Attenuation of experimental asthma by mycobacterial protein combined with CpG requires a TLR9-dependent IFN-γ-CCR2 signalling circuit.

BACKGROUND: Allergic asthma is a chronic pulmonary disease characterized by a Th2 inflammatory response. The modulation of a Th2 immune response based on immune deviation to a Th1 pattern or induction and migration of regulatory T cells to the lungs constitutes one of the major therapeutic approaches that is being investigated for the treatment of allergic asthma. The potentials of Mycobacterium leprae 65-kD heat-shock protein or Toll-like receptor 9 ligand (CpG oligodeoxynucleotides) as immune modulators for the treatment of airway allergic disease have been studied individually. OBJECTIVE: Mycobacterial protein combined with CpG was used as immunotherapy for airway allergy. METHODS: Using an ovalbumin-induced asthma model, mice were sensitized and challenged, and then treated with mycobacterial heat-shock protein (Hsp65) combined with CpG. RESULTS: The treatment of mice with established allergy led to the attenuation of eosinophilia, Th2 cytokines and airway hyperresponsiveness. Hsp65 plus CpG treatment also induced an increase in OVA-specific IFN-γ levels and in the frequency of lung inflammatory monocytes. Moreover, we show that the reduction of eosinophilia and the recruitment of inflammatory monocytes to the lungs required early triggering of TLR9, IFN-γ and CCR2 by immunotherapy components. CONCLUSION: In addition to immune deviation to a Th1 response in the modulation of Th2 allergic inflammation, our findings also attribute an important role to the innate response mediated by TLR9, associated with the recruitment of CCR2-dependent monocytes. CLINICAL RELEVANCE: Our findings show that the Hsp65/CpG treatment is a promising strategy for consideration in translational studies.

A synthetic holliday junction is sandwiched between two tetrameric Mycobacterium leprae RuvA structures in solution: new insights from neutron scattering contrast variation and modelling.

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium leprae RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RG values for the free and complexed protein in 65% 2H2O, where the DNA component is "invisible", showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (x10(-5)), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1-2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.

Both the basal and inducible transcription of the tyrosine hydroxylase gene are dependent upon a cAMP response element.

The cAMP response element (CRE) mediates cAMP responsiveness in many eukaryotic genes (Roesler, W. J., Vandenbark, G. R., and Hansen, R. W. (1988) J. Biol. Chem. 263, 9063-9066). The tyrosine hydroxylase gene (TH) contains a single copy of a consensus CRE at -45 to -38 base pair (bp) upstream of the transcription initiation site. Deletional and mutational analyses of the upstream 2400-base pair region of the rat TH gene using transient transfection assay demonstrated that the CRE was essential for both cAMP-mediated induction and basal transcription of the TH gene. Another domain between -365 and -151 bp, containing the AP1 site, contributed to transcription to a smaller degree. Thus, the CRE appears to play an important dual role as a basal promoter element and an inducible enhancer for TH transcription. Interactions between the DNA binding factors in nuclear extract and CRE-containing oligonucleotides were investigated by gel retardation and competition assays. Oligonucleotides corresponding to the CRE regions of the TH or somatostatin gene gave rise to a pair of distinct protein-DNA complexes with identical mobilities in the gel retardation assay, suggesting that similar nuclear factor(s) might bind to the CREs of the TH and somatostatin genes. This study emphasizes a fundamental role of the CRE in transcriptional activation of the TH gene in catecholaminergic cells.

Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65-kDa heat-shock protein: selective in vivo expansion of T cells bearing defined receptors.

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65-kDa heat-shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo. DR3-restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF-derived clones tested expressed either V beta 5.2 or a closely related beta chain, V beta 5.6. The alpha chains expressed by V beta 5.2+ and V beta 5.6+ clones were from different families, V alpha 2.4 and V alpha 23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either V beta 5.2 or V beta 5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of V beta 5. Conservation of the beta chain third complementarity-determining region (CDR3) sequence was not evident, however. Sequencing alpha and beta chains of representative V beta 5.2+ and V beta 5.6+ PB-derived clones revealed TcR which were identical to those utilized by the SF-derived clones, showing that the repertoire for recognition of this antigen is stable over time. Similar studies of TcR expression were carried out on hsp65-specific, DP4-restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of alpha chain usage, since all clones expressed a member of the V alpha 1 family, but again CDR3 sequence conservation was not apparent. beta chain usage was not restricted since different clones expressed V beta 6.7, V beta 22.3 and V beta 12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical alpha and beta TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.

Sequence and immunological characterization of a serine-rich antigen from Mycobacterium leprae.

Sera from lepromatous leprosy patients were used to screen a Mycobacterium leprae lambda gt11 library. Three positive plaques were picked, and lysogens were constructed. Immunoblot analysis showed that all of the lysogens expressed an apparently identical beta-galactosidase fusion protein which reacted strongly with the sera. The 1.7-kbp insert from one clone was subcloned into the lacZ gene in pUR290; sequence analysis of the end fused to lacZ revealed an open reading frame with no significant homology to previously published sequences. The insert was used to screen an M. leprae cosmid library, and five clones were isolated. The insert was also found to hybridize to clones expressing the M. leprae antigen which had previously been designated class III and 25L. A 1.8-kbp HindIII fragment was subcloned from one of the cosmids and sequenced. The sequence revealed a 1,227-bp open reading frame, encoding a 408-amino-acid protein with a predicted molecular mass of 42,466 Da. The protein contains amino- and carboxy-terminal hydrophobic domains and a hydrophilic central domain; the amino-terminal domain shows some homology to a 51-kDa hypothetical antigen of Mycobacterium tuberculosis, while the hydrophilic region contains a high proportion of serine residues, and we have therefore designated the protein serine-rich antigen (Sra). Some repeated motifs are present in the protein, but their significance is unknown. Seventy-eight percent of serum samples from multibacillary leprosy patients and 68% of serum samples from paucibacillary leprosy patients recognized the fusion protein, showing that this is a major M. leprae antigen. In contrast, all serum samples from endemic controls were negative, while 26% of serum samples from tuberculosis patients were weakly positive.

Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes.

The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes.

Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria.

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.

Selection of T lymphocytes bearing limited T-cell receptor beta chains in the response to a human pathogen.

Delayed-type hypersensitivity (DTH) is a classic measure of T-cell responsiveness to foreign antigen. To estimate the extent of the T-cell repertoire in the DTH response to a human pathogen, we measured T-cell receptor (TCR) beta-chain variable-region (V beta) gene usage in reversal reactions in leprosy. Reversal reactions represent naturally occurring DTH responses in leprosy, in which augmentation of T-cell responses to Mycobacterium leprae is concomitant with clearance of bacilli from lesions. T cells using the V beta 6-, V beta 12-, V beta 14-, and V beta 19-encoded TCRs were strikingly overrepresented in the lesions of patients as compared to blood and pre-DTH lesions from the same individuals. Furthermore, these data indicate a possible association between the predominant expression of a V beta gene segment in lesions and the major histocompatibility complex class II haplotype of the individual. V beta 6 was prominent in the lesions of four patients who were DR15, a marker of resistance in leprosy infection. Sequence analysis of V beta 6 TCRs showed frequent use of V beta 6.1 and J beta 2.7 gene segments and a conserved amino acid motif in the V-J junction in a reversal-reaction lesion, but not in blood from the same patient. The limited TCR repertoire expressed by the infiltrating T cells suggests that a limited set of antigens is recognized in the DTH response to a human pathogen. We suggest that the mechanism by which major histocompatibility complex haplotype influences DTH in this disease involves the presentation of specific peptides, with subsequent selection of specific TCRs followed by local oligoclonal expansion.

T-cell receptor beta-chain gene usage in the T-cell recognition of Mycobacterium leprae antigens in one tuberculoid leprosy patient.

The beta chain of the T-cell antigen receptor present on 20 T-cell clones isolated from a tuberculoid leprosy patient was studied by gene rearrangement and PCR analysis. These T-cell clones all responded to Mycobacterium leprae-encoded protein antigens, and 8 of them specifically recognized peptides of the mycobacterial 65-kDa heat shock polypeptide (65hsp). All T-cell clones studied were HLA-DR-restricted (DR2 or -3). In the DR3-restricted group, 7 of 10 used a beta-chain variable region V beta 5 gene family member, whereas in the DR2-restricted group, 2 of 10 T-cell clones used a V beta 5 gene segment and 5 used the V beta 18 gene segment. The deduced amino acid sequences of the beta chain from 8 T-cell clones have revealed that 3 of 4 DR3-restricted T-cell clones expressed the V beta 5.1 gene segment whereas the fourth DR3-restricted T-cell clone employed a V beta 5 family member not previously described. The V beta 5.1-positive T-cell clones all recognized the same 65hsp peptide from residues 2 to 12. The N-D-N segment (where D is diversity) of the junctional region of these T-cell clones was very similar, despite different beta-chain joining gene segments. Of the 4 DR2-restricted T-cell clones investigated, 3 used the V beta 18 gene segment and recognized the 65hsp peptide from residues 418 to 427. In conclusion, within this panel of M. leprae-reactive T-cell clones, the DR3-restricted T-cell clones mainly used a V beta 5 gene segment, whereas the DR2-restricted clones employed preferentially the V beta 18 gene segment.

Cytokine patterns of immunologically mediated tissue damage.

Reactional states in leprosy are produced by different immunologic mechanisms and are responsible for a major component of tissue damage of the disease. Reversal reactions exhibit increased CD4 T cell infiltration in lesions and augmented cell-mediated immune reactivity to Ag of Mycobacterium leprae that can rapidly produce nerve damage. Erythema nodosum leprosum (ENL) reactions also have CD4 T cell infiltration but appear to be associated with the formation of immune complexes that are responsible for panniculitis, arthritis, vasculitis, and nerve injury. Because these reactional states may serve as paradigms for other types of human immunologically mediated tissue damage, this study sought to characterize the dynamic changes in cytokines associated with these reactions. Expression of cytokine mRNA in lesions of leprosy reactional states were measured by PCR. In reversal reactions, IL-1 beta, TNF-alpha, IL-2, and IFN-gamma mRNA were prominent and found to increase during the reaction, concomitant with decreases in expression of mRNA for IL-4, IL-5, and IL-10. In ENL, selective increases in the expression of IL-6, IL-8, and IL-10 mRNA was observed, with persistent expression of IL-4 and IL-5 mRNA. Reversal reactions represent naturally occurring delayed-type hypersensitivity reactions that favor macrophage activation and protective immunity, but which can engender concomitant cell injury. In contrast, ENL lesions represent immediate-type hypersensitivity reactions reflecting the selective stimulation of cytokines that attract neutrophils, stimulate antibody production, and down-regulate macrophage activation. The analysis of cytokine dynamics within different inflammatory responses can provide insights into immune mechanisms of tissue damage, and provide a useful framework for developing strategies for therapeutic intervention.

Heat shock protein Hsp60-reactive gamma delta cells: a large, diversified T-lymphocyte subset with highly focused specificity.

Previously, we detected a subset of gamma delta T cells in the newborn mouse thymus that responded to the mycobacterial heat shock protein Hsp60, as well as with what seemed to be a self-antigen. All of these cells expressed V gamma 1, most often in association with V delta 6+. It was not clear, however, whether similar, mature gamma delta cells with Hsp60 reactivity are common outside of the thymus, or rather, whether they are largely eliminated during development. From the data presented here, we estimate that gamma delta cells responding to Hsp60 comprise 10-20% of normal splenic and lymph node gamma delta T cells. Such cells, derived from adult spleen, always express a V gamma 1-J gamma 4-C gamma 4 gamma chain, although not all cells with this gamma chain show Hsp60 reactivity. Many of these V gamma 1+ cells also express V delta 6-J delta 1-C delta, though fewer than in V gamma 1+ cells from the newborn thymus. Extensive diversity is evident in both the gamma and delta chain junctional amino acids of the receptors of these cells, indicating that they may largely develop in the thymus of older animals or undergo peripheral expansion. Finally, we found that all such cells responding to both a putative self-antigen and to mycobacterial Hsp60 respond to a 17-amino acid synthetic peptide representing amino acids 180-196 of the Mycobacterium leprae Hsp60 sequence. This report demonstrates that a large subset of Hsp60-reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common heat shock protein.