Rickvinder Besla

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.

Inflammation rapidly reorganizes mouse bone marrow B cells and their environment in conjunction with early IgM responses

Systemic inflammation perturbs the bone marrow environment by evicting resident B cells and favoring granulopoiesis over lymphopoiesis. Despite these conditions, a subset of marrow B cell remains to become activated and produce potent acute immunoglobulin M (IgM) responses. This discrepancy is currently unresolved and a complete characterization of early perturbations in the B-cell niche has not been undertaken. Here, we show that within a few hours of challenging mice with adjuvant or cecal puncture, B cells accumulate in the bone marrow redistributed away from sinusoid vessels. This response correlates with enhanced sensitivity to CXC chemokine ligand 12 (CXCL12) but not CXCL13 or CC chemokine ligand 21. Concurrently, a number of B-cell survival and differentiation factors are elevated to produce a transiently supportive milieu. Disrupting homing dynamics with a CXC chemokine receptor 4 inhibitor reduced the formation of IgM-secreting cells. These data highlight the rapidity with which peripheral inflammation modifies the marrow compartment, and demonstrate that such modifications regulate acute IgM production within this organ. Furthermore, our study indicates that conversion to a state of emergency granulopoiesis is temporally delayed, allowing B cells opportunity to respond to antigen.

Mesenchymal Stromal (Stem) Cell Therapy Fails to Improve Outcomes in Experimental Severe Influenza

Rationale Severe influenza remains a major public health threat and is responsible for thousands of deaths annually. Increasing antiviral resistance and limited effectiveness of current therapies highlight the need for new approaches to influenza treatment. Extensive pre-clinical data have shown that mesenchymal stromal (stem) cell (MSC) therapy can induce anti-inflammatory effects and enhance repair of the injured lung. We hypothesized that MSC therapy would improve survival, dampen lung inflammation and decrease acute lung injury (ALI) in a murine model of severe influenza. Methods C57Bl/6 mice were infected with influenza A/PuertoRico/8/34 (mouse-adapted H1N1) or influenza A/Mexico/4108/2009 (swine-origin pandemic H1N1) and administered human or mouse MSCs via the tail vein, either pre- or post- infection. MSC efficacy was evaluated as both an independent and adjunctive treatment strategy in combination with the antiviral agent, oseltamivir. Weight loss and survival were monitored. Inflammatory cells, cytokine/chemokines (IFN-γ, CXCL10, CCL2 and CCL5) and markers of ALI (total protein and IgM), were measured in bronchoalveolar lavage fluid and lung parenchyma. Results Administration of murine MSCs or human MSCs in a prophylactic or therapeutic regimen failed to improve survival, decrease pulmonary inflammation/inflammatory cell counts or prevent ALI in influenza virus-infected mice. MSCs administered in combination with oseltamivir also failed to improve outcomes. Conclusions Despite similarities in the clinical presentation and pathobiology of ALI and severe influenza, our findings suggest that MSC therapy may not be effective for prevention and/or treatment of acute severe influenza.

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor &kgr; light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)–derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. Objective: To define the role of endogenous miR-146a during atherogenesis. Methods and Results: Paradoxically, Ldlr−/− (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr−/−;miR-146a−/− mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr−/− mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1(Sort1), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.

Effect of removing Kupffer cells on nanoparticle tumor delivery

Significance Nanomaterials are developed for treating and diagnosing cancer, but only 0.7% (median) are delivered to a solid tumor. To address this delivery problem, we are examining each biological barrier to determine its impact on tumor delivery. Because the liver sequesters up to 70% of nanomaterials, in this study, we asked, if liver Kupffer cells were removed, what is the impact on tumor delivery? While we demonstrate that the tumor delivery increased up to 150 times, we achieved 2% for nanomaterials of different size, material, and tumor type. This suggests the need to focus on tumor pathophysiology to increase delivery efficiency, since this approach led to a greater availability of nanoparticles in the blood, but 98% did not accumulate in solid tumors. 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.

Circulating Cell Death Biomarkers May Predict Survival in Human Lung Transplantation

RATIONALE Immediate graft performance after lung transplantation is associated with short- and long-term clinical outcomes. However, the biologic mechanism that determines outcomes is not fully understood. OBJECTIVES To investigate the impact of cell death signals at 24 and 48 hours after lung transplantation on short- and long-term clinical outcomes. METHODS Plasma samples were collected pretransplantation and at 24 and 48 hours after transplant from 60 bilateral lung transplant recipients. Ten patients had primary graft dysfunction (PGD) grade 3 (PaO2/FiO2 ratio <200 or on extracorporeal membrane oxygenation support) at 72 hours after transplant (PGD group). The remaining 50 patients were defined as the control group. Levels of plasma M30 (signifying epithelial apoptosis), M65 (signifying epithelial apoptosis plus necrosis), and high-mobility group box 1 protein (HMGB-1; signifying necrosis of all cell types) were measured by ELISA and correlated with clinical outcomes. Survival analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. Prediction accuracy of markers was assessed by calculated area under the curve of receiver operating characteristic graph. MEASUREMENTS AND MAIN RESULTS The PGD group had significantly higher M30 and M65 levels at 24 and 48 hours after transplant compared with the control group. There was no significant difference in HMGB-1. Area under the curve for 1-year survival was 0.86, 0.93, and 0.51 for M30, M65, and HMGB-1 at 48 hours, respectively. Survival analysis showed that higher M30 and M65 levels at 24 and 48 hours were significantly associated with worse survival. M65 at 48 hours remained significant even after adjustment for PGD. HMGB-1 was not significantly associated with survival. CONCLUSIONS Recipient plasma concentration of epithelial cell death markers (M30, M65) after lung transplantation is negatively correlated with early graft performance and long-term survival.

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.

S-Nitrosoglutathione Reductase Deficiency Confers Improved Survival and Neurological Outcome in Experimental Cerebral Malaria

ABSTRACT Artesunate remains the mainstay of treatment for cerebral malaria, but it is less effective in later stages of disease when the host inflammatory response and blood-brain barrier integrity dictate clinical outcomes. Nitric oxide (NO) is an important regulator of inflammation and microvascular integrity, and impaired NO bioactivity is associated with fatal outcomes in malaria. Endogenous NO bioactivity in mammals is largely mediated by S-nitrosothiols (SNOs). Based on these observations, we hypothesized that animals deficient in the SNO-metabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), which exhibit enhanced S-nitrosylation, would have improved outcomes in a preclinical model of cerebral malaria. GSNOR knockout (KO) mice infected with Plasmodium berghei ANKA had significantly delayed mortality compared to WT animals (P < 0.0001), despite higher parasite burdens (P < 0.01), and displayed markedly enhanced survival versus the wild type (WT) when treated with the antimalarial drug artesunate (77% versus 38%; P < 0.001). Improved survival was associated with higher levels of protein-bound NO, decreased levels of CD4+ and CD8+ T cells in the brain, improved blood-brain barrier integrity, and improved coma scores, as well as higher levels of gamma interferon. GSNOR KO animals receiving WT bone marrow had significantly reduced survival following P. berghei ANKA infection compared to those receiving KO bone barrow (P < 0.001). Reciprocal transplants established that survival benefits of GSNOR deletion were attributable primarily to the T cell compartment. These data indicate a role for GSNOR in the host response to malaria infection and suggest that strategies to disrupt its activity will improve clinical outcomes by enhancing microvascular integrity and modulating T cell tissue tropism.

Macrophage JAK2 deficiency protects against high-fat diet-induced inflammation

During obesity, macrophages can infiltrate metabolic tissues, and contribute to chronic low-grade inflammation, and mediate insulin resistance and diabetes. Recent studies have elucidated the metabolic role of JAK2, a key mediator downstream of various cytokines and growth factors. Our study addresses the essential role of macrophage JAK2 in the pathogenesis to obesity-associated inflammation and insulin resistance. During high-fat diet (HFD) feeding, macrophage-specific JAK2 knockout (M-JAK2−/−) mice gained less body weight compared to wildtype littermate control (M-JAK2+/+) mice and were protected from HFD-induced systemic insulin resistance. Histological analysis revealed smaller adipocytes and qPCR analysis showed upregulated expression of some adipogenesis markers in visceral adipose tissue (VAT) of HFD-fed M-JAK2−/− mice. There were decreased crown-like structures in VAT along with reduced mRNA expression of some macrophage markers and chemokines in liver and VAT of HFD-fed M-JAK2−/− mice. Peritoneal macrophages from M-JAK2−/− mice and Jak2 knockdown in macrophage cell line RAW 264.7 also showed lower levels of chemokine expression and reduced phosphorylated STAT3. However, leptin-dependent effects on augmenting chemokine expression in RAW 264.7 cells did not require JAK2. Collectively, our findings show that macrophage JAK2 deficiency improves systemic insulin sensitivity and reduces inflammation in VAT and liver in response to metabolic stress.

Hepatic JAK2 protects against atherosclerosis through circulating IGF-1

Atherosclerosis is considered both a metabolic and inflammatory disease; however, the specific tissue and signaling molecules that instigate and propagate this disease remain unclear. The liver is a central site of inflammation and lipid metabolism that is critical for atherosclerosis, and JAK2 is a key mediator of inflammation and, more recently, of hepatic lipid metabolism. However, precise effects of hepatic Jak2 on atherosclerosis remain unknown. We show here that hepatic Jak2 deficiency in atherosclerosis-prone mouse models exhibited accelerated atherosclerosis with increased plaque macrophages and decreased plaque smooth muscle cell content. JAK2s essential role in growth hormone signalling in liver that resulted in reduced IGF-1 with hepatic Jak2 deficiency played a causal role in exacerbating atherosclerosis. As such, restoring IGF-1 either pharmacologically or genetically attenuated atherosclerotic burden. Together, our data show hepatic Jak2 to play a protective role in atherogenesis through actions mediated by circulating IGF-1 and, to our knowledge, provide a novel liver-centric mechanism in atheroprotection.