ABSTRACT
BACKGROUND AND PURPOSE: PD-1/PD-L1 inhibitors have become a promising therapy. However, the response rate is lower than 30% in patients with cervical cancer (CC), which is related to immunosuppressive components in tumor microenvironment (TME). Tumor-associated macrophages (TAMs), as one of the most important immune cells, are involved in the formation of tumor suppressive microenvironment. Therefore, it will provide a theoretical basis for curative effect improvement about the regulatory mechanism of TAMs on PD-L1 expression. METHODS: The clinical data and pathological tissues of CC patients were collected, and the expressions of PD-L1, CD68 and CD163 were detected by immunohistochemistry. Bioinformatics was used to analyze the macrophage subtypes involved in PD-L1 regulation. A co-culture model was established to observe the effects of TAMs on the morphology, migration and invasion function of CC cells, and the regulatory mechanism of TAMs on PD-L1. RESULTS: PD-L1 expression on tumor cells could predict the poor prognosis of patients. And there was a strong correlation between PD-L1 expression with CD163+TAMs infiltration. Similarly, PD-L1 expression was associated with M1/M2-type TAMs infiltration in bioinformatics analysis. The results of cell co-culture showed that M1/M2-type TAMs could upregulate PD-L1 expression, especially M2-type TAMs may elevate the PD-L1 expression via PI3K/AKT pathway. Meanwhile, M1/M2-type TAMs can affect the morphological changes, and enhance migration and invasion abilities of CC cells. CONCLUSIONS: PD-L1 expression in tumor cells can be used as a prognostic factor and is closely related to CD163+TAMs infiltration. In addition, M2-type TAMs can upregulate PD-L1 expression in CC cells through PI3K/AKT pathway, enhance the migration and invasion capabilities, and affect the tumor progression.
Subject(s)
B7-H1 Antigen , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Tumor-Associated Macrophages , Uterine Cervical Neoplasms , Humans , B7-H1 Antigen/metabolism , B7-H1 Antigen/genetics , Tumor-Associated Macrophages/metabolism , Tumor-Associated Macrophages/immunology , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/immunology , Female , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Tumor Microenvironment/immunology , Up-Regulation , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Middle Aged , Antigens, CD/metabolism , Antigens, CD/genetics , Prognosis , Gene Expression Regulation, Neoplastic , Cell Movement , Receptors, Cell SurfaceABSTRACT
BACKGROUND Tumor-infiltrating immune cells (TIICs) are implicated in the survival of ovarian cancer (OVCA) patients, but their prognostic significance in advanced or metastatic OVCA patients treated with neoadjuvant chemotherapy (NCAT) has not been well documented, particularly in the Chinese population. MATERIAL AND METHODS A total of 31 advanced or metastatic OVCA patients who underwent NACT were included. The density and positive rate of tumor-infiltrating immune cells (TIICs) within cancer cell nests and in cancer stroma were explored. The correlations of pre- or post-NACT TIICs with the efficacy of NACT and the changes in TIIC subpopulation with NACT were examined. RESULTS Compared with patients with partial benefit from NACT, significantly decreased pre-NACT intratumoral CD68âºCD163⺠cells (P=0.0043) and increased pre-NACT intratumoral CD56⺠cells (P=0.038) were observed in patients with benefit. The high level of pre-NACT intratumoral CD68âºCD163â» M1 macrophage (P=0.075) and stromal CD3âºPD-1⺠cells (P=0.085) predicated improved progression-free survival, respectively. Increased post-NACT stromal CD68âºCD163â» M1 macrophage (P=0.01), stromal CD8⺠T cells (P=0.073), and stromal CD8âºPD-1⺠cells (P=0.072) were associated with benefit from NACT. Moreover, NACT increased intratumoral CD3⺠(P=0.031), CD8+ (P=0.031), and CD3âºCD8⺠cells (P=0.031). CONCLUSIONS High intratumoral CD68âºCD163â», intratumoral CD56⺠cells, and stromal CD3âºPD-1⺠cells pre-NACT predicted good prognosis. Intratumoral CD3âº, CD8âº, and CD3âºCD8⺠cells were increased after NACT. Evaluation of immune profiles may help to identify patients who might benefit from NACT and allow us to further stratify advanced or metastatic OVCA patients treated with NACT for disease management.
Subject(s)
Lymphocytes, Tumor-Infiltrating , Neoadjuvant Therapy , Ovarian Neoplasms , Humans , Female , Neoadjuvant Therapy/methods , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Middle Aged , Prognosis , Retrospective Studies , Lymphocytes, Tumor-Infiltrating/immunology , Adult , Aged , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Neoplasm Metastasis , Treatment Outcome , Tumor Microenvironment/immunology , China , Receptors, Cell SurfaceABSTRACT
Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Macrophages, Alveolar , Receptors, Cell Surface , Animals , Cattle , Macrophages, Alveolar/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, CD/metabolism , Receptors, Cell Surface/metabolism , Phenotype , Mycobacterium bovis/immunology , Flow Cytometry , Tuberculosis, Bovine/metabolism , Tuberculosis, Bovine/immunology , Tuberculosis, Bovine/microbiology , Immunophenotyping , Bronchoalveolar Lavage FluidABSTRACT
Preeclampsia is a disorder of pregnancy characterized by endothelial dysfunction, abnormal placentation, systemic inflammation, and altered immune reaction. The aim of this study was to investigate the immune checkpoint molecules TIM-3 and Gal-9 on macrophages and Hofbauer cells (HBC) in the placenta of preeclampsia patients. Immunohistochemistry and Immunofluorescence was used to characterize the expression of the macrophage markers CD68 and CD163, CK7 and the proteins TIM-3 and Gal-9 in the placentas of preeclampsia patients comparing it to the placentas of healthy pregnancies. Double immunofluorescence staining (TIM-3 with CD3/CD19/CD56) was used to analyze the TIM-3 expression on other immune cells (T cells, B cells, NK cells) within the chorionic villi. The expression of TIM-3 on decidual macrophages did not significantly differ between the preeclamptic and the control group (p = 0.487). When looking at the different offspring we saw an upregulation of TIM-3 expression on decidual macrophages in preeclamptic placentas with female offspring (p = 0.049). On Hofbauer cells within the chorionic villi, the TIM-3 expression was significantly downregulated in preeclamptic cases without a sex-specific difference (p < 0.001). Looking at the protein Gal-9 the expression was proven to be downregulated both, on decidual macrophages (p = 0.003) and on Hofbauer cells (p = 0.002) within preeclamptic placentas compared to healthy controls. This was only significant in male offspring (p < 0.001 and p = 0.013) but not in female offspring (p = 0.360 and p = 0.068). While TIM-3 expression within the extravillious trophoblast and the syncytiotrophoblast was significantly downregulated (p < 0.001 and p = 0.012) in preeclampsia, the expression of Gal-9 was upregulated in (p < 0.001 and p < 0.001) compared to healthy controls. The local variations of the immune checkpoint molecules TIM-3 and Gal-9 in the placenta may contribute to the inflammation observed in preeclamptic patients. It could therefore contribute to the pathogenesis and be an important target in the treatment of preeclampsia.
Subject(s)
Galectins , Hepatitis A Virus Cellular Receptor 2 , Macrophages , Placenta , Pre-Eclampsia , Humans , Pregnancy , Hepatitis A Virus Cellular Receptor 2/metabolism , Female , Pre-Eclampsia/immunology , Pre-Eclampsia/metabolism , Pre-Eclampsia/pathology , Macrophages/immunology , Macrophages/metabolism , Galectins/metabolism , Adult , Placenta/immunology , Placenta/metabolism , Placenta/pathology , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Receptors, Cell Surface/metabolism , Chorionic Villi/immunology , Chorionic Villi/metabolism , Chorionic Villi/pathology , MaleABSTRACT
BACKGROUND: Immunotherapy has revolutionized the treatment of various types of tumors, but there has been no breakthrough in the treatment of gliomas. The aim of this study is to discover valuable immunotherapy target in glioma, analyze its expression in glioma and the related microenvironment, explore potential immunotherapy strategies, and propose new possibilities for the treatment of gliomas. METHODS: Immunohistochemistry (IHC) and multiplex fluorescence immunohistochemistry (mIHC) were used to analyze the expression of common immune markers and checkpoints in 187 glioma patients from Sun Yat-sen University Caner Center (SYSUCC). Bioinformatics analysis was used to examine the expression of TIM-3 in different macrophages using the Chinese Glioma Genome Atlas (CGGA) single-cell sequencing database. The Kaplan-Meier curve was used to predict the prognostic value of samples with high TIM-3 and CD68 expression. The R package was used to analyze the somatic mutation status and the sensitivity of small molecule inhibitors in TIM-3/CD68 double-high expression samples. RESULTS: TIM-3 is a relatively highly expressed immune checkpoint in glioma. Unlike other tumors, TIM-3 is mainly expressed on macrophages in the glioma microenvironment. TIM-3/CD68 double-high expression suggests poor survival in glioma and may be a new upgrade marker in both IDH-mutant glioma and IDH-wildtype low-grade glioma (LGG) glioma (P < 0.01). Exploring the combination of TIM-3 inhibitors and p38 MAPK inhibitor may be a potential treatment direction for TIM-3/CD68 double high expression gliomas in the future. CONCLUSIONS: The combination of TIM-3 and CD68 holds significant importance as a potential target for both prognosis and therapeutic intervention in glioma.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Biomarkers, Tumor , Brain Neoplasms , Glioma , Hepatitis A Virus Cellular Receptor 2 , Tumor Microenvironment , Humans , Hepatitis A Virus Cellular Receptor 2/metabolism , Hepatitis A Virus Cellular Receptor 2/genetics , Glioma/metabolism , Glioma/therapy , Glioma/genetics , Glioma/mortality , Glioma/diagnosis , Brain Neoplasms/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/therapy , Brain Neoplasms/mortality , Prognosis , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, CD/metabolism , Antigens, CD/genetics , Tumor Microenvironment/immunology , Female , Male , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Middle Aged , Immunotherapy/methods , Adult , Macrophages/immunology , Macrophages/metabolism , Gene Expression Regulation, Neoplastic , CD68 MoleculeABSTRACT
Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the KD value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death.
Subject(s)
Adhesins, Bacterial , Antigens, CD , Antigens, Differentiation, Myelomonocytic , Bacterial Adhesion , Host-Pathogen Interactions , Lectins , Humans , Adhesins, Bacterial/metabolism , Adhesins, Bacterial/genetics , Antigens, CD/metabolism , Antigens, CD/genetics , Lectins/metabolism , Lectins/genetics , Lectins/immunology , Animals , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Protein Binding , Mice , CHO Cells , Cricetulus , Neisseria meningitidis/genetics , Neisseria meningitidis/metabolism , Neisseria meningitidis/immunology , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/genetics , Epithelial Cells/microbiology , Epithelial Cells/metabolism , Epithelial Cells/immunology , Meningococcal Infections/microbiology , Meningococcal Infections/immunology , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/genetics , Neisseria meningitidis, Serogroup B/genetics , Neisseria meningitidis, Serogroup B/immunology , Neisseria meningitidis, Serogroup B/metabolismABSTRACT
The poor healing characteristics of diabetic foot ulcers are partially attributed to diabetes-induced pro-inflammatory wounds. Our previous study reported that both miR-146a-5p and miR-200b-3p decrease endothelial inflammation in human aortic endothelial cells and db/db diabetic mice. Although miR-146a-5p has been reported to improve diabetic wound healing, the role of miR-200b-3p is not clear. This study compared the roles of these miRNAs in diabetic wound healing. Two 8-mm full-thickness wounds were created in 12-week-old male db/db mice on the left and right back. After surgery, 100 ng miR-146a-5p, miR-200b-3p, or miR-negative control (NC) was injected in each wound. Full-thickness skin samples were harvested from mice at the 14th day for real-time polymerase chain reaction and immunohistochemistry analyses. At the 14th day, the miR-200b-3p group showed better wound healing and greater granulation tissue thickness than the miR-146a-5p group. The miR-200b-3p group showed a significant decrease of IL-6 and IL-1ß gene expression and a significant increase of Col3α1 gene expression compared to those in the miR-NC group. The miR-200b-3p group had the lowest gene expression of TGF-ß1, followed by the miR-146a-5p and miR-NC groups. Our findings suggest that the miR-200b-3p group had better healing characteristics than the other two groups. Immunohistochemical staining revealed that CD68 immunoreactivity was significantly decreased in both the miR-146a-5p and miR-200b-3p groups compared with that in the miR-NC group. In addition, CD31 immunoreactivity was significantly higher in the miR-200b-3p group than in the miR-146a-5p group. In conclusion, these results suggest that miR-200b-3p is more effective than miR-146a-5p in promoting diabetic wound healing through its anti-inflammatory and pro-angiogenic effects.
Subject(s)
MicroRNAs , Wound Healing , MicroRNAs/genetics , MicroRNAs/metabolism , Animals , Wound Healing/genetics , Male , Mice , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/genetics , Diabetic Foot/genetics , Diabetic Foot/metabolism , Diabetic Foot/pathology , Neovascularization, Physiologic/genetics , Interleukin-6/metabolism , Interleukin-6/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Interleukin-1beta/metabolism , Interleukin-1beta/genetics , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Antigens, CD/genetics , Antigens, CD/metabolism , Skin/metabolism , Skin/pathology , Inflammation/genetics , Inflammation/pathology , Inflammation/metabolism , Mice, Inbred C57BL , CD68 MoleculeABSTRACT
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.
Subject(s)
Antibodies, Neutralizing , Antigens, CD , Antigens, Differentiation, Myelomonocytic , Peptides , Porcine respiratory and reproductive syndrome virus , Receptors, Cell Surface , Single-Domain Antibodies , Porcine respiratory and reproductive syndrome virus/immunology , Porcine respiratory and reproductive syndrome virus/drug effects , Animals , Single-Domain Antibodies/immunology , Single-Domain Antibodies/pharmacology , Single-Domain Antibodies/chemistry , Swine , Antigens, Differentiation, Myelomonocytic/immunology , Antigens, Differentiation, Myelomonocytic/metabolism , Receptors, Cell Surface/immunology , Antigens, CD/immunology , Antigens, CD/metabolism , Antibodies, Neutralizing/immunology , Peptides/chemistry , Peptides/pharmacology , Peptides/immunology , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/prevention & control , Mice , Virus Replication/drug effects , Cell LineABSTRACT
Porcine reproductive and respiratory syndrome (PRRS), which poses substantial threats to the global pig industry, is primarily characterized by interstitial pneumonia. Cluster of differentiation 163 (CD163) is the essential receptor for PRRSV infection. Metalloproteinase-mediated cleavage of CD163 leads to the shedding of soluble CD163 (sCD163), thereby inhibiting PRRSV proliferation. However, the exact cleavage site in CD163 and the potential role of sCD163 in inflammatory responses during PRRSV infection remain unclear. Herein, we found that PRRSV infection increased sCD163 levels, as demonstrated in primary alveolar macrophages (PAMs), immortalized PAM (IPAM) cell lines, and sera from PRRSV-infected piglets. With LC-MS/MS, Arg-1041/Ser-1042 was identified as the cleavage site in porcine CD163, and an IPAM cell line with precise mutation at the cleavage site was constructed. Using the precisely mutated IPAM cells, we found that exogenous addition of sCD163 protein promoted inflammatory responses, while mutation at the CD163 cleavage site suppressed inflammatory responses. Consistently, inhibition of sCD163 using its neutralizing antibodies reduced PRRSV infection-triggered inflammatory responses. Importantly, sCD163 promoted cell polarization from M2 to M1 phenotype, which in turn facilitated inflammatory responses. Taken together, our findings identify sCD163 as a novel proinflammatory mediator and provide valuable insights into the mechanisms underlying the induction of inflammatory responses by PRRSV infection.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Inflammation , Macrophages, Alveolar , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Receptors, Cell Surface , Animals , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/immunology , Antigens, Differentiation, Myelomonocytic/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Porcine respiratory and reproductive syndrome virus/immunology , Swine , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, CD/immunology , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/virology , Macrophages, Alveolar/virology , Macrophages, Alveolar/immunology , Inflammation/virology , Cell LineABSTRACT
ABSTRACT: Mantle cell lymphoma (MCL) is dependent on a supportive tumor immune microenvironment (TIME) in which infiltration of CD163+ macrophages has a negative prognostic impact. This study explores how abundance and spatial localization of CD163+ cells are associated with the biology of MCL, using spatial multiomic investigations of tumor and infiltrating CD163+ and CD3+ cells. A total of 63 proteins were measured using GeoMx digital spatial profiling in tissue microarrays from 100 diagnostic MCL tissues. Regions of interest were selected in tumor-rich and tumor-sparse tissue regions. Molecular profiling of CD163+ macrophages, CD20+ MCL cells, and CD3+ T-cells was performed. To validate protein profiles, 1811 messenger RNAs were measured in CD20+ cells and 2 subsets of T cells. Image analysis was used to extract the phenotype and position of each targeted cell, thereby allowing the exploration of cell frequencies and cellular neighborhoods. Proteomic investigations revealed that CD163+ cells modulate their immune profile depending on their localization and that the immune inhibitory molecules, V-domain immunoglobulin suppressor of T-cell activation and B7 homolog 3, have higher expression in tumor-sparse than in tumor-rich tissue regions and that targeting should be explored. We showed that MCL tissues with more abundant infiltration of CD163+ cells have a higher proteomic and transcriptional expression of key components of the MAPK pathway. Thus, the MAPK pathway may be a feasible therapeutic target in patients with MCL with CD163+ cell infiltration. We further showed the independent and combined prognostic values of CD11c and CD163 beyond established risk factors.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Lymphoma, Mantle-Cell , Macrophages , Receptors, Cell Surface , Tumor Microenvironment , Humans , Lymphoma, Mantle-Cell/immunology , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/pathology , Receptors, Cell Surface/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Macrophages/metabolism , Macrophages/immunology , Antigens, CD/metabolism , Tumor Microenvironment/immunology , Signal TransductionABSTRACT
Despite viral suppression by effective combined antiretroviral therapy, HIV-1-infected individuals have an increased risk of non-AIDS-related overall morbidity, which is due to the persistent chronic inflammation exemplified by the activation of monocytes, such as increased CD16high subset, and elevated plasma level of soluble CD163 (sCD163) and soluble CD14 (sCD14). Here, we show that IL-10, which has been recognized as anti-inflammatory, induces these activated phenotypes of monocytes in vitro. IL-10 increased CD16high monocytes, which was due to the upregulation of CD16 mRNA expression and completely canceled by an inhibitor of Stat3. Moreover, IL-10 increased the production of sCD163 and sCD14 by monocytes, which was consistent with the upregulation of cell surface expression of CD163 and CD14, and mRNA expression of CD163. However, unlike the IL-10-indeuced upregulation of CD16, that of CD14 was minimally affected by the Stat3 inhibitor. Furthermore, the IL-10-induced upregulation of CD163 protein and mRNA was partially inhibited by the Stat3 inhibitor, but completely canceled by an inhibitor of AMPK, an upstream kinase of Stat3 and PI3K/Akt/mTORC1 pathways. In this study, we also found that HIV-1 pathogenic protein Nef, which is known to persist in plasma of virally-suppressed individuals, induced IL-10 production in monocyte-derived macrophages. Our results may suggest that IL-10, which is inducible by Nef-activated macrophages, is one of drivers for activated phenotypes of monocytes in virally-suppressed individuals, and that IL-10 induces the increased CD16high monocytes and elevated level of sCD163 and sCD14 through the activation of different signaling pathways.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , HIV Infections , HIV-1 , Interleukin-10 , Monocytes , Receptors, Cell Surface , Humans , Interleukin-10/metabolism , Monocytes/metabolism , Monocytes/immunology , HIV Infections/immunology , HIV Infections/virology , HIV Infections/metabolism , HIV Infections/blood , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/genetics , Antigens, CD/metabolism , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Receptors, IgG/metabolism , Lipopolysaccharide Receptors/metabolism , STAT3 Transcription Factor/metabolism , Phenotype , Up-Regulation , Cells, CulturedABSTRACT
OBJECTIVE: To investigate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute liver injury induced by lipopolysaccharide (LPS) and D-GalN in mice. METHODS: Adult male C57BL/6J mice with or without LPS/D-GaIN-induced acute liver injury were given intraperitoneal injections of rSj-Cys or PBS 30 min after modeling (n=18), and serum and liver tissues samples were collected from 8 mice in each group 6 h after modeling. The survival of the remaining 10 mice in each group within 24 h was observed. Serum levels of ALT, AST, TNF-α and IL-6 of the mice were measured, and liver pathologies was observed with HE staining. The hepatic expressions of macrophage marker CD68, Bax, Bcl-2 and endoplasmic reticulum stress (ERS)-related proteins were detected using immunohistochemistry or immunoblotting, and TUNEL staining was used to detect hepatocyte apoptosis. RESULTS: The survival rates of PBS- and rSj-Cys-treated mouse models of acute liver injury were 30% and 80% at 12 h and were 10% and 60% at 24 h after modeling, respectively; no death occurred in the two control groups within 24 h. The mouse models showed significantly increased serum levels of AST, ALT, IL-6 and TNF-α and serious liver pathologies with increased hepatic expressions of CD68 and Bax, lowered expression of Bcl-2, increased hepatocyte apoptosis, and up-regulated expressions of ERS-related signaling pathway proteins GRP78, CHOP and NF-κB p-p65. Treatment of the mouse models significantly lowered the levels of AST, ALT, IL-6 and TNF-α, alleviated liver pathologies, reduced hepatic expressions of CD68, Bax, GRP78, CHOP and NF-κB p-p65, and enhanced the expression of Bcl-2. In the normal control mice, rSj-Cys injection did not produce any significant changes in these parameters compared with PBS. CONCLUSION: rSj-Cys alleviates LPS/D-GalN-induced acute liver injury in mice by suppressing ERS, attenuating inflammation and inhibiting hepatocyte apoptosis.
Subject(s)
Apoptosis , Cystatins , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress , Hepatocytes , Inflammation , Mice, Inbred C57BL , Schistosoma japonicum , Animals , Mice , Endoplasmic Reticulum Stress/drug effects , Apoptosis/drug effects , Male , Hepatocytes/metabolism , Hepatocytes/drug effects , Cystatins/pharmacology , Liver/pathology , Liver/metabolism , Lipopolysaccharides , Tumor Necrosis Factor-alpha/metabolism , Interleukin-6/metabolism , Recombinant Proteins/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2-Associated X Protein/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Galactosamine , Antigens, CD/metabolism , Chemical and Drug Induced Liver Injury/drug therapy , CD68 MoleculeABSTRACT
Porcine reproductive and respiratory syndrome (PRRS) is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus (PRRSV), resulting in substantial economic losses in the swine industry. Modifying the CD163 SRCR5 domain, either through deletion or substitution, can eff1ectively confer resistance to PRRSV infection in pigs. However, large fragment modifications in pigs inevitably raise concerns about potential adverse effects on growth performance. Reducing the impact of genetic modifications on normal physiological functions is a promising direction for developing PRRSV-resistant pigs. In the current study, we identified a specific functional amino acid in CD163 that influences PRRSV proliferation. Viral infection experiments conducted on Marc145 and PK-15 CD163 cells illustrated that the mE535G or corresponding pE529G mutations markedly inhibited highly pathogenic PRRSV (HP-PRRSV) proliferation by preventing viral binding and entry. Furthermore, individual viral challenge tests revealed that pigs with the E529G mutation had viral loads two orders of magnitude lower than wild-type (WT) pigs, confirming effective resistance to HP-PRRSV. Examination of the physiological indicators and scavenger function of CD163 verified no significant differences between the WT and E529G pigs. These findings suggest that E529G pigs can be used for breeding PRRSV-resistant pigs, providing novel insights into controlling future PRRSV outbreaks.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Point Mutation , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Receptors, Cell Surface , Animals , Swine , Porcine Reproductive and Respiratory Syndrome/genetics , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/physiology , Porcine respiratory and reproductive syndrome virus/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, CD/genetics , Antigens, CD/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Animals, Genetically Modified/genetics , Cell LineABSTRACT
Fish gut is a versatile organ serving as the primary pathway for invasion by pathogens, particularly parasites, playing a crucial role in modulating the intestinal adaptive immune response. This study aimed to investigate the cellular-mediated reaction, mucosal acidity, and the expression of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), and CD68 in the intestines of catfish, Clarias gariepinus, naturally infected with helminths. Forty catfish were collected from the Nile River and examined for intestinal parasites. The intestinal tissues of the control and infected fish were fixed for histochemical and immunohistochemical studies. Two groups of helminths were found: cestodes Tetracampos ciliotheca and Polyonchobothrium clarias, and nematodes Paracamallanus cyathopharynx, with a prevalence rate of 63.63%, 18.0%, and 18.0%, respectively. Our results showed that the infected fish had a statistically significant rise in the activity of immune cells, including mast cells, eosinophil granular cells, and dendritic cells. This correlated with upregulation in the expressions of PCNA, VEGF, and CD68. Histochemical analyses demonstrated a marked increase in acidic mucus production, Sudan black B, and bromophenol mercury blue. This study enriches our understanding of the evolution of vertebrate immunity in combating intestinal parasitic infections and the host's adaptive responses.
Subject(s)
Catfishes , Fish Diseases , Helminthiasis, Animal , Intestinal Mucosa , Animals , Catfishes/parasitology , Fish Diseases/parasitology , Fish Diseases/immunology , Intestinal Mucosa/parasitology , Intestinal Mucosa/immunology , Helminthiasis, Animal/parasitology , Immunity, Cellular , Vascular Endothelial Growth Factor A/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Intestinal Diseases, Parasitic/veterinary , Intestinal Diseases, Parasitic/parasitology , Intestinal Diseases, Parasitic/immunology , Antigens, CD/metabolismABSTRACT
BACKGROUND: Cluster of differentiation 47 (CD47) has been identified as a new immune checkpoint. The exact role of CD47 in prognosis of breast cancer remains unclear. This study aims to evaluate immunohistochemical (IHC) expression of CD47 in breast cancer, and to measure the density of tumor associated macrophages (TAMs) infiltration by CD68 IHC staining. Furthermore, assessing the relations of CD47 and CD68 expression to different clinicopathological variables and evaluating the prognostic role of CD47 and CD68 in breast cancer cases. METHODS: This retrospective cohort study included 200 diagnosed primary breast cancer cases who underwent surgical resection at the Oncology Center of Mansoura University (OCMU), Faculty of Medicine, Egypt. Clinicopathological and survival data were collected. IHC for CD47 and CD68 was performed. RESULTS: Among 200 breast cancer cases, high CD47 expression was detected in 89 cases (44.5%). CD47 high expression was significantly associated with presence of distant metastasis (P=0.04), advanced TNM stage (P=0.02), ER & PR negativity (P=0.04 & 0.004 respectively), and molecular subtype (P=0.03). Their was a statistically significant association between CD47 and CD68 expression (P=0.002). CD47 high expression was found to predict poor overall survival, but it is not considered alone as independent poor prognostic factor by multivariate analysis. Multivariate analysis spotted combined high expression of CD47 and CD68 as an independent prognostic predictor for shorter OS in breast cancer patients (P=0.002). CONCLUSION: CD47 high expression is related to poor prognosis in breast cancer patients especially when associated with high CD68+TAMs infiltration. Therefore, CD47 is a promising prognostic and therapeutic target in breast carcinoma that may direct selection of patients for immunotherapy.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Biomarkers, Tumor , Breast Neoplasms , CD47 Antigen , Humans , Female , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , CD47 Antigen/metabolism , Prognosis , Retrospective Studies , Middle Aged , Biomarkers, Tumor/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, CD/metabolism , Adult , Follow-Up Studies , Survival Rate , Immunohistochemistry , Tumor-Associated Macrophages/metabolism , Tumor-Associated Macrophages/pathology , CD68 MoleculeABSTRACT
In this study, we examined how systemic inflammation affects repair of brain injury. To this end, we created a brain-injury model by stereotaxic injection of ATP, a damage-associated molecular pattern component, into the striatum of mice. Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS-ip). An analysis of magnetic resonance images showed that LPS-ip reduced the initial brain injury but slowed injury repair. An immunostaining analysis using the neuronal marker, NeuN, showed that LPS-ip delayed removal of dead/dying neurons, despite the fact that LPS-ip enhanced infiltration of monocytes, which serve to phagocytize dead cells/debris. Notably, infiltrating monocytes showed a widely scattered distribution. Bulk RNAseq analyses showed that LPS-ip decreased expression of genes associated with phagocytosis, with PCR and immunostaining of injured brains confirming reduced levels of Cd68 and Clec7a, markers of phagocytic activity, in monocytes. Collectively, these results suggest that systemic inflammation affects properties of blood monocytes as well as brain cells, resulting in delay in clearing damaged cells and activating repair processes.
Subject(s)
Brain , Inflammation , Lipopolysaccharides , Mice, Inbred C57BL , Monocytes , Phagocytosis , Animals , Phagocytosis/drug effects , Monocytes/metabolism , Inflammation/pathology , Brain/pathology , Male , Lipopolysaccharides/pharmacology , Brain Injuries/pathology , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Neurons/metabolism , Neurons/pathology , Neurons/drug effects , Lectins, C-Type/metabolism , Wound Healing , Mice , Adenosine Triphosphate/metabolism , CD68 MoleculeABSTRACT
BACKGROUND: Preeclampsia, especially early-onset preeclampsia (EO-PE), is a pregnancy complication that has serious consequences for the health of both the mother and the fetus. Although abnormal placentation due to mitochondrial dysfunction is speculated to contribute to the development of EO-PE, the underlying mechanisms have yet to be fully elucidated. METHODS: The expression and localization of Siglec-6 in the placenta from normal pregnancies, preterm birth and EO-PE patients were examined by RT-qPCR, Western blot and IHC. Transwell assays were performed to evaluate the effect of Siglec-6 on trophoblast cell migration and invasion. Seahorse experiments were conducted to assess the impact of disrupting Siglec-6 expression on mitochondrial function. Co-IP assay was used to examine the interaction of Siglec-6 with SHP1/SHP2. RNA-seq was employed to investigate the mechanism by which Siglec-6 inhibits mitochondrial function in trophoblast cells. RESULTS: The expression of Siglec-6 in extravillous trophoblasts is increased in placental tissues from EO-PE patients. Siglec-6 inhibits trophoblast cell migration and invasion and impairs mitochondrial function. Mechanismly, Siglec-6 inhibits the activation of NF-κB by recruiting SHP1/SHP2, leading to increased expression of GPR20. Notably, the importance of GPR20 function downstream of Siglec-6 in trophoblasts is supported by the observation that GPR20 downregulation rescues defects caused by Siglec-6 overexpression. Finally, overexpression of Siglec-6 in the placenta induces a preeclampsia-like phenotype in a pregnant mouse model. CONCLUSIONS: This study indicates that the regulatory pathway Siglec-6/GPR20 has a crucial role in regulating trophoblast mitochondrial function, and we suggest that Siglec-6 and GPR20 could serve as potential markers and targets for the clinical diagnosis and therapy of EO-PE.
Subject(s)
Cell Movement , Mitochondria , Pre-Eclampsia , Receptors, G-Protein-Coupled , Trophoblasts , Up-Regulation , Pre-Eclampsia/metabolism , Pre-Eclampsia/genetics , Pre-Eclampsia/pathology , Humans , Pregnancy , Female , Mitochondria/metabolism , Up-Regulation/genetics , Trophoblasts/metabolism , Animals , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Cell Movement/genetics , Lectins/metabolism , Placenta/metabolism , Mice , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Antigens, Differentiation, B-Lymphocyte/genetics , AdultABSTRACT
BACKGROUND: To develop an inflammation-related immunohistochemistry marker-based algorithm that confers higher diagnostic ability for idiopathic inflammatory myopathies (IIMs) than IIM-related histopathologic features. METHODS: Muscle biopsy tissues from 129 IIM patients who met the 2017 EULAR/ACR criteria and 73 control tissues from patients with non-inflammatory myopathies or healthy muscle specimens were evaluated for histological features and immunostaining results of CD3, CD4, CD8, CD20, CD68, CD163, MX1, MHC class I, MHC class II, and HLA-DR. Diagnostic algorithms for IIM were developed based on the results of the classification and regression tree (CART) analysis, which used immunostaining results as predictor variables for classifying patients with IIMs. RESULTS: In the analysis set (IIM, n = 129; control, n = 73), IIM-related histopathologic features had a diagnostic accuracy of 87.6% (sensitivity 80.6%; specificity 100.0%) for IIMs. Notably, muscular expression of CD163 (99.2% vs. 20.8%, p < 0.001) and MHC class I (87.6% vs. 23.1%, p < 0.001) was significantly higher in the IIM group than in controls. Based on the CART analysis results, we developed an algorithm combining CD163 and MHC class I expression that conferred a diagnostic accuracy of 95.5% (sensitivity 96.1%; specificity 94.5%). In addition, our algorithm was able to correctly diagnose IIM in 94.1% (16/17) of patients who did not meet the 2017 EUALR/ACR criteria but were diagnosed as having IIMs by an expert physician. CONCLUSIONS: Combination of CD163 and MHC class I muscular expression may be useful in diagnosing IIMs.
Subject(s)
Antigens, CD , Antigens, Differentiation, Myelomonocytic , Biomarkers , Histocompatibility Antigens Class I , Myositis , Receptors, Cell Surface , Humans , Antigens, Differentiation, Myelomonocytic/metabolism , Female , Male , Myositis/diagnosis , Myositis/metabolism , Middle Aged , Antigens, CD/metabolism , Biomarkers/analysis , Biomarkers/metabolism , Adult , Histocompatibility Antigens Class I/analysis , Receptors, Cell Surface/analysis , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/metabolism , Aged , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Immunohistochemistry , AlgorithmsABSTRACT
Tuberculosis (TB) remains a leading cause of mortality among individuals coinfected with HIV, characterized by progressive pulmonary inflammation. Despite TB's hallmark being focal granulomatous lung lesions, our understanding of the histopathological features and regulation of inflammation in HIV & TB coinfection remains incomplete. In this study, we aimed to elucidate these histopathological features through an immunohistochemistry analysis of HIV & TB co-infected and TB patients, revealing marked differences. Notably, HIV & TB granulomas exhibited aggregation of CD68 + macrophage (Mφ), while TB lesions predominantly featured aggregation of CD20+ B cells, highlighting distinct immune responses in coinfection. Spatial transcriptome profiling further elucidated CD68+ Mφ aggregation in HIV & TB, accompanied by activation of IL6 pathway, potentially exacerbating inflammation. Through multiplex immunostaining, we validated two granuloma types in HIV & TB versus three in TB, distinguished by cell architecture. Remarkably, in the two types of HIV & TB granulomas, CD68 + Mφ highly co-expressed IL6R/pSTAT3, contrasting TB granulomas' high IFNGRA/SOCS3 expression, indicating different signaling pathways at play. Thus, activation of IL6 pathway may intensify inflammation in HIV & TB-lungs, while SOCS3-enriched immune microenvironment suppresses IL6-induced over-inflammation in TB. These findings provide crucial insights into HIV & TB granuloma formation, shedding light on potential therapeutic targets, particularly for granulomatous pulmonary under HIV & TB co-infection. Our study emphasizes the importance of a comprehensive understanding of the immunopathogenesis of HIV & TB coinfection and suggests potential avenues for targeting IL6 signaling with SOCS3 activators or anti-IL6R agents to mitigate lung inflammation in HIV & TB coinfected individuals.
Subject(s)
Coinfection , Granuloma , HIV Infections , Lung , Macrophages , Receptors, Interleukin-6 , STAT3 Transcription Factor , Humans , Coinfection/virology , Coinfection/immunology , Coinfection/microbiology , HIV Infections/complications , HIV Infections/immunology , Macrophages/immunology , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Granuloma/immunology , Lung/pathology , Lung/immunology , Receptors, Interleukin-6/metabolism , Receptors, Interleukin-6/genetics , Suppressor of Cytokine Signaling 3 Protein/metabolism , Suppressor of Cytokine Signaling 3 Protein/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, CD/metabolism , Antigens, CD/genetics , Signal Transduction , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/complications , Male , Tuberculosis/immunology , Tuberculosis/microbiology , Tuberculosis/complications , Female , Adult , Interleukin-6/metabolism , Interleukin-6/genetics , CD68 MoleculeABSTRACT
BACKGROUND: Epstein-Barr virus (EBV) is a double-stranded DNA oncogenic virus. Several types of solid tumors, such as nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and lymphoepithelioma-like carcinoma of the lung, have been linked to EBV infection. Currently, several TCR-T-cell therapies for EBV-associated tumors are in clinical trials, but due to the suppressive immune microenvironment of solid tumors, the clinical application of TCR-T-cell therapy for EBV-associated solid tumors is limited. Figuring out the mechanism by which EBV participates in the formation of the tumor immunosuppressive microenvironment will help T cells or TCR-T cells break through the limitation and exert stronger antitumor potential. METHODS: Flow cytometry was used for analyzing macrophage differentiation phenotypes induced by EBV-infected and EBV-uninfected tumors, as well as the function of T cells co-cultured with these macrophages. Xenograft model in mice was used to explore the effects of M2 macrophages, TCR-T cells, and matrix metalloprotein 9 (MMP9) inhibitors on the growth of EBV-infected tumors. RESULTS: EBV-positive tumors exhibited an exhaustion profile of T cells, despite the presence of a large T-cell infiltration. EBV-infected tumors recruited a large number of mononuclear macrophages with CCL5 and induced CD163+M2 macrophages polarization through the secretion of CSF1 and the promotion of autocrine IL10 production by mononuclear macrophages. Massive secretion of MMP9 by this group of CD163+M2 macrophages induced by EBV infection was an important factor contributing to T-cell exhaustion and TCR-T-cell therapy resistance in EBV-positive tumors, and the use of MMP9 inhibitors improved the function of T cells cocultured with M2 macrophages. Finally, the combination of an MMP9 inhibitor with TCR-T cells targeting EBV-positive tumors significantly inhibited the growth of xenografts in mice. CONCLUSIONS: MMP9 inhibitors improve TCR-T cell function suppressed by EBV-induced M2 macrophages. TCR-T-cell therapy combined with MMP9 inhibitors was an effective therapeutic strategy for EBV-positive solid tumors.