Human amniotic mesenchymal stem cell-conditioned medium inhibited LPS-induced cell injury and inflammation by modulating CD14/TLR4-mediated signaling pathway in monocytes

It has been 100 years since the amniotic membrane was first used in skin transplantation in 1910 (Parolini et al., 2008). Subsequently, it has been widely applied to treat a variety of conditions, such as wound healing and ophthalmology et al. Since cell therapy was successfully performed in the past fifty years (Copelan, 2006), mesenchymal stem cells (MSCs) isolated from various tissues were widely used in clinical and preclinical studies, due to their multipotent differentiation potential and pro-regenerative capability. Human amniotic membrane-derived MSCs (hAMSCs) have gained attention for their unique advantages. Firstly, since human amniotic membrane is regarded as post-labor medical waste, hAMSCs have easily obtained with minimal ethical problem. Secondly, a large amount of hAMSCs is isolated from one single amnion after enzyme digestion, which might yield up to a density of 107 (Alviano et al., 2007; Zhang et al., 2011a). Thirdly, hAMSCs can be easily expanded to 18–20 passages without visible morphological alterations. Some studies also found that the expansion potential of hAMSCs was greater than that of human bone marrow MSCs (hBMSCs) (Alviano et al., 2007, Anker and Scherjon, 2004). Finally, but more importantly, hAMSCs do not express telomerase, which makes them exclude the risk of tumor formation after transplantation. Therefore, hAMSCs are superior as a cell transplantation source compared with other MSCs.

Although MSCs have many promising outcomes in the treatment of diverse diseases, they engraft and differentiate into other cell types at a very low frequency (Noiseux et al., 2006). There is compelling evidence that the paracrine properties of MSCs determine their therapeutic effects (Kusuma et al., 2017, Li et al., 2019). The extra-cellular vehicles, such as cytokines and extracellular matrix, play a pivotal role in tissue repair and regeneration. Particularly, these cell-free products are not only more convenient for transportation and storage, but also exclude the risks of cellular embolism and tumorigenicity. On the ground of these, stem cell-related de-cellular modalities have attained much attention. Numerous basic research supported that the use of conditioned medium (CM) derived from MSCs has become a promising therapeutic option and has more advantages than conventional stem cell therapy. Considering the advantages of hAMSCs compared to other MSCs, hAMSCs-conditioned medium (AM-CM) is a promising candidate for cell therapy and regenerative medicine.

Inflammation, an evolutionarily conserved host immune response to harmful stimuli, is associated with diseases, including type II diabetes, heart disease, and non-alcoholic steatohepatitis (NASH). Therefore, the control of inflammation is crucial to reduce the risk of tissue damage and disease relapse. Growing evidence has demonstrated that CM could ameliorate oxidative damage, modulate the immune system, and suppress inflammation in various experimental models. The therapeutic effects of AM-CM have also been confirmed in various disease models. For example, AM-CM could restrain high-fat diet-induced obesity (Tan et al., 2021) and limit infarct size (Nazarinia et al., 2021, Danieli et al., 2015). It has been reported that a variety of highly expressed proteins appeared in AM-CM during cell culture (Li et al., 2019, Ramalingam et al., 2022). Ra K et al. found that superoxide dismutase (SOD) and catalase in AM-CM were more active than those in adipose-derived MSC-CM (Ra et al., 2021). However, the exact mechanisms of AM-CM against inflammation still need to be fully elucidated for its use in the future.

It is well known that lipopolysaccharide (LPS) from gram-negative bacteria, ubiquitous in the environment, stimulates the innate immune response (Gangloff et al., 2005). Thus, LPS-induced inflammation in the in vitro cell lines represents a standard paradigm for studying inflammation. Two important members of its receptor group, cluster of differentiation (CD) 14 and toll-like receptor 4 (TLR4) play a pivotal role in LPS-mediated signaling by activating immunocompetent cells and inducing cytokines (Li et al., 2019, Frede et al., 2006). Excessive TLR4 activation leads to enhanced cell death and systemic inflammatory responses (Lafferty et al., 2010, Tesse et al., 2011). Meanwhile, the LPS-mediated signal transduction pathway is complex, including the activation of several protein kinases and transcriptional factors, such as mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB). Moreover, LPS induces the activation of NOD-like receptor protein 3 (NLRP3) inflammasome resulting in IL-1β release after caspase-1 maturation (He et al., 2016). However, the relative roles of LPS receptor subunits and their related pathways in the protective effects of AM-CM on monocytes are poorly understood.

In the present study, we isolated hAMSCs from human amniotic membranes and characterized their morphology, phenotypic profile, and pluripotency. We evaluated the protective potential of AM-CM in LPS-stimulated THP-1 monocyte in vitro. We observed that the proper concentrations of AM-CM treatment protected THP-1 cells against LPS-induced dysfunction. The results showed that AM-CM concentration-dependently inhibited LPS-activated MAPK and NF-κB signaling via CD14/TLR4, which in turn leads to the inactivation of NLRP3 inflammasome and the decrease of inflammatory mediators. Findings from the present study further suggest the salutary role of AM-CM in rescuing LPS-induced inflammatory effects.

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