PAM3 supports the generation of M2-like macrophages from lupus patient monocytes and improves disease outcome in murine lupus

Abstract

Systematic Lupus Erythematosus (SLE) is an autoimmune syndrome of unclear etiology. While T and B cell abnormalities contribute to disease pathogenesis, recent work suggests that inflammatory M1-like macrophages also play a role. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1like macrophages. This raised the possibility of PAM3 being used to normalize the M1:M2 ratio in SLE. Consistent with that possibility, monocytes from lupus patients differentiated into M2-like macrophages when treated with PAM3 in vitro. Furthermore, lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Normalization of the M2 macrophage frequency was associated with delayed disease progression, decreased autoantibody and inflammatory cytokine synthesis, reduced proteinuria and prolonged survival in NZB x NZW F1 mice. The ability of PAM3 to bias monocyte differentiation in favor of immunosuppressive macrophages may represent a novel approach to the therapy of SLE. not support inflammatory responses. 'Non-classical' monocytes express a CD14 low CD16 + phenotype and are inflammatory. An 'intermediate' or transitional monocyte population also exists that is CD14 + CD16 + and has both phagocytic and inflammatory properties [7]. In response to stimulation, monocytes differentiate into macrophages with diverse phenotypes and functions. These range from 'classical' pro-inflammatory M1-like macrophages that protect the host from infection to 'alternatively activated' M2like macrophages that are immunosuppressive, support tissue remodeling, and phagocytose apoptotic cells [8-10]. Recent studies suggest that monocytes and macrophages can contribute to the pathogenesis of SLE. Indeed, lupus flares are associated with a relative increase in the frequency of M1 vs M2 macrophages [11]. The frequency of inflammatory monocytes is also elevated in SLE patients and the concentration of serum cytokines that promote macrophage differentiation (such as CSF-1 and MCP-1) correlates with disease severity [12]. Normalizing the M1:M2 ratio by adoptive transfer of M2 macrophages is of benefit

PAM3 protects against DSS-induced colitis by altering the M2:M1 ratio

“…2C). Consistent with previous findings, the inflammation induced by DSS caused the frequency of inflammatory M1 macrophages (which were identified as CD206 − F480 + cells based on previous work) 10,23 to rise in PBS treated mice. PAM3 reversed that effect, as shown by changes in both the absolute number and ratio of M2:M1 macrophages (Fig.…”

“…Cells were washed, re-suspended in 2% BSA-PBS and analyzed using LSR Fortessa (BD Biosciences). As previously documented, M2 macrophages were identified based on their co-expression of F480 and CD206 whereas M1 macrophages did not express CD206 10,23 .…”

“…PAM3 induces murine monocytes to mature into M2 macrophages over four days in vivo {data not shown, section 4.2}. Of note, BMDM cultured with PAM3 for four days in vitro primarily generate M1 macrophages [30][31][32] .This differs from the effect of PAM3 on peripheral blood human monocytes ex vivo and murine monocytes in vivo, where this TLR agonist preferentially generates M2 rather than M1 macrophages 10,18,19 . Other TLR 2/1 agonists also induce the generation of immunosuppressive macrophages whereas ligands that interact with TLR2/6 heterodimers (such as FSL-1) do not (data not shown).…”

“…Flow Cytometry. Single cell suspensions prepared from peritoneal cells and colon were incubated with fluorochrome conjugated Abs specific for murine CD45, F480, CD206 and MHCII for 20 min in 2% BSA-PBS on ice after blocking with Fc-block (Biolegend) for 15 min and then stained 10 . Cells were washed, re-suspended in 2% BSA-PBS and analyzed using LSR Fortessa (BD Biosciences).…”

“…ELISA. ELISA was performed based on the protocol described previously 10 . Immunol 2HB-microtiter plates were coated with mouse anti IL-10 (R&D Systems) or mouse anti-IL12 (BD Biosciences) followed by blocking with 2% BSA-PBS for 2 hr.…”