Fluorinated Lipid Nanoparticles for Enhancing mRNA Delivery Efficiency.

Lipid nanoparticles (LNPs), a nonviral nucleic acid delivery system, have shown vast potential for vaccine development and disease treatment. LNPs assist mRNA to cross physiological barriers such as cell membranes and endosomes/lysosomes, promoting the intracellular presentation of mRNA. However, the endosome escape efficiency and biosafety of currently commercialized LNPs are still unsatisfactory, resulting in underutilization of mRNA. Herein, we report that fluorinated modification of the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (PEG-DSPE), termed as FPD, in the LNPs can improve the delivery efficiency of mRNA. FPD accounts for only 1.5% of lipids in LNPs but could mediate a 5-fold and nearly 2-fold enhancement of mRNA expression efficiency in B16F10 tumor cells and primary dendritic cells, respectively. Mechanism studies reveal that FPD promotes the cellular internalization of LNPs as well as endosome escape. In vivo studies substantiate that FPD can augment overall mRNA expression at least 3-fold, either by intravenous or intraperitoneal injection, compared to LNPs prepared with nonfluorinated PEG-lipids at a relatively low mRNA dose. Besides, with the introduction of FPD, mRNA expression in the spleen augmented compared to that of the DMG-PEG commercial formulations. Benefiting from a prudent dosage of fluorine, the fluorinated LNPs display favorable biosafety profiles at cellular and zoological levels.

Patient's age with papillary thyroid cancer: Is it a key factor for cervical lymph node metastasis?

BACKGROUND: Age is one of the important prognostic indicators of papillary thyroid cancer (PTC). However, the distinct metastatic patterns and prognosis of age-related lymph node metastasis (LNM) are unclear. This study aims to investigate the impact of age on LNM. METHODS: We conducted two independent cohort studies to assess age-nodal disease association using logistic regression analysis and a restricted cubic splines model. A multivariable Cox regression model was utilized to test the impact of nodal disease on cancer-specific survival (CSS) after age stratification. RESULTS: For this study, we included 7572 and 36,793 patients with PTC in Xiangya and SEER cohorts, respectively. After adjustment, advanced age was linearly associated with decreasing risk of central LNM. Patients of age ≤18 years (OR = 4.41, P < 0.001) and 19-45 years (OR = 1.97, P = 0.002) had a higher risk of developing lateral LNM than patients of age >60 years in both cohorts. Furthermore, CSS is significantly reduced in N1b disease (P < 0.001), not N1a disease, regardless of age. The incidence of high-volume LNM (HV-LNM) was significantly higher in patients of age ≤18 years and 19-45 years than in those of age >60 years (P < 0.001), in both cohorts. In addition, CSS was compromised in patients with PTC of age 46-60 years (HR = 1.61, P = 0.022) and those of age >60 (HR = 1.40, P = 0.021) after developing HV-LNM. CONCLUSIONS: Patient age is significantly associated with LNM and HV-LNM. Patients with N1b disease or patients with HV-LNM of age >45 years have significantly shorter CSS. Age can, thus, be a useful guide for determining treatment strategies in PTC.

An mRNA vaccine elicits STING-dependent antitumor immune responses.

Lipid-formulated RNA vaccines have been widely used for disease prevention and treatment, yet their mechanism of action and individual components contributing to such actions remain to be delineated. Here, we show that a therapeutic cancer vaccine composed of a protamine/mRNA core and a lipid shell is highly potent in promoting cytotoxic CD8+ T cell responses and mediating anti-tumor immunity. Mechanistically, both the mRNA core and lipid shell are needed to fully stimulate the expression of type I interferons and inflammatory cytokines in dendritic cells. Stimulation of interferon-ß expression is exclusively dependent on STING, and antitumor activity from the mRNA vaccine is significantly compromised in mice with a defective Sting gene. Thus, the mRNA vaccine elicits STING-dependent antitumor immunity.

Engineered anti-PDL1 with IFNα targets both immunoinhibitory and activating signals in the liver to break HBV immune tolerance.

OBJECTIVE: The purpose of this study is to develop an anti-PDL1-based interferon (IFN) fusion protein to overcome the chronic hepatitis B virus (HBV)-induced immune tolerance, and combine this immunotherapy with a HBV vaccine to achieve the functional cure of chronic hepatitis B (CHB) infection. DESIGN: We designed an anti-PDL1-IFNα heterodimeric fusion protein, in which one arm was derived from anti-PDL1 antibody and the other arm was IFNα, to allow targeted delivery of IFNα into the liver by anti-PDL1 antibody. The effect of the anti-PDL1-IFNα heterodimer on overcoming hepatitis B surface antigen (HBsAg) vaccine resistance was evaluated in chronic HBV carrier mice. RESULTS: The anti-PDL1-IFNα heterodimer preferentially targeted the liver and resulted in viral suppression, the PD1/PDL1 immune checkpoint blockade and dendritic cell activation/antigen presentation to activate HBsAg-specific T cells, thus breaking immune tolerance in chronic HBV carrier mice. When an HBsAg vaccine was administered soon after anti-PDL1-IFNα heterodimer treatment, we observed strong anti-HBsAg antibody and HBsAg-specific T cell responses for efficient HBsAg clearance in chronic HBV carrier mice that received the combination treatment but not in those that received either single treatment. CONCLUSIONS: Targeting the liver with an engineered anti-PDL1-IFNα heterodimer can break HBV-induced immune tolerance to an HBsAg vaccine, offering a promising translatable therapeutic strategy for the functional cure of CHB.

Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri-implantation period.

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1+ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single-cell RNA sequencing (scRNA-seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue-resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function-related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA-seq and cytotoxicity assays confirmed that CYP26A1+ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal-foetal interface by regulating the activity of NK cells.

Antitumor Immunity from Abdominal Flap-Embedded Therapeutic Cancer Vaccine.

BACKGROUND: Abdominal flaps are routinely performed in clinic after primary mastectomy of breast cancer. However, cancer patients can still develop cancer recurrence and metastasis after surgery. In this study, we evaluated the feasibility of concurrent abdominal flap reconstruction and vaccine inoculation in the tissue for prevention and treatment of HER2-positive breast cancer. METHODS: A murine model of metastatic HER2-positive breast cancer was generated by inoculating HER2-expressing TUBO tumor cells into both the mammary gland fat pad and left ventricle. Mammary gland fat pad with primary tumor was resected by mastectomy, and superficial inferior epigastric (SIE) vessel-based abdominal flap was performed for abdominal reconstruction. During the surgery, mice also received a single intra-flap treatment of a microparticulate-based cancer vaccine. Popliteal (Pop) and inguinal (Ing) lymph nodes (LN) were collected at different time points after vaccination, and activation of dendritic cells and T lymphocytes was evaluated with flow cytometry. ELISpot was also performed to measure HER2-specific T cells in splenocytes. In addition, infiltration of CD3+ T cells in brain metastatic nodules was analyzed with immunohistochemistry. RESULTS: Flow cytometry detected increased number of activated dendritic cells in lymph nodes in mice treated with cancer vaccine. ELISpot revealed abundant IFN-γ-expressing T cells in the spleen. Mice treated with abdominal flap-embedded cancer vaccine extended median survival by 9 days over the control group (p<0.05). CONCLUSION: Abdominal flap-embedded cancer vaccine effectively stimulated systemic immune response and inhibited tumor progression in a murine model of HER2-positive breast cancer.

Virus-Mimic mRNA Vaccine for Cancer Treatment.

An effective therapeutic cancer vaccine should be empowered with the capacity to overcome the immunosuppressive tumor microenvironment. Here, the authors describe a mRNA virus-mimicking vaccine platform that is comprised of a phospholipid bilayer encapsulated with a protein-nucleotide core consisting of antigen-encoding mRNA molecules, unmethylated CpG oligonucleotides and positively charged proteins. In cell culture, VLVP potently stimulated bone marrow-derived dendritic cells (BMDCs) to express inflammatory cytokines that facilitated dendritic cell (DC) maturation and promoted antigen processing and presentation. In tumor-bearing mice, VLVP treatment stimulated proliferation of antigen-specific CD8+T cells in the lymphatic organs and T cell infiltration into the tumor bed, resulting in potent anti-tumor immunity. Cytometry by time of flight (CyTOF) analysis revealed that VLVP treatment stimulated a 5-fold increase in tumor-associated CD8+DCs and a 4-fold increase in tumorinfiltrated CD8+T cells, with concurrent decreases in tumor-associated bone marrow-derived suppressor cells and arginase 1- expressing suppressive DCs. Finally, CpG oligonucleotide is an essential adjuvant for vaccine activity. Inclusion of CpG not only maximized vaccine activity but also prevented PD-1 expression in T cells, serving the dual roles as a potent adjuvant and a checkpoint blockade agent.

The Sympathetic Nervous System Modulates Cancer Vaccine Activity through Monocyte-Derived Cells.

The sympathetic nervous system (SNS) is an important regulator of immune cell function during homeostasis and states of inflammation. Recently, the SNS has been found to bolster tumor growth and impair the development of antitumor immunity. However, it is unclear whether the SNS can modulate APC function. Here, we investigated the effects of SNS signaling in murine monocyte-derived macrophages (moMФ) and dendritic cells (DCs) and further combined the nonspecific ß-blocker propranolol with a peptide cancer vaccine for the treatment of melanoma in mice. We report that norepinephrine treatment dramatically altered moMФ cytokine production, whereas DCs were unresponsive to norepinephrine and critically lack ß2-adrenergic receptor expression. In addition, we show that propranolol plus cancer vaccine enhanced peripheral DC maturation, increased the intratumor proportion of effector CD8+ T cells, and decreased the presence of intratumor PD-L1+ myeloid-derived suppressor cells. Furthermore, this combination dramatically reduced tumor growth compared with vaccination alone. Taken together, these results offer insights into the cell-specific manner by which the SNS regulates the APC immune compartment and provide strong support for the use of propranolol in combination with cancer vaccines to improve patient response rates and survival.

Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global crisis, urgently necessitating the development of safe, efficacious, convenient-to-store, and low-cost vaccine options. A major challenge is that the receptor-binding domain (RBD)-only vaccine fails to trigger long-lasting protective immunity if used alone for vaccination. To enhance antigen processing and cross-presentation in draining lymph nodes (DLNs), we developed an interferon (IFN)-armed RBD dimerized by an immunoglobulin fragment (I-R-F). I-R-F efficiently directs immunity against RBD to DLNs. A low dose of I-R-F induces not only high titers of long-lasting neutralizing antibodies (NAbs) but also more comprehensive T cell responses than RBD. Notably, I-R-F provides comprehensive protection in the form of a one-dose vaccine without an adjuvant. Our study shows that the pan-epitope modified human I-R-F (I-P-R-F) vaccine provides rapid and complete protection throughout the upper and lower respiratory tracts against a high-dose SARS-CoV-2 challenge in rhesus macaques. Based on these promising results, we have initiated a randomized, placebo-controlled, phase I/II trial of the human I-P-R-F vaccine (V-01) in 180 healthy adults, and the vaccine appears safe and elicits strong antiviral immune responses. Due to its potency and safety, this engineered vaccine may become a next-generation vaccine candidate in the global effort to overcome COVID-19.

The influence of nodule size on the aggressiveness of thyroid carcinoma varies with patient's age.

BACKGROUND: Thyroid nodule size is one of the key parameters that determines the operative approach for thyroid carcinoma. It is necessary to evaluate the influence of nodule size on the aggressiveness of thyroid carcinoma. The eighth edition of staging system has updated the prognostic age cutoff from 45 to 55 years old. It is needed to re-evaluate the difference in aggressiveness of thyroid carcinoma between younger (<55 years old) and older (≥55 years old) patients. Importantly, whether the influence of nodule size on the aggressiveness of thyroid carcinoma varies according to the new age stratification remains to be explored. METHODS: Medical records from patients were retrospectively reviewed. Patients with a documented thyroid ultrasonography (US), US-guided fine needle aspiration (FNA) and histopathology were included. The risks of unfavorable events such as central-compartment neck lymph node (CLN) metastasis, lateral-compartment neck lymph node (LLN) metastasis and gross extrathyroidal extension (ETE) were analyzed in four subsets of patients according to size and age. RESULTS: Large nodule size (≥10 mm) significantly increased the frequencies of CLN metastasis, LLN metastasis and gross ETE (P<0.05). The frequency of CLN metastasis was significantly higher in younger patients compared with that in older ones. Logistic regression analysis recognized large nodule size as an independent risk factor for all CLN metastasis (OR: 3.304, 95% CI: 2.473-4.415), LLN metastasis (OR: 9.673, 95% CI: 4.542-20.597), and gross ETE (OR: 2.430, 95% CI: 1.508-3.916). Secondly, in younger patients, frequencies of all CLN metastasis, LLN metastasis and gross ETE were significantly higher in nodules ≥10 mm than in nodules <10 mm (P<0.001). However, in older patients, no significant difference was found in the frequencies of LLN metastasis or gross ETE between nodules <10 mm and ≥10 mm. Logistic regression analysis showed, in younger patients, large nodule size was an independent risk factor for all CLN metastasis (OR: 3.241, 95% CI: 2.393-4.389), LLN metastasis (OR: 12.495, 95% CI: 5.281-29.562), and gross ETE (OR: 2.591, 95% CI: 1.519-4.419), while in older patients large nodule size was recognized as an independent risk factor for CLN metastasis (OR: 3.924, 95% CI: 1.413-10.899) but not for LLN metastasis or gross ETE. CONCLUSIONS: Large nodule size is significantly related to high aggressiveness of thyroid carcinoma. The correlation between large nodule size and high aggressiveness varies according to patient's age, indicating that the presence of unfavorable events has different clinical significance for patients of varied ages. These findings contribute to accurately assessing the prognosis of individual patient and developing a better management strategy.

HSF1 functions as a key defender against palmitic acid-induced ferroptosis in cardiomyocytes.

Palmitic acid (PA)-induced myocardial injury is considered a critical contributor to the development of obesity and type 2 diabetes mellitus (T2DM)-related cardiomyopathy. However, the underlying mechanism has not been fully understood. Here, we demonstrated that PA induced the cell death of H9c2 cardiomyoblasts in a dose- and time-dependent manner, while different ferroptosis inhibitors significantly abrogated the cell death of H9c2 cardiomyoblasts and primary neonatal rat cardiomyocytes exposed to PA. Mechanistically, PA decreased the protein expression levels of both heat shock factor 1 (HSF1) and glutathione peroxidase 4 (GPX4) in a dose- and time-dependent manner, which were restored by different ferroptosis inhibitors. Overexpression of HSF1 not only alleviated PA-induced cell death and lipid peroxidation but also improved disturbed iron homeostasis by regulating the transcription of iron metabolism-related genes (e.g., Fth1, Tfrc, Slc40a1). Additionally, PA-blocked GPX4 protein expression was evidently restored by HSF1 overexpression. Inhibition of endoplasmic reticulum (ER) stress rather than autophagy contributed to HSF1-mediated GPX4 expression. Moreover, GPX4 overexpression protected against PA-induced ferroptosis, whereas knockdown of GPX4 reversed the anti-ferroptotic effect of HSF1. Consistent with the in vitro findings, PA-challenged Hsf1-/- mice exhibited more serious ferroptosis, increased Slc40a1 and Fth1 mRNA expression, decreased GPX4 and TFRC expression and enhanced ER stress in the heart compared with Hsf1+/+ mice. Altogether, HSF1 may function as a key defender against PA-induced ferroptosis in cardiomyocytes by maintaining cellular iron homeostasis and GPX4 expression.

Magnesemia: an independent risk factor of hypocalcemia after thyroidectomy.

BACKGROUND: This study was designed to investigate the impact of serum magnesium (Mg) levels on hypocalcemia after thyroidectomy. PATIENTS AND METHODS: In total, 242 patients with differentiated thyroid cancer were retrospectively analyzed. RESULTS: Multivariate regression analysis showed hypomagnesemia was an independent risk factor for hypocalcemia (P<0.001). While Mg in low levels (0.66 mmol/L ≤ Po-Mg ≤0.74 mmol/L) increased the risk of hypocalcemia, postoperative serum Ca (Po-Ca) levels were significantly lower in patients with hypomagnesemia than in patients with normomagnesemia (P=0.01), and the former patients suffered significant decreases in serum Ca (P=0.02). Compared to patients with a mild decline of serum Mg after surgery (ΔMg <0.17), serum Ca decline significantly increased (P<0.001) in patients with a severe decline of serum Mg (ΔMg ≥0.17), while the change in amounts of parathyroid hormone (PTH) after surgery was similar between the two groups (P>0.05). In patients with normal Po-Ca levels, hypomagnesemia increased the risk of symptoms related to hypocalcemia by 4.478 times (OR =5.478, 95% CI 1.724-17.403). CONCLUSION: Hypomagnesemia, or even a low serum Mg level within the normal range, can increase the risk of hypocalcemia. After excluding the potential effects of PTH on serum magnesium and calcium, serum Mg reduction is one of the most important factors that influences postoperative serum Ca reduction. What's more, hypomagnesemia is closely linked with symptoms.

Treatment optimization and prognostic considerations for primary squamous cell carcinoma of the thyroid.

BACKGROUND: Primary squamous cell carcinoma of the thyroid (PSCCT) is a rare aggressive malignancy that usually presents in an advanced stage and has a poor prognosis. Our study aimed to investigate the clinical characteristics, treatment, and prognosis of PSCCT. METHODS: We retrospectively reviewed the medical information of patients with PSCCT diagnosed from January 2006 to May 2018 at Xiangya Hospital. Survival analysis was conducted using the Kaplan-Meier method, and Log-Rank tests were performed for statistical testing. RESULTS: We identified 12 patients with PSCCT (nine males and three females), accounting for only 0.19% of all thyroid cancer diagnosed during this time period. The median age of these patients was 59.5 years old and their symptoms included neck masses (n=5), hoarseness (n=2), dyspnea (n=1), dysphagia (n=1) and neck pain (n=1). Four patients were in stage IVA, five were stage IVB, and three patients were stage IVC. Six patients underwent comprehensive treatment (surgery + radiotherapy or surgery + radiotherapy + chemotherapy) and the remaining patients received radiotherapy and/or chemotherapy. The 6-month survival rate was 66.7%, compared to a 1-year survival rate of 25.0%, with a median overall survival time was 10.5 months. Kaplan-Meier analysis showed that the comprehensive treatment was superior to radiotherapy and/or chemotherapy (P=0.003). CONCLUSIONS: PSCCT is a rare type of thyroid cancer that is highly invasive and has a poor prognosis. We show that a comprehensive treatment plan can significantly improve patient survival.

Hashimoto's thyroiditis elicits decreased diagnostic efficacy of thyroid nodule ultrasound-guided fine needle aspiration.

BACKGROUND: False negative (FN) or false positive (FP) results of thyroid ultrasound-guided fine needle aspiration (US-guided FNA) cause missed diagnosis of thyroid cancer or unnecessary thyroidectomy. PURPOSE: To explore the impact of Hashimoto's thyroiditis (HT) on the diagnostic efficacy of US-guided FNA and to analyze the differences in diagnostic efficacy between US-guided FNA and thyroid ultrasonography (US) in patients with HT. METHOD: Medical records were reviewed retrospectively. Patients with and without Hashimoto's thyroiditis (HT) were included in the exposure and non-exposure group, respectively. RESULTS: HT was not an independent risk factor for thyroid cancer. The percentage of undetermined results of US-guided FNA (Bethesda I, III, IV) in the exposure group was significantly higher. The US-guided FNA's diagnostic sensitivity, specificity, and accuracy were significantly lower, and FP rate (FPR) and FN rate (FNR) were higher in the exposure group. In the exposure group, US tended to give higher diagnostic sensitivity, accuracy, PPV, NPV, and lower FPR and FNR. Receiver operating characteristic (ROC) curve analysis showed that, in the exposure group the diagnostic efficacy of thyroid US was significantly higher than of US-guided FNA. CONCLUSION: HT tends to cause undetermined results and elicit lower diagnostic performance of US-guided FNA. In patients with HT, the diagnostic efficacy of thyroid US is, at least, not inferior to US-guided FNA.

Prediction of permanent hypoparathyroidism by parathyroid hormone and serum calcium 24 h after thyroidectomy.

PURPOSE: To assess the value of 24-hour intact parathyroid hormone (iPTH), serum calcium, and decreases in both were evaluated against preoperative values (iPTH and serum calcium decline) and used to determine the existence of permanent hypoparathyroidism (pHPP) after total thyroidectomy (TT). MATERIALS AND METHODS: The clinical data of patients who underwent total thyroidectomy in our hospital between September 2014 and July 2015 were retrospectively analyzed. RESULTS: There were 42 cases with normal parathyroid function, 58 cases with temporary HPP, and 10 cases with pHPP. When iPTH and serum calcium were administered at 24 h after surgery, iPTH decline and calcium decline differed significantly among the three groups above (P < .01). The accuracy and positive predictive value of 24 h iPTH for pHPP were higher than any one of the others. The sensitivity, specificity, false positive rate, and accuracy were 100%, 95%, 33.33%, and 94.45%, respectively. The AUC was 0.982 when 24-hour iPTH was equal to or <3.15 pg/mL. The use of blood calcium equal to or <2.03 mmol/L (8.12 mg/dL) pointed to a diagnosis of pHPP, with a sensitivity of 100%, specificity of 63%, false positive rate of 78.72%, and accuracy of 66.36%. CONCLUSIONS: Measurement of the postoperative 24-h intact parathyroid hormone and serum calcium concentration can predict the occurrence of permanent hypoparathyroidism and the former is more advantageous. Postoperative 24-h intact parathyroid hormone equal to or <3.15 pg/mL is a reliable index, and it is suitable for the prediction of postoperative permanent hypoparathyroidism.

Cytochrome P450 26A1 modulates natural killer cells in mouse early pregnancy.

Cytochrome P450 26A1 (CYP26A1) has a spatiotemporal expression pattern in the uterus, with a significant increase in mRNA and protein levels during peri-implantation. Inhibiting the function or expression of CYP26A1 can cause pregnancy failure, suggesting an important regulatory role of CYP26A1 in the maintenance of pregnancy. However, little is known about the exact mechanism involved. In this study, using a pCR3.1-cyp26a1 plasmid immunization mouse model and a Cyp26a1-MO (Cyp26a1-specific antisense oligos) knockdown mouse model, we report that the number of Dolichos biflorus agglutinin (DBA) lectin-positive uterine natural killer (uNK) cells was reduced in pCR3.1-cyp26a1 plasmid immunized and Cyp26a1-MO-treated mice. In contrast, the percentage of CD3- CD49b+ NK cells in the uteri from the treatment group was significantly higher than that of the control group in both models. Similarly, significantly up-regulated expression of CD49b (a pan-NK cell marker), interferon gamma, CCL2, CCR2 (CCL2 receptor) and CCL3 were detected in the uteri of pCR3.1-cyp26a1- and Cyp26a1-MO-treated mice. Transcriptome analysis suggested that CYP26A1 might regulate NK cells through chemokines. In conclusion, the present data suggest that silencing CYP26A1 expression/function can decrease the number of uNK cells and significantly increase the percentage of CD3- CD49b+ NK cells in the uteri of pregnant mice. These findings provide a new line of evidence correlating the deleterious effects of blocking CYP26A1 in pregnancy with the aberrant regulation of NK cells in the uterus.

The Balance between Conventional DCs and Plasmacytoid DCs Is Pivotal for Immunological Tolerance during Pregnancy in the Mouse.

Dendritic cells (DCs), which can shape their functions depending on the microenvironment, are crucial for the delicate balance of immunity and tolerance during pregnancy. However, the mechanism underlying the microenvironment-educated plasticity of DC differentiation during pregnancy remains largely unclear. Here, we found that the differentiation of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) is regulated in a tissue-specific manner during pregnancy. The ratio of cDCs and pDCs remained constant in the spleen. However, the ratio changed in the para-aortic lymph nodes (LNs), where cDC percentages were significantly reduced concurrent with an increase in pDCs from E8.5 to E16.5. Moreover, the expansion of pDCs and T regulatory (Treg) cells was correlated in the para-aortic LNs, and pDCs had more potential to induce regulatory T cells (Tregs) compared with cDCs (independent of IDO expression). Notably, the balance between cDCs and pDCs is disrupted in IFN-γ-induced abnormal pregnancy, accompanied by lower Treg percentages in the para-aortic LNs and decidua. To further identify the underlying mechanism, we found that elevated IFN-γ can increase the levels of GM-CSF to alter the differentiation of pDCs into cDCs in vivo. Therefore, we provide a novel regulatory mechanism underlying pregnancy-related immune tolerance that involves the balance of DC subsets, which may offer a new target for the prevention of human spontaneous abortion.

Seminal plasma induces inflammation in the uterus through the γδ T/IL-17 pathway.

After insemination, a large number of leukocytes migrate into the uterus, which is accompanied by intense inflammation. However, the details of how seminal plasma interacts with the uterus are still not very clear. Here, we present that neutrophils migrate and accumulate around the uterine epithelium following insemination, which is accompanied by an increase in interleukin (IL) 17A levels. Additionally, we find that γδ T cells are the major source of IL-17A, and the seminal plasma could induce the γδ T cells to secret IL-17A. Blocking IL-17A could reduce the number of neutrophils in the uterus and prevent them from migrating to the epithelium by decreasing the chemokines CXCL1, CXCL2 and CXCL5. Blocking IL-17A did not affect the Th1/Th2 balance but actually diminished the inflammation in the uterus by reducing the expression of IL-1ß and TNF-α. In summary, we found a new mechanism by which seminal plasma could influence the inflammation in the uterus through the γδ T/IL-17 pathway to regulate the expression of various chemokines and cytokines.

IFN-γ modulates Ly-49 receptors on NK cells in IFN-γ-induced pregnancy failure.

We have previously shown that interferon gamma (IFN-γ) induces aberrant CD49b(+) natural killer (NK) cell recruitment by regulating CX3CL1 and eventually provokes foetal loss. In this study, we show that IFN-γ also modulates Ly-49 receptors on NK cells during pregnancy failure. The percentages of Ly-49A(+) and Ly-49G2(+) NK cells in the uteri of the IFN-γ-treated group were significantly lower than those observed in the control group. Moreover, the median fluorescence intensity (MFI) values of Ly-49A and Ly-49G2 expression on NK cells in the uteri of the IFN-γ-treated group were significantly lower than those of the control group. Using isolated spleen leucocytes, we further found that IFN-γ significantly reduced the percentage of Ly-49A(+) NK cells in vitro. However, CX3CL1 was not involved in the modulation of Ly-49 receptors, and the expression of CX3CR1 was not regulated by IFN-γ in spleen leucocytes. Collectively, our data indicate that IFN-γ can modulate Ly-49 receptors on NK cells and this process may play a role in IFN-γ-induced pregnancy failure. Thus, we provide a new line of evidence correlating the deleterious effects of IFN-γ with its role in regulating NK cell Ly-49 receptors during pregnancy failure.