Manuela Pardini

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.

Activity of human beta-defensin 3 alone or combined with other antimicrobial agents against oral bacteria

ABSTRACT The in vitro activities of human β-defensin 3 (hBD-3) alone or combined with lysozyme, metronidazole, amoxicillin, and chlorhexidine were investigated with the oral bacteria Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus, Lactobacillus acidophilus, Actinobacillus actinomycetemcomitans, and Porphyromonas gingivalis. hBD-3 showed bactericidal activity against all of the bacterial species tested. The bactericidal effect was enhanced when the peptide was used in combination with the antimicrobial agents mentioned above.

Cell wall-associated alpha-glucan is instrumental for Mycobacterium tuberculosis to block CD1 molecule expression and disable the function of dendritic cell derived from infected monocyte

We previously described an escape mechanism exploited by Mycobacterium tuberculosis (Mtb) to prevent the generation of fully competent dendritic cells (DC). We have now tested the effect of isolated mycobacterial components on human monocyte differentiation into DC and demonstrated that cell wall (CW)‐associated alpha‐glucan induces monocytes to differentiate into DC (Glu‐MoDC) with the same altered phenotype and functional behaviour of DC derived from Mtb‐infected monocytes (Mt‐MoDC). In fact, Glu‐MoDC lack CD1 molecule expression, fail to upregulate CD80 and produce IL‐10 but not IL‐12. We also showed that Glu‐MoDC are not able to prime effector T cells or present lipid antigens to CD1‐restricted T‐cell clones. Thus, we propose a mechanism of Mtb–monocyte interaction mediated by CW‐associated alpha‐glucan, which allows the bacterium to evade both innate and acquired immune responses.

Human CD56bright and CD56dim Natural Killer Cell Subsets Respond Differentially to Direct Stimulation with Mycobacterium bovis Bacillus Calmette‐Guérin

Mycobacterium bovis bacillus Calmette‐Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin‐12 and professional antigen presenting cells. To assess the contribution of two main human NK‐cell subsets (CD56dim and CD56bright) to the overall in vitro NK‐cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool‐adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU+ cells were found among the CD56bright subset than the CD56dim subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon‐γ (IFN‐γ) revealed that CD56bright cells were those mainly involved in IFN‐γ production in response to BCG. In contrast, the CD56dim subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis‐mediated cytotoxicity, than the CD56bright subset. Although 16–20‐h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56dim, but not in the CD56bright subset, following 4‐h incubation with the NK‐sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG‐stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56dim subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56bright and CD56dim human NK‐cell subsets exert different functional activities in response to a live bacterial pathogen.

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette‐Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte‐depleted nylon wool non‐adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3– cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon‐γ (IFN‐γ)‐producing cells were NK cells, with a peak IFN‐γ production at 24–30 hr. Interleukin (IL)‐2 and IL‐4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL‐12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non‐stimulated NW cells, the NW cells incubated for 16–20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK‐sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN‐γ production and cytotoxic activity, on negatively selected CD56+ CD3− cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK‐cell functions and suggest a possible alternative mechanism of NK‐cell activation as the first line of defence against mycobacterial infections.

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.

In vitro activity of protegrin-1 and beta-defensin-1, alone and in combination with isoniazid, against Mycobacterium tuberculosis

The antimicrobial peptide protegrin-1 (PG-1) inhibited the growth in vitro of drug-susceptible and multidrug-resistant Mycobacterium tuberculosis; a lower activity was shown by human beta-defensin-1 (HBD-1) against both strains. The combination of PG-1 or HBD-1 with isoniazid significantly reduced M. tuberculosis growth in comparison with the peptides or isoniazid alone.

IFN‐β improves BCG immunogenicity by acting on DC maturation

Given the variable protective efficacy provided by Mycobacterium bovis bacillus Calmette‐Guérin (BCG), there is an urgent need to develop new vaccines against tuberculosis. As dendritic cells (DC) play a critical role in initiating and regulating a protective T cell response against the pathogens, the comprehension of mycobacterium‐induced modulation of DC functions is critical to pinpoint new, immunological strategies. To this end, a comparative analysis of the effect induced by BCG and Mycobacterium tuberculosis (Mtb) infection on the DC immunophenotype indicated that BCG is less efficient in inducing DC maturation than Mtb. In addition, BCG‐infected DC poorly expressed IFN‐β and displayed a reduced production of IL‐12 as compared with Mtb‐stimulated cells. The impaired expression of IL‐12p35 and IFN‐β is likely a result of the inability of BCG to induce the activation of the IFN regulatory factor‐3. Taking into account these data, we sought to investigate whether the exogenous addition of IFN‐β, a cytokine that exerts important effects on the immune system, could enhance the Th1‐polarizing capacity of BCG‐infected DC. Interestingly, when DC infected by BCG were pretreated in vitro with IFN‐β, they displayed a fully mature phenotype and released a significant amount of bioactive IL‐12p70, which resulted in an enhanced Th1 response. This study demonstrates that IFN‐β potentiates DC immunological functions following BCG infection, thus suggesting IFN‐β as a possible candidate as vaccine adjuvant.

The LTK63 adjuvant improves protection conferred by Ag85B DNA-protein prime-boosting vaccination against Mycobacterium tuberculosis infection by dampening IFN-γ response

T helper type-1 response is essential to control Mycobacterium tuberculosis (MTB) infection but excessive antigen-mediated inflammation concurs to pathology. In mice challenged with MTB, the protection elicited by an Ag85B-encoding DNA vaccine, was lost when mice were boosted with Ag85B-protein in the absence of adjuvant. This effect was due to the expansion of a set of IFN-gamma secreting-CD4+ T cells highly responsive to Ag85B-protein but which lost the ability to interact with MTB-infected macrophages and control MTB growth. Ag85B-protein co-administration with the adjuvant LTK63 reduced the expansion of Ag85B-protein-responding CD4+ T cells and allowed the survival of those protective Ag85B-specific CD4+ T cells induced by the Ag85B-encoding DNA vaccine. Consequently, the protection against MTB-infection was restored. LTK63 caused also a marked augmentation of Ag85B-specific antibodies, in particular those belonging to the IgG2b isotype. The recovery of protection through a down-modulation of antigen-specific IFN-gamma response by an adjuvant is a novel finding which could be of relevance in tuberculosis vaccination.