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

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.

The Association of Medications and Vaccination with Risk of Pneumonia in Inflammatory Bowel Disease.

BACKGROUND: Patients with inflammatory bowel disease (IBD) are at increased risk for pneumonia, and corticosteroids are reported to amplify this risk. Less is known about the impact of corticosteroid-sparing IBD therapies on pneumonia risk or the efficacy of pneumococcal vaccination in reducing all-cause pneumonia in real-world IBD cohorts. METHODS: We performed a population-based study using an established Veterans Health Administration cohort of 29,957 IBD patients. We identified all patients who developed bacterial pneumonia. Cox survival analysis was used to determine the association of corticosteroids at study entry and as a time-varying covariate, corticosteroid-sparing agents (immunomodulators and antitumor necrosis-alpha [TNF] inhibitors), and pneumococcal vaccination with the development of all-cause pneumonia. RESULTS: Patients with IBD who received corticosteroids had a greater risk of pneumonia when controlling for age, gender, and comorbidities (hazard ratio [HR] 2.21; 95% confidence interval [CI], 1.90-2.57 for prior use; HR = 3.42; 95% CI, 2.92-4.01 for use during follow-up). Anti-TNF inhibitors (HR 1.52; 95% CI, 1.02-2.26), but not immunomodulators (HR 0.91; 95% CI, 0.77-1.07), were associated with a small increase in pneumonia. A history of pneumonia was strongly associated with subsequent pneumonia (HR = 4.41; 95% CI, 3.70-5.27). Less than 15% of patients were vaccinated against pneumococcus, and this was not associated with a reduced risk of pneumonia (HR = 1.02; 95% CI, 0.80-1.30) in this cohort. CONCLUSION: In a large US cohort, corticosteroids were confirmed to increase pneumonia risk. Tumor necrosis-alpha inhibitors were associated with a smaller increase in the risk of pneumonia. Surprisingly, pneumococcal vaccination did not reduce all-cause pneumonia in this population, though few patients were vaccinated.

Inhaled GM-CSF in neonatal mice provides durable protection against bacterial pneumonia.

Pneumonia poses profound health threats to preterm infants. Alveolar macrophages (AMs) eliminate inhaled pathogens while maintaining surfactant homeostasis. As AM development only occurs perinatally, therapies that accelerate AM maturation in preterms may improve outcomes. We tested therapeutic rescue of AM development in mice lacking the actin-bundling protein L-plastin (LPL), which exhibit impaired AM development and increased susceptibility to pneumococcal lung infection. Airway administration of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to LPL-/- neonates augmented AM production. Airway administration distinguishes the delivery route from prior human infant trials. Adult LPL-/- animals that received neonatal GM-CSF were protected from experimental pneumococcal challenge. No detrimental effects on surfactant metabolism or alveolarization were observed. Airway recombinant GM-CSF administration thus shows therapeutic promise to accelerate neonatal pulmonary immunity, protecting against bacterial pneumonia.

L-Plastin promotes podosome longevity and supports macrophage motility.

Elucidating the molecular regulation of macrophage migration is essential for understanding the pathophysiology 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.

Klebsiella pneumoniae FimK Promotes Virulence in Murine Pneumonia.

Klebsiella pneumoniae, a chief cause of nosocomial pneumonia, is a versatile and commonly multidrug-resistant human pathogen for which further insight into pathogenesis is needed. We show that the pilus regulatory gene fimK promotes the virulence of K. pneumoniae strain TOP52 in murine pneumonia. This contrasts with the attenuating effect of fimK on urinary tract virulence, illustrating that a single factor may exert opposing effects on pathogenesis in distinct host niches. Loss of fimK in TOP52 pneumonia was associated with diminished lung bacterial burden, limited innate responses within the lung, and improved host survival. FimK expression was shown to promote serum resistance, capsule production, and protection from phagocytosis by host immune cells. Finally, while the widely used K. pneumoniae model strain 43816 produces rapid dissemination and death in mice, TOP52 caused largely localized pneumonia with limited lethality, thereby providing an alternative tool for studying K. pneumoniae pathogenesis and control within the lung.

Prevalence of nasopharyngeal pneumococcal colonization in children and antimicrobial susceptibility profiles of carriage isolates.

Nasopharyngeal (NP) pneumococcal carriage predisposes children to pneumococcal infections. Defining the proportion of pneumococcal isolates that are antibiotic-resistant enables the appropriate choice of empiric therapies. The antibiogram of NP carriage isolates derived from a pediatric population following the introduction of the 13-valent pneumococcal conjugate vaccine was defined in this study.

Gelsolin overexpression alters actin dynamics and tyrosine phosphorylation of lipid raft-associated proteins in Jurkat T cells.

Upon T cell receptor engagement, both the actin cytoskeleton and substrates of tyrosine phosphorylation are remodeled to create a signaling complex at the interface of the antigen-presenting cell and responding T cell. While T cell signaling has been shown to regulate actin reorganization, the mechanisms by which changes in actin dynamics affect early T cell signaling have not been fully explored. Using gelsolin, an actin-binding protein with capping and severing activities, and latrunculin, an actin-depolymerizing agent, we have further investigated the interplay between actin dynamics and the regulation of T cell signaling. Overexpression of gelsolin altered actin dynamics in Jurkat T cells, and alteration of actin dynamics correlated with dysregulation of tyrosine phosphorylation of raft-associated substrates. This perturbation of tyrosine phosphorylation was correlated with inhibition of activation-dependent signaling pathways regulating Erk-1/2 phosphorylation, NF-AT transcriptional activation and IL-2 production. Modification of actin by the depolymerizing agent latrunculin also altered the tyrosine phosphorylation patterns of proteins associated with lipid rafts, and pre-treatment with latrunculin inhibited anti-CD3 mAb-mediated NF-AT activation. Thus, our data indicate that actin cytoskeletal dynamics modulate the tyrosine phosphorylation of raft-associated proteins and subsequent downstream signal transduction.