Key Points• A key transition from the prealveolar macrophage precursor to mature alveolar macrophage is impaired in neonatal mice lacking LPL.• Genetic impairment of neonatal alveolar macrophage development associates with impaired clearance of a pulmonary pathogen in adult animals. ) exhibited significant reductions in alveolar macrophages and failed to effectively clear pulmonary pneumococcal infection, showing that immunodeficiency results from reduced alveolar macrophage numbers. We next identified the phase of alveolar macrophage development requiring LPL. In mice, fetal monocytes arrive in the lungs during a late fetal stage, maturing to alveolar macrophages through a prealveolar macrophage intermediate. LPL was required for the transition from prealveolar macrophages to mature alveolar macrophages. The transition from prealveolar macrophage to alveolar macrophage requires the upregulation of the transcription factor peroxisome proliferatoractivated receptor-g (PPAR-g), which is induced by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite abundant lung GM-CSF and intact GM-CSF receptor signaling, PPAR-g was not sufficiently upregulated in developing alveolar macrophages in LPL 2/2 pups, suggesting that precursor cells were not correctly localized to the alveoli, where GM-CSF is produced. We found that LPL supports 2 actin-based processes essential for correct localization of alveolar macrophage precursors: (1) transmigration into the alveoli, and (2) engraftment in the alveoli. We thus identify a molecular pathway governing neonatal alveolar macrophage development and show that genetic disruption of alveolar macrophage development results in immunodeficiency. (Blood. 2016;128(24):2785-2796

B cell development is exquisitely sensitive to location within specialized niches in the bone marrow and spleen. Location within these niches is carefully orchestrated through chemotactic and adhesive cues. Here we demonstrate the requirement for the actin-bundling protein L-plastin (LPL) in B cell motility towards the chemokines CXCL12 and CXCL13 and the lipid chemoattractant sphingosine-1-phosphate, which guide normal B cell development. Impaired motility of B cells in LPL−/− mice correlated with diminished splenic maturation of B cells, with a moderate (40%) loss of follicular B cells and a profound (>80%) loss of marginal zone B cells. Entry of LPL−/− B cells into the lymph nodes and bone marrow of mice was also impaired. Furthermore, LPL was required for the integrin-mediated enhancement of transwell migration but was dispensable for integrin-mediated lymphocyte adhesion. These results suggest that LPL may participate in signaling that enables lymphocyte transmigration. In support of this hypothesis, the phosphorylation of Pyk-2, a tyrosine kinase that integrates chemotactic and adhesive cues, is diminshed in LPL−/− B cells stimulated with chemokine. Finally, a well-characterized role of marginal zone B cells is the generation of a rapid humoral response to polysaccharide antigens. LPL−/− mice exhibited a defective antibody response to Streptococcus pneumoniae, indicating a functional consequence of defective MZ B cell development in LPL−/− mice.

We report that mice deficient for the hematopoietic-specific, actin-bundling protein L-plastin (LPL) succumb rapidly to intratracheal pneumococcal infection. The increased susceptibility of LPL ؊/؊ mice to pulmonary pneumococcal challenge correlated with reduced numbers of alveolar macrophages, consistent with a critical role for this cell type in the immediate response to pneumococcal infection. LPL ؊/؊ mice demonstrated a very early clearance defect, with an almost 10-fold-higher bacterial burden in the bronchoalveolar lavage fluid 3 h following infection. Clearance of pneumococci from the alveolar space in LPL ؊/؊ mice was defective compared to that in Rag1 ؊/؊ mice, which lack all B and T lymphocytes, indicating that innate immunity is defective in LPL ؊/؊ mice. We did not identify defects in neutrophil or monocyte recruitment or in the production of inflammatory cytokines or chemokines that would explain the early clearance defect. However, efficient alveolar macrophage regeneration following irradiation required LPL. We thus identify LPL as being key to alveolar macrophage development and essential to an effective antipneumococcal response. Further analysis of LPL ؊/؊ mice will illuminate critical regulators of the generation of alveolar macrophages and, thus, effective pulmonary innate immunity.

Elucidating the molecular regulation of macrophage migration is essential for understanding the patho-physiology of multiple human diseases, including host responses to infection and autoimmune disorders. Macrophage migration is supported by dynamic rearrangements of the actin cytoskeleton, with formation of actin-based structures such as podosomes and lamellipodia. Here we provide novel insights into the function of the actin-bundling protein l-plastin (LPL) in primary macrophages. We found that podosome stability is disrupted in primary resident peritoneal macrophages from LPL−/− mice. Live-cell imaging of F-actin using resident peritoneal macrophages from LifeACT-RFP+ mice demonstrated that loss of LPL led to decreased longevity of podosomes, without reducing the number of podosomes initiated. Additionally, macrophages from LPL−/− mice failed to elongate in response to chemotactic stimulation. These deficiencies in podosome stabilization and in macrophage elongation correlated with impaired macrophage transmigration in culture and decreased monocyte migration into murine peritoneum. Thus, we have identified a role for LPL in stabilizing long-lived podosomes and in enabling macrophage motility.

Summary Germinal center development, critical for long-term humoral immunity, requires the trafficking of T and B lymphocytes to defined tissues and locations after antigenic challenge. The molecular mechanisms that support lymphocyte trafficking through the linkage of extracellular chemotactic and adhesive cues to the actin cytoskeleton are not yet fully defined. We have previously identified the actin-bundling protein L-plastin (LPL) as a requisite intermediary in both naive B and T lymphocyte migration and in T cell activation. We tested the hypothesis that humoral immunity would require LPL. We show that mice lacking LPL demonstrated defective germinal center formation and reduced production of T cell-dependent antibodies. T cells from LPL−/− mice exhibited defective expansion of the follicular helper T (Tfh) population. Reduced expansion of LPL−/− Tfh cells correlated with impaired trafficking to or retention of cells in the spleen following challenge, highlighting the importance of initial lymphocyte recruitment to the eventual success of the immune response. Furthermore, LPL−/− B cells demonstrated cell-intrinsic defects in population expansion and in differentiation into germinal center B cells. LPL thus modulates both T and B cell function during the germinal center reaction and the production of T cell-dependent antibody responses.