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.

α1-antitrypsin production by proinflammatory and antiinflammatory macrophages and dendritic cells.

α(1)-Antitrypsin (AAT) acts as an important neutrophil elastase inhibitor in the lung. Although the hepatocyte is considered to be the primary source of AAT, local production by monocytes, macrophages, and epithelial cells may contribute to the formation of an antielastase screen. Because monocytes can differentiate into a heterogeneous population of macrophages with subpopulations ranging from proinflammatory properties (MΦ-1) to antiinflammatory properties (ΜΦ-2) and into dendritic cells (DCs), we studied whether LPS, TNF-α, and oncostatin M (OSM) enhance AAT production differentially in cultured ΜΦ-1, ΜΦ-2, and DCs. Monocytes from healthy blood donors were cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor, or GM-CSF with IL-4 to obtain ΜΦ-1, ΜΦ-2, and immature (i)DCs, respectively. Cells were stimulated with LPS, TNF-α, or OSM, and AAT synthesis was assessed by quantitative RT-PCR, immunocytochemistry, and ELISA. Spontaneous release of AAT was higher in ΜΦ-1 than in ΜΦ-2 and iDCs, and only LPS significantly increased AAT production in ΜΦ-1, ΜΦ-2, and DC. TNF-α and OSM did not affect AAT secretion. The secretion levels of the related protease inhibitors α-1 antichymotrypsin and secretory leukocyte proteinase inhibitor were below the limits of detection by ELISA. In contrast to the protein data, analysis by quantitative RT-PCR showed that 24-hour LPS exposure caused a maximal 2.1-fold AAT mRNA increase in ΜΦ-1, a 21-fold increase in ΜΦ-2, and an 11-fold increase in DCs. These data suggest that cellular differentiation is a regulator of local AAT production.

Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation.

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1ß activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

IFN-α cannot substitute lack of IFN-γ responsiveness in cells of an IFN-γR1 deficient patient.

Patients with complete IFN-γR deficiency are unable to respond to IFN-γ and have impaired Th1-immunity and recurrent, severe infections with weakly virulent Mycobacteria. Since IFN-α and IFN-γ share signalling pathways, treatment with IFN-α has been proposed in complete IFN-γR deficiency. We stimulated cells from healthy controls and from a patient lacking IFN-γR1 with IFN-α and IFN-γ, to establish whether IFN-α would substitute for IFN-γ effects. IFN-α induced STAT1 phosphorylation in monocytes of the IFN-γR1(-/-) patient, but did not prime for LPS-induced IL-12p70, IL-12p40, IL-23 or TNF production. In control cells, IFN-α inhibited the priming effect of IFN-γ on LPS-induced pro-inflammatory cytokine release. Finally, IFN-γ but not IFN-α induced killing of M. smegmatis in cultured macrophages. In conclusion, no evidence was found to support the use of IFN-α in IFN-γR-deficient patients as intervention against mycobacterial infection; on the contrary, treatment of individuals with IFN-α may even adversely affect host defence against Mycobacteria.

Induction of regulatory T cells by macrophages is dependent on production of reactive oxygen species.

The phagocyte NAPDH-oxidase complex consists of several phagocyte oxidase (phox) proteins, generating reactive oxygen species (ROS) upon activation. ROS are involved in the defense against microorganisms and also in immune regulation. Defective ROS formation leads to chronic granulomatous disease (CGD) with increased incidence of autoimmunity and disturbed resolution of inflammation. Because regulatory T cells (Tregs) suppress autoimmune T-cell responses and are crucial in down-regulating immune responses, we hypothesized that ROS deficiency may lead to decreased Treg induction. Previously, we showed that in p47(phox)-mutated mice, reconstitution of macrophages (Mph) with ROS-producing capacity was sufficient to protect the mice from arthritis. Now, we present evidence that Mph-derived ROS induce Tregs. In vitro, we showed that Mph ROS-dependently induce Treg, using an NADPH-oxidase inhibitor. This finding was confirmed genetically: rat or human CGD Mph with mutated p47(phox) or gp91(phox) displayed hampered Treg induction and T-cell suppression. However, basal Treg numbers in these subjects were comparable to those in controls, indicating a role for ROS in induction of peripheral Tregs. Induction of allogeneic delayed-type hypersensitivity with p47(phox)-mutated Mph confirmed the importance of Mph-derived ROS in Treg induction in vivo. We conclude that NAPDH oxidase activity in Mph is important for the induction of Tregs to regulate T cell-mediated inflammation.

Anti-inflammatory M2 type macrophages characterize metastasized and tyrosine kinase inhibitor-treated gastrointestinal stromal tumors.

We have made a detailed inventory of the immune infiltrate of gastrointestinal stromal tumors (GISTs), which originate from mesenchymal cells in the intestinal tract. These sarcomas are heavily infiltrated with macrophages and T cells, while immune cells of other lineages were much less abundant. Dissecting the functional subtypes of T cells with multicolor fluorescent microscopy revealed substantial populations of cytotoxic T cells, helper T cells and FoxP3(+) regulatory T cells. The balance of cytotoxic T cells and FoxP3(+) T cells was toward immune suppression. Analysis of the macrophage population also showed a dominance of anti-inflammatory cells, as the M2 type scavenger receptor CD163 was abundantly present. Other subsets of macrophages (CD14(+)CD163(-)) were occasionally detected. M2 type CD163(+) macrophages were associated with the number of infiltrating FoxP3(+) regulatory T cells and twice as many macrophages were found in metastatic GIST compared to primary lesions. Most metastatic GISTs had been treated with the tyrosine kinase inhibitors imatinib and sunitinib, but the high macrophage infiltrate was not related to this treatment. However, imatinib and sunitinib did induce secretion of anti-inflammatory IL-10 in macrophage cultures, indicating that treatment with these inhibitors might contribute to an immune suppressive microenvironment in GIST. Overall, our data reveal a picture of GIST as an active site of tumor-immune interaction in which suppressive mechanisms overrule potential antitumor responses. Tyrosine kinase inhibitors might promote this negative balance.

The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response.

The ESX-5 secretion system of pathogenic mycobacteria is responsible for the secretion of various PPE and PE-PGRS proteins. To better understand the role of ESX-5 effector proteins in virulence, we analyzed the interactions of Mycobacterium marinum ESX-5 mutant with human macrophages (Mphi). Both wild-type bacteria and the ESX-5 mutant were internalized and the ESX-5 mutation did not affect the escape of mycobacteria from phagolysosomes into the cytosol, as was shown by electron microscopy. However, the ESX-5 mutation strongly effected expression of surface Ags and cytokine secretion. Whereas wild-type M. marinum actively suppressed the induction of appreciable levels of IL-12p40, TNF-alpha, and IL-6, infection with the ESX-5 mutant resulted in strongly induced production of these proinflammatory cytokines. By contrast, infection with M. marinum wild-type strain resulted in a significant induction of IL-1beta production as compared with the ESX-5 mutant. These results show that ESX-5 plays an essential role in the modulation of immune cytokine secretion by human Mphi. Subsequently, we show that an intact ESX-5 secretion system actively suppresses TLR signaling-dependent innate immune cytokine secretion. Together, our results show that ESX-5 substrates, directly or indirectly, strongly modulate the human Mphi response at various critical steps.

Human anti-inflammatory macrophages induce Foxp3+ GITR+ CD25+ regulatory T cells, which suppress via membrane-bound TGFbeta-1.

CD4(+) T cell differentiation and function are critically dependent on the type of APC and the microenvironment in which Ag presentation occurs. Most studies have documented the effect of dendritic cells on effector and regulatory T cell differentiation; however, macrophages are the most abundant APCs in the periphery and can be found in virtually all organs and tissues. The effect of macrophages, and in particular their subsets, on T cell function has received little attention. Previously, we described distinct subsets of human macrophages (pro- and anti-inflammatory, m phi1 and m phi2, respectively) with highly divergent cell surface Ag expression and cytokine/chemokine production. We reported that human m phi1 promote, whereas m phi2 decrease, Th1 activation. Here, we demonstrate that m phi2, but not m phi1, induce regulatory T cells with a strong suppressive phenotype (T(m phi2)). Their mechanism of suppression is cell-cell contact dependent, mediated by membrane-bound TGFbeta-1 expressed on the regulatory T cell (Treg) population since inhibition of TGFbeta-1 signaling in target cells blocks the regulatory phenotype. T(m phi2), in addition to mediating cell-cell contact-dependent suppression, express typical Treg markers such as CD25, glucocorticoid-induced TNF receptor (GITR), and Foxp3 and are actively induced by m phi2 from CD25-depleted cells. These data identify m phi2 cells as a novel APC subset capable of inducing Tregs. The ability of anti-inflammatory macrophages to induce Tregs in the periphery has important implications for understanding Treg dynamics in pathological conditions where macrophages play a key role in inflammatory disease control and exacerbation.

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1.

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.

Identification of a human CD8+ regulatory T cell subset that mediates suppression through the chemokine CC chemokine ligand 4.

Regulatory T cells (Treg) comprise multiple subsets and are important in controlling immunity and inflammation. However, the induction and mode of action of the various distinct Treg subsets remain ill defined, particularly in humans. Here, we describe a human CD8+ lymphocyte activation gene-3 (LAG-3)+CD25+FoxP3+ Treg subset, which suppresses T cells partly through the secretion of CC chemokine ligand 4 (CCL4), which can inhibit T cell activation by interfering with T cell receptor signaling. CD8+ Tregs are expanded by antigen in in vivo-primed donors, and can be detected in pathogen-infected human tissue. This CD8+LAG-3+CD25+FoxP3+CCL4+ Treg subset thus may play a role in immunoregulation in humans, including infectious diseases.