The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta4) on NFkappaB protein levels, activation, phosphorylation, and nuclear translocation in a model of tumor necrosis factor (TNF)-alpha-mediated corneal inflammation. Transformed and primary (HCET and HCEC) human corneal epithelial cells were stimulated with the pro-inflammatory cytokine TNF-alpha and treated or not with Tbeta4. Nuclear NFkappaB p65 subunit protein levels were assayed using ELISA, and activity was measured by determining NFkappaB binding to consensus oligonucleotides. NFkappaB p65 protein phosphorylation was also measured by ELISA. Nuclear translocation of NFkappaB p65 subunit was assayed by immunofluorescence microscopy. Compared to non-treated controls, Tbeta4 treatment significantly decreased nuclear NFkappaB protein levels, NFkappaB activity and p65 subunit phosphorylation in corneal epithelial cells after TNF-alpha stimulation. In TNF-alpha-stimulated corneal epithelial cells, NFkappaB p65 subunit translocation to the nucleus was observed using immunofluorescence microscopy. In contrast, Tbeta4 blocked nuclear translocation of the NFkappaB p65 subunit in TNF-alpha-stimulated corneal epithelial cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NFkappaB, thus both are known mediators of the inflammatory process. Tbeta4, a protein with diverse cellular functions including wound healing and suppression of inflammation, inhibits the activation of NFkappaB in TNF-alpha-stimulated cells. These results have important clinical implications for the potential role of Tbeta4 as a corneal anti-inflammatory agent.
Tbeta4 treatment decreases corneal inflammation and modulates the MMP/TIMP balance and thereby promotes corneal wound repair and clarity after alkali injury. These results suggest that Tbeta4 may be useful clinically to treat severe inflammation-mediated corneal injuries.
SummaryCitrate, a central component of cellular metabolism, is a widely used anticoagulant due to its ability to chelate calcium. Adenosine triphosphate (ATP)-citrate lyase, which metabolizes citrate, has been shown to be essential for inflammation, but the ability of exogenous citrate to impact inflammatory signalling cascades remains largely unknown. We hypothesized that citrate would modulate inflammatory responses as both a cellular metabolite and calcium chelator, and tested this hypothesis by determining how clinically relevant levels of citrate modulate monocyte proinflammatory responses to lipopolysaccharide (LPS) in a human acute monocytic leukaemia cell line (THP-1). In normal medium (0Á4 mM calcium), citrate inhibited LPS-induced tumour necrosis factor (TNF)-a and interleukin (IL)-8 transcripts, whereas in medium supplemented with calcium (1Á4 mM), TNF-a and IL-8 levels increased and appeared independent of calcium chelation. Using an IL-8-luciferase plasmid construct, the same increased response was observed in the activation of the IL-8 promoter region, suggesting transcriptional regulation. Tricarballylic acid, an inhibitor of ATP-citrate lyase, blocked the ability of citrate to augment TNF-a, linking citrate's augmentation effect with its metabolism by ATP-citrate lyase. In the presence of citrate, increased histone acetylation was observed in the TNF-a and IL-8 promoter regions of THP-1 cells. We observed that citrate can both augment and inhibit proinflammatory cytokine production via modulation of inflammatory gene transactivation. These findings suggest that citrate anti-coagulation may alter immune function through complex interactions with the inflammatory response.
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