Increase in kidney Dh-S1P is not prevented by ABC294640 in lupus mice.

<p>After 10 weeks of ABC294640 or vehicle administration, kidneys were collected from MRL/MpJ or MLR/lpr mice. Kidney tissue was homogenized and analyzed for sphingolipid content by the Lipidomics Shared Resource at MUSC, <b>A</b>) sphingosine, <b>B</b>) S1P, <b>C</b>) Dh-sphingosine and <b>D</b>) Dh-S1P. Data represent mean ± SEM, n≥10; *p<0.05, **p<0.01, ***p<0.001 treated vs. MPJ.</p

Spleen weight and lymphocyte counts are not significantly altered by ABC294640.

<p>MRL/MpJ mice were treated with vehicle and MLR/lpr mice were treated with vehicle or ABC294640 for 10 weeks. <b>A</b>) Spleen weight was measured following euthanasia from mice at 20 weeks of age. <b>B</b>) Total B and <b>C</b>) T cell populations were analyzed using FACs flow cytometry. Data represent mean ± SEM, n≥10; **p<0.01, ***p<0.001 treated vs. MPJ.</p

ABC294640 does not prevent increases in urine thromboxane or albumin levels.

<p>After 10 weeks of ABC294640 or vehicle administration, 24-hour urine samples were collected from MRL/MpJ and MRL/lpr mice. <b>A</b>) Urine thromboxane and <b>B</b>) urine albumin levels were measured by ELISA. Data represent mean ± SEM, n≥10; *p<0.05 treated vs. MPJ.</p

ABC294640 prevents accumulation of Dh-S1P and S1P in circulation of lupus mice.

<p>After 10 weeks of ABC294640 or vehicle administration, whole blood was collected from MRL/MpJ and MRL/lpr mice. Blood was analyzed for sphingolipid content by the Lipidomics Shared Resource at MUSC, <b>A</b>) sphingosine, <b>B</b>) S1P, <b>C</b>) Dh-sphingosine and <b>D</b>) Dh-S1P. Data represent mean ±SEM, n≥10; *p<0.05, **p<0.01, ***p<0.001 treated vs. MPJ, unless otherwise indicated.</p

Glomerular pathology is not altered by treatment with ABC294640.

<p>MRL/MpJ mice were treated with vehicle and MLR/lpr mice were treated with vehicle or ABC294640 for 10 weeks. Kidneys were collected and sectioned for H&E staining. A blinded pathologist scored kidneys for <b>A</b>) vasculitis, interstitial <b>B</b>) focal or <b>C</b>) intensity, and glomerular <b>D</b>) focal or <b>D</b>) <b>i</b>ntensity scores. Data represent mean ± SEM, n≥10; *p<0.05, **p<0.01, ***p<0.001 treated vs. MPJ.</p

Lupus mice have elevated Dh-S1P levels in circulation.

<p>Serum was collected from MRL/lpr and MRL/MpJ mice and analyzed for sphingolipid content by the Lipidomics Shared Resource at MUSC, <b>A</b>) sphingosine, <b>B</b>) S1P, <b>C</b>) Dh-sphingosine and <b>D</b>) Dh-S1P. Data represent mean ± SEM, n = 6.</p

Sphingoid bases are elevated in kidney tissue from lupus mice.

<p>At 20 weeks of age, kidney tissue was collected from MRL/lpr and MRL/MpJ mice, homogenized and analyzed for sphingolipid content by the Lipidomics Shared Resource at MUSC, <b>A</b>) sphingosine, <b>B</b>) S1P, <b>C</b>) Dh-sphingosine and <b>D</b>) Dh-S1P. Data represent mean ± SEM, n = 6.</p

<i>Iqgap2</i>^<i>-/-</i> colons are characterized by diminished production of IL-6 in response to DSS treatment.

<p><b>A.</b> IL-6 (left) and IL-10 (right) mRNA cytokine levels as quantified by qRT-PCR in colons from WT and <i>Iqgap2</i>^<i>-/-</i> mice from the three groups: untreated, treated with DSS, and treated with DSS and allowed to recover for 7 days, N = 3 per group per genotype. The levels of IL-6 mRNA in DSS-treated <i>Iqgap2</i>^<i>-/-</i> colons were beyond the sensitivity of the method used. Data are presented as a transcript fold change relative to actin mRNA transcript levels. <b>B.</b> IF showing reduced IL-6 production (red) in DSS-treated <i>Iqgap2</i>^<i>-/-</i> colons (panel c) compared to DSS-treated WT (panel a). White arrows indicate representative IL-6-positive cells. Panels b and d show corresponding DAPI staining. Magnification is 200 X. Images are representative of N = 3 per genotype. <b>C.</b> Quantification of IF IL-6 positive cells in colons from WT and <i>Iqgap2</i>^<i>-/-</i> mice from the three groups: untreated, treated with DSS, and treated with DSS and allowed to recover for 7 days. Data represent an average of ten randomly selected fields per sample ± SD. N = 3 per genotype/treatment. P-values indicating statistically significant differences are shown. <b>D.</b> IHC for phospho-STAT3(Tyr705) in WT and <i>Iqgap2</i>^<i>-/-</i> colons before and after DSS treatment, N = 5 per group. Representative pSTAT3-positive cells are pointed with black arrows. Magnification is 200 X.</p

IQGAP2 levels in colon specimens of patients with IBD.

<p><b>A.</b> Two cases of ulcerative colitis (UC): panels a and b represent Case #1, and panels c and d–Case #2. <b>B.</b> Two cases of Crohn’s disease (CD), panels are designated as above. Images are representative of the total of 7 IBD patient cases. Magnification is 200 X.</p

IQ Motif-Containing GTPase-Activating Protein 2 (IQGAP2) Is a Novel Regulator of Colonic Inflammation in Mice

<div><p>IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca²⁺/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking <i>Iqgap2</i> gene (<i>Iqgap2^-/-</i> mice) were resistant to chemically-induced colitis. Unlike wild-type controls, <i>Iqgap2^-/-</i> mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in <i>Iqgap2^-/-</i> mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.</p></div