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, 144 (1), 202-10

Activation of Nuclear factor-κB in Acinar Cells Increases the Severity of Pancreatitis in Mice

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Activation of Nuclear factor-κB in Acinar Cells Increases the Severity of Pancreatitis in Mice

Haojie Huang et al. Gastroenterology.

Abstract

Background & aims: Nuclear factor-κB (NF-κB) is activated during early stages of pancreatitis. This transcription factor regulates genes that control many cell activities, including inflammation and survival. There is evidence that activation of NF-κB protects against pancreatitis, and, in other cases, that it promotes this disease. We compared the effects of NF-κB in different mouse models of pancreatitis to understand these complications.

Methods: To model constitutive activation of NF-κB, we expressed a transgene that encodes its p65 subunit or the inhibitor of κB kinase (IKK)2 in pancreatic acinar cells of mice. We analyzed effects on pancreatic tissues and levels of NF-κB target genes in these mice and compared them with mice that did not express transgenic p65 or IKK2 (controls).

Results: Transgenic expression of p65 led to compensatory expression of the inhibitory subunit IKB-α and, therefore, no clear phenotype. However, p65 transgenic mice given injections of cerulein, to induce acute pancreatitis, had higher levels of NF-κB activity in acinar cells, greater levels of inflammation, and more severe outcomes than control mice. In contrast, constitutive expression of IKK2 directly increased the activity of NF-κB in acinar cells and induced pancreatitis. Prolonged activity of IKK2 (3 months) resulted in activation of stellate cells, loss of acinar cells, and fibrosis, which are characteristics of chronic pancreatitis. Co-expression of IKK2 and p65 greatly increased the expression of inflammatory mediators and the severity of pancreatitis, compared with control mice.

Conclusions: The level of NF-κB activation correlates with the severity of acute pancreatitis in mice. Longer periods of activation (3 months) lead to chronic pancreatitis. These findings indicate that strategies to inactivate NF-κB might be used to treat patients with acute or chronic pancreatitis.

Conflict of interest statement

Disclosures: All authors have no conflict of interest to disclose.

Figures

Figure 1
Figure 1
Transgenic expression of p65 led to a compensatory response. A. Pancreatic p65 expression increased after conditional transgenic mice (LSL-p65) were crossed with mice harboring pancreatic Ela-CreERT (LSL-p65/Cre, 3 days after last TM induction). NF-κB inhibitory subunit IκB-α was unregulated in LSL-p65/Cre mice. B. No obvious phenotypic changes in the pancreata of LSL-p65/Cre mice were observed (3 days after last TM induction).
Figure 2
Figure 2
High-dose caerulein caused enhanced NF-κB activity and increased severity of pancreatitis in mice with high basal p65 expression. A. In an EMSA assay, no NF-κB DNA binding changes were observed in the pancreata of control and LSL-p65/Cre (p65) mice. 1 hour after caerulein (50µg/kg, i.p.) treatment, NF-κB DNA binding activity was increased in control mice (Control+Caerulein) and further upregulated in LSL-p65/Cre mice (p65+Caerulein). B. Real time RT-PCR revealed that cytokines IL-1β, IL-6 and TNF-α dramatically increased in the pancreas of mice with transgenic p65 expression (p65) compared to control mice 1 hour after a single dose of caerulein administration (50µg/kg, i.p.). C. Pancreata were removed 1 hour after 12 hourly repeated caerulein treatments. More severe pancreatic damages were observed in Ela-CreERT × LSL-p65 mice with transgenic p65 overexpression (p65+Caerulein) than controls (Control+Caerulein).
Figure 3
Figure 3
Caerulein treatment increased severity of pancreatitis parameters. A. After 12 repeated caerulein treatments, serum amylase was higher in Ela-CreERT × LSL-p65 mice with pancreatic expression of p65 (p65+Caerulein). B. Water content of the pancreata with high basal p65 expression was much higher. C. In pancreata with transgenic expression of p65 (p65+Caerulein), there were more CD45 inflammatory cell infiltration. (*p<0.05 vs control, #p<0.05 vs control+caerulein).
Figure 4
Figure 4
Repeated caerulein treatment causes chronic pancreatitis in mice with high basal p65 expression. A. Control mice and Ela-CreERT × LSL-p65mice were treated with 5 hourly ip injection of caerulein, once a week for 5 consecutive weeks. Pancreata were harvested 2 weeks after last injection. B. Pancreata of control mice (control+caerulein) showed no obvious damages while focal chronic lesions were observed in most (5/6) of the Ela-CreERT × LSL-p65 mice (p65+caerulein).
Figure 5
Figure 5
Acinar specific expression of constitutively active IKK2 led to NF-κB p65 nuclear translocation and pancreatitis. A. Immunohistochemical staining with antibody against p65 did not detect p65 nuclear translocation in control mice. B. Transgenic p65 expression in Ela-CreERT × LSL-p65 (LSL-p65/Cre) mice led to higher basal p65 level (brown) without nuclear translocation (arrow). C. Pancreatic specific expression of constitutively active IKK2 in Ela-CreERT × LSLIKK2 (LSL-IKK2/Cre) mice caused p65 nuclear translocation (arrow). D. Pancreatic acinar damage and inflammatory cell infiltration were obvious seven day after TM induction of IKK2 expression. E. Pancreatic acinar damage, inflammatory cell infiltration and fibrosis were evident 3 months after TM induction of IKK2 expression. E. Co-expression of IKK2 and p65 further increased the severity of pancreatitis in Ela-CreERT × LSL-IKK2 × LSL-p65 (LSL-IKK2/LSL-p65/Cre) mice (7 days after TM).
Figure 6
Figure 6
Pancreatic specific expression of active IKK2 or combination of active IKK2 and p65 increased pancreatitis parameters. A. pancreata from control mice (Control), B. pancreata from Ela-CreERT × LSL-IKK2 mice with pancreatic acinar specific expression of IKK2(IKK) and C. pancreata from Ela-CreERT × LSL-IKK2 × LSL-p65 mice with pancreatic acinar specific expression of both IKK2 and p65 (IKK/p65) were stained with CD45 antibody by immunohistochemistry (7 days after TM). D. Semi-quantitative pathological scores evaluation including edema, inflammatory cells infiltration and cell death were compared (note: normal tissues were assigned a total score of 3).
Figure 7
Figure 7
Elevated NF-κB led to increased mRNAs expression of cytokines and profibrogenic factors. A. TGF-β, B. IL-1β, C. fibronectin and D. alpha smooth muscle actin (alpha-SMA) were upregulated in the pancreata of mice expressing IKK2 (IKK) or in combination of IKK2 and p65 (IKK/p65) but not in control mice (control) and mice expressing p65 (p65) 3 days and 9 days after last TM induction.

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