Asha Anandaiah

MyD88-dependent TLR4 signaling is selectively impaired in alveolar macrophages from asymptomatic HIV persons

Alveolar macrophages (AMs) are the predominant effector cell in the lungs and contribute to a critical first line of defense against bacterial pathogens through recognition by pattern recognition receptors such as Toll-like receptor 4 (TLR4). TLR4-mediated tumor necrosis factor alpha (TNFalpha) release is significantly impaired in HIV(+) macrophages, but whether HIV impairs myeloid differentiation factor 88 (MyD88)-dependent and/or MyD-independent TLR4 signaling pathways in human macrophages is not known. Comparing human U937 macrophages with HIV(+) U1 macrophages (HIV-infected U937 subclone), the current study shows that HIV infection is associated with impaired macrophage TLR4-mediated signaling, specifically targeting the MyD88-dependent TLR4-mediated signaling pathway (reduced MyD88-interleukin-1 receptor-associated kinase [IRAK] interaction, IRAK phosphorylation, nuclear factor [NF]-kappaB nuclear translocation, and TNFalpha release) while preserving the MyD88-independent TLR4-mediated signaling pathway (preserved STAT1 phosphorylation, interferon regulatory factor [IRF] nuclear translocation, and interleukin-10 [IL-10] and RANTES release). Extracellular TLR4 signaling complex was intact (similar levels of CD14 and MD2), and similar patterns of response were observed in clinically relevant AMs from healthy and asymptomatic HIV(+) persons at high clinical risk of pneumonia. Taken together, these data support the concept that chronic HIV infection is associated with specific and targeted disruption of critical macrophage TLR4 signaling, which in turn may contribute to disease pathogenesis of bacterial pneumonia.

Epigenetic Regulation of Tumor Necrosis Factor α (TNFα) Release in Human Macrophages by HIV-1 Single-stranded RNA (ssRNA) Is Dependent on TLR8 Signaling

Background: TLR7/TLR8 recognize viral ssRNA, but in human macrophages recognition of HIV-1 ssRNA is not known. Results: In human macrophages, HIV-1 ssRNA promotes TNFα release and requires endocytosis through TLR8, MyD88, and histone modification. Conclusion: TLR8 recognizes HIV-1 ssRNA and promotes TNFα release through chromatin remodeling. Significance: Targeting macrophage TLR8 signaling may serve as a novel therapeutic strategy to modify HIV infection. Human macrophages at mucosal sites are essential targets for acute HIV infection. During the chronic phase of infection, they are persistent reservoirs for the AIDS virus. HIV virions gain entry into macrophages following ligation of surface CD4-CCR5 co-receptors, which leads to the release of two copies of HIV ssRNA. These events lead to reverse transcription and viral replication initiation. Toll-like receptors TLR7 and TLR8 recognize specific intracellular viral ssRNA sequences, but in human alveolar macrophages, their individual roles in TLR-mediated HIV ssRNA recognition are unclear. In the current study, HIV-1 ssRNA induced TNFα release in a dose-dependent manner in adherent human macrophages expressing both intracellular TLR7 and TLR8. This response was reduced by inhibiting either endocytosis (50 μm dynasore) or endosomal acidification (1 μg/ml chloroquine). Either MYD88 or TLR8 gene knockdown with relevant siRNA reduced HIV-1 ssRNA-mediated TNFα release, but silencing TLR7 had no effect on this response. Furthermore, HIV-1 ssRNA induced histone 4 acetylation at the TNFα promoter as well as trimethylation of histone 3 at lysine 4, whereas TLR8 gene knockdown reduced these effects. Taken together in human macrophages, TLR8 binds and internalizes HIV ssRNA, leading to endosomal acidification, chromatin remodeling, and increases in TNFα release. Drugs targeting macrophage TLR8-linked signaling pathways may modulate the innate immune response to acute HIV infection by reducing viral replication.

Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway

Purpose To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. Methods Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA. Results HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis. Conclusion Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.

Vitamin D Rescues Impaired Mycobacterium tuberculosis-Mediated Tumor Necrosis Factor Release in Macrophages of HIV-Seropositive Individuals through an Enhanced Toll-Like Receptor Signaling Pathway In Vitro

ABSTRACT Mycobacterium tuberculosis disease represents an enormous global health problem, with exceptionally high morbidity and mortality in HIV-seropositive (HIV+) persons. Alveolar macrophages from HIV+ persons demonstrate specific and targeted impairment of critical host cell responses, including impaired M. tuberculosis-mediated tumor necrosis factor (TNF) release and macrophage apoptosis. Vitamin D may promote anti-M. tuberculosis responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin, and autophagy mechanisms, but whether vitamin D promotes anti-M. tuberculosis mechanisms in HIV+ macrophages is not known. In the current study, human macrophages exposed to M. tuberculosis demonstrated robust release of TNF, IκB degradation, and NF-κB nuclear translocation, and these responses were independent of vitamin D pretreatment. In marked contrast, HIV+ U1 human macrophages exposed to M. tuberculosis demonstrated very low TNF release and no significant IκB degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these critical responses. The vitamin D-mediated restored responses were dependent in part on macrophage CD14 expression. Importantly, similar response patterns were observed with clinically relevant human alveolar macrophages from healthy individuals and asymptomatic HIV+ persons at high clinical risk of M. tuberculosis infection. Taken together with the observation that local bronchoalveolar lavage fluid (BALF) levels of vitamin D are severely deficient in HIV+ persons, the data from this study demonstrate that exogenous vitamin D can selectively rescue impaired critical innate immune responses in vitro in alveolar macrophages from HIV+ persons at risk for M. tuberculosis disease, supporting a potential role for exogenous vitamin D as a therapeutic adjuvant in M. tuberculosis infection in HIV+ persons.

Novel Developments in the Epidemic of Human Immunodeficiency Virus and Tuberculosis Coinfection

Tuberculosis (TB) disease remains one of the highest causes of mortality in HIV-infected individuals, and HIV-TB coinfection continues to grow at alarming rates, especially in sub-Saharan Africa. Surprisingly, a number of important areas regarding coinfection remain unclear. For example, increased risk of TB disease begins early in the course of HIV infection; however, the mechanism by which HIV increases this risk is not well understood. In addition, there is lack of consensus on the optimal way to diagnose latent TB infection and to manage active disease in those who are HIV infected. Furthermore, effective point-of-care testing for TB disease remains elusive. This review discusses key areas in the epidemiology, pathogenesis, diagnosis, and management of active and latent TB in those infected with HIV, focusing attention on issues related to high- and low-burden areas. Particular emphasis is placed on controversial areas where there are gaps in knowledge and on future directions of study.

Maintenance and Repair of the Lung Endothelium Does Not Involve Contributions from Marrow-Derived Endothelial Precursor Cells

Lung endothelium is believed to be a quiescent tissue with the potential to exhibit rapid and effective repair after injury. Endothelial progenitor cells derived from the bone marrow have been proposed as one source of new endothelial cells that may directly contribute to pulmonary endothelial cell homeostasis and repair. Here we use bone marrow transplantation models, using purified hematopoietic stem cells (HSCs) or unfractionated whole marrow, to assess engraftment of cells in the endothelium of a variety of tissues. We find scant evidence for any contribution of bone marrow-derived cells to the pulmonary endothelium in the steady state or after recovery from hyperoxia-induced endothelial injury. Although a rare population of CD45-/CD31+/VECadherin+ bone marrow-derived cells, originating from HSCs, can be found in lung tissue after transplantation, these cells are not readily found in anatomic locations that define the pulmonary endothelium. Moreover, by tracking transplanted bone marrow cells obtained from donor transgenic mice containing endothelial lineage-selective reporters (Tie2-GFP), no contribution of bone marrow-derived cells to the adult lung, liver, pancreas, heart, and kidney endothelium can be detected, even after prolonged follow-up periods of 11 months or after recovery from hyperoxic pulmonary endothelial injury. Our findings argue against any significant engraftment of bone marrow-derived cells in the pulmonary vascular endothelium.

Recombinant human mannose-binding lectin dampens human alveolar macrophage inflammatory responses to influenza A virus in vitro.

IAV pneumonia remains a serious global health problem, and preventative and therapeutic strategies remain limited. AM are critical effector cells in the control of influenza, impairing IAV replication, promoting IAV clearance, and promoting efferocytosis and resolution of lung inflammation. MBL, an innate immune pattern recognition molecule, present in the lungs, binds IAV, and plasma MBL deficiency is associated with increased susceptibility to IAV, although the mechanism remains incompletely understood, and the influence of MBL on the IAV‐AM interaction has not been established. In the current study, focusing on human macrophages (U937 cell line and clinically relevant human AM), data demonstrated that unopsonized IAV is readily internalized, induced release of TNF and ROS, and promoted macrophage apoptosis. In contrast, IAV, opsonized with rhMBL, reduced IAV uptake and macrophage apoptosis and dramatically reduced TNF release and ROS. Macrophage host‐defense responses were reduced further in the presence of MASPs. Taken together, these data support the concept that rhMBL may serve a protective innate host response and a critical biological response modifier function by limiting AM inflammation, oxidative injury, and AM apoptosis, which may allow effective IAV clearance while limiting collateral damage to vital organs, such as the lungs.

Mammalian Target of Rapamycin Inhibition in Macrophages of Asymptomatic HIV+ Persons Reverses the Decrease in TLR-4–Mediated TNF-α Release through Prolongation of MAPK Pathway Activation

TLR-4–mediated signaling is significantly impaired in macrophages from HIV+ persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4–mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4–mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R–associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4–mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV+ macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4–mediated TNF-α release in HIV+ macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV+ persons.

Twelve tips for teaching the informed consent conversation

Abstract Obtaining informed consent has been traditionally viewed as a mundane task, learned on the job and often relegated to an inexperienced member of the healthcare team. In reality, the process of obtaining informed consent is complex, challenging, and warrants focused teaching, observation and feedback. There are few published standards for what should be included in a high-quality informed consent conversation, and little or no guidance regarding how to best teach the process of conducting this type of shared decision-making conversation. The following twelve tips provide a roadmap for teaching the essential components of how to obtain informed consent, including both content and communication skills, with a focus on common pitfalls for trainees, and strategies to address them.

Teaching Residents How to Talk About Death and Dying: A Mixed-Methods Analysis of Barriers and Randomized Educational Intervention:

Objectives: We present a pilot study exploring the effects of a brief, 30-minute educational intervention targeting resident communication surrounding dying in the intensive care unit (ICU). We sought to determine whether simulation or didactic educational interventions improved resident-reported comfort, preparation, and skill acquisition. We also sought to identify resident barriers to using the word “dying.” Methods: In this mixed-methods prospective study, second- and third-year medical residents were randomized to participate in a simulation-based communication training or a didactic session. Residents completed a pre–post survey after the sessions evaluating the sessions and reflecting on their use of the word “dying” in family meetings. Results: Forty-five residents participated in the study. Residents reported increases in comfort (Mean [M]-pre = 3.3 [standard deviation: 0.6], M-post = 3.7 [0.7]; P < .01, Cohen d = 0.75) and preparation (M-pre = 3.4 [0.7], M-post = 3.9 [0.6]; P < .01, d = 1.07) using the word “dying” after both the simulation and didactic versions. Residents randomized to the simulation reported they were more likely to have learned new skills as compared to residents in the didactic (M-simulation = 2.2 [0.4], M-didactic = 1.9 [0.3]; P = .015, d = 0.80). They estimated that they used the word “dying” in 50% of their end-of-life (EOL) conversations and identified uncertain prognosis as the main barrier to explicitly stating the word “dying.” Conclusion: A 30-minute educational intervention improves internal medicine residents’ self-reported comfort and preparation in talking about death and dying in the ICU. Residents in simulation-based training were more likely to report they learned new skills as compared to the didactic session. Residents report multiple barriers to using the word “dying” EOL conversations.