Brucella is a Gram-negative bacterium responsible for brucellosis, a prevalent zoonotic disease globally (About et al., 2023). Conservative estimates indicate that the annual incidence rate of brucellosis is approximately 2.1 million cases with reported instances of human and animal infections in over 170 countries (Laine et al., 2023). In China, the average annual incidence rate of brucellosis is 3 cases per 100,000 people, reflecting an average annual increase of 31.8% (Tao et al., 2021). From 2004 to 2021, the incidence of human brucellosis in China has shown a sustained upward trend, with the predominant transmission route being direct contact with infected livestock, such as sheep, goats and cattle (Tian et al., 2024). Humans can contract Brucella through the consumption of raw dairy products and meat contaminated with this pathogen, or through direct contact with infected animal, particularly via broken mucosal wounds (Moreno, 2014). Brucella has the capacity to infect any organ or system within the human body, initially manifesting as mild flu-like symptoms, including fever, excessive sweating, discomfort, myalgia, arthralgia, loss of appetite, and weight loss (Cerqueira et al., 2018). If left untreated, the condition can progress from the acute to the chronic stage, leading to multiple complications across various systems. Among these, bone and joint involvement is the most prevalent complication, with a reported prevalence rate ranging from approximately 2–77%, typically presenting as spondylitis, sacroiliitis, and peripheral arthritis. Additionally, hepatosplenomegaly is observed in about 50% of patients with Brucella melitensis. The disease is characterized by a prolonged course and frequent recurrences, significantly impacting patients’ quality of life (Jin et al., 2023; Willems et al., 2022).

Myeloid-Derived Suppressor Cells (MDSCs) represent a highly heterogeneous subpopulation of myeloid cells characterized by their potent inhibitory effects on immune responses, particularly those mediated by natural killer (NK) cells, T cells, and B cells. Research has demonstrated the presence of MDSCs in the peripheral blood of patients suffering from various diseases (Cassetta et al., 2020). In humans, MDSCs are typically identified by the markers CD11b⁺CD33⁺HLA-DR⁻. In murine models, the MDSC phenotype is generally defined by the expression of CD11b⁺Gr1⁺ of MDSCs (Veglia et al., 2021). The immunosuppressive function of MDSCs on T-cell functions is predominantly mediated by inducible nitric oxide synthase (iNOS) and arginase 1(Arg1) (Aintablian et al., 2023; Draghiciu et al., 2015). In the hypoxic tumor microenvironment, the expression levels of Arg1 and iNOS are significantly upregulated (Kumar et al., 2016).

Toll-like receptor 4 (TLR4) is a type of Pattern Recognition Receptor (PRR) that rapidly recognizes pathogen-associated molecular patterns (PAMPs) during innate immune responses and plays a crucial role in the pathogenesis of both acute and chronic inflammation (Zhang et al., 2021; Zamyatina and Heine, 2020). In the context of Brucella infection, TLRs interact with MyD88 to initiate cellular signal transduction, ultimately activating the transcription factor NF-κB and regulating the expression of various inflammation-related genes (Heine and Zamyatina, 2022). The number of MDSCs in the peripheral blood of patients with chronic brucellosis is increased, and levels of Th2 cytokines, such as IL-6 and IL-10, as well as Arg1, are significantly elevated (Hou et al., 2024). This study aims to further investigate the association between the presence of TLR4⁺MDSCs in acute and chronic Brucella infections and their associated inflammatory changes.

Programmed death ligand 1 (PD-L1) is an inhibitory ligand that binds to programmed death 1(PD-1), activating the co-inhibitory pathway of T cells to maintain self-tolerance, thereby functioning as an immune checkpoint under physiological conditions. Immune checkpoints are a series of regulatory mechanisms in the immune system, typically employed to maintain the balance of the immune response. However, under certain pathological conditions, such as tumor development, immune checkpoints can inhibit the immune system’s attack on tumor cells, enabling these cells to evade immune surveillance. In the MDSCs of tumor-bearing mice, PD-L1 is highly expressed within the tumor microenvironment, promoting tumor progression (Lu et al., 2016). Additionally, studies have indicated that the gene expression of PD-L1 is upregulated in the whole blood of patients with brucellosis (Pellegrini et al., 2025). This study aims to investigate the presence of PD-L1⁺MDSC in the progression of acute and chronic brucellosis and to elucidate its role in disease progression.

The current literatures on the research of the expression of TLR4 and PD-1 on MDSCs in brucellosis patients at different stages are rare or unreported. In order to further clarify the role of potential biomarkers for assessing the severity and progression of brucellosis, we explored the TLR4⁺MDSCs and PD-L1⁺MDSCs in patients and mouse model with brucellosis. Establishing a robust theoretical foundation for the subsequent development of clinical treatment strategies.

Brucellosis is a zoonotic disease. The symptoms of this infection are non-specific and persistent, leading to chronic infection within the body. It is commonly associated with the consumption of infected dairy products and has resulted in significant economic losses and personal injuries worldwide (Xie et al., 2025). Due to the non-specific nature of its clinical symptoms, brucellosis is often misdiagnosed, which can result in a progression to the chronic stage. More importantly, if appropriate treatment is not administered during the acute stage, various complications may arise. All brucellosis patients enrolled in this study were confirmed to be infected with Brucella melitensis, which is the predominant pathogenic species of brucellosis in the Xinjiang region (Zhang et al., 2015; Yang et al., 2024). The diagnosis of brucellosis was based on typical clinical features and positive Rose Bengal Precipitation Test (RBPT) and/or Serum tube Agglutination Test (SAT) (≥1:100) And/or isolate Brucella from blood, other tissues or body fluids, and combine it with epidemiological history.

Among 154 patients diagnosed with brucellosis, 21.43% exhibited abnormalities in the liver and spleen, 18.83% presented with lumbar spine infections, and 17.63% experienced knee joint infections. These proportions are consistent with most previous reports (Zheng et al., 2019). Feng et al.’s study indicates that among patients with brucellosis complicated by additional conditions, 41.46% exhibit lumbar spine lesions, while 7.01% present with splenomegaly and lymphadenopathy (Qiangsheng et al., 2023). The infection poses a significant threat to the liver, spleen, and skeletal system of affected patients. However, to date, the pathogenesis of brucellosis remains inadequately understood.

MDSCs exhibit potent immunosuppressive activity by inhibiting effective T-cell immune responses, thereby facilitating pathogen persistence and the development of chronic infections (Arshad et al., 2024). MDSCs also serve as critical drivers of tumor progression, exerting significant impacts on tumor angiogenesis, metastasis, and multidrug resistance (MDR)(Lasser et al., 2024; Dhar et al., 2023). In the context of gastric cancer research, MDSCs have been identified as novel targets for early diagnosis, intervention, and precision treatment of this malignancy (Li et al., 2024). High-virulence Klebsiella pneumoniae (hvKp) infection drives MDSC infiltration into lung tissues, where MDSCs inhibit T-cell proliferation and induce apoptosis via indoleamine 2,3-dioxygenase 1 (IDO1)-mediated tryptophan metabolism, leading to lymphopenia and persistent infection (Xu et al., 2025). Furthermore, investigations have demonstrated that MDSCs also exert immunomodulatory effects in other infectious diseases, such as infections caused by Staphylococcus aureus, hepatitis B virus, and Candida albicans (Sanchez-Pino et al., 2021; Bruger et al., 2019). MDSCs promote immunosuppression through the upregulation of immunosuppressive mediators, including Arg1 and iNOS, which leads to antigen-presenting cell (APC) dysfunction, reduced activation of T effector cells (Teff), and enhanced recruitment, survival, and functionality of regulatory T cells (Tregs)(Weber et al., 2021; Saleh et al., 2020).

In this study, we analyzed peripheral blood MDSCs in healthy controls and patients with acute and chronic brucellosis using flow cytometry. We evaluated the expression of TLR4 and PD-L1 on MDSCs. The results indicated that both the percentage and absolute number of MDSCs in patients during the chronic phase were significantly higher than those in the acute phase, suggesting that MDSCs may play a more critical role in chronic brucellosis. Furthermore, the numbers of TLR4⁺MDSCs and PD-L1⁺MDSCs in chronic phase patients were also elevated compared to those in acute phase patients. TLR4, a pattern recognition receptor, is pivotal in innate immune responses, as its activation can promote inflammatory responses and enhance the activation of immune cells (Zhang et al., 2020; Strobl et al., 2022). However, persistent activation of TLR4 may lead to immune system fatigue, thus facilitating the accumulation of MDSCs and resulting in immunosuppression (Sun et al., 2018; Shime et al., 2017). PD-L1, an essential immune checkpoint molecule, exhibits high expression on MDSCs, potentially augmenting their immunosuppressive function and aiding Brucella in evading immune surveillance. Therefore, the elevated expression of TLR4 and PD-L1 on MDSCs may represent a significant mechanism underlying the immunosuppressive status observed in patients during the chronic phase.

MDSCs exert a role in liver injury: they can either exacerbate liver damage and drive fibrogenesis via orchestrating inflammatory responses and immune suppression, or confer hepatoprotection by constraining excessive inflammatory cascades in specific pathological settings (Lu et al., 2025; Luo et al., 2022). ALT and AST are widely utilized in clinical diagnostics to evaluate liver health and tissue damage (Choi et al., 2024). The correlation analysis between the levels of ALT and AST and the cell counts of TLR4⁺MDSCs and PD-L1⁺MDSCs indicates that following Brucella infection, an increase in the numbers of TLR4⁺MDSCs and PD-L1⁺MDSCs corresponds with elevated ALT and AST levels, leading to liver damage. This suggests that these subsets of MDSCs may play a critical role in liver injury associated with Brucella infection. Furthermore, this study reveals that the levels of inflammatory factors, such as Arg1 and iNOS, are directly proportional to the fluctuations in the numbers of TLR4⁺MDSCs and PD-L1⁺MDSCs. During the course of infection, these cell subsets are rapidly activated and express relevant cytokine, thereby exacerbating Brucella infection and liver injury.

Through the determination of organ bacterial load, as well as HE and IHC staining in a Brucella mouse model, we systematically evaluated the pathological changes in the liver, lung, and spleen tissues, along with the colonization of Brucella in the infected mice. In the chronic stage of the infection, the inflammatory response in the organs became more pronounced, indicating severe organ involvement. Results from the mIF experiment revealed a significant increase in MDSC cells within the liver, spleen, and lung tissues of the infected mice. Furthermore, with the progression of the infection, both the proliferation and activation levels of MDSCs gradually increased. This finding suggests that MDSCs may play a crucial role in the progression of Brucella infection, with their heightened activation closely associated with the chronicity of the disease. The flow cytometry results of mouse cells indicated that the frequencies of TLR4⁺MDSCs and PD-L1⁺MDSCs during the chronic phase were significantly higher than those observed in both the acute phase and the healthy control group. Although this suggests that TLR4⁺MDSCs and PD-L1⁺MDSCs may represent key cellular subsets in chronic brucellosis infection, playing a crucial role in regulating immune responses and promoting the persistence of the infection, this study has several limitations. It should be noted that the increased frequency of TLR4⁺ and PD-L1⁺ MDSCs along with disease chronicity may not be solely driven by disease progression per se, but also modulated by multiple confounding factors including systemic inflammatory status, tissue bacterial burden, host immune baseline heterogeneity, and potentially therapeutic interventions.

In summary, this research underscore the significant roles of the two functional subsets, TLR4⁺and PD-L1⁺MDSCs, in chronic brucellosis, thereby offering a novel perspective on the disease’s chronicity. These subsets may serve as potential biomarkers for evaluating the severity and progression of the disease. Furthermore, targeting the immunosuppressive properties of MDSC subsets may dramatically enhance the host’s antimicrobial immune responses, presenting a novel therapeutic avenue for brucellosis treatment. Future research can further explore the specific mechanism of action of MDSCs in Brucella immune escape and chronic infection.