Innate immunity represents the first line of host defense and is characterized by rapid, non-specific responses mediated by innate immune cells, such as macrophages and DCs, through PRRs that sense conserved microbial structures (Li and Wu, 2021). Within this context, accumulating evidence supports the hypothesis that Lactobacilli-derived peptidoglycan modulates innate immune signaling pathways, particularly by regulating cytokine production. In this sense, accumulating evidence supports the concept that Lactobacillus-derived peptidoglycan exerts context-dependent regulation of IL-12 production, rather than a uniform pro- or anti-inflammatory effect. Several studies have demonstrated that purified peptidoglycan from Lactobacillus species suppresses macrophage IL-12 production predominantly through TLR2- and NOD2-dependent mechanisms. In particular, Shida et al. (2009) reported that peptidoglycan from Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus johnsonii significantly inhibited IL-12 secretion by macrophages by downregulating IL-12p40 expression, thereby limiting the formation of bioactive IL-12p70. This inhibitory effect was mediated mainly through TLR2 signaling, with additional contribution from NOD2 recognition of peptidoglycan-derived MDP.

Although peptidoglycan-mediated inhibition was largely dependent on TLR2 signaling, reduced IL-12 production was also observed in TLR2-deficient macrophages, pointing to additional TLR2-independent mechanisms. Watanabe et al. (2004) showed that MDP inhibits IL-12 production via recognition by the intracellular receptor NOD2. In the same way, Shida et al. (2009) noted that L18-MDP, a derivative of MDP, is also likely to be involved in IL-12 suppression. These findings indicate that peptidoglycan inhibits IL-12 production both in its native state through TLR2 and in its processed state (MDP) through NOD2.

In contrast, IL-12 induction reflects distinct experimental conditions, including stimulation with whole bacteria or intracellular digestion-resistant Lactobacillus cell wall structures rather than purified peptidoglycan alone (Watanabe et al., 2004; Shida et al., 2009). Whole bacterial cells can engage additional PRRs beyond TLR2, including non-TLR receptors that recognize higher-order cell wall architecture, resulting in enhanced IL-12p35 transcription and apparent Th1 polarization. Importantly, peptidoglycan alone induced markedly lower IL-12 production compared with intact bacteria, even in wild-type macrophages, and this effect was further diminished in TLR2-deficient cells.

Studies investigating L. casei–derived peptidoglycan have reported both TLR2/TLR9-dependent and TLR2/TLR9-independent immunomodulatory effects, which may appear inconsistent at first glance (Takeda and Akira, 2005). However, these differences largely reflect variations in experimental context rather than conflicting biological outcomes. TLR2- and TLR9-dependent signaling has been predominantly observed in studies employing intact L. casei cells or complex cell wall preparations, where multiple pattern-recognition receptors can be engaged simultaneously at the cell surface or within endosomal compartments. Peptidoglycan, lipoproteins, and Gram-positive bacterial genomic DNA of Lactobacilli are reported to activate TLR2 and TLR9, resulting in the release of proinflammatory cytokines (Takeda and Akira, 2005). In contrast, other studies determined that macrophages that were TLR2- or TLR9-deficient or wild-type continue to induce IL-12 when exposed to certain Lactobacillus species in the L. casei group (Shida et al., 2006; Ichikawa et al., 2007). Unlike peptidoglycan alone, peptidoglycan stimulated low IL-12 in wild-type and even lower in TLR2-deficient macrophages (Shida et al., 2009). These results indicate that TLR2 is involved in the impaired IL-12 production caused by peptidoglycan or intracellular digestion-resistant Lactobacillus strains. Conversely, yet-to-be-characterized receptors aside from TLRs are likely to recognize the three-dimensional fold of the intracellular digestion-resistant Lactobacillus bacterial cell wall and induce augmented IL-12 production. Peptidoglycan-derived fragments are recognized by the NOD1 and NOD2 proteins, which are members of the NLR family of PRRs (Ting et al., 2008; Kawai and Akira, 2010). Both receptors are well-characterized NLRs, with NOD1 recognizing meso-diaminopimelic acid and NOD2 recognizing MDP (Girardin et al., 2003a,b). Though both receptors are highly expressed in immune cells, they are expressed differently in the intestinal epithelium; NOD1 is uniformly expressed in the epithelium, while NOD2 is mainly expressed in Paneth cells in the small intestine (Lala et al., 2003). The PepT1 di/tripeptide transporter, which is highly expressed in the small intestine, is required for the transport of NOD1 and NOD2-activating ligands, such as MDP and tripeptide mesoDAP, into epithelial cells (Dalmasso et al., 2010). NOD1 and NOD2 can also recognize peptidoglycan fragments that have been processed within the phagosome or phagolysosome of antigen-presenting cells (APCs), though translocation transporters for them into the cytoplasm remain to be identified (Philpott and Girardin, 2010).

Fernandez et al. (2011) indicated that peptidoglycan from Lactobacillus may augment anti-inflammatory effects through recognition by NOD2. Treatment with Ls33 was found to have protective effects and was linked to enhanced IL-10 production in the colon, a characteristic that is lacking in NOD2-deficient mice. Since NOD2 is a prominent member of the NLR family capable of recognizing peptidoglycan, Fernandez et al. (22) evaluated whether it was involved in the peptidoglycan anti-inflammatory activity of Ls33. A single systemic injection of Ls33 peptidoglycan exacerbated colitis in rats by reducing pro-inflammatory cytokines [TNF-α, Interleukin 1 (IL-1)-1β, and Interleukin 6 (IL-6)] with augmented expression of genes involved in the Indoleamine 2,3-dioxygenase (IDO) immunosuppressive pathway in the colon. Although a reduction in Il10 gene expression was observed in mice treated with L. salivarius Ls33–derived peptidoglycan, a localized increase in IL-10 protein levels within the intestinal tissue was consistently associated with the protective effect against colitis (Fernandez et al., 2011). This apparent discrepancy likely reflects the rapid and transient kinetics of Il10 transcription, coupled with post-transcriptional regulation and localized cytokine secretion at sites of inflammation. Importantly, the enrichment of IL-10 protein in the colonic microenvironment underscores its functional relevance in mediating immune tolerance and tissue protection. Collectively, these findings indicate that probiotic-derived peptidoglycan confers anti-inflammatory effects primarily through localized, IL-10–dependent immunoregulation rather than sustained systemic cytokine expression, highlighting the importance of spatial cytokine dynamics in probiotic-mediated immune modulation.

Treg cells, especially CD4 + CD25 + FoxP3 + subsets, are essential for immune response modulation and tolerance (Coombes et al., 2005). Fernandez et al. (2011) showed that Ls33 peptidoglycan treatment was strongly associated with the increase of CD4 + FoxP3 + Treg cells and CD103 + DCs in mesenteric lymph nodes. Enhanced Ifng and Ido expression and CD11c + DC enrichment in mesenteric lymph nodes (MLNs) were also found. Although IFN-γ is generally associated with pro-inflammatory Th1 responses, increased production in trinitrobenzene sulphonic acid (TNBS)-induced colitis has also been associated with anti-inflammatory activity. In addition, IFN-γ is a major inducer of the IDO-mediated immunosuppressive pathway (Gurtner et al., 2003). Recent research defines the role of the IDO pathway in CD103 + intestinal DCs as the selective proliferation of CD4 + FoxP3 + Tregs (Gurtner et al., 2003). Together, these findings indicate that Ls33 peptidoglycan induces a tolerogenic immune response.

Shida et al. (2009) have already described that peptidoglycan from certain Lactobacillus species can suppress macrophage production of IL-12, in favor of the hypothesis that Lactobacilli-derived peptidoglycan has functional anti-inflammatory activity. In addition, Watanabe et al. (2004) have shown that MDP activation of NOD2 can suppress TLR2-mediated signaling and Th1 responses. In another study, Watanabe et al. (2008) observed that synthetic MDP cured colitis in mice in a NOD2-dependent fashion. Their finding is in agreement with the hypothesis that NOD2 signaling is the cause of the resolution of inflammation and that NOD2 mutations should worsen IBD by enhancing TLR2-mediated cytokine responses. Interestingly, although it established the binding of NOD2 by Ls33 and NCFM peptidoglycans, as well as by M-tri-N, alone, only Ls33 peptidoglycan had protective activity (Watanabe et al., 2004). New evidence indicates that the transport of NOD2 ligands following the internalization of bacteria and processing in phagolysosomes is facilitated by specific transporters like PEPT1 and PEPT2 (Charrière et al., 2010). It is therefore possible that only the tripeptide M-tri-Lys, a part of Ls33 peptidoglycan, is efficiently transported to the cytosol for recognition by NOD2. While TLR2 activation in peptidoglycan recognition has been disputed, evidence now indicates that peptidoglycan is translocated in a complex with peptidoglycan recognition protein PGLYRP-3 and subsequently activates TLR2-induced phagocytosis (Bu et al., 2010). Phagosome maturation is controlled by TLR through adaptor Myeloid Differentiation Primary Response 88 (MYD88) (Blander, 2007). This can account for the need for MyD88 in protective Ls33 peptidoglycan activity but NOD2 signaling dependency in M-tri-Lys action. The findings indicate that Ls33 exhibits greater peptidoglycan turnover compared to NCFM, which can allow the accumulation and release of M-tri-Lys, hence explaining the greater protective efficacy of Ls33 peptidoglycan (Watanabe et al., 2004). Mechanistically, engagement of TLR2 by Lactobacillus-derived peptidoglycan initiates MyD88-dependent signaling that not only regulates cytokine transcription but also promotes cytoskeletal rearrangement and phagocytic uptake of bacterial material (Blander, 2007). Following internalization, peptidoglycan is enzymatically processed within phagolysosomes, leading to the release of defined muropeptides, including M-tri-Lys, which subsequently translocate into the cytosol (Watanabe et al., 2004). These intracellular peptidoglycan fragments are then recognized by NOD2, triggering RIP2-dependent signaling pathways that modulate NF-κB and MAPK activation. Importantly, NOD2 signaling acts as a regulatory checkpoint, attenuating excessive TLR2-driven inflammatory responses while promoting balanced cytokine production, including IL-10 induction.

Huang et al. (2020) confirmed that intact peptidoglycan from L. rhamnosus MLGA enhances β-defensin production in a dose-responsive manner in chicken embryo jejunum, ileum, and cecum, and in peripheral blood mononuclear cells (PBMCs), splenocytes, thymocytes, and hepatocytes. Notably, peptidoglycan hydrolysates prepared by lysozyme digestion also stimulated avian β-defensin 9 (AvBD9) induction by PBMCs. Additionally, L. rhamnosus MLGA peptidoglycan treatment remarkably enhanced antibacterial activity in PBMC and splenocyte lysates in a dose-responsive manner (Huang et al., 2020). These results indicate that peptidoglycan from certain probiotic bacteria plays a role in host immune modulation and antimicrobial defense through the induction of defensin expression in immune cells and gut tissue. While peptidoglycan fragments also increased AvBD9 expression in PBMCs, their impact was less than the impact of intact peptidoglycan from L. rhamnosus MLGA (Huang et al., 2020). In addition, splenocytes also had decreased AvBD9 induction to peptidoglycan hydrolysates, and this could be due to varying levels of PRR expression among immune cell types.

While NOD1 is a cytosolic PRR that senses meso-diaminopimelic acid–containing peptidoglycan motifs predominantly associated with Gram-negative bacteria, NOD2 is the principal intracellular sensor relevant to Gram-positive Lactobacillus species, including L. rhamnosus MLGA. Huang et al. (2020) demonstrated that peptidoglycan derived from L. rhamnosus MLGA enhances innate immune defense by inducing AvBD9 expression in intestinal tissues and peripheral immune cells. This response was accompanied by increased antimicrobial activity, indicating that probiotic-derived peptidoglycan can potentiate host defense mechanisms beyond cytokine modulation (Huang et al., 2020). These findings highlight NOD1 as a functional mediator of probiotic-induced antimicrobial immunity and reinforce the broader role of peptidoglycan fragments in strengthening mucosal and systemic innate immune responses.

Tejada-Simon and Pestka (1999) showed that L. infantis peptidoglycan was able to stimulate the RAW 264.7 macrophage cell line to produce TNF-α and nitric oxide (NO) at high levels in vitro. Consistent with these results, Wang L. S. et al. (2001) showed that the fluorescence intensity of IL-1, IL-6, and TNF-α, and NO content in nude mice peritoneal macrophages were significantly increased following intraperitoneal injection of B. bifidum peptidoglycan. These findings show that B. bifidum peptidoglycan can efficiently stimulate macrophages to produce large amounts of cytotoxic effector molecules.

Мokrozub et al. (2015) findings showed that the Lactobacillus delbrueckii subsp. bulgaricus IMV B-7281 strain with the more elastic cell wall was processed more efficiently by macrophages. The strain was more functionally active, secreted NO, and could accumulate reactive oxygen species than the stiffer cell wall-containing strains L. casei IMV B-7280 and L. acidophilus IMV B-7279 (Мokrozub et al., 2015). Bacterial substances produced as a consequence of digestion by macrophages seem to be of vital importance in triggering the oxygen-dependent bacteriolytic activity of macrophages and the production of NO against Lactobacilli. Interestingly, induction of production of IL-12 was higher in macrophages treated with L. casei IMV B-7280 or L. acidophilus IMV B-7279, having stronger cell walls compared to L. delbrueckii subsp. bulgaricus IMV B-7281 (Мokrozub et al., 2015). Additionally, Мokrozub et al. (2015) found L. casei IMV B-7280 was more effective as an inducer of IFN-γ production. This indicates that interaction between Lactobacilli cell wall constituents and TLR- and/or NOD receptors is the key to inducing cytokine production and activating macrophages. These findings provide proof of the correlation between the plasticity of cell walls in Bifidobacteria and LAB and their ability to trigger the macrophage component of the immune response.

Adaptive immunity represents the antigen-specific arm of the immune system, characterized by immunological memory and mediated primarily by T and B lymphocytes, which coordinate cellular and humoral immune responses (Chi et al., 2024). Adaptive immune modulation by probiotics-derived peptidoglycan occurs predominantly through APCs rather than through direct stimulation of lymphocytes. Intracellular sensing of peptidoglycan-derived muramyl dipeptide by NOD2, and to a lesser extent NOD1, requires RIP2 and cooperates with TLR2- and TLR4-mediated signaling under antigen-presenting conditions. While NOD1 recognizes meso-diaminopimelic acid–containing peptidoglycan fragments typically associated with Gram-negative bacteria, NOD2 is the principal intracellular sensor relevant to Gram-positive Lactobacillus-derived peptidoglycan. Within this framework, Kobayashi et al. (2005) created Nod2 knockout mice and observed that the mice had a decreased level of IgG1 production following intraperitoneal immunization with human serum albumin and MDP (Kobayashi et al., 2005). This deficiency was not because of an intrinsic or developmental impairment of the adaptive immune response, since Nod2-deficient mice developed a normal response to TLR7 ligands (Kobayashi et al., 2005). Elaborating on these observations, Magalhaes et al. (2008) showed that activation of NOD2 alone evokes a Th2-type response by inducing Th2-inducing cytokines like MCP-1. NOD2 was also found to play a critical role in the induction of Th1 responses following concurrent stimulation of TLR and NOD2, such as would happen in the scenario of infection (Magalhaes et al., 2008). Disruption of NOD2 or RIP2 signaling impairs this synergistic interaction with TLRs, resulting in defective APC activation and altered cytokine production (Magalhaes et al., 2008). This loss of synergy, rather than isolated Nod2deficiency, explains the inability of such mice to mount effective Th1 and Th17 responses following microbial or antigenic stimulation.

Through APC-mediated cytokine modulation, Lactobacillus-derived peptidoglycan influences T-helper cell polarization (Fritz et al., 2007). Suppression of IL-12 and IL-23, coupled with enhanced IL-10 production, limits excessive Th1 and Th17 differentiation while preventing pathological inflammation. At the same time, reduced IL-4 availability leads to restrained Th2 skewing, explaining the decrease in IgG1 production, which is primarily dependent on Th2-driven class switching. Fritz et al. (2007) further elucidated the function of Nod1 in the activation of adaptive immune responses. Nod1-deficient mice exhibited abnormalities in the induction of systemic Th1, Th2, Th17, and antibody responses following Complete Freund’s adjuvant (CFA) + ovalbumen immunization. Moreover, intraperitoneal administration of ovalbumen and FK156 (Nod1 ligand) enormously triggered Th2 T-cell activation and IgG1 secretion in a NOD1-dependent process (Fritz et al., 2007). The inability of such mice to develop Th1 and Th17 T-cell immunity was due to interference with synergy with TLR activation. Interestingly, Fritz et al. (2007) demonstrated that Nod1-deficient mice’s failure to trigger adaptive immunity was not caused by intrinsic defects in B or T cells. Additionally, they demonstrated that the expression of Nod1 in non-hematopoietic cells was crucial for the triggering of the Th2 response (Fritz et al., 2007). RIP2, a crucial adaptor molecule downstream of Nod1 and Nod2, is crucial in facilitating adaptive immune responses. Rip2-deficient mice developed aberrant cytokine responses to Nod1 and Nod2 ligands, defective macrophage and neutrophil recruitment, and failure to activate Cd11cintCd11b + splenic DCs following stimulation by NOD1 and NOD2 (Magalhaes et al., 2011). Rip2-deficient mice also failed to induce a Th2 response to ovalbumen challenges with FK156 or MDP but not with ovalbumen with alum (Fritz et al., 2007). These experiments demonstrate the critical role of peptidoglycan in controlling adaptive immunity. Without TLR ligands, both NOD1 and NOD2 stimulate Th2 responses, indicating that systemic probiotic peptidoglycan release can induce homeostatic Th2 responses (Fritz et al., 2007). Under conditions of bacterial infection, in the presence of both peptidoglycan and TLR ligands, peptidoglycan recognition synergizes with TLR signaling to augment Th1, Th2, and Th17 responses. Although the adaptive immune consequences of NOD1 and NOD2 activation are comparable at the systemic level, their distinct tissue expression patterns make it necessary to further investigate the particular roles of NOD1- and NOD2-regulation of the adaptive immune response in various pathologic settings.

DCs are critical regulators of immunogenic as well as tolerogenic responses based on the nature of encountered antigens, as they can decode the local microenvironment accurately (Coombes and Powrie, 2008). DCs sample directly or indirectly the luminal gastrointestinal tract content, including bacteria and their derived products and metabolites (Coombes and Powrie, 2008). The special ability of DCs to trigger naïve T-cells is attributed to inducing adaptive immunity or tolerance to microbiota constituents (Tóth et al., 2022). Therefore, interaction between LAB, e.g., Lactobacilli, and DCs has been the subject of extensive research. In a study, Tóth et al. (2022) analyzed the impact of peptidoglycan alteration in L. casei BL23 cell wall on monocyte-derived DCs (moDCs) activation and moDC capacity for T-cell polarization. They demonstrated that the ΔLc-p75 mutant L. casei was capable of triggering moDC maturation in parallel with the inhibition of moDC cytokine production (Tóth et al., 2022). Tóth et al. (2022) found that the Lc-p75-deleted L. casei mutant would induce weaker Th1 and Th17 responses than the wild-type strain. In contradiction to expectation, peptidoglycans of the wild-type and mutant L. casei BL23 strains had a similar immunomodulatory effect on moDCs. It has long been known that Pathogen-associated molecular patterns (PAMPs) are recognized by PRRs on host cells, which will result in the activation of the latter.

Th1-type differentiated cells secrete IFN-γ, which is an important cytokine during the initial phase of bacterial infection (Lemme-Dumit et al., 2021). IFN-γ enhances phagocyte-mediated protective immunity and suppresses Th2-type humoral immunity (Pashine et al., 1999; Gordon et al., 2005). Lemme-Dumit et al. (2021) findings showed that whole probiotic bacteria and probiotic cell walls induced similar levels of IFN-γ. This indicates that probiotics are able to stimulate the Th1-type cell-mediated immune response, thereby suppressing the replication and dissemination of enteric pathogens. Therefore, L. casei Lc431 treatment elevated the ratios of IFN-γ- and IL-10-secreting cells in the lamina propria and their level in intestinal fluid (Castillo et al., 2013). Moreover, as a response compatible with the Th1-predominant type elicited by cell wall constituents and whole bacteria of the probiotic strain, Lemme-Dumit et al. (2021) noticed inhibition of IL-4 production. These results indicate the immunomodulatory action of sub-cellular fractions of probiotic bacteria, i.e., the probiotic cell wall, which is seen to stimulate the acquired immune system through a Th1-type cell-mediated pathway.

Lactobacillus peptidoglycan can potentially affect the cytokine and other immunomodulatory factors secreted by macrophages, which have a pivotal role to play in maintaining Th1 and Th2 cell homeostasis (Sun et al., 2005). One was observed to result in mild inflammation in this research by eliciting a dose of peptidoglycan, whereas three doses of peptidoglycan had the potential to boost antigen presentation as well as T-cell differentiation (Sun et al., 2005). Lactobacillus peptidoglycan induced expression of IL-12 with a single dose, but three doses upregulated the expression of IFN-γ, indicating that the immune response might be altered to a Th1-type [cell-mediated immunity (CMI)] response. CMI response is critical in tumor inhibition (Sun et al., 2005). It was also shown here that Lactobacillus peptidoglycan could slow down CT26 colon cancer growth by eliciting increased CMI.

Enhanced phagocytic activity, natural killer cell activity, T- and B-cell immune response, and antigen-specific antibody production are responsible for enhanced resistance to tumor growth (Chester et al., 2015). Activation of macrophages is required for all types of tumoricidal activity (Gordon, 2003). PAMPs interact with PRRs on the cell surface, activating macrophages (Chen et al., 2025). Studies have identified that cell fragments of Lactobacillus murinus and the latter itself migrate through the wall of the intestine, either through penetration of the epithelial lining or via Peyer’s patches (Plant and Conway, 2001). Resident flora like Lactobacilli can pass through the intestinal mucosal barrier and remain in the spleen or other organs for a few days, where they increase phagocytosis activity (Plant and Conway, 2001).

It also observed that varying doses of Lactobacillus peptidoglycan influenced the expression of TNF receptor-associated factor 6 (TRAF6) and Toll-interacting protein (Tollip), implying that cytokine expression is regulated by activating or inhibiting these molecules (Sun et al., 2005). The results suggest that the increased activation of TLR-Nuclear factor kappa B (NF-κB) signaling by Lactobacillus peptidoglycan is due to enhanced expression of TRAF6 (Sun et al., 2005). While different strains of Lactobacillus are commonly applied, they can induce different systemic or mucosal immune responses based on the dose administered (Xin and Zhang, 2002). Maassen et al. (2003) showed that the growth phase of orally given Lactobacillus strains can influence the subclass distribution of antigen-specific antibodies like IgG1 and IgG2a. This heterogeneity in antibody response is a result of variations in bacterial cell compositions, which vary with the growth stage (Link-Amster et al., 1994).

Sun et al. (2005) determined that Lactobacillus peptidoglycan triggered varied immunological reactions and established that variations in cationic antimicrobial peptide (CAMP) compositions and the quantity delivered to macrophages determine the immune stimulatory activity of Lactobacilli. Finally, Lactobacillus peptidoglycan-induced immune response is Th1 type and dose-independent (Cross, 2002; Sun et al., 2005). Lactobacillus peptidoglycan influences the regulation of regulatory signaling molecules involved in the modulation of cytokine profiles and lymphocyte activation. Pro-Th1 immune signals produced by probiotic Lactobacilli at the gut mucosal interface can induce systemic cell-mediated immunity in the context of cancer immunotherapy (Cross, 2002).

Probiotics have strain-specific anti-inflammatory activities (Bibiloni et al., 2005; Lammers et al., 2005). Anti-inflammatory effects of Lactobacillus-derived peptidoglycan are primarily mediated through host innate immune cells, particularly macrophages and DCs, rather than through direct bacterial suppression of cytokines (Katz, 2003; Hanauer and Sparrow, 2004; Kruis et al., 2004). In recent work, Foligne et al. (2007) established the role of NOD2 in L. rhamnosus strain Ls33’s protective activities in a murine model of TNBS-induced colitis. Protection correlated with local IL-10 expression. NOD2 is capable of recognizing peptidoglycan-derived muropeptides, including MDP, and this is the reason behind the protective role of Ls33 peptidoglycan, which exhibited anti-inflammatory activity in vivo and was determined to activate NOD2 signaling (Foligne et al., 2007). The protective effect that was observed was correlated with a marked decrease in pro-inflammatory genes in the colon. Systemic and oral administration were both capable of inducing this effect, and this indicates that it could be through the induction of regulatory immune responses (Foligne et al., 2007).

Shida et al. (2009) demonstrated that although peptidoglycan stimulation of macrophages can induce transcription of IL-12p35, it simultaneously suppresses IL-12p40 expression, thereby preventing the secretion of biologically active IL-12p70. This regulatory effect was particularly evident for peptidoglycan derived from L. plantarum and L. casei, supporting the notion that peptidoglycan does not uniformly promote Th1 immunity. Because IL-12p40 is also a shared subunit of Interleukin 23 (IL-23), peptidoglycan-mediated suppression of IL-12p40 may additionally limit Th17 expansion, providing a mechanistic explanation for the observed anti-inflammatory effects of probiotic peptidoglycan in models of IBD and autoimmunity (Haskó and Szabó, 1999; Peluso et al., 2006). Recent research also points towards the central role of IL-17-producing Th17 cells in inflammation in these diseases (McKenzie et al., 2006; Bettelli et al., 2007). Since IL-12p40 is a component of IL-23, a key cytokine for Th17 cell proliferation and survival, peptidoglycan can potentially suppress these inflammatory disorders by blocking IL-12 and IL-23 production (Shida et al., 2009). Differences in IL-12 outcomes among Lactobacillus species can be attributed to strain-specific variations in peptidoglycan composition, intracellular processing, and receptor engagement. Peptidoglycan fragments that efficiently activate NOD2 signaling preferentially suppress TLR-mediated IL-12 production, whereas digestion-resistant cell wall structures derived from intact bacteria may promote IL-12 induction through alternative receptor pathways. These findings collectively indicate that probiotic peptidoglycan functions as a fine-tuner of cytokine responses rather than a fixed inducer of inflammation.

Excess production of iNOS may result in the generation of excessive amounts of NO, which is toxic to inflammatory cells (Song et al., 2021). Compared with the wild-type L. plantarum AR113 strain, the overexpression-modified strains showed significantly increased expression of iNOS. The incorporation of D-Ala-ended peptidoglycan into the bacterial cell wall led to elevated iNOS gene expression (Song et al., 2021). The cell wall of the L. plantarum even has immunologic stimulating potential by becoming bound with the TLR2 and the other macrophage receptors to induce cell signaling pathways and evoke downstream immunity. Although it had been found to stimulate TLR2, TLR4, and the MAPK pathway through D-Ala, its potential to support TRIF and the IKK (IκB Kinase) pathways was not present. This indicates that strains bound to TLR2 and TLR4 on the surface of macrophages entered cells and stimulated the MyD88-dependent pathway (Song et al., 2021). Of strains tested, L. plantarum AR113△ldhL△ldhD, with the highest ability to bind D-Ala-ended peptidoglycan precursors, showed the highest iNOS expression, more than fourfold higher than that of wild-type L. plantarum AR113. The pathway-specific proteins examined via polymerase chain reaction (PCR) for the macrophages confirmed that only TLR2 and TLR4 showed expressed DNA bands, thus proving the activation of the said two pathways. The findings substantiated that the four strains (AR113, AR113-DdlLc, AR113△ldhL△ldhD, and AR113△ldhL△ldhD-DdlLc) activated TLR2 and TLR4 to induce activation of the TRAF6/MAPK pathway while suppressing the activation of the TRIF and IKK pathways (Song et al., 2021). The DdlLc overexpression raised the level of D-Ala at peptidoglycan ends, markedly enhancing macrophage proinflammatory cytokine release in co-culture (Song et al., 2021). The previous research showed that D-Ala removal from LTA branch chains of L. plantarum caused some anti-inflammatory effects, which indicates that D-Ala was pro-inflammatory (Song et al., 2021).

Recent advances in peptidoglycan profiling, enabled by the peptidoglycan _MS2 in silico MS/MS library, have revealed a remarkable diversity of peptidoglycan chemotypes in gut bacteria and uncovered bioactive peptidoglycan forms with anti-inflammatory properties (Kwan et al., 2024). Through these efforts, an unexpectedly high abundance of anhydro-N-acetylmuramic acid (anhydro-NAM)-bearing peptidoglycan produced through the actions of lytic transglycosylase (LT) enzymes was discovered among Bifidobacterium bacteria, which comprise model probiotic microorganisms (Kwan et al., 2024). In functional terms, these anhydro-peptidoglycans have strong anti-inflammatory effects. Evidence has emerged to suggest that pre-treatment with Bifidobacterium-derived anhydro-peptidoglycan can strongly inhibit pro-inflammatory cytokines in response to LPS in murine macrophages (Kwan et al., 2024). Biochemical analyses revealed the presence of three enzymes in Bifidobacterium that produce LT, named MltG, RpfB-FL, and RpfB-truncated, which possess complementary activities that together account for the abundance of anhydro-peptidoglycan (Kwan et al., 2024). This structural adaptation underlines the particular function by which probiotic bacteria produce peptidoglycan fragments that promote immune tolerance over immune activation. In summary, these findings show that anhydro-peptidoglycans derived from probiotic bacteria play an important role in maintaining immune homeostasis by suppressing excessive immune activation.

Studies involving L. rhamnosus indicate that its protective effects during infection are primarily mediated through modulation of host inflammatory responses rather than direct reduction of pathogen burden. Peptidoglycan represents one of several bioactive bacterial components capable of shaping innate and adaptive immunity, acting in concert with other cell wall–associated molecules (Kolling et al., 2015). Kolling et al. (2015) findings showed that L. rhamnosus CRL1505 peptidoglycan can be used as an effective mucosal immunomodulator for increasing protection against respiratory infections. Additionally, identification of the mechanisms by which L. rhamnosus CRL1505’s peptidoglycan mechanisms of action can assist in the rational design of effective and safe therapies to reinforce respiratory immunity in immunocompromised patients (Kolling et al., 2015).

Like active L. rhamnosus CRL1505, its purified peptidoglycan (PG1505) intake before nasal infection with poly(I: C) strongly increased the production of type I IFNs, especially IFN-β, in bronchoalveolar lavage (BAL) (Clua et al., 2017). In turn, PG1505 enhanced the expression of several interferon-stimulated genes that suppress viral replication. Moreover, PG1505 also regulated production of pro-and anti-inflammatory cytokines in response to TLR3 stimulation, the cytokines whose production is pivotal in establishing an effective antiviral defense (Clua et al., 2017). PG1505 enhanced the production of certain cytokines (TNF-α, IL-10) but reduced the production of others (IL-6, MIP-1), indicating it regulates inflammation. PG1505 treatment enhanced the capacity to regulate respiratory syncytial virus (RSV) replication in the lung and inflammatory damage in lung tissue (Clua et al., 2017). The effect was mimicked by viable L. rhamnosus CRL1505, because PG1505 enhanced the frequencies of CD3 + CD4 + IFN-γ+, MHCII+CD11b^lowCD103+, and MHC-II + CD11b^highCD103- DCs, as well as augmented the respiratory levels of IFN-γ to indicate the induction of a Th1 response, which is a critical mechanism of immunity against viral infection of the respiratory tract (Clua et al., 2017). The findings point to peptidoglycan as one of the significant components of bacteria responsible for antiviral and immunomodulatory activities of L. rhamnosus CRL1505.

Notably, in some infection models, improved survival occurs despite only modest or insignificant reductions in pathogen load. This suggests that protection is achieved through enhanced immune regulation and limitation of immunopathology, rather than pathogen elimination alone. Increased production of IFN-γ likely supports effective antimicrobial immune activation, while concurrent elevation of IL-10 serves to restrain excessive inflammation and tissue damage. One of the most important findings to emerge from the current study is that heat-killed (HK1505) and PG1505 differentially modulated the behavior of infant mice towards secondary pneumococcal pneumonia induced by poly(I: C) or by RSV exposure (Clua et al., 2017). Intranasal administration of the live Lactococcus lactis NZ9000 into adult and neonatal mice led to the reduction by approximately 2 logs of cell counts of Streptococcus pneumoniae from the lungs (Medina et al., 2008). Also, non-viable L. casei CRL431, non-viable L. rhamnosus CRL1505, or their peptidoglycan permitted immunocompromised, undernourished mice to completely eradicate S. pneumoniae from the lungs (Villena et al., 2009; Kolling et al., 2015).

Although small on respiratory pathogen burden, PG1505 treatment had a dramatic impact on lung tissue damage and dissemination to the blood. These findings are important, as human studies and animal models of RSV-S. pneumoniae superinfection revealed that increased lung injury and enhanced bacteremia are defining factors in regulating infection severity and mortality rates (Hament et al., 2005; Weinberger et al., 2013; Smith et al., 2014; Cebey-López et al., 2016). Infant mice immunized with PG1505 showed better survival after superinfection with S. pneumoniae and RSV. RSV superinfection by S. pneumoniae stimulated vigorous inflammation, with enhanced accumulation of infiltrating neutrophils and heightened tissue levels of pro-inflammatory mediators in the lung, relative to mice infected with either pathogen alone (Stark et al., 2006).

Clua et al. (2017) found a minimum of three cytokines—IL-10, IFN-β, and IFN-γ—in playing a part in the HK1505- and PG1505-mediated immunomodulatory protective effect of respiratory superinfection. Precisely, IL-10 plays a protective role against inflammation-induced damage, and IFN-γ plays a role in the repression of pneumococcal multiplication in the lungs. These findings are in agreement with a previous study, where it was shown that the protection of infant mice from poly(I: C) or RSV infection by live L. rhamnosus CRL1505 was IL-10 and IFN-γ-dependent (Chiba et al., 2013). In addition, HK1505 and PG1505 also augmented the populations of CD3 + CD4 + IFN-γ + and CD3 + CD4 + IL-10 + T cells in baby mice’s lungs, again higher than controls after pneumococcal infection, indicating their protection against rechallenge bacterial pneumonia (Clua et al., 2017).

Kolling et al. (2018) investigated the impact of L. rhamnosus peptidoglycan treatment on the numbers and function of effector cells and adaptive immune system mediators for respiratory infections. Malnutrition can significantly alter the bone marrow, thymus, spleen, and lung pools of T cells (Kolling et al., 2018). T-cell reconstitution is required for host defense because they are the ones that produce cytokines, which coordinate and control different mechanisms of immunity and control B-cell antibody production. In Kolling et al. (2018) study, BCD + PG05-treated mice had higher counts of double-negative CD25 + and double-positive T cells following infection, which reflects active thymopoiesis. Additionally, PG05 treatment enhanced the maturation of CD4 T cells in the thymus after pneumococcal challenge, as well as increased the CD3 + CD4 + population in the spleen and sustained high levels of CD3 + CD4 + in the lungs relative to other groups. These treatments, including live L. rhamnosus CRL1505, could play an essential role in protection against S. pneumoniae infection (Kolling et al., 2018).

PG05 therapy itself might influence cytokine response after respiratory challenge with S. pneumoniae. There was marked upregulation of the Th2 cytokine IL-4 in the BAL and serum of undernourished mice treated with BCD + PG05 after pneumococcal infection relative to PG06 or PG534 treatments. This result is consistent with high levels of Immunoglobulin (Ig) A, IgG, and IgM anti-pneumococcal antibodies in the BAL and serum of the BCD + PG05 group, as in other studies (Kolling et al., 2018). Moreover, PG05 treatment induced the greatest response of IL-10 in the BAL after infection, which can be linked to less tissue damage and neutrophilic invasion of the airways in this group (Kolling et al., 2018). In addition, only PG05 caused the secretion of IL-2, which is a key cytokine in CD4 + T-cell response to pneumococcal antigens (Kolling et al., 2018). Malnutrition greatly suppresses BAL and serum anti-pneumococcal antibodies and blunts the humoral immunity of malnourished hosts. Also, the opsonophagocytic activity of IgG antibodies is strongly compromised in starved mice, showing failure in the B cell population within bone marrow, while their capacity to form antibodies is unaffected (Kolling et al., 2018). Kolling et al. (2018) confirmed that PG05-treated malnourished mice exhibited a restored immune response to respiratory disease. The BCD + PG05 group also presented with more immature and mature B cells in the lungs and bone marrow, and mature B lymphocytes in the spleen. These are consistent with the elevated local and systemic anti-pneumococcal antibody production in the BCD + PG05 group (Kolling et al., 2018). Peptidoglycan from the probiotic L. rhamnosus CRL1505 induced an increase in mature B cells in the lungs, which was reflected in increased IgG in the BAL. BCD + PG534 therapy did not increase the number or function of the B cells, as shown by these findings. This experiment shows that nutritional supplementation of malnourished mice by nasal administration of peptidoglycan of L. rhamnosus CRL1505 increases innate immunity and respiratory and systemic adaptive immunity (Kolling et al., 2018).

The worldwide emergence of multidrug-resistant (MDR) bacteria is an important concern for modern healthcare, especially in view of severe coinfections like methicillin-resistant Staphylococcus aureus (MRSA), and this significantly fuels morbidity and mortality (Birlutiu and Birlutiu, 2025). Hence, new approaches using the immune system rather than antibiotics are needed urgently. In this sense, probiotic-derived peptidoglycan, specifically in the form of bacterial-like particles derived from LAB, offered great promise as an immune-modulating approach that can be used for the stimulation of immunity against infection through the concept of trained immunity. Trained immunity is defined as the ability of innate immune cells to enter into a memory-like state after the primary exposure, which leads to an accelerated and robust response to subsequent infections with different pathogens. Unlike traditional adaptive immunity, trained immunity is mediated by macrophages, monocytes, and other innate immune cells due to epigenetic and metabolic reprogramming. Recently available evidence suggests that the peptidoglycan backbone of probiotic bacteria (BLP) is an efficient inducer of trained immunity, offering broad-spectrum protection against bacterial infections, including S. aureus, in the absence of distinct antigens (Niu et al., 2025). Mechanistically, the peptidoglycan fraction of probiotics is mediated mainly through the pattern recognition receptor TLR2, which is prominently expressed on cells of the innate immune system. Signaling downstream of TLR2 receptor engagement by BLP is mediated through several pathways, including JAK-STAT3, mammalian target of rapamycin (mTOR), and hypoxia inducible factor 1 subunit alpha (HIF-1α), which work in concert to coordinate the metabolic and functional repolarization of macrophages (Niu et al., 2025). Stimulation of these pathways enhances the transition to aerobic glycolysis, as is characteristic of trained immunity, as well as macrophage effector responses such as cytokine secretion and phagocytosis. Transcriptomic studies also showed that genes encoding inflammation, antigen presentation, metabolism, and epigenetic functions are all induced by BLP-stimulated cells to maintain the trained state (Niu et al., 2025). Besides its independent protective roles, there is immense translational potential for the probiotic-derived component, peptidoglycan, as an immunopotentiator and vaccine carrier. With subunit and multi-epitope vaccines, the BLP exerts a profound protective effect on MRSA infection by potently accelerating the activation of the innate immune system and the subsequent establishment of the adaptive immune response (Niu et al., 2025). Compared to conventional adjuvants, there exist numerous benefits in the use of BLPs as vaccine adjuvants. For example, these adjuvants possess excellent safety and stability properties and can be easily produced. Furthermore, they can act as antigen-delivery systems using surface display methods. Significantly, probiotic-derived peptidoglycan-based therapeutic approaches are quite in line with trained immunity-based vaccines that can serve to provide early, overall protection against various pathogens without relying on antibiotics. To some extent, BLPs can serve to provide an added or preventive modality against infections by making it possible to resist secondary infections by various pathogens. In summary, peptidoglycan derived from probiotics is a strong innate immune modulator that can generate trained immunity via a TLR2-mediated pathway, with the capacity to provide heterologous protection and function independently of specific antigens, making it a highly attractive tool for the prevention and treatment of MDR bacterial infections. Future investigation into this substance will, therefore, be critical for optimizing and harnessing this immune modulator into a productive tool for use in managing bacterial infections.

Earlier research has indicated that the probiotic L. casei strain LcS is capable of suppressing various autoimmune disease models in mice, such as arthritis, type I diabetes, murine lupus, and chronic IBD (Matsumoto et al., 2009). In a study, Matsumoto et al. (2009) discovered that LcS and its whole cell wall fraction suppressed IL-6 production in LPS-stimulated LPMCs of mice with chronic IBD. They initially determined the inhibitory activity of various PSPGs of LcS and other Lactobacillus species against IL-6 production in LPS-stimulated LPMCs from IBD mice. Matsumoto et al. (2009) validated that PSPG from LcS strongly inhibited IL-6 production. The PSPG in LcS is composed of two fractions: PSPG-I and PSPG-II. Their findings strongly suggest that PSPG-I is responsible for the anti-IL-6 activity of LcS. Purified PSPG-I repressed IL-6 production in LPS-activated RAW cells by repressing NF-kB phosphorylation. Additionally, Matsumoto et al. (2009) revealed that PSPG-I repressed IjB kinase phosphorylation in LPS-activated RAW cells. These findings indicate that PSPG-I represses IL-6 production in inflammatory macrophages, and PSPG-I-positive L. casei strain inhibited the induction of IBD and CAC effectively. Earlier studies have indicated that TLR2 and TLR4 are not implicated in LcS anti-IL-6 activity (Matsumoto et al., 2005). Matsumoto et al. (2009) also noted strong induction of Nod2 in RAW cells exposed to LcS. NOD2 activation by MDP suppressed pro-inflammatory cytokine production following TLR2 and TLR4 ligands dramatically (Hedl et al., 2007). These findings highlight the importance of PSPG-I and Nod2 stimulation in mediating the anti-inflammatory activities of L. casei LcS, and beneficially guide its application as a therapeutic agent in inflammatory disease.

Fernandez et al. (2011) found the therapeutic application of Lactobacillus strain Ls33 GN in producing anti-inflammatory activities through the activation of NOD2 in IBD. Intraperitoneal administration of Ls33 peptidoglycan caused a significant elevation of IL-10 in the colon, which failed to occur in Nod2-deficient mice, and points towards the involvement of NOD2 as a mediator of these protective functions (Fernandez et al., 2011). A single systemic injection of Ls33 peptidoglycan exerted a strong suppressive effect on colitis, along with decreases in key inflammatory cytokines (TNFα, IL-1β, and IL-6) and overexpression of IDO immunosuppressive pathway-related genes in the colon (Fernandez et al., 2011). In spite of reduced Il10 gene expression in Ls33 peptidoglycan -treated mice, localized augmentation of IL-10 protein was directly responsible for the protective effect of peptidoglycan. The inconsistency is most probably caused by the fast kinetics of Il10 gene expression after peptidoglycan treatment. Notably, protection was completely abolished in IL-10-deficient mice, confirming IL-10 as the master regulator of the anti-inflammatory response (Fernandez et al., 2011). Tregs, CD4 + CD25 + FoxP3 + cells, are essential for immune homeostasis and tolerance. In their research, the protective roles of Ls33 peptidoglycan were correlated with enhanced CD4 + FoxP3 + Tregs and CD103 + DCs in the MLNs of peptidoglycan-treated mice. Elevated levels of IFN-γ and IDO were also detected in CD11c + DCs in the MLNs (Fernandez et al., 2011). While IFN-γ is most often associated with pro-inflammatory Th1 responses, it can also be anti-inflammatory in some situations, e.g., TNBS-induced colitis. IFN-γ is a key inducer of the IDO-dependent immunosuppressive cascade, which enhances CD4 + FoxP3 + Treg proliferation, especially by CD103 + intestinal DCs (Fernandez et al., 2011).

One of the primary ways by which probiotic-derived peptidoglycan protects against colitis involves stimulating the signaling pathway of NOD2. The NOD2 recognizes the MDP structural component of peptidoglycan (Gao et al., 2022). The association of genetic polymorphisms of NOD2 with Crohn’s disease underscores its significance in the etiology of intestinal inflammation. Besides its significance in human genetics, evidence suggests that the lack of microbiota-derived MDP or NOD2 signaling plays a critical role in the development of colitis (Gao et al., 2022). In this regard, Gao et al. (2022) found that LPH, a probiotic-derived bifunctional peptidoglycan hydrolase with both N-acetyl-β-D-muramidase and D, L-endopeptidase activities, is required for the efficient generation of MDP from various bacterial peptidoglycans. By strongly triggering NOD2 signaling, LPH inhibits inappropriate inflammation and improves epithelial barrier function and immune tolerance in the gut. Moreover, a diminished expression level of DL-endopeptidase genes was found in patients with Crohn’s disease; supplementation with the enzymes corrected the deficiency and protected against DSS-driven colitis in mouse models (Gao et al., 2022). From a functional standpoint, activation of NOD2 by peptidoglycan has regulatory effects on biological processes related to colitis, such as immune conditioning, inhibition of pro-inflammatory cytokines, autophagy, and epithelial regeneration (Gao et al., 2022). These biological processes indirectly modulate colitis by reducing mucosal and intestinal inflammation.

In a DSS-induced murine colitis model by Li et al. (2025), dietary supplementation with peptidoglycan alleviated key disease symptoms, including weight loss, colon shortening, and elevated disease activity index (DAI) scores. Histopathological analyses revealed improved epithelial barrier integrity, restored crypt morphology, and reduced immune cell infiltration, indicating a direct protective effect on intestinal tissue. The anti-inflammatory effects of peptidoglycan are mediated, in part, through modulation of pro-inflammatory cytokines. Peptidoglycan intervention reduced circulating levels of IL-6, IL-1β, and TNF-α, reflecting suppression of the inflammatory cascade driven by APCs and macrophages (Li et al., 2025). Mechanistically, peptidoglycan likely interacts with PRRs on immune cells, modulating downstream signaling pathways such as NF-κB, thereby limiting excessive cytokine production while maintaining immune homeostasis. Furthermore, in Li et al. (2025) study, treatment with peptidoglycan enhanced microbial richness and diversity, increased the abundance of beneficial taxa, including Lactobacillus, Lachnospiraceae, Dubosiella, and Clostridia, and restored the Firmicutes/Bacteroidetes ratio. These microbial shifts promoted the production of short-chain fatty acids (SCFAs), particularly propionate, butyrate, and pentanoic acid, which are critical for intestinal barrier integrity, Treg cell differentiation, and suppression of pro-inflammatory responses (Li et al., 2025).

In addition, Ls33 peptidoglycan treatment triggered IL-10-secreting partially mature DCs, which, through adoptive transfer, reduced colitis in mice (Fernandez et al., 2011). The protective effect was, however, inhibited when DCs were isolated from Nod2 knockout animals, proving the absolute reliance on NOD2 signaling for Ls33 peptidoglycan-induced tolerogenic effects by activating regulatory DCs (Fernandez et al., 2011). These results were in agreement with earlier research, such as that of Shida et al. (2009), which had established that peptidoglycan from some Lactobacilli was able to suppress the secretion of IL-12 by macrophages, and Watanabe et al. (2004) who had established that stimulation of NOD2 by MDP was able to suppress TLR2 signaling and Th1-type inflammation. Fernandez et al. (2011) results also support the hypothesis that NOD2 signaling is important to counteract inflammation, and NOD2 mutations can induce overactive IBD by facilitating abnormal TLR2-dependent cytokine release. In conclusion, these results indicate that the peptidoglycan of certain Lactobacillus strains, e.g., Ls33, is capable of inducing tolerogenic immune responses via NOD2 signaling and, therefore, is a promising candidate for identifying therapeutic approaches to IBD. Structural and turnover studies of LAB peptidoglycan, including the creation of isogenic mutant pairs, may be useful for obtaining valuable information and selecting the most appropriate strains for potential prophylactic or therapeutic use in the treatment of IBD (Fernandez et al., 2011).

Probiotics also contribute to the control of allergic disorders through immunological mechanisms, including enhancement of mucosal IgA production and modulation of allergen-specific T- and B-cell responses (Eslami et al., 2020). These processes are highly intricate and involve coordinated regulation of multiple genes, PRRs such as TLRs, intracellular signaling pathways, and strengthened IgA-mediated immune activity within the intestinal mucosa (Abreu et al., 2005; Toh et al., 2012). Evidence from numerous in vivo and in vitro investigations, together with findings from human clinical studies, supports a beneficial role for probiotics in allergic conditions. In particular, randomized controlled trials have demonstrated that administration of L. rhamnosus GG to expectant mothers with a strong familial predisposition to eczema, allergic rhinitis, or asthma—and continued supplementation in their infants during the first six months of life—was associated with a marked reduction in the risk of atopic dermatitis (Eslami et al., 2020). The reported decreases in disease incidence were approximately 50, 44, and 36% at two, four, and 7 years of age, respectively (Bager et al., 2003; Illi et al., 2004).

Li A. L. et al. (2017) examined the role of L. acidophilus peptidoglycan on immune modulation in the β-lactoglobulin (β-lg) mouse model allergy model with specific reference to the TLR2/NF-κB pathway. Previous work documented an imbalance in the ratio of Treg/Th17 and correlated it with allergic symptomology, wherein Th17-dominated responses play a causative role in allergy. In Li A. L. et al. (2017) study, it was demonstrated that allergy to β-lg enhanced Th17 predominance and that oral administration of the L. acidophilus KLDS 1.0738 strain successfully down-regulated such Th17 immunopathologies. This research followed up on that by investigating the function of TLR2 in macrophage recognition of Lactobacillus peptidoglycan. Li A. L. et al. (2017) results indicated that peptidoglycan administration triggered macrophage TLR2 expression, which activated downstream NF-κB signaling. Interestingly, inhibition of TLR2 impaired the production of critical cytokines, including IFN-γ, transforming growth factor-β (TGF-β), and IL-10, that are typically induced by peptidoglycan. This observation is consistent with earlier reports, e.g., Rakoff-Nahoum et al. (2004) which have documented the protective and restorative functions of TLR signaling in macrophages. Li A. L. et al. (2017) also investigated the function of TLR2 in immunoinhibitory responses induced by Lactobacillus peptidoglycan in a β-lg allergy mouse model. Pre-treatment with Lactobacillus peptidoglycan resulted in decreased allergy symptoms and elevated levels of TLR2 and NF-κB p65, suggesting a protective function. Notably, MyD88 (a central adapter protein in TLR2 signaling) expression was hardly affected by Lactobacillus peptidoglycan, whereas other research has demonstrated that TLR2 ligands also induce MyD88-independent signaling, resulting in NF-κB activation and cytokine induction (Takeuchi et al., 2000; Wang Q. et al., 2001; Kenny et al., 2009). This indicates that Lactobacillus peptidoglycan can modulate its immunological activity through MyD88-dependent and independent pathways (Takeuchi et al., 2000).

In addition, intraperitoneal instillation of Lactobacillus peptidoglycan in sensitized mice induced concentration-dependent increases in Treg-related cytokines (TGF-β) and transcription factors (Foxp3, CD25), and decreased IL-17A and Retinoid-Related Orphan Receptor gamma t (RORγt) levels (Takeuchi et al., 2000). This indicates that Lactobacillus peptidoglycan induces a Treg-biased immune response, which can suppress IgE-mediated allergy. The correlation between TLR2 activation and the balance of Treg/Th17 was also observed, where TLR2 and NF-κB levels were higher with higher Foxp3 and lower RORγt in the high-dose Lactobacillus peptidoglycan group than in the allergy group (Takeuchi et al., 2000). In summary, Li A. L. et al. (2017) results indicate that Lactobacillus peptidoglycan, via TLR2/NF-κB signaling, enhances the activity of Treg and inhibits Th17-induced inflammation in β-lg-sensitized mice. This study contributes to the literature attesting to the preventive efficacy of probiotics, especially Lactobacillus, to reduce allergic inflammation by establishing a Treg-dominant immune microenvironment. In allergic models, signaling pathways downstream of TLRs, including MyD88 and NF-κB, exert context-dependent effects rather than uniformly promoting inflammation. MyD88-dependent signaling can contribute to allergic inflammation when strongly activated; however, under controlled stimulation conditions, it may also support immune regulation by promoting tolerogenic APC functions and regulatory cytokine production.

Probiotics are increasingly studied in oncology—not as stand-alone cures, but as adjuncts that influence tumor biology, immunity, and therapy response (Jiang et al., 2024). He et al. (2017) showed the immense anticancer impact of peptidoglycan derived from probiotics and selenylated peptidoglycan (Se-peptidoglycan) on the proliferation and apoptosis of cells in human colon cancer. The studies affirm the production of a dose-dependent antiproliferative action of peptidoglycan and Se-peptidoglycan, with Se-peptidoglycan having an inhibitory activity much greater than that of peptidoglycan. The effectiveness boost by Se-peptidoglycan over peptidoglycan results from the modification of peptidoglycan with selenium, which enhances its anticancer activity (He et al., 2017). Past research has established peptidoglycan’s ability to stimulate an immune response since macrophages stimulated by Bifidobacterium-derived peptidoglycan secrete cytokines such as IL-1, IL-6, and TNF-α that enhance the killing of tumor cells (Wang L. S. et al., 2001). Choi et al. (2006) also found that Lactobacillus-derived peptidoglycan suppressed the growth of colon cancer cells in a time- and dose-dependent manner. Current research furthered the above results by proving that Se-peptidoglycan had three times more inhibitory rate to HT-29 cell growth compared to peptidoglycan, supporting the hypothesis that selenium modification increases the antiproliferative efficacy of peptidoglycan. Besides, the induction of apoptosis is an essential mechanism of cancer cell removal. He et al. (2017) research once again proved that peptidoglycan and Se-peptidoglycan induced apoptosis in HT-29 cells. Apoptotic cell death was also marked by mitochondrial depolarization, intracellular calcium increase, activation of caspase-3, and membrane scrambling. Analysis revealed a significantly greater proportion of cells in the late apoptotic and necrotic phases in the Se-peptidoglycan-treated group than in the peptidoglycan group, suggesting that Se-peptidoglycan exerts a more potent pro-apoptotic effect (He et al., 2017). This implies that selenylation of peptidoglycan increases its capacity to induce apoptosis in cancer cells, and this further confirms the notion that selenium modification enhances the anticancer potency of peptidoglycan (Choi et al., 2006). In summary, these results offer evidence that Se-peptidoglycan, with its increased antiproliferative and pro-apoptotic activities, can be a therapeutic drug for colon cancer treatment. The selenium modification of peptidoglycan is found to remarkably enhance its anticancer activity, and thus it is a valuable research area in cancer immunotherapy and treatment.

Wang et al. (2018) centered on the induction of apoptosis in HT-29 human colon cancer cells by Lactobacillus paracasei subsp. paracasei M5 strain peptidoglycan. The result showed that peptidoglycan treatment results in striking changes in the expression levels of apoptosis genes such as Bcl-xl, Bax, Bad, caspase-3, and Cyto-C (Wang et al., 2018). In a more detailed mechanism, treatment-induced expression of the pro-apoptotic genes Bax and Bad, as well as repression of the anti-apoptotic Bcl-xl gene, is evident. This is indicative of the fact that peptidoglycan tilts pro- and anti-apoptotic signals towards apoptosis. The study further established that peptidoglycan induced typical morphological changes, typical of apoptosis, such as cell shrinking and membrane blebbing, and this was linked to caspase-3 activation. These findings are consistent with earlier results by Yu et al. (2004) as they validated the hypothesis that peptidoglycan induces apoptosis through the activation of caspase-dependent pathways. Apart from that, peptidoglycan treatment caused the release of Cyto-C from the mitochondria into the cytosol, a necessary step in the intrinsic apoptotic pathway (Wang et al., 2018). The release of Cyto-C allows for the activation of caspase-9, which in turn activates caspase-3, a key executioner in apoptosis. This series of events demonstrates the intricate molecular mechanisms in peptidoglycan-induced apoptosis in HT-29 cells (Yu et al., 2004). Overall, the findings indicate that peptidoglycan derived from L. paracasei M5 strain causes apoptosis in colon cancer cells via a signal transduction pathway where Bcl-2 family protein regulation and the Cyto-C/caspase-3 pathway are implicated (Yu et al., 2004). These findings point towards the therapeutic role of probiotic-derived peptidoglycan as a cancer treatment.

Nedzvetsky et al. (2020) revealed a dose-dependent action of MPP in glioblastoma cells, which proved that MPP notably increases the expression of PARP1 (Poly(ADP-ribose) polymerase 1) and NF-κB. Muropeptides like MPP have been found to induce innate immune responses and greatly influence various cellular processes such as cytokine secretion, cell survival, and apoptosis (Nedzvetsky et al., 2020). Nedzvetsky et al. (2020) found that MPP treatment in glioblastoma U373MG cells triggers both PARP1- and NF-κB-dependent signaling. They also observed an appreciable loss of migratory capacity and Nicotinamide adenine dinucleotide reduced (NADH) content in the treated cells. NADH reduction in MPP-treated glioblastoma cells is linked to PARP activation, a process that is reported to lower NADH during poly-ADP-ribosylation, a protein-targeting modification that reorganizes DNA (Nedzvetsky et al., 2020). Since sustained activation of PARP can lead to cellular energy homeostasis derangements, activation of PARP-dependent responses can induce cellular dysfunction or death via NF-κB signaling participation (Nedzvetsky et al., 2020). Therefore, the overexpression of PARP and NF-κB reported may have a central function in the augmented reactivity of glial cells in response to MPP treatment. These findings imply that exposure to MPP induces depletion of NADH, hinders migration of cells, and enhances PARP1 and NF-κB expression in glioblastoma cells (Nedzvetsky et al., 2020). Additional studies need to be done to investigate the tumoricidal activity of MPP in glial-derived neoplasms.