Hyaluronan fragments induce IFNβ via a novel TLR4-TRIF-TBK1-IRF3-dependent pathway

Abstract

Background: The extracellular matrix plays a critical role in insuring tissue integrity and water homeostasis. However, breakdown products of the extracellular matrix have emerged as endogenous danger signals, designed to rapidly activate the immune system against a potential pathogen breach. Type I interferons play a critical role in the immune response against viral infections. In the lungs, hylauronan (HA) exists as a high molecular weight, biologically inert extracellular matrix component that is critical for maintaining lung function. When lung tissue is injured, HA is broken down into lower molecular weight fragments that alert the immune system to the breach in tissue integrity by activating innate immune responses. HA fragments are known to induce inflammatory gene expression via TLR-MyD88-dependent pathways.

Methods: Primary peritoneal macrophages from C57BL/6 wild type, TLR4 null, TLR3 null, MyD88 null, and TRIF null mice as well as alveolar and peritoneal macrophage cell lines were stimulated with HA fragments and cytokine production was assessed by rt-PCR and ELISA. Western blot analysis for IRF3 was preformed on cell lysates from macrophages stimulate with HA fragments

Results: We demonstrate for the first time that IFNβ is induced in murine macrophages by HA fragments. We also show that HA fragments induce IFNβ using a novel pathway independent of MyD88 but dependent on TLR4 via TRIF and IRF-3.

Conclusions: Overall our findings reveal a novel signaling pathway by which hyaluronan can modulate inflammation and demonstrate the ability of hyaluronan fragments to induce the expression of type I interferons in response to tissue injury even in the absence of viral infection. This is independent of the pathway of the TLR2-MyD88 used by these matrix fragments to induce inflammatory chemokines. Thus, LMW HA may be modifying the inflammatory milieu simultaneously via several pathways.

Figure 1

HA fragments induce IFNβ mRNA and protein. (a) MH-S alveolar macrophages were stimulated with low molecular weight (LMW) HA fragments, RNA extracted and Real-Time PCR performed for IFNβ normalized to 18 s. HA fragments induce IFNβ mRNA in a dose dependent fashion with peak induction at 3 h with a dose 1000 μg/ml. (b) HA fragments induce IFNβ protein in RAW 264.7 macrophages in a dose dependent fashion with peak induction at 6 h with a dose of 500 μg/ml. (c,d) HA fragments induce IFNβ RNA and protein in peritoneal macrophage cell line in a time dependent fashion with peak induction mRNA at 3 h and protein at 6 h in macrophages; cells were stimulated with 200 μg/ml of HA fragments. (e) Thioglycollate elicited primary mouse macrophages from C57BL/6 WT mice were stimulated with HA fragments (200 μg/ml) for 6 h and IFNβ protein secretion was measured by ELISA. These figures are representative of at least 3-4 identical experiments done in triplicate. ^★ p ≤ 0.05 vs. unstimulated.

Figure 2

HA fragments induce IFNβ in a specific fashion. RAW 264.7 macrophages were stimulated with LMW HA fragments (200 μg/ml) for 3 h, RNA extracted and Real-Time PCR performed for IFNβ. HA fragments but not HMW HA, HA disaccharides, chondroitan sulfate A (CSA) or heparin induce IFNβ mRNA. This figure is representative of at least 3 identical experiments done in triplicate. ^★ p ≤ 0.01 vs. unstimulated.

Figure 3

HA fragment induction of IFNβ is TLR-4 dependent. (a) Thioglycollate elicited peritoneal macrophages from C57BL/6 WT, TLR2 receptor null or TLR4 receptor null mice were stimulated with 200 ug/mL LMW HA fragments for 3 hours; cells were collected in Trizol, RNA extracted, and cDNA was analyzed with real-time PCR. HA fragment-induced MIP1α requires TLR2; HA induction of IFNβ was independent of TLR2 but required TLR4. Data demonstrate IFNβ induction compared to WT unstimulated levels; ^★ p < 0.05 compared to WT unstimulated; # = p <0.05 compared to genotype specific unstimulated). (b) Pharmacological inhibition of TLR4 significantly decreases HA fragment activation of IFNβ. RAW 264.7 macrophages were prestimulated with 1 ug/mL CLI-095 for thirty minutes, prior to HA fragments (200 ug/ml) or LPS (100 ng/ml) for 3 hours. Cells were collected in Trizol, RNA extracted, and cDNA was analyzed with real-time PCR. Blocking the TLR4 receptor with CLI-095 significantly inhibited both HA fragment and LPS induction of IFNβ. Values shown are mean fold induction over WT unstimulated; These figures are representative of at least 3-4 identical experiments done in triplicate ^★ p < 0.05 unstimulated stimulated.

Figure 4

HA fragment induction of IFNβ is independent of MyD88, and TLR3 but dependent upon TRIF and TBK1. Thioglycollate elicited peritoneal macrophages from WT, MyD88 null, TLR3, TRIF null mice were stimulated with 200 ug/mL LMW HA fragments, LPS 10 ng/ml or p(I:C) 5 ug/ml for 3 hours; cells were collected in Trizol, RNA extracted, and cDNA was analyzed with real-time PCR (a) HA fragments require MyD88 to induce MIP1α but NOT IFNβ. (b) TLR3 is not necessary for HA induced IFNβ mRNA. (c) In the absence of TRIF, HA fragments induced significantly less IFNβ. (d,e) RAW macrophages were stimulated with 200 ug/mL HA fragments for 3 hours +/- the TBK1 inhibitor BX795; RNA was isolated and Real-time PCR performed. HA fragments and p(I:C) induced significantly less IFNβ in the presence of the TBK1 inhibitor but HA induced MIP1α was not significantly inhibited. Data reported as fold induction over WT unstimulated; data are mean of 3-7 experiments run in triplicate; * p < 0.05 HA vs US; # p < 0.05 HA vs HA + BX795; p < 0.05 p(I:C) vs p(I:C) + BX795.

Figure 5

HA fragments activate IRF-3 phosphorylation and IFNβ gene activity. (a) MH-S macrophages were stimulated with LMW HA fragments, nuclear extracts were collected and analyzed for phosphorylated IRF-3 via Western blot. (b) RAW 264.7 macrophages were transfected with human IFNβ promoter driven luciferase construct and stimulated with HA fragments for 18 hours prior to luciferase measurement. HA fragments induce the IFNβ promoter by over 2 fold. These data are representative of three identical experiments. * p < 0.02 vs unstimulated.

Figure 6

HA fragment induced inflammatory gene via distinct pathways. Schema of the pathways by which LMW HA fragments induces inflammatory genes via TLR2-MyD88-IRAK1-TRAF6-PKCζ-NK-κB or via TLR4-TRIF-TBK1-IRF3.