Lanfranco Fattorini

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response.

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-α, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-γ-inducing cytokines, IL-12 and IFN-α, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-γ production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-α, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-α and IL-18 play a significant role in enhancing IFN-γ synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.

Towards tuberculosis elimination: an action framework for low-incidence countries

This paper describes an action framework for countries with low tuberculosis (TB) incidence (<100 TB cases per million population) that are striving for TB elimination. The framework sets out priority interventions required for these countries to progress first towards “pre-elimination” (<10 cases per million) and eventually the elimination of TB as a public health problem (less than one case per million). TB epidemiology in most low-incidence countries is characterised by a low rate of transmission in the general population, occasional outbreaks, a majority of TB cases generated from progression of latent TB infection (LTBI) rather than local transmission, concentration to certain vulnerable and hard-to-reach risk groups, and challenges posed by cross-border migration. Common health system challenges are that political commitment, funding, clinical expertise and general awareness of TB diminishes as TB incidence falls. The framework presents a tailored response to these challenges, grouped into eight priority action areas: 1) ensure political commitment, funding and stewardship for planning and essential services; 2) address the most vulnerable and hard-to-reach groups; 3) address special needs of migrants and cross-border issues; 4) undertake screening for active TB and LTBI in TB contacts and selected high-risk groups, and provide appropriate treatment; 5) optimise the prevention and care of drug-resistant TB; 6) ensure continued surveillance, programme monitoring and evaluation and case-based data management; 7) invest in research and new tools; and 8) support global TB prevention, care and control. The overall approach needs to be multisectorial, focusing on equitable access to high-quality diagnosis and care, and on addressing the social determinants of TB. Because of increasing globalisation and population mobility, the response needs to have both national and global dimensions. Action framework for countries with low tuberculosis incidence: a coherent approach for eliminating tuberculosis http://ow.ly/H03ZZ

Mycobacterium tuberculosis Strains with Highly Discordant Rifampin Susceptibility Test Results

ABSTRACT The objectives of this study were to investigate the origin of highly discordant rifampin (rifampicin) (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing. Nine Supra-National Tuberculosis Reference Laboratories tested the RMP susceptibilities of 19 selected M. tuberculosis strains, using standard culture-based methods. The strains were classified as definitely resistant (R) (n = 6) or susceptible (S) (n = 2) or probably resistant (PR) (n = 8) or susceptible (PS) (n = 3) based on rpoB mutations and treatment outcome. All methods yielded a susceptible result for the two S and three PS strains lacking an rpoB mutation and a resistant result for one R strain with a Ser531Leu mutation and one PR strain with a double mutation. Although the remaining 12 R and PR strains had rpoB mutations (four Asp516Tyr, three Leu511Pro, two Leu533Pro, one each His526Leu/Ser, and one Ile572Phe), they were all susceptible by the radiometric Bactec 460TB or Bactec 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen medium and two on Middlebrook 7H10 agar. Low-level but probably clinically relevant RMP resistance linked to specific rpoB mutations is easily missed by standard growth-based methods, particularly the automated broth-based systems. Further studies are required to confirm these findings, to determine the frequency of these low-level-resistant isolates, and to identify technical improvements that may identify such strains.

Mycobacterium tuberculosis subverts the differentiation of human monocytes into dendritic cells

Intracellular pathogens have developed strategies for evading elimination by the defenses of the host immune system. Here we describe an escape mechanism utilized by Mycobacterium tuberculosis that involves the interference with the generation of fully competent DC from monocytes. We show that monocytes infected with live M. tuberculosis differentiated into mature, CD83+ and CCR7+ DC (Mt‐MoDC), but were characterized by a selective failure in the expression of the family of CD1 molecules. These cells also showed levels of MHC class II and CD80 (B7.1) that were reduced in comparison with LPS‐matured DC. In addition, Mt‐MoDC produced TNF‐α and IL‐10, but were unable to secrete IL‐12. The generation of Mt‐MoDC required the infection of monocytes with live M. tuberculosis, since infection with heat‐killed bacteria partially abrogated the effects on monocyte differentiation. Interestingly, Mt‐MoDC revealed an impaired antigen‐presentation function as assessed by the reduced capability to induce proliferation of cord blood T lymphocytes. Further, naive T lymphocytes expanded by Mt‐MoDC were unable to secrete cytokines, in particular IL‐4 and IFN‐γ, suggesting that they could be ineffective in helping the macrophage‐mediated killing of intracellular mycobacteria. Our results suggest that the interference with monocyte differentiation into fully competent DC is an evasion mechanism of M. tuberculosis that could contribute to its intracellular persistence by avoiding immune recognition.

The Extra Cytoplasmic Function Sigma Factor σE Is Essential for Mycobacterium tuberculosis Virulence in Mice

ABSTRACT The virulence of a Mycobacterium tuberculosis H37Rv sigE mutant was studied in immunodeficient and immunocompetent mice. The mutant was strongly attenuated in both animal models and induced formation of granulomas with different characteristics than those induced by the wild-type strain.

Whole Genome Sequencing Reveals Complex Evolution Patterns of Multidrug-Resistant Mycobacterium tuberculosis Beijing Strains in Patients

Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.

An Anti-Inflammatory Role for Vα14 NK T cells in Mycobacterium bovis Bacillus Calmette-Guérin-Infected Mice

The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the Vα14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of Vα14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1+ Vα14 NKT population preferentially producing IFN-γ predominated at an early stage (day 8), which was substituted by an NK1.1− population preferentially producing IL-4 at later stages (day 30). Despite the fact that Vα14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in Vα14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Vα14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-α in liver and lungs of Vα14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-α mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for Vα14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.

Evaluation of a New Line Probe Assay for Rapid Identification of gyrA Mutations in Mycobacterium tuberculosis

ABSTRACT Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 × 103M. tuberculosis CFU per ml of sputum.

Mycobacterium bovis Bacillus Calmette-Guérin infects DC-SIGN– dendritic cell and causes the inhibition of IL-12 and the enhancement of IL-10 production

The only available vaccine against tuberculosis is Mycobacterium bovis Bacillus Calmette Guérin (BCG), although its efficacy in preventing pulmonary tuberculosis is controversial. Early interactions between dendritic cells (DC) and BCG or Mycobacterium tuberculosis (Mtb) are thought to be critical for mounting a protective antimycobacterial immune response. Recent studies have shown that BCG and Mtb target the DC‐specific C‐type lectin intercellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN) to infect DC and inhibit their immunostimulatory function. This would occur through the interaction of the mycobacterial mannosylated lipoarabinomannan to DC‐SIGN, which would prevent DC maturation and induce the immunosuppressive cytokine interleukin (IL)‐10 synthesis. Here, we confirm that DC‐SIGN is expressed in DC derived from monocytes cultured in granulocyte macrophage‐colony stimulating factor (GM‐CSF) and IL‐4 and show that it is not expressed in DC derived from monocytes cultured in GM‐CSF and interferon‐α (IFN‐α). We also demonstrate that DC‐SIGN– DC cultured in GM‐CSF and IFN‐α are able to phagocytose BCG and to undergo a maturation program as well as DC‐SIGN+ DC cultured in IL‐4 and GM‐CSF. We also show that BCG causes the impairment of IL‐12 and the induction of IL‐10 secretion by DC, irrespective of DC‐SIGN expression. Finally, we demonstrate that the capacity to stimulate a mixed leukocyte reaction of naïve T lymphocytes is not altered by the treatment of both DC populations with BCG. These data suggest that DC‐SIGN cannot be considered as the unique DC receptor for BCG internalization, and it is more interesting that the mycobacteria‐induced immunosuppression cannot be attributed to the engagement of a single receptor.

Mycobacterium tuberculosis diverts alpha interferon-induced monocyte differentiation from dendritic cells into immunoprivileged macrophage-like host cells.

ABSTRACT Dendritic cells (DCs) are critical for initiating a pathogen-specific T-cell response. During chronic infections the pool of tissue DCs must be renewed by recruitment of both circulating DC progenitors and in loco differentiating monocytes. However, the interaction of monocytes with pathogens could affect their differentiation. Mycobacterium tuberculosis has been shown to variably interfere with the generation and function of antigen-presenting cells (APCs). In this study we found that when alpha interferon (IFN-α) is used as an inductor of monocyte differentiation, M. tuberculosis inhibits the generation of DCs, forcing the generation of immunoprivileged macrophage-like cells instead. Cells derived from M. tuberculosis-infected monocyte-derived macrophages (M. tuberculosis-infected MoMφ) retained CD14 without acquiring CD1 molecules and partially expressed B7.2 but did not up-regulate B7.1 and major histocompatibility complex (MHC) class I and II molecules. They synthesized tumor necrosis factor alpha and interleukin-10 (IL-10) but not IL-12. They also showed a reduced ability to induce proliferation and functional polarization of allogeneic T lymphocytes. Thus, in the presence of IFN-α, M. tuberculosis may hamper the renewal of potent APCs, such as DCs, generating a safe habitat for intracellular growth. M. tuberculosis-infected MoMφ, in fact, showed reduced expression of both signal 1 (CD1, MHC classes I and II) and signal 2 (B7.1 and B7.2), which are essential for mycobacterium-specific T-lymphocyte priming and/or activation. These data further suggest that M. tuberculosis has the ability to specifically interfere with monocyte differentiation. This ability may represent an effective M. tuberculosis strategy for eluding immune surveillance and persisting in the host.