**2.2. Anti-citrullinated proteins antibodies (ACPA) in RA**

Some other names used to describe ACPA are anti-keratin, antiperinuclear factor antibodies, antifilaggrin antibodies, or anti-Sa [39]. ACPA have been associated with human pathology [40] as well as preclinical disease [16, 18, 41]. Latest ELISA assays, exhibited higher specificity (∼98%) and sensitivity between 40 and 76% (depending on disease stage) [42]. Recently proved that there is a potential association of ACPA with conditions like psoriatic arthritis [43], periodontitis [44], and osteoarthritis [45]. The key difference between ELISA assays was in the antigens used to detect ACPA. Thus, the diagnostic value of ACAP was established by demonstrating the significance of using appropriate citrullinated peptide [39, 46, 47]. Consequentially, a highly sensitive noncommercial ELISA, based on protein targets identified as reactive with ACPA in synovial tissue such as alpha and beta fibrinogen was therefore developed [48]. The positivity of ACPA for one or both to these two citrullinated peptides covered all reactivity in RA sera [49].

Citrullinated peptides are generated in response to a posttranslational modification mediated by peptidyl-arginine deiminase (PAD) enzymes. Multiple antibody isotypes including IgG, IgA, and IgM directed against these citrullinated peptides are detected in RA [50]. Citrullinated proteins are present in the synovial fluid of inflamed RA joints, exhibiting that ACPA could bind to these antigens in the joint and possibly increase local inflammation [51]. A protein that is commonly targeted by ACPA is Vimentin. In collagen-induced arthritis, mouse models passive transfer of ACPA cannot cause synovitis, although it can worsen preexistent synovitis [52]. Therefore, it is suggested that multiple events are necessary for the development of RA.

ACPA causes inflammation via binding to Fc receptors or complement activation. Autoantibodies are usually glycoproteins that means both Fc and Fab region of the antibody bind to the carbohydrate chains, which is essential for immune effector functions. Compared to IgG antibodies, the Fc region of ACPA has a lower level of galactosylation and sialylation against recall antigens [53]. The decreased sialylation of IgG in immune complexes can drive osteoclastogenesis, both *in vitro* and *in vivo*, through altered FcγR signaling. Moreover, it has been found that RA patients with low levels of ACPA-IgG Fc sialylation displayed lower bone volumes and trabecula numbers [54]. Thus, disease pathophysiology could be influenced by the specific Fc glycan signature of ACPA.

### **2.3. Autoantibodies against type II collagen**

Type II collagen (CII) are abundantly present in joint cartilage [55]. Native CII protein consists of a triple-helix structure containing three identical α chains. The collagen fibrils contribute to cartilage integrity by resisting stretching forces caused by hydrophilic proteoglycan molecules in extracellular matrix of articular cartilage, [56]. The degradation of CII leads to the degeneration of cartilage and consequent loss of function in RA patients [57]. Denaturation of CII also causes separation of α chains and loss of antigenic sites (epitopes) present in the molecule which are altered due to the disruption of its three dimensional structure [58]. Autoantibodies to both, native and denatured CII, have been reported in RA [59–62]. Varying levels of anti-CII antibodies were detected in the same patient at different times and also between patients, suggesting that these antibodies might be associated with specific events during arthritis development.

Increased anti-CII antibody levels may degrade CII molecules leading to acute inflammation which is mediated by anti-CII antibody containing surface-bound immune complexes (ICs) [60], which activates complement system activation and enhanced the production of proinflammatory cytokines (TNFα, IL-1β and IL-8) [63], which leads to the inflammation of the joints and hence cartilage damage. Antibodies have been detected against major CII epitopes at the site of inflammation, serum and synovial fluid samples from RA patients, supporting the concept of an increased local immune response to CII in the joints [61].

CII is found to be arthritogenic in animals and an injection of native CII in adjuvant induces collagen-induced arthritis (CIA) characterized by antibodies to CII and inflammatory polyarthritis [64]. Variability in expression of arthritis is linked to the expression of particular class II major histocompatibility (MHC) alleles [65] and also depends on an intact immune system. For example, B cell deficient animals [66] or complement deficient ones [67] are protected. Moreover, monoclonal antibodies (mAb) to CII derived from mice with CIA can induce collagen antibody-induced arthritis (CAIA) in naïve mice. CAIA is a condition characterized by inflammation, formation of pannus and erosions of bone similar to that observed in RA [68]. This model disease does not require the help pf T cells and has proven to be an informative model to better understand how antibodies lead to development of arthritis. Not only is arthritis not MHC-restricted, but it can be induced in most strains of mice and represents a model of the effector arm of CIA. However, it depends on the specificity of the antibodies used. A triplet of arginine-glycine-hydrophobic acids, is a common amino acid motif, shared by these arthritogenic mAb, which recognize epitopes on CII. These map to surface-exposed regions on the collagen fibrils that are accessible for antibody binding [68]. These epitopes are conserved, and are also recognized by antibodies from rats [69–71] and from humans with RA [70, 72]. Amino acid arginine molecules are present on the surface of the major epitopes on the collagen fibrils can also become citrullinated [73] and mAb reactive with these citrullinated epitopes may be arthritogenic themselves, or induce more severe arthritis when injected with subclinical doses of anti-CII [74]. Antibodies to major CII epitopes could be useful as markers for the biomonitoring of joint destruction in some patients.
