CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract

Background: Adaptive immune cells, including CD4 + CD69 + and CD4 + CD25 + FoxP3 + regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4 + CD25 + FoxP3 + Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4 + CD69 + and CD4 + CD25 + FoxP3 + T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4 + CD69 + T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4 + CD69 + T cells and reduced numbers of CD4 + CD25 + FoxP3 + Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Metformin improves FOXP3 mRNA expression through suppression of interferon gamma levels in pristane-induced murine models of lupus

“…Furthermore, it seems that the reduction of IFNγ explains the increase in FOXP3 mRNA expression rather strongly, as shown by the R 2 value of 0.616 (p=0.001). The above result was consistent with several studies in pristane induced models; a decrease in FOXP3+ T-cells and increase in CD4+CD69+ T-cells coincide with an increase in IFNγ levels in intraperitoneal fluid 20 , 23 .…”

“…The expression of FOXP3 mRNA in pristane induced murine models is a marker for Treg cell activity. In a study by Peixoto et al ., it was shown that on day 90 and 120 post induction there was a decrease in CD4+CD25+FOXP3+ (Treg) cell count in peripheral blood samples 20 . These authors also showed that the reduction in Treg count was correlated with an increase in IFNγ (p=0.017), TNFα (p=0.043) and TGF-β1 (p=0.038).…”

Interferon Genes Are Influenced by 17β-Estradiol in SLE

“…In the present study, we showed that 17β-estradiol treatment increased the expression of early activation marker CD69 and IFNγ on CD3 + T cells ( Figure 7 ). Others have shown increased numbers of CD4 + CD69 + T cells with altered expression of interleukins and suggested a correlation with loss of self-tolerance in lupus mice ( 61 ). Of clinical significance, a human study in lupus patients also found that CD69 + T cells are increased and defective in function ( 62 ).…”

Impaired Differentiation of Highly Proliferative ICOS+-Tregs Is Involved in the Transition from Low to High Disease Activity in Systemic Lupus Erythematosus (SLE) Patients

“…As both immunosuppressive CD4 + -Tregs and stimulating CD4 + -Tresps are involved [13,15,33,34], it seems likely that a misguided differentiation of either CD4 + -Tregs or CD4 + -Tresps or probably of both CD4 + -T-cell subsets causes an imbalance in the ratio of CD4 + -Tregs to CD4 + -Tresps in these patients. This balance has been intensively studied in recent years, providing contradictory results, and was shown to depend on the selection of the respective Treg/Tresp subsets [35][36][37][38][39].…”