Correction: Insights into the genetic epidemiology of Crohn's and rare diseases in the Ashkenazi Jewish population.

[This corrects the article DOI: 10.1371/journal.pgen.1007329.].

Insights into the genetic epidemiology of Crohn's and rare diseases in the Ashkenazi Jewish population.

As part of a broader collaborative network of exome sequencing studies, we developed a jointly called data set of 5,685 Ashkenazi Jewish exomes. We make publicly available a resource of site and allele frequencies, which should serve as a reference for medical genetics in the Ashkenazim (hosted in part at https://ibd.broadinstitute.org, also available in gnomAD at http://gnomad.broadinstitute.org). We estimate that 34% of protein-coding alleles present in the Ashkenazi Jewish population at frequencies greater than 0.2% are significantly more frequent (mean 15-fold) than their maximum frequency observed in other reference populations. Arising via a well-described founder effect approximately 30 generations ago, this catalog of enriched alleles can contribute to differences in genetic risk and overall prevalence of diseases between populations. As validation we document 148 AJ enriched protein-altering alleles that overlap with "pathogenic" ClinVar alleles (table available at https://github.com/macarthur-lab/clinvar/blob/master/output/clinvar.tsv), including those that account for 10-100 fold differences in prevalence between AJ and non-AJ populations of some rare diseases, especially recessive conditions, including Gaucher disease (GBA, p.Asn409Ser, 8-fold enrichment); Canavan disease (ASPA, p.Glu285Ala, 12-fold enrichment); and Tay-Sachs disease (HEXA, c.1421+1G>C, 27-fold enrichment; p.Tyr427IlefsTer5, 12-fold enrichment). We next sought to use this catalog, of well-established relevance to Mendelian disease, to explore Crohn's disease, a common disease with an estimated two to four-fold excess prevalence in AJ. We specifically attempt to evaluate whether strong acting rare alleles, particularly protein-truncating or otherwise large effect-size alleles, enriched by the same founder-effect, contribute excess genetic risk to Crohn's disease in AJ, and find that ten rare genetic risk factors in NOD2 and LRRK2 are enriched in AJ (p < 0.005), including several novel contributing alleles, show evidence of association to CD. Independently, we find that genomewide common variant risk defined by GWAS shows a strong difference between AJ and non-AJ European control population samples (0.97 s.d. higher, p<10-16). Taken together, the results suggest coordinated selection in AJ population for higher CD risk alleles in general. The results and approach illustrate the value of exome sequencing data in case-control studies along with reference data sets like ExAC (sites VCF available via FTP at ftp.broadinstitute.org/pub/ExAC_release/release0.3/) to pinpoint genetic variation that contributes to variable disease predisposition across populations.

The role of neutrophils in the pathogenesis of Crohn's disease.

Crohn's disease (CD) is caused by a trigger, almost certainly enteric infection by one of a multitude of organisms that allows faeces access to the tissues, at which stage the response of individuals predisposed to CD is abnormal. In CD the failure of acute inflammation results in the failure to recruit neutrophils to the inflammatory site, as a consequence of which the clearance of bacteria from the tissues is defective. The retained faecal products result in the characteristic chronic granulomatous inflammation and adaptive immune response. Impaired of digestion of bacteria and fungi by CGD neutrophils can result in a similar pathological and clinical picture. The neutrophils in CD are normal and their inadequate accumulation at sites of inflammation generally results from diminished secretion of proinflammatory cytokines by macrophages consequent upon disordered vesicle trafficking.

A New Look at Familial Risk of Inflammatory Bowel Disease in the Ashkenazi Jewish Population.

BACKGROUND AND AIMS: The inflammatory bowel diseases (IBD) are particularly common among the Ashkenazi Jewish (AJ) population. Population-specific estimates of familial risk are important for counseling; however, relatively small cohorts of AJ IBD patients have been analyzed for familial risk to date. This study aimed to recruit a new cohort of AJ IBD patients, mainly from the UK, to determine the familial occurrence of disease. METHODS: A total of 864 AJ IBD patients were recruited through advertisements, hospital clinics, and primary care. Participants were interviewed about their Jewish ancestry, disease phenotype, age of diagnosis, and family history of disease. Case notes were reviewed. RESULTS: The 864 probands comprised 506 sporadic and 358 familial cases, the latter with a total of 625 affected relatives. Of the UK cases, 40% had a positive family history with 25% having at least one affected first-degree relative. These percentages were lower among those recruited through hospital clinics and primary care (33% for all relatives and 22% among first-degree relatives). Examining all probands, the relative risk of IBD for offspring, siblings, and parents was 10.5, 7.4, and 4, respectively. Age of diagnosis was significantly lower in familial versus sporadic patients with Crohn's disease. CONCLUSIONS: This study reports familial risk estimates for a significant proportion of the AJ IBD population in the UK. The high rate of a positive family history in this cohort may reflect the greater genetic burden for IBD among AJs. These data are of value in predicting the likelihood of future recurrence of IBD in AJ families.

Genetic Complexity of Crohn's Disease in Two Large Ashkenazi Jewish Families.

BACKGROUND & AIMS: Crohn's disease (CD) is a highly heritable disease that is particularly common in the Ashkenazi Jewish population. We studied 2 large Ashkenazi Jewish families with a high prevalence of CD in an attempt to identify novel genetic risk variants. METHODS: Ashkenazi Jewish patients with CD and a positive family history were recruited from the University College London Hospital. We used genome-wide, single-nucleotide polymorphism data to assess the burden of common CD-associated risk variants and for linkage analysis. Exome sequencing was performed and rare variants that were predicted to be deleterious and were observed at a high frequency in cases were prioritized. We undertook within-family association analysis after imputation and assessed candidate variants for evidence of association with CD in an independent cohort of Ashkenazi Jewish individuals. We examined the effects of a variant in DUOX2 on hydrogen peroxide production in HEK293 cells. RESULTS: We identified 2 families (1 with >800 members and 1 with >200 members) containing 54 and 26 cases of CD or colitis, respectively. Both families had a significant enrichment of previously described common CD-associated risk variants. No genome-wide significant linkage was observed. Exome sequencing identified candidate variants, including a missense mutation in DUOX2 that impaired its function and a frameshift mutation in CSF2RB that was associated with CD in an independent cohort of Ashkenazi Jewish individuals. CONCLUSIONS: In a study of 2 large Ashkenazi Jewish with multiple cases of CD, we found the genetic basis of the disease to be complex, with a role for common and rare genetic variants. We identified a frameshift mutation in CSF2RB that was replicated in an independent cohort. These findings show the value of family studies and the importance of the innate immune system in the pathogenesis of CD.

Combinatorial Conflicting Homozygosity (CCH) analysis enables the rapid identification of shared genomic regions in the presence of multiple phenocopies.

BACKGROUND: The ability to identify regions of the genome inherited with a dominant trait in one or more families has become increasingly valuable with the wide availability of high throughput sequencing technology. While a number of methods exist for mapping of homozygous variants segregating with recessive traits in consanguineous families, dominant conditions are conventionally analysed by linkage analysis, which requires computationally demanding haplotype reconstruction from marker genotypes and, even using advanced parallel approximation implementations, can take substantial time, particularly for large pedigrees. In addition, linkage analysis lacks sensitivity in the presence of phenocopies (individuals sharing the trait but not the genetic variant responsible). Combinatorial Conflicting Homozygosity (CCH) analysis uses high density biallelic single nucleotide polymorphism (SNP) marker genotypes to identify genetic loci within which consecutive markers are not homozygous for different alleles. This allows inference of identical by descent (IBD) inheritance of a haplotype among a set or subsets of related or unrelated individuals. RESULTS: A single genome-wide conflicting homozygosity analysis takes <3 seconds and parallelisation permits multiple combinations of subsets of individuals to be analysed quickly. Analysis of unrelated individuals demonstrated that in the absence of IBD inheritance, runs of no CH exceeding 4 cM are not observed. At this threshold, CCH is >97% sensitive and specific for IBD regions within a pedigree exceeding this length and was able to identify the locus responsible for a dominantly inherited kidney disease in a Turkish Cypriot family in which six out 17 affected individuals were phenocopies. It also revealed shared ancestry at the disease-linked locus among affected individuals from two different Cypriot populations. CONCLUSIONS: CCH does not require computationally demanding haplotype reconstruction and can detect regions of shared inheritance of a haplotype among subsets of related or unrelated individuals directly from SNP genotype data. In contrast to parametric linkage allowing for phenocopies, CCH directly provides the exact number and identity of individuals sharing each locus. CCH can also identify regions of shared ancestry among ostensibly unrelated individuals who share a trait. CCH is implemented in Python and is freely available (as source code) from http://sourceforge.net/projects/cchsnp/ .

Disruption of macrophage pro-inflammatory cytokine release in Crohn's disease is associated with reduced optineurin expression in a subset of patients.

Crohn's disease (CD) is a complex and highly heterogeneous chronic inflammatory disorder, primarily affecting the gastrointestinal tract. Genetic and functional studies have highlighted a key role for innate immunity in its pathogenesis. Profound systemic defects in innate immunity and acute inflammation are understood to result in markedly delayed clearance of bacteria from the tissues, leading to local chronic granulomatous inflammation and compensatory adaptive immunological changes. Macrophages, key orchestrators of acute inflammation, are likely to play an important role in the initial impaired innate immune response. Monocyte-derived macrophages from CD patients stimulated with Escherichia coli were shown to release attenuated levels of tumour necrosis factor and interferon-γ with normal secretion of interleukin-8 (IL-8), IL-10 and IL-6. In controls, the secretion of these cytokines was strongly positively correlated, which was not seen with CD macrophages. The transcriptomes of CD and control macrophages were examined in an attempt to understand the molecular basis of this defect. There were no differentially expressed genes identified between the two groups, consistent with genetic heterogeneity; however, a number of molecules were found to be under-expressed in subgroups of CD patients. The most common of these was optineurin (OPTN) which was under-expressed in approximately 10% of the CD patients. Reduced OPTN expression coincided with lower intracellular protein levels and diminished cytokine secretion after bacterial stimulation both in the patients and with small interfering RNA knockdown in THP-1 cells. Identifying and studying subgroups of patients with shared defective gene expression could aid our understanding of the mechanisms underlying highly heterogeneous diseases such as CD.

What is wrong with granulocytes in inflammatory bowel diseases?

The neutrophil plays a central role in the acute inflammatory response, a crucial mechanism required for the efficient clearance of invading microorganisms and antigenic material. Patients with primary immunodeficiencies of neutrophil function, particularly chronic granulomatous disease, are predisposed to develop bowel inflammation that is indistinguishable from Crohn's disease (CD) on the basis of clinical, endoscopic and histopathological features. The intrinsic function of the neutrophil is normal in the vast majority of patients with CD; however, there is clear evidence of an impairment of neutrophil recruitment to sites of trauma and bacterial infection. This is associated with an inability to adequately clear bacteria that have penetrated the tissues, resulting in the formation of granulomata, the histological hallmark of the disease, and the subsequent initiation of a chronic adaptive immune response. The reduced secretion of proinflammatory cytokines by macrophages, most notably TNF-α, may account for the attenuated neutrophil recruitment observed in CD. Stimulation of the innate immune system in CD, particularly in patients in remission, may be an alternative therapeutic strategy that could reduce the risk of future disease relapses.

Identifying high-impact variants and genes in exomes of Ashkenazi Jewish inflammatory bowel disease patients.

Inflammatory bowel disease (IBD) is a group of chronic digestive tract inflammatory conditions whose genetic etiology is still poorly understood. The incidence of IBD is particularly high among Ashkenazi Jews. Here, we identify 8 novel and plausible IBD-causing genes from the exomes of 4453 genetically identified Ashkenazi Jewish IBD cases (1734) and controls (2719). Various biological pathway analyses are performed, along with bulk and single-cell RNA sequencing, to demonstrate the likely physiological relatedness of the novel genes to IBD. Importantly, we demonstrate that the rare and high impact genetic architecture of Ashkenazi Jewish adult IBD displays significant overlap with very early onset-IBD genetics. Moreover, by performing biobank phenome-wide analyses, we find that IBD genes have pleiotropic effects that involve other immune responses. Finally, we show that polygenic risk score analyses based on genome-wide high impact variants have high power to predict IBD susceptibility.

Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

BACKGROUND: The molecular cause of inflammatory bowel disease is largely unknown. METHODS: We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient. RESULTS: In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful. CONCLUSIONS: Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.

G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction.

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.

The neutrophil respiratory burst and bacterial digestion in Crohn's disease.

BACKGROUND: Neutrophils are a key part of the innate immune defence against microbes, using the respiratory burst (RB) to optimise killing and digestion. Previous studies of the neutrophil RB in Crohn's disease (CD) have yielded conflicting results. METHODS: Superoxide production in response to phorbol-myristyl acetate (PMA) was measured in neutrophils from 100 patients with CD compared to 50 healthy controls (HCs) and 50 patients with ulcerative colitis (UC). A further 22 CD and 10 HCs were studied using f-Met-Leu-Phe (fMLP), and digestion of E. coli by neutrophils was also evaluated. RESULTS: The mean ± SEM PMA-stimulated RB (nmol O(2)/10(6) cells/min) was 10.86 ± 0.26 in HCs, 9.76 ± 0.23 in CD (P=0.02) and 10.04 ± 0.28 in UC (P=0.09 vs HC and 0.47 vs CD). No significant effect of age, gender or medication was observed. The RB in three patients with presumed CD was found to be in the range expected in patients with inherited neutrophil disorders. Stimulation with fMLP was calcium dependent and attenuated in patients on 5-ASA. Digestion of E. coli by neutrophils was not different in HC vs CD (21.6 vs 20.53%, P=0.60). CONCLUSION: The significant reduction in neutrophil RB in CD does not appear to result in defective bacterial digestion and is therefore unlikely play a major role in pathogenesis. Three patients in this cohort of patients with presumed idiopathic CD were found to have a profound defect of the neutrophil RB. A high index of suspicion for such patients is prudent, as their prognosis can be improved by altering or augmenting the conventional treatment regimens employed for CD.

Genetic analysis of four consanguineous multiplex families with inflammatory bowel disease.

BACKGROUND: Family studies support a genetic predisposition to inflammatory bowel diseases (IBD), but known genetic variants only partially explain the disease heritability. Families with multiple affected individuals potentially harbour rare and high-impact causal variants. Long regions of homozygosity due to recent inbreeding may increase the risk of individuals bearing homozygous loss-of-function variants. This study aimed to identify rare and homozygous genetic variants contributing to IBD. METHODS: Four families with known consanguinity and multiple cases of IBD were recruited. In a family-specific analysis, we utilised homozygosity mapping complemented by whole-exome sequencing. RESULTS: We detected a single region of homozygosity shared by Crohn's disease cases from a family of Druze ancestry, spanning 2.6 Mb containing the NOD2 gene. Whole-exome sequencing did not identify any potentially damaging variants within the region, suggesting that non-coding variation may be involved. In addition, affected individuals in the families harboured several rare and potentially damaging homozygous variants in genes with a role in autophagy and innate immunity including LRRK1, WHAMM, DENND3, and C5. CONCLUSION: This study examined the potential contribution of rare, high-impact homozygous variants in consanguineous families with IBD. While the analysis was not designed to achieve statistical significance, our findings highlight genes or loci that warrant further research. Non-coding variants affecting NOD2 may be of importance in Druze patients with Crohn's disease.

CO binding and ligand discrimination in human myeloperoxidase.

Despite the fact that ferrous myeloperoxidase (MPO) can bind both O(2) and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous-CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, k(on) and k(off) were 0.14 mM(-1) x s(-1) and 0.23 s(-1), respectively, yielding an unusually high K(D) of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O(2). The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pK(a) of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm(-1). Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pK(a) approximately 7.4) with the 1927 cm(-1) form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state.

The large-conductance Ca2+-activated K+ channel is essential for innate immunity.

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.

Subproteome analysis of the neutrophil cytoskeleton.

Neutrophils play a key role in the early host-defense mechanisms due to their capacity to migrate into inflamed tissues and phagocytose microorganisms. The cytoskeleton has an essential role in these neutrophil functions, however, its composition is still poorly understood. We separately analyzed different cytoskeletal compartments: cytosolic skeleton, phagosome membrane skeleton, and plasma membrane skeleton. Using a proteomic approach, 138 nonredundant proteins were identified. Proteins not previously known to associate with the skeleton were: n-acetylglucosamine kinase, phosphoglycerate mutase 1, prohibitin, ficolin-1, phosphogluconate dehydrogenase, glucosidase, transketolase, major vault protein, valosin-containing protein, aldehyde dehydrogenase, and lung cancer-related protein-8 (LCRP8). The majority of these proteins can be classified as energy metabolism enzymes. Such a finding was interesting because neutrophil energy metabolism is unusual, mainly relying on glycolysis. The enrichment of phosphoglycerate mutase in cytosolic skeleton was additionally indicated by the use of Western blotting. This is the broadest subcellular investigation to date of the neutrophil cytoskeletal proteome and the first proteomic analysis in any cell type of the phagosome skeleton. The association of metabolic enzymes with cytoskeleton is suggestive of the importance of their localized enrichment and macromolecular organization in neutrophils.

Impaired macrophage function following bacterial stimulation in chronic granulomatous disease.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical for phagocyte anti-microbial activity and plays a major role in innate immunity. Defects in genes coding for components of the NADPH oxidase enzyme system are responsible for chronic granulomatous disease (CGD), a rare primary neutrophil immunodeficiency associated with recurrent, life-threatening bacterial and fungal infections. Microbial killing and digestion within the neutrophil phagosomal compartment are defective in these patients. NADPH oxidase activity is also crucial for optimal macrophage and dendritic cell function and has recently been implicated in both cross-presentation and T-cell priming. We present evidence of impaired macrophage function in CGD, with attenuated pro-inflammatory cytokine and increased interleukin-10 secretion following bacterial stimulation. These results highlight additional abnormalities in macrophage function associated with CGD and the importance of NADPH oxidase activity in immunity.

Inflammatory bowel disease in CGD reproduces the clinicopathological features of Crohn's disease.

OBJECTIVES: Patients with chronic granulomatous disease (CGD), a rare congenital disorder characterized by defective neutrophil function, frequently develop an inflammatory bowel disease similar to Crohn's disease. The clinical presentations and concordance between the features of the bowel disease in these two conditions have never been formally evaluated. METHODS: Retrospective case note analysis of all adult patients with CGD treated at a tertiary care hospital. RESULTS: A total of 25 eligible patients were identified. Of these, 14 (56%) had experienced gastrointestinal symptoms in the preceding 3 years; and 11 (44%) had documented gastrointestinal inflammation not secondary to infection, manifesting throughout the alimentary canal including the upper gastrointestinal tract (45%), small intestine (27%), colon (73%), and rectum (73%). All had discontinuous inflammation and perianal involvement, and approximately half (55%) demonstrated epithelioid granulomata on histology. All patients fulfilled the Lennard-Jones criteria for the diagnosis of Crohn's disease. Therapeutic responses were observed in five patients to 5-aminosalicylates, and in individual patients to thalidomide, interferon-gamma, azathioprine, infliximab, and intestinal resection. CONCLUSIONS: There are striking clinical and pathological resemblances between the bowel diseases observed in CGD and Crohn's disease, supporting the possibility of mechanistic similarities in their pathogenesis. Patients with CGD appear particularly prone to developing perianal disease.

Variations in the Phagosomal Environment of Human Neutrophils and Mononuclear Phagocyte Subsets.

The phagosome microenvironment maintains enzyme activity and function. Here we compared the phagosomal pH of human neutrophils, monocytes, dendritic cells (DC), and monocyte-derived cells. An unexpected observation was the striking difference in phagosomal environment between the three monocytes subsets. Classical monocytes and neutrophils exhibited alkaline phagosomes, yet non-classical monocytes had more acidic phagosomes, while intermediate monocytes had a phenotype in-between. We next investigated the differences between primary naïve DC vs. in vitro monocyte-derived DC (MoDC) and established that both these cells had acidic phagosomal environments. Across all phagocytes, alkalinization was dependent upon the activity of the NADPH oxidase activity, demonstrated by the absence of NADPH oxidase from a patient with chronic granulomatous disease (CGD) or the use of a pharmacological inhibitor, diphenylene iodonium (DPI). Interestingly, MoDC stimulated with bacterial lipopolysaccharide had increased phagosomal pH. Overall, the increase in alkalinity within the phagosome was associated with increased oxidase activity. These data highlight the heterogeneous nature and potential function of phagocytic vacuoles within the family of mononuclear phagocytes.