Modulation of Dendritic Cell Activation and Subsequent Th1 Cell Polarization by Lidocaine.

Dendritic cells play an essential role in bridging innate and adaptive immunity by recognizing cellular stress including pathogen- and damage-associated molecular patterns and by shaping the types of antigen-specific T cell immunity. Although lidocaine is widely used in clinical settings that trigger cellular stress, it remains unclear whether such treatment impacts the activation of innate immune cells and subsequent differentiation of T cells. Here we showed that lidocaine inhibited the production of IL-6, TNFα and IL-12 from dendritic cells in response to toll-like receptor ligands including lipopolysaccharide, poly(I:C) and R837 in a dose-dependent manner. Notably, the differentiation of Th1 cells was significantly suppressed by the addition of lidocaine while the same treatment had little effect on the differentiation of Th17, Th2 and regulatory T cells in vitro. Moreover, lidocaine suppressed the ovalbumin-specific Th1 cell responses in vivo induced by the adoptive transfer of ovalbumin-pulsed dendritic cells. These results demonstrate that lidocaine inhibits the activation of dendritic cells in response to toll-like receptor signals and subsequently suppresses the differentiation of Th1 cell responses

Programmed Delay of a Virulence Circuit Promotes Salmonella Pathogenicity

To accomplish successful infection, pathogens must operate their virulence programs in a precise, time-sensitive, and coordinated manner. A major question is how pathogens control the timing of virulence gene expression during infection. Here we report that the intracellular pathogen Salmonella controls the timing and level of virulence gene expression by using an inhibitory protein, EIIANtr. A DNA binding master virulence regulator, PhoP, controls various virulence genes inside acidic phagosomes. Salmonella decreases EIIANtr amounts at acidic pH in a Lon- and PhoP-dependent manner. This, in turn, promotes expression of the PhoP-activated virulence program because EIIANtr hampers activation of PhoP-regulated genes by interfering with PhoP binding to DNA. EIIANtr enables Salmonella to impede the activation of PhoP-regulated gene expression inside macrophages. Our findings suggest that Salmonella achieves programmed delay of virulence gene activation by adjusting levels of an inhibitory factor.Signal transduction systems dictate various cellular behaviors in response to environmental changes. To operate cellular programs appropriately, organisms have sophisticated regulatory factors to optimize the signal response. The PhoP/PhoQ master virulence regulatory system of the intracellular pathogen Salmonella enterica is activated inside acidic macrophage phagosomes. Here we report that Salmonella delays the activation of this system inside macrophages using an inhibitory protein, EIIANtr (a component of the nitrogen-metabolic phosphotransferase system). We establish that EIIANtr directly restrains PhoP binding to its target promoter, thereby negatively controlling the expression of PhoP-activated genes. PhoP furthers its activation by promoting Lon-mediated degradation of EIIANtr at acidic pH. These results suggest that Salmonella ensures robust activation of its virulence system by suspending the activation of PhoP until a sufficient level of active PhoP is present to overcome the inhibitory effect of EIIANtr. Our findings reveal how a pathogen precisely and efficiently operates its virulence program during infection

Inhibition of dendritic cell-mediated antigen-specific Th1 cell responses by lidocaine <i>in vivo</i>.

<p>Bone marrow-derived dendritic cells were pulsed with OVA_323-339 in the presence of lidocaine or vehicle before being transferred into OT-II TcR transgenic mice (n = 3~4). (A and B) The frequencies of IFN- γ producers among Vα2⁺ cells. (C) The amounts of the indicated cytokines in the supernatant of splenocyte stimulated with OVA_323-339 were measured by ELISA. Data represent two independent experiments. Data shown are mean ± SEM. *<i>p</i><0.05.</p

Lidocaine inhibits dendritic cell-mediated Th1 cell differentiation while having little effects on dendritic cell-mediated Th2, Th17, regulatory T cell differentiation <i>in vitro</i>.

<p>Naïve CD4⁺ T cells were co-cultured with bone marrow-derived dendritic cells with Th1, Th17, Th2 or regulatory T cell differentiation conditioned-media or cultured with plate-coated anti-CD3 and anti-CD28 with supernatant of dendritic cells stimulated with LPS in the presence of lidocaine (0.2 mg/ml or indicated dose) or vehicle. (A-D) The frequencies of IFN-γ, IL–17, IL–4/5 or Foxp3 positive cells among CD4⁺ population were measured by flow cytometer. (E) The level of IFN-γ was measured using co-cultured supernatants from Th1 differentiation condition. Data represent at least two independent experiments. Data shown are mean ± SEM. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; NS, not significant.</p

Lidocaine inhibits dendritic cell-mediated Th1 cell differentiation <i>in vitro</i>.

<p>Naïve CD4⁺ T cells were either co-cultured with bone marrow-derived dendritic cells in the presence of soluble anti-CD3 and LPS, or in anti-CD3, CD28 pre-coated plates in the presence of IL–2 and IL–12 for Th1 cell differentiation. Lidocaine was added at a concentration of 0.2 mg/ml. (A & B) The frequencies of IFNγ or IL–17 producing cells among CD4⁺ T cells. (C) The mRNA levels of the indicated genes. (D) The levels of IFN-γ in the cultured supernatants of naïve CD4⁺ T cells cultured with vehicle- or lidocaine-conditioned media. Data represent at least three independent experiments. Data shown are mean ± SEM. **<i>p</i><0.01; ***<i>p</i><0.001; NS, not significant.</p

Effects of lidocaine on the expression of various cytokines upon LPS stimulation.

<p>Bone marrow-derived dendritic cells were stimulated with 100 ng/ml of LPS in the presence of vehicle (EtOH) or 0.2 mg/ml lidocaine for 4 h and 24 h to examine mRNA expression and cytokine production, respectively. (A) The mRNA levels of the indicated genes were analyzed by quantitative RT-PCR. (B) The amounts of each cytokine produced were measured by ELISA. All experiments were performed at least three times. Data shown are mean ± SEM. *<i>p</i><0.05; ***<i>p</i><0.001; ND, not detected.</p

Lidocaine regulates the expression of cytokines and NF-κB signaling pathway in a dose-dependent manner.

<p>(A) Bone marrow-derived dendritic cells were stimulated with 100 ng/ml of LPS together with increasing concentrations of lidocaine for 4 h. The mRNA levels of the indicated genes were analyzed by quantitative RT-PCR. (B) Raw 264.7 cells were treated with increasing doses of lidocaine for 2 h and stimulated with LPS for 20 min. The expression of IκB-α was examined by western blot. All experiments were performed at least three times. Data shown are mean ± SEM. *<i>p</i><0.05; **<i>p</i><0.01.</p

Regulation of cytokines expression in dendritic cells in response to various TLR ligands by lidocaine.

<p>(A & B) Bone marrow-derived dendritic cells were stimulated with LPS (100 ng/ml), poly(I:C) (1 μg/ml) or R837 (1 μg/ml) in the presence of lidocaine (0.4 mg/ml) or vehicle. The amounts of IL–6 and TNF-α in the supernatant were measured by ELISA. (C) The mRNA levels of the indicated genes were analyzed by quantitative RT-PCR. Data represent at least two independent experiments. Data shown are mean ± SEM. *<i>p</i><0.05; **<i>p</i><0.01; ND, not detected.</p

Critical regulation of follicular helper T cell differentiation and function by G alpha(13) signaling

GPCR-G alpha protein-mediated signal transduction contributes to spatiotemporal interactions between immune cells to fine-tune and facilitate the process of inflammation and host protection. Beyond this, however, how G alpha proteins contribute to the helper T cell subset differentiation and adaptive response have been underappreciated. Here, we found that G alpha 13 signaling in T cells plays a crucial role in inducing follicular helper T (Tfh) cell differentiation in vivo. T cell-specific G alpha 13-deficient mice have diminished Tfh cell responses in a cell-intrinsic manner in response to immunization, lymphocytic choriomeningitis virus infection, and allergen challenges. Moreover, G alpha 13-deficient Tfh cells express reduced levels of Bcl-6 and CXCR5 and are functionally impaired in their ability to adhere to and stimulate B cells. Mechanistically, G alpha 13-deficient Tfh cells harbor defective Rho-ROCK2 activation, and Rho agonist treatment recuperates Tfh cell differentiation and expression of Bcl-6 and CXCR5 in Tfh cells of T cell-specific G alpha 13-deficient mice. Conversely, ROCK inhibitor treatment hampers Tfh cell differentiation in wild-type mice. These findings unveil a crucial regulatory role of G alpha 13-Rho-ROCK axis in optimal Tfh cell differentiation and function, which might be a promising target for pharmacologic intervention in vaccine development as well as antibody-mediated immune disorders.Y