Dendritic Cell-Derived TSLP Negatively Regulates HIF-1α and IL-1ß During Dectin-1 Signaling.

Thymic stromal lymphopoietin (TSLP) is a functionally pleotropic cytokine important in immune regulation, and TSLP dysregulation is associated with numerous diseases. TSLP is produced by many cell types, but has predominantly been characterized as a secreted factor from epithelial cells which activates dendritic cells (DC) that subsequently prime T helper (TH) 2 immunity. However, DC themselves make significant amounts of TSLP in response to microbial products, but the functional role of DC-derived TSLP remains unclear. We show that TSLPR signaling negatively regulates IL-1ß production during dectin-1 stimulation of human DC. This regulatory mechanism functions by dampening Syk phosphorylation and is mediated via NADPH oxidase-derived ROS, HIF-1α and pro-IL-1ß expression. Considering the profound effect TSLPR signaling has on the metabolic status and the secretome of dectin-1 stimulated DC, these data suggest that autocrine TSLPR signaling could have a fundamental role in modulating immunological effector responses at sites removed from epithelial cell production of TSLP.

Surveillance study on the tolerability and safety of Flebogamma® DIF (10% and 5% intravenous immunoglobulin) in adult and pediatric patients.

Direct comparisons of tolerability and safety of concentrated intravenous immunoglobulin (IVIG) versus less concentrated products are scarce. In this postauthorization, prospective, observational, multicenter study, a systematic comparison of 10% and 5% concentrations of Flebogamma® DIF IVIG was performed in both adult and pediatric patients treated with the studied IVIG products according to the approved indications under routine conditions. Dose of product administered, adverse events (AEs), physical assessments, laboratory tests, and concomitant therapy were analyzed. Patient recruitment in the 10% and 5% product groups was, respectively, 34 (32 analyzed, 13 of them children, receiving 130 IVIG infusions) and 35 (34 analyzed, receiving 135 IVIG infusions). Twenty-four infusions (18.5%; 95% CI: 11.8, 25.1) with the 10% product and 3 (2.2%; 95% CI: -0.3, 4.7) with the 5% product were associated with potentially treatment-related AEs (P < 0.0001). Nine patients (28.1%) infused with the 10% product and 3 (8.8%) infused with the 5% product presented, respectively, 33 and 8 treatment-related AEs (of which 7 and 6, respectively, were serious AEs, experienced by only three hypersensitive patients). The profile of AEs occurring with the infusion of 10% and 5% products were comparable. The most frequent treatment-related AEs were headache (n = 17, 3 patients; 15 episodes, 1 patient) and pyrexia (n = 6, 4 patients). In conclusion, no unpredictable risk was detected for both Flebogamma DIF 10% and 5% concentrations, which were therefore deemed as safe and well-tolerated IVIG in the studied population. The frequency of infusions associated with treatment-related AEs was lower with the 5% concentration.

Targeted Gene Panel Sequencing for Early-onset Inflammatory Bowel Disease and Chronic Diarrhea.

BACKGROUND: In contrast to adult-onset inflammatory bowel disease (IBD), where many genetic loci have been shown to be involved in complex disease etiology, early-onset IBD (eoIBD) and associated syndromes can sometimes present as monogenic conditions. As a result, the clinical phenotype and ideal disease management in these patients often differ from those in adult-onset IBD. However, due to high costs and the complexity of data analysis, high-throughput screening for genetic causes has not yet become a standard part of the diagnostic work-up of eoIBD patients. METHODS: We selected 28 genes of interest associated with monogenic IBD and performed targeted panel sequencing in 71 patients diagnosed with eoIBD or early-onset chronic diarrhea to detect causative variants. We compared these results to whole-exome sequencing (WES) data available for 25 of these patients. RESULTS: Target coverage was significantly higher in the targeted gene panel approach compared with WES, whereas the cost of the panel was considerably lower (approximately 25% of WES). Disease-causing variants affecting protein function were identified in 5 patients (7%), located in genes of the IL10 signaling pathway (3), WAS (1), and DKC1 (1). The functional effects of 8 candidate variants in 5 additional patients (7%) are under further investigation. WES did not identify additional causative mutations in 25 patients. CONCLUSIONS: Targeted gene panel sequencing is a fast and effective screening method for monogenic causes of eoIBD that should be routinely established in national referral centers.

Gene therapy for Wiskott-Aldrich syndrome in a severely affected adult.

Until recently, hematopoietic stem cell transplantation was the only curative option for Wiskott-Aldrich syndrome (WAS). The first attempts at gene therapy for WAS using a ϒ-retroviral vector improved immunological parameters substantially but were complicated by acute leukemia as a result of insertional mutagenesis in a high proportion of patients. More recently, treatment of children with a state-of-the-art self-inactivating lentiviral vector (LV-w1.6 WASp) has resulted in significant clinical benefit without inducing selection of clones harboring integrations near oncogenes. Here, we describe a case of a presplenectomized 30-year-old patient with severe WAS manifesting as cutaneous vasculitis, inflammatory arthropathy, intermittent polyclonal lymphoproliferation, and significant chronic kidney disease and requiring long-term immunosuppressive treatment. Following reduced-intensity conditioning, there was rapid engraftment and expansion of a polyclonal pool of transgene-positive functional T cells and sustained gene marking in myeloid and B-cell lineages up to 20 months of observation. The patient was able to discontinue immunosuppression and exogenous immunoglobulin support, with improvement in vasculitic disease and proinflammatory markers. Autologous gene therapy using a lentiviral vector is a viable strategy for adult WAS patients with severe chronic disease complications and for whom an allogeneic procedure could present an unacceptable risk. This trial was registered at www.clinicaltrials.gov as #NCT01347242.

ß-Glucan Size Controls Dectin-1-Mediated Immune Responses in Human Dendritic Cells by Regulating IL-1ß Production.

Dectin-1/CLEC7A is a pattern recognition receptor that recognizes ß-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. ß-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate ß-glucans are thought to be poor activators of innate immunity. We show that ß-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large ß-glucan-stimulated DC generate significantly more IL-1ß, IL-6, and IL-23 compared to those stimulated with the smaller ß-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to ß-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1ß production determined by ß-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that ß-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.

Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection.

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1ß processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1ß processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

IRE1α mediates PKR activation in response to Chlamydia trachomatis infection.

Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-ß production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses.

EBV-driven diffuse large B-cell lymphoma confined to the liver in a patient with a history of idiopathic CD4 lymphocytopenia.

Idiopathic CD4 lymphocytopenia (ICL) is a rare immunodeficiency disorder. We describe a 49-year-old woman with a history of ICL who developed hepatic Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL). ICL was diagnosed at a time of her presentation with varicella-zoster virus (VZV) meningoencephalitis and chorioretinitis. Her CD4 count subsequently improved but remained at the lower limits of the normal range. Five years later she presented with cough, fever and night-sweat. She was found to have multiple liver nodules on MRI, fluorodeoxyglucose (FDG) avid on the positron emission tomography (PET) CT, histologically defined as DLBCL, EBV positive and of non-germinal centre type. To our knowledge this is the first reported case of EBV-positive DLBCL localised to the liver in the context of ICL. EBV-positive DLBCL typically occurs in immunocompromised individuals. The corticosteroid therapy she received for VZV meningoencephalitis may have contributed to the EBV reactivation with subsequent EBV-driven malignant transformation of B-cells.

Clinical outcome in IL-10- and IL-10 receptor-deficient patients with or without hematopoietic stem cell transplantation.

BACKGROUND: Inherited deficiencies of IL-10 or IL-10 receptor (IL-10R) lead to immune dysregulation with life-threatening early-onset enterocolitis. OBJECTIVES: We sought to gather clinical data of IL-10/IL-10R-deficient patients and devise guidelines for diagnosis and management, including hematopoietic stem cell transplantation (HSCT). METHODS: We enrolled 40 patients with early-onset enterocolitis and screened for mutations in IL10/IL10R using genetic studies, functional studies, or both of the IL-10 signaling pathway. Medical records of IL-10/IL-10R-deficient patients were reviewed and compiled. RESULTS: Of 40 patients, we identified 7 with novel mutations, predominantly in consanguineous families with more than 1 affected member. IL-10/IL-10R-deficient patients had intractable enterocolitis, perianal disease, and fistula formation. HSCT was carried out in 2 patients with IL-10 deficiency and 1 patient with IL-10R α chain deficiency and proved to be an effective therapy, leading to rapid improvement of clinical symptoms and quality of life. CONCLUSION: Because the defect in patients with IL-10/IL-10R deficiency resides in hematopoietic lineage cells and their colitis is resistant to standard immunosuppressive therapy, HSCT should be considered early as a potentially curative therapeutic option.

Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.

Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.

Influence of cytomegalovirus infection on immune cell phenotypes in patients with common variable immunodeficiency.

BACKGROUND: A subset of patients with common variable immunodeficiency (CVID) have debilitating inflammatory complications strongly associated with cytomegalovirus (CMV) infection and a hyperproliferative CMV-specific T-cell response. OBJECTIVES: We studied the T-cell response to CMV and the global effect of this virus on immune effector cell populations in patients with CVID. METHODS: Antibody staining, peptide stimulation, and proliferation assays were used to profile CMV-specific T-cell function. RESULTS: CMV infection drives the CD4/CD8 ratio inversion that is characteristic of CVID. The late effector CD8(+) T-cell subset is expanded in CMV-infected patients with CVID. This expansion is largely attributable to CMV-specific cells and correlates with inflammatory disease; within the CMV-specific population, the frequency of late effector cells correlates inversely with the frequency of cells expressing programmed death 1. Supernatants from proliferating CMV-specific CD8(+) cells from patients with inflammatory disease can confer proliferative potential on cells from patients with noninflammatory CVID and healthy subjects. Blocking experiments showed that this proliferation is mediated in part by IFN-γ and TNF-α. CONCLUSIONS: These data strengthen the association of CMV with inflammatory pathology in patients with CVID, explain some of the well-known T-cell abnormalities associated with this condition, and provide a plausible mechanism for the documented therapeutic activity of anti-TNF-α and antiviral chemotherapy in managing CVID-associated inflammatory disease.

Inflammation in common variable immunodeficiency is associated with a distinct CD8(+) response to cytomegalovirus.

BACKGROUND: Common variable immunodeficiency is the most common primary immunodeficiency. A subset of patients has debilitating inflammatory complications. OBJECTIVES: We investigated the role of cytomegalovirus (CMV), and the T-cell response targeted at this virus, in this inflammatory disease. METHODS: Phenotypic and functional assays were used to profile CMV-specific T cells in patients with common variable immunodeficiency with and without inflammatory complications. Highly sensitive immunohistochemistry was used to detect CMV antigens at sites of inflammation. RESULTS: Cytomegalovirus was significantly associated with inflammatory disease, which occurred in 31 of 43 (72%) virus-exposed patients and 8 of 31 (26%) naive patients (P = .0001). CMV pp65-NLVPMVATV epitope-specific CD8(+) T-cell frequencies were significantly elevated in inflammatory patients, but these cells did not show evidence of exhaustion, with low levels of programmed death-1 and high T-cell receptor avidity. Rather, they showed features consistent with high in vivo functionality and proliferative activity including reduced levels of the anti-inflammatory marker CD73 (1.67% of NLV(+) cells were CD73(+) vs 42.01% in noninflammatory patients; P = .004) and increased Ki-67 expression (37% vs 2% in noninflammatory patients; P < .0001). In vitro, the CMV-specific T cells showed high antigen-specific proliferative potential compared with cells from noninflammatory patients. By using sensitive immunohistochemistry, we detected for the first time viral antigen at the sites of inflammation, indicative of active viral replication. CONCLUSION: Our data strongly support a direct role for CMV and a hyperreactive CMV-specific immune response in the debilitating chronic inflammatory complications of common variable immunodeficiency.

Serum trough IgG level and annual intravenous immunoglobulin dose are not related to body size in patients on regular replacement therapy.

Therapeutic regimens of intravenous immunoglobulin are currently based on actual body weight. The relationship between immunoglobulin dose and serum IgG level in relation to body size was retrospectively explored in patients on replacement therapy. Data were collected as part of a national audit on immunoglobulin therapy in patients with common variable immunodeficiency. 107 patients received immunoglobulin titrated to optimum effect. Correlations were sought between body mass index, trough IgG levels, infusion frequency and total annual dose. The mean (±SD) trough IgG level was 8.4±1.6 g/L and annual immunoglobulin dose received was 456.8±129.4 g. There was no relationship between annual dose and trough IgG level, regardless of infusion frequency, or adjustment for weight or body mass index. These results support the clinical practice of immunoglobulin prescription by clinical outcome rather than fixed dose by body weight. Future studies exploring immunoglobulin efficacy should include treatment arms with dosages based on both ideal and actual body weight, as ideal body weight-based prescribing would save significant amounts of product.

Common variable immunodeficiency with coexisting central nervous system sarcoidosis: case report and literature review with implications for diagnosis and pathogenesis.

We describe a patient with a history of longstanding primary generalised epilepsy, on anticonvulsant therapy, who presented with fever, headache, worsening seizures and hallucinations. Among various investigations, the patient had high CSF protein and ACE levels, leptomeningeal nodular enhancement on MRI brain and non-caseating granulomas in the brain and meninges on the biopsy. The patient was diagnosed with neurosarcoidosis. Subsequently, he was found to be panhypogammaglobulinaemic and was diagnosed with probable common variable immunodeficiency (CVID). The coexistence of common variable immunodeficiency and neurosarcoidosis is rare. Typically, non-caseating granulomas in CVID patients are localised in the lymphatic tissue and solid organs. To our knowledge, there are only five reports of the granulomas of the central nervous system (CNS) in CVID. We discuss the diagnostic difficulties in this case and review the literature.

Impaired macrophage function following bacterial stimulation in chronic granulomatous disease.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical for phagocyte anti-microbial activity and plays a major role in innate immunity. Defects in genes coding for components of the NADPH oxidase enzyme system are responsible for chronic granulomatous disease (CGD), a rare primary neutrophil immunodeficiency associated with recurrent, life-threatening bacterial and fungal infections. Microbial killing and digestion within the neutrophil phagosomal compartment are defective in these patients. NADPH oxidase activity is also crucial for optimal macrophage and dendritic cell function and has recently been implicated in both cross-presentation and T-cell priming. We present evidence of impaired macrophage function in CGD, with attenuated pro-inflammatory cytokine and increased interleukin-10 secretion following bacterial stimulation. These results highlight additional abnormalities in macrophage function associated with CGD and the importance of NADPH oxidase activity in immunity.

Hypogammaglobulinaemia.

This article reviews the primary immunodeficiencies that result in hypogammaglobulinemia or predominantly antibody deficiency disorders. This group makes up the largest proportion of patients with primary immunodeficiency. Significant advances have been made in understanding the molecular basis and clinical characteristics of patients with the more severe forms of antibody deficiency in the last 6 years. Recognition of these disorders remains poor with significant diagnostic delay. The milder forms of antibody deficiency disorders, especially those with normal total serum immunoglobulin G levels, remain poorly characterized and understood. Further work remains to be done in understanding and recognizing these syndromes to benefit patient care and foster further knowledge of the immune system.