No abstract

Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and ␤-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival. Staphylococcus aureus is the primary cause of skin and soft tissue infections (SSTIs) in humans. In the United States alone, approximately 14 million people seek medical treatment each year for SSTIs associated with methicillin-sensitive or methicillin-resistant S. aureus (MSSA or MRSA, respectively) (1), and over 50% of cases are caused by MRSA strains (2). MRSA infections can persist and disseminate to deeper sites in the host, causing diseases such as endocarditis, osteomyelitis, or bacteremia, and are estimated to cause over 18,000 deaths per year in the United States (3). High hospitalization and mortality rates associated with MRSA are attributed to the bacterium's increasing drug resistance; MRSA strains are resistant to the beta-lactam drugs penicillin, methicillin, and oxacillin, and the emergence of vancomycin-resistant strains (4) means that few treatment options remain.S. aureus was once recognized primarily as a hospital-acquired (HA) pathogen that gained access to the host via indwelling medical devices. However, strains of community-acquired MRSA (CA-MRSA) have emerged that infect healthy individuals with no predisposing risk factors f...

The migration of mature dendritic cells (DCs) into the draining lymph node (dLN) is thought to depend solely on the chemokine receptor CCR7. CD301b DCs migrate into the dLN after cutaneous allergen exposure and are required for T helper 2 (Th2) differentiation. We found that CD301b DCs poorly upregulated CCR7 expression after allergen exposure and required a second chemokine signal, mediated by CCR8 on CD301b DCs and its ligand CCL8, to exit the subcapsular sinus (SCS) and enter the lymph node (LN) parenchyma. After allergen exposure, CD169SIGN-R1 macrophages in interfollicular regions produced CCL8, which synergized with CCL21 in a Src-kinase-dependent manner to promote CD301b DC migration. In CCR8-deficient mice, CD301b DCs remained in the SCS and were unable to enter the LN parenchyma, resulting in defective Th2 differentiation. We have defined a CCR8-dependent stepwise mechanism of DC-subset-specific migration through which LN CD169SIGN-R1 macrophages control the polarization of the adaptive immune response.

Blood-brain barrier (BBB) dysfunction is a feature of many neurodegenerative disorders. The mechanisms and interactions between astrocytes, extracellular matrix and vascular endothelial cells in regulating the mature BBB are poorly understood. We have previously shown that transitory GFAP-astrocyte loss, induced by systemic administration of 3-chloropropanediol, leads to reversible disruption of tight junction complexes and BBB integrity to a range of markers. However, early restoration of BBB integrity to dextran (10-70 kDa) and fibrinogen was seen in the absence of paracellular tight junction proteins claudin-5 and occludin. In the present study we show that in the GFAP-astrocyte lesioned rat inferior colliculus, paracellular expression of adherens junction proteins (VE-cadherin and β-catenin) was maintained in vascular endothelial cells that lacked paracellular claudin-5 expression and which showed reversible post-translational occludin modification. Claudin-1 expression paralleled the loss and recovery of claudin-5, while claudin -3 or -12 immunoreactivity was not detected. In addition, the extracellular matrix, as visualized by laminin and fibronectin, underwent extensive reversible remodeling and perivascular CD169 macrophages become abundant throughout the lesioned inferior colliculus. At a time that GFAP-astrocytes repopulated the lesion area and tight junction proteins were returned to paracellular domains, the extracellular matrix and leukocyte profiles normalized and resembled profiles seen in control tissue. This study supports the hypothesis that a combination of paracellular adherens junctional proteins, remodeled basement membrane and the presence perivascular leukocytes provide a temporary barrier to limit extravasation of macromolecules and potentially neurotoxic substances into the brain parenchyma until tight junction proteins are restored to paracellular domains.

Signaling mechanisms involved in regulating blood-brain barrier (BBB) integrity during central nervous system (CNS) inflammation remain unclear. We show that an imbalance between pro-/anti-inflammatory cytokines/chemokines alter claudin-5 expression. In vivo, gliotoxin-induced changes in glial populations and an imbalance between pro-/anti-inflammatory cytokine/chemokine expression occurred as BBB integrity was compromised. The balance was restored as BBB integrity was reestablished. In vitro, TNF-α, IL-6, and MCP-1 induced paracellular claudin-5 expression loss. TNF-α- and IL-6-, effects were mediated through the PI3K pathway and IL-10 attenuated TNF- α’s effect. This study shows that pro-/anti-inflammatory modulators play a critical role in BBB integrity during CNS inflammation.

Blood-brain barrier (BBB) integrity is compromised in many central nervous system disorders. Complex astrocyte and vascular endothelial cell interactions that regulate BBB integrity may be disturbed in these disorders. We previously showed that systemic administration of 3-chloropropanediol [(S)-(1)-3-chloro-1,2-propanediol] induces a transitory glial fibrillary acidic proteinastrocyte loss, reversible loss of tight junction complexes, and BBB integrity disruption. However, the intracellular signaling mechanisms that induce BBB integrity marker loss are unclear. We hypothesize that 3-chloropropanediol-induced modulation of tight junction protein expression is mediated through the phosphoinositide-3-kinase (PI3K)/AKT pathway. To test this hypothesis, we used a mouse brain endothelial cell line (bEnd.3) exposed to 3-chloropropanediol for up to 3 days. Results showed early reversible loss of sharp paracellular claudin-5 expression 90, 105, and 120 minutes after 3-chloropropanediol (500 mM) treatment. Sharp paracellular claudin-5 profiles were later restored, but lost again by 2 and 3 days after 3-chloropropanediol treatment. Western blot and immunofluorescence studies showed increased p85-PI3K expression and transitory increased AKT (Thr308) phosphorylation at 15 and 30 minutes after 3-chloropropanediol administration. PI3K inhibitors LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride; 2.5-25 mM] and PI-828 [2-(4-morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one; 0.1-10 mM] prevented the 3-chloropropanediol-induced AKT (Thr308) phosphorylation and both early and late loss of paracellular claudin-5. However, AKT inhibitors only prevented the early changes in claudin-5 expression. This mechanistic study provides a greater understanding of the intracellular signaling pathways mediating tight junction protein expression and supports a hypothesis that two independent pathways triggered by PI3K mediate early and late loss of paracellular claudin-5 expression.

Dendritic cells (DCs) of the cDC2 lineage are necessary for the initiation of the allergic immune response and in the dermis are marked by their expression of CD301b.CD301b + dermal DCs respond to allergens encountered in vivo, but not in vitro. This suggests that another cell in the dermis may sense allergens and relay that information 5 to activate and induce the migration of CD301b + DCs to the draining lymph node. Using a model of cutaneous allergen exposure, we show that allergens directly activate TRPV1 + sensory neurons leading to itch and pain behaviors. Allergen-activated sensory neurons release the neuropeptide Substance P, which stimulates proximally located CD301b + DCs through MRGPRA1. Substance P induces CD301b + DC migration to the 10 draining lymph node where they initiate Th2 differentiation. Thus, sensory neurons act as primary sensors of allergens, linking exposure to activation of allergic-skewing DCs and the initiation of the allergic immune response. 15

RATIONALE: We demonstrated that cord blood human progenitor cells (HPC) from high allergic risk newborns expressed Toll-like receptors (TLR) less abundantly than low allergic risk infants. We also have recently found a key role for thymic stromal lymphopoietin (TSLP) in human eosinophil and basophil differentiation from HPC in allergic subjects. However, TLR or TLR-induced TSLP receptor (TSLPR) expression by HPC has never been studied in allergic asthmatic subjects. METHODS: The study group comprised 10 healthy and 11 allergic asthmatic subjects. Asthmatics underwent diluent-controlled bronchial allergen challenge. Peripheral blood (PB) was collected from both healthy and asthmatic subjects (before and 24 hours after diluent/allergen challenge). The PB HPC-enriched cell population was stimulated ex vivo with TLR-2 (lipoteichoic acid, LTA), TLR-4 (lipopolysaccharide, LPS) or TLR-9 (ODN2006) ligands. HPC TLR and TSLPR expression after TLR ligation were examined pre-and post-bronchial allergen challenge by flow cytometry. RESULTS: Asthmatic HPC expressed significantly less TLR-2 and TLR-9, compared to non-asthmatic HPC (p<0.05), with a similar trend for TLR-4 (p50.057). TLR-4 stimulation of asthmatic HPC yielded significantly higher expression of TSLPR, compared with HPC from healthy controls (22.51 6 4.24% vs 11.6 6 1.34%, respectively; p<0.05), with a similar trend for asthmatic HPC after TLR-9 stimulation (p50.054). Allergen challenge led to a further increase in TSLPR expression by LPS-stimulated HPC, compared to diluent (32.37 6 4.14% vs 18.25 6 4.94%, respectively; p<0.05). CONCLUSIONS: These findings support the involvement of HPC in the initiation and persistence of airways inflammation during infection-driven asthma exacerbations.