Kynurenine

Start with at least 1 × 10⁶ cells per condition, to obtain at least 200,000 events analysed by flow cytometry at the end of the protocol. Conditions should include: the test samples (treated with kynurenine); background fluorescence control samples (matched samples, not treated with kynurenine, thus allowing for identification of cells exhibiting fluorescence above background); specificity controls such as System L blocked samples (BCH-treated, or Leu-treated), or uptake performed on ice (4 °C) (to determine transported kynurenine as opposed to surface binding); positive controls such as cell treatments driving high expression of System L transport. If required, surface cell antibody staining should be performed prior to uptake assay protocol. This may be performed at room temperature or 4 °C, however test samples must be warmed to 37 °C, e.g. in water bath, prior to kynurenine uptake. (As with all multi-parameter flow cytometry, appropriate antibody staining controls must also be performed.) Samples can be fixed immediately after uptake assay by addition of 4% (vol/vol) paraformaldehyde (PFA; to a final concentration of 1%) for 30 min at room temperature.
For kynurenine uptake assay; pre-warm kynurenine (800 μM, in HBSS), BCH (40 mM, in HBSS) and lysine (20 mM, HBSS) and HBSS to 37 °C. After surface antibody staining of samples, resuspend cells in 200 μl warmed HBSS (1–5 × 10⁶ cells in FACS tubes, or scale accordingly into plates). Keep cells in water bath at 37 °C. Add 100 μl of HBSS, or BCH or lysine to appropriate samples. Add 100 μl HBSS to no kynurenine controls (final volume 400 μl). Finally, add 100 μl kynurenine to appropriate samples. Stop uptake after 4 min by adding 125 μl 4% PFA for 30 min at room temperature, in the dark. The final concentrations for uptake assay are: 200 μM kynurenine; +/-10 mM BCH; +/-5 mM lysine. After fixation, wash cells twice in PBS/0.5% BSA and resuspend in PBS/0.5% BSA prior to acquisition on flow cytometer. The 405 nm laser and 450/50 BP filter are used for kynurenine fluorescence detection.
To monitor kynurenine uptake in live cells, acquire data on flow cytometer immediately following addition of kynurenine and plot fluorescence against time.

Detailed methods for anthropometric measurements, assessment of blood, stool, and urine biomarkers and statistical methods are provided in S1 File. As shown in Table 1, 375 children were enrolled and had initial anthropometry and were invited to provide a fecal specimen, have a L/M absorption test and to have blood obtained for testing potential biomarkers of intestinal barrier function, intestinal and systemic inflammation and injury repair. Shown in Table 2 are the 13 plasma, 4 fecal and urinary tests with at least 274 valid results obtained from within 1 month of enrollment. Markers of barrier function included urinary L/M absorption, fecal A1AT, Reg-1, and plasma LPS (acute levels, by neutralization luminescence [LUM] assay), IgG and IgA anti-LPS anti-FliC, zonulin, I-FABP and, with limited amounts of plasma available, claudin-15. Fecal MPO and neopterin were tested to assess intestinal inflammation, and plasma SAA, sCD14, LBP, citrulline, tryptophan and kynurenine were measured to assess systemic inflammatory responses and as potential predictors of intestinal injury repair. These 18 markers of intestinal barrier disruption and inflammation had adequate samples for assay in at least 274 (to 321) children at enrollment for study of associations with enrollment stunting (or wasting). Of several fecal biomarkers that have been used to assess intestinal inflammation, we selected myeloperoxidase (MPO) as our main test fecal marker of acute neutrophilic intestinal inflammation because of its ready availability, relatively less influence by age or breast-feeding and its potential use in murine models of enteropathy. In a separate analysis comparing fecal MPO, lactoferrin, calprotectin and lipocalin-2, we found that they correlate well with each other [33 (link)].
In addition, followup anthropometry at 2–6 months after initial sampling enabled us to assess these biomarkers as predictors of subsequent growth. We also assessed their associations with each other as well as with separately studied comparisons of fecal lactoferrin, calprotectin and lipocalin, and plasma hsCRP, and claudin-15. Repeated Measures MANOVA analyses were used to model mean growth from Study Start to follow-up anthropometric measurement obtained within 2 to 6 months later by children with low and high levels of each biomarker while controlling for child age and gender. Interaction effects of gender with biomarker were also assessed for each model. Similarly interaction effects of study start growth status (stunting present or not) of biomarkers on growth were assessed, although these results were not significant.
Missing data analysis (Pearson correlations between known characteristics of participants retained or failed to return for followup or were missing biomarker results because of sample limitations) showed that children with caregivers having fewer years of education were missing more of three biomarkers (A1AT, Reg1, MPO, r ≤ -0.18) compared to their more educated counterparts. Boys were disproportionately missing A1AT results (r = 0.15 p = 0.003), older children were more likely to be missing Neopterin (r = 0.12 p = 0.017) while nonbreastfeeding (r = -0.10, p = 0.37) and more wasted children (r = -0.12, p = 0.026) are missing more L/Ms. To account for bias in followup, multiple regression was used to impute missing data for all biomarkers with at least 274 (73% of 375) complete data. Principle Components Analyses (PCA) were then used to combine related combinations of 11 barrier function, 2 intestinal inflammation and then 7 systemic biomarkers. These related sets of biomarkers were then correlated among each other as well as to children’s growth status and growth trajectory initially using Partial Pearson correlations and ultimately using Multiple Regression. All analyses predicting anthropometric status and growth trajectories included controlling for child age and gender.

As our co-culture assays were performed in the presence of NK-92 complete medium, we measured the concentration of L-KYN when GES-1 or SGC-7901 cells were cultured with this medium, which showed no influence on the viability and proliferation of these cells (data not shown). 5 × 10⁵ GES-1 or SGC-7901 cells were seeded in the plate with 1 ml NK-92 complete medium. Five milligram per milliliter tryptophan (half of the content in MEMα medium) was supplemented into the culture medium every 24 h after beginning. Then the concentration of L-KYN in the culture supernatant was measured with the Kynurenine ELISA Kit (abcam), following the manufacture’s instruction.

L-Kynurenine (L-KYN), CH-223191, ferrostatin-1 (Fer-1), necrostatin-1 (Nec-1), Z-VAD-FMK (Z-VAD), VX-765 and GPX4-IN-3 were all purchased from MedChemExpress. We conducted preliminary experiments to determine the effective concentration and safety of each inhibitor on NK cells. For the culture supernatant treatment assays, the GES-1 or SGC-7901 cells were changed to be cultured with NK-92 complete culture medium for 24 h, which contained the highest concentration of L-KYN as determined in Fig. 1A. The supernatants were collected and mixed with 20% fresh medium, which were then used to treat NK cells for 24 or 48 h.Fig. 1

IDO produced L-KYN from GC cells impairs NK viability in vitro. A The concentration of L-KYN in the culture supernatant of SGC-7901, MGC-803 or GES-1 cells measured by ELISA. B The ratio of the remaining NK-92 cell number relative to the originally seeded cell number in the non-contract co-culture system with GES-1, MGC-803 or SGC-7901 cells. The control group referred to that the NK-92 cells were seeded into the co-culture wells alone. C The flow cytometric analysis for the proportion of FVD⁺ NK-92 cells in the co-culture system. The pictures above showed the representative results. D The flow cytometric results that showed the proportion of FVD⁺ NK-92 cells when stimulated with the culture supernatant of GES-1, MGC-803 or SGC-7901 cells. The control group referred to the medium without culturing with cells. E The proportion of FVD⁺ NK-92 cells when treated with gradient concentrations of L-KYN for 24 or 48 h. F and G The proportion of FVD⁺ NK-92 cells when co-cultured with GES-1, SGC-7901^Con, SGC-7901^IDO-KO (F), or treated with the culture supernatant of SGC-7901^Con or SGC-7901^IDO-KO (G) for 48 h. The control group in (F) indicated the NK-92 cells were only treated with medium. H and I The proportion of FVD⁺ primary human NK (hNK) cells when treated with gradient concentrations of L-KYN for 48 h (H) or the culture supernatant from GES-1, SGC-7901^Con and SGC-7901^IDO-KO cells (I). All the results were replicated in 3 (H and I) or 4 (A - G) independent experiments. * refers to the p-value of group SGC-7901 vs GES-1 and # refers to the group SGC-7901 vs MGC-803 (A - D). * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001