Autism spectrum disorders (ASD) are defined as a group of neurodevelopmental conditions of childhood with environmental causes that are still not fully understood. Most environmental factors during the perinatal stage appear to converge into a series of inflammatory conditions, such as bacterial and viral infections and inflammatory bowel disease (1, 2), suggesting that inflammatory responses could be an underlying factor in the etiology of ASD. Large population-based epidemiological studies have linked ASD with autoimmune disease and abnormal blood levels of various inflammatory cytokines and immunological biomarkers (3, 4). For instance, previous studies on inflammatory biomarkers have found increased concentrations of pro-inflammatory factors IL-1β, IL-4, IL-6, IL-8, and TNF-α (5–8), as well as decreased concentrations of anti-inflammatory factors IL-10 and IL-1Ra (9) in the peripheral blood of patients with ASD. These abnormal inflammatory cytokine levels are linked to greater impairments in language function and social interaction in children with ASD (6, 8). Therefore, it is necessary to further identify inflammatory biomarkers in ASD and uncover the causality between ASD and changes in the levels of inflammatory factors.

Observational studies are often susceptible to confounding, reverse causation, and multiple biases, which can lead to unreliable findings regarding the causal effects of exposures on outcomes. The Mendelian randomization (MR) method provides an alternative approach to investigate causality in epidemiological research, by utilizing genetic variants as instrumental variables to determine whether a risk factor has a causal effect on a health outcome. As an individual’s genotype is determined at conception and cannot be altered, this method avoids the reverse causality between genotype and outcome. In general, MR analysis rests on 3 assumptions: (1) genetic variants are associated with the risk factor; (2) genetic variants are not associated with confounders; and (3) genetic variants affect the outcome only through the risk factor (10). The advent of large-scale genome-wide association studies (GWAS) increases the accessibility of single-nucleotide polymorphisms (SNPs) as instrumental variables to infer causality in MR studies.

Based on the GWAS summary statistics, previous studies have examined causal relationships between 41 circulating inflammatory factors (11) and various complex diseases, including depression (12), epilepsy (13), and Alzheimer’s disease (14). Recently, Zhao et al. (15) extended previous works by conducting a genome-wide protein quantitative trait locus study which identified the genetic architecture of 91 circulating inflammatory factors in 14,824 European-ancestry participants. Several recent studies have included these 91 inflammatory factors for MR analysis, which further extends the use of inflammatory factors in MR studies (16, 17). Although previous observational studies have found a strong association between changes in levels of some circulating inflammatory proteins and ASD (5–9), their causal relationships remain undefined. The number of inflammatory factors analyzed in these studies is also relatively limited. Based on the knowledge above, we conducted the first bidirectional two-sample MR analysis (SNPs associated with the exposure and outcome are individually obtained from two independent samples) to determine the causal relationship between 91 inflammatory factors and ASD. The inflammatory biomarkers identified in this work may provide the basis for an objective test for early and accurate diagnosis of ASD and may shed light on the etiology and pathogenesis of ASD.

Although recent observational studies have suggested an association between inflammatory factors and ASD (27–29), it is important to note that these results may be influenced by confounding factors, and the causal relationship between the two remains inconclusive. Using novel data and approaches, this bidirectional MR study offers a genetic insight into the potential causal relationship between circulating inflammatory factors and ASD. The findings of this study suggest that levels of SULT1A1, CD244, CD5, FLT3LG, and TNFSF10 are positively associated with the risk of ASD, while levels of IL-7, IL2Rβ, and IL-2 are inversely associated with the risk of ASD. Furthermore, the genetic susceptibility to ASD exhibited suggestive evidence of increased levels of MMP10 and CASP8, and decreased levels of TNFSF11 and CCL19. Importantly, sensitivity analyses supported the robustness of these results.

Individuals with ASD show alterations in circulating inflammatory factors along with abnormal peripheral blood levels of lymphocytes and macrophages across the lifespan (30). Inflammatory factor profiling can reveal the progression and status of immune system dysregulation in ASD, offering therapeutic targets for improving core autistic symptoms. Current results in the forward MR analysis demonstrated a strong causal association between a decreased level of circulating IL-7 and a higher liability of ASD based on the Bonferroni correction. IL-7 is mainly produced by stromal cells in lymphoid tissues and has pleiotropic effects on the development of T and B cells, as well as T-cell homeostasis. The administration or neutralization of IL-7 may enable the modulation of immune function in individuals with lymphocyte depletion or autoimmunity (31). Data from several studies have shown that plasma levels of IL-7 were higher in children with ASD than those observed in typically developing controls, but these differences did not reach statistical significance after correction for multiple comparisons (32–34). By contrast, Napolioni and colleagues (35) found that plasma levels of IL-7 were inversely correlated with full intelligence quotient in children with ASD using Spearman’s rank correlation analysis. Similarly, a large observational study on the risk of psychopathology found that decreased IL-7 levels in cord serum were linked to emotional symptoms and abnormal pro-social behavior in 5-year-old children (36). Overall, this MR analysis based on large populations replicates and extends these findings, highlighting a causal protective role of genetically encoded higher IL-7 levels against ASD.

Other results did not show robust causality after Bonferroni correction, which can only be regarded as suggestive evidence of potential causality. IL-2 functions as an essential immunoregulatory factor produced primarily by T cells, exerting its effects via binding to the high-affinity IL-2R comprising of α (IL-2Rα), β (IL-2Rβ), and γ (IL-2Rγ) subunits. Both IL-2 and IL-2Rβ have been implicated in clonal expansion and functional differentiation of T cells and natural killer cells (37, 38). Vojdani et al. have found that children with ASD appeared to suffer from decreased blood natural killer cell activity due to their low intracellular IL-2 levels (39). In patients with ASD, the proportion of DR+ (activated) T lymphocytes is abnormally increased, whereas the proportion of IL-2 receptor+ lymphocytes remains unchanged or even decreases. This is inversely proportional to the severity of autistic symptoms and similar to that seen in autoimmune diseases (40–42). In addition, previous studies have observed lower mRNA expression levels of IL-2 and percentages of IL-2 synthesizing CD4+ and CD8+ T cells in the peripheral blood of ASD children as compared to controls (43, 44). To sum up, our findings further support these observations and provide evidence of immune dysfunction and autoimmunity in patients with ASD.

Preliminary results suggest that there may be a positive association between levels of SULT1A1, CD244, CD5, FLT3LG, and TNFSF10 and risk of ASD. However, much of the research up to now has not dealt with the relationship between these inflammatory factors and ASD. SULT1A1 is responsible for the sulfonation of xenobiotics and has been implicated in several cancers by activating carcinogens (45). During autoimmune neuroinflammation, SULT1A is highly expressed in astrocytes, hindering the anti-inflammatory activity of endogenous estrogens (46). CD244 is an immune regulation receptor presented in all NK cells, which can stimulate NK cell cytotoxicity and IFN-γ production by interacting with CD48 on neighboring lymphocytes (47, 48). It has previously been observed that children with ASD had significantly higher serum and plasma levels of CD5 than those of normal controls, which is positively correlated with Childhood Autism Rating Scale score (49, 50). As a pan T cell marker, CD5 is highly expressed in a variety of autoimmune diseases, and this MR study provides new evidence that elevated levels of circulating CD5 may directly promote the development of ASD. In premature infants following respiratory viral infections, hyperoxia-induced high FLT3LG expression can lead to expansion and activation of lung CD103+ dendritic cells. The FLT3LG level is positively correlated with the level of proinflammatory cytokines (51). In addition, chronic HIV-1 patients also displayed significantly high levels of FLT3LG expression (52). TNFSF10 a proapoptotic member of the tumor necrosis factor family, has been shown to be highly up-regulated in patients with inflammatory bowel disease (53) and neurodegenerative diseases (54). The previous findings suggest a positive outcome for our MR results, indicating the need for further research on the current topic.

The occurrence of ASD may increase the levels of MMP10 and CASP8 and decrease the levels of TNFSF11 and CCL19 in the results of reverse MR analyses, suggesting that they may act as downstream factors in ASD. A recent proteomics analysis on plasma inflammation-related protein changes found that MMP-10 expression was significantly up-regulated in the ASD group compared with healthy children (29). Mild cognitive impairment individuals with elevated cerebrospinal fluid levels of MMP-10 had a higher likelihood of progression to Alzheimer’s type dementia and faster cognitive decline (55). CASP8 is a protease with both pro-death and pro-survival functions by mediating extrinsic apoptosis and suppressing necroptosis. Postmortem analysis results showed that the apoptosis was increased in the prefrontal cortex, hippocampus, and cerebellum of the autistic brain, as characterized by significantly increased levels of cleaved CASP8 (56). TNFSF11 exerts essential roles in lymph node organogenesis, cellular immunity, and osteoclastogenesis. For example, TNFSF11 can signal the augmentation of IFN-γ secretion and inhibit apoptosis of human monocyte-derived dendritic cells (57, 58). CCL19 has shown significant potential in the regulation of adaptive immune responses by coordinating dendritic cell migration and increasing interactions between dendritic cells, T cells, and B cells in secondary lymphoid tissues (59, 60). Hence, ASD-induced decreases in peripheral blood CCL19 and TNFSF11 levels may further exacerbate immune system disorders.

This MR study employed a large sample size and instrumental variables obtained from the GWAS database, ensuring statistical robustness in estimating causal associations and enhancing the credibility of results. By addressing the bias introduced by confounding factors and reverse causality through MR analysis, this study provides stronger evidence for assessing the causal relationship between inflammatory factor levels and the risk of ASD compared to traditional observational studies. Nevertheless, several limitations of the present study should be considered. Firstly, the present MR study can only provide statistical evidence for the causal association between circulating inflammatory factors and ASD, and further research is needed to investigate the potential mechanisms involved. Secondly, although current sensitivity analyses did not reveal any significant pleiotropy between SNPs, the effect of pleiotropy on the MR results cannot be completely ruled out. Thirdly, this MR study utilized pooled data from the GWAS database and did not analyze stratified risk factors related to ASD duration, severity, treatment, and comorbidities. Finally, the genetic data were mainly collected from individuals of European descent, and it is uncertain whether these findings are applicable to individuals of other ancestries.

Overall, this study provide novel ideas that IL-7, SULT1A1, IL2Rβ, CD244, CD5, IL-2, FLT3LG, and TNFSF10 may be upstream factors in the pathogenesis of ASD, while levels of MMP10, CASP8, TNFSF11, and CCL19 may act as downstream factors in ASD. These inflammatory factors could potentially serve as biomarkers for early diagnosis and treatment of ASD.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and the institutional requirements.

JL: Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation, Formal Analysis, Data curation, Conceptualization. HD: Writing – review & editing, Writing – original draft, Visualization, Software, Investigation, Formal Analysis. WS: Writing – review & editing, Visualization, Software, Methodology, Investigation. MM: Writing – review & editing, Validation, Resources, Methodology. HZ: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Conceptualization.