**5. The role of citrullination**

The mechanism underlying the switch from DAMPs that initiate controlled tissue repair, to those that mediate chronic, uncontrolled inflammation is still unclear, but recent evidence suggests that the process of citrullination may play a key role in this event. Citrullination is a post-translational event whereby peptidyl arginine deaminase enzymes convert arginine residues on susceptible molecules to citrulline.

Fibrinogen, a DAMP and TLR4 agonist, has been shown to be citrullinated in the RA joint (Sebbag et al., 2006; van Beers et al., 2010), which potentiates its activation of TLR4 and enhances its activity within immune complexes (Sokolove et al., 2011). Moreover, immunization with citrullinated, but not native, fibrinogen induces a T cell dependent murine arthritis (Ho et al., 2010; Yue et al., 2010). In addition, citrullination modifies the antigenicty of fibrinogen by creating new epitopes preferentially recognized by HLA DR (James et al., 2010). Whilst the accumulation of citrullinated proteins is a hallmark of many autoimmune diseases, unique to RA is the loss of tolerance to these epitopes. Anticitrullinated protein antibodies (ACPAs) are present in ~65% of RA patients but are found in only <1% of individuals who do not have RA. Appearing before any evident symptoms, they correlate with poor prognosis; progressive joint destruction and low remission (Scott et al., 2010). Largely used diagnostically, emerging evidence suggests that ACPAs actively contribute to disease pathogenesis as their adoptive transfer enhances experimental murine arthritis (Kuhn et al., 2006; Uysal et al., 2009). Investigation of which DAMPs are

(Ho et al., 2010; Yue

(Gondokaryono et al.,

(Pullerits et al., 2003)

(Midwood et al.,

(Midwood et al.,

(Andersson et al.,

(Rice et al., 2008)

(Kakimoto et al.,

et al., 2010)

2007)

2009)

2009)

2004)

1995)

**DAMP Effect of intra-articular administration in mice Reference** 

Fibrinogen Induced joint inflammation that is inhibited by

HMGB1 Induced synovial inflammation, some pannus

Tenascin-C Induced TLR4 dependent joint inflammation and

**Effect of targeted deletion in mice**  Tenas*c*in-C Protected from persistent synovial inflammation,

HMBG1 Polyclonal antibodies or the DNA binding box A

HSP90 SNX-7081 (inhibitor) ameliorated disease, joints

in collagen induced arthritis

disease in collagen induced arthritis

joint erosion and tissue destruction in antigen

domain reduced severity of established joint

returned to normal in collagen induced arthritis

Table 4. Evidence supporting the role of specific DAMPs in driving inflammation in RA

The mechanism underlying the switch from DAMPs that initiate controlled tissue repair, to those that mediate chronic, uncontrolled inflammation is still unclear, but recent evidence suggests that the process of citrullination may play a key role in this event. Citrullination is a post-translational event whereby peptidyl arginine deaminase enzymes convert arginine

Fibrinogen, a DAMP and TLR4 agonist, has been shown to be citrullinated in the RA joint (Sebbag et al., 2006; van Beers et al., 2010), which potentiates its activation of TLR4 and enhances its activity within immune complexes (Sokolove et al., 2011). Moreover, immunization with citrullinated, but not native, fibrinogen induces a T cell dependent murine arthritis (Ho et al., 2010; Yue et al., 2010). In addition, citrullination modifies the antigenicty of fibrinogen by creating new epitopes preferentially recognized by HLA DR (James et al., 2010). Whilst the accumulation of citrullinated proteins is a hallmark of many autoimmune diseases, unique to RA is the loss of tolerance to these epitopes. Anticitrullinated protein antibodies (ACPAs) are present in ~65% of RA patients but are found in only <1% of individuals who do not have RA. Appearing before any evident symptoms, they correlate with poor prognosis; progressive joint destruction and low remission (Scott et al., 2010). Largely used diagnostically, emerging evidence suggests that ACPAs actively contribute to disease pathogenesis as their adoptive transfer enhances experimental murine arthritis (Kuhn et al., 2006; Uysal et al., 2009). Investigation of which DAMPs are

ONO-5046 (inhibitor) reduced incidence and severity of disease, ablated cartilage destruction

**Effect of blockade in murine disease model** 

swelling, cytokine synthesis, synovial

FNEDA Induced TLR4 dependent transient ankle

CTLA4-Ig

formation

inflammation

tissue erosion

induced arthritis

Neutrophil elastase

**5. The role of citrullination** 

residues on susceptible molecules to citrulline.

that drive autoimmunity; thereby shedding light on RA disease pathogenesis. In summary therefore, the presence of DAMPs within the RA synovia or their elevated levels within the peripheral circulation of patients, implicates their involvement in disease pathology. This hypothesis is now underscored by evidence in animal models of RA that includes the effects on disease after targeted deletion of DAMPs, the induction of disease by administration of DAMPS as well as the manipulation of DAMP function / expression. However, whilst targeted deletion of a particular DAMP is possible in the mouse it is clearly not a viable therapeutic option in the clinic. A more achievable goal is to target the receptors or signalling pathways involved in DAMP activity, an approach that requires a detailed understanding of both.
