Controlled release of cytokines using silk-biomaterials for macrophage polarization
Graphical abstract
Introduction
The innate immune system is the first line of defense against pathogens upon infection or wounding. Macrophages clear dead cell debris from a wound, help fight pathogens, and present pathogen-associated antigens to T-cells to activate the adaptive immune response. While macrophages are extremely important for immune defense and wound healing, chronic activation of different types of macrophages has been associated with several diseases, including atherosclerosis [1], [2], insulin resistance or metabolic syndrome [3], [4], [5], and cancer [6]. Treating these diseases by targeting macrophages systemically is not ideal, as the treatment can impair the immune system as a whole. Localized delivery of treatments for chronic macrophage activation is therefore highly desired. Furthermore, implanted devices or tissue engineered constructs can be functionally hampered by the foreign body response, which involves macrophage-derived giant cell formation and development of granulation tissue surrounding the implants. A film or thin coating on the device that can control macrophage response would be highly beneficial.
Acute inflammation occurs in a wound or at an implant site just after the deposition of fibrinogen and blood clotting. Following neutrophil infiltration monocytes are recruited to the site of the wound within the first 24 h to start clearing away dead cell debris and phagocytose bacteria that may have entered the wound [7], [8]. As the monocytes infiltrate the wound site they begin to differentiate into macrophages [9]. Once at the wound site macrophages polarize into specialized cells for different purposes. Interferon-gamma (IFN-γ) is a cytokine that activates macrophages to polarize along the “classical pathway,” which generates inflammatory macrophages, termed M1 macrophage due to the concurrent emergence of type 1 helper T-cells (Th1) [10]. M1 macrophages incite further inflammation by secreting cytokines such as interleukin-1β (IL-1β), IL-6, IL-12 and tumor necrosis factor alpha (TNF-α) [11]. Several markers of M1 macrophage differentiation have been found including C–C chemokine receptor type-7 (CCR7) [12]. These CCR7+ M1 macrophages help to defend against bacterial infections, contribute to phagocytosis of dead cells, and recruit Th1 cells to the site of the infection. Diseases associated with chronic activation of M1 macrophages include atherosclerosis [2], obesity [13], [14], and inflammatory bowel disease [15], [16].
Following the acute inflammatory phase, M2 macrophages emerge at the wound site, corresponding to the emergence of Th2 cells [17]. M2 macrophages are considered anti-inflammatory because they secrete IL-10, cytokine ligand 13 (CCL13), and transforming growth factor-beta (TGF-β), which promote tissue regeneration [18], [19]. The M2 macrophages are polarized through the “alternative pathway” by activation of the IL-4 receptor with either IL-4 or IL-13 [20]. The M2 macrophages are commonly delineated from M1 macrophages by the expression of the mannose receptor CD206 [20], as well as CD163 and CD209 [21], [22], [23], [24]. M2 macrophages have been implicated in promoting tissue regeneration and proliferation [25]. In addition, tumor associated macrophages (TAM) are believed to take on M2-like characteristics, including the M2 marker CD206, as they promote tumor progression [26], [27], [28].
One of the interesting properties of the different macrophage phenotypes is their plasticity. The ability for an M1 macrophage to become M2 or vice versa has been observed in vitro and in vivo [2], [29], [30], [31]. This leads to the possibility that macrophages can be converted using implantable biomaterials to control a specific disease. For instance, conversion of M1 macrophages that are found abundantly in atherosclerotic plaques into M2 macrophages might result in a reduction of plaque obstruction of blood flow by inducing tissue remodeling surrounding the blood vessel. Similarly, conversion of TAMs to M1 macrophages may allow them to better attack the tumors rather than aiding in their proliferation. Materials that can locally release cytokines to differentiate macrophages without systemically affecting the immune system could be very beneficial for the treatment of the macrophage associated diseases.
Recently, decellularized bone scaffolds with IL-4 bound via biotin-streptavidin interactions were used to promote vascular genesis in vivo and to control macrophage polarization in vitro [32]. That study demonstrates the potential for cytokine manipulation of host macrophages responding to implanted scaffolds. Future materials that can be coated onto any scaffold or medical device and that can release cytokines for macrophage control may have broad applications and implications for the medical industry.
Silk fibroin is a protein derived from the cocoons of the Bombyx mori silk worms and has been used for centuries as a suturing material. Silk has more recently been used as a versatile material for other biomedical purposes such as for fabricating scaffolds for cells, and therapeutics [33], [34], and for localized drug delivery into wounds [35], [36], [37]. Silk fibers can be solubilized and processed into biomaterials that have an advantage over other commonly used implantable materials because they have the ability to stabilize drugs and proteins [38], have a slow and controllable degradation rate, and the degradation products are non-toxic [39], [40].
The goal of the present study was to use thin layers of silk protein as films to modulate the release of cytokines to control macrophage responses. In the long run this approach is anticipated to help with the incorporation of tissue engineered constructs and medical device implants into the body and well as improving wound healing. Silk films were utilized in the study as they can be tailored to sustain the release of cytokines and also be processed in aqueous, ambient conditions, to preserve the functionality of the cytokines for these types of in vitro studies. The goal to control the polarization of macrophages would have value in fundamental in vitro inquiries of macrophage functions, as well as in chronic macrophage associated diseases. If successful, these approaches could be useful for treating a number of diseases from atherosclerosis to cancer by converting the M1 into M2 or vice versa.
Section snippets
Silk degumming and dissolving
Silkworm cocoons from B. mori were purchased from Tajima Shoji Co., LTD (Yokohama, Japan) and are processed into pure silk fibroin solution as previously described [41]. Briefly, cocoons are cut into small pieces and placed into boiling 0.02 mM Na2CO3 for 30 min. After boiling, the fibers are washed in deionized water and left to dry in a fume hood overnight. The next day a solution of 9.3 M LiBr is added to the silk fibers at a ratio of 1:4 weight (g) to volume (mL) and then allowed to
Solubility and cytokine release profile of silk films
Treatment of silk films with 90% methanol (MeOH) and water induces rapid β-sheet content, which causes the films to become insoluble [41]. However, solvents can cause conformational changes to proteins that lead to inactivity of the protein and thus solvents were avoided for the creation of silk films that contained the cytokines. The process of water annealing (WA) allows control of the β-sheet content of silk films, avoids brittleness [42] and avoids solvent-induced protein inactivity. The
Discussion
One goal for biomaterial and tissue engineered implants is to control the biological response, optimizing regenerative needs while reducing inflammatory pathways. Following implantation of a biomaterial, scaffold, or tissue, serum proteins will begin to adsorb to the surface allowing for immune cells to adhere and develop a response to the implant [50], [51]. Monocytes and macrophages are innate immune cells that mount part of the early response to implanted materials. Macrophages have been
Conclusions
This study investigated the ability of silk films to release cytokines and control THP-1 macrophage polarization. Silk films loaded with either IFN-γ or IL-4 were able to polarize macrophages to the M1 or M2 macrophage phenotype, respectively. Release of the cytokines from the films was found to be correlated with the solubility of the silk films, which was in turn dependant on the percentage of β-sheet content in the films. Insoluble films were also able to direct macrophage polarization after
Funding sources
We thank the NIH (P41 EB002520) for support of this research.
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