Mesenchymal stem cell derived exosomes mitigate COVID-19 cytokine storm via Annexin A1 and TGF-β mediated MAPK pathway inhibition

Abstract

Background: Severe COVID-19 is marked by a dysregulated inflammatory response, known as a cytokine storm, resulting in acute respiratory distress syndrome (ARDS) and multiple organ failure. Mesenchymal stem cell-derived exosomes (MSC-Exos) have demonstrated potential as immunomodulatory agents. This work investigates the possibility of MSC-Exos to mitigate excessive inflammation in COVID-19 by targeting the mitogen-activated protein kinase (MAPK) signalling pathway. Methodology: We integrated molecular docking analysis between TGF-β and Annexin A1 as exosomal proteins and key component proteins of the MAPK pathway (p38, ERK1/2, JNK1). The in-silico results were then validated in vivo using a Syrian hamster model of SARS-CoV-2 infection. Quantitative PCR (qPCR), western blotting, and histological examination were employed to evaluate the effects of MSC-Exos therapy on MAPK pathway activation, cytokine production, and lung tissue pathology. Results: The in-silico study revealed extensive hydrogen bonding and hydrophobic interactions at the protein–protein interfaces between exosomal proteins and MAPK components. These interactions suggest that exosomal proteins may modulate MAPK signaling pathways. In vivo, MSC-Exos administration led to marked downregulation of pivotal genes in the MAPK signaling pathway (MEKK1, MEKK2, MEKK3), diminished phosphorylation of JNK1, p38, and ERK1/2, and lowered production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Histopathological examination demonstrated ameliorated lung tissue structure, characterized by diminished alveolar wall thickness and decreased immune cell infiltration. Conclusion: MSC-Exos elicit immunomodulatory effects in SARS-CoV-2-Infected hamsters, partially by directly targeting and blocking the MAPK signaling pathway. These findings offer a compelling justification for the clinical assessment of MSC-Exos as a therapeutic approach to alleviate the cytokine storm and enhance outcomes in severe COVID-19 by targeting the ACE2-Independent pathway.

1. Introduction

The COVID-19 pandemic, induced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has created an extraordinary worldwide health emergency [1]. Although vaccination initiatives have demonstrated substantial efficacy in mitigating severe illness, the rapid emergence of viral variants capable of evading neutralizing antibodies underscores the need for alternative and adjunctive therapeutic strategies [2]. Severe COVID-19 is now recognized as the consequence of a multifactorial pathogenic process in which an exaggerated inflammatory response (cytokine storm) represents one prominent component alongside endothelial dysfunction, coagulopathy, and metabolic dysregulation, collectively contributing to acute respiratory distress syndrome (ARDS), multiorgan injury, and death [3]. One of the central signaling networks governing host responses to viral infection is the mitogen-activated protein kinase (MAPK) pathway, which encompasses the ERK1/2, p38, and JNK branches [4]. This pathway integrates multiple extracellular stress and immune signals and plays a pivotal role in regulating cytokine production, apoptosis, and immune cell activation [4, 5]. In the context of SARS-CoV-2 infection, aberrant activation of MAPK signaling, particularly through the p38 MAPK, ERK1/2, and JNK branches [6, 7]. However, accumulating evidence indicates that MAPK signaling is not unidirectionally pro-inflammatory; rather, its functional output is highly context, cell type, and time-dependent. In myeloid and other innate immune cells, ERK1/2 activation downstream of pattern-recognition and cytokine receptors clearly promotes the production of classical pro-inflammatory mediators, including TNF, IL-1β, IL-6 and IL-12, and is frequently upregulated in chronically inflamed tissues [8]. At the same time, the ERK1/2 module can support anti-inflammatory or pro-resolving programs, for example by enhancing IL-10 expression, restraining excessive IL-12 production, and modulating T-cell differentiation in a way that limits sustained Th1-skewed responses. In non-immune compartments, ERK1/2 signaling has likewise been implicated in cytoprotection and limitation of tissue damage, as shown in experimental models of acute myocardial injury and other inflammatory settings [9]. Systems-level analyses of the MAPK network further underscore that distinct combinations and temporal patterns of ERK, p38 and JNK activation, driven by specific MAP3Ks, can switch the net outcome of MAPK signaling from inflammatory amplification toward survival, repair and resolution. These observations collectively support a more nuanced view of MAPK as a pleiotropic regulator of inflammation and tissue homeostasis, which is particularly relevant when considering MAPK-targeted or MAPK-modulating interventions [6, 7]. During the viral infection, MAPK activation can arise as part of the host’s intrinsic antiviral and stress-response machinery, for example by coordinating interferon-stimulated gene expression, promoting apoptosis of infected cells, and supporting tissue repair and barrier restoration. At the same time, numerous viruses, including SARS-CoV-2, can take over p38, ERK1/2, and JNK modules to support their own replication, amplify pro-inflammatory cytokine production, and subvert type I interferon signaling, thereby tipping the balance from controlled antiviral defense toward pathogenic hyperinflammation and tissue injury [6]. This duality is particularly relevant for therapeutic targeting, because interventions that modulate MAPK activity must account for both its protective host functions and its virus-exploited, disease-promoting roles in COVID-19. SARS-CoV-2 infection causing inflammatory changes in various organs, highlighting the systemic nature of COVID-19 immunopathology. So, indicating the need for therapies that modulate host responses alongside antiviral mechanisms [10].

Exosomes derived from mesenchymal stem cells (MSC-Exos) have a great attention as a promising cell-free therapy for COVID-19, mainly due to their immunomodulatory effect via its cargo which contain varieties of molecules including TGF-β, Annexin A1, IL-10, and many microRNAs [11]. These nanovesicles modulating the host immune responses, suppressing the proinflammatory signaling, and promoting the tissue repair [12]. Supporting this, studies on conditioned medium-enriched MSCs from umbilical cord have shown that MSC secretomes containing several bioactive factors capable of modulating the inflammation and promoting tissue regeneration in preclinical studies of injury [13]. In early