Search

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 (sst1(S)) 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) sst1(S) 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-β/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 sst1(S) 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 sst1(S) 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.

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)-1β and IL-1-receptor knock-out mice are susceptible to mycobacterial infections; however, the role of inflammasome-driven interleukin (IL)-1β 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)-1β 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-1β 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-1β was not significantly lower. In surveying the activation of IL-1β, 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-1β 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.

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.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.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.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.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.

Necrosis in the tuberculous granuloma is a hallmark of tuberculosis that enables pathogen survival and transmission. Susceptibility to tuberculosis and several other intracellular bacteria is controlled by a mouse genetic locus, sst1, and mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human TB granulomas. Our previous work established that increased disease severity in sst1S mice reflects dysfunctional macrophage effector or tolerance mechanisms, but the molecular mechanisms have remained unclear. Here we demonstrate that sst1S macrophages develop aberrant, biphasic responses to TNF characterized by super-induction of stress and type I interferon pathways after prolonged TNF stimulation with this late-stage response being initiated by oxidative stress and Myc activation and driven via a JNK - IFNβ - PKR circuit. This circuit leads to induction of the integrated stress response (ISR) mediated by eIF2α phosphorylation and the subsequent hyper-induction of ATF3 and ISR-target genes Chac1, Trib3, 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 revealing that induction of the ISR and the locked-in state of escalating stress driven by type I IFN pathway in sst1S macrophages plays a causal role in the development of necrosis. Our data support a generalizable paradigm in intracellular pathogen-host interactions wherein host susceptibility emerges within inflammatory tissue due to imbalanced macrophage responses to growth, differentiation, activation and stress stimuli. Successful pathogens such as M. tuberculosis may exploit this aberrant response in susceptible hosts to induce necrotic lesions that favor long-term pathogen survival and transmission. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel therapeutic strategies.

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.

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-β, CD8+LAP+ cells produce IFN-γ, and these cells suppress EAE that is dependent on both TGF-β and IFN-γ. 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-γ production in vivo. Furthermore, in vivo neutralization of IFN-γ and studies with IFN-γ-deficient mice demonstrate an important role for IFN-γ 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.

Tuberculosis remains a significant global health problem: one-third of the human population is infected with Mycobacterium tuberculosis (MTB) and 10% of those are at lifetime risk of developing tuberculosis. Although an important role of genetic variation of host in outcomes of tuberculosis infection is well documented, genetic determinants of susceptibility in immunocompetent individuals remain largely unknown due to complex multigenic control and significant impact of genes - environment interactions. Natural genetic variation of host resistance to MTB in immunocompetent inbred mice reflects heterogeneity among humans and allows stepwise dissection of the genetic control using a mouse model. Previously we characterized a mouse super-susceptibility locus (sst1) and mapped additional quantitative trait loci (QTLs) controlling anti-tuberculosis immunity, among which a QTL on chromosome 7 was most prominent. In this study we addressed a specific role of the chromosome 7 QTL and found that the chromosome 7 and sst1 loci independently control distinct mechanisms of host resistance to MTB, but the phenotypic expression of the chromosome 7 locus is significantly influenced by interactions with the sst1. While the sst1 locus is especially important in the lungs and the effect of the chromosome 7 locus is systemic, both loci affect macrophage-mediated control of virulent MTB in vivo. Their combined effect accounts for half of the dramatic difference in survival between the susceptible and resistant parental strains. Further genetic and functional dissection of the chromosome 7 locus to identify causal genetic variation will help untangle the genetic basis of tuberculosis susceptibility in immunocompetent hosts.

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 TGFβ, and express both cell surface TGFβ and surface receptors for TGFβ. 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 TGFβ 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 TGFβ-dependent manner and has greater in vivo suppressive properties. Our work helps elucidate the ambiguity concerning the role of TGFβ in CD4+CD25+ Treg-mediated suppression and indicates that LAP is an authentic marker able to identify a TGFβ-expressing CD4+CD25+ Treg subset.

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 sst1S macrophages responded to stimulation with IFN-γ 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 sst1S 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.

Maltose-binding protein (MBP) encoded by malE is essential for the energy-dependent translocation of maltose through the cytoplasmic membrane of bacteria. Its property of specific binding to maltose has been used in constructing fusion proteins for easy affinity purification. A monoclonal antibody named MAb SC1D7 was produced against Escherichia coli MBP. This MAb also bound to MBP-containing recombinant proteins in both Western blotting and immunoprecipitation analysis. As a result, this MAb can be a useful probe for tracing MBP-fusion proteins in various applications. Furthermore, intrinsic MBPs from E. coli, Shigella dysenteriae, Salmonella typhimurium, Enterobacter cloacae, and Klebsiella pneumoniae were also detected by this MAb. No reaction was observed with the total proteins from Serratia marcescens, Aeromonas hydrophila and Plesiomonas shigelloides. These observations suggest that the MAb SC1D7-defined epitope is conserved among some enteric bacteria, but not the others. The results strengthen the phylogenetic positions of these closely related bacteria previously placed by other means.

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.

Tuberculosis remains a significant public health problem: one-third of the human population is infected with virulent Mycobacterium tuberculosis (MTB) and 10% of those are at risk of developing tuberculosis during their lifetime. In both humans and experimental animal models, genetic variation among infected individuals contributes to the outcome of infection. However, in immunocompetent individuals (the majority of patients), genetic determinants of susceptibility to tuberculosis remain largely unknown. Mouse models of MTB infection, allowing control of exposure and other potential environmental contributors, have proven extremely useful for examining this genetic component. In a cross of C3HeB/FeJ (susceptible) by C57BL/6J (resistant) inbred mouse strains, we have previously identified one major genetic locus, sst1, the susceptible allele of which did not confer an overt immunodeficiency, but rather specifically affected progression of lung tuberculosis. Having generated and tested the sst1 congenic strains, we have observed that this locus only partially explained the difference in susceptibility of the parental strains to MTB. We now present further studies controlling for the effect of the sst1, identify four additional tuberculosis susceptibility loci and characterize their effects by testing an independent cross, knockout or congenic mice.

Epidemiological, clinical, and experimental approaches have convincingly demonstrated that host resistance to infection with intracellular pathogens is significantly influenced by genetic polymorphisms. Using a mouse model of infection with virulent Mycobacterium tuberculosis (MTB), we have previously identified the sst1 locus as a genetic determinant of host resistance to tuberculosis. In this study we demonstrate that susceptibility to another intracellular pathogen, Listeria monocytogenes, is also influenced by the sst1 locus. The contribution of sst1 to anti-listerial immunity is much greater in immunodeficient scid mice, indicating that this locus controls innate immunity and becomes particularly important when adaptive immunity is significantly depressed. Similar to our previous observations using infection with MTB, the resistant allele of sst1 prevents formation of necrotic infectious lesions in vivo. We have shown that macrophages obtained from sst1-resistant congenic mice possess superior ability to kill L. monocytogenes in vitro. The bactericidal effect of sst1 is dependent on IFN-γ activation and reactive oxygen radical production by activated macrophages after infection, but is independent of NO production. It is possible that there is a single gene that controls common IFN-dependent macrophage function, which is important in the pathogenesis of infections caused by both MTB and L. monocytogenes. However, host resistance to the two pathogens may be controlled by two different polymorphic genes encoded within the sst1 locus. The polymorphic gene(s) encoded within the sst1 locus that controls macrophage interactions with the two intracellular pathogens remains to be elucidated.

Six monoclonal antibodies (MAbs) were raised in mice against the 26-kDa glutathione S-transferase (GST) of the parasite Schistosoma japonicum. These MAbs were originally selected for their specific binding to the recombinant GST (r-GST) generated in E. coli by an enzyme-linked immunosorbent assay. A further study demonstrated that all these MAbs bound to plate-coated GST affinity-purified from the parasite Schistosoma japonicum. However, in Western blotting analysis only a single monoclonal antibody (MAb Y3D7) yielded positive binding. The binding of MAb Y3D7 on Western blotting was further characterized; specific binding was found on other GST fusion proteins and on the authentic 26-kDa GST but not the 28-kDa GST in the total soluble worm proteins from Schistosoma japonicum. Using protein-A-mediated immunoprecipitation, MAbs Y3D7 and Y5D5 precipitated r-GST while in parallel experiments the remaining MAbs did not generate r-GST precipitation. In an alternative co-precipitation experiment, r-GST was first bound to glutathione (GSH) Sepharose beads and subsequently tested for interaction with the MAbs. In this manner, all MAbs except MAb Y5D5 were co-precipitated with the complexes. Thus, these select MAbs readily reacted with GST although their binding characteristics were different. Because GST has been widely used in the generation of fusion proteins for various purposes and is a potential vaccine candidate in controlling schistosomiasis, these MAbs should prove valuable for their application to molecular biology and parasitology.