Shaobin Shang

Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection

Azithromycin is a potent macrolide antibiotic with poorly understood antiinflammatory properties. Long-term use of azithromycin in patients with chronic inflammatory lung diseases, such as cystic fibrosis (CF), results in improved outcomes. Paradoxically, a recent study reported that azithromycin use in patients with CF is associated with increased infection with nontuberculous mycobacteria (NTM). Here, we confirm that long-term azithromycin use by adults with CF is associated with the development of infection with NTM, particularly the multi-drug-resistant species Mycobacterium abscessus, and identify an underlying mechanism. We found that in primary human macrophages, concentrations of azithromycin achieved during therapeutic dosing blocked autophagosome clearance by preventing lysosomal acidification, thereby impairing autophagic and phagosomal degradation. As a consequence, azithromycin treatment inhibited intracellular killing of mycobacteria within macrophages and resulted in chronic infection with NTM in mice. Our findings emphasize the essential role for autophagy in the host response to infection with NTM, reveal why chronic use of azithromycin may predispose to mycobacterial disease, and highlight the dangers of inadvertent pharmacological blockade of autophagy in patients at risk of infection with drug-resistant pathogens.

Phenotypic Definition of Effector and Memory T-Lymphocyte Subsets in Mice Chronically Infected with Mycobacterium tuberculosis

ABSTRACT The bacterium Mycobacterium tuberculosis remains one of the worlds most successful pathogens, a situation that is aggravated by the fact that the existing vaccine, Mycobacterium bovis BCG, is not effective in adults. As with any vaccine, the purpose of giving BCG vaccination is to establish a long-lived state of memory immunity, but whether this is successfully completely established is still unclear. It is generally accepted that memory T cells can be divided into central and effector memory populations by function and by phenotype; however, the majority of data supporting this division have been generated using transgenic mouse models or mice that have recovered from acute viral infections. Tuberculosis, on the other hand, represents a persistent, chronic state of immunity in which the presence of memory T cells is far less well defined. We show here that mice vaccinated with BCG or chronically infected with M. tuberculosis establish antigen-specific populations of cells within the lungs that predominantly express a cellular phenotype consistent with their being effector or effector memory cells. In contrast, cells with a central memory phenotype exist in much lower numbers in the lungs but can be found in significantly larger numbers in the spleen, where they may represent a potential reservoir. These data suggest that the effector-to-central-memory T-cell transition may well be minimal in these persisting mycobacterial infections, and they support a novel hypothesis that this may explain the fundamental basis of the failure of the BCG vaccine in humans.

Evaluation of Standard Chemotherapy in the Guinea Pig Model of Tuberculosis

ABSTRACT The purpose of this study was 2-fold. First, we evaluated standard chemotherapy in the guinea pig model of tuberculosis to determine if this animal species could productively be used for this purpose. Second, given the similarities of the pathology of disease in guinea pigs and humans, we wished to evaluate additional parameters, including magnetic resonance imaging, microscopy, and cytokine expression and lymphocyte phenotypes, in response to an infection treated with drug therapy. This study shows that conventional rifampin-isoniazid-pyrazinamide chemotherapy significantly decreased the numbers of the highly virulent Erdman K01 strain of Mycobacterium tuberculosis, with most of the bacilli being eliminated in a month. Despite this result, bacteria could still be detected in the lungs and other tissues for at least another 3 to 4 months. Resolution of the nonnecrotic granulomas in the lungs and lymph nodes could be clearly visualized by magnetic resonance imaging at the macroscopic level. Microscopically, the majority of the pulmonary and extrapulmonary inflammation resolved spontaneously, leaving residual lesions composed of dystrophic calcification and fibrosis marking the site of necrosis of the primary lesion. Residual calcified lesions, which were also associated with pulmonary lymphangitis, contained acid-fast bacilli even following aggressive chemotherapy. The presence of intact extracellular bacilli within these lesions suggests that these could serve as the primary sites of disease reactivation. The chemotherapy reduced the level of T-cell influx into infected tissues and was accompanied by a large and sustained increase in TH1 cytokine expression. Chemotherapy also prevented the emergence in lung tissues of high levels of interleukin-10 and Foxp3-positive cells, known markers of regulatory T cells.

Beijing sublineages of Mycobacterium tuberculosis differ in pathogenicity in the guinea pig.

ABSTRACT The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human populations.

Cigarette Smoke Increases Susceptibility to Tuberculosis—Evidence From In Vivo and In Vitro Models

BACKGROUND Cigarette smoke (CS) exposure is an epidemiological risk factor for tuberculosis, although the biological basis has not been elucidated. METHODS We exposed C57BL/6 mice to CS for 14 weeks and examined their ability to control an aerosol infection of Mycobacterium tuberculosis Erdman. RESULTS CS-exposed mice had more M. tuberculosis isolated from the lungs and spleens after 14 and 30 d, compared with control mice. The CS-exposed mice had worse lung lesions and less lung and splenic macrophages and dendritic cells (DCs) producing interleukin12 and tumor necrosis factor α (TNF-α). There were significantly more interleukin 10-producing macrophages and DCs in the spleens of infected CS-exposed mice than in non-CS-exposed controls. CS-exposed mice also showed a diminished influx of interferon γ-producing and TNF-α-producing CD4(+) and CD8(+) effector and memory T cells into the lungs and spleens. There was a trend toward an increased number of viable intracellular M. tuberculosis in macrophages isolated from humans who smoke compared with nonsmokers. THP-1 human macrophages and primary human alveolar macrophages exposed to CS extract, nicotine, or acrolein showed an increased burden of intracellular M. tuberculosis. CONCLUSION CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.

Mycobacterium bovis BCG-mediated protection against W-Beijing strains of Mycobacterium tuberculosis is diminished concomitant with the emergence of regulatory T cells.

ABSTRACT Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study, we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly protective against both strains, but by day 60 of the assay, this protection was diminished. Histological examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues demonstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice infected with HN878, but these diminished in number after day 30 of the infections concomitant with increased CD4+ Foxp3+ T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant decrease in effector immunity and continued expansion of regulatory Foxp3+ cells observed here, it is reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious impediment to the efficacy of BCG-based vaccines.

Activities of TMC207, Rifampin, and Pyrazinamide against Mycobacterium tuberculosis Infection in Guinea Pigs

ABSTRACT The experimental compound TMC207 is showing promise against infections caused by Mycobacterium tuberculosis both in a variety of animal studies and in the field. In this study, we used the guinea pig model, a species that shows several similarities to human tuberculosis, including the hallmark of primary granuloma necrosis, to determine the efficacy of a combination regimen combining TMC207 with rifampin and pyrazinamide. This drug regimen rapidly reduced the bacterial load in the lungs to undetectable levels by 8 weeks of treatment. This reduction was associated with a substantial improvement in lung pathology, but despite this effect areas of residual necrosis still remained. In the draining lymph nodes, however, tissue damage was rapid and not significantly reversed by the drug treatment. Approximately 10 to 11 months after the treatment had ended, the animals began to trigger a Karnovsky scale indicating bacterial regrowth and potential relapse, an event confirmed by the new development of both pulmonary and extrapulmonary granulomatous lesions. Interestingly, a similar rate of relapse was also seen in animals receiving 24 weeks of rifampin, pyrazinamide, and isoniazid standard chemotherapy. These data indicate that TMC207 could be a useful addition to current treatment regimens for tuberculosis.

Increased Foxp3 expression in guinea pigs infected with W-Beijing strains of M. tuberculosis.

There is increasing evidence that clinical isolates of Mycobacterium tuberculosis that belong to the W-Beijing genotype of newly emerging strains are often of very high virulence when tested in small animal models, including the mouse and guinea pig. In this report we provide further evidence to support this contention, and show that two W-Beijing strains are of very high virulence when introduced by low dose aerosol into outbred guinea pigs. In addition to severe lung pathology, each of these infections was associated with large influxes of activated CD4 and CD8 T cells into the lungs. Large influxes of macrophages were also observed, but the fraction of these showing evidence of activation by Class-II expression was relatively low. A progressive increase in neutrophils was also seen, with highest levels accumulating in the lungs of the W-Beijing infected animals. In the case of these two infections mRNA levels for TH1 cytokines was elevated early, but these then declined, and were replaced by increasing levels of message encoding for Foxp3, IL-10, and TGFβ. These observations support the hypothesis that W-Beijing strains are potent inducers of regulatory T cells, and that this event may enhance survival and transmission of these bacilli.

T lymphocyte surface expression of exhaustion markers as biomarkers of the efficacy of chemotherapy for tuberculosis

Predictive biomarkers illustrating the effectiveness of chemotherapeutic regimens for tuberculosis still remain elusive. To date, most are predicated on assays using sputum or serum; as a result, if not predictive, treatment failure in patients may not be evident for some time. We report here the results of a simple screening study in which T cell surface markers were examined in mice infected with Mycobacterium tuberculosis and then treated with drugs. These studies identified certain markers, the exhaustion markers PD-1 and TIM-3, as well as the marker KLRG-1, particularly on CD8 T cells, that changed in concert with reduction of the bacterial load in the lungs. While there is no guarantee these changes would also be seen on T cells in the blood, this approach should be further investigated.

Increased virulence of an epidemic strain of Mycobacterium massiliense in mice.

Background Chronic pulmonary disease and skin/soft tissue infections due to non-tuberculous mycobacteria (NTM) of the Mycobacterium chelonae-abscessus-massiliense group is an emerging health problem worldwide. Moreover, the cure rate for the infections this group causes is low despite aggressive treatment. Post-surgical outbreaks that reached epidemic proportions in Brazil recently were caused by M. massiliense isolates resistant to high-level disinfection with glutaraldehyde (GTA). Understanding the differences in the virulence and host immune responses induced by NTM differing in their sensitivity to disinfectants, and therefore their relative threat of causing outbreaks in hospitals, is an important issue. Methodology/Principal Finding We compared the replication and survival inside macrophages of a GTA-susceptible reference Mycobacterium massiliense clinical isolate CIP 108297 and an epidemic strain from Brazil, CRM-0019, and characterized the immune responses of IFNγ knockout mice exposed to a high dose aerosol with these two isolates. CRM-0019 replicated more efficiently than CIP 108297 inside mouse bone marrow macrophages. Moreover, the animals infected with CRM-0019 showed a progressive lung infection characterized by a delayed influx of CD4+ and CD8+ T cells, culminating in extensive lung consolidation and demonstrated increased numbers of pulmonary CD4+ Foxp3+ regulatory T cells compared to those infected with the reference strain. Immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of NTM disease by preventing the induction of specific protective immune responses. Conclusions/Significance These results provide the first direct evidence of the increased virulence in macrophages and mice and pathogenicity in vivo of the Brazilian epidemic isolate and the first observation that NTM infections can be associated with variable levels of regulatory T cells which may impact on their virulence and ability to persist in the host.