Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced Into a Sepsis Phenotype

Abstract

Objectives: Sepsis is a leading cause of death in the United States. Putative targets to prevent systemic inflammatory response syndrome include antagonism of toll-like receptors 2 and 4 and CD44 receptors in vascular endothelial cells. Proteoglycan-4 is a mucinous glycoprotein that interacts with CD44 and toll-like receptor 4 resulting in a blockade of the NOD-like receptor pyrin domain-containing-3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less interleukin-6 expression after recombinant human proteoglycan 4 treatment in vitro. Design: Enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction to measure interleukin-6 protein and gene expression. Setting: Research laboratory. Subjects: Human umbilical vascular endothelial cells, human lung microvascular endothelial cells, and transgenic mouse (wild type) (Cd44 +/+/Prg4 +/+), Cd44 –/– (Cd44 tm1Hbg Prg4 +/+), Prg4 GT/GT (Cd44 +/+ Prg4 tm2Mawa/J), and double knockout (Cd44 tm1Hbg Prg4 tm2Mawa/J) lung microvascular endothelial cells. Interventions: Cells were treated with 100 or 250 ng/mL lipopolysaccharide-Escherichia coli K12 and subsequently treated with recombinant human proteoglycan 4 after 30 minutes. Interleukin-6 levels in conditioned media were measured via enzyme-linked immunosorbent assay and gene expression was measured via reverse transcriptase-quantitative polymerase chain reaction with ΔΔ–Ct analysis. Additionally, human umbilical vascular endothelial cells and human lung microvascular endothelial cells were treated with 1:10 diluted plasma from 15 patients with sepsis in culture media. After 30 minutes, either 50 or 100 µg/mL recombinant human proteoglycan 4 was administered. Interleukin-6 protein and gene expression were assayed. Proteoglycan 4 levels were also compared between control and sepsis patient plasma. Measurements and Main Results: Human umbilical vascular endothelial cell, human lung microvascular endothelial cell, and mouse lung microvascular endothelial cell treated with lipopolysaccharide had significantly increased interleukin-6 protein compared with controls. Recombinant human proteoglycan-4 significantly reduced interleukin-6 in human and mouse endothelial cells. Interleukin-6 gene expression was significantly increased after lipopolysaccharide treatment compared with controls. This response was reversed by 50 or 100 µg/mL recombinant human proteoglycan-4 in 80% of sepsis samples in human umbilical vascular endothelial cells and in 60–73% in human lung microvascular endothelial cells. In Cd44 –/– genotypes of the mouse lung microvascular endothelial cells, recombinant human proteoglycan-4 significantly reduced interleukin-6 protein levels after lipopolysaccharide treatment, indicating that Cd44 is not needed for recombinant human proteoglycan-4 to have an effect in a toll-like receptor 4 agonist inflammation model. Patient sepsis samples had higher plasma levels of native proteoglycan-4 than controls. Interpretation and Conclusions: Recombinant human proteoglycan-4 is a potential adjunct therapy for sepsis patients and warrants future in vivo model studies.

Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood–Brain Barrier after Traumatic Brain Injury

“…PRG4 potently inhibits post-traumatic activation of NF-jB and ERK1/2 signaling pathways, which is in line with our previous observations that PRG4 interferes with both TLR2/4 and CD44 signaling. [31][32][33][34] PRG4 appeared to have a stronger inhibitory effect on activity of ERK1/2 than on that of the NF-jB signaling pathway, and this effect lasted for at least 48 h after a single rhPRG4 injection at 1 h post-TBI. This protracted inhibitory activity of PRG4 could be associated with a prolonged presence in the injured brain of its 60-and/or 12-kDa degradation products (Fig.…”

“… 29 , 30 PRG4 exerts its anti-inflammatory effects by binding to TLR2 and TLR4, and interfering with their signaling. 31 , 32 In addition to its inhibitory effect on TLR2/4 signaling, PRG4 competes with HA for binding to cluster of differentiation 44 (CD44), 33 , 34 a cell-surface glycoprotein playing an important role in inflammation. 35 Thus, PRG4 may inhibit the post-traumatic activation of both TLR2/4 and CD44 in response to proinflammatory low-molecular-weight fragments of HA, 36 which are generated in the injured brain.…”

Recombinant Human Proteoglycan 4 Regulates Phagocytic Activation of Monocytes and Reduces IL-1β Secretion by Urate Crystal Stimulated Gout PBMCs

“…This activity may explain rhPRG4’s disease-modifying activity seen in pre-clinical osteoarthritis models ( 58 , 59 ). The anti-inflammatory activity of rhPRG4 was also demonstrated in an in vitro sepsis model, where rhPRG4 at 50 or 100μg/mL reduced interleukin-6 (IL-6) secretion from murine and human endothelial cells ( 60 ). rhPRG4 was also shown to reduce cytokine and chemokine secretions from immortalized corneal epithelial cells at 300μg/mL in vitro and inflammation in an experimental dry eye disease model ( 61 ).…”

Lubricin as a Therapeutic and Potential Biomarker in Sepsis

“…Results from our lab-Due to lubricin's role in the inflammatory pathways involving CD44, TLR4 and NLRP3 as discussed above, our lab has identified lubricin as a possible therapeutic for patients with sepsis due to its ability to lower both protein levels and gene expression of IL-6 in human umbilical vascular endothelial cells (HUVECs) and in human lung microvascular endothelial cells (HLMVECs) 84 . HUVECs and HLMVECs were treated with LPS to induce a high inflammatory state, resulting in significantly increased IL-6 protein levels and gene expression, both of which were reversed to control levels with 50, 100 and 150 μg/mL rhPRG4 treatment 84 . Moreover, HLMVECs were treated with plasma from septic patients to induce an inflammatory response and rhPRG4 significantly lowered IL-6 protein levels in almost 75% of the patients 84 .…”

“…HUVECs and HLMVECs were treated with LPS to induce a high inflammatory state, resulting in significantly increased IL-6 protein levels and gene expression, both of which were reversed to control levels with 50, 100 and 150 μg/mL rhPRG4 treatment 84 . Moreover, HLMVECs were treated with plasma from septic patients to induce an inflammatory response and rhPRG4 significantly lowered IL-6 protein levels in almost 75% of the patients 84 . Because lubricin biosynthesis is downregulated by various cytokines including IL-1β and TNF-alpha, it is possible lubricin could not only serve as a therapeutic but also as a biomarker of inflammation and infections, including sepsis 61 .…”