Introduction: The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding proteins regulate these rapid rearrangements to induce actin-based shape changes and to generate force. L-plastin (LPL) is a leukocyte-specific, actin-bundling protein that is regulated in part by phosphorylation of the Ser-5 residue. LPL deficiency in macrophages impairs motility, but not phagocytosis; we recently found that expression of LPL in which the S5 residue is converted to a non-phosphorylatable alanine (S5A-LPL) resulted in diminished phagocytosis, but unimpaired motility.Methods: To provide mechanistic insight into these findings, we now compare the formation of podosomes (an adhesive structure) and phagosomes in alveolar macrophages derived from wild-type (WT), LPL-deficient, or S5A-LPL mice. Both podosomes and phagosomes require rapid remodeling of actin, and both are force-transmitting. Actin rearrangement, force generation, and signaling rely upon recruitment of many actin-binding proteins, including the adaptor protein vinculin and the integrin-associated kinase Pyk2. Prior work suggested that vinculin localization to podosomes was independent of LPL, while Pyk2 was displaced by LPL deficiency. We therefore chose to compare vinculin and Pyk2 co-localization with F-actin at sites of adhesion of phagocytosis in AMs derived from WT, S5A-LPL or LPL−/− mice, using Airyscan confocal microscopy.Results: As described previously, podosome stability was significantly disrupted by LPL deficiency. In contrast, LPL was dispensable for phagocytosis and was not recruited to phagosomes. Recruitment of vinculin to sites of phagocytosis was significantly enhanced in cells lacking LPL. Expression of S5A-LPL impeded phagocytosis, with reduced appearance of ingested bacteria-vinculin aggregates.Discussion: Our systematic analysis of the regulation of LPL during podosome vs. phagosome formation illuminates essential remodeling of actin during key immune processes.
Despite the recent introduction of pneumococcal polysaccharide and conjugate vaccines, Streptococcus pneumoniae infection remains a leading cause of illness and death worldwide. In particular, infants in Papua New Guinea are at increased risk of severe pneumococcal pneumonia compared to infants in similar countries. We sought to determine if a novel genetic variant could explain this increased susceptibility. Whole exome sequencing revealed a single nucleotide variant (D308Y) in the gene encoding COQ6 (COQ6DY), a monooxygenase required for CoQ10 biosynthesis. We have utilized both a Saccharomyces cerevisiae model and a mouse model of COQ6DY to show that despite adequate production of CoQ10, this variant directly causes increased susceptibility to S. pneumoniae. This variant represents a previously unknown function of the CoQ10 biosynthetic complex that does not exert its effects through CoQ10 deficiency but rather through alterations of mitochondrial function and metabolism. These mitochondrial deficits in COQ6DY macrophages are sufficient to abrogate macrophage killing of S. pneumoniae and alter the coordination of the downstream immune response. In conclusion, we have identified a novel susceptibility allele to S. pneumoniae infection that exerts its effects via alterations in macrophage mitochondrial function. Supported by NIH (R21 AI142723), SLCH Children's Discovery Institute (PD-II-2018-742)
To identify immune variants predisposing to severe pneumonia, we performed whole exome sequencing in a pediatric population highly susceptible to acute lower respiratory infections, identifying a candidate novel variant in the ubiquinone (CoQ10) biosynthetic pathway. To evaluate the effect of this variant on immune function during bacterial pneumonia, we generated a mouse line using CRISPR-Cas9 that expresses the homologous aspartate to tyrosine variant in the enzyme COQ6. Intra-tracheal S. pneumoniae infection leads to increased bacteremia and mortality in mice homozygous for the variant despite similar numbers of immune cells in the lung. Mechanistic studies show that macrophages expressing the variant have decreased mitochondrial activity at the ubiquinone-dependent reduction of cytochrome c by complex III, as well as decreased maximum respiratory capacity in response to acute stimulation. Variant-expressing macrophages also exhibit impaired generation of mitochondrial reactive oxygen species (mROS) causing a direct, intrinsic defect in intracellular killing of internalized bacteria. Thus, the novel variant in CoQ10 biosynthesis leads to changes in macrophage mitochondria and an intrinsic inability to kill internalized bacteria. As alveolar macrophages are the first responders in the lung to bacterial challenge, the inability of these macrophages to mount a sufficient immune response can explain the observed increase in mortality following bacterial pneumonia. Because variants in CoQ10 biosynthesis can be supplemented with CoQ10, a readily available therapy may be able to correct this defect and improve survival in children with this variant
To identify immune variants predisposing to severe pneumonia, we performed whole exome sequencing in a pediatric population highly susceptible to acute lower respiratory infections, identifying a candidate novel variant in the CoQ biosynthetic pathway. To evaluate the effect of this variant on immune function during bacterial pneumonia, we generated a mouse line using CRISPR-Cas9 that expresses the homologous variant in the enzyme COQ6. Interestingly, we found that the variant does not result in CoQ deficiency, as other known variants in biosynthetic proteins do, however intra-tracheal S. pneumoniae infection leads to increased bacteremia and mortality in mutant mice. Mechanistic studies show that mutant macrophages have reduced pneumococcal killing in vitro, showing an intrinsic defect in innate immune function conferred by the COQ6 mutation. Variant macrophages have decreased mitochondrial respiratory capacity both at baseline and following stimulation with LPS, as well as an inability to induce mitochondrial reactive oxygen species (mROS) in response to stimulation despite increased mROS at baseline. Thus, the novel variant in a CoQ biosynthetic enzyme leads to changes in macrophage mitochondrial function and an intrinsic inability to kill internalized bacteria. As alveolar macrophages are the first responders in the lung to bacterial challenge, the inability of these macrophages to mount a sufficient immune response leads to the observed increase in mortality following bacterial pneumonia. This work describes a novel susceptibility locus to severe childhood pneumonia, and also represents the first known pathogenic variant in a CoQ biosynthetic protein that does not cause pathology resulting from CoQ deficiency.
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