Mesenchymal stromal cells dampen trained immunity in house dust mite-primed macrophages expressing human MIF polymorphism

Trained immunity (TI) results in long-term immunological memory, provoking a faster and greater immune response when innate immune cells encounter a secondary, often heterologous stimuli. We have previously shown that HDM-induced innate training was amplified in mice expressing the human macrophage migration inhibitory factor (MIF)


Introduction
The concept of innate immune memory has significantly developed over the past decade.
Innate immune cells including macrophages can undergo a process termed trained immunity (TI) whereby various stimuli including pathogenic agents such as β-glucan can alter the cells to produce increased levels of pro-inflammatory cytokines in response to subsequent stimuli.This TI is associated with a rest period between the initial and secondary heterologous stimulus, inducing epigenetic remodelling (1).Although TI can boost the immune system and provide non-specific protection against pathogens, it can also be maladaptive, contributing to a hyperinflammatory state leading to autoimmunity and chronic inflammatory disease (2).
To investigate the pathophysiology of allergic asthma, preclinical models involving repetitive intranasal administration of the clinically relevant aeroallergen house dust mite (HDM) are commonly used, as this model reproduces many aspects of human allergic asthma such as eosinophil infiltration and goblet cell hyperplasia (3)(4)(5)(6).Recently, the capacity for HDM to induce TI in mouse and human macrophages has been identified, where macrophages from HDM-allergic mice and HDM-allergic asthma patients displayed a significantly elevated proinflammatory phenotype with epigenetic changes (3,(7)(8)(9).
The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is associated with a range of inflammatory diseases including sepsis, autoimmune diseases and severe asthma (10)(11)(12).The MIF CATT 7 allele correlates with high MIF expression (4,13).We have demonstrated exacerbated HDM-induced airway inflammation in mice expressing the human MIF CATT 7 allele (4,6).Recently, using in vitro assays and an in vivo model of trained immunity, we have identified a novel role for the high expression MIF CATT 7 allele in significantly enhancing HDM-induced TI in mouse BMDMs (5).

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic efficacy in inflammatory
diseases, such as in a HDM-induced model of allergic airway inflammation, where we have demonstrated that MSCs significantly attenuated airway inflammation, cellular infiltration and cytokine production in a COX-2 dependent manner (6).MSC therapeutic efficacy requires the presence of macrophages (14,15), where a bi-directional, complex crosstalk between these cell populations can determine the inflammatory fate of their resident microenvironment.MSCs can modulate macrophages through active (secretion of TSG-6 and PGE2) and passive (being phagocytosed, secreting miRNA containing exosomes and mitochondrial transfer) means (16,17).Moreover, MSCs are primarily known to polarise macrophages to an M2, anti-inflammatory phenotype (18)(19)(20)(21)(22)(23)(24), which in turn, triggers macrophages to secrete mediators which can act to license MSCs and further boost their immunomodulatory efficacy (16).Conversely, there is also evidence that MSCs can polarise macrophages to an M1 phenotype (25,26).However, to date we have not investigated the impact of MSCs on macrophages in the high MIF expressing CATT 7 mice challenged with HDM.
This novel research describes the immunosuppressive capacity of human MSCs to block MIF enhanced M1 priming in macrophages from HDM-challenged mice, in a COX-2 partially dependent manner.Importantly, we show for the first time that MSCs can dampen HDMinduced TI in vitro and in vivo.

Ethical approval and HPRA compliance
Ethical approval for all work was granted by the ethics committee of Maynooth University (BRESC-2018-13).Project Authorisation was received from the HPRA (AE19124/P022), whereby the terms of the animal experiments within this project were outlined and adhered to.

Preclinical model of acute allergic airway inflammation
Human MIF expressing CATT7 mice and WT littermate controls were challenged with 25μg of HDM intranasally, as previously described (4,6).

In vitro innate priming assay (BMDMs)
After HDM challenge in vivo, bone marrow was isolated from CATT 7 and WT mice 4 hours after last challenge.1.5x10 6 bone marrow cells were seeded in cRPMI supplemented with 20% M-CSF into tissue culture grade 6 well plates.Media was changed on days 3 and 6 to remove non-adherent cells.On day 7, differentiated BMDMs were stimulated for 24hrs with LPS (from E. coli O111:B4) (100ng/ml) (Sigma-Aldrich, Wicklow, Ireland) to drive M1 polarisation, or murine IL-4 (10ng/ml) (R&D Systems, Abingdon, UK) to drive M2 polarisation.Cells and supernatants were harvested and stored at -20C for RNA and protein analysis.

In vitro innate training assay (HSPCs)
Naive CATT 7 or WT bone marrow was isolated from femurs and tibiae and centrifuged at 300g for 5 minutes.Whole bone marrow containing hematopoietic stem and progenitor cells (HSPCs) were seeded at 1.5x10 6 per well in 6 well non-tissue culture grade plates.After stimulating with 10μg of HDM for 24hr, HDM was washed out.Cells were cultured in 20% M-CSF supplemented cRPMI until day 6, when media was changed to non-supplemented cRPMI to facilitate a rest period.Differentiated macrophages were stimulated with 100ng/ml of LPS on day 10 for 24hrs.Supernatants were harvested for ELISA.

Analysis of Gene Expression
Total RNA was extracted using TRIzol (Ambion Life Sciences, Cambridgeshire, UK).RNA concentrations were equalised to 100ng/μl.cDNA synthesis was performed using manufacturer's instructions (Quantabio cDNA synthesis kit).Real Time-Polymerase Chain Reaction (RT-PCR) was carried out using PerfeCta SYBR Green FastMix (Quantabio, MA, USA).Expression was quantified in relation to the housekeeper gene HPRT using the ΔCT method.The fold change in the relative gene expression was determined by calculating the 2 - ΔΔCT values.

Statistical Analysis
Mice were randomised to control or treatment groups by a researcher blinded to the experimental protocol and end-points.Observers assessing end-points were blinded to group

Declaration of Interest
The authors declare no conflict of interest.

HDM-challenge in CATT 7 mice enhances pro-inflammatory macrophage polarisation
Previously, we elucidated the inflammatory status of WT and CATT 7 mice exposed to HDM or PBS control, measuring cytokine levels in lung homogenates (5).However to capture the effects of HDM-training on the bone marrow niche, we focused on BMDMs for this study.
CATT7 mice challenged with HDM three times a week for three weeks exhibited a boosted M1 phenotype, with significantly elevated expression of genes associated with classically activated M1 macrophages (Figure 1).Compared to naive BMDMs, tnf expression was significantly upregulated in both WT and CATT 7 BMDMs challenged with HDM in vivo and stimulated with LPS in vitro (Figure 1B).Interestingly, BMDMs from HDM-CATT7 mice demonstrated significantly elevated il-6 (Figure 1C), il-1β (Figure 1D) and nos2 (Figure 1E) expression compared to naive CATT7 mice, after a secondary heterologous LPS stimulation.
In HDM-challenged WT BMDMs, nos2 was significantly increased but ~4 fold less than that of HDM-CATT 7 BMDMs (Figure 1E).This effect for other M1 genes was not observed in BMDMs from HDM-WT mice, as no significant difference in il-6 (Figure 1C) or il-1β (Figure 1D) was observed.Although, not strictly associated with the M1 phenotype, il-10 was significantly increased in HDM-challenged, LPS stimulated WT BMDMs, albeit ~5 fold lower than in CATT 7 BMDMs (Figure 1F).In contrast, M2 polarisation induced by IL-4 stimulation led to significantly increased levels of arg1 and retnla in HDM-challenged WT BMDMs, but not in CATT 7 BMDMs (Figure 1G-H).
hBM-MSCs block HDM macrophage priming effect in a COX-2 partially-dependent manner MSCs are known for their ability to calm an over-zealous immune response through the secretion of paracrine immunomodulatory factors (17,(27)(28)(29)(30).Here we investigated the capacity for MSCs to block HDM-priming of a M1 phenotype in BMDMs from CATT 7 mice (Figure 2A).Using a transwell system, hBM-MSCs co-cultured with differentiated-CATT 7 BMDMs on day 7 significantly reduced M1 marker gene expression after LPS stimulation (Figure 2B-F).MSCs significantly decreased tnfα gene expression in CATT 7 and WT mice.
However MSCs significantly decreased TNF protein production in CATT 7 mice, but not WT mice (Figure 2B).Furthermore, when co-cultured with BMDMs from CATT 7 mice, MSCs significantly reduced il-6 (Figure 2C) and il-1β (Figure 2D) gene expression and protein production in CATT 7 BMDMs, but not in WT BMDMs.Following this trend, MSCs significantly decreased nos2 expression in CATT 7 mice, however MSCs had no significant effect on the expression of nos2 in BMDMs from WT mice (Figure 2E).MSCs also significantly reduced il-10 expression in CATT 7 , but not WT BMDMs (Figure 2F).MSC co-culture with CATT 7 BMDMs had no significant effect on arg1 expression after IL-4 stimulation (Figure 2G).MSCs co-cultured with HDM-WT BMDMs significantly decreased arg1 gene expression to levels comparable to those expressed by HDM-CATT 7 BMDMs (Figure 2G).Lastly, as CATT 7 BMDMs did not exhibit increased expression of retnla after HDM priming, it is not surprising that no effect was seen with this gene after MSC co-culture (Figure 2H).MSC co-culture with HDM-WT BMDMs had no significant effect on retnla expression (Figure 2H).
We next sought to elucidate the mechanism by which MSCs suppressed the pro-inflammatory signature in HDM-CATT 7 BMDMs.As cyclo-oxygenase (COX) is known to facilitate MSC's immunosuppressive capabilities (6), we investigated the role of these enzymes in MSC suppression of CATT 7 BMDM pro-inflammatory cytokine production (Figure 2A).

COX-2 was found to have a mechanistic role in MSC-mediated suppression of TNF, as
MSCs pre-treated with the COX-2 inhibitor NS-398 prior to co-culture with CATT 7 BMDMs could no longer significantly suppress TNF production (Figure 2I).However other factors may play a role in the suppression of CATT 7 pro-inflammatory markers, as COX-2 inhibition had no effect on MSC suppression of IL-6 production by CATT 7 BMDMs (Figure 2J).

hBM-MSCs block HDM-induced trained immunity in macrophage from CATT 7 mice in vitro and in vivo
We used an in vitro HDM-induced model of TI to investigate if MSCs could also block TI in HSPCs within whole bone marrow cells (Figure 3A).Bone marrow cells from CATT 7 mice trained with HDM in vitro on day 0 followed by a rest period and subsequent stimulation with LPS produced significantly increased levels of TNF (Figure 3B) and higher levels of IL-6 (Figure 3C) compared to cells exposed to HDM alone or LPS alone, indicative of a TIphenotype.Importantly, we have previously demonstrated that human MIF expression in the CATT 7 mice plays a key role in enhancing HDM-induced TI with evidence of epigenetic remodelling (5).Strikingly, CATT 7 BMDMs derived from HSPCs that were co-cultured with MSCs on day 0, illustrated a significant reduction in TNFα production (Figure 3B).MSC transwell co-culture also decreased the level of IL-6 protein production by trained CATT 7 BMDMs, however this was not statistically significant (Figure 3C).MSCs had no significant effects on TNFα and IL-6 production by CATT 7 BMDMs that only received LPS stimulation, proving the importance of having both a primary HDM stimulus on day 0 and a rest period followed by a secondary LPS stimulus in this innate immunity in vitro assay.
Next we translated these findings in an in vivo model of HDM-induced innate immune training in humanised MIF CATT 7 mice.CATT 7 mice were HDM-challenged on day 0, 8, 9, 10 and 11.MSCs were administered i.v.day 10 and 11 (Figure 3D).After a rest period of 7 days, bone marrow was harvested on day 18 and BMDMs were differentiated as described.
No significant differences were seen in TNFα production between groups (Figure 3E).However BMDMs from HDM-CATT 7 had significantly increased levels of IL-6 production after LPS stimulation, compared to PBS-CATT 7 BMDMs (Figure 3F).Moreover, MSC administration significantly decreased levels of IL-6 production after LPS stimulation, compared to those that did not receive MSC (Figure 3F).CCL17, a known marker of HDMinduced TI (7), increased (although not significantly) in BMDMs from HDM-CATT 7 mice after LPS stimulation, compared to PBS control mice (Figure 3G).MSCs decreased CCL17 production in HDM-CATT 7 BMDMs compared to those that did not received MSC treatment (Figure 3G).

Discussion
Previously, we have demonstrated the immunomodulatory effects of hBM-MSCs in a model of HDM-induced airway inflammation (6).In this study, we illustrate the ability of MSCs to modulate HDM-induced trained immunity in vitro and in vivo.hBM-MSCs co-cultured with CATT7 BMDMs using transwells significantly suppressed the M1 pro-inflammatory signature after HDM priming.Furthermore, MSCs significantly reduced M2 marker arg1 in BMDMs from WT mice.When co-cultured with CATT7 BMDMs, MSCs had no significant effect on these M2 markers.COX-2 inhibition abolished MSCs' ability to significantly suppress TNFα production.Interestingly, MSC-COX-2 activity was not involved in MSC suppression of BMDM IL-6 production suggesting that other unidentified soluble factors may be involved in MSC suppression of other M1 associated pro-inflammatory cytokines produced by BMDMs.
MSCs can communicate with HSPCs within the bone marrow niche through extracellular soluble mediators and exosomes (31,32), but also by intracellular means through organelles called migrasomes (33).Therefore hBM-MSCs were co-cultured with HSPCs from CATT7 mice using transwell inserts to elucidate if these immunomodulatory cells could block or suppress HDM-induced TI in macrophages.Strikingly, MSCs were able to significantly reduce TNFα and reduce IL-6 production by CATT7 BMDMs trained with HDM, when cocultured with HSPCs from day 0. MSCs had no significant effect in HSPC-derived BMDMs that only received the secondary LPS stimulus.It is important to note that MSCs are present in the transwell co-culture with HSPCs trained with HDM from day 0 to day 6, however the LPS stimulus is added on day 10 in the absence of MSCs.These data clearly illustrate the ability of MSCs to block HDM-imprinting on HSPCs on day 0, and leave a lasting immunomodulatory memory seen in HSPC-derived macrophages on day 11 (34).Other groups have illustrated MSCs ability to block TI in models of ischemic stroke (35) and pulmonary bacterial infection (36), however this manuscript is the first to demonstrate the ability of MSCs to block this HDM-induced innate training in vivo in mice expressing the human MIF 7-7 polymorphism.
The potential ability of MSCs to inhibit epigenetic modifications in response to HDM training within CATT7 HSPCs has clinical implications.By disrupting the epigenetic reprogramming of immune cells, MSCs can exert immunomodulatory effects and reduce amplified inflammatory responses associated with a trained immune system.These findings strengthen the argument that MSC-based therapies could be beneficial for individuals with conditions characterised by these aberrant immune responses, such as asthma, specifically in the context of our HDM-induced model of allergic airway inflammation, with 50-85% of asthmatics allergic to HDM (3,9,(37)(38)(39).These data further demonstrate the universal therapeutic efficacy of MSCs at different time points of disease progression, where they can not only calm an established immune response (6), but also prophylactically prevent HDMinduced epigenetic re-wiring, thus modulating the immune response against future infection or immunological insults.
assignment.Data for individual animals and independent experiments are presented as individual symbols.All data are presented as mean ± SEM. Results of two or more groups were compared by one-way analysis of variance (ANOVA) followed by the post-hoc Tukey's multiple comparison test.GraphPad Prism (GraphPad Software Inc, San Diego, CA, USA) was used for all statistical analyses.HD performed research, data analysis, study design and wrote the manuscript.IJH performed research, data analysis and study design.CT performed research.ENMcN provided reagents and contributed to study design.DJW contributed to study design and interpretation of the data.SCD & MEA provided reagents, contributed to study design and data analysis.KE designed and supervised the study and wrote the manuscript.All authors approved the final manuscript.

Figure 1 .
Figure 1.HDM primes BMDMs from CATT 7 mice, increasing their relative M1 gene expression.A Model of allergic airway inflammation to investigate HDM-induced trained immunity.Bone marrow was isolated from naive or HDM-challenged WT and CATT 7 mice 4hr after last challenge, and were differentiated with M-CSF over 7 days.B-F M1 and G-H M2 macrophage marker expression in BMDMs polarised with LPS or IL-4 on day 7 for 24hr was measured by PCR.Data are presented as mean ± SEM; N=3 mice per group, with each data point representing a single animal.ns = non significant, *p<0.05,**p<0.01,***p<0.001,****p<0.0001.

Figure 2 .
Figure 2. hBM-MSCs block HDM-induced priming effect in CATT 7 mice, decreasing their M1 macrophage gene expression.A Bone marrow cells containing HSPCs were isolated from HDM-challenged WT and CATT 7 mice 4hr after last challenge, and differentiated with M-CSF over 7 days.On day 7, hBM-MSCs were co-cultured with BMDMs using 0.4μM transwells at time of polarisation with LPS or IL-4, for 24hr.B-F Gene expression and/or protein production of M1 macrophage markers in BMDMs, measured by PCR or ELISA G-H Gene expression of M2 markers arg1 and retnla I-J To investigate the

Figure 3 .
Figure 3. hBM-MSCs block HDM-induced innate immune training effect in HSPCs from CATT 7 mice in vitro and in vivo.A Experimental protocol for in vitro trained immunity assay.Bone marrow containing HSPCs were isolated on day 0 and co-cultured with MSCs in transwells during training window with HDM (24hr).HDM stimulus was washed out on day 1.On day 6, MSC transwells were removed from co-culture and differentiated BMDMs entered a rest period.BMDMs received a second stimulus of LPS on