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.

Induction of Autonomous Memory Alveolar Macrophages Requires T Cell Help and Is Critical to Trained Immunity.

Innate immune memory is an emerging area of research. However, innate immune memory at major mucosal sites remains poorly understood. Here, we show that respiratory viral infection induces long-lasting memory alveolar macrophages (AMs). Memory AMs are programed to express high MHC II, a defense-ready gene signature, and increased glycolytic metabolism, and produce, upon re-stimulation, neutrophil chemokines. Using a multitude of approaches, we reveal that the priming, but not maintenance, of memory AMs requires the help from effector CD8 T cells. T cells jump-start this process via IFN-γ production. We further find that formation and maintenance of memory AMs are independent of monocytes or bone marrow progenitors. Finally, we demonstrate that memory AMs are poised for robust trained immunity against bacterial infection in the lung via rapid induction of chemokines and neutrophilia. Our study thus establishes a new paradigm of immunological memory formation whereby adaptive T-lymphocytes render innate memory of mucosal-associated macrophages.

BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis.

The dogma that adaptive immunity is the only arm of the immune response with memory capacity has been recently challenged by several studies demonstrating evidence for memory-like innate immune training. However, the underlying mechanisms and location for generating such innate memory responses in vivo remain unknown. Here, we show that access of Bacillus Calmette-Guérin (BCG) to the bone marrow (BM) changes the transcriptional landscape of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), leading to local cell expansion and enhanced myelopoiesis at the expense of lymphopoiesis. Importantly, BCG-educated HSCs generate epigenetically modified macrophages that provide significantly better protection against virulent M. tuberculosis infection than naïve macrophages. By using parabiotic and chimeric mice, as well as adoptive transfer approaches, we demonstrate that training of the monocyte/macrophage lineage via BCG-induced HSC reprogramming is sustainable in vivo. Our results indicate that targeting the HSC compartment provides a novel approach for vaccine development.

A Hematogenous Route for Medulloblastoma Leptomeningeal Metastases.

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma.

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.

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.

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.

CCL19-CCR7-dependent reverse transendothelial migration of myeloid cells clears Chlamydia muridarum from the arterial intima.

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions.

Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

A CD103+ Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.

Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific T cell responses during subclinical viral myocarditis.

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.

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.

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.

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.

Effect of removing Kupffer cells on nanoparticle tumor delivery.

A recent metaanalysis shows that 0.7% of nanoparticles are delivered to solid tumors. This low delivery efficiency has major implications in the translation of cancer nanomedicines, as most of the nanomedicines are sequestered by nontumor cells. To improve the delivery efficiency, there is a need to investigate the quantitative contribution of each organ in blocking the transport of nanoparticles to solid tumors. Here, we hypothesize that the removal of the liver macrophages, cells that have been reported to take up the largest amount of circulating nanoparticles, would lead to a significant increase in the nanoparticle delivery efficiency to solid tumors. We were surprised to discover that the maximum achievable delivery efficiency was only 2%. In our analysis, there was a clear correlation between particle design, chemical composition, macrophage depletion, tumor pathophysiology, and tumor delivery efficiency. In many cases, we observed an 18-150 times greater delivery efficiency, but we were not able to achieve a delivery efficiency higher than 2%. The results suggest the need to look deeper at other organs such as the spleen, lymph nodes, and tumor in mediating the delivery process. Systematically mapping the contribution of each organ quantitatively will allow us to pinpoint the cause of the low tumor delivery efficiency. This, in effect, enables the generation of a rational strategy to improve the delivery efficiency of nanoparticles to solid tumors either through the engineering of multifunctional nanosystems or through manipulation of biological barriers.