Phenotype and Response to PAMPs of Human Monocyte-Derived Foam Cells Obtained by Long-Term Culture in the Presence of oxLDLs.

Cholesterol-laden, foam macrophages constitute the most characteristic component of human atherosclerotic plaques. Persistent uptake of oxLDLs results in accumulation of lipid bodies inside the cells and determines their phenotype and subsequent functions. In this work, we describe the phenotype of human monocyte-derived foam cells obtained by differentiation in the constant presence of oxLDLs for 30 days (prolonged-hMDFCs). Although neither the total cellular nor the cell surface expression of Toll-like receptors (TLR) was regulated by oxLDLs, the prolonged-hMDFCs changed dramatically their responsiveness to TLR ligands and inactivated bacteria. Using multiplex technology, we observed an acute decline in cytokine and chemokine production after surface and endosomal TLR stimulation with the exception of TLR2/6 triggering with agonists Pam2CSK4 and MALP-2. We also noted significant reduction of some surface receptors which can have accessory function in recognition of particulate antigens (CD47, CD81, and CD11b). In contrast, the prolonged-hMDFCs responded to inflammasome activation by LPS/nigericin with extensive, necrotic type cell death, which was partially independent of caspase-1. This pyroptosis-like cell death was aggravated by necrostatin-1 and rapamycin. These findings identify a potential contribution of mature foam cells to inflammatory status by increasing the immunogenic cell death burden. The observed cross-talk between foam cell death pathways may lead to recognition of a potential new marker for atherosclerosis disease severity. Overall, our study demonstrates that, in contrast to other cellular models of foam cells, the prolonged-hMDFCs acquire a functional phenotype which may help understanding the role of foam cells in the pathogenesis of atherosclerosis.

Bilateral slipped capital femoral epiphysis as first manifestation of primary hyperparathyroidism in a 15-year-old boy.

ABSTRACT: Primary hyperparathyroidism (PHP) in children is a rare condition and has a very dynamic course with nonspecific symptoms, what complicates the diagnosis and delays PHP treatment. CASE PRESENTATION: A 15-year-old boy was admitted to the Orthopedic Ward with the diagnosis of juvenile bilateral slipped capital femoral epiphysis and valgus deformities. Gait disturbances, limb pains and valgus knee deformities, polyuria, polydipsia and weight loss, have been increasing for 8 months. Despite the hypercalcemia found in laboratory tests and bone destruction demonstrated in computed tomography of the hips, orthopedic correction was performed. In histopathological examination -brown bone tumors. The PTH concentration was determined (PTH - 589.1 pg/ml; (N: 10-60) and the child was referred to the Department of Pediatric Endocrinology, where severe hypercalcemia (Ca - 4.07 mmol/l, N: 2.2-2.84) and hypophosphatemia (P - 0.68 mmol/l; N: 0.95-1.75) and adenoma of the left lower parathyroid gland was diagnosed. Forced diuresis, loop diuretics and pamidronic acid were used to obtain normocalcemia. The complications of hypercalcemia were excluded; diagnostic management excluding multiple endocrine neoplasia type 1 and 2a (MEN 1 and MEN 2A) syndrome were performed. The child was referred to the Department of Endocrinological Surgery, where the adenoma of the left inferior parathyroid gland was resected. CONCLUSIONS: 1) Patients with PHP should be diagnosed of the condition based on clinical symptoms. In patients with specific symptoms, it is necessary to determine serum Ca levels, especially prior to the surgical procedures. 2) In each case of PHP, determinations should be made of blood PTH, Ca and P and detection of MEN 1 and MEN 2A syndromes. 3) Patients with hyperparathyroidism require management of multiorgan complications of hypercalcemia. 4) Following surgical treatment of parathyroid adenoma, long-term endocrinological follow-up is necessary.

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages.

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on Mϕ. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived Mϕs (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (α and ß) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria Staphylococcus aureus (SA) and Porphyromonas gingivalis (PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a "signaling complex chaperone," with activity that is essential for cytokine response but not for target engulfment by Mϕ.

Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720.

The sphingolipid analog fingolimod is known to induce apoptosis of tumor cells and lymphocytes. Its effect on neutrophils has not been investigated so far. Here, we describe a fingolimod-induced atypical cell death mechanism in human neutrophils, characterized by rapid translocation of heat shock protein 27 to the cell surface, extensive cell swelling and vacuolization, atypical chromatin staining and nuclear morphology, and phosphorylation of mixed lineage kinase domain-like protein. Fingolimod also induces typical apoptotic features, including rapid externalization of phosphatidylserine and activation of caspase-8. Fingolimod-induced neutrophil death is independent of sphingosine-1-phosphate receptors and positively regulated by protein phosphatase A. Externalization of phosphatidylserine and heat shock protein 27 can be partially inhibited by inhibitors of caspase-8 [Z-Ile-Glu(O-Me)-Thr-Asp(O-Me)-fluoromethyl ketone], receptor-interacting protein kinase 1 (necrostatin-1), receptor-interacting protein kinase 3 (necrosulfonamide), and heat shock protein 90 [geldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin]. Furthermore, NADPH oxidase 1 inhibition with diphenyleneiodonium chloride protects neutrophils against fingolimod-mediated cell death. Overall, these observations suggest that fingolimod acts through a mechanism involving the necrosome signaling complex and the oxidative stress machinery.

Oxidized LDLs inhibit TLR-induced IL-10 production by monocytes: a new aspect of pathogen-accelerated atherosclerosis.

It is widely accepted that oxidized low-density lipoproteins and local infections or endotoxins in circulation contribute to chronic inflammatory process at all stages of atherosclerosis. The hallmark cells of atherosclerotic lesions-monocytes and macrophages-are able to detect and integrate complex signals derived from lipoproteins and pathogens, and respond with a spectrum of immunoregulatory cytokines. In this study, we show strong inhibitory effect of oxLDLs on anti-inflammatory interleukin-10 production by monocytes responding to TLR2 and TLR4 ligands. In contrast, pro-inflammatory tumor necrosis factor secretion was even slightly increased, when stimulated with lipopolysaccharide from Porphyromonas gingivalis-an oral pathogen associated with atherosclerosis. The oxLDLs modulatory activity may be explained by altered recognition of pathogen-associated molecular patterns, which involves serum proteins, particularly vitronectin. We also suggest an interaction between vitronectin receptor, CD11b, and TLR2. The presented data support a novel pathway for pathogen-accelerated atherosclerosis, which relies on oxidized low-density lipoprotein-mediated modulation of anti-inflammatory response to TLR ligands.