skin tissue integrity is a crucial factor to maintain the homeostasis generated through physical barriers , separating the organism from the environment .
every disruption of dermal integrity triggers a complicated cascade of events , including rapid blood clot formation , inflammatory response , and wound healing , leading to the restoration of the integrity and formation of new tissue .
however , repaired structures , known as scars , are nonfunctioning , tight , and tense masses of fibrotic tissue that maintain 7080% of normal strength , with even less flexibility .
inflammatory responses are necessary for wound healing , preventing multiple infection and contamination and stimulating the proliferation , revascularization , and remodeling of the extracellular matrix [ 2 , 3 ] .
nevertheless , wound healing might become uncontrolled and , combined with the inflammatory response , results in massive fibrotic tissue formation called fibrosis . in this review , we will focus on the molecular mechanisms underlying skin fibrosis as a post - wound - healing pathological disorder and the impact of bone marrow - derived cells and inflammation on the formation of scars .
fibrosis is a pathological process that occurs in many different organs ( organ specific fibrosis ) , such as skin , kidney , heart , lung , and liver , which might also take the form of systemic sclerosis ( ssc ) , a global , progressive , and autoimmune disorder , characterized by an extremely poor prognosis and high mortality [ 5 , 6 ] . according to the united states government , every year , in the usa
although the etiology and triggering cascade might differ , fibrosis is characterized by the increased production and deposition of extracellular matrix ( ecm ) components , including collagen type i , fibronectin , hyaluronan , and elastin , and the accumulation of activated , sma - positive , and collagen - secreting fibroblasts , called myofibroblasts [ 4 , 8 , 9 ] .
many authors refer to fibrosis in the context of  uncontrolled  or exceeded wound healing , as an effect of long - term inflammation or mechanical irritation .
this process can be divided into four overlapping phases : coagulation , inflammation , proliferation , and remodeling .
skin injury results , inter alia , in the disruption of endothelial and epithelial cells integrity .
damaged cells release inflammatory mediators that trigger the coagulation cascade , platelet recruitment , and blood clot formation .
degranulated and activated platelets present in the blood clot release multiple chemokines and growth factors ( tgf-1 , pdgf ) , which recruit inflammatory cells .
platelets also participate in the chemotaxis and recruitment of fibroblasts and endothelial cells [ 1113 ] .
the first two phases are often treated as one phase , representing the inflammatory stage .
the blood clot comprises cross - linked fibrin and extracellular matrix proteins , such as fibronectin , vitronectin , and thrombospondin .
this structure serves as a physical barrier that closes the blood vessel , a reservoir of growth factors , and a matrix on which regenerated tissue is formed [ 12 , 14 ] . the next phase , proliferation , results from hypoxic conditions and reactive nitrogen species ( rns ) production from macrophages [ 15 , 16 ] . during this phase
, angiogenesis occurs , forming new capillaries and facilitating the delivery of nutrients to the wound .
in addition to nutrients , collagen - secreting myofibroblasts are recruited to the wound microenvironment .
as previously described , myofibroblasts are activated , sma - positive , and collagen - secreting fibroblasts that deposit new ecm components , primarily fibronectin and collagen type i , to replace the clot - formed matrix , often forming a scar [ 10 , 18 ] . in physiological wound healing
, remodeling is the final phase . during this phase , myofibroblasts and some vascular cells
moreover , the synthesis of ecm components is reduced but not fully terminated , and remodeling is primarily regulated through different matrix metalloproteinases ( mmps ) and their inhibitors ( tissue inhibitor of metalloproteinases , timps ) . after the degradation of the overexpressed ecm components , scarring is reduced and an equilibrium between synthesis and catabolism is reached [ 13 , 20 ] . in fibrosis ,
. the increased deposition of collagen type i and fibronectin stiffens and damages the surrounding tissue .
in addition , connective tissue cells replace the original cells , creating a scar that in some cases might take an extremely severe form [ 13 , 21 , 22 ] .
the wound healing process is shown in figure 1 . activated fibroblasts / myofibroblasts accumulate in the fibrotic tissue environment by three different , simultaneous mechanisms .
first , these cells are derived from preexisting fibroblasts in the affected tissue through activation due to specific , profibrotic , and proproliferative mediators released from infiltrating inflammatory cells , such as t cells [ 2325 ] .
second , myofibroblasts are recruited through bone marrow - derived fibroblast resembling cells , such as fibrocytes , cd45 and cd34 positive cells .
fibrocytes transmigrate to the fibrotic environment , and in a tgf-1-controlled process , these cells undergo transdifferentiation into myofibroblasts . finally , fibroblasts / myofibroblasts accumulate through the transition from endothelial or epithelial cells to mesenchymal fibroblast - like cells [ 5 , 11 ] .
during the endothelial and/or epithelial to mesenchymal transition ( endmt and emt , resp . ) , cells lose their origin markers , polarity , and cell - cell connections and gain promigratory phenotypes and mesenchymal markers [ 2830 ] . both endmt and emt are physiological processes that occur during embryonic organogenesis and wound healing .
epithelial and endothelial cells establish close cell - cell contacts with a certain cell polarity , forming a solid barrier that maintains homeostasis .
this barrier is formed through desmosomes and tight and adherent junctions [ 28 , 31 ] . in contrast
, mesenchymal cells are spindle - shaped solitary cells , possessing migratory and ecm remodeling abilities .
these cells produce and secrete ecm components , such as collagen type i and fibronectin [ 13 , 32 ] . during tissue development or regeneration
therefore , after undergoing endmt / emt , these cells gain the migratory abilities of fibroblasts , facilitating the recruitment of these cells to certain locations .
cells do not typically undergo full transitions , often terminating in intermediate phenotypes between endothelial or epithelial and mesenchymal , and maintaining some cell - cell contacts to perform group migration rather than single cell migration [ 28 , 33 ] .
the endothelial to mesenchymal transition was first observed and described as the leaking and proliferation of endothelial cells during the development of chick and rat endocardial cushions ( cardiac mesenchyme ) .
endmt is closely associated with dermal , renal , cardiac , pulmonary , intestinal , and cystic fibrosis through the establishment of fibroblasts and myofibroblasts [ 35 , 36 ] .
emt is reversible , and fibroblasts might regain epithelial phenotypes ( mesenchymal to epithelial transition , met ) , whereas endmt reversibility is not well understood .
the reversal of endmt ( mesenchymal to endothelial transition ) has been recently observed in cardiac fibroblasts that rapidly adopt an endothelial - cell - like phenotype after acute ischemic cardiac injury .
however , more additional evidence suggests that endmt is irreversible , and transformed cells can not regain endothelial phenotypes , even after the removal of endmt inducing factors .
therefore , endmt , which is not terminated at a certain time , could lead to the accumulation of collagen type i secreted from myofibroblasts and the irreversible transformation into fibrotic tissue .
both endmt and emt are regulated through the zinc finger transcription factor snail family ( snail1 , snail2 , and snail3 ) .
after activation , on the molecular level , snail1 stabilizes the quantity of twist1 transcription factor , and in cooperation , both of these proteins upregulate zeb1 gene expression [ 40 , 41 ] . as a repressor ,
snail proteins downregulate the expression of genes encoding junction proteins , such as claudin , occludin , e - cadherin ( in epithelial cells ) , ve - cadherin , and pecam1 ( in endothelial cells ) .
it is not clear whether snail upregulates the genes encoding mesenchymal markers , as observed in the upregulation of myosin va in some highly metastatic cancer cell lines , such as human lung carcinoma cell lines ( a549 , pg , and calu6 ) , human colon cancer cell lines ( lovo and sw480 ) , human breast cancer cell lines ( bicr - h1 and mcf7 ) , and prostate cancer cell lines with the same genetic background ( pg3m-1e8 and pg3m-2b4 ) , or represses epithelial / endothelial genes and therefore indirectly upregulates mesenchymal markers .
nevertheless , mesenchymal cell proteins , such as vimentin , fibronectin , collagen type i , sma , sm22 ( transgelin ) , n - cadherin , calponin , and fsp-1 ( fibroblast specific protein 1 ) , are expressed during and after the transition [ 4346 ] .
the microrna profile also changes during mesenchymal transition , revealing the significant upregulation of mir-125 , let-7c , let7 g , mir21 , mir30b , and mir195 and downregulation of mir122a , mir127 , mir196 , and mir375 .
a previous study reported that the accumulation of snail in colorectal cancer cells and in mice utricle sensory epithelia cells , after blocking the degradation of this protein through the glycogen synthase kinase-3 ( gsk-3 ) , via lithium chloride treatment or the overexpression of snail , might trigger the transition into mesenchymal - like cells [ 43 , 48 , 49 ] .
however , this transition is typically induced through a variety of proinflammatory cytokines and growth factors secreted from leukocytes , which act synergistically .
the most important proinflammatory / profibrotic molecules are transforming growth factors -1 and -2 ( tgf-1 and tgf-2 ) , tumor necrosis factor- ( tnf- ) , interleukins il-1 , il-6 , il-8 , and il-11 , and fibroblast growth factor-2 ( fgf-2 ) [ 38 , 39 , 5054 ] .
it has been suggested that tgf- receptor is essential for mesenchymal transition signal transduction , and the overexpression of snail might be an insufficient factor .
the inhibition of tgf- receptor accompanied by simultaneous upregulation of snail does not lead to endmt in mouse embryonic stem cell - derived endothelial cells ( mesecs ) .
however , the upregulation of the transcription factor snail directly upregulates profibrotic and proinflammatory cytokines , such as il-8 .
the secretion of tgf- into the fibrotic microenvironment during inflammation is the most important snail inducer .
however , studies on skin cancer formation have shown that smad2 inhibits endmt , whereas smad3 acts as an activator .
the binding of tgf- to tgf- receptor type ii ( trii ) triggers heterodimerization through the activation of the tgf- receptor type i kinase ( tri ) , which activates activin - like kinase 5 ( alk5 ) and transduces a signal through the smad2/3 complex with smad4 , which activates the expression of snail .
the inhibition of either alk5 or alk2 results in the inhibition of endmt [ 29 , 57 ] .
tgf- also activates snail in a non - smad pathway , involving wnt and noch , via the sequestration of gsk-3 and akt2 , through the transcriptional repression of the mir-200 superfamily and the activation of the inflammatory transcription factor nfb [ 30 , 46 , 50 , 58 ] .
as previously discussed , chronic inflammation is one of the main factors triggering fibrosis , particularly endmt - based fibrosis , as endmt is an irreversible process .
constant inflammation leads to the production of a variety of proinflammatory cytokines and growth factors secreted from different leukocytes present in the fibrotic microenvironment . however , fibrosis formation is a multidimensional and multistage process that not only involves endmt .
leukocyte recruitment triggers many different mechanisms and pathways that might lead to disordered wound healing , myofibroblasts and collagen type 1 accumulation , scarring , and fibrosis .
the recruitment of these cells is initiated immediately after activated platelets degranulate and release tgf-1 and pdgf .
tnf- , il-1 , and il-8 released from endothelial cells also stimulate neutrophil recruitment , leading to selectin - mediated rolling adhesion towards the chemoattractant gradient . in the next phase , tight adhesion to endothelial cells occurs via integrin 2 , followed by transmigration through the endothelial tissue .
when necessary , neutrophils cross the ecm barrier along fibroblasts and transmigrate through epithelial cells to enter the wound .
neutrophils begin phagocytosing invading bacteria and damaged necrotic cells to clear the wound , preparing it for the regeneration of homeostasis through scar formation .
however , fetal wounds heal without scar formation , and fetal neutrophils are physiologically distinct from adult neutrophils , as these cells are less adept than adult cells , producing less cytokines and presenting lower contributions to the inflammatory response [ 60 , 61 ] .
neutrophil serine protease , elastase , is secreted into the microenvironment , increasing il-8 expression in the surrounding cells .
il-8 not only is responsible for leukocyte recruitment but also might trigger endmt and increase the survival and proliferation of endothelial - derived fibroblasts / myofibroblasts , leading to fibrosis [ 6 , 63 ] .
additionally , elastase is also believed to cleave the il-8 receptor cxcr1 , interfering with neutrophil functions and antibacterial abilities , thereby prolonging inflammation , which in turn increases additional fibrosis - based changes .
prolonged inflammation might also occur through the elastase - mediated degradation of complement , releasing the strong neutrophil chemoattractant , c5a .
moreover , neutrophil derived oxidative burst , leading to the formation of hocl from h2o2 catalyzed through myeloperoxidase , induces injury to epithelial cells , thereby implicating the switch to fibrotic tissue deposition [ 66 , 67 ] .
two different populations of neutrophils have been observed to enter the wound in mice after the induction of acute inflammation : one population has a proinflammatory function , and the second population is responsible for anti - inflammatory responses .
these cells differ in size , granularity , and the expression of cd11b and ly6 g . respectively ,
the anti - inflammatory neutrophil response is strongly associated with the secretion of the anti - inflammatory cytokine il-10 . moreover ,
a certain population of mature neutrophils , characterized as cd11c / cd62l / cd11b / cd16 , have been reported to suppress t cell proliferation via the expression of the integrin mac-1 ( m2 ) .
the impact of neutrophils on fibrosis has been observed in pulmonary fibrotic disorders , as these cells transmigrate to pulmonary fluids ( such as bronchoalveolar lavage fluid ) and recruit other leukocytes [ 71 , 72 ] .
however , only a few clinical studies have successfully established anti - inflammatory strategies in patients with pulmonary fibrosis .
inhibition of neutrophil derived elastase as strategy of downregulation of self - destructive process of neutrophil derived protease activity as well as elastase derived il-8 expression is currently being elucidated and brings more questions than answers .
clinically useful concepts have only just started to evolve and bring promising , but not yet convincing , answers [ 59 , 73 ] .
macrophages appear as the second type of bone marrow - derived cells invading the wound site , and three to five days after injury , they become the dominant leukocyte type .
similar to neutrophils , different populations of macrophages have been reported , depending on the activation path through different chemokines and growth factors , as shown in figure 2 .
classical macrophage activation , or m1 , is obtained , in particular , through the combination of interferon gamma ( ifn- ) and tumor necrosis factor- ( tnf- ) signaling pathways .
alternative activation , or m2 , is far more complex , leading to the formation of regulatory and wound - healing macrophages .
regulatory macrophages release anti - inflammatory cytokines il-10 and tgf- , which downregulate inflammation , and also lead to the endothelial to mesenchymal transition and increase the fibroblast number at the wound site .
its abilities might shift , depending on other cytokines availability and cell type [ 76 , 77 ] .
it was shown that tgf- administered to animals with infection or inflammation reduces severity of disease and production of proinflammatory il-1 and tnf .
a second group of m2 macrophages , wound - healing macrophages , are derived through il-4 induction .
these cells secrete cc chemokine ligands , including ccl2 , ccl17 , ccl18 , and ccl22 .
wound - healing macrophages are extremely profibrotic , as these cells produce high levels of fibronectin and through ccl18 activation promote collagen production from fibroblasts / myofibroblasts .
moreover , arginase activation in m2 macrophages , stimulated through il-4 , leads to the conversion of arginine to ornithine , a precursor of collagen [ 74 , 80 ] .
blocking il-4 with specific antibodies significantly decreases wound - healing , macrophage accumulation , and fibrosis formation .
recent studies showed that overexpression of mmp9 in macrophage might attenuate bleomycin induced pulmonary fibrosis . respectively , production of mmp13 by kuppfer cells was shown to be sufficient in preventing pig serum - induced rat liver fibrosis .
these data suggest that high level of mmps might play key role in fibrosis reversibility .
macrophages are the main sources of mmps that facilitate ecm degradation during remodeling phase in wound healing process ; they also phagocytose apoptotic myofibroblasts and cellular debris preventing advance in the fibrotic process .
however , some authors suggest strong profibrotic role of macrophage derived mmp13 , as they observed that liver fibrosis was suppressed , along with fibrotic markers and inflammatory mediator expression , in mmp13-deficient mice during cholestasis - induced liver fibrosis .
surpassingly , prolonging inflammation and recruitment of activated macrophages might be involved in fibrosis reversing process , as accumulating evidence strongly correlates macrophages and the macrophages derived mmps ( mmp1 , mmp2 , mmp8 , mmp9 , and mmp13 ) with this process .
after arrival to the wound site , these cells produce lymphokines , which in turn activate other inflammatory cells , such as macrophages . among all lymphocyte subpopulations , th1 and th2
are most relevant for tissue fibrosis . th1 and th2 lymphocytes contribute different responses to wounded tissue .
th1 acts as an antifibrotic , releasing il-10 , and th2 acts as a profibrotic . studies using mouse models have shown that the polarized th2 response leads to massive collagen deposition and increased fibrosis formation .
however , the th1 response activates the genes responsible for apoptosis and acute - phase reactions [ 86 , 87 ] . among all cytokines released from th2 ,
the two most important and most profibrotic cytokines are il-4 and il-13 . both il-4 and il-13 share
functional similarities , as these molecules transduce signal via the il-4r / stat6 pathway [ 8890 ] .
as previously described , il-4 activates m2 wound - healing macrophages , resulting in collagen production and deposition .
moreover , il-4 stimulates in the dose - dependent manner collagen synthesis in fibroblasts and is two times more effective than tgf- . the scleroderma mouse model ( tight - skin mutant mouse tsk/+ ) presented
treatment with an anti - il-4 antibody resulted in collagen downregulation and provided less fibrosis - based pathological changes .
the th2-mediated secretion of il-4 and il-13 enhanced fibrocyte differentiation from cd14-positive precursors , thereby leading to increased fibroblast recruitment and potential fibrosis .
the impact of th2-derived il-5 on fibrosis is strongly associated with the recruitment and activation of eosinophils .
activated eosinophils secrete inflammatory factors , such as il-13 and tgf-1 , into wounded tissue , resulting in fibrosis development , as shown for dermal fibrosis in a mouse model of skin allograft rejection .
both il-13 and tgf-1 might induce collagen secretion from fibroblasts present in the wound ; however , tgf-1 is strongly associated with endmt- and emt - based fibrosis [ 95 , 96 ] . as discussed above , th1 and th2 lymphocytes affect differently fibrotic tissue .
recently , it was shown that patients with cystic fibrosis and p. aeruginosa infection present an age - dependent dysregulation of lymphocyte t response that shifts towards th2 lymphocyte , resulting in enhanced fibrotic tissue deposition .
as previously described , these cells are both cd45- and cd34-positive cells that transdifferentiate into myofibroblasts .
the name fibrocyte represents a combination of shared features of these cells : fibroblast and monocyte . circulating fibrocytes
subsequently , tgf-1 triggers the transdifferentiation of these cells into sma - positive myofibroblasts that express collagen type i , fibronectin , and vimentin and increase the amount and deposition of ecm components [ 98 , 99 ] .
normally , circulating fibrocytes comprise less than 1% of all leukocyte populations , but during fibrotic changes derived from inflammation , the amount of these cells systematically increases .
fibrocytes can be distinguished in at least 4-day - old skin wounds , and the quantity of these cells raises with time and increasing wound age . for the differentiation of fibrocyte precursors ,
the induction of fibrosis through fibrocytes is primarily based on the deposition of ecm components , as discussed above .
nevertheless , we can not omit a variety of proinflammatory cytokines secreted from fibrocytes into the wound , namely , tnf- , il-6 , il-8 , il-10 , and macrophage inflammatory protein 1/ ( mip 1/ ) .
moreover , it has been demonstrated that the fibrocytes in burned patients secrete tgf-1 , which activates myofibroblasts from existing fibroblasts   or triggers differentiation toward fibroblasts - like cells from surrounding endothelial or epithelial tissues through endmt or emt .
however , due to dynamic nature of fibrocytes and constantly changing phenotype and functions of these cells , during their migration , some serious inconsistency appeared on the exact definition and identification of fibrocytes .
these discrepancies are related to different methodology used to investigate fibrocytes involvement in variety of fibrotic disorders on variable stages .
it has been suggested that one must categorize fibrocytes as functionally different depending on the isolation condition .
what is more , it is still unclear whether fibrocytes contribute only to worsening or improving tissue repair , as they possibly represent  the wrong cells in the wrong time  .
fibrosis is a complicated and composed process , leading to severe pathological disorders . the scarce formation of fibrotic tissue , comprising excess amounts of collagen type i , fibronectin , and other ecm components , deregulates normal tissue functions .
inflammation and inflammation - associated bone marrow leukocyte recruitment in wounded tissue trigger a cascade of events , leading to wound enclosure , scar formation , and , in case of prolonged inflammation , massive fibrosis .
the impact of leukocytes on fibrosis formation might be generally divided into direct and indirect effects as shown in figure 3 .
the direct impact is strongly associated with the production and excess deposition of ecm components .
this effect is primarily observed with fibrocytes and alternatively activated , wound - healing macrophages .
the indirect impact is far more complicated , as this effect is multistaged and associated with the activation and recruitment ( including cells transdifferentiation and emt / endmt fibroblasts formation ) of collagen - secreting cells , such as macrophages and myofibroblasts , and increased myofibroblast survivability through the downregulation of proapoptotic signals and increased inflammatory response times .
the most common , and likely , best - known indirect impact is correlated with tgf- family proteins , as these molecules trigger both the endothelial and epithelial to mesenchymal transition and activate myofibroblasts from fibroblasts and fibrocytes , thereby increasing the production of ecm components .
however , although this mechanism is well known , no antifibrotic therapy , based on tgf- deactivation , has been implicated without disruption of the physiological function of this molecule .
several drugs for the downregulation of tgf- transcription or signal transduction have been examined in the last stages of clinical trials . nevertheless , the impact of il-6 , il-8 , il-4 , il-13 , or tnf- can not be neglected .
th2-derived il-4 and il-13 are primarily responsible for macrophage collagen deposition and fibrocyte generation , whereas tnf- , il-6 , and il-8 are strongly associated with emt and endmt and myofibroblast survival and stimulation of collagen production .
thus , leukocyte interactions with wounded tissue cells and other leukocytes are extremely complicated and complex , bringing more questions than answers .
 skin tissue scar formation and fibrosis are often characterized by the increased production and deposition of extracellular matrix components , accompanied by the accumulation of a vast number of myofibroblasts . 
 scaring is strongly associated with inflammation and wound healing to regain tissue integrity in response to skin tissue injury . 
 however , increased and uncontrolled inflammation , repetitive injury , and individual predisposition might lead to fibrosis , a severe disorder resulting in the formation of dense and stiff tissue that loses the physical properties and physiological functions of normal tissue . 
 fibrosis is an extremely complicated and multistage process in which bone marrow - derived leukocytes act as both pro- and antifibrotic agents , and therefore , few , if any , effective therapies are available for the most severe and lethal forms of fibrosis . 
 herein , we discuss the current knowledge on the multidimensional impact of leukocytes on the induction of fibrosis , focusing on skin fibrosis . 
1. Introduction
2. Fibrosis and Wound Healing: Two Faces of the Same Story
3. The Endothelial and Epithelial to Mesenchymal Transition as a Key Factor in Fibrosis
4. Leukocytes in Fibrosis: Unanswered Questions
5. Concluding Remarks