NLRX1 Regulates Effector and Metabolic Functions of CD4+ T Cells

Abstract

Nucleotide oligomerization domain-like receptor X1 (NLRX1) has been implicated in viral response, cancer progression, and inflammatory disorders; however, its role as a dual modulator of CD4⁺ T cell function and metabolism has not been defined. The loss of NLRX1 results in increased disease severity, populations of Th1 and Th17 cells, and inflammatory markers (IFN-γ, TNF-α, and IL-17) in mice with dextran sodium sulfate-induced colitis. To further characterize this phenotype, we used in vitro CD4⁺ T cell-differentiation assays and show that NLRX1-deficient T cells have a greater ability to differentiate into an inflammatory phenotype and possess greater proliferation rates. Further, NLRX1^-/- cells have a decreased responsiveness to immune checkpoint pathways and greater rates of lactate dehydrogenase activity. When metabolic effects of the knockout are impaired, NLRX1-deficient cells do not display significant differences in differentiation or proliferation. To confirm the role of NLRX1 specifically in T cells, we used an adoptive-transfer model of colitis. Rag2^-/- mice receiving NLRX1^-/- naive or effector T cells experienced increased disease activity and effector T cell populations, whereas no differences were observed between groups receiving wild-type or NLRX1^-/- regulatory T cells. Metabolic effects of NLRX1 deficiency are observed in a CD4-specific knockout of NLRX1 within a Citrobacter rodentium model of colitis. The aerobic glycolytic preference in NLRX1^-/- effector T cells is combined with a decreased sensitivity to immunosuppressive checkpoint pathways to provide greater proliferative capabilities and an inflammatory phenotype bias leading to increased disease severity.

Figure 1. DSS colitis induces greater inflammatory T cell populations in NLRX1−/−

Disease activity index of wild type and NLRX1−/− on DSS for 7 days followed by three days of water (A). Colonic lamina propria (B) and spleen (E) cell numbers of Th1 (CD4+NK1.1-CD8-Tbet+IFNy+). Colonic lamina propria (C) and spleen (F) cell numbers of Th17 (CD4+NK1.1-CD8-RORyT+IL17+). Colonic lamina propria (D) and spleen (G) cell numbers of Treg (CD4+NK1.1-CD8-CD25+FOXP3+IL10+). mRNA expression of Ifny (H), Tnfa (I) and IL17A (J) in colons of mice on day 7 of DSS challenge. Asterisks mark significance (P<0.05, n=12).

Figure 2. DSS colitis induces greater inflammatory T cell populations with T cell specific deletion of NLRX1

Disease activity index of wild type and NLRX1fl/fl;CD4cre+ on DSS for 7 days (A). Colonic lamina propria (B) and spleen (E) cell numbers of Th17 (CD4+NK1.1-CD8-RORyT+IL17+). Colonic lamina propria (C) and spleen (F) cell numbers of Th1 (CD4+NK1.1-CD8-Tbet+IFNy+). Colonic lamina propria (D) and spleen (G) cell numbers of Treg (CD4+NK1.1-CD8-CD25+FOXP3+IL10+). Asterisks mark significance (P<0.05, n=12).

Figure 3. NLRX1−/− CD4+ T cells have greater inflammatory and proliferation potential

Proportion of Th17 differentiated cells in vitro in Th17 differentiating media (A). Proportion of Treg differentiated cells in vitro in Treg differentiating media (B). Proportion of Treg differentiated cells in vitro in Th17 differentiating media with and without addition of PD-L1 (C). Th17 proliferation index by CFSE staining with and without addition of PD-L1 (D). Data is a result of three experiments with triplicate samples (p<0.05, n=9).

Figure 4. NLRX1−/− CD4+ T cells have altered metabolic behavior

mRNA expression of Cpt1a (A), Fabp4 (B), and Glut1 (C). Activity of lactate dehydrogenase with and without addition of PD-L1 (D). Incomplete fatty acid oxidation as a measure of acid soluble metabolite production (E). Total fatty acid oxidation with addition of PD-L1 (F). Extracellular acidification (G), and oxygen consumption rates (H) from differentiated CD4+ Th17 cells. Cells were measured over a time course and exposed to glucose oxidase, carbonyl cyanide-4-phenylhydrazone (FCCP), and 2-deoxy-D-glucose (2DG) at the indicated time periods. Carbon dioxide production from radiolabeled [¹⁴C]-glucose in WT and NLRX1−/− differentiated Th17 cells (I) Differentiation (J) and proliferation (K) of Th17 cells in presence of sodium oxamate. Differentiation (L) and LDH activity (M) of Th17 cells in presence of PDL1 and metformin. Differentiation (N) and LDH activity (O) of Th17 cells in microaerophilic conditions. mRNA expression of Hif1a (P). Data is a result of three experiments with triplicate samples (P<0.05, n=9).

Figure 5. Adoptive transfer of naïve NLRX1−/− T cells results in increased disease severity

Disease activity index of Rag2−/− transferred wild type and NLRX1−/− naïve CD4+ T cells (A). Percentage (B) and absolute number (C) of CD4+ T cell populations in colonic lamina propria eight weeks after transfer. Summarized histology scores in leukocytic infiltration and epithelial erosion (D). Percentage (E) and absolute number (F) of CD4+ T cell populations in spleen eight weeks after transfer. Representative photomicrographs of control (G), wild-type transferred (H), and NLRX1−/− transferred (I) Rag2−/− colons eight weeks post-transfer. Asterisks mark significance (P<0.05, n=10).

Figure 6. Adoptive transfer of NLRX1−/− effector T cells increases disease severity

Disease activity index of Rag2−/− transferred wild type and NLRX1−/− (A) effector and regulatory CD4+ T cells. Spleen (B) and colonic lamina propria (C) cell numbers of Treg (CD4+NK1.1-CD8-CD25+FOXP3+IL10+). Spleen (D) and colonic lamina propria (E) cell numbers of Th1 (CD4+NK1.1-CD8-Tbet+IFNy+). Spleen (F) and colonic lamina propria (G) cell numbers of Th17 (CD4+NK1.1-CD8-RORyT+IL17+). Asterisks mark significance between effector and regulatory groups and number signs mark significance between genotypes (p<0.05, n=10).

Figure 7. Citrobacter rodentium challenge of T cell specific knockouts of NLRX1 results in greater inflammation

Colonic lamina propria (A) and spleen (B) cell numbers of Th1 (CD4+NK1.1-CD8-Tbet+IFNy+). Colonic lamina propria (C) and spleen (D) cell numbers of Tfh (CD4+NK1.1-CD8-Bcl6+IL21+). Colonic lamina propria (E) and spleen (F) cell numbers of Th17 (CD4+NK1.1-CD8-RORyT+IL17+). Colonic lamina propria cell numbers of Treg (CD4+NK1.1-CD8-CD25+FOXP3+IL10+) (G) and Tr1 (H) cells. LDH activity (I) and mRNA expression of Glut1 (J) of sorted CD4+ T cells from colons of mice 12 days post infection. Re-isolation of C. rodentium from fecal samples at 12 days post-infection (K). Histological scores of epithelial erosion, leukocytic infiltration and mucosal thickness of colonic sections at 12 days post-infection (L). Asterisks mark significance (P<0.05, n=10).