Ronnie Chee

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.u2003tuberculosis 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.u2003tuberculosis‐triggered necrosis. More, importantly, the M.u2003 tuberculosisΔRD1 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.u2003tuberculosis 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

BACKGROUNDnA 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.nnnOBJECTIVESnWe studied the T-cell response to CMV and the global effect of this virus on immune effector cell populations in patients with CVID.nnnMETHODSnAntibody staining, peptide stimulation, and proliferation assays were used to profile CMV-specific T-cell function.nnnRESULTSnCMV 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.nnnCONCLUSIONSnThese 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

BACKGROUNDnCommon variable immunodeficiency is the most common primary immunodeficiency. Axa0subset of patients has debilitating inflammatory complications.nnnOBJECTIVESnWe investigated the role of cytomegalovirus (CMV), and the T-cell response targeted at this virus, in this inflammatory disease.nnnMETHODSnPhenotypic 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.nnnRESULTSnCytomegalovirus was significantly associated with inflammatory disease, which occurred in 31 of 43 (72%) virus-exposed patients and 8 of 31 (26%) naive patients (Pxa0= .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 inxa0vivo 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; Pxa0= .004) and increased Ki-67 expression (37% vs 2% in noninflammatory patients; Pxa0<xa0.0001). 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.nnnCONCLUSIONnOur 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.

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-1β 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-1β 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-β 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.

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

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

An important diagnosis to consider in recurrent meningitis

Meningitis, a potentially life threatening illness, requires prompt recognition and treatment. Recurrent meningitis necessitates detailed investigations to identify the underlying cause. We describe two adult patients with recurrent meningitis due to an underlying skull base abnormality.

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.

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.

Repeatedly red faced

A 59-year-old man was referred to our department in February, 2011, after nine episodes of erysipelas aff ecting the right cheek within the previous 6 years. During the same period he had developed cellulitis aff ecting a fi nger and right arm, an abscess on the face, and a furuncle on the left cheek. There was no history of infection of other organs. He had a history of type 2 diabetes complicated by retinopathy, hypercholesterol-aemia, and renal colic. Medications included pioglita-zone, metformin, and atorvastatin. Repeated blood cultures and skin swabs taken during the infective episodes were negative. Empir ical antibiotics prescribed acutely to cover streptococcal and staphylococcal infec-tions required prolonged courses (4–6 weeks) before resolu tion. Continuous 12-month prophylactic erythro-mycin and topical antibacterial washes did not change the frequency or severity of infection. Between episodes, the facial skin was normal and examination did not show any other dermatosis.An immunodefi ciency state was suspected because of the history. Laboratory tests showed a homozygous mannose binding lectin (MBL) defi ciency of <0·05 mg/L (normal range 1·0–4·0 mg/L). Full blood count, immunoglobulin concen trations, T -cell and B-cell counts, and lymphocyte subsets were normal, ruling out a granulocyte, T-cell, or B-cell defect. Although he had normal specifi c antibodies to tetanus and pneumococcus,