Novel Mouse Model Reveals That Serine Phosphorylation of L-Plastin Is Essential for Effective Splenic Clearance of Pneumococcus

Abstract

Asplenia imparts susceptibility to life-threatening sepsis with encapsulated bacteria, such as the pneumococcus. However, the cellular components within the splenic environment that guard against pneumococcal bacteremia have not been defined. The actin-bundling protein L-plastin (LPL) is essential for the generation of marginal zone B cells and for anti-pneumococcal host defense, as revealed by a mouse model of genetic LPL deficiency. In independent studies, serine phosphorylation of LPL at residue 5 (S5) has been described as a key “switch” in regulating LPL actin binding and subsequent cell motility, although much of the data are correlative. To test the importance of S5 phosphorylation in LPL function, and to specifically assess the requirement of LPL S5 phosphorylation in anti-pneumococcal host defense, we generated the “S5A” mouse, expressing endogenous LPL bearing a serine-to-alanine mutation at this position. S5A mice were bred to homozygosity, and LPL was expressed at levels equivalent to wild-type, but S5 phosphorylation was absent. S5A mice exhibited specific impairment in clearance of pneumococci following i.v. challenge, with 10-fold-higher bacterial bloodstream burden 24 h after challenge compared with wild-type or fully LPL-deficient animals. Defective bloodstream clearance correlated with diminished population of marginal zone macrophages and with reduced phagocytic capacity of multiple innate immune cells. Development and function of other tested leukocyte lineages, such as T and B cell motility and activation, were normal in S5A mice. The S5A mouse thus provides a novel system in which to elucidate the precise molecular control of critical immune cell functions in specific host–pathogen defense interactions.

L-plastin enhances NLRP3 inflammasome assembly and bleomycin-induced lung fibrosis

“…NLRP3-activated gasdermin-D and IL-1β processing after nigericin or ATP treatment in cells from S5A mice was also equivalent to the WT ( Figure S2F ). These results are in agreement with our previous observation that S5A mice showed no defect in pneumococcal clearance in our lung infection model ( Anaya et al, 2021 ).…”

“…NLRP3-activated gasdermin-D and IL-1b processing after nigericin or ATP treatment in cells from S5A mice was also equivalent to the WT (Figure S2F). These results are in agreement with our previous observation that S5A mice showed no defect in pneumococcal clearance in our lung infection model (Anaya et al, 2021). LPL À/À mice are resistant to bleomycin-induced lung injury and fibrosis NLRP3 drives multiple inflammatory diseases characterized by ongoing tissue injury (Kuipers et al, 2011;Nystrom and Bruckner-Tuderman, 2018).…”

“…Using a new mouse model in which the endogenous LPL locus was modified to substitute the non-phosphorylatable alanine for a serine residue at position 5 (S5A), we recently showed that Ser-5 phosphorylation of LPL is required for pneumococcal clearance by splenic macrophages. However, homozygous S5A mice were able to clear pneumococci from the lungs ( Anaya et al, 2021 ). To evaluate whether S5 phosphorylation of LPL regulates NLRP3 activation, we evaluated IL-1β production in BMDMs derived from WT or S5A mice after NLRP3 activation ( Figure S2F ).…”

“…Because Ser5 is heavily phosphorylated in multiple immune cell lineages after stimulation ( Wang et al, 2010 ; Hagi et al, 2006 ; Freeley et al, 2012 ; De Clercq et al, 2013 ), most work has focused on Ser5 phosphorylation of LPL in regulating cell activation and motility. We reported recently that Ser5 phosphorylation is required for efficient splenic clearance of blood-stream infection with Streptococcus pneumoniae , myeloid cell phagocytosis, and generation of splenic marginal-zone macrophages ( Anaya et al, 2021 ). However, we have not noted defects in macrophage podosome formation or NLRP3 inflammasome activation in macrophages from S5A mice ( Anaya et al, 2021 ).…”

“…We reported recently that Ser5 phosphorylation is required for efficient splenic clearance of blood-stream infection with Streptococcus pneumoniae , myeloid cell phagocytosis, and generation of splenic marginal-zone macrophages ( Anaya et al, 2021 ). However, we have not noted defects in macrophage podosome formation or NLRP3 inflammasome activation in macrophages from S5A mice ( Anaya et al, 2021 ). Further work using these mice will illuminate the specific requirements for Ser5 phosphorylation and how modification at other sites may compensate for or coordinate with this regulatory feature.…”