TLR2 on CD4+ and CD8+ T cells promotes control of Mycobacterium tuberculosis infection.

Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen.

CD40 in Endothelial Cells Restricts Neural Tissue Invasion by Toxoplasma gondii.

Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40+ endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.

Mycobacterium tuberculosis Membrane Vesicles Inhibit T Cell Activation.

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 reduced ability to proliferate, confirming a state of T cell anergy. Furthermore, lipoarabinomannan was associated with T cells after their incubation with infected macrophages in vitro and when T cells were isolated from lungs of M. tuberculosis-infected mice, confirming the occurrence of lipoarabinomannan trafficking to T cells in vivo. These studies demonstrate a novel mechanism for the direct regulation of CD4+ T cells by M. tuberculosis lipoglycans conveyed by BVs that are produced by M. tuberculosis and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion.

Toll like Receptor 2 engagement on CD4+ T cells promotes TH9 differentiation and function.

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 prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4+ T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells.

Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis Induces CD4+ T Cell Anergy via GRAIL.

Mycobacterium tuberculosis cell wall glycolipid, lipoarabinomannan, can inhibit CD4(+) T cell activation by downregulating the phosphorylation of key proximal TCR signaling molecules: Lck, CD3ζ, ZAP70, and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an Ag encounter, yet remain viable and hyporesponsive. We tested whether mannose-capped lipoarabinomannan (LAM)-induced inhibition of CD4(+) T cell activation resulted in CD4(+) T cell anergy. The presence of LAM during primary stimulation of P25 TCR-transgenic murine CD4(+) T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25 TCR-transgenic CD4(+) T cells primed in the presence of LAM also exhibited decreased response upon restimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hyporesponsiveness to restimulation was not due to apoptosis, generation of Foxp3-positive regulatory T cells, or inhibitory cytokines. Acquisition of the anergic phenotype correlated with upregulation of gene related to anergy in lymphocytes (GRAIL) protein in CD4(+) T cells. Inhibition of human CD4(+) T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA-mediated knockdown of GRAIL before LAM treatment abrogated LAM-induced hyporesponsiveness. In addition, exogenous IL-2 reversed defective proliferation by downregulating GRAIL expression. These results demonstrate that LAM upregulates GRAIL to induce anergy in Ag-reactive CD4(+) T cells. Induction of CD4(+) T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition.

Proteomics and Network Analyses Reveal Inhibition of Akt-mTOR Signaling in CD4+ T Cells by Mycobacterium tuberculosis Mannose-Capped Lipoarabinomannan.

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 costimulator CD28 are engaged, we performed label-free quantitative 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. 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. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF-κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x-Akt-mTOR pathway. Thus, ManLAM induced global changes in the CD4+ T-cell proteome by affecting Akt-mTOR signaling, resulting in broad functional impairment of CD4+ T-cell activation beyond inhibition of proximal TCR-CD3 signaling.

Toll-like receptor 3 activation selectively reverses HIV latency in microglial cells.

BACKGROUND: 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. RESULTS: 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 cells. LPS (TLR4 agonist), flagellin (TLR5 agonist), and FSL-1 (TLR6 agonist) reactivated HIV to a lesser extent, while Pam3CSK4 (TLR2/1 agonist) and HKLM (TLR2 agonist) only weakly reversed HIV latency in these cells. While agonists for TLR2/1, 4, 5 and 6 reactivated HIV through transient NF-κB induction, poly (I:C), the TLR3 agonist, did not activate NF-κB, and instead induced the virus by a previously unreported mechanism mediated by IRF3. The selective induction of IRF3 by poly (I:C) was confirmed by chromatin immunoprecipitation (ChIP) analysis. In comparison, in latently infected rat-derived microglial cells (hT-CHME-5/HIV, clone HC14), poly (I:C), LPS and flagellin were only partially active. The TLR response profile in human microglial cells is also distinct from that shown by latently infected monocyte cell lines (THP-1/HIV, clone HA3, U937/HIV, clone HUC5, and SC/HIV, clone HSCC4), where TLR2/1, 4, 5, 6 or 8, but not for TLR3, 7 or 9, reactivated HIV. CONCLUSIONS: TLR signaling, in particular TLR3 activation, can efficiently reactivate HIV transcription in infected microglia, but not in monocytes or T cells. The unique response profile of microglial cells to TLR3 is fundamental to understanding how the virus responds to continuous microbial exposure, especially during inflammatory episodes, that characterizes HIV infection in the CNS.

TLR2 engagement on CD4(+) T cells enhances effector functions and protective responses to Mycobacterium tuberculosis.

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4(+) T cells and upregulate TCR-triggered IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4(+) T-cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T-cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4(+) T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T-cell TLR2 in priming of CD4(+) T cells in vivo, naive MTB Ag85B-specific TCR transgenic CD4(+) T cells (P25 TCR-Tg) were adoptively transferred into Tlr2(-/-) recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3 Cys-SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN-γ-secreting P25 TCR-Tg T cells 1 week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4(+) T cells increases MTB Ag-specific responses and may contribute to protection against MTB infection.

Human phagosome processing of Mycobacterium tuberculosis antigens is modulated by interferon-γ and interleukin-10.

Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen-MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide-MHC-II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide-MHC-II complexes in M. tuberculosis-infected human monocyte-derived macrophages (MDMs) using autologous M. tuberculosis-specific CD4(+) T cells. The MDMs were pre-treated with either IFN-γ or IL-10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M. tuberculosis -specific CD4(+) T cells. Our results demonstrated that in MDMs pre-treated with IFN-γ, M. tuberculosis peptide-MHC-II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN-γ, the complexes were detected in the endosomal fractions. In MDMs pre-treated with IL-10, the M. tuberculosis peptide-MHC-II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA-DR at the cell surface only in the IFN-γ-treated MDMs, suggesting that IFN-γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL-10 favours the trafficking of Ag85B to vesicles that do not contain LAMP-1. Therefore, IFN-γ and IL-10 play a role in the formation and trafficking of M. tuberculosis peptide-MHC-II complexes.

Induction of serine protease inhibitor 9 by Mycobacterium tuberculosis inhibits apoptosis and promotes survival of infected macrophages.

Our recent microarray analysis of infected human alveolar macrophages (AMs) found serine protease inhibitor 9 (PI-9) to be the most prominently expressed of a cluster of apoptosis-associated genes induced by virulent Mycobacterium tuberculosis. In the current study, we show that induction of PI-9 occurs within hours of infection with M. tuberculosis H37Rv and is maintained through 7 days of infection in both AMs and blood monocytes. Inhibition of PI-9 by small inhibitory RNA decreased M. tuberculosis-induced expression of the antiapoptotic molecule Bcl-2 and resulted in a corresponding increase in production of caspase 3, a terminal effector molecule of apoptosis. Further, PI-9 small inhibitory RNA mediated a significant reduction in the subsequent survival of M. tuberculosis within AMs. Thus PI-9 induction within human mononuclear phagocytes by virulent M. tuberculosis serves to protect these primary targets of infection from elimination by apoptosis and thereby promotes intracellular survival of the organism.

Mycobacterium tuberculosis ManLAM inhibits T-cell-receptor signaling by interference with ZAP-70, Lck and LAT phosphorylation.

Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust CD4(+) T cell responses. We have shown previously that M. tuberculosis cell wall glycolipids, including mannose capped lipoarabinomannan (ManLAM), directly inhibit polyclonal murine CD4(+) T cell activation by blocking ZAP-70 phosphorylation. We extended these studies to antigen-specific murine CD4(+) T cells and primary human T cells and found that ManLAM inhibited them as well. Lck and LAT phosphorylation also were inhibited by ManLAM without affecting their localization to lipid rafts. Inhibition of proximal TCR signaling was temperature sensitive, suggesting that ManLAM insertion into T cell membranes was required. Thus, M. tuberculosis ManLAM inhibits antigen-specific CD4(+) T cell activation by interfering with very early events in TCR signaling through ManLAM's insertion in T cell membranes.

Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection.

The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.

Mycobacterium tuberculosis lipoproteins directly regulate human memory CD4(+) T cell activation via Toll-like receptors 1 and 2.

The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4(+) T cells. In the absence of APCs, activation of memory CD4(+) T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4(+) T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4(+) T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.

Mycobacterium tuberculosis cell wall glycolipids directly inhibit CD4+ T-cell activation by interfering with proximal T-cell-receptor signaling.

Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust adaptive CD4(+) T-cell responses. We have previously shown that M. tuberculosis can 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 if M. tuberculosis could 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 of M. tuberculosis and 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 of M. tuberculosis. Fractionation identified that M. tuberculosis cell wall glycolipids, specifically, phosphatidylinositol mannoside and mannose-capped lipoarabinomannan, were potent inhibitors. Glycolipid-mediated inhibition was not dependent on Toll-like receptor signaling and could be bypassed through stimulation with phorbol 12-myristate 13-acetate and ionomycin. ZAP-70 phosphorylation was decreased in the presence of M. tuberculosis glycolipids, indicating that M. tuberculosis glycolipids directly inhibited CD4(+) T-cell activation by interfering with proximal T-cell-receptor signaling.

CD4+ CD25(high) Foxp3+ regulatory T cells downregulate human Vdelta2+ T-lymphocyte function triggered by anti-CD3 or phosphoantigen.

Vdelta2+ T cells, the major circulating T-cell receptor-gammadelta-positive (TCR-gammadelta+) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vdelta2+ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4+ T cells. CD4+ CD25(high) Foxp3+ T cells (Tregs) comprise 5-10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-alphabeta+ lymphocytes, Tregs suppress Vdelta2+ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vdelta2+ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vdelta2+ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-gamma secretion by Vdelta2+ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vdelta2+ T-cell interferon-gamma responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vdelta2+ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vdelta2+ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vdelta2+ T-cell functional deficiencies observed in TB.

Mycobacterium tuberculosis Induces Expansion of Foxp3 Positive CD4 T-cells with a Regulatory Profile in Tuberculin Non-sensitized Healthy Subjects: Implications for Effective Immunization against TB.

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 3H-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+CD25hi+ 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+CD25hi+ T-reg were TGFß positive (p<0.05). Further, MTB H37RvL induced CD4+CD25hi+ Foxp3+ iT-reg suppressed 3H-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 2,3-dideoxygenase (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 TGFß by siRNA reduced Foxp3 mRNA expression in CD4 T cells (p<0.05). CONCLUSION: Therefore, MTB and its components expand functional iT-reg in human mononuclear cells from MTB non-sensitized subjects. Also, MTB-induced iT-reg expansion depends on mononuclear phagocyte expression of both TGFß and IDO.

Novel high throughput pooled shRNA screening identifies NQO1 as a potential drug target for host directed therapy for tuberculosis.

UNLABELLED: 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. NAD(P)H: 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-1ß in response to infection. This suggests that mycobacteria hijacks NQO1 to down-regulate pro-inflammatory and anti-bacterial functions. The competitive inhibitor of NQO1 dicoumarol synergized with rifampin to promote intracellular killing of mycobacteria. Thus, NQO1 is a new host target in mycobacterial infection that could potentially be exploited to increase antibiotic efficacy in vivo. Our findings also suggest that pooled shRNA libraries could be valuable tools for genome-wide screening in the search for novel druggable host targets for adjunctive TB therapies.

Canaliculitis / Canaliculitis

RESUMEN La canaliculitis es una entidad rara, con frecuencia mal diagnosticada por su similitud con otras enfermedades. Se reporta una paciente femenina, de 56 años de edad, remitida a la Consulta de Oculoplastia del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", por secreciones purulentas y epífora del ojo izquierdo. Al examen se observó hiperemia conjuntival, secreción purulenta, punto lagrimal inferior hiperémico, dilatado, y se constató salida de concreciones por este al comprimir el canalículo. Se confirmó el diagnóstico de canaliculitis aguda supurada con concreciones. Se indicó tratamiento quirúrgico, que consistió en la canaliculotomía con remoción de las concreciones. Un examen clínico detallado, con adecuado conocimiento de la vía lagrimal excretora, permitió el diagnóstico certero, con un tratamiento quirúrgico eficaz y una evolución satisfactoria(AU)

ABSTRACT Canaliculitis is an uncommon infectious disease. It is often misdiagnosed due to its overlapping presentation to other common entities. A 56-year-old female patient is reported. She was referred to Ramón Pando Ferrer Cuban Ophthalmologic Institute, Ocular Plastic Surgery consultation, suffering from punctal swelling, discharge, and epiphora. At ocular examination was described conjunctival hyperemia, pouting punctum and mucopurulent discharge. Punctal regurgitation of concretions appears under syringing. It was confirmed acute canaliculitis with concretions in the left eye. A canaliculotomy was performed, and the concretions were removed. Routine clinical examinations helped to get a right diagnosis of canaliculitis and the surgical result was satisfactory(AU)

Human immunity to M. tuberculosis: T cell subsets and antigen processing.

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.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

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.