Immunologic and Genetic Contributors to CD46-Dependent Immune Dysregulation.

Mutations in CD46 predispose to atypical hemolytic uremic syndrome (aHUS) with low penetrance. Factors driving immune-dysregulatory disease in individual mutation carriers have remained ill-understood. In addition to its role as a negative regulator of the complement system, CD46 modifies T cell-intrinsic metabolic adaptation and cytokine production. Comparative immunologic analysis of diseased vs. healthy CD46 mutation carriers has not been performed in detail yet. In this study, we comprehensively analyzed clinical, molecular, immune-phenotypic, cytokine secretion, immune-metabolic, and genetic profiles in healthy vs. diseased individuals carrying a rare, heterozygous CD46 mutation identified within a large single family. Five out of six studied individuals carried a CD46 gene splice-site mutation causing an in-frame deletion of 21 base pairs. One child suffered from aHUS and his paternal uncle manifested with adult-onset systemic lupus erythematosus (SLE). Three mutation carriers had no clinical evidence of CD46-related disease to date. CD4+ T cell-intrinsic CD46 expression was uniformly 50%-reduced but was comparable in diseased vs. healthy mutation carriers. Reconstitution experiments defined the 21-base pair-deleted CD46 variant as intracellularly-but not surface-expressed and haploinsufficient. Both healthy and diseased mutation carriers displayed reduced CD46-dependent T cell mitochondrial adaptation. Diseased mutation carriers had lower peripheral regulatory T cell (Treg) frequencies and carried potentially epistatic, private rare variants in other inborn errors of immunity (IEI)-associated proinflammatory genes, not found in healthy mutation carriers. In conclusion, low Treg and rare non-CD46 immune-gene variants may contribute to clinically manifest CD46 haploinsufficiency-associated immune-dysregulation.

Systematic single-cell pathway analysis to characterize early T cell activation.

Pathway analysis is a key analytical stage in the interpretation of omics data, providing a powerful method for detecting alterations in cellular processes. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package single-cell pathway analysis (SCPA). We demonstrate the effectiveness of SCPA over commonly used methods, generate a scRNA-seq T cell dataset, and characterize pathway activity over early cellular activation. This reveals regulatory pathways in T cells, including an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems-level characterization of pathway activity in T cells across multiple tissues also identifies alpha-defensin expression as a hallmark of bone-marrow-derived T cells. Overall, this work provides a widely applicable tool for single-cell pathway analysis and highlights regulatory mechanisms of T cells.

Mitochondrial C5aR1 activity in macrophages controls IL-1ß production underlying sterile inflammation.

While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the "complosome," functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1ß production, both at the transcriptional level and processing of pro­IL-1ß. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1ß produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

Complement Has Brains-Do Intracellular Complement and Immunometabolism Cooperate in Tissue Homeostasis and Behavior?

The classical liver-derived and serum-effective complement system is well appreciated as a key mediator of host protection via instruction of innate and adaptive immunity. However, recent studies have discovered an intracellularly active complement system, the complosome, which has emerged as a central regulator of the core metabolic pathways fueling human immune cell activity. Induction of expression of components of the complosome, particularly complement component C3, during transmigration from the circulation into peripheral tissues is a defining characteristic of monocytes and T cells in tissues. Intracellular complement activity is required to induce metabolic reprogramming of immune cells, including increased glycolytic flux and OXPHOS, which drive the production of the pro-inflammatory cytokine IFN-γ. Consequently, reduced complosome activity translates into defects in normal monocyte activation, faulty Th1 and cytotoxic T lymphocyte responses and loss of protective tissue immunity. Intriguingly, neurological research has identified an unexpected connection between the physiological presence of innate and adaptive immune cells and certain cytokines, including IFN-γ, in and around the brain and normal brain function. In this opinion piece, we will first review the current state of research regarding complement driven metabolic reprogramming in the context of immune cell tissue entry and residency. We will then discuss how published work on the role of IFN-γ and T cells in the brain support a hypothesis that an evolutionarily conserved cooperation between the complosome, cell metabolism and IFN-γ regulates organismal behavior, as well as immunity.

Complement and human T cell metabolism: Location, location, location.

The complement system represents one of the evolutionary oldest arms of our immune system and is commonly recognized as a liver-derived and serum-active system critical for providing protection against invading pathogens. Recent unexpected findings, however, have defined novel and rather "uncommon" locations and activities of complement. Specifically, the discovery of an intracellularly active complement system-the complosome-and its key role in the regulation of cell metabolic pathways that underly normal human T cell responses have taught us that there is still much to be discovered about this system. Here, we summarize the current knowledge about the emerging functions of the complosome in T cell metabolism. We further place complosome activities among the non-canonical roles of other intracellular innate danger sensing systems and argue that a "location-centric" view of complement evolution could logically justify its close connection with the regulation of basic cell physiology.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Nanoparticles prepared from porcine cells support the healing of cutaneous inflammation in mice and wound re-epithelialization in human skin.

Previous reports have demonstrated that cell-derived nanoparticles (CDNPs) composed of bovine or porcine protein complexes exerted therapeutic effects against viral infections and cancer in mice and humans. Based on these observations, we asked whether CDNPs would improve inflammatory skin disorders. To address this, we utilized two distinct mouse models of cutaneous inflammation: the autoimmune skin-blistering disease epidermolysis bullosa acquisita (EBA) as an example of an autoantibody-induced cutaneous inflammation, and Leishmania major (L. major) infection as an example of a pathogen-induced cutaneous inflammation. In both models, we observed that CDNPs increased mRNA expression of the Th2 cytokine IL-4. Clinically, CDNPs decreased inflammation due to EBA and increased L. major-specific IgG1 levels without major effects on infected skin lesions. In addition, CDNPs supported the growth of keratinocytes in human skin cultures. In vitro studies revealed that CDNPs were taken up predominantly by macrophages, leading to a shift towards the expression of anti-inflammatory cytokine genes. Altogether, our data demonstrate that treatment with porcine CDNPs may be a new therapeutic option for the control of autoimmune-mediated inflammatory skin disorders.

Cell-Derived Nanoparticles are Endogenous Modulators of Sepsis With Therapeutic Potential.

Cell-derived nanoparticles (CDNPs) containing cytosolic proteins and RNAs/DNAs can be isolated from stressed eukaryotic cells. Previously, CDNPs isolated from cultured cells exerted immunomodulatory activities in different infections. Here, we sought to elucidate the role of CDNPs using a murine model of cecal ligation and puncture (CLP). We hypothesized that CDNPs influence the immune response at the site of infection, where severe cellular stress occurs. We observed early CDNP accumulation in the peritoneum after 4 h and continued CDNP presence 24 h after CLP. To determine whether CDNPs influence the host response to sepsis, we isolated CDNPs from a murine fibroblast cell line stressed by nutrient-deprivation, and injected them into septic mice. CDNP-treated mice demonstrated decreased peritoneal interleukin 6 levels and an approximately 2-log lower bacterial load compared with control mice 24 h after CLP. Additionally, a 20% CFU reduction was observed when incubating CDNPs with Pseudomona aeroginosa, indicating that CDNPs are bactericidal. To identify CDNP-responsive cells, CFSE-labeled CDNPs were injected into mice at the time of CLP. We observed that CDNPs were preferentially ingested by F4/80 macrophages, and to a lesser degree, associated with inflammatory monocytes and neutrophils. Strikingly, CDNP-ingesting cells demonstrated elevated CD11b and MHCII expression compared with control cells. Altogether, our data indicate that CDNPs enhance the immune response at the site of infection and promote bacterial clearance, by direct bacterial killing and increasing phagocyte activation. Thus, CDNPs represent a novel, unexplored endogenous sepsis modulator with therapeutic potential.

Neutrophil derived microparticles increase mortality and the counter-inflammatory response in a murine model of sepsis.

Although advances in medical care have significantly improved sepsis survival, sepsis remains the leading cause of death in the ICU. This is likely due to a lack of complete understanding of the pathophysiologic mechanisms that lead to dysfunctional immunity. Neutrophil derived microparticles (NDMPs) have been shown to be the predominant microparticle present at infectious and inflamed foci in human models, however their effect on the immune response to inflammation and infection is sepsis has not been fully elucidated. As NDMPs may be a potential diagnostic and therapeutic target, we sought to determine the impact NDMPs on the immune response to a murine polymicrobial sepsis. We found that peritoneal neutrophil numbers, bacterial loads, and NDMPs were increased in our abdominal sepsis model. When NDMPs were injected into septic mice, we observed increased bacterial load, decreased neutrophil recruitment, increased expression of IL-10 and worsened mortality. Furthermore, the NDMPs express phosphatidylserine and are ingested by F4/80 macrophages via a Tim-4 and MFG-E8 dependent mechanism. Finally, upon treatment, NDMPs decrease macrophage activation, increase IL-10 release and decrease macrophage numbers. Altogether, these data suggest that NDMPs enhance immune dysfunction in sepsis by blunting the function of neutrophils and macrophages, two key cell populations involved in the early immune response to infection. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

The role of microfibrillar-associated protein 4 (MFAP4) in the formation and function of splenic compartments during embryonic and adult life.

Microfibrillar-associated protein 4 (MFAP4) is an extracellular protein belonging to the fibrinogen-related protein superfamily and is recognized as an integrin ligand with suggested functions in pulmonary and vascular tissue homeostasis. MFAP4 expression in the spleen is increased during infections; however, the significance of MFAP4 for the function of the spleen is unknown. Immunohistochemistry, morphometry and real-time RT-PCR were used to analyze wild-type and MFAP4-deficient spleens. In addition, they were compared with splenic tissue, which was newly formed 8 weeks after avascular implantation into adult mice in order to obtain information about the role of MFAP4 in the formation of splenic tissue during ontogeny and adult life. The present study shows that MFAP4 is co-localized with laminin in the B- and T-cell zones of the spleen, in addition to capsular and trabecular expression. MFAP4 is most likely produced by fibroblastic reticulum cells and follicular dendritic cells of the spleen but can also be imported via the blood from other tissues. The development of splenic tissue is not disturbed in MFAP4-deficient mice. However, in splenic tissue regenerating under MFAP4-deficient conditions, the number of FDCs is significantly decreased but is corrected by MFAP4 imported from other tissues. No differences were observed for lymphocyte numbers or splenic structure. The data indicate that MFAP4 promotes FDC development in regenerating splenic tissue and warrant further investigations regarding the MFAP4 dependency of splenic B-cell maturation.

Impact of caspase-8 and PKA in regulating neutrophil-derived microparticle generation.

The morbidity and mortality from sepsis continues to remain high despite extensive research into understanding this complex immunologic process. Further, while source control and antibiotic therapy have improved patient outcomes, many immunologically based therapies have fallen short. Microparticles (MPs) are intact vesicles that serve as mediators of intercellular communication as well as markers of inflammation in various disease processes. We have previously demonstrated that MPs can be produced at the infected foci during sepsis, are predominantly of neutrophil derivation (NDMPs) and can modulate immune cells. In this study, we sought to elucidate the molecular mechanisms underlying NDMP generation. Using thioglycolate (TGA) to recruit and activate neutrophils, we first determined that intra-peritoneal TGA increase NDMP accumulation. We next utilized TGA-elicited neutrophils in vitro to investigate signaling intermediates involved in NDMP production, including the intrinsic and extrinsic caspase pathways, cAMP dependent PKA and Epac activation as well as the role myosin light chain kinase (MLCK) as a final mediator of NDMP release. We observed that NDMP generation was dependent on the extrinsic caspase apoptotic pathway (caspase 3 and caspase 8), cAMP activation of PKA but not of Epac, and on activation of MLCK. Altogether, these data contribute to an overall framework depicting the molecular mechanisms that regulate NDMP generation.

Dose-dependent induction of murine Th1/Th2 responses to sheep red blood cells occurs in two steps: antigen presentation during second encounter is decisive.

The differentiation of CD4 T cells into Th1 and Th2 cells in vivo is difficult to analyze since it is influenced by many factors such as genetic background of the mice, nature of antigen, and adjuvant. In this study, we used a well-established model, which allows inducing Th1 or Th2 cells simply by low (LD, 10(5)) or high dose (HD, 10(9)) injection of sheep red blood cells (SRBC) into C57BL/6 mice. Signature cytokine mRNA expression was determined in specific splenic compartments after isolation by laser-microdissection. LD immunization with SRBC induced T cell proliferation in the splenic T cell zone but no Th1 differentiation. A second administration of SRBC into the skin rapidly generated Th1 cells. In contrast, HD immunization with SRBC induced both T cell proliferation and immediate Th2 differentiation. In addition, splenic marginal zone and B cell zone were activated indicating B cells as antigen presenting cells. Interestingly, disruption of the splenic architecture, in particular of the marginal zone, abolished Th2 differentiation and led to the generation of Th1 cells, confirming that antigen presentation by B cells directs Th2 polarization. Only in its absence Th1 cells develop. Therefore, B cells might be promising targets in order to therapeutically modulate the T cell response.

Basic symptoms in the general population and in psychotic and non-psychotic psychiatric adolescents.

OBJECTIVE: Cognitive-perceptive 'basic symptoms' are used complementary to ultra-high-risk criteria in order to predict onset of psychosis in the pre-psychotic phase. The aim was to investigate the prevalence of a broad selection of 'basic symptoms' in a representative general adolescent population sample (GPS; N=96) and to compare it with adolescents first admitted for early onset psychosis (EOP; N=87) or non-psychotic psychiatric disorders (NP; N=137). METHODS: Subjects were assessed with the Bonn Scale for the Assessment of Basic Symptoms (BSABS). Prevalence of at least one 'basic symptom' and mean numbers were compared across the three groups. Logistic regression was used to predict group membership by BSABS subscales; risk ratios were calculated to identify 'basic symptoms' which best discriminated between groups. RESULTS: The prevalence of at least any one 'basic symptom' was 30.2% in GPS compared to 81% in NP and 96.5% in EOP. Correct classification of EOP when compared to GPS was high (94.0%) and lower when compared to NP (78.6%). Cognitive symptoms discriminated best between EOP and NP. CONCLUSION: Alike other prodromal- and psychotic-like experiences, 'basic symptoms' are prevalent in the general adolescent population, yet at a lower rate compared to EOP and NP. The usage of 'at least one basic symptom' as a screening criterion for youth at risk of developing a psychotic disorder is not recommended in the general population or in unselected psychiatrically ill adolescents. However, particularly cognitive 'basic symptoms' may be a valuable criteria to be included in future 'at risk' studies in adolescents.