Detectable serum flagellin and lipopolysaccharide and upregulated anti-flagellin and lipopolysaccharide immunoglobulins in human short bowel syndrome

Abstract

Gut barrier dysfunction may occur in short bowel syndrome (SBS). We hypothesized that systemic exposure to flagellin and lipopolysaccharide (LPS) in SBS might regulate specific immune responses. We analyzed serial serum samples obtained from parenteral nutrition (PN)-dependent patients with SBS versus non-SBS control serum. Serum from 23 adult SBS patients was obtained at baseline and 4, 8, 12, 16, 20, and 24 wk in a trial of modified diet with or without growth hormone. Control serum was obtained from 48 healthy adults and 37 adults requiring PN during critical illness. Serum flagellin was detected by an ELISA recognizing an array of gram-negative flagellins, and LPS was detected by limulus assay. Serum flagellin- and LPS-specific immunoglobulin levels (IgM, IgA, and IgG) were determined by ELISA. Serum flagellin and LPS were undetectable in control subjects. In contrast, serum flagellin, LPS, or both were detected in 14 SBS patients (61%) during one or more time points [flagellin alone, 5/23 (22%); LPS alone, 6/23 (26%); or flagellin + LPS, 3/23 (13%)]. Flagellin-specific serum IgM, IgA, and IgG levels were markedly increased in SBS patients compared with both control populations and remained elevated during the 6-mo study period. LPS-specific IgA was significantly higher in SBS patients compared with healthy controls; LPS-specific IgM, IgA, and IgG levels each decreased over time in association with PN weaning. We conclude that adults with PN-dependent SBS are systemically exposed to flagellin and LPS, presumably from the gut lumen. This likely regulates innate and adaptive immune responses to these specific bacterial products.

Fig. 1

Patients with short bowel syndrome (SBS) demonstrate markedly elevated flagellin-specific immunoglobulin levels in serum. Flagellin (Flag)-specific immunoglobulins M, A, and G (IgM, IgA, and IgG) were quantitated in serum obtained from clinically stable adults with parenteral nutrition (PN)-dependent severe SBS (n = 23) and compared with samples obtained from 48 healthy adults (control) and 37 critically ill patients requiring PN and without SBS, as outlined in methods. Serum levels of flagellin-specific IgM, IgA, and IgG were markedly increased in SBS patients compared with values in control subjects (each P < 0.001).

Fig. 2

Patients with SBS demonstrate elevated lipopolysaccharide (LPS)-specific IgA levels in serum. LPS-specific IgM, IgA, and IgG were quantitated in serum obtained from clinically stable adults with PN-dependent severe SBS (n = 23) and compared with samples obtained from 48 healthy adults, as outlined in methods. Serum titers of LPS-specific IgA were modestly, but significantly, increased in SBS patients compared with control subjects (P < 0.006). In contrast, anti-LPS IgG and IgM levels in serum were similar in SBS patients and control subjects.

Fig. 3

Theoretical schema of gut barrier dysfunction and translocation of flagellin in human SBS. Various events common in SBS, such as gut mucosal inflammation, small bowel bacterial overgrowth, endotoxemia, cytokinemia, malnutrition, parenteral feeding, malnutrition, and/or relative lack of enteral food stimulation may theoretically cause movement of flagellated luminal bacteria via transcellular pathways, or paracellular movement following disruption of tight junction and adherens junction proteins may result in translocation of bacteria to the basolateral membrane of gut epithelial cells. Alternatively, flagellin itself may traverse these barriers. Flagellin may interact with basolateral toll-like receptor-5 (TLR5) to induce an innate immune response in which gut epithelial cells secrete cytokines and chemokines [e.g., interleukin-8 (IL-8)] after activation of nuclear factor-κB (NF-κB). This, in turn, may recruit polymorphonuclear neutrophils (PMNs) to mediate local inflammation at the epithelial level and may contribute to further gut barrier dysfunction. Flagellin also stimulates an adaptive immune response via antigen-presenting cells and T cells, resulting in B cell production of flagellin-specific IgM, IgA, and IgG.