Receptor CD36 Research Articles

Macrophage-mediated tissue response evoked by subchronic inhalation of lead oxide nanoparticles is associated with the alteration of phospholipases C and cholesterol transporters

BackgroundInhalation of lead oxide nanoparticles (PbO NPs), which are emitted to the environment by high-temperature technological processes, heavily impairs target organs. These nanoparticles pass through the lung barrier and are distributed via the blood into secondary target organs, where they cause numerous pathological alterations. Here, we studied in detail, macrophages as specialized cells involved in the innate and adaptive immune response in selected target organs to unravel their potential involvement in reaction to subchronic PbO NP inhalation. In this context, we also tackled possible alterations in lipid uptake in the lungs and liver, which is usually associated with foam macrophage formation.ResultsThe histopathological analysis of PbO NP exposed lung revealed serious chronic inflammation of lung tissues. The number of total and foam macrophages was significantly increased in lung, and they contained numerous cholesterol crystals. PbO NP inhalation induced changes in expression of phospholipases C (PLC) as enzymes linked to macrophage-mediated inflammation in lungs. In the liver, the subchronic inhalation of PbO NPs caused predominantly hyperemia, microsteatosis or remodeling of the liver parenchyma, and the number of liver macrophages also significantly was increased. The gene and protein expression of a cholesterol transporter CD36, which is associated with lipid metabolism, was altered in the liver. The amount of selected cholesteryl esters (CE 16:0, CE 18:1, CE 20:4, CE 22:6) in liver tissue was decreased after subchronic PbO NP inhalation, while total and free cholesterol in liver tissue was slightly increased. Gene and protein expression of phospholipase PLCβ1 and receptor CD36 in human hepatocytes were affected also in in vitro experiments after acute PbO NP exposure. No microscopic or serious functional kidney alterations were detected after subchronic PbO NP exposure and CD68 positive cells were present in the physiological mode in its interstitial tissues.ConclusionOur study revealed the association of increased cholesterol and lipid storage in targeted tissues with the alteration of scavenger receptors and phospholipases C after subchronic inhalation of PbO NPs and yet uncovered processes, which can contribute to steatosis in liver after metal nanoparticles exposure.Graphical abstract

A Phase Ib Dose-finding Study of Panobinostat and Ruxolitinib in Myelofibrosis.

A Phase Ib Dose-finding Study of Panobinostat and Ruxolitinib in Myelofibrosis.

Angiofibroma of soft tissue: Current status of pathology and genetics.

Angiofibroma of soft tissue (AFST) is a new soft tissue tumor entity described in the 2020 World Health Organization Classification of Soft Tissue and Bone Tumors. It most often arises in the lower extremities of middle-aged adults and pursues a benign clinical course with a low rate of non-destructive local recurrence. Histologically, the lesion consists of uniform bland spindle cells in a fibromyxoid stroma with a prominent vascular network. The vascular component forms a complex arrangement of small, thin-walled branching blood vessels. By immunohistochemistry, AFST is variably positive for epithelial membrane antigen, desmin, smooth muscle actin, CD34, CD68, CD163 and estrogen receptor. The exact etiology of AFST remains unknown, but it appears genetically distinct, with a balanced t(5;8)(p15;q13) translocation resulting in a fusion of aryl hydrocarbon receptor repressor (AHRR) and nuclear receptor coactivator 2 (NCOA2). Knowledge of this recently described entity is important because it can mimic a variety of intermediate and malignant soft tissue tumors, including solitary fibrous tumor, low-grade fibromyxoid sarcoma, myxoid liposarcoma and low-grade myxofibrosarcoma. We review AFST, with an emphasis on the diagnostic spectrum, recent molecular genetic features and the differential diagnosis.

Donor Heterogeneity in the Human Macrophage Response to a Biomaterial Under Hyperglycemia In Vitro.

Macrophages have a commanding role in scaffold-driven in situ tissue regeneration. Depending on their polarization state, macrophages mediate the formation and remodeling of new tissue by secreting growth factors and cytokines. Therefore, successful outcomes of material-driven in situ tissue vascular tissue engineering depend largely on the immuno-regenerative potential of the recipient. A large cohort of patients requiring vascular replacements suffers from systemic multifactorial diseases, such as diabetes, which gives rise to a hyperglycemic and aggressive oxidative inflammatory environment that is hypothesized to hamper a well-balanced regenerative process. Here, we aimed at fundamentally exploring the effects of hyperglycemia, as one of the hallmarks of diabetes, on the macrophage response to three-dimensional (3D) electrospun synthetic biomaterials for in situ tissue engineering, in terms of inflammatory profile and tissue regenerative capacity. To simulate the early phases of the in situ regenerative cascade, we used a bottom-up in vitro approach. Primary human macrophages (n = 8 donors) were seeded in two-dimensional (2D) culture wells and polarized to pro-inflammatory M1 and anti-inflammatory M2 phenotype in normoglycemic (5.5 mM glucose), hyperglycemic (25 mM), and osmotic control (OC) conditions (5.5 mM glucose, 19.5 mM mannitol). Unpolarized macrophages and (myo)fibroblasts were seeded in mono- or co-culture in a 3D electrospun resorbable polycaprolactone bisurea scaffold and exposed to normoglycemic, hyperglycemic, and OC conditions. The results showed that macrophage polarization by biochemical stimuli was effective under all glycemic conditions and that the polarization states dictated expression of the receptors SCL2A1 (glucose transporter 1) and CD36 (fatty acid transporter). In 3D, the macrophage response to hyperglycemic conditions was strongly donor-dependent in terms of phenotype, cytokine secretion profile, and metabolic receptor expression. When co-cultured with (myo)fibroblasts, hyperglycemic conditions led to an increased expression of fibrogenic markers (ACTA2, COL1, COL3, IL-1β). Together, these findings show that the hyperglycemic and hyperosmotic conditions may, indeed, influence the process of macrophage-driven in situ tissue engineering, and that the extent of this is likely to be patient-specific. Impact Statement Success or failure of cell-free bioresorbable in situ tissue-engineered vascular grafts hinges around the immuno-regenerative response of the recipient. Most patients requiring blood vessel replacements suffer from additional multifactorial diseases, such as diabetes, which may compromise their intrinsic regenerative potential. In this study, we used a bottom-up approach to study the effects of hyperglycemia, a hallmark of diabetes, on important phases in the in situ regenerative cascade, such as macrophage polarization and macrophage-myofibroblast crosstalk. The results demonstrate a relatively large donor-to-donor variation, which stresses the importance of taking scaffold-independent patient-specific factors into account when studying in situ biomaterial-driven tissue engineering.

Soluble LPS receptor CD14 is increased in saliva of patients with geographic tongue

Objectives The soluble bacterial pattern recognition receptor, sCD14 augments inflammatory responses in oral cavity. The aim of the study was to investigate whether patients with geographic tongue (GT) with and without fissured tongue (FT) have impaired inflammatory regulation, manifesting as increased levels of sCD14 in the saliva. Material and Methods An enzyme-linked immunosorbent assay was used to measure the amount of sCD14 in whole and parotid saliva of patients diagnosed with GT (GT whole, n = 21; GT parotid, n = 23) and control subjects (GT whole, n = 25; GT parotid, n = 18). The levels of sCD14 were also evaluated according to our previous clinical assessment of GT based on the number of lesions detected on the tongue, as ‘mild’ (a single lesion), ‘moderate’ (2–5 lesions), or ‘severe’ (≥6 lesions). Diagnosis of FT was established when multiple grooves or fissures were observed on the dorsal and lateral surfaces of the tongue. Results GT patients had significantly higher sCD14 levels in whole (p<.05) and parotid saliva (p<.001), compared with controls. GT patients with FT had significantly increased sCD14 levels only in parotid saliva. A gradual increase in sCD14 levels in parotid and unstimulated saliva was seen in GT patients with multiple tongue lesions compared with single lesions. Conclusions GT patients had increased sCD14 in both parotid and unstimulated saliva. sCD14 seems to increase local inflammatory responses, which suggests its involvement in the pathophysiology of GT.

P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES

Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab. Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines. In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance.

The Good and the Bad: Monocytes' and Macrophages' Diverse Functions in Inflammation.

Monocytes and macrophages are central players of the innate immune response and play a pivotal role in the regulation of inflammation. Thereby, they actively participate in all phases of the immune response, from initiating inflammation and triggering the adaptive immune response, through to the clearance of cell debris and resolution of inflammation. In this review, we described the mechanisms of monocyte and macrophage adaptation to rapidly changing microenvironmental conditions and discussed different forms of macrophage polarization depending on the environmental cues or pathophysiological condition. Therefore, special focus was placed on the tight regulation of the pro- and anti-inflammatory immune response, and the diverse functions of S100A8/S100A9 proteins and the scavenger receptor CD163 were highlighted, respectively. We paid special attention to the function of pro- and anti-inflammatory macrophages under pathological conditions.

Soluble mannose receptor CD206 and von Willebrand factor are early biomarkers to identify patients at risk for severe or necrotizing acute pancreatitis

BackgroundIn acute pancreatitis (AP), microcirculatory dysfunction and leukocyte activation contribute to organ damage, inflammation, and mortality. Given the role of macrophage activation, monocyte recruitment, and microthrombus formation in the early pathogenesis of AP, we examined the macrophage activation marker soluble mannose receptor (sCD206) and the endothelial function marker von Willebrand factor (vWF) in patients admitted for AP.MethodsIn an exploratory analysis, serum sCD206 and plasma vWF were prospectively analyzed on day 1 and day 3 in 81 patients with AP admitted to the hospital. In addition, blood samples from 59 patients with early AP admitted to the intensive care unit and symptom onset < 24 h were retrospectively analyzed. Patients were dichotomized as per study protocol into two groups: (i) “non-severe edematous AP” including patients with mild AP without organ failure and patients with transient organ failure that resolves within 48 h and (ii) “severe/necrotizing AP” including patients with severe AP and persistent organ failure > 48 h and/or patients with local complications.ResultsIn the prospective cohort, 17% developed severe/necrotizing pancreatitis compared with 56% in the ICU cohort. Serum concentrations of sCD206 on admission were higher in patients with severe/necrotizing AP than in patients with non-severe edematous AP (prospective: 1.57 vs. 0.66 mg/l, P = 0.005; ICU: 1.76 vs. 1.25 mg/l, P = 0.006), whereas other inflammatory markers (leukocytes, C-reactive protein, procalcitonin) and disease severity (SOFA, SAPS II, APACHE II) did not show significant differences. Patients with severe/necrotizing AP had a greater increase in sCD206 than patients with non-severe edematous AP at day 3 in the prospective cohort. In contrast to routine coagulation parameters, vWF antigen levels were elevated on admission (prospective cohort: 375 vs. 257%, P = 0.02; ICU cohort: 240 vs. 184%, P = 0.03). When used as continuous variables, sCD206 and VWF antigen remained predictors of severe/necrotizing AP after adjustment for etiology and age in both cohorts.ConclusionssCD206 identifies patients at risk of severe AP at earlier timepoints than routine markers of inflammation and coagulation. Prospective studies are needed to investigate whether incorporating early or repeated measurements into the existing scoring system will better identify patients at increased risk for complications of AP.

Flow Cytometric Analysis of Leukocyte Populations in the Lung Tissue of Dromedary Camels.

Respiratory tract infections are among the most common infections in dromedary camels, with a high impact on animal health, production, and welfare. Tissue-specific distribution of immune cells is one of the important factors that influence the nature and outcome of the immune response to pathogens. Several protocols have recently been described for the flow cytometric analysis of immune cells in the lung tissue of several species. However, no such protocol currently exists for dromedary camels. The aim of the present study was, therefore, to establish a flow cytometric protocol for the identification of immune cell populations in the camel lung tissue and the evaluation of some of their phenotypic and functional properties. Combined staining of camel lung leukocytes with monoclonal antibodies to the pan-leukocyte marker CD45 and the myeloid cell marker CD172a allowed the identification of myeloid cells (CD45+CD172a+) and lymphoid cells (CD45+CD172a−) in the lung of healthy camels. The cell adhesion molecules CD11a and CD18 were found in a higher abundance on myeloid cells compared to lymphoid cells. Based on their differential expression of the LPS receptor CD14, macrophages (CD172a+CD14high cells) were identified as the most abundant immune cell population in the camel lung tissue. In contrast to their dominance in camel peripheral blood, granulocytes (CD172a+CD14low) presented only a minor population in the lung tissue. The higher frequency of γδ T cells in the lung tissue than in peripheral blood suggests a role for these cells in the pulmonary immune system. Flow cytometric analysis of bacterial phagocytosis and ROS production upon bacterial stimulation revealed high antimicrobial activity of camel lung phagocytes, which was comparable with the antimicrobial activity of blood granulocytes. Comparative analysis of immune cell distribution between the cranial and caudal lobes of the camel lung revealed a higher frequency of granulocytes and a lower frequency of macrophages in the cranial compared to the caudal lung lobe. In addition, the higher frequency of cells expressing the M2 macrophage marker CD163 in the caudal lung tissue, with a slightly higher fraction of MHCII-positive cells (M1 phenotype) in the cranial lung tissue, may suggest the distribution of different macrophage subtypes in the different lobes of the camel lung. Such differences between lung lobes could influence the effectiveness of the immune response to infection or vaccination with respiratory pathogens. Collectively, the present study identified some similarities and differences between camels and other farm animals regarding the distribution of the main immune cell populations in their lungs. Further studies are required for comprehensive immunophenotyping of the cellular pulmonary immune system in camels.

412-P: Acute Hyperglycemia-Induced Endoplasmic Reticulum and Oxidative Stress Increases Monocyte Cholesterol Content and Cytokine Secretion

Background: Cardiovascular disease (CVD) is an inflammatory disorder enhanced by diabetes. The mechanisms by which recurrent acute hyperglycemia promotes inflammation and CVD are unclear. We showed that acute hyperglycemia mediates increased monocyte cholesterol uptake and cytokine release, but the underlying signaling pathways are unknown. We hypothesized that acute hyperglycemia-induced oxidative and ER stress in circulating monocytes increases cholesterol uptake and a pro-inflammatory phenotype. Methods: Circulating monocytes from 35 healthy subjects were isolated and exposed to euglycemic (5mM) or hyperglycemic (16.7 mM) conditions with or without inhibitors of ER stress (4 phenyl butyric acid) or oxidative stress (N-Acetyl cysteine) for 2 or 6 hrs to assess changes in LDL or oxLDL uptake and cytokine (TNF-α, IL1-β) release. Results: We found that acute hyperglycemia-induced ER and oxidative stress increased total and free cholesterol content via enhanced LDL (18% and 26%, p&amp;lt;0.05) and oxLDL (56% and 30%, p&amp;lt;0.05) uptake as a result of enhanced CD36 and LDL receptor expression. Conversely, inhibiting monocyte oxidative stress or ER stress attenuated monocyte LDL and oxLDL uptake. Finally, blocking monocyte ER stress decreased acute hyperglycemia-induced monocyte TNF-α (87% and 40%, p&amp;lt;0.05) and IL1-β (41% and 44%, p&amp;lt;0.05) secretion after 2 and 6 hrs. Conclusion: Recurrent acute hyperglycemia promotes inflammation by increasing monocyte ER and oxidative stress, which accelerates cholesterol uptake, cholesterol deposition, and cytokine secretion. Future studies are needed to test if acute hyperglycemia-induced monocyte cholesterol deposition is a mechanism to contribute to increased CVD in diabetes. Disclosure R.Zhang: Research Support; American Heart Association. J.Oh: None. G.A.Kutz: None. M.Bambouskova: None. A.S.Dusso: None. C.Bernal-mizrachi: None.

Microbial antigen in human milk: a natural vaccine?

Microbial antigen in human milk: a natural vaccine?

Inflammatory gene silencing in activated monocytes by a cholesterol tagged-miRNA/siRNA: a novel approach to ameliorate diabetes induced inflammation.

There is a major unmet need for the development of effective therapies for diabetes induced inflammation. Increased adenosine-uridine rich elements (AREs) containing mRNAs of inflammatory molecules are reported in inflamed monocytes. Destabilizing these inflammatory mRNAs by the miR-16 could reduce inflammation. DNA microarrays andin vitrocell studies showed that exogenous miR16 and its mimic treatment, in LPS/PMA induced monocytes, significantly downregulated several ARE containing inflammatory cytokine mRNAs similar to those seen in the normal monocytes. Ingenuity pathway analyses showed exogenous miR-16 or its synthetic mimic treatment alleviates inflammatory responses. To selectively target uptake, especially to inflamed cells, one of the CD36 substrate cholesterol was tagged tomiR16/siRNA. Cholesterol tagged miR-16/ARE-siRNA showed enhanced uptake in CD36 expressing inflamed cells. In LPS or PMA, treated monocytes, candidate genes expressions levels such as IL-6, IL-8, IL-12β, IP-10, and TNF-α mRNA were increased, as measured by RT-qPCR as seen in primary monocytes of diabetes patients. Exogenous miR16 or ARE-siRNA transfection reduced mRNAs of pro-inflammatory cytokines levels in monocyte, and its adhesion. Increased uptake of cholesterol tagged miR-16 through the CD36 receptor was observed. This destabilizes numerous inflammatory ARE containing mRNAs and alleviates inflammatory responses. Cholesterol-tagged miR-16 and its mimic are novel anti-inflammatory molecules that can be specifically targeted to, via through CD36 expressing, "inflamed" cells and thus serve as therapeutic candidates to alleviate inflammatory diseases.

Loss of CD226 protects apolipoprotein E-deficient mice from diet-induced atherosclerosis

CD226 is a costimulatory molecule that regulates immune cell functions in T cells, natural killer cells, and macrophages. Because macrophage-derived foam cell formation is a crucial factor contributing to the development of atherosclerosis, we aimed to evaluate the potential roles of CD226 in the pathogenesis of atherosclerosis. The effects of CD226 on atherosclerosis were investigated in CD226 and apolipoprotein E double-knockout (CD226−/− ApoE−/−) mice fed with a high-cholesterol atherogenic diet. CD226 expression in macrophages was evaluated using flow cytometry. Histopathological analysis was performed to evaluate the atherosclerotic lesions. Inflammatory cell infiltration was detected using immunofluorescence staining. Bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PEMs) were isolated from the mice and used to explore the mechanism in vitro. The in vivo results indicated that CD226 knockdown protected against atherosclerosis in ApoE−/− mice, evidenced by reduced plaque accumulation in the brachiocephalic artery, aortic roots, and main aortic tree. CD226 gene-deficient macrophages showed reduced foam cell formation under ox-low density lipoprotein stimulation compared with wild-type (WT) cells. CD226 deficiency also decreased the expression of CD36 and scavenger receptor (SR)-A (responsible for lipoprotein uptake) but increased the expression of ATP-binding cassette transporter A1 and G1 (two transporters for cholesterol efflux). Therefore, loss of CD226 hinders foam cell formation and atherosclerosis progression, suggesting that CD226 is a promising new therapeutic target for atherosclerosis.

CD18 Mediates Neutrophil Imperviousness to the Aggregatibacter actinomycetemcomitans JP2 Clone in Molar-Incisor Pattern Periodontitis.

IntroductionMolar-incisor pattern periodontitis (MIPP) in the absence of significant local risk factors or systemic disease, is a rare, early onset periodontal disease phenotype, with 0.5% to 2.5% global prevalence. The condition is characterized by impaired neutrophil function and persistent Aggregatibacter actinomycetemcomitans (JP2 clone) infection. The aim of this study was to characterize neutrophil functional responses to JP2 and to investigate the neutrophil receptors involved.Materials and MethodsNeutrophils were obtained from whole blood samples of periodontally healthy and MIPP subjects and incubated with the JP2 clone or a non-JP2 clone of A. actinomycetemcomitans. Bacterial survival was tested by blood agar culture; neutrophil death was tested with propidium iodide and flow cytometry; Reactive oxygen production (ROS) was measured with 2′,7′-dichlorofluorescein diacetate and a fluorescence plate reader; the cytokinome was analysed using an array profiler, ELISA and RT-PCR. Receptors binding to JP2 were isolated using a novel immunoprecipitation assay and validated functionally using specific blocking antibodies.ResultsJP2 and non-JP2 survival was comparable between all the neutrophil groups. Resistance to neutrophil necrosis following exposure to JP2 was significantly lower in the MIPP group, than in all the other groups (p<0.0001). Conversely, MIPP neutrophils showed lower levels of ROS production in response to JP2 infection compared with that of healthy neutrophils (p<0.001). Furthermore, significantly lower levels of cytokines, such as IL8, IL10 and TNFα, were observed during JP2 incubation with MIPP neutrophils than upon incubation with periodontally healthy neutrophils. Various proteins expressed on neutrophils bind to JP2. Of these, CD18 was found to mediate neutrophil necrosis. The CD18 receptor on MIPP neutrophils acts differently from that on periodontally healthy patients neutrophils, and appears to reflect differential neutrophil reactions to JP2.ConclusionThis study portrays a fundamental difference in neutrophil response to JP2 infection between periodontally healthy and MIPP patients. This was evident in the resistance to necrosis, and lower ROS and cytokine production, despite the persistent presence of viable JP2. Whilst in periodontally healthy neutrophils, JP2 binds to CD18 on cell surfaces, this is not the case in MIPP neutrophils, suggesting a potential role for CD18 in the periodontal susceptibility of MIPP patients.

The zinc finger transcription factor Sall1 is required for the early developmental transition of microglia in mouse embryos.

Microglia play many critical roles in neural development. Recent single‐cell RNA‐sequencing studies have found diversity of microglia both across different stages and within the same stage in the developing brain. However, how such diversity is controlled during development is poorly understood. In this study, we first found the expression of the macrophage mannose receptor CD206 in early‐stage embryonic microglia on mouse brain sections. This expression showed a sharp decline between E12.5 and E13.5 across the central nervous system. We next tested the roles of the microglia‐expressed zinc finger transcription factor SALL1 in this early transition of gene expression. By deleting Sall1 specifically in microglia, we found that many microglia continued to express CD206 when it is normally downregulated. In addition, the mutant microglia continued to show less ramified morphology in comparison with controls even into postnatal stages. Thus, SALL1 is required for early microglia to transition into a more mature status during development.

Single-cell genomics identifies distinct B1 cell developmental pathways and reveals aging-related changes in the B-cell receptor repertoire

BackgroundB1 cells are self-renewing innate-like B lymphocytes that provide the first line of defense against pathogens. B1 cells primarily reside in the peritoneal cavity and are known to originate from various fetal tissues, yet their developmental pathways and the mechanisms underlying maintenance of B1 cells throughout adulthood remain unclear.ResultsWe performed high-throughput single-cell analysis of the transcriptomes and B-cell receptor repertoires of peritoneal B cells of neonates, young adults, and elderly mice. Gene expression analysis of 31,718 peritoneal B cells showed that the neonate peritoneal cavity contained many B1 progenitors, and neonate B cell specific clustering revealed two trajectories of peritoneal B1 cell development, including pre-BCR dependent and pre-BCR independent pathways. We also detected profound age-related changes in B1 cell transcriptomes: clear difference in senescence genetic program was evident in differentially aged B1 cells, and we found an example that a B1 subset only present in the oldest mice was marked by expression of the fatty-acid receptor CD36. We also performed antibody gene sequencing of 15,967 peritoneal B cells from the three age groups and discovered that B1 cell aging was associated with clonal expansion and two B1 cell clones expanded in the aged mice had the same CDR-H3 sequence (AGDYDGYWYFDV) as a pathogenically linked cell type from a recent study of an atherosclerosis mouse model.ConclusionsBeyond offering an unprecedent data resource to explore the cell-to-cell variation in B cells, our study has revealed that B1 precursor subsets are present in the neonate peritoneal cavity and dissected the developmental pathway of the precursor cells. Besides, this study has found the expression of CD36 on the B1 cells in the aged mice. And the single-cell B-cell receptor sequencing reveals B1 cell aging is associated with clonal expansion.

The Impact of the CTHRSSVVC Peptide Upon Experimental Models of Trypanosoma cruzi Infection.

Chagas disease (CD), caused by the hemoflagellate protozoan Trypanosoma cruzi, affects more than six million people worldwide and presents an unsatisfactory therapy, based on two nitroderivatives, introduced in clinical medicine for decades. The synthetic peptide, with CTHRSSVVC sequence (PepA), mimics the CD163 and TNF-α tripeptide “RSS” motif and binds to atheromatous plaques in carotid biopsies of human patients, spleen tissues, and a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. CD163 receptor is present on monocytes, macrophages, and neutrophils, acting as a regulator of acute-phase processes and modulating aspects of the inflammatory response and the establishment of infections. Due to the potential theranostic role of PepA, our aim was to investigate its effect upon T. cruzi infection in vitro and in vivo. PepA and two other peptides with shuffled sequences were assayed upon different binomials of host cell/parasite, including professional [as peritoneal mouse macrophages (PMM)] and non-professional phagocytes [primary cultures of cardiac cells (CM)], under different protocols. Also, their impact was further addressed in vivo using a mouse model of acute experimental Chagas disease. Our in-vitro findings demonstrate that PepA and PepB (the peptide with random sequence retaining the “RS” sequence) reduced the intracellular parasitism of the PMM but were inactive during the infection of cardiac cells. Another set of in-vitro and in-vivo studies showed that they do not display a trypanocidal effect on bloodstream trypomastigotes nor exhibit in-vivo efficacy when administered after the parasite inoculation. Our data report the in-vitro activity of PepA and PepB upon the infection of PMM by T. cruzi, possibly triggering the microbicidal arsenal of the host professional phagocytes, capable of controlling parasitic invasion and proliferation.

MO663: No Translocation of Intact Intestinal Bacteria During Intermittent Dialysis Therapies

Abstract BACKGROUND AND AIMS Accumulating evidence shows that online post-dilution haemodiafiltration (HDF), especially when a high convection volume is achieved, is associated with a lower mortality risk than haemodialysis (HD) [1]. The mechanism behind this effect, however, is unclear. In this respect, a superior intradialytic organ perfusion and hence, less intestinal tissue damage, permitting the transfer of bacteria from the gut to the blood, might play an important role. Therefore, we assessed whether differences exist in the intradialytic translocation of intact bacteria between four dialysis modalities. METHOD A randomized cross-over trial was performed (NCT03249532, ClinicalTrials.gov) in 11 prevalent dialysis patients, who were exposed to four dialysis strategies: HD with standard dialysate temperature (Td) 36.5°C and cool (Td 35.5°C) dialysate, and HDF with a high (≥23 L/1.73 m2/session) and low (15 L/1.73 m2/session) convection volume. Microbial DNA (mDNA), soluble CD14, high-sensitivity CRP and IL-6 receptor were measured during each modality, as secondary endpoints of this study. Quantitative assessment of mDNA was performed by 16S-23S interspace profiling after DNA isolation [2, 3]. In the current analysis, the quantitative difference in circulating mDNA between modalities was investigated. In addition, differences in the acute phase reaction (APR), as measured by soluble CD14, high-sensitivity CRP and IL-6 receptor and potential relations between mDNA and the APR were analysed. RESULTS DNA of intact bacteria could not be demonstrated in blood samples of the patients in any dialysis strategy. Yet, in all modalities a similar increase in IL-6 receptor, high-sensitivity CRP and soluble CD14 was observed (pre- versus post-dialysis: P-values &amp;lt;.05) (Figures 1A-B). CONCLUSION i) The appearance of DNA from intact bacteria in the blood of dialysis patients could not be demonstrated, despite the use of a sensitive state-of-the-art technique, which is free from contamination by human DNA; ii) during all modalities a comparable and significant APR was observed; iii) hence, the intradialytic APR does not seem to result from translocated intact bacteria.

MO587: Chronic Kidney Disease Induces Endotoxin-Related Activation of The Innate Immune System

Abstract BACKGROUND AND AIMS Chronic inflammation is common in patients with chronic kidney disease (CKD) and is associated with poor outcomes. Although the etiology is only partly understood, the gut–kidney axis is considered an important contributor [1]. Lipopolysaccharide (LPS), also known as endotoxin, is a well-characterized pyrogen found in the exterior cell membrane of most of the gram-negative bacteria. It plays an important role in promoting intestinal inflammatory responses. When absorbed through the intestinal epithelium, LPS induces inflammation by activating macrophages and monocytes. Due to short half-life and semi-quantitative assay characteristics of the Limulus Amebocyte Lysate (LAL) assay, direct quantification of LPS is not suitable to quantify activation of the innate immune system. Lipoprotein-binding protein (LBP), a more stable biomarker, is an acute phase protein produced mostly by the hepatocytes and intestinal epithelial cells and is an essential component of an effective innate immune response to LPS. Circulating LBP significantly enhances the sensitivity of CD14 + cells (mostly monocytes and macrophages) to stimulation by LPS. LBP has been shown to facilitate binding of LPS to CD14 receptor. Levels of LBP peak in serum shortly after endotoxemia and remain increased up to 72 h later. An increased plasma LBP indicates gut epithelial barrier dysfunction [2]. Whether LBP is altered by CKD is not known. METHOD We analyzed the effects of CKD on LBP plasma concentrations. We used samples from the Leuven mild-to-moderate CKD cohort (NCT00441623). To study causality, we used animal models of experimentally induced CKD. To exclude model-related bias, two different rat models of CKD, i.e. 5/6th nephrectomy and adenine supplementation, were used. LBP was measured using commercially available ELISA kits (Hycultbiotech, The Netherlands). RESULTS In a cohort of 460 patients with CKD, we found a significant increase in LBP levels across the different stages of CKD (ANOVA, P: 0.001). When analyzed as a continuous variable, LBP is significantly inversely correlated with eGFR (P &amp;lt; 0.001; Spearman, r: −0.221), and positively correlated with CRP (P &amp;lt; 0.001; Spearman, r: 0.592). During a median follow-up of 56 (IQR, 53–59) months, 70 patients died, with more deaths observed among patients with LBP in higher tertiles [tertiles 1–3: 11, 22, and 37 events, respectively (Fig. 1)]. In univariate Cox proportional hazard analysis, plasma LBP was significantly associated with mortality [hazard ratio (HR) of 1.032; 95% confidence interval (95% CI): 1.014– 1.046; P &amp;lt; 0.001). This association remained significant in multivariate models with adjustment for age, sex and BMI. In two different rat models, induction of CKD resulted in a significant increase in LBP (P &amp;lt; 0.001). In these animals, we observed a significant inverse association between eGFR (and measured creatinine clearance) and LBP concentrations (P &amp;lt; 0.001; Spearman, r: −0.460). CONCLUSION Patients with CKD have higher levels of LBP, with higher levels of plasma LBP present in patients with more advanced CKD. Plasma LBP is an independent predictor of mortality. Experimentally induced CKD equally results in significantly increased LBP. Our data suggest that CKD leads to increased passage of LPS across the intestinal barrier. These findings strengthen the hypothesis of the gut–kidney axis as a source of chronic inflammation.

Abstract 391: Dissecting The Role Of Macrophage Cd36 In Resolution Of Inflammation

Monocytes and macrophages are implicated in cardiovascular disease, including atherosclerotic plaque progression and regression. The scavenger receptor CD36, which is highly expressed in macrophages, mediates lipid uptake into macrophages and promotes clearance of apoptotic and dead cells. Moreover, CD36 as multi-ligand scavenger receptor binds both fatty acids and lipoproteins, which differ in their effects on macrophage phenotype. Some in vitro experiments have reported that CD36-mediated uptake of lipids convert macrophages to an inflammatory phenotype, whereas other studies show that lipid uptake converts macrophages to an alternatively activated state. To determine the role of this receptor in vivo where cells are exposed to both lipoproteins and free fatty acids, we created macrophage-specific CD36 knockout mice. Using zymosan-induced peritonitis to challenge immune cells, we found a broad heterogeneity of resident and infiltrated immune cells, primarily macrophages, in the peritoneum 72h after the initial stimuli. Macrophage CD36 deficient cells had increased expression of pathways related to innate immunity, antigen presentation and oxidative phosphorylation; whereas pathways related to efferocytosis adaptive immunity and B cell activation were downregulated. Gene signatures related to pro-inflammatory features were evident only in CD36 deficient macrophages. Macrophage efferocytosis capacity and B cell antibody production did not differ between control and CD36 depleted mice. Our data show that CD36 expression allows macrophages to convert to a less inflammatory state.