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Mechanical checkpoint regulates monocyte differentiation in fibrotic niches


Myelofibrosis is a progressive bone marrow malignancy associated with monocytosis, and is believed to promote the pathological remodelling of the extracellular matrix. Here we show that the mechanical properties of myelofibrosis, namely the liquid-to-solid properties (viscoelasticity) of the bone marrow, contribute to aberrant differentiation of monocytes. Human monocytes cultured in stiff, elastic hydrogels show proinflammatory polarization and differentiation towards dendritic cells, as opposed to those cultured in a viscoelastic matrix. This mechanically induced cell differentiation is blocked by inhibiting a myeloid-specific isoform of phosphoinositide 3-kinase, PI3K-γ. We further show that murine bone marrow with myelofibrosis has a significantly increased stiffness and unveil a positive correlation between myelofibrosis grading and viscoelasticity. Treatment with a PI3K-γ inhibitor in vivo reduced frequencies of monocyte and dendritic cell populations in murine bone marrow with myelofibrosis. Moreover, transcriptional changes driven by viscoelasticity are consistent with transcriptional profiles of myeloid cells in other human fibrotic diseases. These results demonstrate that a fibrotic bone marrow niche can physically promote a proinflammatory microenvironment.

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Fig. 1: Interpenetrating fibrillar collagen and click-modified alginate network hydrogels tune mechanical resistance of ECM.
Fig. 2: Viscoelastic properties of ECM differentially regulate monocyte secreted cytokines and patterns of gene expression.
Fig. 3: Viscoelasticity of ECM controls dendritic cell differentiation and immature monocytes expressing pSTAT3 and an endogenous inhibitor of non-canonical NF-κB, IκBζ.
Fig. 4: Elasticity promotes dendritic cell differentiation through PI3K-γ.
Fig. 5: Elasticity of ECM is associated with inflammation in MPNs and is targetable by PI3K-γ inhibition.
Fig. 6: Single-cell transcriptional profile of myeloid cells in IPF and liver cirrhosis was associated with differential gene expression regulated by viscoelasticity in vitro.

Data availability

Digital data supporting the findings of this article are available at In vitro mouse bulk RNA-seq and human in vitro and patient-derived nanoString datasets are available at the NCBI GEO database, GEO accession code GSE206773. The following scRNA-seq datasets were utilized for analysis in this study: liver cirrhosis, GEO accession GSE136103; IPF, GEO accession GSE122960.


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At the Wyss Institute, we thank T. Ferrante for assistance with imaging, and group members D. Zhang and A. Gonzalez-Pujana for their assistance and feedback on this work. At Massachusetts General Hospital, we thank D. Lagares for his feedback and editing. At the Massachusetts Institute of Technology, the authors thank T. Gierahn and the BioMicroCenter for assistance with bulk RNA-sequencing. At Dana-Farber Cancer Research Institute, we thank C. Laurore for technical assistance. Part of this work was performed at the Bauer Core Facility at Harvard University. We thank S. Reinke and his Cell Harvest Unit of the Berlin Institute of Health Center for Regenerative Therapies and the Charité for supply of the human fracture haematoma samples. We acknowledge Sysmex America, M. Sola-Visner and her laboratory members E. Nolton and P. Davenport (Boston Children’s Hospital) for kindly sharing Sysmex equipment for complete blood counts. Research reported in this manuscript was supported by the National Cancer Institute of the National Institutes of Health under award number U01CA214369 (D.J.M.), the National Institute of Dental & Craniofacial Research of the National Institutes of Health under award numbers K08DE025292 and K99DE030084 (K.H.V.), the Federal Drug Administration R01FD006589 (D.J.M.), and the Harvard University Materials Research Science and Engineering Center (grant DMR 1420570). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Parts of the work were supported by the Einstein foundation and the German Research Foundation in the context of the DFG FOR 2165 grant and CRC 1444 grants (G.N.D.). A.E.M. was supported by a long-term fellowship from the European Molecular Biology Organization (EMBO, ALTF 268-2017) and Horizon award from the US Department of Defense (W81XWH-20-1-0904). A.M. is a Scholar of the Leukaemia and Lymphoma Society (LLS). O.P. was supported by a Brigham and Women’s Hospital Faculty Career Development Award and by the Brigham Research Institute and the Center for Faculty Development and Diversity’s Office for Research Careers Microgrant Program.

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Authors and Affiliations



K.H.V., A.E.M., K.W. and D.J.M conceptualized and designed research. F.S.H. advised K.H.V. K.H.V. and D.J.M wrote the original draft. K.H.V., A.E.M., K.A.-B., C.M.T., J.M.G., W.J.W., O.P., M.S., A.M. and A.S. performed research. K.H.V., A.E.M., K.A.B. and Y.L. analysed and visualized data. W.J.W. provided histology images of patient biopsies. G.N.D. provided data on human bone marrow mechanics. W.J.W., O.P., A.E.M. and A.M. provided expertise on MPN and myelofibrosis, as well as mouse JAK2-V617F and wild-type monocytes and femurs. A.E.M. performed mouse experiments. All authors contributed review and/or editing of the manuscript.

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Correspondence to Kai W. Wucherpfennig or David J. Mooney.

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D.J.M. has sponsored research from Novartis, and consults and/or has stock options/stock in J&J, Samyang Corp., Lyell, Attivare, IVIVA, and Revela; none of these relate to the topic of this manuscript. Within the past 12 months, A.M. has consulted for Janssen, PharmaEssentia, Constellation and Relay Therapeutics and receives research support from Janssen and Actuate Therapeutics. The authors confirm that there are no known conflicts of interest associated with this publication and there has been no notable financial support for this work that could have influenced its outcome. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Related to Fig. 2. Nested scatterplots of normalized gene expression of markers upregulated in cells in viscous and soft-elastic gels (A) and stiff-elastic gels (B).

Each set of data points represents biological replicates from a particular donor (the two sets of data points at each experimental condition represent cells from the n = 2 donors). Soft ~ 0.75 kPa, Stiff ~ 7.5 kPa elastic moduli. P-values indicate statistical significance (p < 0.05) compared to stiff-elastic gels (*, asterix) of nested one-way ANOVA with Tukey’s multiple comparisons tests. All analyses were performed at 3 days after encapsulation.

Extended Data Fig. 2 Related to Fig. 3. Nested scatterplots of flow cytometry performed on samples from n = 3 donors.

HLA-DR mean fluorescence intensity (MFI), fraction of live cells expressing dendritic cell differentiation markers CD11c + CD1c + , CD80 + , and PDL1 + gated on live-HLA-DR + cells for soft viscous (blue), stiff viscous (red), soft elastic (green), and stiff elastic (purple) gels. P-values indicate statistical significance (p < 0.05) of n = 3 biological replicates of two-way ANOVA with Tukey’s multiple comparisons tests compared to stiff elastic gel within each donor (asterix). All analyses were performed at 3 days after encapsulation.

Extended Data Fig. 3 Related to Fig. 3. STAT3 transcription factor is associated with differentially expressed genes in stiff viscous hydrogels.

A) Unsupervised hierarchical clustering of differentially expressed genes from bulk RNA sequencing. Cells from soft and stiff viscous gels are compared to cells in non-adherent tissue culture with or without calcium control. The calcium control are cells exposed to free calcium released from an empty hydrogel. Markers were separated into 11 clusters associated with patterns of gene expression. B) Clusters 5, 6, 7, 8, and 11 were associated with genes upregulated in stiff viscous conditions. These markers were put into the ENCODE ChIP-Seq database, which identified STAT3 as one of the top ranked transcription factors involved in regulating these genes. C) Flow cytometry of HLA-DR MFI and CD11b + CD11c + cells (gated on live-HLA-DR + ) with blocking anti-IL6R antibody (1μg/ml), n = 3 biological replicates. D) Normalized gene expression of NFKBIZ at Day 0 prior to encapsulation and after 3 days in viscous and elastic gels of soft (0.75 kPa) and stiff (7.5 kPa) elastic modulus. Data points indicate biological replicates from n = 2 donors for viscous and elastic gels, and n = 1 donor for day 0 control. P-values<0.05 indicate statistical significance of one-way ANOVA with Tukey multiple comparisons test. Experiments were repeated with at least two donors. Data are presented as mean values + /- SEM.

Extended Data Fig. 4 Related to Fig. 4. F-actin in elastic gels is independent of collagen adhesive ligands and actomyosin contractility.

A) Pan-actin staining of cells in viscous and elastic gels. B) F-actin staining of cells in elastic gels without collagen. C) F-actin staining with or without inhibition of actomyosin contractility by non-muscle myosin-II by blebbistatin (10 μM). D) Flow cytometry for dendritic cell differentiation markers with or without blebbistatin. Images are maximum intensity projections of representative cells. Scale bar 10 um. Data points indicate n = 3 biological replicates from 1 donor. P-values<0.05 indicate statistically significant difference by ordinary one-way ANOVA with Tukey multiple comparisons test. All analyses were performed at 3 days after encapsulation. Data are presented as mean values + /- SEM.

Extended Data Fig. 5 Related to Fig. 5. Viscoelasticity impacts Jak2 signaling in Jak2-V167F monocytes.

A) Volcano plot of global transcriptome of differentially expressed genes comparing mouse Jak2-V617F monocytes in stiff, elastic versus stiff, viscous gels. Bulk RNA-sequencing was performed in duplicate. Genes marked in red are above thresholds of both fold change = 21 and p-value = 10-2 threshold (dotted lines). Green indicates genes above fold change threshold, but below the p-value threshold. Blue indicates genes above the p-values threshold, but below the fold change threshold. Grey indicates genes that are not significantly differentially expressed. Data was obtained from n = 2 biological replicates. B) Normalized expression of JAK-STAT pathway genes in Jak2-V617F monocytes cultured in elastic versus viscous hydrogels, with linear regression fit (black line). Genes enriched in elastic gels are marked in red and appear above the linear regression (viscous in blue). Data points indicate mean normalized expression of n = 2 biological replicates from a single experiment (one of each gender). C) IL6 and LIF levels of conditioned media from WT or Jak2-V617F monocytes in viscous (blue) or elastic (red) hydrogels. Data shown from a single experiment with n samples as individual data points collected from n = 2 biological replicates (one of each gender). P-value<0.05 indicates statistically significant difference by Brown–Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons test.

Extended Data Fig. 6 Related to Fig. 5. Pathologic grading of model of MF and flow cytometry analysis of BM neutrophils.

A) Representative MF grading of histologic sections from femurs of recipient retroviral Jak2-V617F mice, repeated n = 3 independent experiments. 60x objective. B) Flow cytometry to determine the fraction of bone marrow Ly6C + LY6G + neutrophils in mouse models of fibrotic (pMEGIX Jak2-V617F) and non-fibrotic (Mx1-cre Jak2-V617F) MPNs, gated on CD11b + cells. In the left panel, mice were untreated. Values were normalized to empty-vector controls, and data shown is from n = 3 independent cohorts of mice. In the right panel, cells were analysed from fibrotic pMEGIX Jak2-V617F recipient mice and treated with either vehicle control (-) or IPI-549 (+) daily for 2 weeks with oral gavage of 15 mg/kg IPI-549 10% DMSO/5%NMP/85% PEG400. Data points indicate n biological replicates from 1 independent experiment. P-values <0.05 indicate statistically significant differences of one-way ANOVA with Tukey multiple comparisons test or unpaired two-tailed Student’s t test. Data are presented as mean values + /- SEM.

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Supplementary methods, Figs. 1–8, Tables 1–9 and references.

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Supplementary Data 1

Custom human nanoString elements panel.

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Vining, K.H., Marneth, A.E., Adu-Berchie, K. et al. Mechanical checkpoint regulates monocyte differentiation in fibrotic niches. Nat. Mater. 21, 939–950 (2022).

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