Accumulation Of M2 Macrophages Research Articles

Abstract P6-06-12: Upregulation of beige adipocytes in African-American triple negative breast tumors induce tumorigenesis by promoting anti-inflammatory microenvironment

Abstract Background: The complexity of breast cancer (BC) development and progression is heavily influenced by the cross-talk of critical cell types within the tumor microenvironment. While there have been many advancements in BC treatment, triple negative breast cancer (TNBC) still exhibits the lowest rate of response and the nature of its aggressive properties remain poorly understood. African American (AA) women are disproportionally affected and have worse overall survival. Our lab previously reported important role of beige adipocytes in breast tumor growth. Interestingly, we found beige adipocytes were highly up regulated in AA TN breast tumors when compared to TNBC from Caucasian (CA) women. In this study, we have further investigated the mechanisms that increase beige adipocytes in AA TN breast tumors and their influence on breast tumor growth. Methods: Human tumor samples provided by Cooperative Human Tissue Network and National Disease Research Institute were subjected to immunohistochemistry (IHC) and western blot analysis to examine key beige adipocyte markers. To further investigate the mechanisms by which beige adipocytes may contribute tumor growth, xenografts were established from AA TNBC cell lines HCC70, HCC1806 and MDA-MB 468. Tumors excised at various time points were examined for beige adipocyte markers and their key regulators using validated species specific primers to distinguish between host and tumor cell contributions. Boyden chamber assays and ex-vivo experiments were performed to examine whether beige adipocytes were recruited from the host or induced in xenografts, and test whether paracrine secretion of cytokines or contact between various cell types were necessary for adipose browning in these tumors. Possible involvement of lactic acid-mediated adipose browning in these tumors was tested by inhibiting lactate dehydrogenase using sodium oxamate. We further examined whether increased browning in these tumors was associated with increase in anti-inflammatory cytokines that promote M2 macrophage phenotype and angiogenesis, both of which are highly implicated in tumor progression. Results: Both quantitative IHC and western blot analysis showed higher expression of beige adipocyte markers CD137 and TMEM26 in AA TNBC compared to CA TNBC. Expression of beige marker CD137 was detected in all xenografts from AA TNBC by IHC. Quantitative gene expression analysis (qPCR) revealed that host contributed to the origin of beige adipocytes in these tumors. We demonstrate using Boyden Chamber assay that contact between tumor cells and host adipocytes was important for adipose browning. Ex-vivo experiments also showed that breast cancer cells in contact with either host adipocytes or 3T3-L1 mouse adipocyte cell line increased the expression of beige markers that could be attenuated by lactate dehydrogenase inhibitor. We further show that increase in beige adipocytes in AA TNBC was associated with high anti-inflammatory cytokines IL13/IL4 expression, as well as anti-inflammatory pro-tumorigenic M2 macrophages and high angiogenesis. Conclusion: Key beige markers CD137 and TMEM26 are significantly upregulated in AA TNBC compared to the CA TNBC. Host microenvironment contributed to beige adipocyte characteristics in response to lactic acid-mediated adipose browning of tumor cells. Furthermore, beige adipocytes increased anti-inflammatory cytokines that facilitate accumulation of M2 macrophages and promote angiogenesis. Our results implicate that beige adipocytes are key mediators of aggressive tumor development in AA TNBC and could potentially be targeted for therapeutic purposes. Citation Format: Brandis A. Moore, Briana Osorio, Rajan Singh, Shehla Pervin. Upregulation of beige adipocytes in African-American triple negative breast tumors induce tumorigenesis by promoting anti-inflammatory microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-12.

M1 macrophages accelerate renal glomerular endothelial cell senescence through reactive oxygen species accumulation in streptozotocin-induced diabetic mice

Cellular senescence is a fundamental aging mechanism leading to tissue dysfunction. Accumulation of senescent cells is observed in the context of diabetes, which plays an important role in the pathogenesis of diabetes and its complications. Macrophages, the most prevalent leucocytes found in diabetic kidney, have been implicated in the modulation of cellular senescence; however, their role and mechanism in cellular senescence of diabetic kidney have not been determined. In this study, we found trends of cellular senescence in the glomeruli of streptozotocin-induced diabetic mice. The onset of glomerular senescence was confirmed by increased SA-β-gal staining, the upregulation of p16INK4a, p21, and p53 protein levels and the increased expression of SASP RNA. The senescent cells in the glomeruli were mainly endothelial cells. We next confirmed that M1 macrophages accumulated in the glomeruli, occurred just shortly before glomerular senescence. Therefore, we examined whether M1 macrophage accumulation is associated with glomerular endothelial cell senescence. Thus, an in vitro co-culture model was established using human renal glomerular endothelial cells (HRGECs) and M1-polarized THP-1 macrophages. Indeed, M1 macrophages induced senescence in HRGECs. Furthermore, intracellular ROS levels and p38 MAPK signalling activation were significantly increased in HRGECs and reducing ROS generation significantly abolished M1 macrophage-mediated endothelial senescence and p38 MAPK activation, suggesting that M1 macrophage-mediated endothelial senescence is largely dependent on ROS. Thus, our results demonstrate that kidney M1 macrophage accumulation is in connection with endothelial cell senescence and strategy to modulate M1 macrophages accumulation is promising to be a new target for immunotherapy for diabetic kidney disease and other age-related diseases.

TSG-6 Inhibits Oxidative Stress and Induces M2 Polarization of Hepatic Macrophages in Mice With Alcoholic Hepatitis via Suppression of STAT3 Activation.

Tumor necrosis factor (TNF)-α-stimulated protein 6 (TSG-6) is a secreted protein with diverse tissue protective and anti-inflammatory properties. We aimed to investigate its effects in treating mice with alcoholic hepatitis (AH) and the associated mechanisms. AH was induced in 8–10 week female C57BL/6N mice by chronic-binge ethanol feeding for 10 days. Intraperitoneal (i.p.) injection of recombinant mouse TSG-6 or saline were performed in mice on day 10. Blood samples and hepatic tissues were collected on day 11. Biochemistry, liver histology, flow cytometry, and cytokine measurements were conducted. Compared to the normal control mice, the AH mice had significantly increased liver/body weight ratio, serum alanine aminotransferase (ALT) and aspartate aminotransferases (AST), hepatic total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), hepatic macrophage infiltration, serum and hepatic interleukin (IL)-6, and tumor necrosis factor (TNF)-α, which were markedly reduced by i.p. injection of rmTSG-6. Compared to the normal control mice, the hepatic glutathione (GSH), accumulation of M2 macrophages, serum, and hepatic IL-10 and TSG-6 were prominently reduced in the AH mice, which were significantly enhanced after i.p. injection of rmTSG-6. Compared to the normal control mice, hepatic activation of signal transducer and activator of transcription 3 (STAT3) was significantly induced, which was markedly suppressed by rmTSG-6 treatment. TSG-6 was effective for the treatment of AH mice, which might be associated with its ability in inhibiting hepatic oxidative stress and inducing hepatic M2 macrophages polarization via suppressing STAT3 activation.

The immunomodulatory effects of mesenchymal stem cells on long term pulmonary complications in an animal model exposed to a sulfur mustard analog

IntroductionSulfur Mustard (SM) is one of the most lethal chemicals with major complications manifested in the lungs. Although the pathogenesis behind SM-induced lung injury still remains poorly understood, prolonged activation and the imbalance of two major macrophage populations (M1 and M2) have been suggested to be involved. Here, we tried to investigate the effectiveness of adipose-derived mesenchymal stem cells (AD-MSC) on long-term lesions induced by CEES, an SM analog. The modulation of pulmonary immune cells and alveolar macrophage phenotype alteration was studied in the animal model used. MethodsHistopathological changes were investigated in the lungs and analysis of surface markers of alveolar macrophages as well as their cytokine expression in the BAL fluid was carried out by flow cytometry and ELISA, respectively. ResultsTreatment of mice with AD-MSC after intraperitoneal administration of CEES (10 mg/kg) reduces progressive histopathologic changes in the lung. Flow cytometric analysis of isolated alveolar macrophages in the bronchoalveolar lavage showed that the accumulation of both M1 and M2 macrophages in response to CEES was reduced by MSC administration. AD-MSCs caused a marked reduction in the CD86- and CD206-expressing macrophages compared to the untreated groups. The modulating effect of AD-MSCs in the M1-subset was much more significant compared to M2.These findings suggest that AD-MSCs understand their environment and restore the balance in disorders associated with Th1 or Th2 imbalance. Our results indicate that MSCs may represent an effective approach to repair lung injury induced by mustards.

A Combination Therapy with Dendritic Cells, Pomalidomide and Programmed Death-Ligand 1 Blockade Exerts a Potent Antitumor Immunity in a Murine Model of Multiple Myeloma

Multiple myeloma (MM) is the second-most-common hematologic malignancy, and develops from clonal malignant plasma cells within bone marrow. Despite tremendous improvements in therapeutic strategies (e.g. stem cell transplantation, immune-modulatory drugs (IMiDs), proteasome inhibitors, and, more recently, immunotherapy), which have led to improved responses to treatment and overall survival, most patients eventually relapse. We have previously shown that the immunization with tumor antigen-loaded dendritic cells (DCs) and pomalidomide/dexamethasone synergistically potentiates the enhancing the antitumor immunity in a myeloma mouse model. In the present study, we investigated whether a DC-based vaccine combined with pomalidomide and PD-L1 blockade has a synergistic effect in a murine MM model. MOPC-315 cell lines were injected subcutaneously to establish MM-bearing mice. Four test groups were used to mimic the clinical protocol: (1) PBS control, (2) DCs + pomalidomide/dexamethasone, (3) pomalidomide/dexamethasone + PD-L1 blockade, and (4) DCs + pomalidomide/dexamethasone + PD-L1 blockade. After treatment, preclinical response and in vitro immunological responses were evaluated. The study was designed to closely mimic the clinical MM treatment protocol and clearly demonstrated that combination treatment with DCs + pomalidomide with dexamethasone + PD-L1 blockade more strongly inhibited MM tumor growth. Consequently, the mice treated with DCs + pomalidomide with dexamethasone + PD-L1 blockade displayed markedly induced tumor regression and significantly prolonged survival, as well as very strong anti-myeloma CTL responses and increased numbers of effector cells (such as CD4+ T cells, CD8+ T cells, memory T cells, NK cells and M1 macrophages) associated with antitumor effects. This treatment also effectively decreased the proportions of suppressor cells, including MDSCs, Tregs and M2 macrophages, in the spleen and tumor microenvironment of treated mice. Tregs, MDSCs and M2 macrophages play crucial roles in immunosuppression and tolerance, which are mediated by tumor-secreted cytokines. The inhibition of Tregs, MDSC and M2 macrophage accumulation may enhance systemic cell-mediated immunity through the activation of DCs or CTLs. Importantly, treatment with pomalidomide with dexamethasone + PD-L1 blockade led to decreased expression of PD-L1 and CTLA-4 in treated mice, which further induced effector cell infiltration of the tumor microenvironment. Moreover, DCs + pomalidomide with dexamethasone + PD-L1 blockade induced the activation of cell-mediated immunity by increasing Th1-specific immune responses, as evidenced by the increased production of IFN-γ and a decrease in the regulatory-specific immune response, as evidenced by the decreased production of TGF-β, IL-10 and VEGF in the spleen and tumor microenvironment. These findings show that inducing the systemic immune response represent a means of treating myeloma. Immunotherapy clearly represents a revolution in cancer care, and promising responses have been shown to various treatments, particularly immune checkpoint inhibitors, IMiDs, DCs and CAR T cells. However, not all patients are responsive to current immunotherapies, and among those patients who do respond, the effects are not always long-lasting. Thus, combination approaches are a cornerstone of cancer therapy for improving patient outcomes in MM. This study demonstrated that the combination of DC vaccination + pomalidomide with dexamethasone + PD-L1 blockade synergistically enhances myeloma immune responses to inhibit tumor growth, restores and enhances host immune effector cells, and reduces the generation of immune suppressor cells in MM. This study provides a framework for developing and understanding the role of immunotherapeutic modalities employing DCs, pomalidomide and PD-L1 blockade to inhibit tumor growth and restore immune function in myeloma-bearing mice. Figure Disclosures No relevant conflicts of interest to declare.

Alpha-hemolysin of uropathogenic Escherichia coli induces GM-CSF-mediated acute kidney injury

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), inducing acute pyelonephritis and may result in permanent renal scarring and failure. Alpha-hemolysin (HlyA), a key UPEC toxin, causes serious tissue damage; however, the mechanism through which HlyA induces kidney injury remains unclear. In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by renal epithelial cells was upregulated by HlyA in vitro and in vivo, which induced M1 macrophage accumulation in kidney, and ADAM10 was found involved in HlyA-induced GM-CSF. Macrophage elimination or GM-CSF neutralization protected against acute kidney injury in mice, and increased GM-CSF was detected in urine of patients infected by hlyA-positive UPEC. In addition, HlyA was found to promote UPEC invasion into renal epithelial cells by interacting with Nectin-2 in vitro. However, HlyA did not affect bacterial titers during acute kidney infections, and HlyA-induced invasion did not contribute to GM-CSF upregulation in vitro, which indicate that HlyA-induced GM-CSF is independent of bacteria invasion. The role of GM-CSF in HlyA-mediated kidney injury may lead to novel strategies to treat acute pyelonephritis.

MTORC1-mediated polarization of M1 macrophages and their accumulation in the liver correlate with immunopathology in fatal ehrlichiosis

A polarized macrophage response into inflammatory (M1) or regenerative/anti-inflammatory (M2) phenotypes is critical in host response to multiple intracellular bacterial infections. Ehrlichia is an obligate Gram-negative intracellular bacterium that causes human monocytic ehrlichiosis (HME): a febrile illness that may progress to fatal sepsis with multi-organ failure. We have shown that liver injury and Ehrlichia-induced sepsis occur due to dysregulated inflammation. Here, we investigated the contribution of macrophages to Ehrlichia-induced sepsis using murine models of mild and fatal ehrlichiosis. Lethally-infected mice showed accumulation of M1 macrophages (iNOS-positive) in the liver. In contrast, non-lethally infected mice showed polarization of M2 macrophages and their accumulation in peritoneum, but not in the liver. Predominance of M1 macrophages in lethally-infected mice was associated with expansion of IL-17-producing T, NK, and NKT cells. Consistent with the in vivo data, infection of bone marrow-derived macrophages (BMM) with lethal Ehrlichia polarized M0 macrophages into M1 phenotype under an mTORC1-dependent manner, while infection with non-lethal Ehrlichia polarized these cells into M2 types. This work highlights that mTORC1-mediated polarization of macrophages towards M1 phenotype may contribute to induction of pathogenic immune responses during fatal ehrlichiosis. Targeting mTORC1 pathway may provide a novel aproach for treatment of HME.

An anti-inflammatory phenotype in visceral adipose tissue of old lean mice, augmented by exercise

Visceral adipose tissue is an immunogenic tissue, which turns detrimental during obesity by activation of proinflammatory macrophages. During aging, chronic inflammation increases proportional to visceral adipose tissue (VAT) mass and associates with escalating morbidity and mortality. Here, we utilize a mouse model to investigate the inflammatory status of visceral adipose tissue in lean aging mice and assess the effects of exercise training interventions. We randomized adult (11 months; n = 21) and old (23 months; n = 27) mice to resistance training (RT) or endurance training (ET), or to a sedentary control group (S). Strikingly, we observed an anti-inflammatory phenotype in the old mice, consisting of higher accumulation of M2 macrophages and IL-10 expression, compared to the adult mice. In concordance, old mice also had less VAT mass and smaller adipocytes compared to adult mice. In both age groups, exercise training enhanced the anti-inflammatory phenotype and increased PGC1-α mRNA expression. Intriguingly, the brown adipose tissue marker UCP1 was modestly higher in old mice, while remained unchanged by the intervention. In conclusion, in the absence of obesity, visceral adipose tissue possesses a pronounced anti-inflammatory phenotype during aging which is further enhanced by exercise.

Alcohol-induced adipose tissue macrophage phenotypic switching is independent of myeloid Toll-like receptor 4 expression.

Alcoholic liver disease results from a combination of immune and metabolic pathogenic events. In addition to liver injury, chronic alcohol consumption also causes adipose tissue inflammation. The specific immune mechanisms that drive this process are unknown. Here, we sought to determine the role of the innate immune receptor Toll-like receptor 4 (TLR4) in alcohol-induced adipose tissue inflammation. Using a model of chronic, multiple-binge alcohol exposure, we showed that alcohol-mediated accumulation of proinflammatory adipose tissue macrophages was absent in global TLR4 knockout mice. Proinflammatory macrophage accumulation did not depend on macrophage TLR4 expression; LysMCre-driven deletion of Tlr4 from myeloid cells did not affect circulating endotoxin or the accumulation of M1 macrophages in adipose tissue following alcohol exposure. Proinflammatory cytokine/chemokine production in the adipose stromal vascular fraction also occurred independently of TLR4. Finally, the levels of other adipose immune cells, such as dendritic cells, neutrophils, B cells, and T cells, were modulated by chronic, multiple-binge alcohol and the presence of TLR4. Together, these data indicate that TLR4 expression on cells, other than myeloid cells, is important for the alcohol-induced increase in proinflammatory adipose tissue macrophages.

Decreased M1 macrophage polarization in dabigatran-treated Ldlr-deficient mice: Implications for atherosclerosis and adipose tissue inflammation

Background and aimsThe non-vitamin K oral anticoagulant dabigatran etexilate (dabigatran) is increasingly prescribed to patients with non-valvular atrial fibrillation and venous thromboembolism. Adipose tissue (AT) inflammation during obesity plays a crucial role in the development of insulin resistance, type II diabetes and atherogenesis. The aim of the present study was to investigate the effects of thrombin inhibition by dabigatran in a combined model of diet-induced obesity and atherosclerosis. MethodsFemale Low density lipoprotein receptor knockout (Lldr−/−) mice were fed a high-fat diet containing 5 mg/g dabigatran or matching control for 20 weeks. ResultsDabigatran-treated animals showed increased adipocyte hypertrophy, but reduced numbers of pro-inflammatory M1-polarized macrophages in the adipose tissue. Abundance of pro-inflammatory M1 macrophages was also decreased in the aortic wall of dabigatran-fed mice. Multiple circulating cytokines were reduced, indicating an effect in systemically relevant secretory compartments such as the AT. ConclusionsDabigatran treatment reduces pro-inflammatory M1 macrophages in atherosclerotic lesions, thereby contributing to plaque stabilizing and atheroprotective effects of the thrombin inhibitor. This finding is not restricted to the vascular wall but is also present in AT where dabigatran treatment reduced the release of pro-inflammatory cytokines and accumulation of M1 macrophages.

Metabolic remodeling contributes towards an immune-suppressive phenotype in glioblastoma.

Glioblastoma (GBM) is one of the most aggressive tumors. Numerous studies in the field of immunotherapy have focused their efforts on identifying various pathways linked with tumor-induced immunosuppression. Recent research has demonstrated that metabolic reprogramming in a tumor can contribute towards immune tolerance. To begin to understand the interface between metabolic remodeling and the immune-suppressive state in GBM, we performed a focused, integrative analysis coupling metabolomics with gene-expression profiling in patient-derived GBM (n = 80) and compared them to low-grade astrocytoma (LGA; n = 28). Metabolic intermediates of tryptophan, arginine, prostaglandin, and adenosine emerged as immuno-metabolic nodes in GBM specific to the mesenchymal and classical molecular subtypes of GBM. Integrative analyses emphasized the importance of downstream metabolism of several of these metabolic pathways in GBM. Using CIBERSORT to analyze immune components from the transcriptional profiles of individual tumors, we demonstrated that tryptophan and adenosine metabolism resulted in an accumulation of Tregs and M2 macrophages, respectively, and was recapitulated in mouse models. Furthermore, we extended these findings to preclinical models to determine their potential utility in defining the biologic and/or immunologic consequences of the identified metabolic programs. Collectively, through integrative analysis, we uncovered multifaceted ways by which metabolic reprogramming may contribute towards immune tolerance in GBM, providing the framework for further investigations designed to determine the specific immunologic consequence of these metabolic programs and their therapeutic potential.

Whole Body Hyperthermia Ameliorates Inflammation via Regulating Pro‐inflammatory but not Anti‐inflammatory Macrophages in Tenotomized Rat Skeletal Muscle

Following disuse condition, macrophages play significant role in inflammatory responses during muscle degeneration and repairing processes. Pro‐inflammatory (M1 phenotype) macrophage becomes classically activated during an initial phase of muscle degeneration. Contrariwise, anti‐inflammatory (M2 phenotype) macrophage abrogates inflammation by deactivating M1 macrophage and promotes muscle growth in repairing stage. Previously, we demonstrated that heat treatment alleviates the loss of muscle mass via enhancing intramuscular capillarization and remodeling extracellular matrix in disuse muscle. Of these, the effect of heat stress on inflammatory responses in tenotomy‐induced atrophy is currently unknown. In this study, the effect of hyperthermia on M1 and M2 macrophage populations as well as the responses of inflammatory cytokine protein expression in the soleus muscle following Achilles tendon transection (tenotomy) were examined. Male Wistar rats were designed into sedentary control (CON), tenotomy without heat treatment (TEN), and tenotomy plus heat treatment (TEN+HEAT) groups. Whole body hyperthermia was maintained in TEN+HEAT group with animal core body temperature at 40.5–41.5°C, 30 min for 7 days. After 8 days of tenotomy, soleus muscle demonstrated a reduction in fiber size and tremendous infiltration of inflammatory macrophages. These occurrences coincided with an increase in pro‐inflammatory cytokine, particularly tumor necrosis factor‐alpha (TNF‐α). Whilst tenotomy had no effect on interleukin‐10 (IL‐10) which is an anti‐inflammatory cytokine. Interestingly, whole body hyperthermia diminished accumulation of M1 macrophages and associated with reduction of TNF‐α protein expression in tenotomized muscle. Nevertheless, heat stress affected neither M2 macrophage phenotype invasion nor IL‐10 protein level following tenotomy. Collectively, heat stress relieved tenotomy‐induced inflammatory response, partly by inhibiting M1 macrophage invasion. Our findings provide a foundation for therapeutic strategy of heat stress on fiber inflammation during skeletal muscle atrophy.Support or Funding InformationThe research was supported by the grant from Srinakharinwirot University (to MH).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA Sequencing of Intestinal Biopsies Expands the T Cell-Centric Paradigm of Steroid-Refractory Acute Graft-Versus-Host Disease

Background Steroid-refractory acute GVHD (aGVHD) is a fatal disease with little known regarding its pathophysiology at the tissue level. We studied the gene expression profiles of onset and steroid-refractory aGVHD intestinal biopsies, including both supervised analyses of tolerance (indoleamine 2,3 dioxygenase [IDO1]), repair factors (amphiregulin [AREG]), and immune cell composition, plus unsupervised analyses to define the transcriptome profile. Patients and Methods We performed ribonucleic acid sequencing (RNA-seq) on archival rectal biopsies from 22 patients with clinical stage 3-4 GI aGVHD (a) at the onset of GI aGVHD and (b) in the same patients at the diagnosis of steroid-refractory GI aGVHD, compared to 10 healthy controls at the University of Minnesota. We compared differential gene expression using Limma package, analyzed pathways using Enrichr®, and estimated immune cell composition using CIBERSORT®. Results Several immune- and damage-relevant genes differed between GVHD and normal samples. The mostly highly differentially expressed gene in aGVHD compared to normal was chitinase 3-L-1 (CHI3L1, fold-change 3.4, adjusted p=0.003), with known pathogenic roles in neovascularization, macrophage recruitment, and bacterial adhesion. The most significantly decreased gene in aGVHD was aquaporin-8 (AQP8, fold change -5.0, adjusted p Discussion While we observed distinctions between normal and onset aGVHD samples (increased CHI3L1 and decreased AQP8), we did not identify differences in T-cell driven inflammation in onset versus steroid-refractory aGVHD. Steroid-refractory aGVHD may be characterized by increased stress-induced MT expression, loss of host tissue tolerance signals, and accumulation of M2 macrophages. Our results expand the paradigm of T cell-centric therapies for steroid-refractory GI aGVHD and highlight new mechanisms for therapeutic exploration.

Impact of uterine macrophage phenotype on placental retention in dairy cows

Reproductive diseases affect 25% of dairy cows in the US and often develop from retention of the placenta. It is well established that expulsion of the placenta is a highly regulated inflammatory process, but the mechanisms by which dysregulation of uterine immune responses impair this process are poorly understood. In healthy non-ruminants, pro-inflammatory M1 macrophages are predominant in uterine tissue after parturition. However, macrophage phenotype in the postpartum bovine uterus is unknown. Our study compared macrophage phenotypes in the uterine caruncles of multiparous dairy cows that during the first day postpartum either retained (RET, n = 5) or had normal expulsion (NOR, n = 5) of placenta. Immune cells were sorted magnetically from the caruncular endometrial cell fraction using the CD172a marker and monocyte/macrophage population was characterized using flow cytometry. Transcriptional and protein expression studies were performed on uterine caruncles. Compared to NOR, RET samples showed a lower CD14+/CD16+ expression (P < 0.05) in caruncle monocyte/macrophage population. As opposed to NOR, RET further demonstrated greater expression of anti-inflammatory M2 macrophage associated genes CD206, C-type lectin domain family 7 member A (CLEC7A), and RNASE6. In addition, caruncles from RET showed decreased signal transducer and activator of transcription 3 (STAT3) activation, an important promoter of proteolytic activity, compared to NOR. Our studies demonstrate that there is an overall lower number of macrophage populations in the caruncle of cows with RET placenta and these are polarized towards M2 phenotype. Excessive accumulation of M2 macrophages may lead to reduced trafficking of immune cells into the caruncle thus impairing the inflammatory, phagocytic and proteolytic processes that lead to placental expulsion.

Prostate cancer induces C/EBPβ expression in surrounding epithelial cells which relates to tumor aggressiveness and patient outcome.

Implantation of rat prostate cancer cells into the normal rat prostate results in tumor-stimulating adaptations in the tumor-bearing organ. Similar changes are seen in prostate cancer patients and they are related to outcome. One gene previously found to be upregulated in the non-malignant part of tumor-bearing prostate lobe in rats was the transcription factor CCAAT/enhancer-binding protein-β (C/EBPβ). To explore this further, we examined C/EBPβ expression by quantitative RT-PCR, immunohistochemistry, and Western blot in normal rat prostate tissue surrounding slow-growing non-metastatic Dunning G, rapidly growing poorly metastatic (AT-1), and rapidly growing highly metastatic (MatLyLu) rat prostate tumors-and also by immunohistochemistry in a tissue microarray (TMA) from prostate cancer patients managed by watchful waiting. In rats, C/EBPβ mRNA expression was upregulated in the surrounding tumor-bearing prostate lobe. In tumors and in the surrounding non-malignant prostate tissue, C/EBPβ was detected by immunohistochemistry in some epithelial cells and in infiltrating macrophages. The magnitude of glandular epithelial C/EBPβ expression in the tumor-bearing prostates was associated with tumor size, distance to the tumor, and metastatic capacity. In prostate cancer patients, high expression of C/EBPβ in glandular epithelial cells in the surrounding tumor-bearing tissue was associated with accumulation of M1 macrophages (iNOS+) and favorable outcome. High expression of C/EBPβ in tumor epithelial cells was associated with high Gleason score, high tumor cell proliferation, metastases, and poor outcome. This study suggest that the expression of C/EBP-beta, a transcription factor mediating multiple biological effects, is differentially expressed both in the benign parts of the tumor-bearing prostate and in prostate tumors, and that alterations in this may be related to patient outcome.

Enhancement of Antitumor Immunity Using Dendritic Cells Combined with Lenalidomide and Programmed Death Ligand-1 Blockade in Multiple Myeloma Mouse Model

Background: The therapeutic efficacy of dendritic cell (DC)-based immunotherapy may be potentiated in combination with other anticancer therapies that enhance DC function by modulating immune responses and the tumor microenvironment. In this study, we investigated the efficacy of DC vaccination in combination with lenalidomide and programmed death (PD)-1 blockade in a model of murine myeloma.Materials &Methods: MOPC-315 cell lines were injected subcutaneously to establish myeloma-bearing mice and the following five test groups were established: PBS control, DCs, DCs + lenalidomide, DCs + PD-1 blockade, and DCs + lenalidomide + PD-1 blockade. On day 0, mice were injected subcutaneously in the right flank with 5 × 105 MOPC-315 cells in a volume of 0.1 mL. After tumor growth, lenalidomide (0.5 mg/kg/day) was administrated orally once a day for 25 days with a 3-day break after the first 11-day dosing period. Each dose of DCs (1 × 106/mouse) was injected subcutaneously into the left flank of BALB/c mice in a volume of 0.1 mL PBS on days 11, 15, 25, and 29; anti-PD-1 (250 µg/mouse) was injected intraperitoneally in a 0.1 mL volume on the same days as DC vaccination.Results: This study showed that DC vaccination combined to the lenalidomide and PD-1 blockade regiment further inhibited MM tumor growth, consequently prolonging the survival of tumor-bearing mice. These effects were associated with a significant increase in IFN-γ-secreting splenocytes against MOPC-315 and YAC-1 cells, as well as the significantly increased the number of effector CD4+ T cells, CD8+ T cells, effector memory T cells, effector NK cells, and M1 macrophages while effectively discouraging suppressor cells, such as myeloid-derived suppressor cells (MDSCs), M2 macrophages, and regulatory T cells (Tregs) in the systemic immune compartment. These findings evidence the induction of systemic immune response potentially being able to eradicate disseminated diseases. In this study, DCs combined with lenalidomide and PD-1 blockade also heightened the anti-myeloma cell mediate immunity by inducing the Th1 polarization, as evidenced by the high-level production of IFN-γ in the spleen, and by suppressing Th2 immune responses, as evidenced by the low-level production of IL-10 and TGF-β in the spleen and tumor site. Tregs, MDSCs, and M2 macrophages are major elements molding the potent immunosuppressive environment in tumor tissues. The inhibition of Treg, MDSC, and M2 macrophage accumulation in the spleen should further contribute to effective anti-myeloma cell mediate immunity in the systemic immune compartment by reciprocally activating DCs or cytotoxic T lymphocytes.Conclusion: This study suggests that lenalidomide plus PD-1 blockade treatment synergistically enhances the efficacy of DC vaccination in a murine myeloma model by inhibiting the generation of immunosuppressive cells and the Th2 immune response and enhancing effector cells and the Th1 immune responses. We hereby propose a framework for a more efficacious DC-based vaccination strategy against MM with the combination of immunomodulatory drug lenalidomide and anti-PD-1 antibody. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Radiation-Induced Pulmonary Fibrosis (RIPF) Is Mediated By Interleukin 13 Receptor Subunit Alpha 2 (IL13Ra2)

Radiation induced pulmonary fibrosis (RIPF) is a late toxicity of thoracic radiotherapy. Type 2 immune responses driven by IL-13 have recently been implicated in the pathogenesis of RIPF, in part through interaction with the heterodimeric IL-13Ra1/IL4Rα receptor complex. Although its mechanism is not clearly understood, IL-13Rα2 is a transmembrane receptor thought to function as a decoy receptor for IL-13. In this study, we sought to characterize the role of IL13Rα2. Recent evidence implicates IL-13Rα2 in signaling of chitinases produced in type 2 inflammatory responses, which play a role in RIPF initiation and progression. The 10 week old C57BL6/NcR (WT) and IL13Ra2 deficient [C57BL/6-[Ko]IL13Rα2 (IL-13Rα2-/-)] mice were exposed to 5 daily 6 Gy fractions of thoracic radiation (RT). Lung tissue was collected at 16 weeks after RT. Collagen accumulation was measured with hydroxyproline content and accumulation of total and alternatively activated (M2) macrophages was assessed with immunohistochemistry. Bone marrow derived macrophages (BMDM) from each strain were treated with IL-13. Gene expression in RNA isolated from lung tissue and BMDM was evaluated with quantitative RT-PCR and PCR array. Deficiency of IL13Rα2 significantly extended survival significantly after thoracic irradiation compared to WT mice (19 vs. 21 weeks, p=0.03 log rank). WT mice developed confluence of fibrosis, increased hydroxyproline content, and accumulation of M2 macrophages after RT. IL-13Ra2-/- mice had no observable increase in hydroxyproline content after RT and had no evidence of macrophage accumulation when compared to unirradated controls. The expression of fibrosis-associated cytokines and chemokines (Ccl2, Timp1, and IL-6) was suppressed in irradiated IL-13Ra2-/-lung compared to WT. WT macrophages isolated from whole lung expressed increased levels both soluble and membrane bound isoforms of IL13Rα2 after radiation. BMDM from IL-13Ra2-/- mice treated with IL-13 had suppressed expression of Ccl11, Timp1, and Pdgfb compared to BMDM from WT mice. Our data show that IL-13Rα2 deficiency attenuates RIPF, and that this protection is accompanied by decreased accumulation of macrophages and altered production of inflammatory cytokines and chemokines by macrophages. Whether the protective benefit of IL-13Rα2 deficiency is attributable to absent decoy-receptor function is currently being investigated.

Deficiency in interleukin‐10 production by M2 macrophages in eosinophilic chronic rhinosinusitis with nasal polyps

M2 macrophages are characterized by high interleukin-10 (IL-10) expression and are critical for resolving inflammation. Although increased accumulation of M2 macrophages has been demonstrated in chronic rhinosinusitis with nasal polyps (CRSwNP), particularly the eosinophilic type, their functional relevance in CRSwNP remains poorly understood. M1 and M2 macrophages and IL-10 expression in sinonasal tissues were detected by double-immunofluorescence staining. THP-1 cells, a human monocytic leukemia cell line, were stimulated with various cytokines to study macrophage polarization and IL-10 expression. Polyp size, computed tomography (CT) scans, and symptom severity were scored. Compared with numbers in control tissues, the numbers of total CD68+ macrophages, interferon regulatory factor 5-positive and CD68+ M1 macrophages, and CD163+ CD68+ and CD206+ CD68+ M2 macrophages were increased in both eosinophilic and non-eosinophilic polyps. However, compared with non-eosinophilic polyps, eosinophilic polyps contained fewer M1 macrophages and more M2 macrophages. Consistent with this, the M1/M2 macrophage ratio was increased in non-eosinophilic polyps, whereas it decreased in eosinophilic polyps. Strikingly, the numbers of IL-10+ CD68+ macrophages and the percentage of IL-10+ CD68+ macrophages relative to the total number of macrophages were decreased in eosinophilic polyps, despite the upregulation of M2 macrophages in this type of polyp. The number of IL-10+ CD68+ M2 macrophages correlated negatively with total symptoms scores, polyp sizes, total CT scores, and the total number of inflammatory cells in patients with eosinophilic CRSwNP. Poly I:C downregulated IL-10 expression in M2 macrophages differentiated from THP-1 cells in vitro. Impaired IL-10 production by M2 macrophages may contribute to sustained inflammation in eosinophilic CRSwNP.vv.

Obesity-induced insulin resistance and macrophage infiltration of the adipose tissue: A vicious cycle.

Obesity-induced insulin resistance and macrophage infiltration of the adipose tissue: A vicious cycle.

Inhibition of IL-18 reduces renal fibrosis after ischemia-reperfusion

Acute kidney injury induced by ischemia-reperfusion injury (IRI) is a high risk factor in the progression towards chronic kidney disease, which is featured by renal interstitial fibrosis. Interleukin (IL)-18 is produced by T cells and macrophages and has been involved in the pathophysiology of IRI. However, the role of IL-18 in IRI-induced renal fibrosis is poorly understood. In the present study, we showed that interleukin (IL)-18 was significantly up-regulated after IRI stress. Mice treated with IL-18 Bp, a natural inhibitor of IL-18, presented less severe fibrotic response in the kidneys following IRI compared with vehicle-treated mice. Inhibition of IL-18 decreased myofibroblasts formation in the kidneys in response to IRI, which was associated with reduction of fibronectin and collagenⅠproteins. Moreover, inhibition of IL-18 impaired infiltration of CD3+ T cells and F4/80+ macrophages in the kidneys of mice after IRI. Treatment with IL-18 Bp reduces the levels of profibrotic molecules in the kidneys of mice following IRI. Finally, administration of IL-18 Bp impedes the transition of M2 macrophages to myofibroblasts and suppressed the accumulation of bone marrow-derived M2 macrophages. Adoptive transfer of M2 macrophages abolished the anti-fibrotic effect of IL-18 Bp. In summary, our results suggest that IL-18 plays an important role in the progression of IRI-induced renal fibrosis via modulating inflammation cells infiltration, the expression of inflammatory cytokines and chemokines, and the transition of bone marrow-derived M2 macrophages to myofibroblasts.