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.

The role of plasmacytoid dendritic cells in the regulation of adaptive immunity

Plasmacytoid dendritic cells (pDCs) selectively express TLR7 which allows them to respond to RNA viruses and TLR9 which allows them to respond to DNA viruses and CpG oligonucleotides. Upon exposure to virus pDCs produce vast amounts of type I interferon (IFN) directly inhibiting viral replication and contributing to the activation of other immune cells. The ability of pDCs to promote B and T cell differentiation through type I IFN has been well documented although the role of additional factors including tumor necrosis factor (TNF) family members has not been thoroughly addressed. Here the expression of selected TNF family members in pDCs was examined and the role of TNF receptor-ligand interactions in the regulation of B and T lymphocyte growth and differentiation by pDCs was investigated. Upon stimulation with CpG-B, pDCs exhibit strong and stable expression of CD70, a TNF family ligand that binds to its receptor CD27 on memory B cells and promotes plasma cell differentiation and Ig secretion. Using an in vitro pDC/B cell co-culture system, it was determined that CpG-B-stimulated pDCs induce the proliferation of CD40L-activated human peripheral B cells and Ig secretion. This occurs independently of IFN and residual CpG, and requires physical contact between pDCs and B cells. CpG-stimulated pDCs induce the proliferation of both naive and memory B cells although Ig secretion is restricted to the memory subset. Blocking the interaction of CD70 with CD27 using an antagonist anti-CD70 antibody reduces the induction of B cell proliferation and IgG secretion by CpG-B-stimulated pDCs. Published studies have also indicated an important role for CD70 in promoting the expansion of CD4+ and CD8+ T cells and the development of effector function. CpG-B-stimulated pDCs induce naïve CD4+ T cell proliferation and production of multiple cytokines including IFN-γ, TNF-α, IL-10, IL-4, IL-5 and IL-13. Blocking the function of CD70 with an antagonist anti-CD70 antibody significantly reduced the induction of naïve CD4+ T cell proliferation by CpG-B-stimulated pDCs and the production of IL-4 and IL-13. Collectively these data indicate an important role for CD70 in the regulation of B and T lymphocyte growth and differentiation by pDCs. ^

ALLERGEN SENSITIZATION AND IMMUNOMODULATION BY SYNTHETIC LIGANDS, PUL-042, IN AN ALLERGEN-INDUCED ASTHMA MURINE MODEL

Allergen-induced asthma is the leading form of asthma and a chronic condition worldwide. Common allergens are known to contribute to the pathogenesis of this disease. Murine models of allergic asthma have mostly used an intraperitoneal route of sensitization (not airway) to study this disease. Allergic asthma pathophysiology involves the activation of TH2-specific cells, which triggers production of IgE antibodies, the up-regulation of TH2-specific cytokines (i.e. IL-4, IL-5, IL-9 and IL-13), increased airway eosinophilia, and mucin hypersecretion. Although there are several therapeutics currently treating asthmatic patients, some of these treatments can result in drug tolerance and may be linked to increased mortality. CpG oligodeoxynucleotides (ODNs) is a synthetic ligand that targets Toll-like Receptor (TLR) 9. It has been evaluated as a therapeutic agent for the treatment of cancer, infectious diseases, and for treating allergy and asthma. PUL-042 is also a synthetic TLR ligand and is composed of two agonists against TLR2/6 heterodimer and TLR9. Previous studies have evaluated PUL-042 for its ability to confer resistance against bacterial and viral lung infection. These findings, combined with studies performed using CpG ODNs, led to speculation that PUL-042 dampens the immune response in allergen-induced asthma. My thesis research investigated airway route sensitization and airway delivery of PUL-042 to evaluate its effects in reducing an allergen-induced asthma phenotype in a murine model. The results of this study contribute to the foundation for future investigations to evaluate the efficacy of PUL-042 as a novel therapy in allergic-asthma disease.