Abstract
Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during bacterial uptake. The participation of essential host cell signaling kinases in pneumococcal phagocytosis was deciphered for the kinase Akt, ERK1/2, and p38 and phosphoimmunoblots showed an increased phosphorylation and thus activation upon infection with pneumococci. Taken together, this study deciphers host cell kinases in innate immune cells that are induced upon infection with pneumococci and interfere with bacterial clearance after phagocytosis.
Highlights
Streptococcus pneumoniae is a common colonizer of the upper respiratory tract of human, with increased colonization rates in children and the elderly (Garenne et al, 1992; Bogaert et al, 2004b; Hussain et al, 2005)
Monocytic THP-1 cells were seeded in 24-well plates (2 × 105 cells per well in RPMI-1640 supplemented with 10% heat inactivated FCS in a volume of 1 ml) and differentiation was stimulated by the addition of 200 nmol/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich)
To assess the impact of the bacterial growth medium on pneumococcal phagocytosis by PMA-differentiated THP-1 cells, S. pneumoniae were grown in complex medium (THY) or chemically defined medium (RPMImod)prior to infection
Summary
Streptococcus pneumoniae is a common colonizer of the upper respiratory tract of human, with increased colonization rates in children and the elderly (Garenne et al, 1992; Bogaert et al, 2004b; Hussain et al, 2005). Recognition of bacteria via PAMPs and sensing of opsonized bacteria leads to the initiation of signal transduction cascades catalyzed by different protein kinases ending up in the activation of proteins involved in actin remodeling and phagocytosis (Freeman and Grinstein, 2014). Blocking of the PI3K leads not to an inhibition of opsonized particle binding or initial actin polymerization but seems to be required for membrane extension and fusion during engulfment (Araki et al, 1996; Cox et al, 1999) Another important component of intracellular signaling processes is the protein kinase B (Akt), a serine/threonine (Ser/Thr) protein kinase involved in a wide variety of signaling pathways concerning such as cell growth, survival, or cellular metabolism (del Peso et al, 1997; Wullschleger et al, 2006; Manning and Cantley, 2007). Cell lysates from different time points of infected THP-1 cells were analyzed by immunoblot analysis to identify the participation of important cellular protein kinases involved in cell signaling during pneumococcal phagocytosis
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