Ionizable Lipid with Supramolecular Chemistry Features for RNA Delivery In Vivo.

Lipid nanoparticles (LNPs) and ribonucleic acid (RNA) technology are highly versatile tools that can be deployed for diagnostic, prophylactic, and therapeutic applications. In this report, supramolecular chemistry concepts are incorporated into the rational design of a new ionizable lipid, C3-K2-E14, for systemic administration. This lipid incorporates a cone-shaped structure intended to facilitate cell bilayer disruption, and three tertiary amines to improve RNA binding. Additionally, hydroxyl and amide motifs are incorporated to further enhance RNA binding and improve LNP stability. Optimization of messenger RNA (mRNA) and small interfering RNA (siRNA) formulation conditions and lipid ratios produce LNPs with favorable diameter (<150 nm), polydispersity index (<0.15), and RNA encapsulation efficiency (>90%), all of which are preserved after 2 months at 4 or 37 °C storage in ready-to-use liquid form. The lipid and formulated LNPs are well-tolerated in animals and show no deleterious material-induced effects. Furthermore, 1 week after intravenous LNP administration, fluorescent signal from tagged RNA payloads are not detected. To demonstrate the long-term treatment potential for chronic diseases, repeated dosing of C3-K2-E14 LNPs containing siRNA that silences the colony stimulating factor-1 (CSF-1) gene can modulate leukocyte populations in vivo, further highlighting utility.

A comparative gene expression matrix in Apoe-deficient mice identifies unique and atherosclerotic disease stage-specific gene regulation patterns in monocytes and macrophages.

BACKGROUND AND AIMS: Atherosclerosis is a systemic and chronic inflammatory disease propagated by monocytes and macrophages. Yet, our knowledge on how transcriptome of these cells evolves in time and space is limited. We aimed at characterizing gene expression changes in site-specific macrophages and in circulating monocytes during the course of atherosclerosis. METHODS: We utilized apolipoprotein E-deficient mice undergoing one- and six-month high cholesterol diet to model early and advanced atherosclerosis. Aortic macrophages, peritoneal macrophages, and circulating monocytes from each mouse were subjected to bulk RNA-sequencing (RNA-seq). We constructed a comparative directory that profiles lesion- and disease stage-specific transcriptomic regulation of the three cell types in atherosclerosis. Lastly, the regulation of one gene, Gpnmb, whose expression positively correlated with atheroma growth, was validated using single-cell RNA-seq (scRNA-seq) of atheroma plaque from murine and human. RESULTS: The convergence of gene regulation between the three investigated cell types was surprisingly low. Overall 3245 differentially expressed genes were involved in the biological modulation of aortic macrophages, among which less than 1% were commonly regulated by the remote monocytes/macrophages. Aortic macrophages regulated gene expression most actively during atheroma initiation. Through complementary interrogation of murine and human scRNA-seq datasets, we showcased the practicality of our directory, using the selected gene, Gpnmb, whose expression in aortic macrophages, and a subset of foamy macrophages in particular, strongly correlated with disease advancement during atherosclerosis initiation and progression. CONCLUSIONS: Our study provides a unique toolset to explore gene regulation of macrophage-related biological processes in and outside the atheromatous plaque at early and advanced disease stages.

Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity.

During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes by in vivo and ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD in mice. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, Ly6Chigh skewing was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.

Colony stimulating factor-1 producing endothelial cells and mesenchymal stromal cells maintain monocytes within a perivascular bone marrow niche.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1op/op) causes severe musculoskeletal defects that may indirectly affect hematopoiesis. Indeed, we show here that osteolineage-derived Csf1 prevented developmental abnormalities but had no effect on monopoiesis in adulthood. However, ubiquitous deletion of Csf1 conditionally in adulthood decreased monocyte survival, differentiation, and migration, independent of its effects on bone development. Bone histology revealed that monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular mesenchymal stromal cells (MSCs). Targeted deletion of Csf1 from sinusoidal ECs selectively reduced Ly6C- monocytes, whereas combined depletion of Csf1 from ECs and MSCs further decreased Ly6Chi cells. Moreover, EC-derived CSF-1 facilitated recovery of Ly6C- monocytes and protected mice from weight loss following induction of polymicrobial sepsis. Thus, monocytes are supported by distinct cellular sources of CSF-1 within a perivascular BM niche.

BCG vaccination provides protection against IAV but not SARS-CoV-2.

Since the vast majority of species solely rely on innate immunity for host defense, it stands to reason that a critical evolutionary trait like immunological memory evolved in this primitive branch of our immune system. There is ample evidence that vaccines such as bacillus Calmette-Guérin (BCG) induce protective innate immune memory responses (trained immunity) against heterologous pathogens. Here we show that while BCG vaccination significantly reduces morbidity and mortality against influenza A virus (IAV), it fails to provide protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In contrast to IAV, SARS-CoV-2 infection leads to unique pulmonary vasculature damage facilitating viral dissemination to other organs, including the bone marrow (BM), a central site for BCG-mediated trained immunity. Finally, monocytes from BCG-vaccinated individuals mount an efficient cytokine response to IAV infection, while this response is minimal following SARS-CoV-2. Collectively, our data suggest that the protective capacity of BCG vaccination is contingent on viral pathogenesis and tissue tropism.

Macrophage Jak2 deficiency accelerates atherosclerosis through defects in cholesterol efflux.

Atherosclerosis is a chronic inflammatory condition in which macrophages play a major role. Janus kinase 2 (JAK2) is a pivotal molecule in inflammatory and metabolic signaling, and Jak2V617F activating mutation has recently been implicated with enhancing clonal hematopoiesis and atherosclerosis. To determine the essential in vivo role of macrophage (M)-Jak2 in atherosclerosis, we generate atherosclerosis-prone ApoE-null mice deficient in M-Jak2. Contrary to our expectation, these mice exhibit increased plaque burden with no differences in macrophage proliferation, recruitment or bone marrow clonal expansion. Notably, M-Jak2-deficient bone marrow derived macrophages show a significant defect in cholesterol efflux. Pharmacologic JAK2 inhibition with ruxolitinib also leads to defects in cholesterol efflux and accelerates atherosclerosis. Liver X receptor agonist abolishes the efflux defect and attenuates the accelerated atherosclerosis that occurs with M-Jak2 deficiency. Macrophages of individuals with the Jak2V617F mutation show increased efflux which is normalized when treated with a JAK2 inhibitor. Together, M-Jak2-deficiency leads to accelerated atherosclerosis primarily through defects in cholesterol efflux from macrophages.

Adventitial recruitment of Lyve-1- macrophages drives aortic aneurysm in an angiotensin-2-based murine model.

OBJECTIVE: Aortic macrophage accumulation is characteristic of the pathogenesis of abdominal aortic aneurysm (AAA) but the mechanisms of macrophage accumulation and their phenotype are poorly understood. Lymphatic vessel endothelial receptor-1 (Lyve-1+) resident aortic macrophages independently self-renew and are functionally distinct from monocyte-derived macrophages recruited during inflammation. We hypothesized that Lyve-1+ and Lyve-1- macrophages differentially contribute to aortic aneurysm. Approach and results: Angiotensin-2 and ß-aminopropionitrile (AT2/BAPN) were administered to induce AAA in C57BL/6J mice. Using immunohistochemistry (IHC), we demonstrated primarily adventitial accumulation of aortic macrophages, and in association with areas of elastin fragmentation and aortic dissection. Compared with controls, AAA was associated with a relative percent depletion of Lyve-1+ resident aortic macrophages and accumulation of Lyve-1- macrophages. Using CD45.1/CD45.2 parabiosis, we demonstrated aortic macrophage recruitment in AAA. Depletion of aortic macrophages in CCR2-/- mice was associated with reduced aortic dilatation indicating the functional role of recruitment from the bone marrow. Depletion of aortic macrophages using anti-macrophage colony-stimulating factor 1 receptor (MCSF1R)-neutralizing antibody (Ab) reduced the incidence of AAA. Conditional depletion of Lyve-1+ aortic macrophages was achieved by generating Lyve-1wt/cre Csf1rfl/fl mice. Selective depletion of Lyve-1+ aortic macrophages had no protective effects following AT2/BAPN administration and resulted in increased aortic dilatation in the suprarenal aorta. CONCLUSIONS: Aortic macrophage accumulation in AAA derives from adventitial recruitment of Lyve-1- macrophages, with relative percent depletion of Lyve-1+ macrophages. Selective targeting of macrophage subtypes represents a potential novel therapeutic avenue for the medical treatment of AAA.

Dj1 deficiency protects against atherosclerosis with anti-inflammatory response in macrophages.

Inflammation is a key contributor to atherosclerosis with macrophages playing a pivotal role through the induction of oxidative stress and cytokine/chemokine secretion. DJ1, an anti-oxidant protein, has shown to paradoxically protect against chronic and acute inflammation. However, the role of DJ1 in atherosclerosis remains elusive. To assess the role of Dj1 in atherogenesis, we generated whole-body Dj1-deficient atherosclerosis-prone Apoe null mice (Dj1-/-Apoe-/-). After 21 weeks of atherogenic diet, Dj1-/- Apoe-/-mice were protected against atherosclerosis with significantly reduced plaque macrophage content. To assess whether haematopoietic or parenchymal Dj1 contributed to atheroprotection in Dj1-deficient mice, we performed bone-marrow (BM) transplantation and show that Dj1-deficient BM contributed to their attenuation in atherosclerosis. To assess cell-autonomous role of macrophage Dj1 in atheroprotection, BM-derived macrophages from Dj1-deficient mice and Dj1-silenced macrophages were assessed in response to oxidized low-density lipoprotein (oxLDL). In both cases, there was an enhanced anti-inflammatory response which may have contributed to atheroprotection in Dj1-deficient mice. There was also an increased trend of plasma DJ-1 levels from individuals with ischemic heart disease compared to those without. Our findings indicate an atheropromoting role of Dj1 and suggests that targeting Dj1 may provide a novel therapeutic avenue for atherosclerosis treatment or prevention.

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation.

RATIONALE: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT. OBJECTIVE: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms. METHODS AND RESULTS: Ldlr-/- mice underwent total body γ-irradiation, bone marrow reconstitution, and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared with mice without BMT. In BMT mice, neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet for 3, 6, and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation, and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL (low-density lipoprotein) accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70 kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr-/- relative to Ldlr+/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single-cell resolution, was performed to give insights into pathways modulated by BMT. CONCLUSIONS: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single-cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.

Interrupting reactivation of immunologic memory diverts the allergic response and prevents anaphylaxis.

BACKGROUND: IgE production against innocuous food antigens can result in anaphylaxis, a severe life-threatening consequence of allergic reactions. The maintenance of IgE immunity is primarily facilitated by IgG+ memory B cells, as IgE+ memory B cells and IgE+ plasma cells are extremely scarce and short-lived, respectively. OBJECTIVE: Our aim was to investigate the critical requirements for an IgE recall response in peanut allergy. METHODS: We used a novel human PBMC culture platform, a mouse model of peanut allergy, and various experimental readouts to assess the IgE recall response in the presence and absence of IL-4Rα blockade. RESULTS: In human PBMCs, we have demonstrated that blockade of IL-4/IL-13 signaling aborted IgE production after activation of a recall response and skewed the cytokine response away from a dominant type 2 signature. TH2A cells, identified by single-cell RNA sequencing, expanded with peanut stimulation and maintained their pathogenic phenotype in spite of IL-4Rα blockade. In mice with allergy, anti-IL-4Rα provided long-lasting suppression of the IgE recall response beyond antibody treatment and fully protected against anaphylaxis. CONCLUSION: The findings reported here advance our understanding of events mediating the regeneration of IgE in food allergy.

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering.

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression.

A durable murine model of spleen transplantation with arterial and venous anastomoses.

The spleen is a large lymphoid organ located in the abdomen that filters blood and regulates the immune system. The extent of mobilization of splenic immune cells to peripheral tissues in health and disease, however, remains poorly understood. This is due, in large part, to a lack of in vivo, spleen-specific lineage tagging strategies. Here, we describe a detailed practical protocol of spleen transplantation and its evaluation for long-term graft survival. Unlike implantation of splenic morsels in the great omentum, our approach uses arterial and venous anastomoses which rapidly restores blood flow and facilitates long-term survival of the graft. The use of congenic mouse strains permits the use of immunofluorescence and flow cytometry-based methodologies to unambiguously track the migration of spleen-derived cells to peripheral tissues.

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

Publisher Correction: Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

In the version of this article initially published, the equal contribution of the third author was omitted. The footnote links for that author should be "Sara Nejat1,11" and the correct statement is as follows: "11These authors contributed equally: Sarah A. Dick, Jillian A. Macklin, Sara Nejat." The error has been corrected in the HTML and PDF versions of the article.

B-Cell Deficiency Lowers Blood Pressure in Mice.

The proto-oncogene c-myb (and corresponding nuclear transcription factor, c-Myb) regulates the proliferation and differentiation of hematologic and vascular smooth muscle cells; however, the role of c-Myb in blood pressure regulation is unknown. Here, we show that mice homozygous for a hypomorphic c-myb allele ( c-myb h/h) conferring reduced c-Myb activity manifest reduced peripheral blood and kidney B220+ B-cells and have decreased systolic (104±2 versus 120±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 83±1 mm Hg; P<0.0001) compared with WT (wild type) mice. Additionally, c-myb h/h mice had lower susceptibility to deoxycorticosterone acetate-salt experimental hypertension. Although cardiac (echocardiography) and resistance artery (perfusion myography) functions were normal, metabolic cage studies revealed that c-myb h/h mice had increased 24-hour urine output and sodium excretion versus WT. Reconstitution of WT mice with c-myb h/h bone marrow transplant and chimeric bone marrow transplant using mice lacking B-cells ( J H T; h/h>WT and h/h:J H T>WT, respectively) decreased blood pressure and increased 24-hour urine output compared with controls ( WT>WT; WT:J H T>WT). J H T mice also had decreased systolic (103±2 versus 115±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 79±1; P<0.01) and increased 24-hour urine output versus WT. Real-time quantitative reverse transcription polymerase chain reaction of kidney medulla revealed reduced V2R (vasopressin receptor 2) expression in c-myb h/h and J H T mice. These data implicate B-cells in the regulation of V2R and its associated effects on salt and water handling and blood pressure homeostasis.

Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4+LYVE1+MHC-IIloCCR2- resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4-LYVE1-MHC-IIhiCCR2- macrophages and fully replaced TIMD4-LYVE1-MHC-IIhiCCR2+ macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4+ and TIMD4- resident macrophage abundance, whereas CCR2+ monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.