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\title{M13~ phage coated surface promotes anti\_inflammatory responses of
Balb/c peritoneal~ macrophages~~~~~~~~~~~~}



\author[1]{Zohreh Safari}%
\author[1]{Majid Sadeghizadeh}%
\author[1]{Golareh Asgaritarghi}%
\author[2]{Hassan Bardania}%
\author[1]{Dina Sadeghizadeh}%
\author[3]{Sara Soudi}%
\affil[1]{Tarbiat Modares University Faculty of Biological Sciences}%
\affil[2]{Yasuj University of Medical Sciences}%
\affil[3]{Tarbiat Modares University Faculty of Medical Sciences}%


\vspace{-1em}



  
  \date{April 25, 2026}


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\selectlanguage{english}
\begin{abstract}
The largest populations of the human virome belong to bacteriophages.
M13 filamentous bacteriophages can survive, circulate and being cleared
within the body. The M13 phage has been developed as a new therapeutic
application due to its ability. Being detected by innate immune cells,
M13 phage triggers an immune response. Hereupon, macrophages are counted
as the first immune cells stimulated by phage particles. The possible
interference between immune responses with the phage-induced treatment
process, necessitates determining M13 phage effect on immune responses.
For this purpose, M13 phage with RGD were used as substrates for
macrophage culture. Gelatin and uncoated wells were used as controls. 2
and 7 days post culture on the coated surfaces, peritoneal macrophages
were analyzed in terms of morphological characteristics and metabolic
profile and \emph{measurement~}of cytokines production. Macrophages on
M13 phage containing surfaces secreted anti-inflammatory cytokines,
increased efferocytosis activity and show metabolic profile of an M2
macrophage. The results demonstrate that M13 phage can modulate immune
responses by polarization of macrophages toward anti-inflammatory
phenotype over time. Moreover, the combination of RGD peptide motif with
M13 phage was able to stimulate macrophage polarization more effectively
which may introduce an Immune-modulating technology for therapy and
biocontrol.%
\end{abstract}%



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\par\null

\href{https://www.authorea.com/browse-all?tags=\%5B\%22anti-inflammatory\%22\%5D}{ANTI-INFLAMMATORY}\href{https://www.authorea.com/browse-all?tags=\%5B\%22immuno-modulating\%22\%5D}{IMMUNO-MODULATING}



\textbf{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-\selectlanguage{greek}α\selectlanguage{english}) are involved in initial cellular responses
in inflammation occurred after immunological responses against foreign
objects as delivery or implantable biomaterials utilized for therapy or
\textbf{} 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- \selectlanguage{greek}β\selectlanguage{english}){[}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.

\textbf{Materials and methods}

\textbf{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\selectlanguage{ngerman}°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 \selectlanguage{greek}μ\selectlanguage{english}L 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.

\textbf{Large-scale amplification of and preparation coated surfaces}

phage was grown and purified following standard biochemical protocol. An
amount of 500 mL E. coli TG1 culture was grown in 2ytx media to mid-log
phase and infected with 1 mL wild-type bacteriophage
(10\textsuperscript{12}~PFU/ mL). The culture was incubated at 37\selectlanguage{ngerman}°C with
shaking for five to six hours and centrifuged at 8000g for 30 minutes to
remove bacterial cells; and then, the virus was collected by subsequent
centrifuging at 20000 g for 150 minutes. The resultant pellet was
suspended in 500 \selectlanguage{greek}μ\selectlanguage{english}L PBS and concentration of the isolated bacteriophage
was determined spectrophotometrically using an extinction coefficient of
3.84 cm\textsuperscript{2}/mg at 269 nm.

\par\null

\textbf{Preparation of samples for TEM analysis}

A solution of isolated M13 phage (20 \selectlanguage{greek}μ\selectlanguage{english}L) was adsorbed into a 300-mesh
carbon-coated copper grid (AGS160-3) for two minutes. For transmission
electron microscopy (TEM) analysis, the grid was then stained with 2\%
uranyl acetate. A Zeiss - EM10C - 80 KV (Oberkochen, Germany)
transmission electron microscope was employed for visualizing the
prepared samples.

\textbf{Macrophage preparation and characterization}

Twenty female BALB/c mice were obtained at six to eight weeks of age
from the Pasteur Institute of Iran, and maintained in the animal
laboratory in accordance with the Ethical Commission of Tarbiat Modares
University guidelines. For preparation of peritoneal macrophages, 3 ml
of 4\% w/v thioglycollate medium was injected into the peritoneal cavity
of the BALB/c mice. After four days, the macrophages were harvested by
injecting; and thereafter, harvesting the fresh cold DMEM from the
peritoneal cavity near the fat region of the lower abdominal area took
place. Harvested peritoneal fluid was centrifuged for five minutes at
350\selectlanguage{ngerman}×g and the resultant cell pellets were seeded
(1×10\textsuperscript{6} cells/ ml) to 4 or 24 well plates for
downstream analysis and incubated in a humidified incubator (37\selectlanguage{english}@C and
5\% CO2). The medium of newly isolated macrophages was changed after six
hours for separating the non-adhesive cells. Phenotypic analysis of
isolated cells was performed by flow cytometry for CD14+ and CD11b+
markers (Figure S2, Supporting Information ) as described previously
{[}26{]}.

\textbf{Cell viability assay}

To investigate the biocompatibility of a selected concentration of M13
phage (10\textsuperscript{12} 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 \selectlanguage{ngerman}×
10\textsuperscript{4} cells/well), MTT solution (20 \selectlanguage{greek}μ\selectlanguage{english}L, 5 mg/ml) was
added to cell culture media and plates were incubated at 37\selectlanguage{ngerman}°C. After
four hours, the medium was removed and 200 \selectlanguage{greek}μ\selectlanguage{english}L 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).

\textbf{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.

\textbf{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).

\subsection*{RNA preparation, cDNA synthesis, and
qRT-PCR}

{\label{739635}}

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. RT\textsuperscript{2}~SYBR
Green High ROX Master mix was used for qRT-PCR and data were quantified
using [?]CT method.

\textbf{Cytokine measurement}

Supernatants of the macrophages in experimental groups were collected
after two and seven days and stored at -20@\selectlanguage{english}C. The presence of IL-6,
TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-10 and TGF- \selectlanguage{greek}β \selectlanguage{english}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.

\subsection*{NO production}

{\label{607737}}

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 \selectlanguage{greek}μ\selectlanguage{english}L 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.

\textbf{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 \selectlanguage{greek}μ\selectlanguage{english}M) in serum-free culture media for 45~minutes at 37@\selectlanguage{english}C,
washed twice with PBS, and finally, analyzed by flow cytometry (BD
FACSCanto II, BD Bioscience, San Diego, CA, USA).

\subsection*{Phagocytosis assay}

{\label{295869}}

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@\selectlanguage{english}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.~

\textbf{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 \selectlanguage{ngerman}±
standard error of means. P \selectlanguage{english}? 0.05 was considered statistically
significant.

\textbf{Results}

\textbf{Characterization of M13} \textbf{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.

\textbf{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 \textbf{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 (\textbf{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 (\textbf{Figure 1D}).

\textbf{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- \selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}cytokines.
Gene expression and cytokine production of IL-6 and TNF-\selectlanguage{greek}α \selectlanguage{english}were
upregulated in macrophages of M13 phage containing surfaces in two days;
but after seven days, the expression of TNF-\selectlanguage{greek}α \selectlanguage{english}showed a downtrend as
compared to control surface (\textbf{Figure 2 A and B}). Accordingly,
levels of IL-10 and TGF- \selectlanguage{greek}β \selectlanguage{english}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 (\textbf{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-\selectlanguage{greek}α, \selectlanguage{english}TGF- \selectlanguage{greek}β \selectlanguage{english}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-\selectlanguage{greek}α\selectlanguage{english}). Interestingly, exposure to LPS could not increase
inflammatory cytokines of macrophages in M13 phage containing surfaces.
(\textbf{Fig. 2E}).

\textbf{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 (\textbf{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
(\textbf{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 (\textbf{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 (\textbf{Figure 3D}).

\textbf{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 (\textbf{Figure 4}).

\textbf{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
(\textbf{Figure 5A, B}).

\textbf{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-\selectlanguage{greek}α, \selectlanguage{english}IL-6, TGF- \selectlanguage{greek}β \selectlanguage{english}and IL-10 at 2 days after culture. A time-dependent
increase in M2 macrophage markers (IL-10 and TGF- \selectlanguage{greek}β\selectlanguage{english} {[}36{]}) and
decrease in M1 macrophage marker (TNF-\selectlanguage{greek}α\selectlanguage{english} {[}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.

\textbf{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.

\par\null

\subsection*{Conflict of interest}

All the Authors declare no conflict of interest.

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\par\null\par\null\par\null

\emph{}

~\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Untitled/Untitled}
\caption{{\textbf{Figure 1:} Outgrown morphology of Macrophage stimulated by
Phage-RGD tissue matrices. (A) Fluorescent microscopy images of outgrown
Macrophage after 2 and 7 days culture. The cells are stained by PKH
(red), DAPI (blue) (B) representative scanning electronic micrographs
images of the macrophages on different surfaces at 2 and 7 days (C)
Quantification of the cell areas on m13 phage, m13 phage-RGD and gelatin
film and control after 2 and 7 days culture. Two to three fields of
views were taken to analyze each image (cell n = 3-6 per one field of
view, (D) MTT cell proliferation assays on different types of film. MTT
assay result shows that cells all groups have a similar viability
pattern (*; Significant at P-value\textless{}0.05, **; Significant at
P-value\textless{}0.01, ***; Significant at P-value\textless{}0.001).
(scale bar = 2000 \selectlanguage{greek}μ\selectlanguage{english}m).
{\label{800544}}%
}}
\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/real-time/real-time}
\caption{{This is a caption
{\label{524426}}%
}}
\end{center}
\end{figure}

\textbf{Figure 2:} quantitative analysis. (A-D) quantitative analysis of
IL-10, IL-6, Tnf- \selectlanguage{greek}α \selectlanguage{english}transcript levels and Secretion of IL-10, Tnf- \selectlanguage{greek}α \selectlanguage{english}and
IL-6 in macrophage on four surfaces after 2 and 7 days.

(E) quantitative analysis of CXCL9, CXCL10, IL-10, IL-22, Tnf- \selectlanguage{greek}α, \selectlanguage{english}TGF- \selectlanguage{greek}β
\selectlanguage{english}in macrophages after culture on each surface in the absence or presence
of LPS for 2 days.

Value for genes~mRNA~expression~were~normalized~to~GAPDH~mRNA~levels for
each experimental condition. (*; Significant at P-value\textless{}0.05,
**; Significant at P-value\textless{}0.01, ***; Significant at
P-value\textless{}0.001) (n=3)

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/inos-3-3/inos-3-3}
\caption{{\textbf{Figure 3:} iNOS and ARG1 expression, nitric oxide (NO)
production in peritoneal macrophages from 4 groups experimental. (A)
Amplicons densitometrically quantified; bars represent the relative
amounts of amplified arginase-1 mRNA normalized against GAPDH, RT-PCR
analysis for the expression of arginase-1 mRNA. (B) Amplicons were
densitometrically quantified; bars represent relative amounts of
amplified iNOS mRNA normalized against GAPDH. (C)Comparison of NO
production, in macrophages at 2 and 7 days. (D)
Comparison~of~levels~of~expression~of~iNOS~with ARG1 mRNAs at 2 and 7
days. Graph shows collective data (*; Significant at
P-value\textless{}0.05, **; Significant at P-value\textless{}0.01, ***;
Significant at P-value\textless{}0.001). (n=3)
{\label{401745}}%
}}
\end{center}
\end{figure}

\textbf{}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/4-Ros1/4-Ros1}
\caption{{\textbf{Figure 4:} intracellular ROS level in peritoneal macrophages
from 4 groups surfaces. The effect of different surfaces on the
intracellular ROS level was determined by flow cytometry analysis at 2
and 7 days. Graph shows collective data (*; Significant at
P-value\textless{}0.05, **; Significant at P-value\textless{}0.01, ***;
Significant at P-value\textless{}0.001). (n=3)
{\label{629428}}%
}}
\end{center}
\end{figure}

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/phagooo/phagooo}
\caption{{This is a caption
{\label{519753}}%
}}
\end{center}
\end{figure}

\textbf{Figure 5:} Flow cytometric analyses of phagocytosis and
efferocytosis. Unstained macrophage cells culture on control, M13phage,
M13phage-RGD and gelatin surfaces for 2 and 7 days after co-cultured for
1 hours with stained yeast (PE-red) or stained apoptotic cells (PE-red)
and fluorescence images of analyses of phagocytosis and efferocytosis.
Unstained macrophage cells culture on control, M13phage, M13phage-RGD
and gelatin surfaces for 2 and 7 days after co-cultured for 1 hours with
stained yeast (DAPI-blue) or stained apoptotic cells (DAPI-blue).

(A) Flow cytometric and fluorescence images analyses of macrophages
efferocytosis. Representative histogram shows the macrophage marker PE
indicating efferocytosis.

(B) Flow cytometric fluorescence images analyses of macrophages
phagocytosis. Representative histogram shows the macrophage marker PE
indicating Phagocytosis.





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