Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease

Abstract

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This 'experiment of nature' indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

Figure 1

CYBB mutations encoding Q231P and T178 substitutions in XR-MSMD-2. (a) Pedigrees of the families with XR-MSMD-2, including only those selected for the X-chromosome scan. I–V (left margin) indicate generations; black symbols indicate patients with BCG disease (P2–P7); gray symbols indicate patients with tuberculosis (P1 and H1); arrows indicate probands; black dots indicate heterozygous female subjects (n = 15); vertical bars indicate the two founders who must have carried the CYBB mutation but did not show any mycobacterial phenotype; ‘E?’ indicates those whose genetic status could not be evaluated; white indicates all other family members (wild-type CYBB). (b) Automated sequencing profile showing the CYBB mutations encoding the Q231P and T178P substitutions in cDNA extracted from EBV-B cells from patients (P) and a control (C+). The mutations were confirmed in genomic DNA and cDNA for seven patients. (c) Topology model of gp91^phox regions corresponding to the extracellular (EC), transmembrane (TM) and intracellular (IC) regions. Q231P is in the third extracellular loop and T178P is in the transmembrane region (red dots).

Figure 2

NADPH oxidase activity in PMNs and monocytes. (a) O₂⁻ generation in PMNs and monocytes from healthy controls (C+; n = 5 (PMNs) and n = 6 (monocytes)), a patient with CGD (CGD; n = 1) and patients with the CYBB mutations (P-Q231P (n = 4) and P-T178P (n = 1)), assessed after the addition of PMA (40 ng/ml) with or without catalase (ten conditions: six for PMNs and four for monocytes), measured by an assay for reduction of cytochrome c that can be inhibited by superoxide dismutase. Each symbol represents an individual subject. P = 0.01 (not significant), healthy controls versus patients with the Q231P substitution (nonparametric Wilcoxon exact test, accounting for multiple testing). (b) Fluorometric quantification of H₂O₂ release from PMNs and monocytes from healthy controls (n = 11 (PMNs) and n = 12 (monocytes)) and in patients with CGD or the CYBB mutations, assessed with the fluorogenic substrate ADHP (N-acetyl-3,7–dihydroxyphenoxazine) after the addition of PMA. (c) Flow cytometry of intracellular H₂O₂ production (dihydrorhodamine 123 (DHR) assay) in PMNs and monocytes from healthy controls, a patient with X-linked CGD and a patient with the Q231P substitution, assessed before (dotted lines) and after (solid lines) stimulation with PMA (400 ng/ml). (d) Flow cytometry (dihydrorhodamine 123 assay) of PMNs from healthy controls, patients with CGD, a patient with the Q231P substitution and a female heterozygous for the allele encoding Q231P (H), left untreated (NS) or treated with various combinations (above plots) of tumor necrosis factor (TNF), IL-1β, cytochalasin b (Cyt b) and formyl-Met-Leu-Phe (fMLF). (e) Killing of S. aureus by granulocytes from healthy controls (n = 4; 1–4 in parentheses), patients with CGD (n = 3; 1–3 in parentheses) and a patient from kindred A (Q231P; n = 1) and one from kindred B (T178P; n = 1). CFU, colony-forming units. Data are representative of three experiments (a,b; mean of duplicates in b), two experiments (c,e) or two independent experiments (d).

Figure 3

NADPH oxidase activity in MDMs. Release of H₂O₂ from MDMs (MΦ) obtained from healthy controls (n = 4), a patient with X-linked CGD (n = 1), patients with the CYBB mutations (Q231P (n = 1) and T178P (n = 1)), and females heterozygous for the CYBB mutations (n = 4), then left untreated (NS) or treated with M-CSF and IL-4 and then activated by incubation for 18 h with live BCG (10:1 (BCG:MDM); left), PPD (1 mg/ml; right) or IFN-γ (1 × 10⁵ IU/ml; bottom), followed by no trigger or by treatment with PMA (400 ng/ml) to trigger H₂O₂ release (+PMA). Each symbol represents an individual subject. Data are representative of two independent experiments.

Figure 4

NADPH oxidase activity in EBV-B cells. (a) O₂⁻ production (cytochrome c reduction) by EBV-B cells (1 × 10⁶) obtained from healthy controls (n = 22), patients with XR-CGD (n = 4) and patients with the CYBB mutations (Q231P (n = 4) and T178P (n = 3)), then activated by incubation for 2 h with PMA (400 ng/ml). Each symbol represents an individual subject. (b) H₂O₂ release by EBV-B cells obtained from a healthy control, patients P1–P7 and a patient with XR-CGD, activated with various doses of PMA (key) at four time points (horizontal axis). (c) NBT reduction by EBV-B cells from a healthy control, patients P1–P6 and a patient with X-linked CGD, assessed before (NS) and after (PMA) activation with PMA (400 ng/ml). Original magnification, ×100. (d) NADPH oxidase function in EBV-B cells from a patient with XR-CGD transduced with retroviral particles encoding wild-type gp91^phox (WT), T178P gp91^phox and Q231P gp91^phox, and EBV-B cells obtained from patients with the T178P substitution and a healthy control transduced with retroviral particles encoding wild-type gp91^phox, assessed as H₂O₂ release after 2 h of PMA activation (400 ng/ml). Data are representative of two independent experiments (a,d) or three experiments (b,c).

Figure 5

Expression of gp91^phox and flavocytochrome b₅₅₈. (a) Immunoblot analysis of PMNs and monocytes from a healthy control, a patient with no gp91^phox protein (phox0) and patients with the CYBB mutations, probed with three antibodies to gp91^phox (53 BD, 54.1 and 7A2) and an antibody to GAPDH (glyceraldehyde phosphate dehydrogenase; loading control). Right margin, molecular size in kDa. (b) Immunoblot analysis of PMNs, monocytes and MDMs (treated with M-CSF alone or M-CSF plus IL-4) from a healthy control, a patient with CGD and no gp91^phox0 (CGD-phox0) and patients with the CYBB mutations, probed with mAb 54.1 to gp91^phox and an antibody to the transcription factor STAT1 (loading control). (c) Immunoblot analysis of lysates of EBV-B cells from a healthy control, a patient with XR-CGD and patients with the CYBB mutations (left), EBV-B cells from the same patient with XR-CGD transduced with wild-type gp91^phox, T178P gp91^phox or Q231P gp91^phox retroviral particles (middle), or EBV-B cells as described for the far left blot, left untransduced (left) or transduced (right; except XR-CGD) with wild-type gp91^phox retroviral particles (right); blots were probed with mAb 54.1 to gp91^phox and an antibody to p22^phox; β-actin serves as a loading control. (d,e) Flow cytometry of PMNs and monocytes (d) and EBV-B cells (e) from healthy controls (d,e), patients with no gp91^phox0 (gp91 −/y; d,e), patients with the CYBB mutations (d) and patients P1–P7 (e), stained on the surface with mAb 7D5 to gp91^phox and isotype-matched control antibody (immunoglobulin G1 (IgG1)). Data are representative of two independent experiments (a,d,e), two experiments (b) or three experiments (c).

Figure 6

Expression and function of mutant gp91^phox in cell lines. (a) Immunoblot analysis of CHO cells and CHO22 cells (CHO cells generated for the stable expression of untagged p22^phox) transduced with retroviral particles encoding wild-type gp91^phox, T178P gp91^phox or Q231P gp91^phox, then selected with puromycin; lysates were evaluated for gp91^phox (mAb 54.1), p22^phox (mAb NS5) and β-actin (loading control). (b) Flow cytometry of cell surface gp91^phox in untransduced CHO22 cells (top) and CHO22 cells expressing wild-type gp91^phox, Q231P gp91^phox or T178P gp91^phox, detected with mAb 7D5. (c) Immunoblot analysis of lysates (amount, above lanes) of wild-type PLB-985 cells (PLB WT) and PLB XR-CGD cells left untransduced (X) or transduced and selected as in a (X + gp91), then left undifferentiated (−) or differentiated into granulocytes by 6 d of induction with DMF (+); blots were probed for gp91^phox (mAb 54.1), p67^phox (to verify differentiation) and β-actin (loading control). (d) Immunoblot analysis of lysates (20 μg per well) of PLB-985 cells transduced as in c and differentiated into monocyte-like cells with vitamin D (Vit D) or into macrophage-like cells with a combination of vitamin D and PMA (below lanes); blots were probed for gp91^phox (mAb 54.1) and p67^phox (to verify differentiation). Far right, lysates of human PMNs (2.5 μg) serve as a control. Data are representative of three independent experiments.