Oral Cell Lysates Reduce the Inflammatory Response of Activated Macrophages.

Necrotic cell damage occurs as a consequence of invasive dental procedures. Loss of membrane integrity being the hallmark of necrotic cells leads to the release of cytoplasmic and membranous components. Macrophages are predestined to respond to lysates originating from necrotic cells. Here, we implement necrotic lysates from human gingival fibroblasts, HSC2, and TR146 oral epithelial cell lines, and RAW264.7 macrophage cell lines to be tested for their potential to modulate the inflammatory response of macrophages. To this aim, necrotic cell lysates were prepared by sonication or freezing/thawing of the respective cell suspension. Necrotic cell lysates were tested for their potential to modulate the lipopolysaccharide (LPS)-induced expression of inflammatory cytokines using RAW264.7 macrophages as a bioassay. We show here that all necrotic cell lysates, independent of the origin and the preparation way, reduced the expression of IL1 and IL6 in LPS-induced RAW264.7 macrophages, most obviously shown for TR146 cells. This finding was supported in a bioassay when macrophages were exposed to poly (I:C) HMW, an agonist of TLR-3. Consistently, all necrotic lysates from gingival fibroblasts, HSC2, TR146, and RAW264.7 cells reduced the nuclear translocation of p65 in LPS-exposed macrophages. This screening approach supports the overall concept that necrotic cell lysates can modulate the inflammatory capacity of macrophages.

Blocking of Caspases Exerts Anti-Inflammatory Effects on Periodontal Cells.

Periodontitis is an inflammatory process that is associated with caspase activity. Caspases could thus become molecular targets for the modulation of the inflammatory response to harmful factors, such as lipopolysaccharides (LPS) and TNFα. Here, the impact of the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methyl ketone) on the modulation of the LPS-induced inflammatory response of murine RAW 264.7 cells and primary macrophages was examined. Moreover, the inflammatory responses of human gingival fibroblasts, HSC2 oral squamous carcinoma cells and murine ST2 mesenchymal fibroblasts when exposed to TNFα were studied. Data showed that Z-VAD-FMK significantly lowered the inflammatory response of RAW 264.7 cells and primary macrophages, as indicated by the expression of IL1 and IL6. In murine ST2 mesenchymal fibroblasts, the TNFα-induced expression of CCL2 and CCL5 was significantly reduced. In human gingival fibroblasts and HSC2 cells, Z-VAD-FMK considerably reduced the TNFα-induced expression of CXCL8 and CXCL10. These findings suggest that pharmacological blocking of caspases in an inflammatory environment lowers the expression of cytokines and chemokines in periodontal cells.

Disruption of mitochondrial membrane integrity induced by amyloid aggregates arising from variants of SOD1.

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for copper-zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS (fALS), aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. A growing body of evidence suggests that fALS-causing mutations destabilize the native structure of SOD1, leading to aberrant protein interactions for aggregation. In this work, we show that wild-type human SOD1 and two of its mutants (D101N, G72S) form amyloid like fibrils under destabilizing condition (in the presence of KSCN 0.2 M and DTT 50 mM) at 37 °C, pH 7.4. The formation of the aggregates was monitored by their ability to enhance the fluorescence of Thioflavin T (ThT) and their morphology was assessed by transmission electron microscopy (TEM). Furthermore, interaction of SOD1 aggregates with mitochondrial membrane of rat brain, as an in vitro biological model, with the aim of gaining an insight into possible mechanism of cytotoxicity at the membrane level was verified. Release of mitochondrial enzyme, malate dehydrogenase (MDH), upon exposure to SOD1 aggregates demonstrates that these aggregates could affect membrane permeability.