Osteocalcin and regulatory cytokine imbalance in children with congenital cleft lip and palate

Abstract

The aim of the work was a comprehensive assessment of the cytokine system and peripheral blood osteocalcin with the establishment of features of their interconnections in children with congenital cleft lip and palate (CCLP) in comparison with corresponding controls at different age periods. Levels of IL17, IL4, IL6, IL1b, IFNc and osteocalcin were analyzed by enzyme immunoassay in the peripheral blood of 80 children (0-12 months, 1-3 years, 4-9 years, 10-15 years) with CCLP and age-appropriate control of healthy individuals (40 people). An analysis of the obtained data shows that in children with CCLP we revealed significant differences between pro-inflammatory (IL1b, IL6, IL17), regulatory (IFNc), anti-inflammatory (IL4) cytokines and osteocalcin compared with controls. Differences were found in the content of IL17, IFNc, IL4 and osteocalcin in healthy children and in children with CCLP in postnatal ontogenesis. Cytokine deregulation of immunosteogenesis in CCLP, leading to a significant deficit of osteocalcin in the first year of life due to imbalance of the cytokine profile: discordant IL17, IFNc and IL4 were detected. Obtained data are undoubtedly important in the future for developing new strategies for targeted therapy aimed at normalizing osteocalcin levels at different age periods in children with CCLP.

Orofacial clefts lead to increased pro-inflammatory cytokine levels on neonatal oral mucosa

“…Another study investigating lip tissue from infants with OFC (4-14 months) during cleft surgery without a control group, found overall highest concentrations of TNF compared to other measured cytokines in lip tissue from neonates with OFC ( 23 ). Considering systemic cytokine levels (in peripheral blood), infants with OFC (0-12 months) presented significantly increased levels of IL-17 and INF-γ compared to an age-matched control group, however, levels of IL-6 and IL-8 were similar in both groups ( 63 ), which is in contrast to our results found in tongue smear samples. TNF is a primary pro-inflammatory cytokine ( 37 ) and promotes vasodilatation, edema formation, leukocyte adhesion, regulation of blood coagulation ( 28 ).…”

“…So far, two invasive methods were used to analyse cytokine concentrations in infants with OFC: 1) Two previous studies ( 23 , 24 ) collected lip tissue during cleft surgery in infants (3-18 months of age) to investigate cytokine concentrations, but lacked an adequate control group since surgical collection of lip tissue from healthy neonates would be ethically inacceptable. 2) One study investigated cytokine concentrations and osteocalcin in peripheral blood samples collected from children with OFC (0-12 months, 1-3 years, 4-9 years, 10-15 years; n=80) compared to an age-matched control group (n=10/per age group) ( 63 ). The authors detected significantly higher pro-inflammatory cytokine concentrations in OFC children and distinct age-related correlations between IL-4 and osteocalcin with a focus on immune-skeletal interactions and postnatal osteogenesis ( 63 ), however, they did not detect differences between different cleft phenotypes and did not correlate their results to oral parameters.…”

“…2) One study investigated cytokine concentrations and osteocalcin in peripheral blood samples collected from children with OFC (0-12 months, 1-3 years, 4-9 years, 10-15 years; n=80) compared to an age-matched control group (n=10/per age group) ( 63 ). The authors detected significantly higher pro-inflammatory cytokine concentrations in OFC children and distinct age-related correlations between IL-4 and osteocalcin with a focus on immune-skeletal interactions and postnatal osteogenesis ( 63 ), however, they did not detect differences between different cleft phenotypes and did not correlate their results to oral parameters. With respect to non-invasive methods to investigate cytokine concentrations in the oral cavity, several methods were used in patients with OFC: 1) Collection of stimulated saliva ( 64 ) or unstimulated saliva ( 65 ) using the spitting method: patients are asked to collect saliva in their mouth in an upright position with the head slightly tilted forward (unstimulated saliva: without moving the head or regurgitation) and to spit the collected saliva in a sterile tube several times until the required amount of saliva is collected ( 65 ); 2) Usage of sterile swabs to collect smear of defined areas, e.g., wiping over the tongue or the palate several times (areas with low saliva flow rate) or wiping over the cheek or sublingual area (high saliva flow rate) ( 25 ); 3) Sampling of gingival crevicular fluid by putting sterile paper strips in gingival pockets for a defined amount of time (in dentulous individuals) ( 25 , 66 – 70 ); 4) Gathering of dental biofilm [supragingival plaque ( 25 ) or subgingival plaque ( 71 )] with sterile dental instruments (in dentulous individuals).…”

“…Further, a significant increase of IL-2 and IL-4 from T0 to T1 was found resulting in highest levels of IL-4 at T1 in neonates without OFC. Regarding age-related changes of cytokine levels (in peripheral blood), children without OFC were characterized by a significant increase of IL-4 between 1 year and 3 years of age ( 63 ). IL-2 is an important regulator in communication of innate and adaptive immunity, e.g., by activation of T/B/NK cells ( 32 ); promotes tissue integrity, defense and tolerance in wound healing processes ( 50 ) and is a key regulator of regulation of oral mucosal inflammation ( 51 ).…”

Evaluating the Expression of Candidate Homeobox Genes and Their Role in Local-Site Inflammation in Mucosal Tissue Obtained from Children with Non-Syndromic Cleft Lip and Palate

“…Additionally, proinflammatory cytokines like IL17 and IL6, regulatory cytokine IFN-γ (interferon gamma) and anti-inflammatory cytokine IL4 modulate immunosteogenesis by actively participating in the process of osteogenesis [23]. Children with cleft lip and palate show disturbances in the immunosteogenesis during embryogenesis which affects the characteristics and the course of the disease postnatally [25]. These disturbances also create certain difficulties in the rehabilitation of such patients, which makes it essential to investigate the role of the gene-gene interactions, especially between causative genes and cytokine regulators.…”