M. leprae HSP18 suppresses copper (II) mediated ROS generation: Effect of redox stress on its structure and function.

Mycobacterium leprae, causative organism of leprosy, is known to counter redox stress generated by reactive oxygen species (ROS) during its survival inside host macrophages. But, the involvement of any antigenic protein(s) for countering such redox stress is still unknown. Interestingly, M. leprae HSP18, an important antigenic protein that helps in the growth and survival of M. leprae pathogen inside host macrophages, is induced under redox stress. Moreover, HSP18 also interacts with Cu2+. Copper (II) can induce redox stress via Fenton reaction. But, whether HSP18 suppresses Cu2+ mediated ROS generation, is still far from clear. Also, the effect of redox stress on its structure and function is not known. In this study, we show that HSP18 efficiently suppresses Cu2+ mediated generation of ROS and also prevents the redox mediated aggregation of a client protein (γD-crystallin). Upon exposure to substantial redox stress, irreversible perturbation in the secondary and tertiary structure of HSP18 and the tryptophan and tyrosine oxidation are evidenced. Interestingly, HSP18 retains a considerable amount of functionality even after being exposed to substantial redox stress. Perhaps, the redox scavenging ability as well as the chaperone function of HSP18 may possibly help M. leprae pathogen to counter redox stress inside host macrophages.

Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages.

OBJECTIVE: To evaluate the efficacy of erythrocytes loaded with the haemolytic toxin listeriolysin O against Mycobacterium avium replication within human macrophages. METHODS: Recombinant listeriolysin O was loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to allow them to be recognized and taken up by human macrophages infected with M. avium. The antimycobacterial activity of the erythrocytes loaded with listeriolysin O was evaluated by supernatant and intracellular cfu counts on days 4 and 7 post-erythrocyte administration. RESULTS: Recombinant listeriolysin O was encapsulated in human erythrocytes to reach final concentrations ranging from 1 to 4 ng/mL of erythrocytes. Erythrocytes loaded with increasing quantities of recombinant protein were able to reduce (at most by 50%) M. avium replication in a dose-dependent fashion when administered to infected macrophages. CONCLUSIONS: Erythrocytes loaded with listeriolysin O are effective against M. avium replication within macrophages. We are confident that the strategy presented could be useful against mycobacteria other than M. avium (such as Mycobacterium tuberculosis and Mycobacterium leprae) by itself or as part of an antimycobacterial treatment.

Effect of microbial heat shock proteins on airway inflammation and hyperresponsiveness.

Microbial heat shock proteins (hsp) have been associated with the generation and induction of Th1-type immune responses. We tested the effects of treatment with five different microbial hsp (Mycobacterium leprae, Streptococcus pneumoniae, Helicobacter pylori, bacillus Calmette-Guérin, and Mycobacterium tuberculosis) in a murine model of allergic airway inflammation and airway hyperresponsiveness (AHR). Mice were sensitized to OVA by i.p. injection and then challenged by OVA inhalation. Hsp were administered to each group by i.p. injection before sensitization and challenge. Sensitized and challenged mice developed increased serum levels of OVA-specific IgE with significant airway eosinophilia and heightened responsiveness to methacholine when compared with nonsensitized animals. Administration of M. leprae hsp prevented both development of AHR as well as bronchoalveolar lavage fluid eosinophilia in a dose-dependent manner. Treatment with M. leprae hsp also resulted in suppression of IL-4 and IL-5 production in bronchoalveolar lavage fluid, while IL-10 and IFN-gamma production were increased. Furthermore, M. leprae hsp treatment significantly suppressed OVA-specific IgE production and goblet cell hyperplasia/mucin hyperproduction. In contrast, treatment with the other hsp failed to prevent changes in airway responsiveness, lung eosinophilia, or cytokine production. Depletion of gamma/delta T lymphocytes before sensitization and challenge abolished the effect of M. leprae hsp treatment on AHR. These results indicate selective and distinctive properties among the hsp, and that M. leprae hsp may have a potential therapeutic role in the treatment of allergic airway inflammation and altered airway function.

T and B cell responses to mycobacterial 65-kDa heat-shock protein in sheep infected with maedi visna virus.

Sheep infected with maedi visna virus were tested for immune reactivity to recombinant HSP65 and tuberculin PPD from mycobacteria. The results showed that both naturally and experimentally infected animals had elevated IgM but not IgG or IgA antibodies to HSP65 from Mycobacterium leprae or M. bovis. In experimentally infected animals, the elevated IgM antibodies appeared in blood from about 3 to 4 weeks postinfection. Increased T cell proliferative responses to HSP65 and PPD were also found in both naturally and experimentally infected sheep. The T cell responses to HSP65 were substantially inhibited by antibodies to ovine major histocompatibility complex class II molecules, indicating that the responses were class II restricted. Increased expression of a putative HSP65 molecule was observed in synovial membranes from sheep infected with maedi visna virus and goats infected with the related, caprine arthritis encephalitis virus. The results thus show that lentivirus infection induces T and B cell anti-HSP65 immune responses and suggest that synovial inflammation may be due, at least in part, to T and B cell recognition of HSP65-like molecules expressed in joints.

Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures.

Bacterial heat shock proteins (hsp) have been shown to be important immunogens stimulating both T cells and B cells. However, little is known concerning the direct interactions between hsp and macrophages. In this study, we demonstrated that treatment of macrophage cultures with purified bacterial hsp, including Legionella pneumophila hsp60, Escherichia coli GroEL, Mycobacterium tuberculosis hsp70, Mycobacterium leprae hsp65, and Mycobacterium bovis BCG hsp65, increased the steady-state levels of cytokine mRNA for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor as well as supernatant IL-1 secretion. This effect was shown not to be due to contamination of the hsp preparations with bacterial lipopolysaccharide. However, not all hsp induced cytokines; M. tuberculosis hsp10 showed minimal activity in our study. These results suggest that bacterial hsp might modulate immunity by rapidly and directly increasing cytokine production in macrophages.

The Mycobacterium tuberculosis 71-kDa heat-shock protein induces proliferation and cytokine secretion by murine gut intraepithelial lymphocytes.

Murine intraepithelial lymphocytes (IEL) respond poorly to T cell mitogens and to monoclonal antibody stimulation of T cell receptor (TCR)- and CD3- associated molecules. In contrast, we found that a soluble extract of Mycobacterium tuberculosis (Mtb), but not purified protein derivative of tuberculin, induced significant proliferative responses in IEL cultures. The active component was apparently a heat shock protein (HSP), since recombinant 71-kDa HSP from Mtb induced IEL to proliferate, while 65-kDa HSP from M. bovis and M. leprae did not. Both alpha/beta and gamma/delta TCR-enriched IEL gave proliferative responses to 71-kDa HSP. Further, culture supernatants from IEL stimulated with 71-kDa HSP contained elevated levels of interleukin-(IL)-3/granulocyte-macrophage colony-stimulating factor, interferon-gamma and IL-6, but not IL-2, IL-4, IL-5 or transforming growth factor-beta. Finally, several IEL T cell clones have been maintained for up to 6 weeks, when stimulated with 71-kDa HSP, IL-2 and feeder cells. Our results show that the 71-kDa HSP of Mtb induces IEL T cells to divide and to secrete cytokines and this may offer a model for cloning and study of IEL T cells in vitro.

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.