P247 Novel therapeutic peptides targeting CD206 modulate ATP-induced release of inflammatory and B cell activating factors in lupus macrophages
Authors: Shah Shivanee , Smillie Robert , Abdi Bahja Ahmed , Stratton Richard J 
Published: April 2022

Background/Aims

Whilst plasmacytoid dendritic cells, B-cells and damaged tissue resident cells are established as key players in lupus pathogenesis, macrophages have received limited attention. However, in the disease microenvironment these cells might release inflammatory cytokines, thus contributing to the tissue damage. Moreover, macrophages have the potential to synthesise factors that stimulate B-cell proliferation and promote polyclonal immunoglobulin synthesis, underlying the autoantibody component. There is evidence that anti-inflammatory peptides derived from endogenous host defence proteins can modulate the macrophage activation state. Here, we measure the capacity for RP832c, one such peptide that targets cells via the CD206 receptor, to inhibit ATP-stimulated lupus macrophages.

Methods

Peripheral blood monocytes from SLE patients and healthy controls (HC) (both n = 4) were cultured with 4ng/ml of M-CSF for 7 days, in order to derive macrophages. These cells were stimulated with 2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) 0.1mM, a selective P2X7 receptor agonist, to model the effect of damaged tissue, with or without RP832c (10µM). After 24 hours media were removed for assay of inflammatory factors (IL-6), and B-cell promoting growth factors (IL-10 and BAFF) by ELISA, and cells lysed for RNA extraction, profiled by qPCR for CD206 (M2-like), CD86 (classical M1-like), relative to TBP.

Results

In SLE macrophages there was lower basal IL-6 release than in healthy controls (Mean±SEM: HC 74±44, SLE 4±0.7, P-value &lt;0.029). BzATP induced IL-6 release (basal HC 74±44 vs BzATP stimulated HC 187±63, p-value NS; basal SLE 4±0.7 vs BzATP stimulated SLE 104±46, p-value 0.037). The addition of RP832c treatment inhibted BzATP induced IL-6 (BzATP stimulated HC 187±63 vs BzATP+RP832c HC 126±45, p NS; BzATP stimulated SLE 104±46 vs BzATP+RP832c SLE 27±10, p &lt; 0.078). IL-10 release in SLE lines followed a similar pattern, induced by BzATP and suppressed by co-added RP832c whereas IL-10 release in HC lines did not change with the various treatments. Most interestingly, BAFF was detectable only in BzATP treated lupus macrophage media, and was subsequently suppressed in 3 out of 4 lines by co-addition of RP832c. In certain lupus macrophage lines, BzATP treatment led to induction of CD206, inhibited by RP832c (basal 6.84±0.29, BzATP treated 12.41±1.8, BzATP+RP832c 9.08±1.48, RP832c alone 9.11±1.48 relative expression P &lt; 0.025 for effect of BzATP, P &lt; 0.13 for effect of RP832c), whereas the RP832c treatment appeared to enhance CD86 expression in lupus (enhanced in 3 out of 4 lupus lines, 0 of 4 HC lines).

Conclusion

These data confirm macrophages as a potential source of inflammatory mediators and B cell-activating factors in lupus. These properties are enhanced by exposure to an ATP-like stimulus. In particular, lupus but not control macrophages had the capacity to synthesis BAFF when exposed to the DAMP stimulus. Moreover, we observed a trend towards suppression of pathogenic responses by RP832c, of potential benefit to patients.

Disclosure

S. Shah: None. R. Smillie: None. B. Ahmed Abdi: None. R.J. Stratton: None.