Suppressed Th17 levels correlate with elevated PIAS3, SHP2, and SOCS3 expression in CD4 T cells during acute simian immunodeficiency virus infection

Abstract

T helper 17 (Th17) cells play an important role in mucosal immune homeostasis and maintaining the integrity of the mucosal epithelial barrier. Loss of Th17 cells has been extensively documented during human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. The lack of effective repopulation of Th17 cells has been associated with chronic immune activation mediated by the translocation of microbial products. Using ex vivo analysis of purified peripheral blood CD4 T cells from SIV-infected rhesus macaques, we show that the suppression of interleukin-17 (IL-17) expression correlated with upregulated expression of negative regulatory genes PIAS3, SHP2, and SOCS3 in CD4 T cells. Suppressed Th17 expression was accompanied by elevated levels of soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP) in the plasma during early stages of infection. Plasma viral loads rather than sCD14 or LBP levels correlated with acute immune activation. Additionally, we observed a significant increase in the expression of CD14 on peripheral blood monocytes that correlated with IL-23 expression and markers of microbial translocation. Taken together, our results provide new insights into the early events associated with acute SIV pathogenesis and suggest additional mechanisms playing a role in suppression of Th17 cells.

Fig 1

Plasma sCD14 and LBP levels are significantly elevated during acute SIV infection. (a) Plasma viral loads at days 7, 14, and 35 p.i. The limit of detection was 30 copies/ml of plasma. (b) Percentage of total CD3⁺ CD4⁺ memory T cells in peripheral blood at days 0, 7, and 35 p.i. Memory CD4 T cells were identified on the basis of the expression of CD28 and CD95, with all memory CD4 T cells expressing CD95. (c and d) Plasma sCD14 (c) and plasma LBP (d) levels at days 0, 7, and 35 p.i.

Fig 2

IL-17 expression in CD4 T cells is significantly suppressed during acute SIV infection. (a) Gating strategy used to sort pure populations of peripheral blood CD4 T cells by fluorescence-activated cell sorting. PBMCs were labeled with anti-CD20, anti-CD8, and anti-CD14 and negatively sorted for CD4 T cells. An aliquot of the sorted cells was stained with anti-CD4 to confirm purity. SSC, side scatter. (b) IL-17 mRNA expression in purified peripheral blood CD4 T cells after short-term stimulation for 15 min with recombinant human IL-6. The fold change at days 7 and 35 p.i. relative to the levels for unstimulated control cells is shown. (c and d) IL-17 expression negatively correlates with LBP (c) and sCD14 (d) levels in plasma.

Fig 3

Expression of PIAS3, SHP2, and SOCS3 is significantly upregulated in CD4 T cells during acute SIV infection. (a) STAT3, PIAS3, SHP2, and SOCS3 mRNA expression in purified peripheral blood CD4 T cells at days 7 and 35 p.i. after short-term stimulation for 15 min with recombinant human IL-6. The fold change at days 7 and 35 p.i. relative to the levels for unstimulated control cells is shown. (b to d) IL-17 expression negatively correlates with PIAS3 (b), SHP2 (c), and SOCS3 (d) levels in peripheral blood CD4 T cells.

Fig 4

CD14⁺ monocytes upregulate the density of CD14 expression and IL-23 mRNA levels during acute SIV infection. (a) Ex vivo expression of IL-6, IL-21, IL-23, and TGF-β in total PBMCs at day 7 and day 35 p.i. The fold change relative to the level at day 7 p.i. is shown. The line indicates day 7 p.i. as the baseline. (b) Gating strategy used to sort purified populations of CD14⁺ monocytes by fluorescence-activated cell sorting. (c) Ex vivo expression of TLR4 and IL-23 expression in purified populations of CD14⁺ monocytes at days 7 and 35 p.i. The fold change relative to the level at day 7 p.i. is shown. The line indicates day 7 p.i. as the baseline. (d) Histogram showing the density of CD14 expression from a representative animal at days 7 and 35 p.i. and data showing the MFI of CD14 expression on CD14⁺ monocytes at days 7 and 35 p.i. (e to g) The correlations between the plasma levels of sCD14 and IL-23 expression on CD14⁺ monocytes (e), plasma levels of LBP and IL-23 expression on CD14⁺ monocytes (f), and plasma levels of LBP and MFI of CD14 expression on CD14⁺ monocytes (g) at days 7 and 35 p.i. are shown.

Fig 5

CD3⁺ CD8⁺ Ki-67⁺ memory T cells significantly correlate with plasma viral loads but not sCD14 or LBP levels in plasma. (a) Percentage of CD3⁺ CD8⁺ Ki-67⁺ memory T cells in peripheral blood. (b to e) The correlations between the percentage of CD3⁺ CD8⁺ Ki-67⁺ memory T cells in peripheral blood and plasma sCD14 levels (b), plasma LBP levels (c), IL-17 expression in CD4 T cells (d), and plasma viral loads (e) are shown.