Immunomodulation of airway epithelium cell activation by mesenchymal stromal cells ameliorates house dust mite-induced airway inflammation in mice.

AJRCMB Articles in Press. Published on 19-March-2015 as 10.1165/rcmb.2014-0431OC

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Immunomodulation of airway epithelium cell activation by mesenchymal stromal cells ameliorates HDM-induced airway inflammation in mice.

Khang M. Duong*, Jaisy Arikkatt*, M.Ashik Ullah†, Jason P. Lynch*, Vivian Zhang*, Kerry Atkinson§∞, Peter D. Sly‡ and Simon Phipps*.

* The School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia † The Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, QLD, Australia ‡ The Queensland Children’s Medical Research Institute, The University of Queensland, Brisbane, QLD, Australia § The Queensland University of Technology at the Translational Research Institute, Brisbane, QLD, Australia ∞The University of Queensland Centre for Clinical Research, Brisbane, QLD, Australia

Author contributions Conception and design: K. M. D, P.D.S., K.A., and S.P. Analysis and interpretation: K.M.D., J.A., A.U., J.P.L., V.Z., P.D.S., K.A., and S.P. Drafting the manuscript for important intellectual content: K.M.D., P.D.S., K.A., J.P.L., and S.P. 1

Copyright © 2015 by the American Thoracic Society


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Corresponding author Dr. Simon Phipps, The School of Biomedical Sciences, The University of Queensland, QLD, Australia, St. Lucia, QLD, Australia. [email protected]

phone 61(0)733652785

fax 61(0)733651766.

Funding sources

This work was supported by the Asthma Foundation of Queensland, an NHMRC Project Grant to S.P., and an Australian Research Council Future Fellowship to S.P.

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Abstract

Allergic asthma is underpinned by T helper 2 (Th2) inflammation. Redundancy in Th2 cytokine function and production by both innate and adaptive immune cells suggests that strategies aimed at immunomodulation may prove more beneficial. Hence, we sought to determine whether administration of mesenchymal stromal cell (MSCs) to house dust mite (HDM)-sensitised mice would suppress the development of Th2 inflammation and airway hyperresponsiveness (AHR) following HDM challenge. We report that the intravenous administration of allogeneic donor MSCs one hour prior to allergen challenge significantly attenuated the features of allergic asthma, including tissue eosinophilia, Th2 cytokine (IL-5 and IL-13) levels in bronchoalveolar lavage fluid, and AHR. The number of infiltrating type 2 innate lymphoid cells was not affected by MSC transfer, suggesting that MSCs may modulate the adaptive arm of Th2 immunity. The effect of MSC administration was long-lasting: all features of allergic airways disease were significantly suppressed in response to a second round of HDM challenge 4 weeks after MSC administration. Further, we observed that MSCs decreased the release of epithelial cell-derived alarmins IL-1α and high mobility group box-1 in an IL-1 receptor antagonist-dependent manner. This in turn, significantly decreased the expression of the pro-Th2 cytokine IL-25 and reduced the number of activated and antigenacquiring CD11c+CD11b+ dendritic cells in the lung and mediastinal lymph nodes. Our findings suggest that MSC administration can ameliorate allergic airway inflammation by blunting the amplification of epithelial-derived inflammatory cytokines induced by HDM exposure and may offer long term protection against Th2-mediated allergic airway inflammation and AHR.

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Keywords Asthma, house dust mite, IL-1a, HMGB1, IL-25, mesenchymal stromal cells, mouse, alarmin.

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Introduction

Asthma is a heterogeneous inflammatory disease of the airways commonly associated with sensitization to common aero-allergens. Although the use of inhaled corticosteroids is beneficial in the majority of patients with few systemic side effects, it is not ideal for patients that require long-term treatment with high doses to control their asthma or for those that have steroid-resistant phenotypes. In addition, it is now clear that the targeting of a single inflammatory cytokine involved with allergen-induced inflammation is not an effective strategy for improving lung function, which may suggest that an underlying inflammatory response is not being adequately regulated by therapy (1, 2). Systemic immune-modulating therapies may be more effective at switching off immune responses to allergens, reducing asthma exacerbations and preventing the consequences of chronic inflammation, such as airway remodelling and airway hyper-responsiveness (AHR).

Mesenchymal stem/stromal cells (MSCs) are a heterogeneous population of plastic adherent and highly proliferative cells, which have been observed to have widespread immunomodulatory properties in vitro (3). It is now apparent that MSCs can be expanded in culture from most adult tissues and that bone marrow-derived MSCs have increased expression of immuno-regulatory associated genes (4). Studies in mice have shown that local or systemic administration of MSCs can increase the regeneration of damaged tissue, diminish disease pathology in a range of inflammatory disorders, and prevent the rejection of grafts after organ transplantation. Of particular note, intravenously delivered MSCs have been shown to be beneficial in preclinical models of sepsis (5), graft-versus-host disease (6), acute myocardial infarction (7), type 1 diabetes (8), organ transplantation (9) and degenerative disc disease 5



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(10). Allogeneic MSC therapy is currently approved as an anti-inflammatory and toleranceenhancing treatment for children with severe graft-versus-host disease occurring after allogeneic hematopoietic stem cell transplantation in Canada and New Zealand.

In the acute ovalbumin (OVA)/aluminium hydroxide model of allergic airway inflammation, MSC administration prior to OVA challenge ameliorates the magnitude of allergic airway inflammation, Th2 cytokine production and AHR (11-15). However, the canonical acute OVA model induces sensitization by methods different from those that occur naturally in humans with allergic asthma. Moreover, the use of adjuvants to promote sensitization to chemically inert proteins such as OVA skews towards a T cell-dominated response in the challenge phase (16), whereas in human allergic asthma, allergens activate the airway epithelium to produce factors that regulate the underlying dendritic cell (DC) network. For example, house dust mite (HDM) activates the toll-like receptor 4 (TLR4) on the airway epithelium to initiate a cytokine cascade that includes IL-1α, high mobility group box 1 (HMGB1), IL-25 and IL-33 (17-19), leading to the downstream activation of type 2 innate lymphoid cells (ILC) and CD4+ Th2 cells.

Although some investigators have used natural allergens such as ragweed, cockroach extract and house dust mite, none of these studies has evaluated DC and epithelial cell interactions after mucosal sensitisation and allergen challenge (14, 20, 21). Hence, the primary mode of MSC-mediated suppression of allergic inflammation has been ascribed to the promotion of anti-inflammatory macrophages and regulatory T cells (11, 13, 14). Additionally the majority of studies have evaluated the efficacy of MSC in acute models of allergic inflammation. Although MSCs were shown to reduce AHR and features of airway remodelling in a chronic 6


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HDM model, the underlying mechanism of action was not elucidated (22, 23). In this study we sought to characterise the anti-inflammatory properties of allogeneic bone marrow-derived MSCs in an acute and semi-chronic model of HDM-induced allergic airway inflammation.

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Materials and Methods Extended methods are available in the online supplement. Model of HDM-induced allergic airway inflammation. Female BALB/c mice (H2d) were sensitised and challenged to HDM (Dermatophagoides pteronyssinus) extract (Greer Labs, Lenoir, NC) or PBS (vehicle control group) as per the study design in Figure 1A and as described previously (24). The hallmark features of allergic airway inflammation were measured at 3 or 24 hours (day 18) post final challenge. Please refer to the online supplement for further details. Donor MSCs (1x 106) from wildtype or IL-1 receptor antagonist-deficient C57BL/6 mice (H2b) or vehicle (fresh MSC culture medium) was infused via the tail-vein to HDM-sensitised mice immediately prior to the first allergen challenge (day 14). In this allogeneic system donor and recipient mice were thus two haplotype-mismatched with each other. To ensure that our results were not influenced by genetic differences, in one study, MSCs from C57BL/6 mice (H2b) were transferred to recipient HDM-sensitised C57BL/6 mice (H2b) prior to challenge, as described above. Where indicated, DQ-OVA (25 µg; Molecular Probes, Eugene, OR) was administered (i.n. route) simultaneously with HDM at day 14. In one experiment, mice were exposed to a second round of HDM challenge at day 42, 43, 44 and 45, and allergic airway inflammation assessed at 3 or 24 hours after the last HDM challenge. All studies were approved by the University of Queensland Animal Ethics Committee.

Isolation and expansion of MSCs. Femurs were dissected, crushed, and cells cultured for 48 hours. Adherent cells were then removed, and FACS-purified MSCs expanded until passage 6 8



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for experimentation use. Please refer to the online supplement for further details. To verify that the MSCs were multipotent, MSCs were induced into adipocyte, osteoblast, and chrondrocyte lineages in vitro (see Figure E1 in the online supplement) as described previously (25).

Flow cytometry, differential counts in BAL, BALF and serum collection, measurement of cytokines and IgE. All performed as described previously (26, 27). Please refer to the online supplement for further details.

Immunohistochemical detection of HMGB1. Tissue sections were probed for HMGB1 with rabbit anti-mouse HMGB1 (1:400 dilution; Abcam PLC, Cambridge, MA) as described previously (19). Please refer to the online supplement for further details.

Quantification of mucus cell hyperplasia, mast cells and tissue eosinophils. Lung tissue representing the central and peripheral airways was fixed in 10% phosphate-buffered formalin, sectioned, and stained with periodic acid-Schiff (PAS), toluidine blue or chromotrope 2R to enumerate mucin-producing goblet cells, mast cells or eosinophils respectively, and as described previously (24, 26). Please refer to the online supplement for further details.

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Assessment of AHR. AHR in response to increasing concentrations of methacholine (MCh) was measured as previously described (28).

Statistical analysis. Data are presented as the mean ± 1 SD or SEM as appropriate. All presented data are from one representative experiment (n = 5-6 mice/group) of at two. Statistical analysis was undertaken using non-parametric Mann-Whitney t test or two-way ANOVA with post-hoc Bonferroni test, as appropriate. Statistical significance was accepted at the 5% level.

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Results MSC treatment suppresses HDM-induced allergic airway inflammation HDM challenge in sensitized mice significantly increased the total number of cells recovered in the BALF at day 17 (Figure 1B), which, as expected, consisted of increased numbers of lymphocytes, neutrophils and eosinophils but not monocytes (Figure 1C). This was accompanied by increased numbers of eosinophils, type 2 innate lymphoid cells, mast cells and mucus-producing airway epithelial cells (AEC) in the airway wall (Figure 1D-G). Additionally total IgE production (HDM-specific IgE was not detectable) in serum and Th2 (IL-5, IL-13) but not Th1 (IFN-γ) cytokines in the BALF were elevated following HDM challenge (Figure 1H and I). Intravenous administration of allogeneic MSC prior to challenge significantly diminished the total cell count, and the numbers of airway lymphocytes, neutrophils and eosinophils, but not monocytes, in response to HDM challenge at day 17 (Figure 1B-C). Further, MSC administration diminished peribronchial eosinophilia (Figure 1D), mast cells numbers (Figure 1F), mucus hyper-production (Figure 1G), total IgE production in the serum (Figure 1H). Similar findings were observed with a semi-quantitative assessment of lung pathology (see Figure E2 in the online supplement). In contrast, HDMspecific IgG1 was unaffected (data not shown). Consistent with an attenuated inflammatory response, MSC administration significantly diminished the production of IL-5 (p=0.002) and IL-13 (p=0.002), but not IFN-γ in the BALF (Figure 1I); however, MSC administration did not diminish type 2 ILC recruitment (Figure 1E). Importantly, syngeneic MSC therapy [MSCs from C57BL/6 mice (H2b) transferred to recipient HDM-sensitised C57BL/6 mice (H2b)] also ameliorated key features of allergic inflammation (see Figure E3 in the online supplement), suggesting that the effects were not influenced by genetic differences. 11



MSCs diminish airway hyperresponsiveness HDM challenge significantly increased the responsiveness of the airways to metacholine (MCh) (Figure 2A-C). HDM-induced airway hyperresponsiveness, tissue damping and elastance was significantly diminished in mice pre-treated with allogeneic MSCs. The immunomodulatory effects of MSCs persist for up to 4 weeks We next assessed whether the effect of allogeneic MSC administration at day 14 would persist following a second round of HDM challenge 4 weeks later (Figure 3A). HDM challenge induced features of allergic inflammation, including airway and tissue eosinophilia, IL-13 production and AHR that were of similar magnitude to responses when mice were challenged at day 14-17 post sensitization (Figure 3). As with the acute model, the total number of BAL cells (Figure 3B), airway lymphocytes, neutrophils and eosinophils (Figure 3C), and tissue eosinophils (Figure D) were significantly decreased in mice that had received MSCs at day 14. The long lasting effect of MSC treatment was also evident with regards to mucus hypersecretion, IgE in serum, IL-13 secretion in the BALF, and lung pathology (Figure 3E-G, and Figure E2 in the online supplement). Most notably, MSC administration at day 14 attenuated HDM-induced AHR at day 45 following secondary challenge (Figure 3H). MSC treatment decreases dendritic cell activation in response to HDM challenge We next assessed whether MSC treatment modulated the DC response in the challenge phase of the acute model. At day 16, the number of CD11b+ DCs in the lung was significantly increased in response to HDM challenge compared to vehicle controls (Figure 4A). MSC administration significantly diminished the total number of CD11b+ DCs in the lung and the number of these DCs co-expressing MHC class II and the co-stimulatory molecule CD86 12


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(Figure 4A-B). DC numbers in the mediastinal lymph nodes (MLN) were increased in HDM challenged mice compared to the vehicle control group (Figure 4C). MSC administration did not significantly lower the total number of CD11b+ DC or MHCII+CD86+ activated CD11b+ DCs in the MLN (Figure 4C-D). To address whether MSCs affected DC antigen acquisition and processing, mice were co-exposed to both DQ-OVA and HDM at day 14. MSC treatment significantly decreased the number of mature DQ-OVA+ DC in both the lung and MLN (Figure 4E-F). HDM-mediated activation of the airway epithelium is decreased by MSC administration In response to HDM exposure, the airway epithelium is known to rapidly produce a cascade of alarmins, including IL-1α and HMGB1, which recruit and activate DCs (18, 19). Consistent with previous reports, IL-1α expression was significantly increased at day 15 (i.e. 24 hr after HDM challenge) of HDM challenge and remained elevated at day 16 (Figure 5A)(18, 19). Similarly, HMGB1 nuclear to cytoplasmic translocation in airway epithelial cells, which occurs downstream of IL-1α (19), was also increased in response to HDM challenge at day 15 and 16. Critically, MSC treatment decreased the expression of IL-1α and significantly attenuated the translocation of HMGB1 at day 15 (Figure 5A-B). Expression of IL-25 and IL-33, both Th2-instructive cytokines, is partly dependent on HMGB1-RAGE signalling (19). As expected, both IL-25 and IL-33 were elevated in the lung at day 16; however, only the expression of IL-25 was significantly diminished by administration of MSCs (Figure 5C-D). The expression of thymic stromal lymphopoietin, another Th2instructive cytokine, was not increased by HDM in this model (data not shown).

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MSC-derived IL-1 receptor antagonist contributes to airway epithelial and DC activation. In light of the effects of MSC treatment on IL-1α expression, and our previous findings that HDM-induced HMGB1 release is ablated with anakinra (19), an IL-1 receptor antagonist (IL1ra), we hypothesised that the effects of MSCs may be in part mediated by this naturally occurring anti-inflammatory molecule. Whereas the transfer of IL-1ra-sufficient MSCs significantly reduced the number of airway epithelial cells with cytoplasmic HMGB1, IL-1radeficient MSCs had no effect on HMGB1 translocation (Figure 6A). Similarly, whereas IL1ra-sufficient MSCs significantly diminished the production of IL-25 and the numbers of activated DCs, IL-1ra-deficient MSC had no effect (Figure 6B-D).

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Discussion The results of the present study have demonstrated the ability of a single intravenous administration of MSCs to suppress the characteristic features of allergic airway inflammation. In a conventional acute HDM model, administration of MSCs one hour prior to HDM challenge in sensitized mice reduced inflammatory cells and Th2 cytokines in BAL, reduced mucus cell metaplasia and tissue eosinophilia and prevented the development of AHR. The protection afforded by a single administration of MSC (at day 14) was still present when the allergen challenge was repeated 4 weeks later. Mechanistically, we determined that MSCs attenuate HDM-induced activation of the airway epithelium via the production of IL1ra. This anti-inflammatory mediator broke the IL-1α autocrine loop and amplification of downstream cytokines (i.e. HMGB1 and IL-25), to prevent the recruitment, antigen acquisition/processing and maturation of DCs in the lung during the early phases after HDM challenge. Previous reports have shown that MSC treatment is able to ameliorate allergic airway inflammation in murine models of acute asthma (11-13, 15, 20, 23, 29). However, all of these studies with the exception of two (23, 29), sensitised the recipient mice via the peritoneum and used the adjuvant alum to prime towards a Th2 immune response. Although this protocol has been valuable in elucidating many mechanisms of Th2 inflammation, it is less informative with regards to understanding the contribution of innate immune processes, and in particular the airway epithelium, in initiating acute exacerbations of disease since the challenge phase relies on an inert antigen (30, 31). Critically, the airway epithelium is now known to be a central player in recognising environmental insults and shaping innate and adaptive immunity (32). For example, using chimeric mice, Hammad et al. demonstrated that TLR4 on non15



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haematopoietic cells rather than haematopoietic cells is necessary for the development of type 2 inflammation in an acute model of HDM-induced asthma (17). It has subsequently been shown that the activation of TLR4 on airway epithelial cells induces the rapid release of constitutively expressed IL-1α, which acts in an autocrine feed-forward loop to amplify itself, as well as promoting the release of another alarmin, HMGB1, and the downstream production of IL-25 and IL-33, both pro-Th2 inducing cytokines (18, 19). Although these studies have primarily been performed during sensitisation, in this study we show that low dose HDM challenge induces IL-1α within 24 hours, followed by nuclear to cytoplasmic HMGB1 translocation and the production of IL-25. We show for the first time that MSC treatment decreases the translocation of HMGB1 from the nucleus to the cytoplasm, which we have previously shown to closely associate with its later release into the airway lumen (19), where it can act as a chemokine or cytokine depending on its redox state (33). Posttranslational modifications also regulate its mobilisation inside the cell, which can be induced by type I interferons via activation of the JAK/STAT1 pathway (34). Although previous studies have shown that the inflammasome contributes to the release of cytoplasmic HMGB1, our results demonstrating that IL-1ra-deficient MSCs are unable to protect against HDM-induced HMGB1 translocation, together with our previous data demonstrating that anakinra blocks HMGB1 translocation (19), suggests that IL-1α, either directly or indirectly, can also promote the translocation of HMGB1. In the canonical OVA/alum model, DCs are essential for the effector phase of allergic airways inflammation (35), although emerging data from other more clinically relevant models has demonstrated that many features of allergic inflammation can be induced by IL-13-producing type 2 ILCs in the absence of T or B cells, and hence would not require the presence of DCs 16


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(36). In the present study, we observed that MSC administration attenuated the production of Th2 cytokines in the BALF. Although this was associated with a reduction in T cells and not type 2 ILCs in the lung, it is possible that the production of type 2 cytokines by both cell types was diminished by MSC therapy. An additional limitation of our study was that we only assessed type 2 ILCs at day 17. Chiesa et al have shown that by decreasing the expression of lymph node homing receptors, MSCs are able to reduce DC migration to draining lymph nodes, and hence affect the expansion of antigen-specific T cells (37). In view of the decreased production of epithelial alarmins and Th2 cytokines in the absence of an increase in regulatory T cells (data not shown), we hypothesised that MSC administration was impacting on the licensing of DCs. Consistent with this theory, we found that DC recruitment, acquisition of antigen, and maturation were all decreased following MSC administration. Again, this phenotype was absent when IL-1ra-deficient MSCs were adoptively transferred, although it remains to be determined whether IL-1a acts directly on DCs or indirectly via an intermediary cell, such as the airway epithelium. It is noteworthy that MSCs have also been shown to ameliorate bleomycin-induced pulmonary fibrosis via the production of IL-1ra, although whether this involves pro-fibrotic HMGB1 remains unknown. The precise mechanism(s) remain elusive, but may relate to the ability of IL-1α to induce epithelialderived granulocyte-macrophage colony-stimulating factor, which is known to recruit and support DC survival (18). Intriguingly, extracellular HMGB1 was recently shown to bind to CD24 on a population of CD103+ DCs, where it was ‘presented’ to the pattern recognition receptor for advanced glycation endpoints (RAGE) on T cells (38). Whether this pathway is operational in response to HDM remains unknown; however, we and others have previously shown that Th2 priming is attenuated in RAGE-deficient mice (19, 33). This phenotype was mediated in part because HMGB1-RAGE signalling is upstream of IL-25 and IL-33 (19), 17



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which can promote Th2 immunity (18, 39-44). It was therefore unexpected that IL-25 but not IL-33 expression was diminished by MSC administration. Athough we did not identify the cellular source of IL-33 by immunohistochemistry, we and others have previously shown that IL-25 is expressed in airway epithelial cells and DCs (45). The need for frequent infusions of MSCs would make MSCs a less attractive option for the treatment of chronic inflammatory diseases such as asthma. Remarkably, we found that a single delivery of MSCs significantly attenuated the magnitude of allergic airways inflammation and airway hyperresponsiveness for up to 4 weeks. The only other study to evaluate the long term effects of MSC employed a chronic model of asthma (22), dosing with 100 µg of HDM three times per week for up to 6 weeks. Marinas-Pardo et al were unable to detect Th2 cytokines, most likely due to sampling 72 hours after the final allergen challenge (46). Although airway eosinophils were decreased by MSC therapy when assessed 14 days post-MSC administration, there was no effect on total IgE levels in serum. However, in contrast to our findings, Marinas-Pardo et al used a 20-fold higher dose of HDM, which may have influenced the type of DC acquiring and presenting antigen, most notably by promoting the involvement of CD11b+ monocyte-derived DCs (47). Nevertheless, we confirmed that MSC ameliorate airway smooth muscle remodelling (data not shown), possibly related to the decreased release of HMGB1 which can promote airway remodelling in chronic models of allergic asthma (48), and additionally demonstrated that MSC treatment has long-term effects on AHR. Critically, we were unable to show the presence of MSCs in the lung after day 15 (data not shown). Similarly, others have found that MSCs do not persist in the lungs after intravenous administration (13, 14), although this is not a consistent finding (15, 22, 49, 50). Clearly our 18


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data indicate that MSCs decrease the early innate inflammatory response, and that this has long term consequences. The mechanism by which MSCs exhibited their prolonged effects requires further investigation. The simplest explanation is that the transfer of MSCs affected the expansion of memory CD4+ Th2 cells during primary challenge, and that the diminished pool of these cells limited their expansion upon secondary challenge. However, it is also possible that the MSCs induced persistent changes in the airway epithelium, for example by altering the transcriptome or through epigenetic modifications. It will be important to interrogate these concepts, although this was beyond the scope of the present study. One attraction of the present study is that we used allogeneic MSCs, and non-HLA-matched MSCs from volunteer unrelated human donors are currently being used in over 400 clinical trials worldwide (ClinicalTrials.gov/mesenchymal stem cells and mesenchymal stromal cells). However, to ensure that strain differences did not contribute to our results, confirmatory studies were performed using a syngeneic model, e.g. transfer of MSCs isolated from C57Bl/6 mice into HDM-sensitized C57Bl/6 mice. Similar to our findings in BALB/c mice, syngeneic MSC treatment significantly dampened all features of allergic airways inflammation including eosinophilia, mucus hyper-production, and the expression of IgE. A limitation of the study is that we did not assess whether IL-1ra-deficiency affected AHR, however our primary aim was to elucidate the mechanistic processes by which MSCs dampen innate inflammation. In summary, we observed that MSC administration ameliorated allergic airways inflammation and AHR by producing IL-1ra to prevent the amplification of a cytokine cascade at the airway epithelium. This in turn ablated the recruitment, activation and maturation of DCs in the lung and the re-activation of Th2-cytokine-producing CD4+ Th2 cells. Significantly, the effects of MSCs were persistent, lasting up to 4 weeks after a single treatment, suggesting that MSCs 19



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may offer a viable alternative or add-on therapy to conventional therapies for asthma, in particular in severe disease (to reduce corticosteroid dosage) and asthmatics who are resistant to conventional treatment.

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Acknowledgments IL-1 receptor antagonist-deficient mice were kindly supplied by Prof. Ian Frazer (University of Queensland).

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29



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Figure Legends Figure 1. Allogeneic MSC administration decreases HDM-induced allergic airway inflammation. A. Study design. B. Total cells in BAL at day 18. C. Differential cell counts in BAL. D. Peribronchial eosinophils. E. Type 2 innate lymphoid cells (ILC2) in the lung. F. Mast cells in the subepithelial mucosa. G. Mucus secreting airway epithelial cells (AEC). H. Concentration of total IgE in serum. I. Inflammatory cytokines in BAL. All data are from one representative experiment (n = 5-6 mice/group) of at least three. C and I, ‘-‘ indicates PBS, ‘+’ indicates HDM. A-C, E, H, and I: Data are mean ±SD. D, F and G: Data are mean ±SD. ***P< .001, **P< .01, *P< .05 vs PBS vehicle control.

###

P< .0001,

##

P< .001, #P< .05 vs

HDM sensitized and challenged mice. Figure 2. MSC inhibit HDM-induced airway hyper-responsiveness. A. Resistance of the central airways (Raw) and parameters of the lower airways. B. Coefficient of tissue damping (GL). C. Coefficient of tissue elastance (HL). All data are from one representative experiment (n = 6-12 mice/group) of at least three. Data are mean ±SD. Statistical analysis using 2 way ANOVA; ***P< .001, **P< .01, *P< .05 vs PBS vehicle control. ###P< .001, ##P< .01, #P< .05 vs HDM sensitized and challenged mice. Figure 3. Immune modulation of HDM-induced allergic airway inflammation is persistent after MSC administration. A. Study design of semi-chronic model. B. Total cell number in BAL. C. Differential cell count of BAL; ‘-’ indicates PBS, ‘+’ indicates HDM. D. Peribronchial eosinophils. E. Mucus secreting airway epithelial cells (AECs). F. Concentration of total IgE in serum. G. IL-13 in BALF. H. Airway hyperresponsiveness. All data are from one representative experiment (n = 5-6 mice/group) of at least two. B-C, F-H:

30


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Data are mean ±SD. D-E: Data are mean ±SEM. ***P< .0001, **P< .001, *P< .05 vs PBS vehicle control. ###P< .001, ##P< .01, #P< .05 vs HDM sensitized and challenged mice. Figure 4. MSC treatment decreases CD11b+ dendritic cells (DCs) in the lung and mediastinal lymph nodes (MLN). A and B: The total number of DCs (A) and MHCII+CD86+CD11b+ DCs (B) in the lung. C and D: The total number of CD11b+ DCs (C) and MHCII+CD86+CD11b+ DC (D) in the MLN. E and F: DQ-OVA+ activated DCs in the lung (E) and MLN (F). All data were obtained at day 16. All data are from one representative experiment (n = 5-6 mice/group) of at least two. Data are mean ±SD. ***P< .001, **P< .01, *P< .05 vs PBS vehicle control. ###P< .001, ##P< .01, #P< .05 vs HDM sensitized and challenged mice.

Figure 5. MSC treatment dampens innate cytokine production. A. IL-1α concentration in the lung at day 15 and 16. B. Representative photomicrographs of HMGB1 immunoreactivity; scale bar, 100 micrometers. Cytoplasmic HMGB1 expression in the airway epithelium at day 15 and 16. C-D. IL-25 (C) IL-33 (D) concentration in the lung at day 16. All data are from one representative experiment (n = 5-6 mice/group) of at least two. A,C, D: Data are mean ±SD. B: Data are mean ±SEM. **P< .001, *P< .05 vs PBS vehicle control. ##P< .01, #P< .05 vs HDM sensitized and challenged mice.

Figure 6. IL-1 receptor antagonist (IL-1ra) contributes in part to the immunomodulatory effects of MSCs on the airway epithelium and underlying dendritic cell (DC) network. A. Cytoplasmic HMGB1 expression in the airway epithelium at day 16. B. IL-25 concentration in the lung. C and D. CD11b+ DCs (C) and MHCII+CD86+CD11b+ DCs (D) in the lung. All data are from one representative experiment (n = 5 mice/group). A: Data are mean ±SEM, and B-D: Data are mean ±SD. *P< .05 vs HDM sensitized and challenged mice. 31



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Figure 1

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SUPPLEMENTAL INFORMATION

Immunomodulation of the airway epithelium and DC cell activation by mesenchymal stromal cells ameliorates HDM-induced allergic airway inflammation in mice.

Khang M. Duong, Jaisy Arikkatt, M.Ashik Ullah, Jason P. Lynch, Vivian Zhang, Kerry Atkinson, Peter D. Sly and Simon Phipps.


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Page 39 of 46

MATERIALS AND METHODS Animals Female 6-8 week old Balb/c mice were obtained from Animal Research Centre, Perth. All mice were specific pathogen free and were housed under 12 hour light-dark with food and water ad libitum in individually ventilated cages. MSCs were derived from C57BL/6 mice (also from Animal Research Centre) or IL-1 receptor antagonist-deficient mice on the same background, kindly supplied by Prof. Ian Frazer (University of Queensland). All studies conform to the principles outlined in the Australian Animal Care and Protection Act (2001) and were approved by the University of Queensland Animal Ethics Committee. Model of HDM-induced allergic airway inflammation Female BALB/c mice (H2d) were sensitised and challenged to HDM as described previously (E1). Briefly, adult mice were exposed to HDM (Dermatophagoides pteronyssinus) extract (40 µg in 50 μL; Greer Labs, Lenoir, NC) or PBS (vehicle control group) via intranasal delivery at day 0, 1 and 2. At day 14, one million donor MSCs from wildtype or IL-1 receptor antagonist-deficient C57BL/6 mice (H2b) or vehicle (fresh MSC culture medium) was infused via the tail-vein to HDM-sensitised mice immediately prior to allergen challenge. In this allogeneic system donor and recipient mice were thus two haplotype-mismatched with each other. Mice were challenged intranasally with HDM (5 μg in 50 µL) or PBS control at day 14, 15, 16 and 17 (Figure 1A). The hallmark features of allergic airway inflammation were measured at 3 or 24 hours (day 18) after the final challenge, again as indicated in Figure 1. Inflammation was analysed at day 15 and 16. In one experiment, mice were exposed to a second round of HDM challenge at day 42, 43, 44 and 45, and allergic airway inflammation assessed at 3 or 24 hours after the last HDM challenge.



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Isolation and expansion of MSCs Femurs were dissected and crushed in a mortar and pestle. The bone fragments were treated with type 1 collagenase and recovered cells cultured in a humidified incubator at 37°C, 5% CO2 for 48 hours, after which time the non-adherent cells were discarded. The adherent cells were removed with TrypLE™ Select (Gibco) and the CD45-Sca-1+ FACS-purified MSCs seeded at 104 cells/cm2. MSC were expanded until passage 6, then used for experimentation. To verify that the MSCs were multipotent, MSCs were induced into adipocyte, osteoblast, and chrondrocyte lineages in vitro (see Figure E1 in the online supplement) as described previously (E2). Broncho-alveolar lavage fluid (BALF) and serum collection Serum was collected via cardiac puncture and centrifuged twice at 13000 rpm for 10 mins to separate cells from the plasma. BALF was collected by performing a tracheotomy to cannulate the exposed trachea. The cannula was tied into the trachea for stability before a single volume of 600 µl of PBS was slowly injected to flush the airways three times. The BALF was centrifuged at 300 x g for 5 mins and supernatant collected for cytokine analysis and differential cell counts. The single cell suspensions were treated with red blood cell lysis buffer (Biolegend, San Diego, CA) and the cell number was calculated using a glass haemocytometer. Cells were spun at 600 x g for 5 mins on to positively charged cytospin slide. Measurement of cytokines and IgE The concentration of total IgE in serum, IFN- (BD Biosciences, Franklin Lakes, NJ), IL-5, IL-13 in BALF, and IL-25, IL-33 (R&D Systems, Minneapolis, MN) and IL-1α (Biolegend,



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San Diego, CA) in lung homogenate was quantified by ELISA as per the manufacturer’s instructions. Histological analysis of airway mucus, eosinophil, and mast cells The left lung lobe was dissected and fixed in 10% buffered formalin for 24 hours and stored in 70% ethanol until paraffin embedding and sectioning. Paraffin-embedded lung sections were stained with periodic acid-Schiff reagent to enumerate mucus secreting cells in the airways. Mast cells were detected by toluidine blue staining and Lendrum’s carbol chromotrope 2R was used to identify eosinophils. The three most inflamed airways (assessed by peribronchial cuffing) with a diameter greater than 200 µm were assessed per mouse (one section/mouse). Peribronchial eosinophils and mast cells were expressed as cells/100 m of epithelial basement membrane (BM) length. In addition, we performed a semi-quantitative assessment of histologic injury, identical to that described by Livraghi et al (3). In brief, severity of the five features (airway obstruction, airway obstruction, airspace enlargement, lymphoid hyperplasia, and airway inflammation) was graded on a scale from 0 to 3, then a mean score obtained for each mouse. Immunohistochemical detection of HMGB1 Paraffin-embedded lung sections were dewaxed, followed by antigen retrieval with acetate buffer. Tissue sections were probed for HMGB1 with rabbit anti-mouse HMGB1 (1:400 dilution; Abcam PLC, Cambridge, MA) and developed with fast red as described previously (Sigma, St. Louis, MO) (E4). To quantify HMGB1 expression in the cytoplasm of AECs, The three most inflamed airways (assessed by peribronchial cuffing) were selected. For each airway, all AECs were assessed as cyto-HMGB1 negative or positive to obtain a percentage, and a mean of the three values (airways) generated for each mouse.



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Quantification of type 2 innate lymphoid cells and dendritic cells by flow cytometry Lung or mediastinal lymph nodes (MLNs) samples were mechanically digested and red blood cells were lysed with RBC Lysis Buffer solution (Biolegend, San Diego, CA). Nonspecific binding to CD16/CD32 was blocked with Fc receptor neutralising antibodies. Cells were incubated with a cocktail of antibodies from Biolegend and analysed with the BD LSRFortessa™ flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Type-2 innate

lymphoid cells in the left lung as defined by CD45+ CD25+ CD90.2+ SCA-1+ lung cells and negative for the lineage markers; CD2, CD3e, CD4, CD11c, CD11b, CD19, GR-1 and B220 at day 17 of the acute model of HDM-induced allergic airway inflammation, as described previously (E5). CD11b+ CD11c+ dendritic cells were analysed for co-expression of MHC class II and CD86 (all antibodies from Biolegend, San Diego, CA). To detect antigen acquisition and processing, mice were exposed to DQ-Ovalbumin (25 µg; Molecular Probes, Eugene, OR) co-administered with HDM at day 14. Antigen acquired and activated DCs in the lung and mediastinal lymph nodes (MLNs) were enumerated at day 16.



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REFERENCES 1. Phipps S, Lam CE, Kaiko GE, Foo SY, Collison A, Mattes J, Barry J, Davidson S, Oreo K, Smith L, Mansell A, Matthaei KI, Foster PS. Toll/il-1 signaling is critical for house dust mite–specific th1 and th2 responses. American Journal of Respiratory and Critical Care Medicine 2009;179:883-893. 2. Cook MM, Futrega K, Osiecki M, Kabiri M, Kul B, Rice A, Atkinson K, Brooke G, Doran M. Micromarrows--three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells. Tissue Eng Part C Methods 2012;18:319-328. 3. Livraghi A, Grubb BR, Hudson EJ, Wilkinson KJ, Sheehan JK, Mall MA, O'Neal WK, Boucher RC, Randell SH. Airway and lung pathology due to mucosal surface dehydration in {beta}-epithelial na+ channel-overexpressing mice: Role of tnf-{alpha} and il4r{alpha} signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment. J Immunol 2009;182:4357-4367. 4. Ullah MA, Loh Z, Gan WJ, Zhang V, Yang H, Li JH, Yamamoto Y, Schmidt AM, Armour CL, Hughes JM, Phipps S, Sukkar MB. Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation. J Allergy Clin Immunol 2014. 5. Kaiko GE, Loh Z, Spann K, Lynch JP, Lalwani A, Zheng Z, Davidson S, Uematsu S, Akira S, Hayball J, Diener KR, Baines KJ, Simpson JL, Foster PS, Phipps S. Toll-like receptor 7 gene deficiency and early-life pneumovirus infection interact to predispose toward the development of asthma-like pathology in mice. J Allergy Clin Immunol 2013;131:13311339 e1310.



A

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Osteoblast

Adipocyte

Figure E1. Morphology, phenotype, mesodermal differentiation potential and immunoregulatory properties of bone marrow-derived Mesenchymal Stromal Cells (MSCs). A, MSCs have a spindle shape and fibroblast like morphology in log phase growth (scale bar: 100μm). B, MSCs do not express CD45 and CD11b and are Sca-1 and CD44 positive cells. C MSCs differentiate in to Chondrogenic (x80 magnification), Osteogenic (x80 magnification) and Adipogenic (x160 magnification) lineages following 3 weeks in culture with specific induction media.



B ##

***

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2

1

3

#

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***

2

1

M

SC M

H D M

PB S

SC

0

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H D M

Pathology score

3

PB S

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Pathology score

Page 45 of 46

Figure E2. MSC administration significantly reduces lung pathology in an acute (A) and semi-chronic (B) model of HDM-induced asthma. Lung pathology was scored using a semi-quantitative method as described in the online supplement. All data are from one representative experiment (n = 5-6 mice/group) of at least two. ***P< .001 vs PBS vehicle control. ##P< .001, #P< .05 vs HDM sensitized and challenged mice.



B **

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C

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Ig E in s e r u m ( n g /m l)

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Page 46 of 46

** 800

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400 #

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Figure E3. Donor MSCs from C57BL/6 mice are intravenously administered in to MHC class II matching HDM- sensitised C57BL/6 mice. A, total number of BAL cells. B, absolute numbers of lymphocytes, neutrophils and eosinophils. C, percent of mucus secreting airway epithelial cells. D, total IgE in serum. All data are from one

representative experiment (n = 5-6 mice/group) of at least two. ***P< .001, **P< .001, *P< .05 vs PBS vehicle control.

###P

-) mice.

Schistosoma japonicum infection downregulates house dust mite-induced allergic airway inflammation in mice.

β2 adrenergic agonist attenuates house dust mite-induced allergic airway inflammation through dendritic cells.

Protective Roles for RGS2 in a Mouse Model of House Dust Mite-Induced Airway Inflammation.

Biosignature for airway inflammation in a house dust mite-challenged murine model of allergic asthma.

Investigating the Effects of Particulate Matter on House Dust Mite and Ovalbumin Allergic Airway Inflammation in Mice.

Systemic Administration of Human Bone Marrow-Derived Mesenchymal Stromal Cell Extracellular Vesicles Ameliorates Aspergillus Hyphal Extract-Induced Allergic Airway Inflammation in Immunocompetent Mice.

Dectin-2 sensing of house dust mite is critical for the initiation of airway inflammation.

Antisense MicroRNA Therapy of Airway Remodeling in House Dust Mite-sensitized Mice.

Epithelial NF-κB orchestrates house dust mite-induced airway inflammation, hyperresponsiveness, and fibrotic remodeling.

Activation of the Prostaglandin E2 receptor EP2 prevents house dust mite-induced airway hyperresponsiveness and inflammation by restraining mast cells' activity.

Mesenchymal stromal cells mediate Aspergillus hyphal extract-induced allergic airway inflammation by inhibition of the Th17 signaling pathway.

Effect of P2X4R on airway inflammation and airway remodeling in allergic airway challenge in mice.

Influence of Asian dust particles on immune adjuvant effects and airway inflammation in asthma model mice.

Cell Jamming in the Airway Epithelium.

Dectin-2 promotes house dust mite-induced T helper type 2 and type 17 cell differentiation and allergic airway inflammation in mice.

Anti-HMGB1 neutralizing antibody ameliorates neutrophilic airway inflammation by suppressing dendritic cell-mediated Th17 polarization.

Raw Cow's Milk Prevents the Development of Airway Inflammation in a Murine House Dust Mite-Induced Asthma Model.

Cystic fibrosis airway epithelium remodelling: involvement of inflammation.

Vaccination against IL-33 Inhibits Airway Hyperresponsiveness and Inflammation in a House Dust Mite Model of Asthma.

Interleukin-33 from Monocytes Recruited to the Lung Contributes to House Dust Mite-Induced Airway Inflammation in a Mouse Model.

[Regeneration of airway epithelium].

Long-Term Exposure to House Dust Mite Leads to the Suppression of Allergic Airway Disease Despite Persistent Lung Inflammation.

Apyrase protects against allergic airway inflammation by decreasing the chemotactic migration of dendritic cells in mice.