The immune system serves as the body's primary defense mechanism against pathogens, malignancies, and foreign substances [1]. Dysregulation of the immune system can lead to a wide range of diseases. However, cyclophosphamide (CTX), a widely used chemotherapeutic agent, is commonly employed in the treatment of various types of malignancies [2]. Despite its therapeutic efficacy, CTX can disrupt immune homeostasis and induce immunosuppression [3]. One of its main characteristics is the suppression of immune cell proliferation [4], which significantly increases the risk of infections, weakens the body's ability to combat disease, and delays treatment progress. Furthermore, many immunomodulatory drugs are not suitable for long-term or prophylactic administration. For example, treatment with levamisole hydrochloride may cause adverse effects such as leukopenia and organ damage [5]. Therefore, the development of strategies to enhance immune function or of adjunctive therapies that preserve immune integrity during cancer treatment holds significant potential for improving patients' quality of life and clinical outcomes.

Alterations in the microbiota composition, or dysbiosis, can lead to immune dysfunction and subsequently cause inflammation [6]. The intricate mechanisms of interaction between the immune system and the gut microbiota enable the gut microbiome to play a significant role in regulating systemic immunity, which is crucial for preventing inflammation and maintaining gut homeostasis [7,8]. Modulating the gut microbiome is considered a promising approach to support normal immune function. Gut commensal bacteria may regulate immune responses through several pathways. For instance, short-chain fatty acids (SCFAs) produced by bacterial fermentation can interact with G protein-coupled receptor 43 (GPR43) and influence T helper 1 (Th1) cells, or inhibit histone deacetylases (HDACs), thereby exerting an immunotherapeutic effect. Additionally, SCFAs can suppress Toll-like receptor signaling, inhibiting the NF-κB-driven inflammatory cascade [[9], [10], [11]].

Akkermansia muciniphila is a gut symbiont abundantly present in the human intestine, playing a critical role in maintaining intestinal barrier integrity and modulating immune responses [12]. A. muciniphila interacts with host immune cells through various mechanisms, including the production of bioactive metabolites and activation of specific signaling pathways. These interactions facilitate the regulation of inflammation, T cell differentiation, and antibody production, underscoring A. muciniphila's association with beneficial effects in metabolic cancer immunotherapy. Inactivated A. muciniphila has garnered attention for its enhanced safety profile and potential to retain immunomodulatory properties. Notably, pasteurized A. muciniphila is more effective than its live counterpart at improving gut health by enhancing intestinal integrity and sustaining immune homeostasis [13]. Similarly, treatment with pasteurized forms has been shown to more effectively upregulate tight junctions and genes related to immune response modulation in the Caco-2 cell line. Recent studies have revealed that heat-inactivated A. muciniphila can drive M2 macrophage polarization by upregulating IL-4 protein levels at inflammatory sites [14]. Post-pasteurization, A. muciniphila retains the activity of its outer membrane protein Amuc_1100, a key TLR2 agonist crucial for immune modulation, positioning inactivated A. muciniphila as a safer candidate for tumor therapy compared to live probiotics [15]. Moreover, pasteurized A. muciniphila enhances the proliferation of beneficial gut bacteria, increases SCFA production, and inhibits intestinal inflammation [16], while also ameliorating immune dysfunction via activation of the TLR2/NF-κB signaling pathway [17]. Therefore, compared to live bacteria, inactivated A. muciniphila reduces concerns related to potential pathogenicity, offering greater stability during storage and gastrointestinal transit, thereby emerging as a promising therapeutic alternative. Despite extensive research on the immunomodulatory roles of live A. muciniphila, the efficacy of inactivated A. muciniphila in mitigating immune suppression remains less understood.

We previously isolated an Akkermansia muciniphila strain (designated as Akkermansia muciniphila AKK PROBIO) from healthy adult intestine. Whole-genome sequencing and toxicological assessments confirmed its safety [18]. Animal studies demonstrated its efficacy in preventing acute gout and alleviating colorectal cancer [19,20]. This study aims to investigate the efficacy of inactivated A. muciniphila AKK PROBIO in alleviating CTX-induced immunosuppression and restoring immune function. We examined whether supplementation mitigates CTX-induced immune suppression and associated toxicities through modulation of gut microbiota composition, enhancement of intestinal barrier integrity, and restoration of systemic immune homeostasis. Comprehensive assessments included hematological profiling, serum immunoglobulins (IgA/IgG/IgM) for humoral immunity, and cytokine analysis (e.g., ILs, IFN-γ, TNF-α) to characterize inflammatory responses. Structural alterations in spleen/thymus were evaluated histologically, while in vivo functional assays (ear swelling test, carbon clearance) assessed cell-mediated immunity and phagocytic activity. To elucidate underlying mechanisms, we analyzed NF-κB signaling pathway activation and gut microbiota-associated changes, including short-chain fatty acid (SCFA) production, to determine the role of the gut-immune axis in mediating beneficial effects.