Decursinol Angelate Mitigates Sepsis Induced by Methicillin-Resistant Staphylococcus aureus Infection by Modulating the Inflammatory Responses of Macrophages

The herbal plant Angelica gigas (A. gigas) has been used in traditional medicine in East Asian countries, and its chemical components are reported to have many pharmacological effects. In this study, we showed that a bioactive ingredient of A. gigas modulates the functional activity of macrophages and investigated its effect on inflammation using a sepsis model. Among 12 different compounds derived from A. gigas, decursinol angelate (DA) was identified as the most effective in suppressing the induction of TNF-α and IL-6 in murine macrophages. When mice were infected with a lethal dose of methicillin-resistant Staphylococcus aureus (MRSA), DA treatment improved the mortality and bacteremia, and attenuated the cytokine storm, which was associated with decreased CD38+ macrophage populations in the blood and liver. In vitro studies revealed that DA inhibited the functional activation of macrophages in the expression of pro-inflammatory mediators in response to microbial infection, while promoting the bacterial killing ability with an increased production of reactive oxygen species. Mechanistically, DA treatment attenuated the NF-κB and Akt signaling pathways. Intriguingly, ectopic expression of an active mutant of IKK2 released the inhibition of TNF-α production by the DA treatment, whereas the inhibition of Akt resulted in enhanced ROS production. Taken together, our experimental evidence demonstrated that DA modulates the functional activities of pro-inflammatory macrophages and that DA could be a potential therapeutic agent in the management of sepsis

Mammalian Target of Rapamycin Protein Complex 2 Regulates Differentiation of Th1 and Th2 Cell Subsets via Distinct Signaling Pathways

SummaryMany functions of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) have been defined, but relatively little is known about the biology of an alternative mTOR complex, mTORC2. We showed that conditional deletion of rictor, an essential subunit of mTORC2, impaired differentiation into T helper 1 (Th1) and Th2 cells without diversion into FoxP3+ status or substantial effect on Th17 cell differentiation. mTORC2 promoted phosphorylation of protein kinase B (PKB, or Akt) and PKC, Akt activity, and nuclear NF-κB transcription factors in response to T cell activation. Complementation with active Akt restored only T-bet transcription factor expression and Th1 cell differentiation, whereas activated PKC-θ reverted only GATA3 transcription factor and the Th2 cell defect of mTORC2 mutant cells. Collectively, the data uncover vital mTOR-PKC and mTOR-Akt connections in T cell differentiation and reveal distinct pathways by which mTORC2 regulates development of Th1 and Th2 cell subsets

Extracellular Release of CD11b by TLR9 Stimulation in Macrophages.

CpG-DNA upregulates the expression of pro-inflammatory cytokines, chemokines and cell surface markers. Investigators have shown that CD11b (integrin αM) regulates TLR-triggered inflammatory responses in the macrophages and dendritic cells. Therefore, we aimed to identify the effects of CpG-DNA on the expression of CD11b in macrophages. There was no significant change in surface expression of CD11b after CpG-DNA stimulation. However, CD11b was released into culture supernatants after stimulation with phosphorothioate-backbone modified CpG-DNA such as PS-ODN CpG-DNA 1826(S). In contrast, MB-ODN 4531 and non-CpG-DNA control (regardless of backbone type and liposome-encapsulation) failed to induce release of CD11b. Therefore, the context of the CpG-DNA sequence and phosphorothioate backbone modification may regulate the effects of CpG-DNA on CD11b release. Based on inhibitor studies, CD11b release is mediated by p38 MAP kinase activation, but not by the PI3K and NF-κB activation. CD11b release is mediated by lysosomal degradation and by vacuolar acidification in response to CpG-DNA stimulation. The amount of CD11b in the exosome precipitant was significantly increased by CpG-DNA stimulation in vivo and in vitro depending on TLR9. Our observations perhaps give more insight into understanding of the mechanisms involved in CpG-DNA-induced immunomodulation in the innate immunity

Cdc42 promotes host defenses against fatal infection

The small Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, invasion, migration, differentiation, and morphogenesis. As the role of Cdc42-dependent signaling in fibroblasts in vivo is unknown, we attempted to specifically delete it in these cells by crossing the Cdc42(fl/fl) mouse with an fibroblast-specific protein 1 (FSP1)-Cre mouse, which is thought to mediate recombination exclusively in fibroblasts. Surprisingly, the FSP1-Cre;Cdc42(fl/fl) mice died at 3 weeks of age due to overwhelming suppurative upper airway infections that were associated with neutrophilia and lymphopenia. Even though major aberrations in lymphoid tissue development were present in the mice, the principal cause of death was severe migration and killing abnormalities of the neutrophil population resulting in an inability to control infection. We also show that in addition to fibroblasts, FSP1-Cre deleted Cdc42 very efficiently in all leukocytes. Thus, by using this nonspecific Cre mouse, we inadvertently demonstrated the importance of Cdc42 in host protection from lethal infections and suggest a critical role for this small GTPase in innate immunity

CpG-DNA-induced release of CD11b is linked to p38 MAP kinase activation, lysosomal degradation, and vacuolar acidification.

<p>RAW 264.7 cells were pretreated with each inhibitor. The cells were then stimulated for 24 h with CpG-DNA 1826(S) (5 μg/ml). Then, the cell culture media and cell lysates were collected, and the amount of CD11b was monitored by Western blotting. β-actin expression level was used as a control. One representative experiment of three is shown. (A, graph) The band intensity of CD11b in the cell lysates of the media control was taken as 1.0 and relative intensity of the CD11b protein bands in the supernatant and cell lysates compared with the control was calculated. * p < 0.01 (Significant decrease).</p

Induction of CD11b release by CpG-DNA stimulation is associated with exosome.

<p>(A) RAW 264.7 cells were treated with PBS, CpG-DNA 1826(S) (1826(S)), or non-CpG-DNA 2041(S) (2041(S)) for 24 h. The cell culture media and cell lysates (left panel) were collected, and the CD11b was monitored by Western blotting. β-actin expression level was used as a control. Exosome precipitant (right panel) was collected with ExoQuick-TC^™ exosome precipitation solution, and the CD11b was monitored by Western blotting. CD81 was used as an exosome control protein. (B) Macrophages obtained from the peritoneal cavity of BALB/c mice and TLR9 knockout mice were treated with PBS, CpG-DNA 1826(S) (1826(S)), or non-CpG-DNA 2041(S) (2041(S)) for 24 h. The cell lysates (left panel) and exosome precipitant (right panel) were collected, and the CD11b was monitored by Western blotting. (C) BALB/c mice and TLR9 knockout mice were i.p. injected with CpG-DNA 1826(S) (1826(S)), non-CpG-DNA 2041(S) (2041(S)), incomplete Freund’s adjuvants, or alum. After 24 h, peritoneal cavity fluids and peritoneal cavity macrophages were collected. CD11b expression in the cell lysates and exosome precipitant was monitored by Western blotting. One representative experiment of three is shown.</p

Effect of CpG-DNA on the expression of CD11b and CD18 in RAW 264.7 cells.

<p>RAW 264.7 cells were treated with MB-ODN 4531 (4531(O), 4531(S)), CpG-DNA 1826 (1826(O), 1826(S)), non-CpG-DNA 2041 (2041(O), 2041(S)) or LPS for 24 h. After stimulation, RAW 264.7 cells were harvested and the expressions of CD11b (A) and CD18 (B) were analyzed by flow cytometry. Each bar indicates the Mean ± SD of average mean fluorescence intensities (log scale) from three independent experiments. (O), phosphodiester CpG-DNA, (S) phosphorothioate-modified backbone CpG-DNA. For example, the phosphorothioate version of MB-ODN 4531(O) is MB-ODN 4531(S).</p