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Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E2 (PGE2) secreted by hAM-MSCs on neutrophil extracellular trap (NET) release and to characterize the role of its receptors (EP2/EP4) in PAD-4 and NFκB activity in neutrophils. Human peripheral blood neutrophils were ionomycin-stimulated in the presence of hAM-MSC conditioned medium (CM) treated or not with the selective PGE2 inhibitor MF-63, PGE2, EP2/EP4 agonists, and the selective PAD-4 inhibitor GSK-484. NET release, PAD-4, and NFκB activation were analyzed. Ionomycin induced NET release, which was inhibited in the presence of hAM-MSC-CM, while CM from hAM-MSCs treated with MF-63 prevented NET release inhibition. PGE2 and EP2/EP4 agonists, and GSK-484 inhibited NET release. EP2/EP4 agonists and GSK-484 inhibited H3-citrullination but did not affect PAD-4 protein expression. Finally, PGE2 and EP2/EP4 agonists and GSK-484 increased NFκB phosphorylation. Taken together, these results suggest that hAM-MSC exert their immunomodulatory activities through PGE2, inhibiting NET release in a PAD-4-dependent pathway. This research proposes a new mechanism by which hAM-MSC exert their activities when modulating the innate immune response and inhibiting NET release.

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Inflammatory responses are terminated by the clearance of dead cells, a process termed efferocytosis. A consequence of efferocytosis is the synthesis of the antiinflammatory mediators TGF-ß, PGE2, and IL-10; however, the efferocytosis of infected cells favors Th17 responses by eliciting the synthesis of TGF-ß, IL-6, and IL-23. Recently, we showed that the efferocytosis of apoptotic Escherichia coli-infected macrophages by dendritic cells triggers PGE2 production in addition to pro-Th17 cytokine expression. We therefore examined the role of PGE2 during Th17 differentiation and intestinal pathology. The efferocytosis of apoptotic E. coli-infected cells by dendritic cells promoted high levels of PGE2, which impaired IL-1R expression via the EP4-PKA pathway in T cells and consequently inhibited Th17 differentiation. The outcome of murine intestinal Citrobacter rodentium infection was dependent on the EP4 receptor. Infected mice treated with EP4 antagonist showed enhanced intestinal defense against C. rodentium compared with infected mice treated with vehicle control. Those results suggest that EP4 signaling during infectious colitis could be targeted as a way to enhance Th17 immunity and host defense.

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OBJECTIVE: This study investigated the effect of Low Intensity-pulsed Ultrasound (LIPUS) on the repair process of ruptured Achilles tendon using a rat model and also examined the regulation of a biological molecule that may contribute to this in vitro and in vitro. METHODS: To investigate the effect of LIPUS and its biological mechanism ofpromoting Achilles tendon repair after acute injury, ninety-eight male Sprague-Dawley (SD) rats (mean body weight, 258 ±9.8 g) aged 12 weeks were used in this study. To create the model, the Achilles tendon attachment site and musculotendinous junction were ruptured under direct vision. The leg on one side was exposed to LIPUS (frequency at 1.5 MHz, the repetition cycle at 1.0 kHz, the burst width at 200 msec and the power output at 45 mW/cm2), for 20 minutes daily with a 0.7 mm diameter probe. Results:Low Intensity-pulsed Ultrasound treatment accelerated the repair of the Achilles tendon compared to the untreated group, judged by electron microscopy. Both cyclo-oxygenase (COX)-2* and EP4* expressions were over-expressed in the LIPUS treated group in the inflammatory period, and TGFJ31* expression was markedly induced in LIPUS treated groups followed by collagen I* and III* expression in the repair and reconstitution process. CONCLUSION: These findings suggest that LIPUS is potentially able to accelerate the repair of acute ruptured Achilles tendon in several ways: by exaggerating inflammation by inducing COX-2 and EP4 and reconstituting tissue by inducing TGFJ31 followed by collagen I and III. (*: p < 0.05, **: 0.001).

OBJETIVO: Este estudio estuvo encaminado a investigar el efecto de los ultrasonidos pulsados de baja intensidad (LIPUS) sobre el proceso de reparación del tendón de Aquiles tras una ruptura, usando un modelo de rata. Asimismo, se examinó la regulación de una molécula biológica que puede contribuir a este proceso in vitro e in vitro. MÉTODOS: Con el fin de investigar el efecto de LIPUS y el mecanismo biológico por el cual este efecto promueve la reparación del tendón de Aquiles tras una lesión aguda, noventa y ocho ratas machos Sprague-Dawley (SD) (peso corporal promedio, 258 ± 9.8 g) de 12 semanas de edad fueron usadas en este estudio. Para crear el modelo, el sitio de ligazón microbiológica del tendón de Aquiles y la unión músculo-tendinosa fueron desgarrados bajo visión directa. La pierna de un lado fue expuesta a LIPUS (frecuencia de 1.5 MHz, ciclo de repetición de 1.0 kHz, ancho de ruptura de 200 msec, y potencia de salida de 45 mW/cm2), por 20 minutos diariamente con una sonda de 0.7 mm diámetro. RESULTADOS: El tratamiento de ultrasonidos pulsados de baja intensidad aceleró la reparación del tendón de Aquiles, en comparación con el grupo no tratado, según se apreció mediante el microscopio electrónico. Tanto la ciclo-oxygenasa (COX)-2* como las expresiones EP4* estuvieron sobe-expresadas en el grupo tratado con LIPUS en el periodo inflamatorio, y la expresión TGFfi1* fue marcadamente inducida en los grupos tratados con LIPUS seguidos por la expresión de colágeno I* y III* en el proceso de reparación y reconstitución. CONCLUSIÓN: Estos resultados sugieren que LIPUS puede potencialmente acelerar la reparación del tendón de Aquiles luego de un desgarramiento, de varias maneras: exagerando la inflamación mediante inducción de COX-2 y EP4 y reconstituyendo el tejido induciendo TGFfil seguido por colágeno I y III. (*: p < 0.05, **: 0.001).

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