Migration Of Peripheral Blood Mononuclear Cells Research Articles

AGEs-RAGE-KCa3.1 pathway mediates palmitic acid-induced migration of PBMCs from patients with type 2 diabetes

Type 2 diabetes mellitus (T2DM) is characterized by chronic low-grade systemic inflammation. Tissue infiltration by monocyte migration contributes to the pathogenesis of vascular complications in T2DM. We studied the role of intermediate-conductance Ca2+-activated K+ (KCa3.1) channels in the palmitic acid (PA)-induced migration of peripheral blood mononuclear cells (PBMCs) from T2DM patients and the influence of advanced glycation endproducts (AGEs). A total of 49 T2DM patients and 33 healthy subjects was recruited into this study. Using flow cytometry and Western blotting analysis as well as cell migration assay, we found that there was a significant decrease in frequency of T lymphocytes and monocytes in CD45+ leukocyte population. PA at 100 μM stimulated migration of PBMCs from T2DM individuals, which was inhibited by the specific KCa3.1 channel blocker TRAM-34 (1 μM). The PBMC migration was positively correlated with glycosylated hemoglobin A1 chain (HbA1c) level of T2DM patients, an indicator of AGEs, and PBMCs with higher level of HbA1c showed upregulated expression of toll-like receptor (TLR) 2/4 and KCa3.1 channels. In THP-1 cells, AGEs at 200 μg/ml increased protein expression of TLR 2/4 and KCa3.1 channels, and were synergistically involved in PA-induced migration through receptors of AGEs (RAGE)-mediated KCa3.1 upregulation. In conclusion, in PBMCs of T2DM patients, AGEs promotes PA-induced migration via upregulation of TLR2/4 and KCa3.1 channels.

HLA class II antibody activation of endothelial cells induces M2 macrophage differentiation in peripheral blood.

Donor-specific human leukocyte antigen (HLA) class II antibodies (HLA-II Abs) combined with allogeneic endothelial cells (ECs) mediate high-risk rejection in kidney transplant patients. Macrophage accumulation is a significant histological feature of antibody-mediated rejection (AMR) in kidney transplant patients. Here, we further investigated the effect of HLA-II Abs on macrophage phenotypes to provide theoretical basis for clinical treatment of AMR. We prepared an experimental model containing HLA-II Ab-stimulated microvascular ECs and peripheral blood mononuclear cells (PBMCs) co-culture and explored the potential relationship of HLA-II Ab, ECs activation, and macrophage differentiation. Immune phenotype of macrophage subsets was analyzed and quantified by flow cytometry. HLA-II Ab activation of ECs induces M2 macrophage differentiation signal pathways which were investigated by qPCR and western blotting. The stimulation of ECs by F(ab')2 fragment of HLA-II Abs led to phosphorylation of PI3K, Akt, and mTOR, which mediated IL-10, ICAM-1, VCAM-1 secretion. The enhanced ICAM-1 and IL-10 promoted the migration of PBMCs and their differentiation into CD68+ and CD163+ (M2-type) macrophages, respectively, but not CD86+ macrophages. These findings revealed the PI3K/Akt/mTOR signal pathways activated by HLA-II Abs in ECs and the immune regulation ability of HLA-II Abs to induce PBMC differentiation.

Abstract 3509: Immune modulation in immune co-cultures of pancreatic ductal adenocarcinoma by small molecule CLK/WNT pathway inhibitor SM04690

Abstract Pancreatic ductal adenocarcinoma (PDAC) shows little to no symptoms and is often detected at later stages which makes it difficult to treat resulting in a very low survival rate. PDAC is resistant to most chemotherapy mostly due to a high amount of stroma and low vascularity. Additionally, PDAC is resistant to the newer immunotherapies due to the lack of immune cells and/or the presence of exhausted T-cells. Expression of WNT pathway genes allows cancer to avoid the immune system by affecting immune cell activation and migration into the tumor microenvironment. In this study, we investigate Immune activation and migration by co-culturing peripheral blood mononuclear cells (PBMCs) with PDAC spheroids in the presence of SM04690 (CLK/WNTi). SM04690 is a potent CLK and downstream WNT signaling inhibitor. The migration of PBMCs into PDAC spheroids were analyzed by FLOW cell cytometry and fluorescence confocal microscopy. The total CD3+ T cell population remained unchanged in the presence of SM04690, however, T cell subpopulations were affected by WNT inhibition. The total CD4+ T cell population in the spheroid co-culture was significantly increased while the CD8+ population was significantly reduced. Among the CD4+ T cell population, there was a significant increase in CCR7+CD45RA- central memory cells and CCR7-CD45RA- effector memory cells, while there was a significant decrease in FoxP3+CD25+ regulatory T cells. Surface expression of PD-L1 and HLA-DR on PDAC cells were also significantly increased compared to the untreated co-culture control. Fluorescent labeled PDAC and PBMCs allow clear quantification of immune infiltration through spheroid and Matrigel organoid systems. Genes associated with b-catenin destruction complex (AXIN1 and 2, APC, GSK3A) and transcription factors associated with gene expression (TCF7 and TCF7L1) were also significantly reduced in the drug treatment. Additional studies are ongoing investigating the role cancer-associated fibroblast (CAFs) play in the tumor microenvironment in the presence of SM04690 (CLK/WNTi). WNT inhibition can be a potent immune modulator and influence surface marker expression on the tumor. This can lead to future drug combinations with immune checkpoint inhibitors or drug combinations taking advantage of expression changes. Future directions involve utilizing PRO-seq to identify enhancer RNAs and immediate transcriptional effects of WNT inhibition, identify novel targets for combination therapy, and in vivo PDAC humanized animal models studying the effects in the presence of an autologous immune system. Citation Format: Adrian TA Dominguez, Adreanna G. Real, Cecilia B. Levandowski, Matthew S. Lewis, Sarah Hartman, Betelehem Yacob, Stephen Smoots, Anna Schreiber, Jordi Lanis, Pragya Nepal, Jennifer Diamond, Chris Lieu, Wells Messersmith, Julie Lang, Todd Pitts. Immune modulation in immune co-cultures of pancreatic ductal adenocarcinoma by small molecule CLK/WNT pathway inhibitor SM04690 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3509.

Porcine Epidemic Diarrhea Virus Infection Disrupts the Nasal Endothelial Barrier To Favor Viral Dissemination.

ABSTRACTCrossing the endothelium from the entry site and spreading in the bloodstream are crucial but obscure steps in the pathogenesis of many emerging viruses. Previous studies confirmed that porcine epidemic diarrhea virus (PEDV) caused intestinal infection by intranasal inoculation. However, the role of the nasal endothelial barrier in PEDV translocation remains unclear. Here, we demonstrated that PEDV infection causes nasal endothelial dysfunction to favor viral dissemination. Intranasal inoculation with PEDV compromised the integrity of endothelial cells (ECs) in nasal microvessels. The matrix metalloproteinase 7 (MMP-7) released from the PEDV-infected nasal epithelial cells (NECs) contributed to the destruction of endothelial integrity by degrading the tight junctions, rather than direct PEDV infection. Moreover, the proinflammatory cytokines released from PEDV-infected NECs activated ECs to upregulate ICAM-1 expression, which favored peripheral blood mononuclear cells (PBMCs) migration. PEDV could further exploit migrated cells to favor viral dissemination. Together, our results reveal the mechanism by which PEDV manipulates the endothelial dysfunction to favor viral dissemination and provide novel insights into how coronavirus interacts with the endothelium.IMPORTANCE The endothelial barrier is the last but vital defense against systemic viral transmission. Porcine epidemic diarrhea virus (PEDV) can cause severe atrophic enteritis and acute viremia. However, the mechanisms by which the virus crosses the endothelial barrier and causes viremia are poorly understood. In this study, we revealed the mechanisms of endothelial dysfunction in PEDV infection. The viral infection activates NECs and causes the upregulation of MMP-7 and proinflammatory cytokines. Using NECs, ECs, and PBMCs as in vitro models, we determined that the released MMP-7 contributed to the destruction of endothelial barrier, and the released proinflammatory cytokines activated ECs to facilitate PBMCs migration. Moreover, the virus further exploited the migrated cells to promote viral dissemination. Thus, our results provide new insights into the mechanisms underlying endothelial dysfunction induced by coronavirus infection.

Abstract PR-001: Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma

Abstract Introduction: Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12) axis is a key immunosuppressive pathway where CXCL12 released by cancer-associated fibroblasts (CAF) binds to its receptor CXCR4 expressed by cytotoxic CD8+ T cells (CTL) which results in their decreased abundance within tumors. Encouraging preclinical and clinical data from mouse PDAC models and human PDAC tumor sections have shown T cell tumor accumulation after co-inhibition of CXCR4 and PD1/PDL1, however number of tumor regressions were limited. Results: To further explore the mechanism by which inhibition of the CXCR4/CXCL12 pathway modulates the immune TME in triggering an anti-tumor immune response in human PDAC, we generated an autologous co-culture system with patient-derived tumor organoids (PDTO) and peripheral blood mononuclear cells (PBMC). PDAC tumor tissue from surgical resection was used to generate lines of PDTO that recapitulate the histological and genetic characteristics of the original tumor. We demonstrate that tumor-specific reactive T cells can be obtained in PDAC by co-culture with autologous PDTO, with up to 5% of CD8+ T cells producing IFNγ after tumor organoid stimulation. No T cell reactivity was observed against normal pancreatic organoids, and blocking tumor MHC I/MHC II decreased the proportion of activated T cells. Ex vivo migration assays were established to better understand the role of CXCL12 in PDAC tumor T cell exclusion. Tumor organoids attracted autologous PBMC and addition of recombinant CXCL12 decreased PBMC migration, while treatment of PBMC with the CXCR4 inhibitor AMD3100, rescued the number of PBMCs that were able to migrate towards the tumor organoids. PBMC infiltrated PDTO-containing but not empty Matrigel domes. Addition of AMD3100 increased PBMC infiltration within the Matrigel dome containing PDTOs. Finally, we found that pre-treatment of PBMC with AMD3100 increased T cell reactivity against PDTO, and that T cell activation was further increased when stimulated with PDTO pre-treated with chemotherapy. Conclusions: We found that CXCL12 modulates the migration of T cells towards tumor organoids generated from pancreatic cancer tissue, and that inhibition of CXCR4 not only increases the migration potential, but also increases tumor-specific T cell activation. The use of ex vivo autologous co-culture and migration assays provide a unique strategy to identify modulators that may enhance T cell mediated neoplastic cell death to guide therapeutic intervention in PDAC. Citation Format: Emily Alouani, Ilenia Pellicciotta, Winston Wong, Alexander S. Thomas, Michael D. Kluger, Anna M. Chiarella, Anil K. Rustgi, Gulam A. Manji. Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-001.

Oestrogen deprivation induces chemokine production and immune cell recruitment in in vitro and in vivo models of oestrogen receptor-positive breast cancer

BackgroundOestrogen receptor-positive (ER+) breast cancer is commonly treated using endocrine therapies such as aromatase inhibitors which block synthesis of oestradiol, but the influence of this therapy on the immune composition of breast tumours has not been fully explored. Previous findings suggest that tumour infiltrating lymphocytes and immune-related gene expression may be altered by treatment with aromatase inhibitors. However, whether these changes are a direct result of impacts on the host immune system or mediated through tumour cells is not known. We aimed to investigate the effect of oestrogen deprivation on the expression of chemokines and immune infiltration in vitro and in an ER+ immunocompetent mouse model.MethodsRT-qPCR and a bead-based Bioplex system were used to investigate the expression of chemokines in MCF-7 breast cancer cells deprived of oestrogen. A migration assay and flow cytometry were used to measure the migration of human peripheral blood mononuclear cells (PBMCs) to MCF-7 cells grown without the main biologically active oestrogen, oestradiol. Using flow cytometry and immunohistochemistry, we examined the immune cell infiltrate into tumours created by injecting SSM3 ER+ breast cancer cells into wild-type, immunocompetent 129/SvEv mice.ResultsThis study demonstrates that oestrogen deprivation increases breast cancer secretion of TNF, CCL5, IL-6, IL-8, and CCL22 and alters total human peripheral blood mononuclear cell migration in an in vitro assay. Oestrogen deprivation of breast cancer cells increases migration of CD4+ T cells and decreases migration of CD11c+ and CD14+ PBMC towards cancer cells. PBMC migration towards breast cancer cells can be reduced by treatment with the non-steroidal anti-inflammatory drugs, aspirin and celecoxib. Treatment with endocrine therapy using the aromatase inhibitor letrozole increases CD4+ T cell infiltration into ER+ breast cancer tumours in immune competent mice.ConclusionsThese results suggest that anti-oestrogen treatment of ER+ breast cancer cells can alter cytokine production and immune cells in the area surrounding the cancer cells. These findings may have implications for the combination and timing of anti-oestrogen therapies with other therapies.

Decreased Astrocytic CCL2 Accounts for BAF-312 Effect on PBMCs Transendothelial Migration Through a Blood Brain Barrier in Vitro Model

Disruption of the blood brain barrier (BBB) is a common event in several neurological diseases and in particular, in multiple sclerosis (MS), it contributes to the infiltration of the central nervous system by peripheral inflammatory cells. Sphingosine-1-phosphate (S1P) is a bioactive molecule with pleiotropic effects. Agonists of S1P receptors such as fingolimod and siponimod (BAF-312) are in clinical practice for MS and have been shown to preserve BBB function in inflammatory conditions. Using an in vitro BBB model of endothelial-astrocytes co-culture exposed to an inflammatory insult (tumor necrosis factor-α and interferon-γ; T&I), we show that BAF-312 reduced the migration of peripheral blood mononuclear cells (PBMCs) through the endothelial layer, only in the presence of astrocytes. This effect was accompanied by decreased expression of the adhesion molecule ICAM-1. BAF-312 also reduced the activation of astrocytes, by controlling NF-kB and NLRP3 induction and preventing the increase of proinflammatory cytokine and chemokines. Reduction of CCL2 by BAF-312 may be responsible for the observed effects and, accordingly, addition of exogenous CCL2 was able to counteract BAF-312 effects and rescued T&I responses on PBMC migration, ICAM-1 expression and astrocyte activation. The present results further point out BAF-312 effects on BBB properties, suggesting also the key role of astrocytes in mediating drug effects on endothelial function.Graphical abstract

Chemokine-Induced PBMC and Subsequent MSC Migration Toward Decellularized Heart Valve Tissue.

Enhancing the recellularization of a decellularized heart valve in situ may lead to an improved or ideal heart valve replacement. A promising approach is leveraging the immune response for inflammation-mediated recellularization. However, this mechanism has not been previously demonstrated in vitro. This study investigated loading the chemokine MCP-1 into decellularized porcine heart valve tissue and measured the migration of human peripheral blood mononuclear cells (PBMCs) and mesenchymal stem cells (MSCs) toward the chemokine loaded valve tissue. The results of this study demonstrate that MCP-1-loaded tissues increase PBMC migration compared to non-loaded tissues. Additionally, we demonstrate MCP-1-loaded tissues that have recruited PBMCs lead to increased migration of MSCs compared to decellularized tissue alone. The results of this study provide evidence for the inflammation-mediated recellularization mechanism. Furthermore, the results support the use of such an approach for enhancing the recellularization of a decellularized heart valve.

Regulation of toll-like receptors expression in muscle cells by exercise-induced stress.

ObjectiveThis study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them.MethodsThe expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H2O2), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells.ResultsThe qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H2O2, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells.ConclusionIn this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H2O2, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

Reciprocal Interplay Between Astrocytes and CD4+ Cells Affects Blood-Brain Barrier and Neuronal Function in Response to β Amyloid.

Background: In Alzheimer’s disease (AD) neuronal degeneration is associated with gliosis and infiltration of peripheral blood mononuclear cells (PBMCs), which participate in neuroinflammation. Defects at the blood-brain barrier (BBB) facilitate PBMCs migration towards the central nervous system (CNS) and in particular CD4+ T cells have been found in areas severely affected in AD. However, the role of T cells, once they migrate into the CNS, is not well defined. CD4+ cells interact with astrocytes able to release several factors and cytokines that can modulate T cell polarization; similarly, astrocytic properties are modulated after interaction with T cells.Methods: In in vitro models, astrocytes were primed with β-amyloid (Aβ; 2.5 μM, 5 h) and then co-cultured with magnetically isolated CD4+ cells. Cytokines expression was evaluated both in co-cultured CD4+ cells and astrocytes. The effects of this crosstalk were further evaluated by co-culturing CD4+ cells with the neuronal-like SH-SY5Y cell line and astrocytes with endothelial cells.Results: The pattern of cytokines and trophic factors expressed by CD4+ cells were strongly modulated in the presence of Aβ-primed astrocytes. Specifically, the percentage of IL-4+ and IFNγ+ CD4+ cells was significantly increased and reduced, respectively. Further, increased BDNF mRNA levels were observed in CD4+ cells. When SH-SY5Y cells were co-cultured with astrocyte-conditioned CD4+ cells and exposed to Aβ, the reduction of the presynaptic protein synaptophysin was prevented with a BDNF-dependent mechanism. In astrocytes co-cultured with CD4+ cells, reduced mRNA levels of inflammatory cytokines and VEGF were observed. This was paralleled by the prevention of the reduction of claudin-5 when astrocytes were co-cultured with endothelial cells.Conclusion: Following Aβ exposure, there exists reciprocal crosstalk between infiltrating peripheral cells and astrocytes that in turn affects not only endothelial function and thus BBB properties, but also neuronal behavior. Since astrocytes are the first cells that lymphocytes interact with and are among the principal players in neuroinflammation occurring in AD, understanding this crosstalk may disclose new potential targets of intervention in the treatment of neurodegeneration.

Caveolin-1 Regulates CCL5 and PPARγ Expression in Nthy-ori 3-1 Cells: Possible Involvement of Caveolin-1 and CCL5 in the Pathogenesis of Hashimoto's Thyroiditis.

Hashimoto's thyroiditis (HT) is characterized by lymphocytic infiltration of the thyroid parenchyma, which ultimately leads to tissue destruction and loss of function. Caveolin-1 (Cav-1) is an essential structural constituent of lipid rafts in the plasma membrane of cells and is reported to be significantly reduced in thyrocytes from HT patients. However, the mechanism of Cav-1 involvement in HT pathogenesis is still largely unclear. Cav-1 expression in thyroid tissues from HT patients and euthyroid nodular goiter tissues was detected by immunohistochemistry staining. Cav-1 knockdown and overexpression were constructed by lentiviral transfection in the human thyroid follicular epithelial cell (TFC) line of Nthy-ori 3-1. The mRNA expression levels of chemokines in TFCs were determined by quantitative real-time PCR (qPCR). Cav-1 and peroxisome proliferator-activated receptor gamma (PPARγ) levels were analysed by qPCR and Western blot analysis. The migration ability of peripheral blood mononuclear cells (PBMCs) was detected by the Transwell assay. In this study, Cav-1 and PPARγ expression was reduced in the thyroid tissues from HT patients. In vitro experiments showed that the expressions of chemokine (C-C motif) ligand 5 (CCL5) and migration of PBMCs were markedly increased, while the level of PPARγ was significantly decreased after the lentivirus-mediated knockdown of Cav-1 in Nthy-ori 3-1 cells. Interestingly, pioglitazone, a PPARγ agonist, not only upregulated PPARγ and Cav-1 proteins significantly, but also effectively reversed the Cav-1-knockdown-induced upregulation of CCL5 in Nthy-ori 3-1 cells and reduced the infiltration of lymphocytes. The inhibition of Cav-1 upregulated the CCL5 expression and downregulated the PPARγ expression in TFC while pioglitazone, a PPARγ agonist, reversed the detrimental consequence. This outcome might be a potential target for the treatment of lymphocyte infiltration into the thyroid gland and HT development.

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro.

During host-parasite interactions, binding of excretory/secretory proteins (ESPs) on the host immune cells is considered the fundamental phase for regulation of immune responses. In this study, gene encoding Haemonchus contortus tropomyosin (Hc-TpMy), was successfully cloned and expressed, and the recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells (PBMCs) in vitro. The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy. The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs. Furthermore, immunoregulatory roles of rHc-TpMy on cytokines expression, PBMC proliferation, migration, nitric oxide (NO) production, apoptosis and monocytes phagocytosis were observed. The results showed that expression of IL-4 and IFN-γ cytokines, cell proliferation, NO production and PBMC migration were significantly suppressed by goat PBMCs after co-incubation with rHc-TpMy protein. However, the productions of IL-10, IL-17 and TGF-β1 cytokines, PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner. Our findings indicated that rHc-TpMy is an important ES binding protein exhibit distinct immuno-suppressive roles on goat PBMCs which might be a potential molecular target to control haemonchosis in future.

Abstract P3-01-29: Impact of heparan sulphate binding domain of chemokine CCL21 to migration of breast cancer cells

Abstract Lymph node metastasis constitutes a key event in breast cancer progression. Chemokines are small proteins, which can promote metastatic spread by inducing cancer cell migration and invasion. Chemokine function is dependant upon their binding to both cell surface heparan sulphate (HS) molecules and to their specific receptor. Our group has demonstrated a significant increase in chemokine receptor CCR7 expression in cancerous breast epithelia compared to healthy controls. This study is designed to test the hypothesis that a non-HS binding forms of chemokine CCL21 can disrupt the normal response to CCL21, therefore reducing the metastasis of CCR7-expressing cancer cells. Truncated CCL21 chemokine (Δ98-134 c-terminal basic extension), was synthesised to investigate a possible linkage between chemokine binding capacity and cell activation. Wild type (WT) and mutant-CCL21 were tested for their ability to stimulate a dose-dependent increase in intracellular-free calcium in peripheral blood mononuclear cell (PBMC) and breast cancer epithelial cells MDA-MB-231. Mutant-CCL21 at concentrations 5 and 10nM showed potential to mobilise Ca2+ at levels similar to that produced by WT-CCl21. A series of experiments was performed to determine how deletion of the HS-binding site altered the ability of CCL21 to stimulate chemotaxis within a concentration gradient generated by free solute diffusion. PBMC stimulated to migrate by wild-type CCL21 was not significantly different from that stimulated by mutant (P> 0.05). Similar results were observed in assays using MDA-MB-231 cells. A further series of experiments was performed to compare the potential of WT and mutant-CCL21 to stimulate the migration of cells across endothelium. In contrast to results for trans-filter migration, it was found that the non HS-binding mutant stimulated no increased in transendothelial cell migration above the background at each of the tested concentrations, 10, 30 and 50 nM respectively (P>0.05). However, WT-CCL21 stimulated significant increased PBMC migration at each of the tested concentration (all P <0.001). Furthermore, the effect of heparin on chemotactic properties of WT and mutant- CCL21 was examined. Interestingly, heparin (250 µg/ml) completely inhibit the chemotaxis mediated by WT-CCL21 (5nM) (P < 0.001), whereas it did not inhibit the chemotaxis at concentrations 100, 250 & 500 µg/ml in response to mutant CCL21 (5nM) (P > 0.05). Similar assay will be performed using MDA-MB-231 cells. Work is ongoing to characterise the biophysical properties of mutant-CCL21 and determine its potential role for a therapeutic blockade of the migration of breast cancer cells in-vivo. Our primarily data showed that mutant CCL21 in xenograft brain tumor models showed substantial inhibition of tumour growth. Our results indicate that truncated CCL21 chemokine might be a potential preventive biofactor for human breast cancer metastasis by targeting chemokine receptor genes. Citation Format: Mohammed Imad Malki. Impact of heparan sulphate binding domain of chemokine CCL21 to migration of breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-29.

Astrocytes Modify Migration of PBMCs Induced by β-Amyloid in a Blood-Brain Barrier in vitro Model.

BackgroundThe brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer’s disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-β (Aβ).MethodsWe used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aβ, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs).ResultsIn the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aβ treatment. On the contrary, in endothelial monocultures, Aβ induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aβ. This modulation by Aβ was not observed in endothelial monocultures. In addition, endothelial expression of β-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aβ only in the co-culture model. miR-200b, increased in astrocytes following Aβ treatment and may represent one of the factors involved in the control of Gnt-III expression.ConclusionThese data point out that, at least in the early phases of Aβ exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer.

Peficitinib Inhibits the Chemotactic Activity of Monocytes via Proinflammatory Cytokine Production in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Background: This study was performed to examine the effects of the Janus kinase (JAK) inhibitor peficitinib on fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA). Methods: To examine the expression of JAK1, JAK2, and JAK3 in RA synovial tissue (ST) and FLS, immunohistochemistry was performed. We investigated the effects of peficitinib on interleukin 6 and IL-6 receptor responses in RA FLS. Phosphorylation of STAT was determined by western blot. To examine the functional analysis of peficitinib, we performed a proliferation and chemotaxis assays with FLS using THP-1 and peripheral blood mononuclear cells (PBMC). The inflammatory mediator expression of FLS was estimated by enzyme-linked immunosorbent assay. Results: JAK1, JAK2, and JAK3 were expressed in RA STs and FLS. Phosphorylation of STAT1, STAT3, and STAT5 in RA FLS was suppressed by peficitinib in a concentration-dependent manner. Peficitinib-treated RA FLS-conditioned medium reduced THP-1 and PBMC migration (p < 0.05) and proliferation of RA FLS (p < 0.05). Peficitinib suppressed the secretion of MCP-1/CCL2 in the RA FLS supernatant (p < 0.05). Conclusion: Peficitinib suppressed the JAK-STAT pathway in RA FLS and also suppressed monocyte chemotaxis and proliferation of FLS through inhibition of inflammatory cytokines.

Semaphorin 3F Promotes Transendothelial Migration of Leukocytes in the Inflammatory Response After Survived Cardiac Arrest.

Leukocyte transmigration through the blood vessel wall is a fundamental step of the inflammatory response and requires expression of adhesion molecule PECAM-1. Accumulating evidence implicates that semaphorin (Sema) 3F and its receptor neuropilin (NRP) 2 are central regulators in vascular biology. Herein, we assess the role of Sema3F in leukocyte migration in vitro and in vivo. To determine the impact of Sema3F on leukocyte recruitment in vivo, we used the thioglycollate-induced peritonitis model. After the induction of peritonitis, C57BL/6 mice were intraperitoneally (i.p.) injected daily with recombinant Sema3F or solvent for 3days. Compared with solvent-treated controls, leukocyte count was increased in the peritoneal lavage of Sema3F-treated mice indicating that Sema3F promotes leukocyte extravasation into the peritoneal cavity. In line with this observation, stimulation of human endothelial cells with Sema3F enhanced the passage of peripheral blood mononuclear cells (PBMCs) through the endothelial monolayer in the transwell migration assays. Conversely, silencing of endothelial Sema3F by siRNA transfection dampened diapedesis of PBMCs through the endothelium in vitro. xMechanistically, Sema3F induced upregulation of adhesion molecule PECAM-1 in endothelial cells and in murine heart tissue shown by immunofluorescence and western blotting. The inhibition of PECAM-1 by blocking antibody HEC7 blunted Sema3F-induced leukocyte migration in transwell assays. SiRNA-based NRP2 knockdown reduced PECAM-1 expression and migration of PBMCs in Sema3F-treated endothelial cells, indicating that PECAM-1 expression and leukocyte migration in response to Sema3F depend on endothelial NRP2. To assess the regulation of Sema3F in human inflammatory disease, we collected serum samples of patients from day 0 to day 7 after survived out-of-hospital cardiac arrest (OHCA, n = 41). First, we demonstrated enhanced migration of PBMCs through endothelial cells exposed to the serum of patients after OHCA in comparison to the serum of patients with stable coronary artery disease or healthy volunteers. Remarkably, serum samples of OHCA patients contained significantly higher Sema3F protein levels compared with CAD patients (CAD, n = 37) and healthy volunteers (n = 11), suggesting a role of Sema3F in the pathophysiology of the inflammatory response after OHCA. Subgroup analysis revealed that elevated serum Sema3F levels after ROSC are associated with decreased survival, myocardial dysfunction, and prolonged vasopressor therapy, clinical findings that determine the outcome of post-resuscitation period after OHCA. The present study provides novel evidence that endothelial Sema3F controls leukocyte recruitment through a NRP2/PECAM-1-dependent mechanism. Sema3F serum concentrations are elevated following successful resuscitation suggesting that Sema3F might be involved in the inflammatory response after survived OHCA. Targeting the Sema3F/NRP2/PECAM-1 pathway could provide a novel approach to abolish overwhelming inflammation after resuscitation.

Varicella zoster virus differentially alters morphology and suppresses proinflammatory cytokines in primary human spinal cord and hippocampal astrocytes

BackgroundVaricella zoster virus (VZV) is a ubiquitous alphaherpesvirus that produces varicella and zoster. VZV can infect multiple cell types in the spinal cord and brain, including astrocytes, producing myelopathy and encephalopathy. While studies of VZV-astrocyte interactions are sparse, a recent report showed that quiescent primary human spinal cord astrocytes (qHA-sps) did not appear activated morphologically during VZV infection. Since astrocytes play a critical role in host defenses during viral infections of the central nervous system, we examined the cytokine responses of qHA-sps and quiescent primary human hippocampal astrocytes (qHA-hps) to VZV infection in vitro, as well as the ability of conditioned supernatant to recruit immune cells.MethodsAt 3 days post-infection, mock- and VZV-infected qHA-sps and qHA-hps were examined for morphological changes by immunofluorescence antibody assay using antibodies directed against glial fibrillary acidic protein and VZV. Conditioned supernatants were analyzed for proinflammatory cytokines [interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-gamma, and tumor necrosis factor-α] using the Meso Scale Discovery multiplex ELISA platform. Finally, the ability of conditioned supernatants to attract peripheral blood mononuclear cells (PBMCs) was determined using a chemotaxis assay. Quiescent primary human perineurial cells (qHPNCs) served as a control for VZV-induced cytokine production and PBMC migration. To confirm that the astrocytes have the ability to increase cytokine secretion, qHA-sps and qHA-hps were treated with IL-1β and examined for morphological changes and IL-6 secretion.ResultsVZV-infected qHA-sps displayed extensive cellular processes, whereas VZV-infected qHA-hps became swollen and clustered together. Astrocytes had the capacity to secrete IL-6 in response to IL-1β. Compared to mock-infected cells, VZV-infected qHA-sps showed significantly reduced secretion of IL-2, IL-4, IL-6, IL-12p70, and IL-13, while VZV-infected qHA-hps showed significantly reduced IL-8 secretion. In contrast, levels of all 10 cytokines examined were significantly increased in VZV-infected qHPNCs. Consistent with these results, conditioned supernatant from VZV-infected qHPNCs, but not that from VZV-infected qHA-sps and qHA-hps, recruited PBMCs.ConclusionsVZV-infected qHA-sps and qHA-hps have distinct morphological alterations and patterns of proinflammatory cytokine suppression that could contribute to ineffective viral clearance in VZV myelopathy and encephalopathy, respectively.

The pervasive effects of recombinant Fasciola gigantica Ras-related protein Rab10 on the functions of goat peripheral blood mononuclear cells

BackgroundFasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats.MethodsWe cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0.ResultsThe FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-β) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway.ConclusionsrFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite.

IL-1β promotes transendothelial migration of PBMCs by upregulation of the FN/α5β1 signalling pathway in immortalised human brain microvascular endothelial cells

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain.The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the β1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether β1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1β (IL-1β)-induced inflammatory in vitro BBB model. We demonstrate that IL-1β increases cell-matrix adhesion and induces a redistribution of active β1 integrins to the basal surface. In particular, binding of α5β1 integrin to its ligand fibronectin is enhanced and α5β1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5β1 integrin reduces the IL-1β-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs).These data imply that IL-1β-induced inflammation not only destabilizes tight junctions but also increases α5β1 integrin-dependent cell-matrix adhesion to fibronectin.

Fingolimod reduces circulating tight-junction protein levels and in vitro peripheral blood mononuclear cells migration in multiple sclerosis patients

There are no data on the effects of fingolimod, an immunomodulatory drug used in treatment of multiple sclerosis (MS), on circulating tight-junction (TJ) protein levels as well as on peripheral blood mononuclear cells (PBMC) migration. Serum TJ protein [occludin (OCLN), claudin-5 (CLN-5) and zonula occludens-1 (ZO-1)] levels, sphingosine-1 phosphate 1 (S1P1) receptor expression on circulating leukocyte populations as well as in vitro PBMC migration were longitudinally assessed in 20 MS patients under 12-months fingolimod treatment and correlated with clinical and magnetic resonance imaging (MRI) parameters. After 12 months of treatment, a significant reduction of mean relapse rate as well as number of active lesions at MRI was found. TJ protein levels significantly decreased and were associated with reduction of S1P1 expression as well as of PBMC in vitro migratory activity. A significant correlation of CLN-5/OCLN ratio with new T2 MRI lesions and a significant inverse correlation of CLN-5/ZO-1 ratio with disability scores were found. These findings support possible in vivo effects of fingolimod on the blood-brain barrier (BBB) functional activity as well as on peripheral cell trafficking that could result in avoiding passage of circulating autoreactive cells into brain parenchyma. Circulating TJ protein levels and respective ratios could be further studied as a novel candidate biomarker of BBB functional status to be monitored in course of fingolimod as well as of other immunomodulatory treatments in MS.