Inflammatory profile of lower risk myelodysplastic syndromes.

The precise link between inflammation and pathogenesis of myelodysplastic syndrome (MDS) is yet to be fully established. We developed a novel method to measure ASC/NLRP3 protein specks which are specific for the NLRP3 inflammasome only. We combined this with cytokine profiling to characterise various inflammatory markers in a large cohort of patients with lower risk MDS in comparison to healthy controls and patients with defined autoinflammatory disorders (AIDs). The ASC/NLRP3 specks were significantly elevated in MDS patients compared to healthy controls (p < 0.001) and these levels were comparable to those found in patients with AIDs. The distribution of protein specks positive only for ASC was different to ASC/NLRP3 ones suggesting that other ASC-containing inflammasome complexes might be important in the pathogenesis of MDS. Patients with MDS-SLD had the lowest levels of interleukin (IL)-1ß, tumour necrosis factor (TNF), IL-23, IL-33, interferon (IFN) γ and IFN-α2, compared to other diagnostic categories. We also found that inflammatory cytokine TNF was positively associated with MDS progression to a more aggressive form of disease and IL-6 and IL-1ß with time to first red blood cell transfusion. Our study shows that there is value in analysing inflammatory biomarkers in MDS, but their diagnostic and prognostic utility is yet to be fully validated.

Anti-inflammatory effects of elexacaftor/tezacaftor/ivacaftor in adults with cystic fibrosis heterozygous for F508del.

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1ß (p<0.0013, p = 0.0476), IL-6 (p = 0.0109, p = 0.0216) and TNF (p = 0.0028, p = 0.0033) levels in CF serum and following PBMCs stimulation respectively. The corresponding mRNA levels were also found to be reduced in stimulated PBMCs, as well as reduced ASC specks and caspase-1 levels, indicative of NLRP3-mediated production of pro-inflammatory cytokines, IL-1ß and IL-18. While ETI therapy is highly effective at reducing sweat chloride and improving lung function, it also displays potent anti-inflammatory properties, which are likely to contribute to improved long-term clinical outcomes.

Novel Targeted Therapies for Rheumatoid Arthritis Based on Intracellular Signalling and Immunometabolic Changes: A Narrative Review.

Rheumatoid arthritis (RA) is a relatively common systemic autoimmune disease with an estimated prevalence of approximately 1% worldwide. Patients present predominantly with symmetrical small joint inflammatory arthritis, which involves dysregulated immune responses, leading to bone and cartilage deformities due to extensive erosive damage. The introduction of biological based therapies for the management of this life-altering condition, over the past three decades, has led to marked improvements in patients' quality of life. A wide range of both innate and adaptive immune cells are involved in the pathogenesis of RA, with a complex interplay of cytokines, T-cells, B-cells, and dendritic cells. Some of these cells have been successfully targeted in the treatment of RA by the use of biologics-based therapies. For example, rituximab therapy blocks B cell activation and abatacept effectively blocks T cell activation in patients with RA. Despite these advances, there remain some patients who are resistant to all current therapeutic options, which has encouraged further research into understanding the primary signal transduction pathways that mediate the disease. In this review we discuss the roles of the main signalling pathways, including metabolic reprogramming that have been implicated in RA disease progression, in order to develop a conceptual framework for more precise deployment of existing therapies, and to provide a rationale for producing molecular inhibitors of these pathways. Improved knowledge of the many intracellular signalling pathways in RA will complement current precision medicine strategies, particularly for the patients with difficult-to-treat RA, and especially in those with multidrug resistance disease.

Inhibition of IRE1 RNase activity modulates tumor cell progression and enhances the response to chemotherapy in colorectal cancer.

Drug resistance is one of the clinical challenges that limits the effectiveness of chemotherapy. Recent reports suggest that the unfolded protein response (UPR) and endoplasmic reticulum stress-adaptation signalling pathway, along with increased activation of its inositol-requiring enzyme 1α (IRE1α) arm, may be contributors to the pathogenesis of colorectal cancer (CRC). Here, we aimed to target the IRE1α/XBP1 pathway in order to sensitise CRC cells to the effects of chemotherapy. The CT26 colorectal cell line was treated with tunicamycin, and then was exposed to different concentrations of 5-fluorouracil (5-FU), either alone and/or in combination with the IRE1α inhibitor, 4µ8C. An MTT assay, flow cytometry and RT-PCR were performed to determine cell growth, apoptosis and IRE1α activity, respectively. In vivo BALB/c syngeneic colorectal mice received chemotherapeutic drugs. Treatment responses, tumour sizes and cytotoxicity were assessed via a range of pathological tests. 4µ8C was found to inhibit the growth of CRC, at a concentration of 10 µg/ml, without detectable cytotoxic effects and also significantly enhanced the cytotoxic potential of 5-FU, in CRC cells. In vivo experiments revealed that 4µ8C, at a concentration of 50 µM/kg prevented tumour growth without any cytotoxic or metastatic effects. Interestingly, the combination of 4µ8C with 5-FU remarkably enhanced drug responses, up to 40-60% and also lead to significantly greater inhibition of tumour growth, in comparison to monotherapy, in CRC mice. Targeting the IRE1α/XBP1 axis of the UPR could enhance the effectiveness of chemotherapy in both in vitro and in vivo models of CRC.

Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism.

The pyrin inflammasome acts as a guard of RhoA GTPases and is central to immune defenses against RhoA-manipulating pathogens. Pyrin activation proceeds in two steps. Yet, the second step is still poorly understood. Using cells constitutively activated for the pyrin step 1, a chemical screen identifies etiocholanolone and pregnanolone, two catabolites of testosterone and progesterone, acting at low concentrations as specific step 2 activators. High concentrations of these metabolites fully and rapidly activate pyrin, in a human specific, B30.2 domain-dependent manner and without inhibiting RhoA. Mutations in MEFV, encoding pyrin, cause two distinct autoinflammatory diseases pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). Monocytes from PAAND patients, and to a lower extent from FMF patients, display increased responses to these metabolites. This study identifies an unconventional pyrin activation mechanism, indicates that endogenous steroid catabolites can drive autoinflammation, through the pyrin inflammasome, and explains the "steroid fever" described in the late 1950s upon steroid injection in humans.

Inflammasome activation: from molecular mechanisms to autoinflammation.

Inflammasomes are assembled by innate immune sensors that cells employ to detect a range of danger signals and respond with pro-inflammatory signalling. Inflammasomes activate inflammatory caspases, which trigger a cascade of molecular events with the potential to compromise cellular integrity and release the IL-1ß and IL-18 pro-inflammatory cytokines. Several molecular mechanisms, working in concert, ensure that inflammasome activation is tightly regulated; these include NLRP3 post-translational modifications, ubiquitination and phosphorylation, as well as single-domain proteins that competitively bind to key inflammasome components, such as the CARD-only proteins (COPs) and PYD-only proteins (POPs). These diverse regulatory systems ensure that a suitable level of inflammation is initiated to counteract any cellular insult, while simultaneously preserving tissue architecture. When inflammasomes are aberrantly activated can drive excessive production of pro-inflammatory cytokines and cell death, leading to tissue damage. In several autoinflammatory conditions, inflammasomes are aberrantly activated with subsequent development of clinical features that reflect the degree of underlying tissue and organ damage. Several of the resulting disease complications may be successfully controlled by anti-inflammatory drugs and/or specific cytokine inhibitors, in addition to more recently developed small-molecule inhibitors. In this review, we will explore the molecular processes underlying the activation of several inflammasomes and highlight their role during health and disease. We also describe the detrimental effects of these inflammasome complexes, in some pathological conditions, and review current therapeutic approaches as well as future prospective treatments.

An Atypical Autoinflammatory Disease Due to an LRR Domain NLRP3 Mutation Enhancing Binding to NEK7.

The NLRP3 inflammasome is a vital mediator of innate immune responses. There are numerous NLRP3 mutations that cause NLRP3-associated autoinflammatory diseases (NLRP3-AIDs), mostly in or around the NACHT domain. Here, we present a patient with a rare leucine-rich repeat (LRR) domain mutation, p.Arg920Gln (p.R920Q), associated with an atypical NLRP3-AID with recurrent episodes of sore throat and extensive oropharyngeal ulceration. Unlike previously reported patients, who responded well to anakinra, her oral ulcers did not significantly improve until the PDE4 inhibitor, apremilast, was added to her treatment regimen. Here, we show that this mutation enhances interactions between NLRP3 and its endogenous inhibitor, NIMA-related kinase 7 (NEK7), by affecting charge complementarity between the two proteins. We also demonstrate that additional inflammatory mediators, including the NF-кB and IL-17 signalling pathways and IL-8 chemokine, are upregulated in the patient's macrophages and may be directly involved in disease pathogenesis. These results highlight the role of the NLRP3 LRR domain in NLRP3-AIDs and demonstrate that the p.R920Q mutation can cause diverse phenotypes between families.

Neurodegenerative Disease and the NLRP3 Inflammasome.

The prevalence of neurodegenerative disease has increased significantly in recent years, and with a rapidly aging global population, this trend is expected to continue. These diseases are characterised by a progressive neuronal loss in the brain or peripheral nervous system, and generally involve protein aggregation, as well as metabolic abnormalities and immune dysregulation. Although the vast majority of neurodegeneration is idiopathic, there are many known genetic and environmental triggers. In the past decade, research exploring low-grade systemic inflammation and its impact on the development and progression of neurodegenerative disease has increased. A particular research focus has been whether systemic inflammation arises only as a secondary effect of disease or is also a cause of pathology. The inflammasomes, and more specifically the NLRP3 inflammasome, a crucial component of the innate immune system, is usually activated in response to infection or tissue damage. Dysregulation of the NLRP3 inflammasome has been implicated in the progression of several neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases. This review aims to summarise current literature on the role of the NLRP3 inflammasome in the pathogenesis of neurodegenerative diseases, and recent work investigating NLRP3 inflammasome inhibition as a potential future therapy.

Identification of Critical Transcriptomic Signaling Pathways in Patients with H Syndrome and Rosai-Dorfman Disease.

Biallelic mutations in SLC29A3 cause histiocytosis-lymphadenopathy plus syndrome, also known as H syndrome (HS). HS is a complex disorder, with ~ 25% of patients developing autoinflammatory complications consisting of unexplained fevers, persistently elevated inflammatory markers, and unusual lymphadenopathies, with infiltrating CD68+, S100+, and CD1a- histiocytes, resembling the immunophenotype found in Rosai-Dorfman disease (RDD). We investigated the transcriptomic profiles of monocytes, non-activated (M0), classically activated (M1), and alternatively activated macrophages (M2) in two patients with HS, one without autoinflammatory (HS1) and one with autoinflammatory complications (HS2). RNA sequencing revealed a dysregulated transcriptomic profile in both HS patients compared to healthy controls (HC). HS2, when compared to HS1, had several differentially expressed genes, including genes associated with lymphocytic-histiocytic predominance (e.g. NINL) and chronic immune activation (e.g. B2M). The transcriptomic and cytokine profiles of HS patients were comparable to patients with SAID with high levels of TNF. SERPINA1 gene expression was found to be upregulated in all patients studied. Moreover, higher levels of IFNγ were found in the serum of both HS patients when compared to HC. Gene ontology (GO) enrichment analysis of the DEGs in HS patients revealed the terms "type I IFN," "IFNγ signaling pathway," and "immune responses" as the top 3 most significant terms for monocytes. Gene expression analysis of lymph node biopsies from sporadic and H syndrome-associated RDD suggests common underlying pathological process. In conclusion, monocytes and macrophages from both HS patients showed transcriptomic profiles similar to SAIDs and also uniquely upregulated IFNγ signature. These findings may help find better therapeutic options for this rare disorder.

COVID-19 meets Cystic Fibrosis: for better or worse?

Cystic fibrosis (CF) is one of the most common autosomal recessive life-limiting conditions affecting Caucasians. The resulting defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) results in defective chloride and bicarbonate secretion, as well as dysregulation of epithelial sodium channels (ENaC). These changes bring about defective mucociliary clearance, reduced airway surface liquid and an exaggerated proinflammatory response driven, in part, by infection. In this short article we explore the overlap in the pathophysiology of CF and COVID-19 infection and discuss how understanding the interaction between both diseases may shed light on future treatments.

Inflammasome inhibition under physiological and pharmacological conditions.

Inflammasomes are key regulators of the host response against microbial pathogens, in addition to limiting aberrant responses to sterile insults, as mediated by environmental agents such as toxins or nanoparticles, and also by endogenous danger signals such as monosodium urate, ATP and amyloid-ß. To date at least six different inflammasome signalling platforms have been reported (Bauernfeind & Hornung, EMBO Mol Med. 2013;5:814-26; Broz & Dixit, Nat Rev Immunol. 2016;16:407). This review focuses on the complex molecular machinery involved in activation and regulation of the best characterised inflammasome, NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3), and the development of molecular agents to modulate NLRP3 inflammasome function. Activation of the NLRP3 inflammasome induces inflammation via secretion of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) proinflammatory cytokines, with orchestration of pyroptotic cell death, to eliminate invading microbial pathogens. This field has gradually moved from an emphasis on monogenic autoinflammatory conditions, such as cryopyrin-associated periodic syndromes (CAPS), to the broad spectrum of innate immune-mediated disease. NLRP3 inflammasome activation is also linked to a range of common disorders in humans including type 2 diabetes (Krainer et al., J Autoimmun. 2020:102421), cystic fibrosis (Scambler et al., eLife. 2019;8), myocardial infarction, Parkinson's disease, Alzheimer's disease (Savic et al., Nat Rev Rheumatol. 2020:1-16) and cancers such as mesotheliomas and gliomas (Moossavi et al., Mol Cancer. 2018;17:158). We describe how laboratory-based assessment of NLRP3 inflammasome activation is emerging as an integral part of the clinical evaluation and treatment of a range of undifferentiated systemic autoinflammatory disorders (uSAID) (Harrison et al., JCI Insight. 2016;1), where a DNA-based diagnosis has not been possible. In addition, this review summarises the current literature on physiological inhibitors and features various pharmacological approaches that are currently being developed, with potential for clinical translation in autoinflammatory and immune-mediated conditions. We discuss the possibilities of rational drug design, based on detailed structural analyses, and some of the challenges in transferring exciting preliminary results from trials of small-molecule inhibitors of the NLRP3 inflammasome, in animal models of disease, to the clinical situation in human pathology.

Dysregulated signalling pathways in innate immune cells with cystic fibrosis mutations.

Cystic fibrosis (CF) is one of the most common life-limiting recessive genetic disorders in Caucasians, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF is a multi-organ disease that involves the lungs, pancreas, sweat glands, digestive and reproductive systems and several other tissues. This debilitating condition is associated with recurrent lower respiratory tract bacterial and viral infections, as well as inflammatory complications that may eventually lead to pulmonary failure. Immune cells play a crucial role in protecting the organs against opportunistic infections and also in the regulation of tissue homeostasis. Innate immune cells are generally affected by CFTR mutations in patients with CF, leading to dysregulation of several cellular signalling pathways that are in continuous use by these cells to elicit a proper immune response. There is substantial evidence to show that airway epithelial cells, neutrophils, monocytes and macrophages all contribute to the pathogenesis of CF, underlying the importance of the CFTR in innate immune responses. The goal of this review is to put into context the important role of the CFTR in different innate immune cells and how CFTR dysfunction contributes to the pathogenesis of CF, highlighting several signalling pathways that may be dysregulated in cells with CFTR mutations.

Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis.

Previously, we showed that serum and monocytes from patients with CF exhibit an enhanced NLRP3-inflammasome signature with increased IL-18, IL-1ß, caspase-1 activity and ASC speck release (Scambler et al. eLife 2019). Here we show that CFTR modulators down regulate this exaggerated proinflammatory response following LPS/ATP stimulation. In vitro application of ivacaftor/lumacaftor or ivacaftor/tezacaftor to CF monocytes showed a significant reduction in IL-18, whereas IL-1ß was only reduced with ivacaftor/tezacaftor. Thirteen adults starting ivacaftor/lumacaftor and eight starting ivacaftor/tezacaftor were assessed over three months. Serum IL-18 and TNF decreased significantly with treatments, but IL-1ß only declined following ivacaftor/tezacaftor. In (LPS/ATP-stimulated) PBMCs, IL-18/TNF/caspase-1 were all significantly decreased and IL-10 was increased with both combinations. Ivacaftor/tezacaftor alone showed a significant reduction in IL-1ß and pro-IL-1ß mRNA. This study demonstrates that these CFTR modulator combinations have potent anti-inflammatory properties, in addition to their ability to stimulate CFTR function, which could contribute to improved clinical outcomes.

Moving towards a systems-based classification of innate immune-mediated diseases.

Autoinflammation as a distinct disease category was first reported in 1999 as a group of monogenic disorders characterized by recurrent episodes of systemic and organ-specific inflammation, known as periodic fever syndromes. Since this original description, the focus has shifted considerably to the inclusion of complex multifactorial conditions with an autoinflammatory basis. Furthermore, the boundaries of what are considered to be autoinflammatory disorders are constantly evolving and currently encompass elements of immunodeficiency and autoimmunity. Notable developments in the intervening 20 years include substantial progress in understanding how the different inflammasomes are activated, how infection is sensed by the innate immune system and how intracellular signalling systems are consequently activated and integrated with many different cellular functions in the autoinflammatory process. With these developments, the field of autoinflammation is moving from a gene-centric view of innate immune-mediated disease towards a systems-based concept, which describes how various convergent pathways, including pyrin and the actin cytoskeleton, protein misfolding and cellular stress, NF-κB dysregulation and interferon activation, contribute to the autoinflammatory process. The development and adoption of a systems-based concept of systemic autoinflammatory diseases is anticipated to have implications for the development of treatments that target specific components of the innate immune system.

TRAP1 chaperone protein mutations and autoinflammation.

We identified a consanguineous kindred, of three affected children with severe autoinflammation, resulting in the death of one sibling and allogeneic stem cell transplantation in the other two. All three were homozygous for MEFV p.S208C mutation; however, their phenotype was more severe than previously reported, prompting consideration of an oligogenic autoinflammation model. Further genetic studies revealed homozygous mutations in TRAP1, encoding the mitochondrial/ER resident chaperone protein tumour necrosis factor receptor associated protein 1 (TRAP1). Identification of a fourth, unrelated patient with autoinflammation and compound heterozygous mutation of TRAP1 alone facilitated further functional studies, confirming the importance of this protein as a chaperone of misfolded proteins with loss of function, which may contribute to autoinflammation. Impaired TRAP1 function leads to cellular stress and elevated levels of serum IL-18. This study emphasizes the importance of considering digenic or oligogenic models of disease in particularly severe phenotypes and suggests that autoinflammatory disease might be enhanced by bi-allelic mutations in TRAP1.

ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR-mediated chloride and bicarbonate transport, with dysregulation of epithelial sodium channels (ENaC). These changes alter fluid and electrolyte homeostasis and result in an exaggerated proinflammatory response driven, in part, by infection. We tested the hypothesis that NLRP3 inflammasome activation and ENaC upregulation drives exaggerated innate-immune responses in this multisystem disease. We identify an enhanced proinflammatory signature, as evidenced by increased levels of IL-18, IL-1ß, caspase-1 activity and ASC-speck release in monocytes, epithelia and serum with CF-associated mutations; these differences were reversed by pretreatment with NLRP3 inflammasome inhibitors and notably, inhibition of amiloride-sensitive sodium (Na+) channels. Overexpression of ß-ENaC, in the absence of CFTR dysfunction, increased NLRP3-mediated inflammation, indicating that dysregulated, ENaC-dependent signalling may drive exaggerated inflammatory responses in CF. These data support a role for sodium in modulating NLRP3 inflammasome activation.

Metabolic Reprograming of Cystic Fibrosis Macrophages via the IRE1α Arm of the Unfolded Protein Response Results in Exacerbated Inflammation.

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress, with activation of the IRE1α-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however, the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress, mainly affecting neutrophils, monocytes, and macrophages. An overactive IRE1α-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state, with increased glycolytic rates and mitochondrial function, associated with exaggerated production of TNF and IL-6. This hyper-metabolic state, seen in CF macrophages, is reversed by inhibiting the RNase domain of IRE1α, thereby decreasing the increased glycolic rates, mitochondrial function and inflammation. Altogether, our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover, the IRE1α-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages, which is associated with the exaggerated inflammatory response. Modulating ER stress, metabolism and inflammation, by targeting IRE1α, may improve the metabolic fitness of macrophages, and other immune cells in CF and other immune-related disorders.

TNF receptor signalling in autoinflammatory diseases.

Autoinflammatory syndromes are a group of disorders characterized by recurring episodes of inflammation as a result of specific defects in the innate immune system. Patients with autoinflammatory disease present with recurrent outbreaks of chronic systemic inflammation that are mediated by innate immune cells, for the most part. A number of these diseases arise from defects in the tumour necrosis factor receptor (TNFR) signalling pathway leading to elevated levels of inflammatory cytokines. Elucidation of the molecular mechanisms of these recently defined autoinflammatory diseases has led to a greater understanding of the mechanisms of action of key molecules involved in TNFR signalling, particularly those involved in ubiquitination, as found in haploinsufficiency of A20 (HA20), otulipenia/OTULIN-related autoinflammatory syndrome (ORAS) and linear ubiquitin chain assembly complex (LUBAC) deficiency. In this review, we also address other TNFR signalling disorders such as TNFR-associated periodic syndrome (TRAPS), RELA haploinsufficiency, RIPK1-associated immunodeficiency and autoinflammation, X-linked ectodermal dysplasia and immunodeficiency (X-EDA-ID) and we review the most recent advances surrounding these diseases and therapeutic approaches currently used to target these diseases. Finally, we explore therapeutic advances in TNF-related immune-based therapies and explore new approaches to target disease-specific modulation of autoinflammatory diseases.