Immune recognition of self nucleic acids driven by endogenous antimicrobial peptides: role in autoimmunity

Innate immune recognition of extracellular host-derived self-DNA and self-RNA is prevented by endosomal seclusion of the Toll-like receptors (TLRs) in the dendritic cells (DCs). However, in psoriasis plasmacytoid dendritic cells have been found to be able to sense self-DNA molecules in complex with the endogenous cationic antimicrobial peptide LL37, which are internalized into the endosomal compartments and thus can access TLR9. We investigated whether this endogenous peptide can also interact with extracellular self-RNA and lead to DC activation. We found that LL37 binds self-RNA as well as self-DNA going into an electrostatic interaction; forms micro-aggregates of nano-scale particles protected from enzymatic degradation and transport it into the endosomal compartments of both plasmacytoid and myeloid dendritic cells. In the plasmacytoid DCs, the self-RNA-LL37 complexes activate TLR7 and like the self-DNA-LL37 complexes, trigger the production of IFN-α in the absence of induction of maturation or production of IL-6 and TNF-α. In contrast to the self-DNA-LL37 complexes, the self-RNA-LL37 complexes are also internalized into the endosomal compartments of myeloid dendritic cells and trigger activation through TLR8, leading to the production of TNF-α and IL-6, and the maturation of the myeloid DCs. Furthermore, we found that these self nucleic acid-LL37 complexes can be found in vivo in the skin lesions of the cutaneous autoimmune disease psoriasis, where they are associated with mature mDCs in situ. On the other hand, in the systemic autoimmune disease systemic lupus erythematosus, self-DNA-LL37 complexes were found to be a constituent of the circulating immune complexes isolated from patient sera. This interaction between the endogenous peptide with the self nucleic acid molecules present in the immune complexes was found to be electrostatic and it confers resistance to enzymatic degradation of the nucleic acid molecules in the immune complexes. Moreover, autoantibodies to these endogenous peptides were found to trigger neutrophil activation and release of neutrophil extracellular traps composed of DNA, which are potential sources of the self nucleic acid-LL37 complexes present in SLE immune complexes. Our results demonstrate that the cationic antimicrobial peptide LL37 drives the innate immune recognition of self nucleic acid molecules through toll-like receptors in human dendritic cells, thus elucidating a pathway for innate sensing of host cell death. This pathway of autoreactivity was found to be pathologically relevant in human autoimmune diseases psoriasis and SLE, and thus this study provides new insights into the mechanisms autoimmune diseases.

Maintenance of phosphatidylserine asymmetry in erythroid cells during differentiation, aging, and its significance for macrophage recognition

Phosphatidylserine (PS) is distributed almost entirely in the inner leaflet of the erythrocyte membrane bilayer, and appears to be maintained by a 32 kDa integral membrane protein (PS translocase). The expression of PS on the outer leaflet may serve as a recognition signal for macrophages, since insertion of PS into erythrocytes enhances their adherence to macrophages and clearance from the circulation. Therefore I have hypothesized that erythroid cells display PS on their outer leaflet early in differentiation and upon aging. Analysis of murine erythroleukemia cells (MELC, undifferentiated erythroid progenitor cells) showed high levels of PS on the outer leaflet that decreased during differentiation, correlating with the pattern of macrophage adherence. The activity of the PS translocase during differentiation appears to be unchanged although the equilibrium distribution of PS differs. This difference may be due to qualitative changes in the PS translocase. $\sp{125}$I-Bolton/Hunter-labeled-pyridyldithioethylamine ($\sp{125}$I-B/H-PDA), a radiolabeled probe for the PS translocase, labeled a 32 kDa protein in mature erythrocytes whereas in MELC a 45 kDa protein as well as a 32 kDa protein was identified. The abundance of the 45 kDa protein in relation to the 32 kDa protein declined during differentiation, possibly indicating this protein was a precursor of the 32 kDa protein. Analysis of the 45 kDa protein by N-glycosidase F and endoproteinase cleavage suggested this protein was not a glycosylated form of the 32 kDa protein but appeared to share some structural homology. Aged murine erythrocytes had elevated levels of PS on their outer leaflet, as well as decreased PS translocase activity. $\sp{125}$I-B/H-PDA labeled a 32 kDa protein in both normal and aged erythrocytes. However, the latter cells also contained a 28 kDa protein. Experimental evidence suggests that the appearance of the 28 kDa protein may be due to increased oxidation of aged erythrocytes. Examination of PS distribution showed that the levels of PS on the outer leaflet were elevated early in differentiation, decreased during the mature state, and returned to high levels as the erythrocyte aged. In conclusion,the levels of outer leaflet PS correlated with the differentiation status and macrophage recognition of erythroid cells. ^