In a recent study presented in Nature communications, the researchers created mice that carry a gain-of-function (GoF) mutation in the gene encoding the inhibitor of nuclear factor kappa-β kinase beta subunit (IKBKB), known as the IKBKB gene encoding IKK2. This was done to investigate how this mutation works.
Study: IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a mouse gain-of-function model. Image credit: Gorodenkoff/Shutterstock.com
Record
Loss-of-function mutations demonstrate the importance of P3-positive (Foxp3+) regulatory T cells (Tregs) within the forkhead box in immune 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 requires efficient signaling downstream of the T-cell receptor (TCR), particularly the CARD11-BCL10-MALT1 (CBM) complex.
Mice lacking specific genes have a Treg deficiency that causes a selective loss of differentiation pool 4-positive (CD4+) Helios+ thymic T cells.
Twigs move between lymphoid organs according to adhesion molecule expression. The presence of an activated or active phenotype (eTreg) in circulating Tregs increases the risk of disease.
About the study
The present study examined mice with an Ikbkb GoF mutation homologous to a defective human IKBKB GoF variant.
The researchers followed a group of mice with various Ikbkb genotypes and recorded the age at which the 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 development within the pathological lesions. They created mixed bone marrow chimeras with allotype-tagged donor cells from WT and mutant mice.
They isolated primary CD4+ T cells from murine splenocyte suspensions and activated them with Th17-inducing conditions. The researchers then measured IL-17+ Tregs ex vivo and labeled them for cytokine production after Foxp3 entry.
The researchers extracted them from WT mice and cocultured them with CTV-labeled pure conventional WT T cells to explore the traditional immunosuppressive activity of Tregs. They followed up with an in vivo test to suppress mutant Treg.
They analyzed mice for signs of systemic immune dysregulation and generated mutualistic bone marrow (BM) chimeras to study the cell-intrinsic effects of Ikbkbmut on the Treg phenotype.
The team obtained serum from recipient mice to analyze a panel of cytokines. They isolated green fluorescent protein (GFP)-tagged Foxp3+ Tregs from Ikbkbmut donors and implanted them into Ikbkbmut x Rag1−/− or IkbkbWT x Rag1−/− animals to determine the cause of the disease as proinflammatory Treg activity.
The researchers used six-week- to 12-month-old mice for analysis. They performed flow cytometry, flow cytometry cell sorting, ex vivo PMA/ionomycin stimulation for cytokine production, T-cell polarization, in vitro Treg suppression experiment, cell tracer violet (CTV) labeling, and monocyte and tumor ribonucleic acid (RNA) sequencing studies.
Results
Normal overactivity of NF-κB led to the development of pathogenic, NF-κB-dependent, and nonlymphatic skin-modified Tregs. Mice heterozygous for the Ikbkb GoF mutation developed psoriasis, and Ikbkb-mut mice contained IL-17-producing Tregs.
These animals retained 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 retained suppressive function.
The study further examined the effects of doubling the dosage of the IkbkbGoF/GoF gene in psoriatic arthritis, characterized by spondylitis, dactylitis and distinct nail abnormalities.
IkbkbGoF mice showed selective expansion of CD25+ and Foxp3+ Treg, with a fraction expressing IL-17. These transformed Tregs were present in inflamed tissues, spleen and blood, and their transfer was sufficient to cause disease without ordinary T lymphocytes.
Phenotyping and transcriptional investigations of individual T cells showed non-lymphocyte tissue proliferation of Treg-expressing Th17-associated genes, Helios, tissue-associated markers such as CD69 and CD103, and a major nuclear factor kappa B (NF-κB) transcript .
Overactive IKK2 induced cutaneous Treg accumulation and psoriasis. Heterozygous (Ikbkbmut/+) and homozygous (Ikbkbmut/mut) mutant mice developed skin diseases with histopathological similarities to psoriasis.
People who are heterozygous for IKBKBV203I have combined immunodeficiency, but their Treg numbers are increased. Ikbkbmut has a similar phenotype, with gene dose-dependent lymphopenia caused by depletion of αβ and γδ T cells in homozygous mice.
The study also found an increase in Th17 CD4+ T cells, strongly associated with psoriasis. Spleen of Ikbkbmut/mut 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 mediates an expansion of the IL-17-producing Foxp3+ Treg population.
Foxp3 deficiency and Treg functional abnormalities were associated with early onset and severe extensive lymphadenopathy unrelated to the Ikbkbmut mutation.
conclusion
The study linked psoriasis and psoriatic arthritis to NF-κB dysfunction, which causes non-specific leukocytes to take on an effector-like function, resulting in disease. The primary finding is a pathway that drives tissue-resident Foxp3+CD4+ Tregs to become proinflammatory and pathogenic.
In vivo, an altered Treg population emerges due to enhanced activity of the normal NF-κB pathway. This pathway controls Treg abundance, increases tissue-resident Tregs, and mediates end-organ pathologies.
