Naimish R. Patel

Cell Elasticity Determines Macrophage Function

Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function.

Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro.

Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystis pneumonia. Recognition of unopsonized Pneumocystis organisms by human AM is mediated predominantly via mannose receptors and results in phagocytosis, release of reactive oxygen species, and activation of the nuclear transcription factor (NF)‐κB. However, the AM host defense genes activated by Pneumocystis have not been defined. In the present study, incubation of AM with unopsonized Pneumocystis organisms was not associated with release of interleukin (IL)‐1β, IL‐6, or tumor necrosis factor (TNF)‐α (important cytokines in the host response to Pneumocystis) and did not induce IL‐1β, IL‐6, or TNF‐α mRNA transcripts. These findings were not attributed to Pneumocystis‐induced cytopathic changes, as these same AM released IL‐8 and matrix metalloproteinase‐9 in response to Pneumocystis. NF‐κB‐mediated IL‐8 release was independent of Pneumocystis phagocytosis. The observed response was specific, as IL‐1β, IL‐6, and TNF‐α release and mRNA induction were preserved in response to lipopolysaccharide or serum‐opsonized Pneumocystis. The absence of IL‐1β, IL‐6, and TNF‐α release in response to Pneumocystis was predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF‐α release in response to unopsonized Pneumocystis organisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystis organisms reduced Toll‐like receptor 4‐mediated TNF‐α release. Taken together, these data suggest that mannose receptors on human AM may suppress select proinflammatory cytokine release and may serve to regulate the innate inflammatory responses to infectious challenge in the lungs.

HIV Impairs TNF-α Mediated Macrophage Apoptotic Response to Mycobacterium tuberculosis

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV+ persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-α. In contrast, apoptosis of differentiated HIV+ human U1 macrophages (HIV+ U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-α release, whereas apoptosis and TNF-α release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-α release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-κB nuclear translocation were reduced in HIV+ U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV+ persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-α release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV+ persons.

Pneumocystis-mediated IL-8 release by macrophages requires coexpression of mannose receptors and TLR2.

Interaction with the unique fungus Pneumocystis (Pc) promotes IL‐8 release by human alveolar macrophages (AM), although the receptor(s) mediating IL‐8 release have not been identified. TLR2 recognizes fungal components and mediates release of host defense cytokines and chemokines, although whether TLR2 mediates signaling in response to Pc is not known. In the current study, Pc induced IL‐8 release by human AM, and AM pretreatment with anti‐TLR2 neutralizing antibody reduced IL‐8 release. However, in nonphagocytic human embryonic kidney (HEK)293 cells transfected with human TLR2 cDNA, incubation with Pc did not induce IL‐8 release, whereas these same cells released IL‐8 in response to the TLR2 agonist lipoteichoic acid. Targeted gene silencing of AM mannose receptors (MR; phagocytic receptors for Pc) using small interfering RNA also reduced Pc‐mediated IL‐8 release in human AM. However, HEK293 cells transfected with human MR cDNA alone did not release IL‐8 in response to Pc. In contrast, HEK293 cells cotransfected with human TLR2 and human MR cDNA released IL‐8 in response to Pc. In human AM, Pc promoted direct interaction of MR and TLR2, IL‐8 release was reduced markedly upon simultaneous blocking of TLR2 and gene silencing of MR, and IL‐8 release was dependent in part on transcription factor NF‐κB and ERK1/2 and JNK MAPKs. These studies demonstrate that Pc‐mediated IL‐8 release by human AM requires the coexpression of MR and TLR2 and further supports the concept that combinatorial interactions of macrophage innate receptors provide specificity of host defense cell responses to infectious challenge.

The Influence of Diagnostic Bronchoscopy on Clinical Outcomes Comparing Adult Autologous and Allogeneic Bone Marrow Transplant Patients

STUDY OBJECTIVES To review our experience with diagnostic bronchoscopy in the evaluation of pulmonary infiltrates in adult hematopoietic stem cell transplantation (HSCT) recipients in the era of Pneumocystis prophylaxis and cytomegalovirus antigen testing. The study focused on diagnostic yields and the influence of bronchoscopic findings on pharmacologic therapy and mortality, comparing allogeneic (allo) HSCT patients to autologous (auto) HSCT patients. DESIGN Case series review. SETTING Tertiary care academic urban medical centers. PATIENTS All adult allo-HSCT and auto-HSCT patients undergoing bronchoscopy for the evaluation of pulmonary infiltrates from January 1997 to September 2001. MEASUREMENTS AND RESULTS The review identified 169 bronchoscopies that had been performed on HSCT patients, representing 12.5% of all HSCT patients (allo-HSCT patients, 125 bronchoscopies; auto-HSCT patients, 44 bronchoscopies). Bronchoscopy was requested more often in allo-HSCT patients (18.7%) compared to auto-HSCT patients (6.6%). Findings at bronchoscopy provided a specific diagnosis more frequently in allo-HSCT patients (50%) compared to auto-HSCT patients (34%). For both allo-HSCT and auto-HSCT patients, most diagnoses were obtained by BAL alone, whereas transbronchial biopsy (TBBx) provided additional specific information in < 10% of cases. For select patients (n = 27), surgical lung biopsy following bronchoscopy provided unique diagnoses in 47 to 50% of cases. Information from bronchoscopy influenced clinical decisions more often in allo-HSCT patients (50%) than in auto-HSCT patients (36%), and allowed for the discontinuation or addition of antimicrobial, corticosteroid, or antineoplastic agents to treatment. Complications from bronchoscopy occurred in 9% of all HSCT patients (n = 15), and were associated with higher in-hospital mortality rates in allo-HSCT patients (82%; n = 9) compared to auto-HSCT patients (50%; n = 2). The overall in-hospital mortality rates for allo-HSCT and auto-HSCT patients having bronchoscopy was similar (38% vs 27%, respectively; p = 0.25), and establishing a specific diagnosis by bronchoscopy did not improve the in-hospital mortality rate for allo-HSCT or auto-HSCT patients. CONCLUSIONS Bronchoscopy may provide clinically useful information in the evaluation of adult allo-HSCT and auto-HSCT recipients with pulmonary infiltrates. The results of testing BAL fluid samples alone suggested an etiology in most cases, whereas the findings of TBBx provided unique diagnoses infrequently. Further studies are warranted to improve the utility of diagnostic bronchoscopy in the evaluation of HSCT patients.

Impaired M. tuberculosis-mediated apoptosis in alveolar macrophages from HIV+ persons: potential role of IL-10 and BCL-3

The mechanism of increased MTb disease susceptibility in HIV+ persons remains poorly understood. Apoptosis of macrophages in response to MTb represents a critical host defense response, and decreased apoptosis may represent a mechanism of increased susceptibility to MTb in HIV. In the current study, MTb‐mediated apoptosis of human AM was reduced in HIV+ subjects compared with healthy subjects in a TNF‐α‐dependent manner. IL‐10 levels in BALF from HIV+ persons were significantly elevated compared with HIV– persons, and exogenous IL‐10 reduced MTb‐mediated apoptosis in healthy AM, suggesting that IL‐10 could mediate decreased apoptosis observed in HIV. Further study showed that IL‐10 reduced TNF release in response to MTb in AM through a reduction in TNF mRNA levels, and exogenous TNF could partially reverse IL‐10‐associated effects on AM apoptosis. IL‐10 did not influence p‐IRAK, IκB degradation, or NF‐κB p65 nuclear translocation in response to MTb, but IL‐10 did increase levels of AM BCL‐3, an inhibitor of NF‐κB nuclear activity. BCL‐3 knockdown in human macrophages increased MTb‐mediated TNF release. Importantly, BCL‐3 levels in AM from HIV+ subjects were higher compared with healthy subjects. Taken together, these data suggest that elevated lung levels of IL‐10 may impair MTb‐mediated AM apoptosis in HIV through a BCL‐3‐dependent mechanism. BCL‐3 may represent a potential therapeutic target to treat or prevent MTb disease in HIV+ persons.

Constitutive Activation of Phosphatidylinositol 3-Kinase Signaling Pathway Down-regulates TLR4-mediated Tumor Necrosis Factor-α Release in Alveolar Macrophages from Asymptomatic HIV-positive Persons in Vitro

Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinse (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 μg/ml) resulted in lower TNF-α release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-α release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3β (GSK-3β) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3β phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3β (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-α release in HIV+ human macrophages and impair host cell response to infectious challenge.

Pneumocystis jiroveci Pneumonia in Adult Patients with AIDS

Pneumocystis jiroveci (P. carinii) is an opportunistic pathogen that has gained particular prominence since the onset of the AIDS epidemic. Among several important advances in diagnosis and management, appropriately targeting chemoprophylaxis to HIV-infected patients at high clinical risk for P. jiroveci pneumonia and the introduction of effective combination anti-retroviral therapy (including highly active antiretroviral therapy [HAART]) have contributed to the reduced incidence of P. jiroveci pneumonia. Despite the success of these clinical interventions, P. jiroveci pneumonia remains the most common opportunistic pneumonia and the most common life-threatening infectious complication in HIV-infected patients. Trimethoprim/sulfamethoxazole (cotrimoxazole) remains the first-line agent for effective therapy and chemoprophylaxis, and corticosteroids represent an important adjunctive agent in the treatment of moderate-to-severe P. jiroveci pneumonia. However, problems of chemoprophylaxis and treatment failures, high rates of adverse drug reactions and drug intolerance to first-line antimicrobials, high rates of relapse or recurrence with second-line agents, and newer concerns about the development of P. jiroveci drug resistance represent formidable challenges to the management and treatment of AIDS-related P. jiroveci pneumonia. With the expanding global problem of HIV infection, the intolerance or unavailability of HAART to many individuals and limited access to healthcare for HIV-infected patients, P. jiroveci pneumonia will remain a major worldwide problem in the HIV-infected population. New drugs under development as anti-Pneumocystis agents such as echinocandins and pneumocandins, which inhibit β-glucan synthesis, or sordarins, which inhibit fungal protein synthesis, show promise as effective agents. Continued basic research into the biology and genetics of P. jiroveci and host defense response to P. jiroveci will allow the development of newer antimicrobials and immunomodulatory therapeutic agents to more effectively treat life-threatening pneumonia caused by this organism.

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.

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.