Clare L V Westhorpe

The macrophage: the intersection between HIV infection and atherosclerosis

HIV‐infected individuals are at increased risk of coronary artery disease (CAD) with underlying mechanisms including chronic immune activation and inflammation secondary to HIV‐induced microbial translocation and low‐grade endotoxemia; direct effects of HIV and viral proteins on macrophage cholesterol metabolism; and dyslipidemia related to HIV infection and specific antiretroviral therapies. Monocytes are the precursors of the lipid‐laden foam cells within the atherosclerotic plaque and produce high levels of proinflammatory cytokines such as IL‐6. The minor CD14+/CD16+ “proinflammatory” monocyte subpopulation is preferentially susceptible to HIV infection and may play a critical role in the pathogenesis of HIV‐related CAD. In this review, the central role of monocytes/macrophages in HIV‐related CAD and the importance of inflammation and cholesterol metabolism are discussed.

Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus

In contrast with many capillary beds, the glomerulus readily supports leukocyte recruitment. However, little is known regarding the actions of leukocytes following their recruitment to glomeruli. We used multiphoton confocal microscopy to examine leukocyte behavior in the glomerular microvasculature. In normal glomeruli, neutrophils and monocytes were retained in capillaries for several minutes, remaining static or migrating intravascularly. Induction of glomerular inflammation resulted in an increase in the duration of retention of static and migratory leukocytes. In response to immune complex deposition, both static and migratory neutrophils generated oxidants in inflamed glomeruli via a Mac-1–dependent mechanism. Our results describe a new paradigm for glomerular inflammation, suggesting that the major effect of acute inflammation is to increase the duration of leukocyte retention in the glomerulus. Moreover, these findings describe a previously unknown form of multicellular intravascular patrolling that involves both monocytes and neutrophils, which may underlie the susceptibility of the glomerulus to inflammation.

Differential Expression of CD163 on Monocyte Subsets in Healthy and HIV-1 Infected Individuals

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16− monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16− monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16− subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16− monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals.

Associations between surface markers on blood monocytes and carotid atherosclerosis in HIV-positive individuals

Chronic HIV infection is associated with increased risk of cardiovascular disease (CVD), including in patients with virological suppression. Persistent innate immune activation may contribute to the development of CVD via activation of monocytes in these patients. We investigated whether changes in monocyte phenotype predict subclinical atherosclerosis in virologically suppressed HIV‐positive individuals with low cardiovascular risk. We enroled 51 virologically suppressed HIV‐positive individuals not receiving protease inhibitors or statins and 49 age‐matched uninfected controls in this study. Carotid artery intima‐media thickness (cIMT) was used as a surrogate marker for CVD, and traditional risk factors, including Framingham risk scores, were recorded. Markers of monocyte activation (CD14, CD16, CCR2, CX3CR1, CD38, HLA‐DR and CD11b) were measured in whole‐blood samples by flow cytometry. Associations were assessed using univariate and multivariate median regressions. Median cIMT was similar between HIV‐positive and HIV‐negative participants (P=0.3), although HIV‐positive patients had significantly higher Framingham risk score (P=0.009) and systemic inflammation. Expression of two monocyte markers, CD11b and CX3CR1, independently predicted carotid artery thickness in HIV‐positive individuals after controlling for Framingham risk score (P=0.025 and 0.015, respectively). These markers were not predictive of carotid artery thickening in controls. Our study indicates that monocyte surface markers may serve as novel predictors of CVD in HIV‐positive individuals and is consistent with an important role for monocyte activation in the progression of HIV‐related cardiovascular pathology.

Effects of HIV‐1 infection in vitro on transendothelial migration by monocytes and monocyte‐derived macrophages

Monocytes constitutively migrate from the bloodstream across the vascular endothelium for systemic immune surveillance and maintenance of macrophage populations. They also perform reverse transendothelial migration (TEM) across the endothelium, which is required for entry of tissue monocytes/macrophages into the lymphatics or back into the bloodstream. We have modeled these processes previously using HUVEC monolayers grown on three‐dimensional collagen matrices. The aim of the present study was to determine whether HIV‐1 infection of monocytes/macrophages in vitro affects TEM. Purified primary human monocytes and monocyte‐derived macrophages (MDM) expressed important TEM proteins such as CD62L, CD18, PECAM‐1, CCR2, and CCR8. Purified monocytes underwent efficient forward and reverse TEM across HUVEC, and this function was maintained by MDM after up to 15 days of culture. Monocytes exposed to HIV‐1 for 2 days had unaltered forward or reverse TEM. However, HIV‐1 infection of MDM for 7 days decreased reverse TEM by an average of 66.5% compared with mock‐infected MDM (n=9 independent donors; P=0.004), without affecting forward TEM. Decreased reverse TEM by HIV‐infected MDM required viral RT and was not a result of alterations in surface expression of CCR8 or p‐glycoprotein or a general impairment in mobility, as assessed by migration toward fMLP. This study indicates that HIV‐1 infection of macrophages reduces their capacity to emigrate from the subendothelial extracellular matrix in vitro, which could result in defective cell‐mediated immune responses to infections and promote establishment of viral reservoirs of HIV in tissue macrophages in vivo.

Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus

Significance Nonclassical monocytes patrol the microvascular lumen in numerous organs in behavior thought to represent a form of immune surveillance. However, the mechanisms whereby they promote inflammatory responses are unclear. Here, we show using in vivo imaging that, in the unique microvasculature of the glomerulus in the kidney, monocytes constitutively undergo interactions with intravascular migratory neutrophils. Upon induction of glomerular inflammation, neutrophils that interact with monocytes show increased retention in glomerular capillaries and increased propensity to generate reactive oxygen species, leading to renal injury. These findings of immune cell interactions occurring within the glomerular microvasculature indicate that cell–cell contact between neutrophils and nonclassical monocytes is a previously unrecognized intravascular inflammatory mechanism underpinning glomerular injury. Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using β2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil-dependent tissue injury.

Imaging inflammatory leukocyte recruitment in kidney, lung and liver—challenges to the multi-step paradigm

Intravital microscopy has been essential in establishing the multi‐step paradigm that describes how leukocytes in the bloodstream interact with the blood vessel wall during the process of leukocyte recruitment. Much of this work has been performed in readily‐visualized tissues such as the mesentery and the cremaster muscle, where leukocyte‐endothelial cell interactions are restricted to postcapillary venules. However, the microvasculatures of the liver, lung and renal glomerulus differ markedly from these conventionally structured microvascular beds. Moreover, the liver, lung and kidney can be the target of life‐threatening leukocyte‐mediated inflammation. Therefore, a clear understanding of the mechanisms of leukocyte recruitment to these sites is critical. In this review, we examine the advances made in the understanding of leukocyte recruitment in the liver, lung and glomerulus, as determined using intravital microscopy. We describe how leukocyte recruitment to these sites occurs via mechanisms distinct from the conventional rolling/adhesion/transmigration paradigm, and in some cases involves adhesion molecules with minimal roles in conventional postcapillary venules. In addition, we describe how advanced forms of in vivo imaging in combination with novel approaches for labeling immune cell subsets is revealing new complexities in leukocyte function and immune cell interactions in these specialized microvascular beds.

Endothelial cell activation promotes foam cell formation by monocytes following transendothelial migration in an in vitro model

Foam cells are a pathological feature present at all stages of atherosclerosis. Foam cells develop from monocytes that enter the nascent atheroma and subsequently ingest modified low density lipoproteins (LDL). The regulation of this process has previously been studied in vitro using cultured macrophage fed modified LDL. We used our existing in vitro model of transendothelial migration (TEM) to study this process in a more physiologically relevant setting. In our model, monocytes undergo TEM across a primary endothelial monolayer into an underlying three-dimensional collagen matrix in the presence of 20% human serum. Foam cells were detected by Oil Red O staining for intracellular lipid droplets. We demonstrate that sub-endothelial monocytes can develop into foam cells within 48 h of TEM across TNF-α activated endothelium, in the absence of additional lipids. Our data indicate a role for both monocyte-endothelial interactions and soluble factors in the regulation of foam cell development, including oxidation of LDL in situ from lipid present in culture medium following TNF-α stimulation of the endothelial cells. Our study provides a simple model for investigating foam cell development in vitro that mimics cell migration in vivo, and demonstrates the critical role of inflammation in regulating early atherogenic events.

Regulatory T Cells Dynamically Regulate Selectin Ligand Function during Multiple Challenge Contact Hypersensitivity

Regulatory T cells (Tregs) play critical roles in restricting T cell–mediated inflammation. In the skin, this is dependent on expression of selectin ligands required for leukocyte rolling in dermal microvessels. However, whether there are differences in the molecules used by Tregs and proinflammatory T cells to undergo rolling in the skin remains unclear. In this study, we used spinning disk confocal microscopy in Foxp3-GFP mice to visualize rolling of endogenous Tregs in dermal postcapillary venules. Tregs underwent consistent but low-frequency rolling interactions under resting and inflamed conditions. At the early stage of the response, Treg adhesion was minimal. However, at the peak of inflammation, Tregs made up 40% of the adherent CD4+ T cell population. In a multiple challenge model of contact hypersensitivity, rolling of Tregs and conventional CD4+ T cells was mostly dependent on overlapping contributions of P- and E-selectin. However, after a second challenge, rolling of Tregs but not conventional CD4+ T cells became P-selectin independent, and Tregs showed reduced capacity to bind P-selectin. Moreover, inhibition of E-selectin at this time point resulted in exacerbation of inflammation. These findings demonstrate that in this multiple challenge model of inflammation, Treg selectin binding capacity and the molecular basis of Treg rolling can be regulated dynamically.

Enhanced monocyte Fc phagocytosis by a homologue of interleukin-10 encoded by human cytomegalovirus

Human cytomegalovirus (HCMV) expresses several homologues of human interleukin 10 (hIL-10) possessing immunomodulatory properties which may promote viral infection by modulating the function of myeloid cells. We examined the phenotype and phagocytic capability of human monocytes exposed to hIL-10, an HCMV-encoded hIL-10 homologue expressed during the productive phase of infection (cmvIL-10), and a differentially spliced form of cmvIL-10 expressed during latent and productive phases of infection, (LAcmvIL-10). hIL-10 and cmvIL-10 upregulated expression of Fcgamma receptors, stimulated phagocytosis of IgG-opsonised erythrocytes and decreased MHC class II (HLA-DR) expression on purified monocytes within 24 h. In contrast, LAcmvIL-10 decreased HLA-DR expression at later times (48 h and 72 h) but did not increase Fcgamma receptor expression. We conclude that cmvIL-10 promotes differentiation of monocytes towards a pro-phagocytic phenotype and that LAcmvIL-10 does not affect monocytes by the same mechanism as cmvIL-10. The significance of these properties to cytomegalovirus pathogenesis is discussed.