Highly Purified Mycobacterial Phosphatidylinositol Mannosides Drive Cell-Mediated Responses and Activate NKT Cells in Cattle

Abstract

M embers of the mycobacterial genus are renowned for their waxy, lipid-rich outer envelope. Under physiological conditions, this outer layer is likely to be the first point of contact between the bacterial cell and the host's immune system, and the outcome of this interaction is pivotal in the establishment of infection. One of the most important groups of membrane bound lipids consists of the phosphatidylinositol mannosides (PIMs). Interest in PIMs was stimulated since it was shown that phosphatidylinositol dimannoside (PIM 2 ) forms the phosphoglycolipid anchor which tethers a large array of glycolipids and lipoglycans, including lipomannan (LM) and lipoarabinomannan (LAM), to the cellular membrane (1). PIMs have been shown to interact with a variety of immune components and mediate significant effects on the host. Ever since the realization that NKT cells could respond to lipid antigens (2-4) and the subsequent discovery of the CD1d-restricted lipid antigen ␣-galactosylceramide (␣-GalCer) (5, 6), much research effort has been concentrated on understanding lipid antigens. Although work was initially focused on invariant NKT cells, it has since been shown that great diversity exists in the lipid-responsive T cell receptor (TCR) repertoire (7-9) and that these diverse NKT cells contribute to the Th1/Th2 balance (7, 10). It has been shown that CD1d-restricted NKT cells are capable of recognizing a variety of lipid antigens, including phospholipids (11). More recently, a CD1b-restricted subset of T cells has been found (12). Similarly to CD1d-restricted invariant NKT (iNKT) cells, the CD1b-restricted variant cells require CD1B for their development and produce proinflammatory cytokines in response to CD1b-expressing dendritic cells (DCs) (12). It is clear that NKT-like cells have a significant role to play in lipid-mediated responses, and the hunt for their antigens has continued (13-15).Given the ability of lipid molecules to generate responses in peripheral blood mononuclear cells (PBMC), we decided to assess the ability of individual, highly purified natural PIMs to activate bovine lymphocytes. In this study, we developed a novel extraction method which allowed us to extract and highly purify a variety of PIM molecules from virulent Mycobacterium tuberculosis H37Rv. The ability of these molecules to induce lymphocyte responses in Mycobacterium bovis-infected cattle was investigated by measuring lymphocyte proliferation and gamma interferon (IFN-␥) production. Furthermore, flow cytometry techniques were utilized to characterize responding cell populations. MATERIALS AND METHODS Extraction of PIMs.Using the novel methodology outlined below, highly pure phosphatidylinositol dimannoside (PIM 2 ), acylphosphatidylinositol dimannoside (AcPIM 2 ), diacyl-phosphatidylinositol dimannoside (Ac 2 PIM 2 ), acylphosphatidylinositol hexamannoside (AcPIM 6 ), and diacylphosphatidylinositol hexamannoside (Ac 2 PIM 6 ) were successfully isolated. Individual PIM molecules were analyzed by electrospray ionization mass spectrometr...

CD335 (NKp46)+ T-Cell Recruitment to the Bovine Upper Respiratory Tract during a Primary Bovine Herpesvirus-1 Infection

“…NKT cells also recognize lipid ligands complexed with the non-MHC surface molecule, CD1d, and were also referred to as “CD1d-restricted” T-cells ( 20 ). CD1d is absent in cattle ( 21 ) but bovine T-cells co-expressing CD335 do recognize lipid ligands by a CD1d-independent mechanism ( 22 ).…”

“…Previous studies demonstrated that non-MHC-restricted lymphocytes were recruited to the lungs of calves following BHV-1 infection ( 7 ) but it was not determined whether these cells were conventional NK cells or non-conventional T-cells. It is not known if non-conventional T-cells may be recruited to bovine mucosal surfaces since previous studies of bovine non-conventional T-cells were limited to cells isolated from blood ( 12 , 21 , 22 ).…”

The Role of Phosphatidylinositol Mannosides in the Serological Diagnosis of Mycobacterial Infections

“…However, the comparison to PPD-A may not be appropriate in animals or regions where colonizing or sensitizing NTMs are only distantly or not at all related to M. avium [7]. Moreover, the sensitivity and specificity of the SIT and SICCT could be affected in MAP infected herds [48]. Therefore, our results suggest that PIMs generate a non-specific immune response that inhibits mycobacterial diagnostic test specificity when using complex antigen preparations.…”

“…Although the humoral PIM response is too unspecific for the implementation in diagnostic assays, the CD1 based T-cell response to lipid extracts of naturally infected MAP and MB infected animals is highly specific, and exclusively detected in infected animals [36]. Among these lipids, PIMs, and specifically AcPIM 6 , are recognized by CD3 receptors, resulting in natural killer T-cell activation and induced interferon gamma production in bovine lymphocytes [48]. This suggests that PIMs elicit a specific cell mediated immune response, and that PIMs could be suitable antigen candidates for novel (non-classical) T cell-based tests.…”

Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of Streptococcus suis

“…The chromatography was performed in a TLC chamber saturated with C/M 94:6. Afterwards, the TLC plate was stained with Hanessian's stain [92], baked for 1 min at 150 • C, and scanned at 300 dpi. Finally, the signal was quantified using the software LabImage 1D (Intas, Göttingen, Germany).…”