Gut inflammation and indoleamine deoxygenase inhibit IL-17 production and promote cytotoxic potential in NKp44+ mucosal NK cells during SIV infection

Abstract

Natural killer (NK) cells are classically viewed as effector cells that kill virus-infected and neoplastic cells, but recent studies have identified a rare mucosal NK- cell subpopulation secreting the TH17 cytokine IL-22. Here, we report identification of 2 distinct lineages of mucosal NK cells characterized as NKG2A(+)NFIL3(+)RORC(-) and NKp44(+)NFIL3(+)RORC(+). NKG2A(+) NK cells were systemically distributed, cytotoxic, and secreted IFN-γ, whereas NKp44(+) NK cells were mucosae-restricted, noncytotoxic, and produced IL-22 and IL-17. During SIV infection, NKp44(+) NK cells became apoptotic, were depleted, and had an altered functional profile characterized by decreased IL-17 secretion; increased IFN-γ secretion; and, surprisingly, increased potential for cytotoxicity. NKp44(+) NK cells showed no evidence of direct SIV infection; rather, depletion and altered function were associated with SIV-induced up-regulation of inflammatory mediators in the gut, including indoleamine 2,3-dioxygenase 1. Furthermore, treatment of NKp44(+) NK cells with indoleamine 2,3-dioxygenase 1 catabolites in vitro ablated IL-17 production in a dose-dependent manner, whereas other NK-cell functions were unaffected. Thus lentiviral infection both depletes and modifies the functional repertoire of mucosal NK cells involved in the maintenance of gut integrity, a finding that highlights the plasticity of this rare mucosal NK-cell population.

Figure 1

Gut-associated lymphoid tissues contain 2 distinct lineages of NK cells. (A) Representative gating strategy to identify NKG2A⁺ and NKp44⁺ NK cells among live mononuclear cells in tissues in rectal mucosa specimens. (B) Flow cytometry plots demonstrating disparate expression of CD56 and CD16 on NKG2A⁺ and NKp44⁺ NK cells. (C) Representative histogram overlays depicting expression of NKp46, CD8α, CD117, CD127, CCR6, and CXCR3 on the 2 NK-cell subpopulations. Isotype-matched controls are shown in gray. RT-PCR analysis was used to quantify mRNA transcripts of transcription factors (D) and regulatory cytokines IL-22 and CCL20 (E) in NKG2A⁺ and NKp44⁺ NK cells sorted from rectal mucosa of normal macaques. Mann-Whitney U test (*P < .05). (F) Flow cytometry plots depicting CD107a and intracellular cytokine responses in the 2 NK-cell subpopulations after mitogen stimulation. Polychromatic flow cytometry and ICS experiments are representative of 8-16 animals per group; molecular analyses show geometric means ± SEM of 4 animals per group. (G) Flow cytometry plots comparing distribution of NKG2A⁺ and NKp44⁺ NK cells in blood and various tissues of rhesus macaques using the gating strategy shown in panel A; data are representative of > 12 animals. MLN indicates mesenteric lymph node; and PLN, peripheral lymph node.

Figure 2

Chronic SIV infection induces numeric and phenotypic alterations in gut-associated NK cells. (A) Frequencies of NKp44⁺ and NKG2A⁺ NK cells among live CD45⁺CD3⁻ mononuclear cells in rectal biopsies of naive and SIV-infected macaques. Horizontal lines indicate medians of 16-18 animals per group. (B) Absolute numbers of NKp44⁺, NKG2A⁺, and CD4⁺ and CD8⁺ T cells per milligram of rectal biopsy tissue in naive and chronically SIV-infected macaques were enumerated using a bead-normalized flow cytometry assay. (C) Expression of intracellular Ki67 and caspase-3 or cell surface annexin V binding was determined on NKp44⁺ and NKG2A⁺ NK cells. (D) Median fluorescence intensities (MFI) of cell surface molecules on NKG2A⁺ and NKp44⁺ NK cells in naive and SIV-infected macaques. Bars represent means ± SEM of 8-14 animals per group. Asterisks above columns reflect naïve versus SIV statistical comparisons, whereas those below the axes reflect NKG2A⁺ versus NKp44⁺ NK cells statistical comparisons in normal animals. Mann-Whitney U tests were used for naïve versus SIV comparisons and Wilcoxon matched pairs tests were used to compare NKG2A⁺ and NKp44⁺ NK cells (*P < .05, **P < .01, ***P < .001).

Figure 3

Mucosal NK cells are not infected by SIV in vivo. (A) Cell surface expression of CD4 and CCR5 on mucosal NKG2A⁺ and NKp44⁺ NK cells. Bars represent means ± SEM of 8 animals per group. Mann-Whitney U tests were used for naïve versus SIV comparisons and Wilcoxon matched pairs tests were used to compare NKG2A⁺ and NKp44⁺ NK cells (*P < .05, **P < .01, ***P < .001). (B) RT-PCR analysis was used to quantify SIV RNA in NKG2A⁺ and NKp44⁺ NK cells and CD4⁺ T cells sorted from rectal mucosa of normal and SIV-infected macaques. The lower limit of detection of the assay was 3 SIV RNA copies/1000 cells and is indicated by the dashed bar.

Figure 4

Chronic SIV infection alters functional profiles of gut NK cells. (A) Colorectal mononuclear cells were stimulated with phorbol 12-myristate 13-acetate/ionomycin for 12 hours and then IL-17, IFN-γ, and TNF-α production and CD107a expression were measured on NKG2A⁺ and NKp44⁺ NK cells in naive and SIV-infected macaques. The monofunctional profiles of each subpopulation were determined by expressing each response as a proportion of the total cell population. The means ± SEM for 12 animals per group are shown. (B) Multiparametric analyses on the data shown in Figure 6A were performed with SPICE 5.0 software. Pies indicate means of 12 animals per group. Tables show results of 1-sided permutation tests comparing each of the pies as calculated by SPICE; P < .05 are considered significant and are highlighted in yellow. (C) Intracellular perforin expression was determined in NK cells ex vivo; bars represent means ± SEM for 13-16 animals per group. (D) Expression of CCL4 (MIP-1β) was determined by ICS. (E) Quantitative RT-PCR analysis of constitutively expressed CCL3 (MIP-1α) and CCL5 (RANTES) in NKG2A⁺ and NKp44⁺ NK cells sorted from rectal biopsies of normal macaques. Mann-Whitney U tests were used for naïve versus SIV comparisons, and Wilcoxon matched pairs tests were used to compare NKG2A⁺ and NKp44⁺ NK cells (*P < .05, **P < .01, ***P < .001. MFI indicates median fluorescence intensity.

Figure 5

IDO1 is up-regulated in the gut mucosa of SIV-infected macaques and is associated with a loss of NKp44⁺ NK cells. (A) Quantitative RT-PCR analysis of cytokine transcripts was performed on rectal biopsies collected from naive and SIV-infected macaques. Mann-Whitney U tests were used for naïve versus SIV comparisons (*P < .05, **P < .01, ***P < .001). (B) Relative expression of IDO1 in rectosigmoid biopsies was correlated with plasma viral loads (top left) and absolute numbers of NKp44⁺ and NKG2A⁺ NK and CD4⁺ and CD8⁺ T cells quantified in biopsy specimens of the same animals as shown in Figure 4B. P values < .05 are considered significant.

Figure 6

Increased IDO1 expression in the gut mucosa is associated with modulation of IL-17 and IFN-γ secretion by NKp44⁺ cells. (A) Monofunctional analyses of stimulated NKp44⁺ NK cells as shown in Figure 6A were correlated with relative expression values of IDO in whole biopsy specimens of the same animals. P values < .05 are considered significant. (B) Multiparametric analyses of NKp44⁺ NK cells function using SPICE 5.0 for individual naive and SIV-infected animals. Corresponding relative expression (RE) of IDO1 and plasma viral loads (VL) of infected animals are shown.

Figure 7

3-HAA suppresses IL-17 production by NKp44⁺ NK cells in vitro in a dose-dependent manner. Mononuclear cells isolated from mesenteric lymph nodes of SIV-naive rhesus macaques were cultured in the presence of increasing concentrations of 3-HAA for 24 hours and then mitogen-stimulated. NKG2A⁺ (A) and NKp44⁺ (B) NK cells were analyzed functionally as shown in Figures 1 and 6. Means ± SEM for 3 animals are shown.