In a recent study featured in Nature Communications, researchers created mice that carry a gain-of-function (GoF) mutation in the gene encoding the inhibitor of nuclear factor kappa-b kinase subunit beta (IKBKB), known as the IKK2-encoding IKBKB gene. This was done to explore how this mutation works.
Study: IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model. Image Credit: Gorodenkoff/Shutterstock.com
Background
Loss-of-function mutations demonstrate the importance of forkhead box P3-positive (Foxp3+) regulatory T cells (Tregs) in immunological control. Tregs mediate dominant tolerance and protect against autoimmune disorders.
They undergo positive selection in the thymus, and interleukin-2 (IL-2) protects them from apoptosis. Treg formation needs effective signaling downstream of the T-cell receptor (TCR), particularly the CARD11-BCL10-MALT1 (CBM) complex.
Mice lacking particular genes have a Treg deficit that causes a selective loss of cluster of differentiation 4-positive (CD4+) Helios+ thymic T cells.
Tregs move between lymphoid organs according to adhesion molecule expression. The presence of an activated or effector phenotype (eTreg) in recirculating Tregs increases disease risk.
About the study
The present study examined mice with an Ikbkb GoF mutation homologous to a problematic human IKBKB GoF variation.
The researchers tracked a cohort of mice with various Ikbkb genotypes and recorded the age at which skin disease appeared. Animal house technicians were unaware of the mouse genotype and identified abnormal Ikbkbmut/+ and Ikbkbmut/mut animals. The researchers examined the transcriptomes of tails and ears from Ikbkbmut/mut and Ikbkb+/+ mice.
The team investigated the inflammatory infiltrate in skin lesions and the nature of Treg growth inside pathological lesions. They created mixed bone marrow chimeras with allotype-marked donor cells from WT and mutant mice.
They isolated naïve CD4+ T cells from mouse splenocyte suspensions and activated them with Th17-inducing conditions. The researchers then counted IL-17+ Tregs ex vivo and labeled them for cytokine production after gating on Foxp3.
The researchers extracted them from WT mice and cocultured them with pure WT conventional T cells labeled with CTV to explore Tregs' traditional immunosuppressive activity. They followed up with an in vivo test of mutant Treg suppression.
They analyzed mice for signs of systemic immunological dysregulation and created reciprocal bone marrow (BM) chimeras to study Ikbkbmut's cell-intrinsic effects on the Treg phenotype.
The team obtained serum from recipient mice to analyze a panel of cytokines. They isolated green fluorescent protein (GFP)-labeled Foxp3+ Tregs from Ikbkbmut donors and implanted them into Ikbkbmut x Rag1−/− or IkbkbWT x Rag1−/− animals to establish disease cause as pro-inflammatory Treg activity.
The researchers used mice aged six weeks to 12 months for analysis. They performed flow cytometry, flow cytometric cell sorting, ex vivo PMA/ionomycin stimulation for cytokine production, T-cell polarization, an in vitro Treg suppression experiment, cell trace violet (CTV) labeling, and single-cell and bulk ribonucleic acid (RNA) sequencing studies.
Results
Canonical NF-κB overactivity led to the growth of pathogenic, NF-κB-dependent, and modified non-lymphoid tissue skin Tregs. Mice with Ikbkb GoF mutation heterozygosity developed psoriasis, and Ikbkb-mut mice included IL-17-producing Tregs.
These animals maintained suppressive function, indicating that normal CD4+ T cells are not the source of IL-17 in Ikbkb mutant mice. Foxp3+ CD4+ T cells from Ikbkb mutant mice maintained suppressive function.
The study additionally examined the effects of doubling the IkbkbGoF/GoF gene dosage on psoriatic arthritis, characterized by spondylitis, dactylitis, and distinctive nail abnormalities.
IkbkbGoF mice showed selective CD25+ and Foxp3+ Treg expansion, with a fraction expressing IL-17. These transformed Tregs were present in inflamed tissues, spleen, and blood, and their transfer was sufficient to cause illness without ordinary T lymphocytes.
Single-cell phenotyping and transcriptional investigations of isolated regulatory T cells indicated the non-lymphocytic tissue proliferation of Treg expressing Th17-associated genes, Helios, tissue-related markers such as CD69 and CD103, and a significant nuclear factor kappa B (NF-κB) transcriptome.
Overactive IKK2 caused dermal Treg accumulation and psoriasis. Heterozygous (Ikbkbmut/+) and homozygous (Ikbkbmut/mut) mutant mice developed skin illnesses with histopathological similarities to psoriasis.
Humans heterozygous for IKBKBV203I have combined immune insufficiency, but their Treg count increased. Ikbkbmut has a similar phenotype, with gene-dose-dependent lymphopenia caused by a decrease in αβ and γδ T cells in homozygous mice.
The study also found an increase in Th17 CD4+ T cells, strongly associated with psoriasis. Ikbkbmut/mut mice spleen Tregs produced more IL-17 than wild-type mice.
Interferon-gamma (IFNγ) production by Tregs was similar between WT and mutant animals, indicating that Ikbkbmut imparts an expansion of the IL-17-producing Foxp3+ Treg population.
Foxp3 deficiency and Treg functional abnormalities were associated with early-onset and severe widespread lymphadenopathy unrelated to the Ikbkbmut mutation.
Conclusion
The study linked psoriasis and psoriatic arthritis to NF-κB malfunction, which causes non-specific leukocytes to acquire an effector-like function, resulting in disease. The primary finding is a route that leads Foxp3+CD4+ tissue-resident Tregs to turn pro-inflammatory and pathogenic.
In vivo, a modified Treg population emerges owing to enhanced activity of the canonical NF-κB pathway. This route controls Treg abundance, increases tissue-resident Tregs, and mediates end-organ pathologies.
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