Activation of Toll-Like Receptor 2 (TLR2) and TLR4/MD2 by Neisseria Is Independent of Capsule and Lipooligosaccharide (LOS) Sialylation but Varies Widely among LOS from Different Strains

Abstract

Lipooligosaccharide (LOS) structure and capsular polysaccharide of Neisseria meningitidis each greatly influence the virulence of the organism and the quality of host innate immune responses. In this study, we found that production of the proinflammatory cytokine tumor necrosis factor (TNF) by a human monocytederived cell line (THP-1) exposed to strains of N. meningitidis lacking capsule and/or with truncated LOS was similar to that elicited by the isogenic wild-type strain. These mutants also exhibited no difference in induction of the interleukin-8 (IL-8) promoter in a transfected HeLa cell system of Toll-like receptor 2 (TLR2) and TLR4/MD2 signaling. However, purified LOS from diverse strains of Neisseria (both N. meningitidis and N. gonorrhoeae) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells. These data suggest that although modification of the oligosaccharide chain of LOS and/or absence of capsule do not affect cell signaling mediated by TLR4/MD2, finestructural differences in the LOS do influence signaling through TLR4/MD2 and, through this pathway, influence some of the proinflammatory responses elicited by Neisseria.Neisseria meningitidis is an important cause of sepsis syndrome and meningitis, the severity of which is correlated with the release of proinflammatory cytokines (50, 51). The lipooligosaccharide (LOS) of N. meningitidis, like other gram-negative bacteria, is a potent stimulator of the proinflammatory response, and plasma LOS levels correlate with the severity of disease (3). Neisserial LOS is composed of a hydrophobic lipid A portion that anchors it to the bacterial outer membrane, and this is linked to a hydrophilic oligosaccharide core, which is exposed on the surface. However, it does not possess the repeating O-polysaccharide region that is seen in the lipopolysaccharide (LPS) of many other bacteria. The outer core of the LOS is highly variable, and this forms the basis of strain immunotyping (41). The lipid A structure of both N. meningitidis (29) and N. gonorrhoeae (44) consists of a ␤-D-glucosaminyl-(1Ј36)-D-glucosamine disaccharide backbone with variable patterns of phosphorylation and fatty acid acylation (23).Addition of sialic acid to the terminal galactose moiety of the lacto-N-neotetraose of meningococcal LOS and the polysialic acid capsule of serogroup B organisms both determine virulence through the regulation of complement (10,20,22) and also by modification of a number of innate host immune responses, including expression of phagocyte adhesion molecules (26) and nonopsonic macrophage phagocytosis of the organism (38).The Toll-like receptors (TLRs) have been shown to initiate innate proinflammatory signal transduction in response to a number of pathogen-associated molecular patterns (33,34,39). TLR4, with its cofactor MD2, has been shown to respond to LPS (30,42), and TLR2 responds to lipoproteins (2) and peptidoglycan (45). TLR3, TLR5, and TLR9 respond to double-stranded RNA (1), fla...

TREM-2 binds to lipooligosaccharides of Neisseria gonorrhoeae and is expressed on reproductive tract epithelial cells

“…This protein could act as a soluble receptor for LOS and Opa on bacterial membranes if produced by cells in the local environment of gonococcal infection. The effect of soluble TREM-2 binding to these molecules remains to be investigated, but we can speculate that it might modulate their interaction with human cells as both LOS and Opa function as adhesins for several epithelial cell receptors (7,(44)(45)(46)(47). In addition, the binding of soluble TREM-2 to LOS and Opa may influence the binding and function of soluble proteins galectin-3 and mannose-binding lectin known to target LOS and Opa, respectively (27,32).…”

“…Six strains of N. gonorrhoeae (F62, 1291, GC56, and MS11 mk LOS variants C [MkC], A [MkA], and MkC Δ1-5) were studied and have been described previously (7,26,27). MkC Δ1-5 is an isogenic mutant of MkC expressing LOS containing a truncated OS α chain (26).…”

“…Cervical carcinoma cell lines ME-180 and HeLa, monocytic cell line THP-1, and three HPV16/ E6E7 immortalized reproductive tract epithelial cell lines, endocervical End1/E6E7, ectocervical Ect1/E6E7, and vaginal Vk2/E6E7, were obtained from the American Type Culture Collection (Manassas, VA) and have been characterized previously (7,10,12). The E6E7 cell lines were maintained in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) supplemented with 0.05mg/ml bovine pancreatic extract, 0.1 ng/ml recombinant human epidermal growth factor, and 0.44 μg/ml CaCl 2 .…”

“…Our previous work and that of other investigators has shown that gonococci can initiate proinflammatory cytokine expression through activation of NFκB. In particular, gonococci engage innate immune receptors TLR4 and TLR2 with lipooligosaccharide (LOS) engaging TLR4, and PorB and lipoprotein Lip functioning as agonists for TLR2 (7)(8)(9). In urogenital epithelial cell lines, gonococcal infection induces upregulation of a variety of proinflammatory and inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-8 (10)(11)(12).…”

“…Among the important virulence factors involved in the pathogenesis of gonococcal infections, the LOS has been identified as a major component inducing the host proinflammatory cytokine response to the organisms (7). Gonococcal LOS is structurally related to the lipopolysaccharide (LPS) expressed by enteric Gram-negative bacteria, although LOS does not possess the repeating O-polysaccharide regions (15,16).…”