Publications by authors named "John L van Hamme"

21 Publications

  • Page 1 of 1

Autophagy-enhancing drugs limit mucosal HIV-1 acquisition and suppress viral replication ex vivo.

Sci Rep 2021 Feb 26;11(1):4767. Epub 2021 Feb 26.

Amsterdam UMC, University of Amsterdam, Department of Experimental Immunology, Amsterdam institute for Infection & Immunity, Meibergdreef 9, Amsterdam, The Netherlands.

Current direct-acting antiviral therapies are highly effective in suppressing HIV-1 replication. However, mucosal inflammation undermines prophylactic treatment efficacy, and HIV-1 persists in long-lived tissue-derived dendritic cells (DCs) and CD4 T cells of treated patients. Host-directed strategies are an emerging therapeutic approach to improve therapy outcomes in infectious diseases. Autophagy functions as an innate antiviral mechanism by degrading viruses in specialized vesicles. Here, we investigated the impact of pharmaceutically enhancing autophagy on HIV-1 acquisition and viral replication. To this end, we developed a human tissue infection model permitting concurrent analysis of HIV-1 cellular targets ex vivo. Prophylactic treatment with autophagy-enhancing drugs carbamazepine and everolimus promoted HIV-1 restriction in skin-derived CD11c DCs and CD4 T cells. Everolimus also decreased HIV-1 susceptibility to lab-adapted and transmitted/founder HIV-1 strains, and in vaginal Langerhans cells. Notably, we observed cell-specific effects of therapeutic treatment. Therapeutic rapamycin treatment suppressed HIV-1 replication in tissue-derived CD11c DCs, while all selected drugs limited viral replication in CD4 T cells. Strikingly, both prophylactic and therapeutic treatment with everolimus or rapamycin reduced intestinal HIV-1 productive infection. Our findings highlight host autophagy pathways as an emerging target for HIV-1 therapies, and underscore the relevancy of repurposing clinically-approved autophagy drugs to suppress mucosal HIV-1 replication.
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http://dx.doi.org/10.1038/s41598-021-84081-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910550PMC
February 2021

Neutrophil specific granule and NETosis defects in gray platelet syndrome.

Blood Adv 2021 Jan;5(2):549-564

Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, The Netherlands.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.
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http://dx.doi.org/10.1182/bloodadvances.2020002442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839360PMC
January 2021

Synthetic Abortive HIV-1 RNAs Induce Potent Antiviral Immunity.

Front Immunol 2020 23;11. Epub 2020 Jan 23.

Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

Strong innate and adaptive immune responses are paramount in combating viral infections. Dendritic cells (DCs) detect viral infections via cytosolic RIG-I like receptors (RLRs) RIG-I and MDA5 leading to MAVS-induced immunity. The DEAD-box RNA helicase DDX3 senses abortive human immunodeficiency virus 1 (HIV-1) transcripts and induces MAVS-dependent type I interferon (IFN) responses, suggesting that abortive HIV-1 RNA transcripts induce antiviral immunity. Little is known about the induction of antiviral immunity by DDX3-ligand abortive HIV-1 RNA. Here we synthesized a 58 nucleotide-long capped RNA (HIV-1 Cap-RNA) that mimics abortive HIV-1 RNA transcripts. HIV-1 Cap-RNA induced potent type I IFN responses in monocyte-derived DCs, monocytes, macrophages and primary CD1c DCs. Compared with RLR agonist poly-I:C, HIV-1 Cap-RNA induced comparable levels of type I IFN responses, identifying HIV-1 Cap-RNA as a potent trigger of antiviral immunity. In monocyte-derived DCs, HIV-1 Cap-RNA activated the transcription factors IRF3 and NF-κB. Moreover, HIV-1 Cap-RNA induced DC maturation and the expression of pro-inflammatory cytokines. HIV-1 Cap-RNA-stimulated DCs induced proliferation of CD4 and CD8 T cells and differentiated naïve T helper (T) cells toward a T2 phenotype. Importantly, treatment of DCs with HIV-1 Cap-RNA resulted in an efficient antiviral innate immune response that reduced ongoing HIV-1 replication in DCs. Our data strongly suggest that HIV-1 Cap-RNA induces potent innate and adaptive immune responses, making it an interesting addition in vaccine design strategies.
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http://dx.doi.org/10.3389/fimmu.2020.00008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990453PMC
February 2021

Vaginal dysbiosis associated-bacteria Megasphaera elsdenii and Prevotella timonensis induce immune activation via dendritic cells.

J Reprod Immunol 2020 04 22;138:103085. Epub 2020 Jan 22.

Amsterdam UMC, University of Amsterdam, Department of Experimental Immunology, Amsterdam Institute for Infection & Immunity, Meibergdreef 9, Amsterdam, The Netherlands. Electronic address:

Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria leads to bacterial vaginosis (BV) which is associated with increased inflammation in the genital mucosa. Moreover, BV increases susceptibility to sexual transmitted infections (STIs) and is associated with adverse pregnancy outcomes. It remains unclear how specific vaginal aerobic and anaerobic bacteria affect health and disease. We selected different vaginal bacteria ranging from true commensals to species associated with dysbiosis and investigated their effects on activation of dendritic cells (DCs). Commensal Lactobacilli crispatus did not induce DC maturation nor led to production of pro-inflammatory cytokines. In contrast, BV-associated bacteria Megasphaera elsdenii and Prevotella timonensis induced DC maturation and increased levels of pro-inflammatory cytokines. Notably, DCs stimulated with Prevotella timonensis suppressed Th2 responses and induced Th1 skewing, typically associated with preterm birth. In contrast, Lactobacillus crispatus and Megasphaera elsdenii did not affect Th cell polarization. These results strongly indicate that the interaction of vaginal bacteria with mucosal DCs determines mucosal inflammation and we have identified the anaerobic bacterium Prevotella timonensis as a strong inducer of inflammatory responses. Specifically targeting these inflammation-inducing bacteria might be a therapeutic strategy to prevent BV and associated risks in STI susceptibility and preterm birth.
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http://dx.doi.org/10.1016/j.jri.2020.103085DOI Listing
April 2020

Sexually transmitted founder HIV-1 viruses are relatively resistant to Langerhans cell-mediated restriction.

PLoS One 2019 19;14(12):e0226651. Epub 2019 Dec 19.

Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands.

A single HIV-1 variant establishes infection of the host after sexual contact. Identifying the phenotypic characteristics of these Transmitted Founder (T/F) viruses is important to understand the restriction mechanisms during transmission. Langerhans cells (LCs) are the mucosal dendritic cell subset that has been shown to have a protective role in HIV-1 transmission. Immature LCs efficiently capture and degrade HIV-1 via langerin-mediated restriction. Here we have investigated the capacity of T/F HIV-1 strains to infect mucosal Langerhans cells (LCs). Notably, most T/F variants efficiently infected immature LCs derived from skin and vaginal tissue in contrast to chronic HIV-1 laboratory strains. Next we screened a panel of T/F viruses and their matched 6-month consensus sequence viruses. Interestingly most T/F variants infected immature LCs whereas donor-matched 6-month consensus sequence viruses had lost the ability to infect LCs. However, we also identified 6-month consensus sequence viruses that had retained an ability to infect LCs similar to that of the donor-matched T/F virus. Moreover, some T/F viruses and 6-month consensus sequence viruses were unable to infect immature LCs. Further analyses indicated that T/F viruses are less sensitive to langerin-mediated restriction. These data suggest that T/F HIV-1 variants have the ability to infect immature LCs, which will facilitate transmission.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922402PMC
April 2020

Inherited p40phox deficiency differs from classic chronic granulomatous disease.

J Clin Invest 2018 08 6;128(9):3957-3975. Epub 2018 Aug 6.

Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.
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http://dx.doi.org/10.1172/JCI97116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6118590PMC
August 2018

RIG-I-like receptor activation by dengue virus drives follicular T helper cell formation and antibody production.

PLoS Pathog 2017 Nov 29;13(11):e1006738. Epub 2017 Nov 29.

Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

Follicular T helper cells (TFH) are fundamental in orchestrating effective antibody-mediated responses critical for immunity against viral infections and effective vaccines. However, it is unclear how virus infection leads to TFH induction. We here show that dengue virus (DENV) infection of human dendritic cells (DCs) drives TFH formation via crosstalk of RIG-I-like receptor (RLR) RIG-I and MDA5 with type I Interferon (IFN) signaling. DENV infection leads to RLR-dependent IKKε activation, which phosphorylates IFNα/β receptor-induced STAT1 to drive IL-27 production via the transcriptional complex ISGF3. Inhibiting RLR activation as well as neutralizing antibodies against IL-27 prevented TFH formation. DENV-induced CXCR5+PD-1+Bcl-6+ TFH cells secreted IL-21 and activated B cells to produce IgM and IgG. Notably, RLR activation by synthetic ligands also induced IL-27 secretion and TFH polarization. These results identify an innate mechanism by which antibodies develop during viral disease and identify RLR ligands as potent adjuvants for TFH-promoting vaccination strategies.
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http://dx.doi.org/10.1371/journal.ppat.1006738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724900PMC
November 2017

DCs facilitate B cell responses against microbial DNA via DC-SIGN.

PLoS One 2017 4;12(10):e0185580. Epub 2017 Oct 4.

Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

Microbial DNA is highly immunostimulatory and is sensed by endosomal pattern recognition receptors after release from internalized microbes. It is unclear how extracellular DNA released from dead microbes is delivered to endosomal PRRs to induce immune responses. Here we have investigated the ability of DCs to bind and internalize extracellular E.coli DNA as well as synthetic DNA. DCs internalized E.coli and synthetic DNA, which was dependent on the C-type lectin receptor DC-SIGN. Notably, endosomal uptake of DNA by DCs enhanced TLR9-dependent responses of B cells against DNA. Hence, we have identified DC-SIGN as a cell surface receptor for DNA that facilitates immune responses directed against DNA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185580PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627929PMC
November 2017

RIG-I-like Receptor Triggering by Dengue Virus Drives Dendritic Cell Immune Activation and T1 Differentiation.

J Immunol 2017 06 15;198(12):4764-4771. Epub 2017 May 15.

Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands; and

Dengue virus (DENV) causes 400 million infections annually and is one of several viruses that can cause viral hemorrhagic fever, which is characterized by uncontrolled immune activation resulting in high fever and internal bleeding. Although the underlying mechanisms are unknown, massive cytokine secretion is thought to be involved. Dendritic cells (DCs) are the main target cells of DENV, and we investigated their role in DENV-induced cytokine production and adaptive immune responses. DENV infection induced DC maturation and secretion of IL-1β, IL-6, and TNF. Inhibition of DENV RNA replication abrogated these responses. Notably, silencing of RNA sensors RIG-I or MDA5 abrogated DC maturation, as well as cytokine responses by DENV-infected DCs. DC maturation was induced by type I IFN responses because inhibition of IFN-α/β receptor signaling abrogated DENV-induced DC maturation. Moreover, DENV infection of DCs resulted in CCL2, CCL3, and CCL4 expression, which was abrogated after RIG-I and MDA5 silencing. DCs play an essential role in T cell differentiation, and we show that RIG-I and MDA5 triggering by DENV leads to T1 polarization, which is characterized by high levels of IFN-γ. Notably, cytokines IL-6, TNF, and IFN-γ and chemokines CCL2, CCL3, and CCL4 have been associated with disease severity, endothelial dysfunction, and vasodilation. Therefore, we identified RIG-I and MDA5 as critical players in innate and adaptive immune responses against DENV, and targeting these receptors has the potential to decrease hemorrhagic fever in patients.
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http://dx.doi.org/10.4049/jimmunol.1602121DOI Listing
June 2017

Dexamethasone promotes granulocyte mobilization by prolonging the half-life of granulocyte-colony-stimulating factor in healthy donors for granulocyte transfusions.

Transfusion 2017 03 28;57(3):674-684. Epub 2016 Dec 28.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam.

Background: Granulocyte transfusion (GTX) is a potential approach to correcting neutropenia and relieving the increased risk of infection in patients who are refractory to antibiotics. To mobilize enough granulocytes for transfusion, healthy donors are premedicated with granulocyte-colony-stimulating factor (G-CSF) and dexamethasone. Granulocytes have a short circulatory half-life. Consequently, patients need to receive GTX every other day to keep circulating granulocyte counts at an acceptable level. We investigated whether plasma from premedicated donors was capable of prolonging neutrophil survival and, if so, which factor could be held responsible.

Study Design And Methods: The effects of plasma from G-CSF/dexamethasone-treated donors on neutrophil survival were assessed by annexin-V, CD16. and CXCR4 staining and nuclear morphology. We isolated an albumin-bound protein using α-chymotrypsin and albumin-depletion and further characterized it using protein analysis. The effects of dexamethasone and G-CSF were assessed using mifepristone and G-CSF-neutralizing antibody. G-CSF plasma concentrations were determined by Western blot and Luminex analyses.

Results: G-CSF/dexamethasone plasma contained a survival-promoting factor for at least 2 days. This factor was recognized as an albumin-associated protein and was identified as G-CSF itself, which was surprising considering its reported half-life of only 4.5 hours. Compared with coadministration of dexamethasone, administration of G-CSF alone to the same GTX donors led to a faster decline in circulating G-CSF levels, whereas dexamethasone itself did not induce any G-CSF, demonstrating a role for dexamethasone in increasing G-CSF half-life.

Conclusion: Dexamethasone increases granulocyte yield upon coadministration with G-CSF by extending G-CSF half-life. This observation might also be exploited in the coadministration of dexamethasone with other recombinant proteins to modulate their half-life.
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http://dx.doi.org/10.1111/trf.13941DOI Listing
March 2017

Receptor usage dictates HIV-1 restriction by human TRIM5α in dendritic cell subsets.

Nature 2016 12 7;540(7633):448-452. Epub 2016 Dec 7.

Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

The most prevalent route of HIV-1 infection is across mucosal tissues after sexual contact. Langerhans cells (LCs) belong to the subset of dendritic cells (DCs) that line the mucosal epithelia of vagina and foreskin and have the ability to sense and induce immunity to invading pathogens. Anatomical and functional characteristics make LCs one of the primary targets of HIV-1 infection. Notably, LCs form a protective barrier against HIV-1 infection and transmission. LCs restrict HIV-1 infection through the capture of HIV-1 by the C-type lectin receptor Langerin and subsequent internalization into Birbeck granules. However, the underlying molecular mechanism of HIV-1 restriction in LCs remains unknown. Here we show that human E3-ubiquitin ligase tri-partite-containing motif 5α (TRIM5α) potently restricts HIV-1 infection of LCs but not of subepithelial DC-SIGN DCs. HIV-1 restriction by TRIM5α was thus far considered to be reserved to non-human primate TRIM5α orthologues, but our data strongly suggest that human TRIM5α is a cell-specific restriction factor dependent on C-type lectin receptor function. Our findings highlight the importance of HIV-1 binding to Langerin for the routeing of HIV-1 into the human TRIM5α-mediated restriction pathway. TRIM5α mediates the assembly of an autophagy-activating scaffold to Langerin, which targets HIV-1 for autophagic degradation and prevents infection of LCs. By contrast, HIV-1 binding to DC-SIGN DCs leads to disassociation of TRIM5α from DC-SIGN, which abrogates TRIM5α restriction. Thus, our data strongly suggest that restriction by human TRIM5α is controlled by C-type-lectin-receptor-dependent uptake of HIV-1, dictating protection or infection of human DC subsets. Therapeutic interventions that incorporate C-type lectin receptors and autophagy-targeting strategies could thus provide cell-mediated resistance to HIV-1 in humans.
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http://dx.doi.org/10.1038/nature20567DOI Listing
December 2016

Proinflammatory cytokine response toward fungi but not bacteria in chronic granulomatous disease.

J Allergy Clin Immunol 2016 09 5;138(3):928-930.e4. Epub 2016 May 5.

Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1016/j.jaci.2016.03.035DOI Listing
September 2016

Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components.

Haematologica 2016 05 22;101(5):587-96. Epub 2016 Jan 22.

Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of antibacterial activity, but less is known about their fungal killing capacity. We investigated the neutrophil-mediated cytotoxic response against C. albicans and A. fumigatus in detail. Whereas G-CSF/dexamethasone-mobilized neutrophils appeared less mature as compared to neutrophils from untreated controls, these cells exhibited normal ROS production by the NADPH oxidase system and an unaltered granule mobilization capacity upon stimulation. G-CSF/dexamethasone-mobilized neutrophils efficiently inhibited A. fumigatus germination and killed Aspergillus and Candida hyphae, but the killing of C. albicans yeasts was distinctly impaired. Following normal Candida phagocytosis, analysis by mass spectrometry of purified phagosomes after fusion with granules demonstrated that major constituents of the antimicrobial granule components, including major basic protein (MBP), were reduced. Purified MBP showed candidacidal activity, and neutrophil-like Crisp-Cas9 NB4-KO-MBP differentiated into phagocytes were impaired in Candida killing. Together, these findings indicate that G-CSF/dexamethasone-mobilized neutrophils for transfusion purposes have a selectively impaired capacity to kill Candida yeasts, as a consequence of an altered neutrophil granular content.
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http://dx.doi.org/10.3324/haematol.2015.136630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004374PMC
May 2016

Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

J Immunol 2016 Feb 30;196(3):1272-83. Epub 2015 Dec 30.

Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands; Department of Pediatric Hematology, Immunology, and Infectious Diseases, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands;

Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response.
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http://dx.doi.org/10.4049/jimmunol.1501811DOI Listing
February 2016

ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells.

PLoS One 2015 2;10(12):e0143613. Epub 2015 Dec 2.

Department of Experimental Immunology, Academic Medical Center (AMC), University of Amsterdam (UvA), Amsterdam, The Netherlands.

Unlike resting CD4+ T cells, activated CD4+T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4+T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4+T cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143613PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667845PMC
June 2016

Impaired microbial killing by neutrophils from patients with protein kinase C delta deficiency.

J Allergy Clin Immunol 2015 Nov 30;136(5):1404-7.e1-10. Epub 2015 Jul 30.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1016/j.jaci.2015.06.016DOI Listing
November 2015

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

Sci Rep 2014 Oct 24;4:6690. Epub 2014 Oct 24.

1] Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children's Hospital, Academic Medical Centre (AMC), University of Amsterdam (UvA), Amsterdam, The Netherlands [2] Department of Experimental Immunology, AMC, UvA, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1038/srep06690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208038PMC
October 2014

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects.

Blood 2014 Jul 19;124(4):590-7. Epub 2014 Jun 19.

Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.
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http://dx.doi.org/10.1182/blood-2014-01-551473DOI Listing
July 2014

Defects in neutrophil granule mobilization and bactericidal activity in familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) syndrome caused by STXBP2/Munc18-2 mutations.

Blood 2013 Jul 17;122(1):109-11. Epub 2013 May 17.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by genetic defects in cytotoxic granule components or their fusion machinery, leading to impaired natural killer cell and/or T lymphocyte degranulation and/or cytotoxicity. This may accumulate into a life-threatening condition known as macrophage activation syndrome. STXBP2, also known as MUNC18-2, has recently been identified as the disease-causing gene in FHL type 5 (FHL-5). A role for STXBP2 in neutrophils, and for neutrophils in FHL in general, has not been documented thus far. Here, we report that FHL-5 neutrophils have a profound defect in granule mobilization, resulting in inadequate bacterial killing, in particular, of gram-negative Escherichia coli, but not of Staphylococcus aureus, which rather depends on intact reduced NAD phosphate oxidase activity. This impairment of bacterial killing may contribute to the apparent susceptibility to gastrointestinal tract inflammation in patients with FHL-5.
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http://dx.doi.org/10.1182/blood-2013-03-494039DOI Listing
July 2013

Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency.

Blood 2013 Mar 18;121(13):2385-92. Epub 2013 Jan 18.

Sanquin Research, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1182/blood-2012-08-450551DOI Listing
March 2013

Memory CD4(+)CCR5(+) T cells are abundantly present in the gut of newborn infants to facilitate mother-to-child transmission of HIV-1.

Blood 2012 Nov 1;120(22):4383-90. Epub 2012 Oct 1.

Departments of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

Despite potential clinical importance, target cells for mother-to-child transmission of HIV-1 have not yet been identified. Cord blood-derived CD4(+) T cells are largely naive and do not express CCR5, the mandatory coreceptor for transmitted HIV-1 R5 strains in infants. In the present study, we demonstrate that in the human fetal and infant gut mucosa, there is already a large subset of mucosal memory CD4(+)CCR5(+) T cells with predominantly a Th1 and Th17 phenotype. Using next-generation sequencing of the TCRβ chain, clonally expanded T cells as a hallmark for memory development predominated in the gut mucosa (30%), whereas few were found in the lymph nodes (1%) and none in cord blood (0%). The gut mucosal fetal and infant CD4(+) T cells were highly susceptible to HIV-1 without any prestimulation; pol proviral DNA levels were similar to infected phytohemagglutinin-stimulated adult PBMCs. In conclusion, in the present study, we show that extensive adaptive immunity is present before birth and the gut mucosa is the preferential site for memory CD4(+) T cells. These CD4(+)CCR5(+) T cells in the infant mucosa provide a large pool of susceptible cells for ingested HIV-1 at birth and during breastfeeding, indicating a mucosal route of mother-to-child transmission that can be targeted in prevention strategies.
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http://dx.doi.org/10.1182/blood-2012-06-437566DOI Listing
November 2012