Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis.

RATIONALE: The development of host-targeted, prophylactic, and therapeutic interventions against tuberculosis requires a better understanding of the immune mechanisms that determine the outcome of infection with Mycobacterium tuberculosis. OBJECTIVES: To identify T-cell-dependent mechanisms that are protective in tuberculosis. METHODS: Multicolor flow cytometry, cell sorting and growth inhibition assays were employed to compare the frequency, phenotype and function of T lymphocytes from bronchoalveolar lavage or the peripheral blood. MEASUREMENTS AND MAIN RESULTS: At two independent study sites, bronchoalveolar lavage cells from donors with latent tuberculosis infection limited the growth of virulent Mycobacterium tuberculosis more efficiently than those in patients who developed disease. Unconventional, glycolipid-responsive T cells contributed to reduced mycobacterial growth because antibodies to CD1b inhibited this effect by 55%. Lipoarabinomannan was the most potent mycobacterial lipid antigen (activation of 1.3% T lymphocytes) and activated CD1b-restricted T cells that limited bacterial growth. A subset of IFN-γ-producing lipoarabinomannan-responsive T cells coexpressed the cytotoxic molecules perforin, granulysin, and granzyme B, which we termed polycytotoxic T cells. Taking advantage of two well-defined cohorts of subjects latently infected with Mycobacterium tuberculosis or patients who developed active disease after infection, we found a correlation between the frequency of polycytotoxic T cells and the ability to control infection (latent tuberculosis infection, 62%; posttuberculosis patients, 26%). CONCLUSIONS: Our data define an unconventional CD8(+) T-cell subset (polycytotoxic T cells) that is based on antigen recognition and function. The results link clinical and mechanistic evidence that glycolipid-responsive, polycytotoxic T cells contribute to protection against tuberculosis.

Hypoxia promotes Mycobacterium tuberculosis-specific up-regulation of granulysin in human T cells.

Oxygen tension affects local immune responses in inflammation and infection. In tuberculosis mycobacteria avoid hypoxic areas and preferentially persist and reactivate in the oxygen-rich apex of the lung. Oxygen restriction activates antimicrobial effector mechanisms in macrophages and restricts growth of intracellular Mycobacterium tuberculosis (M.Tb). The effect of oxygen restriction on T cell-mediated antimicrobial effector mechanisms is unknown. Therefore we determined the influence of hypoxia on the expression of granulysin, an antimicrobial peptide of lymphocytes. Hypoxia increased the antigen-specific up-regulation of granulysin mRNA and protein in human CD4(+) and CD8(+) T lymphocytes. This observation was functionally relevant, because oxygen restriction supported the growth-limiting effect of antigen-specific T cells against virulent M.Tb residing in primary human macrophages. Our results provide evidence that oxygen restriction promotes the expression of granulysin and suggest that this effect-in conjunction with additional T cell-mediated immune responses-supports protection against mycobacteria. The therapeutic modulation of oxygen availability may offer a new strategy for the host-directed therapy of infectious diseases with intracellular pathogens.

O-antigen delays lipopolysaccharide recognition and impairs antibacterial host defense in murine intestinal epithelial cells.

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.

Role of Salmonella enterica lipopolysaccharide in activation of dendritic cell functions and bacterial containment.

In contrast to nonpathogenic bacteria, the Gram-negative pathogen Salmonella enterica is not eradicated, but persists in murine dendritic cells (DC). The molecular basis of this phenotype is unknown. We set out to characterize bacterial and DC functions that are involved in Salmonella persistence. Our data prove that neither bacterial nor host cell de novo protein biosynthesis is required for Salmonella persistence in DC. We identified the Salmonella O-Ag of the LPS of Salmonella as an important factor for controlling the intracellular fate of Salmonella in DC. A Salmonella strain with entirely absent O-Ag showed an increased rate of uptake by DC, altered intracellular processing, and increased degradation, and also boosted the activation of immune functions of DC. These novel findings demonstrate that in addition to the multiple functions of the bacterial LPS in adaptation to the intestinal environment and protection against innate immune function, this molecule also has an important role in interaction of Salmonella with DC.

Stabilization of Hypoxia-Inducible Factor Promotes Antimicrobial Activity of Human Macrophages Against Mycobacterium tuberculosis.

Hypoxia-inducible factor (HIF) is a key oxygen sensor that controls gene expression patterns to adapt cellular metabolism to hypoxia. Pharmacological inhibition of prolyl-hydroxylases stabilizes HIFs and mimics hypoxia, leading to increased expression of more than 300 genes. Whether the genetic program initialized by HIFs affects immune responses against microbial pathogens, is not well studied. Recently we showed that hypoxia enhances antimicrobial activity against Mycobacterium tuberculosis (Mtb) in human macrophages. The objective of this study was to evaluate whether the oxygen sensor HIF is involved in hypoxia-mediated antimycobacterial activity. Treatment of Mtb-infected macrophages with the prolyl-hydroxylase inhibitor Molidustat reduced the release of TNFα and IL-10, two key cytokines involved in the immune response in tuberculosis. Molidustat also interferes with the p38 MAP kinase pathway. HIF-stabilization by Molidustat also induced the upregulation of the Vitamin D receptor and human ß defensin 2, which define an antimicrobial effector pathway in human macrophages. Consequently, these immunological effects resulted in reduced proliferation of virulent Mtb in human macrophages. Therefore, HIFs may be attractive new candidates for host-directed therapies against infectious diseases caused by intracellular bacteria, including tuberculosis.

Functional analysis of the Salmonella pathogenicity island 2-mediated inhibition of antigen presentation in dendritic cells.

Salmonella enterica is a facultative intracellular pathogen that is able to modify host cell functions by means of effector proteins translocated by the type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2). The SPI2-T3SS is also active in Salmonella after uptake by murine bone marrow-derived dendritic cells (BM-DC). We have previously shown that intracellular Salmonella interfere with the ability of BM-DC to stimulate antigen-dependent T-cell proliferation in an SPI2-T3SS-dependent manner. We observed that Salmonella-mediated inhibition of antigen presentation could be restored by external addition of peptides on major histocompatibility complex class II (MHC-II). The processing of antigens in Salmonella-infected cells was not altered; however, the intracellular loading of peptides on MHC-II was reduced as a function of the SPI2-T3SS. We set out to identify the effector proteins of the SPI2-T3SS involved in inhibition of antigen presentation and demonstrated that effector proteins SifA, SspH2, SlrP, PipB2, and SopD2 were equally important for the interference with antigen presentation, whereas SseF and SseG contributed to a lesser extent to this phenotype. These observations indicate the presence of a host cell-specific virulence function of a novel subset of SPI2-effector proteins.

Liposomal delivery of lipoarabinomannan triggers Mycobacterium tuberculosis specific T-cells.

Lipoarabinomannan (LAM) is a major cell wall component of Mycobacterium tuberculosis (Mtb). LAM specific human T-lymphocytes release interferon-γ (IFNγ) and have antimicrobial activity against intracellular Mtb suggesting that they contribute to protection. Therefore the induction of LAM-specific memory T-cells is an attractive approach for the design of a new vaccine against tuberculosis. A prerequisite for the activation of LAM-specific T-cells is the efficient uptake and transport of the glycolipid antigen to the CD1 antigen presenting machinery. Based on the hydrophobicity of LAM we hypothesized that packaging of LAM into liposomes will support the activation of T-lymphocytes. We prepared liposomes containing phosphatidylcholine, cholesterol, stearylated octaarginine and LAM via thin layer hydration method (LIPLAM). Flow cytometry analysis using fluorescently labelled LIPLAM showed an efficient uptake by antigen presenting cells. LAM delivered via liposomes was biologically active as demonstrated by the down-regulation of peroxisome proliferator activated receptor gamma (PPARγ) protein expression. Importantly, LIPLAM induced higher IFNγ production by primary human T-lymphocytes than purified LAM (2-16 times) or empty liposomes. These results suggest that the delivery of mycobacterial glycolipids via liposomes is a promising approach to promote the induction of M. tuberculosis specific T-cell responses.

Identification of minor inner-membrane components of the Shigella type III secretion system 'needle complex'.

Type III secretion systems (T3SSs or secretons) are central virulence factors of many Gram-negative bacteria, used to inject protein effectors of virulence into eukaryotic host cells. Their overall morphology, consisting of a cytoplasmic region, an inner- and outer-membrane section and an extracellular needle, is conserved in various species. A portion of the secreton, containing the transmembrane regions and needle, has been isolated biochemically and termed the 'needle complex' (NC). However, there are still unsolved questions concerning the nature and relative arrangement of the proteins assembling the NC. Until these are resolved, the mode of function of the NC cannot be clarified. This paper describes an affinity purification method that enables highly efficient purification of Shigella NCs under near-physiological conditions. Using this method, three new minor components of the NC were identified by mass spectrometry: IpaD, a known component of the needle tip complex, and two predicted components of its central inner-membrane export apparatus, Spa40 and Spa24. A further minor component of the NC, MxiM, is only detected by immunoblotting. MxiM is a 'pilotin'-type protein for the outer-membrane 'secretin' ring formed of MxiD. As expected, it localized to the outer rim of the upper ring of NCs, validating the other findings.

The type III secretion system needle tip complex mediates host cell sensing and translocon insertion.

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.

The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus.

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned "on." In a further set, it was "constitutively on" but experimentally "uninducible." Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to "sense" host cells.