Interplay of cytokines in preterm birth.

Preterm infants (i.e., born before <37 wk of gestation) are at increased risk of morbidity and mortality and long-term disabilities. Global prevalence of preterm birth (PTB) varies from 5 to 18 per cent. There are multiple aetiological causes and factors associated with PTB. Intrapartum infections are conventionally associated with PTB. However, maternal genotype modulates response to these infections. This review highlights the association of cytokine gene polymorphisms and their levels with PTB. Varying PTB rates across the different ethnic groups may be as a result of genetically mediated varying cytokines response to infections. Studies on genetic variations in tumour necrosis factor-alpha, interleukin-1 alpha (IL-1α), IL-1β, IL-6, IL-10 and toll-like receptor-4 genes and their association with PTB, have been reviewed. No single polymorphism of the studied genes was found to be associated with PTB. However, increased maternal levels of IL-1β and IL-6 and low levels of IL-10 have been found to be associated with PTB.

About 27 per cent of neonatal mortality has been reported to be related to complications of preterm birth (PTB)1 or delivery before 37 wk of gestation. In India, the incidence of PTB is about 21 per cent23, which translates into 3.6 million births annually. This corresponds to 23.6 per cent of global annual PTB burden which is estimated to be 15 million4.

PTB can be medically induced when there is an indication either related to the mother such as pre-eclampsia, eclampsia or foetus such as foetal distress. On the other hand, PTB can occur spontaneously due to multiple aetiologies such as uterine overdistension, as in multiple gestation, infection or inflammation56. Other risk factors for PTB are poor maternal nutritional status as evident by low maternal body mass index, periodontal disease and racial disparity (as reported higher risk is seen in African American than European American)7. Increased levels of inflammatory cytokines, such as toll-like receptor 4 (TLR4), tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6 have been reported in serum and/or amniotic fluid of women with spontaneous preterm labour (PTL)8. This review reports existing evidence on association of genetic variations in TNF-α, IL-1α, IL-1β, IL-6, IL-10 and TLR-4 with PTB.

Cytokines and preterm birth

PTB and spontaneous PTL (PTL is defined as ‘regular contractions of the uterus resulting in changes in the cervix that start before 37 wk of pregnancy’)9 have been shown to be associated with infections such as bacterial vaginosis and chorioamniotis61011. Infection leads to inflammation as evident by increased levels of TLR4, TNF-α, IL-1 and IL-6 in the amniotic fluid. The release of pro-inflammatory cytokines is followed by leucocytosis which results in apoptosis, preterm premature rupture of membrane along with cervical ripening and onset of premature labour. Since specific genes regulate corresponding cytokines, genetic polymorphisms in mother have been investigated to assess their association with PTB1213.

Inflammatory signalling is a highly complex pathway (Figure). This pathway can be modulated by external as well as the internal signals. The balance between pro-inflammatory and anti-inflammatory cytokines is crucial for implantation of the foetus, preparation of placenta and pregnancy outcome. While the T-helper 1 (Th1) cytokine is responsible for inflammation, the Th2 cytokine manages the anti-inflammation counter-regulatory pathway. The dominance of Th2 cytokine expression plays an important role in reducing inflammation and prevents allograft dismissal of the foetus1415.

Figure

Factors initiating onset of labour. Schematic diagram showing that labour can be induced by various factors (i) infection, (ii) surfactant protein/lipid, (iii) corticotropin-releasing hormone (CRH), (iv) uterine stretch. These activate inflammatory cytokines (IL-1, 6, 8) and metalloproteinases (mainly MMP-9) cause cervical ripening. Increased Cox-2 levels in myometrium cause prostaglandin synthesis and initiate labour. These inflammatory cytokines are counter balanced by anti-inflammatory cytokine IL-10. Cox-2, cyclooxygenases-2; IL, interleukin; MMP, matrix metalloproteinases protein.

Genetic Factors

Familial and twin studies have reported that PTB is sometimes heritable16171819. It has been observed that women with PTB have higher chances for recurrent PTB7. There seems to be a genetic predisposition to the PTB. Therefore, it seems plausible that polymorphisms in maternal genes regulating cytokine expression are related to PTB171819. Tables I and II summarize the genes associated with inflammatory pathway and therefore, PTB202122232425262728293031323334353637383940414243444546. It has been reported that altered production of pro-inflammatory cytokines mainly IL-1β, TNF-α and interferon (IFN)-lambda at the maternal-foetal interface results in PTB. On the contrary, IL-10 downregulates the secretion as well as expression of pro-inflammatory cytokines by other cells4748. The present review focussed only on polymorphisms in the coding or promoter regions of genes listed in Tables I and II.

Table I

Pooled data for the association of toll-like receptor-4 and tumour necrosis factor-alpha polymorphisms with preterm birth in different populations

Table II

Pooled data for the association of interleukin (IL)-1, interleukin-6 and interleukin-10 polymorphisms with preterm birth in different populations

Toll-like receptor (TLR)

Location, function and regulation

TLR-4 gene is located on chromosome 9q33.1. Its alternative name is cluster of differentiation 284. TLR family has 13 distinctive proteins (TLR-1 to TLR-13). These are capable of recognizing microbial agents and initiating early immune response by activating various downstream pathways, such as transduction of nuclear-kappa β pathway which regulates expression of genes secreting pro-inflammatory cytokines49.

TLR-2 and TLR-4 genes have been extensively studied and their role has been identified in pathogen recognition and initiation of immune response. TLR4 regulates innate immune response during pregnancy and thus directly affects the duration of gestation. It is mainly expressed in human placenta50.

TLR-4 pathway

It has been reported that most variations in TLR-4 are seen in the third exon20. TLR-4 signal pathway includes enrolment of some signal transducer adapter proteins (MyD88, IRAK1/4 and TRAF6), rapid activation of intermediate kinases (RIP1, TAB2/3, TAK1 and IKK α/β) and phosphorylation/degradation of the chaperone protein (Iκβ)51. Activation of immune system by endogenous and exogenous ligands such as heat shock proteins and bacterial lipopolysaccharides (LPS) is mediated through TLR-4. TLR-4 signalling activates the pro-inflammatory cytokines (IL-1, IL-6, IL-8) cascade which increases the level of prostaglandin (mostly PG-E and PG-F) and thus stimulates PTL causing PTB50. TLR-4 is expressed by macrophages located in placental villi and in intermediate trophoblast of the placenta. Increased expression of TLR-4 was found in placentas of patients with chorioamnionitis50, an independent risk factor for PTL. Hence, it is extrapolated that increased levels of TLR-4 may be associated with PTB. However, TLR4 expression has been studied in the placenta in vitro only50. Corresponding serum levels have not been assessed.

Polymorphism of TLR-4 gene

TLR-4 is located on long arm of chromosome 9. The polymorphic site rs4986790 is present on position 896. This A/G transition causes substitution of amino acid aspartic acid by glycine at position of 299 (i.e. Asp299Gly). This polymorphism has also been found to be associated with increased risk of severe disease due to respiratory syncytial virus and Gram-negative bacterial infection in children52. Thus, it can be hypothesized that substitution of aspartic acid by glycine in TLR-4 gene at position 299 can exaggerate the chances of infection and thus inflammation during pregnancy leading to PTB.

Many studies were conducted to determine the association of TLR-4 and PTB. Table I summarizes the studies of TLR4 and PTB. Lorenz et al20 reported significant association of PTB with TLR4 Asp299Gly in infants but not in mothers and this was supported by other studies also212253. On the contrary, other groups50525354 reported increased expression of TLR-4 in chorioamniotic membranes of patients with histologic chorioamnionitis regardless of their gestational status and in mothers with PTL, respectively. Equivocal results have been found for the association of polymorphism of TLR-4 gene and PTB.

Tumour necrosis factor-alpha (TNF-α)

TNF-α is located on chromosome 6p21.3. It is a pro-inflammatory cytokine, which promotes the production of collagen-degrading matrix metalloproteinases, and suppresses biosynthesis of tissue inhibitors of metalloproteinases5556. The metalloproteinases act on foetal membrane collagen resulting in loss of tensile strength. It also impairs the progesterone stimulating receptor B thus blocking the progesterone release. Both these actions promote onset of PTL56.

Polymorphism of TNF-α

Increased level of TNF-α was linked with various reproductive diseases such as frequent spontaneous abortions, pre-eclampsia, infections or endometriosis57. Elevated levels of TNF-α can change the delicate equilibrium between the anti-inflammatory and pro-inflammatory cytokines and thus induce PTB. Till date, two polymorphisms, -238G/A and -308G/A, present on promoter region have been studied. Table I lists the studies which analyzed the association of TNF-α and PTB. The TNF-α-238 G allele was reported to be associated with high transcriptional activity2358. Significant association of TNF-α (-308G/A) polymorphism has been reported with PTB2425262759. Interaction between infection, stress, obesity and TNF-α (-308G/A) polymorphism has also been reported, and all of these increase the risk of PTB59. However, in contradiction to these studies, negative or no associations were also reported2829303132333435363738. A meta-analysis which included all studies from 1990 to 2005 found no association between TNF-α (-308G/A) and PTB (odds ratio=1.41; 95% confidence interval=0.90-2.19)39. Hence, association of polymorphisms of TNF-α with PTB is equivocal till date.

Interleukin-1 (IL-1)

The IL-1 gene is located on long arm of chromosome 2 (2q14). IL-1 is a pro-inflammatory cytokine. Its secretion is controlled by IL-1 gene which has two subunits, IL-1α and IL-1β. On the same chromosome, IL-1 receptor antagonist (IL-1RA) gene is also located which is a competitive inhibitor of IL-1β. IL-1β is the most investigated candidate gene of the pro-inflammatory cytokine family. The activity of pro-inflammatory IL-1β is counterbalanced by the action of IL-1RA which inhibits the binding of circulating IL-1β to cell surface receptors6061. Therefore, IL-1RA helps in terminating the acute inflammation response but gets activated late during the course of an inflammatory event60.

Polymorphisms in IL-1 gene complex

There are many reported polymorphisms and microsatellites in the IL-1 gene complex, and the most studied polymorphisms are summarized in Table II. The promoter site of IL-1α consists of two polymorphisms; +4845G/T and -899C/T. IL-1β consists of three polymorphisms, namely, -31T/C, -511C/T and +3954C/T. Studies have reported a microsatellite in intron 2 of the IL-1RA6061. This polymorphism results in five alleles. The most common allele is allele 2 (IL1RN*2) with the recurrence of 4-26 per cent, whereas alleles 3, 4 and 5 are in <5 per cent of population. Allele 2 has been associated with various chronic inflammatory conditions. IL1RA polymorphism appears to affect both IL-1 and IL-1RA gene expression. The T allele of a polymorphism at position 31 (IL1β-31T) is in a transcriptional start site and is likewise connected with a decrease in IL-1β production. This may be a consequence of the underlying link between IL1RN*2 and IL1β-31T. Carriers of rare alleles of IL-1β polymorphisms (IL-1β-511T and -31C) have shown higher levels of IL-1RA than individuals with wild-type IL-1β genotypes626364656667686970717273.

IL-1β has consistently been associated with increased risk of spontaneous preterm delivery. A study conducted on European population by Puchner et al74 reported that with a unit increase in IL-1β level in women, there was 7.2 times increased risk of PTB. Thus, it may serve as predictive marker of PTL.

In a case-control study conducted on European27 and Japanese40 population, significant association was found between IL-1 (+4845G/T) and PTB. Others reported the significant association of IL-1β (+3953/3954)with enhanced production of IL-1β4166. On the contrary, inconsistent results were reported in case of IL-1β (-511C/T) and IL-1β (-31C/T) polymorphisms27324041727374. Various studies have reported inconsistent association of different polymorphisms of IL-1α and β with PTB. However, increased IL-1β levels are found consistently associated with PTB.

Interleukin-6 (IL-6)

Gene for IL-6 is located on 7q21 and commonly known as IL-6, IFN β-2 or rarely as hybridoma growth factor or hepatocytes-stimulating factor or B-cell stimulatory factor-2. IL-6 is a pro-inflammatory cytokine causing induction of T-lymphocytes, C-reactive protein synthesis and B-cell differentiation. It is widely expressed in the decidual tissue, placenta, foetal membrane and amniotic fluid. It mainly functions in embryo implantation and placental development, as well as in the immune adaptations, which are required for continuing pregnancy75. IL-6 production is stimulated by various factors, namely, IL-1, TNF-α and LPS. Increased levels of IL-6 are found in unexplained infertility, recurrent miscarriage, pre-eclampsia and preterm delivery. Altered systemic IL-6 trans-signalling in women can lead to recurrent miscarriage. IL-6 inhibits the generation of CD4+ T regulatory cells required for pregnancy tolerance3743767778.

Polymorphism in IL-6 gene

At position -174 in the IL-6 gene, C>G substitution (i.e. Cytosine to Guanine) causes higher transcriptional activity in response to IL-1 and LPS stimuli. A polymorphism at the -174 position (G/C) in the promoter region of the IL-6 gene results in decreased cytokine production and therefore, decreased risk of PTB37.

Table II shows the polymorphisms of IL-6 and their association with PTB. Sugita et al43 reported a significant association of IL-6 (-6572 G/C) in PTB in the Japanese population. Moura et al37 found strong evidence for the association of IL-6 (-174G/C) with the PTB in the European population. Menon et al79 compared amniotic fluid concentrations of IL-6 in cases of PTB and term births and found significant association (P=0.003). On the contrary, Kalinka and Bitner32 reported no association between IL-6 (-174G/C)and PTB but found an increased incidence of PTB with combined GG+GC genotype. Harper et al30 carried out a study on 834 women with high risk of PTB and assessed the IL-6 (-174 G/C)polymorphisms but was unable to detect any association with PTB. A study by Karakaş et al80 found this polymorphism protective against PTB, while others reported that maternal IL-6 (-174G/C) polymorphism was associated with chorioamnionitis818283.

Inconsistent results were found for the association of IL-6 polymorphism with PTB. However, increased IL-6 levels have been reported in chorioamnionitis458485 which in turn leads to PTB. Further translation research in this area may be able to identify therapeutic agents to prevent PTB.

Interleukin-10 (IL-10)

The IL-10 gene is located on chromosome 1q31-1q32. It is also known as cytokine synthesis inhibitory factor or T-cell growth factor inhibitor. IL-10 is an anti-inflammatory cytokine produced mainly by monocytes and to a lesser extent by lymphocytes. Being pleiotropic in nature, it modulates both immune regulation and inflammation. It reduces Th1 cytokines by reducing the MHC class II antigens on macrophages and thus enhances B-cell survival, proliferation and antibody production. IL-10 can hinder NF-kappa B activity, which is a key mediator of the JAK-STAT signalling pathway86.

Polymorphism in IL-10 gene

Table II summarizes the studied polymorphisms and their outcome in PTB. Polymorphisms located at the promoter region of IL-10 gene are -1082G/A, -819C/T and -592C/A. Studies conducted on Caucasian population found polymorphism (rs1800896) associated with PTB4687. Moura et al37 conducted two independent studies on Brazilian population and found no association between polymorphisms (IL-10-1082G/A, 1L-10-819C/T and IL-10-592C/A) and PTB. Similar findings were reported by other studies also36374447.

Thus, IL-10 was not consistently found to be associated with PTB. However, low levels of IL-10 were reported to be associated with PTB4784858889.

Conclusion

Since PTB rate has remained almost static over the past few years in the developed countries90, researchers are now looking into possible genetic aetiology. The concept of involvement of cytokines-stimulating prostaglandin production resulting in PTB has been widely accepted. Many studies have been conducted in different populations to find out the association of TLR-4, IL-1α, IL-1β, IL-6 and IL-10 gene polymorphisms with PTB, yet the results are inconclusive. This can be due to differences in the ethnic groups studied or the influence of environmental factors. Further genome-wide and gene expression studies are needed that are also capable of assessing interactions with infections and environment. Accurate prediction of risk of PTB by molecular methods may help in planning appropriate antenatal care in women at risk.