[en] Introduction and objectives: Macrophages present in the synovial lining of the joints are involved in tissue inflammation, thus participating in the progression of osteoarthritis. Specifically, there is an increase in the percentage of macrophages type M1 (pro-inflammatory) over those of type M2 (anti-inflammatory). Hydrogen sulfide (H2S), a small gaseous molecule that is synthesized among other tissues in the joint, is involved in the polarization of macrophages maintaining the M1/M2 homeostatic balance. However, recent studies indicate a reduction in the enzymatic synthesis of H2S in OA patients, and specifically in their metabolic phenotype. In this work we evaluated whether a stress due to high glucose levels could favor the polarization of macrophages towards type M1, and if the administration of an exogenous source of H2S could attenuate these effects. Methods: An in vitro model of osteoarthritis associated with diabetes (DB) in macrophages using the TPH-1 monocyte cell line was performed. First, cells were differentiated to mature macrophages with phorbol myristate acetate (PMA). Cells were then stimulated with lipopolysaccharide (LPS) under normal (GN; 1g/l) or high glucose (GA; 4,5 g/l) conditions in the presence or absence of an exogenous source of H2S (Gyy-4137, a slow gas releaser). Macrophage polarization was monitored by gene and protein expression studies evaluating surface markers using flow cytometry. In addition, cell proliferation and release of the pro-inflammatory mediator interleukin 6 (IL- 6) were determined. Finally, we evaluated the expression of the sulfur synthionizing enzyme cystathionine γ-lyase (CSE) in synovial tissue samples from osteoarthritic patients with or without a diagnosis of type II DB (OA vs OA-DB). Results: Incubation of LPS activated macrophages in high glucose conditions increased the expression of mediators associated with the M1 phenotype, both at gene and protein levels. These results were confirmed by the increase of IL-6 levels and proliferative capacity in these cells. In addition, we observed that the activation of macrophages was associated with a reduced gene expression of the H2S synthesis machinery. It was noted that co-treatment of the cells with Gyy-4137 attenuated the effects of stress from high glucose levels, possibly by activating the anti-oxidant response mediated by the Nrf-2/HO-1 signaling pathway. In addition, OA-DB patients showed lower levels of CSE expression in synovial tissue than non-DB OA patients. Conclusions: Our study seems to reinforce the key role of H2S in OA and specifically its metabolic phenotype, as well as to point out its effects on the polarity of synovial macrophages, opening new therapeutic perspectives in the management of this pathology.

[gl] Introducción y objetivos: Macrófagos presentes no revestimento sinovial das articulacións están involucrados na inflamación do tecido, participando así na progresión da artrose (OA). En concreto, ten lugar un incremento na porcentaxe de macrófagos tipo M1 (pro-inflamatorios) sobre os de tipo M2 (anti- inflamatorios). O sulfuro de hidróxeno (H2S), unha pequena molécula gasosa que se sintetiza entre outros tecidos na articulación, está implicado na polarización de macrófagos mantendo o equilibrio homeostático M1/M2. Porén, estudos recentes sinalan unha redución na síntese encimática do H2S nos pacientes artrósicos, e especificamente no seu fenotipo metabólico. Deste xeito, neste traballo avaliamos se un estrés por altos niveis de glucosa podería favorecer a polarización de macrófagos facía o tipo M1, e se a administración dunha fonte exóxena de H2S podería atenuar estes efectos. Metodoloxía: Realizouse un modelo in vitro de OA asociada a diabete (DB) en macrófagos, empregando a liña celular de monocitos TPH-1. En primeiro lugar, as células diferenciaronse a macrófagos maduros con ester de alcohol forbol (PMA). As células foron logo estimuladas con lipopolisacárido (LPS) baixo condicións normais (GN; 1g/l) ou altas de glucosa (GA; 4,5 g/l) na presenza ou ausencia dunha fonte exóxena de H2S (Gyy-4137, un liberador lento do gas). A polarización dos macrófagos monitorizouse mediante estudos de expresión xénica e proteica avaliando marcadores de superficie utilizando citometría de fluxo. Ademais, determinouse a proliferación celular e a liberación do mediador pro-inflamatorio interleucina 6 (IL-6). Finalmente, avaliouse a expresión da encima sintetizadora de sulfuro cistationina γ-liasa (CSE) en mostras de tecido sinovial de pacientes artrósicos con ou sen diagnóstico de DB tipo II (OA vs OA-DB). Resultados: A incubación en altos niveis de glucosa de macrófagos activados con LPS incrementou a expresión de mediadores asociados ao fenotipo M1, tanto a nivel xénico como proteico. Estes resultados foron confirmados mediante a detección dunha maior liberación de IL-6 e capacidade proliferativa nestas células. Ademais, tamén detectamos que a activación dos macrófagos estaba asociada a unha menor expresión xénica da maquinaría de síntese de H2S. A destacar, o co-tratamento das células con Gyy-4137 atenuou os efectos do estrés por altos niveis de glucosa, posiblemente ao activar a resposta anti-oxidante mediada pola vía de sinalización Nrf-2/HO-1. En relación, os pacientes OA-DB mostraban menores niveis de expresión de CSE no tecido senónvial que os pacientes OA non DB. Conclusións: O noso estudo parece reforzar o papel clave do H2S na OA e concretamente no seu fenotipo metabólico, así como sinalar os seus efectos na polaridade de macrófagos senónviais, abrindo novas perspectivas terapéuticas no manexo desta patoloxía.