Many patients with chronic autoimmune diseases such as systemic lupus erythematosus (SLE) develop autoantibodies to ubiquitous host cellular molecules, such as Sm small ribonucleoproteins (snRNPs) containing the small RNAs U1, U2, U4-6, and U5 RNAs. These small RNAs are highly structured molecules containing extensive regions of duplex (double-stranded, ds) RNA. High concentrations of dsRNA produced by replicating RNA viruses activate innate immunity by binding to dsRNA binding proteins such as TLR3 and/or the dsRNA dependent protein kinase (PKR), causing increased production of type I interferon (IFN) leading to an antiviral response. The purpose of this project is to elucidate the mechanism by which snRNP activates innate immunity in systemic lupus erythematosus (SLE). Mononuclear cells from SLE patients with increased anti-snRNPs autoantibodies express higher levels of genes usually induced by type-I IFN, an innate cytokine that plays a key role in SLE. Additional preliminary data show that double-stranded (ds) U1 RNA from snRNP stimulates mononuclear cells to activate dsRNA-induced protein kinase (PKR), a hallmark of the IFN response. It is hypothesized that snRNP triggers IFN production by engaging Toll-like receptor 3 (TLR3) in innate immune cells via U1 RNA. Preliminary studies indicated that peripheral blood mononuclear cells from a subset of patients with autoantibodies against the small ribonucleoproteins nRNP/Sm and Ro/La express higher levels of IFN inducible genes than healthy controls or the subset of lupus patients who are negative for these autoantibodies. Moreover, it was found that purified authentic U1 RNA and synthetic stem-loop II of U1 are potent stimulators of IFN-inducible gene expression and PKR autophosphorylation. Interestingly, type I IFNs also have been implicated in the pathogenesis of SLE. We hypothesize that the small RNAs associated with U1 snRNPs promote autoimmune responses to the protein components of UI snRNPs by stimulating signaling via TLR3 and/or PKR. By stimulating type I IFN production, highly structured small RNAs of cellular origin may promote dendritic cell maturation and activation of autoreactive T cells and/or the activation and maturation of autoreactive B cells specific for snRNPs.
Three Specific Aims are proposed.
Aim I is to examine how U1 RNA stimulates an antiviral (IFN) response by investigating whether the IFN response is mediated by TLR3 signaling, internalization of the U1 RNA followed by binding and activation of PKR, or some other pathway. The portion(s) of U1 RNA responsible for the immunostimulatory effect will be identified and the possibility that immunoinhibitory dsRNAs also exist will be examined.
Aim 2 is to determine what normally prevents endogenous U1 RNA from activating the IFN system. It is hypothesized that proteins bound to structured regions of the RNA prevent it from engaging TLR3 or PKR. The ability of series of partially disrupted U1 snRNP fractions to stimulate IFN responses or PKR activation will be examined.
Aim 3 is to identify the cell type that becomes activated in response to U1 RNA. The effects of U1 RNA on dendritic cell maturation, macrophage activation, and B cell development will be determined. These studies may lead to a better understanding of why autoimmune responses in SLE selectively target the Sm (U series) and other snRNPs. By triggering innate immunity, the highly structured, RNAs contained in these molecules could promote autoimmunity by promoting dendritic cell maturation and/or the activation of autoreactive B or T lymphocytes.
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