Sandra M. Newton

IFN-gamma- and TNF-independent vitamin D-inducible human suppression of mycobacteria: the role of cathelicidin LL-37.

Vitamin D deficiency is associated with susceptibility to tuberculosis, and its biologically active metabolite, 1α,25 dihydroxyvitamin D3 (1α,25(OH)2D3), has pleiotropic immune effects. The mechanisms by which 1α,25(OH)2D3 protects against tuberculosis are incompletely understood. 1α,25(OH)2D3 reduced the growth of mycobacteria in infected human PBMC cultures in a dose-dependent fashion. Coculture with agonists or antagonists of the membrane or nuclear vitamin D receptors indicated that these effects were primarily mediated by the nuclear vitamin D receptors. 1α,25(OH)2D3 reduced transcription and secretion of protective IFN-γ, IL-12p40, and TNF in infected PBMC and macrophages, indicating that 1α,25(OH)2D3 does not mediate protection via these cytokines. Although NOS2A was up-regulated by 1α,25(OH)2D3, inhibition of NO formation marginally affected the suppressive effect of 1α,25(OH)2D3 on bacillus Calmette Guérin in infected cells. By contrast, 1α,25(OH)2D3 strongly up-regulated the cathelicidin hCAP-18 gene, and some hCAP-18 polypeptide colocalized with CD14 in 1α,25(OH)2D3 stimulated PBMC, although no detectable LL-37 peptide was found in supernatants from similar 1α,25(OH)2D3-stimulated PBMC cultures. A total of 200 μg/ml of the active peptide LL-37, in turn, reduced the growth of Mycobacterium tuberculosis in culture by 75.7%. These findings suggest that vitamin D contributes to protection against TB by “nonclassical” mechanisms that include the induction of antimicrobial peptides.

Neutrophil-mediated innate immune resistance to mycobacteria

Neutrophils contain antimicrobial peptides with antituberculous activity, but their contribution to immune resistance to tuberculosis (TB) infection has not been previously investigated to our knowledge. We determined differential white cell counts in peripheral blood of 189 adults who had come into contact with patients diagnosed with active TB in London, United Kingdom, and evaluated them for evidence of TB infection and capacity to restrict mycobacterial growth in whole-blood assays. Risk of TB infection was inversely and independently associated with peripheral blood neutrophil count in contacts of patients diagnosed with pulmonary TB. The ability of whole blood to restrict growth of Mycobacterium bovis bacille Calmette Guérin and Mycobacterium tuberculosis was impaired 7.3- and 3.1-fold, respectively, by neutrophil depletion. In microbiological media, human neutrophil peptides (HNPs) 1-3 killed M. tuberculosis. The neutrophil peptides cathelicidin LL-37 and lipocalin 2 restricted growth of the organism, the latter in an iron-dependent manner. Black African participants had lower neutrophil counts and lower circulating concentrations of HNP1-3 and lipocalin 2 than south Asian and white participants. Neutrophils contribute substantially to innate resistance to TB infection, an activity associated with their antimicrobial peptides. Elucidation of the regulation of neutrophil antimicrobial peptides could facilitate prevention and treatment of TB.

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.

Effect of Treatment of Latent Tuberculosis Infection on the T Cell Response to Mycobacterium tuberculosis Antigens

Most cases of latent tuberculosis infection (LTBI) do not cause symptoms during the lifetime of the infected person. Longitudinal analysis of the immune response of healthy Mycobacterium tuberculosis-infected people might, therefore, give insight into the basis of protective immunity. In a longitudinal study, we documented the effect that treatment had on the T cell response to M. tuberculosis antigens in 33 healthy people with LTBI. Preventive treatment of LTBI resulted in a 1.8-fold average increase in the numbers of interferon (IFN)- gamma -producing T cells within 26 +/- 4 days (P = .006), followed by a decrease by the end of the treatment period (82 +/- 6 days; P = .004). There was no significant overall change in the T cell response to any antigen in a control group (n = 8) of patients who elected radiological follow-up. Using live M. tuberculosis strain H37Rv as a stimulant in an enzyme-linked immunospot assay in sensitized individuals, we showed that isoniazid, but not rifampin, led to an increase in the number of IFN- gamma -producing cells. These results suggest that the integrity of the bacterial cell wall is important for M. tuberculosis in avoiding immune recognition by T cells and favor a dynamic model of LTBI.

1α,25-dihydroxyvitamin D3 inhibits matrix metalloproteinases induced by Mycobacterium tuberculosis infection

Matrix metalloproteinases (MMP) can degrade all components of pulmonary extracellular matrix. Mycobacterium tuberculosis induces production of a number of these enzymes by human macrophages, and these are implicated in the pathogenesis of pulmonary cavitation in tuberculosis. The active metabolite of vitamin D, 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3], has previously been reported to inhibit secretion of MMP‐9 in human monocytes (MN), but its influence on the secretion and gene expression of MMP and tissue inhibitors of MMP (TIMP) in M. tuberculosis‐infected cells has not previously been investigated. We therefore determined the effects of 1α,25(OH)2D3 on expression, secretion and activity of a number of MMP and TIMP in M. tuberculosis‐infected human leucocytes; we also investigated the effect of 1α,25(OH)2D3 on the secretion of interleukin‐10 (IL‐10) and prostaglandin E2 (PGE2), both transcriptional regulators of MMP expression. We found that M. tuberculosis induced expression of MMP‐1, MMP‐7 and MMP‐10 in MN and MMP‐1 and MMP‐10 in peripheral blood mononuclear cells (PBMC). 1α,25(OH)2D3 significantly attenuated M. tuberculosis‐induced increases in expression of MMP‐7 and MMP‐10, and suppressed secretion of MMP‐7 by M. tuberculosis‐infected PBMC. MMP‐9 gene expression, secretion and activity were significantly inhibited by 1α,25(OH)2D3 irrespective of infection. In contrast, the effects of 1α,25(OH)2D3 on the expression of TIMP‐1, TIMP‐2 and TIMP‐3 and secretion of TIMP‐1 and TIMP‐2 were small and variable. 1α,25(OH)2D3 also induced secretion of IL‐10 and PGE2 from M. tuberculosis‐infected PBMC. These findings represent a novel immunomodulatory role for 1α,25(OH)2D3 in M. tuberculosis infection.

Association between Gc genotype and susceptibility to TB is dependent on vitamin D status

Group-specific component (Gc) variants of vitamin D binding protein differ in their affinity for vitamin D metabolites that modulate antimycobacterial immunity. We conducted studies to determine whether Gc genotype associates with susceptibility to tuberculosis (TB). The following subjects were recruited into case–control studies: in the UK, 123 adult TB patients and 140 controls, all of Gujarati Asian ethnic origin; in Brazil, 130 adult TB patients and 78 controls; and in South Africa, 281 children with TB and 182 controls. Gc genotypes were determined and their frequency was compared between cases versus controls. Serum 25-hydroxyvitamin D (25(OH)D) concentrations were obtained retrospectively for 139 Gujarati Asians, and case-control analysis was stratified by vitamin D status. Interferon (IFN)-γ release assays were also performed on 36 Gujarati Asian TB contacts. The Gc2/2 genotype was strongly associated with susceptibility to active TB in Gujarati Asians, compared with Gc1/1 genotype (OR 2.81, 95% CI 1.19–6.66; p = 0.009). This association was preserved if serum 25(OH)D was <20 nmol·L−1 (p = 0.01) but not if serum 25(OH)D was ≥20 nmol·L−1 (p = 0.36). Carriage of the Gc2 allele was associated with increased PPD of tuberculin-stimulated IFN-γ release in Gujarati Asian TB contacts (p = 0.02). No association between Gc genotype and susceptibility to TB was observed in other ethnic groups studied.

A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion.

Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.

The stress-responsive chaperone alpha-crystallin 2 is required for pathogenesis of Mycobacterium tuberculosis.

Mycobacterium tuberculosis has two members of the α‐crystallin (Acr) family of molecular chaperones. Expression of Acr1 is induced by exposure to hypoxia or nitric oxide and is associated with bacterial persistence in a non‐replicating state. Expression of Acr2 is induced by heat shock, oxidative stress, and uptake by macrophages. We have shown that Acr2 continues to be expressed at a high level during both acute and chronic infection in the mouse model, with an increased ratio of acr2:acr1 mRNA in the persistent phase. Deletion of the acr2 gene resulted in a decrease in the resistance of M. tuberculosis to oxidative stress but did not impair growth in mouse bone marrow macrophages. There was no difference in bacterial load in mice infected with an acr2 deletion mutant, but a marked alteration in disease progression was evident from reduced weight loss over a prolonged infection. This correlated with reduced recruitment of T‐cells and macrophages to the lungs of mice infected with the acr2 mutant and reduced immune‐related pathology. These findings demonstrate that both α‐crystallins contribute to persistent infection with M. tuberculosis and suggest that manipulation of acr expression can influence the host response to infection.

Infection Biology of a Novel α-Crystallin of Mycobacterium tuberculosis: Acr2

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the α-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Δacr2) was unimpaired in log phase growth and persisted in IFN-γ-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-γ-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell‐based screening of FDA‐approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug‐resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR‐independent pathway controlled by cellular depletion of myo‐inositol. While strain‐specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug‐resistant mycobacterial infection.