sepsis is one of the most common infectious conditions during the neonatal period , and it is still a significant cause of morbidity and mortality , despite the outstanding development of neonatology in recent years . described as systemic inflammatory response ( sirs ) associated with a suspected or proven infection , the sepsis is an infectious disease of varied etiology , which determines degrees of inflammatory and metabolic responses [ 2 , 3 ] .
tumor necrosis factor ( tnf ) , interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , and interleukin-8 ( il-8 ) are proinflammatory cytokines , whereas interleukin-10 ( il-10 ) and transforming growth factor - beta ( tgf- ) are known as anti - inflammatory cytokines , both produced rapidly in the setting of neonatal sepsis . in the past years , several authors have supported the use of cytokines in the diagnosis of both early and late sepsis .
previous results from our study , in which cytokines were measured in the plasma and in the umbilical cord blood at birth , support the idea that increased levels of either proinflammatory cytokines ( tnf- ) or anti - inflammatory cytokines ( il-10 ) in neonates at birth change throughout the infectious process and there is also a positive and significant correlation between the levels of these cytokines . in the present review , we will cover the actual role of cytokines in the pathogenesis of neonatal sepsis and its value as a diagnostic criterion .
neonatal sepsis is a systemic infection that occurs in newborns up to 28 days of age and it is a major cause of morbidity and mortality in newborns . according to world health organization , in 2010 , 3.7 million newborns died before reaching 28 days of age in the united states , and 37% were due to infectious causes . as proposed by the international pediatric sepsis consensus conference in 2002 , specific definitions for pediatric sirs and sepsis present important differences related to clinical signs and laboratory biomarkers specific to adults .
the major differences between adults and children are that the diagnosis of pediatric sirs requires lower values for heart rate , leukocyte count , and systolic blood pressure and upper values for heart rate , respiration rate , or leukocyte count . in sepsis , to confirm the diagnosis , it is necessary the presence of bacterial infection suspected or confirmed by culture or other methods . taken together , some clinical findings can also help the diagnosis process such as : petechiae and purpura ( in the setting of hemodynamic instability ) ; fever , cough , and hypoxemia ( in the setting of leukocytosis and pulmonary infiltrates ) . in adults
, sepsis is defined as a complex clinical syndrome of severe systemic inflammatory response syndrome ( sirs ) with multiple physiological and immunological abnormalities , which is usually associated with bacterial or fungal infections [ 2 , 810 ] .
sepsis pathogenesis is associated with hemodynamic changes , disturbances of microcirculation and cellular changes that cause imbalance between blood flow and metabolic tissue requirements , leading to multiple - organ dysfunctions , which is responsible for the severe and often fatal form of the disease . neonatal sepsis and sepsis in adults
are very different conditions , with implications for the epidemiology and pathophysiology and even in clinical management .
in addition , differences between neonates and adults directly impact the involvement of cytokines during development of sepsis and the use of assessment of these mediators in clinical routine [ 1214 ] .
although our focus here is neonatal sepsis , as far as possible we compare results of studies involving adults from those obtained from studies involving neonates .
sepsis development can be initiated through recognition of one or more components of invading organism , including structural elements such as gram - negative endotoxins or secreted exotoxins that stimulate the local and systemic release of endogenous inflammatory mediators . among the inflammatory mediators
are cytokines such as tnf- , ifn- , il-1 , il-6 , and il-8 , which act favoring the migration and activation of immune cells [ 15 , 16 ] .
stimulation of polymorphonuclear leukocytes , histiocytes , platelets , and endothelial cells leads to the production of biologically active mediators , including platelet activating factor ( paf ) , arachidonic acid metabolites , histamine , bradykinin , complement proteins , vasoactive peptides , and oxide nitric ( no ) .
the production and release of these proinflammatory mediators can induce a systemic inflammatory response characteristic of the initial phase of sepsis [ 17 , 18 ] . for a long time
, it was believed that sepsis was caused by an exacerbated inflammatory response generated from innate immune response triggered by bacterial infections .
however , researchers eventually described the presence and importance of  compensatory anti - inflammatory response syndrome ,  cars , which often occurs after hyperinflammatory phase , especially in patients who develop severe sepsis . in neonates ,
severe sepsis is characterized by the persistence and prevalence of proinflammatory mediators up to the third day after diagnosis , whereas these groups of cytokines are prevalent in sepsis , with good clinical evolution , just on the day of diagnosis .
the study of inflammatory mediators and cytokines as biomarkers of neonatal sepsis are really important for syndrome diagnosis .
recent research has pointed to the role of damage - associated molecular patterns ( damps ) , which are intracellular proteins released in response to cell injury , such as high mobility group box protein 1 ( hmgb-1 ) .
damps act as endogenous danger signals to activate and amplify the function of receptors such as receptor for advanced glycation end products ( rage ) , hence perpetuating the inflammatory response .
this may be particularly important for neonatal sepsis , because the maternal - fetal barrier is compromised by the inflammatory response , leading to translocation of damps into the fetus .
the study of proinflammatory cytokines il-6 , tnf- , il-1 , and il-8 and anti - inflammatory cytokines il-10 and tgf- as reliable biomarkers of neonatal infection has been shown to be potentially useful for early sepsis diagnosis and to predict the severity of disease at early stages of the infection [ 2326 ] .
cytokines are relatively small molecules with short serum half - life ( from minutes to a few hours ) and play a central role in immune response in neonates with sepsis . during sepsis , cytokine levels
may be observed in picograms per milliliter of plasma or in nanograms or even micrograms per milliliter . in the 1990s , sepsis was believed to be associated with an exacerbated release of mainly proinflammatory cytokines , such as tumor necrosis factor ( tnf- ) , interleukin ( il-1 , il-6 , and il-12 ) , interferon- ( ifn- ) , and macrophage migration inhibitory factor ( mif ) . the expression
however , recent research on the pathophysiologic mechanisms underlying sepsis indicates that the profound proinflammatory response is counteracted by certain anti - inflammatory cytokines , including il-10 , transforming growth factor ( tgf- ) , and il-4 , which attempt to restore immunological balance [ 18 , 29 , 30 ] .
il-6 is a cytokine that shows early response to infection , preceding the increase in c - reactive protein and followed by tnf- release .
it is synthesized by mononuclear phagocytes , endothelial cells , fibroblasts , and the decidua , chorion , amnion , and trophoblast cells soon after stimulation by microbial products [ 31 , 32 ] .
il-6 acts as a signal in the activation of t cells , and it induces the secretion of antibodies by b cells and the differentiation of cytotoxic t cells .
it stimulates the release of other cytokines , particularly tnf- and il-1. il-6 is an early marker in the diagnosis of neonatal sepsis , increasing several hours before the increase in c - reactive protein .
the sensitivity of these tests together can reach values close to 100% , hence the importance of these markers .
it is capable of interfering with the production of c - reactive protein , so it can be detected earlier than c - reactive protein during bacterial infection .
the c - reactive protein has both anti - inflammatory and proinflammatory effects during infection , since it mediates the elimination of pathogens , despite also inhibiting the interaction between endothelial cells and leukocytes .
as the secretion starts 46 hours after stimulation and it peaks at 36 hours after infection , it is often used in the diagnosis of infection .
biomarker to have a very short half - life , approximately 100 minutes in patients with meningococcal infection ; additionally , the circulating levels decrease or return to basal levels 24 hours after appropriate treatment in late - onset neonatal sepsis .
several studies have reported sensitivity for detection of il-6 in 75 to 90% of circulating serum in the first 24 hours of infection , with a marked reduction in the diagnostic effectiveness 48 hours after the onset of symptoms and suspected sepsis [ 23 , 25 , 37 , 38 ] .
moreover , il-6 has been correlated with maternal chorioamnionitis and used in the initial diagnosis of early neonatal sepsis when detected at high levels in umbilical cord blood .
similar results were found by our research group when analyzing liver of perinatal deaths diagnosed with fetal inflammatory response syndrome ( firs ) using immunoperoxidase method ; we observed that il-6 and c - reactive protein were overexpressed . when detected in the umbilical cord blood of newborns at term and without risk factors
, il-6 does not have significant clinical utility in differentiating infected and healthy newborns . on the other hand , in newborns with premature rupture of membranes , il-6 showed high sensitivity and specificity in predicting funisitis and positive cord blood culture .
the difference in il-6 levels in cord blood and blood of the newborn infant at birth is due to the kinetics of il-6 .
therefore , sample collection time is an important factor for the detection of high levels of il-6 in neonates .
release of tnf- may occur approximately 30 minutes after lps injection , and the circulating levels reach their peak in approximately one and a half hour , with an estimated half - life of about 70 minutes [ 30 , 32 ] .
tumor necrosis factor ( tnf- ) is the prime mediator of septic shock in neonates and widespread tissue injury , and it regulates the secretion of il-1 [ 37 , 38 ] .
high levels of tnf- appear to be related to the severity of the disease , although some studies in adults do not confirm this relationship [ 44 , 45 ] .
the peak plasma concentration of tnf- is reached after an hour of experimental endotoxemia , with near - zero levels for three hours .
there are evidences that tnf- is found free in plasma concomitant with the appearance of signs and symptoms of bacterial infection .
systemic release of tnf- can cause vasodilation and increased vascular permeability leading to systemic edema , with decreased blood volume and hypoproteinemia that can progress to shock .
there was stimulus for leukocyte and platelet adhesion , with clots formation in small vessels and consumption of coagulation proteins that may lead to disseminated intravascular coagulation .
this condition may also progress to multiple - organ failure and death in early - onset neonatal [ 47 , 48 ] .
in addition , tnf- and il-1 were identified as crucial cytokines to development of fever and , therefore , belong to a group of pyrogenic cytokines [ 30 , 49 ] .
tnf- induces increased adherence of neutrophils and integrins to endothelial tissues and upregulates the endothelial expression of procoagulant proteins . moreover , together with il-1 , tnf- was one of the first mediators identified in inflammatory sites .
synergically , tnf- and il-1 amplify the inflammatory signals by activating macrophages to produce proinflammatory cytokines such as il-6 and il-8 , as well as lipid mediators and reactive oxygen species   leading to sepsis - induced organ dysfunction .
tnf- acts through two receptors , tnfr1 ( tnf receptor-1 ) and tnfr2 ( tnf receptor-2 ) , resulting in immune cell activation and release of several immunoregulatory mediators .
the role of tnf- as a marker for the prediction of early - onset neonatal sepsis has been suggested .
additionally , when used in combination with il-6 , tnf- may achieve up to 98.5% sensitivity . on the other hand , santana and colleagues   demonstrated that tnf- was not significantly different between sick and healthy neonates .
discrepancy between the studies could be due to the fact that the kinetics of tnf- production is not fully understood in early life .
furthermore , due to the short half - life of tnf- and its interaction with soluble receptor , the detection becomes difficult .
thus , tnf- as a reliable biomarker for sepsis is compromised . in contrast , some studies , especially by pickler and colleagues , reported that high levels of tnf- , il-6 , and il-1 are often associated with profiles of sepsis in neonates .
these authors also confirmed the low sensitivity and specificity of tnf- to differentiate sepsis in infected newborns .
experimental studies have shown that the injection of tnf causes a syndrome which is indistinguishable from septic shock .
it was observed that the infusion of recombinant tnf- in humans results in sirs [ 5557 ] .
it is believed that because this role in the inflammatory process results in severe clinical conditions of sepsis , tnf- may have a direct correlation with the severity of sepsis and the mortality rate during the development of sepsis in newborns at risk for infections .
il-1 is a proinflammatory cytokine released by timely activated macrophages similar to those of tnf-. it signals through two distinct receptors , called il-1 receptor type i ( il-1r1 ) and il-1r2 , and has many effects on immune cells [ 30 , 59 ] . in the inflammatory response cascade ,
namely , il-1 can be produced by the central nervous system , particularly in the hypothalamus , and also can be induced by infectious agents ( bacterial endotoxins , viruses , fungi , and parasite antigens ) and c5a complement , usually in one hour , reaching peak levels in 510 hours [ 61 , 62 ] . during sepsis , il-1 seems to induce fever , coagulation , and hematopoiesis , promoting the extravasation of inflammatory cells .
therefore , it has been noticed that il-1 is significantly increased in most patients with sepsis , and it has been associated with the severity of sepsis [ 63 , 64 ] .
moreover , persistently elevated levels of this cytokine have been correlated with the development of multiple - organ failure and with a worse prognosis in adults [ 45 , 64 ] .
interleukin-1 has been described as a marker of neonatal sepsis , although its diagnostic efficacy is lower than that of il-6 and tnf- .
the diagnostic value of il-6 , tnf- , and il-1 is limited by the time of blood sample collection , which should be as early as possible if neonatal sepsis is suspected , since these cytokines have very short half - life [ 46 , 65 ] .
therefore , analysis of a panel of cytokines , and not only the isolated measurement of a single cytokine , is required in order to characterize the inflammatory response in sepsis .
il-8 belongs to the class of proinflammatory chemokines and it is produced by the placental cells , fetal monocytes / macrophages , and cells .
il-8 is a chemokine which follows a course of time similar to that of il-6 .
this characteristic limits , to a great extent , the role of il-6 and il-8 as clinically useful biomarkers for all stages of sepsis , although they may be useful early in the disease prior to treatment in neonates .
recently , the association of high levels of il-8 in the presence of retinopathy was determined in seventy - four very low birth weight preterm infants with clinical criteria of early infection whose cytokines were obtained during the first three days of life .
the cut - off points for il-6 > 357  pg / ml , il-8 > 216  pg / ml , and tnf- > 245  pg / ml were significantly associated with the development of retinopathy .
placental cells , fetal monocytes / macrophages , and endothelial cells are able to produce il-8 after infectious process originated in the uterus
. il-8 levels increase about 90 minutes after infection and peak at about 120 minutes in septic neonates , whereas its circulating concentration decreases significantly 48 hours after birth , likewise the kinetics of il-6 .
only preterm infants less than 32 weeks of gestation may have increased levels of il-8 due to gestational age .
data by dembinski and colleagues   showed that il-8 levels were undetectable in the umbilical cord blood of healthy newborns . in the past years
, il-8 has been extensively investigated as a predictive biomarker of early - onset neonatal sepsis   that is corroborated by dllner and colleagues , who observed increased il-8 levels in the umbilical cord blood of infected preterm neonates .
the disadvantage of il-8 measurement in comparison with il-6 levels is the limit of detection .
the il-6 serum detection limit is > 0.7  pg / ml , whereas the serum detection limit of il-8 is > 10  pg / ml . anti - inflammatory cytokines , such as il-10 and tgf- , are important inflammatory mediators , since they play a major role in preventing excess proinflammatory response during sepsis .
il-10 is produced by different types of immune system cells such as monocytes , macrophages , t and b lymphocytes , and nk cells .
this cytokine suppresses the production of proinflammatory mediators including tnf- , il-1 , il-6 , ifn- , and gm - csf in cells of the immune system .
high levels of il-10 have been correlated with poor prognosis of sepsis in adults , shown to be a useful predictor of severity in septic shock and death [ 76 , 77 ] . however , it was shown that the appropriate response of il-10 may have a protective effect on sirs and that high il-6/il-10 ratio was found in patients with a worse prognosis .
similarly , a high il-10/tnf ratio has also been associated with severe late - onset neonatal sepsis [ 26 , 75 ] . in an experimental model
, it was shown that the administration of recombinant murine il-10 protects from lethal endotoxemia , even when il-10 was injected 30 minutes after lps administration .
in contrast , the immunoneutralization of il-10 led to increased levels of tnf and il-6 in circulating mice , and it also reversed the ability of il-10 to protect mice from lethal endotoxin . despite these clear protective effects of il-10 in lps - induced diseases , il-10 actions are not always beneficial .
the effects of il-10 appear to depend on the time of administration in case of neutralizations .
furthermore , the authors reported that il-10-deficient mice showed an earlier onset of lethality after experimental sepsis induced by cecal ligation and puncture as compared with wild type mice .
tgf- , as well as il-10 , is a member of the growth factor family and it is an important anti - inflammatory cytokine .
tgf- was shown to play a role in tissue repair and fibrosis , as well as in sepsis - induced immunosuppression . in vitro
, tgf- suppresses the release of proinflammatory mediators such as il-1 and tnf- from monocytes and macrophages .
tgf- also inhibits t - lymphocyte functions , such as il-2 , and the secretion of t cell proliferation , as well as promoting the development of t regulatory cells .
studies involving evaluation of in vitro assays , experimental models of sepsis , and human clinical evaluations support the anti - inflammatory actions of tgf-. these experiments have shown that treatment with tgf- blocked endotoxin - induced hypotension , probably inhibiting the hypotensive effects of no and improved survival in a rat model of salmonella endotoxin - induced septic shock [ 72 , 88 ] .
another study reported that adult patients at the onset of sepsis presented high levels of tgf- even though these levels were not correlated with severity or prognosis of disease .
recent data have demonstrated that tgf- reverses the depression of cardiac myocyte contraction , which is induced by cytokines such as tnf- and il-1 and by the serum of patients with septic shock .
hence , it is suggested that tgf- may have cardioprotective effects in sepsis - induced cardiac injury .
recently , il-7 , which is a hematopoietic growth factor , has been reported to have antiapoptotic roles , essential for lymphocyte survival and growth [ 91 , 92 ] .
in addition to its antiapoptotic properties , it induces proliferation of cd4 and cd8 t cells .
one of the characteristic features of sepsis is the profound loss of t cells in various lymphoid organs . during experimental sepsis ,
il-7 decreases cell apoptosis through the expression of antiapoptotic bcl-2 gene [ 94 , 95 ] .
another study showed that septic mice treated with recombinant il-7 ( rhil-7 ) increase the local and systemic production of neutrophils and il-17 , thus recruiting more neutrophils to the site of infection .
nevertheless , further studies are necessary to understand the real role of il-7 in the pathogenesis of neonatal sepsis .
another cytokine which has been studied and seems to be really involved in the pathogenesis of sepsis is il-22 . in a small group of hospitals within a single health center
it is believed that il-22 that is produced during sepsis may contribute to host defense and to stabilizing mucosal barrier functions under systemic infection conditions .
however , the adverse effects of il-22 are also described in a model of polymicrobial peritonitis , in which the levels of il-22 and its receptor in the spleen and kidney were very high .
the biological activity of il-22 is modulated by its antagonist , il-22 pb . treatment of mice with il-22 pb before sepsis led to increased accumulation of neutrophils and mononuclear phagocytes , as well as a reduction in bacterial burden at the site of infection .
thus , the beneficial effects of il-22 are mediated by promoting tissue protection , whereas ill effects are connected with exacerbated inflammation .
this dual role of il-22 implies that it participates in the pathophysiology of experimental sepsis .
il-33 can induce t helper cells , mast cells , eosinophils , and basophils to produce th2 cytokines .
il-33 mediates its biological effects through interaction with its receptors and with associated proteins abundantly expressed on the surface of th2 cells and mast cells .
il-33 also functions as a chemotactic mediator for th2 cells [ 100 , 101 ] . in mast cells
, il-33 triggers the production and release of proinflammatory cytokines , promotes maturation , and induces degranulation .
furthermore , il-33 amplifies the polarization of alternatively activated macrophages and it enhances tlr4-mediated cytokine production by macrophages .
st2 exists in different splice variants , which results in a cell membrane - bound form and in a soluble form .
the soluble form , sst2 , is generated by alternative splicing and does not induce signaling , thus acting as a receptor for il-33 .
high levels of sst2 have been associated with the pathogenesis of sepsis , so sst2 may be a potential marker of poor prognosis [ 95 , 105 ] .
il-33 has beneficial effects in experimental sepsis , enhancing the accumulation of neutrophils through the upregulation of cxcr2 via grk2-dependent pathways at the site of infection and reducing the inflammatory response in systemic sites .
however , it has not yet been determined whether the administration of il-33 actually represents a therapeutic strategy .
the proinflammatory cytokine il-17a is mainly produced by th17 cells and is involved in the mediation of proinflammatory responses , hence triggering the production of many other cytokines such as il-1 , il-6 , and tnf- .
it has recently been shown that increased il-17a levels have adverse effects during experimental sepsis in clp - induced sepsis models . whereas the blocking of il-17a was associated with reduced levels of bacteremia , proinflammatory cytokines and an increased survival rates of animals .
it causes an increased production of il-4 itself and of other anti - inflammatory cytokines , suppressing the secretion of monocyte - derived proinflammatory cytokines .
experimental studies have shown that il-4 increases the survival rates of mice exposed to lethal lps doses .
similarly , in humans , mrna expression of il-4 was associated with survival of patients with severe sepsis . nonetheless , il-4 plasma levels of septic patients on the day of hospital admission were not different between the patients who survived and those who did not survive sepsis .
it was recently suggested that polymorphisms in the il-4 gene promoters may affect the balance of th1 and th2 response and , consequently , predispose trauma patients to develop sepsis .
although there are several studies suggesting that il-4 plays an important role in the pathogenesis of sepsis , its real role in the course of the disease remains unknown .
procalcitonin ( pct ) is the hormone calcitonin , which is normally produced by the c cells of the thyroid gland , leading to massive release of pct into the bloodstream depending on the severity of sepsis .
assicot and colleagues   were the first to describe pct as a potential biomarker of sepsis and infection .
the authors showed a more favorable pct kinetic profile than the profile of c - reactive protein and cytokines .
pct circulating levels decrease within about 24 hours , when the infection is sufficiently treated .
decreasing pct levels are , thus , associated with improved survival , whereas increased or persistently increasing pct levels are predictive of an unfavorable outcome [ 113 , 114 ] .
discrimination between infectious and noninfectious conditions for pct and decreasing pct levels in properly treated patients raised the hypothesis that pct levels can help in determining the antimicrobial therapy .
pct serum concentrations may be increased in medullary thyroid carcinoma in the absence of bacterial infections and in conditions such as severe trauma , surgery , or postcardiac arrest , heat shock , stress birth , and various types of immunotherapies and some autoimmune diseases .
however , as any other biomarker , pct levels must be evaluated within the clinical context of the patient .
understanding the neonatal sepsis pathogenesis still remains a challenge , given its complexity and the inherent immunological characteristics of the newborn .
cytokines seem to be one of the major mediators involved in the outcome of this entity .
the imbalance between proinflammatory and anti - inflammatory cytokines appears to be related to both the severity and prognosis of neonatal sepsis .
therefore , the use of cytokines as biomarkers of neonatal sepsis seems plausible and necessary , since the early diagnosis of neonatal sepsis directly influences the therapy and prognosis .
 neonatal sepsis is a major cause of morbidity and mortality and its signs and symptoms are nonspecific , which makes the diagnosis difficult . 
 the routinely used laboratory tests are not effective methods of analysis , as they are extremely nonspecific and often cause inappropriate use of antibiotics . 
 sepsis is the result of an infection associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators . 
 cytokines are potent inflammatory mediators and their serum levels are increased during infections , so changes from other inflammatory effector molecules may occur . 
 although proinflammatory and anti - inflammatory cytokines have been identified as probable markers of neonatal infection , in order to characterize the inflammatory response during sepsis , it is necessary to analyze a panel of cytokines and not only the measurement of individual cytokines . 
 measurements of inflammatory mediators bring new options for diagnosing and following up neonatal sepsis , thus enabling early treatment and , as a result , increased neonatal survival . by taking into account the magnitude of neonatal sepsis , 
 the aim of this review is to address the role of cytokines in the pathogenesis of neonatal sepsis and its value as a diagnostic criterion . 
1. Introduction
2. Neonatal Sepsis
3. Cytokines
4. New Perspectives of Biomarkers in Sepsis
5. Conclusion