Claudia Günther

Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn’s disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn’s disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8ΔIEC) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8ΔIEC mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8ΔIEC mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn’s disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-α-induced necroptotic cell death.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Human slan (6-sulfo LacNAc) dendritic cells are inflammatory dermal dendritic cells in psoriasis and drive strong Th17/Th1 T-cell responses

BACKGROUND Psoriasis is a chronic inflammatory skin disease that is considered to result from activated T cells stimulated by a population of inflammatory dermal dendritic cells (DCs). The origin and identity of these inflammatory dermal DCs are largely unknown. OBJECTIVE We previously identified slanDCs (6-sulfo LacNAc) DCs as a rich source of TNF-α and as the early major source of IL-12. Here we studied the relevance of slanDCs as inflammatory dermal DCs in psoriasis. METHODS Psoriasis skin samples were stained for the presence of activated slanDCs. Functional studies were carried out to determine the cytokine production of slanDCs, their T(h)17/T(h)1 T-cell programming, and their migration behavior. RESULTS Large numbers of IL-23, TNF-α, and inducible nitric oxide synthase expressing slanDCs were found in psoriatic skin samples, which can be recruited by C5a, CX3CL1, and CXCL12. SlanDCs isolated from blood produced high levels of IL-1ß, IL-23, IL-12, and IL-6. Compared with classic CD1c(+) DCs, slanDCs were far more powerful in programming T(h)17/T(h)1 T cells that secrete IL-17, IL-22, TNF-α, and IFN-γ, yet CD1c(+) DCs induced a higher IL-10 production of T cells. Self-nucleic acids complexed to cathelicidin LL37 trigger endosomal Toll-like receptor (TLR) signaling (TLR7, TLR8, TLR9) and are key factors for the activation of DCs in psoriasis. We show that slanDCs respond particularly well to complexes formed of self-RNA and LL37. Similarly, slanDCs stimulated with a synthetic TLR7/8 ligand produced high levels of proinflammatory cytokines. CONCLUSION Our study defines slanDCs as inflammatory dermal DCs in psoriasis and identifies their strong capacity to induce T(h)17/T(h)1 responses.

Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium

Intestinal epithelial cells (IEC) are organised as a single cell layer which covers the intestine. Their primary task is to absorb nutrients present in the intestinal lumen. However, IEC also play an important role in the immune defence of our body by building a barrier that separates the bowel wall from potentially hazardous bacteria present in the gut lumen. The life cycle of IEC is determined by the time span in which cells migrate from their place of origin at the crypt base to the villus tip, from where they are shed into the lumen. Cell death in the intestinal epithelium has to be tightly regulated and irregularities might cause pathologies. Excessive cell death has been associated with chronic inflammation as seen in patients with Crohns disease and ulcerative colitis. While until recently apoptosis was discussed as being essential for epithelial turnover and tissue homeostasis in the intestinal epithelium, recent data using gene deficient mice have challenged this concept. Moreover, an apoptosis-independent mode of programmed cell death, termed necroptosis, has been identified and described in the intestinal epithelium. The following article reviews previous studies on cell death regulation in IEC and a potential role of necroptosis for gut homeostasis.

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

Norovirus triggered microbiota-driven mucosal inflammation in interleukin 10-deficient mice.

Background:Infection may trigger clinically overt mucosal inflammation in patients with predisposition for inflammatory bowel disease. However, the impact of particular enteropathogenic microorganisms is ill-defined. In this study, the influence of murine norovirus (MNV) infection on clinical, histopathological, and immunological features of mucosal inflammation in the IL10-deficient (Il10−/−) mouse model of inflammatory bowel disease was examined. Methods:C57BL/6J and C3H/HeJBir wild-type and Il10−/− mice kept under special pathogen-free conditions and devoid of clinical and histopathological signs of mucosal inflammation were monitored after MNV infection for structural and functional intestinal barrier changes by in situ MNV reverse transcription PCR, transgene reporter gene technology, histology, flux measurements, quantitative real-time PCR, immunohistology, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. In addition, the influence of the enteric microbiota was analyzed in MNV-infected germfree Il10−/− mice. Results:Although MNV-infected wild-type mice remained asymptomatic, mucosal inflammation was noted in previously healthy Il10−/− mice 2 to 4 weeks after infection. MNV-induced changes in Il10−/− mice included increased paracellular permeability indicated by increased mucosal mannitol flux, reduced gene expression of tight junction molecules, and an enhanced rate of epithelial apoptosis. MNV-induced reduction of tight junction protein expression and inflammatory lesions were absent in germfree Il10−/− mice, whereas epithelial apoptosis was still observed. Conclusions:Despite its subclinical course in wild-type animals, MNV causes epithelial barrier disruption in Il10−/− animals representing a potent colitogenic stimulus that largely depends on the presence of the enteric microbiota. MNV might thus trigger overt clinical disease in individuals with a nonsymptomatic predisposition for inflammatory bowel disease by impairment of the intestinal mucosa.

Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis

Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts.

Aicardi–Goutières syndrome: a model disease for systemic autoimmunity

Systemic autoimmunity is a complex disease process that results from a loss of immunological tolerance characterized by the inability of the immune system to discriminate self from non‐self. In patients with the prototypic autoimmune disease systemic lupus erythematosus (SLE), formation of autoantibodies targeting ubiquitous nuclear antigens and subsequent deposition of immune complexes in the vascular bed induces inflammatory tissue injury that can affect virtually any organ system. Given the extraordinary genetic and phenotypic heterogeneity of SLE, one approach to the genetic dissection of complex SLE is to study monogenic diseases, for which a single gene defect is responsible. Considerable success has been achieved from the analysis of the rare monogenic disorder Aicardi–Goutières syndrome (AGS), an inflammatory encephalopathy that clinically resembles in‐utero‐acquired viral infection and that also shares features with SLE. Progress in understanding the cellular and molecular functions of the AGS causing genes has revealed novel pathways of the metabolism of intracellular nucleic acids, the major targets of the autoimmune attack in patients with SLE. Induction of autoimmunity initiated by immune recognition of endogenous nucleic acids originating from processes such as DNA replication/repair or endogenous retro‐elements represents novel paradigms of SLE pathogenesis. These findings illustrate how investigating rare monogenic diseases can also fuel discoveries that advance our understanding of complex disease. This will not only aid the development of improved tools for SLE diagnosis and disease classification, but also the development of novel targeted therapeutic approaches.

Familial chilblain lupus due to a gain-of-function mutation in STING

Objectives Familial chilblain lupus is a monogenic form of cutaneous lupus erythematosus caused by loss-of-function mutations in the nucleases TREX1 or SAMHD1. In a family without TREX1 or SAMHD1 mutation, we sought to determine the causative gene and the underlying disease pathology. Methods Exome sequencing was used for disease gene identification. Structural analysis was performed by homology modelling and docking simulations. Type I interferon (IFN) activation was assessed in cells transfected with STING cDNA using an IFN-β reporter and Western blotting. IFN signatures in patient blood in response to tofacitinib treatment were measured by RT-PCR of IFN-stimulated genes. Results In a multigenerational family with five members affected with chilblain lupus, we identified a heterozygous mutation of STING, a signalling molecule in the cytosolic DNA sensing pathway. Structural and functional analyses indicate that mutant STING enhances homodimerisation in the absence of its ligand cGAMP resulting in constitutive type I IFN activation. Treatment of two affected family members with the Janus kinase (JAK) inhibitor tofacitinib led to a marked suppression of the IFN signature. Conclusions A heterozygous gain-of-function mutation in STING can cause familial chilblain lupus. These findings expand the genetic spectrum of type I IFN-dependent disorders and suggest that JAK inhibition may be of therapeutic value.

Cellular FLICE-like inhibitory protein secures intestinal epithelial cell survival and immune homeostasis by regulating caspase-8.

BACKGROUND & AIMS The intestinal epithelium generates a barrier that protects mammals from potentially harmful intestinal contents, such as pathogenic bacteria. Dysregulation of epithelial cell death has been implicated in barrier dysfunction and in the pathogenesis of intestinal inflammation. We investigated mechanisms of cell-death regulation in the intestinal epithelium of mice. METHODS Conditional knockout mice (either inducible or permanent) with deletion of cellular FLICE-inhibitory protein (cFlip) or caspase-8 in the intestinal epithelium were analyzed by histology and high-resolution endoscopy. We assessed the effects of cFlip or caspase-8 deficiency on intestinal homeostasis. RESULTS Expression of cFlip in the intestinal epithelium was required for constitutive activation of caspase-8 under steady-state conditions. Intestinal expression of cFlip was required for development; disruption of the gene encoding cFlip from the intestinal epithelium (cFlip(fl/fl) VillinCre(+) mice) resulted in embryonic lethality. When cFlip was deleted from the intestinal epithelium of adult mice (cFlip(iΔIEC) mice), the animals died within a few days from severe tissue destruction, epithelial cell death, and intestinal inflammation. Death of cFlip-depleted intestinal epithelial cells was regulated extrinsically and required the presence of death receptor ligands, such as tumor necrosis factor-α and CD95 ligand, but was independent of receptor-interacting protein 3. cFlip deficiency was associated with strong up-regulation of caspase-8 and caspase-3 activity and excessive apoptosis in intestinal crypts. CONCLUSIONS cFlip is required for intestinal tissue homeostasis in mice. It controls the level of activation of caspase-8 to promote survival of intestinal epithelial cells.