Functional neutrophil disorders: Chronic granulomatous disease and beyond

Since their description by Metchnikoff in 1905, phagocytes have been increasingly recognized to be the entities that traffic to sites of infection and inflammation, engulf and kill infecting organisms, and clear out apoptotic debris all the while making antigens available and accessible to the lymphoid organs for future use. Therefore, phagocytes provide the gateway and the first check in host protection and immune response. Disorders in killing and chemotaxis lead not only to infection susceptibility, but also to autoimmunity. We aim to describe chronic granulomatous disease and the leukocyte adhesion deficiencies as well as myeloperoxidase deficiency and G6PD deficiency as paradigms of critical pathways.

heterodimer.Upon phagocyte activation, three cytosolic proteins p47 phox (encoded by NCF1), p67 phox (encoded by NCF2) and p40 phox (encoded by NCF4) are phosphorylated, aggregate and translocate to cytochrome b 558 along with RAC1/RAC2 at the membrane surface.The critical interaction is the binding of the p47 phox /p67 phox complex to p22 phox5 .RAC1/RAC2 are GTPases which are bound to the complex, with RAC2 being the predominant phagocyte form. 5ectron transfer from NADPH to molecular oxygen results in a superoxide anion (O 2 − ), which is converted to hydrogen peroxide (H 2 O 2 ) by superoxide dismutase (SOD).H 2 O 2 is then converted to HOCl (hypochlorous acid; bleach in neutrophils because the halide is chlorine) by myeloperoxidase (MPO).The intravacuolar generation of superoxide changes the intravacuolar electrical charge, eliciting a K + flux that enhances intracellular killing through proteases which had been delivered by the primary granule 6 (Figure 1).Another genetic cause of CGD is due to a deficiency of the chaperone protein required for the transport of the gp91 phox /p22 phox complex from the endoplasmic reticulum into a plasma membrane, named essential for reactive oxidant signaling (EROS, encoded by CYBC1). 7Neutrophil extracellular traps (NETs) appear to be dependent on ROS, therefore are also defective in CGD, which may also affect microbial killing. 8,9Ts are released decondensed nuclear chromatin coated with histones and proteases that require MPO for protease activation after nuclear elastases promote decondensation of chromatin.Defects of microbial killing via NETosis in CGD cells have been reversed with the addition of tamoxifen, which induced NET formation in a ROS independent manner and neutrophils treated with tamoxifen showed significant improvement in microbial killing. 9NET formation was restored in a patient, who underwent gene therapy for CGD. 10

| G ENE TI C S OF CG D
The most common affected gene causing CGD in the United States is CYBB, located at Xp21.1, encoding gp91 phox .There are 681 mutations identified in CYBB so far. 11Overall, mortality is inversely linked to ROS production, 12 with survival increasing in patients with mutations that allow more residual ROS production.Early missense mutations before amino acid 310 (except for His 222) tend to result in an intact protein with some residual ROS production and higher rates of overall survival, while those mutations after amino acid 310, which are in the NADPH, FAD, and heme binding domain, tend to be unable to support ROS production.In contrast, nonsense mutations, deletions and some splice mutations result in no protein production and have the lowest residual ROS production and lower overall survival. 12Additionally, liver abscess and liver disease as evidenced by platelet decline of >9000/mm 3 /year and elevation in alkaline phosphatase by >0.25 U/L/year have been independently linked to mortality. 13Given its location on Xp21, large deletions involving CYBB may also encompass other genes: telomeric deletions involving KX lead to McLeod syndrome (Kell antigen deletion), as CYBB and KX are <50 kbp apart.In this deletion, initial red blood cell transfusion can result in formation of anti-Kx and anti-Km antibodies making future transfusions extremely dangerous. 14Duchenne muscular dystrophy (DMD) and X-linked retinitis pigmentosa (RPGR) can also be impacted by large deletions in Xp21.1 as part of a contiguous deletion.6][17] This is why chromosomal microarrays to detect the boundaries of large deletions are important in X-linked CGD.
The second most common CGD mutation in the US is in NCF1, the recessive gene encoding p47 phox and accounting for ~25% of cases.The vast majority of mutations are homozygous for a GT deletion at the start of exon 2. 18 Of ~900 mutations in NCF1 worldwide, about 80% were homozygous for the GT deletion.This common deletion occurs because most individuals carry two pseudogenes, each of which carries the deletion and flanks the functional gene.
Recombination events in which a GT-deleted pseudogene replaces the functional NCF1 result in early truncation and no protein production. 18,19Despite this lack of protein, p47 phox deficient CGD phagocytes typically produce some residual ROS, albeit not normal amounts, and can result in a milder infection susceptibility phenotype.

F I G U R E 1
Assembly of the nicotinamide adenine dinucleotide phosphate (NADPH) complex leads to both the production of HOCl and the influx of potassium as a result of the net negative intraphagosomic charge resulting in activation of proteases.This leads to killing in intracellular organisms to which the patients with CGD are susceptible.
The second subunit of the cytochrome b 558 is AR p22 phox (encoded by CYBA at 16q24) accounts for 7% of the mutations leading to US CGD. 18Because it is required for gp91phox expression and stability, mutations in CYBA are clinically almost indistinguishable from mutations in CYBB, except that CYBA is AR, leading to females with the X-linked phenotype.p67 phox is another AR cytosolic component (encoded by NCF2, at 1q25.3), and the critical cytosolic factor for NADPH activation.The P47 phox is the partner protein crucial for getting p67 phox into the NADPH complex, partly explaining why p67 phox deficiency is usually more severe than p47phox deficiency.
The p40 phox protein (encoded by NCF4 at 22q12.3) is another AR member of the NADPH oxidase complex that serves a more regulatory role.The standard functional test, Dihydrorhodamine-1,2,3 (DHR) oxidation, may be normal to PMA and ionomycin but abnormal to testing with formyl-methionyl-leucyl-phenylalanine (fMLF) or lipopolysaccharide (LPS).This form of CGD should be considered in patients with significant inflammatory bowel disease, even without infections. 20,21This diagnosis should also be considered in patients severe inflammatory bowel disease, and in particular with very early onset inflammatory bowel disease.EROS is encoded by CYBC1 at 17q25.3 and is the critical chaperone for the gp91 phox / p22 phox complex to get out of the endoplasmic reticulum (ER) and into the secondary granule membrane.It is AR and may also present with inflammatory bowel disease. 7,22,23

| Infection
5][26][27] Infections typically occur in infancy or early childhood.However, later diagnoses occur in patients with significant residual superoxide production, which can be missed when the phenotype is less severe.Characteristic sites of infection include lungs, liver, skin, and bone. 24,25The most common pathogens in the US are Staphylococcus aureus, Burkholderia cepacia complex, Serratia marcescens, Nocardia spp., and Aspergillus spp.In other parts of the world Salmonella spp, Mycobacterium tuberculosis and Bacille Calmette-Guerin are also important.9][30][31][32] Both Chromobacterium and Francisella are gram negative organisms that are found in warm brackish water and can cause sepsis in CGD.Chromobacterium and Francisella are both effectively treated with carbapenems.
Granulibacter, a fastidious gram-negative rod, can present as chronic or recurrent necrotizing lymphadenitis and can be particularly difficult to eradicate, and often responds to ceftriaxone. 30,31In the US, there has been one case of fatal sepsis secondary to G. bethesdensis. 33European strains of Granulibacter have been more severe in presentation. 34Granulibacter requires special culture media, such as charcoal yeast extract and can be identified by 16S sequencing. 30,35 some instances, after parenteral therapy with ceftriaxone, extended therapy with cefdinir has been effective as this can be a difficult organism to eradicate.
Staphylococcus aureus is a gram-positive bacteria that tends to cause deep seated infections in CGD including lymph nodes, skin, liver and bone.Liver abscess was previously treated with surgery and antibiotics.This approach was complicated by high rates of relapse and recurrence.The liver abscesses of CGD are not very amenable to percutaneous drainage as the abscess is often fibrocaseous and septated. 36,37The seemingly counterintuitive approach of simultaneous therapy with steroids and antibiotics for staphylococcal liver abscess has resulted in decreased need for surgery lower morbidity and mortality. 36However, diagnosis of the microbiological cause of liver abscess still requires definitive determination, such as percutaneous aspiration. 36fections caused by Burkholderia spp often cause pneumonia, but bacteremia can result in both sepsis and hemophagocytic lymphohistiocytosis. 38 B. gladioli and B. pseudomallei have also been reported in CGD. 39,40pergillus and other mold infections can be fatal in CGD.With the advent of highly effective antifungal prophylaxis and newer azoles, A. fumigatus is largely curable.However, the non-fumigatus Aspergillus species can be intrinsically resistant to amphotericin and newer antifungal therapies and can be invasive, eroding through tissue planes. 41In a review of non-Aspergillus infections in CGD the most frequent offenders were Rhizopus spp.and Trichosporon spp.and overall mortality was 26%. 42Other molds that have been described to cause invasive and often fatal disease in CGD include A. viridinutans, A. nidulans, A. tanneri, Paecilomyces variotii, P. lilacinus, Neosartorya udagawae, and Phellinus spp 41,[43][44][45][46] In these cases, if there is isolated disease then surgical resection may be advisable.
Because of the stark therapeutic divide, it is critical to definitively diagnose invasive fungal disease to the species level as medical therapies are not always sufficient and adjunctive treatments take time to arrange (hematopoietic stem cell transplant, allogeneic granulocyte infusions).
8][49][50][51] The history often includes exposure to decaying matter such as mulching, landscaping, demolition, lawn debris, or animal feed.Patients may present for dyspnea with what is called community acquired pneumonia.Left untreated, patients invariably return in respiratory distress with abnormal x-ray and widespread diffuse nodules on CT.This condition is life threatening.

| INFL AMMATORY COMPLIC ATIONS
Gastrointestinal complications occur in about 50% of patients with X-linked CGD, appearing usually by 7 years, whereas in p47 phox CGD the rate is about 40% but the age of onset is 22 years. 52Signs and symptoms can range from failure to thrive, irritability and diarrhea in the very young, to urgency, frequency, tenesmus, iron deficiency anemia, diarrhea, and hematochezia in adults.Functional gastric, esophageal, and duodenal obstruction have also been described. 53,54Common radiographic findings can include peri-colonic fat stranding, mesenteric adenopathy, and bowel wall thickening. 55D-related IBD does not affect overall mortality, but it does cause significant morbidity. 12,56IBD may be the initial presentation of any of the genotypes of CGD.In particular NCF4 deficiency often presents as IBD only, without infections. 18,20,21Typically, the colon is most affected, with the perirectal region most involved.IBD can progress from distal to proximal affecting the entirety of the colon. 53,57Disease is morphologically similar to Crohn disease (CD) with ulcerations, friability, and erythema of the mucosa with acute colitis, and crypt abscesses. 58,59There is a high rate of perianal disease, with fistula formation and fissures. 59In addition to the colonic disease, video capsule endoscopy in CGD found that 85% had some sort of small bowel involvement, not predicted by radiographic appearance. 60Therefore, comprehensive staging of CGD patients with IBD is indicated.
CGD IBD does not correlate with residual superoxide production. 12,53In addition, within families who all share the same mutation rates of IBD are discordant.A comparison of patients with CGD IBD against those with IBD without CGD found that patients with CGD IBD had many of the same genetic risks as in the general IBD population, but their burden was lower overall, consistent with the idea that ROS deficiency is per se an important contributor to the development of CGD IBD. 61Inflammasome activation and defective autophagy in the setting of elevated IL-1beta has also been implicated in a mouse model of CGD as a cause of CGD IBD. 62Treatment of CGD IBD with IL-1 blockade has shown some small improvements in disease, but has not been sustained in small reports. 62,63The intestinal microbiome regulates immunity and inflammasome activation and may be important in the development of CGD IBD. 64The microbiota of CGD patients with and without IBD show different levels of alpha diversity in CGD patients compared with healthy controls. 64,65Moreover, the beta diversity of the fecal microbiome can distinguish CGD patients from healthy controls. 65The microbiome in symptomatic CGD IBD is distinct from that in CGD without IBD and can shift from one to the other upon institution of an elemental diet. 64,66There are ongoing trials targeting the microbiome in CGD, both through the use of fecal transplant and also using elemental diets.
Medical therapy for CGD IBD is directed at the dysregulated inflammation, ideally without increasing the risk of infection.
Traditionally, the mainstay of therapy has been corticosteroids and lumenal non-steroidal gastrointestinal medications such as mesalamine, or sulfasalazine; for more refractory cases azathioprine is usually added. 53,67Unfortunately, long term steroids can lead to aseptic necrosis and osteopenia. 53Therapies aimed at Crohn IBD have been utilized with varying degrees of success.Despite improvement in fistulizing disease with TNFα blockade in CGD IBD, fatal infections and increased infection susceptibility have been associated with their use. 68,69The humanized monoclonal antibody that targets the integrin α4β7, vedolizumab, has shown modest improvement in CGD IBD.Although it did not show significant sustained improvement, it may have a role as a bridge to more definitive therapy. 70,71Ustekinumab, a monoclonal antibody targeting IL-12/IL-23 through binding of the p40 subunit has been reported to improve CGD IBD but has been associated with some increase in infection susceptibility.However, it can take several months to see histopathological effects and disease may recur if monoclonal therapy is discontinued. 72,73We use a long steroid taper until the effects of ustekinumab can be established.There is still not an optimal immunomodulatory therapy that is both safe and 100% effective for severe CGD IBD.The only definitive therapy for CGD IBD is hematopoietic stem cell transplantation (HSCT). 74,75couragingly, CGD IBD largely resolves with transplant and its presence does not impact transplant outcomes.[76][77] The liver is another part of the GI tract that can be affected by dysregulated inflammation, with complications including non-cirrhotic portal hypertension, nodular regenerative hyperplasia, and hepatosplenomegaly resulting in sequestration and thrombocytopenia. 13,78proximately one quarter of patients with CGD exhibit signs of pulmonary inflammatory complications. 79,80These can be severe enough to result in hypoxemia and pulmonary hypertension.In one European cohort, upwards of 77% of their patients had pulmonary abnormalities on computed tomography scanning, including 43% with bronchiectasis or fibrosis. 81Unpublished data suggest that some of this may resolve with transplantation.Urological manifestations of CGD can include eosinophilic cystitis as well as obstructive uropathy.Both are effectively treated with corticosteroids; however, the episodes can recur and may require low dose steroids over a prolonged period. 54,82orioretinitis, uveitis, and ocular granulomata have also been described in CGD, most of which do not progress.However, there are reports of loss of vision sometimes resulting in enucleation.The etiology of this complication is poorly understood and no clear effective treatments have been described. 79,83,84Patients with CGD should be screened and followed routinely for this inflammatory complication.

| CUR ATIVE THER APY
Much of the therapy for CGD is aimed at prevention and treatment of both infections and inflammatory complications.6][87] Resolution of inflammatory bowel disease and full engraftment with both matched related and unrelated donors have been reported, making transplant a viable option even in the setting of ongoing infection or inflammatory complications.Transplant has also been reported in highly Lyonized carriers, either in the setting of incurable infection or for the same inflammatory complications of the GI tract as in X-linked boys.Gene therapy for X-linked CGD has been performed with success, but has been complicated in some cases by loss of gene expression and in rare cases by development of myelodysplasia or leukemia, as has been the case with some other early efforts at gene therapy. 88However, some of the newer vectors and approaches are promising.Whether gene therapy can fully control the inflammatory complications of CGD remains to be demonstrated.Newer ex-vivo using technologies like CRISPR/Cas9 have shown promise and are on the cusp of trials in humans. 89,90

| MYELOPEROXIDA S E DEFICIEN C Y
After the superoxide free radical is converted by SOD to H 2 O 2 , MPO is the heme-containing enzyme necessary for its conversion to hypochlorous acid (HOCl).MPO is expressed early in myeloid differentiation and resides in the azurophilic (primary) granules of neutrophils and the lysosomes of monocytes.Located at 17q23, this is the most common inherited neutrophil disorder, occurring in approximately 1/2000-4000.It is rarely associated with infection despite its demonstrated role in oxidation of phagocytosed organisms. 913][94] It is important to keep in mind that MPO deficiency is another of the causes of an abnormal DHR assay, which can on occasion be quite misleading.More commonly, MPO has been associated with inflammatory disorders given its role as a mediator of inflammation. 95Recent work in mouse models suggests that MPO is involved in mediating apoptosis and that in its absence inflammation is enhanced, offering a possible overlap between MPO deficiency, in which HOCl is not generated because of an enzyme defect and CGD, in which HOCl is not generated because of a substrate defect. 96

| G LUCOS E-6 -PHOS PHATE DEHYDROG ENA S E DEFICIEN C Y (G6PD)
Glucose-6-phosphate dehydrogenase deficiency (G6PD) belongs in the category of neutrophil disorders, as it is one of the first two reactions of the hexose monophosphate shunt pathway that generates NADPH.G6PD deficiency affects about 400 million persons worldwide with an increased prevalence in Africa, Asia and the Middle East, making it the most common X-linked disorder. 97The amount of NADPH available for use in a number of reactions, including for the NADPH oxidase, is dependent on G6PD, and therefore less or less effective enzyme results in insufficient NADPH and reviewed recently. 11,98G6PD deficiency is most commonly associated with hemolysis of red blood cells.Located at Xq23, G6PD deficiency can range from mild to severe, with 5 different classes of variants.Infections occur in the most severe form of G6PD deficiency and generally phenocopy CGD susceptibility. 99G6PD deficiency TA B L E 1 Genetic testing for clinicians who suspect a neutrophil disorder and the genes affected.should be considered in a patient with severe hemolysis and CGDassociated infections (Table 1).

| LEUKOC Y TE ADHE S ION DEFICIEN C Y
Defects in migration resulting in infection susceptibility and inflammatory complications can be due to one of the three types of leukocyte adhesion deficiency (LAD).These defects can result in variable phenotypes as a result of impaired adhesion to the endothelium or transendothelial migration out of the bloodstream. 100utrophil and monocyte adhesion to the endothelium, other cells and to some pathogens is mediated in part by molecules col- the surface of all leukocytes that is essential for neutrophil recruitment to sites of tissue inflammation, but also for processes such as homotypic adhesion leading to T cell activation. 101Mac-1 or complement receptor (CR) 3 is essential in responding to chemotactic factors, such as the complement fragment C5a, IL-8, leukotriene B4 (LTB4), or the bacterial product fMLF and leukocyte adhesion to the endothelium. 100,101CR4 (also known as p150,95) mediates binding to LPS directly as well as fibrinogen.It is also highly expressed on monocyte-derived dendritic cells. 102CD18/CD11d is an integrin whose expression is more complexly regulated and whose role in human health and disease is less well understood. 102There is a bidirectional interplay between the neutrophil (integrin expression) and the endothelium (ICAM expression) required for appropriate adhesion and extravasation.Neutrophil integrin expression is entirely dependent on the common subunit of all 4 integrins, CD18 encoded by ITGB2 at 21q22. 103Therefore, defects in CD18 expression or function lead to defects in any or all of the CD11 family molecules and therefore defects in adhesion, trafficking and killing.Disease severity in LAD-1 is determined by the amount of CD18 expression and categorized as severe (<2%), moderate (2%-30%), or mild (>30%).Inherited in an AR manner, patients with LFA-1 severe mutations frequently present with delayed umbilical cord separation, poor wound healing, neutrophilia, oral ulcers, and aggressive gingivitis with accelerated apical bone loss. 104,105The severe periodontitis typically results in loss of all secondary teeth by early adulthood.The mechanism of the inflammation associated with LAD1 is somewhat counterintuitive, as the inability of neutrophils to exit the vascular space leads to tissue neutropenia, which is associated with hypoinflammatory lesions, such as in neutropenia.
However, tissue resident macrophages are constantly monitoring for adequate neutrophil levels in the tissue, and when they decline, macrophages produce IL-23 which eventually leads to IL-17 and G-CSF production.Therefore, it is the local production of high levels of IL-17 as a result of macrophage IL-23 that leads to the severe inflammatory complications of LAD1, including severe gingivitis and inflammatory skin lesions. 106This latter observation led to the use of ustekinumab in the treatment of gingivitis and inflammation in LAD1. 107HSCT is curative for LAD1 but results are complicated by high rates of graft failure and graft versus host disease. 108In view of the persisting difficulties of HSCT there has been significant effort for gene therapy for LAD1, which has so far been successful with survival of all recipients past 1 year. 109D-2 is due to defect in the fucosylation of the protein CD15s (sialyl Lewis X , SLe X ) on neutrophils, thus impairing the early lowavidity rolling step of neutrophil adhesion which is mediated by selectins on the endothelium.The molecular defect is in the guanine diphosphate-fucose transporter-1 (FUCT1 or SLC35C1).This defect in glycosylation places LAD2 within the congenital disorders of glycosylation (CDG), making this CDGIIc.Inherited in an AR manner, patients with LAD2 have the Bombay blood group phenotype, which is due to the absence of the H antigen, which is also dependent on fucosylation. 104There are many fucoslylated proteins, all of which are affected, so patients often present with short stature and cognitive deficits.The infection phenotype in LAD2 appears to improve with age and is usually less prominent in adulthood, but the number of cases reports is small.There may be a subset of cases who respond to fucose supplementation, as well. 110Because the integrin, phagocyte killing and T-cell receptor are still functional in this LAD2, the infectious complications tend to be less severe. 105itially thought to be a variant of LAD-1, LAD-III was first described in a Turkish patient with mutations in FERMT3, which encodes KINDLIN3, an adaptor protein expressed in hematopoietic cells that regulates integrin activation through binding to the short intracellular tails of β2 integrins. 111The process of KINDLIN3 binding to the intracellular portion of the integrins is called inside-out signaling, since the KINDLIN3 interaction strengthens integrin binding.3][114] Patients typically have a purpuric ("mulberry") rash at birth and may have early fatal infections.
LAD-III platelets have decreased binding to soluble fibrinogen, and do not respond properly to thrombin via thrombin receptors, resulting in improper platelet granule secretion via integrin activation and hence the purpuric rash.[117]

CO N FLI C T O F I NTE R E S T
No conflict of interests for either authors.

Funding for this review
was provided in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government.
May be difficult given that there are two pseudo genes which may miss the typical GT deletion.
a b If clinical suspicion is high-request DHR with serum-opsonized zymosan or E.coli rather than PMA.However direct sequencing is indicated for high clinical suspicion.cPatient may erroneously be diagnosed with CGD, however direct visualization of neutrophils when stained for peroxidase is more specific.dAbnormal flow cytometry for CD11a, CD11b, CD11c, and CD18 in combination with clinical suspicion fitting the phenotype.