Introduction:
Going beyond the primary application of bacteriophages since their discovery, phage particles as a tool have commenced to a new field of technology not only for drug delivery and drug discovery but also for therapy, biocontrol and biomedical science [1]. Although bacteriophages are bacterial viruses, they naturally come in contact with mammalian immune cells through microbiome [2]. Phages can interact with immune cells after being phagocytosis or direct contact with immune cell receptors. Both protein and nucleic acid structure of the phage can stimulate different pathogen recognition receptors of immune cells and turn on the relevant signaling pathways [3]. Moreover, phage-immune system interaction influences immune responses to environmental stimuli that leads to anti-inflammatory condition [4]. Accordingly, modulation of immune response is fundamental to regenerate multiple tissue types and in immunotherapy. Immunomodulatory drugs are usually used systemically and represent to have poor effect and being quickly lost or having toxic side effects alongside. To overcome these impediments, immunomodulatory materials can be placed at required sites to locally control immune responses[5, 6]. In recent times, biocontrol agents have been commonly used by researchers in order to control immune responses which some sort of them represent to have anti-inflammatory ability. Phages can easily be modified and include different parameters for biocontrol which can be considered as an Immunomodulatory biomaterial for therapy avoiding unwanted immune responses. [13]. It is noteworthy to mention that the formation of nanofiber structures, self-assembly, eligible size, interaction with different materials and the capability of chemical and genetic modifications of the filamentous M13 phage protein coats for expressing desired exogenous peptides have extended the application of this viral nanoparticles as a safe therapeutic tool for gene delivery, immunotherapy, tissue engineering and vaccine development[7-10]. Over and above that several researchers have illustrated the role of filamentous phages as ECM-mimicking nanofibers in enhancing cell adhesion, proliferation, and differentiation[11-13].
In the light of this notion that bacteriophages cooperate to maintain the immune homeostasis and support the immune system, [14, 15].To assess this concept, M13 phage was used as a substrate for macrophage culture and the outcome of attached macrophage responses was investigated. Macrophages was considered for the reason that they are counted as key cells in the early interaction between the material and the immune system. The characteristics of macrophages make them to have a fundamental role in the immunomodulation of the other immune cells involving in immunotherapy, drug delivery as well as regenerative process[14]The biological activity of macrophages, as crucial players in regulating immune system responses, make them mediate innate immunity and initiate responses in adaptive immunity [16-18]. One of their remarkable features is that, macrophages have functional phenotype plasticity and they react to microenvironment by changing their phenotype. As follows, they can change their polarization toward anti-inflammation (M2) or inflammation (M1) in response to microenvironment stimulus [19]. The modulation of immune responses by macrophage cells are tied to the balance of their phenotype(M1/M2)[20].Macrophages as an important source of chemokines and inflammatory molecules such as interleukin-6 and tumor necrosis factor alpha (TNF-α) are involved in initial cellular responses in inflammation occurred after immunological responses against foreign objects as delivery or implantable biomaterials utilized for therapy or tissue regeneration [21].After the initial inflammation responses, macrophage characteristics should go toward the anti-inflammatory state of the M2 macrophages, which exert their effects through the secretion of cytokines such as interleukin 10 and transforming growth factor beta (TGF- β)[22].
We hypothesize that M13 phage alter immune cell responses. It is known that the type of biomaterial exerted on macrophage cell can directly affect their phenotype and their cytokine profile[23]. The aim of the present study is to suggestion a novel application of M13 phage as an Immuno-modulating biomaterial for therapy and biocontrol. So, their bioactivity and immunostimulatory properties in encountering with macrophages were assessed in in-vitro condition. Then, the efficacy of M13 phage was measured on different macrophage immune responses including phagocytosis, efferocytosis, inflammatory and anti-inflammatory cytokine production, oxidative stress, and nitric oxide production.
Materials and methods
2.1. Large-scale amplification of M13 phage
M13 phage was grown and purified as reported previously[24, 25]. An amount of 500 mL E. coli TG1 culture was grown in 2ytx medium to mid-log phase and infected with 1 mL wild-type M13 bacteriophage (1012 PFU/ mL) was applied. The culture was incubated at 37°C with shaking for five to six hours and centrifuged at 8000g for 30 minutes to remove bacterial cells; subsequently, the virus was collected by subsequent CsCl density gradient centrifugation at 40,000 g for 2 hours. The resultant pellet was suspended in 500 μL Phosphate-buffered saline (PBS) and concentration of the isolated bacteriophage was determined spectrophotometrically using an extinction coefficient of 3.84 cm2/mg at 269 nm. This study contained 3 biological replicates (3 scaffolds of each group) and each group was replicated 3 times.