Functional domains of SP110 that modulate its transcriptional regulatory function and cellular translocation.

BACKGROUND: SP110, an interferon-induced nuclear protein, belongs to the SP100/SP140 protein family. Very recently, we showed that SP110b, an SP110 isoform, controls host innate immunity to Mycobacterium tuberculosis infection by regulating nuclear factor-κB (NF-κB) activity. However, it remains unclear how the structure of SP110 relates to its cellular functions. In this study, we provide experimental data illustrating the protein domains that are responsible for its functions. METHODS: We examined the effects of SP110 isoforms and a series of deletion mutants of SP110 on transcriptional regulation by luciferase reporter assays. We also employed confocal microscopy to determine the cellular distributions of enhanced green fluorescent protein-tagged SP110 isoforms and SP110 mutants. In addition, we performed immunoprecipitation and Western blotting analyses to identify the regions of SP110 that are responsible for protein interactions. RESULTS: Using reporter assays, we first demonstrated that SP110 isoforms have different regulatory effects on NF-κB-mediated transcription, supporting the notion that SP110 isoforms may have distinct cellular functions. Analysis of deletion mutants of SP110 showed that the interaction of the N-terminal fragment (amino acids 1-276) of SP110 with p50, a subunit of NF-κB, in the cytoplasm plays a crucial role in the down-regulation of the p50-driven tumor necrosis factor-α (TNFα) promoter activity in the nucleus, while the middle and C-terminal regions of SP110 localize it to various cellular compartments. Surprisingly, a nucleolar localization signal (NoLS) that contains one monopartite nuclear localization signal (NLS) and one bipartite NLS was identified in the middle region of SP110. The identification of a cryptic NoLS in the SP110 suggests that although this protein forms nuclear speckles in the nucleoplasm, it may be directed into the nucleolus to carry out distinct functions under certain cellular conditions. CONCLUSIONS: The findings from this study elucidating the multidomain structure of the SP110 not only identify functional domains of SP110 that are required for transcriptional regulation, cellular translocation, and protein interactions but also implicate that SP110 has additional functions through its unexpected activity in the nucleolus.

SP110b Controls Host Immunity and Susceptibility to Tuberculosis.

RATIONALE: How host genetic factors affect Mycobacterium tuberculosis (Mtb) infection outcomes remains largely unknown. SP110b, an IFN-induced nuclear protein, is the nearest human homologue to the mouse Ipr1 protein that has been shown to control host innate immunity to Mtb infection. However, the function(s) of SP110b remains unclear. OBJECTIVES: To elucidate the role of SP110b in controlling host immunity and susceptibility to tuberculosis (TB), as well as to identify the fundamental immunological and molecular mechanisms affected by SP110b. METHODS: Using cell-based approaches and mouse models of Mtb infection, we characterized the function(s) of SP110b/Ipr1. We also performed genetic characterization of patients with TB to investigate the role of SP110 in controlling host susceptibility to TB. MEASUREMENTS AND MAIN RESULTS: SP110b modulates nuclear factor-κB (NF-κB) activity, resulting in downregulation of tumor necrosis factor-α (TNF-α) production and concomitant upregulation of NF-κB-induced antiapoptotic gene expression, thereby suppressing IFN-γ-mediated monocyte and/or macrophage cell death. After Mtb infection, TNF-α is also downregulated in Ipr1-expressing mice that have alleviated cell death, less severe necrotic lung lesions, more efficient Mtb growth control in the lungs, and longer survival. Moreover, genetic studies in patients suggest that SP110 plays a key role in modulating TB susceptibility in concert with NFκB1 and TNFα genes. CONCLUSIONS: These results indicate that SP110b plays a crucial role in shaping the inflammatory milieu that supports host protection during infection by fine-tuning NF-κB activity, suggesting that SP110b may serve as a potential target for host-directed therapy aimed at manipulating host immunity against TB.

Shisa3 is associated with prolonged survival through promoting ß-catenin degradation in lung cancer.

RATIONALE: Despite advances in treatment and prognosis of non-small cell lung cancer (NSCLC), patient outcomes are still unsatisfactory. OBJECTIVES: To reduce the morbidity and mortality of patients with NSCLC, a more comprehensive understanding of mechanisms involved in cancer progression is urgently needed. METHODS: By comparison of gene expression profiles in the cell line pair with differential invasion ability, CL1-0 and CL1-5, we found that Shisa3 was highly expressed in the low invasive cells. The effect of Shisa3 on invasion, migration, proliferation, apoptosis, epithelial-mesenchymal transition, and anchorage-independent growth activities in vitro and on tumor growth and metastasis in mice models were examined. The underlying mechanism of Shisa3 was explored by microarray and pathway analysis. Finally, the correlation of Shisa3 expression and clinical outcome was also calculated. MEASUREMENTS AND MAIN RESULTS: We identified Shisa3 as a novel tumor suppressor, which induces ß-catenin degradation resulting in suppression of tumorigenesis and invasion in vitro. Shisa3 decreased the tumor growth in mice with subcutaneous implantation and reduced the number of metastatic nodules in mice with tail vein injection and orthotopic implantation. Shisa3 performs the tumor suppression activity through WNT signaling predicted by microarray analysis. Our data found that Shisa3 accelerates ß-catenin degradation and was positively associated with overall survival and progression-free survival of NSCLC. CONCLUSIONS: Our results reveal that Shisa3 acts as a tumor suppressor by acceleration of ß-catenin degradation and provide new insight for cancer prognosis and therapy.

Sorafenib relieves cell-intrinsic and cell-extrinsic inhibitions of effector T cells in tumor microenvironment to augment antitumor immunity.

Sorafenib, a multitargeted antiangiogenic tyrosine kinase inhibitor, is the standard of care for patients with advanced hepatocellular carcinoma (HCC). Cumulating evidence suggests that sorafenib differentially affects immune cells; however, whether this immunomodulatory effect has any impact on antitumor immune responses is unknown. Using an orthotopic mouse model of HCC and tumor-free mice, we investigated the effects of sorafenib on antitumor immunity and characterized the underlying mechanisms. Sorafenib treatment inhibited tumor growth and augmented antitumor immune responses in mice bearing established orthotopic HCC. The tumor-specific effector T cell functions were upregulated, while the proportion of PD-1-expressing CD8(+) T cells and regulatory T cells (Tregs) was reduced in tumor microenvironment of sorafenib-treated mice. Mechanistically, the sorafenib-mediated effects on Tregs could be independent of its direct tumor-suppressing activities. Sorafenib treatment reduced Treg numbers by inhibiting their proliferation and inducing apoptosis. Moreover, sorafenib inhibited the function of Tregs, characterized by diminished expression of Treg-associated molecules important for their function and by their impaired suppressive capacity. These data reveal that sorafenib treatment enhanced functions of tumor-specific effector T cells as well as relieved PD-1-mediated intrinsic and Treg-mediated non-cell-autonomous inhibitions in tumor microenvironment leading to effective antitumor immune responses. In addition to the well-known tumor-inhibiting activity of sorafenib, its enhancement of antitumor immunity may also contribute to the clinical efficacy. Our findings uncover a previously unrecognized mechanism of action of sorafenib and indicate that sorafenib represents a potential targeted agent suitable to be combined with immunotherapeutic approaches to treat cancer patients.

Gut microbiota dysbiosis-related susceptibility to nontuberculous mycobacterial lung disease.

The role of gut microbiota in host defense against nontuberculous mycobacterial lung disease (NTM-LD) was poorly understood. Here, we showed significant gut microbiota dysbiosis in patients with NTM-LD. Reduced abundance of Prevotella copri was significantly associated with NTM-LD and its disease severity. Compromised TLR2 activation activity in feces and plasma in the NTM-LD patients was highlighted. In the antibiotics-treated mice as a study model, gut microbiota dysbiosis with reduction of TLR2 activation activity in feces, sera, and lung tissue occurred. Transcriptomic analysis demonstrated immunocompromised in lung which were closely associated with increased NTM-LD susceptibility. Oral administration of P. copri or its capsular polysaccharides enhanced TLR2 signaling, restored immune response, and ameliorated NTM-LD susceptibility. Our data highlighted the association of gut microbiota dysbiosis, systematically compromised immunity and NTM-LD development. TLR2 activation by P. copri or its capsular polysaccharides might help prevent NTM-LD.

Inhibition of the Mycobacterium tuberculosis reserpine-sensitive efflux pump augments intracellular concentrations of ciprofloxacin and enhances susceptibility of some clinical isolates.

BACKGROUND/PURPOSE: Active efflux is known to play a major role in the resistance of many bacteria to antibiotics. To evaluate the possibility of overcoming resistance by suppressing the efflux, we determined the effect of reserpine, an efflux pump inhibitor. METHODS: Intracellular accumulations and the minimal inhibitory concentrations (MICs) of ciprofloxacin in M. tuberculosis H37Rv and 16 clinical isolates were determined, compared, and analyzed. Nine of the clinical isolates were resistant to isoniazid and rifampin (multiple-drug resistant MDR). Five of these were resistant to ciprofloxacin. RESULTS: A reserpine-inhibited efflux system was identified in the H37Rv control and 10:1 (90.9%) of ciprofloxacin-susceptible and 4:1 (80%) of ciprofloxacin-resistant clinical isolates. The MIC of ciprofloxacin decreased in the presence of reserpine in 3/10 (30%) of the ciprofloxacin-susceptible and 2/4 (50%) of the MDR ciprofloxacin-resistant strains that expressed efflux pumps. Two of the efflux-positive, ciprofloxacin-resistant strains in which the MIC of ciprofloxacin was not decreased by reserpine were found to carry a D94A gyrA mutation. In contrast, two strains with the D94G gyrA mutation were susceptible to ciprofloxacin in the presence of reserpine. An efflux-negative strain, highly resistant to multiple antibiotics, was found to have a novel G247S mutation that differs from known mutations in the QRDR region of the gyrA gene. CONCLUSION: These findings indicate t hat reserpine can increase intracellular concentrations of ciprofloxacin, but is unable to overcome other mechanisms of resistance in clinical isolates.

Susceptibility of Mycobacterium tuberculosis to sulfamethoxazole, trimethoprim and their combination over a 12 year period in Taiwan.

OBJECTIVES: This study was designed to determine the susceptibility of clinical isolates of multidrug-resistant (MDR) and non-MDR Mycobacterium tuberculosis to sulfamethoxazole, trimethoprim and trimethoprim/sulfamethoxazole over a 12 year period in Taiwan. PATIENTS AND METHODS: We examined a total of 117 clinical isolates of M. tuberculosis collected from Southern Taiwan, 116 from 1995 to 2006 and an extensively drug-resistant (XDR) isolate in 2009. These included 28 isolates susceptible to all four first-line agents, 52 MDR isolates and 36 isolates with a mixed combination of drug resistance patterns other than MDR and 1 XDR isolate. RESULTS: Sulfamethoxazole inhibited 80% growth of all 117 isolates regardless of their susceptibility to the first-line agents at an MIC(90) of 9.5 mg/L. The concentration required to inhibit 99% growth was 38 mg/L. There were no significant changes in the MIC(50) or MIC(90) of sulfamethoxazole over a 12 year period. All 117 isolates were resistant to trimethoprim at >8 mg/L. The combination of trimethoprim/sulfamethoxazole at a ratio of 1:19 had no additive or synergistic effects. CONCLUSIONS: Sulfamethoxazole inhibited the growth of clinical isolates of M. tuberculosis at achievable concentrations in plasma after oral administration. Susceptibility to sulfamethoxazole remained constant over a 12 year period. Trimethoprim was inactive against M. tuberculosis and trimethoprim/sulfamethoxazole provided no additional activity. Although the current and prior studies demonstrate that sulfamethoxazole is active against M. tuberculosis the search needs to continue for more active, lipid-soluble sulphonamides that are better absorbed into tissues and have improved therapeutic efficacy.

Ipr1 gene mediates innate immunity to tuberculosis.

An estimated eight million people are infected each year with the pathogen Mycobacterium tuberculosis, and more than two million die annually. Yet only about 10% of those infected develop tuberculosis. Genetic variation within host populations is known to be significant in humans and animals, but the nature of genetic control of host resistance to tuberculosis remains poorly understood. Previously we mapped a new genetic locus on mouse chromosome 1, designated sst1 (for supersusceptibility to tuberculosis 1). Here we show that this locus mediates innate immunity in sst1 congenic mouse strains and identify a candidate gene, Intracellular pathogen resistance 1 (Ipr1), within the sst1 locus. The Ipr1 gene is upregulated in the sst1 resistant macrophages after activation and infection, but it is not expressed in the sst1 susceptible macrophages. Expression of the Ipr1 transgene in the sst1 susceptible macrophages limits the multiplication not only of M. tuberculosis but also of Listeria monocytogenes and switches a cell death pathway of the infected macrophages from necrosis to apoptosis. Our data indicate that the Ipr1 gene product might have a previously undocumented function in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death and pathogenesis.

The integrated stress response mediates necrosis in murine Mycobacterium tuberculosis granulomas.

The mechanism by which only some individuals infected with Mycobacterium tuberculosis develop necrotic granulomas with progressive disease while others form controlled granulomas that contain the infection remains poorly defined. Mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human tuberculosis (TB) granulomas, which are linked to macrophage dysfunction, while their congenic counterpart (B6) mice do not. In this study we report that (a) sst1S macrophages developed aberrant, biphasic responses to TNF characterized by superinduction of stress and type I interferon pathways after prolonged TNF stimulation; (b) the late-stage TNF response was driven via a JNK/IFN-ß/protein kinase R (PKR) circuit; and (c) induced the integrated stress response (ISR) via PKR-mediated eIF2α phosphorylation and the subsequent hyperinduction of ATF3 and ISR-target genes Chac1, Trib3, and Ddit4. The administration of ISRIB, a small-molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis-infected sst1S mice and concomitantly reduced the bacterial burden. Hence, induction of the ISR and the locked-in state of escalating stress driven by the type I IFN pathway in sst1S macrophages play a causal role in the development of necrosis in TB granulomas. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel host-directed therapy strategies.

Novel CD8+ Treg suppress EAE by TGF-beta- and IFN-gamma-dependent mechanisms.

Although CD8+ Treg-mediated suppression has been described, CD8+ Treg remain poorly characterized. Here we identify a novel subset of CD8+ Treg that express latency-associated peptide (LAP) on their cell surface (CD8+LAP+ cells) and exhibit regulatory activity in vitro and in vivo. Only a small fraction of CD8+LAP+ cells express Foxp3 or CD25, although the expression levels of Foxp3 for these cells are higher than their LAP- counterparts. In addition to TGF-beta, CD8+LAP+ cells produce IFN-gamma, and these cells suppress EAE that is dependent on both TGF-beta and IFN-gamma. In an adoptive co-transfer model, CD8+LAP+ cells suppress myelin oligodendrocyte glycoprotein (MOG)-specific immune responses by inducing or expanding Foxp3+ cells and by inhibiting proliferation and IFN-gamma production in vivo. Furthermore, in vivo neutralization of IFN-gamma and studies with IFN-gamma-deficient mice demonstrate an important role for IFN-gamma production in the function of CD8+LAP+ cells. Our findings identify the underlying mechanisms that account for the immunoregulatory activity of CD8+ T cells and suggest that induction or amplification of CD8+LAP+ cells may be a therapeutic strategy to help control autoimmune processes.

Dominant role of the sst1 locus in pathogenesis of necrotizing lung granulomas during chronic tuberculosis infection and reactivation in genetically resistant hosts.

Significant host heterogeneity in susceptibility to tuberculosis exists both between and within mammalian species. Using a mouse model of infection with virulent Mycobacterium tuberculosis (Mtb), we identified the genetic locus sst1 that controls the progression of pulmonary tuberculosis in immunocompetent hosts. In this study, we demonstrate that within the complex, multigenic architecture of tuberculosis susceptibility, sst1 functions to control necrosis within tuberculosis lesions in the lungs; this lung-specific sst1 effect is independent of both the route of infection and genetic background of the host. Moreover, sst1-dependent necrosis was observed at low bacterial loads in the lungs during reactivation of the disease after termination of anti-tuberculosis drug therapy. We demonstrate that in sst1-susceptible hosts, nonlinked host resistance loci control both lung inflammation and production of inflammatory mediators by Mtb-infected macrophages. Although interactions of the sst1-susceptible allele with genetic modifiers determine the type of the pulmonary disease progression, other resistance loci do not abolish lung necrosis, which is, therefore, the core sst1-dependent phenotype. Sst1-susceptible mice from tuberculosis-resistant and -susceptible genetic backgrounds reproduce a clinical spectrum of pulmonary tuberculosis and may be used to more accurately predict the efficacy of anti-tuberculosis interventions in genetically heterogeneous human populations.

Differed IL-1 Beta Response between Active TB and LTBI Cases by Ex Vivo Stimulation of Human Monocyte-Derived Macrophage with TB-Specific Antigen.

BACKGROUND: The difference of macrophage-specific interleukin-1 beta (IL-1b) response between latent tuberculosis infection (LTBI) and active tuberculosis (TB) remains less studied. METHOD: We performed this prospective study and recruited active TB patients, contacts with LTBI, and uninfected contacts. The gene and protein expression of human monocyte-derived macrophage (hMDM) after ex vivo stimulation by early secretory antigenic target-6KD (ESAT-6) and tuberculin purified protein derivatives (PPD) was studied by real-time PCR and flow cytometry. The effect of caspase-1 inhibitor was also studied. RESULT: The IL-1b gene expression after 6 hr ESAT-6 1 µg/ml stimulation was different among active TB patients (n = 12), LTBI cases (n = 12), and uninfected contacts (n = 23) (log fold change: 0.98 ± 1.26 vs. 2.20 ± 0.96 vs. 2.20 ± 0.96, P = 0.013). The IL-1b gene expression at 24 hours was higher than that at 6 hours in LTBI cases (n = 4) and uninfected contacts (n = 6). After 24 hr ESAT-6 1 µg/ml stimulation, the percentage of IL-1b-expressed hMDM was borderline lower in the active TB patients (n = 9) than in the LTBI cases (n = 10) (14.0 ± 11.2% vs. 31.6 ± 22.5%, P = 0.065). Compared with ESAT-6 1 µg/ml stimulation but without the addition of caspase-1 inhibitor (CasI) (55.6 ± 16.3%), the percentage of IL-1b-positive hMDMs decreased after addition of CasI (50 µg/ml CasI: 49.8 ± 18.2%, P = 0.078; 100 µg/ml CasI: 46.6 ± 20.8%, P = 0.030; 150 µg/ml CasI: 33.7 ± 15.5%, P = 0.016). CONCLUSIONS: This study revealed that macrophage-specific IL-1b response differed among different stages of Mycobacterium tuberculosis infection. The role of IL-1b and inflammasome in the process of LTBI progressing to active TB warrants further investigation.

NLRP3 inflammasome is attenuated in patients with Mycobacterium avium complex lung disease and correlated with decreased interleukin-1ß response and host susceptibility.

The incidence of nontuberculous mycobacteria lung disease (NTM-LD) is increasing in patients without human immunodeficiency virus. Mycobacterium avium complex (MAC) is one of the most common pathogenic species. The presence of MAC has a clinical relevance of around 35~42%, indicating the possibility of host susceptibility. Previous studies have shown that interleukin (IL)-1ß and IL-1-receptor knock-out mice are susceptible to mycobacterial infections; however, the role of inflammasome-driven interleukin (IL)-1ß has not been studied in MAC-LD. We enrolled patients with MAC-LD and healthy controls. Peripheral blood mononuclear cells (PBMCs), monocytes, and monocyte-derived macrophages were stimulated by MAC bacilli. The responses of interleukin(IL)-1ß and the expression of inflammasome and toll-like receptors (TLRs) were measured. Single nucleotide polymorphisms (SNPs) were also examined for NLRP3 and TLR2 genes. In the patients with MAC-LD, the IL-1ß responses decreased in PBMCs, monocytes, and macrophages assayed by MAC bacilli in comparison to the healthy controls. In addition, the level of caspase-1 after stimulation was lower in the MAC-LD group, although the mRNA level of IL-1ß was not significantly lower. In surveying the activation of IL-1ß, the MAC-LD group had an attenuated mRNA level of NLRP3 but similar levels of AIM2 and ASC compared with the controls. The SNPs rs3806268 and rs34298354 in NLRP3 for females and rs3804100 in TLR2 for males were associated with MAC-LD. In conclusion, our patients with MAC-LD had attenuated IL-1ß production, which may have been due to lower activation of the NLRP3-caspase-1 axis. Two SNPs of NLRP3 and one of TLR2 were correlated with MAC-LD, possibly indicating host susceptibility.

Isoniazid Concentration and NAT2 Genotype Predict Risk of Systemic Drug Reactions during 3HP for LTBI.

Weekly rifapentine and isoniazid therapy (known as 3HP) for latent tuberculosis infection (LTBI) is increasingly used, but systemic drug reactions (SDR) remain a major concern. Methods: We prospectively recruited two LTBI cohorts who received the 3HP regimen. In the single-nucleotide polymorphism (SNP) cohort, we collected clinical information of SDRs and examined the NAT2, CYP2E1, and AADAC SNPs. In the pharmacokinetic (PK) cohort, we measured plasma drug and metabolite levels at 6 and 24 h after 3HP administration. The generalised estimating equation model was used to identify the factors associated with SDRs. Candidate SNPs predicting SDRs were validated in the PK cohort. A total of 177 participants were recruited into the SNP cohort and 129 into the PK cohort, with 14 (8%) and 13 (10%) in these two cohorts developing SDRs, respectively. In the SNP cohort, NAT2 rs1041983 (TT vs. CC+CT, odds ratio [OR] [95% CI]: 7.00 [2.03-24.1]) and CYP2E1 rs2070673 (AA vs. TT+TA, OR [95% CI]: 3.50 [1.02-12.0]) were associated with SDR development. In the PK cohort, isoniazid level 24 h after 3HP administration (OR [95% CI]: 1.61 [1.15-2.25]) was associated with SDRs. Additionally, the association between the NAT2 SNP and SDRs was validated in the PK cohort (rs1041983 TT vs. CC+CT, OR [95% CI]: 4.43 [1.30-15.1]). Conclusions: Isoniazid played a role in the development of 3HP-related SDRs. This could provide insight for further design of a more optimal regimen for latent TB infection.

SP110 Polymorphisms Are Genetic Markers for Vulnerability to Latent and Active Tuberculosis Infection in Taiwan.

One-fourth of the human population is estimated to have been exposed to Mycobacterium tuberculosis (Mtb) and carries the infection in its latent form. This latent infection presents a lifelong risk of developing active tuberculosis (TB) disease, and persons with latent TB infection (LTBI) are significant contributors to the pool of active TB cases. Genetic polymorphisms among hosts have been shown to contribute to the outcome of Mtb infection. The SP110 gene, which encodes an interferon-induced nuclear protein, has been shown to control host innate immunity to Mtb infection. In this study, we provide experimental data demonstrating the ability of the gene to control genetic susceptibility to latent and active TB infection. Genetic variants of the SP110 gene were investigated in the Taiwanese population (including 301 pulmonary TB patients, 68 LTBI individuals, and 278 healthy household contacts of the TB patients), and their association with susceptibility to latent and active TB infection was examined by performing an association analysis in a case-control study. We identified several SNPs (rs7580900, rs7580912, rs9061, rs11556887, and rs2241525) in the SP110 gene that are associated with susceptibility to LTBI and/or TB disease. Our studies further showed that the same SNPs may have opposite effects on the control of susceptibility to LTBI versus TB. In addition, our analyses demonstrated that the SP110 rs9061 SNP was associated with tumor necrosis factor-α (TNFα) levels in plasma in LTBI subjects. The results suggest that the polymorphisms within SP110 have a role in controlling genetic susceptibility to latent and active TB infection in humans. To the best of our knowledge, this is the first report showing that the SP110 variants are associated with susceptibility to LTBI. Our study also demonstrated that the identified SP110 SNPs displayed the potential to predict the risk of LTBI and subsequent TB progression in Taiwan.

Rapid Sputum Multiplex Detection of the M. tuberculosis Complex (MTBC) and Resistance Mutations for Eight Antibiotics by Nucleotide MALDI-TOF MS.

The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) adds further urgency for rapid and multiplex molecular testing to identify the MTB complex and drug susceptibility directly from sputum for disease control. A nucleotide matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed to identify MTB (MTBID panel) and 45 chromosomal mutations for resistance to eight antibiotics (MTBDR panel). We conducted a 300 case trial from outpatients to evaluate this platform. An MTBID panel specifically identified MTB with as few as 10 chromosome DNA copies. The panel was 100% consistent with an acid-fast stain and culture for MTB, nontuberculous mycobacteria, and non-mycobacteria bacteria. The MTBDR panel was validated using 20 known MDR-MTB isolates. In a 64-case double-blind clinical isolates test, the sensitivity and specificity were 83% and 100%, respectively. In a 300-case raw sputum trial, the MTB identification sensitivity in smear-negative cases using MALDI-TOF MS was better than the COBAS assay (61.9% vs. 46.6%). Importantly, the failure rate of MALDI-TOF MS was better than COBAS (11.3% vs. 26.3%). To the best of our knowledge, the test described herein is the only multiplex test that predicts resistance for up to eight antibiotics with both sensitivity and flexibility.

Enterovirus-induced miR-141 contributes to shutoff of host protein translation by targeting the translation initiation factor eIF4E.

Viruses rely on the host translation machinery to complete their life cycles. Picornaviruses use an internal ribosome entry site to initiate cap-independent protein translation and in parallel host cap-dependent translation is shut off. This process is thought to occur primarily via cleavage of host translation initiation factors eIF4GI and eIF4GII by viral proteases. Here we describe another mechanism whereby miR-141 induced upon enterovirus infection targets the cap-dependent translation initiation factor, eIF4E, for shutoff of host protein synthesis. Knockdown of miR-141 reduces viral propagation, and silencing of eIF4E can completely reverse the inhibitory effect of the miR-141 antagomiR on viral propagation. Ectopic expression of miR-141 promotes the switch from cap-dependent to cap-independent translation. Moreover, we identified a transcription factor, EGR1, which is partly responsible for miR-141 induction in response to enterovirus infection. Our results suggest that upregulation of miR-141 upon enterovirus infection can facilitate viral propagation by expediting the translational switch.

Latency-associated peptide identifies a novel CD4+CD25+ regulatory T cell subset with TGFbeta-mediated function and enhanced suppression of experimental autoimmune encephalomyelitis.

CD4(+)CD25(+) regulatory T cells (Tregs) are essential for maintaining self-tolerance and immune homeostasis. Here we characterize a novel subset of CD4(+)CD25(+) Tregs that express latency-associated peptide (LAP) on their cell surface (CD4(+)CD25(+)LAP(+) cells). CD4(+)CD25(+)LAP(+) cells express elevated levels of Foxp3 and Treg-associated molecules (CTLA4, glucocorticoid-induced TNFR-related gene), secrete TGFbeta, and express both cell surface TGFbeta and surface receptors for TGFbeta. In vitro, the suppressive function of CD4(+)CD25(+)LAP(+) cells is both cell contact and soluble factor dependent; this contrasts with CD4(+)CD25(+)LAP(-) cells, which are mainly cell contact dependent. In a model of experimental autoimmune encephalomyelitis, CD4(+)CD25(+)LAP(+) cells exhibit more potent suppressive activity than CD4(+)CD25(+)LAP(-) cells, and the suppression is TGFbeta dependent. We further show that CD4(+)CD25(+)LAP(+) cells suppress myelin oligodendrocyte glycoprotein-specific immune responses by inducing Foxp3 and by inhibiting IL-17 production. Our findings demonstrate that CD4(+)CD25(+) Tregs are a heterogeneous population and that the CD4(+)CD25(+) subset that expresses LAP functions in a TGFbeta-dependent manner and has greater in vivo suppressive properties. Our work helps elucidate the ambiguity concerning the role of TGFbeta in CD4(+)CD25(+) Treg-mediated suppression and indicates that LAP is an authentic marker able to identify a TGFbeta-expressing CD4(+)CD25(+) Treg subset.

Heme oxygenase-1-derived carbon monoxide induces the Mycobacterium tuberculosis dormancy regulon.

The mechanisms that allow Mycobacterium tuberculosis (Mtb) to persist in human tissue for decades and to then abruptly cause disease are not clearly understood. Regulatory elements thought to assist Mtb to enter such a state include the heme two-component sensor kinases DosS and DosT and the cognate response regulator DosR. We have demonstrated previously that O(2), nitric oxide (NO), and carbon monoxide (CO) are regulatory ligands of DosS and DosT. Here, we show that in addition to O(2) and NO, CO induces the complete Mtb dormancy (Dos) regulon. Notably, we demonstrate that CO is primarily sensed through DosS to induce the Dos regulon, whereas DosT plays a less prominent role. We also show that Mtb infection of macrophage cells significantly increases the expression, protein levels, and enzymatic activity of heme oxygenase-1 (HO-1, the enzyme that produces CO), in an NO-independent manner. Furthermore, exploiting HO-1(+/+) and HO-1(-/-) bone marrow-derived macrophages, we demonstrate that physiologically relevant levels of CO induce the Dos regulon. Finally, we demonstrate that increased HO-1 mRNA and protein levels are produced in the lungs of Mtb-infected mice. Our data suggest that during infection, O(2), NO, and CO are being sensed concurrently rather than independently via DosS and DosT. We conclude that CO, a previously unrecognized host factor, is a physiologically relevant Mtb signal capable of inducing the Dos regulon, which introduces a new paradigm for understanding the molecular basis of Mtb persistence.

Progression of pulmonary tuberculosis and efficiency of bacillus Calmette-Guérin vaccination are genetically controlled via a common sst1-mediated mechanism of innate immunity.

Using a mouse model for genetic analysis of host resistance to virulent Mycobacterium tuberculosis, we have identified a genetic locus sst1 on mouse chromosome 1, which controls progression of pulmonary tuberculosis. In vitro, this locus had an effect on macrophage-mediated control of two intracellular bacterial pathogens, M. tuberculosis and Listeria monocytogenes. In this report, we investigated a specific function of the sst1 locus in antituberculosis immunity in vivo, especially its role in control of pulmonary tuberculosis. We found that the sst1 locus affected neither activation of Th1 cytokine-producing T lymphocytes, nor their migration to the lungs, but rather controlled an inducible NO synthase-independent mechanism of innate immunity. Although the sst1(S) macrophages responded to stimulation with IFN-gamma in vitro, their responsiveness to activation by T cells was impaired. Boosting T cell-mediated immunity by live attenuated vaccine Mycobacterium bovis bacillus Calmette-Guérin or the adoptive transfer of mycobacteria-activated CD4(+) T lymphocytes had positive systemic effect, but failed to improve control of tuberculosis infection specifically in the lungs of the sst1(S) animals. Thus, in the mouse model of tuberculosis, a common genetic mechanism of innate immunity mediated control of tuberculosis progression in the lungs and the efficiency of antituberculosis vaccine. Our data suggest that in immunocompetent humans the development of pulmonary tuberculosis and the failure of the existing vaccine to protect against it, in some cases, may be explained by a similar defect in a conserved inducible NO synthase-independent mechanism of innate immunity, either inherited or acquired.