CD36 Expression In Macrophages Research Articles

From LAL-D to MASLD: Insights into the role of LAL and Kupffer cells in liver inflammation and lipid metabolism

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver pathology worldwide, closely associated with obesity and metabolic disorders. Increasing evidence suggests that macrophages play a crucial role in the development of MASLD. Several human studies have shown an inverse correlation between circulating lysosomal acid lipase (LAL) activity and MASLD. LAL is the sole enzyme known to degrade cholesteryl esters (CE) and triacylglycerols in lysosomes. Consequently, these substrates accumulate when their enzymatic degradation is impaired due to LAL deficiency (LALD). This study aimed to investigate the role of hepatic LAL activity and liver-resident macrophages (i.e., Kupffer cells (KC)) in MASLD. To this end, we analyzed lipid metabolism in hepatocyte-specific (hep)Lal−/− mice and depleted KC with clodronate treatment. When fed a high-fat/high-cholesterol diet (HF/HCD), hepLal−/− mice exhibited CE accumulation and an increased number of macrophages in the liver and significant hepatic inflammation. KC were depleted upon clodronate administration, whereas infiltrating/proliferating CD68-expressing macrophages were less affected. This led to exacerbated hepatic CE accumulation and dyslipidemia, as evidenced by increased LDL-cholesterol concentrations. Along with proteomic analysis of liver tissue, these findings indicate that hepatic LAL-D in HF/HCD-fed mice leads to macrophage infiltration into the liver and that KC depletion further exacerbates hepatic CE concentrations and dyslipidemia.

The Role of Soluble CD163 (sCD163) in Human Physiology and Pathophysiology.

Soluble CD163 (sCD163) is a circulating inflammatory mediator, indicative of acute and chronic, systemic and non-systemic inflammatory conditions. It is the cleavage outcome, consisting of almost the entire extracellular domain, of the CD163, a receptor expressed in monocytic lineages. Its expression is proportional to the abundance of CD163+ macrophages. Various mechanisms trigger the shedding of the CD163 receptor or the accumulation of CD163-expressing macrophages, inducing the sCD163 concentration in the circulation and bodily fluids. The activities of sCD163 range from hemoglobin (Hb) scavenging, macrophage marker, decoy receptor for cytokines, participation in immune defense mechanisms, and paracrine effects in various tissues, including the endothelium. It is an established marker of macrophage activation and thus participates in many diseases, including chronic inflammatory conditions, such as atherosclerosis, asthma, and rheumatoid arthritis; acute inflammatory conditions, such as sepsis, hepatitis, and malaria; insulin resistance; diabetes; and tumors. The sCD163 levels have been correlated with the severity, stage of the disease, and clinical outcome for many of these conditions. This review article summarizes the expression and role of sCD163 and its precursor protein, CD163, outlines the sCD163 generation mechanisms, the biological activities, and the known underlying molecular mechanisms, with an emphasis on its impact on the endothelium and its contribution in the pathophysiology of human diseases.

Expression of CD68 by macrophages and histopathology of the placenta in COVID-19: association with obstetric and neonatal complications

Introduction. COVID-19 causes direct and indirect placental damage through inflammatory monocytes/ macrophages, increasing the risk of infectious and inflammatory diseases in newborns.Aim. To analyze the expression of CD68 by macrophages and structural changes in the placenta, and to assess the risks of obstetric and neonatal complications in cases of moderate COVID-19 during the third trimester of pregnancy.Materials and methods. A histological examination was conducted on 33 placentas from women with moderate COVID-19 in the third trimester of pregnancy and 30 placentas from women not infected with SARS-CoV-2. Tissue sections were analyzed using an automated microscopy system for histological studies. CD68 expression on placental macrophages was studied using flow cytometry. Detection of SARS-CoV-2 RNA in placental tissue samples, as well as in nasopharyngeal swabs from newborns, was carried out by real-time reverse transcription polymerase chain reaction (RT-PCR).Results. An increase in the number of CD68-positive macrophages was observed in the placentas from women with COVID-19 (80.8 (78.2; 83.5) %; p<0.0001) compared to samples from women not infected with SARS-CoV-2 (19.6 (17.2; 21.8) %). All placental samples and nasopharyngeal swabs from newborns of mothers with COVID-19 tested negative for SARS-CoV-2, indicating a low risk of vertical transmission. Histological analysis revealed signs of infectious involvement of both maternal and fetal placental parts, including focal deciduitis, villitis, and histiocytic intervillositis. Circulatory disorders were characterized by decidual vasculopathy, accelerated villous maturation, perivillous fibrin deposition, thrombosis, villous necrosis, and an increase in syncytial knots. A link was established between structural placental abnormalities and the development of chronic placental insufficiency (relative risk (RR)=2.33), preterm birth and premature rupture of membranes (RR=4.26), and fetal growth restriction. Newborns from mothers with COVID-19 exhibited complications such as cerebral ischemia and respiratory distress syndrome.Conclusion. Moderate COVID-19 in the third trimester of pregnancy can induce structural changes in the placenta, reflecting both compensatory-adaptive responses and inflammation driven by CD68-expressing macrophages. These alterations are associated with a high risk of obstetric and neonatal complications.

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1530.

Abstract B101: Obesity mediates altered triple-negative breast cancer tumor biology in African American women independent of ancestry

Abstract African American (AA) breast cancer (BC) is associated with a higher incidence of aggressive BC phenotypes at younger age, higher grade of tumor as compared to BCs of age-matched patients of European American (EA). For AA women, there is a higher frequency of the more aggressive TNBC subtype and of the PAM50-molecular basal subtype. AA tumors are enriched for inflammatory and immune pathways, implicating inherited susceptibility associated with African ancestry. Obesity induces a state of low-grade inflammation. AA women tend to be more obese than EA and have elevated a inflammatory markers, including CRP, IL6, and IL1. Since inflammation related to obesity is a reflection of dysfunctional adipocyte biology, we used dataset GSE88837 to perform in silico analysis of gene expression changes in visceral adipose tissue for AAs and EAs. AA adipose tissue is more inflamed than EAs at the same level of obesity (BMI>30). There was a high expression of secreted mediators causing systemic inflammation, including heat shock protein 60 (HSPD1), CXCL1, S100A9, and SPP1. VEGFA, S100A9, and SPP, tumor-promoting angiogenic mediators, were higher in obese AAs than EAs. These data indicate that obesity enhances inflammation in visceral adipose tissue of AAs. We performed in silico analysis of dataset GSE78958, of 400 BC patients annotated for race, BMI, and hormone receptor expression. We analyzed BC patients for differential gene expression, PAM50 subtype, tumor stage and grade, based on race and BMI. AA women have a higher BMI (48.9%) than EA women (32.3%) and a higher percentage of poorly differentiated tumors (51.1%) than EAs (40.6%). AA women with BMI >30, had more basal-like tumors (34.8%) than EA women (19.3%). These analyses confirm that excess adiposity promotes the basal BC phenotype. With BMI >30 as a criterion for obesity, the GSE78958 dataset containing BCs for 96 AA and 200 EA patients was analyzed for differences in gene expression. DEGs showed upregulation of T cell exhaustion marker PTPN22 and LAIR2. The metastasis and proliferation promoting chemokines CXCL1, CCL2, CXC12, CXCR4, and S100A9 were higher in the BCs of obese AAs, than BCs of non-obese AAs. Stemness related genes, STAT1, SOX4, and KLF4, were higher in BCs of obese AAs. CD163-expressing M2 macrophages were higher in BCs of obese AAs. Our group showed that there was a considerable overlap of genes among African and AA cases, but there were some non-overlapping genes indicating that ancestry alone cannot explain the genetic diversity in the tumor phenotype of AA patients. The divergence was largely driven by an upregulated gene signature found among AAs and not seen among African patients. GSE78958 (obesity BC dataset) was mapped for ancestral correlation of altered genes in the AA BC dataset. Ancestry mapping showed an inverse correlation of HSPD1 with ancestry. Obesity upregulated HSPD1 in BCs of AAs as well as in AA adipose tissue and decreased the probability of survival for women of AA ethnicity, suggesting delineation of ancestry-specific and race-specific effects. Citation Format: Deepa Bedi, Rachel Martini, Melissa B. Davis, Clayton Yates. Obesity mediates altered triple-negative breast cancer tumor biology in African American women independent of ancestry [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B101.

2-(2-Phenylethyl)chromone-enriched extract of Chinese agarwood (Aquilaria sinensis) inhibits atherosclerosis progression through endoplasmic reticulum stress-mediated CD36 expression in macrophages

Ethnopharmacological relevanceChinese agarwood, derived from the Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), has a long history of use in Traditional Chinese Medicine for the management of cardiovascular disease. However, the specific active ingredients responsible for its impact on atherosclerosis are yet to be fully understood.Aim of the study: The aim of this study is to investigate the anti-atherosclerotic effectiveness of the 2-(2-phenylethyl)chromone-enriched extract derived from Chinese agarwood (CPE) through the ER stress-mediated CD36 pathway. Materials and methodsTo assess the effectiveness of CPE, an atherosclerotic mouse model was established using ApoE−/− mice with a high-fat diet. Then we assessed the impact of CPE on lipid accumulation in THP-1 macrophages that were exposed to oxLDL. Subsequently, the effect of CPE on the expression of CD36 and markers related to ER stress was characterized. ResultsOur in vivo research confirmed that CPE effectively reduces the formation of aortic plaques in atherosclerotic ApoE−/− mice. Additionally, our in vitro study observed that CPE inhibits the uptake of oxLDL and hinders the generation of foam cells. This effect is achieved by downregulating the level of CD36 in macrophages. Furthermore, our study revealed that the increase in CD36 expression, resulting from oxLDL exposure, is governed by the activation of JNK1/2/3 signaling pathways and the initiation of ER stress. ConclusionCPE demonstrated significant efficacy to inhibit the atherosclerosis. The ER stress/P-JNK/PPARγ/CD36 signaling pathway plays critical involvement in modulating the foam cell formation in vitro and in vivo. These findings underscore the efficacy of CPE as a viable therapeutic intervention for the treatment of atherosclerosis.

Morphological Distribution Patterns and Neuroimmune Communication of Ganglia in Molly Fish (Poecilia sphenops, Valenciennes 1846)

Twenty-four adult molly fish (Poecilia sphenops, Valenciennes 1846) were collected to study the morphology and distribution of ganglia using histological, immunohistochemical, and electron microscopy and focusing on their relation to the immune cells. The ganglia were classified spatially into cranial and spinal, and functionally into sensory and autonomic. Spinal ganglia (dorsal root ganglia, DRG) contained large close ganglionic cells, enclosed by satellite cells, as well as bundles of both myelinated and non-myelinated nerve fibers. There are glial cells, immune cells and telocytes close to the ganglion. In addition, oligodendrocytes were closely related to myelinated axons. Glial fibrillary acidic protein (GFAP) expression was confined to the glia cells and the nerve fibers in the cervical ganglia next to the gills, and surprisingly, in the large ganglionic cells of the DRG. The vestibular ganglia were large, connected to the hind brain, and contained numerous neurons packed in columns. The cervical ganglia were large and observed around the pseudobranch, head kidney, and thymus. Their neurons are randomly distributed, and nerve fibers are peripherally situated. CD3-positive T-lymphocytes, dendritic cells, and CD68-positive macrophages were in close contact with the ganglia. Furthermore, the ganglia around the head kidney showed positive Iba1-expressing cells. Most ganglion cells and nerve fibers in the DRG, autonomic, and vestibular ganglia showed moderate to strong S-100 immunoreactivity. The enteric glia, CD68-expressing macrophages, and acetylcholine (Ach)-expressing neurons were observed along the muscular layer of the intestinal wall. In conclusion, different ganglia of molly fish displayed direct communication with immune cells which support and maintain healthy ganglionic cells.

CD163-Expressing Porcine Macrophages Support NADC30-like and NADC34-like PRRSV Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a strict cell tropism. In addition to the primary alveolar macrophages, PRRSV is strictly cytotropic to African green monkey kidney cells, such as MARC-145 cells; however, MARC-145 cells are not infected by most NADC30-like and NADC34-like PRRSV strains. The essential scavenger receptor CD163 has been proved to mediate productive infection of PRRSV in various non-permissive cell lines. In this study, we systematically tested the porcine CD163 stably expressing 3D4/21 cells for infections with various PRRSV strains. The results showed that the porcine CD163-expressing macrophages support the infections of PRRSV2 of lineages 1, 5, and 8, as evidenced by Western blotting, immunofluorescence assay, quantitative PCR, and virus titration assay. Considering the current prevalence of NADC30-like and NADC34-like PRRSV2 of lineage 1 in China, the CD163-expressing macrophages are very useful for PRRSV research and disease management.

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer

BackgroundTumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance.MethodsStage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma.ResultsTYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09–2.56; p < 0.05).ConclusionsTYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Pregnane X Receptor Mediates Atherosclerosis Induced by Dicyclohexyl Phthalate in LDL Receptor-Deficient Mice.

Plastic-associated endocrine disrupting chemicals (EDCs) have been implicated in the etiology of cardiovascular disease (CVD) in humans, but the underlying mechanisms remain elusive. Dicyclohexyl phthalate (DCHP) is a widely used phthalate plasticizer; whether and how exposure to DCHP elicits adverse effects in vivo is mostly unknown. We previously reported that DCHP is a potent ligand of the pregnane X receptor (PXR) which acts as a xenobiotic sensor to regulate xenobiotic metabolism. PXR also functions in macrophages to regulate atherosclerosis development in animal models. In the current study, LDL receptor-deficient mice with myeloid-specific PXR deficiency (PXRΔMyeLDLR−/−) and their control littermates (PXRF/FLDLR−/−) were used to determine the impact of DCHP exposure on macrophage function and atherosclerosis. Chronic exposure to DCHP significantly increased atherosclerotic lesion area in the aortic root and brachiocephalic artery of PXRF/FLDLR−/− mice by 65% and 77%, respectively. By contrast, DCHP did not affect atherosclerosis development in PXRΔMyeLDLR−/− mice. Exposure to DCHP led to elevated expression of the scavenger receptor CD36 in macrophages and increased macrophage form cell formation in PXRF/FLDLR−/− mice. Our findings provide potential mechanisms underlying phthalate-associated CVD risk and will ultimately stimulate further investigations and mitigation of the adverse effects of plastic-associated EDCs on CVD risk in humans.

Prognostic significance of CD163- and CD68-expressing macrophages in nodular sclerosis Hodgkin lymphoma

The objective of the study was to determine the clinical and morphological criteria and prognostic significance of CD163- and CD68-expressing macrophages in formalin-fixed paraffin-embedded (FFPE) lymph node samples of nodular sclerosis Hodgkin lymphoma.Materials and methods. We have studied clinical and laboratory characteristics and treatment results of 52 patients with newly diagnosed nodular sclerosis Hodgkin lymphoma. The treatment efficacy was assessed after 2–4 courses of chemotherapy by positron emission tomography combined with computed tomography scan (PET-CT) according to the Deauville scale.Results. In FFPE lymph node samples, differences in the localization and relative number of CD163-positive macrophages in patients of both study groups were determined. Morphometric counting of CD68-positive cells did not reveal statistically significant intergroup differences. The threshold of CD163-positive cells has been established. The relationship between the CD68 expression level and the response to frontline therapy was not revealed. The ratio of tumorassociated macrophages in each patient was assessed by the CD163/CD68 index. The threshold of the index was determined, which allows predicting the course of nodular sclerosis Hodgkin lymphoma.Conclusion. The following parameters could be used as additional morphological criteria for predicting the response to frontline therapy in patients with nodular sclerosis Hodgkin lymphoma: tissue distribution pattern and quantitative calculation of macrophage-histiocytic microenvironment subpopulation composition. The results could be used to risk group stratification of patients with nodular sclerosis Hodgkin lymphoma in order to predict the response to initial phase of therapy, as well as to determine personalized treatment approaches.

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

COVID-19-induced “acute respiratory distress syndrome” (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

Differential Expression of IL-17 Correlates with Clinical and Pathological Features of ITP

Introduction:Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder, which is characterized by megakaryocyte (MgK) inhibition/destruction in the bone marrow (BM) and/or platelet in the spleen and liver. Abnormal MgKs, which is recognized by autoantibodies, are phagocytosed by macrophages. Recently, it has been demonstrated that interleukin-(IL-)17-mediated cells contribute to the imbalance of cellular immunity in the pathogenesis of ITP. Studies of pathological findings in BM of patients with ITP using immunohistochemistry (IHC) are limited. Therefore, we examined samples of BM clots to test the hypothesis that IL-17-mediated immunological changes are involved in the BM of patients with ITP.Methods:We enrolled 35 patients with a median age of 61 years (range, 19-91 years), who were retrospectively selected at random and were referred to our hospital between 2005 and 2016. All patients were diagnosed with chronic ITP. Twenty-five patients underwent first- or second-line therapies such as glucocorticosteroids, cyclosporine A, azathioprine, danazol and intravenous immunoglobulin and/or splenectomy as conventional therapies. Twenty-three patients who could not undergo those therapies were administered with eltrombopag. BM clots were obtained before treatment and stained with hematoxylin and eosin for morphological examination. Formalin-fixed and paraffin-embedded specimens were used for IHC using antibodies for the following markers: CD3, CD4, CD8, CD20 (L26), CD25, CD68, CD163, and IL-17. Pathological findings and clinical information including laboratory data were compared between the patients with ITP and control subjects, which were obtained from 11 untreated malignant lymphoma patients without the infiltration of lymphoma cells in BM. We also compared those between 2 groups: an IL-17-high-expression group and IL-17-low-expression group.Results:A univariate analysis revealed increased cells expressing the following markers in patients with ITP than in the control subjects: CD20 (5.80 ± 3.46% vs. 3.06 ± 1.90%; p= 0.02), CD68 (11.3 ± 11.1% vs. 3.14 ± 3.23%; p= 0.02), CD163 (5.10 ± 2.74% vs. 2.13 ± 1.10%; p= 0.001), and IL-17 (3.13 ± 1.48% vs. 1.49 ± 1.00%; p= 0.002) and decreased cells expressing following markers: CD4 (0.33 ± 0.71% vs. 1.96 ± 1.13%; p < 0.0001) and CD25 (0.09 ± 0.18% vs. 0.23 ± 0.10%; p= 0.01). The difference of cells expressing CD3 (p= 0.96) and CD8 (p= 0.41) were not statistically significant. There were significant correlations between CD68 and CD163 expressions (r= 0.61). The expressions of both CD68 and CD163 showed correlations with IL-17 expressions (r= 0.61, 0.51). We found significant increased expressions of CD3 (3.98 ± 2.96% vs. 6.37 ± 3.33%; p= 0.03), CD25 (0.05 ± 0.08% vs. 0.17 ± 0.22%; p= 0.03), CD68 (7.84 ± 5.09% vs. 17.3 ± 15.6%; p= 0.01), and CD163 (4.23 ± 2.37% vs. 6.57 ± 2.77%; p= 0.01) in the IL-17-high-expression group (N = 13) compared with IL-17-low-expression group (N = 22). The expressions of CD4 (p= 0.98), CD8 (p= 0.54), and CD20 (p= 0.85) were not statistically significant. Responses to eltrombopag were significantly better in the IL-17-low-expression group than in the IL-17-high-expression group (p= 0.03).Conclusions:By IHC analysis, we report for the first time increased CD68- and/or CD163-expressing cells in the BM of patients with ITP. Those are possibly associated with the pathogenesis of ITP. Our results indicate that CD68- and/or CD163-expressing macrophages and monocytes, but not lymphocytes are involved in deregulated expression of IL-17 in ITP. We demonstrate that the response of eltrombopag is significantly better in the IL-17-low-expression group. These results indicate that patients with ITP, achieving remission early, might belong to the category of the low-IL-17, CD68, and CD163 expressions. We suggest that IHC staining in patients with ITP may be a potential indicator to understand the mechanism of ITP and to predict the prognosis for treatment. DisclosuresNo relevant conflicts of interest to declare.

Acute Lymphoblastic Leukaemia (ALL) Cells and Commonly-Used Chemotherapy Drugs Can Induce Bone Marrow-Derived Mesenchymal Stromal Cells to Become Cancer Associated Fibroblasts (CAF), Leading to Enhanced ALL Proliferation and Treatment Resistance

To investigate the role of mesenchymal stromal cells (MSCs) in treatment resistance in acute lymphoblastic leukaemia (ALL) we systematically isolated and studied, ISCT-confirmed, bone-marrow-derived MSCs isolated from 151 adults (22-69 years) with ALL registered on the UKALL14 and ALL60+ trials both at diagnosis and during therapy.CD11b, CD68 and CD163-expressing tumour-associated macrophages were observed in 34/75 (45%) of MSC cultures from follow-up samples (termed M+MSC). By contrast, macrophages were absent (M-MSC) from all diagnostic (0/56) and healthy donor specimens (0/7) tested. ELISA on supernatant from expanded M+MSC showed significantly higher levels of pro-inflammatory cytokines IL8 (4961 pg/ml vs 1093 pg/ml, p = 0.01) and CXCL2 (106 pg/ml vs 36 pg/ml, p = 0.03) than that from expanded M-MSC. The B-ALL cell line Nalm6 proliferated more rapidly when co-cultured with primary M+MSCs than with primary M-MSC (1.93 fold, p = 0.03). Importantly, primary M+MSCs strongly expressed α-smooth muscle actin (αSMA) by immunostaining, consistent with a cancer associated fibroblast (CAF) phenotype.To establish the role of ALL cells and/or chemotherapy in the induction of CAFs, we used the MSC cell line HS27a to model CAF generation in-vitro . Co-culture of HS27a with B-ALL cell lines Nalm6, SD1 and 697 cells induced increased aSMA expression and cytoskeleton remodeling including increased actin stress fibers in the HS27a consistent with a CAF phenotype. These changes were not observed in HS27a in co-culture with healthy donor B-cells or the B-ALL cell line SEM. A targeted RQPCR gene panel showed up-regulation of CAF-defining genes (FAPA 2-4.4.6 fold, COX2 5.9 - 32.1 fold, IL6 11.2 - 495 fold, FN 2.2 - 3.0 fold, TGFβ1 2.0 - 4.6 fold, IL8 15.5 - 85.2 fold, CCL2 10.6 - 33.3 fold, VEGF 2.9 - 13.4 fold, FGF2 2.9 - 14.6 fold) and expected down-regulation of CAV1 (1.8 - 5.4 fold) when HS27a were cultured with SD1 and 697 cells compared to HS27a alone. HS27a exposed to healthy donor B-cells or SEM cells in contrast did not induce differential regulation of CAF defining genes. In a flow-based, cytokine bead assay, SD1 cells induced HS27a to secrete pro-inflammatory cytokines (IL8, IL6, CCL2, CXCL10 and CCL5) and growth factor bFGF at several fold higher levels than normal B-cells or SEM cells consistent with a CAF secretion profile. Similar morphological changes, gene expression profile and cytokine profile were observed when cells were co-cultured in a transwell system, suggesting a soluble mechanism of CAF induction. TGFβ1 and reactive oxygen species, known to induce CAFs in solid tumors, were both increased in all B-ALL cell lines able to induce CAFs compared to SEM cells, suggesting a probable mechanism behind CAF induction.Therapeutically-relevant concentrations of chemotherapy agents cytarabine and daunorubicin - but not vincristine and dexamethasone - induced gross morphological changes in HS27a cells with an increase in actin stress fibres and αSMA. RQPCR of cytarabine-treated HS27a showed upregulation of several genes consistent with a CAF phenotype and cytokine bead assay showed increased secretion of IL8 and CCL2 compared to untreated HS27a (973 pg/ml vs 430pg/ml, p < 0.0001 and 1089pg/ml vs 717.3pg/ml p = 0.02 respectively).To examine the functional significance of CAF on ALL cell proliferation and chemoresistance, we pre-treated HS27a with cytarabine to induce a CAF phenotype and then assessed the impact on SEM (selected for their lack of CAF induction) proliferation and chemosensitivity. Pretreatment with vincristine or dexamethasone and untreated HS27a comprised the controls. Cytarabine-generated, HS27a-derived CAF increased SEM cell proliferation compared to both the untreated and vincristine and dexamethasone pre-treated HS27a (1.2 fold p = 0.002, 1.3 fold p = 0.0004 and 1.2 fold p = 0.0002 respectively). Cytarabine-generated, HS27a-derived CAF also reduced the sensitivity of SEM cells to both dexamethasone and cytarabine compared to both the untreated and dexamethasone or vincristine pre-treated HS27a.We have demonstrated for the first time in both primary cells and cell-line models that bone-marrow derived MSCs can adopt a CAF phenotype in response to both ALL cells and ALL therapies. The functional impact is to enhance both ALL cell proliferation and chemotherapy resistance. Ongoing work will delineate the precise nature of the interaction between MSC and ALL cells. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Sexual Dimorphism in Outcomes of Non–muscle-invasive Bladder Cancer: A Role of CD163+ Macrophages, B cells, and PD-L1 Immune Checkpoint

BackgroundNon–muscle-invasive bladder cancer (NMIBC) is over three times as common in men as it is in women; however, female patients do not respond as well to immunotherapeutic treatments and experience worse clinical outcomes than their male counterparts. Based on the established sexual dimorphism in mucosal immune responses, we hypothesized that the tumor immune microenvironment of bladder cancer differs between the sexes, and this may contribute to discrepancies in clinical outcomes. ObjectiveTo determine biological sex-associated differences in the expression of immune regulatory genes and spatial organization of immune cells in tumors from NMIBC patients. Design, setting, and participantsImmune regulatory gene expression levels in tumors from male (n = 357) and female (n = 103) patients were measured using whole transcriptome profiles of tumors from the UROMOL cohort. Multiplexe immunofluorescence was performed to evaluate the density and spatial distribution of immune cells and immune checkpoints in tumors from an independent cohort of patients with NMIBC (n = 259 males and n = 73 females). Outcome measurements and statistical analysisTranscriptome sequencing data were analyzed using DESeq2 in R v4.0.1, followed by application of the Kruskal-Wallis test to determine gene expression differences between tumors from males and females. Immunofluorescence data analyses were conducted using R version 3.5.3. Survival analysis was performed using survminer packages. Results and limitationsHigh-grade tumors from female patients exhibited significantly increased expression of B-cell recruitment (CXCL13) and function (CD40)-associated genes and the immune checkpoint genes CTLA4, PDCD1, LAG3, and ICOS. Tumors from female patients showed significantly higher infiltration of PD-L1+ cells and CD163+ M2-like macrophages than tumors from male patients. Increased abundance of CD163+ macrophages and CD79a+ B cells were associated with decreased recurrence-free survival. ConclusionsThese novel findings highlight the necessity of considering sexual dimorphism in the design of future immunotherapy trials in NMIBC. Patient summaryIn this study, we measured the abundance of various immune cell types between tumors from male and female patients with non–muscle-invasive bladder cancer. We demonstrate that tumors from female patients have a significantly higher abundance of immunosuppressive macrophages that express CD163. Higher abundance of tumor-associated CD163-expressing macrophages and B cells is associated with shorter recurrence-free survival in both male and female patients.

Postoperative elevation in the plasma CCL2 level is a predictive biomarker of colorectal cancer recurrence.

There is currently no adequate biomarker for predicting colorectal cancer (CRC) recurrence. Chemokine (C-C motif) ligand 2 (CCL2) induces macrophages and fibroblasts to occupy metastatic niches in distant organs. The purpose of this study was to examine CCL2 as a potential predictive biomarker for CRC recurrence. Plasma samples (n = 402) were collected from 80 stage II/III/IV CRC cases and the relationship between CCL2 profiles and recurrence was investigated. The tumor immune response genes associated with CCL2 mRNA expression in a subgroup of 8 stage I/II CRC cases with 12 recurrent sites and The Cancer Genome Atlas database were also analyzed retrospectively. Sixteen stage II/III/IV postoperative recurrent CRC cases experienced a significant increase in plasma CCL2 levels 6months after surgery and continuously increased even after R0-1 resection. The 6-month postoperative CCL2 levels in recurrent cases of ≥ 1year were significantly higher than in non-recurrent cases and recurrent cases of < 1year. The CCL2 level in the primary tumor cases significantly correlated with the cytolytic activity, thus indicating a tumor immune response from the CD163-expressing macrophages. Plasma CCL2 was found to be a predictive biomarker of postoperative CRC recurrence. CCL2 in metastatic sites derives from metastatic niches that surpass the host immune response.

Monocyte-macrophage polarization and recruitment pathways in the tumour microenvironment of B-cell acute lymphoblastic leukaemia.

B-cell acute lymphoblastic leukaemia (B-ALL) reprograms the surrounding bone marrow (BM) stroma to create a leukaemia-supportive niche. To elucidate the contribution of immune cells to the leukaemic microenvironment, we investigated the involvement of monocyte/macrophage compartments, as well as several recruitment pathways in B-ALL development. Immunohistochemistry analyses showed that CD68-expressing macrophages were increased in leukaemic BM biopsies, compared to controls and predominantly expressed the M2-like markers CD163 and CD206. Furthermore, the "non-classical" CD14+ CD16++ monocyte subset, expressing high CX3CR1 levels, was significantly increased in B-ALL patients' peripheral blood. CX3CL1 was shown to be significantly upregulated in leukaemic BM plasma, thus providing an altered migratory pathway possibly guiding NC monocyte recruitment into the BM. Additionally, the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) strongly increased in leukaemic BM plasma, possibly because of the interaction of leukaemic cells with mesenchymal stromal cells and vascular cells and due to a stimulatory effect of leukaemia-related inflammatory mediators. C5a, a macrophage chemoattractant and M2-polarizing factor, further appeared to be upregulated in the leukaemic BM, possibly as an effect of PTX3 decrease, that could unleash complement cascade activation. Overall, deregulated monocyte/macrophage compartments are part of the extensive BM microenvironment remodelling at B-ALL diagnosis and could represent valuable targets for novel treatments to be coupled with classical chemotherapy.

Obeticholic acid ameliorates non-alcoholic steatohepatitis and atherosclerosis in SHRSP5/Dmcr rats

Background and Aims: Obeticholic acid (OCA) is the farnesoid X receptor (FXR) agonists and expected as a therapeutic medicine of non-alcoholic steatohepatitis (NASH) because it inhibits lipid and bile acid synthesis. FXR is activated by bile acids and expresses in various tissues. Activation of macrophage-FXR is reported to suppress the expression of CD36 in macrophages. CD36 is a scavenger receptor which is important for phagocytosis of oxidized LDL. We investigated whether OCA ameliorates NASH and atherosclerosis using SHRSP5/Dmcr rats.

Enhanced expression of coxsackievirus and adenovirus receptor in lipopolysaccharide-induced inflammatory macrophages is through TRIF-dependent innate immunity pathway

AimsInflammatory macrophages have been proposed as a therapeutic target for joint disorders caused by inflammation. This study aimed to investigate the expression and regulation of coxsackievirus-adenovirus receptor (CAR) in lipopolysaccharide (LPS)-stimulated inflammatory macrophages whereby to evaluate the feasibility of virus-directed enzyme prodrug therapy (VDEPT). Main methodsMacrophage cell lines (RAW264.7 and J774A.1) and primary macrophage cells derived from rat spleen were used to evaluate the expression of CAR protein or CAR mRNA. Specific inhibitors for TLR4 pathway were used to investigate the regulation of CAR expression. CAR expression in rat joints was documented by immunohistochemistry. Conditionally replicating adenovirus, CRAd-EGFP(PS1217L) or CRAd-NTR(PS1217H6), and non-replicating adenovirus CTL102 were used to transduce genes for enhanced green fluorescent protein (EGFP) or nitroreductase (NTR), respectively. The expression of EGFP, NTR, and the toxicity induced by CB1954 activation were evaluated. Key findingsThe in vitro experiments revealed that CAR upregulation was mediated through the TLR4/TRIF/IRF3 pathway in LPS-stimulated inflammatory macrophage RAW264.7 and J774A.1 cells. The inflammatory RAW264.7 cells upregulated CAR expression following LPS stimulation, leading to higher infectability, increased NTR expression, and enhanced sensitization to CB1954. In animal experiments, the induction of CAR expression was observed in the CD68-expressing primary macrophages and in the CD68-expressing macrophages within joints following LPS stimulation. SignificanceIn conclusion, we report an enhanced CAR expression in inflammatory macrophages in vitro and in vivo through the immune response elicited by LPS. Thus, the TLR4/TRIF/IRF3 pathway of macrophages, when activated, could facilitate the therapeutic application of adenovirus-mediated VDEPT.

CD163 deficiency increases foam cell formation and plaque progression in atherosclerotic mice.

Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/- /CD163-/- compared with ApoE-/- /CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression.