The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria

<div><p>Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with <i>Citrobacter rodentium</i>, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, <i>Slc7a2</i>^–/–mice infected with <i>C</i>. <i>rodentium</i> had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected <i>Slc7a2</i>^–/–mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking <i>Slc7a2</i> exhibited reduced adherence of <i>C</i>. <i>rodentium</i> to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of <i>C</i>. <i>rodentium</i> and induction of inflammatory response was confirmed <i>in vitro</i>, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of <i>Slc7a2</i> or <i>Tln1</i>. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of <i>C</i>. <i>rodentium</i> and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic <i>Escherichia coli</i>. Overexpression of <i>SLC7A2</i> in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that <i>C</i>. <i>rodentium</i> enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.</p></div

Immunophenotyping of innate immune cells.

<p>Cells isolated from the lamina propria of <i>C</i>. <i>rodentium</i>-infected mice (<i>C</i>. <i>rod</i>) or of uninfected animals were analyzed by flow cytometry for the following markers: (<b>A</b>) F4/80, (<b>B</b>) GR1, (<b>C</b>) CD11c. For each marker, representative flow plots and combined data from <i>n</i> = 4 Ctrl and <i>n</i> = 6 infected mice are shown **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Effect of SLC7A2 on intimate attachment of <i>C</i>. <i>rodentium</i>.

<p>(<b>A</b>) Phalloidin staining in HEK 293T cells transfected with pLX304 or p<i>Slc7a2</i> and infected for 4 h with <i>C</i>. <i>rodentium</i>. *<i>P</i> < 0.05; representative images of 3 independent experiments are shown. Scale bar, 20 μm. (<b>B</b>) <i>CXCL8</i> mRNA in transfected HEK 293T cells in response to <i>C</i>. <i>rodentium</i>. ***<i>P</i> < 0.001; <i>n</i> = 3. (<b>C</b>) YAMC cells were transduced with Ctrl or <i>Slc7a2</i> shRNA, infected for 4 h with <i>C</i>. <i>rodentium</i> and stained for phalloidin. **<i>P</i> < 0.01; representative images of 3 independent experiments are shown. Scale bar, 10 μm. (<b>D</b>) <i>Cxcl1</i> and <i>Cxcl2</i> mRNA levels in YAMC cells with Ctrl or <i>Slc7a2</i> shRNA. **<i>P</i> < 0.01, ***<i>P</i> < 0.001; <i>n</i> = 3. (<b>E</b>) FAS test in YAMC cells infected for 4 h with <i>C</i>. <i>rodentium</i> in the presence or absence of L-Lys. *<i>P</i> < 0.05; representative images of 3 independent experiments are shown. Scale bar, 20 μm. (<b>F</b>) <i>Cxcl1</i> and <i>Cxcl2</i> mRNA expression in YAMC cells in response to <i>C</i>. <i>rodentium</i> ± L-Lys. ***<i>P</i> < 0.001; <i>n</i> = 3 independent experiments.</p

Expression and role of SLC7A2 in human CECs.

<p><i>SLC7A2</i> mRNA levels (<b>A</b>), FAS test (<b>B</b>), and <i>CXCL8</i> mRNA levels (<b>C</b>) in Caco-2 cells transfected with control (Ctrl) or <i>SLC7A2</i> siRNA and infected for 4 h with EPEC. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001; <i>n</i> = 3 independent experiments. Scale bar, 20 μm. (<b>D-E</b>) Caco-2 cells were infected for 4 h with EPEC ± L- Lys and stained for phalloidin (<b>D</b>) or assessed <i>CXCL8</i> expression (<b>E</b>). ***<i>P</i> < 0.001; <i>n</i> = 3 independent experiments. In (B) and (D): scale bar, 20 μm.</p

Analysis of focal contact proteins.

<p>WT or <i>Slc7a2</i>^–/–mice were infected with <i>C</i>. <i>rodentium</i> (<i>C</i>. <i>rod</i>) for 14 days. (<b>A</b>) ACTN1, Talin-1 and β-actin were detected in lysates of whole colonic tissues by Western blotting; each lane is from a different mouse. (<b>B</b>) Longitudinal sections of the colon were used for immunofluorescence for bacterial EspB (red), Talin-1 (green), and nuclei (blue); the merge of red and green is depicted in yellow. Representative images from 3 animals in each group are shown. Scale bar = 20 μm. (<b>C</b>) Expression of <i>Tln1</i> mRNA in whole colon tissue and CECs. **<i>P</i> < 0.01; <i>n</i> = 3 mice for Ctrl, and <i>n</i> = 5 for infected animals.</p