Douglas B. Kuhns

Common Severe Infections in Chronic Granulomatous Disease

BACKGROUNDnChronic granulomatous disease (CGD) is due to defective nicotinamide adenine dinucleotide phosphate oxidase activity and characterized by recurrent infections with a limited spectrum of bacteria and fungi as well as inflammatory complications. To understand the impact of common severe infections in CGD, we examined the records of 268 patients followed at a single center over 4 decades.nnnMETHODSnAll patients had confirmed diagnoses of CGD, and genotype was determined where possible. Medical records were excerpted into a standard format. Microbiologic analyses were restricted to Staphylococcus, Burkholderia, Serratia, Nocardia, and Aspergillus.nnnRESULTSnAspergillus incidence was estimated at 2.6 cases per 100 patient-years; Burkholderia, 1.06 per 100 patient-years; Nocardia, 0.81 per 100 patient-years; Serratia, 0.98 per 100 patient-years, and severe Staphylococcus infection, 1.44 per 100 patient-years. Lung infection occurred in 87% of patients, whereas liver abscess occurred in 32%. Aspergillus incidence was 55% in the lower superoxide-producing quartiles (quartiles 1 and 2) but only 41% in the higher quartiles (rate ratio, <0.0001). Aspergillus and Serratia were somewhat more common in lower superoxide producing gp91phox deficiency. The median age at death has increased from 15.53 years before 1990 to 28.12 years in the last decade. Fungal infection carried a higher risk of mortality than bacterial infection and was the most common cause of death (55%). Gastrointestinal complications were not associated with either infection or mortality.nnnCONCLUSIONSnFungal infections remain a major determinant of survival in CGD. X-linked patients generally had more severe disease, and this was generally in those with lower residual superoxide production. Survival in CGD has increased over the years, but infections are still major causes of morbidity and mortality.

Chromothriptic Cure of WHIM Syndrome

Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patients cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

A phase 1 clinical trial of long-term, low-dose treatment of WHIM syndrome with the CXCR4 antagonist plerixafor

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder caused by gain-of-function mutations in the G protein-coupled chemokine receptor CXCR4. The CXCR4 antagonist plerixafor, which is approved by the US Food and Drug Administration (FDA) for stem cell mobilization in cancer and administered for that indication at 0.24 mg/kg, has been shown in short-term (1- to 2-week) phase 1 dose-escalation studies to correct neutropenia and other cytopenias in WHIM syndrome. However, long-term safety and long-term hematologic and clinical efficacy data are lacking. Here we report results from the first long-term clinical trial of plerixafor in any disease, in which 3 adults with WHIM syndrome self-injected 0.01 to 0.02 mg/kg (4% to 8% of the FDA-approved dose) subcutaneously twice daily for 6 months. Circulating leukocytes were durably increased throughout the trial in all patients, and this was associated with fewer infections and improvement in warts in combination with imiquimod; however, immunoglobulin levels and specific vaccine responses were not fully restored. No drug-associated side effects were observed. These results provide preliminary evidence for the safety and clinical efficacy of long-term, low-dose plerixafor in WHIM syndrome and support its continued study as mechanism-based therapy in this disease. The ClinicalTrials.gov identifier for this study is NCT00967785.

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 -/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 -/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.

Single amino acid charge switch defines clinically distinct proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1)–associated inflammatory diseases

BACKGROUNDnHyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin).nnnOBJECTIVEnWe sought to characterize the genetic cause and clinical spectrum of Hz/Hc.nnnMETHODSnProline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA.nnnRESULTSnThirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 μg/mL) compared with those with PAPA syndrome (116 ± 74 μg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1.nnnCONCLUSIONnMutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.

X-linked carriers of chronic granulomatous disease: Illness, lyonization, and stability

Background: Chronic granulomatous disease (CGD) is characterized by recurrent life‐threatening bacterial and fungal infections and aberrant inflammation. Mutations in CYBB cause X‐linked CGD and account for 65% to 70% of cases in Western countries. Objective: We sought to understand the clinical manifestations associated with the X‐linked CGD carrier state. Methods: We undertook a comprehensive retrospective study of 162 affected female subjects. We examined dihydrorhodamine 123 (DHR) oxidation data for percentage of X‐chromosome inactivation. We correlated lyonization (%DHR+) with clinical features. Where possible, we followed %DHR+ values over time. Results: Clinical data were available for 93 female subjects: %DHR+ values were 46% (mean) and 47% (median; SD, 24). Using the %DHR+ value as the criterion for X inactivation, 78% of patients had levels of inactivation of 20% to 80%, suggesting random inactivation that was independent of age. In contrast, carriers with CGD‐type infections had median %DHR+ values of 8% (n = 14; range, 0.06% to 48%), and those with only autoimmune or inflammatory manifestations had median %DHR+ values of 39% (n = 31; range, 7.4% to 74%). Those with both infections and autoimmunity had low %DHR+ values (n = 6; range, 3% to 14%). A %DHR+ value of less than 10% was strongly associated with infections (odds ratio, 99). Strong association persisted when %DHR+ values were less than 20% (odds ratio, 12). Autoimmunity was not associated with %DHR+ values. In 2 sets of identical twins, the %DHR+ populations tracked closely over time. Although the %DHR+ populations were very similar between sisters, those between mothers and daughters were unrelated. Conclusions: A low %DHR+ value strongly predicts infection risk in X‐linked CGD carriers, and the carrier state itself is associated with autoimmunity.

Colitis susceptibility in p47(phox-/-) mice is mediated by the microbiome.

BackgroundChronic granulomatous disease (CGD) is caused by defects in nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) complex subunits (gp91phox (a.k.a. Nox2), p47phox, p67phox, p22phox, p40phox) leading to reduced phagocyte-derived reactive oxygen species production. Almost half of patients with CGD develop inflammatory bowel disease, and the involvement of the intestinal microbiome in relation to this predisposing immunodeficiency has not been explored.ResultsAlthough CGD mice do not spontaneously develop colitis, we demonstrate that p47phox−/− mice have increased susceptibility to dextran sodium sulfate colitis in association with a distinct colonic transcript and microbiome signature. Neither restoring NOX2 reactive oxygen species production nor normalizing the microbiome using cohoused adult p47phox−/− with B6Tac (wild type) mice reversed this phenotype. However, breeding p47phox+/− mice and standardizing the microflora between littermate p47phox−/− and B6Tac mice from birth significantly reduced dextran sodium sulfate colitis susceptibility in p47phox−/− mice. We found similarly decreased colitis susceptibility in littermate p47phox−/− and B6Tacxa0mice treated with Citrobacter rodentium.ConclusionsOur findings suggest that the microbiome signature established at birth may play a bigger role than phagocyte-derived reactive oxygen species in mediating colitis susceptibility in CGD mice. These data further support bacteria-related disease in CGD colitis.

Isolation and Functional Analysis of Human Neutrophils.

This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll‐Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described.

Genetic Risk for Inflammatory Bowel Disease Is a Determinant of Crohn's Disease Development in Chronic Granulomatous Disease.

Background:Approximately, one-third to one-half of children with chronic granulomatous disease (CGD) develop gastrointestinal inflammation characteristic of idiopathic inflammatory bowel disease (IBD), usually Crohns disease. We hypothesized that the overall IBD genetic risk, determined by IBD genetic risk score (GRS), might in part determine IBD development in CGD. Methods:We reviewed medical records to establish IBD diagnoses in CGD subjects seen at NIAID. IBD risk single nucleotide polymorphism genotypes were determined using the Immunochip, and GRS were estimated by Mangrove. Results:Among 157 white patients with CGD, 55 were confirmed, 78 excluded, and 24 were uncertain for IBD. Two hundred one established, independent European IBD risk single nucleotide polymorphisms passed quality control. After sample quality control and removing non-IBD CGD patients with perianal disease, mean GRS for 40 unrelated patients with CGD-IBD was higher than 53 CGD non-IBD patients (in log2-scale 0.08 ± 1.62 versus −0.67 ± 1.64, P = 0.026) but lower than 239 IBD Genetics Consortium (IBDGC) young-onset Crohns disease cases (0.76 ± 1.60, P = 0.025). GRS for non-IBD CGD was similar to 609 IBDGC controls (−0.69 ± 1.60, P = 0.95). Seven established IBD single nucleotide polymorphisms were nominally significant among CGD-IBD versus CGD non-IBD, including those near LACC1 (P = 0.005), CXCL14 (P = 0.007), and TNFSF15 (P = 0.016). Conclusions:The weight of the common IBD risk alleles are significant determinants of IBD in CGD. However, IBD risk gene burden among CGD children with IBD is significantly lower than that in nonsyndromic pediatric Crohns disease, congruent with the concept that defective superoxide production in CGD is also a major IBD risk factor. Individual IBD genes might interact with the CGD defect to cause IBD in CGD.

Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) Txa0cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR Txa0cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR Txa0cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.