Selective inhibition of soluble TNF using XPro1595 relieves pain and attenuates cerulein-induced pathology in mice

Abstract

Background: Symptoms associated with acute pancreatitis can be debilitating, and treatment remains a challenge. This study aimed to investigate the efficacy of selectively inhibiting the soluble form of TNF (solTNF) using the biologic XPro1595 in a mouse model of acute pancreatitis.

Methods: Acute pancreatitis was induced in adult male C57Bl/6J mice by administering cerulein (8 injections of 50 µg/kg I.P., spaced an hour apart), with XPro1595 (10 mg/kg, S.C.) or vehicle being administered approximately 18 h after the last injection. Serum was collected 6 or 18 h after the last cerulein injection, pancreatic tissue was collected 2 and 7 days post-induction, and brain hippocampal tissue was collected at 7 days post-induction. The animal's pain level was assessed 3, 5 and 7 days post-induction.

Results: The induction of acute pancreatitis promoted a strong increase in serum amylase levels, which had receded back to baseline levels by the next morning. XPro1595 treatment began after amylase levels had subsided at 18 h, and prevented pancreatic immune cell infiltration, that subsequently prevented tissue disruption and acinar cell death. These improvements in pathology were associated with a significant reduction in mechanical hypersensitivity (neuropathic pain). XPro1595 treatment also prevented an increase in hippocampal astrocyte reactivity, that may be associated with the prevention of neuropathic pain in this mouse model.

Conclusion: Overall, we observed that selectively inhibiting solTNF using XPro1595 improved the pathophysiological and neurological sequelae of cerulein-induced pancreatitis in mice, which provides support of its use in patients with pancreatitis.

Keywords: Acute pancreatitis; Cerulein; Cytokines; Inflammation; Mice; Neuropathic pain; TNF; TNFR1.

Fig. 1

Cerulein administration promotes induction of pancreatitis. Acute pancreatitis was induced by 8 intraperitoneal injections of cerulein (50 µg/kg) or vehicle to mice, spaced an hour apart each, and serum amylase concentrations were measured 6 and 18 h after the last cerulein injection. Cerulein-treated mice displayed a temporary spike in serum amylase concentration at 6 h, that had reduced back to baseline levels by the next morning (n = 3 per group). NC non-cerulein, C cerulein, **p < 0.01, ***p < 0.001

Fig. 2

XPro1595 treatment attenuates cerulein-induced pancreatic inflammatory infiltrates. Photomicrographs illustrate hematoxylin and eosin stained sections of mouse pancreas in non-cerulein (a, d) and cerulein (b, c, e, f) mice, 2 days after cerulein/vehicle administration. Vehicle-treated non-cerulein mice had an absence of inflammatory infiltrates (a) (n = 5), but cerulein treatment promoted an influx of inflammatory cells within 2 days (b) (n = 3), which had resolved by 7 days (c, d) (n = 4). XPro1595 treatment significantly reduced the immune cell infiltration at 2 days (e) (n = 3), that was even further reduced by 7 days (f) (n = 4). A week following pancreatitis induction the circulating macrophage population was also minimal in cerulein-treated mice, independent of vehicle or XPro1595 treatment (n = 4 each). C cerulein, V vehicle, XP XPro1595, **p < 0.01, scale bars = 100 µm

Fig. 3

Selective inhibition of solTNF in mice with acute pancreatitis prevents pancreatic tissue degradation at 7 days after induction. Photomicrographs illustrate H&E stained pancreatic sections from mice with acute pancreatitis (cerulein treated) with either vehicle—a (n = 4) or XPro1595-treatment b (n = 4), where quantification shows vehicle-treated pancreatitis mice have significant deterioration of tissue integrity (c), compared to pancreatitis mice treated with XPro1595 (n = 4), or non-pancreatitis mice (n = 3–4). In accordance, the vehicle-treated pancreatitis mice also display an exacerbated level of pancreatic acinar cell atrophy (d) that is not observed in the XPro1595 treated pancreatitis group (e, f). A week after induction of pancreatitis the intralobular ducts in the vehicle-treated group appeared to be invaded by groups of acini spreading apart (g), although this was less prominent in the XPro1595-treated group (h, i). NC non-cerulein, C cerulein, V vehicle, XP XPro1595, *p < 0.05, **p < 0.01, ***p < 0.001, scale bar in a and b = 500 µm, scalebar in d and e = 25 µm, scalebar in g and h = 500 µm

Fig. 4

Soluble TNF activity reduces hindpaw mechanical hyper-sensitivity following induction of acute pancreatitis in mice. Graph shows mechanical hypersensitivity of the hindpaw when touched by von Frey filaments, as a percentage of pre-cerulein baseline data for each mouse. No differences were observed in hypersensitivity of non-pancreatitis mice between baseline and on days 3, 5 and 7, independent of treatment (n = 6–8). In contrast, vehicle-treated pancreatitis mice displayed significantly more hypersensitivity than non-pancreatitis control mice at each time point assessed during the first week (n = 9). Treating mice with XPro1595 prevented this hypersensitivity at all time-points tested (n = 9), and this group displayed significantly less hypersensitivity than the vehicle-treated pancreatitis group on the last 2 days of testing. NC non-cerulein, C cerulein, V vehicle, XP XPro1595, *p < 0.05, ***p < 0.001

Fig. 5

Hippocampal GFAP expression is more pronounced in cerulein-treated mice without XPro1595 administration. CA1 hippocampal GFAP expression was semi-quantitated 7 days following induction of pancreatitis in mice. Non-pancreatitis mouse groups, independent of vehicle or XPro1595 treatment, did not display reactive astrogliosis (a, c) (n = 3–4). In contrast, the vehicle-treated pancreatitis tissue (b) had numerous reactive astrocytes and when quantitated displayed a tendency for increased GFAP expression (n = 4), compared to the non-pancreatitis groups, but was not statistically significantly different (e). XPro1595 treated pancreatitis mice prevented the induction of reactive astrocytes (d) (n = 4). NC non-cerulein, C cerulein, V vehicle, XP XPro1595, scalebar = 100 µm