Machiel H. Jansen

Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule in the cytosol of myeloid cells, required for induction of T-helper cells producing interleukin-17 (Th17 cells) and important in antifungal immunity. In a patient suffering from Candida dubliniensis meningoencephalitis, mutations in the CARD9 gene were found to result in the loss of protein expression. Apart from the reduced numbers of CD4(+) Th17 lymphocytes, we identified a lack of monocyte-derived cytokines in response to Candida strains. Importantly, CARD9-deficient neutrophils showed a selective Candida albicans killing defect with abnormal ultrastructural phagolysosomes and outgrowth of hyphae. The neutrophil killing defect was independent of the generation of reactive oxygen species by the reduced NAD phosphate oxidase system. Taken together, this demonstrates that human CARD9 deficiency results in selective defect in the host defense against invasive fungal infection, caused by an impaired phagocyte killing.

Human NLRP3 inflammasome activation is Nox1-4 independent

The NLRP3 inflammasome can be activated by pathogen-associated molecular patterns or endogenous danger-associated molecular patterns. The activation of the NLRP3 inflammasome results in proteolytic activation and secretion of cytokines of the interleukin-1 (IL-1) family. The precise mode of activation of the NLRP3 inflammasome is still elusive, but has been postulated to be mediated by reactive oxygen species (ROS) generated by an NADPH oxidase. Using primary cells from chronic granulomatous disease (CGD) patients lacking expression of p22(phox), a protein that is required for the function of Nox1-4, we show that cells lacking NADPH oxidase activity are capable of secreting normal amounts of IL-1beta. Thus, we provide evidence that activation of the NLRP3 inflammasome does not depend on ROS generated from an NADPH oxidase.

Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for β-glucan-bearing particles

We investigated the role of the beta-glucan receptor, Dectin-1, in the response of human neutrophils to unopsonized Saccharomyces cerevisiae and its major beta-glucan-containing capsular constituent, zymosan. Although reported to be indispensable for yeast phagocytosis in murine phagocytes, human Dectin-1 was not involved in the phagocytosis of S. cerevisiae or zymosan by human neutrophils. Phagocytosis of yeast particles proved to be completely dependent on CD11b/CD18, also known as complement receptor 3 (CR3). The findings were supported by data with neutrophils from a patient suffering from Leukocyte-Adhesion Deficiency type-1 (LAD-1) syndrome lacking CD11b/CD18. In addition, neither the priming by zymosan of the fMLP-induced NADPH-oxidase activity in human neutrophils nor the secretion of IL-8 by human neutrophils in response to zymosan preparations was affected by blocking anti-Dectin-1 antibodies or laminarin as a monovalent inhibitor. As shown by neutrophils from an IRAK-4-deficient patient, the zymosan-induced IL-8 release was also independent of TLR2. In summary, our data show that Dectin-1, although indispensable for recognition of beta-glucan-bearing particles in mice, is not the major receptor for yeast particles in human neutrophils.

Idiopathic CD4+ T lymphopenia without autoimmunity or granulomatous disease in the slipstream of RAG mutations

A girl presented during childhood with a single course of extensive chickenpox and moderate albeit recurrent pneumonia in the presence of idiopathic CD4+ T lymphocytopenia (ICL). Her clinical condition remained stable over the past 10 years without infections, any granulomatous disease, or autoimmunity. Immunophenotyping demonstrated strongly reduced naive T and B cells with intact proliferative capacity. Antibody reactivity on in vivo immunizations was normal. T-cell receptor-Vβ repertoire was polyclonal with a very low content of T-cell receptor excision circles (TRECs). Kappa-deleting recombination excision circles (KRECs) were also abnormal in the B cells. Both reflect extensive in vivo proliferation. Patient-derived CD34+ hematopoietic stem cells could not repopulate RAG2(-/-)IL2Rγc(-/-) mice, indicating the lymphoid origin of the defect. We identified 2 novel missense mutations in RAG1 (p.Arg474Cys and p.Leu506Phe) resulting in reduced RAG activity. This report gives the first genetic clue for ICL and extends the clinical spectrum of RAG mutations from severe immune defects to an almost normal condition.

The human EGF-TM7 receptor EMR3 is a marker for mature granulocytes

EMR3 is a member of the epidermal growth factor‐seven‐transmembrane (EGF‐TM7) family of adhesion class TM7 receptors. This family also comprises CD97, EMR1, EMR2, and EMR4. To characterize human EMR3 at the protein level, we generated Armenian hamster mAb. Using the mAb 3D7, we here demonstrate that EMR3, like other EGF‐TM7 receptors, is expressed at the cell surface as a heterodimeric molecule consisting of a long extracellular α‐chain, which possesses at its N‐terminus EGF‐like domains and a membrane‐spanning β‐chain. Flow cytometric analysis revealed that all types of myeloid cells express EMR3. In peripheral blood, the highest expression of EMR3 was found on granulocytes. More mature CD16+ monocytes express high levels of EMR3, and CD16– monocytes and myeloid dendritic cells (DC) are EMR3dim/low. Lymphocytes and plasmacytoid DC are EMR3–. It is interesting that in contrast with CD97 and EMR2, CD34+CD33–/CD38– committed hematopoietic stem cells and CD34+CD33+/CD38+ progenitors in bone marrow do not express EMR3. In vitro differentiation of HL‐60 cells and CD34+ progenitor cells revealed that EMR3 is only up‐regulated during late granulopoiesis. These results demonstrate that the expression of EGF‐TM7 receptors on myeloid cells is differentially regulated. EMR3 is the first family member found mainly on granulocytes.

Toll-like receptor responses in IRAK-4-deficient neutrophils.

Human neutrophils were found to express all known Toll-like receptors (TLRs) except TLR3 and TLR7. IRAK-4-deficient neutrophils were tested for their responsiveness to various TLR ligands. Essentially all TLR responses in neutrophils, including the induction of reactive oxygen species generation, adhesion, chemotaxis and IL-8 secretion, were found to be dependent on IRAK-4. Surprisingly, the reactivity towards certain established TLR ligands, imiquimod and ODN-CpG, was unaffected by IRAK-4 deficiency, demonstrating their activity is independent of TLR. TLR-4-dependent signaling in neutrophils was totally dependent on IRAK-4 without any major TRIF-mediated contribution. We did not observe any defects in killing capacity of IRAK-4-deficient neutrophils for Staphylococcus aureus, Escherichia coli and Candida albicans, suggesting that microbial killing is primarily TLR independent.

Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1

Absolute numbers of lymphocytes are decreased in uninfected infants born to HIV-1-infected women (HIV-1-exposed). Although the exact mechanism is unknown, fetal exposure to maternal HIV-1-infection could prime the immune system and affect T cell trafficking. We compared the expression of chemokine receptors on cord blood CD4+ T cells from HIV-1-exposed children and healthy controls. At baseline CD4+ T cells had a largely naïve phenotype. However, stimulation with cytokines resulted in an upregulation of inflammatory response-related chemokine receptors on CD4+ T cells, with HIV-1-exposed infants having a significantly higher frequency of CD4+ T cells expressing, in particularly Th2 associated chemokine receptors (CCR3 p < 0.01, CCR8 p = 0.03). Numbers of naive CCR7+ CD4+ T cells were reduced (p = 0.01) in HIV-1-exposed infants. We further assessed whether the inflammatory phenotype was associated with susceptibility to HIV-1 and detected higher levels of p24 upon in in vitro infection of stimulated CD4+ T cells of HIV-1-exposed infants. In summary, fetal exposure to HIV-1 primes the immune system in the infant leading to an enhanced immune activation and altered T cell homing, with potential ramifications regarding T cell responses and the acquisition of HIV-1 as an infant.

Chronic exposure of astrocytes to interferon-α reveals molecular changes related to Aicardi-Goutieres syndrome

Aicardi-Goutières syndrome is a genetically determined infantile encephalopathy, manifesting as progressive microcephaly, psychomotor retardation, and in ∼25% of patients, death in early childhood. Aicardi-Goutières syndrome is caused by mutations in any of the genes encoding TREX1, RNASEH2-A, -B, -C and SAMHD1, with protein dysfunction hypothesized to result in the accumulation of nucleic acids within the cell, thus triggering an autoinflammatory response with increased interferon-α production. Astrocytes have been identified as a major source of interferon-α production in the brains of patients with Aicardi-Goutières syndrome. Here, we study the effect of interferon-α treatment on astrocytes derived from immortalized human neural stem cells. Chronic interferon-α treatment promoted astrocyte activation and a reduction in cell proliferation. Moreover, chronic exposure resulted in an alteration of genes and proteins involved in the stability of white matter (ATF4, eIF2Bα, cathepsin D, cystatin F), an increase of antigen-presenting genes (human leukocyte antigen class I) and downregulation of pro-angiogenic factors and other cytokines (vascular endothelial growth factor and IL-1). Interestingly, withdrawal of interferon-α for 7 days barely reversed these cellular alterations, demonstrating that the interferon-α mediated effects persist over time. We confirmed our in vitro findings using brain samples from patients with Aicardi-Goutières syndrome. Our results support the idea of interferon-α as a key factor in the pathogenesis of Aicardi-Goutières syndrome relating to the observed leukodystrophy and microangiopathy. Because of the sustained interferon-α effect, even after withdrawal, therapeutic targets for Aicardi-Goutières syndrome, and other interferon-α-mediated encephalopathies, may include downstream interferon-α signalling cascade effectors rather than interferon-α alone.

Aicardi–Goutières syndrome harbours abundant systemic and brain-reactive autoantibodies

Objectives Aicardi–Goutières syndrome (AGS) is an autoimmune disorder that shares similarities with systemic lupus erythematous. AGS inflammatory responses specially target the cerebral white matter. However, it remains uncertain why the brain is the most affected organ, and little is known about the presence of autoantibodies in AGS. Here, we aim to profile specific autoantibodies in AGS and to determine whether these autoantibodies target cerebral epitopes. Methods Using a multiplex microarray, we assessed the spectrum of serum autoantibodies in 56 genetically confirmed patients with AGS. We investigated the presence of immunoglobulins in AGS brain specimens using immunohistochemistry and studied the reactivity of sera against brain epitopes with proteomics. Results Serum from patients exhibited high levels of IgGs against nuclear antigens (gP210, Nup62, PCNA, Ro/SSA, Sm/RNP complex, SS-A/SS-B), components of the basement membrane (entactin, laminin), fibrinogen IV and gliadin. Upon testing whether antibodies in AGS could be found in the central nervous system, IgGs were identified to target in vivo endothelial cells in vivo and astrocytes in brain sections of deceased patients with AGS. Using a proteomics approach, we were able to confirm that IgGs in serum samples from AGS patients bind epitopes present in the cerebral white matter. Conclusions Patients with AGS produce a broad spectrum of autoantibodies unique from other autoimmune diseases. Some of these autoantibodies target endothelial cells and astrocytes in the brain of the affected patients, perhaps explaining the prominence of neurological disease in the AGS phenotype.

Phenotypic variation in Aicardi-Goutières syndrome explained by cell-specific IFN-stimulated gene response and cytokine release

Aicardi–Goutières syndrome (AGS) is a monogenic inflammatory encephalopathy caused by mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, or MDA5. Mutations in those genes affect normal RNA/DNA intracellular metabolism and detection, triggering an autoimmune response with an increase in cerebral IFN-α production by astrocytes. Microangiopathy and vascular disease also contribute to the neuropathology in AGS. In this study, we report that AGS gene silencing of TREX1, SAMHD1, RNASEH2A, and ADAR1 by short hairpin RNAs in human neural stem cell–derived astrocytes, human primary astrocytes, and brain-derived endothelial cells leads to an antiviral status of these cells compared with nontarget short hairpin RNA–treated cells. We observed a distinct activation of the IFN-stimulated gene signature with a substantial increase in the release of proinflammatory cytokines (IL-6) and chemokines (CXCL10 and CCL5). A differential impact of AGS gene silencing was noted; silencing TREX1 gave rise to the most dramatic in both cell types. Our findings fit well with the observation that patients carrying mutations in TREX1 experience an earlier onset and fatal outcome. We provide in the present study, to our knowledge for the first time, insight into how astrocytic and endothelial activation of antiviral status may differentially lead to cerebral pathology, suggesting a rational link between proinflammatory mediators and disease severity in AGS.