IFNγ-induced IRF1 synergizes with TLR7 signals to tune the IRF4-IRF8 axis and drive pathogenic effector B cell fate

Keywords

● B cells

● Systemic Lupus Erythematosus

● Irf1

Main Findings

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of anti-nuclear antibodies (ANAs), implicating auto-reactive B cells at the centre of the disease. Generation of pathogenic antibody secreting cells (ASCs) from autoreactive B cells involves Interferon (IFN) gamma and Toll Like Receptor (TLR) 7 signals which converge through activation of downstream transcription factors (TFs), including interferon regulatory factors (IRFs). While IRF1 is the prototypic TF downstream of type 1 and type 2 IFN signalling and is located in a susceptibility locus for SLE, little is known about IRF1’s role in the differentiation of pathogenic ASCs in the context of autoimmune disease. In this preprint, Owiredu et al. explored whether IRF1 is involved in the core ASC differentiation pathway through the integration of IFNg and TLR7 signals.

The authors used a YaaFC mouse model of SLE, which combines two key aspects to initiate and accelerate autoimmunity: (1) a knockout of the inhibitory FcgRIIB receptor leading to increased B cell activation and autoantibody production, and (2) a yaa mutation resulting in translocation of Tlr7 to the Y chromosome leading to its overexpression. This TLR7 dependent mouse model of SLE was combined with global IRF1 knockout (KO) and B cell specific IRF1 KO to examine the effects of IRF1 on systemic autoimmunity. The authors show that IRF1 modulates the composition of the splenic and lymph node B cell compartment. Deletion of IRF1 reduces the severity of key features of the lupus-like phenotype in this mouse model (kidney involvement, autoantibody production, survival). These results point toward a role for B cell intrinsic IRF1 in generating pathogenic ASC responses and promoting systemic autoimmunity.

Single cell sequencing and ATAC-sequencing of B cell nuclei from B-cell specific IRF1 KO and IRF1 sufficient mice reveals transcriptional skewing of several B cell populations within the entire pool of B cells. ASCs from IRF1-sufficient mice exhibit secretory/activated gene profiles, increased chromatic accessibility surrounding the Irf4 locus and increased Irf4 gene expression. The IRF4:IRF8 ratio is implicated in B cell biology, with IRF4 promoting ASC development and IRF8 constraining it. The authors reason that this pathogenic skewing of the ASCs may occur prior to commitment to the ASC lineage. Indeed, they show that age-associated B cells (ABCs), germinal centre B cells (GCBs) and memory B cells (MBCs) collectively exhibit similar transcriptional deviations. In general, these precursor populations show increased Irf4 expression, decreased Irf8 expression, increased Irf4 chromatin accessibility and pathogenic phenotypes prone to ASC differentiation. In support of the sequencing results, stimulation of MBC and ABC precursor populations with IFNg and TLR7 agonists supports B cell proliferation and differentiation to an ASC fate in the IRF1 sufficient mice. Taken together, these results indicate a role for IRF1 in skewing the B cell response toward a pathogenic, pre-ASC fate capable of readily differentiating to ASCs in response to IFNg and TLR7 stimulation.

Finally, Owiredu et al., interrogate publicly available datasets to examine whether a similar IRF1-driven mechanism is present in human B cells. Compared to healthy controls, SLE patients exhibit higher gene expression of Irf1, increased chromatin accessibility around an IRF1-binding site and increasedIrf4 expression, suggesting a similar coordinate expression of IRF1/IRF4 in humans. Finally, the authors corroborate the difference in fate potential that was observed in murine B cells. Stimulation of human B cells with IFNg and TLR7 is sufficient to induce Irf1and Irf4 expression, which results in increased B cell proliferation and subsequent ASC differentiation.

Altogether, this paper implicates IRF1 in the IFNg-TLR7 driven ASC differentiation pathway. Mechanistically, the authors show that IRF1 tips the IRF4:IRF8 balance towards IRF4, promoting a pathological ASC fate in the context of systemic autoimmunity.

Limitations

● The experiments involving mice used a TLR7 dependent mouse model of SLE (YaaFc) that spontaneously develop disease around 4-5 months of age. Validation of the effects of IRF1 on B cell differentiation to ASC in additional mouse models of lupus that do not rely on TLR7 overexpression (e.g., MRL/lpr) would have strengthened the findings. Additionally, since the YaaFc model relies on overexpression of Tlr7 on the Y chromosome in male mice, alternative mouse models would have allowed the authors to explore this pathway in female mice, which is highly relevant given the predominance of SLE in women. Alternatively, induced mouse models of SLE would have allowed for further exploration into age-associated effects on this B cell differentiation pathway.

● B cells examined in this paper were primarily derived from the spleen. Given that much of the immune involvement in SLE occurs in organs like the kidney, it would be interesting to investigate whether a similar IRF1 mediated skew of IRF4:IRF8 in B cells exists in tissue niches.

● All experiments in this paper used a homozygous knockout of IRF1. It would be interesting to investigate the effects on B cells and the disease phenotype using a heterozygous IRF1 knockout to determine whether there is a gene dosage effect. To support the role of IRF4:IRF8 ratio skewing, it would be informative to show that a reduction of IRF8 (e.g., through heterozygous knockout) in IRF1 deficient mice was sufficient to reverse the observed protective effects.

● The authors show that global IRF1 deletion and B cell specific IRF1 deletion generated similar changes to the B cell compartment and the disease phenotype. However, IRF1 is involved in the maturation, development, and function of both innate and adaptive immune cells. It would be interesting to further investigate the effects of IRF1 deletion on myeloid cells and T cells in the context of this mouse model. Additionally, whether B cell specific IRF1 deficiency influences B cell development in steady state in the C57BL/6 background was not investigated but would be an informative additional experiment.

Significance/Novelty

With this preprint, the authors uncover a role for IRF1 in driving IFNg and TLR7 induced ASC differentiation in the context of systemic autoimmunity for the first time. They provide an integrative framework in both murine and human B cells, in which IRF1 modulates chromatin accessibility around the Irf4 locus and skews the IRF4:IRF8 ratio toward a pathogenic B cell fate. Taken together, this paper provides mechanistic insight into the development of the pathogenic B cell population at the centre of SLE. While treatment by IFNAR1 blockade has recently proven successful for SLE, anti-IFNgblockade has shown little clinical benefit. Importantly, these results highlight a novel opportunity for therapeutic targeting downstream of type 2 IFNs through the IRF1/IR4:IRF8 axis in SLE.

Credit

Reviewed by Carmen Ucciferri as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.