Roxana Apaza Rojas - Academia.edu (original) (raw)

Papers by Roxana Apaza Rojas

The Journal of Immunology

For MTB to survive in the face of robust innate and adaptive immune responses multiple immune eva... more For MTB to survive in the face of robust innate and adaptive immune responses multiple immune evasion mechanisms are required. These mechanisms and the MTB molecules responsible for them are beginning to be elucidated. Apart from interfering with the innate and adaptive immune functions of macrophages, recent studies suggest that MTB also can directly modulate CD4+ T cell function. The current study was undertaken to identify MTB molecules and mechanisms for direct inhibition of CD4+ T cell activation. Murine CD4+ T cells were purified from spleens by a combination of negative selection with mAb-coated beads and flow sorting. Purified T cells were activated for 24–48 hrs. with plate-bound anti-CD3 and soluble anti-CD28 mAbs with whole bacilli or subcellular fractions of MTB. Multiple measures of activation were used including IL-2 production by ELISA, proliferation by thymidine incorporation, and cell surface receptor expression by flow cytometry. MTB bacilli decreased T cell activa...

Infection and Immunity, 1999

ABSTRACTMycobacterium tuberculosisis the etiologic agent of human tuberculosis and is estimated t... more ABSTRACTMycobacterium tuberculosisis the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control ofM. tuberculosisrequires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor β [TGF-β]) cytokines. IL-10 and TGF-β are produced byM. tuberculosis-infected macrophages. The effect of IL-10 and TGF-β onM. tuberculosis-reactive human CD4+and γδ T cells, the two major human T-cell subsets activated byM. tuberculosis, was investigated. Both IL-10 and TGF-β inhibited proliferation and gamma interferon production by CD4+and γδ T cells. IL-10 was a more potent inhibitor than TGF-β for both T-cell subsets. Combinations of IL-10 and TGF-β did not result in additive or synergistic inhibition. IL-10 inhibited γδ and CD4+T...

Proteomics, Jan 10, 2017

Mycobacterium tuberculosis (Mtb) cell wall glycolipid Mannose-capped Lipoarabinomannan (ManLAM) i... more Mycobacterium tuberculosis (Mtb) cell wall glycolipid Mannose-capped Lipoarabinomannan (ManLAM) inhibits CD4(+) T cell activation by inhibiting proximal T cell receptor (TCR) signaling when activated by anti-CD3. To understand the impact of ManLAM on CD4(+) T cell function when both the TCR-CD3 complex and major co-stimulator CD28 are engaged, we performed label-free quantitative mass spectrometry (MS) and network analysis. Mixed effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti-CD3 and anti-CD28 activated CD4(+) T cells. Crosstalker(™) , a novel network analysis tool identified dysregulated translation, TCA cycle and RNA metabolism network modules. Proliferating Cellular Nuclear Antigen (PCNA), Akt, mTOR and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. We...

European journal of immunology, Sep 30, 2017

We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD... more We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4(+) T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4(+) T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4(+) T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4(+) T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4(+) T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a pro...

Retrovirology, Feb 6, 2017

Multiple toll-like receptors (TLRs) are expressed in cells of the monocytic lineage, including mi... more Multiple toll-like receptors (TLRs) are expressed in cells of the monocytic lineage, including microglia, which constitute the major reservoir for human immunodeficiency virus (HIV) infection in the brain. We hypothesized that TLR receptor mediated responses to inflammatory conditions by microglial cells in the central nervous system (CNS) are able to induce latent HIV proviruses, and contribute to the etiology of HIV-associated neurocognitive disorders. Newly developed human microglial cell lines (hµglia), obtained by immortalizing human primary microglia with simian virus-40 (SV40) large T antigen and the human telomerase reverse transcriptase, were used to generate latently infected cells using a single-round HIV virus carrying a green fluorescence protein reporter (hµglia/HIV, clones HC01 and HC69). Treatment of these cells with a panel of TLR ligands showed surprisingly that two potent TLR3 agonists, poly (I:C) and bacterial ribosomal RNA potently reactivated HIV in hμglia/HIV ...

Journal of immunology (Baltimore, Md. : 1950), Jan 25, 2017

Mycobacterium tuberculosis utilizes multiple mechanisms to evade host immune responses, and inhib... more Mycobacterium tuberculosis utilizes multiple mechanisms to evade host immune responses, and inhibition of effector CD4(+) T cell responses by M. tuberculosis may contribute to immune evasion. TCR signaling is inhibited by M. tuberculosis cell envelope lipoglycans, such as lipoarabinomannan and lipomannan, but a mechanism for lipoglycans to traffic from M. tuberculosis within infected macrophages to reach T cells is unknown. In these studies, we found that membrane vesicles produced by M. tuberculosis and released from infected macrophages inhibited the activation of CD4(+) T cells, as indicated by reduced production of IL-2 and reduced T cell proliferation. Flow cytometry and Western blot demonstrated that lipoglycans from M. tuberculosis-derived bacterial vesicles (BVs) are transferred to T cells, where they inhibit T cell responses. Stimulation of CD4(+) T cells in the presence of BVs induced expression of GRAIL, a marker of T cell anergy; upon restimulation, these T cells showed ...

Journal of Clinical & Cellular Immunology, 2016

Objective-Infection by MTB or exposure to MTB constituents is associated with intense microbial s... more Objective-Infection by MTB or exposure to MTB constituents is associated with intense microbial stimulation of the immune system, through both antigenic and TLR components, and induction of a milieu that is rich in pro-inflammatory/anti-inflammatory cytokines. Here, we addressed the basis of induced regulatory T-cell (iT-reg) expansion in response to MTB stimulation, in the absence of prior T cell antigen responsiveness. Methods-PBMC from HIV-1 un-infected TST negative and TST positive control subjects were stimulated by virulent MTB H37Rv lysate (L), a French press preparation of MTB that includes all bacterial components. Phenotype of MTB H37RvL induced iT-reg was assessed using immunostaining and flow cytometry. Functional capacity of iT-reg was assessed using 3 H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in the presence or absence of iT-reg in corresponding culture supernatants in response to TCR stimulation. Realtime PCR was used to assess IDO and FoxP3 mRNA expression. Results-The capacity of MTB H37RvL to induce CD4 + CD25 hi+ Foxp3 + T-cells in PBMC from TST negative subjects was robust (p<0.001), and in fact comparable to induction of iT-reg in PBMC from TST positive subjects. MTB-induced CD4 + CD25 hi+ T-reg were TGFβ positive (p<0.05). Further, MTB H37RvL induced CD4 + CD25 hi+ Foxp3 + iT-reg suppressed 3 H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in response to TCR stimulation. MTB H37RvL induction of iT-reg was significantly stronger (p<0.01) than that by TLR-2, TLR-4, TLR-9 ligands, or combination of all TLR ligands. MTB H37RvL inducted indoleamine 2,3dideoxygenase (IDO) mRNA expression in monocytes (p<0.001), and co-culture with the IDO inhibitor, D-1MT, decreased frequencies of T-reg (p<0.05). Inhibition of TGFβ by siRNA reduced Foxp3 mRNA expression in CD4 T cells (p<0.05). This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Scientific reports, Jun 14, 2016

Chemical regulation of macrophage function is one key strategy for developing host-directed adjuv... more Chemical regulation of macrophage function is one key strategy for developing host-directed adjuvant therapies for tuberculosis (TB). A critical step to develop these therapies is the identification and characterization of specific macrophage molecules and pathways with a high potential to serve as drug targets. Using a barcoded lentivirus-based pooled short-hairpin RNA (shRNA) library combined with next generation sequencing, we identified 205 silenced host genes highly enriched in mycobacteria-resistant macrophages. Twenty-one of these "hits" belonged to the oxidoreductase functional category. quinone oxidoreductase 1 (NQO1) was the top oxidoreductase "hit". NQO1 expression was increased after mycobacterial infection, and NQO1 knockdown increased macrophage differentiation, NF-κB activation, and the secretion of pro-inflammatory cytokines TNF-α and IL-1β in response to infection. This suggests that mycobacteria hijacks NQO1 to down-regulate pro-inflammatory and...

Cytometric Cellular Analysis

... gd T cells represent only 1±5% of peripheral CD3 cells in primates and rodents, they are abun... more ... gd T cells represent only 1±5% of peripheral CD3 cells in primates and rodents, they are abundant in the avian immune system and they account for up to 70% of circulating CD3 T cells in young ruminants, decreasing to 5±25% with aging (Cooper et al., 1989; Hein and Mackay ...

The Journal of experimental medicine, Jan 2, 2014

Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding ... more Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

Tuberculosis, 2003

A hallmark of M. tuberculosis infection is the ability of most (90-95%) healthy adults to control... more A hallmark of M. tuberculosis infection is the ability of most (90-95%) healthy adults to control infection through acquired immunity, in which antigen specific T cells and macrophages arrest growth of M. tuberculosis bacilli and maintain control over persistent bacilli. In addition to CD4+ T cells, other T cell subsets such as, gammadelta, CD8+ and CD1-restricted T cells have roles in the immune response to M. tuberculosis. A diverse T cell response allows the host to recognize a wider range of mycobacterial antigens presented by different families of antigen-presenting molecules, and thus greater ability to detect the pathogen. Macrophages are key antigen presenting cells for T cells, and M. tuberculosis survives and persists in this central immune cell. This is likely an important factor in generating this T cell diversity. Furthermore, the slow growth and chronic nature of M. tuberculosis infection results in prolonged exposure to antigens, and hence further T cell sensitization. The effector mechanisms used by T cells to control M. tuberculosis are poorly understood. To survive in macrophages, M. tuberculosis has evolved mechanisms to block immune responses. These include modulation of phagosomes, neutralization of macrophage effector molecules, stimulating the secretion of inhibitory cytokines, and interfering with processing of antigens for T cells. The relative importance of these blocking mechanisms likely depends on the stage of M. tuberculosis infection: primary infection, persistence, reactivation or active tuberculosis. The balance of the host-pathogen interaction in M. tuberculosis infection is determined by the interaction of T cells and infected macrophages. The outcome of this interaction results either in control of M. tuberculosis infection or active disease. A better understanding of this interaction will result in improved approaches to treatment and prevention of tuberculosis.

Nature Structural & Molecular Biology, 2010

Knockout of lprG results in decreased virulence of Mycobacterium tuberculosis (Mtb) in mice. Mtb ... more Knockout of lprG results in decreased virulence of Mycobacterium tuberculosis (Mtb) in mice. Mtb lipoprotein LprG has TLR2 agonist activity, thought to be dependent on its N-terminal triacylation. Surprisingly, here we find that non-acylated LprG retains TLR2 activity. Moreover, we show LprG association with triacylated glycolipid TLR2 agonists lipoarabinomannan, lipomannan and phosphatidylinositol mannosides (which share core structures). Binding of triacylated species was specific to LprG (not LprA) and increased LprG TLR2 agonist activity; conversely, association of glycolipids with LprG enhanced their recognition by TLR2. The crystal structure of LprG in complex with phosphatidylinositol mannoside revealed a hydrophobic pocket that accommodates the three alkyl chains of the ligand. In conclusion, we demonstrate a glycolipid binding function of LprG that enhances recognition of triacylated Mtb glycolipids by TLR2 and may affect glycolipid assembly or transport for bacterial cell wall biogenesis. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The Journal of Infectious Diseases, 2005

Background. Vg9 + Vd2 + gd T cells (Vd2 + T cells) are activated by Mycobacterium tuberculosis an... more Background. Vg9 + Vd2 + gd T cells (Vd2 + T cells) are activated by Mycobacterium tuberculosis and secrete interferon (IFN)-g. Vd2 + T cells recognize phosphoantigens, such as bromohydrin pyrophosphate (BrHPP), and link innate and adaptive immunity. Methods. A whole-blood assay was developed that used IFN-g secretion in response to BrHPP as a measurement of Vd2 + T cell function. Results. Peak IFN-g levels were detected after stimulating whole blood with BrHPP for 7-9 days. IFN-g production in whole blood in response to BrHPP paralleled IFN-g production and Vd2 + T cell expansion of peripheral-blood mononuclear cells. The assay was used to evaluate Vd2 + T cell function in subjects in the United States () and Uganda () who were or were not infected with M. tuberculosis and/or human im-n p 24 n p 178 munodeficiency virus (HIV) type 1. When 50 mmol/L BrHPP was used, 100% of healthy subjects produced IFNg. The Vd2 + T cell response was independent of the tuberculin skin test response. In Uganda, Vd2 + T cell responses were decreased in patients with tuberculosis () compared with responses in household contacts (). n p 73 n p 105 HIV-1-positive household contacts had lower responses than did HIV-1-negative household contacts. HIV-1positive patients with tuberculosis had the lowest Vd2 + T cell responses. Conclusions. Tuberculosis and HIV-1 infection are associated with decreased Vd2 + T cell function. Decreased Vd2 + T cell function may contribute to increased risk for tuberculosis in HIV-1-positive patients. gd T cells represent 1%-5% of circulating human T cells [1, 2]. Vg9 + Vd2 + gd T cells (Vd2 + T cells), the dominant circulating gd T cell subset in adults, are activated in response to intracellular pathogens, such as Mycobacterium tuberculosis [3-7]. Vd2 + T cells secrete

The Journal of Infectious Diseases, 2011

The Journal of Immunology, 2006

The pathological hallmark of the host response to Mycobacterium tuberculosis is the granuloma whe... more The pathological hallmark of the host response to Mycobacterium tuberculosis is the granuloma where T cells and macrophages interact with the extracellular matrix (ECM) to control the infection. Recruitment and retention of T cells within inflamed tissues depend on adhesion to the ECM. T cells use integrins to adhere to the ECM, and fibronectin (FN) is one of its major components. We have found that the major M. tuberculosis cell wall glycolipid, phosphatidylinositol mannoside (PIM), induces homotypic adhesion of human CD4+ T cells and T cell adhesion to immobilized FN. Treatment with EDTA and cytochalasin D prevented PIM-induced T cell adhesion. PIM-induced T cell adhesion to FN was blocked with mAbs against α5 integrin chain and with RGD-containing peptides. α5β1 (VLA-5) is one of two major FN receptors on T cells. PIM was found to bind directly to purified human VLA-5. Thus, PIM interacts directly with VLA-5 on CD4+ T lymphocytes, inducing activation of the integrin, and promotin...

Journal of Immunological Methods, 2009

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an... more Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4 + T cells isolated either by IMACS (IMACS-CD4 +) or by IMACS followed by FACS (IMACS/FACS-CD4 +). As expected, IMACS-CD4 + were less pure than IMACS/FACS-CD4 + (92.5% +/− 1.4% versus 99.7% +/− 0.2%, respectively). Consequently, IMACS-CD4 + proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4 +. In addition IMACS-CD4 + but not IMACS/FACS-CD4 + responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4 + and highly purified IMACS-/FACS-CD4 +. Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.

Infection and Immunity, 2009

ABSTRACTImmune evasion is required forMycobacterium tuberculosisto survive in the face of robust ... more ABSTRACTImmune evasion is required forMycobacterium tuberculosisto survive in the face of robust adaptive CD4+T-cell responses. We have previously shown thatM. tuberculosiscan indirectly inhibit CD4+T cells by suppressing the major histocompatibility complex class II antigen-presenting cell function of macrophages. This study was undertaken to determine ifM. tuberculosiscould directly inhibit CD4+T-cell activation. Murine CD4+T cells were purified from spleens by negative immunoaffinity selection followed by flow sorting. Purified CD4+T cells were activated for 16 to 48 h with CD3 and CD28 monoclonal antibodies in the presence or absence ofM. tuberculosisand its subcellular fractions. CD4+T-cell activation was measured by interleukin 2 production, proliferation, and expression of activation markers, all of which were decreased in the presence ofM. tuberculosis. Fractionation identified thatM. tuberculosiscell wall glycolipids, specifically, phosphatidylinositol mannoside and mannose...

Infection and Immunity, 2006

ABSTRACTMycobacterium tuberculosisresides in phagosomes inside macrophages. In this study, we ana... more ABSTRACTMycobacterium tuberculosisresides in phagosomes inside macrophages. In this study, we analyzed the kinetics and location ofM. tuberculosispeptide-major histocompatibility complex class II (MHC-II) complexes inM. tuberculosis-infected human macrophages.M. tuberculosispeptide-MHC-II complexes were detected with polyclonal autologousM. tuberculosis-specific CD4+T cells or F9A6 T hybridoma cells specific forM. tuberculosisantigen (Ag) 85B (96-111). Macrophages processed heat-killedM. tuberculosismore rapidly and efficiently than liveM. tuberculosis. To determine whereM. tuberculosispeptide-MHC-II complexes were formed intracellularly, macrophages incubated with heat-killedM. tuberculosiswere homogenized, and subcellular compartments were separated on Percoll density gradients analyzed with T cells. In THP-1 cells,M. tuberculosisAg 85B (96- 111)-DR1 complexes appeared initially in phagosomes, followed by MHC class II compartment (MIIC) and the plasma membrane fractions. In monocy...

Infection and Immunity, 2003

Mycobacterium tuberculosis survives in macrophages in the face of acquired CD4 + T-cell immunity,... more Mycobacterium tuberculosis survives in macrophages in the face of acquired CD4 + T-cell immunity, which controls but does not eliminate the organism. Gamma interferon (IFN-γ) has a central role in host defenses against M. tuberculosis by activating macrophages and regulating major histocompatibility complex class II (MHC-II) antigen (Ag) processing. M. tuberculosis interferes with IFN-γ receptor (IFN-γR) signaling in macrophages, but the molecules responsible for this inhibition are poorly defined. This study determined that the 19-kDa lipoprotein from M. tuberculosis inhibits IFN-γ-regulated HLA-DR protein and mRNA expression in human macrophages. Inhibition of HLA-DR expression was associated with decreased processing and presentation of soluble protein Ags and M. tuberculosis bacilli to MHC-II-restricted T cells. Inhibition of HLA-DR required prolonged exposure to 19-kDa lipoprotein and was blocked with a monoclonal antibody specific for Toll-like receptor 2 (TLR-2). The 19-kDa l...

Infection and Immunity, 2002

ABSTRACTVγ9Vδ2+T cells (γδ T cells) are activated byMycobacterium tuberculosisand recognize mycob... more ABSTRACTVγ9Vδ2+T cells (γδ T cells) are activated byMycobacterium tuberculosisand recognize mycobacterial nonpeptide phosphoantigens. The role of antigen-presenting cells in the processing and presentation of phosphoantigens to Vγ9Vδ2+T cells is not understood. We analyzed the role of macrophages for activation of γδ T cells by a new synthetic phosphoantigen bromohydrin pyrophosphate (BrHPP) andM. tuberculosis. Macrophages greatly increased γδ T-cell activation by both BrHPP andM. tuberculosis. Fixation of macrophages before infection demonstrated that uptake ofM. tuberculosiswas required for presentation to γδ T cells. Antigens ofM. tuberculosisremained stably associated with macrophage surface and were not removed by paraformaldehyde fixation or washing. Macrophages processedM. tuberculosisfor γδ T cells through a brefeldin A-insensitive pathway, suggesting that transport through the endoplasmic reticulum and Golgi complex of a putative presenting molecule is not important in the ...