Cell viability assay
To investigate the biocompatibility of a selected concentration of M13 phage (1012 pfu/ml), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used for examining the viability of peritoneal macrophages seeded onto M13 phage, M13 phage-RGD, and gelatin-coated and control plates. Two or seven days after culture of cells in a 96 well plate (1 × 104 cells/well), MTT solution (20 μL, 5 mg/ml) was added to cell culture media and plates were incubated at 37°C. After four hours, the medium was removed and 200 μL dimethyl sulfoxide (Sigma-Aldrich, USA) was added to each well for eluting the formazan crystals, and optical density was measured at 490 nm with a microplate reader (BioTek, USA).
Morphological analysis
Macrophages were cultured on the M13 phage, M13 phage-RGD, and gelatin-coated and control 24-well plates for 2 and 7 days. Then, the cells were stained by using a PKH26 Fluorescent Cell Linker Kit (Red, Sigma-Aldrich) according to the manufacturer’s recommendations. The cells were fixed with 4% paraformaldehyde and their nuclei were stained with DAPI. Finally, fluorescently labeled macrophages were imaged with an inverted fluorescence microscope (Olympus) and analyzed by ImageJ 1.8.0.
Scanning electron microscopy
Next, the morphology of macrophages from each experimental group was observed using SEM in order to evaluate the induced morphological changes. Briefly, cells were cultured on precoated tissue culture grade coverslips and after 2 and 7 days the coverslips were air-dried. Then, the samples were gold-coated and after that they were visualized using a scanning electron microscope (KYKY-EM3200, 26KV).
RNA preparation, cDNA synthesis, and qRT-PCR
Macrophages of experimental groups were harvested for RNA extraction after two or seven days in culture. Total RNA was extracted from freshly harvested macrophages using RiboEX (GeneAll) according to the manufacturer’s protocol. After Dnase treatment (Thermo), RNA samples were subjected to cDNA synthesis and qRT-PCR. RT2 SYBR Green High ROX Master mix was used for qRT-PCR and data were quantified using ∆CT method.
Cytokine measurement
Supernatants of the macrophages in experimental groups were collected after two and seven days and stored at -20˚C. The presence of IL-6, TNF-α, IL-10 and TGF- β cytokines were assessed using ELISA kits (eBioscience) following the manufacturer’s instructions. Each sample was dispensed in triplicate. The optical density of each well was determined at 450 nm.
NO production
NO production was measured according to the accumulation of NO2 in culture supernatants after 2 and 7 days culture using the Griess reagent, as previously described [27]. Briefly, 100 μL of Griess reagent was mixed with equal volumes of culture supernatants from each experimental group for 10 minutes at room temperature. Then, the absorbance at 540 nm was measured using a microplate reader. Standard curve was established using a graded solution of NO2. Results were presented as mean values from three separate samples.
Determination of intracellular ROS
The accumulation of intracellular ROS in each experimental group was evaluated by using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). This molecule de-acetylates after entry into the cells and then oxidizes with intracellular ROS to form fluorescently reactive DCF[28]. To determine ROS production, the experimental groups were incubated with DCFH-DA (10 μM) in serum-free culture media for 45 minutes at 37˚C, washed twice with PBS, and finally, analyzed by flow cytometry (BD FACSCanto II, BD Bioscience, San Diego, CA, USA).
Phagocytosis assay
For phagocytosis assay, primary macrophages were seeded on M13 phage, M13 phage-RGD, and gelatin-coated and control 24 well plates; after 2 and 7 days, they were evaluated for their ability to phagocyte the labeled Saccharomyces cerevisiae (yeast) and thymus. The yeast and thymus cells were induced to apoptosis by autoclaving and UV light exposure, respectively; they were counted and stained by PI or DAPI (1mg/ml in DMEM). After staining, the cells were washed twice and resuspended in DMEM. DAPI-stained Saccharomyces cerevisiae and thymus cells were added to each well at a ratio of 5:1 (5 yeast or thymus cells:1 macrophage). Then, macrophages were incubated for one hour at 37˚C and 5% CO2, and washed carefully to remove all thymus cells and yeast particles. The phagocytosis ability of macrophages in experimental groups was detected using a flow cytometer and fluorescence microscopy after terminating the reaction with cold PBS containing 2% fetal bovine serum (FBS) and washing with PBS containing 2% paraformaldehyde.
Statistical analysis
Differences between the two experimental groups were estimated by using Student's t-test. For more than two groups, significance was estimated by using one-way analysis of variance (ANOVA). Statistical analyses were performed by a GraphPad Prism (Version 6). Data were presented as mean ± standard error of means. P ⩽ 0.05 was considered statistically significant.
Results
Characterization of M13 bacteriophage and RGD peptide
The morphology of M13 phage was first confirmed by TEM imaging. As shown in Figure S3 (Supporting Information), TEM micrographs represent the direct observation of native M13 bacteriophage nanofilaments by diameter of 6.6 nm. Subsequently, the synthesized RGD peptide was verified by LC-MS analysis by determining the exact mass of peptide. Figure S4 (Supporting Information) shows the structure along with mass spectrum of the RGD peptide. The calculated exact mass (m/z) for peptide was reported 604.28, while the measured exact mass was 605.
Different coatings change cellular morphology and viability
To determine the effects of different coatings on cellular morphology and viability, primary mouse macrophages were placed directly on the M13, M13-RGD, and gelatin-coated and control plates. Cell morphology on each surface was observed and analyzed after two and seven days using PKH dye labeling and scanning electron microscopy (SEM) as represented in Figure 1 A and B. According to the fluorescent microscope images and SEM micrographs, the macrophages exhibited the more pronounced outgrowth and well-spread morphology on the M13 phage containing surfaces. It should be noted that as compared to other coated surfaces, macrophages seeded on the control plates occupied the least area. Macrophages on the gelatin surface also exhibited a well-spread morphology but their cell sizes were smaller than those of M13 phage containing surfaces (Figure 1C). To investigate the effects of different coatings on the survival and viability of primary macrophages, cells were subjected to MTT assay on the second and seventh days. Results of MTT assay suggested that mouse macrophages in each experimental group displayed a similar viability pattern, and the survival rate was not significantly different among non-coated or coated surfaces at any point of time (Figure 1D).
Different surfaces alter gene expression and cytokine secretion of M1-M2 macrophage markers
In this study, to further confirm the effects of different coatings on macrophage characteristics and paracrine secretion, the gene and protein secretion of the master regulatory cytokines related to macrophage polarization were determined in both two and seven days after culture. To examine the effects of different surfaces on macrophage characteristics, we conducted qRT-PCR and ELISA assays on the second and seventh days after culture for IL-6, IL-10, TGF- β and TNF-α cytokines. Gene expression and cytokine production of IL-6 and TNF-α were upregulated in macrophages of M13 phage containing surfaces in two days; but after seven days, the expression of TNF-α showed a downtrend as compared to control surface (Figure 2 A and B). Accordingly, levels of IL-10 and TGF- β gene expression and cytokine secretion were much higher in M13 phage containing surfaces as compared to the control surface in both 2 and 7 days (Figure 2C, D). Furthermore, macrophages from gelatin surface showed the same gene expression and cytokine production patterns as control surface.
To confirm the macrophage polarization pattern, primary murine macrophages in each experimental group were either treated or not treated with LPS. 2 days later, the expression of IL-10, IL-22, CCL22, CXCL10, TNF-α, TGF- β genes were investigated. According to the results obtained, non-LPS treated macrophages cultured on M13 phage containing surfaces showed higher levels of anti-inflammatory gene expression and cytokine production as compared to gelatin and control surfaces (except for TNF-α). Interestingly, exposure to LPS could not increase inflammatory cytokines of macrophages in M13 phage containing surfaces. (Fig. 2E).
Different surfaces alter gene expressions of iNOS and ARG1, NO secretion
To investigate the role of selected surfaces on polarization state of macrophages, cultured cells from each experimental group were analyzed for ARG1 and iNOS gene expression and NO secretion. Our results demonstrated that the transcript level of ARG1 in the M13 phage containing surfaces was higher than the control surface at 2 days. During the 7-day culture of cells, the gene expression of ARG1 was significantly upregulated only in the cells cultured on M13 phage-RGD surface compared to control surface (Figure 3A). In addition, iNOS gene expression was significantly upregulated in the M13 phage containing and Gelatin surfaces compared to their control counterparts after 2 days. While, the expression of iNOS was significantly downregulated after 7 days in M13 phage containing surfaces (Figure 3B). The results of qRT-PCR for iNOS gene expression were confirmed by the results of NO production in the culture supernatant of macrophages from each group (Figure 3C). For determining the M1/M2 balance of cultured macrophages, the ARG1/iNOS mRNA expression ratio was measured. There was a significant increase in the ratio of ARG1/iNOS mRNA in M13 phage-RGD surface compared to this ratio in control surface after 7 days cultured (Figure 3D).
Different surfaces alter cellular redox potential in macrophages
The redox potential of cultured macrophages was assessed using the determination of intracellular ROS production. Here, culturing of macrophages on M13 phage containing surfaces dramatically decreased the intracellular ROS level as compared to control surface in both 2 and 7 days (Figure 4).
Different surfaces alter phagocytosis and efferocytosis of cultured primary macrophages
To define the functional effects of selected surfaces on macrophage phagocytosis and efferocytosis, we analyzed the uptake of labeled yeasts and apoptotic cells by cultured cells. In this study, the capacity of macrophages for internalization of yeasts and apoptotic cells was evaluated by both fluorescent microscopy and flow cytometry. We found significantly increased uptake of apoptotic cells by macrophages cultured on M13 phage containing surfaces on the second and seventh days after culture. However, macrophages from control and gelatin surfaces exhibited increased level of phagocytic activity in two and seven days (Figure 5A, B).
Discussion:
In recent past, the importance of the biomaterial ability utilized in diverse areas including implantation, immunotherapy, drug delivery and vaccination has changed from ‘’immune evasive” to ‘’immune interactive” modulating the immune system responses in favor of mentioned biotechnological approaches [29].Bacteriophage M13 has be considered as new biomaterial for using in the regenerative processes, delivery and immunotherapy. M13 phage has specific characteristics making it to be a potential biomaterial to control the immune system responses. This filamentous bacteriophage has naturally contacted with mammalian cells. Recent studies have revealed that bacteriophages can stimulate immune cells and modulate both innate and adaptive immune responses in the host[4, 30]. Macrophages according to the environment stimulus are able to polarize into pro-inflammatory and anti-inflammatory phenotypes. Besides, they affect the other cell responses and phenotypes directly and indirectly. These abilities have made them a key regulator of immune system. Biomaterials by controlling macrophage polarization are able to mediate tolerance and modulation of the immune response[31, 32].
In the present study, we demonstrated that the M13 phage as a biomaterial is able to modulate macrophage responses. For this purpose, we examined M13 phage interaction with macrophages as a representative of tissue resident immune cells in vitro. we illustrated that M13 phage containing surfaces, change macrophage response and alter cytokine profile and polarization.
Previous reports demonstrated that RGD peptide plays a critical role in the spread of cells through focal adhesion [46]. Supporting previous studies, our immunofluorescence and scanning electron microscopy analysis represent the most dramatic increase in the cell number and contact areas of cultured macrophages on M13 phage surface and its RGD modification. Therefore, phage coated surfaces can provide better structural support for adhesion that facilitate more cell proliferation and migration[33, 34] [35]. Furthermore, our findings indicated that the M13 phage alone and the M13 phage in combination with RGD peptide are both non-toxic and biocompatible.
It has been well documented that macrophages contribute to modulate immune responses through their paracrine secretion [36]. During the early stages of normal wound healing, M1 macrophages infiltrate the wound to promote inflammation and to stimulate the wound healing process. M2 macrophages begin to accumulate around the third or fourth day after injury, while the level of M1 macrophages decreases. M2 macrophages generate in several ways including direct shift of M1 type to M2 type macrophages, polarization of newly migrating macrophages toward M2 phenotype, and proliferation of other M2 macrophages [37]. Developing a phage-based strategy modulating the host innate immune and inflammatory responses, herein, we showed that the M13 phage, especially when combined with RGD, had the ability to reprogram naive peritoneal macrophages toward M2-like phenotype. By the same token, our results demonstrated that M13 phage and RGD peptide stimulate the secretion of TNF-α, IL-6, TGF- β and IL-10 at 2 days after culture. A time-dependent increase in M2 macrophage markers (IL-10 and TGF- β [36]) and decrease in M1 macrophage marker (TNF-α [37]) was observed as well. Over and above that, cytokine analysis at both gene expression and protein level showed polarization of macrophages to M2 phenotype after interaction with M13 phage containing surfaces. Our findings indicated a significant increase in IL-6 in M13 phage containing surfaces after 2 days, while after 7 days we observed no significant changes in the level of IL-6 compared to control surface and the basal level of IL-6 remained constant after 7 days. Since IL-6 has been shown to enhance polarization of M2 macrophages [40], we postulated that the M13 phage containing surfaces could modulate the polarization of macrophages toward anti-inflammation M2 phenotype. To examine whether M13 phage containing surfaces can suppress LPS-induced inflammatory responses (LPS tolerance), we stimulated the experimental groups with LPS. Cytokine analysis demonstrated that M13 phage and RGD peptide coated surfaces polarize phenotypic characteristics similar to M2 macrophage; and also, M13 phage containing surfaces induce tolerance by inhibiting LPS-induced M2 to M1 macrophage phenotypic shift. Former investigations have adduced evidences that LPS tolerance is essential for the reduction of tissue damage and innate immune response against infection which terminated to cell proliferation and migration [41, 42].
Moreover, M2 macrophages are metabolically different from M1 macrophages and the metabolic patterns of each are directly related to their immune-modulating functions. It has been accepted that the Inducible nitric oxide synthase (iNOS) gene and NO are highly expressed in M1 macrophages, while the upregulation of arginase1 (ARG1) was observed in M2 macrophages [43].Notably, L-arginine metabolic pathway of macrophages determines the polarization status toward M1 or M2 phenotype [44]. Whilst NO is a key molecule produced by M1 macrophages to exert their role in immune defense. High level production of NO is generated from the oxidation of L-arginine. ARG1 is responsible for another metabolic pathway for L-arginine in macrophages, and this pathway produces L-ornithine for the biosynthesis of polyamine and collagen. These products help M2 macrophages to modulate biomaterial-immune system interaction [45, 46]. It is noteworthy to mention that the competition between ARG1 and NOS enzymes determine the M1/M2 phenotypic shift in macrophages. We found that the ratio of ARG1/iNOS transcript levels in the M13 phage-RGD surface was much higher than this ratio in control surface during 7 days culturing. This finding further indicated the potency of M13 phage-RGD in M2 phenotype polarization.
It has been proposed that redox potential can play a complex role in the determination of macrophage cellular fate [47]. A previous study demonstrated that ROS level is closely related to essential signaling pathways, which regulate M1 macrophage polarization [48]. It is interesting to note that interactions between ROS and NO are responsible for the regulation of cellular inflammatory conditions [49]. The decreased redox potential of macrophages on M13 phage containing surfaces in our study further emphasized the potential role of these biomaterials in reducing inflammatory responses of macrophages.
Other than that, macrophages are responsible for the clearance of microbial pathogens (phagocytosis) and apoptotic cells (efferocytosis) [50]. [48]. Likewise, alternatively activated M2 macrophages have enhanced efferocytic capability while M1 macrophages are known to have low efferocytic properties [48, 49]. Therefore, to ask if selected surfaces in the current study could alter the functional properties of macrophages, we analyzed the phagocytic and efferocytic activity of each experimental group. Our results indicated that the M13 phage containing surfaces induce macrophages to uptake the apoptotic cells, while control and gelatin surfaces lead to increased phagocytic activity by macrophages. These evidences are indicative of the fact that M13 phage and RGD peptide could functionally alter the polarization state of macrophages toward M2 phenotype.
Conclusion:
Biomaterials are most important part of strategies for biomedical technologies like drug delivery, therapy and biocontrol. Modulation of immune system and reducing inflammatory responses are one of the most important properties of immune-interactive biomaterials. Here, we demonstrated that M13 phage stimulates macrophage polarization toward anti-inflammatory phenotype, alters their cytokine profile and functional property. More interestingly, our study pointed out that the combination of well-characterized RGD peptide motif embedded in M13 phage nanostructures may enhance the macrophage responses more effectively than M13 phage surface. In conclusion, we believe that the M13 phage can be introduced as new immuno-modulating biomaterial for bionanotechnological approaches.
Conflict of interest
All the Authors declare no conflict of interest.
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