LITAF mediation of increased TNF-α secretion from inflamed colonic lamina propria macrophages.

Dysregulation of TNF-α in lamina propria macrophages (LPM) is a feature of inflammatory bowel diseases (IBD). LPS-Induced-TNF-Alpha-Factor (LITAF) is a transcription factor that mediates TNF-α expression. To determine whether LITAF participates in the mediation of TNF-α expression in acutely inflamed colonic tissues, we first established the TNBS-induced colonic inflammation model in C57BL/6 mice. LPM were harvested from non-inflamed and inflamed colonic tissue and inflammatory parameters TNF-α and LITAF mRNA and protein levels were measured ex-vivo. LPM from TNBS-treated mice secreted significantly more TNF-α at basal state and in response to LPS than LPM from untreated mice (p<0.05). LITAF mRNA and protein levels were elevated in LPM from TNBS compared with untreated animals and LPS further increased LITAF protein levels in LPM from inflamed tissue (P<0.05). To further confirm the role of LITAF in acutely inflamed colonic tissues, TNBS-induced colonic inflammation was produced in LITAF macrophage specific knockout mice (LITAF mac -/- mice) and compared to wild type (WT) C57BL/6. Twenty four hours following TNBS administration, colonic tissue from LITAF mac -/- mice had less MPO activity and reduced colonic TNF-α mRNA then WT C57BL/6 mice (p<0.05). LPM harvested from LITAF mac -/- secreted significantly less TNF-α in response to LPS than wild type (WT) C57BL/6 (p<0.05). This study provides evidence that LITAF contributes to the regulation of TNF-α in LPM harvested following acute inflammation or LPS treatment paving the way for future work focusing on LITAF inhibitors in the treatment of TNF-α-mediated inflammatory conditions

LPM harvested from LITAF macrophage specific knockout mice (LITAF mac -/- mice) produce less TNF-α than LPM harvested from C57BL/6 mice.

<p><b><i>A</i></b>) Western blot of LITAF and Actin confirming the knockout of LITAF protein in peritoneal macrophages harvested from LITAF mac -/- and C57BL/6 mice. <b><i>B</i></b>) TNF-α secretion from inflamed LPM isolated from colonic tissues of LITAF mac -/- (N = 6) or wildtype C57BL/6 (N = 12) following LPS stimulation (24 hours). Data are expressed as % Inflamed C57BL/6 group ± SEM. * Statistical difference (p<0.05), # statistical difference (p<0.001).</p

LITAF protein levels are increased in LPM harvested from inflamed colonic tissue and further increased in inflamed LPM following LPS (1 ug/mL) stimulation (N = 5).

<p><b><i>A</i></b>) Western blot of LITAF levels and GAPDH levels in freshly harvested LPM from Non-Inflamed and Inflamed colonic tissue. The graph shows the optical density (OD) of LITAF levels normalized to GAPDH levels. * Statistical difference from Non-Inflamed group (p<0.05). B) Western blot of LITAF levels and β-Actin levels in LPM harvested from inflamed colonic tissue stimulated with LPS ex vivo compared to LPM harvested from inflamed colonic tissue, but not treated with LPS (Non-treated). The graph show OD of LITAF levels normalized to β-Actin levels. * Statistical difference (p<0.05).</p

TNF-α message levels from inflamed colonic tissue harvested from LITAF macrophage specific mice (LITAF mac -/- mice) is reduced compared to C57BL/6 mice.

<p>TNF-α message levels were measured in colonic tissue following TNBS administration (0.3 mg) for 24 hrs in LITAF mac -/- mice (N = 6) and C57BL/6 mice (N = 12). Data are expressed as % Non-Inflamed control ± SEM. * Statistical difference (p<0.05).</p

LITAF macrophage specific knockout mice (LITAF mac -/-) show reduced changes in bodyweight and MPO following TNBS administration.

<p><b><i>A</i></b>) Following TNBS (3.0 mg) administration to LITAF mac -/- mice (N = 6) and C57BL/6 mice (N = 6), bodyweights were measured 24 hours later. Data are expressed as % initial (day 0) bodyweight. * Statistical difference (p<0.05), # statistical difference (p<0.001). B) Following TNBS (3.0 mg) administration to LITAF mac -/- mice (N = 6) and C57BL/6 mice (N = 6), colonic tissue was collected and assessed for MPO activity. Data are expressed as % Non-inflamed MPO activity. ^# Statistical difference (p<0.001).</p

TNBS-induced inflammation in C57BL/6 Mice.

<p>Inflammation was induced by TNBS (1.5 mg, in 50% ethanol solution) and animals were monitored for: A) Colonic tissue was collected and assessed for MPO activity (N = 4, except Day 1, N = 3). Data are expressed as % Day 0 Control ± SEM. ^# Statistical difference (p<0.001). <b><i>B</i></b>) Representative histological images of colonic tissue from C57BL/6 mice 24 hours following TNBS administration showing colonic inflammation (Mag 100×).</p

TNBS-induced histological damage is not reduced in LITAF mac -/- mice.

<p><b><i>A</i></b>) Representative histological images of colonic inflammation: Untreated Control; TNBS 24 hours; and, TNBS 48 hours following TNBS administration. (N = 3) images (Mag 100×). <b><i>B</i></b>) Histological score (0–4) ± SEM (N = 5) of colonic tissue 24 and 48 hours following TNBS administration.</p

Secreted TNF-α levels from LPM harvested from inflamed colonic tissue are reduced with the administration of signaling inhibitors.

<p><b><i>A</i></b>) Secreted TNF-α from LPM harvested from inflamed colonic tissue are reduced when administered p38 inhibitor (SB-202190) prior to, and throughout LPS (1 ug/mL) stimulation (24 hours) (N = 8). Data are expressed as % Inflamed DMSO vehicle ±SEM. * Statistical difference (p<0.05), ^# statistical difference (p<0.001). B) Secreted TNF-α from LPM harvested from inflamed colonic tissue are reduced when administered NF-κB inhibitor (BAY 11-7082) prior to, and throughout LPS (1 ug/mL) stimulation (24 hours) (N = 6). Data are expressed as % Inflamed DMSO Vehicle group ± SEM. ^# Statistical difference (p<0.001).</p

Secreted TNF-α levels and LITAF message are increased in LPM harvested from inflamed colonic tissue compared to LPM harvested from non-inflamed colonic tissue.

<p><b><i>A</i></b>) Following TNBS administration, LPM were harvested and stimulated with LPS (1 ug/mL) <i>ex vivo</i> (24 hours) and TNF-α secretion was measured (N = 8). Data are presented as % Non-Inflamed Non-treated control ± SEM. * Statistical difference (p<0.05), # statistical difference (p<0.001). B) LITAF message levels are increased in LPM harvested from inflamed colonic tissue following LPS (1 ug/mL) stimulation (1 hour) compared to LPM harvested from non-inflamed colonic tissue (N = 7 for Non-treated, N = 8 LPS treated). Data were normalized to β-Actin message and presented as % Non-Inflamed Non-treated group ± SEM. * Statistical difference (p<0.05).</p