Human TLR8 induces inflammatory bone marrow erythromyeloblastic islands and anemia in SLE-prone mice.

Anemia commonly occurs in systemic lupus erythematosus, a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause erythroid cell death, while sparing other hematopoietic cell lineages. Whereas chronic inflammation is involved in this mechanism, less is known about the impact of systemic lupus erythematosus on the BM erythropoietic niche. We discovered that expression of the endosomal ssRNA sensor human TLR8 induces fatal anemia in Sle1.Yaa lupus mice. We observed that anemia was associated with a decrease in erythromyeloblastic islands and a block in differentiation at the CFU-E to proerythroblast transition in the BM. Single-cell RNAseq analyses of isolated BM erythromyeloblastic islands from human TLR8-expressing mice revealed that genes associated with essential central macrophage functions including adhesion and provision of nutrients were down-regulated. Although compensatory stress erythropoiesis occurred in the spleen, red blood cell half-life decreased because of hemophagocytosis. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation.

Combination CTLA4Ig and Anti-CD40 Ligand Treatment Modifies T and B Cell Metabolic Profiles and Promotes B Cell Receptor Remodeling in a Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti-CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

Context-dependent induction of autoimmunity by TNF signaling deficiency.

TNF inhibitors are widely used to treat inflammatory diseases; however, 30%-50% of treated patients develop new autoantibodies, and 0.5%-1% develop secondary autoimmune diseases, including lupus. TNF is required for formation of germinal centers (GCs), the site where high-affinity autoantibodies are often made. We found that TNF deficiency in Sle1 mice induced TH17 T cells and enhanced the production of germline encoded, T-dependent IgG anti-cardiolipin antibodies but did not induce GC formation or precipitate clinical disease. We then asked whether a second hit could restore GC formation or induce pathogenic autoimmunity in TNF-deficient mice. By using a range of immune stimuli, we found that somatically mutated autoantibodies and clinical disease can arise in the setting of TNF deficiency via extrafollicular pathways or via atypical GC-like pathways. This breach of tolerance may be due to defects in regulatory signals that modulate the negative selection of pathogenic autoreactive B cells.

Rubicon promotes rather than restricts murine lupus and is not required for LC3-associated phagocytosis.

NADPH oxidase deficiency exacerbates lupus in murine models and patients, but the mechanisms remain unknown. It is hypothesized that NADPH oxidase suppresses autoimmunity by facilitating dead cell clearance via LC3-associated phagocytosis (LAP). The absence of LAP reportedly causes an autoinflammatory syndrome in aged, nonautoimmune mice. Prior work implicated cytochrome b-245, ß polypeptide (CYBB), a component of the NADPH oxidase complex, and the RUN and cysteine-rich domain-containing Beclin 1-interacting protein (RUBICON) as requisite for LAP. To test the hypothesis that NADPH oxidase deficiency exacerbates lupus via a defect in LAP, we deleted Rubicon in the B6.Sle1.Yaa and MRL.Faslpr lupus mouse models. Under this hypothesis, RUBICON deficiency should phenocopy NADPH oxidase deficiency, as both work in the same pathway. However, we observed the opposite - RUBICON deficiency resulted in reduced mortality, renal disease, and autoantibody titers to RNA-associated autoantigens. Given that our data contradict the published role for LAP in autoimmunity, we assessed whether CYBB and RUBICON are requisite for LAP. We found that LAP is not dependent on either of these 2 pathways. To our knowledge, our data reveal RUBICON as a novel regulator of SLE, possibly by a B cell-intrinsic mechanism, but do not support a role for LAP in lupus.