Transcriptional profiling of circulating mononuclear cells from patients with chronic obstructive pulmonary disease receiving mesenchymal stromal cell infusions

Abstract Chronic obstructive pulmonary disease (COPD) is an inflammatory airways disease with limited therapeutic options. We have previously shown that mesenchymal stromal cell (MSC) infusions are well tolerated in patients with COPD and reduce circulatory biomarkers associated with systemic inflammation and oxidative stress. This study aimed to delineate the underlying mechanisms further by characterizing the transcriptional networks in these patients and to explore the role of MSC‐derived paracrine factors in regulating these pathways. Allogeneic, bone marrow‐derived MSCs were systemically administered into patients with stable COPD (n = 9). Gene expression profiles from peripheral blood mononuclear cells (PBMCs) were analyzed across the first week after infusion. Paracrine mechanisms associated with these transcriptional changes were explored further by culturing patient PBMCs with MSC‐conditioned medium (MSC‐CM) or post‐MSC infusion (PI) plasma to measure the regulatory effects of soluble factors that may be derived from MSCs. MSC‐CM and PI‐plasma were characterized further to identify potential immunoregulatory candidates. MSC infusion elicited a strong but transient transcriptional response in patient PBMCs that was sustained up to 7 days. MSC infusion strongly downregulated transcriptional pathways related to interleukin (IL)‐8 and IL‐1β, which were also significantly inhibited in vitro following co‐culture of PBMCs with MSC‐CM and PI‐plasma. MSC‐derived soluble tumor necrosis factor receptor‐1, transforming growth factor‐β1, and extracellular vesicle‐associated microRNAs were identified as potential mechanisms promoting these changes, but depletion of these individual candidates revealed inconsistent results. MSC‐derived paracrine factors modulate important inflammatory pathways that are relevant to COPD pathogenesis. These data strengthen the hypothesis that therapies using MSCs and their secreted products may be beneficial to patients with COPD.


Lessons learned
• Mesenchymal stromal cells (MSCs) attenuate important pathogenic immunological pathways that underlie chronic airways disease.
• These effects were highly transient (waning by 7 days), suggesting that frequent, 1-week doses may be important in achieving clinical benefit.
• MSC-derived soluble factors may be responsible for these changes in vivo, suggesting new therapeutic options harnessing the MSC secretome.

Significance statement
Mesenchymal stromal cells are an emerging stem cell therapy that can be used to relieve chronic inflammation. As part of a phase I clinical study, this article reports for the first time that these stem cells modulate important pathological pathways that drive inflammation in patients with chronic airways disease. This study provides novel insights that may guide further investigation of mesenchymal stromal cell therapy for the treatment of patients with chronic airway disease.

| INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is among the top five causes of global morbidity and mortality. COPD is characterized by airway obstruction, inflammation, and irreversible lung remodeling. 1 Systemic inflammation and oxidative stress are further features of COPD associated with comorbidities such as cardiovascular disease. 2 Smoking is the commonest cause of COPD, although genetic predisposition and air pollution are other etiological factors. Despite optimal treatment and the cessation of precipitants (eg, smoking), chronic inflammation (evidenced by increased activation of T cells and elevation of pulmonary and systemic proinflammatory cytokines) and oxidative stress persist in most patients, 3 highlighting the need to develop novel immunomodulatory strategies to address this condition.
Mesenchymal stromal cells (MSCs) are obtained from a variety of sources such as bone marrow and exert a range of anti-inflammatory and reparative effects. 4 These are largely mediated through paracrine mechanisms such as the release of anti-inflammatory cytokines, growth factors, and extracellular vesicles. 5 Hence, the use of MSCs in a broad range of inflammatory and immune-mediated diseases is increasingly being investigated. 6 Preclinical studies of MSCs in COPD demonstrate efficacy in alleviating inflammation and reducing emphysema following either systemic or intratracheal administration in rodent models; however, these findings have translated poorly in human studies. 7 A large clinical trial, despite showing no significant clinical improvement in patients with stable COPD following a regimen of four monthly intravenous MSC infusions, demonstrated a reduction in circulating C-reactive protein (CRP) after 1 month, suggesting that intravenous MSC infusion may alter systemic inflammation in patients with COPD. 8 More recently, a trial using MSCs to minimize localized inflammation in response to one-way endobronchial valve insertion in patients with COPD showed similar reductions in circulating CRP between 1 and 3 months after treatment. 9 The reasons for a lack of efficacy in clinical trials to date are unclear, and this is underscored by a lack of mechanistic studies in humans. To delineate these mechanisms, we characterized the systemic immunological changes of patients with stable COPD (n = 9) receiving radiolabeled MSCs over 1 week. We previously week after infusion. 10 In the present study, we performed whole transcriptome sequencing of peripheral blood mononuclear cells (PBMCs) isolated from these patients with COPD before and after MSC infusion. These findings provide novel insights into the mechanisms of MSC-mediated systemic immunomodulation, as these short-term dynamics have not previously been described in human studies.

| Patient cohort and sample collection
A single-site, phase I study (Australian clinical trials registry no. 12614000731695) was conducted to initially determine the effects of MSC infusion on clinical and safety endpoints in patients with stable COPD (described further in the supplemental online data). Nine patients with stable COPD received two doses of 2 Â 10 6 allogenic bone marrow-derived MSCs per kilogram patient weight, 1 week apart. 10  was collected prior to MSC infusion (baseline) and 1 hour, 1 day, 2 days, and 7 days after the first infusion, followed by a collection 1 hour after the second infusion. Plasma was first isolated and stored at À80 C. PBMCs were isolated by Ficoll density gradient centrifugation and cryopreserved in liquid nitrogen.

| RNA sequencing and analysis
Details on RNA extraction, sequencing, and preprocessing are described in the supplemental online data. Gene clustering was performed using the signed weighted gene coexpression network analysis (WGCNA) pipeline ( Figure S1). 11        Quantification of EVs after depletion ( Figure S2) was performed using imaging flow cytometry as previously described. 14 Figure 4C). Levels of IL-1β were unchanged in unstimulated conditions ( Figure 4D); however, the MSC-CM significantly reduced IL-1β secretion by LPS-stimulated PBMCs (P = .01; Figure 4E). Interestingly, the presence of MSC-CM or control medium stimulated potent IL-8 production, whereas IL-1β was slightly reduced, suggesting that common components of the culture medium (ie, 1% HSA/DMEM) play a role in modulating cytokine production.

| Quantification of plasma and MSC-EV microRNAs
Culturing of PBMCs with patient post-MSC infusion plasma (PIplasma) revealed that IL-8 production was significantly attenuated in the presence of 2-and 7-day PI-plasma compared with baseline plasma (P = .08, P = .02; Figure 4C). Similarly, culturing with 2-day PIplasma showed a reduction in IL-1β compared with baseline plasma, although this was only marginally significant (P = .06; Figure 4F).
Notably, the production of IL-8 and IL-1β by PBMC was greatly reduced in the presence of patient plasma (at any time points) compared with plasma-free cultures, suggesting an abundance of   gene hubs within the inflammation module (IL-8, CD44, NFKB1,   STAT3, and TLR2; Figure 5E). Of these 14 candidates, 11 were detectable in plasmas samples from our cohort, and four were also detectable in MSC-EVs ( Figure 5F). To delineate the role of MSC-EVs, TGF-β1, and sTNFR1 in attenuating IL-8, we co-cultured patient PBMCs with MSC-CM that was depleted for these mediators; however, we observed no significant changes in IL-8 expression compared with PBMCs cultured with undepleted MSC-CM ( Figure S2).

| DISCUSSION
The present study describes changes in PBMC gene expression profiles following MSC infusion, using unbiased correlation networks to identify novel pathways of immunomodulation. We also describe that these pathways can be potentially attenuated by MSC-derived paracrine factors in vitro. In the same cohort of patients, our previous work has characterized the biodistribution of intravenously infused MSCs, where there is an early breakdown of MSCs (within 7 days) that are subsequently taken up by the reticuloendothelial system. 10 This rapid loss of MSCs is likely to impact the longevity of the beneficial effects of MSC infusion. Nevertheless, numerous preclinical studies in COPD have demonstrated that MSC infusions can successfully attenuate inflammation and repair damaged lung tissue. 7 Despite this, results from clinical trials in COPD have been disappointing, 8,9 highlighting the need to better understand these mechanisms in patients with COPD.
In this study, the transcriptional response in PBMCs to MSC infusion was shown to be potent yet transient, with a lack of DEGs observed by 7 days after infusion. This is in line with a recent phase I pilot study in patients with septic shock receiving MSC infusions, where plasma inflammatory cytokine levels (IL-1β, IL-8, IL-6, MCP-1, and IL-2) are most prominently reduced around 12-24 hours after infusion before reverting to baseline levels in the subsequent days. 17 These effects are likely a consequence of the rapid breakdown of MSCs following intravenous infusion.
Network analysis reveals that genes within the downregulated module were associated with inflammatory cytokines pathways such as IL-8 and IL-1β. These pathways are especially important in COPD pathogenesis, as elevated levels of IL-8 and IL-1β promote chronic inflammation and decline in pulmonary function. 18,19 Both these cytokines are potent proinflammatory mediators that contribute to systemic and airway inflammation; however, IL-8 in particular is a major mediator of neutrophil activation and chemotaxis, leading to a high oxidative stress burden and infiltration of neutrophils into the lungs. 20 These findings are in line with our previous work, where MSC infusion reduced levels of circulating proinflammatory and oxidative stress biomarkers. 10 Other work has also shown that MSCs can strongly attenuate respiratory burst in neutrophils, either directly or through the actions of mononuclear cells preconditioned by MSCs such as regulatory T cells. [21][22][23] Current medications such as corticosteroids remain ineffective at targeting this axis of COPD pathogenesis, 24 and clinical trials using biologics against both IL-1β and IL-8 have lacked efficacy or failed to achieve safety outcomes. 25 we showed that PI-plasma was also capable of suppressing the production of these cytokines, suggesting these same MSC-derived soluble factors may potentially be released systemically following MSC infusion. Although it is plausible that these changes may reflect a reduction in circulating proinflammatory mediators that dampen PBMC cytokine production, we identified common molecular drivers such as sTNFR1 and TGF-β1 that are produced by MSCs and elevated in plasma following MSC infusion. Indeed, sTNFR1 and TGF-β1 are well described anti-inflammatory paracrine mediators produced by MSCs. [28][29][30][31] Additional in vitro experiments confirmed that patient PBMC isolated from day 2 or 7 after infusion did not significantly increase sTNFR1 and TGF-B1 production compared with baseline PBMC, suggesting that these changes in plasma are more likely driven directly by MSCs (data not shown). Of note, MSC-CM also contained high amounts of IL-6, which is a well-known pleiotropic cytokine that has been shown to downregulate IL-1 and TNF activity in specific models. 32,33 Although we have not explored the effects of IL-6 in our study, further investigation is warranted given that IL-6 has been known to regulate other MSC paracrine mechanisms such as prostaglandin E2 secretion. 34 Our study also explored the EV compartment of the MSC secretome, as EV cargo such as miRNAs are known to regulate immune responses. 35 We discovered a panel of miRNAs that may be responsible for the inhibition of IL-8 and IL-1β, including miR-23a-3p, miR-21-5p, and miR-199a-5p, which were elevated in plasma following MSC infusion and detectable in MSC-EVs. Notably, these miRNAs are known to exert immunoregulatory functions. 36 Figure S2). Although different mechanisms may be acting in each patient, it is also plausible that these candidates act in concert to attenuate these inflammatory pathways, highlighting the therapeutic potential of "whole" MSC-CM rather than its individual constituents. Moreover, although we speculate that changes in concentrations of inflammatory mediators in plasma alter the inflammatory response of PBMCs, more mechanistic studies are required to determine whether PBMCs are the cause or effect of these changes in plasma composition.

| CONCLUSION
We show that MSC infusion downregulated several pathogenically relevant transcriptional pathways in patients with stable COPD. Notably, the variability of responses was not attributed to MSC donor type, mainly due to most patients (n = 8) receiving MSCs from a single donor. Our exploratory study has also outlined several potential paracrine mechanisms that may be exerting these effects, demonstrating