case_images: PMC10637399_01
case_images: Inflammation of the femur. The anteroposterior radiograph is indicative of osteomyelitis (arrow) in the right (Dx) femur of the patient at age 14 years, prior to adalimumab treatment.
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case_images: X-ray was normal and interferon gamma-assay (QuantiFERON ) was negative. X-ray examination showed bilateral inflammation of the femur ( Figure 1 ) and a bone biopsy indicated unspecific inflammation without granuloma. However, we could not detect any altered production of TNFalpha, IL1beta or IL8 as compared to control cells ( Supplementary Figure 1 ), indicating that there is no direct link between MPO deficiency in phagocytic cells and their capacity to produce TNFalpha that could be of potential importance for regulating the CNO-associated systemic inflammation.
case_images: Neutrophil plasma membrane expression of CD11b, CD62L and cytochrome b
  558. (A) Peripheral blood samples from the patient and controls were incubated at room temperature without or at 37 C with TNFalpha (10 ng/mL) for 20 min after which the neutrophil surface expression of CD11b and CD62L was analyzed by flow cytometry. (B) Isolated neutrophils from the patient and controls were incubated on ice or at 37 C in the presence of TNFalpha (10 ng/mL) for 20 min, after which the surface expression of cytochrome b
  558 was evaluated by flow cytometry. Patient samples: +adalimumab (on adalimumab treatment), -adalimumab (withdrawal of adalimumab). Control samples: healthy adult donors.
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case_images: The patient neutrophils displayed a resting phenotype with similar surface expression of these markers as control neutrophils, both during treatment withdrawal and after reintroduction of adalimumab ( Figure 2A ). When the analyses were performed nine weeks after adalimumab withdrawal, the patient neutrophils responded by altered surface marker exposure ( Figure 2A ), indicating that the residual level of adalimumab was not sufficient to sequester added TNFalpha and that the patient's neutrophils exhibited normal regulation of TNFalpha-induced cell activation. TNFalpha treatment and then measuring cell surface levels of mobilizable markers, for this assay the cytochrome b 558 was used, adalimumab-independent TNFalpha-induced degranulation could be investigated. The isolated neutrophils displayed increased surface expression of cytochrome b 558 in response to TNFalpha in a similar way as the control neutrophils ( Figure 2B ), supporting that the patient neutrophils were fully capable of responding to TNFalpha in an in vitro setting.
case_images: Neutrophil production and processing of ROS, NET formation, elastase activity, and viability. (A) Neutrophil intra (IC)- and extracellular (EC) ROS production was measured (mega counts per minute; Mcpm) by luminol- or isoluminol-enhanced chemiluminescence (CL), respectively, upon stimulation with PMA (50 nM; added at arrow after 5 min pre-incubation of cells at 37 C). (B) Neutrophils were stained with DCFDA for 30 min at 37 C in the absence or presence of PMA (50 nM) during the final 15 min. Thereafter ROS production was evaluated by flow cytometry. ROS production is given as geometric mean fluorescent intensity (MFI). (A, B) + adalimumab (on adalimumab treatment), - adalimumab (withdrawal of adalimumab). (C) PHPA-oxidation by hydrogen peroxide (H2O2) was used for MPO-independent evaluation of neutrophil ROS production. Neutrophils were incubated with HRP and PHPA and stimulated with PMA (50 nM) after which oxidation of PHPA was monitored by fluorometry (arbitrary units, AU) over time (min). Total H2O2 production was measured in the presence of azide (1 mM, solid lines, left graph) to abrogate H2O2 consumption by intracellular MPO and catalase, while extracellular H2O2 was measured in the absence of azide (dashed lines, left graph). The bar graph to the right shows the PHPA fluorescence representing intracellular ROS (arbitrary units, AU; calculated by subtracting the values of EC ROS from total ROS after 30 min stimulation) in the patient neutrophils (age 17 years +/- adalimumab). The horizontal dotted line shows mean IC ROS of control neutrophils after 30 min stimulation (n=2, mean 215.6 +/- SD 49.1). (D) To investigate the formation of NETs, neutrophils were incubated with the cell impermeable DNA stain Sytox green before stimulation with PMA (50 nM) or PSMalpha-2 (2.5 muM) and fluorescence (arbitrary units, AU) was monitored over time (min). (E) Human neutrophil elastase (HNE) was measured by incubating lysates (corresponding to 3125 neutrophils/sample) from the patient (age 23 years, + adalimumab) and one control with an elastase-specific substrate while fluorescence was monitored over time. Mean fluorescence +- SD of triplicates are shown (arbitrary units; AU). (F) Neutrophil apoptosis (Annexin V positive cells) was evaluated by flow cytometry after incubation of the cells with buffer, LPS (100 ng/ml) or FASL (anti-CD95 ab, 10 mug/ml) for 20 h at 37 C (number of apoptotic cells are given as fraction of total cells, %). Patient samples: +adalimumab (on adalimumab treatment), -adalimumab (withdrawal of adalimumab); Control samples: healthy adult donors.
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case_images: The two first techniques are dependent of MPO for the detection of intracellular ROS, and hence these assays gave an expected negative result for this parameter in the MPO-deficient patient neutrophils when stimulated with the standard agonist phorbol myristate acetate (PMA), regardless of adalimumab treatment or not ( Figures 3A, B ). The production of intracellular ROS was instead proven by PHPA-oxidation, showing normal levels in the patient cells both during withdrawal and after reintroduction of adalimumab, as compared to controls ( Figure 3C ). Extracellular release of ROS was also similar ( Figure 3A ), or even higher ( Figure 3C ), in patient cells as compared to controls, which is corroborated by other studies showing increased/prolonged production of hydrogen peroxide by MPO-deficient cells. We here confirmed those previously published data with novel data showing that also when symptoms were alleviated by adalimumab therapy, NET formation in response to PMA (but not to PSMalpha-2) was defective, in line with the MPO deficiency of the patient ( Figure 3D ). Apart from MPO-processed ROS, neutrophil elastase activity is critical for PMA-induced NET formation but patient neutrophil lysates obtained when the patient was on adalimumab therapy (at age 23 years) displayed normal elastase activity ( Figure 3E ). Yet, for this patient with CNO, neutrophil apoptosis was normal, including responses to pro- and anti-apoptotic stimulation, both during the inflammatory flare caused by treatment withdrawal and after reintroduction of treatment ( Figure 3F ), suggesting that regulation of apoptosis by ROS per se was unaltered in the patient leukocytes.