IL-17-producing innate lymphoid cells are restricted to mucosal tissues and are depleted in SIV-infected macaques - PubMed (original) (raw)

IL-17-producing innate lymphoid cells are restricted to mucosal tissues and are depleted in SIV-infected macaques

H Xu et al. Mucosal Immunol. 2012 Nov.

Abstract

Innate lymphoid cells (ILCs) are an emerging subset of lymphocytes involved in surveillance against virally infected cells. Here, we show CD3(-)CD8(high) lymphocytes in macaque blood include major subsets of ILCs including natural killer (NK) cells expressing CD16, NKp46, and NKG2A, but also populations of ILCs in mucosal tissues having different properties. One ILC subset secreted interleukin (IL)-17 (ILC17), but these were restricted to mucosal tissues. Some mucosal ILC17 cells expressed classical NK-cell markers, but little NKG2A or NKG2D. Some ILC17 cells secreted IL-22 and tumor necrosis factor-α, but few produced interferon (IFN)-γ or contained granzyme B. IL-17 production by ILCs was induced by IL-6, transforming growth factor-β, and IL-23. Further, simian immunodeficiency virus (SIV) infection resulted in a significant loss of ILC17 cells, especially in the jejunum, which persisted throughout SIV infection. These findings indicate that ILC17 cells may be involved in innate mucosal immune responses, and their loss may contribute to loss of intestinal mucosal integrity and disease progression in human immunodeficiency virus (HIV)/SIV infection.

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Figures

Figure 1

Figure 1. Phenotypic characterization of innate lymphoid cells in peripheral blood of normal rhesus macaques

(a) Representative gating strategy to define ILCs in PBMCs. Macaque ILCs were defined as CD3−CD8αhigh gated lymphocytes. (b) Representative dot plot depicting the expression of CD20, HLA-DR, CD14, CD11c, CD163 and CD11b+CD11int on CD3−CD8αhigh (R1) as compared to CD3−CD8αlow/− (R3) subsets. (c) Representative histogram showing expression of classical NK markers, CD16, CD56, NKp46, NKG2A and NKG2D on CD3−CD8αhigh (R1), CD3+CD8+ (R2), CD3-CD8−/intermediate (R3), and CD3+CD4+ (R4) subsets from peripheral blood. (d) Expression of classical NK markers on CD3−CD8αhigh (R1), CD3+CD8+ (R2), CD3-CD8−/intermediate (R3), and CD3+CD4+ (R4) subsets from peripheral blood (n=8). Examples are representative of 8 naive animals. *, p<0.001; **, p<0.05, compared between CD3−CD8high and other three subsets, respectively.

Figure 2

Figure 2. Comparison of anti-CD16 antibody clones for cross-reactivity with rhesus macaques, and distribution of NK cells in lymphoid tissues

(a) Cross-reactivity and comparison of whole blood (WB) and washed PBMC from the same animals using different anti-CD16 antibody clones in chronically SIV infected rhesus macaques (n=8). Note only the DJ130c clone does not demonstrate differential staining between washed PBMC and WB. (b) Tissue distribution of classical NK cell subsets (CD16+, CD56+, NKG2A+) in naïve rhesus macaques (n=5). *, p<0.05 between using 3G8 and DJ130c clones in PBMC samples (a) or blood compared with other tissues (b); **, p<0.01 between using 3G8 and DJ130c clones in whole blood; #, p<0.01, compared with peripheral blood; ##. p<0.05 between liver and other tissues (except blood), respectively.

Figure 3

Figure 3. Distinct expression of surface molecules on innate lymphoid cells between mucosal tissues and peripheral blood

(a) Representative histogram displaying different expression of molecules on ILCs from PBMCs and jejunum lamina propria. (b) Statistical comparison of expression between ILCs from PBMCs and jejunum lamina propria in naïve rhesus macaques (n=12). *, p<0.05.

Figure 4

Figure 4. Innate lymphoid cells that secrete IL-17 are restricted to mucosal tissues in rhesus macaques

(a) Jejunum dot plots gated on IL-17-secreting lymphocytes (left) contain both CD3+ and CD3− subsets in jejunum after PMA/Ionomycin stimulation. (b) Comparison of IL-17-producing CD4+ (Th17), CD8+ T cells, and CD3−CD8high (ILCs) in various lymphoid tissues, including peripheral blood, jejunum, colon, spleen, tonsil and duodenum. Noted that IL-17-secreting ILCs are restricted to mucosal tissues, with little to no expression of IL-17 from PBMC or spleen-derived ILCs.

Figure 5

Figure 5. Detection of ILC17 in jejunum tissues of normal rhesus macaques by confocal microscopy

(a) Jejunum from a normal macaque showing CD3+ (red), IL-17 (green) and CD8+ (blue) cells. The white arrows show ILC17 cells (CD3−CD8+IL-17+); the yellow arrows demonstrate Th17 cells (CD3+CD8−IL-17+), and the blue arrows show Tc17 cells (CD3+CD8+IL-17+). Scale bar = 10 μm. (b) Relative percentages of Th17 , Tc17, and ILC17 cells gated through total IL-17+ lymphocytes in the jejunum of normal animals as assessed by flow cytometry.

Figure 6

Figure 6. Phenotyping IL-17-producing ILCs for classical NK cell markers and cytokine secretion in peripheral blood and mucosal tissues of normal rhesus macaques

(a) Expression of classical NK cell markers on ILC17 cells in the peripheral blood, jejunum and colon. Note there was little to no production of IL-17 from NKG2A+, or NKG2D+ ILCs. (b) Cytokine secretion of ILC17 cells isolated from jejunum after PMA/ionomycin stimulation. Note ILC17 cells also secrete pro-inflammatory (TNF-α), and innate (IL-22) cytokines, but express little to no IFN-γ or Granzyme B. (c) Response of jejunum ILC17 cells to various cytokine and LPS stimulation.

Figure 7

Figure 7. Effects of SIV infection on blood and jejunum-derived innate lymphoid cells (CD3−CD8high) or its subpopulations in rhesus macaques

(a and b) Prospective analysis of changes in percentage (a) and absolute numbers (b) of total CD3−CD8high ILCs in peripheral blood after SIV infection. (c, e–g, and i–k) Dynamic of ILCs and their subpopulation in blood and jejunum ILCs after SIV infection (Naïve, n=55; 7dpi, n=30; 14dpi, n=23; 21dpi, n=26; 28dpi, n=26; 35dpi, n=18; chronic, n=23). (d, h and i) Proliferation (Ki-67+) of circulating or intestinal ILCs after SIV infection (Naïve, n=24; 7dpi, n=5; 14dpi, n=5; 21dpi, n=4; 28dpi, n=3; 42dpi, n=4; chronic, n=14). *, p<0.05, compared with uninfected normal animals.

Figure 8

Figure 8. Reduction of intestinal mucosal ILC17 cells after SIV infection of rhesus macaques

(a) Dot plot demonstrating ILC17 cells after gating on CD3−CD8high subsets from the jejunum and colon from a representative normal and AIDS macaque. (b) Loss of intestinal ILCs occurs after SIV infection (Normal, n=10; acute, n=6; chronic, 12; AIDS, n=4). *, P<0.05. (c) Correlation between percentages of ILC17 cells and CD4+ T cells or (d) Th17 cells in jejunum during SIV infection (c, n=18; d, n=28).

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