Effects of Azithromycin on Gene Expression Profiles of Proinflammatory and Anti-inflammatory Mediators in the Eyelid Margin and Conjunctiva of Patients With Meibomian Gland Disease

Studies on rat testicular macrophages (TMs) have indicated that these cells play an important role in testis function by supporting the immunosuppressive environment that protects developing germ cells and by responding to pathogens. By comparison, mouse TMs are essentially uncharacterised due to difficulties in isolating sufficient cells for study. We have established a technique for isolating 95% pure TMs from adult mice by differential adherence. Mouse TMs were cultured for 3h with saline, 10 or 100 ng/mL lipopolysaccharide (LPS) and compared with resident peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMMs). Expression of inflammatory regulators was determined using real-time Q-PCR and AgilentTM microarray analysis. Microarray analysis indicated that each macrophage type displayed very distinct gene expression profiles. There were 526 genes uniquely expressed in TMs at basal levels compared with the other macrophages and 268 genes uniquely expressed by TMs after LPS treatment. Q-PCR determined that LPS induced expression of the anti-inflammatory cytokine interleukin (IL)-10 in each of the macrophage types, with BMMs the strongest responders. LPS stimulated IL-10 mRNA approximately 100-fold in TMs, but only 20-fold in PMs. The anti-inflammatory transforming growth factor-β1 was not significantly induced at this time-point in any macrophage type. In terms of pro-inflammatory mediators, the TM response to LPS was always lower compared to the BMMs. Compared to PMs, the responses of TMs were similar for the hallmark pro-inflammatory cytokine tumour necrosis factor- a, but 40% less for IL-1β. TMs were also deficient in production of IL-6 and cyclooxygenase-2 and IL-12. TMs were therefore relatively strong responders to LPS in terms of IL-10, but weak responders in terms of pro-inflammatory mediators, indicating an immunosuppressive phenotype. The isolation and gene measurement methods established in this study will allow us to use knockout and transgenic mouse models to determine the role for TMs in testicular inflammation/fibrosis models.

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Abstract 149: Regulation of Pathogen-Associated Molecular Pattern Recognition by the Apo A-I Mimetic Peptide 4F

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a149 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Geeta Datta ◽

David K Crossman ◽

Lesley E Smythies ◽

M N Palgunachari ◽

Manjula Chaddha ◽

...

Keyword(s):

Gene Expression ◽

Inflammatory Responses ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Rna Isolation ◽

Mimetic Peptide ◽

M1 Macrophage ◽

Vehicle Treatment ◽

Anti Inflammatory ◽

Pre Treatment

The apolipoprotein A-I (apoA-I) mimetic peptide 4F displays prominent anti-inflammatory properties, including the ability to reduce vascular macrophage content. Macrophages are a heterogenous group of cells that are represented by two principal phenotypes, the classically-activated M1 macrophage and an alternatively-activated M2 phenotype. We previously reported that apoA-I and 4F favor the differentiation of human monocytes to an anti-inflammatory phenotype similar to that displayed by M2 macrophages. Further, 4F treatment attenuated LPS-induced inflammatory responses in monocyte-derived macrophages (MDMs). In the current study, we investigated effects of 4F and vehicle on LPS-induced gene expression in human MDMs by microarray analysis. RNA isolation, labeling and hybridization were performed, and the transcriptional profile was examined using the Human Gene ST 1.0 Affymetrix chip. Analysis of MDM gene expression profiles revealed that 4F modulated mRNA expression for 1099 genes (± 2-fold change, p<0.05), of which 149 genes regulated inflammatory responses. LPS treatment of MDMs significantly up-regulated genes encoding Toll-like receptors (TLR1, 2, 4, 6, and 8) compared to vehicle treatment. These responses were attenuated by 4F treatment. MyD88, CD14, IRAK4, TRAF6, TRAF3, MALT1 and IKBKB, genes that modulate NF-κB activation and subsequent cytokine synthesis, were also reduced by 4F. Corroborating this, FACS analyses showed that pre-treatment of MDMs with 4F reduced the LPS-dependent phosphorylation of NF-κB by 70% compared to vehicle treatment. These 4F-induced responses were also associated with a reduction in TNF-α and IL-6 secretion. These data suggest that an important anti-inflammatory mechanism of 4F action may be to down-regulate genes involved in the TLR signaling pathway, thus attenuating the responsiveness of macrophages to LPS and other pathogen-associated molecular patterns (PAMPs).

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SIRT1 Exerts Anti-Inflammatory Effects and Improves Insulin Sensitivity in Adipocytes

Molecular and Cellular Biology ◽

10.1128/mcb.00705-08 ◽

2008 ◽

Vol 29 (5) ◽

pp. 1363-1374 ◽

Cited By ~ 284

Author(s):

Takeshi Yoshizaki ◽

Jill C. Milne ◽

Takeshi Imamura ◽

Simon Schenk ◽

Noriyuki Sonoda ◽

...

Keyword(s):

Gene Expression ◽

Glucose Uptake ◽

Insulin Signaling ◽

Energy Homeostasis ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Serine Phosphorylation ◽

Cellular Functions ◽

Necrosis Factor Alpha ◽

Anti Inflammatory

ABSTRACT SIRT1 is a prominent member of a family of NAD+-dependent enzymes and affects a variety of cellular functions ranging from gene silencing, regulation of the cell cycle and apoptosis, to energy homeostasis. In mature adipocytes, SIRT1 triggers lipolysis and loss of fat content. However, the potential effects of SIRT1 on insulin signaling pathways are poorly understood. To assess this, we used RNA interference to knock down SIRT1 in 3T3-L1 adipocytes. SIRT1 depletion inhibited insulin-stimulated glucose uptake and GLUT4 translocation. This was accompanied by increased phosphorylation of JNK and serine phosphorylation of insulin receptor substrate 1 (IRS-1), along with inhibition of insulin signaling steps, such as tyrosine phosphorylation of IRS-1, and phosphorylation of Akt and ERK. In contrast, treatment of cells with specific small molecule SIRT1 activators led to an increase in glucose uptake and insulin signaling as well as a decrease in serine phosphorylation of IRS-1. Moreover, gene expression profiles showed that SIRT1 expression was inversely related to inflammatory gene expression. Finally, we show that treatment of 3T3-L1 adipocytes with a SIRT1 activator attenuated tumor necrosis factor alpha-induced insulin resistance. Taken together, these data indicate that SIRT1 is a positive regulator of insulin signaling at least partially through the anti-inflammatory actions in 3T3-L1 adipocytes.

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