Elicited and pre‐existing anti‐Neu5Gc antibodies differentially affect human endothelial cells transcriptome

Abstract

Humans cannot synthesize N‐glycolylneuraminic acid (Neu5Gc) but dietary Neu5Gc can be absorbed and deposited on endothelial cells (ECs) and diet‐induced anti‐Neu5Gc antibodies (Abs) develop early in human life. While the interaction of Neu5Gc and diet‐induced anti‐Neu5Gc Abs occurs in all normal individuals, endothelium activation by elicited anti‐Neu5Gc Abs following a challenge with animal‐derived materials, such as following xenotransplantation, had been postulated. Ten primary human EC preparations were cultured with affinity‐purified anti‐Neu5Gc Abs from human sera obtained before or after exposure to Neu5Gc‐glycosylated rabbit IgGs (elicited Abs). RNAs of each EC preparation stimulated in various conditions by purified Abs were exhaustively sequenced. EC transcriptomic patterns induced by elicited anti‐Neu5Gc Abs, compared with pre‐existing ones, were analyzed. qPCR, cytokines/chemokines release, and apoptosis were tested on some EC preparations. The data showed that anti‐Neu5Gc Abs induced 967 differentially expressed (DE) genes. Most DE genes are shared following EC activation by pre‐existing or anti‐human T‐cell globulin (ATG)‐elicited anti‐Neu5Gc Abs. Compared with pre‐existing anti‐Neu5Gc Abs, which are normal component of ECs environment, elicited anti‐Neu5Gc Abs down‐regulated 66 genes, including master genes of EC function. Furthermore, elicited anti‐Neu5Gc Abs combined with complement‐containing serum down‐regulated most transcripts mobilized by serum alone. Both types of anti‐Neu5Gc Abs‐induced a dose‐ and complement‐dependent release of selected cytokines and chemokines. Altogether, these data show that, compared with pre‐existing anti‐Neu5Gc Abs, ATG‐elicited anti‐Neu5Gc Abs specifically modulate genes related to cytokine responses, MAPkinase cascades, chemotaxis, and integrins and do not skew the EC transcriptome toward a pro‐inflammatory profile in vitro.

Challenging the Role of Diet-Induced Anti-Neu5Gc Antibodies in Human Pathologies

“…Since unambiguously observing such deposits using anti-Neu5Gc chicken polyclonal Ab staining on frozen or fixedhistological tissue samples was difficult, we used flow cytometry to assess the binding of anti-Neu5Gc chicken Abs on living ECs from large arteries of brain-dead donors (9). Although we confirmed a faint signal on gated ECs in four samples, three other preparations were found to be negative (9). A roughly similar proportion was found in eight additional living EC preparations that were tested after sorting, of which, two were positive, two were negative, and four had extremely faint or negative staining (unpublished data).…”

“…Few studies have investigated Neu5Gc loading among human cells (8,9); however, further studies are warranted to establish whether indeed a substantial fraction of humans actually lack the fundamental component of the theoretical basis of the xenosialitis model. Although Neu5Gc exists at trace levels on ECs of positive individuals, we must also consider the myriad of surface glycoproteins or lipoproteins that display Neu5Gc and the high diversity of Neu5Gc terminal residues, which result in a huge dispersion of cell-surface epitopes that are potentially recognized by A-GcAbs (15).…”

“…A second study (9) used affinity-purified A-GcAbs from either normal sera (diet-induced Abs) or sera of those highly immunized by rabbit polyclonal IgGs (elicited Abs) (18). In addition, large artery ECs that undergo physiological loading levels of Neu5Gc were used (9).…”

The Possible Role of Anti-Neu5Gc as an Obstacle in Xenotransplantation

“…Compared to sera without anti-Neu5Gc, human sera containing high titer of ATG-elicited anti-Neu5Gc antibodies induce an increase in transcripts (qRT-PCR) encoding for IL-1β, IL-6, and IL-8 pro-inflammatory cytokines in human endothelial cells (57). However, elicited anti-Neu5Gc antibodies do not seem to induce an inflammatory transcriptomic profile in human endothelial cells loaded with a "physiological" Neu5Gc concentration similar to this obtained from diet (109). It has been also described that Neu5Gc and anti-Neu5Gc antibodies may contribute to exacerbate atherosclerotic cardiovascular disease mediated by xenosialitis (37,106,117), infectious mononucleosis (118), early serum sickness disease (57,113,119), and autoimmunity (120).…”

“…Evoked anti-Neu5Gc antibodies ( Table 1), which persist for a long time and are characterized by a diversification of their recognition repertoire, could mediate biological effects different from pre-existing antibodies. The transcriptome of human endothelial cells exhibiting "physiological" amounts of Neu5Gc is differentially affected by diet-derived and elicited anti-Neu5Gc antibodies (109). Of note, the involvement in xenotransplantation of elicited antibodies specific for Neu5Gc will be probably different depending on the individual as animal products, cells, tissues and organs do not induce anti-Neu5Gc antibodies in all treated humans and these antibodies show an individual-related variable affinity and specificity for the multiple Neu5Gc-containing antigens (74,75,92,101,110).…”