Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background: Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood. Methodology/Principal Findings: We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c + cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c + cells from acute granulomas. As a consequence of their phenotype, CD11c + cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85Bspecific CD4 + IFNc + T cells or induce an IFNc response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c + cells from chronic lesions to stimulate a protective IFNc T cell response. Conclusions/Significance: Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explai

Wistin Exerts an Anti-Inflammatory Effect via Nuclear Factor-κB and p38 Signaling Pathways in Lipopolysaccharide-Stimulated RAW264.7 Cells

Inflammation is an immune response to cellular damage caused by various stimuli (internal or external) and is essential to human health. However, excessive inflammatory responses may be detrimental to the host. Considering that the existing drugs for the treatment of inflammatory diseases have various side effects, such as allergic reactions, stomach ulcers, and cardiovascular problems, there is a need for research on new anti-inflammatory agents with low toxicity and fewer side effects. As 4′,6-dimethoxyisoflavone-7-O-β-d-glucopyranoside (wistin) is a phytochemical that belongs to an isoflavonoid family, we investigated whether wistin could potentially serve as a novel anti-inflammatory agent. In this study, we found that wistin significantly reduced the production of nitric oxide and intracellular reactive oxygen species in lipopolysaccharide-stimulated RAW 264.7 cells. Moreover, wistin reduced the mRNA levels of pro-inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1β and IL-6) and significantly reduced the protein expression of pro-inflammatory enzymes (iNOS and COX-2). Furthermore, wistin reduced the activation of the nuclear factor-κB and p38 signaling pathways. Together, these results suggest that wistin is a prospective candidate for the development of anti-inflammatory drugs

Anti-inflammatory effects of TP1 in LPS-induced Raw264.7 macrophages

Abstract Inflammation is an essential defense mechanism in health; however, excessive inflammation contributes to the pathophysiology of several chronic diseases. Although anti-inflammatory drugs are essential for controlling inflammation, they have several side effects. Recent findings suggest that naturally derived compounds possess physiological activities, including anti-inflammatory, antifungal, antiviral, anticancer, and immunomodulatory activities. Therefore, this study aimed to investigate the anti-inflammatory effects and molecular mechanisms of 2,5,6-trimethoxy-p-terphenyl (TP1), extracted from the Antarctic lichen Stereocaulon alpinum, using in vitro models. TP1 treatment decreased the production of nitric oxide (NO) and reactive oxygen species (ROS) in LPS-stimulated Raw264.7 macrophages. Additionally, TP1 treatment significantly decreased the mRNA levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and the mRNA and protein levels of the pro-inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2). Moreover, TP1 suppressed lipopolysaccharide-induced phosphorylation of the NF-κB and MAPK signaling pathways in Raw264.7 macrophages. Conclusively, these results suggest that TP1 ameliorates inflammation by suppressing the expression of pro-inflammatory cytokines, making it a potential anti-inflammatory drug for the treatment of severe inflammatory diseases

Surface expression of activating and inhibitory costimulatory molecules and chemokine receptors is different on CD11c⁺ cells in acute and chronic granulomas.

<p>CD11c⁺ cells from granuloma single cell suspensions obtained from three, six and ten week systemically infected C57BL/6 mice were phenotyped using flow cytometry. A, Histograms represent fluorochrome surface staining with monoclonal antibodies against MHCII, activating costimulatory molecules (CD40, CD80 and CD86) and inhibitory costimulatory molecules (PD-L1 and PD-L2). Black-dashed histograms represent costimulatory molecule expression on naïve splenic CD11c⁺CD11b⁺ cells. Blacks arrows indicate directional shift in MHCII and costimulatory molecule expression compared to 3-week expression levels at time points where substantial change is observed. Histograms representative of 3 independent experiments with 3–7 mice per group. B, Average fold change between three and ten week time points in mean fluorescent intensity of MHCII and costimulatory molecules. Average generated from fold change in three independent experiments.</p

A portion of BCG is sustained within CD11c⁺ cells, both in acute and chronic granulomas.

<p>A, <i>Left</i>, Fluorescent images of CD11c-EYFP⁻ (left panels) and CD11c-EYFP⁺ cells (right panels) with dsRED BCG at three and ten weeks. Images magnified from 1000× images. DAPI nuclear stain (blue), dsRED BCG (red rods) and cytoplasmic CD11c-EYFP cells (green). <i>Right</i>, Average number of viable dsRED BCG rods per cell type (CD11c-EYFP+ or non-fluorescent) within the granuloma at three- and ten-weeks after infection. Data are represented as mean +/− SEM, <i>P<0.0001</i>. B, FACS plots generated from CD11c⁺ gate (left plot) and CD11b⁺CD11c⁻ gate (right plot). Boxed gate shows percentage of cells containing GFP-BCG. Gate placement was made from non-fluorescent-BCG infected CD11b+ and CD11c+ cells, <i>not shown</i>. Plots representative of 3- and 10-week time points with 3, 8 and 8 mice per group, respectively. C, Histograms generated from 10 week CD11c⁺ (1.17) and CD11b⁺CD11c⁻ (8.61) GFP BCG+ gates in (B).</p

Location of DCs in acute and chronic mycobacterium-granulomas is different.

<p>A, Schematic outline of DCs in the center, periphery or outside of the granuloma. B, Distribution of DC location from stained sections. 42, 43 and 17 lesions combined from three mice per time point were observed for CD11c⁺ location for three, six and ten weeks, respectively. C, CD11c-EYFP mice were systemically infected with dsRED BCG for three, six and ten weeks. EYFP expression (green cells) and DAPI nuclear stain (blue). Granulomas are outlined with white dashed lines and shown at 1000× magnification.</p