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<p>Interactions among the gut microbiome, dysregulated immune responses, and genetic factors contribute to the pathogenesis of inflammatory bowel disease (IBD). Nlrx1^−/− mice have exacerbated disease severity, colonic lesions, and increased inflammatory markers. Global transcriptomic analyses demonstrate enhanced mucosal antimicrobial defense response, chemokine and cytokine expression, and epithelial cell metabolism in colitic Nlrx1^−/− mice compared to wild-type (WT) mice. Cell-specificity studies using cre-lox mice demonstrate that the loss of NLRX1 in intestinal epithelial cells (IEC) recapitulate the increased sensitivity to DSS colitis observed in whole body Nlrx1^−/− mice. Further, organoid cultures of Nlrx1^−/− and WT epithelial cells confirm the altered patterns of proliferation, amino acid metabolism, and tight junction expression. These differences in IEC behavior can impact the composition of the microbiome. Microbiome analyses demonstrate that colitogenic bacterial taxa such as Veillonella and Clostridiales are increased in abundance in Nlrx1^−/− mice and in WT mice co-housed with Nlrx1^−/− mice. The transfer of an Nlrx1^−/−-associated gut microbiome through co-housing worsens disease in WT mice confirming the contributions of the microbiome to the Nlrx1^−/− phenotype. To validate NLRX1 effects on IEC metabolism mediate gut–microbiome interactions, restoration of WT glutamine metabolic profiles through either exogenous glutamine supplementation or administration of 6-diazo-5-oxo-l-norleucine abrogates differences in inflammation, microbiome, and overall disease severity in Nlrx1^−/− mice. The influence NLRX1 deficiency on SIRT1-mediated effects is identified to be an upstream controller of the Nlrx1^−/− phenotype in intestinal epithelial cell function and metabolism. The altered IEC function and metabolisms leads to changes in barrier permeability and microbiome interactions, in turn, promoting greater translocation and inflammation and resulting in an increased disease severity. In conclusion, NLRX1 is an immunoregulatory molecule and a candidate modulator of the interplay between mucosal inflammation, metabolism, and the gut microbiome during IBD.</p

Network model of <i>Helicobacter pylori</i>-host interactions.

<p>Systems biology markup language (SBML)-compliant network diagram of four compartment <i>H</i>. <i>pylori</i> response model. Cell types include <i>H</i>. <i>pylori</i> (HP), tolerogenic bacteria (TolB), epithelial cells (E), damaged epithelial cells (Edamaged), immature dendritic cells (iDC), effector dendritic cells (eDC), tolerogenic dendritic cells (tDC), monocytes (Monocytes), regulatory macrophages (M_reg), naïve T cells (nT), T helper 1 (Th1), T helper 17 (Th17), induced T regulatory (iTreg), type 1 regulatory (Tr1), IFNγ (IFNg), and IL10 (IL10).</p

Simulation, analysis and validation of intracellular macrophage network model.

<p>Global sensitivity analysis displaying parameter sensitivity ranges on the differentiation of regulatory macrophages (A). Parameters are grouped by associated model species: NFkB-associated (purple), LANCL2-associated (red), PKA-associated (blue), IL10-associated (green). Predicted impairment of regulatory macrophage differentiation following simulated loss of LANCL2 (B). Fold changes in molecule production or activation following simulated loss of LANCL2 compared to wild-type simulation (C). mRNA expression of LANCL2 in whole stomach (D) and cultured bone marrow derived macrophages (E), normalized to expression of beta-actin. mRNA expression of Ncor2 (F) and Foxp1 (G), Klf4 (H), and Il10 (I) within the stomach of <i>H</i>. <i>pylori</i> SS1-infected and uninfected LANCL2fl/fl; LysCre- and LANCL2fl/fl LysCre+ mice at three weeks post-infection. NFkB p65 activity in whole stomach tissue homogenate of <i>H</i>. <i>pylori</i> SS1-infected and uninfected LANCL2fl/fl; LysCre- and LANCL2fl/fl LysCre+ mice at three weeks post-infection (J). <i>p</i>-values less than 0.05 are considered significant and marked by an asterisk (*), (n = 7).</p

Simulation and analysis of <i>Helicobacter pylori</i> colonization.

<p>Time course simulation with calibrated <i>H</i>. <i>pylori</i>-host interaction model displaying <i>H</i>. <i>pylori</i> burden (A), macrophage and cytokine responses (B) and T cell responses (C). Local sensitivity analysis of model displaying positive and negative effects of parameters on model species H. pylori (D), IL10 (E), damaged epithelial cells (F). Boxed region contains parameters associated with Mreg model species. Specific model species of interest are indicated by symbols: & marks column associated with HP model species, * marks column associated with IL10 model species and # marks column associated with Edamaged model species. Global sensitivity analysis displaying parameter sensitivity ranges on the differentiation of regulatory macrophages (G). Parameters associated with cytokines are indicated by (*) and parameters associated with <i>H</i>. <i>pylori</i> are indicated by (#).</p

Network of intracellular signaling pathways controlling regulatory macrophage differentiation.

<p>Systems Biologt Markup Language (SBML)-compliant network diagram displaying the three main pathways, CSF1R, CX3CR1, and IL-10R, controlling cytokine production and macrophage phenotype commitment. While interconnected, the three signaling receptors have dominant effects on arms within the network. The CSF1R/M-CSF pathway integrates DAP12 and NFAT activity and is closely associated with the inflammatory Traf/NF-kB pathway as well. The IL-10R pathway is modified by calcium signaling and the transcriptional activities of FOXP1 and NCOR2. The CX3CR1 pathway combines cAMP/PKA pathways with modification to Akt and inflammatory signaling.</p

Binding of eleostearic acid (ESA) with cNLRX1 wildtype and mutant.

<p>A) Chemical structure of ESA. B) Interactions between ESA and NLRX1 predicted by molecular docking. ESA is in green ball-and-stick representation surrounded by molecular surface of the binding site with coloring by element. The free energy of binding is -6.2 kcal/mol. C) SPR sensograms for the binding of varying concentrations of ESA (40.0, 20.0, 10.0, 5.0, 2.5, 1.25 and 0.0 μM) to immobilized cNLRX1 (WT) and ASP677, PHE680, PHE681, and GLU684 to alanine mutant (Mutant). The equilibrium constant of dissociation, K_D, of ESA is 1.33 × 10⁻⁵ M for WT and 1.70 × 10⁻⁴ M for Mutant. D) Strength of association plot showing maximum response units for captured cNLRX1 WT or Mutant for a given concentration of ESA.</p

Binding of punicic acid (PUA) with cNLRX1 wildtype and mutant.

<p>A) Chemical structure of PUA. B) Interactions between PUA and cNLRX1 predicted by molecular docking. PUA is in cyan ball-and-stick representation surrounded by molecular surface of the binding site with coloring by element. The free energy of binding is -6.2 kcal/mol. C) SPR sensograms for the binding of varying concentrations of PUA (40.0, 20.0, 10.0, 5.0, 2.5, 1.25 and 0.0 μM) to immobilized cNLRX1 wildtype (WT) and ASP677, PHE680, PHE681, and GLU684 to alanine mutant (Mutant). The equilibrium constant of dissociation, K_D, of PUA is 1.46 × 10⁻⁵ M for WT and 3.38 × 10⁻⁴ M for Mutant. D) Strength of association plot showing maximum response units for captured cNLRX1 WT or Mutant for a given concentration of PUA</p

Effect of NLRX1 disruption on NF-κB activation in Bone marrow derived macrophage (BMDM).

<p>BMDMs were isolated from hind legs obtained fromm wild type and <i>Nlrx1-/-</i> in sterile conditions and cultured for 7 days After stimulated with LPS (1μg/ml) for 12 hours, cells were treated with control (medium), 40 μM PUA, and 40 μM DHA for 12 hours. Nuclear extraction was performed on colon homogenates using the Active Motif Nuclear Extraction Kit (Carlsbad, CA). ELISA was performed on both cytoplasmic and nuclear fractions using the Active Motif TransAM^® NF-κB p65 kit according to the manufacturer’s instructions. Letter superscripts indicate significant (P-value < 0.05) differences by ANOVA.</p

Effect of PUA on WT and <i>Nlrx1-/-</i> mice in DSS-induced colitis (10 mice per group).

<p>A) Average disease activity of treatment groups scored 0–4 daily for six days. B) mRNA expression of TNFα in whole colon on day six of DSS treatment. C) Histopathological assessment of colons by epithelial erosion (EE), leukocytic infiltration (LI), and mucosal thickening (MT). Asterisk indicates P-value < 0.05 by ANOVA.</p

Binding of docosahexaenoic acid (DHA) with cNLRX1 wildtype and mutant.

<p>A) Chemical structure of DHA. B) Interactions between DHA and cNLRX1 predicted by molecular docking. DHA is in orange ball-and-stick representation surrounded by molecular surface of the binding site with coloring by element. The free energy of binding is -8.0 kcal/mol. C) SPR sensograms for the binding of varying concentrations of DHA (100.0, 50.0, 25.0, 12.5, 6.23, and 3.125 μM) to immobilized cNLRX1 wild-type (WT) and ASP677, PHE680, PHE681, and GLU684 to alanine mutant (Mutant). The equilibrium constant of dissociation, KD, of DHA is 2.3 × 10–6 M for WT and 75.9 × 10–6 M for Mutant. D) Strength of association plot showing maximum response units for captured cNLRX1 WT or Mutant for a given concentration of DHA.</p