RIP2-deficiency induces inflammation in response to SV40 Large T induced genotoxic stress through altered ROS homeostasis.

Deciphering signaling pathways that regulate the complex interplay between inflammation and cell death is a key challenge in understanding innate immune responses. Over recent years, receptor interacting protein (RIP) kinases have been described to regulate the interplay between inflammation and cell death. Whereas RIP1 and 3, the most well described members of the RIP kinase family, play important roles in necroptosis, RIP2's involvement in regulating inflammation, cell death processes and cancer is less well described and controversially discussed. Here, we demonstrate that RIP2 exerts immune regulatory functions by regulating mitochondrial damage and mitochondrial superoxide production in response to SV40 LT-induced genotoxic stress by the induction of ULK1-phosphorylation, therefore regulating the expression of interferon stimulated genes (ISGs) and NLRP3-inflammasome dependent IL-1ß release. Because RIP2 is upregulated and/or activated in autoimmune/inflammatory disease and cancer, observations from this study promise implications of RIP kinases in human disease.

Cyclic AMP Response Element Modulator-α Suppresses PD-1 Expression and Promotes Effector CD4+ T Cells in Psoriasis.

Effector CD4+ T lymphocytes contribute to inflammation and tissue damage in psoriasis, but the underlying molecular mechanisms remain poorly understood. The transcription factor CREMα controls effector T cell function in people with systemic autoimmune diseases. The inhibitory surface coreceptor PD-1 plays a key role in the control of effector T cell function and its therapeutic inhibition in patients with cancer can cause psoriasis. In this study, we show that CD4+ T cells from patients with psoriasis and psoriatic arthritis exhibit increased production of IL-17 but decreased expression of IL-2 and PD-1. In genetically modified mice and Jurkat T cells CREMα expression was linked to low PD-1 levels. We demonstrate that CREMα is recruited to the proximal promoter of PDCD1 in which it trans-represses gene expression and corecruits DNMT3a-mediating DNA methylation. As keratinocytes limit inflammation by PD-1 ligand expression and, in this study, reported reduced expression of PD-1 on CD4+ T cells is linked to low IL-2 and high IL-17A production, our studies reveal a molecular pathway in T cells from people with psoriasis that can deserve clinical exploitation.

cAMP Response Element Modulator α Induces Dual Specificity Protein Phosphatase 4 to Promote Effector T Cells in Juvenile-Onset Lupus.

Effector CD4+ T cells with increased IL-17A and reduced IL-2 production contribute to tissue inflammation and organ damage in systemic lupus erythematosus (SLE). Increased expression of the transcription factor cAMP response element modulator (CREM) α promotes altered cytokine expression in SLE. The aim of this study was to investigate CREMα-mediated events favoring effector CD4+ T cells in health and disease. Using CRISPR/Cas9 genome editing and lentiviral transduction, we generated CREMα-deficient and CREMα-overexpressing Jurkat T cells. Gene expression and regulatory events were assessed using luciferase reporter assays and chromatin immunoprecipitation. Interaction between CREMα and p300 was investigated using proximity ligation assays, coimmunoprecipitation, and knockdown of p300. Gene expression profiles of modified cells were compared with CD4+ T cells from patients with juvenile-onset SLE. We show that CREMα induces dual specificity protein phosphatase (DUSP) 4 in effector CD4+ T cells through corecruitment of p300. The transcriptional coactivator p300 mediates histone acetylation at DUSP4, prompting increased gene expression. Using DUSP4 transfection models and genetically modified CREM-deficient and CREMα-overexpressing T cells, we demonstrate the molecular underpinnings by which DUSP4 induces IL-17A while limiting IL-2 expression. We demonstrate that CD4+ T cells from patients with juvenile-onset SLE share phenotypical features with CREMα-overexpressing CD4+ T cells, including increased DUSP4 expression and imbalanced IL-17A and IL-2 production. Taken together, we describe CREMα-mediated mechanisms that involve the transcriptional upregulation of DUSP4, leading to imbalanced cytokine production by effector T cells. Our findings identify the CREMα/DUSP4 axis as a promising candidate in the search for biomarkers and therapeutic targets in SLE.

CASP1 variants influence subcellular caspase-1 localization, pyroptosome formation, pro-inflammatory cell death and macrophage deformability.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1ß release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.

Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa).

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

Chronic Recurrent Multifocal Osteomyelitis (CRMO): Presentation, Pathogenesis, and Treatment.

PURPOSE OF REVIEW: Chronic non-bacterial osteomyelitis (CNO) with its most severe form chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder. We summarize the clinical presentation, diagnostic approaches, most recent advances in understanding the pathophysiology, and available treatment options and outcomes in CNO/CRMO. RECENT FINDINGS: Though the exact molecular pathophysiology of CNO/CRMO remains somewhat elusive, it appears likely that variable defects in the TLR4/MAPK/inflammasome signaling cascade result in an imbalance between pro- and anti-inflammatory cytokine expressions in monocytes from CNO/CRMO patients. In this context, we present previously unpublished data on cytokine and chemokine expression in monocytes and tissues. CNO/CRMO is an autoinflammatory bone disorder resulting from imbalanced cytokine expression from innate immune cells. Though the exact molecular pathophysiology remains unclear, variable molecular defects appear to result in inflammasome activation and pro-inflammatory cytokine expression in monocytes from CNO/CRMO patients. Recent advances suggest signaling pathways and single molecules as biomarkers for CNO/CRMO as well as future treatment targets.

The molecular pathophysiology of chronic non-bacterial osteomyelitis (CNO)-a systematic review.

Chronic non-bacterial osteomyelitis (CNO) belongs to the growing spectrum of autoinflammatory diseases and primarily affects the skeletal system. Peak onset ranges between 7 and 12 years of age. The clinical spectrum of CNO covers sometimes asymptomatic inflammation of single bones at the one end and chronically active or recurrent multifocal osteitis at the other.Despite the intense scientific efforts, the exact molecular mechanisms of CNO remain unknown. Recent data suggest CNO as a genetically complex disorder with dysregulated TLR4/MAPK/inflammasome signaling cascades resulting in an imbalance between pro- and anti-inflammatory cytokine expression, leading to osteoclast activation and osteolytic lesions.In this manuscript, the current understanding of molecular patho-mechanisms in CNO will be discussed.

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division.

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1ß in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1ß secretion. The apparent paradox of reduced IL-1ß secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1ß and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1ß-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.

Fluorescent tags influence the enzymatic activity and subcellular localization of procaspase-1.

Subcellular localization studies and life cell imaging approaches usually benefit from fusion-reporter proteins, such as enhanced green fluorescent protein (EGFP) and mCherry to the proteins of interest. However, such manipulations have several risks, including protein misfolding, altered protein shuttling, or functional impairment when compared to the wild-type proteins. Here, we demonstrate altered subcellular distribution and function of the pro-inflammatory enzyme procaspase-1 as a result of fusion with the reporter protein mCherry. Our observations are of central importance to further investigations of subcellular behavior and possible protein-protein interactions of naturally occurring genetic variants of human procaspase-1 which have recently been linked to autoinflammatory disorders.

High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli.

BACKGROUND: For their application in the area of diagnosis and therapy, single-domain antibodies (sdAbs) offer multiple advantages over conventional antibodies and fragments thereof in terms of size, stability, solubility, immunogenicity, production costs as well as tumor uptake and blood clearance. Thus, sdAbs have been identified as valuable next-generation targeting moieties for molecular imaging and drug delivery in the past years. Since these probes are much less complex than conventional antibody fragments, bacterial expression represents a facile method in order to produce sdAbs in large amounts as soluble and functional proteins. RESULTS: By the combined use of high cell density cultivation media with a genetically engineered E. coli mutant strain designed for the cytoplasmic formation of proper disulfide bonds, we achieved high level of intracellular sdAb production (up to 200 mg/L). Due to a carboxyterminal hexahistidine epitope, the soluble recombinant sdAbs could be purified by one-step immobilized metal affinity chromatography to apparent homogeneity and easily radiolabeled with 99mTc within 1 h. The intradomain disulfide bridge being critical for the stability and functionality of the sdAb molecule was shown to be properly formed in ~96% of the purified proteins. In vitro binding studies confirmed the high affinity and specificity of the expressed sdAb 7C12 towards its molecular target. CONCLUSIONS: Our study demonstrates an efficient cultivation and expression strategy for the production of substantial amounts of soluble and functional sdAbs, which may be adopted for high-yield production of other more complex proteins with multiple disulfides as well.