Fig 2: Pseudomonas aeruginosa depletion restores CRAMP production and dampens GIE AAnimal protocol.BThe levels of CRAMP in serum (n = 8).CThe levels of CRAMP in duodenum (n = 6).DRepresentative images of intestinal damage by H&E staining. Scale bar: 200 µm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6).EWestern blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6).FIntestinal permeability was assessed by measuring FITC-Dextran (n = 6).GThe frequency of NKG2D+ IELs among CD3+CD4+ population (n = 8).HThe frequency of CX3CR1+ macrophages among CD45+CD11b+ population (n = 8).IThe percentages of three subsets of CD45+CD11c+ DCs (n = 8).J–LThe percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) ROR?t+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data (B–F) were representative and were the mean ± SD from three independent experiments. Data (G–L) were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Source data are available online for this figure.

Fig 3: CRAMP modulates intestinal immune disorder in GIE A Il15 expression in duodenal epithelial cells ex vivo (n = 8).BThe frequency of NKG2D+ IELs among CD3+CD4+ population (n = 8).CThe frequency of CX3CR1+ macrophages in CD45+CD11b+ population and ALDH activity in CD45+CD11b+CX3CR1+ macrophages in lamina propria (n = 8).DThe percentages of three subsets of CD45+CD11c+ DCs and ALDH activity in CD45+CD11c+ DCs in lamina propria (n = 8).E–GThe percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) ROR?t+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data in A were representative and were the mean ± SD from two independent experiments. Data (B–G) were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Source data are available online for this figure.

Fig 4: NKG2D Antibody Block Attenuates Effect of IL-2 Complex Treatment on Sensory Recovery after Partial Sciatic Nerve Crush, Related to Figure 6(A) Adult male C57BL/6 mice received a total of 200 µg anti-mouse NKG2D (CX5 clone) (n = 3) or IgG1k isotype control (n = 3) via retroorbital injection (i.v.) over 4 days (Ogasawara et al., 2004): 50 µg (Day 0), 50 µg (Day 2), 100 µg (Day 4). One day later (Day 5) PBMCs were isolated and labeled with PE-conjugated anti-mouse NKG2D or isotype controls, as well as cell surface NK cell markers NKp46 and DX5. Flow cytometry data were gated on NKp46+DX5+ lymphocytes from 70,000 events.(B) Histogram of NKG2D-PE fluorescence labeling on NKP46+DX5+ gated lymphocytes relative to isotype-PE controls (gray filled). Blue, IgG1k isotype control injection; Red, NKG2D blocking antibody injection.(C) Quantification of mean NKG2D immunofluorescence (IgG isotype subtracted) in NKp46+DX5+ cell population. Student’s t test, t = 9.763, p = 0.0006; n = 3 mice per treatment.(D) Daily pinprick responses in wild-type mice which received either blocking anti-NKG2D antibody (n = 12) or IgGk1 isotype control (n = 7) on days 0, 2 and 4 following partial (moderate) crush of the sciatic nerve. All mice were treated with IL-2/anti-IL-2 antibody complex i.p. daily for four consecutive days from the evening of day 2 (arrows). Two-way ANOVA. Effect of antibody: F(1,187) = 8.03, **p = 0.0051).(E) Area under the curve measurements. Days 1-4: t = 0.1739, p = 0.8640; Days 5-10: t = 2.13, p = 0.0451; Days 11-15: t = 1.059, p = 0.3044. Student’s unpaired t test.(F) Mechanical sensitivity thresholds of ipsilateral (injured: U = 12.00 p = 0.0122, Mann-Whitney U test) and contralateral (uninjured: U = 39.00, p = 0.8303, Mann-Whitney U test) hind paw 16 days after partial crush injury.

Fig 5: Isolation, Enrichment, and Activation of NK Cells from Adult Mouse Spleen and Cytotoxicity of IL-2-Stimulated NK Cells against Embryonic DRG Neurons, Related to Figure 1(A) Intracellular staining of granzyme B of freshly isolated (left) and IL-2 stimulated (right) NK cells.(B) Flow cytometry plot of splenic lymphocytes. NKp46+DX5+ NK cells (top-right quadrant) in whole spleen homogenate (left) and after MACS enrichment by negative selection (right). NK cells require cell contact to mediate toxicity against embryonic DRG neurons.(C) Representative immunostaining images of embryonic DRG neurons (ß-tubulin III, magenta) after 4 h co-culture with IL-2 stimulated NK cells (NKp46, green) either in direct contact or separated by a transwell membrane (0.4 µm pores).(D) ELISA detection of granzyme B in media of NK cell-DRG co-cultures. One way ANOVA, F(2,6) = 0.7248, p = 0.5225. n = 3 experimental repeats. Granzyme B was not detected in cultures containing unstimulated NK cells (data not shown).(E) NKG2D receptor contributes to NK cell-mediated lysis of embryonic DRG neurons. Cytotoxicity of IL-2 stimulated NK cells against embryonic DRG neurons after 4 h co-culture in the presence of blocking anti-NKG2D antibody or IgG control antibody. Two-way ANOVA: Effect of antibody treatment, F(1,4) = 21.21, p = 0.0100. n = 3 experimental repeats.