The impact of toll-like receptor 7 and 8 (TLR7/8)-mediated neutrophil activation on B cell responses and clinical and immunological features of systemic lupus erythematosus (SLE)

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder in which recruitment and activation of neutrophils by immune complexes (ICs) formed between autoantibodies and self-antigens are responsible for tissue injury. Toll-like receptors 7/8 (TLR7/8) recognize single-stranded RNA and are expressed in the endosomal compartment of the cells of innate immunity. Neutrophils have been shown to constitutively express all TLRs except for TLR3 and TLR7. Upon stimulation, neutrophils form neutrophil extracellular trap (NET), which displays granule proteins and chromatin-DNA complexes that are thought to stimulate innate antigen presenting cells to produce inflammatory cytokines. Neutrophils in SLE patients show certain abnormalities including inefficient phagocytosis, poor NADPH oxidase activity, and an increased frequency of low density granulocytes (LDGs), which are a distinct subset with activated phenotypes. Activated neutrophils produce high levels of BAFF (B cell activating factor) and APRIL (a proliferation inducing ligand) in SLE patients with active disease suggesting the role of neutrophils in B cell responses. In addition, LDGs stimulated with nucleosome-containing ICs from SLE patients produce TNF, IL-6 and IL-8 at higher levels suggesting that LDGs are activated via TLR7/8/9. Most of these studies have used stimulants (e.g. GM-CSF or PMA) that do not involve stimulation of TLR7/8 except for one study, which showed that direct triggering of TLR7/8 in neutrophils resulted in a loss of phagocytic capability due to decreasing Fc?RIIA, and investigations of the effects of neutrophil activation by direct trigger of TLR7/8 have been sparse. It has been shown that higher proportions of Omani SLE patients have significant levels of autoantibodies to DNA and RNA-associated proteins compared with patient from other ethnic groups (e.g. European Caucasians, African-Americans). The high levels of autoantibodies to RNA-associated proteins may suggest TLR7/8-mediated activation of immune cells including neutrophils may occur more frequently in Omani SLE patients. Thus, the goal of this study is to delineate the impact of TLR7/8-mediated activation of neutrophils by examining: 1) the level of TLR7/8 expression in neutrophils and LDGs in SLE patients and controls; 2) the effects of TLR7/8-mediated activation of neutrophils on cytokine production, NET formation, and contents displayed in NET; 3) the role of activated neutrophils in altering B cell responses by assessing B cell proliferation, upregulation of co-stimulatory molecules, and antibody production in neutrophil-B cell co-cultures; and 4) whether any relationship exists between TLR7/8-induced neutrophil and B cell functions and the features of SLE. Findings from this study may provide important insights into the effects of high frequencies of autoantibodies to RNA-associated proteins on mechanisms of pathogenesis in Omani SLE patients. Better understanding of the role of TLR7/8 in neutrophil activation and functions and their impact on B cell responses and features of the disease may contribute toward the development of more effective therapeutic strategy that could be specifically targeted for the local population.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder in which recruitment and activation of neutrophils by immune complexes (ICs) formed between autoantibodies and self-antigens are responsible for tissue injury. Toll-like receptors 7/8 (TLR7/8) recognize single-stranded RNA and are expressed in the endosomal compartment of the cells of innate immunity. Neutrophils have been shown to constitutively express all TLRs except for TLR3 and TLR7. Upon stimulation, neutrophils form neutrophil extracellular trap (NET), which displays granule proteins and chromatin-DNA complexes that are thought to stimulate innate antigen presenting cells to produce inflammatory cytokines. Neutrophils in SLE patients show certain abnormalities including inefficient phagocytosis, poor NADPH oxidase activity, and an increased frequency of low density granulocytes (LDGs), which are a distinct subset with activated phenotypes. Activated neutrophils produce high levels of BAFF (B cell activating factor) and APRIL (a proliferation inducing ligand) in SLE patients with active disease suggesting the role of neutrophils in B cell responses. In addition, LDGs stimulated with nucleosome-containing ICs from SLE patients produce TNF, IL-6 and IL-8 at higher levels suggesting that LDGs are activated via TLR7/8/9. Most of these studies have used stimulants (e.g. GM-CSF or PMA) that do not involve stimulation of TLR7/8 except for one study, which showed that direct triggering of TLR7/8 in neutrophils resulted in a loss of phagocytic capability due to decreasing Fc?RIIA, and investigations of the effects of neutrophil activation by direct trigger of TLR7/8 have been sparse. It has been shown that higher proportions of Omani SLE patients have significant levels of autoantibodies to DNA and RNA-associated proteins compared with patient from other ethnic groups (e.g. European Caucasians, African-Americans). The high levels of autoantibodies to RNA-associated proteins may suggest TLR7/8-mediated activation of immune cells including neutrophils may occur more frequently in Omani SLE patients. Thus, the goal of this study is to delineate the impact of TLR7/8-mediated activation of neutrophils by examining: 1) the level of TLR7/8 expression in neutrophils and LDGs in SLE patients and controls; 2) the effects of TLR7/8-mediated activation of neutrophils on cytokine production, NET formation, and contents displayed in NET; 3) the role of activated neutrophils in altering B cell responses by assessing B cell proliferation, upregulation of co-stimulatory molecules, and antibody production in neutrophil-B cell co-cultures; and 4) whether any relationship exists between TLR7/8-induced neutrophil and B cell functions and the features of SLE. Findings from this study may provide important insights into the effects of high frequencies of autoantibodies to RNA-associated proteins on mechanisms of pathogenesis in Omani SLE patients. Better understanding of the role of TLR7/8 in neutrophil activation and functions and their impact on B cell responses and features of the disease may contribute toward the development of more effective therapeutic strategy that could be specifically targeted for the local population.