Mastitis is a painful benign breast disease that can be categorized into lactational and non-lactational mastitis. Lactational mastitis (LM) is the most common inflammatory disease of the mammary gland in primiparous women, usually occurring 3-4 weeks postpartum. Global incidence ranges from 3% to 33%, affecting breastfeeding and fetal health (1, 2). Mastitis can also occur in non-breastfeeding women, and rarely in men. Non-lactational mastitis is refractory including periductal mastitis (PDM) and granulomatous lobular mastitis (GLM) (2).

LM happens mostly due to milk stasis or nipple damage, bacteria that colonize the skin invade breasts along the lymphatic and milk duct, and subsequent infections in stagnant lactiferous ducts progress with Staphylococcus aureus being the main pathogen (3). Generally, 5% to 9% of reproductive-aged women will have PDM which often influences the subareolar ducts. The cause of periductal mastitis is still unclear while mammary ductal ectasia, plasma cell infiltration and abscess formation have been implicated as basic pathogenesis (4). In addition, more than one study shows immune responses may play a role (5). Alternatively, GLM is a rare autoimmune inflammatory disorder prone to recur that can be clinically similar to breast cancer with the highest risk in parous women ordinarily within 5 years of parturition (6). The etiology of GLM remains ambiguous while Corynebacterium infection may be closely related to the pathogenesis of GLM (7). The most widely accepted theory indicates that specific triggers such as trauma, bacteria, or exosmotic milk induce autoimmune destruction, giving rise to leakage of ductal secretions into the breast tissue and inflammatory cell infiltration, resulting in a granulomatous response. In such cases, patients may benefit from immunosuppressive therapy (8).

While clinical manifestations and etiology of these disorders are with great heterogeneity, inflammation and immune response appear to be the commonalities in the occurrence and development of such diseases, and their causal relationship remains to be verified. Mendelian randomization (MR) analyses aim to investigate causal correlations between exposures and outcomes via genetic variation such as single nucleotide polymorphisms (SNPs) as instrumental variables (IVs), making them less susceptible to environmental confounders and reverse causation (9). MR uses genetic variants as proxies for exposures to assess causal relationships, minimizing confounding and reverse causation. Although MR has been widely used to study the association between inflammatory factors and immune system disorders (10, 11), evidence on the causality of inflammatory cytokines and immune cells with inflammatory disorders of the breast remains limited. It is unclear whether they play a causal role or are merely a consequence of shared risk factors. However, the causal role of these factors in mastitis has not been fully established. To identify circulating inflammatory cytokines and immune cells as potential risk factors for mastitis, this study conducted a comprehensive two-sample Mendelian randomization (TSMR) analysis which explored their causal connection, providing insights into the role of inflammatory cytokines and immune cells in pathogenesis and potential therapeutic interventions of mastitis.

In the two-sample MR analyses, we first conducted a thorough and comprehensive exploration of the causative links when circulating inflammatory cytokines and immune cells were seen as exposures separately and mastitis as the outcome, utilizing publicly accessible genetic data. And it was shown that CX3CL1, CD28- CD8dim %CD8dim and CD45 on CD33br HLA DR+ may suggestively be the upstream causes of mastitis, while heightened levels of CD39+ secreting Treg AC were connected to a reduced mastitis risk. Among immune cells, two risk factors were found to be related to the Treg panel (CD28- CD8dim %CD8dim) and Myeloid cells (CD45 on CD33br HLA DR+). Additionally, the presence of CD39+ secreting Treg AC from the Treg panel showed a negative association.

The mammary gland is a hormonally responsive exocrine gland. Immunoinflammatory response is a common phenomenon observed in individuals with mastitis (32). Also, it has been the subject of recent research providing compelling evidence of the crucial interplay between immunoinflammatory response with mastitis, including Liu, L., et al., noted that PDM patients exhibit a higher level of IFN- γ, and IL-12A compared to controls (5). These cytokines are secreted by T helper 1 cells aiding in eradicating foreign pathogens. Increased expression pattern of these cytokines suggests that immune responses may play a role in the pathogenesis of periductal mastitis. Similarly, the analysis of immune modulation by L. rhamnosus strains on mastitis identified two strains with significant anti-inflammatory potential, which can strongly induce IL-10 and weakly secrete pro-Th1 cytokine (IL-12 and IFN-γ). Therefore, these strains can be considered as a probiotic candidate for the management of infectious mastitis during lactation (33). After being exposed to lipopolysaccharide (LPS) in vitro experiments, untreated bovine mammary epithelial cells demonstrated a noteworthy reduction in the expression of CXCL8, IL1B, CCL20, and CXCL1 upon inflammatory response (34). Barreto, D.S., et al., also indicated autoimmune response is the most widely accepted etiology of GLM (35), and treatment with immunosuppression (steroids combined with Methotrexate) is an effective breast-conserving option. T lymphocyte subsets play a crucial role in maintaining immune homeostasis, with Treg cells exerting an immunosuppressive function. Likewise, Ucaryilmaz, H., et al., confirmed that there was an intrinsic defect of Tregs in patients with idiopathic granulomatous mastitis through flow cytometry. The expression of Foxp3 in Treg cells, and the frequency of non-suppressive Tregs, were found to be significantly lower in patients with idiopathic granulomatous mastitis when compared to healthy controls (36). However, different immunophenotypes in the Treg panel exhibited distinct roles in our results. Specifically, CD28- CD8dim %CD8dim was involved with increased risk while CD39+ secreting Treg AC demonstrated an opposing effect. Increasing evidence suggests that different subtypes of Tregs appear to have distinct roles in regulating the immune system. CD28 is a key co-stimulatory receptor typically expressed on most CD4+ T cells, binding CD80 and CD86 on antigen-presenting cells to promote T-cell activation. Lack of CD28 expression might impair their functional effectiveness or alter their activation state, leading to increased inflammation or reduced regulatory function. CD4+ CD28- T cells are associated with autoimmune diseases like rheumatic arthritis. Huang Y et al. also discovered that the absence of CD28 expression could identify T cell subsets exhibiting terminally differentiated and senescent characteristics, which were accompanied by the deterioration of the disease (37). Decreased CD28 expression can be an indicator of T-cell exhaustion. While CD39 is an ectoenzyme that converts ATP and ADP to AMP, it helps restrain excessive inflammatory responses (38). The immunoregulatory functions contribute to maintaining immune homeostasis and mitigating tissue damage. Chen C et al. demonstrated the immunosuppressive effect of CD39+ Tregs on acute lung injury via autophagy and the ERK/FOS Pathway (39). Our findings indicate a potential role of Tregs in the pathogenesis of idiopathic granulomatous mastitis and suggest a possible immunological basis for the development of this disease. These insights may inform future therapeutic approaches by targeting Treg cells and addressing the underlying immune dysregulation, which could generate novel therapies in the treatment and management of mastitis. Further investigation into the role of Tregs in this condition is warranted in order to develop more effective diagnostic and treatment strategies for patients with granulomatous mastitis. According to Zhao, J., et al., the expression of Th1 and Th17 cytokines was found to be significantly higher in the breast tissues of patients suffering from nonpuerperal mastitis, whereas the levels of Th2 cytokine and Treg cytokines such as IL-10 and transforming growth factor β were observed to be lower (40). In models of both sterile inflammation and bacterial infection, a diverse recruitment of myeloid cells was observed. The number of CD45+ leukocytes within the mammary gland significantly increased during inflammation and infection, which was in agreement with our studies. Among sterile inflammation models, increased numbers of neutrophils, Ly6C low monocytes and CD11b+F4/80+ macrophages were detected. While numbers of DCs and CD206+ macrophages increased among infection models, which was not seen during the sterile inflammation challenge. Our research also found that CD45 on CD33br HLA DR+ from Myeloid cells was linked to an increased risk of mastitis causally. These findings can provide valuable insights into the different immune responses of the mammary gland during inflammation and infection (41). Through flow cytometric analysis of milk, nonspecific mastitis revealed the highest percentage of CD4+ T lymphocytes, the percentage of CD8+ T lymphocytes was found to be highest in infectious bacterial mastitis (42). Our results showed CD28- CD8dim %CD8dim immunophenotype was significantly linked to the risk of mastitis (p = 0.0006). In the light of these observations, limited research on the relationship between immunoinflammatory response and mastitis may present a new perspective for early detection, prevention and monitoring. It is therefore necessary to conduct further research to explore the specific mechanism of these inflammatory cytokines and immune cells in mastitis.

To the best of our knowledge, this is the first Mendelian randomization research to evaluate the causal relationship between 91 inflammatory cytokines and 731 immune cells with mastitis, which provides genetic evidence emphasizing the strong causal relevance between immunoinflammatory response and mastitis. Nevertheless, it is crucial to acknowledge the restrictions of our study. Firstly, the obtained GWAS summary data predominantly consisted of patients of European descent, potentially generating biased estimates so it should be taken with caution if the conclusions would be applied in diverse ethnic groups. Future research should include diverse populations to determine whether these findings are consistent across different ethnic groups, ultimately enhancing the generalizability of the results and providing a more comprehensive understanding of genetic associations. Secondly, subgroup analyses aimed at attaining more precise correlations were not feasible due to the lack of specific demographic information and clinical records of study patients. Finally, owing to the limitations of the MR analysis, the second and third assumptions could not be properly examined and possible violations against these assumptions may exist. Colocalization analyses could be performed to further explain potential causal variation.

To sum up, our TSMR findings suggest that circulating inflammatory cytokines and immune cells might causally influence the risk of mastitis. It can be concluded an upregulation of CX3CL1, CD28- CD8dim %CD8dim and CD45 on CD33br HLA DR+ may contribute to an increased risk of mastitis, while raised levels of CD39+ secreting Treg AC are associated with a lower risk. Furthermore, these biomarkers may act as the initiatives in the onset and progression of mastitis, which could potentially serve as a viable approach for earlier diagnosis and more effective treatment options. They can also serve as candidate molecules for future mechanism exploration.