Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance.

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

Monosodium Urate Crystals Promote Innate Anti-Mycobacterial Immunity and Improve BCG Efficacy as a Vaccine against Tuberculosis.

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination.

In vitro analysis of pyrogenicity and cytotoxicity profiles of flex sensors to be used to sense human joint postures.

Flex sensors can be usefully adopted as mechanical-electrical transducers to measure human joint movements, since their electrical resistance varies proportionally to the angle assumed by the joint under measure. Over time, these sensors have been investigated in terms of mechanical and electrical behavior, but no reports have detailed the possibility of their adoption not just on top but under the human skin of the joint. To this aim, our work investigated in vitro the pyrogenic potential and cytotoxicity of some commercially available flex sensors as a first step toward the necessary requirements regarding their biocompatibility, to predict possible foreign body reactions when used in vivo. Results demonstrated that some specific flex sensors satisfy such requirements.

Janus-faced liposomes enhance antimicrobial innate immune response in Mycobacterium tuberculosis infection.

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1ß, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-ß without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1ß, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.

Lysophosphatidic acid enhances antimycobacterial response during in vivo primary Mycobacterium tuberculosis infection.

Lysophospholipids may play an important protective role during primary infection of Mycobacterium tuberculosis (MTB) by enhancing innate antimycobacterial immune response of both macrophages and alveolar epithelial cells. Here, we show that treatment with lysophosphatidic acid (LPA) of mice aerogenically infected with MTB immediately after infection results in a significant early reduction of pulmonary CFUs and of histopathological damage in comparison with control mice. In contrast, treatment of acute disease does not result in any improvement of both microbiological and histopathological parameters. Altogether, these results show that LPA treatment can exert protective effect if administrated during primary infection, only.

Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells.

Human alveolar epithelial cells actively contribute to the innate immune response in the lung and play an important role in mycobacterial dissemination during primary infection, by undergoing cell death and by releasing mycobacteria. In the present study, we report that natural lysophospholipids, such as lysophosphatidic acid or sphingosine 1-phosphate, reduce Mycobacterium tuberculosis-induced cytotoxicity and enhance anti-mycobacterial activity in the A549 cell line, used as a model of type II alveolar epithelial cells. Intracellular mycobacterial killing was strictly dependent on phagolysosome maturation, which in turn was promoted by the activation of a Ca(2+)dependent phospholipase D. Finally, the restriction of mycobacteria in highly microbiocidal compartments was associated, in vitro, with a significant decrease in mycobacterial dissemination to macrophages. Taken as whole, these results suggest that the pulmonary lysophospholipid microenvironment may play a protective role during the early phases of host-pathogen interaction by enhancing anti-mycobacterial activity in type II alveolar epithelial cells.

Successful simplification of protease inhibitor-based HAART with triple nucleoside regimens in children vertically infected with HIV.

OBJECTIVE: To assess the virological, immunological and metabolic effects of switching from an efficacious first-line protease inhibitor (PI)-based HAART to a simplified triple nucleoside reverse transcriptase inhibitor (NRTI) regimen in children vertically infected with HIV. DESIGN: Prospective, open-label, before-after study of 20 vertically infected children with at least 12 consecutive months of undetectable viral load under a PI-based HAART and no previous history of NRTI treatment. METHODS: At study entry, HAART was shifted to a triple-NRTI combination. RESULTS: The children were aged 2 to 18 years (median, 7.9) and were followed for 96 weeks. All were receiving a PI-based regimen for an average duration of 4 years before enrollment. At study entry, 12 patients (60%) switched to abacavir, 5 (25%) to lamivudine; 2 (10%) to zidovudine and 2 to didanosine (10%). All but one patient maintained plasma HIV RNA < 50 copies/ml during the entire follow-up. No immunological failure was observed at week 96. A trend of normalization (P < 0.001) of T cell receptor Vbeta families of the CD8 cell subset was detected in 19/20 (95%), with an increased HIV-specific CD8 T cell response (P < 0.01) in 17/20 (85%). Dyslipidaemia significantly improved during the follow up (P < 0.001). No new cases of lipodystrophy were detected. CONCLUSIONS: Switching to triple-NRTI regimens in selected HIV-infected children with an extremely low likelihood of harbouring nucleoside-associated mutations maintains viral suppression and immunological function, improving metabolic abnormalities and the effort to take medication for up to 96 weeks.

Sphingosine 1-phosphate promotes antigen processing and presentation to CD4+ T cells in Mycobacterium tuberculosis-infected monocytes.

Sphingosine 1-phosphate (S1P) has recently been described to induce antimycobacterial activity. The present study analyses the role played by S1P in antigen presentation of monocytes and in the next activation of Mycobacterium tuberculosis (MTB)-specific CD4+ T cell response. Results reported herein show that S1P stimulation of MTB-infected monocytes (i) inhibits intracellular mycobacterial growth, (ii) enhances phagolysosome maturation and the transit of mycobacteria in MHC class II compartments, (iii) increases the frequency of MTB-specific CD4+CD69+ T cells, expressing the inflammatory homing receptor CCR5, derived from tuberculosis patients and PPD+, BCG naïve, healthy subjects, and (iv) induces IFN-gamma production in CD4+CD69+CCR5+ T cells derived from PPD+ healthy individuals, only. Altogether, these results show that S1P promotes antigen processing and presentation in monocytes, increases the frequency of MTB-specific CD4+ T cells and can regulate IFN-gamma production by antigen specific CD4+ T cells in the course of active disease.

Does sphingosine 1-phosphate play a protective role in the course of pulmonary tuberculosis?

Sphingosine 1-phosphate (S1P) has recently been reported to induce antimycobacterial activity in vitro and in a mouse model of in vivo Mycobacterium tuberculosis infection. However, its role in the course of pulmonary tuberculosis in humans is still not known. This study shows that S1P levels in airway surface fluid of tuberculosis (TB) patients are significantly less than those observed in non-TB control patients. Moreover, the in vitro stimulation of bronchoalveolar lavage cells coming from TB patients with S1P significantly reduces intracellular growth of endogenous mycobacterial isolates. These results show that, in the course of pulmonary TB, airway epithelial fluid-associated S1P may play a protective role in the containment of intracellular mycobacterial growth and that its decrease may represent a novel pathogenic mechanism through which M. tuberculosis favors its replication.

Induction of apoptosis and release of interleukin-1 beta by cell wall-associated 19-kDa lipoprotein during the course of mycobacterial infection.

Mycobacterium tuberculosis induces apoptosis in human monocyte-derived macrophages (MDMs) during the early stages of infection. We investigated the proapoptotic role of cell wall-associated mycobacterial 19-kDa lipoprotein and the possible association between 19-kDa lipoprotein signaling and production of proinflammatory cytokines. Purified mycobacterial 19-kDa lipoprotein, 19-kDa lipoprotein-expressing M. smegmatis (M. smegmatis 19+), 19-kDa lipoprotein knockout (KO) M. tuberculosis, and 19-kDa lipoprotein KO M. bovis bacille Calmette-Guerin (BCG) strains were analyzed for their ability to induce apoptosis in MDMs. The 19-kDa lipoprotein and infection with M. smegmatis 19+ induced apoptosis in MDMs. M. tuberculosis and BCG KO strains had significantly decreased abilities to induce apoptosis. The 19-kDa lipoprotein proapoptotic signal was mediated by Toll-like receptor 2 but not by tumor necrosis factor-alpha. Only the release of interleukin (IL)-1 beta was decreased after infection with 19-kDa lipoprotein KO strains. These findings indicate that the 19-kDa lipoprotein is the main signal required to trigger both apoptosis and the release of IL-1 beta during the early stages of mycobacterial infection.