Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.

Monocytes and macrophages are central cells of the innate immune system, responding to a diverse repertoire of pathogens. These cells originate from a common myeloid precursor in the bone marrow and while sharing responsibilities during innate immunity, differ greatly in their lifespan. Normally, blood monocytes live for just few days before undergoing apoptosis. Macrophages, in contrast, live up for months. Monocytes' lifespan can switch dramatically, from prolonged survival during inflammation to apoptosis as inflammation resolves. Interestingly, many of the mechanisms mediating survival during inflammation and cancer also operate in monocyte/macrophage differentiation. Differentiation and inflammatory stimuli determine monocyte/macrophage lifespan, by blocking the apoptotic pathway and activating a myriad of survival pathways. How these complicated networks of survival and apoptotic regulators are integrated remains yet to be fully elucidated. The present review summarizes the different monocytes' subpopulations and their function during pathogen recognition. We discuss the role of the caspases and the mechanisms that determine monocytes/macrophages fate highlighting their significance in the regulation of inflammatory diseases.

The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors’ accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo.

Apigenin, a natural plant flavonoid with antiproliferative activity, is emerging as a promising compound for cancer prevention and therapy, but its mechanism of action remains unclear. High expression of the small heat-shock protein-27 (Hsp27) in leukemia contributes to the resistance of these cells to cancer treatments. Changes in Hsp27 phosphorylation have been associated with heat and metabolic stress, but its role in flavonoid anticancer activity has not been investigated. In this study, we examined the effect of apigenin in the regulation of Hsp27 on leukemia. We showed that apigenin does not affect Hsp27 expression but induces a bimodal phosphorylation on Ser78 and Ser82. The phosphorylation at early times was regulated by p38. At later times, Hsp27 phosphorylation was dependent on p38 activity and for some residues on PKCδ. Silencing of p38 expression reduced apigenin-induced phosphorylation on Ser15, Ser78, and Ser82, whereas silencing of PKCδ expression reduced the phosphorylation on Ser15 and Ser82 without affecting Ser78. In addition, we found that apigenin-induced PKCδ activity is mediated by p38. We also showed that the phosphorylation of Hsp27 significantly increased the susceptibility of leukemia cells to apigenin-induced apoptosis. Together, these results identify a complex signaling network regulating the cytotoxic effect of apigenin through Hsp27 phosphorylation.

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase^-1−/− and Nlrc4^−/− allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc^−/− macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.