Inflammatory Markers in Children and Adolescents with Functional Somatic Disorders: A Systematic Review

Functional somatic disorders (FSDs) are common in children and adolescents. Recent findings suggest that low-grade inflammation has a role in the development and maintenance of pediatric FSDs. This systematic review included studies with original data on systemic inflammatory markers in children and adolescents with an FSD compared to individuals without an FSD. The literature search identified 1374 articles. After assessment, a total of 15 studies met the inclusion criteria. In total, 41 serum or plasma cytokines were assayed in a population of 696 children and adolescents. Altered cytokine levels in patients with FSDs were reported in 12 studies, whereas three studies found no significant differences when comparing patients with FSDs and controls. The cytokine levels were significantly elevated in nine studies (i.e., IL-2, IL-6, IL-8, IL-12 (p70), CRP, hsCRP, IP-10, MCP-1, sTIM-3, sCD25 and TNF-α). The findings indicate that inflammatory response may have a role in the pathophysiology of pediatric FSDs. However, the included studies showed limited quality with potential risk of bias, small study populations and a narrow spectrum of included FSDs, which limits the generalizability of the results. To further explore the potential link between inflammatory markers and pediatric FSDs, future research using a longitudinal study design is recommended.


Introduction
Functional somatic disorders (FSDs) are defined by the presence of one or multiple persistent physical symptoms or characteristic symptom patterns that cannot be attributed to a known well-defined somatic or psychiatric disease.The term encompasses a wide range of diagnoses, such as fibromyalgia, chronic pain, irritable bowel syndrome (IBS) and chronic fatigue syndrome (CFS) [1].Mild transient physical symptoms are common among children and adolescents; still, the exact number suffering from FSDs remains unknown.A recent meta-analysis found a prevalence rate of 3.3% for FSDs in children and adolescents, and the prevalence is possibly increasing [2,3].At this severe end of the spectrum, the symptoms typically result in significant disruption of the daily life of the child or adolescent with school absenteeism and social withdrawal and moreover often lead to high levels of health service use [4,5].There is a substantial risk that symptoms sustain into adulthood, and furthermore, FSDs in childhood and adolescence are associated with adverse physical and mental health outcomes later in life [6,7].
FSDs are thought to arise in a complex interaction between biological and psychosocial factors [8].Although the etiology is still not fully understood, it is suggested to involve different patterns of dysregulation of the stress system, including activation of the immune-inflammatory system.These mechanisms are thought to be triggered by continuing, cumulative or overwhelming stress, potentially resulting in a subtle but chronic state of immune-inflammatory activation, which is termed low-grade inflammation.Low-grade inflammation might activate other stress-system components, such as the hypothalamicpituitary-adrenal axis, subsequently inducing increased bodily arousal contributing to the generation and maintenance of functional somatic symptoms [9][10][11].
The aforementioned inflammatory response, a biological response to an infection or non-infectious process, is brought on by a group of inflammatory markers secreted by immune cells.The main group of systemic inflammatory markers is cytokines, which are protein mediators of the immune system produced by both leukocytes and non-immune cells.Cytokines can be classified depending upon their role in the inflammatory process as pro-or anti-inflammatory, while some cytokines serve as both [12].Common proinflammatory cytokines are IL1-alpha, IL1-beta, IL6, CRP and TNF-alpha, whereas IL-1ra, IL-4, IL-10, IL-11, and IL-13 are common anti-inflammatory cytokines [12].
A connection between inflammatory markers and several FSDs has been found in adults; for instance, a study found that levels of high-sensitivity CRP (hsCRP) were significantly elevated for both patients with CSF and patients with fibromyalgia compared to healthy controls [13].Likewise, another study found that an increased expression of inflammatory markers is associated with a variety of chronic pain conditions, including fibromyalgia, which in turn can lead to an increased release or upregulation of pro-inflammatory cytokines [14].Considering another important type of FSD, namely IBS, a study found that the pro-inflammatory cytokines TNF-α and IL-17 were significantly higher in adult patients with this disorder compared to healthy controls, suggesting a possible role for low-grade inflammation also in the pathogenesis of IBS [15].
It has been proposed that this relationship between FSDs and low-grade inflammation in adults might also be present in children and adolescents with FSDs [16].A recent review focused on the local inflammatory response in pediatric IBS and suggested a role of low-grade inflammation in the pathophysiology and symptom generation [17].The high prevalence of children and adolescents presenting with functional somatic symptoms and the multifaceted and not well-understood pathophysiology of FSDs propose a great need for further investigation of this topic.A profile of systemic circulating inflammatory markers in children and adolescents with a broad spectrum of FSDs could potentially generate a better understanding of the complex pathophysiology, ultimately leading to an improvement in early diagnostics and the development of better and more targeted treatment in the future.Thus, the aim of this review is to investigate the possible association between systemic inflammatory markers and FSDs in children and adolescents.

Review Protocol and Search Strategy
The current systematic review was conducted in accordance with the guidelines set out in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [18].The study information was registered a priori on 11 October 2023 in the Open Science Framework (OSF) registry (https://doi.org/10.17605/OSF.IO/32WNJ, accessed on 11 October 2023).The PICO framework was used to establish the search strategy for the study question [19].A systematic literature search was performed in the PubMed, PsycINFO and Embase databases.The literature search included both free-text words and identified search terms in MeSH in PubMed, Thesaurus in PsycINFO and Emtree in Embase.The PubMed search string can be found in Supplementary Materials (Table S1).The web-based software program Covidence was used to manage and organize the selection of articles.The final literature search included publications dating up to 23 November 2023.

Eligibility Criteria, Study Selection and Data Collection Process
Studies were considered eligible for data extraction if they met the following inclusion criteria: (1) children or adolescents (aged 0-17 years, both included), (2) patients diagnosed with an FSD, (3) measurement of systemic inflammatory markers, (4) studies including a control group without an FSD, and (5) studies written in English.The exclusion criteria were (1) animal experiments, (2) in vitro experiments, (3) case studies, (4) studies measuring inflammatory response exclusively in feces, biopsies, and urine (i.e., not systemic markers).
Duplicate references were eliminated, and initial screening was performed based on title and/or abstract.Potentially relevant articles were screened based on the full text.The identification of studies was carried out by two independent evaluators (ASH and KHK), and a third evaluator (CUR) was consulted to resolve discrepancies.All evaluators were experienced clinicians and researchers regarding FSDs in children and adolescents.A kappa estimate of the initial agreement between the two evaluators was calculated for the full text screening.To ensure accurate participant diagnosis, the evaluators assessed that the criteria used to define the FSD diagnoses were within the scope of this study based on the International Classification of Disorders (ICD) and the Diagnostic and Statistical Manual of Mental Disorders (DSM) [20].
Data were extracted from the included studies by the two independent evaluators (ASH and KHK) using a structured data collection form.The data were collated in an Excel spreadsheet and included information on study design, participant characteristics, outcome details regarding the measured inflammatory markers and the results of the comparison between cases and controls.Disagreements were solved through discussion or by consulting the third evaluator (CUR).Finally, a qualitative synthesis of the included data was conducted to summarize and assess the results.A meta-analysis was not applied due to heterogenic data and risk of bias when comparing the cytokine levels across the studies.

Quality Assessment of Studies
The quality of the included studies was assessed using the Newcastle-Ottawa scale (NOS) of quality to identify biases within studies [21].Three broad perspectives of casecontrol and cohort studies are assessed with the NOS using a star system: (1) the selection of the study groups, (2) the comparability of the groups, and (3) the ascertainment of outcome of interest.The total maximum score of these 3 subsets is 9.The quality assessment was conducted independently by two evaluators (ASH and KHK) using the specific NOS criteria (see Supplementary Information S2).A third evaluator (CUR) was consulted to determine final quality scores if discrepancies were not resolved.In the current review a score ≥ 7 was used to categorize high-quality studies based on previously applied cut-offs [22].

Study Selection
A total of 1842 records were identified in the literature search (PubMed n = 856, Embase n = 898, and PsycINFO n = 88).After duplicate removal, 1374 records were screened based on title and/or abstract.A total of 34 reports were assessed after full-text retrieval, of which 20 reports were excluded, and 14 were determined to fulfill the requirements for inclusion (Kappa of initial agreement = 0.87).Finally, a citation screening of the included studies revealed one further study, leading to the inclusion of a total of 15 studies in the review.The selection process is displayed in Figure 1.

Study Characteristics
The study characteristics are outlined in Table 1.The included studies were pub between 2012 and 2023.The number of cases in the studies ranged from 9 to 100, total of 696 cases were included in this review when accounting for overlapping populations in five of the included reports [16,[23][24][25][26].The cases were defined by C 11 studies and functional gastrointestinal disorders (FGID) were identified in four st A total of 611 controls were included in this review when considering overla study populations [16,[23][24][25][26].The controls were defined as healthy controls in 11 s [16,[23][24][25][26][27][28][29][30][31][32], individuals recovered after Epstein-Barr virus (EBV) or infectious mono osis (IM) infection in four studies [24,[33][34][35], with one study also including a gro healthy controls [24], and children and adolescents with an asymptomatic parasitic tion in a single study [36].Three studies used the matching of controls to cases on f like age, sex, Tanner stage and social class [33][34][35].

Study Characteristics
The study characteristics are outlined in Table 1.The included studies were published between 2012 and 2023.The number of cases in the studies ranged from 9 to 100, and a total of 696 cases were included in this review when accounting for overlapping study populations in five of the included reports [16,[23][24][25][26].The cases were defined by CFS in 11 studies and functional gastrointestinal disorders (FGID) were identified in four studies.

Quality Assessment
The included studies displayed quality scores from 3 to 6 out of a maximum score of 9 as displayed in Table 3.None of the included studies were considered high-quality research studies when assessing the study design using the NOS.Regarding the selection of the cases, the studies lacked consensus regarding the criteria used to determine the CFS diagnosis and used a broad range of definitions, whereas the Rome criteria for IBS were used in three out of the four studies investigating cases with FGID [30][31][32]37].Potential bias was especially observed regarding the comparability of cases and controls with none of the included studies explicitly stating that the controls had no history of FSD or controlling for psychiatric comorbidity.Eight studies controlled for relevant covariates, i.e., physical activity and/or body mass index (BMI) [16,[23][24][25][26][27]29,35].Finally, when regarding exposure, all studies reported the measured concentrations of the inflammatory markers and used the same method for measuring the inflammatory markers in cases and controls, and two studies reported non-response rates (i.e., participants not completing the blood sample collection) [27,34].a The NOS used for the quality assessment is outlined in Supplementary Information S2. * A star was awarded if the requirements of the item was rated to be fulfilled.

Discussion
This review aimed to summarize existing published data on systemic inflammatory markers in children and adolescents with FSDs compared to controls.In total, 15 studies were identified covering 696 pediatric cases with FSDs only confined to CFS or FGID.A list of 41 serum or plasma cytokines was assayed in the included studies.Altered cytokine levels in children and adolescents with FSDs were reported in 12 studies, whereas only three studies found no significant difference when comparing the FSD cases and controls.The studies, which reported significantly altered cytokine levels in children and adolescents with FSD, displayed both elevated and decreased levels of the assayed cytokines.The reported elevated cytokines are all considered pro-inflammatory when regarding the existing literature [12,38].However, the reported decreased cytokines capture both pro-inflammatory cytokines (i.e., IL-6, IL-23, hsCRP and IFN-γ) and anti-inflammatory cytokines (i.e., IL-5, IL-10, IL-13) [12,38].This makes it difficult to firmly conclude whether the inflammatory response is expected to be increased or decrease in children and ado-lescents with FSDs.Still, the main part of the reported altered cytokine levels mediates a pro-inflammatory response, indicating a process of low-grade inflammation in children and adolescents with FSDs.
When regarding the existing evidence on adults with FSDs, a recent systematic review by Blundell et al. found no significant differences in cytokine levels between adult CFS cases and healthy controls, except transforming growth factor-beta (TGF-β), across the majority of the 38 included studies [39].Still, a number of studies on adults with other FSDs (e.g., fibromyalgia and chronic pain) point in the direction of an association between inflammation response and FSD [13,14,40].Furthermore, a large register-based cohort study on adult cases with a broad range of FSDs found that former infections leading to hospital contacts were associated with a higher risk of having an FSD, and the number of prior infections increased the risk of an FSD, also indicating an association between FSDs and severe infections [41].
In pediatric FSDs, the TRAILS study by Joncker et al. investigated a population of adolescents with non-neurological functional somatic symptoms [42].The study did not include a control group but compared the measured levels of hsCRP with the reported burden of functional somatic symptoms.A significant association between hsCRP levels and the level of functional somatic symptoms was shown, indicating a role for inflammatory processes associated with the severity of symptoms in pediatric FSD.Furthermore, two studies from Australia by Kozlowska et al. and McInnis et al. investigated children with functional neurological disorders as well as children with chronic functional pain and measured CRP levels [43,44].The studies did not include control groups but compared reference ranges on CRP levels.Both studies reported an upward shift in CRP levels in the FSD cases, suggesting an activation of the inflammatory response system, also indicating a role for inflammatory processes in pediatric FSD.
A skewing of the immune system with altered cytokine production has been suggested to be linked to long-term stress exposure causing an activation of the hypothalamicpituitary-adrenal (HPA) axis and the inflammatory system.In a state of chronic stress, prolonged activation of the HPA axis with elevated production of the stress hormone cortisol can cause impairment of the immune system's sensitivity to cortisol, resulting in elevated levels of inflammatory markers [45].The influence of chronic stress on the HPA axis may change over time, transitioning from heightened to diminished activity, ultimately leading to inadequate regulation of inflammation [46].The relationship between cortisol levels and FSDs has been investigated in prior studies, indicating an altered cortisol response in FSDs [47,48].However, long-term stress can also inhibit the inflammatory response system mediated by elevated levels of stress hormones, causing a shift in the balance of certain T-lymphocyte types (e.g., Th1, Th2 and Th17) resulting in lowered levels of cytokines [49].Thus, the interplay between the stress system and inflammatory response is a complex process, and the development of FSDs may be mediated by an imbalance of both elevated and lowered levels of different cytokines, as also displayed with the results of this current review.Furthermore, the heterogenic alterations in cytokine levels observed in this review may reflect a varying pathophysiology due to the different bio-psycho-social underlying factors involved in the development and maintenance of FSDs, as also displayed in this review including both post-infectious and post-radiation conditions.
To summarize, this review outlined the existing knowledge and displayed a possible association between the level of inflammatory markers and FSDs in children and adolescents.The noteworthy strengths of this paper are the comprehensive systematic literature search and the inclusion of studies reporting on FSD cases compared to controls.However, the evidence level is limited by the quality of the included studies as well as the lack of generalizability of the data due to a narrow spectrum of investigated FSDs as well as different types and numbers of assayed inflammatory markers in these studies.Regarding the measured level of the inflammatory markers, we decided not to conduct a meta-analysis, since the heterogeneity in applied methods for measuring the cytokine levels made it difficult to compare the results across the studies.None of the included studies reported data on the usage of anti-inflammatory medication, although patients with FSDs may occasionally use non-steroidal anti-inflammatory drugs (NSAID)s to relieve pain.Furthermore, the small sample size of the studies and the lack of matching regarding control groups may lead to bias, and it is important to interpret the results with caution.
Future research could aim to apply a consistent methodology in case-control designs investigating a wide range of cytokines and testing the association with subgroups of pediatric FSDs.The studies identified in this systematic review only investigated children and adolescents with either CFS or FGID.Future studies should include a broad spectrum of FSDs to examine levels of systemic inflammatory markers across FSDs.Furthermore, a longitudinal study of cytokine levels before and after the onset of FSDs in children and adolescents, as well as after remission, could provide knowledge regarding the relationship between circulating cytokines and the development and maintenance of FSDs.Possible confounders such as comorbid psychiatric and somatic conditions, physical activity, weight as well as medication should be accounted for to avoid potential bias.

Conclusions
This systematic review aimed to outline the existing body of evidence regarding the level of inflammatory markers in children and adolescents with FSDs.Altered levels of inflammatory markers in pediatric FSDs were reported in most of the included studies, indicating a role for the inflammatory response system in the pathophysiology of pediatric FSDs.However, the review demonstrates that the quality of the existing studies is limited, and further research is recommended to explore the possible role of inflammatory processes in the development and maintenance of pediatric FSDs.

Figure 1 .
Figure 1.PRISMA flow diagram of the study selection process.

Figure 1 .
Figure 1.PRISMA flow diagram of the study selection process.

Table 1 .
Characteristics of the included studies.

Table 1 .
Characteristics of the included studies.

Table 2 .
Main results of the included studies.

Table 3 .
Quality assessment of included studies according to Newcastle-Ottawa scale (NOS) a .