In "Vitro" Lps-Stimulated Sertoli Cells Pre-Loaded With Microparticles: Intracellular Activation Pathways

Abstract

Sertoli cells (SC) are immune privileged cells with the capacity of modulating the immune response by expressing several immune-regulatory factors. SC have the capacity to respond to external stimuli through innate phagocytic and antibacterial activities. This evidence evoked a potential role of SC as drug carriers and therapeutic agents. Such stimuli drive SC towards a still unknown evolution, the clinical relevance of which as yet remains undisclosed. This study sought to investigate the effects of external stimuli in the form of polymeric microparticles (MP) and bacteria derived endotoxins, such as lipopolysaccharides (LPS), in order to identify the pathways potentially involved in cell phenotype modifications. Compared to single stimulation, when combined, MP and LPS provoked a significant increase in the gene expression of IDO, PD-L1, FAS-L, TLR-3, TLR-4, MHC-II, ICAM-1, TFGβ1, BDF123, BDF129, BDF3 and pEP2C. Western Blotting analysis demonstrated up-regulation of the ERK 1-2 and NF-kB p65 phosphorylation ratios. Our study, showing the exponential increase of these mediators upon combined MP and LPS stimulation, suggests a "switch" of SC function from typical cells of the blood-testicular barrier to nonprofessional tolerogenic antigen-presenting cells. Further studies should target the clinical and technological implications of such stimuli-induced SC transformation.

Keywords: Sertoli cells; lipopolysaccharide; microparticles; nonprofessional tolerogenic; pro-inflammatory pathways.

Figure 1

MP characterization and uptake. Dimensional analysis obtained by granulometer (A) and SEM image of MP (B).

Figure 2

Real-Time PCR. Gene expressions of IDO, PD-L1, FAS-L, TFGβ1, TFGβ3 (A) TLR-3, TLR-4, MHC-II, ICAM-1 (B) BDF123, BDF129, BDF3, BDF4 and pEP2C (C) were evaluated by Real Time PCR. See text for more details. Data represent the mean ± S.E.M. (*p < 0.05 and **p < 0.001 respect to untreated SC) of three independent experiments, each performed in triplicate.

Figure 3

WB analysis. Immunoblots of phospho-ERK1-2/ERK1-2 and NF-kB p65 (A). Densitometric analysis of the protein bands of phospho- ERK1-2/ERK1-2 and NF-kB p65 (B). Data represent the mean ± S.E.M. (*p < 0.05 and **p < 0.001, respect to untreated SC) of three independent experiments, each performed in triplicate.