K02288 In addition to attenuating joint inflammation via T c

In addition to attenuating joint K02288 via T cells, Cpd43 could also decrease the severity of CIA/AIA by suppressing the function of innate leukocytes such as neutrophils and macrophages via FPRs, as has been shown in other acute inflammatory models including neutrophil/macrophage-mediated K/B × N arthritis [18,24,64]. For instance, Cpd43 may inhibit joint accumulation of neutrophils, which are pathogenic in CIA [65]. As reported [24], it is possible that FPR ligation by Cpd43 also decreases pro-inflammatory cytokine production by macrophages, which are injurious in multiple models of RA [66]. Cpd43 is likely to exert these effects via FPR2 since our results demonstrate that Cpd43-mediated attenuation of arthritis severity is fully reversed by FPR2 inhibitors. Cpd43 may also attenuate arthritis by affecting FLS. One of the major RA-promoting characteristics of FLS is their expansion and resistance to various apoptotic stimuli including TNF, which is abundant in the inflamed joints [36,67]. This was confirmed here by our results showing decreased FLS apoptosis in response to TNF. Importantly, Cpd43 markedly augmented apoptosis in the presence of TNF, suggesting that the capacity to overcome FLS resistance to TNF-induced cell death contributes to Cpd43-mediated attenuation of arthritis. Cpd43 is likely to mediate its effects on FLS apoptosis via FPR2 rather than FPR1 since FPR2, but not FPR1, is expressed by the synoviocytes [24]. Internalisation of FPR2 following ligand binding is associated with increased apoptosis in other cell types [45]. Similarly, our results suggest that Cpd43 promotes FLS apoptosis by increasing intracellular levels of FPR2. Other potential mechanisms of Cpd43-induced FLS apoptosis include FPR2-mediated regulation of apoptosis-related molecules including TRAIL and resolving D1 [36,68,69]. We further studied the effects of Cpd43 on FLS expansion by assessing their proliferation. Cpd43 increased proliferation in unstimulated, but decreased cell division in TNF-stimulated FLS. The reason for these contrasting effects is not known, but has occurred in other cells. For example, Cpd43 alone activates neutrophils, but it decreases their function in the presence of immune mediators [70]. Despite increased FLS proliferation by Cpd43 alone, FPR2 ligation by this agonist, overall, had a negative effect on FLS expansion in the absence of TNF since it increased their apoptosis about 2 times more than their proliferation. Importantly, Cpd43 decreased FLS proliferation in the presence of TNF which is more clinically relevant since FLS are exposed to an inflammatory environment in arthritic joints. As confirmed here, FLS express endogenous FPR2 ligands such as AnxA1 [24]. Here, silencing AnxA1 mimicked the effect of FPR2 inhibition on FLS proliferation, demonstrating that FPR2 activation by endogenous AnxA1 suppresses FLS proliferation, and it does so in an ERK and NFκB-dependent manner. These findings are supported by other studies showing that AnxA1/FPR2 interaction induces ERK and NFκB in other cell types [43].
Declarations of interests
Funding
Acknowledgements These studies were supported by grants from the National Health and Medical Research Council (1008991) of Australia.
Formyl peptide receptors Bacteria are mostly eliminated by the innate immune system, which recognizes pathogens through receptors known as pattern recognition receptors (PRRs). These receptors sense conserved molecular motifs known as pathogen- or danger-associated molecular patterns (PAMPs, DAMPs), which in turn activate the recruitment and activation of immune cells leading to inflammation (Fig. 1) [1]. N-formyl peptides, such as the Escherichia coli-derived fMet-Leu-Phe (fMLF), are PAMPs recognized by formyl peptide receptors (FPRs) [2]. The three FPRs identified in humans (FPR1–FPR3) are encoded by three different genes clustered on chromosome 19q13.3–19q13.4 (Fig. 1) [3]. FPRs (FPRs) are seven transmembrane G protein-coupled receptors that can be inhibited by pertussis toxin [4], [5], [6], [7], [8], indicating that the G proteins associated with these receptors belong to the Gi family [9]. FPR triggering activates various signalling pathways, including: phospholipase C (PLC)-dependent production of inositol (3,4,5)-trisphosphate (IP3), inducers of Ca2+ increase, and diacyl glycerol (DAG), which in turn activates protein kinase C (PKC); and RAS-dependent activation of the mitogen-activated protein kinases (MAP kinases) cascade [3].