<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

IQGAP2 expression in organs of the digestive system.

<p><b>A.</b> Immunoblot of digestive tract organs from wild-type (WT) and <i>Iqgap2</i>^<i>-/-</i> mice, probed for IQGAP2 and β-actin as a control for equal protein loading. Representative blots of N = 3 per genotype are shown. <b>B.</b> IHC of IQGAP1 and IQGAP2 in WT (panels a, b) and <i>Iqgap2</i>^<i>-/-</i> (panels c, d) mouse colon. Magnification is 200X.</p

Suppression of NF-κB signaling in <i>Iqgap2</i>^<i>-/-</i> colons.

<p><b>A.</b> IHC shows decreased baseline levels of p65 subunit of NF-κB in <i>Iqgap2</i>^<i>-/-</i> colon compared to WT (panels a, d). While DSS treatment resulted in elevated levels of p65 in WT colon, it failed to elicit the same response in <i>Iqgap2</i>^<i>-/-</i> colon (panel b, e). Termination of DSS treatment results in a restoration of the baseline p65 levels within 7 days in both genotypes (panel c, f). <b>B.</b> IHC of TLR4 in WT and <i>Iqgap2</i>^<i>-/-</i> colons before and after DSS treatment. <b>C.</b> IHC of MyD88 in the same samples. Note low levels of both TLR4 and MyD88 in <i>Iqgap2</i>^<i>-/-</i> colons after DSS exposure (panels d). A representative image of N = 5 per genotype is shown for each IHC. Magnification is 200 X.</p

Reduced production of white blood cells (WBC) in <i>Iqgap2</i>^<i>-/-</i> mice.

<p><b>A.</b> Quantification of positive cells as a result of IF shows significantly decreased numbers of infiltrating macrophages (F4/80), neutrophils (Ly-6G) and dendritic cells (CD11c) in <i>Iqgap2</i>^<i>-/-</i> colons compared to WT following DSS treatment. The number of positive fluorescent cells was obtained by counting cells in six randomly selected fields per colon sample 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. <b>B.</b> Complete blood count (CBC) confirms reduced numbers of neutrophils, lymphocytes and monocytes in circulation in <i>Iqgap2</i>^<i>-/-</i> mice treated with DSS. Data are presented as the mean ± SEM. N = 3 per genotype/treatment (<b>A</b>), N = 5 per genotype/treatment (<b>B</b>), p-values indicating statistically significant differences are shown.</p

Physiological and histological evidence of resistance to DSS-induced colitis in <i>Iqgap2</i>^<i>-/-</i> mice.

<p><b>A.</b> Schema of a DSS treatment experiment. DSS at the concentration of 3% was administered in drinking water for up to 13 days. A separate group of mice was treated with 3% DSS for 13 days and then allowed to recover on regular water for 7 days. <b>B.</b> Baseline body weight of untreated WT and <i>Iqgap2</i>^<i>-/-</i> mice. The difference between genotypes is not statistically significant (p = 0.1059). <b>C.</b> In contrast to the WT control group, <i>Iqgap2</i>^<i>-/-</i> mice did not lose weight as a result of DSS treatment (left panel); after a 7-day recovery period, WT mice restored 95% of their baseline weight before DSS treatment (right panel). Data are presented as the mean ± SEM. Statistically significant (p < 0.05) differences between genotypes are indicated with asterisks. Resistance of <i>Iqgap2</i>^<i>-/-</i> mice to experimental colitis is also demonstrated by colon length (<b>D</b>) and low colitis disease activity index (DAI) (<b>E</b>). <b>F.</b> Histological evidence (H&E) of the absence of colitis in <i>Iqgap2</i>^<i>-/-</i> mice. While DSS treatment resulted in an expansive colonic epithelium loss (black arrows) in WT colons (panels a, b), <i>Iqgap2</i>^<i>-/-</i> colons were minimally affected (panels c, d). N = 5 per group per experiment; experiment was repeated three times; the results of one representative experiment are shown.</p

Mice with loss of SK1 in hematopoietic cells exhibit splenomegaly with DSS-induced colitis.

<p>Bone marrow transplants were performed with WT and SK1^−/− mice. Following 6 weeks of rest, mice were administered either regular drinking water or water containing 5% DSS for 5 days. <b>A</b>) Change in body weight was assessed. Data represent mean ±SD, n≥6 for each treatment group; (significance at Day 5 =  *WT^WTBM, **WT^SK1BM, **SK1^WTBM); *p<0.05, **p<0.01, ***p<0.001 vs strain Day 0. <b>B</b>) Spleen weight and <b>C</b>) colon length were assessed following treatment. <b>D</b>) Pathology damage scores were determined by a pathologist in a blinded fashion with H&E sections of colon tissue. Data represent mean ±SD, n≥6 for each treatment group; *p<0.05, **p<0.01, ***p<0.001 vs strain untreated, #p<0.05 as compared to WT^WTBM DSS treated, %p<0.05 as compared to SK1^SK1BM DSS. For the X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Extra-hematopoietic SK1 is necessary for COX2 expression in the colon epithelium.

<p>COX2 expression levels were examined by IHC <b>A–D</b>): untreated mice; <b>E–H</b>) DSS treated mice. Scale bars  = 20 µm. Regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Hematopoietic genotype determines circulating sphingolipid levels.

<p>Whole blood was collected and analyzed for sphingolipid content using ESI/MS/MS. <b>A and C</b>) Ceramide and <b>B and D</b>) S1P levels were normalized to volume. Data represent mean ±SD, n≥6 for each treatment group, *p<0.05, **p<0.01 as compared to both strains of mice with WT bone marrow. X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p