Background/Purpose: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by loss of tolerance to nuclear antigens, immune activation, and organ damage. Loss of NADPH oxidase (NOX2) function exacerbates SLE in murine models and is associated with autoimmunity in humans. Yet, the cell types and mechanisms by which NOX2 exerts its protective effects remain unclear. Here, to identify the cell lineages in which NOX2 deficiency drives SLE, we employed bone marrow (BM) chimera and conditional knockout (KO) approaches to delete a critical component of NOX2, encoded by Cybb, in several hematopoietic cell lineages of MRL.Fas^lpr lupus prone mice.

Methods: Reciprocal BM chimeras were generated by irradiating wild-type (WT) or Cybb^-/- MRL.Fas^lpr mice and reconstituting irradiated recipients with either WT or Cybb^-/- MRL.Fas^lpr BM.  Cybb-flox mice were generated on the MRL.Fas^lpr background using CRISPR-Cas9 technology. Resulting Cybb-flox MRL.Fas^lpr mice were crossed to LysM-Cre (neutrophil and macrophage targeting), MRP8-Cre (neutrophil only targeting), CD4-Cre (T cell targeting), and CD19-Cre (B cell targeting). Global NOX2-deficient mice have higher levels of TLR7-dependent autoantibodies. To test the hypothesis that NOX2 regulates lupus by dampening TLR7-mediated autoimmunity, we crossed Tlr7-deficient mice to global Cybb KOs on the MRL.Fas^lpr SLE-prone background. We also tested whether B cell-intrinsic TLR7 would be important in driving NOX2-deregulated lupus, by deleting Tlr7 in B cells of globally Cybb-deficient MRL.Fas^lpr mice. Cohorts were aged for >16 weeks. Disease severity was evaluated by measuring survival, proteinuria, dermatitis, and renal histopathology. The anti-self-response was assessed by autoantibody ELISAs and ANA assays. Immune composition was determined by flow cytometry. To test for a direct effect of the absence of NOX2 in promoting TLR7 signaling, we measured NF-kB signaling downstream of TLR7 (CL097) or 9 (CpG) stimulation by both phospho-p-65 upregulation and p-65 nuclear translocation in purified WT and Cybb-deficient B cells by conventional or imaging flow cytometry.

Results: Deletion of Cybb in the hematopoietic but not stromal compartments resulted in decreased survival and worse nephritis. The absence of CYBB in macrophages/monocytes exacerbated lupus nephritis, though not to the degree observed in the CYBB global KOs. No differences in clinical or immunological parameters of lupus were observed when CYBB was knocked out in neutrophils or T cells of MRL.Fas^lpr mice. Unexpectedly, the absence of CYBB in B cells resulted in profound glomerulonephritis and interstitial nephritis, rivaling that seen with global deletion. Further, we identified that NOX2 is a key regulator of TLR7, a driver of SLE pathology, both globally and specifically in B cells. This is mediated in part through specific suppression of TLR7, but not TLR9-, mediated NF-kB signaling in B cells.

Conclusion: NOX2’s immunomodulatory effect in SLE is orchestrated not only by its function in the myeloid compartment, but through a pivotal role in B cells by selectively inhibiting TLR7 signaling.

« Back to ACR Convergence 2024

ACR Meeting Abstracts - https://acrabstracts.org/abstract/nadph-oxidase-in-b-cells-and-macrophages-protects-against-murine-lupus-by-regulation-of-tlr7/