IL-6 from cerebrospinal fluid causes widespread pain via STAT3-mediated astrocytosis in chronic constriction injury of the infraorbital nerve.

BACKGROUND: The spinal inflammatory signal often spreads to distant segments, accompanied by widespread pain symptom under neuropathological conditions. Multiple cytokines are released into the cerebrospinal fluid (CSF), potentially inducing the activation of an inflammatory cascade at remote segments through CSF flow. However, the detailed alteration of CSF in neuropathic pain and its specific role in widespread pain remain obscure. METHODS: A chronic constriction injury of the infraorbital nerve (CCI-ION) model was constructed, and pain-related behavior was observed on the 7th, 14th, 21st, and 28th days post surgery, in both vibrissa pads and hind paws. CSF from CCI-ION rats was transplanted to naïve rats through intracisternal injection, and thermal and mechanical allodynia were measured in hind paws. The alteration of inflammatory cytokines in CCI-ION's CSF was detected using an antibody array and bioinformatic analysis. Pharmacological intervention targeting the changed cytokine in the CSF and downstream signaling was performed to evaluate its role in widespread pain. RESULTS: CCI-ION induced local pain in vibrissa pads together with widespread pain in hind paws. CCI-ION's CSF transplantation, compared with sham CSF, contributed to vibrissa pad pain and hind paw pain in recipient rats. Among the measured cytokines, interleukin-6 (IL-6) and leptin were increased in CCI-ION's CSF, while interleukin-13 (IL-13) was significantly reduced. Furthermore, the concentration of CSF IL-6 was correlated with nerve injury extent, which gated the occurrence of widespread pain. Both astrocytes and microglia were increased in remote segments of the CCI-ION model, while the inhibition of astrocytes in remote segments, but not microglia, significantly alleviated widespread pain. Mechanically, astroglial signal transducer and activator of transcription 3 (STAT3) in remote segments were activated by CSF IL-6, the inhibition of which significantly mitigated widespread pain in CCI-ION. CONCLUSION: IL-6 was induced in the CSF of the CCI-ION model, triggering widespread pain via activating astrocyte STAT3 signal in remote segments. Therapies targeting IL-6/STAT3 signaling might serve as a promising strategy for the widespread pain symptom under neuropathological conditions.

Downregulation of CRHBP is associated with trophoblast invasion inhibition in recurrent pregnancy loss.

In brief: Corticotropin-releasing hormone binding protein (CRHBP) is fundamental to the stress response and plays an important role in parturition during pregnancy. This study shows that abnormal CRHBP expression could be an early warning sign of recurrent pregnancy loss and that CRHBP knockdown could suppress HTR8/SVneo cell invasion by the PKC signaling pathway via interacting with CRH receptor 2. Abstract: Trophoblast invasion is critical for placentation and pregnancy success. Trophoblast dysfunction results in many pregnancy complications, including recurrent pregnancy loss (RPL). Corticotropin-releasing hormone binding protein (CRHBP) is fundamental to the stress response and plays an important role in parturition during pregnancy via binding with CRH. To further characterize its function in early pregnancy, we explored the expression of CRHBP in villi during early pregnancy. Compared with normal pregnant women, we demonstrated that the expression of CRHBP decreased in the trophoblasts and villi in RPL patients and that knockdown of CRHBP expression could suppress HTR8/SVneo cell invasion significantly. Our further exploration indicated that the capacity of CRHBP for regulating trophoblast invasion was associated with the PKC signaling pathway via interacting with CRH receptor 2. These findings might provide a new fundamental mechanism for successful pregnancy and a new diagnostic and therapeutic target for RPL.

Tim-3 Coordinates Macrophage-Trophoblast Crosstalk via Angiogenic Growth Factors to Promote Pregnancy Maintenance.

T-cell immunoglobulin mucin-3 (Tim-3) is an important checkpoint that induces maternal-fetal tolerance in pregnancy. Macrophages (Mφs) play essential roles in maintaining maternal-fetal tolerance, remodeling spiral arteries, and regulating trophoblast biological behaviors. In the present study, the formation of the labyrinth zone showed striking defects in pregnant mice treated with Tim-3 neutralizing antibodies. The adoptive transfer of Tim-3+Mφs, rather than Tim-3-Mφs, reversed the murine placental dysplasia resulting from Mφ depletion. With the higher production of angiogenic growth factors (AGFs, including PDGF-AA, TGF-α, and VEGF), Tim-3+dMφs were more beneficial in promoting the invasion and tube formation ability of trophoblasts. The blockade of AGFs in Tim-3+Mφs led to the narrowing of the labyrinthine layer of the placenta, compromising maternal-fetal tolerance, and increasing the risk of fetal loss. Meanwhile, the AGFs-treated Tim-3-Mφs could resolve the placental dysplasia and fetal loss resulting from Mφ depletion. These findings emphasized the vital roles of Tim-3 in coordinating Mφs-extravillous trophoblasts interaction via AGFs to promote pregnancy maintenance and in extending the role of checkpoint signaling in placental development. The results obtained in our study also firmly demonstrated that careful consideration of reproductive safety should be taken when selecting immune checkpoint and AGF blockade therapies in real-world clinical care.

Decidual Stromal Cell Ferroptosis Associated with Abnormal Iron Metabolism Is Implicated in the Pathogenesis of Recurrent Pregnancy Loss.

Iron is necessary for various critical biological processes, but iron overload is also dangerous since labile iron is redox-active and toxic. We found that low serum iron and decidual local iron deposition existed simultaneously in recurrent pregnancy loss (RPL) patients. Mice fed with a low-iron diet (LID) also showed iron deposition in the decidua and adverse pregnancy outcomes. Decreased ferroportin (cellular iron exporter) expression that inhibited the iron export from decidual stromal cells (DSCs) might be the reason for local iron deposition in DSCs from low-serum-iron RPL patients and LID-fed mice. Iron supplementation reduced iron deposition in the decidua of spontaneous abortion models and improved pregnancy outcomes. Local iron overload caused ferroptosis of DSCs by downregulating glutathione (GSH) and glutathione peroxidase 4 levels. Both GSH and cystine (for the synthesis of GSH) supplementation reduced iron-induced lipid reactive oxygen species (ROS) and cell death in DSCs. Ferroptosis inhibitor, cysteine, and GSH supplementation all effectively attenuated DSC ferroptosis and reversed embryo loss in the spontaneous abortion model and LPS-induced abortion model, making ferroptosis mitigation a potential therapeutic target for RPL patients. Further study that improves our understanding of low-serum-iron-induced DSC ferroptosis is needed to inform further clinical evaluations of the safety and efficacy of iron supplementation in women during pregnancy.

Tim-3/CTLA-4 pathways regulate decidual immune cells-extravillous trophoblasts interaction by IL-4 and IL-10.

To obtain a successful pregnancy, the establishment of maternal-fetal tolerance and successful placentation are required to be established. Disruption of this immune balance and/or inadequate placental perfusion is believed to be associated with a lot of pregnancy-related complications, such as recurrent spontaneous abortion, pre-eclampsia, and fetal intrauterine growth restriction. Extravillous trophoblasts (EVTs) have the unique ability to instruct decidual immune cells (DICs) to develop a regulatory phenotype for fetal tolerance. Utilizing immortalized human first trimester extravillous trophoblast cells and primary EVTs, we found that DICs promote EVT function and placental development. We have previously shown that checkpoints T-cell immunoglobulin mucin-3 (Tim-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are important for DIC function. In the present study, we showed that blockade of Tim-3 and CTLA-4 pathways leaded to the abnormal DICs-EVTs interaction, poor placental development, and increased fetal loss. Treatment with IL-4 and IL-10 could rescue the adverse effects of targeting Tim-3 and CTLA-4 on the pregnancy outcome. Hence, the reproductive safety must be a criterion considered in the assessment of immuno-therapeutic agents. In addition, IL-4 and IL-10 may represent novel therapeutic strategies to prevent pregnancy loss induced by checkpoint inhibition.

Altered frequency and function of spleen CTLA-4+Tim-3+ T cells are associated with miscarriage†.

Normal pregnancy is associated with several immune adaptations in both systemic and local maternal-fetal interface to allow the growth of semi-allogeneic conceptus. A failure in maternal immune tolerance to the fetus may result in abnormal pregnancies, such as recurrent spontaneous abortion. The regulation of T-cell homeostasis during pregnancy has important implications for maternal tolerance and immunity. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and T-cell immunoglobulin mucin-3 (Tim-3) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here we described the lower frequency of splenic T cells co-expressing CTLA-4 and Tim-3 accompanied by higher levels of proinflammatory but lower anti-inflammatory cytokines production in abortion-prone mouse model. Blockade of CTLA-4 and Tim-3 pathways leaded to the dysfunction of splenic T cells. By the higher expression during normal pregnancy, CTLA-4 and Tim-3 co-expression on splenic T cells linked to immunosuppressive phenotype. As the spleen is an important site for peripheral immune activation, our data suggest potential noninvasive biomarkers and therapeutic targets for miscarriage.

Decreased level of Eomes+dCD8+ T cells with altered function might be associated with miscarriage.

The T-box transcription factor protein eomesodermin (Eomes) is known for both homeostasis and function of effector and memory CD8+T cells. However, much less is known about the functional regulation of Eomes on CD8+ T cells during pregnancy. In the present study, we concluded the higher Eomes expression dCD8+T cells during normal early pregnancy. The number of Eomes+dCD8+T cells decreased in miscarriage. This Eomes+dCD8+T cell subset also expressed less growth-promoting factors, shifted toward pro-inflammatory phenotype in miscarriage. Primary Trophoblasts and HTR8/SVneo cell line could increase Eomes expression of dCD8+T cells from both normal early pregnancy and miscarriage, which might provide a new strategy for therapy to promote maternal-fetal tolerance and prevent pregnancy loss. These findings indicated that Eomes might be promising early warming targets of miscarriage. In addition, this study suggested that the reproductive safety must be a criterion considered in modulating the dose and function of Eomes in CD8+T cells to reverse T cell exhaustion.

Galectin-9 regulates HTR8/SVneo function via JNK signaling.

To obtain a successful pregnancy, trophoblasts must provide a physical barrier, suppress maternal reactivity, produce immunosuppressive hormones locally, and enhance the production of blocking factors that are able to bind to several antigenic sites. Inadequate placental perfusion has been closely associated with several pregnancy-associated diseases. Galectin-9 (Gal-9) has a wide variety of regulatory functions in innate and adaptive immunity during infection, tumor growth, and organ transplantation. We utilized immortalized human first-trimester extravillous trophoblast cells (HTR8/SVneo) for our functional study and examined the effects of Gal-9 on apoptosis, cytokine production and angiogenesis of HTR8/SVneo cells. Gal-9 inhibited the apoptosis and IFN-γ and IL-17A production, promoted IL-4 production, and coordinated the crosstalk between HTR8/SVneo cells and human umbilical vein endothelial cells via its interaction with Tim-3. Blockade of JNK signaling inhibited Gal-9 activities in HTR8/SVneo cells. In addition, we detected a correlation between low levels of Gal-9 and spontaneous abortion. So Gal-9 could inhibit the apoptosis and proinflammatory cytokine expression, and promote the angiogenesis and IL-4 production in HTR8/SVneo cells via Tim-3 in a JNK dependent manner to help the maintenance of normal pregnancy. These findings possibly identify Gal-9 as a key regulator of trophoblast cells and suggest its potential as a biomarker and target for the treatment of recurrent pregnancy loss.

Trophoblast-derived hyaluronan promotes the regulatory phenotype of decidual macrophages.

There is delicate crosstalk between fetus-derived trophoblasts (Tros) and maternal cells during normal pregnancy. Dysfunctions in interaction are highly linked to some pregnancy complications, such as recurrent spontaneous abortion (RSA), pre-eclampsia and fetal growth restriction. Hyaluronan (HA), the most abundant component of extracellular matrix, has been reported to act as both a pro- and an anti-inflammatory molecule. Previously, we reported that HA promotes the invasion and proliferation of Tros by activating PI3K/Akt and MAPK/ERK1/2 signaling pathways. While lower HA secretion by Tros was observed during miscarriages than that during normal pregnancies, in the present study, we further confirmed that higher secretion of HA by Tros could induce M2 polarization of macrophages at the maternal-fetal interface by interacting with CD44 and activating the downstream PI3K/Akt-STAT-3/STAT-6 signaling pathways. Furthermore, HA could restore the production of IL-10 and other normal pregnancy markers by decidual macrophages (dMφs) from RSA. These findings underline the important roles of HA in regulating the function of dMφs and maintaining a normal pregnancy.

Reduced neurogenesis in human hippocampus with Alzheimer's disease.

Adult hippocampal neurogenesis (AHN), essential for the plasticity of hippocampal structure and function, may be disrupted in Alzheimer's disease (AD). However, the relationship between the changes in AHN and AD-related pathology in humans remains uncertain. By utilizing advanced immunostaining techniques, we could identify multiple biomarkers representing different stages of AHN in postmortem human hippocampal tissue that exhibited various AD-related neuropathological changes. In this study, we observed a significant presence of neurogenic cells in the hippocampus's dentate gyrus (DG) region in 30 individuals, including 14 individuals diagnosed with AD-related neuropathological changes and the remaining 16 individuals without any neurological diseases. Further investigation revealed that patients with AD exhibited pronounced astrogliosis and reduced neurogenesis. Specifically, the number of neuroblasts, immature and early mature granule cells decreased significantly as AD advanced. Although the number of neural stem cells (NSCs) remained unchanged in AD patients compared with mentally healthy individuals, they tended to be more quiescent state regulated by Notch and bone morphogenetic protein (BMP) signaling pathways. These abnormalities were strongly associated with the neuropathological alterations in AD patients. These research findings provide potential insights into the underlying mechanisms that underpin the pathogenesis of AD.

TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein.

Pain has become a major symptom of long COVID-19 without effective therapy. Apart from viral infection pathological process, SARS-CoV-2 membranal proteins (envelope [S2E], spike [S2S] and membrane [S2M]) also present pro-inflammatory feature independently. Here, we aim to uncover the neuroinflammatory mechanism of COVID-pain induced by SARS-CoV-2 membranal proteins. We detected the three proteins in both peripheral sensory ganglions and spinal dorsal horn of COVID-19 donors. After intradermal and intrathecal injection, only S2E triggered pain behaviors, accompanied with upregulated-phosphorylation nuclear factor kappa B (NF-κB), which was significantly attenuated by minocycline in mice. We further identified Toll-like receptor 2 (TLR2) among TLRs as the target of S2E to evoke inflammatory responses leading to COVID-pain. This study identified the nociceptive effect of S2E through directly interacting with macrophage/microglia TLR2 and inducing the following NF-κB inflammatory storm. Clearing away S2E and inhibiting macrophage/microglia TLR2 served as perspective therapeutic strategies for COVID-19 pain.

Rhodojaponin VI indirectly targets Cav2.2 channels via N-ethylmaleimide-sensitive fusion protein to alleviate neuropathic pain.

Neuropathic pain is a chronic disease that severely afflicts the life and emotional status of patients, but currently available treatments are often ineffective. Novel therapeutic targets for the alleviation of neuropathic pain are urgently needed. Rhodojaponin VI, a grayanotoxin from Rhododendron molle, showed remarkable antinociceptive efficacy in models of neuropathic pain, but its biotargets and mechanisms are unknown. Given the reversible action of rhodojaponin VI and the narrow range over which its structure can be modified, we perforwmed thermal proteome profiling of the rat dorsal root ganglion to determine the protein target of rhodojaponin VI. N-Ethylmaleimide-sensitive fusion (NSF) was confirmed as the key target of rhodojaponin VI through biological and biophysical experiments. Functional validation showed for the first time that NSF facilitated trafficking of the Cav2.2 channel to induce an increase in Ca2+ current intensity, whereas rhodojaponin VI reversed the effects of NSF. In conclusion, rhodojaponin VI represents a unique class of analgesic natural products targeting Cav2.2 channels via NSF.

Focal-type, but not Diffuse-type, Amyloid Beta Plaques are Correlated with Alzheimer's Neuropathology, Cognitive Dysfunction, and Neuroinflammation in the Human Hippocampus.

Amyloid beta (Aß) plaques are one of the hallmarks of Alzheimer's disease (AD). However, currently available anti-amyloid therapies fail to show effectiveness in the treatment of AD in humans. It has been found that there are different types of Aß plaque (diffuse and focal types) in the postmortem human brain. In this study, we aimed to investigate the correlations among different types of Aß plaque and AD-related neuropathological and cognitive changes based on a postmortem human brain bank in China. The results indicated that focal plaques, but not diffuse plaques, significantly increased with age in the human hippocampus. We also found that the number of focal plaques was positively correlated with the severity of AD-related neuropathological changes (measured by the "ABC" scoring system) and cognitive decline (measured by the Everyday Cognitive Insider Questionnaire). Furthermore, most of the focal plaques were co-localized with neuritic plaques (identified by Bielschowsky silver staining) and accompanied by microglial and other inflammatory cells. Our findings suggest the potential of using focal-type but not general Aß plaques as biomarkers for the neuropathological evaluation of AD.

Tim-3+ decidual Mφs induced Th2 and Treg bias in decidual CD4+T cells and promoted pregnancy maintenance via CD132.

T-cell immunoglobulin mucin-3 (Tim-3) plays roles in the functional regulation of both adaptive and innate immune cells and is greatly involved in many diseases. However, the precise roles of Tim-3 on macrophages (Mφs) in pregnancy remain unstated. In the current study, we found the higher frequency of Tim-3+ decidual Mφs (dMφs) in response to trophoblasts. The reduced abundance of Tim-3 on Mφs was accompanied by disordered anti- and pro-inflammatory cytokine profiles in miscarriage. Adoptive transfer of Tim-3+Mφs, but not Tim-3-Mφs, relieved murine embryo absorption induced by Mφ depletion. Our flow cytometry results and the extensive microarray analysis confirmed that Tim-3+ and Tim-3-dMφs were neither precisely pro-inflammatory (M1) nor anti-inflammatory (M2) Mφs. However, with higher CD132 expression, Tim-3+dMφs subset induced Th2 and Treg bias in decidual CD4+T cells and promoted pregnancy maintenance. Blockade of Tim-3 or CD132 pathways leaded to the dysfunction of maternal-fetal tolerance and increased fetal loss. These findings underscored the important roles of Tim-3 in regulating dMφ function and maintaining normal pregnancy, and suggested that Tim-3 on Mφs is a potential biomarker for diagnosis of miscarriage. Our study also emphasized the importance of careful consideration of reproductive safety when choosing immune checkpoint blockade therapies in real world clinical care. Though IL-4 treated Tim-3-Mφs could rescue the fetal resorption induced by Mφ depletion, whether IL-4 represent novel therapeutic strategy to prevent pregnancy loss induced by checkpoint inhibition still needs further research.

Functional regulation of decidual macrophages during pregnancy.

A successful pregnancy requires that the maternal immune system recognizes and tolerates the semi-allogeneic fetus without compromising the capability of protecting both mother and fetus from various pathogens. Decidual macrophages present unique phenotypes to play a key role in the establishment of the immunological aspects of maternal-fetal interaction. Dysfunction of decidual macrophages gives rise to pregnancy complications such as preeclampsia, recurrent spontaneous miscarriage, preterm labor and fetal growth restriction. Here, we reviewed the latest knowledge on the origin, differentiation, unique phenotype and function of macrophages in normal pregnancy and in pregnancy complications. We mainly focused on the significant roles of decidual macrophages in the process of extravillous trophoblast invasion, spiral arterial remodeling, decidual stromal cells cultivation and immune tolerance maintenance in normal pregnancy, and their pathological roles in pregnancy-related complications, offering more integrated information in maternal-fetal immunity.

Eomesodermin regulate decidual CD4+T cell function during human early pregnancy.

Decidual CD4+T (dCD4+T) cells play pivotal roles in inducing and maintaining maternal-fetal tolerance. Dysfunctional dCD4+T cells are associated with miscarriage. In the present study, we demonstrated that the T-box transcription factor protein eomesodermin (Eomes) was involved in the functional regulation of dCD4+T cells during early pregnancy. We concluded the higher Eomes expression dCD4+T cells during normal pregnancy, and the Eomes+dCD4+T cells displayed an active status and produced more Th2- and Treg type cytokines. Decreased number and altered function of Eomes+dCD4+T cells were observed in miscarriage. Progesterone, the traditional treatment for miscarriage, had no effect on Eomes expression by dCD4+T cells from normal pregnancy, but increased Eomes expression by dCD4+T cells from miscarriage. We also found the higher frequency of Eomes+dCD4+T cells from miscarriage in response to cyclosporine, tacrolimus, Trophoblasts, and HTR8/SVneo cell line, might provide new strategy for therapy to promote maternal-fetal tolerance and prevent pregnancy loss. These results indicated that Eomes might be promising early warming targets of miscarriage, though further studies are required to determine that the altered number and function of Eomes+dCD4+T cells are the cause or consequence of miscarriage.

Involvement of the Tim-3 Pathway in the Pathogenesis of Pre-Eclampsia.

Current methods of early diagnosis and prevention of pre-eclampsia (PE) are limited; the only available definite treatment is the initiation of delivery and complete removal of the placenta. Inappropriate activation of the immune system is thought to play considerable roles in PE. T cell immunoglobulin mucin-3 (Tim-3) has been reported to regulate immune responses and play important roles in maternal-fetal tolerance during early pregnancy. In this study, we investigated the functional regulation of Tim-3 in the maternal-fetal crosstalk during 3rd-trimester healthy pregnancy and its possible role in the pathogenesis of PE. We found that Tim-3 expression on decidual immune cells was associated with production of anti-inflammatory cytokines. Tim-3 pathway blockade resulted in higher IFN-γ but lower IL-4 and IL-10 production. Using a tube formation assay between HTR8/SVneo cells and human umbilical vein endothelial cells, we found that Tim-3 pathway blockade inhibits tube formation and reversed by addition of recombinant IL-4 and/or IL-10. Pre-eclamptic patients showed reduced Tim-3 expression on both decidual and peripheral immune cells (especially on peripheral CD8+T cells). Therefore, we proposed that abnormal Tim-3 signal resulted in immunological imbalance at the maternal-fetal interface and may be involved in the progress of PE by affecting uterine spiral artery remodeling. Our study expanded the regulatory function of Tim-3 signaling pathway to the 3rd-trimester pregnancy and provided a new target for early warning and therapeutic strategies of PE.

Th17/Treg-cell balance in the peripheral blood of pregnant females with a history of recurrent spontaneous abortion receiving progesterone or cyclosporine A.

A successful pregnancy requires the maternal immune system to accept a fetus expressing allogeneic paternal antigens and provide competent responses to infections. Accordingly, maternal-fetal immune abnormalities may have an important role in the development of recurrent spontaneous abortion (RSA). Ever since the establishment of the association between immunologic abnormalities and RSA, various types of immune therapy to restore normal immune homeostasis have been increasingly developed. Although previous studies have focused on the maternal-fetal interface, non-invasive examination is of great importance in clinical practice. The present study investigated the balance between type-17 T-helper (Th17) and T-regulatory (Treg) cells in the peripheral blood to improve the current understanding of the pathogenesis of RSA. Imbalances in Th17/Treg cells and associated molecular profiles were observed in patients with RSA. Furthermore, it was determined that the immunosuppressant cyclosporine A reduced the proportion of Th17 cells and promoted Treg-cell dominance by upregulating the expression of co-inhibitory molecules in pregnant females with a history of RSA. Progesterone, the traditional maternal-care drug, also had a certain immunomodulatory role through restoring the levels of several co-inhibitory molecules (including T-cell immunoglobulin mucin family member-3, programmed cell death-1 and cytotoxic T-lymphocyte associated protein-4) in the treatment of RSA. Changes in these immune molecules within the maternal peripheral blood may be indicators for monitoring pregnancy and prediction of RSA.

Bidirectional regulation between 1st trimester HTR8/SVneo trophoblast cells and in vitro differentiated Th17/Treg cells suggest a fetal-maternal regulatory loop in human pregnancy.

PROBLEM: During normal pregnancy, delicate crosstalk is established between fetus-derived trophoblasts and maternal immune cells to ensure maternal-fetal tolerance and successful placentation. Dysfunction in these interactions has been highly linked to certain pregnancy complications. METHOD OF STUDY: Naïve CD4+ T cells were cultivated with or without 1st trimester derived trophoblast cell line HTR8/SVneo cells in the absence or presence of T helper 17 (Th17) or regulatory (Treg)cell-inducing differentiation conditions. After 5 days of co-culture, HTR8/SVneo cells and CD4+ T cells were harvested and analyzed using flow cytometry. RESULTS: CD4+ T cells exposed to HTR8/SVneo cells showed enhanced induction of CD4+ Foxp3+ Treg cells with strong expression of TGF-ß1 and inhibitory molecules (cytotoxic T lymphocyte-associated protein-4 [CTLA-4], T-cell immunoglobulin mucin-3 [Tim-3], and programmed cell death-1 [PD-1]). Though not effecting Th17 differentiation, exposure to HTR8/SVneo cells promoted increased expression of proliferative and apoptotic markers on Th17 cells. Co-culture with Th0 cells, or differentiated Th17 or Treg cells, down-regulated Caspase-3 and MMP-9 (but not MMP-2) expression in HTR8/SVneo cells, while promoting Ki67 expression. CONCLUSIONS: HTR8/SVneo cells regulated maternal CD4+ T-cell differentiation, resulting in the expansion of immunosuppressive Treg cells, while CD4+ T cells might promote the growth, and control the invasiveness of HTR8/SVneo cells. Thus, a bidirectional regulatory loop might exist between trophoblasts and maternal immune cell subsets, thereby promoting harmonious maternal-fetal crosstalk.

The appropriate frequency and function of decidual Tim-3+CTLA-4+CD8+ T cells are important in maintaining normal pregnancy.

Maternal decidual CD8+ T (dCD8+ T) cells must integrate the antithetical demands of maternal-fetal tolerance and anti-viral immunity to establish a successful pregnancy. T-cell immunoglobulin mucin-3 (Tim-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are two important co-inhibitory molecules that regulating CD8+ T cells responses during infection and tumor. In the present study, we examined the co-expression of Tim-3 and CTLA-4 on CD8+ T cells during pregnancy and found the higher frequency of Tim-3+CTLA-4+dCD8+ T cells in response to trophoblasts. This Tim-3+CTLA-4+dCD8+ T cells subset showed an active status and produced more anti-inflammatory cytokines. Furthermore, the decreased number and altered function of Tim-3+CTLA-4+dCD8+ T cells correlated to miscarriage. Combined blocking Tim-3 and CTLA-4 pathways were highly effective in inhibiting the production of anti-inflammatory cytokines and were detrimental to the maintenance of pregnancy. Together, these findings supported that Tim-3 and CTLA-4 pathways might play positive roles in the establishment and/or maintenance of maternal-fetal tolerance so to promote the maintenance of normal pregnancy. So the reproductive safety must be considered, especially when anti-Tim-3/CTLA-4 antibody (and other immune checkpoint inhibitors) are used in pregnancy.