Antibodies Recognizing Yersinia enterocolitica Lipopolysaccharides of Various Chemotypes in Synovial Fluids From Patients With Juvenile Idiopathic Arthritis

Yersinia enterocolitica O:3 (YeO3) is considered to be associated with reactive arthritis (ReA), and its lipopolysaccharide (LPS) has been detected in synovial fluids from patients. Interestingly, YeO3 wild-type LPS was processed by host cells, resulting in truncated LPS molecules presenting the core region. Previously, we reported the immunogenicity but not adjuvanticity of YeO3 LPSs of wild (S) type, Ra, Rd, or Re chemotypes in mice. Here, we demonstrate the presence of YeO3 LPS chemotype-specific antibodies in all analyzed synovial fluids (SF) from patients with juvenile idiopathic arthritis (JIA). Interestingly, the high titer of antibodies specific for the Kdo-lipid A region was found in most tested SF. In contrast, only a few were positive for antibodies recognizing O-specific polysaccharides. Western blot analysis revealed the presence of antibodies reacting with fast-migrating LPS fractions and enterobacterial common antigen (ECA) in synovial fluid samples. Our data also suggest the importance of LPS-associated ECA for the antigenicity of endotoxin. Furthermore, we confirmed in vitro that Yersinia LPS processing leads to the exposure of its core region and enhanced potency of complement lectin pathway activation.Peer reviewe

H-Ficolin

H-ficolin is a serum lectin synthesized (as a ~34 kDa polypeptide) predominantly by the liver and lung tissues and is one of the soluble pattern recognition receptors of the innate immune system. It is structurally similar to L- and M- ficolins, but is different in its tissue expression and binding affinities to pathogenic ligands. Ficolins have an amino (N)-terminal cysteine-rich region, a middle stretch of a collagen-like sequence, and a fibrinogen-like domain in the carboxy (C)-terminus. Three identical polypeptides form a structural (triple helical) subunit, with the help of the collagen-like domain. Further oligomerization of this subunit results in different sized H-ficolin molecules in circulation. The polypeptides in the structural subunit are cross-linked by disulphide bonds in the N-terminal region and the fibrinogen-like domain forms a globular structure. Thus, the overall structure of H-ficolin also resembles mannose/mannan- binding lectin (MBL). The primary role of H-ficolin is that of a pattern recognition receptor, recognizing acetylated sugar residues on the cell surface of different bacteria, viruses and other pathogens. There are two pathways by which H-ficolin may participate in a host defense response: 1) It activates the complement lectin pathway, via MBL/ficolin associated serine proteases (MASPs), that converges with the classical complement pathway at the level of complement C4, and 2) it may also act directly as an opsonin, enhancing phagocytosis by binding to cell-surface receptors present on phagocytic cells

H-Ficolin

H-ficolin is a serum lectin synthesized (as a ~34 kDa polypeptide) predominantly by the liver and lung tissues and is one of the soluble pattern recognition receptors of the innate immune system. It is structurally similar to L- and M- ficolins, but is different in its tissue expression and binding affinities to pathogenic ligands. Ficolins have an amino (N)-terminal cysteine-rich region, a middle stretch of a collagen-like sequence, and a fibrinogen-like domain in the carboxy (C)-terminus. Three identical polypeptides form a structural (triple helical) subunit, with the help of the collagen-like domain. Further oligomerization of this subunit results in different sized H-ficolin molecules in circulation. The polypeptides in the structural subunit are cross-linked by disulphide bonds in the N-terminal region and the fibrinogen-like domain forms a globular structure. Thus, the overall structure of H-ficolin also resembles mannose/mannan- binding lectin (MBL). The primary role of H-ficolin is that of a pattern recognition receptor, recognizing acetylated sugar residues on the cell surface of different bacteria, viruses and other pathogens. There are two pathways by which H-ficolin may participate in a host defense response: 1) It activates the complement lectin pathway, via MBL/ficolin associated serine proteases (MASPs), that converges with the classical complement pathway at the level of complement C4, and 2) it may also act directly as an opsonin, enhancing phagocytosis by binding to cell-surface receptors present on phagocytic cells

The Role of Yersinia enterocolitica O : 3 Lipopolysaccharide in Collagen-Induced Arthritis

Yersinia enterocolitica O:3 is mentioned among the most common arthritogenic pathogens. Bacterial components (including lipopolysaccharide (LPS)) may persist in the joint after eradication of infection. Having an adjuvant activity, LPS may enhance production of anticollagen antibodies, involved in the pathogenesis of rheumatoid arthritis. Furthermore, its ability to activate complement contributes to the inflammation. The aim of this work was to investigate whether Yersinia LPS (coinjected with collagen) is associated with arthritis progression or other pathological effects and to elucidate the mechanism of this association. It was demonstrated that murine mannose-binding lectin C (MBL-C) recognizes the inner core heptoses of the Rd1 chemotype LPS of Yersinia. In addition, the Rd1 LPS activates the MBL-associated serine protease 1 (MASP-1) stronger than the S and Ra chemotype LPS and comparable to Klebsiella pneumoniae O:3 LPS. However, in contrast to the latter, Yersinia Rd1 LPS was associated neither with the adjuvancity nor with the enhancement of pathological changes in animal paws/impairment of motility. On the other hand, it seemed to be more hepatotoxic when compared with the other tested endotoxins, while the enlargement of inguinal lymph nodes and drop in hepatic MBL-C expression (at the mRNA level) were independent of LPS chemotype. Our data did not suggest no greater impact Y. enterocolitica O:3 on the development or severity of arthropathy related to anticollagen antibody-induced arthritis in mice, although its interaction with MBL-C and subsequent complement activation may contribute to some adverse effects.Peer reviewe

Interaction of Mannose-Binding Lectin With Lipopolysaccharide Outer Core Region and Its Biological Consequences

Lipopolysaccharide (LPS, endotoxin), the main surface antigen and virulence factor of Gram-negative bacteria, is composed of lipid A, core oligosaccharide, and O-specific polysaccharide (O-PS) regions. Each LPS region is capable of complement activation. We have demonstrated that LPS of Hafnia alvei, an opportunistic human pathogen, reacts strongly with human and murine mannose-binding lectins (MBLs). Moreover, MBL–LPS interactions were detected for the majority of other Gram-negative species investigated. H. alvei was used as a model pathogen to investigate the biological consequences of these interactions. The core oligosaccharide region of H. alvei LPS was identified as the main target for human and murine MBL, especially l-glycero-d-manno-heptose (Hep) and N-acetyl-d-glucosamine (GlcNAc) residues within the outer core region. MBL-binding motifs of LPS are accessible to MBL on the surface of bacterial cells and LPS aggregates. Generally, the accessibility of outer core structures for interaction with MBL is highest during the lag phase of bacterial growth. The LPS core oligosaccharide–MBL interactions led to complement activation and also induced an anaphylactoid shock in mice. Unlike Klebsiella pneumoniae O3 LPS, robust lectin pathway activation of H. alvei LPS in vivo was mainly the result of outer core recognition by MBL; involvement of the O-PS is not necessary for anaphylactoid shock induction. Our results contribute to a better understanding of MBL–LPS interaction and may support development of therapeutic strategies against sepsis based on complement inhibition

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

<div><p>Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of <i>Hafnia alvei</i> PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement <i>in vitro</i>. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions <i>in vitro</i>, it may have numerous consequences <i>in vivo</i>. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3.</p></div

Detection of ficolin-3, MASPs and Ig in plasma-derived ficolin-3 complexes.

<p>Analysis of ficolin-3 preparation (1 μg of final product) fractionated on a 10% polyacrylamide gel under reducing (lanes 1, 2, 5–8) or non-reducing (lanes 3–4) conditions. Coomassie Blue stain (lanes 1, 3) and Western blotting using anti-ficolin-3 (lanes 2, 4), anti-MASP-1 (lane 5), anti-MASP-2 (lane 6), and anti-MASP-3 (lane 7) antibodies. The last lane (8) is the strip 4 re-probed with anti-human IgG, IgA and IgM. MASP bands are marked with asterisks (*). A, b, c–heavy chains of IgM (a), IgA (b), IgG (c); d- ficolin-3 monomer; e- light chains of Ig. The positions of the molecular mass markers are indicated in kDa.</p