Abstract
Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM) and trehalose monomycolate (TMM), the apolar phthiocerol dimycocersates (PDIMs), triacyl glycerol (TAG), pentacyl trehalose (PAT), phenolic glycolipid (PGL), and mono-mycolyl glycerol (MMG). Polar lipids identified included glucose monomycolate (GMM), diphosphatidyl glycerol (DPG), phenylethanolamine (PE) and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs). These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.
Highlights
Bovine tuberculosis (BTB), caused by Mycobacterium bovis (M. bovis), is a zoonotic disease of significant economic, animal and public health burden
Extraction and analysis of lipid from AF 2122/97 In order to identify individual lipid components within the polar and apolar fractions, lipids extracted from the M. bovis reference strain (AF 2122/97) were subjected to 2D Thin Layer Chromatography (TLC) analysis using solvent systems of increasing polarity and subsequently stained with molybdophosphoric acid (MPA)
TLC analysis of the polar lipid fraction using system D showed the presence of trehalose monomycolate (TMM) and trehalose dimycolate (TDM) (Figure 1e) while the most polar solvent system (E, Figure 1f ) enabled identification of the most polar lipids, which included diphosphatidyl glycerol (DPG), phosphatidyl ethanolamine (PE), phosphatidylinositol mannosides (PIMs, integers denote number of mannoside or acyl groups), phosphatidyl inositol (PI) and an unknown phospholipid (P)
Summary
Bovine tuberculosis (BTB), caused by Mycobacterium bovis (M. bovis), is a zoonotic disease of significant economic, animal and public health burden. Consumption of raw or unpasteurised animal products and contact with infected carcasses plays a large role in zoonotic M. bovis infection of humans in Africa and South America [1,2]. This is not a problem solely associated with poverty and less developed countries as evidenced by a recent report that up to 45% of all TB infected children in San Diego were caused by M. bovis [3]. DC are known to possess and utilise these same receptors as well as other, structurally related molecules such as the DC specific C - type lectin (DC - SIGN) which have been heavily implicated in immune recognition of the bacilli [12,13,14]
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