TLR2 and TLR4 bridge physiological and pathological inflammation in the reproductive system.

Numerous studies have indicated that TLR2 and TLR4 are expressed in various parts of the FRT. In humans, TLR1, TLR2, TLR4, and TLR6 have been reported in the fallopian tubes, uterine endometrium, cervix, and ectocervix (expressed in epithelial and leukocytes cells) 11 . However, the intensity of TLR2 and TLR4 expression varies significantly depending on the specific region of the FRT. For TLR2, the highest expression is observed in the fallopian tube and cervical tissues, followed by the endometrium and ectocervix. In contrast, TLR4 expression gradually decreases along the tract, with the highest levels in the fallopian tubes and endometrium, and lower levels in the cervix and ectocervix 11 . This differential expression pattern of TLR2 and TLR4 is attributed to the distinct regulation of inflammation and immunity within different regions of the FRT 11 . Similarly, in other species such as bovine and mice, TLR2 and TLR4 are expressed throughout the FRT 64 – 66 . Regarding their roles, it has been reported that these molecules are critical for immunity in the FRT and directly influence pregnancy. Specifically, the upregulation or downregulation of TLR2 and TLR4 can significantly impact pregnancy outcomes 67 . The dynamic regulation of TLR2/4 expression is essential for normal reproductive processes. The regulation of these TLRs is important for sperm function and interaction with the FRT. Dysregulated TLR2/4 expression, often triggered by microbial infections or other pathological stimuli, can lead to excessive or inappropriate inflammatory responses. This dysregulation can impair immune tolerance, increase susceptibility to infections, and may contribute to adverse pregnancy outcomes such as preterm labor, preeclampsia, and recurrent pregnancy loss 68 . Chronic inflammation due to TLR upregulation can lead to pathologies such as endometriosis. Although the exact factors responsible for the regulation of TLR2 and TLR4 expression remain unclear, it has been suggested that the diversity of bacterial ligands activating TLR2, such as peptidoglycan from Gram-positive bacteria, bacterial lipopeptides, and zymosan, may modulate their expression in cervical tissue to help prevent ascending reproductive tract infections 67 . So far, the exact mechanism behind the differential expression of TLR2 and TLR4 in the cervix during pregnancy has not been fully elucidated. However, one possible factor is that sex hormones, which are associated with pregnancy, may modulate the expression of TLR2 and TLR4 67 . Immunohistochemistry studies have shown that early pregnancy is associated with increased expression of TLR2 and TLR4 receptors in cervical tissue. The upregulation of TLR2 and TLR4 in the lower FRT has been reported to play a pivotal role in modulating innate immune and inflammatory mechanisms in the ectocervix during pregnancy 67 . Additionally, experimental evidence suggests that elevated inflammation mediated via TLR signaling may be implicated in premature birth 67 . Moreover, it can be hypothesized that cytokines and growth factors regulate TLR expression within the FRT. Therefore, it is clear that the up-/down-regulation of TLR2 and TLR4 in the reproductive tract can have significant physiological consequences, influencing both normal immune function and pregnancy outcomes. In this section, we will explain the roles of TLR2 and TLR4 in three main parts of the FRT: the uterus, oviduct, and oocytes. Both TLR2 and TLR4 play critical roles in pathogen recognition within the uterus 14 , 26 , 69 , 70 . In bovine models, this interaction with pathogens can trigger severe inflammation, often leading to tissue damage due to excessive Prostaglandin E2 production via MyD88 and p38 MAPK pathways 71 . In humans, bacterial infections in the endometrium have been effectively treated using TLR2 and TLR4 inhibitors, highlighting their importance in managing such conditions 72 . Additionally, TLR2 and TLR4 contribute to physiological inflammation in the endometrium, although their roles differ by species 63 , 73 . In bovines, physiological inflammation induced by sperm is predominantly mediated by TLR2, while in mice, TLR4 plays a more significant role 63 , 73 . As discussed above, TLR2 requires heterodimerization with TLR1 or TLR6 to induce inflammation. In the endometrium, TLR2/1 activation results in weak, mild, and physiological inflammation, whereas TLR2/6 induces a strong, severe inflammatory response, potentially causing tissue damage (Fig.  3 ) 26 . Fig. 3 The effect of TLR dimerization on functions in the female reproductive tract (FRT). In the uterus, activation of TLR2/6 and TLR4/4 under pathological conditions results in severe and prolonged inflammation, whereas TLR2/1 activation induces mild inflammation (sperm employ TLR2/1 under physiological conditions). During different stages of the estrous cycle, TLR4 expression is significantly downregulated in the follicular phase to protect sperm from immune attack and in the luteal phase (downregulation of TLR4 and TLR2) to support embryo implantation. In the oviduct, TLR4 activation induces pro-inflammatory responses, while sperm employ TLR2/1 to stimulate anti-inflammatory responses. In the ovary and oocyte, TLR2 and TLR4 activation in surface epithelial and granulosa cells triggers inflammation, a major cause of infertility in mammals. However, TLR2/1 and TLR4/4 activation in oocytes promotes sperm capacitation, enhancing fertilization success. The figure has been created using BioRender.com software. In the uterus, activation of TLR2/6 and TLR4/4 under pathological conditions results in severe and prolonged inflammation, whereas TLR2/1 activation induces mild inflammation (sperm employ TLR2/1 under physiological conditions). During different stages of the estrous cycle, TLR4 expression is significantly downregulated in the follicular phase to protect sperm from immune attack and in the luteal phase (downregulation of TLR4 and TLR2) to support embryo implantation. In the oviduct, TLR4 activation induces pro-inflammatory responses, while sperm employ TLR2/1 to stimulate anti-inflammatory responses. In the ovary and oocyte, TLR2 and TLR4 activation in surface epithelial and granulosa cells triggers inflammation, a major cause of infertility in mammals. However, TLR2/1 and TLR4/4 activation in oocytes promotes sperm capacitation, enhancing fertilization success. The figure has been created using BioRender.com software. In human endometritis, elevated gene expression of TLR2 and TLR4 in endometrium has been observed, suggesting their involvement in endometritis 74 . In fact, an association between the increased expression of TLR2 and TLR4 and the development of endometriosis has been demonstrated, indicating that the upregulation of these receptors may contribute to inflammation and, consequently, endometritis 74 . The higher expression of TLR2 and TLR4 in endometritis is attributed to the presence of microbial ligands, including components from both Gram-positive and Gram-negative bacteria, particularly those recognized by TLR2 in the FRT 74 . It has been hypothesized that these microbial components may either directly upregulate TLR expression in resident endometrial cells or promote the recruitment of immune cells that express TLR2 and TLR4, thereby increasing overall expression levels at the tissue level. However, it remains unclear whether this elevated expression is due to upregulation within resident endometrial cells or increased infiltration of immune cells expressing these receptors. This distinction has not been clearly addressed in current studies, and further investigation is needed to determine which mechanism predominates in this pathological context. Similar findings have been reported in other mammals, such as in canines. In canine endometrium, TLR2 and TLR4 expression levels vary across the estrous cycle (Fig.  3 ). Previous studies indicated that TLR4 activation can trigger strong inflammation, compared to TLR2. TLR4 expression is absent during estrus, likely to prevent inflammation during mating and sperm deposition 75 . However, given the limited inflammatory effect of TLR2 activation, its expression during this phase presents no significant complications. During early diestrus, both TLR2 and TLR4 expression levels decrease, possibly to support embryo implantation (Fig.  3 ) 75 . During diestrus, when embryo implantation and uterine receptivity occur, the endometrium must undergo anti-inflammatory regulation 76 . As a result, the downregulation of TLRs, particularly TLR4 and also TLR2, appears to be a strategic adaptation. However, high TLR4 expression, but not TLR2, has been associated with uterine infections in canine 77 . These observations indicate a protective role for TLR2, in maintaining uterine homeostasis by balancing protective and pathological inflammation 77 . TLR2 and TLR4 are also expressed in the oviduct, where they play roles in both physiological and pathological inflammation 11 , 65 , 78 . In bovine models, TLR2 and TLR4 are involved in recognizing pathogen-associated molecules, particularly LPS, to induce pro-inflammatory responses. However, ovarian steroids and luteinizing hormone significantly inhibit LPS-induced TLR4 and TLR2 activation, promoting immune balance and enhancing homeostasis (Fig.  3 ) 65 . A similar mechanism of inflammation induction through TLR4 has been observed in other mammalian species, such as rabbits 79 . In humans, TLR4 inhibition using betulonic acid has been shown to downregulate pro-inflammatory cytokines, leading to enhanced cell proliferation and reduced apoptosis 80 . Interestingly, in bovine oviducts, TLR2, rather than TLR4, plays a crucial role in sperm recognition (Fig.  3 ) 78 . Activation of TLR4 in the oviduct prior to sperm interaction negatively affects sperm motility and function 81 . Unlike the bovine uterus, sperm interaction with the oviduct induces an anti-inflammatory response (Fig.  3 ) 78 . This anti-inflammatory effect is blocked by TLR2/1 antagonists, suggesting that the TLR2/1 signaling pathway in the oviduct contributes to the regulation of inflammation 78 . Both TLR2 and TLR4 are expressed in the ovary and oocytes across various species. In humans and murine ovaries, the expression of these TLRs has been reported, with TLRs being highly expressed in the surface epithelial cells of normal ovaries. Interestingly, their expression is also observed in ovarian epithelial tumors 82 . In murine models, TLR2 and TLR4 expression remain consistent across different stages of the estrous cycle 82 . Similarly, in rabbits, TLR2 and TLR4 expression have been documented, with significant changes noted after LPS treatment 83 . In bovine species, TLR2 and TLR4 are expressed in granulosa cells of the ovary 84 . Activation of these TLRs using synthetic bacterial lipoproteins and LPS in granulosa cells triggers the MAPK14 and MAPK3/1 pathways, leading to increased production of pro-inflammatory cytokines such as IL1-b, IL6, IL10, and TNF 84 . This production of pro-inflammatory cytokines, heightened cytokine activity, can contribute to infertility in cattle (Fig.  3 ) 84 . In oocytes, TLR2 and TLR4 are expressed, and their activation can lead to the release of cytokines (Fig.  3 ). These cytokines, in turn, enhance sperm capacitation, ultimately contributing to improved fertility 85 . It is well-established that TLR activation within the FRT can initiate immune responses, leading to the production of inflammatory mediators. Immune cells are central to these processes, and considerable research has focused on profiling their populations across different FRT compartments to elucidate their roles in reproduction. Complementary to these efforts is the identification of specific TLR ligands and their mechanisms of action. However, studies directly investigating the effects of TLR activation in FRT-resident immune cells and their subsequent impact on reproductive outcomes remain limited. A diverse array of immune cells, including macrophages, DCs, natural killer (NK) cells, neutrophils, mast cells, and T cells, populate the FRT 86 , with their relative abundance varying significantly between different anatomical locations 87 . Furthermore, the immune cell landscape within the reproductive tract exhibits dynamic fluctuations throughout the estrous/menstrual cycle. Notably, many immune cell types reach peak abundance during the estrus and metestrus phases, aligning with their critical roles in responding to seminal fluid components (including potential paternal antigens) and invading pathogens introduced around the time of insemination/intercourse 88 . Studies in women during the menstrual cycle have revealed that T cells, antigen-presenting cells, and neutrophils are prevalent throughout the FRT 87 . In contrast, NK cells are predominantly localized to the uterus and are generally scarce in the vagina, while B cells and monocytes are present at lower levels compared to other immune cell types, particularly T cells 87 (Fig.  4 ). These immune cells perform dual functions: providing immune defense against pathogens in the lower FRT and establishing immune tolerance towards sperm and the developing embryo/fetus in the upper FRT. Fig. 4 Immune cell profiling in the female reproductive tract (FRT). This figure illustrates the distribution of immune cells in the FRT. T cells, antigen-presenting cells (APCs), and neutrophils are abundant throughout the FRT, while natural killer (NK) cells are predominantly found in the uterus. In contrast, B cells are present at much lower levels compared to other immune cells. Regarding cytokine production and TLR activation, TLR4 stimulation in FRT immune cells results in a relatively low level of cytokine production, suggesting limited activation. However, TLR2 activation in NK cells, especially in the uterus, leads to a marked increase in cytokine production. For other regions of the FRT, data on the effects of TLR2 activation and cytokine response remain limited. The number of arrows represents the relative frequency of each immune cell type or level of cytokine production in different regions of the FRT. Arrows with a question mark indicate a hypothesis based on previous studies showing that TLR2 activation in T cells can enhance cytokine production; however, this has not been confirmed in the FRT. A lone question mark indicates that no information is available. Therefore, we propose that activation of TLR2 in FRT-resident T cells may similarly lead to increased cytokine production. This figure illustrates the distribution of immune cells in the FRT. T cells, antigen-presenting cells (APCs), and neutrophils are abundant throughout the FRT, while natural killer (NK) cells are predominantly found in the uterus. In contrast, B cells are present at much lower levels compared to other immune cells. Regarding cytokine production and TLR activation, TLR4 stimulation in FRT immune cells results in a relatively low level of cytokine production, suggesting limited activation. However, TLR2 activation in NK cells, especially in the uterus, leads to a marked increase in cytokine production. For other regions of the FRT, data on the effects of TLR2 activation and cytokine response remain limited. The number of arrows represents the relative frequency of each immune cell type or level of cytokine production in different regions of the FRT. Arrows with a question mark indicate a hypothesis based on previous studies showing that TLR2 activation in T cells can enhance cytokine production; however, this has not been confirmed in the FRT. A lone question mark indicates that no information is available. Therefore, we propose that activation of TLR2 in FRT-resident T cells may similarly lead to increased cytokine production. Regarding TLR responsiveness in FRT immune cells, Benjelloun et al. reported that TLR4 activation in these cells elicits a comparatively moderate cytokine response compared to peripheral blood mononuclear cells, suggesting a tighter regulatory control of TLR4-mediated inflammation within the FRT 87 . While data on TLR2 activation in FRT immune cells (Fig.  4 ) is still emerging, evidence from other tissues indicates differential responsiveness, with T cells and NK cells generally exhibiting a stronger response to TLR2 than TLR4 89 , 90 . Specifically, TLR2 ligation in T cells can enhance cytokine production and act as a costimulatory signal for T cell proliferation 89 . Similarly, NK cells typically express higher levels of TLR2 than TLR4 and are more sensitive to TLR2 stimulation 90 . Notably, uterine NK cells, a dominant immune population in the uterus, strongly express TLR2 and weakly express TLR4, and their activation can lead to the production of cytokines such as IFN-γ 91 . This underscores the intricate interplay between TLR signaling and immune responses within the reproductive tract, highlighting the crucial role of resident immune cells in regulating reproductive processes. TLR-activated signaling pathways in the FRT involve key immune cell types such as macrophages, DCs, and neutrophils, which are integral to various reproductive events: (1) Decidualization and embryo implantation: Macrophages and DCs play critical roles in establishing immune tolerance and facilitating successful implantation by modulating cytokine production and T cell responses 92 . (2) Uterine clearance: Neutrophils and macrophages are essential for the efficient clearance of excess sperm, cellular debris and pathogens post-partum or during menstruation, driven by TLR-mediated activation 26 , 70 . (3) Follicular development and ovulation: TLR signaling in follicular immune cells can influence the local cytokine environment, impacting granulosa cell function and ultimately, ovulation 93 . In this section, we discuss how the activation or inhibition of TLR2 and TLR4 can affect fertility and pregnancy outcomes. In bovine, altering the immune response in any part of the FRT, including the uterus, oviduct, oocyte, and sperm, significantly impacts pregnancy. These areas are critical for sperm cross-talk with FRT, embryo competence, and embryo-maternal interactions. Starting with the uterus, it plays a dual role: facilitating sperm immune cross-talk and supporting embryo implantation. Sperm triggers a mild, transient inflammation by interacting with innate immune receptors, including TLRs 94 . This inflammation triggers epithelial cells to release cytokines, attracting immune cells such as neutrophils and macrophages into the uterine lumen 94 – 96 . These immune cells help clear excess sperm and pathogens, a process known as uterine clearance, which is essential for creating an ideal uterine environment for embryo receptivity and implantation 94 . A balanced, moderate inflammation is crucial for this process. Recent studies have shown that in bovine species, sperm-induced inflammation is mediated through the TLR2/1 signaling pathway, which ensures a mild and transient response 26 . However, in mice, TLR4 has been reported to be involved in sperm-mediated signaling, suggesting species-specific differences in TLR involvement 63 . So far, there is no definitive information on which molecules from sperm or semen activate TLR2/1 signaling to induce weak inflammation. To address this question, several possibilities can be considered. One possibility is that certain sperm-derived molecules, such as lipoproteins, lipopeptides, glycans, triglycerides, and phospholipids, may serve as potential candidates. Additionally, seminal plasma-derived molecules, particularly DAMPs, could also fall into this category. It is also important to note that the ligands and receptors involved may differ across species. In contrast, TLR2/6 or TLR4 activation leads to stronger, prolonged inflammation that can damage uterine tissues (Fig.  3 ) 73 . In artificial insemination, TLR2 is vital for initiating inflammation and aiding uterine clearance. Notably, semen extenders, particularly those containing egg yolk, can induce mild inflammation via the TLR2/1 pathway (activated by triglycerides) without interfering with sperm-induced inflammation 97 . This emphasizes the importance of TLR2/1 activation in promoting mild, transient inflammation while avoiding the severe responses associated with TLR4 or TLR2/6 activation 97 . Such balanced inflammation has been shown to improve pregnancy outcomes. Regarding embryo transfer, activating TLR2/1 signaling in sperm before IVF enhances cleavage and blastocyst rates in cumulus-oocyte complexes or cumulus-free oocytes but not in ZP-free oocytes 61 . In contrast, inhibiting this pathway reduces these rates 61 . Overall, the role of TLR2/1 signaling is critical at multiple stages of the reproductive process to ensure successful pregnancy outcomes. Regarding the expression of TLR2 and TLR4 in uterine cells during embryo/blastocyst communication with the uterus, it has been reported that TLR4 is significantly downregulated in endometrial epithelial cells (Fig.  3 ) 98 . This downregulation plays a crucial role in modulating immune responses during early pregnancy, creating a receptive uterine environment for implantation and protecting the conceptus (embryo/fetus and associated membranes). While immune tolerance in classical immunology is primarily associated with antigen-specific regulatory T cells (adaptive immunity), in pregnancy, innate immune modulation is also essential 99 . This tolerance involves a complex interplay of innate and adaptive immune mechanisms. Beyond its role in preventing excessive inflammatory responses to potential pathogens, TLR4 downregulation may facilitate embryo-maternal communication. One possible mechanism involves extracellular vesicles (EVs) and microRNAs, which are key mediators of embryo–maternal interactions 100 . Evidence suggests that TLR4 can serve as a target for EVs delivering microRNAs, indicating that TLR4 may play a role in regulating communication between the embryo and the maternal immune system via EV-mediated signaling 101 . This observation was made in equine species. In bovines, one study indicated that TLR2 expression might be influenced by the embryo culture medium, suggesting that TLR2 expression is neither markedly upregulated nor downregulated in the uterus during embryo implantation (Fig.  3 ) 102 . TLR4 appears to have a negative impact on embryo implantation, and TLR2/6 activation has also been associated with decreased embryo implantation and impaired uterine receptivity in mouse models 103 . Therefore, it seems that TLR2, TLR4, and TLR6 may negatively affect uterine receptivity and embryo implantation (Fig.  3 ). To investigate the roles of TLRs, particularly TLR2 and TLR4, following embryo implantation and during placental development, studies have revealed distinct effects of their activation in trophoblasts. Activation of TLR4 in these cells has been shown to induce the expression of pro-inflammatory cytokines, while activation of TLR2 can trigger apoptosis 104 . Trophoblasts express TLR1, TLR2, and TLR4, but reportedly lack TLR6 expression, whereas the mature human placenta expresses a broader repertoire, including TLR1, TLR2, TLR4, and TLR6 104 , 105 . Activation of both TLR2 and TLR4 in the placenta has been associated with the release of cytokines and the initiation of local immune responses 106 . Furthermore, accumulating evidence highlights a critical role for TLR4 in driving excessive uterine inflammation, which can be detrimental to the developing fetus, impair its growth, and ultimately contribute to adverse pregnancy outcomes such as preterm birth (Fig.  3 ) 107 . Notably, preclinical studies have demonstrated that blocking TLR4 with specific inhibitors can effectively reduce inflammation and prevent early delivery in certain models 107 . This highlights the potential for prolonged TLR4-mediated inflammation to negatively impact pregnancy progression. While the effects of TLR2 activation in the context of overall placental development and pregnancy outcomes are less definitively characterized in this section, the apoptotic effect on trophoblasts suggests a potential role in placental remodeling or in pathological conditions. Further research is needed to fully elucidate the contributions of different TLRs to both normal placental development and pregnancy complications.

TLR1 and TLR6 play distinct but critical roles in modulating the functions of TLR2 and TLR4, influencing the inflammatory response and tissue homeostasis. TLR1, when forming heterodimers with TLR2 or TLR4, is associated with recognizing triacylated lipopeptides and promoting weak inflammation and homeostasis, specifically in epithelial cells, potentially through beyond MyD88 and NF-κB or Pi3K/Akt pathways or the preferential recruitment of MyD88 over TRIF adapters 114 . This limited signaling might lead to reduced NF-κB activation and the induction of anti-inflammatory cytokines like IL-10 115 . In contrast, TLR6 forms heterodimers with TLR2 or TLR4 to recognize diacylated lipopeptides, triggering severe inflammation and tissue damage. These dimerizations likely recruit both MyD88 and TRIF, amplifying downstream signaling pathways such as NF-κB and IRF3/7, leading to the production of pro-inflammatory cytokines like TNF-α and IL-6, as well as type I interferons. This enhanced response may also involve the generation of ROS and inflammatory mediators that attract neutrophils, exacerbating tissue damage. The difference in signaling strength and outcomes between these dimer pairs may arise from their ligand-binding specificity, interaction with accessory proteins like MD2 and CD14, and recruitment of regulatory molecules like SOCS in TLR1-associated dimers. Future experiments, such as co-immunoprecipitation to study adapter protein interactions, transcriptomics to identify cytokine signatures, and CRISPR-based knockout models for TLR1 or TLR6, could elucidate the precise pathways and mechanisms underlying these distinct dimerization effects.

As we explained earlier, TLR4 and TLR2 share a common signaling pathway for inducing inflammation. Both utilize MyD88 to activate the NF-κB sub-pathway in response to PAMPs. However, TLR4 can also activate the TRIF-dependent pathway, which TLR2 cannot, resulting in the production of interferons. Since both TLRs rely on MyD88, signaling from one receptor could influence the other, possibly due to competition for the availability of the MyD88 adapter. One mechanism that might downregulate TLR-induced inflammation is the Suppressor of Cytokine Signaling 1 (SOCS-1), which degrades Mal-dependent p65 phosphorylation. It has been reported that activation of TLR2 and TLR4 can lead to the production of SOCS-1 108 . Therefore, it can be hypothesized that activation of one TLR may induce SOCS-1, which could, in turn, affect the other TLR (Fig.  5 ) 108 . Another key regulator of TLR function and cross-talk is A20 (Fig.  5 ). A20 plays a crucial role in terminating TLR signaling pathways 109 . It suppresses pro-inflammatory responses by targeting key signaling molecules downstream of TLRs, such as TRAF6 and RIP1, for deubiquitination and degradation 110 . This mechanism ensures inflammation resolution and prevents immune system overactivation. A20 is particularly important for ligand-specific tolerance. For instance, TLR1/2 activation by PAM3 strongly upregulates A20, contributing to tolerance by inhibiting MAPKs (JNK and p38) and NF-κB pathways upon subsequent PAM3 stimulation 109 . Similarly, TLR4 stimulation by LPS induces A20, which mediates cross-tolerance by reducing responses to subsequent TLR1/2 activation 109 . Interestingly, A20 expression is induced not only by TLR ligands but also by pro-inflammatory cytokines like TNF-α and IL-1β, highlighting its broad regulatory role 110 . This cross-regulatory function suggests that TLR-induced A20 expression could influence other TLR pathways, either by modulating shared downstream signaling components or by creating an anti-inflammatory environment. In the bovine uterus, A20 expression has been observed to reduce inflammation associated with endometritis 111 . Upregulation of A20 during LPS treatment in the bovine endometrium inhibited LPS-induced inflammation 111 . These findings suggest that A20 plays a role in inducing tolerance in the endometrium and might mediate cross-talk between TLR2 and TLR4. Fig. 5 Overview of TLR2 and TLR4 signaling pathways and their interplay. Both TLR2 and TLR4 utilize the MyD88-dependent signaling pathway, leading to the production of pro-inflammatory cytokines and inhibitors such as SOCS and A20. These inhibitors modulate MyD88 signaling and contribute to the regulation of the immune response. Additionally, TLR1 and TLR6 influence both the TLR2 and TLR4 systems, playing a role in their interplay and coordination. The figure has been created using BioRender.com software. Both TLR2 and TLR4 utilize the MyD88-dependent signaling pathway, leading to the production of pro-inflammatory cytokines and inhibitors such as SOCS and A20. These inhibitors modulate MyD88 signaling and contribute to the regulation of the immune response. Additionally, TLR1 and TLR6 influence both the TLR2 and TLR4 systems, playing a role in their interplay and coordination. The figure has been created using BioRender.com software. In addition, other factors can mediate the interplay between TLR2 and TLR4. It has been reported that while TLR4 cannot directly sense gram-positive bacteria, it can amplify the inflammatory response initiated by TLR2 112 . It has been indicated that the gram-positive bacterium Mycoplasma pneumoniae activates the MyD88 and NF-κB pathways through TLR2/1 and TLR2/6 signaling, and in this model, bacterial clearance was significantly lower in TLR1−/− and TLR6−/− mice compared to wild-type mice, emphasizing the importance of TLR1 and TLR6 in inflammation and bacterial clearance 113 . Surprisingly, in TLR2−/− mice, accelerated clearance and increased IL-12 production were observed, suggesting that inflammation was heavily dependent on TLR4 113 . In TLR2−/− mice, MyD88-dependent and -independent pathways were upregulated through TLR4. This evidence underscores the critical role of TLR2-TLR4 interplay in controlling cytokine production, possibly mediated by TLR1 and TLR6. As previously explained, TLR1 and TLR6 elicit different cellular responses 47 . TLR1 and TLR6 significantly impact TLR2 and TLR4 function, playing key roles in their interplay (Fig.  5 ). In the bovine uterus, TLR1 is associated with weak, mild, and homeostatic inflammation, while TLR6 generally induces a stronger inflammatory response. Notably, TLR1 expression is higher than TLR6 in the bovine uterus, suggesting that TLR1, possibly via both TLR2/1 and TLR4/1, mediates weak and transient inflammation, promoting homeostasis. Conversely, TLR6 interaction with both TLR2 and TLR4 results in severe inflammation. This suggests that TLR6 may mediate the TLR2-TLR4 interplay, leading to strong inflammatory responses. In conclusion, TLR1 and TLR6 modulate the interplay between TLR2 and TLR4, contributing to either homeostasis or severe inflammation depending on their relative contributions.

Generally, cell membrane TLRs are considered type I integral transmembrane proteins, consisting of three main domains: the N-terminal domain, the transmembrane domain, and the C-terminal domain 20 . The N-terminal domain, located outside the cytoplasm, forms an ectodomain responsible for recognizing pathogen ligands. The transmembrane domain is a single helix embedded in the cell membrane, while the C-terminal domain resides in the cytoplasm and mainly activates signal transduction adapters to initiate downstream signaling 21 . This signaling is mediated through the Toll/IL-1 receptor (TIR) homologous domains. To activate key adapters such as MyD88, MAL/TIRAP, TRIF, TRAM, and SARM, the TIR domains of two TLRs must interact 22 . For this interaction to occur, the TIR domains require close physical association, which necessitates the formation of TLR dimers (either heterodimers or homodimers) 23 – 25 . This dimerization is triggered by the binding of ligands or bridging molecules. The necessity of ligands or bridging molecules arises from the inherent lack of affinity between TLRs in their dimeric forms 20 , 23 – 26 . Bridging ligands stabilize the dimeric structure by interacting with both TLRs and maintaining their closeness, thereby facilitating effective signaling. To activate TLR2 for selective cellular signaling or responses, such as cytokine expression, TLR2 must form dimers. These include heterodimers with TLR1 and TLR6 or homodimers with another TLR2, although homodimerization remains a topic of ongoing debate 27 . Some studies have also reported that TLR2 can form heterodimers with TLR4 and TLR10 28 , 29 . As explained above, due to the lack of strong intrinsic affinity between TLR2 and other TLRs, a bridging ligand is essential to stabilize the dimeric forms. Computational studies combined with binding free energy (BFE) calculations in human, mouse, and bovine species have demonstrated the absence of spontaneous interactions between TLRs in dimer forms 20 , 26 . Conversely, these studies indicated strong interactions between bridging ligands and TLR2 dimers, highlighting the critical role of such molecules in stabilizing these complexes 20 , 26 . The type of ligand determines the specific dimerization of TLR2. For example, triacylated lipopeptides derived from Gram-negative bacteria or mycoplasma are recognized by TLR2/1 heterodimers, while diacylated lipopeptides and lipoteichoic acid from Gram-positive bacteria and mycoplasma are detected by TLR2/6 heterodimers 10 , 30 – 32 . Additionally, low-endotoxic atypical lipopolysaccharides (LPS) have been reported to form TLR2/4 heterodimers, whereas Helicobacter pylori LPS induces TLR2/10 dimerization 33 , 34 . Interestingly, certain TLR2 ligands, such as triacylated and diacylated lipopeptides, also bind to TLR10 to form TLR2/10 heterodimers. Proteoglycans have been identified as ligands for TLR2/2 homodimers 27 . Beyond natural ligands, synthetic ligands can act as bridging molecules to stabilize TLR2 dimers. Pam3CSK4 (PAM3), a synthetic triacylated LP, is a well-known TLR2/1 ligand (i.e., agonist) 23 . Similarly, Pam2CSK4 (PAM2) and fibroblast-stimulating lipopeptide 1 are recognized as TLR2/6 ligands 24 , 35 . Diprovocim is another synthetic ligand that activates both TLR2/1 and TLR2/2 dimers 36 . Regarding the inflammatory response mediated by TLR2 dimerization, both pro-inflammatory and anti-inflammatory responses can be induced depending on the cell type and the origin of the ligand 30 . While the heterodimerization of TLR2 with TLR1 or TLR6 is predominantly associated with pro-inflammatory responses, the anti-inflammatory reaction can act as an immune regulator in certain contexts, particularly in TLR2/6 interactions 37 . DePaolo et al. reported that the virulence factor LcrV from Yersinia pestis activates TLR2/6, leading to the induction of interleukin 10 (IL-10) in DC, which subsequently becomes tolerogenic 37 . In contrast, when TLR2 heterodimerizes with TLR1, it promotes IL-12p40 production (a pro-inflammatory cytokine), resulting in inflammatory DC and T cell differentiation 37 . To activate TLR4 and initiate selective cellular signaling (either MyD88-dependent or -independent), it must form a homodimer. This homodimerization is triggered by ligands such as LPS. Specifically, TLR4 interacts with a co-receptor protein called myeloid differentiation factor 2 (MD2), which is responsible for detecting LPS 25 . LPS is an important component of the outer membrane of Gram-negative bacteria, consisting of three main parts: lipid A (a hydrophobic domain and the primary endotoxin), a core oligosaccharide, and the O-antigen (a repeating hydrophilic polysaccharide) 38 . LPS binds to a hydrophobic pocket in MD2, where five of its six lipid chains fit snugly. The sixth chain interacts directly with TLR4. This binding induces the formation of a symmetrical “M-shaped” complex comprising two TLR4-MD2-LPS units 25 . This multimerization is essential for activating downstream immune signaling. Additionally, LPS undergoes subtle shifts during binding, allowing its phosphate groups to interact with positively charged regions of TLR4 and MD2. Simultaneously, MD2 undergoes minor structural adjustments to stabilize the interaction, ensuring effective immune activation 25 . Similar to TLR2, both pro-inflammatory and anti-inflammatory responses have been reported in TLR4-mediated processes. Numerous studies have shown that activation of TLR4 by a variety of ligands can induce pro-inflammatory cytokines through the NF-κB pathway 39 . Anti-inflammatory responses mediated by TLR4 have also been documented 40 . For instance, certain probiotic bacteria, such as Bacillus subtilis , can modulate the immune response via a TLR4-dependent mechanism. This occurs through the action of exopolysaccharides, which promote the induction of anti-inflammatory M2 macrophages or inhibitory DCs 40 . TLRs can utilize both MyD88-dependent and MyD88-independent pathways 40 , 41 . The MyD88-dependent pathway is crucial for activating NF-κB and MAP kinase pathways in response to TLR signaling 40 . Conversely, the MyD88-independent pathway is associated with IRF-3 and IRF-7 activation, ultimately leading to the production of interferon-beta (IFN-β), which has antiviral effects 42 . Both receptors can utilize the MyD88-dependent pathway to activate NF-κB and MAP kinase pathways for pro-inflammatory cytokine production 43 . This process involves a set of intracellular TIR-domain-containing adapters, such as MyD88 and TIRAP, which are initiated by a direct interaction between these two adapters 44 . The key difference between TLR2 and TLR4 is that TLR4 employs the MyD88-independent pathway. This pathway, unique to TLR4 and TLR3, requires the TIR-domain-containing adapter TRAM 45 . In this signaling cascade, the TRIF adapter plays a central role, enabling TLR4 to mediate IFN-β production. Notably, TLR2 is incapable of inducing IFN-β expression, a function specific to TLR4 46 . There is experimental evidence suggesting that TLR1 and TLR6 interact with both TLR2 and TLR4, although with distinct effects (Fig.  1 ). In epithelial cells, the distinct roles of TLR2/1 and TLR2/6 heterodimers are evident in modulating immune responses and maintaining epithelial integrity during inflammation 47 . TLR2/1 signaling, involving TLR1 as a co-receptor, plays a protective role by limiting excessive inflammation and supporting epithelial homeostasis (Fig.  1 ). Notably, TLR1 deficiency leads to a sharp increase in pro-inflammatory cytokine production (e.g., TNF and IL-1β), resulting in severe tissue damage 47 . In contrast, TLR2/6 signaling, mediated by TLR6, is linked to a robust inflammatory response that, while effective against microbial threats, can worsen tissue injury (Fig.  1 ). Deletion of TLR6 significantly reduces levels of inflammatory cytokines, including TNF, IL-1β, and IL-17A, and mitigates epithelial damage, highlighting its role in driving a pro-inflammatory cascade 47 . Beyond its interaction with TLR2, TLR4 is also proposed to form heterodimers with TLR1 and TLR6. A report in human endothelial cells suggests that TLR1 associates with TLR4 through a distinct mechanism, primarily suppressing TLR4-mediated LPS signaling 48 . This suppression is linked to extracellular, rather than intracellular, signaling. Thus, it can be concluded that TLR1 plays a central role in the interplay between TLR2 and TLR4 (Fig.  1 ). Specifically, TLR1 primarily acts by activating TLR2 to induce a mild inflammatory response while suppressing TLR4 to restrain potentially harmful innate immune activation 26 , 48 . Also, TLR4 can form a heterodimer with TLR6, which plays a significant role in neuroinflammation and is implicated in Alzheimer’s disease 49 . Shmuel-Galia et al. identified the critical involvement of transmembrane domains (TMDs) in the TLR4/6 heterodimer, particularly when stimulated by amyloid-beta peptides 49 . Using a peptide-based interference approach, the authors demonstrated that TMD-derived peptides selectively inhibited TLR4-TLR6 dimerization without affecting TLR2-TLR6 interactions (Fig.  1 ). This disruption effectively reduced microglia-mediated inflammatory responses, highlighting a potential therapeutic strategy for controlling neuroinflammation 49 . Additionally, TLR2 and TLR4 can interact directly as a heterodimer. Studies by Francisco et al. showed that TLR2/4 heterodimers recognize atypical LPS, though the inflammatory response is weaker compared to the TLR4/4 homodimer 34 . In summary, the functional divergence between TLR2 and TLR4 signaling hinges on TLR1’s distinct roles. TLR2 necessitates heterodimerization with TLR1 to activate the MyD88-dependent pathway, driving pro-inflammatory cytokine production 20 , 26 . Conversely, TLR1 appears to exert a regulatory, potentially inhibitory, influence on TLR4-mediated inflammatory responses. Fig. 1 The effect of different TLR dimerizations on immune responses. TLR1 forms dimers with TLR2 and TLR4, resulting in mild inflammation and ultimately promoting cellular homeostasis. In contrast, TLR6, when paired with TLR2 or TLR4, leads to severe inflammation and tissue damage. Additionally, TLR4 homodimerization induces higher levels of inflammation compared to TLR2/TLR4 heterodimerization. TLR1 forms dimers with TLR2 and TLR4, resulting in mild inflammation and ultimately promoting cellular homeostasis. In contrast, TLR6, when paired with TLR2 or TLR4, leads to severe inflammation and tissue damage. Additionally, TLR4 homodimerization induces higher levels of inflammation compared to TLR2/TLR4 heterodimerization.

This review highlights the complex interplay between TLR2 and TLR4 in male and female reproductive biology, emphasizing their shared and distinct signaling mechanisms. TLR2 and TLR4 act as a double-edged sword in reproductive processes. On one hand, they are essential for initiating immune responses to defend against pathogens and for critical functions such as sperm activation, sperm-uterine interaction, and uterine tolerance and immunity. On the other hand, excessive activation can lead to severe inflammation and tissue damage, negatively impacting reproductive health and economic outcomes. Balancing immune responses, particularly the collaboration between TLR2 and TLR4, is vital for maintaining cellular homeostasis. Both TLRs rely on the MyD88-dependent pathway to activate NF-κB and drive inflammatory responses, which creates the potential for mutual influence during activation. However, TLR4 uniquely employs the TRIF-dependent pathway to induce interferon production, a mechanism absent in TLR2. Regulatory factors such as SOCS-1 and A20 play critical roles in modulating these pathways, ensuring balanced immune responses and preventing excessive inflammation. Additionally, TLR1 and TLR6 significantly contribute to the TLR2–TLR4 interplay, influencing the balance between transient and severe inflammatory responses depending on their involvement. Importantly, understanding these mechanisms opens new avenues for therapeutic interventions. TLRs may serve as potential drug targets; for example, selective use of TLR agonizts or inhibitors could modulate fertility or mitigate adverse pregnancy outcomes 107 . However, substantial biological and clinical knowledge gaps remain. For instance, the role of TLRs in innate immune memory within the reproductive tract is not fully understood. Moreover, chronic TLR-mediated activation may have long-term effects, potentially contributing to irregular estrous or menstrual cycles, ovulatory dysfunction, or chronic pelvic pain. Future research addressing these gaps will be essential for developing targeted and safe interventions to support reproductive health in mammalian species. Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Toll-like receptors (TLRs) are well-known ligand-bound transmembrane proteins belonging to the pattern-recognition receptor (PRR) family, playing a central role in the innate immunity 1 . While TLRs are primarily recognized for their role in detecting microbial components, their involvement extends beyond immunity to include diverse functions in health and disease, such as inflammation, tissue homeostasis, and cancer 2 , 3 . In mammals, ten TLRs have been identified and are classified into two groups based on their cellular localization: cell membrane TLRs (TLR1, TLR2, TLR4, TLR5, and TLR6) and intracellular vesicle TLRs (TLR3, TLR7, TLR8, and TLR9) 4 . Additionally, TLR10 is expressed on the cell membrane in certain species such as humans, although its function remains unclear 5 . Other TLRs, like TLR11, TLR12, and TLR13, are not universally expressed across mammalian species 6 . Among these receptors, TLR2 and TLR4 are particularly well-studied due to their unique and broad roles in innate immunity 7 . TLRs can recognize both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Specifically, TLR2 and TLR4 recognize a wide range of microbial components, including those from bacteria, fungi, viruses, and parasites 8 – 10 . Their expression extends across various tissues, including immune cells (e.g., macrophages and dendritic cells (DCs)), epithelial cells, and reproductive tissues 11 , 12 . Beyond their immune functions, these receptors are implicated in non-immune processes such as tissue homeostasis, metabolism, reproduction, and pathogenesis of diseases like cancer and chronic inflammatory conditions 13 – 16 . While TLR2 and TLR4 are often the first lines of defense against extracellular pathogens, intracellular TLRs play a critical role in combating intracellular infections, particularly viral infections 17 . Thus, both extracellular and intracellular TLRs are essential components of the host’s defense against a wide range of pathogens. In addition to PAMPs, TLR2 and TLR4 are activated by a diverse array of endogenous DAMPs released from damaged or stressed cells, to initiate immune signaling. DAMPs include a wide range of molecules, consisting of nucleic acids, proteins, ions, glycans, and metabolites. Several DAMPs have been reported to activate TLRs, and specifically for TLR2 and TLR4, key examples include PAUF, API5, RPS3, HMGB1, DNA-binding protein, heat shock proteins, DJ-1, extra domain A-fibronectin, tenascin C, oxidized lipids in the extracellular matrix, S100 proteins, and cold-inducible RNA-binding protein 18 . These proteins are also expressed in the reproductive tract and can influence TLR signaling 19 . In the context of reproductive biology, DAMPs are likely to play a significant role in several key processes, including ovulation, decidualization, parturition, sperm functions, and sperm–female reproductive tract (FRT) interactions. The precise roles of specific DAMPs in these processes are actively being investigated, highlighting the complex interplay between innate immunity and reproductive physiology. Recent evidence highlights the important role of TLR2 and TLR4 in reproductive health, where both physiological and pathological inflammation play pivotal roles. Physiological inflammation is requisite for processes like uterine clearance and embryo implantation, while excessive or pathological inflammation can lead to severe tissue damage and infertility. The pathways mediating these processes often overlap, making it essential to understand the interplay between physiological and pathological inflammation. While TLR2 and TLR4’s roles in innate immunity are well-characterized, their combined impact on reproductive biology remains underexplored. This review aims to address this gap by examining the roles of TLR2 and TLR4 in reproduction, their interactions within the environment of the reproductive tract and their importance in both inflammatory and immune responses, and reproductive outcomes.

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