SYSTEMIC SCLEROSIS

Targeting the TLR4–MD2 axis in systemic sclerosis

Inappropriate activation of Toll-like receptor 4 (TLR4) on resident fibroblasts, through the binding of damage-associated molecular patterns, is a potential driver of fibrosis in systemic sclerosis. New evidence suggests that targeting fibroblast-specific TLR4 or an accessory molecule MD2 could have therapeutic value.

Refers to Bhattacharyya, S. et al. TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung. JCI Insight 3, e98850 (2018).

Systemic sclerosis (SSc) is an autoimmune idiopathic connective tissue disease characterized by chronic inflammation, vascular defects and fibrosis. Among all the autoimmune rheumatic diseases, SSc has the highest all-cause mortality1. Persistent fibrosis is a hallmark of the disease and is caused by excessive production and accumulation of collagens and other extracellular matrix (ECM) molecules, such as fibronectin, by activated fibroblasts. Inappropriate activation of Toll-like receptors (TLRs) is associated with fibrosis in SSc and increased numbers of TLR molecules are present in patients with SSc2,3. Findings from a new study by Bhattacharyya et al.4 have added considerably to our understanding of the role of TLR4 in the disease and how to target this receptor.

TLR4 is expressed on fibroblasts and seems to be important in the pathogenesis of SSc. Although lipopolysaccharide (LPS) was the first ligand described for TLR4, this receptor has since been shown to bind to other, highly diverse molecules that seem to have no structural similarities to LPS. These molecules are so-called ‘danger signals’ or damage-associated molecular patterns (DAMPs) and are endogenous molecules that bind to TLR4 and instigate signalling that culminates in inflammation via the activation of distinct inflammatory genes. Levels of these endogenous molecules are increased in SSc and these DAMPs seem to bind to TLR4 and promote fibrosis (Fig. 1). Evidence for the role of TLR4 in SSc comes from the fact that Tlr4-global knockout mice develop less severe experimental fibrosis than wild-type controls5 and the fact that mice lacking the gene Tnc, which encodes the TLR4 ligand tenascin C, are also protected from chronic fibrosis6. However, pharmacological targeting of TLR4 has not yet been fully realized.

Fig. 1: Targeting TLR4–MD2 signalling in fibroblast activation.
figure1

Damage-associated molecular patterns (DAMPs) such as tenascin C signal via Toll-like receptor 4 (TLR4), which requires the accessory protein MD2 for signalling to occur, thereby activating various downstream signalling molecules that ultimately lead to the differentiation of fibroblasts to myofibroblasts and to fibrosis. Inhibiting MD2 using T5342126 attenuated fibrosis mediated by TLR4 stimulation. TRAM, TRIF-related adapter molecule; TRIF, TIR domain-containing adapter protein inducing interferon-β.

Molecular profiling of SSc skin biopsy samples has revealed at least four distinct molecular subsets of SSc: an ‘inflammatory intrinsic subset’, a ‘fibroproliferative’ subset, a ‘limited’ subset and a ‘normal-like’ subset, in which the skin resembles that of healthy individuals7. In their study, Bhattacharyya et al.4 first interrogated data from a meta-analysis of skin biopsy samples from 80 patients with SSc (70 with diffuse cutaneous SSc and 10 with limited cutaneous SSc) for these four distinct SSc subsets. To identify TLR4-responsive genes, they characterized the set of genes that are upregulated following ectopic expression of constitutively active TLR4 in primary skin fibroblasts (referred to as the TLR4 gene signature); the results of gene expression microarrays revealed a set of 332 upregulated genes that could be mapped to the gene ontology terms cytokines, cell migration, wound healing and ECM organization. Compared with the other SSc subsets, skin biopsy samples from patients with the inflammatory intrinsic subset of SSc were enriched for this TLR4 gene signature4. The authors also demonstrated that MD2 was increased in the ‘inflammatory’ cohort compared with other cohorts and that TLR4 and MD2 expression correlated with each other4. MD2 is an accessory protein that forms a TLR4–MD2 complex that is indispensable for LPS-mediated signalling8. Prompted by these findings and knowing that TLR4 seems to be important in SSc, the authors set out to find inhibitors for MD2.

Using in silico screening, Bhattacharyya et al.4 identified a new inhibitor of TLR4: a β-amino alcohol derivative called T5342126. This molecule is predicted to compete with MD2 for binding to TLR4 and thus to prevent the formation of a competent TLR4 signalling complex. Initial testing of T5342126 on in vitro cultures of healthy skin fibroblasts revealed that the compound had minimal cellular toxicity and blocked in vitro inflammation mediated by ultra-pure LPS (as measured by levels of pro-inflammatory cytokines); importantly the blocking of pro-inflammatory cytokines by T5342126 was not seen when the in vitro cultures were stimulated with a specific TLR2 agonist that does not require MD2 for downstream signalling4, suggesting that T5342126 is specific for MD2.

In a bleomycin-induced model of fibrosis (a standard inflammation-driven model of skin fibrosis), T5342126 reduced skin fibrosis by 50%, which was accompanied by a reduction in the number of myofibroblasts in the skin4. Treatment with the MD2 inhibitor also reduced collagen and IL-6 gene expression compared with bleomycin treatment alone. In animals with established fibrosis, treatment with T5342126 reduced dermal thickness and collagen gene expression, indicating a ‘reversal’ of fibrosis in an established fibrosis model4. This important finding suggests that targeting the TLR4–MD2 axis might have therapeutic value in SSc.

Importantly, Bhattacharyya et al.4 showed that the MD2 inhibitor reduced the induction of collagen expression in isolated human dermal fibroblasts cultured with tenascin C or fibronectin containing extra domain A5, DAMPs that are critical in the differentiation of fibroblasts to myofibroblasts6,9 and that are implicated in SSc pathogenesis. These DAMPs are particularly intriguing in the context of fibrosis as they are not normally expressed in the adult body during homeostasis. For instance, expression of tenascin C is restricted postnatally, and it is only expressed upon the occurrence of tissue damage. This observation suggests that when tissue damage occurs in SSc, increased secretion of tenascin C helps to repair the damage. Failure to terminate this wound-healing response is speculated to underpin the disease. In other words, a programmed wound healing response that is sensed by TLR4 and mediated through DAMPs is beneficial, but failure to terminate this response leads to SSc. Whether the novel MD2 inhibitor tested here can diminish signalling by other DAMPs remains an open question.

Previously, monocytes and/or macrophages were thought to be the predominant cell type(s) mediating pro-fibrotic effects through TLR4 signalling9. Given that only global Tlr4-knockout mice have previously been used, it has been difficult to delineate the precise role of monocytes and macrophages as opposed to resident fibroblasts in this context. The finding that specific genetic ablation of TLR4 in fibroblasts resulted in attenuated fibrosis4 suggests that fibroblasts are the primary cell type responsible for TLR4-mediated fibrosis.

“targeting the TLR4–MD2 axis might have therapeutic value in SSc”

Interestingly, the small-molecule TLR4 antagonist eritoran failed to demonstrate efficacy in a phase III trial for severe sepsis10, perhaps owing to the biological complexity of sepsis (that is, targeting one specific pathway is insufficient, owing to redundancy). However, using a small-molecule inhibitor of MD2 might yield clinical benefits in SSc owing to the unique reliance of TLR4-mediated fibroblast activation on MD2, regardless of the ligand used, be it LPS or a DAMP (Fig. 1). Furthermore, the use of a specific MD2–TLR4 inhibitor that targets a protein–protein interaction to abolish TLR-mediated fibrosis is a unique approach, as typically, protein–protein interactions are considered difficult drug targets owing to the difficulty of inhibiting multiple interactions with small molecular weight drugs (<500 Da).

Given the fact that the novel MD2 inhibitor identified by Bhattacharyya et al.4 not only blocked but reversed fibrosis in relevant animal models, and the association with the TLR4 gene signature in a specific subset of patients (the inflammatory intrinsic subset), it would seem that targeting TLR4 signalling pathways in these patients could result in the greatest clinical benefit. Disease heterogeneity is notorious in SSc and identifying the appropriate patients for precision medicine approaches seems likely to hold great promise. A strategy such as this, in which patients with SSc can be profiled at the molecular level to guide physicians towards the appropriate treatment, is a welcome addition. Moreover, further lead optimization of the MD2 inhibitor T5342126 might improve its efficacy.

References

  1. 1.

    Elhai, M. et al. Trends in mortality in patients with systemic sclerosis over 40 years: a systematic review and meta-analysis of cohort studies. Rheumatology 51, 1017–1026 (2012).

    Article  Google Scholar 

  2. 2.

    O’Reilly, S. Toll like receptors in systemic sclerosis: an emerging target. Immunol. Lett. 195, 2–8 (2018).

    Article  Google Scholar 

  3. 3.

    Fullard, N. & O’Reilly, S. Role of innate immune system in systemic sclerosis. Sem. Immunopathol. 37, 511–517 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Bhattacharyya, S. et al. TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung. JCI Insight 3, e98850 (2018).

    Article  Google Scholar 

  5. 5.

    Bhattacharyya, S. et al. Toll-like receptor 4 signaling augments transforming growth factor-β responses: a novel mechanism for maintaining and amplifying fibrosis in scleroderma. Am. J. Pathol. 182, 192–205 (2013).

    CAS  Article  Google Scholar 

  6. 6.

    Bhattacharyya, S. et al. Tenascin-C drives persistence of organ fibrosis. Nat. Commun. 7, 11703 (2016).

    CAS  Article  Google Scholar 

  7. 7.

    Milano, A. et al. Molecular subsets in the gene expression signatures of scleroderma skin. PLoS ONE 3, e2696 (2008).

    Article  Google Scholar 

  8. 8.

    Park, B. S. et al. The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex. Nature 458, 1191 (2009).

    CAS  Article  Google Scholar 

  9. 9.

    Dowson, C. et al. Innate immunity in systemic sclerosis. Curr. Rheumatol. Rep. 19, 2 (2017).

    Article  Google Scholar 

  10. 10.

    Opal, S. M. et al. Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: The ACCESS randomized trial. JAMA 309, 1154–1162 (2013).

    CAS  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Steven O’Reilly.

Ethics declarations

Competing interests

The authors declare no competing interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

O’Reilly, S., van Laar, J.M. Targeting the TLR4–MD2 axis in systemic sclerosis. Nat Rev Rheumatol 14, 564–566 (2018). https://doi.org/10.1038/s41584-018-0077-6

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing