In COVID-19 cases of greatest severity, a defective host immune response appears to play a central role. In particular, the acute respiratory distress syndrome (ARDS) and organ failure that typify a severe disease course are accompanied by elevated levels of IL-2, IL-7, G-CSF, IFN-γ, IP-10, MCP-1, MIP1-α, and TNF-α [1]. These characteristics mirror the cytokine release syndrome (CRS)–induced ARDS associated with SARS-CoV and MERS-CoV infection [2]. While cytokine elevation is commonly observed in COVID-19 patients, high serum IL-6 in particular seems to trigger an unfortunate "cytokine storm" [3] that correlates with mortality [4, 5, 6]. This ominous term received attention in the mainstream media during the 2003 H5N1 influenza outbreak and has re-emerged into our discourse in the wake of COVID-19. Cytokine storm, sometimes called hyperinflammation, refers to an excessive and self-perpetuating release of cytokines which culminates in acute vascular leakage, tissue damage and potentially multi-organ failure. This syndrome has been associated with specific viruses such as Dengue, malaria, poxvirus, variola virus, influenza and other coronaviruses [7] however it can also occur as an adverse effect of a therapy [8, 9].

While excessive inflammation always causes some degree of tissue damage, cytokine storm implies a systemic condition rather than a local one. In the context of COVID-19, type II pneumocytes (a cell type within the alveolar epithelial layer) are typically the first cells to be infected. However, if the immune response is dysregulated, the recruitment of T-cells, monocytes, and macrophages to the respiratory epithelium may trigger an uncontrolled amplification of the feedback loop of cytokine secretion. This in turn leads to accumulation of excessive numbers of immune cells at the cell layer. Ultimately the delicate lung infrastructure and vasculature becomes leaky and damaged, allowing immune cells to pass into the bloodstream along with deleterious levels of cytokines which circulate to other organs. This manifests as pulmonary edema, ARDS, and acute cardiac, renal or hepatic injury [10].

Taking Control of the Storm

Prominent among the treatments being explored for COVID-19 are chloroquine, hydroxychoroquine, and the antiviral agents remdesivir and lopinavir-ritonavir in combination. However, to mitigate the most severe outcomes of COVID-19, controlling the immune response may be as important as targeting the virus itself [11]. In clinical trials, several immunosuppressive therapies are being explored which target key cytokines, their receptors, or downstream pathways. Anakinra (IL-1 blocker) previously proved effective for sepsis, tocilizumab (IL-6 receptor blocker) has been approved in clinical trials for COVID-19 in China, and baricinitib (JAK inhibitor) is predicted based on machine learning to have potential for COVID-19 [12]. Corticosteroids, while anti-inflammatory, have been contraindicated [1, 13] due to complications previously observed with SARS and MERS.

Another therapeutic modality has attracted attention for COVID-19 intensive care patients. Mesenchymal stem cells (MSCs, Figure 1), a multipotent cell type, has been extensively studied in clinical trials for treatment of various diseases and injuries, and have proven successful for immune disorders such as graft-versus-host disease (GvHD) and systemic lupus erythematosus [14]. MSC's appear to exert their beneficial effects in at least three ways. First, they have immunomodulatory properties by virtue of their secretory profile which includes cytokines, chemokines, and hormones. Secondly, they potentiate tissue regeneration through production of growth factors, extracellular vesicles, and exosomes [15]. Finally, they have been observed to have antimicrobial properties through a combination of direct and indirect mechanisms [16].

Mesenchymal stem cells
Figure 1: Fluorescent-labeled mesenchymal stem cells

Early in 2020, two key studies in China first explored intravenous transplantation of MSCs as therapy for COVID-19 patients. First, Liang et al reported that in a 65-year-old severely ill COVID-19 patient, human umbilical cord-derived MSCs induced recovery from multi-organ injury [17]. In the second study, seven patients with COVID-19 pneumonia were treated with MSC transplantation. In only 2 days, Leng and colleagues observed a marked improvement in both symptoms and pulmonary function. Additionally, the MSCs induced an increase in IL-10 levels in the patients, while the MSCs themselves expressed TGF-β, HGF, LIF, GAL, NOA1, FGF, VEGF, EGF, BDNF, and NGF. In the most critically ill patients, the MSC therapy reduced the populations of CXCR3+CD4+ T cells, CXCR3+CD8+ T cells, and CXCR3+ NK cells to nearly zero after 6 days [18]. Other studies indicate that in a mouse model of lung fibrosis, bleomycin-induced lung inflammation was significantly reduced by administration of MSCs, as was collagen deposition (fibrosis) and circulating levels of IFN-γ, IL-2, IL-1β, and IL-4 [19, 20].

The strain of MSCs used in the Leng study were ACE2 negative, and thus SARS-COV-2 resistant, making them an appealing treatment option. MSCs in general have other advantages such as being straightforward to expand and able to be isolated from a variety of tissues, including bone marrow, adipose tissue, umbilical cord, dental pulp, and other sources. The disadvantage of MSC therapy is the difficulty of transporting them between distant facilities and the high cost of propagating them. Additionally, they carry a theoretical risk of tumor formation [21].

Do We Really Need the Cells?

While the preliminary data regarding MSC transplantation for COVID-19 treatment is favorable, it is widely accepted that the cells act in a paracrine fashion, suggesting that many of their properties are the result of bioactive factors they secrete/shed [14, 22]. This has led a number of researchers to speculate that the MSC secretome, independent of the cells themselves, confers benefit. [22, 23]. In fact, individual cytokines have been proposed, or are undergoing clinical trials, as COVID-19 therapies [12].

Although the effectiveness of MSC secretome for severe COVID-19 has not been demonstrated at the time of writing this article, there is ample evidence to suggest that components of their secretome can recapitulate their beneficial effects in other contexts. For example, MSC-derived extracellular vesicles can aid recovery from ARDS and mitigate lung damage [15]. Also, conditioned medium (CM) from MSCs, when administered as a concentrated bolus, significantly improved survival rates of rats after hepatocellular injury [24]. In general, the stem cell secretome is thought to have an advantage of safety over transplanted cells: it has low immunogenic potential and carries a lower risk for endogenous tumor formation since it cannot self-replicate [22]. From a practical standpoint it is also more appealing because it can be stored and transported with greater ease.

Progress with Proteomics

Whether live cells or secreted components, MSC-based therapies offer promise for COVID-19 patients but are still a long way from translation to the clinic. The molecular mechanisms of their function and communication with injured lung epithelia are poorly understood and warrant further interrogation to establish safety, predictability, and efficacy. What cellular processes are impacted? What paracrine molecules and vesicles are responsible for altering pathological pathways? Could these factors be used as biomarkers for properly primed stem cells? Proteomic methods of secretome profiling have been extensively utilized to shed light on these questions [25, 26, 27]. Using an antibody array of 200 soluble factors along with bioinformatic analysis, Ragni and colleagues showed that adipose-derived stem cells preconditioned with IFN-γ could promote cell motility and modulate inflammation, noting that the molecules identified by the array may prove useful as potency assays for MSC secretome-based therapies for joint disease [25]. Recently, Denninger et al. used a combination of a 308-target antibody array, mass spectrometry, and transcriptomics to broadly characterize neural stem and progenitor cells [26]. In summary, to realize the promise of MSC based therapies for cytokine storm, it will be critical to gain global perspectives of the numerous, intertwined molecular events underpinning their immunomodulatory properties. Equally important will be monitoring the hyperinduction of multiple cytokines in COVID-19 patients before and during any therapy. Both needs can be addressed by antibody array technologies and mass spectrometry methods, and we expect other diseases to reap similar benefits. For further reading on antibody arrays and mass spectrometry in proteomic applications, please refer to this blog post.

References

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