Temporal expression pattern of GHSR/ghrelin
The temporal expression patterns of GHSR and ghrelin during DSS challenge were examined in colon, mesenteric lymph nodes (MLN) and spleen at 3 time points (day 0, 4 and 7). Our results demonstrated that GSHR mRNA expression in MLN or colon, but not spleen, were increased by ~3 (p < 0.05) or ~1 fold (p < 0.05) at day 7 DSS challenge, respectively (Figure 1A).
Moreover, there was constitutive level of ghrelin mRNA in colon, MLN and spleen. Splenic ghrelin mRNA was induced by ~2 fold (p < 0.01) in MLN at day 7 from DSS-challenged WT mice. However, no obvious change of ghrelin expression was observed in colon or spleen following 7 days DSS treatment (Figure 1B).
Lack of GHSR attenuated DSS-induced acute colitis
There was no obvious body weight change or clinical as well as histopathological abnormalities in mock challenged GHSR−/− and WT mice, suggesting that deficiency of GHSR does not cause spontaneous inflammation in the gastrointestinal system. Significant body weight loss was observed on days 6 and 7 in DSS challenged GHSR−/− and WT mice, compared to that of mock challenge (p < 0.05) (Figure 2A). However there was no significant difference between WT and GHSR−/− mice with DSS challenge at any time point. Disease activity index (DAI) score was used to evaluate the clinical severity of colitis, referring to three parameters, i.e. body weight loss, diarrhoea and faecal blood as descried previously [13]. The body weight change was monitored twice day, each group there were 6 mice. Thus the mean and SD of the bodyweight change were calculated. The DAI score was gradually increased from day 1 to day 7 in both DSS-treated GHSR−/− and WT mice. There was significantly higher DAI in WT with DSS challenge at days 6 (p < 0.01) or 7 (p < 0.05), compared to GHSR−/− mice (Figure 2B), suggesting attenuation of clinical signs in DSS-treated GHSR−/− mice.
Splenic weight at day 7 was measured as it reflects systemic immunity. There was no difference in splenic weight between WT and GHSR−/− mice without challenge. A ~2-fold increase of splenic weight was observed in WT mice with DSS challenge, compared to mock challenged (p < 0.01), but no significant different splenic weight was detected in GHSR−/− mice between DSS and mock treatment. Moreover, splenic weight from DSS-challenged WT was 40% higher than that of GHSR−/− mice (p < 0.05) (Figure 2C).
Colon length is an objective macroscopic criteria in determining severity of colitis [13,14]. No significant difference in colon length was observed between GHSR−/− and WT mice without challenge. However, there was ~25% (p < 0.001) or ~10% (p < 0.05) reduction for the WT or GHSR−/− mice, respectively, with DSS 7 day treatment, compared to unchallenged counterparts. In addition, colon length was significantly shortened in WT compared to that of GHSR−/− mice following 7 day DSS challenge (p < 0.05) (Figure 2D).
Histopathology score, including intestinal inflammation and crypt damage, was applied to determine the severity of colitis, as described [13]. There was no obvious colonic damage from both GHSR−/− and WT mice without challenge. Severe inflammation was observed in the colons from both GHSR−/− and WT mice following 7 days DSS treatment, showing epithelial ulceration, crypt damage, goblet cell loss and infiltration of inflammatory cells. Additionally, in agreement with previous studies [13], the damage/inflammation in transverse/descending colon (Figure 3A, B) was much more severe than had occurred in the ascending colon (Figure 3C).
Significantly higher histopathological score was observed in transverse (WT: 5.7 ± 0.5, GHSR−/−: 4.5 ± 0.5, p < 0.05) and descending colons (WT: 7.0 ± 0.5, GHSR−/−: 4.9 ± 1.0, p < 0.05) from DSS-treated WT, compared to GHSR−/− mice. However, there was no significant difference in histopathological score in ascending colon between DSS-treated WT and GHSR−/− mice (Figure 3A, B, C).
Lack of GHSR inhibits the production of cytokines
There was constitutive level of colonic TNF from both GHSR−/− and WT mice without significant difference, as measured by tissue ELISA. Colonic TNF was significantly upregulated in DSS-challenged WT mice (~2 fold, p < 0.01), but not GHSR−/− mice, compared to mock treated mice. Furthermore, colonic TNF was significantly higher in DSS-challenged WT compared to that of GHSR−/− mice (p < 0.05) (Figure 4A). A similar pattern was observed in colonic IL-6 production, which was markedly increased in DSS-challenged WT mice compared to mock treated WT mice (~2 fold, p < 0.05) or DSS-treated GHSR−/− mice (~2 fold, p < 0.05) (Figure 4A).
Colonic mRNA levels of TNF, IL-1β, IL-6, IFN-γ, M-CSF and GM-CSF were quantified, using rRT-PCR. There were constitutive levels of colonic TNF, IL-1β, IL-6, IFN-γ, M-CSF and GM-CSF without significant difference between GHSR−/− and WT mice. The expressions of TNF, IL-1β, IL-6, M-CSF and GM-CSF were up-regulated by 11 fold, (p < 0.01), 348 fold (p < 0.05), 2 fold (p < 0.05), 2 fold (p < 0.05), 2 fold (p < 0.05), respectively in WT mice following DSS challenge, excepted for and IFN-γ (no significance). These cytokines were also up-regulated significantly in GHSR−/− mice following DSS challenge, but TNF, IL-1β, IL-6, M-CSF or GM-CSF for GHSR−/− mice were 40%, (p < 0.05), 60%, (p < 0.01), 80%, (p < 0.05), 40%, (p < 0.05), 70%, (p < 0.05) lower compared to WT mice following DSS challenge (Figure 4B).
Lack of GHSR decreases TLRs expression
TLR-2 and TLR −4 are two key molecules involved in triggering innate immunity, which plays an important role in DSS-induced acute colitis [9,11]. There was a constitutive level of colonic TLR-2 or TLR-4 expression for both GHSR−/− and WT mice without significant difference. The expression of colonic TLR-2/4 was 1.8 (p < 0.05), or 1.5 (p < 0.01) fold increased, respectively, following 7 days DSS challenge in WT mice. However, there was no obvious change of colonic TLR-2 or TLR-4 for DSS-challenged GHSR−/− mice; which was significantly lower than WT expression of TLR-2 (p < 0.01) or −4 (p < 0.005). Moreover, a similar pattern was observed in MLN expression of TLR-4, but not TLR-2. GHSR−/− showed a ~40% lower expression of TLR-4 than WT (p < 0.05) following 7 days DSS challenge (Figure 4C).
Lack of GHSR reduces macrophage infiltration
There was constitutive level of macrophages (F4/80+ cells) in the lamina propria from GHSR−/− and WT mice without significant difference (Figure 5A and B). Infiltrating macrophages were recruited substantially in the inflamed colon following 7 days DSS challenge from both GHSR−/− and WT mice. Compared to unchallenged, there was a ~5 or ~9 fold increase of colonic F4/80+ cell infiltration in GHSR−/− (p < 0.01), or WT (p < 0.005), respectively. The level of infiltrating colonic macrophages was ~2 fold higher in DSS-treated WT mice compared to GHSR−/− mice at 7 days treatment.
Lack of GHSR suppresses macrophage cytokine production
There was a constitutive level of TNF in the supernatant of cultured primary peritoneal macrophages from both GHSR−/− and WT mice. TNF was induced significantly in LPS-stimulated macrophages from GHSR−/− and WT mice (p < 0.005). However, the level of TNF from LPS-stimulated GHSR−/− macrophages was significantly lower than that of WT (p < 0.05) (Figure 6A). Additionally, similar patterns were observed in IL-6 and IL-12 (p40) productions. The levels of LPS-induced IL-6 or IL-12 (p40) in the supernatant of macrophages from GHSR−/− mice was reduced by ~50% (p < 0.005) and ~35% (p < 0.05) respectively, compared to that of WT mice (Figure 6B, C).
GHSR antagonist alleviates the cytokines released by macrophages
DLS, an antagonist of GHSR, suppressed the LPS-stimulated WT macrophage TNF production by ~70% (p < 0.05) at 100 μM compared to mock treated cells (Figure 7A). Similarly, it was observed that DLS at 20 or 100 μM reduced IL-12 (p40) by >30% or >40%, respectively (p < 0.05) (Figure 7C). However, no inhibitory function was observed for IL-6 (Figure 7B).