Monomeric C-reactive protein as a therapeutic target in inflammatory diseases

C-reactive protein (CRP) is produced by the liver in response to inflammation. The stimulation of CRP synthesis mainly occurs in response to pro-inflammatory cytokines, most notably IL-6 and to a lesser degree IL-1 and tumour necrosis alpha (TNF-α).^{1 2} Therefore, CRP is used as an inflammation marker for clinical purposes. CRP is a dynamic protein, has several conformational statuses, native pentameric CRP forms (pCRP), pentameric symmetrical forms (pCRP*), monomeric CRP forms (mCRP).^{1 2} pCRP can undergo dissociation into mCRP through pCRP*. pCRP binds to the cell surface via interaction with phosphocholine. Cell-bound pCRP undergoes a conformational change that leads to its dissociation to mCRP. For clinical purposes, pCRP is typically quantified rather than mCRP.^{1 2} pCRP does not have inflammatory activity.³ On the other hand, mCRP has pro-inflammatory function through the activation of endothelial cells, leukocytes and platelets via NF-κβ-regulated translation of proteins (e.g. IL-6, IL-8, MCP1), in addition to the complement pathway.^{1 2} This difference in function can be explained by the two isoforms binding to differing types of Fcgamma (Fcγ)-receptor involved in the signalling process. The mCRP uses the low-affinity immune complex binding immunoglobulin G (IgG) receptor called FcγRIIIb (CD16b) on neutrophils and FcγRIIIa (CD16a) on monocytes, while pCRP binds to the low-affinity IgG receptor FcγRIIa (CD32) (3 Khreiss T). Therefore, mCRP is a potential therapeutic target for the treatment of inflammatory diseases. In our previous study, we generated the anti-mCRP mAb to develop an anti-mCRP therapy. Clone 3C specifically recognised mCRP but not pCRP.⁴ Anti-mCRP mAb 3C suppressed arthritis and lupus nephritis in mouse model.⁴ In this study, we evaluated whether anti-mCRP mAb 3C attenuates aortitis in a Lactobacillus casei cell wall extract (LCWE)-induced Kawasaki disease-like vasculitis mouse model, a well-established animal model for vasculitis.⁵,⁷ In the LCWE mouse model, a single intraperitoneal injection of LCWE is sufficient to induce aortic root inflammation and/or coronary arteritis.⁵,⁷

We found that anti-mCRP mAb 3C attenuated aortitis in a LCWE-induced Kawasaki disease-like vasculitis mouse model. Briefly, mice were intraperitoneally injected with 1000 µg of LCWE (n=14) or PBS (n=7) to induce aortitis. After 2 weeks, LCWE-treated mice were divided into two groups and injected intraperitoneally with IgG (n=7) or anti-mCRP mAb (3C) (n=7) weekly for 4 weeks. Mice with LCWE-induced vasculitis exhibited greater infiltration of inflammatory cells into the aortic root compared with PBS-treated mice. Treatment with anti-mCRP mAb attenuated aortitis (p<0.05) (figure 1). Representative microphotographs of LCWE-induced aortitis are also shown in figure 1. Treatment with IgG did not affect the aortitis (online supplemental figure). Experimental methods were described in detail in online supplemental material.

Figure 1. Effect of mAb-3C on LCWE-induced vasculitis. Mice were injected intraperitoneally with PBS (n=7) or LCWE (n=14). After 2 weeks, LCWE-treated mice were further divided into two groups: no treatment (LCWE+IgG, n=7) and mAb-3C (LCWE+mAb-3C, n=7). All mice were sacrificed 14 weeks after LCWE or PBS injection. Representative photographs showing the cross-section of an aortic lesion with H&E. Semiquantitative evaluation of the aorta inflammatory score in mice injected with PBS (n=7), LCWE+IgG (n=7), or LCWE+mAb-3C (n=7). Values are expressed as the mean±SD *p<0.05. LVWE, Lactobacillus casei cell wall extract.

In this study, we found that anti-mCRP mAb 3C attenuated aortitis in an LCWE-induced vasculitis mouse model. To directly target the mCRP may have therapeutic potential. However, we have not performed IHC with a marker for infiltrating immune cells (eg, CD45) in the tissue specimens to evaluate the severity of vessel inflammation yet. A more objective evaluation of the effects of the treatment is needed to strengthen the data. Also, in this study, we have not performed the control experiments using anti-pentameric CRP antibodies. Therefore, it remains a limitation that whether only the monomeric isoform of CRP can be attributed to inflammation such as vasculitis. Some experiments using anti-pentameric CRP antibodies may be helpful for understanding the roles of mCRP and/or pCRP.⁸ Data are preliminary and their findings need to be confirmed in larger studies.