Positive Macrophages Research Articles

Monocyte chemotactic protein (MCP)-1 (CCL2) and its receptor (CCR2) are elevated in chronic heart failure facilitating lung monocyte infiltration and differentiation which may contribute to lung fibrosis.

Dyspnea, the cardinal manifestation of chronic heart failure (CHF), may reflect both pulmonary oedema and pulmonary remodeling resulting in tissue stiffening. Emerging evidence suggests that predominance of distinct phenotypes of alveolar and recruited macrophages, designated M1 and M2, may regulate the course of inflammatory tissue repair and remodeling in the lung. In a CHF rat model, we found fibrotic reinforcement of the extracellular matrix with an increase in monocyte chemotactic protein (MCP)-1/CCL2 in bronchoalveolar lavage (BAL), corresponding to a 3-fold increase in recruited macrophages. In this clinical cross sectional study, we aimed to examine potential mediators of leukocyte activation and lung infiltration in parallel BAL and blood from CHF patients compared to non-CHF controls. Mini-BAL and peripheral blood samples were obtained from hospitalized CHF, acute decompensated CHF and non-CHF patients. CHF patients and decompensated CHF patients demonstrated increases from non-CHF patients in BAL MCP-1, as well as the M2 macrophage cytokines interleukin-10 and transforming growth factor-β. BAL and plasma MCP-1 were significantly correlated; however, MCP-1 was 20-fold higher in epithelial lining fluid in BAL, indicative of an alveolar chemotactic gradient. An increase in transglutaminase 2 positive M2 macrophages in parallel with a decrease in the MCP-1 receptor, CC chemokine receptor 2 (CCR2), was apparent in BAL cells of CHF patients compared to non-CHF. These data suggest a pathway of MCP-1 mediated M2 macrophage prevalence in the lungs of CHF patients which may contribute to pulmonary fibrotic remodeling and consequent increased severity of dyspnea.

Vinpocetine's immunomodulating, anti-oxidant, anti-inflammatory, ant-ifibrotic, and PDE inhibiting potencies ameliorate bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis (PF) is a global health problem with a high economic burden. Intratracheal administration of bleomycin is the best model that resembles the pathogenesis of PF in humans. Recently, vinpocetine proved to have neuroprotective, cardioprotective, hepatoprotective, anti-aging, and antifibrotic effects through its anti-oxidant, immunomodulating, and anti-inflammatory activities. The present study investigated the antifibrotic potentiality of vinpocetine in a rat model of PF induced by intratracheal bleomycin administration. PF induced by a single intratracheal instillation of 5 mg/kg bleomycin in nine-week-old Wister rats. Oral vinpocetine was used at doses of 5, 10, or 20 mg/kg to treat PF for 21 days immediately after the bleomycin instillation. Vinpocetine dose-dependently ameliorates PF induced by bleomycin administration since vinpocetine effectively restored the normal body weight gain rates, pulmonary architecture, and collagen fiber distribution and suppressed the elevated BALF cell count, lymphocytes and neutrophils percentage, BALF, IL-6, TNF-α, and TGF-β1 levels and LDH activity, lung tissue MDA level, PDE activity, hydroxyproline content, immunohistochemical expression of α-SMA and CD68 positive macrophage, and fibrosis score. Meanwhile, it efficiently augmented the reduced BALF macrophage percentage, IL-10 level, lung tissue GSH level, CAT, and SOD activities. Vinpocetine may propose a new promising agent to manage PF.

ATL I, Acts as a SIRT6 Activator to Alleviate Hepatic Steatosis in Mice via Suppression of NLRP3 Inflammasome Formation.

Accumulating evidence has highlighted that sirtuin-6 (SIRT6) plays an important role in hepatic gluconeogenesis and lipogenesis. We aim to investigate the underlying mechanisms and pharmacological interventions of SIRT6 on hepatic steatosis treatment. Herein, our results showed that atractylenolide I (ATL I) activated the deacetylase activity of SIRT6 to promote peroxisome proliferator-activated receptor alpha (PPARα) transcription and translation, while suppressing nuclear factor NF-kappa-B (NFκB)-induced NACHT, LRR, and PYD domains containing protein 3 (NLRP3) inflammasome formation. Together, these decreased the infiltration of F4/80 and CD11B positive macrophages, accompanied by decreased mRNA expression and serum levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL6), and interleukin-1 beta (IL1β). Additionally, these changes decreased sterol regulatory element-binding protein-1c (SREBP-1c) expression, while restoring carnitine O-palmitoyltransferase 1a (Cpt1a) expression, to decrease the size of adipocytes and adipose deposition, which, in turn, reversed high-fat diet (HFD)-induced liver weight and body weight accumulation in C57 mice. SIRT6 knockout or hepatic SIRT6 knockout in C57 mice largely abolished the effect of ATL I on ameliorating hepatic steatosis. Taken together, our results suggest that ATL I acts as a promising compound that activates SIRT6/PPARα signaling and attenuates the NLRP3 inflammasome to ameliorate hepatic inflammation and steatosis.

Liver-adipose tissue crosstalk in alcohol-associated liver disease: The role of mTOR

BackgroundAlcohol-associated liver disease (ALD) is a major chronic liver disease around the world without successful treatment. Acute alcoholic hepatitis is one of the most severe forms of ALD with high mortality, which is often associated with binge drinking. Alcohol drinking dysregulates lipid metabolism, increases adipose tissue lipolysis, and induces liver steatosis and adipose tissue atrophy. Increasing evidence implicates that crosstalk of liver and adipose tissue in the pathogenesis of ALD. Mechanistic target of rapamycin (mTOR) is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that regulates lipid metabolism, cell proliferation and autophagy. However, the role of mTOR in regulating adipose-liver crosstalk in binge drinking-induced organ damage remains unclear. MethodsWe generated liver-specific and adipocyte-specific regulatory-associated protein of mTOR (Rptor) knockout (RptorLKO and RptorAKO) as well as Mtor knockout (MtorLKO and MtorAKO) mice, by crossing Rptorflox and Mtorflox mice with albumin Cre or adiponectin Cre mice, respectively. In addition, we generated liver and adipocyte double deletion of Rptor or Mtor (MtorLAKO and RptorLAKO) mice. The knockout mice with their matched wild-type littermates (RptorWT and MtorWT) were subjected to acute gavage of 7 g/kg ethanol. ResultsMice with adipocyte deletion of Rptor or Mtor developed hepatomegaly and adipose tissue atrophy. Alcohol gavage increased liver injury, hepatic steatosis and inflammation in mouse livers as demonstrated by elevated serum alanine aminotransferase activities, increased hepatic levels of triglyceride and increased hepatic numbers of CD68 positive macrophages in mouse livers after alcohol gavage. Liver injury was further exacerbated by deletion of adipocyte Rptor or Mtor. Serum adipokine array analysis revealed that increased levels of pro-inflammatory cytokines IL-6 and TNFα as well as chemokine MCP-1 following acute alcohol gavage in wild-type mice, which were further increased in adipocyte-specific Mtor or Rptor knockout mice. Conversely, levels of anti-inflammatory cytokine IL-10 decreased in adipocyte-specific Mtor or Rptor knockout mice. The levels of circulating fibroblast growth factor 21 (FGF21) increased whereas levels of circulating adiponectin and fetuin A decreased in wild-type mice after alcohol gavage. Intriguingly, adipocyte-specific Mtor or Rptor knockout mice already had decreased basal level of FGF21 which increased by alcohol gavage. Moreover, adipocyte-specific Mtor or Rptor knockout mice already had increased basal level of adiponectin and decreased fetuin A which were not further changed by alcohol gavage. ConclusionsAdipocyte but not hepatocyte ablation of Mtor pathway contributes to acute alcohol-induced liver injury with increased inflammation. Our results demonstrate the critical role of adipocyte mTOR in regulating the adipose-liver crosstalk in ALD.

Single-Cell Transcriptome Reveals Comprehensive Immune Profiles of T Follicular Helper Cell Lymphoma

Background: Angioimmunoblastic T-cell lymphoma and other nodal lymphomas of T follicular helper (TFH) cell origin (TFH lymphoma) have a poor prognosis and are associated with immune dysregulation. However, comprehensive immune profiles of TFH lymphoma have not been fully investigated. The RHOA G17V mutation (G17V) is a disease-specific mutation found in 60-70% of TFH lymphoma and considered to gain a novel function through binding to VAV1 protein. Nevertheless, the prevalence of homozygous (Homo) and heterozygous (Hetero) G17V mutations and the difference in their oncogenicity due to its zygosity have not been elucidated. Here, we performed single-cell transcriptomic analysis to characterize tumor and immune cells of TFH lymphoma. Methods: We performed single-cell RNA sequencing (scRNA-seq) and T-cell/B-cell receptor sequencing (scTCR/BCR-seq) for 9 lymph nodes (LNs) and 16 peripheral blood (PB) from 14 TFH lymphoma patients (9 at initial diagnosis [ID] and 5 at relapsed/refractory diagnosis [RR]), as well as 7 homeostatic LNs (HLNs) from patients with solid cancers (Fig. 1). Whole-exome sequencing (WES) was also performed for genetic mutation analysis using bulk DNA extracted from tumor tissues and PB of the same cohort as scRNA-seq. scRNA-seq and scTCR/BCR-seq libraries were constructed using the Chromium system (10x Genomics). Publicly available scRNA-seq data from 5 healthy donors (HDs) were also integrated as control. scRNA-seq data analysis was performed by Seurat, Scater, and Batchelor for quality control, integration, and clustering; SingleR for annotation; Immunarch for TCR/BCR repertoire analysis; gene set variation analysis (GSVA) for pathway analysis, and CellPhoneDB for cell-cell interaction analysis. Genomon2 pipeline was used for calling somatic mutations in WES data. Subsequently, VarTrix was applied to estimate mutations detected by WES in scRNA-seq data. Results: In total, 68,738 cells from LNs and 98,891 cells from PB were analyzed. Unsupervised clustering and annotation by canonical markers identified 4 major cell types: CD4/CD8+ T, natural killer (NK), B, and myeloid cells. Tumor cells were identified by the expanded TCR clonality and the TFH-like gene expression signatures. Next, using scRNA-seq data, we reanalyzed a total of 393 somatic mutations discovered by WES and identified 15 mutations in more than 5 cells of scRNA-seq data, of which only RHOA G17V was identified in multiple samples. G17V-mutated cells were detected in 4 LN and 6 PB samples, mostly restricted to tumor cells. G17V-Homo and Hetero cells were identified based on frequency of mutant reads. Differentially expression gene (DEG) analysis and GSVA in G17V-mutated tumor cells of LNs revealed that the regulatory signature was significantly activated in Homo, whereas the proliferation signature was up-regulated in Hetero. Additionally, DEG analysis identified three tumor-specific gene candidates and the expression of cell surface protein PLS3 was validated by flow cytometry and immunohistochemistry. Subsequently, we performed sub-clustering and GSVA of immune cells and found that dysfunctional CD8 T cells were significantly increased in LNs and PB of patients compared to controls and exhibited oligoclonal expansion by TCR repertoire analysis. Notably, TREG, complement component 1q positive macrophages (C1Q+ Mφ), and LAMP3+ dendritic cells (DCs) were significantly increased in LNs from patients with RR diseases than HLNs. DEG analysis and GSVA revealed high expression of PD-L1 and regulatory signatures in LAMP3+ DCs. Moreover, genes related to immune suppression were significantly upregulated in TREG of patients compared to those of controls. Finally, cell-cell interaction analysis uncovered that tumor cells and TREG of RR patients strongly interacted with C1Q+ Mφ and LAMP3+ DCs via CXCR3-CXCL9 and CCR4-CCL17 interactions, respectively, indicating that these myeloid cells not only induced TREG into tumor tissues but also actively interacted with tumor cells. Conclusion: Tumor cells of TFH lymphoma acquired the immunosuppressive signature in the clonal evolution process from the G17V Hetero to Homo. Furthermore, comprehensive immune profiling of TFH lymphoma revealed expansion of cells involved in the immune escape. These results unveiled the enrichment of immune evasive phenotypes in TFH lymphoma and suggested their contribution to treatment resistance in RR patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract C051: Ceramide signaling regulates PDA aggression through exosome reprogramming of the stroma

Abstract Ceramide is a bioactive lipid signaling molecule that regulates multiple cellular processes influencing pancreatic tumor progression and drug response. The pleiotropic role of ceramide signaling in cancer includes modulation of exosome biogenesis and secretion. Smpd3 encodes an enzyme that generates ceramide through hydrolysis of sphingomyelin. Employing the KPC mouse model of pancreatic cancer, we demonstrated that Smpd3 regulates exosome biogenesis in pancreatic ductal adenocarcinoma (PDA) cells and is pro-tumorigenic during PDA progression. Ablation of Smdp3 in KPC mice significantly extends survival by 19% when compared to KPC; Smpd3wt/wt controls. KPC; Smpd3f/f mice display significantly less PanIN and tumor burden compared to KPC; Smpd3wt/wt controls. Lipidomics analysis of epithelial cell lines generated from end-stage pancreatic tumors of KPC; Smpd3f/f and KPC; Smpd3wt/wt mice demonstrated an alteration in hundreds of lipid species including ceramides, triacylglycerides, sphingomyelins, and phosphatidylcholines. Analysis of RNA-seq data of these epithelial cell lines showed a switching of primary tumors from the predominant more aggressive basal-like subtype seen in KPC; Smpd3wt/wt mice to classical in KPC; Smpd3f/f mice. Pathways analysis of our RNA-seq dataset showed an enrichment for genes involved in cellular mechanics and regulation of the tumor microenvironment. To query if Smpd3-generated exosomes have a direct effect on pancreatic tumor progression, we injected KPC; Smpd3wt/wt and KPC; Smpd3f/f mice with exosomes isolated from KPC; Smpd3f/f and KPC; Smpd3wt/wt PDA cell lines. Injection of exosomes derived from KPC; Smpd3f/f mice significantly extended survival of both Smpd3wt/wt and KPC; Smpd3f/f mice when compared to injection of exosomes isolated from KPC; Smpd3wt/wt mice, suggesting an anti-tumorigenic effect of exosomes isolated from Smpd3-deficient PDA cell lines. We observed a decrease in extracellular matrix collagen abundance and fewer activated stellate cells and fibroblasts in KPC; Smpd3f/f compared to control KPC; Smpd3wt/wt pancreata. Abrogation of Smpd3 expression also affected immune cell infiltration, as demonstrated by a significant increase in iNOS+ F4/80+ double positive macrophages in KPC; Smpd3f/f pancreata when compared to KPC; Smpd3wt/wt pancreata. Loss of Smpd3 resulted in a significant reduction in CD31+ endothelial cells in pancreatic tumors of KPC; Smpd3f/f mice when compared to KPC; Smpd3wt/wt mice, which may influence the ability of chemotherapeutics to enter pancreatic tumors. Our patient data demonstrate that high SMPD3 expression in surgically resected, treatment naive PDA significantly correlated with longer patient survival when patients received adjuvant chemotherapy, more than 95% of which was gemcitabine. Collectively, our data show that ceramide-dependent exosomes promote tumorigenesis, specifically activation of stellate cells and fibroblasts – which may in turn induce a stiff, fibrotic, proinflammatory tumor microenvironment that also impedes vasculature formation. Citation Format: Audrey M. Hendley, Atsushi Urano, Xianlu L. Peng, Sudipta Ashe, Natanya R. Kerper, Tuan A. Phu, Martin Ng, Simone Giacometti, David I. Berrios, Gun H. Jang, Jen J. Yeh, Steven Gallinger, David K. Chang, Andrew V. Biankin, Valerie M. Weaver, Grace E. Kim, David W. Dawson, Robert L. Raffai, Matthias Hebrok. Ceramide signaling regulates PDA aggression through exosome reprogramming of the stroma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C051.

Abstract 9570: Different Distribution of CD163 Positive Macrophages in Thrombus Retrieved From Infarct-Related Artery: Atherothrombosis vs. Cardiogenic Thrombosis, Pathological Analyses From the MITO Study

Introduction: Diagnosis of cardiogenic thrombosis is important because anticoagulant therapy is recommended for its secondary prevention. However, it is difficult to make a clear distinction in the pathogenesis between atherothrombosis and cardiogenic thrombosis. Hypothesis: Role of macrophage in thrombus formation is different between atherothrombosis and cardiogenic thrombosis. Methods: The Macrophage in Thrombus (MITO) study is a prospective observational study examining pathological and biological differences of arterial thrombus between atherothrombosis and cardiogenic thrombosis. Patients with ST elevation myocardial infarction (STEMI) or acute ischemic stroke (AIS) whose solid thrombus could be retrieved from the infarct-related artery were enrolled. Patients were divided into 2 groups as follows; STEMI with sinus rhythm: Group A (presumed atherothombosis) and AIS with atrial fibrillation (presumed cardiogenic thrombosis): Group C, and compared macrophage score as follows; 1:almost no macrophages detected byх40; 2:difficult to detect the macrophages in х20 but detected by х40; and 3:easy to detect macrophages in х20 in the thrombus. Serum levels of soluble CD163 were measured all the patients. Results: The score for CD163 positive macrophage of Group C (n=33) was significantly higher than that of Group A (n=32) (p<0.01): Figure. Serum level of soluble CD163 in Group C (median 603 ng/ml, IQR 405, 788) tended to be higher than that in Group A (median 520 ng/ml, IQR: 314, 634); p=0.08. Conclusions: CD163 positive macrophages are more involved in the thrombus formation of cardiogenic thrombus than that of atherothrombosis. Detection of CD163 positive macrophages are likely to help discriminate between atherothrombosis thrombus versus cardiogenic thrombus.

Cutting edges and therapeutic opportunities on tumor-associated macrophages in lung cancer.

Lung cancer is a disease with remarkable heterogeneity. A deep understanding of the tumor microenvironment (TME) offers potential therapeutic strategies against this malignant disease. More and more attention has been paid to the roles of macrophages in the TME. This article briefly summarizes the origin of macrophages, the mutual regulation between anti-tumoral immunity and pro-tumoral statuses derived from macrophage polarization, and the therapeutic opportunities targeting alternately activated macrophages (AAM)-type macrophage polarization. Among them, cellular components including T cells, as well as acellular components represented by IL-4 and IL-13 are key regulators driving the polarization of AAM macrophages. Novel treatments targeting macrophage-associated mechanisms are mainly divided into small molecule inhibitors, monoclonal antibodies, and other therapies to re-acclimate AMM macrophages. Finally, we paid special attention to an immunosuppressive subgroup of macrophages with T cell immunoglobulin and mucin domain-3 (TIM-3) expression. Based on cellular interactions with cancer cells, TIM3+ macrophages facilitate the proliferation and progression of cancer cells, yet this process exposes targets blocking the ligand-receptor recognition. To sum up, this is a systematic review on the mechanism of tumor-associated macrophages (TAM) polarization, therapeutic strategies and the biological functions of Tim-3 positive macrophages that aims to provide new insights into the pathogenesis and treatment of lung cancer.

A-Kinase Anchor Protein 1 deficiency causes mitochondrial dysfunction in mouse model of hyperoxia induced acute lung injury.

Background: Critically ill patients on supplemental oxygen therapy eventually develop acute lung injury (ALI). Reactive oxygen species (ROS) produced during ALI perturbs the mitochondrial dynamics resulting in cellular damage. Genetic deletion of the mitochondrial A-kinase anchoring protein 1 (Akap1) in mice resulted in mitochondrial damage, Endoplasmic reticulum (ER) stress, increased expression of mitophagy proteins and pro-inflammatory cytokines, exacerbating hyperoxia-induced Acute Lung Injury (HALI). Objective: Despite a strong causal link between mitochondrial dysfunction and HALI, the mechanisms governing the disease progression at the transcriptome level is unknown. Methods: In this study, RNA sequencing (RNA-seq) analysis was carried out using the lungs of Akap1 knockout (Akap1 −/−) mice exposed to normoxia or 48 h of hyperoxia followed by quantitative real time PCR and Ingenuity pathway analysis (IPA). Western blot analysis assessed mitochondrial dysfunction, OXPHOS complex (I-V), apoptosis and antioxidant proteins. Mitochondrial enzymatic assays was used to measure the aconitase, fumarase, citrate synthase activities in isolated mitochondria from Akap1 −/− vs. Wt mice exposed to hyperoxia. Results: Transcriptome analysis of Akap1 −/− exposed to hyperoxia reveals increases in transcripts encoding electron transport chain (ETC) and tricarboxylic acid cycle (TCA) proteins. Ingenuity pathway analysis (IPA) shows enrichment of mitochondrial dysfunction and oxidative phosphorylation in Akap1 −/− mice. Loss of AKAP1, coupled with oxidant injury, significantly decreases the activities of TCA enzymes. Mechanistically, a significant loss of dynamin-related protein 1 (Drp1) phosphorylation at the protein kinase A (PKA) site Serine 637 (Ser637), decreases in Akt phosphorylation at Serine 437 (Ser47) and increase in the expression of pro-apoptotic protein Bax indicate mitochondrial dysfunction. Heme oxygenase-1 (HO-1) levels significantly increased in CD68 positive alveolar macrophages in Akap1 −/− lungs, suggesting a strong antioxidant response to hyperoxia. Conclusion: Overall these results suggest that AKAP1 overexpression and modulation of Drp1 phosphorylation at Ser637 is an important therapeutic strategy for acute lung injury.

OA22 A case report of Whipple's arthritis

Introduction/BackgroundWhipple’s disease is rare chronic systemic infection caused by the Tropheryma whipplei. Initially it was thought it can only affect the Gastrointestinal tract but later on it was discovered that it is multisystemic and can affect heart, brain, skin, eyes and lungs. The common presenting features are gastrointestinal tract symptoms like diarrhoea, vomiting, reduced appetite and weight loss. Other common extra intestinal features are joints pain, uveitis, and confusion and memory loss. Rarely, it can present with seizures and other neurological deficits. It predominantly affects the males as compared to females likely with defective T-Lymphocytes particularly TH1 population function.Description/MethodA 69 year old gentleman presented with unexplained weight loss, chronic diarrhoea, reduced appetite and abdominal bloating with travelling history in the past. Background history of epilepsy, hypercholesterolemia and osteoarthritis. Initial blood tests showed a normocytic anaemia along with lymphopenia. CT thorax and abdomen found indeterminate mesenteric lymphadenopathy. Upper GI endoscopy confirmed tiny proximal stomach tiny polyps, polypectomy was done along with multiple biopsies taken from duodenum. The duodenal biopsies histopathology report had shown an infiltration of PAS stain positive foamy macrophages with mixed inflammatory cells within the lamina propria along with broadening of villi. Furthermore, no micro-organisms were seen, including fungi and mycobacteria along with atypical cells. Histopathology is quite consistent with Whipple's disease however tissue biopsy for Whipple’s PCR was negative but serum Whipple’s PCR was detected. On infectious disease team advice he was started on ceftriaxone for two weeks later, switched to oral trimoxazole for 12 months resulted in complete symptoms resolution along with negative Whipple's PCR.Three months later, Rheumatology team was involved as he had developed palindromic migratory polyarthritis along with raised inflammatory markers, involving his right knee, right wrist, left elbow and then left wrist along with active joint inflammation. Blood investigations had shown weakly positive Rheumatoid factor with negative anti CCP and ANA. The plain films of various joints did not show any early inflammatory changes like periarticular osteopenia and erosions. Infectious disease team had suggested a repeat endoscopy reassuringly, he had negative serum and biopsies Whipple's PCR. After couple of weeks he developed a left knee pain with joint effusion which was aspirated with aseptic technique and the left knee synovial fluid came positive for Whipple’s PCR. On Infectious team recommendation he was commenced on hydroxychloroquine and doxycycline for another 12 months.Discussion/ResultsWhipple’s disease arthritis is easily misdiagnosed and approximately 1.58% of patients with unexplained seronegative inflammatory arthritis are positive for Tropheryma whipplei. Furthermore, the diagnosis of Whipple’s disease can be prolonged in patients with no gastrointestinal tract symptoms and in some cases gastrointestinal symptoms occur after extra intestinal features. In 41%to 61% cases of Whipple’s disease arthritis was reported which indicates Whipple's disease is significantly associated with arthritis and even more in Whipple's prodromal disease where the percentages of joint symptoms peak around 40%-80%. With the antibiotics the outcome of Whipple's disease can be fatal, it can be the worst when patients are misdiagnosed and on immunosuppressants.Giving a reference of retrospective single centre cohort study of seven patients was done. All 7 patients presented with symmetrical polyarthritis and 3 patients developed erosive arthritis. The most common joints were wrists, knuckles and knees involved, less commonly joints involvement were hips, elbows and shoulders. All patients were seronegative and out of 7, 6 patients were given disease modifying agents for considering seronegative rheumatoid arthritis but didn’t get good treatment response. All patients were treated with treated with ceftriaxone along with Co –Trimoxazole or Doxycycline/hydroxychloroquine. Both combinations were used as alternatives due to side effects or inefficacy and eventually lead to arthritis remission over the treatment course of 12 - 27 months. Interestingly, 2 out of 6 patients duodenal biopsies were positive for PAS staining and but all patients synovial fluid from one inflamed joint was positive for Whipple's PCR.The desirable investigations for suspected Whipple's arthritis are for suspected Whipple’s disease are PCR with samples of joint fluid, saliva and stools.In our case, Patient joints symptoms resolved with the course of antibiotics without progression of further joint involvement.Key learning points/ConclusionIn conclusion, Whipple’s disease can rarely present like seronegative arthritis with or without GI symptoms. For patients with seronegative inflammatory arthritis not responding to initial DMARDs therapy, Whipple’s arthritis should be excluded. The positive outcome about Whipple’s arthritis is that it is antimicrobial treatment responsive. In the suspected patients diagnostics test to rule out Whipple's arthritis is minimal invasive synovial fluid tap for Whipple's PCR, which can be performed in routine clinical practice.

Crosstalk between tumor-associated macrophages and tumor cells promotes chemoresistance via CXCL5/PI3K/AKT/mTOR pathway in gastric cancer

Background5-fluorouracil (5-FU)-based chemotherapy regimen has been widely used for the treatment of gastric cancer, but meanwhile the development of chemotherapeutic resistance remains a major clinical challenge. Tumor microenvironment (TME) frequently correlates with the development of chemoresistance in human cancer. As a major component of TME, the role of tumor-associated macrophages (TAMs) in the chemoresistance of gastric cancer has not been fully elucidated.MethodsImmunohistochemistry (IHC) was applied to detect the density of TAMs in clinical samples of 103 patients with gastric cancer who had undergone 5-FU-based neoadjuvant chemotherapy. 5-FU-resistant gastric cell lines MKN45-R and HGC27-R were established, macrophages were then separately co-cultured with MKN45-R, HGC27-R cells and their parental cells. The effect of gastric cancer cells on the polarization of macrophages, the biological function of M2-polaried macrophages and the mechanism for promoting 5-FU-resistance were investigated. Then the correlation between the expression of CXC motif chemokine ligand 5 (CXCL5) and the infiltration of hemoglobin scavenger receptor (CD163) positive and mannose receptor (CD206) positive macrophages was analyzed, the prognostic value of CXCL5 expression in clinical samples was further explored.ResultsThe high infiltration of macrophages marked by CD68 in gastric cancer samples was significantly associated with the resistance of gastric cancer to chemotherapy. Gastric cancer cells could modulate macrophages to M2-like polarization through indirect co-culture, and chemoresistant cells were more efficient in inducing macrophages polarization to M2 phenotype. Co-culturing M2-polarized macrophages in turn enhanced 5-FU-resistance of gastric cancer cells, and it was further verified that CXCL5 derived from M2-polarized macrophages promoted chemoresistance through activing the PI3K/AKT/mTOR pathway. Besides, high level of CXCL5 could recruit monocytes to form more M2-polarized macrophages. Clinically, high expression of CXCL5 in gastric cancer samples was associated with the high infiltration of CD163 positive macrophages and CD206 positive macrophages, and patients with high expression of CXCL5 presented lower overall survival (OS) rates than those with low expression of CXCL5.ConclusionInteraction between TAMs and gastric cancer cells promoted chemoresistance in gastric cancer via CXCL5/PI3K/AKT/mTOR pathway. Thus, targeting TAMs and blocking the cell–cell crosstalk between TAMs and gastric cancer cells may represent prospective therapeutic strategies for patients with gastric cancer.

Interplay between cellular changes in the knee joint, circulating lipids and pain behaviours in a slowly progressing murine model of osteoarthritis.

Synovial inflammation has known contributions to chronic osteoarthritis (OA) pain, but the potential role in transitions from early to late stages of OA pain is unclear. The slowly progressing surgical destabilization of the medial meniscus (DMM) murine OA model and sham control, was used in male C57BL/6J mice to investigate the interplay between knee inflammation, plasma pro- and anti-inflammatory oxylipins and pain responses during OA progression. Changes in joint histology, macrophage infiltration, chemokine receptor CX3CR1 expression, weight bearing asymmetry, and paw withdrawal thresholds were quantified 4, 8 and 16 weeks after surgery. Plasma levels of multiple bioactive lipid mediators were quantified using liquid chromatography with tandem mass-spectrometry (LC-MS/MS). Structural joint damage was evident at 8 weeks post-DMM surgery onwards. At 16 weeks post-DMM surgery, synovial scores, numbers of CD68 and CD206 positive macrophages and pain responses were significantly increased. Plasma levels of oxylipins were negatively correlated with joint damage and synovitis scores at 4 and 8 weeks post-DMM surgery. Higher circulating levels of the pro-resolving oxylipin pre-cursor 17-HDHA were associated with lower weight bearing asymmetry at week 16. The transition to chronic OA pathology and pain is likely influenced by both joint inflammation and plasma oxylipin mediators of inflammation and levels of pro-resolution molecules. Using a slow progressing surgical model of osteoarthritis we show how the changing balance between local and systemic inflammation may be of importance in the progression of pain behaviours during the transition to chronic osteoarthritis pain.

Oxidized low-density lipoprotein stimulates CD206 positive macrophages upregulating CD44 and CD133 expression in colorectal cancer with high-fat diet.

BACKGROUNDOxidized low-density lipoprotein (ox-LDL), which is abnormally increased in the serum of colorectal cancer (CRC) patients consuming a high-fat diet (HFD), may be one of the risk factors for the development of CRC. Ox-LDL exerts a regulatory effect on macrophages and may influence CRC through the tumor microenvironment. The role of ox-LDL in CRC remains unclear.AIMTo investigate the role of ox-LDL through macrophages in HFD associated CRC.METHODSThe expression of ox-LDL and CD206 was detected in colorectal tissues of CRC patients with hyperlipidemia and HFD-fed mice by immunofluorescence. We stimulated the macrophages with 20 μg/mL ox-LDL and assessed the expression levels of CD206 and the cytokines by cell fluorescence and quantitative polymerase chain reaction. We further knocked down LOX-1, the surface receptor of ox-LDL, to confirm the function of ox-LDL in macrophages. Then, LoVo cells were co-cultured with the stimulated macrophages to analyze the CD44 and CD133 expression by western blot.RESULTSThe expression of ox-LDL and the CD206 was significantly increased in the stroma of colorectal tissues of CRC patients with hyperlipidemia, and also upregulated in the HFD-fed mice. Moreover, an increased level of CD206 and decreased level of inducible nitric oxide synthase were observed in macrophages after ox-LDL continuous stimulation. Such effects were inhibited when the surface receptor LOX-1 was knocked down in macrophages. Ox-LDL could induce CD206+ macrophages, which resulted in high expression of CD44 and CD133 in co-cultured LoVo cells.CONCLUSIONOx-LDL stimulates CD206+ macrophages to upregulate CD44 and CD133 expression in HFD related CRC.

Visualization of macrophage subsets in the development of the fetal human inner ear.

BackgroundHuman inner ear contains macrophages whose functional role in early development is yet unclear. Recent studies describe inner ear macrophages act as effector cells of the innate immune system and are often activated following acoustic trauma or exposure to ototoxic drugs. Few or limited literature describing the role of macrophages during inner ear development and organogenesis.Material and MethodsWe performed a study combining immunohistochemistry and immunofluorescence using antibodies against IBA1, CX3CL1, CD168, CD68, CD45 and CollagenIV. Immune staining and quantification was performed on human embryonic inner ear sections from gestational week 09 to 17.ResultsThe study showed IBA1 and CD45 positive cells in the mesenchymal tissue at GW 09 to GW17. No IBA1 positive macrophages were detected in the sensory epithelium of the cochlea and vestibulum. Fractalkine (CX3CL1) signalling was initiated GW10 and parallel chemotactic attraction and migration of macrophages into the inner ear. Macrophages also migrated into the spiral ganglion, cochlear nerve, and peripheral nerve fibers and tissue-expressing CX3CL1. The mesenchymal tissue at all gestational weeks expressed CD163 and CD68.ConclusionExpressions of markers for resident and non-resident macrophages (IBA1, CD45, CD68, and CD163) were identified in the human fetal inner ear. We speculate that these cells play a role for the development of human inner ear tissue including shaping of the gracile structures.

Immunohistochemical analysis of MMP-9 and COX-2 expression in carotid atherosclerotic plaques among patients undergoing carotid endarterectomy: A prospective study

BackgroundMatrix metalloproteinase-9 protein (MMP-9) and cyclooxygenase-2 (COX-2) proteins may have a role in remodelling of atherosclerotic plaques. We analysed and compared the radiological, histological and immunohistochemical characteristics of carotid atherosclerotic plaques between symptomatic and asymptomatic patients who underwent carotid endarterectomy (CEA). MethodsThis prospective single-blinded study included 31 patients (70 [64-75] years, 58% males, 42% symptomatic) who underwent CEA and a total of 155 carotid plaque sections that were analysed. Preoperative assessment and multimodality diagnostic imaging with magnetic resonance imaging (MRI) or computed tomography angiography (CTA), histological and immunohistochemical analyses of carotid plaques including the expression of MMP-9 and COX-2 proteins were performed. ResultsSymptomatic and asymptomatic patients did not significantly differ in respect to preoperative characteristics. Unstable plaques were detected in 12/13 (92.3%, p = 0.020) symptomatic patients using MRI or CTA. There was no perioperative mortality and perioperative outcomes were comparable in both groups. A significantly higher expression of MMP-9 in macrophages was observed among symptomatic patients (p = 0.020). ROC curve analysis showed statistically significant associations of both the higher intensity of COX-2 staining in CD68 PG-M1 positive macrophages (area under the curve [AUC]=0.701, p = 0.014) and higher MVD (AUC=0.821, p < 0.001) within the plaque with cerebrovascular symptoms. The expression of COX-2 and the intensity of COX-2 staining in macrophages within the unstable carotid plaques detected by preoperative MRI or CTA were significantly higher (76.1% vs. 40.0%, p = 0.038; 76.2% vs. 30.0%, p = 0.01, respectively). ConclusionsAdvanced non-invasive multimodality diagnostic imaging including MRI or CTA is reliable in differentiating unstable from stable carotid plaques. High expression of MMP-9 and COX-2 in macrophages within the symptomatic plaque is associated with increased risk of cerebrovascular complications. Trial RegistrationThis study has been registered at the ISRCTN registry (ID ISRCTN46536832), isrctn.org Identifier: https://www.isrctn.com/ISRCTN46536832

Sex differences in the therapeutic effect of unaltered versus NFκB sensing IL-4 over-expressing mesenchymal stromal cells in a murine model of chronic inflammatory bone loss

Wear particles from joint arthroplasties induce chronic inflammation associated with prolonged upregulation of nuclear factor kappa-B (NF-κB) signaling in macrophages and osteoclasts, which leads to osteolysis and implant loosening. Mesenchymal stromal cell (MSC)-based therapy showed great potential for immunomodulation and mitigation of osteolysis in vivo, especially in the chronic phase of inflammation. We previously generated genetically modified MSCs that secrete the anti-inflammatory cytokine interleukin 4 (IL-4) in response to NF-κB activation (NFκB-IL-4 MSCs). However, whether the impact of sexual difference in the internal environment can alter the therapeutic effects of IL-4 over-secreting MSCs that simultaneously mitigate prolonged inflammation and enhance bone formation remains unknown. This study investigated the therapeutic effects of unaltered MSCs versus NFκB-IL-4 MSCs in mitigating chronic inflammation and enhancing bone formation in male and female mice. The murine model was established by continuous infusion of polyethylene particles contaminated with lipopolysaccharide (cPE) into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation. Unaltered MSCs or NFκB-IL-4 MSCs were infused into the femoral intramedullary cavity in sex-matched groups beginning 3 weeks after primary surgery. Femurs were harvested at 6 weeks, and bone marrow density was measured with micro-computational tomography. Numbers of osteoclast-like cells, osteoblasts, and macrophages were evaluated with histochemical and immunofluorescence staining. cPE infusion resulted in severe bone loss at the surgery site, increased tartrate-resistant acid phosphatase positive osteoclasts and M1 pro-inflammatory macrophages, and decreased alkaline phosphatase expression. MSC-based therapy effectively decreased local bone loss and polarized M1 macrophages into an M2 anti-inflammatory phenotype. In females, unaltered MSCs demonstrated a larger impact in enhancing the osteogenesis, but they demonstrated similar anti-inflammatory effects compared to NFκB-IL-4 MSCs. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments in a sexually dimorphic manner, which could be an efficacious therapeutic strategy for treatment of periprosthetic osteolysis in both genders.

Molecular Basis of Extramural Vascular Invasion (EMVI) in Colorectal Carcinoma

Extramural vascular invasion (EMVI) is a known poor prognostic factor in colorectal carcinoma; however, its molecular basis has not been defined. This study aimed to assess the expression of molecular markers in EMVI positive colorectal carcinoma to understand their tumor microenvironment. Immunohistochemistry was performed on tissue microarrays of surgically resected colorectal cancer specimens for immunological markers, and BRAFV600E mutation (and on the tissue blocks for mismatch repair proteins). Automated quantification was used for CD8, LAG3, FOXP3, PU1, and CD163, and manual quantification was used for PDL1, HLA I markers (beta-2 microglobulin, HC10), and HLA II. The Wilcoxon rank-sum test was used to compare EMVI positive and negative tumors. A logistic regression model was fitted to assess the predictive effect of biomarkers on EMVI. There were 340 EMVI positive and 678 EMVI negative chemo naïve tumors. PDL1 was barely expressed on tumor cells (median 0) in the entire cohort. We found a significantly lower expression of CD8, LAG3, FOXP3, PU1 cells, PDL1 positive macrophages, and beta-2 microglobulin on tumor cells in the EMVI positive subset (p ≤ 0.001). There was no association of BRAFV600E or deficient mismatch repair proteins (dMMR) with EMVI. PU1 (OR 0.8, 0.7-0.9) and low PDL1 (OR 1.6, 1.1-2.3) independently predicted EMVI on multivariate logistic regression among all biomarkers examined. There is a generalized blunting of immune response in EMVI positive colorectal carcinoma, which may contribute to a worse prognosis. Tumor-associated macrophages seem to play the most significant role in determining EMVI.

Clever-1 positive macrophages in breast cancer

PurposeCommon Lymphatic Endothelial and Vascular Endothelial Receptor 1 (Clever-1) is expressed by a subset of immunosuppressive macrophages and targeting the receptor with therapeutic antibodies has been shown to activate T-cell-mediated anti-cancer immunity. The aim of this research was to study Clever-1 expression in breast cancer. Specifically, how Clever-1 + macrophages correlate with clinicopathologic factors, Tumor Infiltrating Lymphocytes (TILs) and prognosis.MethodsTissue microarray blocks were made from 373 primary breast cancer operation specimens. Hematoxylin and Eosin (H&E-staining) and immunohistochemical staining with Clever-1, CD3, CD4 and CD8 antibodies were performed. Differences in quantities of Clever-1 + macrophages and TILs were analyzed. Clever-1 + cell numbers were correlated with 25-year follow-up survival data and with breast cancer clinicopathologic parameters.ResultsLow numbers of intratumoral Clever-1 + cells were found to be an independent adverse prognostic sign. Increased numbers of Clever-1 + cells were found in high grade tumors and hormone receptor negative tumors. Tumors that had higher amounts of Clever-1 + cells also tended to have higher amounts of TILs.ConclusionThe association of intratumoral Clever-1 + macrophages with better prognosis might stem from the function of Clever as a scavenger receptor that modulates tumor stroma. The association of Clever-1 + macrophages with high number of TILs and better prognosis indicates that immunosuppression by M2 macrophages is not necessarily dampening adaptive immune responses but instead keeping them in control to avoid excess inflammation.

Single-cell transcriptome analysis of fractional CO2 laser efficiency in treating a mouse model of alopecia.

Hair loss, including alopecia, is a common dermatological issue worldwide. At present, the application of fractional carbon dioxide (CO2 ) laser in the treatment of alopecia has been documented; however, the results vary between reports. These varying results may be due to the limited knowledge of cellular action in laser-irradiated skin. The objective of this study was to investigate the molecular and cellular mechanisms of laser treatment under effective conditions for hair cycle initiation. A fractional CO2 laser was applied and optimized to initiate the hair cycle in a mouse model of alopecia. Several cellular markers were analyzed in the irradiated skin using immunofluorescence staining. Cellular populations and their comprehensive gene expression were analyzed using single-cell RNA sequencing and bioinformatics. The effective irradiation condition for initiating the hair cycle was found to be 15 mJ energy/spot, which generates approximately 500 μm depth columns, but does not penetrate the dermis, only reaching approximately 1 spot/mm2 . The proportion of macrophage clusters significantly increased upon irradiation, whereas the proportion of fibroblast clusters decreased. The macrophages strongly expressed C-C chemokine receptor type 2 (Ccr2), which is known to be a key signal for injury-induced hair growth. We found that fractional CO2 laser irradiation recruited Ccr2 positive macrophages, and induced hair regrowth in a mouse alopecia model. These findings may contribute to the development of stable and effective fractional laser irradiation conditions for human alopecia treatment.

Experimental study on effects of berberine combined with 6-shogaol on intestinal inflammation and flora in mice with ulcerative colitis

Cold-heat combination is a common method in the treatment of ulcerative colitis, which is represented by classic drug pair, Coptidis Rhizoma and Zingiberis Rhizoma.The present study explored the synergetic effects of berberine and 6-shogaol, the primary components of Coptidis Rhizoma and Zingiberis Rhizoma, respectively, on intestinal inflammation and intestinal flora in mice with ulcerative colitis to reveal the effect and mechanism of cold-heat combination in the treatment of ulcerative colitis.The ulcerative colitis model was induced by dextran sulfate sodium(DSS) in mice.The model mice were administered with berberine(100 mg·kg~(-1)), 6-shogaol(100 mg·kg~(-1)), and berberine(50 mg·kg~(-1)) combined 6-shogaol(50 mg·kg~(-1)) by gavage, once per day.After 20 days of drug administration, mouse serum, colon tissues, and feces were sampled.Hematoxylin-eosin(HE) staining was used to observe histopathological changes in colon tissues.Alcian blue/periodic acid-Schiff(AB/PAS) staining was used to observe the changes in the mucus layer of colon tissues.Enzyme-linked immunosorbent assay(ELISA) was employed to detect the serum content of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6).Immunohistochemical method was adopted to detect the protein expression of macrophage surface markers F4/80, mucin-2, claudin-1, and zonula occludens-1(ZO-1) in colon tissues.High-throughput Meta-amplicon library sequencing was used to detect changes in the intestinal flora of mice.The results indicated that the 6-shogaol group, the berberine group, and the combination group showed significantly relieved intestinal injury, reduced number of F4/80-labeled positive macrophages in colon tissues, increased protein expression of mucin-2, claudin-1, and ZO-1, and decreased serum le-vels of TNF-α, IL-1β, and IL-6.Shannon, Simpson, Chao, and Ace indexes of the intestinal flora of mice in the 6-shogaol group and the combination group significantly increased, and Chao and Ace indexes in the berberine group significantly increased.As revealed by the bioinformatics analysis of intestinal flora sequencing, the relative abundance of Verrucomicrobia at the phylum, class, and order levels decreased significantly in all treatment groups after drug administration, while that of Bacillibacteria gradually increased.In the 6-shogaol group and the combination group, Akkermansia muciniphila completely disappeared, but acid-producing bacillus still existed in large quantities.As concluded, both 6-shogaol and berberine can inhibit intestinal inflammation, reduce the infiltration and activation of macrophages, relieve intestinal damage, reduce intestinal permeability, improve the structure of flora, and promote intestinal microecological balance.The combined application of berberine and 6-shogaol has a significant synergistic effect.