Intravascular heavy chain-modification of hyaluronan during endotoxic shock

Abstract

During inflammation, the covalent linking of the ubiquitous extracellular polysaccharide hyaluronan (HA) with the heavy chains (HC) of the serum protein inter alpha inhibitor (IαI) is exclusively mediated by the enzyme tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6). While significant advances have been made regarding how HC-modified HA (HC-HA) is an important regulator of inflammation, it remains unclear why HC-HA plays a critical role in promoting survival in intraperitoneal lipopolysaccharide (LPS)-induced endotoxemia while exerting only a modest role in the outcomes following intratracheal exposure to LPS. To address this gap, the two models of intraperitoneal LPS-induced endotoxic shock and intratracheal LPS-induced acute lung injury were directly compared in TSG-6 knockout mice and littermate controls. HC-HA formation, endogenous TSG-6 activity, and inflammatory markers were assessed in plasma and lung tissue. TSG-6 knockout mice exhibited accelerated mortality during endotoxic shock. While both intraperitoneal and intratracheal LPS induced HC-HA formation in lung parenchyma, only systemically-induced endotoxemia increased plasma TSG-6 levels and intravascular HC-HA formation. Cultured human lung microvascular endothelial cells secreted TSG-6 in response to both TNFα and IL1β stimulation, indicating that, in addition to inflammatory cells, the endothelium may secrete TSG-6 into circulation during systemic inflammation. These data show for the first time that LPS-induced systemic inflammation is uniquely characterized by significant vascular induction of TSG-6 and HC-HA, which may contribute to improved outcomes of endotoxemia.

Inter-α-inhibitor Ameliorates Endothelial Inflammation in Sepsis

“…Furthermore, IαI has been shown to bind extracellular histones and neutralize histone-induced adverse effects in sepsis [49]. Finally, we recently described that IαI complexing with hyaluronan in septic animals leading to its rapid clearance and ameliorated sepsis mortality [50,51]. This suggests that IαI is able to reduce the inflammatory cascade in injured endothelia through many pathways, ultimately leading to reduced VCAM-1 and ICAM-1 upregulation and secretion.…”

Inter-α-inhibitor heavy chain-1 has an integrin-like 3D structure mediating immune regulatory activities and matrix stabilization during ovulation

“…Initial SPR screening experiments with the 50-kDa and H120 fragments of fibronectin (see (51)), corresponding to the cell-ITIH1 structure reveals roles in inflammation & ovulation and heparin-binding regions, respectively, indicated that while both bound to rHC1 (in a metal ion-independent manner), the interaction with H120 was of considerably higher affinity than that with the 50-kDa fragment; the H120* fragment (missing the 12-13 type III repeats, but otherwise identical to H120) did not bind to rHC1. Therefore, we expressed fibronectin type III repeats 13-14 (denoted as cFN13- 14) in Escherichia coli, purifying this to homogeneity (see Figure S7 and Experimental Procedures), and showed that this bound rHC1 in an essentially identical fashion to H120; i.e. localizing the high affinity HC1-binding site on fibronectin to the 13-14 type III domains.…”

“…TSG-6 plays a catalytic role in transferring HCs from the CS chains of IαI and PαI onto HA to form HC•HA complexes (8,9); where this is essential for female fertility (10)(11)(12)(13). As well as being expressed by cumulus cells during ovulation, TSG-6 is also produced in the context of inflammation, where it mediates the formation of HC•HAs (14), e.g. when IαI/PαI leak into tissues from the blood circulation (reviewed in (9)).…”

“…But it is unknown whether this, or indeed the altered hydrodynamic properties of the modified HA (26), are part of a protective process or contribute to arthritis pathology (9). In this regard, HC•HA complexes from human amniotic membrane, which are reported to contain only HC1 (27), are potently anti-fibrotic and anti-inflammatory (9,28); HC•HAs also protect against endotoxic shock and sepsis (14,29,30). However, the role of HCs (including HC1) has not been determined in these processes.…”

Bacterial products in donor airways prevent the induction of lung transplant tolerance

“…In select experiments, lipopolysaccharide (LPS) (20 μg) was dissolved in 50 μL of phosphate‐buffered saline and injected into the donor airway before connecting donor and recipient bronchi (Sigma). The concentrations and administration route were based on previous studies examining the impact of Pam 3 Cys 4 and LPS on pulmonary immune responses 12‐14 . For select experiments, 10 × 10 6 CD8 + T cells (Ly‐2 microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany) 15 or 3 × 10 6 monocytes (Monocyte Isolation Kit, Miltenyi Biotec), 16 isolated from the spleen or bone marrow, respectively, of naïve B6 WT mice were injected intravenously into B6 TLR2KO recipients at the time of transplantation.…”