Federico Giannoni

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.

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.

Activities of moxifloxacin alone and in combination with other antimicrobial agents against multidrug-resistant Mycobacterium tuberculosis infection in BALB/c mice

ABSTRACT The activity of moxifloxacin was enhanced by the addition of ethionamide but not by that of cycloserine, thiacetazone, capreomycin, para-aminosalicylic acid, or linezolid in BALB/c mice infected with a strain of Mycobacterium tuberculosis resistant to isoniazid, rifampin, and six other drugs. These observations are important for the therapy of multidrug-resistant tuberculosis.

Prevention of False Resistance Results Obtained in Testing the Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Bactec MGIT 960 System Using a Reduced Inoculum

ABSTRACT The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.

Activities of Drug Combinations against Mycobacterium tuberculosis Grown in Aerobic and Hypoxic Acidic Conditions

ABSTRACT Mycobacterium tuberculosis is exposed to hypoxia and acidity within granulomatous lesions. In this study, an acidic culture model of M. tuberculosis was used to test drug activity against aerobic 5-day-old (A5) and hypoxic 5-, 12-, and 19-day-old (H5, H12, and H19, respectively) bacilli after 7, 14, and 21 days of exposure. In A cultures, CFU and pH rapidly increased, while in H cultures growth stopped and pH increased slightly. Ten drugs were tested: rifampin (R), isoniazid (I), pyrazinamide (Z), ethambutol (E), moxifloxacin (MX), amikacin (AK), metronidazole (MZ), nitazoxanide (NZ), niclosamide (NC), and PA-824 (PA). Rifampin was the most active against A5, H5, H12, and H19 bacilli. Moxifloxacin and AK efficiently killed A5 and H5 cells, I was active mostly against A5 cells, Z was most active against H12 and H19 cells, and E showed low activity. Among nitrocompounds, NZ, NC, and PA were effective against A5, H5, H12, and H19 cells, while MZ was active against H12 and H19 cells. To kill all A and H cells, A5- and H5-active agents R, MX, and AK were used in combination with MZ, NZ, NC, or PA, in comparison with R-I-Z-E, currently used for human therapy. Mycobacterial viability was determined by CFU and a sensitive test in broth (day to positivity, MGIT 960 system). As shown by lack of regrowth in MGIT, the most potent combination was R-MX-AK-PA, which killed all A5, H5, H12, and H19 cells in 14 days. These observations demonstrate the sterilizing effect of drug combinations against cells of different M. tuberculosis stages grown in aerobic and hypoxic acidic conditions.

Endogenous PGE2 promotes the induction of human Th17 responses by fungal β-glucan

The interaction of PAMPs with cells of the innate immune system shapes the adaptive host response. Here, we report that β‐glucan, a major fungal PAMP purified from Candida albicans, stimulates human DCs to secrete a pro‐Th17 cytokine pattern. Notably, β‐glucan induces PGE2 production, which has been shown to play a pivotal role in Th17 cell expansion. Inhibition of PGE2 synthesis or blockade of PGE2 receptors EP2 and EP4 drastically reduces IL‐23 production by β‐glucan‐activated DCs, suggesting that endogenous PGE2 amplifies IL‐23 synthesis in response to the C. albicans PAMP. Moreover β‐glucan promotes the expansion of Th17 cells, which is strongly decreased by EP2 and EP4 receptor blockade on DCs. Our results highlight a novel role for PGE2 in the regulation of innate and adaptive immune response triggered by recognition of a prominent, highly conserved fungal PAMP such as β‐glucan.

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.

The Ag85B protein of Mycobacterium tuberculosis may turn a protective immune response induced by Ag85B‐DNA vaccine into a potent but non‐protective Th1 immune response in mice

Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B‐encoding plasmid DNA (DNA‐85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B‐specific CD4+ T cells producing IFN‐γ and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4+ T cell proliferation and IFN‐γ production in response to Ag85B protein, despite restraint of Th1 response by CD8+ T cell‐dependent mechanisms and activation of CD4+ T cell‐dependent IL‐10 secretion. Importantly, these Ag85B‐responding CD4+ T cells lost the ability to produce IFN‐γ and control MTB intramacrophagic growth in coculture with MTB‐infected macrophages, suggesting that the protein‐dependent expansion of non‐protective CD4+ T cells determined dilution or loss of the protective Ag85B‐specific CD4+ induced by DNA‐85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA‐priming, protein‐booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective anti‐tuberculosis immune response, and actually concur to disease progression.