Reduced substrate stiffness promotes M2-like macrophage activation and enhances peroxisome proliferator-activated receptor γ expression
Introduction
Tissue stiffness represents an important micro-environmental signal that triggers functional alterations of tissue-resident cells. Increased tissue stiffness has been observed in clinical samples marked by underlying chronic inflammation such as atherosclerosis [1], cirrhosis [2], multiple sclerosis [3] and cancer [4]. Sustained inflammation and tissue remodeling induce tissue stiffening by enhancing the crosslinking [5], [6] and production of an extracellular matrix (ECM) [5], [7], proteins, activating fibroblasts to secrete transforming growth factor-β (TGF-β) protein [6], [8], and/or by modifying cytoskeletal proteins in endothelial cells [9] and smooth muscle cells [10]. The pivotal role played by increased tissue stiffness in promoting tumor progression has previously been shown [5]. Levental and colleagues have reported that chronic inflammation-associated ECM remodeling in the tumor microenvironment induces ECM stiffening by collagen crosslinking [5]. This leads to the promotion of focal adhesions, phosphoinositide 3-kinase (PI3K) activation, and the invasion of tumor cell [5], suggesting that ECM stiffness plays an important role in the regulation of cell adhesion, cell growth and tumor malignancy. Following transformation, breast tissue becomes progressively stiffer by ECM-rearrangement while tumor cells become more contractile and hyper-responsive to matrix compliance cues during the early phase [11]. Conversely, tumor cells manipulate their microenvironment to enhance their own survival, thereby creating a positive tumorigenic feedback loop [5], [12], [13]. In addition to tumorigenesis, the cell differentiation of pluripotent mesenchymal stem cells has been directly linked to culture substrate stiffness [14]. Substrate stiffness is the property of a substrate to resist deformation in the response to any applied force and is a function of material property. Engler and colleagues have shown that soft substrates are capable of differentiating mesenchymal stem cells into neuronal-like cells, moderating elasticity promoted myogenic differentiation, and forming a rigid substrate that stimulates osteogenic differentiation [14]. Furthermore, expanding myogenic stem cells on soft hydrogel materials can lead to enhanced self-renewal and improved engraftment into mice [15]. Therefore, alteration of tissue stiffness in ECM remodeling is important to regulating cellular functions.
Macrophages play important roles in ECM remodeling during tumor progression [16], [17], liver injury [18], [19], [20], and atherosclerosis [21], [22]. While driving ECM remodeling, macrophages are affected by the alteration of substrate stiffness that occurs as a consequence of ECM remodeling. It has been shown that macrophages on soft substrate produced less pro-inflammatory cytokines than those on stiff substrate [23], [24], [25]. In addition, soft substrates induced less focal adhesion complexes and filamentous-actin (F-actin) stress fiber formation in macrophages [26], thereby suggesting that soft substrates might suppress macrophage activation. It has been demonstrated that macrophages use αMβ2 integrins to mechano-sense substrate stiffness and, thereby, induce cell spreading on stiff substrates [26], [27].
Macrophages acquire at least two distinct activation phenotypes: classical M1 activation and alternative M2 activation. M1 macrophages are characterized by the expression of high levels of pro-inflammatory cytokines [28], elevated production of reactive nitrogen and oxygen intermediates [29], the promotion of a Th1 response [30], and anti-microbial and anti-tumor activity [29], [31]. In contrast, M2 macrophages are characterized by low production of tumor necrosis factor-α (TNF-α) and high production of interleukin-10 (IL-10) and TGF-β. Thus, they are believed to be involved in parasite clearance [32], the promotion of a Th2 response [30], [33], immunoregulation [32], tissue remodeling and tumor progression [28], [34]. M2-like macrophages are thought to support cell proliferation and matrix deposition, thereby contributing to tissue remodeling and wound healing [28], [35]. A recent study showed that human macrophages in stiffer matrices displayed a wound healing phenotype [36]. Although previous studies have suggested that macrophage activation phenotypes are modulated by substrate stiffness [23], [24], [26], [36], the molecular mechanisms by which substrate stiffness regulates activation phenotypes of macrophages remain to be elucidated. In this study, we have shown that reduced substrate stiffness induces the anti-inflammatory M2-like activation phenotype of macrophages, possibly through the induction of peroxisome proliferator-activated receptor γ (PPARγ) expression.
Section snippets
Cells
A human monocytic THP-1 cell was obtained from the American Type Culture Collection (Manassas, VA, USA), and maintained in RPMI1640 medium supplemented with 10% fetal calf serum (FCS).
Substrate
As the stiffness of healthy and diseased arteries and inflamed endothelial cells has ranged from ̴2 kPa and 100 kPa [7], [37], [38] in previous studies, three types of substrates were used as follows: 1% gel (4 kPa) and 4% gel (15 kPa) were used to model stiffness in normal vessels, and 10% stiff gel (100 kPa) was
The soft substrate suppressed M1-like activation of THP-1 cells
To investigate the impact of substrate stiffness on THP-1 cell activation, we employed 1% and 4% of agarose gel as soft substrates and plastic plate as a stiff substrate. PMA-stimulated THP-1 cells on agarose gels significantly reduced IL-6 and MCP-1 expression compared with those on stiff plastic plate (Supplemental Fig. 1). This reduction depended on substrate stiffness and does not depend on agarose gel concentration. Activation of monocytes with PMA alone does not induce specific
Discussion
Environmental cues including microbial products, damaged cells, activated lymphocytes, and mechanical stress affect distinct functional macrophage phenotypes [46]. In addition, physical properties such as tissue stiffness may influence macrophage function and activation phenotypes [23], [36]. Recently, Friedemann has reported that human macrophages respond to the stiffness of 3-dimensional matrices in terms of polarization toward pro-inflammatory or wound healing phenotypes in vitro [36].
Conclusion
We have found that the soft substrate attenuates M1-like macrophage polarization and predominantly induced the M2-like macrophage polarization through induction of PPARγ expression, suggesting that substrate stiffness dictates a particular macrophage polarization. Thus it is anticipated a common mechanism that stiff substrate facilitates M1-like macrophage polarization whereas soft tissue predominantly induces M2-like macrophage polarization. It would be interesting to explore how alteration of
Acknowledgements
We thank Dr. Taruho Kuroda (Mie University), Nodoka Nago (Mie University), Bie Qingli (Jiangsu University), Dr. Takuma Kato (Mie University), Dr. Sungsoo Na (Indiana University-Purdue University Indianapolis), and Dr. Hiroki Yokota (Indiana University-Purdue University Indianapolis) for their experimental assistance.
Sources of funding
This work was supported in part by Grants-in-Aid for Scientific Research and Grants-in-Aid for Young Scientists from the Japan Society for the Promotion of Science (JSPS) (Nos. 24590579, 25461125, 16K09513, 16K15759).
Declaration of interest
All authors have no conflict of interest to declare.
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