Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction.

Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

Integrating machine learning algorithms and single-cell analysis to identify gut microbiota-related macrophage biomarkers in atherosclerotic plaques.

Objective: The relationship between macrophages and the gut microbiota in patients with atherosclerosis remains poorly defined, and effective biological markers are lacking. This study aims to elucidate the interplay between gut microbial communities and macrophages, and to identify biomarkers associated with the destabilization of atherosclerotic plaques. The goal is to enhance our understanding of the underlying molecular pathways and to pave new avenues for diagnostic approaches and therapeutic strategies in the disease. Methods: This study employed Weighted Gene Co-expression Network Analysis (WGCNA) and differential expression analysis on atherosclerosis datasets to identify macrophage-associated genes and quantify the correlation between these genes and gut microbiota gene sets. The Random Forest algorithm was utilized to pinpoint PLEK, IRF8, BTK, CCR1, and CD68 as gut microbiota-related macrophage genes, and a nomogram was constructed. Based on the top five genes, a Non-negative Matrix Factorization (NMF) algorithm was applied to construct gut microbiota-related macrophage clusters and analyze their potential biological alterations. Subsequent single-cell analyses were conducted to observe the expression patterns of the top five genes and the interactions between immune cells. Finally, the expression profiles of key molecules were validated using clinical samples from atherosclerosis patients. Results: Utilizing the Random Forest algorithm, we ultimately identified PLEK, IRF8, CD68, CCR1, and BTK as gut microbiota-associated macrophage genes that are upregulated in atherosclerotic plaques. A nomogram based on the expression of these five genes was constructed for use as an auxiliary tool in clinical diagnosis. Single-cell analysis confirmed the specific expression of gut microbiota-associated macrophage genes in macrophages. Clinical samples substantiated the high expression of PLEK in unstable atherosclerotic plaques. Conclusion: Gut microbiota-associated macrophage genes (PLEK, IRF8, CD68, CCR1, and BTK) may be implicated in the pathogenesis of atherosclerotic plaques and could serve as diagnostic markers to aid patients with atherosclerosis.

Systematic analysis of mucosal-associated invariant T cells in haematological malignancies.

Mucosal-associated invariant T-cells (MAIT) are unconventional T-cells with cytotoxic and pro-inflammatory properties. Previous research has reported contradictory findings on their role in cancerogenesis with data being even scarcer in haematological malignancies. Here, we report the results of a systematic analysis of MAIT cells in treatment-naïve patients with a broad range of haematological malignancies. We analysed peripheral blood of 204 patients and 50 healthy subjects. The pool of haematological patients had a statistically significant lower both the absolute value (median values, 0.01 × 109/L vs. 0.05 × 109/L) of MAIT cells and their percentage (median values 0.94% vs. 2.56%) among T-cells compared to the control group. Separate analysis showed that the decrease in the absolute number of MAIT cells is significant in patients with acute myeloid leukaemia, myeloproliferative neoplasms, plasma cell myeloma, B-cell non-Hodgkin lymphomas, otherwise not specified, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma compared to the control population. Furthermore, in haematological malignancies, MAIT cells overexpress PD-1 (average values, 51.7% vs. 6.7%), HLA-DR (average values, 40.2% vs. 7%), CD38 (average values, 25.9% vs. 4.9%) and CD69 (average values, 40.2% vs. 9.2%). Similar results were obtained when comparing patients with individual malignancies to the control population. Our data show that the depletion of circulating MAIT cells is a common observation in a broad spectrum of haematological malignancies. In addition to their reduced numbers, MAIT cells acquire an activated/exhausted phenotype.

Gene expression of iron transporters, markers of vascularization and structural integrity in placenta of mothers with and without anemia.

OBJECTIVE: To compare the mRNA expression of placental iron transporters (TfR-1 and FPN), markers of placental vascularization (VEGF and sFLT1) and marker of structural integrity (LMN-A) in term women with and without iron deficiency anemia. MATERIALS AND METHODS: A total of 30 pregnant women were enrolled; 15 cases of iron deficiency anemia (Hb 7-10.9 gm/dL) and 15 gestational age matched healthy controls (Hb ≥ 11 gm/dL). Peripheral venous blood was collected for assessment of hemoglobin levels and serum iron profile. Placental tissue was used for assessing the mRNA expression of TfR-1, FPN, VEGF, sFLT-1 and LMN-A via real time PCR. RESULTS: Placental expression of TfR-1, VEGF and LMN-A was increased in pregnant women with anemia compared to healthy pregnant controls. Placental expression of sFLT-1 was decreased in pregnant women with anemia compared to healthy pregnant controls. There was no change in the placental expression of FPN. CONCLUSION: The increased expression of TfR-1, VEGF and LMN-A in cases of iron deficiency anemia are most likely to be compensatory in nature to help maintain adequate fetal iron delivery. WHAT DOES THIS STUDY ADDS TO THE CLINICAL WORK: Compensatory changes in the placenta aimed at buffering transport of iron to the fetus are seen in pregnant women with anemia compared to healthy pregnant controls.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

CD163+ macrophages in the triple-negative breast tumor microenvironment are associated with improved survival in the Women's Circle of Health Study and the Women's Circle of Health Follow-Up Study.

BACKGROUND: Tumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes. METHODS: We investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women's Circle of Health Study and Women's Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43-12.33). RESULTS: Women with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16-0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12-0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71-1.10). CONCLUSIONS: In contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer. Trial registration Not applicable.

A novel anti-LAG-3/TIGIT bispecific antibody exhibits potent anti-tumor efficacy in mouse models as monotherapy or in combination with PD-1 antibody.

We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.

CLIC4 Function in the Epithelial-Mesenchymal Transition of Epithelial Odontogenic Lesions.

BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.

CD4+T and CD8+T Cells in Uterus Exhibit Both Selective Dysfunction and Residency Signatures.

Unlike T cells in other tissues, uterine T cells must balance strong immune defense against pathogens with tolerance to semiallogeneic fetus. Our previous study fully elucidated the characteristics of γδT cells in nonpregnant uterus and the mechanism modulated by estrogen. However, comprehensive knowledge of the immunological properties of αßT (including CD4+T cells and CD8+T) cells in nonpregnancy uterus has not been acquired. In this study, we fully compared the immunological properties of αßT cells between uterus and blood using mouse and human sample. It showed that most of CD4+T cells and CD8+T cells in murine uterus and human endometrium were tissue resident memory T cells which highly expressed tissue residence markers CD69 and/or CD103. In addition, both CD4+T cells and CD8+T cells in uterus highly expressed inhibitory molecular PD-1 and cytokine IFN-γ. Uterine CD4+T cells highly expressed IL-17 and modulated by transcription factor pSTAT3. Moreover, we compared the similarities and differences between human and murine uterine T cell phenotype. Together, uterine CD4+T cells and CD8+ cells exhibited a unique mixed signature of T cell dysfunction, activation, and effector function which enabled them to balance strong immune defense against pathogens with tolerance to fetus. Our study fully elucidated the unique immunologic properties of uterine CD4+T and CD8+T cells and provided a base for further investigation of functions.

Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia.

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury.

Dynamic changes in B cell subpopulations in response to triple-negative breast cancer development.

Despite presenting a worse prognosis and being associated with highly aggressive tumors, triple-negative breast cancer (TNBC) is characterized by the higher frequency of tumor-infiltrating lymphocytes, which have been implicated in better overall survival and response to therapy. Though recent studies have reported the capacity of B lymphocytes to recognize overly-expressed normal proteins, and tumor-associated antigens, how tumor development potentially modifies B cell response is yet to be elucidated. Our findings reveal distinct effects of 4T1 and E0771 murine tumor development on B cells in secondary lymphoid organs. Notably, we observe a significant expansion of total B cells and plasma cells in the tumor-draining lymph nodes (tDLNs) as early as 7 days after tumor challenge in both murine models, whereas changes in the spleen are less pronounced. Surprisingly, within the tumor microenvironment (TME) of both models, we detect distinct B cell subpopulations, but tumor development does not appear to cause major alterations in their frequency over time. Furthermore, our investigation into B cell regulatory phenotypes highlights that the B10 Breg phenotype remains unaffected in the evaluated tissues. Most importantly, we identified an increase in CD19 + LAG-3 + cells in tDLNs of both murine models. Interestingly, although CD19 + LAG-3 + cells represent a minor subset of total B cells (< 3%) in all evaluated tissues, most of these cells exhibit elevated expression of IgD, suggesting that LAG-3 may serve as an activation marker for B cells. Corroborating with these findings, we detected distinct cell cycle and proliferation genes alongside LAG-3 analyzing scRNA-Seq data from a cohort of TNBC patients. More importantly, our study suggests that the presence of LAG-3 B cells in breast tumors could be associated with a good prognosis, as patients with higher levels of LAG-3 B cell transcripts had a longer progression-free interval (PFI). This novel insight could pave the way for targeted therapies that harness the unique properties of LAG-3 + B cells, potentially offering new avenues for improving patient outcomes in TNBC. Further research is warranted to unravel the mechanistic pathways of these cells and to validate their prognostic value in larger, diverse patient cohorts.

Combined Ferritin Nanocarriers with ICG for Effective Phototherapy Against Breast Cancer.

Introduction: Photodynamic Therapy (PDT) is a promising, minimally invasive treatment for cancer with high immunostimulatory potential, no reported drug resistance, and reduced side effects. Indocyanine Green (ICG) has been used as a photosensitizer (PS) for PDT, although its poor stability and low tumor-target specificity strongly limit its efficacy. To overcome these limitations, ICG can be formulated as a tumor-targeting nanoparticle (NP). Methods: We nanoformulated ICG into recombinant heavy-ferritin nanocages (HFn-ICG). HFn has a specific interaction with transferrin receptor 1 (TfR1), which is overexpressed in most tumors, thus increasing HFn tumor tropism. First, we tested the properties of HFn-ICG as a PS upon irradiation with a continuous-wave diode laser. Then, we evaluated PDT efficacy in two breast cancer (BC) cell lines with different TfR1 expression levels. Finally, we measured the levels of intracellular endogenous heavy ferritin (H-Fn) after PDT treatment. In fact, it is known that cells undergoing ROS-induced autophagy, as in PDT, tend to increase their ferritin levels as a defence mechanism. By measuring intracellular H-Fn, we verified whether this interplay between internalized HFn and endogenous H-Fn could be used to maximize HFn uptake and PDT efficacy. Results: We previously demonstrated that HFn-ICG stabilized ICG molecules and increased their delivery to the target site in vitro and in vivo for fluorescence guided surgery. Here, with the aim of using HFn-ICG for PDT, we showed that HFn-ICG improved treatment efficacy in BC cells, depending on their TfR1 expression. Our data revealed that endogenous H-Fn levels were increased after PDT treatment, suggesting that this defence reaction against oxidative stress could be used to enhance HFn-ICG uptake in cells, increasing treatment efficacy. Conclusion: The strong PDT efficacy and peculiar Trojan horse-like mechanism, that we revealed for the first time in literature, confirmed the promising application of HFn-ICG in PDT.

Tumor microenvironment and clinical efficacy of first line immunotherapy-based combinations in metastatic renal cell carcinoma.

The impact of tumor microenvironment (TME) in influencing clinical response to first-line immune checkpoint inhibitor (ICI)-based treatment in advanced renal cell carcinoma (RCC) is unclear. Immunohistochemistry (IHC) could identify biomarkers related to immune checkpoints and immune cell population. This study retrospectively characterized TME from 28 RCC patients who received first line ICI-based therapy through IHC assessment of selected markers and explored preliminary evidence about their possible correlation with treatment efficacy. We found a significantly higher count of CD80+, CD163+ cells and their ratio in RCC with clear cell component compared to those without clear cell features; additionally, patients with metastatic disease at diagnosis were associated with higher expression of CD163+ cells, while higher count of CD4+ cells and CD4+/CD8+ ratio were found in RCC with sarcomatoid features. Patients achieving partial or complete response were associated with lower expression of CD163+ cells (median 28 vs 47; p = 0.049). Furthermore, lower expression of CD163+ was associated with better PFS (median PFS 20.0 vs 4.7 months; HR 0.22 p = 0.011) and OS (median OS NR vs 14.4 months; HR 0.28 p = 0.036). A longer OS was reported in PD-L1 CPS negative patients (median OS NR vs 11.8 months; HR 0.20 p = 0.024). High infiltration of CD163+ macrophages, who typically present "anti-inflammatory" M2-like phenotype, could identify a subgroup of patients with poor survival after receiving first-line ICI.

F. Nucleatum enhances oral squamous cell carcinoma proliferation via E-cadherin/ß-Catenin pathway.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) is a microbial risk factor whose presence increases the risk of oral squamous cell carcinoma (OSCC) progression. However, whether it can promote the proliferation of OSCC cells remains unknown. METHODS: In this study, we investigated F. nucleatum effect on OSCC cell proliferation using in vitro and in vivo experiments. RESULTS: Our results showed that F. nucleatum promoted OSCC cell proliferation, doubling the cell count after 72 h (CCK-8 assay). Cell cycle analysis revealed G2/M phase arrest. F. nucleatum interaction with CDH1 triggered phosphorylation, upregulating downstream protein ß-catenin and activating cyclinD1 and Myc. Notably, F. nucleatum did not affect noncancerous cells, unrelated to CDH1 expression levels in CAL27 cells. Overexpression of phosphorylated CDH1 in 293T cells did not upregulate ß-catenin and cycle-related genes. In vivo BALB/c nude experiments showed increased tumor volume and Ki-67 proliferation index after F. nucleatum intervention. CONCLUSION: Our study suggests that F. nucleatum promotes OSCC cell proliferation through the CDH1/ß-catenin pathway, advancing our understanding of its role in OSCC progression and highlighting its potential as a therapeutic target.

Exchangeable leaders of collectively migrating glioma abuse N-cadherin trafficking.

Collectively migrating cells consist of leaders and followers with different features. In this issue, Kim et al. (https://doi.org/10.1083/jcb.202401057) characterize the leader and follower cells in collective glioma migration and uncover important roles of YAP1/TAZ-mediated regulation of N-cadherin in the leader cells.

Myeloid cell expression of CD200R is modulated in active TB disease and regulates Mycobacterium tuberculosis infection in a biomimetic model.

A robust immune response is required for resistance to pulmonary tuberculosis (TB), the primary disease caused by Mycobacterium tuberculosis (Mtb). However, pharmaceutical inhibition of T cell immune checkpoint molecules can result in the rapid development of active disease in latently infected individuals, indicating the importance of T cell immune regulation. In this study, we investigated the potential role of CD200R during Mtb infection, a key immune checkpoint for myeloid cells. Expression of CD200R was consistently downregulated on CD14+ monocytes in the blood of subjects with active TB compared to healthy controls, suggesting potential modulation of this important anti-inflammatory pathway. In homogenized TB-diseased lung tissue, CD200R expression was highly variable on monocytes and CD11b+HLA-DR+ macrophages but tended to be lowest in the most diseased lung tissue sections. This observation was confirmed by fluorescent microscopy, which showed the expression of CD200R on CD68+ macrophages surrounding TB lung granuloma and found expression levels tended to be lower in macrophages closest to the granuloma core and inversely correlated with lesion size. Antibody blockade of CD200R in a biomimetic 3D granuloma-like tissue culture system led to significantly increased Mtb growth. In addition, Mtb infection in this system reduced gene expression of CD200R. These findings indicate that regulation of myeloid cells via CD200R is likely to play an important part in the immune response to TB and may represent a potential target for novel therapeutic intervention.

Characterization of innate lymphoid cell subsets infiltrating melanoma and epithelial ovarian tumors.

The innate lymphoid cell (ILC) family is composed of heterogeneous innate effector and helper immune cells that preferentially reside in tissues where they promote tissue homeostasis. In cancer, they have been implicated in driving both pro- and anti-tumor responses. This apparent dichotomy highlights the need to better understand differences in the ILC composition and phenotype within different tumor types that could drive seemingly opposite anti-tumor responses. Here, we characterized the frequency and phenotype of various ILC subsets in melanoma metastases and primary epithelial ovarian tumors. We observed high PD-1 expression on ILC subsets isolated from epithelial ovarian tumor samples, while ILC populations in melanoma samples express higher levels of LAG-3. In addition, we found that the frequency of cytotoxic ILCs and NKp46+ILC3 in tumors positively correlates with monocytic cells and conventional type 2 dendritic cells, revealing potentially new interconnected immune cell subsets in the tumor microenvironment. Consequently, these observations may have direct relevance to tumor microenvironment composition and how ILC subset may influence anti-tumor immunity.

Autophagy-related protein 5 (ATG5) interacts with bone marrow stromal cell antigen 2 (BST2) to stimulate HBV replication through antagonizing the antiviral activity of BST2.

Hepatitis B virus (HBV) infection is a major global health burden with 820 000 deaths per year. In our previous study, we found that the knockdown of autophagy-related protein 5 (ATG5) significantly upregulated the interferon-stimulated genes (ISGs) expression to exert the anti-HCV effect. However, the regulation of ATG5 on HBV replication and its underlying mechanism remains unclear. In this study, we screened the altered expression of type I interferon (IFN-I) pathway genes using RT² Profiler™ PCR array following ATG5 knock-down and we found the bone marrow stromal cell antigen 2 (BST2) expression was significantly increased. We then verified the upregulation of BST2 by ATG5 knockdown using RT-qPCR and found that the knockdown of ATG5 activated the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway. ATG5 knockdown or BST2 overexpression decreased Hepatitis B core Antigen (HBcAg) protein, HBV DNA levels in cells and supernatants of HepAD38 and HBV-infected NTCP-HepG2. Knockdown of BST2 abrogated the anti-HBV effect of ATG5 knockdown. Furthermore, we found that ATG5 interacted with BST2, and further formed a ternary complex together with HBV-X (HBx). In conclusion, our finding indicates that ATG5 promotes HBV replication through decreasing BST2 expression and interacting with it directly to antagonize its antiviral function.

Molecular incompatibility between pig CD200 and human CD200 receptor in in vitro xenogeneic immune responses.

Overexpression of human CD200 (hCD200) in porcine endothelial cells (PECs) has been reported to suppress xenogeneic immune responses of human macrophages against porcine endothelial cells. The current study aimed to address whether the above-mentioned beneficial effect of hCD200 is mediated by overcoming the molecular incompatibility between porcine CD200 (pCD200) and hCD200 receptor or simply by increasing the expression levels of CD200 without any molecular incompatibility across the two species. We overexpressed hCD200 or pCD200 using lentiviral vectors with V5 marker in porcine endothelial cells and compared their suppressive activity against U937-derived human macrophage-like cells (hMCs) and primary macrophages. In xenogeneic coculture of porcine endothelial cells and human macrophage-like cells or macrophages, hCD200-porcine endothelial cells suppressed phagocytosis and cytotoxicity of human macrophages to a greater extent than pCD200-porcine endothelial cells. Secretion of tumor necrosis factor-α, interleukin-1ß, and monocyte chemoattractant protein-1 from human macrophages and expression of M1 phenotypes (inducible nitric oxide synthase, dectin-1, and CD86) were also suppressed by hCD200 to a greater extent than pCD200. Furthermore, in signal transduction downstream of CD200 receptor, hCD200 induced Dok2 phosphorylation and suppressed IκB phosphorylation to a greater extent than pCD200. The above data supported the possibility of a significant molecular incompatibility between pCD200 and human CD200 receptor, suggesting that the beneficial effects of hCD200 overexpression in porcine endothelial cells could be mediated by overcoming the molecular incompatibility across the species barrier rather than by simple overexpression effects of CD200.