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Conditional signaling by Toll-like receptor 4

Gregory J. Brunn^*,, Marlo K. Bungum^*, Geoffrey B. Johnson^*, and Jeffrey L. Platt^*,,,||,1

^* Transplantation Biology and Departments of
Pharmacology and Experimental Therapeutics,
Immunology,
Surgery, and
^|| Pediatrics, Mayo Clinic, Rochester, Minnesota, USA

¹ Correspondence: Transplantation Biology, Mayo Clinic, 200 First St. SW, 2-66 Medical Sciences Building, Rochester, Minnesota 55905, USA. Email: platt.jeffrey{at}mayo.edu

SPECIFIC AIMS

Resistance to infection is thought to be triggered when microorganisms or their products stimulate Toll-like receptors (TLR), a family of proteins homologous to drosophila Toll. As one example, TLR4 is stimulated by subnanomolar concentrations of lipopolysaccharide (LPS), a component of the cell wall of Gram (–) bacteria. While LPS clearly can stimulate TLR4, endogenous substances such as heparan sulfate were also recently found to be effective agonists. If TLR4 responds to normal constituents of extracellular matrix, such as heparan sulfate and hyaluronic acid, we questioned how leukocytes, endothelial cells, and other cells that express TLR4 could maintain quiescence in microenvironments rich in these endogenous activators of the receptor. To determine how cells expressing the TLR4 receptor complex function in microenvironments rich in endogenous agonists, we developed in vitro and in vivo model systems that would allow us to measure TLR4 activation. We found that under quiescent conditions, intact extracellular matrix inhibits TLR4 activation and that degradation of extracellular matrix relieves inhibition of TLR4 and generates activators of the receptor, including components of extracellular matrix containing heparan sulfate.

PRINCIPAL FINDINGS

1. Cells expressing TLR4 and growing in extracellular matrix do not respond to TLR4 activators
HEK 293 cells were stably transfected with components of the TLR4 complex (HEK/TLR4(+) cells) and transiently transfected with a NF-B-luciferase reporter gene to monitor TLR4 activation. The cells responded both to LPS and heparan sulfate. However, when HEK/TLR4(+) cells were cultured on plates coated with extracellular matrix rich in heparan sulfate proteoglycans, cells exhibited a low baseline level of NF-B-luciferase activity, similar to NF-B activity of HEK/TLR4(+) cells cultured in plates coated with fibronectin (which does not stimulate TLR4) (Fig. 1A ). HEK/TLR4(+) cells cultured on extracellular matrix responded only minimally to stimulation with heparan sulfate (Fig. 1A ) or with LPS (Fig. 1B ). Expression of TLR4/MD2 was not different in cells cultured on extracellular matrix or fibronectin (Fig. 1C ). This suggested that TLR4 function was suppressed in cells exposed to extracellular matrix. Inhibition of TLR4 by extracellular matrix was not unique to HEK/TLR4(+) cells as RAW 264.7 macrophages, which naturally express TLR4 complexes, exhibit a blunted response to heparan sulfate or LPS when cultured in extracellular matrix (Fig. 1D, E ).

View larger version (30K): [in this window] [in a new window]   Figure 1. TLR4 signaling is conditioned by extracellular matrix. The impact of extracellular matrix, rich in heparan sulfate proteoglycans, on TLR4 activation was tested in cells cultured in wells coated with extracellular matrix. A, B) Inhibition of NF-B activation by extracellular matrix in HEK/TLR4(+) cells. HEK/TLR4(+) cells were cultured in wells coated with extracellular matrix (ECM) or fibronectin (FN) and transfected with NF-B- and control-luciferase reporter genes. TLR4 signaling in response to increasing amounts of heparan sulfate (A) or LPS (B) was measured 6 h after stimulation. C) Effect of extracellular matrix on cell surface expression of TLR4/MD2. HEK/TLR4(+) cells were cultured in wells coated with fibronectin (FN) or extracellular matrix (ECM). Cells were tested for cell surface expression of the TLR4/MD2 complex by flow cytometry using monoclonal antibodies specific for that complex. As a control, HEK cells that do not express TLR4 were tested using the same monoclonal antibody (No TLR4). TLR4/MD2 expression did not differ in cells cultured in wells coated with extracellular matrix from those cultured in wells coated with FN. D, E) Inhibition of p38 MAP Kinase activation by extracellular matrix in RAW 264 cells. D) RAW 264.7 murine macrophages were cultured in wells coated with ECM or FN. The cells were stimulated with heparan sulfate (left) or LPS (right) for the indicated time (minutes), after which the cells were lysed and activated p38 MAPK (phospho p38) was measured by immunoblotting. E) Densitometry scanning of immunoblot films from panel C indicates that activation of p38 phosphorylation, relative to total p38, was 60% lower in cells cultured in the presence of ECM and stimulated with heparan sulfate. Similar results were obtained for cells stimulated with LPS (not shown). The data shown are representative of 3 separate experiments and indicate that activation of TLR4 signaling is inhibited by extracellular matrix.

2. Degradation of extracellular matrix releases inhibition on TLR4 signaling
If extracellular matrix inhibits TLR4 activation, an important question is how that inhibition is relieved so that immunity and resistance to infection can be mounted. We hypothesized that whereas signaling through TLR4 is constitutively constrained in unperturbed tissues, constraint might be relieved if extracellular matrix is cleaved by proteases. To test this, we asked whether elastase, a protease released by phagocytes, relieves inhibition of TLR4 complexes conferred by extracellular matrix. Consistent with that concept, HEK/TLR4(+) cells in extracellular matrix treated with low concentrations (0.1 U/mL) of elastase responded vigorously to heparan sulfate. Elastase by itself did not stimulate HEK/TLR4(+) cells, but HEK/TLR4(+) cells on extracellular matrix treated with a higher concentration of elastase (0.3 U/mL) responded even without added heparan sulfate. These results indicate that digestion of extracellular matrix relieves constraint on TLR4 activation and generates endogenous agonists for the receptor.

3. Suppression of TLR4 in vivo
To test the possibility that elastase activates TLR4 responses in living tissues, we studied the properties of cells in the spleens of mice injected with TLR4 activators with or without elastase. Injection of a 10 ng of LPS or small amounts (0.01 U) of elastase only modestly increased expression of CD86 (a protein expressed in response to TLR4 signaling) in splenocytes in intact spleens (Fig. 2A ). Similar increases in CD86 were observed in spleens injected with 10 µg of heparan sulfate (Fig. 2A ). Injection of a smaller amount of LPS (1 ng) did not increase expression of CD86 (not shown) but injection of 1 ng LPS along with a small amount of elastase, or injection of increased amounts (0.1U) of elastase alone, profoundly increased expression of CD86 (Fig. 2A, B ). These changes in CD86 expression induced by elastase required TLR4 function, as the increases in CD86 were not observed when elastase, heparan sulfate, or LPS were injected into spleens of mice lacking TLR4 function (Fig. 2) . These results show that extracellular matrix suppresses TLR4 signaling in response to exogenous activators in intact tissues and that cleavage of extracellular matrix facilitates TLR4 responses to exogenous agonists and triggers TLR4 signaling by generating endogenous agonists.

View larger version (50K): [in this window] [in a new window]   Figure 2. Elastase relieves constraint on TLR4 signaling in vivo. Whether TLR4 signaling is constitutively constrained and whether elastase releases that constraint in vivo was tested. Spleens of wild-type mice and mice lacking TLR4 function were injected with LPS with or without a small amount of elastase to relieve constraint on TLR4 signaling. Alternatively, a higher amount of elastase was injected alone to generate endogenous TLR4 activator(s). Expression of CD86 was assayed in the spleen by immunopathology 12 h after administration of elastase. A) Spleens of C57BL/10SnJ mice with wild-type TLR4 (SnJ) were injected with 10 ng LPS, 10 µg of heparan sulfate (HS), 0.01 U elastase (elastase, low), or 0.01 U elastase + 1 ng LPS (elastase+LPS). Spleens of control C57BL/10 ScN mice that lack TLR4 function (ScN) were injected with 10 ng LPS 10 µg of HS or 300 ng CpG DNA. Control mice were injected with vehicle alone, 50 µL of PBS. B) Spleens of mice with wild-type TLR4 (SnJ) or that lack TLR4 function (ScN) were injected with 0.1 U of elastase (elastase, high) or inactive elastase (elastase, boiled). Spleens from normal (SnJ) mice had modestly increased expression of CD86 in response to LPS, heparan sulfate, or low-dose elastase. However, low-dose elastase enhanced expression of CD86 in response to LPS. Higher dose elastase (0.1 U) induced profound expression of CD86. Expression of CD86 was absent in spleens from mice lacking TLR4 (ScN), which responded only to the TLR9 activator CpG DNA. Results show that extracellular matrix limits TLR4 signaling in response to LPS and that cleavage of extracellular matrix both facilitates TLR4 responses to exogenous activators and triggers TLR4 responses by generating endogenous activators in vivo.

4. Degradation of extracellular matrix generates endogenous activators of TLR4
We asked whether one endogenous TLR4 agonist generated by elastase might be heparan sulfate. We tested whether constituents of extracellular matrix liberated by elastase activate HEK/TLR4(+) cells and whether activation is abolished by selective digestion of heparan sulfate. Fragments of extracellular matrix released by elastase activated HEK/TLR4(+) cells to the same extent as direct treatment of the cells on extracellular matrix with elastase or treatment of control cells with a TLR4 agonist. Incubation of extracellular matrix releasate with heparanase decreased the heparan sulfate content by 50% and the ability of the releasate to activate HEK/TLR4(+) cells by 50%. These results demonstrate that heparan sulfate is the major agonist generated when elastase acts on extracellular matrix.

CONCLUSIONS AND SIGNIFICANCE

Our findings should change the concept of how TLR4 functions in health and disease. We show that under quiescent conditions, TLR4 function is strongly constrained by extracellular matrix found in normal tissue. Although large amounts of LPS may incite immunity and the sepsis syndrome, this inhibition may avert unwanted activation of innate immunity and the sepsis syndrome by physiologic or innocuous events. On the other hand, release of proteases (as occurs in infection or tissue injury) degrades extracellular matrix, relieving constraint on TLR4 function and allowing agonists to stimulate the receptor. If sufficient degradation of matrix occurs, endogenous agonists, particularly heparan sulfate, can stimulate TLR4. Thus, in normal tissues the first step in innate and adaptive immunity may not be stimulation of TLR4 as previously thought, but rather the release of TLR4 from constitutive inhibition by extracellular matrix. Since nearly every type of tissue injury, infection, or inflammation causes degradation of heparan sulfate proteoglycans, the TLR4-heparan sulfate interaction is uniquely poised to monitor threats to well-being from exogenous or endogenous origin.

View larger version (15K): [in this window] [in a new window]   Figure 3. TLR4 signaling in normal tissues and in disease. A) Normal Tissues. Cells in tissues with intact extracellular matrix (intact ECM) do not respond to small amounts of TLR4 agonist, preventing inadvertent systemic responses. Large amounts of agonist may overcome the activation threshold and allow cell activation to occur (asterisk). B) Injured tissue. Partial degradation of ECM by proteases, as in injury or infection, lowers the threshold for activation of TLR4. This allows small amounts of LPS or other agonists to stimulate the receptor. C) Massive injury to tissue. Following severe trauma, pancreatitis, or other situations, degradation of ECM may proceed to a degree where the activation threshold is eliminated and TLR4 agonists endogenous to ECM activate signals, and no other agonist is required.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3211fje;

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