Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct;49(1-2):273-80.
doi: 10.1016/j.molimm.2011.08.022. Epub 2011 Sep 23.

Cell type and gender-dependent differential regulation of the p202 and Aim2 proteins: implications for the regulation of innate immune responses in SLE

Ravichandran Panchanathan  1 , Xin Duan, Muthuvel Arumugam, Hui Shen, Hongzhu Liu, Divaker Choubey
Affiliations

Cell type and gender-dependent differential regulation of the p202 and Aim2 proteins: implications for the regulation of innate immune responses in SLE

Ravichandran Panchanathan et al. Mol Immunol. 2011 Oct.

Abstract

Upon sensing cytosolic double-stranded DNA (dsDNA), the murine Aim2 (encoded by the Aim2 gene) protein forms an inflammasome and promotes the secretion of proinflammatory cytokines, such as IL-1β and IL-18. In contrast, the p202 protein (encoded by the Ifi202 gene) does not form an inflammasome. Previously, we have reported that the interferon (IFN) and female sex hormone-induced increased nuclear levels of p202 protein in immune cells are associated with increased susceptibility to develop a lupus-like disease. However, signaling pathways that regulate the expression of Aim2 protein remain unknown. Here we report that the expression of Aim2 gene is induced in bone marrow-derived macrophages (BMDMs) by IFN-α treatment and the expression is, in part, STAT1-dependent. However, treatment of splenic T or B cells with IFN-α or their stimulation, which induced the expression of Ifi202 gene, did not induce the expression of Aim2 gene. Furthermore, treatment of cells with the male hormone androgen increased levels of Aim2 mRNA and protein. Moreover, treatment of murine macrophage cell lines (RAW264.7 and J774A.1) with IFN-α differentially induced the expression of Aim2 and p202 proteins and regulated their sub-cellular localization. Additionally, activation of Toll-like receptors (TLR3, 4, and 9) in BMDMs and cell lines also differentially regulated the expression of Aim2 and Ifi202 genes. Our observations demonstrate that cell type and gender-dependent factors differentially regulate the expression of the Aim2 and p202 proteins, thus, suggesting opposing roles for these two proteins in innate immune responses in lupus disease.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

None

Figures

Fig. 1
Fig. 1
The IFN-inducibility of Aim2 gene is cell type-dependent. (A) Bone marrow-derived macrophages (BMDMs) were either left without any treatment (lane 1) or treated with IFN-α (1,000 u/ml) for the indicated time (h). At the end of the treatment, total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins. Fold change indicates increases in the levels of Aim2 protein. (B) BMDMs as in panel (A) were either left untreated or treated with IFN-α for 14 h. After the treatment, total RNA was analyzed for the levels of the indicated mRNAs by regular RT-PCR using specific primers. (C) Total RNA was prepared from splenocytes isolated from wild-type and age-matched Stat1-deficient male or female mice (age ~9 weeks). Steady state levels of Aim2 mRNA were analyzed by analyzed by quantitative TaqMan real-time PCR using the assay specific to the Aim2 gene. The ratio of the test gene to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the test gene to β2-microglobulin mRNA). The relative steady-state levels of Aim2 mRNA in male wild-type mice are indicated as 1. Results are mean values of triplicate experiments and error bars represent standard deviation. (D) Splenocytes isolated from B6 female mice (age ~9 weeks) were either left untreated or treated with IFN-α for 14 h. Steady state levels of mRNAs corresponding to the Aim2, Ifi202, and Ifi204 genes were analyzed by quantitative TaqMan real-time PCR as described in the panel (C). The relative steady-state levels of mRNAs for all genes in control untreated Splenocytes are indicated as 1. (E) Purified splenic T or B cells from B6 female mice (age ~9 weeks) were either left untreated or treated with IFN-α for 16 h. Steady state levels of mRNAs corresponding to the Aim2 and Ifi202 genes were analyzed by quantitative TaqMan real-time PCR as described in the panel (D). The relative steady-state levels of mRNAs for both genes in control untreated T cells are indicated as 1. Results are mean values of triplicate experiments and error bars represent standard deviation.
Fig. 2
Fig. 2
Stimulation of T and B cells down-regulates the expression of Aim2 gene. (A) Splenic cells (cells pooled from two or more age-matched mice) from the B6 or B6.Nba2 female mice (age ~9-weeks) were either incubated with an isotype control antibody or with α-CD3 and α-CD28 for 22 h. After the incubations, total mRNA were isolated and the steady-state levels of mRNA were analyzed by quantitative TaqMan real-time PCR for Ifi202 and Aim2 genes. The relative steady-state levels of mRNAs for both genes in control isotype treated cells are indicated as 1. Results are mean values of triplicate experiments and error bars represent standard deviation. (B). Splenic cells from B6 or B6.Nba2 female mice as noted in the panel (A) were either incubated with an isotype control antibody or with α-IgM for 20 h and the steady-state levels of mRNA were analyzed by quantitative TaqMan real-time PCR for Ifi202 and Aim2 genes. The relative steady-state levels of mRNAs for both genes in control isotype treated splenic cells are indicated as 1. Results are mean values of triplicate experiments and error bars represent standard deviation.
Fig. 3
Fig. 3
Gender-dependent factors regulate the expression of Aim2 gene. (A) Total RNA was prepared from splenocytes isolated from the indicated strain of male or age-matched female mice (age ~9 weeks). Steady state levels of Aim2 mRNA were analyzed by analyzed by quantitative TaqMan real-time PCR. The ratio of the test gene to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the test gene to β2-microglobulin mRNA). The relative steady-state levels of Aim2 mRNA in male mice of all strains are indicated as 1. Results are mean values of triplicate experiments and error bars represent standard deviation. (B) Total cell lysates prepared from B6 splenic cells isolated from male or age-matched female (age ~8 weeks) mice were analyzed by immunoblotting using antibodies specific to the indicated proteins. (C) Total cell lysates prepared from B6 T or B cells purified from male or age-matched female (age ~8 weeks) mice were analyzed by immunoblotting using antibodies specific to the indicated proteins. (D) Sub-confluent cultures of WT-276 cells were either left untreated (lane 1), treated with 5 nM (lane 2), or 10 nM DHT in phenol-free DMEM medium (supplemented with 10% charcoal stripped fetal bovine serum) for 18 h. Total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins. (E) Sub-confluent cultures of WT-276 cells were either left untreated or treated with DHT as described in panel (D). Total RNA was analyzed for levels of Aim2 MRNA by real-time PCR. The relative steady-state levels of Aim2 mRNA in untreated cells are indicated as 1. Results are mean values of triplicate experiments and error bars represent standard deviation.
Fig. 4
Fig. 4
Differentially regulation of Aim2 and Ifi202 expression by IFNs in macrophage cell lines. (A) Sub-confluent cultures of macrophage RAW264.7 or J774.A1 cell lines were either left untreated or treated with IFN-α (1,000 u/ml) for 16 h. Total RNA was isolated and analyzed by real-time PCR for increases in Aim2 mRNA levels. The relative steady-state levels of Aim2 mRNA in untreated cells are indicated as 1. (B and C) Cells of RAW264.7 (B) or J774.A1 (C) macrophage cell lines were either left untreated (lane 1), treated with IFN-α (lane 2), or IFN-γ (10 ng/ml) for 16 h. Cells were harvested and total cell lysates were analyzed by immunoblotting for the indicated proteins. (D and E) Sub-confluent cultures of RAW264.7 (D) or J774A.1 (E) macrophage cell lines were either left untreated or treated with IFN-α (1,000 u/ml) for 16 h. Cells were fractionated into the cytoplasmic (Cyt) and nuclear (Nu) fractions and fractions containing equal amounts of proteins were analyzed by immunoblotting using specific antibodies to the indicated proteins. Levels of IκBα protein in the cytoplasm and histone H3 in the nucleus served as a quality control for the cell fractionations.
Fig. 5
Fig. 5
Toll-like receptor signaling differentially regulates the expression of Aim2 and Ifi202 genes in BMDMs. (A-C) BMDMs from B6.Nba2 female mice were either left untreated or treated with the indicated TLR ligand for 16 h. Total RNA was isolated and analyzed by real-time PCR (A and B) or regular PCR (C) for steady-state levels of Aim2 and Ifi202 mRNA. Levels of mRNA in untreated cells or cells treated with non-stimulatory TLR9 control ODN are indicated as 1. Results are mean values of triplicate experiments and error bars represent standard deviation.
Fig. 6
Fig. 6
Toll-like receptor signaling differentially regulates the expression of Aim2 and p202 proteins in macrophage cell lines. (A) Sub-confluent cultures of RAW264.7 cells were either left untreated or treated with the indicated TLR ligand for 6 h. After the treatments, cells were harvested and total cell lysates were analyzed by immunoblotting. (B) Raw264.7 cells were either left untreated or treated with the indicated TLR ligand for 6 h. Total RNA was isolated and analyzed by regular PCR. (C and D) Sub-confluent cultures of J774.A1 cells were either left untreated or treated with the TLR3 ligand (polyI:C ; 10 μg/ml) (C) or TLR4 ligand (LPS; 100 ng/ml) for the indicated time. Cells were harvested and total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bolland S, Ravetch JV. Spontaneous autoimmune disease in FcγRIIB-deficient mice results from strain-specific epistasis. Immunity. 2000;13:277–285. - PubMed
    1. Chen J, Panchanathan R, Choubey D. Stimulation of T cells up-regulates expression of Ifi202, an interferon-inducible lupus susceptibility gene, through activation of JNK/c-Jun pathway. Immunol Lett. 2008;118:13–20. - PMC - PubMed
    1. Choubey D, Duan X, Dickerson E, Ponomareva L, Panchanathan R, Shen H, Srivastava R. Interferon-inducible p200-family proteins as novel sensors of cytoplasmic DNA: role in inflammation and autoimmunity. J Interferon Cytokine Res. 2010;30:371–380. - PMC - PubMed
    1. Choubey D, Kotzin BL. Interferon-inducible p202 in the susceptibility to systemic lupus. Front Biosc. 2002;7:e252–262. - PubMed
    1. Choubey D, Lengyel P. Interferon action: cytoplasmic and nuclear localization of the interferon-inducible 52-kD protein that is encoded by the Ifi200 gene from the gene 200 cluster. J Interferon Res. 1993;13:43–52. - PubMed

Publication types

MeSH terms

-