Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia

Abstract

Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. Here we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C (PKC) expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production following PMA stimulation compared to non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent PKC conventional subclass α/β leading to reduced phosphorylation of NADPH oxidase components p47phox & p40phox in comparison to non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared to non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst due to decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia. These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response in order to limit infection.

C/EBP homologous protein–induced loss of intestinal epithelial stemness contributes to bile duct ligation–induced cholestatic liver injury in mice

“…To further identify the specific hepatic cells involved in BDL‐induced activation of the UPR and innate immunity, we isolated primary hepatocytes, Kupffer cells (KCs), cholangiocytes, and HSCs from WT BDL and sham control mice, and purity of the isolated cells was checked by real‐time RT‐PCR with specific hepatic cell markers, CK‐19 for primary cholangiocytes, CYP1A2 for primary hepatocytes, f4/80 for KCs, and Desmin for HSCs (http://onlinelibrary.wiley.com/doi/10.1002/hep.29540/suppinfo). BDL induced CHOP expression in hepatocytes, not in other hepatic cells (http://onlinelibrary.wiley.com/doi/10.1002/hep.29540/suppinfo). However, BDL significantly induced TLR2, TLR4, CD14, IL‐18, and IL‐1β in KCs and hepatocytes (http://onlinelibrary.wiley.com/doi/10.1002/hep.29540/suppinfo).…”

“…S4). (26,27) BDL induced CHOP expression in hepatocytes, not in other hepatic cells (Supporting Fig. S5A).…”

Genome-Wide Association Study of Cryptosporidiosis in Infants Implicates PRKCA

“…We identified a SNP that was associated with decreased expression of PRKCA and thus was less able to mediate the IL-17 immune response during Cryptosporidium infection. PRKCA has also been shown to be associated with numerous other infections, including infections by Staphylococcus aureus (26); with progression of sepsis (27) and toxoplasmosis (28); with Burkholderia cenocepacia infections in cystic fibrosis patients (29); and with hepatitis E virus replication (30).…”

Secondhand smoke alters arachidonic acid metabolism and inflammation in infants and children with cystic fibrosis

“…Early P. aeruginosa acquisition predicts long-term outcomes in children with CF,51 52 thereby predicting that detection and prevention of SHSe in young children with CF can aid in delaying establishment of certain chronic bacterial infections and subsequently worsened outcomes. The lack of impact of SHSe on macrophage-mediated B. cenocepacia clearance may be related to inherent differences in pathogenicity between B. cenocepacia and other pathogens in CF, such as differential regulation of the oxidative burst 53. Likewise, differences in increased clinical acquisition of P. aeruginosa in children with SHSe compared with macrophage-mediated clearance suggests that other immune cells responsible for P. aeruginosa clearance such as neutrophils may be impacted by SHSe.…”