Macrophage Accumulation Research Articles

Abstract 2067: Myeloid Cell Lgr4 Contributes To Atherosclerotic Lesion Formation

Background: R-Spondin 2 (RSPO2), a matricellular protein, primarily functions via binding to cell surface receptors including leucine-rich repeat-containing G protein-coupled receptor (LGR) 4, 5, and 6. We previously identified elevated RSPO2 levels in both human and mouse atherosclerotic arteries. Our preliminary experiments demonstrated increased expression of LGR4 in human atherosclerotic aortic segments, identified Lgr4 as the major RSPO2’s receptor in primary murine macrophages, and atherogenic molecule oxLDL stimulated LGR4 levels in macrophages. However, the precise role of macrophage LGR4 in atherosclerosis development is unknown. Methods and Results: We generated novel myeloid cell-specific Lgr4 knockout mice ( Lgr4 fl/fl LysM Cre +/- , Lgr4 ΔM ) by breeding Lgr4 fl/fl mice with LysM Cre +/- mice. AAV8-h PCSK9 -injected Lgr4 ΔM and littermate control Lgr4 fl/fl ( Lgr4 WT ) were fed a Western diet (16 weeks) to investigate for atherosclerosis. Oil red O (ORO) staining of whole aortas (en face) and aortic root sections showed reduced atherosclerosis in male Lgr4 ΔM mice compared with sex-matched Lgr4 WT mice. Further histochemical staining performed on aortic root sections revealed smaller necrotic cores in Lgr4 ΔM mice. However, there were no significant differences in plasma total cholesterol and body weight. Interestingly, male Lgr4 ΔM mice had decreased fat mass percentage, blood glucose, and epididymal adipose tissue weight. Initial studies with female mice ( n = 4/group) demonstrated no differences in atherosclerosis and metabolic parameters. Additional in vitro studies exhibited that RSPO2 treatment induces oxLDL uptake, reduces efferocytic capacity, and promotes activation of VASP protein (inducer of actin aggregates) in macrophages. Further, the deletion of Lgr4 reduces lipid accumulation in macrophages. Conclusions: Taken together, these findings suggest that deletion of myeloid cell Lgr4 suppresses atherosclerotic lesion formation and identify Lgr4 as a novel therapeutic target for atherosclerosis.

Abstract 3058: Cofilin 1 Mediates Fatty Acid-induced Foam Cell Formation And Inflammation In Mouse Bone Marrow-derived Macrophages

Background: Macrophages contribute to the development of atherosclerosis, in part through the accumulation of lipid droplets and subsequent transformation into highly inflammatory, lipid laden foam cells. Our previous work suggested that the RAGE/DIAPH1 axis plays a key role in regulating lipid metabolism, as deletion of Diaph1 in hepatocytes reduced nuclear translocation of SREBP1 through the actin cytoskeleton. Cofilin 1 (CFL1) is an actin polymerization molecule which has also been linked to the nuclear translocation of SRBP1, as well as triglyceride synthesis through activation of phospholipase D1. In the aforementioned study by our group, Diaph1 deletion was found to increase the Serine 3 phosphorylation (i.e., inactivation) of Cfl1 . Hypothesis: Accordingly, we hypothesized that CFL1 plays a role in lipid droplet accumulation in macrophages, potentially increasing the risk of atherosclerotic development. Methods: To investigate its role in macrophage lipid handling, we silenced Cfl1 in bone marrow derived macrophages (BMDMs) extracted from male C57BL/6J mice, using silencing RNAs and scramble controls. BMDMs were treated with a mixture of fatty acids palmitate and oleate (250 uM). The effects of Cfl1 knockdown on lipid accumulation were assessed using immunohistochemical staining (Bodipy). Quantitative (q)PCR was used to interrogate the mechanisms underlying these effects, and their impact on inflammation. Results: Cfl1- silenced (75% knockdown shown by qPCR) vs. scramble cells exhibited a significant decrease in the ratio of Bodipy to DAPI % area (p=0.0001). By qPCR, compared to scramble controls, BMDMs subjected to silencing of Cfl1 and incubated with fatty acids (palmitate-oleate) displayed significantly lower mRNA expression of Cd36 (p=0.0018) ; Ppargc1a (p=0.004) , Il6 (p<0.0001), and Tnfa (p=0.013). Conclusions: Our results indicate that silencing of Cfl1 leads to a decrease in lipid accumulation and markers of inflammation in BMDMs. Collectively, our data suggest that the effects of Cfl1 silencing on lipid handling result from decreased fatty acid uptake, which limits fatty acid oxidation on account of fatty acid availability in the cell. Studies are underway to probe the implications of these findings in atherosclerosis.

Abstract 1061: The Phospholipase cγ2 And Protein Kinase cα Signaling Axis Is A Key Regulator Of The Extracellular Digestion Of Lipoprotein Aggregates By Macrophages

Atherosclerosis is characterized by cholesterol ester accumulation in macrophages, leading to foam cell formation. In atherosclerotic lesions, most lipoproteins are aggregates (agLDL) that are tightly crosslinked to the extracellular matrix, making digesting them by endocytosis or phagocytosis difficult. Thus, it has been unclear how the cholesteryl esters in the core of agLDL are hydrolyzed. Our laboratory discovered a novel mechanism macrophages use to digest agLDL and generate free cholesterol in an extracellular, acidic, hydrolytic compartment known as the lysosomal synapse. Macrophages form a tight seal around agLDL through actin polymerization and deliver lysosomal contents into this space in a process termed digestive exophagy. Vacuolar ATPase on the plasma membrane lowers the pH of the lysosomal synapse, enabling lysosomal acid lipase (LAL) activity. LAL generates free cholesterol, which accumulates in macrophages and leads to foam cell formation. Our laboratory has begun to characterize the signaling pathways that regulate digestive exophagy, having previously identified TLR4 activation of MyD88/Syk as critical. Here, our goal was to identify digestive exophagy regulators downstream of Syk. Syk activates Bruton’s tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). We tested the hypothesis that Syk phosphorylates and activates PLCγ2 during digestive exophagy. Using pharmacological treatment and genetic manipulation, we found that PLCγ2 and to a lesser extent BTK regulate digestive exophagy. We confirmed that inhibition of Syk or BTK leads to a loss of PLCγ2 activation. PLCγ2 cleaves PI(4,5)P 2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ). Soluble IP 3 then activates the release of Ca 2+ from the endoplasmic reticulum (ER) into the cytoplasm. We demonstrated that Ca 2+ release from the ER is upregulated by agLDL and plays a role in digestive exophagy. Both DAG and Ca 2+ activate protein kinase Cα (PKCα). We also found that PKCα regulates digestive exophagy. In conclusion, we identified new signaling mechanisms regulating digestive exophagy downstream of Syk. We hope that expanding our understanding of the mechanisms of digestive exophagy will be helpful in identifying therapeutics to slow atherosclerosis.

Abstract 2139: Galectin-3 Plays A Critical Intracellular Role In Macrophages By Promoting The Repair, Removal, And Replacement Of Damaged Lysosomes And Protecting Against Atherosclerosis

Galectin-3 (Gal3) is a β-galactoside-binding lectin predominantly known as a secreted inflammatory biomarker in cardiovascular disease, with significantly elevated circulating levels in patients with atherosclerosis and myocardial infarction. However, the molecular mechanisms of Galectin-3 action and whether its intracellular role contributes to atherogenesis remain unclear. Using several databases of bulk and single cell RNAseq, we first show that Gal3 transcripts are upregulated during plaque progression with particular abundance in the myeloid/macrophage lineage and foamy macrophages. Furthermore, Gal3 transcripts correlate significantly with increases in a network of lysosomal genes, suggesting a possible link between macrophage Gal3 and lysosomal function. Indeed, instigation of lysosome membrane damage in primary macrophages via cholesterol crystals and LLOMe triggers robust recruitment of Gal3 to lysosomes by sensing exposed carbohydrate moieties of proteins including Lamp1. Gal3 recruitment initiates a two-pronged lysosomal recovery program composed of either lysosomal repair involving the ESCRT complex or lysosomal removal and replacement involving autophagy/lysophagy and TFEB-mediated lysosomal biogenesis. Gal3-KO macrophages corroborate these findings displaying blunted ESCRT recruitment, diminished autophagy and TFEB activation, and resultant accumulation of damaged lysosomes. Gal3-deficiency also results in enhanced apoptosis and inflammasome/IL-1β activation upon triggering of lysosomal stress in macrophages. These findings are recapitulated in vivo, where Gal3-null bone marrow transplanted in atherogenic LDLR-null mice yields increased lesion size as well as altered plaque composition with macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of the advanced plaque. Taken together, our data implicate unique and previously unrecognized protective function for intracellular Galectin-3 in macrophages and atherosclerosis which are distinct from its traditional role as an inflammatory biomarker in cardiovascular disease.

Abstract 3013: Indoleamine 2,3-dioxygenase-mediated Tryptophan Catabolism Limits Experimental Abdominal Aortic Aneurysms

Background: Indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression. Genetic IDO deficiency limits angiotensin II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic mice. We investigated the influence of IDO inhibition or IDO-catabolized tryptophan catabolite on elastase-induced AAAs in normolipidemic mice. Methods: AAAs were induced in male C57B/6J mice via intra-aortic porcine pancreatic elastase (PPE) infusion. Mice were treated with IDO inhibitor 1-methyl-DL-tryptophan (MT, 2 mg/ml in drinking water), tryptophan metabolite 3-hydroxyanthranilic acid (HAA, 200 mg/kg, oral gavage), or vehicle, starting 3 days prior to PPE infusion for 17 days. AAAs were assessed via ultrasonography and histopathology at sacrifice. Results: The Figure summarizes aortic diameters and histopathology in differentially treated PPE-infused mice. Rapid aortic enlargement was noted in MT-, as compared to vehicle-treated mice following PPE infusion. AAA rupture, unusual for this model, was noted in MT-treated AAA mice. No difference was noted between groups in medial elastin degradation, smooth muscle cell depletion, or macrophage accumulation. However, mural neoangiogenesis and lymphocyte accumulation were remarkably augmented in MT-treated mice. HAA Treatment attenuated aortic enlargement in conjunction with improved medial elastin and smooth muscle cell retention and reduced aortic leukocyte accumulation and neovessels. Conclusion: IDO activity and its tryptophan metabolite HAA limit experimental AAA progression in the PPE model. These results provide further support for investigating tryptophan metabolites in clinical AAA disease suppression.

Mir-146a is required for foreign body response and giant cell formation

The implantation of biomaterials or medical devices often leads to the development of a foreign body response (FBR), an inflammatory condition that can lead to implant failure, which may cause harm to or death of the patient. Hallmarks of the FBR include accumulation of macrophages and destructive foreign body giant cells (FBGCs), and development of fibrous scar tissue encompassing the implant. There are no effective medical treatments for FBR, although improved fundamental understanding of the molecular mechanisms underlying the development of FBR may lead to novel strategies for the amelioration of FBR. MicroRNAs (miRs) are endogenous, small, non-coding RNAs that have emerged as powerful regulators of gene expression in numerous cellular processes including macrophage activation, inflammation, and fibrosis. In this research, we tested the hypothesis that specific miRs play a crucial role in regulating the FBR to biomaterials. In our subcutaneous implantation mouse model, we found that: 1) miR-146a expression levels decreased in the implant-adhered tissues, and the decrease correlated with increased macrophage accumulation, FBGC formation, and collagen accumulation; and 2) miR-146a deletion in mice exacerbated in vivo macrophage accumulation, FBGC formation, and collagen accumulation. In our in vitro model, we found that deficiency of miR-146a function exacerbated key macrophage functions including adhesion on stiff matrix, and FBGC formation in primary mouse macrophages. In essence, our findings suggest that miR-146a might act as a suppressor of FBR. We expect that the results of this study may provide invaluable information and insight regarding the molecular mechanisms mediating the FBR to biomaterials, which may lead to the development of a novel microRNA-based therapeutic strategy for the amelioration of the poorly understood FBR to biomaterials. NIH (R01 AI172086) grant to Shaik O. Rahaman. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2070: Myeloid Smyd2 Deficiency Exacerbates Atherosclerosis Via Promoting Plaque Necrosis And Calcification

Introduction: The accumulation of lipoprotein-laden macrophages within arterial walls marks the earliest phase of atherosclerotic cardiovascular disease, giving rise to foam cells. Here, we aim to define the previously unexplored role of myeloid specific SMYD2 (a histone lysine methyltransferase) in atherosclerosis and plaque formation. Methods: Myeloid-specific Smyd2 knockout mice in ApoE -/- background (Smyd2 mKO ApoE -/- ) and their littermate controls (Smyd2 fl/fl ApoE -/- ) were subjected to Western diet (WD) feeding for up to 26 weeks. Atherosclerotic plaque distribution was examined by en-face Oil Red O (ORO) staining of freshly collected aortas. Hematoxylin & Eosin (H&E) staining on brachiocephalic artery (BCA) tissues were performed to determine total plaque size, lipid pool area, and necrotic core area. Plaque calcification was determined by von Kossa staining and Alizarin Red S staining. Primary bone marrow derived macrophages (BMDMs) isolated from Smyd2 fl/fl ApoE -/- and Smyd2 mKO ApoE -/- mice and cultured RAW 246.7 cells (with Smyd2 knock-down) were used for in vitro studies including calcification assay, PR-PCR, and Western blot analysis. Results: Myeloid Smyd2 deficient mice demonstrated significantly increased aortic plaque burden on ORO staining as compared to control mice following WD feeding, though with slower WD-induced body weight gain relative to Smyd2 fl/fl ApoE -/- control mice. H&E staining of BCA indicated that there was significant reduction in total lumen and lipid core area, while significantly increased total plaque and necrotic core in Smyd2 mKO ApoE -/- mice. Myeloid Smyd2 deletion promotes plaque calcification in mice fed with WD, and in cultured BMDMs and RAW 246.7 cells treated with calcification promoting media. Gene expression analysis of key calcification factors of aortic tissues and cultured cells showed significant upregulation of Sox9 and Msx2 genes in aortas of WD-fed Smyd2 mKO ApoE -/- mice and cultured Smyd2 deficient cells, as compared to controls. Conclusion: We delineated the consequences of Smyd2 deficiency in macrophages on atherosclerotic plaque development, particularly its impact on plaque necrosis and calcification.

Modified Zhenwu Decoction suppresses chronic colitis via targeting macrophage CCR2/Fyn/p38 MAPK signaling axis

BackgroundUlcerative colitis (UC) is associated with intestinal macrophage infiltration due to disruption of the mucosal barrier and bacterial invasion. Therefore, it is crucial to identify therapeutic agents capable of attenuating the macrophage-induced inflammatory response to preserve mucosal homeostasis and immune tolerance. The modified Zhenwu decoction (CDD-2103) is a novel herbal formulation developed based on the principles of Traditional Chinese medicine. To date, there are no clinically approved herbal formulations for UC with a well-known mechanism of action on macrophages. PurposeThe objective of this study was to systematically investigate the inhibitory effect of the active fraction of CDD-2103 in a mouse model of chronic colitis and delineate the mechanisms underlying its inhibitory action. MethodsCDD-2103 was extracted into four fractions using organic solvents with increasing polarity. A chronic 49-day dextran sulfate sodium (DSS)-induced colitis mice model, closely resembling human clinical conditions, was used to examine the effect of CDD-2103 on chronic colitis. To confirm the effect of CDD-2103 on macrophages in this chronic colitis model, adoptive macrophage transfer and CCL2 supplementation were conducted. The mechanisms of action of CDD-2103 were further elucidated utilizing bone marrow-derived macrophages (BMDMs). Transcriptome analysis was conducted to gain insights into the underlying mechanism of action of CDD-2103 in BMDMs. ResultsOur in vitro and in vivo findings demonstrated that the ethanol-enriched fraction of CDD-2103 exhibited significant anti-inflammatory effects, leading to the suppression of colitis severity. This effect was associated with diminished accumulation of colonic macrophages in the lamina propria of CDD-2103-intervened colitis mice. Specifically, CDD-2103 inhibited CCR2/L2-mediated proinflammatory macrophage infiltration into the colon without affecting macrophage proliferation. Mechanistically, CDD-2103 inhibited Fyn expression-mediated p38 MAPK activation and subsequently suppressed CCR2 expression in BMDMs. ConclusionsCollectively, our study supports the potential use of CDD-2103 to limit macrophage infiltration, thereby reducing inflammation during UC treatment. CDD-2103 and the components in the ethanolic fraction are promising candidates for the development of novel drugs for UC management. Additionally, our study underscores Fyn-mediated CCR2 expression as a potential therapeutic target for the management of UC.

Abstract 1015: Knockout Of P27 Kip1 Accelerates Topical Elastase/3-aminopropionitrile Fumarate Salt-induced Abdominal Aortic Aneurysm Rupture In Mice

Background: Abdominal aortic aneurysms (AAAs) are multifactorial diseases, and unknown genetic factors could contribute to aneurysms. The cell cycle inhibitor CDKN1B gene, also known as p27 Kip1 (p27), affects the arterial response to injury. Previous studies have shown that the loss of p27 exacerbates atherosclerosis progression by increasing vascular smooth muscle cells and macrophages. To assess the role of p27 in AAA development, another form of arterial response to inflammatory injury, we conducted experiments on the AAA model using p27 knockout (p27 -/- ) and wild-type (WT) mice. Methods: AAAs were induced in male and female p27 -/- and WT (C57BL/6J) mice by topically applying porcine pancreatic elastase and administrating 0.2% 3-aminopropionitrile fumarate salt (BAPN) in the drinking water. Topical elastase application was divided into active and heat-inactivated elastase groups. The aortic diameter was evaluated by ultrasound examination preoperatively and every 7 days postoperatively ( Fig. A ). Surviving mice were sacrificed after the final ultrasound evaluation, and aortic samples were collected for histological analysis. Results: Fig. B shows the aortic diameter change evaluated by ultrasound examination. AAA rupture occurred in one male p27-/- mice (33%, 1 of 3) and two female p27-/- mice (40%, 2 of 5), but in no WT mice of either gender. Macrophages were assessed using anti-F4/80 antibody. In the elastase-active group, both male and female p27 -/- mice had a significantly higher accumulation of macrophage than WT mice (P<0.01 and P<0.01, respectively), but there was no significant staining of F4/80 in the elastase-inactive group ( Fig. C and D ). Conclusions: Knockout of p27 exacerbated aneurysm rupture in a topical elastase/BAPN-induced AAA model and was associated with increased macrophage infiltration.

Abstract 2010: Atherosclerotic Plaque Macrophages Drive Abdominal Aortic Aneurysm Formation

Introduction: Cigarette smoking (CS) contributes to the most deaths in abdominal aortic aneurysms (AAA) and tobacco use associates with early damage to the abdominal aorta. Associated risk factors, such as hyperlipidemia, link clinical AAA to the atherosclerotic process, suggesting common pathogenetic features with plaque development. We hypothesized that CS exposure exacerbates atherosclerotic disease in the abdominal aorta by increasing the recruitment of inflammatory macrophages that mediate arterial degradation and AAA formation. Methods: Adult male Apoe deficient mice commenced a high cholesterol diet and were concomitantly exposed to CS or room air (RA) for 4, 8, 12, and 16 weeks. As plaque macrophages are highly prevalent in advanced AAA, additional CS exposed mice received a selective CSF1R inhibitor, PLX3397, for up to 16 weeks to deplete macrophages. AAA incidence, atherosclerotic plaque burden and lesion composition were assessed in the aorta by immunofluorescence, Movat, EVG, and Oil Red O staining. Results: CS induced AAA at all timepoints with the highest incidence of 37% at 16 weeks of exposure (n=151 CS, n=75 RA p< 0.05). Aneurysms always coincided with atherosclerosis (n=7 CS, n=7 CS/AAA p<0.05), and severe elastin fragmentation was consistently overlaid by plaque (n=20 CS p<0.05, Fig 1A and B). In some cases, lesions also associated with aortic rupture, causing death in ~11% of animals (n = 35 RA, n = 46 CS p<0.04). CD68+ macrophages associated highly with severe elastin damage but less at intact regions (2.6-fold p<0.0001, Fig 1C). PLX3397-mediated macrophage depletion attenuated plaque development and prevented AAA (n=20 treated, n=27 control p<0.05, Fig 1D). Conclusion: CS exacerbates atherosclerosis and increases the accumulation of macrophages at sites of arterial injury. Depletion of macrophages results in a lack of aneurysm formation, highlighting their capacity to directly injure the aortic wall and necessity to mediate AAA.

Abstract 3076: Cd36 Restricts Lipid-associated Macrophages Accumulation In White Adipose Tissues During Atherogenesis

Visceral white adipose tissue (WAT) regulates systemic lipid metabolism and inflammation. Dysfunctional WAT drives chronic inflammation and facilitates atherosclerosis. Adipose tissue macrophages (ATMs) are the predominant immune cells in WAT, but their heterogeneity and phenotypes are poorly defined during atherogenesis. The scavenger receptor CD36 mediates ATMs crosstalk with other cells, driving chronic inflammation. Here, we conducted single-cell RNA sequencing on major WAT ATM subpopulations using a diet-induced atherosclerotic mouse model ( Apoe -null). We also examined the role of CD36 using Apoe / Cd36 double-null mice. Our results indicate that ATMs are constantly under lipid stress. We identified lipid-associated macrophages (LAMs), which were previously described in obesity. Interestingly, LAMs increased 8.4-fold in Apoe / Cd36 double-null mice on an atherogenic diet, but not in Apoe -null mice. The increase in LAM was accompanied by more ATM lipid uptake, reduced adipocyte hypertrophy, and less inflammation, which indicate a delicate balance between lipid metabolism and inflammation, mediated through CD36.

Glycoprotein VI Platelet Receptor Blockade Attenuates Progression of Established Abdominal Aortic Aneurysms in Murine Models

Objective: Abdominal aortic aneurysms (AAA) frequently feature the formation of a nonocclusive intraluminal thrombus (ILT) along the site of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation via the glycoprotein VI receptor pathway impacts the pathogenesis of AAA. Methods and Results: RNA sequencing was performed on age-matched control human aortic tissue, AAA aortic tissue, and AAA ILT. Platelet transcripts comprised 25% of significantly enriched genes in the comparison of AAA thrombus to AAA tissue and control aortic wall, with GPVI increased by 9.1 Log2-FC (P = 3.21E−08). Circulating GPVI was significantly elevated in platelets isolated from AAA patients versus age-matched controls. Soluble GPVI (sGPVI—indicator of platelet activation), was significantly increased in the plasma of patients with fast-growing AAAs compared to slow-growing AAAs and healthy control subjects. To determine if GPVI blockade was protective from AAA progression, mice underwent either the angiotensin II (AngII) infusion or the topical elastase models of aneurysm formation. Following model initiation, aortic diameter was monitored by weekly ultrasounds. After two weeks, mice which developed an aneurysm (defined as an aortic diameter of >1.2mm) were randomized into control (IgG—50μg) or treatment (JAQ1—50μg, monoclonal GPVI antibody) groups. As expected, platelets isolated from JAQ1 treated mice did not activate in response to the GPVI specific agonist convulxin. Furthermore, JAQ1 treatment attenuated aneurysm progression, increased type I collagen deposition, attenuated macrophage accumulation, and increased survival in both murine AAA models versus IgG control treated mice. Conclusions: Platelets play a critical role in AAA pathophysiology and specific blockade of the GPVI-mediated activation pathway attenuates progression of established aneurysms in two independent murine models. Thus, our work demonstrates that specific targeting of GPVI to blunt platelet activation in AAA could be a potential therapeutic for a pathology with no current pharmacological treatments. R01HL147171-04 and R01HL158801-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transcriptomic and developmental effects of persistent organic pollutants in sentinel fishes collected near an arctic formerly used defense site

Alaska contains over 600 formerly used defense (FUD) sites, many of which serve as point sources of pollution. These sites are often co-located with rural communities that depend upon traditional subsistence foods, especially lipid-rich animals that bioaccumulate and biomagnify persistent organic pollutants (POPs). Many POPs are carcinogenic and endocrine-disrupting compounds that are associated with adverse health outcomes. Therefore, elevated exposure to POPs from point sources of pollution may contribute to disproportionate incidence of disease in arctic communities. We investigated PCB concentrations and the health implications of POP exposure in sentinel fishes collected near the Northeast Cape FUD site on Sivuqaq (St. Lawrence Island), Alaska. Sivuqaq residents are almost exclusively Yupik and rely on subsistence foods. At the request of the Sivuqaq community, we examined differential gene expression and developmental pathologies associated with exposure to POPs originating at the Northeast Cape FUD site. We found significantly higher levels of PCBs in Alaska blackfish (Dallia pectoralis) collected from contaminated sites downstream of the FUD site compared to fish collected from upstream reference sites. We compared transcriptomic profiles and histopathologies of these same blackfish. Blackfish from contaminated sites overexpressed genes involved in ribosomal and FoxO signaling pathways compared to blackfish from reference sites. Contaminated blackfish also had significantly fewer thyroid follicles and smaller pigmented macrophage aggregates. Conversely, we found that ninespine stickleback (Pungitius pungitius) from contaminated sites exhibited thyroid follicle hyperplasia. Despite our previous research reporting transcriptomic and endocrine differences in stickleback from contaminated vs. reference sites, we did not find significant differences in kidney or gonadal histomorphologies. Our results demonstrate that contaminants from the Northeast Cape FUD site are associated with altered gene expression and thyroid development in native fishes. These results are consistent with our prior work demonstrating disruption of the thyroid hormone axis in Sivuqaq residents.

Anguilla anguilla vs Contracaecum rudolphii: Granuloma allows host tolerance and parasite survival

High survival of the nematode Contracaecum rudolphii in European eel, Anguilla anguilla L. despite an extensive recruitment of immune cells in granulomas is described. A total of 39 eel specimens captured in Canneviè lagoon (Northern Adriatic Sea, Italy) was obtained from local fishermen. The fish were examined for the presence of endoparasitic helminths. Twenty-six eels (67%) harbored third-stage larvae (L3) of Contracaecum rudolphii A and the intensity of infection ranged from 1 to 100 parasites per fish (mean 11.4). A total of 296 larvae were counted and 293 were alive and encysted in the serosa (primarily on external surface of the rectum) as conspicuous granulomas, appearing as white nodules. Histological sections showed a granuloma formed around an individual larva and consisted of three concentric cell layers, containing numerous mast cells (MCs), fibroblasts, macrophages, and epithelioid cells. Encysted larvae were also present in other layers of the intestinal wall, including the mucosa, where recruitment of macrophage aggregates in lamina propria was documented. Immunohistochemical investigations, applying eight antibodies raised against immune cell markers, revealed histamine and serotonin in MCs and inducible-nitric oxide synthase and toll-like receptor-2 in macrophages surrounding the parasite larvae. The study indicates that this innate cellular response in the eel has a chronic inflammatory character and keeps the nematode larvae arrested in a state allowing both host tolerance and parasite survival. The European eel serves as prey for birds and the high survival of C. rudolphii A larvae in the eel indicates that this fish species is a good paratenic host for the parasite allowing completion of its life cycle in birds.

PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis

Background and objectiveProgranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment.MethodsImmunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization.ResultsThe IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization.ConclusionsIn summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.

Photosensitive AIEgens sensitize bacteria to oxidative damage and modulate the inflammatory responses of macrophages to salvage the photodynamic therapy against MRSA

The urgent need for antimicrobial agents to combat infections caused by multidrug-resistant bacteria facilitates the exploration of alternative strategies such as photosensitizer (PS)-mediated photoinactivation. However, increasing studies have discovered uncorrelated bactericidal activities among PSs possessing similar photodynamic and pathogen-targeted properties. To optimize the photodynamic therapy (PDT) against infections, we investigated three type-I PSs of D-π-A AIEgens TI, TBI, and TTI. The capacities of reactive oxygen species (ROS) generation of TI, TBI, and TTI did not align with their bactericidal activities. Despite exhibiting the lowest photodynamic efficiency, TI exhibited the highest activities against methicillin-resistant Staphylococcus aureus (MRSA) by impairing the anti-oxidative responses of bacteria. By comparison, TTI, characterized by the strongest ROS production, inactivated intracellular MRSA by potentiating the inflammatory response of macrophages. Unlike TI and TTI, TBI, despite possessing moderate photodynamic activities and inducing ROS accumulation in both MRSA and macrophages, did not exhibit any antibacterial activity. Therefore, relying on the disturbed anti-oxidative metabolism of pathogens or potentiated host immune responses, transient ROS bursts can effectively control bacterial infections. Our study reevaluates the contribution of photodynamic activities of PSs to bacterial elimination and provides new insights into discovering novel antibacterial targets and agents.

Macrophages in guided bone regeneration: potential roles and future directions.

Guided bone regeneration (GBR) is one of the most widely used and thoroughly documented alveolar bone augmentation surgeries. However, implanting GBR membranes inevitably triggers an immune response, which can lead to inflammation and failure of bone augmentation. It has been shown that GBR membranes may significantly improve in vivo outcomes as potent immunomodulators, rather than solely serving as traditional barriers. Macrophages play crucial roles in immune responses and participate in the entire process of bone injury repair. The significant diversity and high plasticity of macrophages complicate our understanding of the immunomodulatory mechanisms underlying GBR. This review provides a comprehensive summary of recent findings on the potential role of macrophages in GBR for bone defects in situ. Specifically, macrophages can promote osteogenesis or fibrous tissue formation in bone defects and degradation or fibrous encapsulation of membranes. Moreover, GBR membranes can influence the recruitment and polarization of macrophages. Therefore, immunomodulating GBR membranes are primarily developed by improving macrophage recruitment and aggregation as well as regulating macrophage polarization. However, certain challenges remain to be addressed in the future. For example, developing more rational and sophisticated sequential delivery systems for macrophage activation reagents; addressing the interference of bone graft materials and dental implants; and understanding the correlations among membrane degradation, macrophage responses, and bone regeneration.

A machine learning algorithm improves the diagnostic accuracy of the histologic component of antibody mediated rejection (AMR-H) in cardiac transplant endomyocardial biopsies

BackgroundPathologic antibody mediated rejection (pAMR) remains a major driver of graft failure in cardiac transplant patients. The endomyocardial biopsy remains the primary diagnostic tool but presents with challenges, particularly in distinguishing the histologic component (pAMR-H) defined by 1) intravascular macrophage accumulation in capillaries and 2) activated endothelial cells that expand the cytoplasm to narrow or occlude the vascular lumen. Frequently, pAMR-H is difficult to distinguish from acute cellular rejection (ACR) and healing injury. With the advent of digital slide scanning and advances in machine deep learning, artificial intelligence technology is widely under investigation in the areas of oncologic pathology, but in its infancy in transplant pathology. For the first time, we determined if a machine learning algorithm could distinguish pAMR-H from normal myocardium, healing injury and ACR. Materials and MethodsA total of 4,212 annotations (1,053 regions of normal, 1,053 pAMR-H, 1,053 healing injury and 1,053 ACR) were completed from 300 hematoxylin and eosin slides scanned using a Leica Aperio GT450 digital whole slide scanner at 40X magnification. All regions of pAMR-H were annotated from patients confirmed with a previous diagnosis of pAMR2 (>50% positive C4d immunofluorescence and/or >10% CD68 positive intravascular macrophages). Annotations were imported into a Python 3.7 development environment using the OpenSlide™ package and a convolutional neural network approach utilizing transfer learning was performed. ResultsThe machine learning algorithm showed 98% overall validation accuracy and pAMR-H was correctly distinguished from specific categories with the following accuracies: normal myocardium (99.2%), healing injury (99.5%) and ACR (99.5%). ConclusionOur novel deep learning algorithm can reach acceptable, and possibly surpass, performance of current diagnostic standards of identifying pAMR-H. Such a tool may serve as an adjunct diagnostic aid for improving the pathologist's accuracy and reproducibility, especially in difficult cases with high inter-observer variability. This is one of the first studies that provides evidence that an artificial intelligence machine learning algorithm can be trained and validated to diagnose pAMR-H in cardiac transplant patients. Ongoing studies include multi-institutional verification testing to ensure generalizability.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

CD226 promotes renal fibrosis by regulating macrophage activation and migration.

It has been found that CD226 plays an important role in regulating macrophage function, but its expression and function in macrophages during renal fibrogenesis have not been studied. Our data demonstrated that CD226 expression in macrophages was obviously upregulated in the unilateral ureteral obstruction model, while CD226 deficiency attenuated collagen deposition in renal interstitium along with fewer M1 within renal cortex and renal medulla and a lower level of proinflammatory factors compared to that of control littermates. Further studies demonstrated that Cd226-/- bone marrow-derived macrophages transferring could significantly reduce the tubular injury, collagen deposition, and proinflammatory cytokine secretion compared with that of Cd226+/+ bone marrow-derived macrophages transferring in the unilateral ureteral obstruction model. Mechanistic investigations revealed that CD226 promoted proinflammatory M1 macrophage accumulation in the kidney via suppressing KLF4 expression in macrophages. Therefore, our results uncovered a pathogenic role of CD226 during the development of chronic kidney disease by promoting monocyte infiltration from peripheral blood into the kidney and enhancing macrophage activation toward the inflammatory phenotype by suppressing KLF4 expression.