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. 2023 Apr 14;24(8):7267.
doi: 10.3390/ijms24087267.

HIV-Differentiated Metabolite N-Acetyl-L-Alanine Dysregulates Human Natural Killer Cell Responses to Mycobacterium tuberculosis Infection

Baojun Yang  1   2   3 , Tanmoy Mukherjee  2   3 , Rajesh Radhakrishnan  2   3 , Padmaja Paidipally  2   3 , Danish Ansari  1   2   3 , Sahana John  1   2   3 , Ramakrishna Vankayalapati  2   3 , Deepak Tripathi  2   3 , Guohua Yi  1   2   3
Affiliations

HIV-Differentiated Metabolite N-Acetyl-L-Alanine Dysregulates Human Natural Killer Cell Responses to Mycobacterium tuberculosis Infection

Baojun Yang et al. Int J Mol Sci. .

Abstract

Mycobacterium tuberculosis (Mtb) has latently infected over two billion people worldwide (LTBI) and caused ~1.6 million deaths in 2021. Human immunodeficiency virus (HIV) co-infection with Mtb will affect the Mtb progression and increase the risk of developing active tuberculosis by 10-20 times compared with HIV- LTBI+ patients. It is crucial to understand how HIV can dysregulate immune responses in LTBI+ individuals. Plasma samples collected from healthy and HIV-infected individuals were investigated using liquid chromatography-mass spectrometry (LC-MS), and the metabolic data were analyzed using the online platform Metabo-Analyst. ELISA, surface and intracellular staining, flow cytometry, and quantitative reverse-transcription PCR (qRT-PCR) were performed using standard procedures to determine the surface markers, cytokines, and other signaling molecule expressions. Seahorse extra-cellular flux assays were used to measure mitochondrial oxidative phosphorylation and glycolysis. Six metabolites were significantly less abundant, and two were significantly higher in abundance in HIV+ individuals compared with healthy donors. One of the HIV-upregulated metabolites, N-acetyl-L-alanine (ALA), inhibits pro-inflammatory cytokine IFN-γ production by the NK cells of LTBI+ individuals. ALA inhibits the glycolysis of LTBI+ individuals' NK cells in response to Mtb. Our findings demonstrate that HIV infection enhances plasma ALA levels to inhibit NK-cell-mediated immune responses to Mtb infection, offering a new understanding of the HIV-Mtb interaction and providing insights into the implication of nutrition intervention and therapy for HIV-Mtb co-infected patients.

Keywords: HIV–Mtb co-infection; Mtb latent infection (LTBI); N-acetyl-L-alanine; interferon-γ; metabolite; natural killer cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Plasma metabolic profiles of HIV+ patients (treatment-naïve and ART-treated): (A) Scatter plot showing partial least-square discriminant analysis (PLS-DA) of plasma metabolomic profiles. The two principal components explaining the highest variance were plotted on X and Y axis; (B) heatmap shows the top 60 differentially abundant metabolites identified in a comparison between healthy donors and HIV patients at 5% FDR; (C) fold change in abundance of 6 selected metabolites computed from metabolome profiles in a comparison between HIV patients and healthy donors. The bars represent log2 fold change in HIV patients compared with healthy donors. The asterisks *, **, and *** denote FDR < 0.05, FDR < 0.01, and FDR < 0.001, respectively.
Figure 2
Figure 2
Effect of metabolites on cytokine production by PBMCs of LTBI+ donors. PBMCs of healthy LTBI+ donors were cultured with or without γMtb (10 μg/mL) and in the presence or absence of different concentrations of ALA and other metabolites, as mentioned in the Methods section. After 72 h, culture supernatants were collected, and cytokine levels were measured using ELISA: (A) IFN-γ, (B) TNF-α, (C) IL-1β, (D) IL-10, (E) IL-13, and (F) IL-17A. In (A–E), six donors were collected, while in (F), five donors were collected. Paired t-tests were used to compare the differences between untreated and treated PBMCs from the same samples. The mean values and SDs are shown, and the significant p values are shown (p < 0.05).
Figure 3
Figure 3
IFN-γ and TNF-α gene expression profile of various immune cells in γMtb-cultured PBMCs of LTBI+ donors. PBMCs of healthy LTBI+ donors were cultured with or without γMtb (10 μg/mL) and in the presence or absence of ALA, as mentioned in the Methods section. After 48 h, various immune cells were isolated via flow sorting, RNA was collected, and real-time PCR analysis was performed to determine IFN-γ and TNF-α gene expression: (A) IFN-γ transcription levels in different cell types; (B) TNF-α transcription levels in different cell types. PBMCs from five LTBI+ donors were used for the study. Paired t-tests were used to compare the differences between untreated and treated PBMCs from the same samples. The significant p values are shown (p < 0.05). ns: not significant.
Figure 4
Figure 4
IFN-γ levels of various immune cells in γMtb-cultured PBMCs of LTBI+ donors. PBMCs of healthy LTBI+ donors were cultured with or without γMtb (10 μg/mL) and in the presence or absence of ALA, as mentioned in the Methods section. After 48 h, intracellular staining was performed to determine IFN-γ levels of various immune cell populations: (A–C) IFN-γ staining of a representative donor PBMC; the top, middle and bottom panels represent CD4+ T cells (A), CD8+ T cells (B), and NK cells (C), respectively. The percentages of IFN-γ-positive cells are shown; (D) collective summary of IFN-γ-positive CD4+, CD8+, and CD56+ cells of five donors. Paired t-tests were used to compare the differences between untreated and treated PBMCs from the same samples. The significant p values are shown in the figure.
Figure 5
Figure 5
ALA inhibits NF-κB, AP1, GZMA, and GZMB gene expression in γMtb-cultured NK cells. PBMCs of healthy LTBI+ donors were cultured with or without γMtb (10 μg/mL) and in the presence or absence of ALA, as mentioned in the Methods section. After 48 h, NK cells were isolated by flow sorting, RNA was collected, and real-time PCR analysis was performed to determine various signaling molecules and transcription factors. PBMCs from six LTBI+ donors were used for this experiment. Paired t-tests were used to compare the differences between untreated and treated PBMCs from the same samples. The significant p values are shown (p < 0.05); ns: not significant, p > 0.05.
Figure 6
Figure 6
ALA inhibits antimicrobial peptide expression and enhances Atg3 expression in γMtb cultured NK cells. PBMCs of healthy LTBI+ donors were cultured with or without γMtb (10 μg/mL) and in the presence or absence of ALA, as mentioned in the Methods section. After 48 h, NK cells were isolated via flow sorting, RNA was collected, and real-time PCR analysis was performed to determine various death pathway gene expressions. PBMCs from six LTBI+ donors were used for this experiment. Paired t-tests were used to compare the differences between untreated and treated PBMCs from the same samples. The p values are shown in the figure.
Figure 7
Figure 7
ALA treatment switches NK cells to an energetically quiescent state. PBMCs of healthy LTBI+ donors were cultured with or without γMtb (10 μg/mL) and in the presence or absence of ALA (3 µM), as mentioned in the Methods section. After 48 h, NK cells were isolated via magnetic cell sorting and subjected to extracellular flux analysis using an Agilent Seahorse XFe96 analyzer. NK cell glycolysis was measured with the sequential addition of glucose, oligomycin, and 2-DG. Similarly, OXPHOS parameters were measured in isolated NK cells after the addition of oligomycin, FCCP, and rotenone/antimycin: (A,B) mitochondrial OCR and (C,D) ECAR were measured; graphs (A,C) show mitochondrial OCR (A) and ECAR (C) in real time as kinetic graphs; graph (B) shows the collective OXPHOS parameters of basal respiration, maximum respiration, ATP production, and spare respiratory capacity as bar graphs. The p values were derived using an unpaired 2-tailed independent t-test. The mean values and SEMs are shown; (D) bar graphs show the collective glycolytic parameters such as basal glycolysis, glycolytic capacity, and glycolytic reserve. In (B,D), for all panels, the data are presented as mean ± SEM (n = 12; 4 statistical replicates from 3 individual donors); each parameter between treatments was compared using independent Student’s t-test: * p < 0.05, ** p < 0.01, and **** p < 0.0001. ns: not significant.
Figure 8
Figure 8
Schematic representation of the potential mechanism of how ALA regulates the immune response of the PBMCs from the LTBI+ individuals. The HIV-differentiated metabolite ALA targets NK cells and functions as a downregulator to reduce NK cell glycolysis, resulting in NK cell autophagy and the reduction in IFN-γ production.
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