Publications by authors named "Mary C Dinauer"

107 Publications

Immunodeficiency and bone marrow failure with mosaic and germline TLR8 gain of function.

Blood 2021 May;137(18):2450-2462

Division of Allergy and Immunology, Department of Pediatrics, University of Arkansas for Medical Sciences and Arkansas Children's Hospital, Little Rock, AR.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.
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http://dx.doi.org/10.1182/blood.2020009620DOI Listing
May 2021

Publisher Correction: Neutrophil swarming delays the growth of clusters of pathogenic fungi.

Nat Commun 2020 05 14;11(1):2492. Epub 2020 May 14.

BioMEMS Resource Center, Massachusetts General Hospital, Boston, MA, 02129, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-16446-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224198PMC
May 2020

Neutrophil swarming delays the growth of clusters of pathogenic fungi.

Nat Commun 2020 04 27;11(1):2031. Epub 2020 Apr 27.

BioMEMS Resource Center, Massachusetts General Hospital, Boston, MA, 02129, USA.

Neutrophils employ several mechanisms to restrict fungi, including the action of enzymes such as myeloperoxidase (MPO) or NADPH oxidase, and the release of neutrophil extracellular traps (NETs). Moreover, they cooperate, forming "swarms" to attack fungi that are larger than individual neutrophils. Here, we designed an assay for studying how these mechanisms work together and contribute to neutrophil's ability to contain clusters of live Candida. We find that neutrophil swarming over Candida clusters delays germination through the action of MPO and NADPH oxidase, and restricts fungal growth through NET release within the swarm. In comparison with neutrophils from healthy subjects, those from patients with chronic granulomatous disease produce larger swarms against Candida, but their release of NETs is delayed, resulting in impaired control of fungal growth. We also show that granulocyte colony-stimulating factors (GCSF and GM-CSF) enhance swarming and neutrophil ability to restrict fungal growth, even during treatment with chemical inhibitors that disrupt neutrophil function.
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http://dx.doi.org/10.1038/s41467-020-15834-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184738PMC
April 2020

NADPH oxidase controls pulmonary neutrophil infiltration in the response to fungal cell walls by limiting LTB4.

Blood 2020 03;135(12):891-903

Department of Pediatrics.

Leukocyte reduced NADP (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyperinflammation induced by fungal cell walls in CGD are still incompletely defined. In this study, we found that CGD (CYBB-) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro after activation with zymosan or immune complexes, compared with wild-type (WT) neutrophils. This finding correlated with increased calcium influx in CGD neutrophils, which was restrained in WT neutrophils by the electrogenic activity of NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared with WT cells, and the effect was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation, focal neutrophil infiltrates were increased in CGD compared with WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor after zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus, LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls, likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation, which could potentially be exploited therapeutically to reduce excessive inflammation in CGD.
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http://dx.doi.org/10.1182/blood.2019003525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082617PMC
March 2020

Neutrophil Defects and Diagnosis Disorders of Neutrophil Function: An Overview.

Authors:
Mary C Dinauer

Methods Mol Biol 2020 ;2087:11-29

Department of Pediatrics (Hematology/Oncology), St. Louis Children's Hospital, Washington University School of Medicine in St. Louis, St. Louis, MO, USA.

Primary disorders of neutrophil function result from impairment in neutrophil responses that are critical for host defense. This chapter summarizes inherited disorders of neutrophils that cause defects in neutrophil adhesion, migration, and oxidative killing. These include the leukocyte adhesion deficiencies, actin defects and other disorders of chemotaxis, hyperimmunoglobulin E syndrome, Chédiak-Higashi Syndrome, neutrophil specific granule deficiency, chronic granulomatous disease, and myeloperoxidase deficiency. Diagnostic tests and treatment approaches are also summarized for each neutrophil disorder.
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http://dx.doi.org/10.1007/978-1-0716-0154-9_2DOI Listing
January 2021

Insights into the NOX NADPH Oxidases Using Heterologous Whole Cell Assays.

Authors:
Mary C Dinauer

Methods Mol Biol 2019 ;1982:139-151

Department of Pediatrics, Washington University in St. Louis School of Medicine , St. Louis, MO, USA.

Assays based on ectopic expression of NOX NADPH oxidase subunits in heterologous mammalian cells are an important approach for investigating features of this family of enzymes. These model systems have been used to analyze the biosynthesis and functional domains of NOX enzyme components as well as their regulation and cellular activities. This chapter provides an overview of the basic principles and applications of heterologous whole cell assays in studying NOX NADPH oxidases.
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http://dx.doi.org/10.1007/978-1-4939-9424-3_9DOI Listing
January 2020

Vav1 inhibits RANKL-induced osteoclast differentiation and bone resorption.

BMB Rep 2019 Nov;52(11):659-664

Laboratory for Leukocyte Signaling Research, Department of Pharmacology, Inha University School of Medicine, Incheon 22212, Korea.

Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1-/-) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1-/- mice than in WT mice. Furthermore, the bone status of Vav1-/- mice was analyzed in situ and the femurs of Vav1-/- mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvβ3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption. [BMB Reports 2019; 52(11): 659-664].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889896PMC
November 2019

Inflammatory consequences of inherited disorders affecting neutrophil function.

Authors:
Mary C Dinauer

Blood 2019 05 21;133(20):2130-2139. Epub 2019 Mar 21.

Department of Pediatrics and Department of Pathology & Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO.

Primary immunodeficiencies affecting the function of neutrophils and other phagocytic leukocytes are notable for an increased susceptibility to bacterial and fungal infections as a result of impaired leukocyte recruitment, ingestion, and/or killing of microbes. The underlying molecular defects can also impact other innate immune responses to infectious and inflammatory stimuli, leading to inflammatory and autoimmune complications that are not always directly related to infection. This review will provide an update on congenital disorders affecting neutrophil function in which a combination of host defense and inflammatory complications are prominent, including nicotinamide dinucleotide phosphate oxidase defects in chronic granulomatous disease and β2 integrin defects in leukocyte adhesion deficiency.
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http://dx.doi.org/10.1182/blood-2018-11-844563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524563PMC
May 2019

Inherited p40phox deficiency differs from classic chronic granulomatous disease.

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

Primary Immunodeficiencies Group, Department of Microbiology and Parasitology, School of Medicine, and.

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

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

Mol Ther 2018 05 10;26(5):1181-1197. Epub 2018 Mar 10.

Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA. Electronic address:

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue.
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http://dx.doi.org/10.1016/j.ymthe.2018.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993968PMC
May 2018

NADPH oxidase activation regulates apoptotic neutrophil clearance by murine macrophages.

Blood 2018 05 4;131(21):2367-2378. Epub 2018 Apr 4.

Department of Pediatrics, Washington University School of Medicine in St. Louis, St. Louis, MO.

The phagocyte reduced NAD phosphate (NADPH) oxidase generates superoxide, the precursor to reactive oxygen species (ROS) that has both antimicrobial and immunoregulatory functions. Inactivating mutations in NADPH oxidase alleles cause chronic granulomatous disease (CGD), characterized by enhanced susceptibility to life-threatening microbial infections and inflammatory disorders; hypomorphic NADPH oxidase alleles are associated with autoimmunity. Impaired apoptotic cell (AC) clearance is implicated as an important contributing factor in chronic inflammation and autoimmunity, but the role of NADPH oxidase-derived ROS in this process is incompletely understood. Here, we demonstrate that phagocytosis of AC (efferocytosis) potently activated NADPH oxidase in mouse peritoneal exudate macrophages (PEMs). ROS generation was dependent on macrophage CD11b, Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response 88 (MyD88), and was also regulated by phosphatidylinositol 3-phosphate binding to the p40 oxidase subunit. Maturation of efferosomes containing apoptotic neutrophils was significantly delayed in CGD PEMs, including acidification and acquisition of proteolytic activity, and was associated with slower digestion of apoptotic neutrophil proteins. Treatment of wild-type macrophages with the vacuolar-type H+ ATPase inhibitor bafilomycin also delayed proteolysis within efferosomes, showing that luminal acidification was essential for efficient digestion of efferosome proteins. Finally, cross-presentation of AC-associated antigens by CGD PEMs to CD8 T cells was increased. These studies unravel a key role for the NADPH oxidase in the disposal of ACs by inflammatory macrophages. The oxidants generated promote efferosome maturation and acidification that facilitate the degradation of ingested ACs.
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http://dx.doi.org/10.1182/blood-2017-09-809004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969376PMC
May 2018

LysMD3 is a type II membrane protein without an role in the response to a range of pathogens.

J Biol Chem 2018 04 1;293(16):6022-6038. Epub 2018 Mar 1.

From the Departments of Pathology and Immunology and.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule .
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http://dx.doi.org/10.1074/jbc.RA117.001246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912457PMC
April 2018

Somatic mutations and clonal hematopoiesis in congenital neutropenia.

Blood 2018 01 1;131(4):408-416. Epub 2017 Nov 1.

Division of Oncology, Department of Internal Medicine.

Severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS) are congenital neutropenia syndromes with a high rate of leukemic transformation. Hematopoietic stressors may contribute to leukemic transformation by increasing the mutation rate in hematopoietic stem/progenitor cells (HSPCs) and/or by promoting clonal hematopoiesis. We sequenced the exome of individual hematopoietic colonies derived from 13 patients with congenital neutropenia to measure total mutation burden and performed error-corrected sequencing on a panel of 46 genes on 80 patients with congenital neutropenia to assess for clonal hematopoiesis. An average of 3.6 ± 1.2 somatic mutations per exome was identified in HSPCs from patients with SCN compared with 3.9 ± 0.4 for healthy controls ( = NS). Clonal hematopoiesis due to mutations in was present in 48% (13/27) of patients with SDS but was not seen in healthy controls (0/17, < .001) or patients with SCN (0/40, < .001). Our SDS cohort was young (median age 6.3 years), and many of the patients had multiple mutations. Conversely, clonal hematopoiesis due to mutations of was present in patients with SCN but was not detected in healthy controls or patients with SDS. These data show that hematopoietic stress, including granulocyte colony-stimulating factor, do not increase the mutation burden in HSPCs in congenital neutropenia. Rather, distinct hematopoietic stressors result in the selective expansion of HSPCs carrying specific gene mutations. In particular, in SDS there is enormous selective pressure to expand -mutated HSPCs, suggesting that acquisition of mutations is an early, likely initiating event, in the transformation to myelodysplastic syndrome/acute myeloid leukemia in patients with SDS.
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http://dx.doi.org/10.1182/blood-2017-08-801985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790127PMC
January 2018

Haploinsufficiency of NADPH Oxidase Subunit Neutrophil Cytosolic Factor 2 Is Sufficient to Accelerate Full-Blown Lupus in NZM 2328 Mice.

Arthritis Rheumatol 2017 08 5;69(8):1647-1660. Epub 2017 Jul 5.

Washington University School of Medicine in St. Louis, St. Louis, Missouri.

Objective: We have previously established that the gene for neutrophil cytosolic factor 2 (NCF-2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE).

Methods: We generated NCF-2-null mice, in which NADPH oxidase activity is absent, on the nonautoimmune C57BL/6 (B6) mouse background and on the NZM 2328 mouse background, a polygenic model in which mice spontaneously develop lupus. Clinical disease, serology, and immunopathology were evaluated.

Results: NCF-2-null mice on the B6 background were susceptible to Aspergillus fumigatus pneumonia characteristic of chronic granulomatous disease, but did not develop systemic lupus disease. In contrast, NCF-2-null and even NCF-2-haploinsufficient mice on the NZM 2328 background developed accelerated full-blown lupus with significantly accelerated lupus kidney disease. This was characterized by more rapid development of hyperactive B cell and T cell immune compartments, increased expression of type I interferon-responsive genes, and generation of neutrophil extracellular traps, which were observed even in the absence of NADPH oxidase activity.

Conclusion: Just as patients with chronic granulomatous disease who lack NADPH oxidase rarely develop SLE, NCF-2-null mice on a nonautoimmune background were susceptible to a chronic granulomatous disease-like opportunistic infection but did not develop lupus. In contrast, on a lupus-prone background, even haploinsufficiency of NCF-2 accelerated the development of full-blown lupus disease. This establishes an interaction between reduced oxidase activity and other lupus-predisposing genes, paralleling human SLE-associated variants predicted to have only reduced NADPH oxidase activity.
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http://dx.doi.org/10.1002/art.40141DOI Listing
August 2017

Laurence Alan Boxer, M.D. (1940-2017).

Authors:
Mary C Dinauer

J Leukoc Biol 2017 05;101(5):1075

Departments of Pediatrics and Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

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http://dx.doi.org/10.1189/jlb.1LT0217-060DOI Listing
May 2017

Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense.

Cell Rep 2017 05;19(5):1008-1021

Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI 53706, USA; Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. Electronic address:

The Fenton-chemistry-generating properties of copper ions are considered a potent phagolysosome defense against pathogenic microbes, yet our understanding of underlying host/microbe dynamics remains unclear. We address this issue in invasive aspergillosis and demonstrate that host and fungal responses inextricably connect copper and reactive oxygen intermediate (ROI) mechanisms. Loss of the copper-binding transcription factor AceA yields an Aspergillus fumigatus strain displaying increased sensitivity to copper and ROI in vitro, increased intracellular copper concentrations, decreased survival in challenge with murine alveolar macrophages (AMΦs), and reduced virulence in a non-neutropenic murine model. ΔaceA survival is remediated by dampening of host ROI (chemically or genetically) or enhancement of copper-exporting activity (CrpA) in A. fumigatus. Our study exposes a complex host/microbe multifactorial interplay that highlights the importance of host immune status and reveals key targetable A. fumigatus counter-defenses.
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http://dx.doi.org/10.1016/j.celrep.2017.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512462PMC
May 2017

Primary immune deficiencies with defects in neutrophil function.

Authors:
Mary C Dinauer

Hematology Am Soc Hematol Educ Program 2016 Dec;2016(1):43-50

Department of Pediatrics, Washington University School of Medicine in St. Louis, St. Louis, MO.

Immune deficiencies resulting from inherited defects in neutrophil function have revealed important features of the innate immune response. Although sharing an increased susceptibility to bacterial and fungal infections, these disorders each have distinctive features in their clinical manifestations and characteristic microbial pathogens. This review provides an update on several genetic disorders with impaired neutrophil function, their pathogenesis, and treatment strategies. These include chronic granulomatous disease, which results from inactivating mutations in the superoxide-generating nicotinamide dinucleotide phosphate oxidase. Superoxide-derived oxidants play an important role in the control of certain bacterial and fungal species, and also contribute to the regulation of inflammation. Also briefly summarized are updates on leukocyte adhesion deficiency, including the severe periodontal disease characteristic of this disorder, and a new immune deficiency associated with defects in caspase recruitment domain-containing protein 9, an adaptor protein that regulates signaling in neutrophils and other myeloid cells, leading to invasive fungal disease.
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http://dx.doi.org/10.1182/asheducation-2016.1.43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142438PMC
December 2016

PI(3)P-p40phox binding regulates NADPH oxidase activation in mouse macrophages and magnitude of inflammatory responses in vivo.

J Leukoc Biol 2017 02 19;101(2):449-457. Epub 2016 Aug 19.

Department of Pediatrics, Washington University School of Medicine in St. Louis, St. Louis, MO, USA; and

Mutations in the leukocyte NADPH oxidase that abrogate superoxide production result in chronic granulomatous disease (CGD), an inherited immunodeficiency associated with recurrent infections and inflammatory complications. The cytosolic regulatory subunit p40 plays a specialized role in stimulating NADPH oxidase activity on intracellular membranes via its phosphatidylinositol 3-phosphate [PI(3)P]-binding domain, as revealed by studies largely focused on neutrophils. Whether PI(3)P-p40-regulated superoxide production contributes to regulating inflammatory responses is not well understood. Here, we report that mice expressing p40 R58A, which lacks PI(3)P binding, had impaired macrophage NADPH oxidase activity and increased sterile inflammation. p40 macrophages exhibited diminished phagosome reactive oxygen species (ROS) in response to certain particulate and soluble ligands, including IgG-opsonized particles and a TLR2 agonist, along with unexpected defects in plasma membrane oxidase activity. Compared with wild-type (WT) mice, p40 mice had elevated numbers of newly recruited neutrophils and monocytes in peritoneal inflammation elicited by zymosan, monosodium urate (MSU) crystals, or sodium periodate. At later time points, higher numbers of inflammatory macrophages in p40 mice were consistent with delayed resolution. Our studies demonstrate a critical role of PI(3)P-p40 binding for optimal activation of the NADPH oxidase in macrophages. Furthermore, selective loss of PI(3)P-regulated NADPH oxidase activity was sufficient to enhance significantly responses to inflammation and delay resolution.
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http://dx.doi.org/10.1189/jlb.3AB0316-139RDOI Listing
February 2017

Homeostatic Control of Innate Lung Inflammation by Vici Syndrome Gene Epg5 and Additional Autophagy Genes Promotes Influenza Pathogenesis.

Cell Host Microbe 2016 Jan;19(1):102-13

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Mutations in the autophagy gene EPG5 are linked to the multisystem human disease Vici syndrome, which is characterized in part by pulmonary abnormalities, including recurrent infections. We found that Epg5-deficient mice exhibited elevated baseline innate immune cellular and cytokine-based lung inflammation and were resistant to lethal influenza virus infection. Lung transcriptomics, bone marrow transplantation experiments, and analysis of cellular cytokine expression indicated that Epg5 plays a role in lung physiology through its function in macrophages. Deletion of other autophagy genes including Atg14, Fip200, Atg5, and Atg7 in myeloid cells also led to elevated basal lung inflammation and influenza resistance. This suggests that Epg5 and other Atg genes function in macrophages to limit innate immune inflammation in the lung. Disruption of this normal homeostatic dampening of lung inflammation results in increased resistance to influenza, suggesting that normal homeostatic mechanisms that limit basal tissue inflammation support some infectious diseases.
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http://dx.doi.org/10.1016/j.chom.2015.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714358PMC
January 2016

NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1α/G-CSF axis.

Blood 2015 Dec 6;126(25):2724-33. Epub 2015 Oct 6.

Department of Pediatrics and Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO; and.

The leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates reactive oxygen species essential in microbial killing and regulation of inflammation. Inactivating mutations in this enzyme lead to chronic granulomatous disease (CGD), associated with increased susceptibility to both pyogenic infections and to inflammatory disorders. The role of the NADPH oxidase in regulating inflammation driven by nonmicrobial stimuli is poorly understood. Here, we show that NADPH oxidase deficiency enhances the early local release of interleukin-1α (IL-1α) in response to damaged cells, promoting an excessive granulocyte colony-stimulating factor (G-CSF)-regulated neutrophilic response and prolonged inflammation. In peritoneal inflammation elicited by tissue injury, X-linked Cybb-null (X-CGD) mice exhibited increased release of IL-1α and IL-1 receptor -mediated G-CSF production. In turn, higher levels of systemic G-CSF increased peripheral neutrophilia, which amplified neutrophilic peritoneal inflammation in X-CGD mice. Dampening early neutrophil recruitment by neutralization of IL-1α, G-CSF, or neutrophil depletion itself promoted resolution of otherwise prolonged inflammation in X-CGD. IL-1β played little role. Thus, we identified an excessive IL-1α/G-CSF response as a major driver of enhanced sterile inflammation in CGD in the response to damaged cells. More broadly, these results provide new insights into the regulation of sterile inflammation, and identify the NADPH oxidase in regulating the amplitude of the early neutrophilic response.
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http://dx.doi.org/10.1182/blood-2015-05-644773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683333PMC
December 2015

Hyper-responsive Toll-like receptor 7 and 9 activation in NADPH oxidase-deficient B lymphoblasts.

Immunology 2015 Dec 29;146(4):595-606. Epub 2015 Sep 29.

Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40(phox) or p47(phox) subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients.
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http://dx.doi.org/10.1111/imm.12530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4693894PMC
December 2015

Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67.

J Clin Cell Immunol 2014 Jun;5(3)

Departments of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN, USA.

Study Background: Chronic granulomatous Disease (CGD) is a rare immunodeficiency caused by a defect in the leukocyte NADPH oxidase. This enzyme generates superoxide, which is needed for the killing of bacteria and fungi by phagocytic leukocytes. Most CGD patients have mutations in , the X-linked gene that encodes gp91, the catalytic subunit of the leukocyte NADPH oxidase. We report here three autosomal recessive CGD patients from two families with a homozygous mutation in , the gene that encodes p67, the activator subunit of the NADPH oxidase.

Methods: Leukocyte NADPH oxidase activity, expression of oxidase components and gene sequences were measured with standard methods. The mutation found in the patients' gene was expressed as Ala202Val-p67 in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67 from the cytosol of the patients' neutrophils to the plasma membrane was measured by confocal microscopy and by Western blotting after membrane purification.

Results: The exceptional feature of the A67 CGD patients reported here is that the p.Ala202Val mutation in the activation domain of p67 was clearly hypomorphic: substantial expression of p67 protein was noted and the NADPH oxidase activity in the neutrophils of the patients was 20-70% of normal, dependent on the stimulus used to activate the cells. The extent of Ala202Val-p67 translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67 in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67.

Conclusion: The mutation found in is the cause of the decreased NADPH oxidase activity and the (mild) clinical problems of the patients. We propose that the p.Ala202Val mutation has changed the conformation of the activation domain of p67, resulting in reduced activation of gp91.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414043PMC
June 2014

Mechanisms of interferon-γ production by neutrophils and its function during Streptococcus pneumoniae pneumonia.

Am J Respir Cell Mol Biol 2015 Mar;52(3):349-64

1 Center for Airways Disease, Department of Medicine.

Bacterial pneumonia is a common public health problem associated with significant mortality, morbidity, and cost. Neutrophils are usually the earliest leukocytes to respond to bacteria in the lungs. Neutrophils rapidly sequester in the pulmonary microvasculature and migrate into the lung parenchyma and alveolar spaces, where they perform numerous effector functions for host defense. Previous studies showed that migrated neutrophils produce IFN-γ early during pneumonia induced by Streptococcus pneumoniae and that early production of IFN-γ regulates bacterial clearance. IFN-γ production by neutrophils requires Rac2, Hck/Lyn/Fgr Src family tyrosine kinases, and NADPH oxidase. Our current studies examined the mechanisms that regulate IFN-γ production by lung neutrophils during acute S. pneumoniae pneumonia in mice and its function. We demonstrate that IFN-γ production by neutrophils is a tightly regulated process that does not require IL-12. The adaptor molecule MyD88 is critical for IFN-γ production by neutrophils. The guanine nucleotide exchange factor CalDAG-GEFI modulates IFN-γ production. The CD11/CD18 complex, CD44, Toll-like receptors 2 and 4, TRIF, and Nrf2 are not required for IFN-γ production by neutrophils. The recently described neutrophil-dendritic cell hybrid cell, identified by its expression of Ly6G and CD11c, is present at low numbers in pneumonic lungs and is not a source of IFN-γ. IFN-γ produced by neutrophils early during acute S. pneumoniae pneumonia induces transcription of target genes in the lungs, which are critical for host defense. These studies underline the complexity of the neutrophil responses during pneumonia in the acute inflammatory response and in subsequent resolution or initiation of immune responses.
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http://dx.doi.org/10.1165/rcmb.2013-0316OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370257PMC
March 2015

Disorders of neutrophil function: an overview.

Authors:
Mary C Dinauer

Methods Mol Biol 2014 ;1124:501-15

Departments of Pediatrics (Hematology/Oncology) and Pathology & Immunology, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO, USA.

Primary disorders of neutrophil function result from impairment in neutrophil responses that are critical for host defense. This chapter summarizes inherited disorders of neutrophils that cause defects in neutrophil adhesion, migration, and oxidative killing. These include the leukocyte adhesion deficiencies, actin defects, and other disorders of chemotaxis, hyperimmunoglobulin E syndrome, Chédiak-Higashi syndrome, neutrophil-specific granule deficiency, chronic granulomatous disease, and myeloperoxidase deficiency. Diagnostic tests and treatment approaches are also summarized for each neutrophil disorder.
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http://dx.doi.org/10.1007/978-1-62703-845-4_30DOI Listing
October 2014

Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic.

ACS Chem Biol 2014 Feb 11;9(2):551-61. Epub 2013 Dec 11.

Center for Women's Infectious Diseases Research, ‡Division of Infectious Diseases, §Department of Internal Medicine, ∥Department of Chemistry, ⊥Department of Pediatrics, and #Department of Pathology and Immunology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.

Many Gram-negative bacteria interact with extracellular metal ions by expressing one or more siderophore types. Among these, the virulence-associated siderophore yersiniabactin (Ybt) is an avid copper chelator, forming stable cupric (Cu(II)-Ybt) complexes that are detectable in infected patients. Here we show that Ybt-expressing E. coli are protected from intracellular killing within copper-replete phagocytic cells. This survival advantage is highly dependent upon the phagocyte respiratory burst, during which superoxide is generated by the NADPH oxidase complex. Chemical fractionation links this phenotype to a previously unappreciated superoxide dismutase (SOD)-like activity of Cu(II)-Ybt. Unlike previously described synthetic copper-salicylate (Cu(II)-SA) SOD mimics, the salicylate-based natural product Cu(II)-Ybt retains catalytic activity at physiologically plausible protein concentrations. These results reveal a new virulence-associated adaptation based upon spontaneous assembly of a non-protein catalyst.
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http://dx.doi.org/10.1021/cb400658kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934373PMC
February 2014

Autophagy proteins control goblet cell function by potentiating reactive oxygen species production.

EMBO J 2013 Dec 1;32(24):3130-44. Epub 2013 Nov 1.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.

Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi-vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3-positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3-positive vacuole-associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion.
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http://dx.doi.org/10.1038/emboj.2013.233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3981139PMC
December 2013

Primary Immune Deficiency Treatment Consortium (PIDTC) report.

J Allergy Clin Immunol 2014 Feb 15;133(2):335-47. Epub 2013 Oct 15.

Wiskott-Aldrich Foundation, Smyrna, Ga.

The Primary Immune Deficiency Treatment Consortium (PIDTC) is a network of 33 centers in North America that study the treatment of rare and severe primary immunodeficiency diseases. Current protocols address the natural history of patients treated for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and chronic granulomatous disease through retrospective, prospective, and cross-sectional studies. The PIDTC additionally seeks to encourage training of junior investigators, establish partnerships with European and other International colleagues, work with patient advocacy groups to promote community awareness, and conduct pilot demonstration projects. Future goals include the conduct of prospective treatment studies to determine optimal therapies for primary immunodeficiency diseases. To date, the PIDTC has funded 2 pilot projects: newborn screening for SCID in Navajo Native Americans and B-cell reconstitution in patients with SCID after hematopoietic stem cell transplantation. Ten junior investigators have received grant awards. The PIDTC Annual Scientific Workshop has brought together consortium members, outside speakers, patient advocacy groups, and young investigators and trainees to report progress of the protocols and discuss common interests and goals, including new scientific developments and future directions of clinical research. Here we report the progress of the PIDTC to date, highlights of the first 2 PIDTC workshops, and consideration of future consortium objectives.
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http://dx.doi.org/10.1016/j.jaci.2013.07.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960312PMC
February 2014

Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis.

Traffic 2013 Nov 16;14(11):1118-31. Epub 2013 Sep 16.

Department of Cell Physiology and Metabolism, University of Geneva, 1 rue Michel-Servet, CH-1211, Geneva 4, Switzerland.

The production of reactive oxygen species (ROS) within immune cell phagosomes is critical for antimicrobial activity and for correct antigen processing, and influences signaling pathways that direct host responses to infection and inflammation. Because excess oxidants can cause tissue damage and oxidative stress, phagocytes must precisely control both the location and timing of NADPH oxidase activity. How differential regulation is achieved at phagosomes is not well understood. Recent studies have revealed that the PI(3)P phosphoinositide plays an important role in locally boosting phagosomal NADPH oxidase activity through its binding to the p40(phox) NADPH oxidase subunit. Furthermore, phox subunit dynamics at phagosomes may regulate the timing of the oxidative burst. Novel elements regulating catalytic core trafficking include Rab27 and SNAP-23. In addition to trafficking events, the activity of the electrogenic oxidase is also governed by ionic fluxes, which are constrained at phagosomes owing to low intraphagosomal volume and dynamic display of channels, transporters, and pumps. New insights on the interdependence of phagosomal pH and ROS have been recently elucidated, and chloride channels important for microbicidal functions, including CFTR, and CLIC family channels, have been identified. Finally, periphagosomal calcium microdomains and calcium-dependent S100A8/9 protein recruitment may help fine-tune spatiotemporal regulation of NADPH oxidase activation for an effective immune response.
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http://dx.doi.org/10.1111/tra.12115DOI Listing
November 2013

From bench to bedside: preclinical evaluation of a self-inactivating gammaretroviral vector for the gene therapy of X-linked chronic granulomatous disease.

Hum Gene Ther Clin Dev 2013 Jun;24(2):86-98

Institute for Biomedical Research, Georg-Speyer-Haus, 60596 Frankfurt, Germany.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.
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http://dx.doi.org/10.1089/humc.2013.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461155PMC
June 2013