Brian T. Saunders

Intravital and Kidney Slice Imaging of Podocyte Membrane Dynamics

In glomerular disease, podocyte injury results in a dramatic change in cell morphology known as foot process effacement. Remodeling of the actin cytoskeleton through the activity of small GTPases was identified as a key mechanism in effacement, with increased membrane activity and motility in vitro However, whether podocytes are stationary or actively moving cells in vivo remains debated. Using intravital and kidney slice two-photon imaging of the three-dimensional structure of mouse podocytes, we found that uninjured podocytes remained nonmotile and maintained a canopy-shaped structure over time. On expression of constitutively active Rac1, however, podocytes changed shape by retracting processes and clearly exhibited domains of increased membrane activity. Constitutive activation of Rac1 also led to podocyte detachment from the glomerular basement membrane, and we detected detached podocytes crawling on the surface of the tubular epithelium and occasionally, in contact with peritubular capillaries. Podocyte membrane activity also increased in the inflammatory environment of immune complex-mediated GN. Our results provide evidence that podocytes transition from a static to a dynamic state in vivo, shedding new light on mechanisms in foot process effacement.

CCR7 and IRF4-dependent dendritic cells regulate lymphatic collecting vessel permeability

Lymphatic collecting vessels direct lymph into and from lymph nodes (LNs) and can become hyperpermeable as the result of a previous infection. Enhanced permeability has been implicated in compromised immunity due to reduced flow of lymph and immune cells to LNs, which are the primary site of antigen presentation to T cells. Presently, very little is known about the molecular signals that affect lymphatic collecting vessel permeability. Here, we have shown that lymphatic collecting vessel permeability is controlled by CCR7 and that the chronic hyperpermeability of collecting vessels observed in Ccr7-/- mice is followed by vessel fibrosis. Reexpression of CCR7 in DCs, however, was sufficient to reverse the development of such fibrosis. IFN regulatory factor 4-positive (IRF4+) DCs constitutively interacted with collecting lymphatics, and selective ablation of this DC subset in Cd11c-Cre Irf4fl/fl mice also rendered lymphatic collecting vessels hyperpermeable and fibrotic. Together, our data reveal that CCR7 plays multifaceted roles in regulating collecting vessel permeability and fibrosis, with one of the key players being IRF4-dependent DCs.

Heart-resident CCR2 + macrophages promote neutrophil extravasation through TLR9/MyD88/CXCL5 signaling

It is well established that maladaptive innate immune responses to sterile tissue injury represent a fundamental mechanism of disease pathogenesis. In the context of cardiac ischemia reperfusion injury, neutrophils enter inflamed heart tissue, where they play an important role in potentiating tissue damage and contributing to contractile dysfunction. The precise mechanisms that govern how neutrophils are recruited to and enter the injured heart are incompletely understood. Using a model of cardiac transplant-mediated ischemia reperfusion injury and intravital 2-photon imaging of beating mouse hearts, we determined that tissue-resident CCR2+ monocyte-derived macrophages are essential mediators of neutrophil recruitment into ischemic myocardial tissue. Our studies revealed that neutrophil extravasation is mediated by a TLR9/MyD88/CXCL5 pathway. Intravital 2-photon imaging demonstrated that CXCL2 and CXCL5 play critical and nonredundant roles in guiding neutrophil adhesion and crawling, respectively. Together, these findings uncover a specific role for a tissue-resident monocyte-derived macrophage subset in sterile tissue inflammation and support the evolving concept that macrophage ontogeny is an important determinant of function. Furthermore, our results provide the framework for targeting of cell-specific signaling pathways in myocardial ischemia reperfusion injury.

CD36 Deficiency Impairs the Small Intestinal Barrier and Induces Subclinical Inflammation in Mice

Background & Aims CD36 has immunometabolic actions and is abundant in the small intestine on epithelial, endothelial, and immune cells. We examined the role of CD36 in gut homeostasis by using mice null for CD36 (CD36KO) and with CD36 deletion specific to enterocytes (Ent-CD36KO) or endothelial cells (EC-CD36KO). Methods Intestinal morphology was evaluated by using immunohistochemistry and electron microscopy. Intestinal inflammation was determined from neutrophil infiltration and expression of cytokines, toll-like receptors, and cyclooxygenase-2. Barrier integrity was assessed from circulating lipopolysaccharide and dextran administered intragastrically. Epithelial permeability to luminal dextran was visualized by using two-photon microscopy. Results The small intestines of CD36KO mice fed a chow diet showed several abnormalities including extracellular matrix accumulation with increased expression of extracellular matrix proteins, evidence of neutrophil infiltration, inflammation, and compromised barrier function. Electron microscopy showed shortened desmosomes with decreased desmocollin 2 expression. Systemically, leukocytosis and neutrophilia were present together with 80% reduction of anti-inflammatory Ly6Clow monocytes. Bone marrow transplants supported the primary contribution of non-hematopoietic cells to the inflammatory phenotype. Specific deletion of endothelial but not of enterocyte CD36 reproduced many of the gut phenotypes of germline CD36KO mice including fibronectin deposition, increased interleukin 6, neutrophil infiltration, desmosome shortening, and impaired epithelial barrier function. Conclusions CD36 loss results in chronic neutrophil infiltration of the gut, impairs barrier integrity, and systemically causes subclinical inflammation. Endothelial cell CD36 deletion reproduces the major intestinal phenotypes. The findings suggest an important role of the endothelium in etiology of gut inflammation and loss of epithelial barrier integrity.

Thermoneutrality but Not UCP1 Deficiency Suppresses Monocyte Mobilization Into BloodNovelty and Significance

Rationale: Ambient temperature is a risk factor for cardiovascular disease. Cold weather increases cardiovascular events, but paradoxically, cold exposure is metabolically protective because of UCP1 (uncoupling protein 1)-dependent thermogenesis. Objective: We sought to determine the differential effects of ambient environmental temperature challenge and UCP1 activation in relation to cardiovascular disease progression. Methods and Results: Using mouse models of atherosclerosis housed at 3 different ambient temperatures, we observed that cold temperature enhanced, whereas thermoneutral housing temperature inhibited atherosclerotic plaque growth, as did deficiency in UCP1. However, whereas UCP1 deficiency promoted poor glucose tolerance, thermoneutral housing enhanced glucose tolerance, and this effect held even in the context of UCP1 deficiency. In conditions of thermoneutrality, but not UCP1 deficiency, circulating monocyte counts were reduced, likely accounting for fewer monocytes entering plaques. Reductions in circulating blood monocytes were also found in a large human cohort in correlation with environmental temperature. By contrast, reduced plaque growth in mice lacking UCP1 was linked to lower cholesterol. Through application of a positron emission tomographic tracer to track CCR2+ cell localization and intravital 2-photon imaging of bone marrow, we associated thermoneutrality with an increased monocyte retention in bone marrow. Pharmacological activation of &bgr;3-adrenergic receptors applied to mice housed at thermoneutrality induced UCP1 in beige fat pads but failed to promote monocyte egress from the marrow. Conclusions: Warm ambient temperature is, like UCP1 deficiency, atheroprotective, but the mechanisms of action differ. Thermoneutrality associates with reduced monocyte egress from the bone marrow in a UCP1-dependent manner in mice and likewise may also suppress blood monocyte counts in man.

Opposing Roles of Dendritic Cell Subsets in Experimental GN

Dendritic cells (DCs) are thought to form a dendritic network across barrier surfaces and throughout organs, including the kidney, to perform an important sentinel function. However, previous studies of DC function used markers, such as CD11c or CX3CR1, that are not unique to DCs. Here, we evaluated the role of DCs in renal inflammation using a CD11c reporter mouse line and two mouse lines with DC-specific reporters, Zbtb46-GFP and Snx22-GFP. Multiphoton microscopy of kidney sections confirmed that most of the dendritically shaped CD11c+ cells forming a network throughout the renal interstitium expressed macrophage-specific markers. In contrast, DCs marked by Zbtb46-GFP or Snx22-GFP were less abundant, concentrated around blood vessels, and round in shape. We confirmed this pattern of localization using imaging mass cytometry. Motility measurements showed that resident macrophages were sessile, whereas DCs were motile before and after inflammation. Although uninflamed glomeruli rarely contained DCs, injury with nephrotoxic antibodies resulted in accumulation of ZBTB46 + cells in the periglomerular region. ZBTB46 identifies all classic DCs, which can be categorized into two functional subsets that express either CD103 or CD11b. Depletion of ZBTB46 + cells attenuated the antibody-induced kidney injury, whereas deficiency of the CD103+ subset accelerated injury through a mechanism that involved increased neutrophil infiltration. RNA sequencing 7 days after nephrotoxic antibody injection showed that CD11b+ DCs expressed the neutrophil-attracting cytokine CXCL2, whereas CD103+ DCs expressed high levels of several anti-inflammatory genes. These results provide new insights into the distinct functions of the two major DC subsets in glomerular inflammation.

The resident macrophages in murine pancreatic islets are constantly probing their local environment, capturing beta cell granules and blood particles

Aims/hypothesisWe studied here the interactions between the resident macrophages of pancreatic islets with beta cells and the blood vasculature. We also examined the immunological consequences of such interactions.MethodsIslets were isolated from C57BL/6 mice expressing CX3C motif chemokine receptor 1–green fluorescent protein (CX3CR-GFP) and examined live by two-photon microscopy. Islets were also examined by electron microscopy to study the relationship of the intra-islet macrophages with the beta cells. In NOD.Rag1−/− mice and young (non-diabetic) male mice, the acquisition of beta cell granules was tested functionally by probing with CD4+ T cells directed against insulin epitopes.ResultsTwo-photon microscopy showed that the islet resident macrophages were in close contact with blood vessels and had extensive filopodial activity. Some filopodia had direct access to the vessel lumen and captured microparticles. Addition of glucose at high concentration reduced the degree of filopodia sampling of islets. This finding applied to in vivo injection of glucose or to in vitro cultures. Ultrastructural examination showed the close contacts of macrophages with beta cells. Such macrophages contained intact dense core granules. Functional studies in NOD mice indicated that the macrophages presented insulin peptides to insulin-reactive T cells. Presentation was increased after glucose challenge either ex vivo or after an in vivo pulse. In agreement with the morphological findings, presentation was not affected by insulin receptor blockade.Conclusions/interpretationIslet resident macrophages are highly active, sampling large areas of the islets and blood contents and capturing beta cell granules. After such interactions, macrophages present immunogenic insulin to specific autoreactive T cells.

Limited Macrophage Positional Dynamics in Progressing or Regressing Murine Atherosclerotic Plaques

Objective— Macrophages play important roles in the pathogenesis of atherosclerosis, but their dynamics within plaques remain obscure. We aimed to quantify macrophage positional dynamics within progressing and regressing atherosclerotic plaques. Approach and Results— In a stable intravital preparation, large asymmetrical foamy macrophages in the intima of carotid artery plaques were sessile, but smaller rounded cells nearer plaque margins, possibly newly recruited monocytes, mobilized laterally along plaque borders. Thus, to test macrophage dynamics in plaques over a longer period of time in progressing and regressing disease, we quantified displacement of nondegradable phagocytic particles within macrophages for up to 6 weeks. In progressing plaques, macrophage-associated particles appeared to mobilize to deeper layers in plaque, whereas in regressing plaques, the label was persistently located near the lumen. By measuring the distance of the particles from the floor of the plaque, we discovered that particles remained at the same distance from the floor regardless of plaque progression or regression. The apparent deeper penetration of labeled cells in progressing conditions could be attributed to monocyte recruitment that generated new superficial layers of macrophages over the labeled phagocytes. Conclusions— Although there may be individual exceptions, as a population, newly differentiated macrophages fail to penetrate significantly deeper than the limited depth they reside on initial entry, regardless of plaque progression, or regression. These limited dynamics may prevent macrophages from escaping areas with unfavorable conditions (such as hypoxia) and pose a challenge for newly recruited macrophages to clear debris through efferocytosis deep within plaque.