Activation of NLRX1 by NX-13 Alleviates Inflammatory Bowel Disease through Immunometabolic Mechanisms in CD4+ T Cells

Abstract

Inflammatory bowel disease (IBD) is a complex autoimmune disease with dysfunction in pattern-recognition responses, including within the NLR family. Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is a unique NLR with regulatory and anti-inflammatory functions resulting in protection from IBD in mouse models. NX-13 is an orally active, gut-restricted novel drug candidate that selectively targets and activates the NLRX1 pathway locally in the gut. In vitro and in vivo efficacy of NLRX1 activation by NX-13 was examined. Oral treatment with NX-13 alleviates disease severity, colonic leukocytic infiltration, and cytokine markers of inflammation in three mouse models of IBD (dextran sulfate sodium, Mdr1a^-/-, and CD45RB^hi adoptive transfer). Treatment of naive CD4⁺ T cells with NX-13 in vitro decreases differentiation into Th1 and Th17 subsets with increased oxidative phosphorylation and decreased NF-κB activation and reactive oxygen species. With stimulation by PMA/ionomycin, TNF-α, or H₂O₂, PBMCs from ulcerative colitis patients treated with NX-13 had decreased NF-κB activity, TNF-α⁺ and IFN-γ⁺ CD4⁺ T cells and overall production of IL-6, MCP1, and IL-8. NX-13 activates NLRX1 to mediate a resistance to both inflammatory signaling and oxidative stress in mouse models and human primary cells from ulcerative colitis patients with effects on NF-κB activity and oxidative phosphorylation. NX-13 is a promising oral, gut-restricted NLRX1 agonist for treating IBD.

Figure 1.

NX-13 binding to NLRX1 in situ. Splenocytes were cultured with NX-13 (1, 10, 50, 100, 200, 500 nM) for 3 h following which cells were exposure to acute thermal stress. Protein was isolated, NLRX1 content was assessed by Western blot and compared at a ratio of soluble to total (A). Melting temperature (B) was calculated by 5-parameter logistic fit of ratio of soluble to insoluble/denatured NLRX1. EC50 estimated to be 58 nM in situ by protection from denaturation.

Figure 2.

In vitro responses to NX-13. Naïve CD4+ T cells were obtained from wild-type spleens and differentiated into Th1 cells in vitro in presence of NX-13 (0 – 0.5 μM) (A). After differentiation LDH activity (B), glucose uptake (C), proliferation (D), NF-κB activity (E) and reactive oxygen species (F) were measured. Representative plots of Th1 populations in untreated (G) and 0.5 μM NX-13 (H). Representative histogram (I) of untreated (orange) and 0.05 μM NX-13 (blue). Expression of mt-Co3 (J), mt-Nd3 (K), Odgh (L), Idh2 (M), Gpx1 (N), Gstm1 (O), Txnrd1 (P), and Eno1 (Q) were measured by qRT-PCR. Data presented as mean ± SEM (n = 6). Asterisks mark significance (p ≤ 0.05).

Figure 3.

Efficacy of NX-13 in DSS colitis. Wild-type mice were challenged with DSS and treated daily with oral NX-13 (0 – 20 mg/kg). Disease activity was assessed daily (A). Colon histology was scored at day 7 of DSS challenge for leukocytic infiltration (B), epithelial erosion (C), and mucosal thickening (D). Fecal calprotectin was assessed in colonic contents on day 7 (E). Th1 (F, CD4+ CD8- Tbet+ IFNγ+), neutrophils (G, Gr1^hi CD11b+), Treg (H, CD4+ CD25+ FOXP3+ IL10+), IL4+ CD4+ (I), and Th17 (J, CD4+ CD8- RORγT+ IL17+) were quantified by flow cytometry on day 7. Representative photomicrographs of H&E stained colon from vehicle (K) and 10 mg/kg NX-13 (L) groups; asterisks mark leukocytic infiltration, arrows mark ulceration (scale bar, 100 μm). Data presented as mean ± SEM (n = 9). Asterisks mark significance (p ≤ 0.05).

Figure 4.

Efficacy of NX-13 in CD45RB^hi adoptive transfer. Rag2−/− were adoptively transferred 4×10⁵ naïve CD4+ T cells and treated daily with oral NX-13 (10 mg/kg). Mice were scored twice weekly for disease activity until eight weeks post-transfer (A). Colon histology was scored at 8 weeks post-transfer for leukocytic infiltration (B), mucosal thickening (C), and epithelial erosion (D). Representative photomicrographs of H&E stained colon from vehicle (E) and 10 mg/kg NX-13 (L) groups; asterisks mark leukocytic infiltration, bars mark mucosal thickening (scale bar, 100 μm). Th1 (G, CD4+ CD8- Tbet+ IFNγ+), Th17 (H, CD4+ CD8- RORγT+ IL17+), Treg (I, CD4+ CD25+ FOXP3+ IL10+), and neutrophils (J, Gr1^hi CD11b+) were quantified by flow cytometry in colon at eight weeks post-transfer. Data presented as mean ± SEM (n = 10). Asterisks mark significance (p ≤ 0.05).

Figure 5.

Efficacy of NX-13 in Mdr1a−/− mice. Mdr1a−/− mice were administered oral NX-13 (20 mg/kg) daily for six weeks during which disease activity was assessed weekly (A). Representative photomicrographs of vehicle (B) and NX-13-treated (C) mice after six weeks of treatment; asterisks mark sites of leukocytic infiltration, brackets mark mucosal thickening (scale bar, 100 μm). Th1 (D, CD4+ CD8- Tbet+ IFNγ+), Treg (E, CD4+ CD25+ FOXP3+ IL10+), and neutrophils (F, Gr1^hi CD11b+) were quantified by flow cytometry in colon after six weeks of treatment. Expression of colonic Ifng (G) and Tnf (H) were measured by qRT-PCR. Data presented as mean ± SEM (n = 9). Asterisks mark significance (p ≤ 0.05).

Figure 6.

In vivo biomarker and metabolic effects of NX-13 in Mdr1a−/− mice. Mdr1a−/− mice were administered oral NX-13 (0, 10, or 20 mg/kg) daily for six weeks. Protein levels (A) of cytokines and chemokines in colon after six weeks were measured by Luminex; single asterisks mark significance relative to vehicle in the 20 mg/kg group and double asterisks mark significance at both 10 and 20 mg/kg. Expression of mt-Nd3 (B), mt-Co3 (C), Odgh (D), Eno1 (E), Gpx1 (F), and Gstm1 (G) were measured by qRT-PCR. Glutathione peroxidase (H) and glutathione (I) were measured in colon after six weeks of treatment. Data presented as mean ± SEM (n = 8). Asterisks mark significance (p ≤ 0.05).

Figure 7.

Efficacy of NX-13 in ulcerative colitis PBMCs. PBMCs were isolated from blood samples of UC donors and cultured ex vivo with NX-13 (0, 0.01, 0.05, 0.1, 0.5 μM) for 24 h. Cells were stimulated with PMA (5 ng/mL) / ionomycin (500 ng/mL), TNF (0.5 ng/mL), or hydrogen peroxide (1 mM) as indicated. TNFα+ (A), IL4+ (B), IL10+ (C), and IFNγ+ (D) CD4+ T cells by flow cytometry. NF-κB activity (E) and reactive oxygen species levels (F) after 24 h treatment. Secreted IL-8 (G), MCP1 (H) and IL-6 (I) protein levels by Luminex. Data presented as mean ± SEM (n = 5). Asterisks mark significance (p ≤ 0.05).