Streptococcus pyogenes is an important human pathogen that causes a variety of diseases including life-threatening invasive diseases, such as toxic shock and deep tissue infections. Although S. pyogenes are classically considered extracellular pathogens, a clinical significance of an intracellular source has been emphasized. In patients with deep tissue infections, an intracellular reservoir of S. pyogenes within macrophages was shown to contribute to prolonged bacterial persistence. Here we demonstrate that intracellular survival of S. pyogenes in macrophages is associated with an M1 protein-dependent intracellular trafficking in the phagosomal-lysosomal pathway, which results in impaired fusion with lysosomes. The phagocytic vacuoles harbouring M1 protein-expressing bacteria not only served as a safe haven for the bacteria, but also as a replicating niche. An M1 protein-dependent modulation of macrophages was further supported by differences in NF-ĸB signalling between cells infected with either the wild-type or M1 protein-deficient strains, thereby indicating a suppressed inflammatory response when M1 protein was involved. Evidence of egress of bacteria out of their host cell and subsequent re-infection of new cells emphasize the importance of intracellular bacteria as a reservoir for dissemination of infection and continued tissue injury.

Control of human tuberculosis (TB) requires induction and maintenance of both macrophage and T cell effector functions. We demonstrate that pulmonary TB patients with a vitamin D deficiency had significantly reduced local levels of the vitamin D-inducible antimicrobial peptide LL-37 in granulomatous lesions compared to distal parenchyma from the infected lung. Instead, TB lesions were abundant in CD3(+) T cells and FoxP3(+) regulatory T cells as well as IgG-secreting CD20(+) B cells, particularly in sputum-smear positive patients with cavitary TB. Mycobacteria-specific serum IgG titers were also elevated in patients with active TB. An up-regulation of the B cell stimulatory cytokine IL-21 correlated with mRNA expression of CD20, total IgG and also IL-10 in the TB lesions. Altogether, vitamin D-deficient TB patients expressed a weak antimicrobial response but an IL-21 associated expansion of IgG-secreting B cells combined with a rise in FoxP3(+) regulatory T cells at the local site of infection.

During the process of differentiation, osteoblasts commit through strictly controlled checkpoints under the influence of several growth factors, cytokines, and extracellular matrix (ECM) proteins. The mineralized tissue-specific ECM component osteoadherin (OSAD) belongs to the small leucine-rich repeat protein family of proteoglycans. Proteoglycans modulate cellular behavior either through the attached glycosaminoglycan chains or by direct protein-protein interactions via the core protein sequences. Leucine-rich repeats have been shown to directly interact with cell-surface receptors such as epidermal growth factor receptor, blocking its ability to bind its ligand. In the present study, we investigated the influence of OSAD on the behavior and maturation of MC3T3E1 osteoblasts. OSAD overexpression and repression clones were created by stably transfecting with plasmids coding for either mouse OSAD cDNA or small-hairpin RNA, targeted against mouse OSAD. Overexpression of OSAD resulted in an increase of osteoblast differentiation features, such as increased alkaline phosphatase (ALP) activity and increased in vitro mineralization, as well as reduced proliferation and migration. Bone sialoprotein (BSP) levels were unchanged, while upregulation of osteocalcin (OC) and osteoglycin (OGN) was observed. Conversely, repression of OSAD expression resulted in increased cell proliferation and migration. BSP and OC were unaffected, while OGN was downregulated. ALP activity was reduced, though no change in in vitro mineralization was observed. We conclude that OSAD overexpression enhanced the differentiation and maturation of osteoblasts in vitro.

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