•Complement deficiencies are increasingly recognized and present an unexpected wide spectrum of diseases.•Modern comprehensive complement analysis allows early detection of complement ...deficiencies.•Advanced molecular analysis sheds new light on the importance of complement in organ-specific severe inflammatory disorders and even developmental processes.
Complement defects are associated with an enhanced risk of a broad spectrum of infectious as well as systemic or local inflammatory and thrombotic disorders. Inherited complement deficiencies have been described for virtually all complement components but can be mimicked by autoantibodies, interfering with the activity of specific complement components, convertases or regulators. While being rare, diseases related to complement deficiencies are often severe with a frequent but not exclusive manifestation during childhood. Whereas defects of early components of the classical pathway significantly increase the risk of autoimmune disorders, lack of components of the terminal pathway as well as of properdin are associated with an enhanced susceptibility to meningococcal infections. The impaired synthesis or function of C1 inhibitor results in the development of hereditary angioedema (HAE). Furthermore, complement dysregulation causes renal disorders such as atypical hemolytic uremic syndrome (aHUS) or C3 glomerulopathy (C3G) but also age-related macular degeneration (AMD). While paroxysmal nocturnal hemoglobinuria (PNH) results from the combined deficiency of the regulatory complement proteins CD55 and CD59, which is caused by somatic mutation of a common membrane anchor, isolated CD55 or CD59 deficiency is associated with the CHAPLE syndrome and polyneuropathy, respectively. Here, we provide an overview on clinical disorders related to complement deficiencies or dysregulation and describe diagnostic strategies required for their comprehensive molecular characterization – a prerequisite for informed decisions on the therapeutic management of these disorders.
The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T ...cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell-keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.
Intestinal epithelial cells (IECs) are constantly exposed to commensal flora and pathogen challenges. How IECs regulate their innate immune response to maintain gut homeostasis remains unclear. ...Interferons (IFNs) are cytokines produced during infections. While type I IFN receptors are ubiquitously expressed, type III IFN receptors are expressed only on epithelial cells. This epithelium specificity strongly suggests exclusive functions at epithelial surfaces, but the relative roles of type I and III IFNs in the establishment of an antiviral innate immune response in human IECs are not clearly defined. Here, we used mini-gut organoids to define the functions of types I and III IFNs to protect the human gut against viral infection. We show that primary non-transformed human IECs, upon viral challenge, upregulate the expression of both type I and type III IFNs at the transcriptional level but only secrete type III IFN in the supernatant. However, human IECs respond to both type I and type III IFNs by producing IFN-stimulated genes that in turn induce an antiviral state. Using genetic ablation of either type I or type III IFN receptors, we show that either IFN can independently restrict virus infection in human IECs. Importantly, we report, for the first time, differences in the mechanisms by which each IFN establishes the antiviral state. Contrary to type I IFN, the antiviral activity induced by type III IFN is strongly dependent on the mitogen-activated protein kinases signaling pathway, suggesting a pathway used by type III IFNs that non-redundantly contributes to the antiviral state. In conclusion, we demonstrate that human intestinal epithelial cells specifically regulate their innate immune response favoring type III IFN-mediated signaling, which allows for efficient protection against pathogens without producing excessive inflammation. Our results strongly suggest that type III IFN constitutes the frontline of antiviral response in the human gut. We propose that mucosal surfaces, particularly the gastrointestinal tract, have evolved to favor type III IFN-mediated response to pathogen infections as it allows for spatial segregation of signaling and moderate production of inflammatory signals which we propose are key to maintain gut homeostasis.
Background
The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe ...enteric neuropathies. Neural crest–derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction.
Methods
Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin‐embedded individuals' specimens were analyzed accordingly.
Key Results
Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles.
Conclusions & Inferences
Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications.
Neural crest–derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms of neurogastrointestinal disorders. However, so far little is known about the detailed cellular composition of enteric neurospheres. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Our data revealed a remarkable molecular complexity of enteric neurospheres and emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors.
In psoriasis and other inflammatory skin diseases, keratinocytes (KCs) secrete chemokines that attract T cells, which, in turn, cause epidermal hyperplasia by secreting proinflammatory cytokines. To ...date, it remains unclear whether skin-homing T cells, particularly memory T cells, can also be activated by direct cell contact with KCs. In this study, we demonstrated the ability of primary human KCs to activate human memory T cells directly by transmitting costimulatory signals through the CD6/CD166/CD318 axis. Interestingly, despite being negative for CD80/CD86, KCs initiate a metabolic shift within T cells. Blockade of the CD6/CD166/CD318 axis prevents mammalian target of rapamycin activation and T cell proliferation but promotes oxidative stress and aerobic glycolysis. In addition, it diminishes formation of central memory T cells. Importantly, although KC-mediated costimulation by CD2/CD58 also activates T cells, it cannot compensate for the lack of CD6 costimulation. Therefore, KCs likely differentially regulate T cell functions in the skin through two distinct costimulatory receptors: CD6 and CD2. This may at least in part explain the divergent effects observed when treating inflammatory skin diseases with antibodies to CD6 versus CD2. Moreover, our findings may provide a molecular basis for selective interference with either CD6/CD166/CD318, or CD2/CD58, or both to specifically treat different types of inflammatory skin diseases.
Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMNs). Here, we describe the development of a subgroup of human PMNs ...expressing CCR5, termed CCR5+ PMNs. Auto- and paracrine tumor necrosis factor (TNF) signaling increases intracellular neutrophil elastase (ELANE) abundance and induces neutrophil extracellular traps formation (NETosis) in CCR5+ PMNs, and triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, known activators of NETosis. In vivo, we find an increased number of CCR5+ PMNs in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ PMNs. In conclusion, we identify a phenotype of pro-NETotic, CCR5+ PMNs present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.
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•Priming induces neutrophil diversification into CCR5− and CCR5+ cells•TNFR2 signaling renders CCR5+ neutrophils pro-NETotic by ELANE upregulation•CCR5+ neutrophils appear in the lamina propria of ulcerative colitis patients•Retrograde TNF signaling induces NETosis of CCR5+ neutrophils
Neuenfeldt et al. show that neutrophil priming induces dichotomous expression of CCR5. TNFR2 signaling increases ELANE expression in CCR5+ neutrophils. CCR5 triggering or reverse signaling of membrane-bound TNF activates ELANE, leading to enhanced NETosis. Pro-NETotic CCR5+ neutrophils in the lamina propria of ulcerative colitis patients may affect the success of anti-TNF therapies.
Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal ...half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (
), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal
. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in
. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type
partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.
Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are ...largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL1B, IL6, IL8, IL23A, TNFA, CXCL2, and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. Furthermore, genes never been previously associated with intestinal inflammation were identified using this model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Introduction
Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. It is at least partially ...mediated by low expression of the innate response receptors CD11b, CD14, CD16 as well as the cystine‐glutamate transporter xCT on these cells. Milieu‐specific mechanisms leading to the down‐regulation of these receptors on circulating monocytes, the precursor cells of resident macrophages, are mostly unknown.
Methods
Here, we addressed the question whether the short chain fatty acid n‐butyrate, a fermentation product of the mammalian gut microbiota exhibiting histone deacetylase inhibitory activity, is able to modulate expression of these receptors in human circulating monocytes.
Results
Exposure to n‐butyrate resulted in the downregulation of CD11b, CD14, as well as CD16 surface expression on circulating monocytes. XCT transcript levels in circulating monocytes were also reduced following exposure to n‐butyrate. Importantly, treatment resulted in the downregulation of protein and gene expression of the transcription factor PU.1, which was shown to be at least partially required for the expression of CD16 in circulating monocytes. PU.1 expression in resident macrophages in situ was observed to be substantially lower in healthy when compared to inflamed colonic mucosa.
Conclusions
In summary, the intestinal microbiota may support symbiosis with the human host organism by n‐butyrate mediated downregulation of protein and gene expression of innate response receptors as well as xCT on circulating monocytes following recruitment to the lamina propria. Downregulation of CD16 gene expression may at least partially be caused at the transcriptional level by the n‐butyrate mediated decrease in expression of the transcription factor PU.1 in circulating monocytes.
Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. The intestinal microbiota may support symbiosis with the human host organism by n‐butyrate mediated downregulation of protein and/or gene expression of innate response receptors as well as xCT on PBMO following recruitment to the lamina propria.
Targeting early molecular events in intestinal inflammation may represent a useful therapeutic strategy for maintaining remission in inflammatory bowel disease. Recently, we established an intestinal ...organ culture model (LEL model), which allows to study the initiation of an intestinal inflammatory response in human tissue. In this model, EDTA-mediated depletion of epithelial cells of colonic mucosa results in an instantaneous inflammatory response in resident lamina propria cells, which shows features of intestinal inflammation in vivo. Furthermore, activated immune cells emigrate from the lamina propria onto the luminal side of the basement membrane.
Here, we standardize the LEL model and explore its suitability for drug testing. To this end, human mucosal punches of defined surface area were prepared, depleted of epithelial cells, and cultured at an optimized ratio of medium volume/punch area. The intra-assay variability of measurements of inflammatory parameters ranged from 13% for cell migration to 19% for secretion and 30% for tissue gene expression, respectively, of the inflammatory mediators IL-8 and IL-6. Importantly, known suppressive effects of dexamethasone, a drug employed for the treatment of inflammatory bowel diseases, on leucocyte migration, IL8, IL6, and TNF-α production as well as CD86 surface expression by myeloid cells were observed in this model.
In conclusion, the present results suggest that the LEL model may represent a useful human experimental system not only for studying initial activation mechanisms in intestinal inflammation but also for evaluating drug compounds for the treatment of mucosal inflammation.
•A human organ culture model of intestinal inflammation (LEL model) was standardized.•The precision of measurements of inflammatory parameters in this model was determined.•Immunomodulatory effects of dexamethasone were observed in this model.•The explant model may be useful for testing drugs for inflammatory bowel disease.
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