ß and interleukin-18. This is typically followed by a form of inflammatory cell death through pyroptosis. Since the discovery of inflammasomes in 2002, they have come to be recognized as central regulators of acute and chronic inflammation, a hallmark of progressive neurological diseases. Indeed, over the last decade, extensive inflammasome activation has been found at the sites of neuropathology in all progressive neurodegenerative diseases. Disease-specific misfolded protein aggregates which accumulate in neurodegenerative diseases, such as alpha synuclein or beta amyloid, have been found to be important triggers of NLRP3 inflammasome activation in the central nervous system. Together, these discoveries have transformed our understanding of how chronic inflammation is triggered and sustained in the central nervous system, and how it can contribute to neuronal death and disease progression in age-related neurodegenerative diseases. Therapeutic strategies around inhibition of NLRP3 activation in the central nervous system are already being evaluated to determine their effectiveness to slow progressive neurodegeneration. This review summarizes current understanding of inflammasomes in the most prevalent neurodegenerative diseases and discusses current knowledge gaps and inflammasome inhibition as a therapeutic strategy.

Pluripotency and the origin of animal multicellularity.

A widely held-but rarely tested-hypothesis for the origin of animals is that they evolved from a unicellular ancestor, with an apical cilium surrounded by a microvillar collar, that structurally resembled modern sponge choanocytes and choanoflagellates1-4. Here we test this view of animal origins by comparing the transcriptomes, fates and behaviours of the three primary sponge cell types-choanocytes, pluripotent mesenchymal archaeocytes and epithelial pinacocytes-with choanoflagellates and other unicellular holozoans. Unexpectedly, we find that the transcriptome of sponge choanocytes is the least similar to the transcriptomes of choanoflagellates and is significantly enriched in genes unique to either animals or sponges alone. By contrast, pluripotent archaeocytes upregulate genes that control cell proliferation and gene expression, as in other metazoan stem cells and in the proliferating stages of two unicellular holozoans, including a colonial choanoflagellate. Choanocytes in the sponge Amphimedon queenslandica exist in a transient metastable state and readily transdifferentiate into archaeocytes, which can differentiate into a range of other cell types. These sponge cell-type conversions are similar to the temporal cell-state changes that occur in unicellular holozoans5. Together, these analyses argue against homology of sponge choanocytes and choanoflagellates, and the view that the first multicellular animals were simple balls of cells with limited capacity to differentiate. Instead, our results are consistent with the first animal cell being able to transition between multiple states in a manner similar to modern transdifferentiating and stem cells.

Landscape of histone modifications in a sponge reveals the origin of animal cis-regulatory complexity.

Combinatorial patterns of histone modifications regulate developmental and cell type-specific gene expression and underpin animal complexity, but it is unclear when this regulatory system evolved. By analysing histone modifications in a morphologically-simple, early branching animal, the sponge Amphimedonqueenslandica, we show that the regulatory landscape used by complex bilaterians was already in place at the dawn of animal multicellularity. This includes distal enhancers, repressive chromatin and transcriptional units marked by H3K4me3 that vary with levels of developmental regulation. Strikingly, Amphimedon enhancers are enriched in metazoan-specific microsyntenic units, suggesting that their genomic location is extremely ancient and likely to place constraints on the evolution of surrounding genes. These results suggest that the regulatory foundation for spatiotemporal gene expression evolved prior to the divergence of sponges and eumetazoans, and was necessary for the evolution of animal multicellularity.

Origin and Evolution of the Sponge Aggregation Factor Gene Family.

Although discriminating self from nonself is a cardinal animal trait, metazoan allorecognition genes do not appear to be homologous. Here, we characterize the Aggregation Factor (AF) gene family, which encodes putative allorecognition factors in the demosponge Amphimedon queenslandica, and trace its evolution across 24 sponge (Porifera) species. The AF locus in Amphimedon is comprised of a cluster of five similar genes that encode Calx-beta and Von Willebrand domains and a newly defined Wreath domain, and are highly polymorphic. Further AF variance appears to be generated through individualistic patterns of RNA editing. The AF gene family varies between poriferans, with protein sequences and domains diagnostic of the AF family being present in Amphimedon and other demosponges, but absent from other sponge classes. Within the demosponges, AFs vary widely with no two species having the same AF repertoire or domain organization. The evolution of AFs suggests that their diversification occurs via high allelism, and the continual and rapid gain, loss and shuffling of domains over evolutionary time. Given the marked differences in metazoan allorecognition genes, we propose the rapid evolution of AFs in sponges provides a model for understanding the extensive diversification of self-nonself recognition systems in the animal kingdom.

Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease.

Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate "real time" gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-ß in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

Human proximal tubule epithelial cells modulate autologous dendritic cell function.

BACKGROUND: We have previously demonstrated that human kidney proximal tubule epithelial cells (PTEC) are able to modulate autologous T and B lymphocyte responses. It is well established that dendritic cells (DC) are responsible for the initiation and direction of adaptive immune responses and that these cells occur in the renal interstitium in close apposition to PTEC under inflammatory disease settings. However, there is no information regarding the interaction of PTEC with DC in an autologous human context. METHODS: Human monocytes were differentiated into monocyte-derived DC (MoDC) in the absence or presence of primary autologous activated PTEC and matured with polyinosinic:polycytidylic acid [poly(I:C)], while purified, pre-formed myeloid blood DC (CD1c(+) BDC) were cultured with autologous activated PTEC in the absence or presence of poly(I:C) stimulation. DC responses were monitored by surface antigen expression, cytokine secretion, antigen uptake capacity and allogeneic T-cell-stimulatory ability. RESULTS: The presence of autologous activated PTEC inhibited the differentiation of monocytes to MoDC. Furthermore, MoDC differentiated in the presence of PTEC displayed an immature surface phenotype, efficient phagocytic capacity and, upon poly(I:C) stimulation, secreted low levels of pro-inflammatory cytokine interleukin (IL)-12p70, high levels of anti-inflammatory cytokine IL-10 and induced weak Th1 responses. Similarly, pre-formed CD1c(+) BDC matured in the presence of PTEC exhibited an immature tolerogenic surface phenotype, strong endocytic and phagocytic ability and stimulated significantly attenuated T-cell proliferative responses. CONCLUSIONS: Our data suggest that activated PTEC regulate human autologous immunity via complex interactions with DC. The ability of PTEC to modulate autologous DC function has important implications for the dampening of pro-inflammatory immune responses within the tubulointerstitium in renal injuries. Further dissection of the mechanisms of PTEC modulation of autologous immune responses may offer targets for therapeutic intervention in renal medicine.

Activated human renal tubular cells inhibit autologous immune responses.

BACKGROUND: Renal proximal tubule epithelial cells (PTEC) respond and contribute to the pathological process in a range of kidney diseases. Within this disease setting, PTEC up-regulate surface antigens which may enable them to act as non-professional antigen-presenting cells and become targets for infiltrating T cells in the context of disease and allograft rejection. In order to define, for the first time, whether PTEC modulate immune responses within the autologous human system, we monitored their interaction with T and B cells in the presence of stimuli which mimic immunological signalling. METHODS: The expression of PTEC surface antigen in response to inflammatory mediators was monitored by flow cytometry. Purified T and B lymphocyte subsets and peripheral blood mononuclear cells were cultured in the presence or absence of autologous activated PTEC, and their responses to specific activators were monitored by proliferation, cytokine secretion and surface antigen expression. Some experiments were performed in the presence of blocking antibodies to PD-L1. RESULTS: The presence of activated primary autologous PTEC resulted in significantly decreased T- and B-cell proliferative responses, which were only partly mediated by programmed death ligand 1. This modulation was not induced by a decrease in activation markers or an increase in T regulatory cells but was accompanied by strong significant skewing of cytokine profiles. Significant decreases in gamma-interferon, interleukin-2 and tumour necrosis factor and increases in interleukin-4 were detected in the presence of PTEC, indicating that these cells induce a shift away from an inflammatory Th1 effector profile to a Th2 type profile. CONCLUSION: Human PTEC do modulate autologous immune responses. We hypothesize that such mechanisms may have developed to help dampen inflammatory responses and macrophage activation seen within kidney interstitium in many immune-mediated kidney diseases.

The effect of larval age on morphology and gene expression during ascidian metamorphosis.

Metamorphosis is both an ecological and a developmental genetic transition that an organism undergoes as a normal part of ontogeny. Many organisms have the ability to delay metamorphosis when conditions are unsuitable. This strategy carries obvious benefits, but may also result in severe consequences for older larvae that run low on energy. In the marine environment, some lecithotrophic larvae that have prolonged periods in the plankton may begin forming postlarval and juvenile structures that normally do not appear until after settlement and the initiation of metamorphosis. This precocious activation of the postlarval developmental program may reflect an adaptation to increase the survival of older, energy-depleted larvae by allowing them to metamorphose more quickly. In the present study, we investigate morphological and genetic consequences of delay of metamorphosis in larvae of Herdmania momus (a solitary stolidobranch ascidian). We observe significant morphological and genetic changes during prolonged larval life, with older larvae displaying significant changes in RNA levels, precocious migration of mesenchyme cells, and changes in larval shape including shortening of the tail. While these observations suggest that the older H. momus larvae are functionally different from younger larvae and possibly becoming more predisposed to undergo metamorphosis, we did not find any significant differences in gene expression levels between postlarvae arising from larvae that metamorphosed as soon as they were competent and postlarvae developing from larvae that postponed metamorphosis. This recalibration, or convergence, of transcript levels in the early postlarva suggests that changes that occur during prolonged larval life of H. momus are not necessarily associated with early activation of adult organ differentiation. Instead, it suggests that an autonomous developmental program is activated in H. momus upon the induction of metamorphosis regardless of the history of the larva.

Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein.

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.