Lung-resident mesenchymal stem cells regulated the inflammatory responses in innate and adaptive immune cells through HVEM-BTLA pathway during ARDS
Introduction
The international panel of the Berlin definition agreed that acute respiratory distress syndrome (ARDS) is severe, diffuse, inflammatory lung injury that can lead to increased pulmonary vascular permeability and lung edema and reduced lung tissue ventilation [1]. Established studies have demonstrated that ARDS is an organ failure syndrome caused by overactivation of the immune system, which is initiated by excessive production of proinflammatory cytokines. Regardless of their different etiologies, the inflammatory responses by innate and adaptive immune cells in ARDS are similar [2]. Despite advances during fifty years of research, there is no real therapy for ARDS based on molecular or pathophysiological data, and management remains supportive [3]. The ARDS mortality rate remains substantial, at 40% in major observational trials, and has not changed much over the last decades [4].
In recent years, as mesenchymal stem cells (MSCs) have become understood, their multiple potentials, including excellent immunomodulatory abilities, make them promising candidates for ARDS therapy. Extensive preclinical and clinical experiments with MSCs for the treatment of ARDS have found these cells to be effective [5]. MSCs can be isolated from a variety of tissue sources, such as the bone marrow, fat, and umbilical cord [6,7]. Current treatments with MSCs generally involve in vitro preparation and reinfusion. However, the preparation processes take several days with many steps, which lack strict evaluation standards and supervision. Variations in production and cryopreservation methods may impart variability in the function of MSCs. Even though there is little evidence at present, people still have concerns regarding whether MSCs are tumorigenic [5]. Studies have shown that a population of endogenous MSCs exists in the lungs, and these cells also have therapeutic effects on ARDS [[8], [9], [10]]. However, the mechanism involving lung-resident mesenchymal stem cells (LRMSCs) in local tissue homeostasis is unclear.
Herpes virus entry mediator (HVEM) is a membrane protein that is a ligand/receptor for the coinhibitory molecule B and T Lymphocyte attenuator (BTLA) [11]. BTLA was the third coinhibitory molecule discovered after programmed death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) [12]. BTLA was initially found to be expressed in lymphocytes and later discovered also to be expressed in innate immune cells such as macrophages and dendritic cells. The intracellular segment of BTLA contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which can act together to recruit Src homology 2-containing protein tyrosine phosphatase (SHP-2) and inhibit the inflammatory response [13,14]. Studies have shown that BTLA can inhibit the activation, survival, and proliferation of immune cells and play important roles in multiple diseases, such as graft-versus-host disease, experimental autoimmune encephalomyelitis, inflammatory bowel disease, and hepatitis [15].
In our published study, we showed that BTLA expression elevated significantly and played important role in reducing inflammation during ARDS [16]. It was reported that bone marrow mesenchymal stem cells with overexpression of HVEM have enhanced immunomodulatory ability to inhibit the production of IL-6 and IL-17 by peripheral blood mononuclear cells (PBMC) and inhibit the proliferation of PBMC [17]. In our preliminary experiments, we found that LRMSCs expressed HVEM, and the expression of HVEM/BTLA was upregulated in pulmonary cells during ARDS. Therefore, we hypothesized that LRMSCs may exert immunomodulatory effects in ARDS through the HVEM/BTLA pathway.
Section snippets
Isolation and culture of LRMSCs
Specific pathogen-free (SPF) Sprague-Dawley rats (4–6 weeks old, male) were anesthetized by intraperitoneal injection of 10% chloral hydrate and then sacrificed by cervical dislocation. Rat lungs were dissected into single lobes, and the lobes were rinsed in a petri dish containing PBS to remove any remaining blood. Lung cells were digested with the Lung Dissociation Kit (130-095-927, Miltenyi Biotec) according to the manufacturer's instructions. Single cells were suspended in sterile PBS and
Coculture with LRMSCs inhibited inflammation-related pathways in immune cells
Based on the previous study [9,10], LRMSCs (Sca-1+CD45−CD31−) were sorted from rat lung cells by flow cytometry (Fig. 1A). After being cultured, the LRMSCs were found to adhere to culture surfaces with a long, thin, stellate morphology resembling that of bone marrow-derived MSCs (Fig. 1B). The sorted cells proliferated rapidly and were passaged every 2 days on average. In addition to Sca-1, CD45, and CD31, other surface markers that characterize MSCs, including CD90 and CD105, were examined by
Discussion
ARDS cases caused by multiple etiologies share a common pathological process, that is, an inappropriate and uncontrolled inflammatory response [19]. Scientists have been looking for an effective way to control this overwhelming inflammation [20]. In recent years, MSCs have been considered to have sound therapeutic effects on ARDS [21]. In addition to their functions of promoting blood-gas barrier repair, regulating pulmonary water transport, and participating in antibacterial activity, the most
Conclusion
In conclusion, our results suggest that Sca-1+CD45−CD31− LRMSCs have immunoregulatory effects on both innate and adaptive immune cells in ARDS and that the immune regulation was carried out through the HVEM-BTLA pathway. The clarification of this immunoregulatory mechanism may provide evidence for ARDS therapy mediated by mobilizing endogenous MSCs in the future.
Funding
This study was supported by the National Natural Science Foundation of China (81900077, 81770075, 81630001, 81570028), Shanghai Municipal Key Clinical Specialty (shslczdzk02201), Shanghai Top-Priority Clinical Key Disciplines Construction Project (2017ZZ02013), and the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (2018ZX09201002-006).
Study approval
All experimental protocols were approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. Animals were handled following the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and efforts were made to minimize the suffering and pain of the animals in our study.
Declarations of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Author contributions
Tingting Cheng: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Yun Feng: Methodology, Writing - original draft, Writing - review & editing. Xiaoyan Chen: Formal analysis, Investigation. Jian Zhou: Writing - review & editing. Yuanlin Song: Supervision, Funding acquisition. Tingting Cheng and Yun Feng contributed equally to this work.
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