Adult, but Not Neonatal, Platelet Transfusions Drive a Monocyte Trafficking Phenotype in Vitro and In Vivo

Although, thrombocytopenia can affect all age groups, neonates, especially pre-term, have an increased incidence of thrombocytopenia. Platelet transfusions may reduce the bleeding risk in neonates, but are also associated with adverse short and long-term immune and inflammatory outcomes. A randomized trial of platelet transfusions in neonates found that transfusion was associated with an increased risk of necrotizing enterocolitis, unilateral/bilateral retinopathy, and bronchopulmonary dysplasia. Past work from our research team found that neonatal platelets expressed lower levels of mRNA for many immune related molecules compared to adult platelets. We therefore sought to determine whether the transfusion of adult platelets to neonates resulted in developmental immune dysregulation, with a focus on platelet and monocyte interactions. To explore the interactions between monocytes and platelets, we isolated monocytes from adult mouse bone marrow and co-incubated monocytes with adult (>8 weeks old) or neonatal mouse platelets (7 days old mice) and determined inflammatory and trafficking monocyte phenotypes by flow cytometry and qRT-PCR. Monocytes treated with adult platelets had an increased inflammatory (Ly6C hi) and trafficking phenotype (CCR2 hi), while monocytes treated with neonatal platelets adopted an inflammatory, but not trafficking phenotype. As expected, adult platelets increased the expression of monocyte inflammatory (Nos2, Cxcl1, Ccl2) and trafficking (Ccr2) mRNA, while neonatal platelets also increased inflammatory mRNA expression, but did not increase Ccr2 expression. Adult platelets express more Selp (P-selectin) than neonatal platelets and P-selectin is a major mediator of platelet and monocyte interactions. We confirmed that adult platelets expressed more P-selectin protein compared to neonatal platelets, and found that blocking P-selectin decreased adult platelet induced CCR2 expression to levels similar to monocytes treated with neonatal platelets. Using a transwell chamber we assessed adult and neonatal platelet effects on monocyte migration towards the CCR2 ligand CCL2. Monocytes were treated with adult platelets had significantly greater monocyte migration compared to monocytes co-incubated with neonatal platelets. To model platelet transfusions in the setting of thrombocytopenia, we used 14d old thrombopoietin receptor knockout mice (TPOR -/-) that have low platelet counts, and infused adult or neonatal platelets. We observed a significant increase in inflammatory and trafficking monocytes in mice transfused with adult platelets compared to those transfused with neonatal platelets. Using an in vivo model of monocyte chemotaxis, mice were treated with CCL2 intraperitoneal after platelet transfusion. Adult platelet transfusions, but not neonatal, increased monocyte peritoneal trafficking to CCL2. These data provide comparative insights as to how adult and neonatal platelet transfusions regulate monocyte functions. Adult platelet transfusions to neonates are associated with an inflammatory and trafficking monocyte phenotype that is platelet P-selectin dependent and may have a major impact on neonatal platelet transfusion complications. Disclosures Palis: Rubius Therapeutics: Consultancy.

MFAP4 Deficiency Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation Through Regulation of Macrophage Infiltration and Activity

Objective: Abdominal aortic aneurysm (AAA) is a common age-related vascular disease characterized by progressive weakening and dilatation of the aortic wall. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein involved in the induction of vascular remodeling. This study aimed to investigate if MFAP4 facilitates the development of AAA and characterize the underlying MFAP4-mediated mechanisms.Approach and Results: Double apolipoprotein E- and Mfap4-deficient (ApoE−/−Mfap4−/−) and control apolipoprotein E-deficient (ApoE−/−) mice were infused subcutaneously with angiotensin II (Ang II) for 28 days. Mfap4 expression was localized within the adventitial and medial layers and was upregulated after Ang II treatment. While Ang II-induced blood pressure increase was independent of Mfap4 genotype, ApoE−/−Mfap4−/− mice exhibited significantly lower AAA incidence and reduced maximal aortic diameter compared to ApoE−/− littermates. The ApoE−/−Mfap4−/− AAAs were further characterized by reduced macrophage infiltration, matrix metalloproteinase (MMP)-2 and MMP-9 activity, proliferative activity, collagen content, and elastic membrane disruption. MFAP4 deficiency also attenuated activation of integrin- and TGF-β-related signaling within the adventitial layer of AAA tissues. Finally, MFAP4 stimulation promoted human monocyte migration and significantly upregulated MMP-9 activity in macrophage-like THP-1 cells.Conclusion: This study demonstrates that MFAP4 induces macrophage-rich inflammation, MMP activity, and maladaptive remodeling of the ECM within the vessel wall, leading to an acceleration of AAA development and progression. Collectively, our findings suggest that MFAP4 is an essential aggravator of AAA pathology that acts through regulation of monocyte influx and MMP production.

Deletion of GPR21 improves glucose homeostasis and inhibits the CCL2-CCR2 axis by divergent mechanisms

IntroductionA potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are complicated by non-selective deletion of Gpr21.Research design and methodsWe hypothesized that a Gpr21-selective knockout mouse model, coupled with type 2 diabetes patient samples, would clarify these issues and enable clear assessment of GPR21 as a potential therapeutic target.ResultsHigh-fat feeding studies in Gpr21−/− mice revealed improved glucose tolerance and modest changes in inflammatory gene expression. Gpr21−/− monocytes and intraperitoneal macrophages had selectively impaired chemotactic responses to monocyte chemoattractant protein (MCP)-1, despite unaltered expression of Ccr2. Further genotypic analysis revealed that chemotactic impairment was due to dysregulated monocyte polarization. Patient samples revealed elevated GPR21 expression in peripheral blood mononuclear cells in type 2 diabetes, which was correlated with both %HbA1c and fasting plasma glucose levels.ConclusionsCollectively, human and mouse data suggest that GPR21 influences both glucose homeostasis and MCP-1/CCL2-CCR2-driven monocyte migration. However, a Gpr21−/− bone marrow transplantation and high-fat feeding study in mice revealed no effect on glucose homeostasis, suggesting that there is no (or limited) overlap in the mechanism involved for monocyte-driven inflammation and glucose homeostasis.

Platelet-Derived PCSK9 Is Associated with LDL Metabolism and Modulates Atherothrombotic Mechanisms in Coronary Artery Disease

Platelets play a significant role in atherothrombosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of LDL metabolism and interacts with platelet function. The effect of PCSK9 in platelet function is poorly understood. The authors of this article sought to characterize platelets as a major source of PCSK9 and PCSK9’s role in atherothrombosis. In a large cohort of patients with coronary artery disease (CAD), platelet count, platelet reactivity, and platelet-derived PCSK9 release were analyzed. The role of platelet PCSK9 on platelet and monocyte function was investigated in vitro. Platelet count and hyper-reactivity correlated with plasma LDL in CAD. The circulating platelets express on their surface and release substantial amounts of PCSK9. Release of PCSK9 augmented platelet-dependent thrombosis, monocyte migration, and differentiation into macrophages/foam cells. Platelets and PCSK9 accumulated in tissue derived from atherosclerotic carotid arteries in areas of macrophages. PCSK9 inhibition reduced platelet activation and platelet-dependent thrombo-inflammation. The authors identified platelets as a source of PCSK9 in CAD, which may have an impact on LDL metabolism. Furthermore, platelet-derived PCSK9 contributes to atherothrombosis, and inhibition of PCSK9 attenuates thrombo-inflammation, which may contribute to the reported beneficial clinical effects.

Tumor-Associated Macrophages in Vestibular Schwannoma and Relationship to Hearing

Objective (1) Characterize the distribution of M1 and M2 macrophages in vestibular schwannomas by hearing status. (2) Develop assays to assess monocyte migration and macrophage polarization in cocultures with vestibular schwannoma cells. Study Design Basic and translational science. Setting Tertiary care center. Methods A retrospective chart review of 30 patients with vestibular schwannoma (VS) was performed. Patients were stratified into serviceable and unserviceable hearing groups. Immunohistochemistry for CD80+ M1 and CD163+ M2 macrophages was conducted. Primary VS cultures (n = 4) were developed and cocultured with monocytes. Immunohistochemistry for macrophage markers was performed to assess monocyte migration and macrophage polarization. Results Although tumors associated with unserviceable hearing had higher levels of CD80 and CD163 than those with serviceable hearing, the relationship was only significant with CD163 ( P = .0161). However, CD163 level did not remain a significant predictor variable associated with unserviceable hearing on multivariate analysis when adjusted for other variables. In vitro assays show that VS cells induced monocyte migration and polarization toward CD80+ M1 or CD163+ M2 macrophage phenotypes, with qualitative differences in CD163+ macrophage morphologies between serviceable and unserviceable hearing groups. Conclusion Vestibular schwannomas express varying degrees of CD80+ M1 and CD163+ M2 macrophages. We present evidence that higher expression of CD163+ may contribute to poorer hearing outcomes in patients with VS. We also describe in vitro assays in a proof-of-concept investigation that VS cells can initiate monocyte migration and macrophage polarization. Future investigations are warranted to explore the relationships between tumor, macrophages, secreted cytokines, and hearing outcomes in patients with VS.

Induction of RAGE-NFkB signalling axis enhances SHP-2 tyrosine phosphatase expression resulting in deviant activation of diabetic monocytes

Purpose Aberrant activation of Type 2 Diabetes mellitus (T2DM) monocytes is an important pathomechanism leading to restricted arteriogenesis and augmented atherosclerosis, hereby, accelerating CAD and PAD. Tyrosine phosphatase SHP-2 was found to be upregulated in T2DM-monocytes. This study aimed to identify the pathways regulating SHP-2 expression in T2DM-monocytes. Methods Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Monocytes were incubated with Methylglyoxal (MG), a highly reactive side product of glycolysis, Receptor for advanced glycation end product (RAGE) ligand AGE-bovine serum (AGE-BSA) or TNFα for 24 hours. Transwell migration assays were used to analyse the migratory potential of monocytes. Western Blot, RT-qPCR and FACS were performed to quantify the expression of relevant molecules. Pharmacological inhibitors were used to study functional relevance of the RAGE-NFκB-SHP-2 signalling axis. Results Significantly enhanced SHP-2 expression was detected in monocytes, which were incubated with TNFα, MG or AGE-BSA, respectively. Co-incubation of these molecules with NFκB-inhibitor blocked SHP-2 upregulation. Pharmacological inhibition of RAGE reversed the MG or AGE-BSA induced SHP-2 expression and activity in monocytes. RAGE expression on monocytes was upregulated after the incubation with MG or AGE-BSA, consistent with enhanced RAGE mRNA levels in T2DM monocytes. Besides, we also detected elevated SHP-2 transcripts in monocytes of T2DM patients which was more pronounced in monocytes with augmented TNFα expression. Furthermore, MG and AGE-BSA provoked the enhanced migration of monocytes which could be significantly reduced after the application of an allosteric SHP-2 inhibitor. Interestingly, pharmacological inhibition of RAGE in these conditions alone was sufficient to block the elevated monocyte migration. Moreover, monocytes isolated from T2DM patients revealed a comparable pro-migratory phenotype, which was completely restored after the pharmacological inhibition of SHP-2. Conclusions This study identified the upstream signalling mediators that contribute to SHP-2 dependent monocyte activation in T2DM conditions. Glucose metabolite (MG) or RAGE ligand (AGE-BSA) alone were sufficient to induce a pro-migratory phenotype in monocytes by upregulating SHP-2. Of note, an inflammatory state seems to accelerate this effect since enhanced TNFα levels were found to be positively correlated with the augmented SHP-2 expression. Moreover, we identified the RAGE-NFκB signalling axis through which the SHP-2 upregulation is conveyed when augmented accumulation of glucose metabolites occur. These findings reveal a basis for potential new therapeutic approaches to prevent accelerated CAD and PAD in diabetic patients since independent pharmacological inhibition of every step in the RAGE-NFκB-SHP-2 axis was sufficient to reset the aberrant monocyte activation. FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): Interdisciplinary Center for Clinical Research of the Medical Faculty of the University of Münster

Functional Characterization of the Wheat Macrophage Migration Inhibitory Factor TaMIF1 in Wheat-Stripe Rust (Puccinia striiformis) Interaction

Macrophage migration inhibitory factor (MIF), named for its role in inhibiting macrophage/monocyte migration, has multiple functions in modulation of inflammation, cell proliferation, angiogenesis, and tumorigenesis in vertebrates. Although homologs of this gene can be found in plants, the function of MIF in plants remains obscure. Here, we characterized TaMIF1 in Triticum aestivum resembling the MIF secreted from Homo sapiens. Transcript analysis revealed that TaMIF1 responded to stripe rust infection of wheat and was upregulated during the infection stage. TaMIF1 was localized to both the cytosol and nuclei in wheat mesophyll protoplast. Additionally, TaMIF1 possessed significant tautomerase activity, indicating conservation of MIFs across kingdoms. Agrobacterium tumefaciens infiltration assay demonstrated that TaMIF1 was capable of suppressing programmed cell death hinting its role in plant immunity. Heterologous expression of TaMIF1 increased fission yeast sensitivity to oxidative stress. Silencing TaMIF1 decreased the susceptibility of wheat to Pst seemingly through increasing reactive oxygen species accumulation. In conclusion, functions of the TaMIF1 were investigated in this study, which provides significant insight into understanding the role of MIFs across kingdoms.

Abstract P328: Regulation Of Monocyte Physiology By Il-4 Stimulated Memory Cd8+ T-cells

Following a myocardial infarction (MI) monocytes and T-cells begin to infiltrate into the ischemic area in effort to remove necrotic debris and initiate formation of scar tissue. Interleukin (IL)-4 has been linked to improved cardiac wound healing via alterations in both the macrophage and T-cell populations. The goal of this study was determine if proteins secreted by IL-4 stimulated CD8+ T-cells would regulate monocyte physiology that would ultimately improve cardiac healing after an MI. Isolated splenic naïve CD8+ T-cells from day 0 (no MI) mice (n=5/sex/stimulation)were cultured in RPMI with either 0.1% FBS (unstimulated) or 0.1% FBS+ IL-4. After 24 hours of stimulation, the cells and media were collected and separated by centrifugation. The cell pellet was stained and analyzed for markers of activation (CD44) and memory (CD27) by flowcytometry. Conditioned media (secretome) was collected for stimulation of bone marrow monocytes (n=4; females only). After stimulation with the secretome, monocytes were analyzed for viability, phagocytosis, and macrophage phenotype by flow cytometry. Migration of the monocytes after stimulation was also measured using electric cell-substrate impedance sensing (ECIS). After IL-4 stimulation, there was a shift from effector (CD44+ CD27-) to the memory phenotype (CD44+ CD27+; p<0.05 vs unstimulated cells). Interestingly, bone marrow monocyte viability was decreased by 15% when stimulated with the secretome of IL-4 treated CD8+ T-cells compared to unstimulated CD8+ T-cells (0.1%). Phagocytosis was slightly elevated though not significant (p=0.07) in monocytes that were stimulated with the secretome from the IL-4 group compared to the unstimulated CD8+ T-cells. No differences were found in expression of macrophage markers F4/80 (p=0.532) or M1 marker CD86 (p=0.471). The secretome of IL-4 stimulated T-cells increased monocyte migration after wounding similar to levels of the positive control (monocytes in 10% FBS only). The data collected showed that IL-4 stimulated CD8+ T-cells were able to upregulate memory marker CD27. These memory-like CD8+ T-cells initiated monocyte phagocytosis and migration but decreased monocyte viability suggesting that they may play a role in regulating macrophage biology post-MI.

Effects of Antimomium crudum 30cH and 200cH on the macrophage – Leishmania (L) amazonensis relation in vitro

Background: Leishmaniasis is a chronic skin and systemic disease, whose treatment is related to important side effects and loss of life quality. In previous results [1], mice treated with Antimonium crudum (AC) 30cH presented reduction in local inflammatory process and modulation of B1 cell-phagocyte differentiation and migration, but also increase of free amastigotes number among the surrounding tissue. Aims: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with AC 30cH and 200cH, in different times. Methodology: The morpho-functional features of macrophages (spreading, phagocytosis and oxidative activity), the number of free promastigotes in the supernatant and the cytokines measurement were evaluated. The spreading and phagocytosis assays were performed in quadruplicate, in three experiments, resulting in 12 datapoints for each dilution/time. Kruskal Wallis (for no parametric variables) and two-way ANOVA test (for parametric variables) were used to verify time to time differences and the interaction between treatment and time respectively, being p≤0.05 considered significant. Animals were not used in this study. Results: The treatment with AC 30cH and AC 200cH resulted in significant but transitory increase in spreading and phagocytosis activity after 2 to 24 hours of incubation, followed by increase in the number of free promastigotes in the supernatant (in AC 30cH treatment) and decrease in CD86 expression (in AC 200cH treatment). GMCSF, alpha-INF, IL1, IL6, IL10 and IL12 were reduced after 48 to 72 hours and CCL4 and RANTES were reduced after 120H, for both potencies. Two peaks of CCL2 were seen in Leishmania sp infected macrophages, at 24 and 120 hours, but only AC30cH inhibited them. A peak of VEGF was observed after 120 hours following the treatment with AC 200cH, together with the increase in the number of multinucleated cells. The morphology of macrophages at fluorescence microscopy after the staining with acridine orange in fresh unfixed cells showed severe reduction of acid vacuoles in AC 30CH treated cells at 2 hours of parasite-macrophage interaction, revealing possible macrophage anergy. No difference in peroxide / NO production and in apoptosis percentage of free promastigotes was seen among groups, in all evaluated times. Conclusions: Both potencies were able to decrease most of cytokines production, specially CCL2 in AC 30cH treated cells, which explains the inhibition of monocyte migration seen in vivo [1]. The late peak of VEGF observed in AC 200cH treated cells suggests a M1 - M2 polarization, whose biological meaning is still under discussion. Acknowledgements: CAPES, UNIP, FAPESP (2014/00967-1)   References Rodrigues de Santana F, de Paula Coelho C, Cardoso TN, Perez Hurtado EC, Roberti Benites N, Dalastra Laurenti M, Villano Bonamin L. Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH. Homeopathy, 2014;103(4):264-74. doi: 10.1016/j.homp.2014.08.006.