Impaired respiratory burst contributes to infections in PKCδ-deficient patients

Abstract

Patients with autosomal recessive protein kinase C δ (PKCδ) deficiency suffer from childhood-onset autoimmunity, including systemic lupus erythematosus. They also suffer from recurrent infections that overlap with those seen in patients with chronic granulomatous disease (CGD), a disease caused by defects of the phagocyte NADPH oxidase and a lack of reactive oxygen species (ROS) production. We studied an international cohort of 17 PKCδ-deficient patients and found that their EBV-B cells and monocyte-derived phagocytes produced only small amounts of ROS and did not phosphorylate p40phox normally after PMA or opsonized Staphylococcus aureus stimulation. Moreover, the patients' circulating phagocytes displayed abnormally low levels of ROS production and markedly reduced neutrophil extracellular trap formation, altogether suggesting a role for PKCδ in activation of the NADPH oxidase complex. Our findings thus show that patients with PKCδ deficiency have impaired NADPH oxidase activity in various myeloid subsets, which may contribute to their CGD-like infectious phenotype.

Graphical abstract

Figure 1.

AR PKCδ deficiency in 10 families. (A) Pedigree of the 10 unrelated kindreds showing familial segregation of the different PRKCD alleles. Generations are indicated by Roman numerals (I–II), and each individual is indicated by an Arabic numeral (1–4). Male and female individuals are represented by squares and circles, respectively. Double horizontal lines indicate consanguinity. Affected patients are represented by closed black symbols, the index cases are indicated by an arrow, and asymptomatic carriers are indicated by a black vertical line. Individuals of unknown genotype are indicated by “E?” Symbols crossed with a black diagonal line indicate deceased individuals. (B) Schematic representation of the PRKCD gene. Coding exons are numbered (3–19). The PKCδ protein is represented with four domains: C2-like, without Ca₂⁺ binding motif; C1, binding to diacylglycerol and phorbitol esters; C3, ATP-binding; and C4, substrate-binding domain. The positions of the variants observed in the patients are indicated by arrows. (C) Minor allele frequency (MAF) and CADD score for all PRKCD variants studied (colored symbols) or found in the homozygous state in gnomAD v2.1.1 (white circles; https://gnomad.broadinstitute.org/) or our in-house cohort (Laboratory of Human Genetics of Infectious Diseases [HGID]; white triangles). The missense and nonsense mutations of the patients are represented as blue lozenges, and essential splice site mutations are indicated by closed red circles. The dotted line corresponds to the MSC of 26.5, with its 95% confidence interval.

Figure S1.

Evolutionary conservation of PKCδ and PRKCD transcripts in cells carrying essential splice site variants. (A) Amino-acid residue conservation in various species, for the four PKCδ missense mutations (indicated by red arrows) found in kindreds A–D. (B) Schematic diagram and proportions of the different splice variants produced in cells from homozygous carriers of the c.1352 + 1G>A or c.517 + 2dup variants and compound heterozygous carriers of the p.Q191* and c.788-2A>G variants. Positions of affected amino acids are indicated by red triangles.

Figure 2.

In vitro characterization of the various PRKCD alleles in an overexpression system. (A) RT-qPCR of cDNA from HEK293T cells nontransfected (NT) or transfected with an empty vector (EV), WT PRKCD, or mutated PRKCD. GUSB was used for normalization (n = 2; mean ± SD). (B) Western blot of total protein extracts from HEK293T cells either NT or transfected with EV, WT PRKCD, or mutated PRKCD, all inserted into pCMV6 with a C-terminal DDK tag. PKCδ was detected with a polyclonal anti-PKCδ antibody and an antibody directed against the C-terminal DDK tag. PKCδ phosphorylation was detected with polyclonal antibodies against the T505 (upper panel) and S643 (lower panel) phosphorylation sites. An antibody against GAPDH was used as a loading control. The results shown are representative of three independent experiments. Transcripts identified by TOPO-TA cloning are indicated in blue. (C) Quantification of phosphorylated PKCδ expression compared with the amount of total PKCδ. All values were normalized to the WT transfected cells (n = 3; mean ± SD).

Figure 3.

PKCδ deficiency in patients’ EBV-B cells. (A) RT-qPCR for PRKCD with a probe spanning the junction between exons 3 and 4 (left) and a probe spanning the junction between exons 17 and 18 (right), in EBV-B cells from healthy controls (Ctrl; n = 10) and patients. GUSB was used for normalization (n = 3; mean ± SD). (B) Western blot of total protein extracts from EBV-B cells from healthy controls (C1, C2) and patients (left). PKCδ was detected with a polyclonal anti-PKCδ antibody. The asterisk indicates nonspecific bands. Quantification of the PKCδ protein normalized against GAPDH (right; n = 3; mean ± SD). The results shown are representative of three independent experiments. (C) Western blot of total protein extract from the EBV-B cells of controls or patients. PKCδ was detected with a polyclonal anti-PKCδ antibody. The autophosphorylation of PKCδ was detected with antibodies against the T505 (upper panel) and S643 (lower panel) phosphorylation sites. The results shown are representative of three independent experiments. (D) Apoptosis of EBV-B cells from healthy controls (Ctrl; n = 10) and patients (n = 8) after 24 h of stimulation with APO-1-1 (APO; 1 µg/ml) or PMA (100 ng/ml). The percentages of live cells were normalized against the number of nonstimulated (NS) cells, after acquisition by flow cytometry over a constant time (n = 3; mean ± SD). A linear mixed model was used to determine whether survival rates were the same for PKCδ-deficient patients and controls after APO or PMA stimulation (n.s., not significant; ***, P < 0.001).

Figure S2.

Phosphorylation of MARCKS in the patients’ EBV-B cells. Phosphorylation of MARCKS was detected by Western blot using whole-cell protein lysates of EBV-B cells of two healthy controls (C1, C2) and PKCδ-deficient patients before (–) and after (+) 30-min PMA activation (upper panel; 400 ng/ml). Antibodies against phospho-MARCKS (p-MARCKS), MARCKS, and PKCδ were used. An antibody against GAPDH was used as loading control. Quantification of pMARCKS expression compared with the amount of total MARCKS (lower panel; n = 2; mean ± SD). The results shown are representative of two independent experiments.

Figure 4.

NADPH oxidase activity and retroviral transduction of the patients’ EBV-B cells. (A) Production of O₂⁻ by EBV-B cells from healthy controls (Ctrl; n = 3), PKCδ-deficient patients (n = 8), p40^phox-deficient patients (n = 2), and gp91^phox-deficient patients (n = 1) after stimulation with PMA stimulation (left; 400 ng/ml) or Pansorbin (right; 2 mg/ml), as assessed by luminol bioluminescence. LU, luminescence units. The results shown are representative of two or three independent experiments. (B) Extracellular H₂O₂ production by EBV-B cells from healthy controls (n = 3), PKCδ-deficient patients (n = 8), p40^phox-deficient patients (n = 2), and gp91^phox-deficient patients (n = 1) after PMA stimulation (400 ng/ml), as assessed with the Amplex Red test. The results shown are representative of three independent experiments. (C) Extracellular H₂O₂ production by EBV-B cells from a healthy control (C1), a gp91^phox-deficient patient, and PKCδ-deficient patients (n = 8) transduced with an empty vector (EV) or PRKCD WT cDNA, after PMA stimulation (400 ng/ml), as assessed with the Amplex Red test (mean ± SD). The results shown are representative of two independent experiments. (D) Production of H₂O₂ by healthy control (C1), and PRKCD^−/− EBV-B cells transduced with EV, WT, or the various mutant PRKCD cDNAs, upon PMA stimulation (400 ng/ml), as assessed with the Amplex Red test. The results shown are representative of two independent experiments.

Figure S3.

Complementation of the patients’ EBV-B cells with WT PRKCD. (A) Surface expression of CD271 on EBV-B cells of a healthy control (C1) and PKCδ-deficient patients after retroviral transduction with the empty pLZRS vector (EV) and WT PRKCD cDNA (gray, isotype; black, surface staining). (B) Extracellular H₂O₂ production by the EBV-B cells of a healthy control (C1), a gp91^phox-deficient patient, and PKCδ-deficient patients either nontransduced (NT) or transduced with the empty pLZRS plasmid (EV) or WT-PRKCD, at various time points after PMA stimulation (400 ng/ml), as assessed with the Amplex Red test. Representative results of two independent experiments (duplicates, mean ± SD).

Figure 5.

NADPH oxidase subunit expression and interaction with PKCδ. (A) Western blot of total protein extracts from the EBV-B cells of healthy controls (C1, C2), PKCδ-deficient patients and CGD patients. Antibodies against gp91^phox, p67^phox, p47^phox, p40^phox, p22^phox, and EROS were used. An antibody against GAPDH was used as a loading control. (B) Coimmunoprecipitation (IP) of protein lysates from HEK293T cells transfected with empty vector (EV) or plasmids encoding PKCδ, p40^phox, or p47^phox. Pulldowns with anti-DDK (PKCδ) and immunoblots (IB) with anti-DDK or specific antibodies (p40^phox, upper panel; p47^phox, lower panel) are shown. (C) IP on protein lysates from HEK293T cells transfected with EV, CYBB, or PRKCD cDNAs. The pulldown of PKCδ (left) and gp91^phox (right) was performed with an anti-DDK antibody. (D) IP on protein lysates from HEK293T cells transfected with the EV, CYBA, or PRKCD cDNAs. The pulldown of PKCδ (left) and p22^phox (right) was performed with an anti-DDK antibody. (E) IP on protein lysates from HEK293T cells transfected with EV, CYBC1, or PRKCD cDNAs. The pulldown of PKCδ (left) and EROS (right) was performed with an anti-DDK antibody. (F and G) Detection of p40^phox (F) and p47^phox (G) phosphorylation by Western blot on cell lysates of HEK293T cells transfected with EV, NCF4, NCF1, and PRKCD cDNAs. Representative images of three independent experiments.

Figure S4.

Expression of NADPH oxidase components in PKCδ-deficient EBV-B cells and their interaction with PKCδ. (A) Extracellular detection of cytochrome b₅₅₈ (cytB) and intracellular staining for gp91^phox, p67^phox, p47^phox, and p22^phox in EBV-B cells from healthy controls (C1, C2), PKCδ-deficient patients, and CGD patients (gray, isotype; black, surface staining). Representative results are shown for three independent experiments. (B) Coimmunoprecipitation (IP) on protein lysates from HEK293T cells transfected with the empty vector (EV), NCF4, or PRKCD cDNAs. The pulldown of p40^phox was performed with an anti-DDK antibody. (C) IP on protein lysates from HEK293T cells transfected with EV, NCF1, or PRKCD cDNAs. The pulldown of p47^phox was performed with an anti-DDK antibody. (D) Co-IP on protein lysates from HEK293T cells transfected with EV, NCF2, or PRKCD cDNAs. The pulldown of PKCδ (upper panel) and p67^phox (lower panel) was performed with an anti-DDK or an anti-V5 antibody, respectively. (E) IP on protein lysates from HEK293T cells transfected with EV, RAC2, or PRKCD cDNAs. The pulldown of PKCδ (upper panel) and Rac2 (lower panel) was performed with an anti-DDK or an anti-V5 antibody, respectively. All results shown are representative of two independent experiments.

Figure 6.

p40^phox and p47^phox phosphorylation in PKCδ-deficient EBV-B cells.(A) Phosphorylation of p40^phox in total protein extracts from EBV-B cells of healthy controls (C1, C2), PKCδ-deficient patients, and a p40^phox-deficient patient, before (–) and after (+) 30 min of PMA stimulation (400 ng/ml), measured by Western blot. (B) Phosphorylation of p40^phox in total protein extracts from EBV-B cells of healthy controls, PKCδ-deficient patients, and a p40^phox-deficient patient, before (–) and after (+) 30 min of Pansorbin stimulation (2 mg/ml), measured by Western blot. (C) Phosphorylation of p47^phox in EBV-B cells of healthy controls, PKCδ-deficient patients, and a p47^phox-deficient patient before (–) and after (+) 30 min of PMA stimulation (400 ng/ml), measured by Western blot. (D) Phosphorylation of p47^phox in total protein extracts from the EBV-B cells of healthy controls, PKCδ-deficient patients, and a p47^phox-deficient patient, before (–) and after (+) 30 min of PMA stimulation (400 ng/ml), measured by Western blot. (E) Phosphorylation of p40^phox in total protein extracts from EBV-B cells of a healthy control, and PKCδ-deficient patients either nontransduced (NT) or transduced with an EV or PRKCD WT cDNA, before (–) and after (+) 30 min of PMA stimulation (400 ng/ml). Total protein extracts from EBV-B cells of a p40^phox-deficient patient were used as controls. All results shown are representative of two to three independent experiments.

Figure 7.

NADPH oxidase activity in primary phagocytic cells from PKCδ-deficient patients. (A) Neutrophil intracellular ROS production, as measured by DHR, upon PMA stimulation, for travel controls (ctrl; n = 11), PKCδ-deficient patients (n = 11), p40^phox-deficient patients (n = 5), and gp91^phox-deficient patients (n = 2), or upon E. coli stimulation, for travel controls (n = 5), PKCδ-deficient patients (n = 5), p40^phox-deficient patients (n = 1), and gp91^phox-deficient patients (n = 2). (B) Monocyte intracellular ROS production, measured by DHR, upon PMA stimulation, for travel controls (n = 11), PKCδ-deficient patients (n = 11), p40^phox-deficient patients (n = 5), and gp91^phox-deficient patients (n = 2), or upon E. coli stimulation, for travel controls (n = 5), PKCδ-deficient patients (n = 5), p40^phox-deficient patients (n = 1), and gp91^phox-deficient patients (n = 1). All values are expressed as a percentage of DHR oxidation normalized against travel controls. (C) Left: Quantification of NET formation for healthy controls (n = 6), PKCδ-deficient patients (n = 3), and a gp91^phox-deficient patient, after PMA stimulation. All values are expressed as a percentage of NET-forming cells. Right: Representative images of PMA-induced NET formation by neutrophils of local and travel controls and a PKCδ-deficient patient (P17). Green represents myeloperoxidase, and blue, DNA (DAPI). Scale bar = 60 µm. (D) Extracellular H₂O₂ production in response to stimulation with IFN-γ, PMA, or both (mean ± SD) for MDMs from local controls (n = 10), travel controls (n = 10), PKCδ-deficient patients (n = 12), p40^phox-deficient patients (n = 3), and gp91^phox-deficient patients (n = 1). (E) Extracellular H₂O₂ production in response to stimulation with LPS, PMA, or both (mean ± SD), in MDDCs from local controls (n = 10), travel controls (n = 10), PKCδ-deficient patients (n = 8), p40^phox-deficient patients (n = 2), and gp91^phox-deficient patients (n = 2). (F) Western blot of MDMs of healthy controls (n = 4) and PKCδ-deficient patients (n = 5), without (–) or with (+) PMA stimulation. Solid bars between blots indicate different regions of the same membrane. In A–E, dots represent individual samples, and bars, the mean and SD. Two-way ANOVA was used in A–D; one-way ANOVA was used in C; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure S5.

NADPH oxidase activity and p47^phox phosphorylation in primary cells of PKCδ-deficient patients.(A and B) Representative flow cytometry images of intracellular ROS production measured by DHR in neutrophils (A) and monocytes (B) of a healthy control and a PKCδ-deficient patient (P14) are shown on the right (gray filled, nonstimulated; black solid line, PMA stimulation; black dotted line, E. coli stimulation). (C) PKCδ expression in neutrophils and monocytes of healthy controls and two PKCδ-deficient patients, measured by Western blot. (D) Representative images of PMA-induced NET formation by neutrophils of controls, three PKCδ-deficient patients, and a gp91^phox-deficient patient. Green represents myeloperoxidase (MPO) and blue DNA (DAPI). The merged images of the local and travel controls and of P17 are the same as displayed in Fig. 7 C. Scale bars, 60 µm. (E) Representative kinetic of extracellular H₂O₂ production after PMA plus IFN-γ stimulation by MDMs of patients (P14, P15) and controls. (F) Western Blot of MDMs of healthy controls (n = 4) and PKCδ-deficient patients (n = 5) before (–) and after (+) 30 min of PMA stimulation (400 ng/ml). Antibodies against PKCδ, phosphoS304-p47^phox, phosphoS315-p47^phox, and p47^phox were used. Anti-GAPDH and anti-Vinculin antibodies served as a loading controls. The control marked with an asterisk is the same as in Fig. 7 D because proteins of P2 (Fig. 7 D) and P13 were blotted on the same membrane. Solid bars between two images indicate different regions of the same membrane.