Recruitment Of Myeloid Cells Research Articles

Polypropylene mesh implantation for hernia repair causes myeloid cell-driven persistent inflammation.

Polypropylene meshes that are commonly used for inguinal hernia repair may trigger granulomatous foreign body reactions. Here, we show that asymptomatic patients display mesh-associated inflammatory granulomas long after surgery, which are dominated by monocyte-derived macrophages expressing high levels of inflammatory activation markers. In mice, mesh implantation by the onlay technique induced rapid and strong myeloid cell accumulation, without substantial attenuation for up to 90 days. Myeloid cells segregated into distinct macrophage subsets with separate spatial distribution, activation profiles, and functional properties, showing a stable inflammatory phenotype in the tissue surrounding the biomaterial and a mixed, wound-healing phenotype in the surrounding stromal tissue. Protein mass spectrometry confirmed the inflammatory nature of the foreign body reaction, as characterized by cytokines, complement activation, and matrix-modulating factors. Moreover, immunoglobulin deposition increased over time around the implant, arguing for humoral immune responses in association with the cell-driven inflammation. Intravital multiphoton microscopy revealed a high motility and continuous recruitment of myeloid cells, which is partly dependent on the chemokine receptor CCR2. CCR2-dependent macrophages are particular drivers of fibroblast proliferation. Thus, our work functionally characterizes myeloid cell-dependent inflammation following mesh implantation, thereby providing insights into the dynamics and mechanisms of foreign body reactions to implanted biomaterials.

Blastemal progenitors modulate immune signaling during early limb regeneration.

Blastema formation, a hallmark of limb regeneration, requires proliferation and migration of progenitors to the amputation plane. Although blastema formation has been well described, the transcriptional programs that drive blastemal progenitors remain unknown. We transcriptionally profiled dividing and non-dividing cells in regenerating stump tissues, as well as the wound epidermis, during early axolotl limb regeneration. Our analysis revealed unique transcriptional signatures of early dividing cells and, unexpectedly, repression of several core developmental signaling pathways in early regenerating stump tissues. We further identify an immunomodulatory role for blastemal progenitors through interleukin 8 (IL-8), a highly expressed cytokine in subpopulations of early blastemal progenitors. Ectopic il-8 expression in non-regenerating limbs induced myeloid cell recruitment, while IL-8 knockdown resulted in defective myeloid cell retention during late wound healing, delaying regeneration. Furthermore, the il-8 receptor cxcr-1/2 was expressed in myeloid cells, and inhibition of CXCR-1/2 signaling during early stages of limb regeneration prevented regeneration. Altogether, our findings suggest that blastemal progenitors are active early mediators of immune support, and identify CXCR-1/2 signaling as an important immunomodulatory pathway during the initiation of regeneration.

CCL15 Recruits Suppressive Monocytes to Facilitate Immune Escape and Disease Progression in Hepatocellular Carcinoma.

Chemokines play a key role in orchestrating the recruitment and positioning of myeloid cells within the tumor microenvironment. However, the tropism regulation and functions of these cells in hepatocellular carcinoma (HCC) are not completely understood. Herein, by scrutinizing the expression of all chemokines in HCC cell lines and tissues, we found that CCL15 was the most abundantly expressed chemokine in human HCC. Further analyses showed that CCL15 expression was regulated by genetic, epigenetic, and microenvironmental factors, and negatively correlated with patient clinical outcome. In addition to promoting tumor invasion in an autocrine manner, CCL15 specifically recruited CCR1+ cells toward HCC invasive margin, approximately 80% of which were CD14+ monocytes. Clinically, a high density of marginal CCR1+ CD14+ monocytes positively correlated with CCL15 expression and was an independent index for dismal survival. Functionally, these tumor-educated monocytes directly accelerated tumor invasion and metastasis through bursting various pro-tumor factors and activating signal transducer and activator of transcription 1/3, extracellular signal-regulated kinase 1/2, and v-akt murine thymoma viral oncogene homolog signaling in HCC cells. Meanwhile, tumor-derived CCR1+ CD14+ monocytes expressed significantly higher levels of programmed cell death-ligand 1, B7-H3, and T-cell immunoglobulin domain and mucin domain-3 that may lead to immune suppression. Transcriptome sequencing confirmed that tumor-infiltrating CCR1+ CD14+ monocytes were reprogrammed to upregulate immune checkpoints, immune tolerogenic metabolic enzymes (indoleamine and arginase), inflammatory/pro-angiogenic cytokines, matrix remodeling proteases, and inflammatory chemokines. Orthotopic animal models confirmed that CCL15-CCR1 axis forested an inflammatory microenvironment enriched with CCR1+ monocytes and led to increased metastatic potential of HCC cells. Conclusion: A complex tumor-promoting inflammatory microenvironment was shaped by CCL15-CCR1 axis in human HCC. Blockade of CCL15-CCR1 axis in HCC could be an effective anticancer therapy.

Interleukin-22 promotes tumor angiogenesis.

TH17 cells play important yet complex roles in cancer development and progression. We previously reported that TH17 cells and IL-17 mediate resistance to anti-VEGF therapy by inducing recruitment of immunosuppressive and proangiogenic myeloid cells to the tumor microenvironment. Here, we demonstrate that IL-22, a key effector cytokine expressed by TH17 cells, directly acts on endothelial cells to promote tumor angiogenesis. IL-22 induces endothelial cell proliferation, survival, and chemotaxis in vitro and neovascularization in an ex vivo mouse choroid explant model. Blockade of IL-22, with a neutralizing antibody, significantly inhibits tumor growth associated with reduced microvascular density. No synergistic effect of IL-22 with VEGF was observed. These results identify IL-22 as a potential therapeutic target for blocking tumor angiogenesis.

Integrin CD11b activation drives anti-tumor innate immunity

Myeloid cells are recruited to damaged tissues where they can resolve infections and tumor growth or stimulate wound healing and tumor progression. Recruitment of these cells is regulated by integrins, a family of adhesion receptors that includes integrin CD11b. Here we report that, unexpectedly, integrin CD11b does not regulate myeloid cell recruitment to tumors but instead controls myeloid cell polarization and tumor growth. CD11b activation promotes pro-inflammatory macrophage polarization by stimulating expression of microRNA Let7a. In contrast, inhibition of CD11b prevents Let7a expression and induces cMyc expression, leading to immune suppressive macrophage polarization, vascular maturation, and accelerated tumor growth. Pharmacological activation of CD11b with a small molecule agonist, Leukadherin 1 (LA1), promotes pro-inflammatory macrophage polarization and suppresses tumor growth in animal models of murine and human cancer. These studies identify CD11b as negative regulator of immune suppression and a target for cancer immune therapy.

How KEAP1 Deletion in Single Cell Mitigates Sickle Cell Pathology

Sickle cell disease (SCD) is one of the most frequent blood disorders caused by a point mutation of β-globin gene. In the pathology of SCD, we often encounter contributions of vascular and cellular components. The activation of vascular endothelial cells promotes adhesion of inflammatory cells and vaso-occlusion responsible for low blood flow, hypoxemia and organ damages. Hemolysis triggers activation and recruitment of myeloid cells to the inflammation site, further sustaining the inflammatory process. Recent progress has created the need to examine targeted induction of the KEAP1-NRF2 system as a potential therapeutic tool for the SCD. We have demonstrated that genetic as well as pharmacological induction of NRF2 in SCD prevent organ damages and suppress inflammation, although it still remains unclear how the NRF2 induction modifies the pathology or which type of cells contribute to the NRF2-mediated improvement of SCD. To address these questions, we conducted targeted deletion of Keap1 gene and induced expression of Nrf2 gene in endothelial cells and macrophages/monocytes using Tie1-Cre and LysM-Cre system, respectively. We found that SCD::Keap1F/F::Tie1-Cre mice (NRF2-increase in endothelial cells) showed low hematocrit and low platelet counts. While severe infiltration of monocytes, lymphocytes and macrophages was observed in the lungs of SCD mice, such phenotype was diminished in the SCD::Keap1F/F::Tie1-Cre mice. In the compound mutant mice, livers showed fewer necrotic zones and less to none accumulation of free iron. On the contrary, imaging MS (iMScope) analyses revealed decrease of free heme in livers and spleens in SCD::Keap1F/F::LysM-Cre mice (NRF2-increase in macrophages/monocytes). Furthermore, upregulations of most known Nrf2-dependent pathways and other yet-to-be-defined pathways are found in an RNA-sequencing analysis of the mice. Importantly, NRF2 induction enhanced heme catabolism and decreases the plasma level of free heme and the quantity of accumulated free heme in the SCD::Keap1F/F::LysM-Cre mouse tissues, whereas the change is not obvious in SCD::Keap1F/F::Tie1-Cre mice. On the contrary, vascular leakage of fluids was decreased in the SCD::Keap1F/F::Tie1-Cre mice. Thus, our results demonstrate that cell-lineage-specific activation of Nrf2 does inhibit the inflammation and organ injury with some particularities to each genotype. DisclosuresNo relevant conflicts of interest to declare.

Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM‐1

Atherosclerosis is a chronic disease characterized by vascular lipid retention and inflammation, and pattern recognition receptors (PRRs) are important contributors in early stages of the disease. Given the implication of the intracellular PRR nucleotide-binding oligomerization domain 1 (NOD1) in cardiovascular diseases, we investigated its contribution to early atherosclerosis. We evidenced NOD1 induction in atherosclerotic human and mouse tissues, predominantly in vascular endothelial cells. Accordingly, NOD1 genetic inactivation in Apoe-/- mice reduced not only atherosclerosis burden, but also monocyte and neutrophil accumulation in atheromata. Of note, in the presence of either peptidoglycan or oxidized LDLs, endothelial NOD1 triggered VCAM-1 up-regulation through the RIP2-NF-κB axis in an autocrine manner, enhancing firm adhesion of both sets of myeloid cells to the inflamed micro- and macrovasculature in vivo. Our data define a major proatherogenic role for endothelial NOD1 in early leukocyte recruitment to the athero-prone vasculature, thus introducing NOD1 as an innovative therapeutic target and potential prognostic molecule.-González-Ramos, S., Paz-García, M., Rius, C., del Monte-Monge, A., Rodríguez, C., Fernández-García, V., Andrés, V., Martínez-González, J., Lasunción, M. A., Martín-Sanz, P., Soehnlein, O., Boscá, L. Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM-1.

Electrochemotherapy with cisplatin increases survival and induces immunogenic responses in murine models of lung cancer and colorectal cancer

Electrochemotherapy is an emerging treatment modality for cancer patients which can effectively reduce tumour burden and induce immunogenic cell death. Electrochemotherapy is most commonly used with bleomycin as the drug of choice, here we examine the efficacy of electrochemotherapy with cisplatin. Electrochemotherapy with cisplatin was found to effectively reduce tumour growth in a range of murine models and induce significant intratumoural recruitment of myeloid and humoral immune cells. Following the observations of immune system mobilisation, we have shown an ability of electrochemotherapy to reduce metastatic potential as determined by tumour burden in the lung, and to exert an abscopal effect by reducing growth at distal untreated secondary tumours.

The Host Microbiota Contributes to Early Protection Against Lung Colonization by Mycobacterium tuberculosis.

Tuberculosis (TB), caused by the airborne bacterial pathogen Mycobacterium tuberculosis, remains a major source of morbidity and mortality worldwide. So far, the study of host-pathogen interactions in TB has mostly focused on the physiology and virulence of the pathogen, as well as, on the various innate and adaptive immune compartments of the host. Microbial organisms endogenous to our body, the so-called microbiota, interact not only with invading pathogens, but also with our immune system. Yet, the impact of the microbiota on host defense against M. tuberculosis remains poorly understood. In order to address this question, we adapted a robust and reproducible mouse model of microbial dysbiosis based on a combination of wide-spectrum antibiotics. We found that microbiota dysbiosis resulted in an increased early colonization of the lungs by M. tuberculosis during the first week of infection, correlating with an altered diversity of the gut microbiota during this time period. At the cellular level, no significant difference in the recruitment of conventional myeloid cells, including macrophages, dendritic cells and neutrophils, to the lungs could be detected during the first week of infection between microbiota-competent and -deficient mice. At the molecular level, microbiota depletion did not impact the global production of pro-inflammatory cytokines, such as interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)-1β in the lungs. Strikingly, a reduced number of mucosal-associated invariant T (MAIT) cells, a population of innate-like lymphocytes whose development is known to depend on the host microbiota, was observed in the lungs of the antibiotics-treated animals after 1week of infection. These cells produced less IL-17A in antibiotics-treated mice. Notably, dysbiosis correction through the inoculation of a complex microbiota in antibiotics-treated animals reversed these phenotypes and improved the ability of MAIT cells to proliferate. Altogether, our results demonstrate that the host microbiota contributes to early protection of lung colonization by M. tuberculosis, possibly through sustaining the function(s) of MAIT cells. Our study calls for a better understanding of the impact of the microbiota on host-pathogen interactions in TB. Ultimately, this study may help to develop novel therapeutic approaches based on the use of beneficial microbes, or components thereof, to boost anti-mycobacterial immunity.

Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells

ABSTRACT The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as in situ cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer in vitro and in vivo. Previously, we have shown that especially high single doses (20 Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes in vivo. Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20 Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses – at least in models of triple-negative breast cancer.

34 - The role of myeloperoxidase in hyperoxia-induced bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is the most common chronic pulmonary disease of premature neonates. While ventilation with supplemental O2 is required for development and survival of the premature neonate, it increases the risk of BPD. Both hyperoxia-induced oxidative stress and transient hypoxia resulting from apneic events increase recruitment and activation of myeloid cells to the lungs. Activated myeloid cells release myeloperoxidase (MPO), which oxidizes chloride ions to hypochlorous acid causing oxidative damage and cell death. If myeloid cell recruitment, activation, and MPO release contribute to the pathogenesis of BPD, then inhibiting MPO oxidant production should protect against hyperoxia-induced lung injury. N-acetyl lysyltyrosylcysteine amide (KYC) is a tripeptide inhibitor of MPO that prevents the generation of toxic oxidants by shuttling high energy peroxy radicals in the iron-heme active site into the glutathione pathway. In order to test the hypothesis that inhibiting MPO prevents hyperoxia-induced lung damage, Sprague-Dawley neonatal rat pups were exposed to ten days (P1-P10) of hyperoxia (>90% O2) and injected twice daily with either 5 mg/kg KYC or vehicle control (PBS). KYC treatment reduced myeloid cell counts and MPO and Cl-Tyr levels in the lungs of hyperoxic neonatal pups. Additionally, KYC-treated pups had increased microvascular and alveolar complexity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels. These data provide evidence that myeloid cell recruitment and activation and MPO-induced oxidative damage are significant contributors to hyperoxia-induced lung injury in the developing lungs, and that KYC is an effective inhibitor of MPO-dependent oxidative damage to neonatal lungs.

Persistence of Systemic Murine Norovirus Is Maintained by Inflammatory Recruitment of Susceptible Myeloid Cells

Viral persistence can contribute to chronic disease and promote virus dissemination. Prior work demonstrated that timely clearance of systemic murine norovirus (MNV) infection depends on cell-intrinsic type I interferon responses and adaptive immunity. We now find that the capsid of the systemically replicating MNV strain CW3 promotes lytic cell death, release of interleukin-1α, and increased inflammatory cytokine release. Correspondingly, inflammatory monocytes and neutrophils are recruited to sites of infection in a CW3-capsid-dependent manner. Recruited monocytes and neutrophils are subsequently infected, representing a majority of infected cells invivo. Systemic depletion of inflammatory monocytes or neutrophils from persistently infected Rag1-/- mice reduces viral titers in a tissue-specific manner. These data indicate that the CW3 capsid facilitates lytic cell death, inflammation, and recruitment of susceptible cells to promote persistence. Infection of continuously recruited inflammatory cells may be a mechanism of persistence broadly utilized by lytic viruses incapable of establishing latency.

Pathologic Fibroblasts in Idiopathic Subglottic Stenosis Amplify Local Inflammatory Signals

To characterize the phenotype and function of fibroblasts derived from airway scar in idiopathic subglottic stenosis (iSGS) and to explore scar fibroblast response to interleukin 17A (IL-17A). Basic science. Laboratory. Primary fibroblast cell lines from iSGS subjects, idiopathic pulmonary fibrosis subjects, and normal control airways were utilized for analysis. Protein, molecular, and flow cytometric techniques were applied in vitro to assess the phenotype and functional response of disease fibroblasts to IL-17A. Mechanistically, IL-17A drives iSGS scar fibroblast proliferation ( P < .01), synergizes with transforming growth factor ß1 to promote extracellular matrix production (collagen and fibronectin; P = .04), and directly stimulates scar fibroblasts to produce chemokines (chemokine ligand 2) and cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor) critical to the recruitment and differentiation of myeloid cells ( P < .01). Glucocorticoids abrogated IL-17A-dependent iSGS scar fibroblast production of granulocyte-macrophage colony-stimulating factor ( P = .02). IL-17A directly drives iSGS scar fibroblast proliferation, synergizes with transforming growth factor ß1 to promote extracellular matrix production, and amplifies local inflammatory signaling. Glucocorticoids appear to partially abrogate fibroblast-dependent inflammatory signaling. These results offer mechanistic support for future translational study of clinical reagents for manipulation of the IL-17A pathway in iSGS patients.

Ibuprofen supports macrophage differentiation, T cell recruitment, and tumor suppression in a model of postpartum breast cancer

BackgroundWomen diagnosed with breast cancer within 5 years postpartum (PPBC) have poorer prognosis than age matched nulliparous women, even after controlling for clinical variables known to impact disease outcomes. Through rodent modeling, the poor prognosis of PPBC has been attributed to physiologic mammary gland involution, which shapes a tumor promotional microenvironment through induction of wound-healing-like programs including myeloid cell recruitment. Previous studies utilizing immune compromised mice have shown that blocking prostaglandin synthesis reduces PPBC tumor progression in a tumor cell extrinsic manner. Given the reported roles of prostaglandins in myeloid and T cell biology, and the established importance of these immune cell populations in dictating tumor growth, we investigate the impact of involution on shaping the tumor immune milieu and its mitigation by ibuprofen in immune competent hosts.MethodsIn a syngeneic (D2A1) orthotopic Balb/c mouse model of PPBC, we characterized the impact of mammary gland involution and ibuprofen treatment on the immune milieu in tumors and draining lymph nodes utilizing flow cytometry, multiplex IHC, lipid mass spectroscopy and cytokine arrays. To further investigate the impact of ibuprofen on programming myeloid cell populations, we performed RNA-Seq on in vivo derived mammary myeloid cells from ibuprofen treated and untreated involution group mice. Further, we examined direct effects of ibuprofen through in vitro bone marrow derived myeloid cell cultures.ResultsTumors implanted into the mammary involution microenvironment grow more rapidly and display a distinct immune milieu compared to tumors implanted into glands of nulliparous mice. This milieu is characterized by increased presence of immature monocytes and reduced numbers of T cells and is reversed upon ibuprofen treatment. Further, ibuprofen treatment enhances Th1 associated cytokines as well as promotes tumor border accumulation of T cells. Safety studies demonstrate ibuprofen does not impede gland involution, impact subsequent reproductive success, nor promote auto-reactivity as detected through auto-antibody and naïve T cell priming assays.ConclusionsIbuprofen administration during the tumor promotional microenvironment of the involuting mammary gland reduces overall tumor growth and enhances anti-tumor immune characteristics while avoiding adverse autoimmune reactions. In sum, these studies implicate beneficial prophylactic use of ibuprofen during the pro-tumorigenic window of mammary gland involution.

2-Arachidonoylglycerol mobilizes myeloid cells and worsens heart function after acute myocardial infarction.

Myocardial infarction (MI) leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischaemic myocardium. These myeloid cells originate from haematopoietic precursors in the bone marrow and are rapidly mobilized in response to MI. We aimed to determine whether endocannabinoid signalling is involved in myeloid cell mobilization and cardiac recruitment after ischaemia onset. Intravenous administration of endocannabinoid 2-arachidonoylglycerol (2-AG) into wild type (WT) C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor 2 knockout mice. In response to MI induced in WT mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid 2-AG 24 h after infarction, but no changes in anandamide, palmitoylethanolamide, and oleoylethanolamide. This was a consequence of an increased expression of 2-AG synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase (MAGL) in infarcted hearts, as determined by quantitative RT-PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of MAGL with JZL184 and thus increased systemic 2-AG levels in WT mice subjected to MI resulted in elevated cardiac CXCL1, CXCL2, and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts 24 h after infarction compared with vehicle-treated mice. Increased post-MI inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to 21 days. Likewise, JZL184-administration in a myocardial ischaemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size. These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function.

Turn Back the TIMe: Targeting Tumor Infiltrating Myeloid Cells to Revert Cancer Progression.

Tumor cells frequently produce soluble factors that favor myelopoiesis and recruitment of myeloid cells to the tumor microenvironment (TME). Consequently, the TME of many cancer types is characterized by high infiltration of monocytes, macrophages, dendritic cells and granulocytes. Experimental and clinical studies show that most myeloid cells are kept in an immature state in the TME. These studies further show that tumor-derived factors mold these myeloid cells into cells that support cancer initiation and progression, amongst others by enabling immune evasion, tumor cell survival, proliferation, migration and metastasis. The key role of myeloid cells in cancer is further evidenced by the fact that they negatively impact on virtually all types of cancer therapy. Therefore, tumor-associated myeloid cells have been designated as the culprits in cancer. We review myeloid cells in the TME with a focus on the mechanisms they exploit to support cancer cells. In addition, we provide an overview of approaches that are under investigation to deplete myeloid cells or redirect their function, as these hold promise to overcome resistance to current cancer therapies.

PD-1 deficiency is not sufficient to induce myeloid mobilization to the brain or alter the inflammatory profile during chronic neurodegeneration

Innate immune activation is a major driver of neurodegenerative disease and immune regulatory pathways could be potential targets for therapeutic intervention. Recently, Programmed cell death-1 (PD-1) immune checkpoint inhibition has been proposed to mount an IFN-γ-dependent systemic immune response, leading to the recruitment of peripheral myeloid cells to the brain and neuropathological and functional improvements in mice with Alzheimer’s disease-like β-amyloid pathology. Here we investigate the impact of PD-1 deficiency on murine prion disease (ME7 strain), a model of chronic neurodegeneration. Although PD-1 was found to be increased in the brain of prion mice, the absence of PD-1 did not cause myeloid cell infiltration into the brain or major changes in the inflammatory profile. However, we observed a slight exacerbation of the behavioural phenotype of ME7 mice upon PD-1 deficiency. These results do not support the possibility of using immune checkpoint blockade as a therapeutic strategy in neurodegenerative disease.

Abstract 5075: Single-cell RNAseq of human prostate cancer-associated fibroblasts reveals distinct subpopulations that may promote inflammatory cell recruitment to the tumor microenvironment

Abstract Introduction: Carcinoma-associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment that can promote tumorigenesis in the prostate. The full extent of heterogeneity in CAF and its consequences are not understood. A more detailed description of the prostate tumor microenvironment could aid in overcoming major obstacles within the field, including defining indolent versus aggressive disease and uncovering novel therapeutic and disease-stabilizing pathways in a step toward personalized therapy. Methods: These studies utilized Fluidgm's C1 Single-Cell Auto Prep System and library construction in preparation for single-cell (sc) RNAseq of human prostate CAF. Bioinformatics analysis was used to generate cell clusters. Further examination of the differentially expressed (DE) gene profiles for each cell cluster was performed to elucidate the function and means of communication among clusters and with other cells, particularly those within the immune/inflammatory network. Communication between CAF and immune cells was also analyzed by in vivo testing of tissue recombinants generated with CAF or normal fibroblasts. The resultant tumors were analyzed using FACS to assess the effects of the CAF on inflammatory cell recruitment. Results: Principal component analysis and unsupervised cell clustering allows for visualization of at least three CAF subpopulations, each with distinct DE gene profiles. Further investigation into the DE genes for each cluster suggests these subpopulations have unique functions within the tumor microenvironment, including a role in immune/inflammatory cell recruitment. For example, one cluster expresses high levels of CCL11 and CXCL1, suggesting a role in the recruitment of neutrophils, myeloid cells, or other inflammatory cells, while a second cluster has decreased expression of chemokines but elevated expression of extracellular matrix-related genes such as COL1A1, MMP16, and FN1, suggesting an alternative, potentially structural, role for this CAF cluster. Previous analysis in vivo demonstrated that CAF drive tumorigenesis in a reporter epithelium. The speculated role of CAF in immune cell recruitment was supported by increased recruitment of myeloid cells, including macrophages and granulocytes, to prostate tissues in the presence of CAF compared to normal fibroblasts. Conclusions: These studies demonstrate that CAF contain a limited number of distinct subpopulations of cells; the relatively small number of identified CAF clusters allows for feasible biologic study and mathematical modeling. Some CAF highly express extracellular mitotic and chemotactic signaling molecules that may be involved in the recruitment of inflammatory cells as well as direct growth regulation of the tumor by promoting an M2-dominant microenvironment. Citation Format: Renee E. Vickman, Meaghan M. Broman, Nadia M. Atallah, Ayu Sudyanti, Omar E. Franco, Timothy L. Ratliff, Simon W. Hayward. Single-cell RNAseq of human prostate cancer-associated fibroblasts reveals distinct subpopulations that may promote inflammatory cell recruitment to the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5075.

Early Targeting of L-Selectin on Leukocytes Promotes Recovery after Spinal Cord Injury, Implicating Novel Mechanisms of Pathogenesis.

L-selectin, a lectin-like receptor on all leukocyte classes, functions in adhesive and signaling roles in the recruitment of myeloid cells from the blood to sites of inflammation. Here, we consider L-selectin as a determinant of neurological recovery in a murine model of spinal cord injury (SCI). Spinal cord-injured, L-selectin knock-out (KO) mice (male) showed improved long-term recovery with greater white matter sparing relative to wild-type (WT) mice and reduced oxidative stress in the injured cord at 72 h post-SCI. There was a partial and transient reduction in accumulation of neutrophils in the injured spinal cords of KOs at 24 h post-injury. To complement these findings with KO mice, we sought a pharmacologic means for lowering L-selectin levels. We found that diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induced the shedding of L-selectin from the cell surface of myeloid subsets, specifically neutrophils and non-classical monocytes, in the blood and the injured spinal cord. Diclofenac administration to injured WT mice enhanced neurological recovery to a level comparable to that of KOs but did not improve recovery in KOs. While diclofenac treatment had no effect on myeloid cell accumulation, there was a reduction in oxidative stress at 72 h post-SCI. These findings implicate L-selectin in secondary pathogenesis beyond a role in leukocyte recruitment and raise the possibility of repurposing diclofenac for the treatment of SCI.

Abstract 2567: Novel therapeutic interventions to alleviate CAR T cell-induced cytokine release syndrome

Abstract CD19 chimeric antigen receptor (CAR) therapy is highly effective for some refractory B cell malignancies. Its broad implementation is limited in part by the occurrence of cytokine release syndrome (CRS), which is characterized by fever, hypotension and respiratory insufficiency that may require intensive care to overcome. CRS is associated with elevated serum cytokines, including interleukin-6 (IL-6), the blockade of which abates CRS in some but not all patients. The biological mechanisms involved in the pathogenesis of CRS are currently unknown, including the specific role of CAR T cells, tumor cells and other host cells. The development of relevant animal models is imperative in order to study the biology of CRS, analyze the role of different cell populations and ultimately identify interventions that not only treat but prevent CRS without reducing the therapeutic efficacy of CAR T cells. Here we present a novel mouse model in which CD19 CAR-induced CRS is predictably and reproducibly elicited and mirrors the rapid onset and acuteness of clinical CRS. Symptoms present within 24 hours post CAR T cell administration, including rapid weight loss and eventual mortality. Survival correlates negatively with pro-inflammatory human (T cell-derived) and murine (host-derived) cytokine levels. A comparison between the cytokine fingerprint in patients with CRS and our mouse model revealed remarkable similarity between the two profiles. Importantly, IL-6 is elevated, as seen in patients receiving CAR therapy, but of mouse origin. In accordance with the demonstrated clinical benefit of tocilizumab, we demonstrate that CRS-associated mortality can be abrogated by murine IL-6 receptor blockade. We further show that CAR T cell - tumor interaction is required for the robust recruitment of myeloid cells to the tumor site, including neutrophils, eosinophils, macrophages, monocytes and dendritic cells. Cytokine measurements and the transcriptomic analysis of myeloid populations from multiple sites reveal that IL-6 is of myeloid cell origin, dependent on CAR T cell - myeloid proximity and interactions at the tumor site. Furthermore, we probe the impact of myeloid involvement by activating or suppressing macrophage functions to show that CRS outcomes are largely determined by macrophage engagement. Probing into the functions of macrophages led us to identify multiple therapeutic interventions that can ameliorate the toxicities of CRS. We have also designed a novel CAR construct that can prevent CRS-associated mortality while maintaining intact antitumor efficacy in a mouse model. Overall, our results suggest an indispensible role for the myeloid system in severe CRS, especially macrophages, representing a cellular compartment that has been largely overlooked in pre-clinical models to date. Moreover, our model should be useful to guide the rational design of novel CAR T cells that can autonomously ameliorate or prevent CRS. Citation Format: Theodoros Giavridis, Sjoukje J. van der Stegen, Justin Eyquem, Mohamad Hamieh, Michel Sadelain. Novel therapeutic interventions to alleviate CAR T cell-induced cytokine release syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2567.