Leishmania guyanensis parasites block the activation of the inflammasome by inhibiting maturation of IL-1ß.

The various symptomatic outcomes of cutaneous leishmaniasis relates to the type and potency of its underlying inflammatory responses. Presence of the cytoplasmic Leishmania RNA virus-1 (LRV1) within Leishmania guyanensis, worsens lesional inflammation and parasite burden, as the viral dsRNA genome acts as a potent innate immunogen stimulating Toll-Like-Receptor-3 (TLR3). Here we investigated other innate pattern recognition receptors capable of reacting to dsRNA and potentially contributing to LRV1-mediated inflammatory pathology. We included the cytoplasmic dsRNA sensors, namely, the RIG-like receptors (RLRs) and the inflammasome-dependent and -independent Nod-like-receptors (NLRs). Our study found no role for RLRs or inflammasome-dependent NLRs in the pathology of L. guyanensis infection irrespective of its LRV1-status. Further, neither LRV1-bearing L. guyanensis (LgyLRV1+) nor LRV1-negative L. guyanensis (LgyLRV1-) activated the inflammasome in vitro. Interestingly, similarly to L. donovani, L. guyanensis infection induced the up-regulation of the A20 protein, known to be involved in the evasion of inflammasome activation. Moreover, we observed that LgyLRV1+ promoted the transcription of inflammasome-independent NLRC2 (also called NOD2) and NLRC5. However, only NLRC2 showed some contribution to LRV1-dependent pathology. These data confirmed that the endosomal TLR3 pathway is the dominant route of LRV1-dependent signalling, thus excluding the cytosolic and inflammasome pathways. We postulate that avoidance of the inflammasome pathways is likely an important mechanism of virulence in Leishmania infection irrespective of the LRV1-status.

Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition.

The gut microbiota regulate susceptibility to multiple human diseases. The Nlrp6-ASC inflammasome is widely regarded as a hallmark host innate immune axis that shapes the gut microbiota composition. This notion stems from studies reporting dysbiosis in mice lacking these inflammasome components when compared with non-littermate wild-type animals. Here, we describe microbial analyses in inflammasome-deficient mice while minimizing non-genetic confounders using littermate-controlled Nlrp6-deficient mice and ex-germ-free littermate-controlled ASC-deficient mice that were all allowed to shape their gut microbiota naturally after birth. Careful microbial phylogenetic analyses of these cohorts failed to reveal regulation of the gut microbiota composition by the Nlrp6- and ASC-dependent inflammasomes. Our results obtained in two geographically separated animal facilities dismiss a generalizable impact of Nlrp6- and ASC-dependent inflammasomes on the composition of the commensal gut microbiota and highlight the necessity for littermate-controlled experimental design in assessing the influence of host immunity on gut microbial ecology.

Silencing of ASC in Cutaneous Squamous Cell Carcinoma.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an important adaptor protein for inflammasome activation, mediating the secretion of protumorigenic innate cytokines. However, ASC is also known to trigger apoptosis in tumor cells, acting as a tumor-suppressor gene, which is lost in several human cancers. The aim of this study was to evaluate the clinical significance of ASC in human cutaneous squamous cell carcinoma (SCC). Initially, ASC expression was immunohistochemically evaluated in non-metastic and metastatic SCC. While ASC expression does not correlate with metastatic potential, it correlates with the degree of dedifferentiation. Using methylation specific PCR we were able to demonstrate ASC silencing by promotor specific methylation and impaired inflammasome function in methylated cell lines, linking epigenetic modifications to innate immune activation in keratinocytes. Interestingly, upon ASC restoration by treatment with demethylating agents, we were able to restore AIM2 and NLRP3 activation. In summary, loss of ASC driven tumor development is counterbalanced in the identical cell by the inhibition of pro-tumorigenic inflammation in the tumor cell itself.

Mammalian Innate Immune Response to a Leishmania-Resident RNA Virus Increases Macrophage Survival to Promote Parasite Persistence.

Some strains of the protozoan parasite Leishmania guyanensis (L.g) harbor a viral endosymbiont called Leishmania RNA virus 1 (LRV1). LRV1 recognition by TLR-3 increases parasite burden and lesion swelling in vivo. However, the mechanisms by which anti-viral innate immune responses affect parasitic infection are largely unknown. Upon investigating the mammalian host's response to LRV1, we found that miR-155 was singularly and strongly upregulated in macrophages infected with LRV1+ L.g when compared to LRV1- L.g. LRV1-driven miR-155 expression was dependent on TLR-3/TRIF signaling. Furthermore, LRV1-induced TLR-3 activation promoted parasite persistence by enhancing macrophage survival through Akt activation in a manner partially dependent on miR-155. Pharmacological inhibition of Akt resulted in a decrease in LRV1-mediated macrophage survival and consequently decreased parasite persistence. Consistent with these data, miR-155-deficient mice showed a drastic decrease in LRV1-induced disease severity, and lesional macrophages from these mice displayed reduced levels of Akt phosphorylation.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays.

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers(1-4). This process is dependent on the release of cyclic adenosine monophosphate (cAMP)(5). cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors(6,7). A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions(8,9). This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)(10). Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities(11,12). Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade(10). The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

The two faces of the inflammasome adaptor ASC in epithelial skin carcinogenesis.

The development of tumours is a multistep process during which cells acquire the capability to sustain proliferation, evade growth suppressors and/or resist cell death. One factor, which is increasingly recognised to influence tumour progression, is the inflammatory environment of the tumour. The responsible molecular mechanisms and signalling pathways are only beginning to emerge. One major pathway able to induce potent inflammation is the activation of the inflammasome and the subsequent secretion of the pro-inflammatory cytokines IL-1ß and IL-18. Both these cytokines have been implicated in tumour-genesis/progression. However, evidence for the role of inflammasomes in this process is still scarce and mainly derived from murine colitis associated tumour models. In this short review we discuss current knowledge on the role of inflammasomes in epithelial cancer of the gut and skin with a special focus on the complex role of the inflammasome adaptor ASC in epithelial skin carcinogenesis.

Complex roles of inflammasomes in carcinogenesis.

The central role of chronic inflammation in the promotion of tumor growth is supported by a broad range of experimental and clinical evidence. However, the molecular mechanisms converting transient inflammatory tissue reactions into a tumor-promoting microenvironment remain largely elusive. Because inflammasomes have been shown to regulate the proinflammatory cytokines interleukin 1ß (IL-1ß) and IL-18, they have been implicated in the relationship between tumor genesis/progression and inflammation. For instance, many cancers have been directly linked to inflammasome-mediated sterile inflammation, where a blockade of IL-1ß and IL-18 has been shown to inhibit tumor growth. On the other hand, inflammasome activation also has potent antitumorigenic effects, where malignant precursor cells are eliminated through pyroptotic cell death. Indeed, inflammasome activity can even increase the efficacy of certain chemotherapies. Here, we review the current understanding on the complex and sometimes contradictory role of inflammasomes in carcinogenesis.

Regulation of interleukin 1α secretion by inflammasomes.

The crucial role of the proinflammatory cytokine interleukin 1ß (IL-1ß) in driving inflammatory disorders, such as Muckle-Wells syndrome and gout, has been extensively characterised. Owing to its high potency to induce inflammation the activation and secretion of IL-1ß is tightly regulated. The sensing of various host 'dangers', including infections and metabolic deregulation, results in the formation of large protein complexes, termed inflammasomes. Formation of the inflammasomes leads to the cleavage and activation of caspase-1, which in turn proteolytically processes its substrates, including pro-IL-1ß. Biologically active IL-1ß is subsequently secreted by the cell. In contrast to IL-1ß, little is known about mechanisms underlying the activation and secretion of its close homologue IL-1α. Moreover, the physiological role of IL-1α is still not well defined. Several studies hypothesise that IL-1α serves as a danger signal, which is passively released from dying cells. However, recent studies suggest a more complex function of this cytokine. Indeed, NLRP3 inflammasome agonists such as uric acid crystal or nigericin induce IL-1α cleavage and secretion, leading to the cosecretion of both IL-1ß and IL-1α. Depending on the type of NLRP3 agonist, release of IL-1α is NLRP3-inflammasome/caspase-1 dependent or independent, but in both cases IL-1α processing depends on calpain protease activity. Taken together, these results suggest that the promotion and progression of inflammatory diseases is not solely due to IL-1ß but also to its close relative IL-1α. This should be considered when IL-1 blockade is applied as a therapeutic strategy for diseases such as cryopyrin-associated periodic syndromes or gout.

Tissue-specific opposing functions of the inflammasome adaptor ASC in the regulation of epithelial skin carcinogenesis.

A chronic inflammatory microenvironment favors tumor progression through molecular mechanisms that are still incompletely defined. In inflammation-induced skin cancers, IL-1 receptor- or caspase-1-deficient mice, or mice specifically deficient for the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) in myeloid cells, had reduced tumor incidence, pointing to a role for IL-1 signaling and inflammasome activation in tumor development. However, mice fully deficient for ASC were not protected, and mice specifically deficient for ASC in keratinocytes developed more tumors than controls, suggesting that, in contrast to its proinflammatory role in myeloid cells, ASC acts as a tumor-suppressor in keratinocytes. Accordingly, ASC protein expression was lost in human cutaneous squamous cell carcinoma, but not in psoriatic skin lesions. Stimulation of primary mouse keratinocytes or the human keratinocyte cell line HaCaT with UVB induced an ASC-dependent phosphorylation of p53 and expression of p53 target genes. In HaCaT cells, ASC interacted with p53 at the endogenous level upon UVB irradiation. Thus, ASC in different tissues may influence tumor growth in opposite directions: it has a proinflammatory role in infiltrating cells that favors tumor development, but it also limits keratinocyte proliferation in response to noxious stimuli, possibly through p53 activation, which helps suppressing tumors.

Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.

Through their capacity to sense danger signals and to generate active interleukin-1ß (IL-1ß), inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1ß, little is known about how IL-1α is regulated. We found that all inflammasome activators also induced the secretion of IL-1α, leading to the cosecretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α was inflammasome dependent or independent. Calcium influx induced by the opening of cation channels was sufficient for the inflammasome-independent IL-1α secretion. In both cases, IL-1α was released primarily in a processed form, resulting from intracellular cleavage by calpain-like proteases. Inflammasome-caspase-1-dependent release of IL-1α and IL-1ß was independent of caspase-1 catalytic activity, defining a mode of action for caspase-1. Because inflammasomes contribute to the pathology of numerous chronic inflammatory diseases such as gout and diabetes, IL-1α antagonists may be beneficial in the treatment of these disorders.

Nanoparticles activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome and cause pulmonary inflammation through release of IL-1α and IL-1ß.

Nanoparticles are increasingly used in various fields, including biomedicine and electronics. One application utilizes the opacifying effect of nano-TiO(2), which is frequently used as pigment in cosmetics. Although TiO(2) is believed to be biologically inert, an emerging literature reports increased incidence of respiratory diseases in people exposed to TiO(2). Here, we show that nano-TiO(2) and nano-SiO(2), but not nano-ZnO, activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome, leading to IL-1ß release and in addition, induce the regulated release of IL-1α. Unlike other particulate Nlrp3 agonists, nano-TiO(2)-dependent-Nlrp3 activity does not require cytoskeleton-dependent phagocytosis and induces IL-1α/ß secretion in nonphagocytic keratinocytes. Inhalation of nano-TiO(2) provokes lung inflammation which is strongly suppressed in IL-1R- and IL-1α-deficient mice. Thus, the inflammation caused by nano-TiO(2) in vivo is largely caused by the biological effect of IL-1α. The current use of nano-TiO(2) may present a health hazard due to its capacity to induce IL-1R signaling, a situation reminiscent of inflammation provoked by asbestos exposure.

The role of the inflammasome in nonmyeloid cells.

Inflammasomes are cytosolic multiprotein complexes that can proteolytically activate caspase-1. Activated caspase-1 is needed for the maturation and secretion of interleukin (IL)-1beta and IL-18. In the past decade, there has been tremendous progress in our knowledge of inflammasome function and IL-1 signaling, mainly in cells of the innate immune system, such as monocytes, macrophages, neutrophils, and dendritic cells. Because nonimmune cells, including keratinocytes, synovial cells, or astrocytes, can form an interface between the body and the environment or a defined compartment (brain, joint), they are important guardians for the detection of danger signals and the consecutive initiation of an inflammatory response. They are present in anatomical compartments that are less accessible to myeloid cells and thus can fulfill tasks usually performed by residential macrophages. This review focuses on recent progress in our understanding of the processing and functional role of IL-1 in epithelial, mesenchymal, and neuronal cells and in conditions such as tissue repair.

SIGIRR/TIR-8 is an inhibitor of Toll-like receptor signaling in primary human cells and regulates inflammation in models of rheumatoid arthritis.

OBJECTIVE: Single-immunoglobulin interleukin-1 receptor-related (SIGIRR), which is also known as Toll/interleukin-1 receptor 8 (TIR-8), is a member of the TIR domain-containing family of receptors and was first characterized as an inhibitor of interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling. In the Dextran sulfate sodium-induced colitis model, SIGIRR(-/-) mice were shown to have increased inflammation and to be more susceptible to endotoxin challenge. Increasing evidence implicates TLR and IL-1R signaling in the pathology of rheumatoid arthritis (RA). Therefore, the purpose of this study was to investigate the involvement of SIGIRR in regulating inflammation in disease-relevant models. METHODS: Primary human monocyte-derived macrophages and dendritic cells (DCs) were used to overexpress SIGIRR as well as to knock down endogenously expressed SIGIRR using small interfering RNAs. SIGIRR was also overexpressed in synovial cells derived from RA patients. To investigate the role of SIGIRR in vivo, zymosan-induced arthritis (ZIA) and collagen antibody-induced arthritis (CAIA) were induced in SIGIRR-knockout mice. RESULTS: SIGIRR overexpression inhibited TLR-induced cytokine production in macrophages and DCs, while SIGIRR knockdown resulted in increased cytokine production following TLR stimulation. Moreover, SIGIRR overexpression inhibited the spontaneous release of cytokines by human RA synovial cells. The role of SIGIRR as an inhibitor of inflammation was confirmed in vivo, since SIGIRR(-/-) mice developed a more severe disease in both the ZIA and CAIA models. CONCLUSION: Our study is the first to show the expression pattern and function of SIGIRR in primary human cells. Furthermore, this investigation defines the role of SIGIRR in disease-relevant cell types and demonstrates that SIGIRR is a potential therapeutic target for RA.

Inflammatory caspases in innate immunity and inflammation.

Caspases are best known for their role in apoptosis. More recently, they have gained prominence as critical mediators of innate immune responses. The so-called 'inflammatory caspases' include human caspase-1, -4, -5 and -12 and murine caspase-1, -11 and -12. Of these, caspase-1 is best characterized and serves as the prototype for our understanding of the processing, activation and function of inflammatory caspases. Like their apoptotic counterparts, inflammatory caspases are produced as inactive zymogens and require activation to become proteolytically active. Caspase-1 is activated within the inflammasome, a large cytosolic protein complex that is induced by a growing number of endogenous, microbial, chemical or environmental stimuli. The importance of caspase-1 in initiating innate immune responses is demonstrated by its role in cleaving pro-IL-1 beta and pro-IL-18 to their biologically active forms. New functions have also been implicated, as these proteases and the mechanisms underlying their activation and regulation emerge as important mediators of human health and disease.

The role of toll-like receptors in chronic inflammation.

The role of Toll-like receptors (TLRs) in innate immunity and their ability to recognise microbial products has been well characterised. TLRs are also able to recognise endogenous molecules which are released upon cell damage and necrosis and have been shown to be present in numerous autoimmune diseases. Therefore, the release of endogenous TLR ligands during inflammation and consequently the activation of TLR signalling pathways may be one mechanism initiating and driving autoimmune diseases. An increasing body of circumstantial evidence implicates a role of TLR signalling in systemic lupus erythematosus (SLE), atherosclerosis, asthma, type 1 diabetes, multiple sclerosis, bowl inflammation and rheumatoid arthritis (RA). Although at present their involvement is not comprehensively defined. However, future therapies targeting individual TLRs or their signalling transducers may provide a more specific way of treating inflammatory diseases without global suppression of the immune system.

Cell signalling in macrophages, the principal innate immune effector cells of rheumatoid arthritis.

Rheumatoid arthritis is a multisystemic auto-inflammatory disease affecting up to 1% of the population and leading to the destruction of the joints. Evidence exists for the involvement of the innate as well as the adaptive immune systems in the pathology of the disease. The success of anti-tumour necrosis factor-alpha indicates the importance of pro-inflammatory mediators produced by innate immune cells in rheumatoid arthritis progression. Therefore, considerable efforts have been made in elucidating the signalling pathways leading to the expression of those mediators. This review will concentrate on the role of signalling pathways in innate immune cells in the context of rheumatoid arthritis.

Could toll-like receptors provide a missing link in chronic inflammation in rheumatoid arthritis? Lessons from a study on human rheumatoid tissue.

In the last decade the development of a number of biological therapies has revolutionised the treatment of rheumatic diseases. The first and most widely used of these approaches, tumour necrosis factor (TNF) blockade (infliximab, entanercept, adalimumab), has now been administered to over a million patients. However, the success of these biological therapies has also highlighted their limitations. None of these treatments has shown a 100% patient response; normally responses are in the 50-70% range. As proteins, these drugs cannot be given orally and they are expensive to produce, a cost ultimately borne by the patient/health provider that can seriously limit the availability of these drugs. Lastly, these treatments, whether involving the systemic neutralisation of a cytokine (eg, TNF or IL6 receptor blockade (tocilizumab)), the ablation of a B cell population (anti-CD20, rituximab), or the potential disruption of important cellular interactions as with CTLA4-Ig (abatacept), can cause major perturbations of the immune system, the long-term effects of which are still unclear. At present, treatments such as TNF blockade can result in an increased infectious risk and the reactivation of tuberculosis can be a major issue in certain populations. As with all therapies, there is an increasing large refractory population over time. Therefore, despite the undoubted success of these therapies, there is room for improvement. Although it might be too much to expect any new treatment to affect a "cure" (all the current biological therapies require repeated administrations), there are definite gains to be made in terms of cost, oral bioavailability and a more selective interference with the immune-inflammatory response.

Key differences in TLR3/poly I:C signaling and cytokine induction by human primary cells: a phenomenon absent from murine cell systems.

TLR3 recognizes double-stranded RNA, a product associated with viral infections. Many details of TLR3-induced mechanisms have emerged from gene-targeted mice or inhibition studies in transformed cell lines. However, the pathways activated in human immune cells or cells from disease tissue are less well understood. We have investigated TLR3-induced mechanisms of human primary cells of the innate immune system, including dendritic cells (DCs), macrophages (MØs), endothelial cells (ECs), and synovial fibroblasts isolated from rheumatoid arthritis joint tissue (RA-SFs). Here, we report that while these cells all express TLR3, they differ substantially in their response to TLR3 stimulation. The key antiviral response chemokine IP-10 was produced by all cell types, while DCs and MØs failed to produce the proinflammatory cytokines TNFalpha and IL-6. Unexpectedly, TNFalpha was found secreted by TLR3-stimulated RA-SF. Furthermore, TLR3 stimulation did not activate NFkappaB, MAPKs, or IRF-3 in DCs and MØs, but was able to do so in ECs and RA-SF. These findings were specific for human cells, thereby revealing a complexity not previously expected. This is the first report of such cell type- and species-specific response for any TLR stimulation and helps to explain important difficulties in correlating murine models of inflammatory diseases and human inflammation.

Toll-like receptors: a new target in rheumatoid arthritis?

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases. It is characterized by chronic inflammation of the joint leading to its destruction. Although the initiating cause remains elusive, environmental factors and genetic background are known to contribute to the etiology of RA. The role of Toll-like receptors (TLRs) in innate immunity and their ability to recognize microbial products has been well characterized. TLRs are able to recognize endogenous molecules released upon cell damage and necrosis, and are present in RA synovial fluid. Although it appears unlikely that a pathogen underlies the pathogenesis or progression of RA, the release of endogenous TLR ligands during inflammation may activate TLRs and perpetuate the disease. An increasing body of circumstantial evidence implicates TLR signaling in RA, although, at present, their involvement is not defined comprehensively. Targeting individual TLRs or their signaling transducers may provide a more specific therapy without global suppression of the immune system.