To the Editor: The enhanced activation of immune cells is a decisive factor for the pathological progress of inflammatory bowel disease (IBD), which can release generous inflammatory cytokines and bioactive molecules suggesting their roles as potential therapeutic targets for IBD. Purinergic receptors are a family of membranous proteins found extensively in many mammalian tissues and organs. As a subtype of purinergic receptors, the expressions of P2X7 receptor (P2X7R) can be detected abundantly in epithelial cells and most immune cells. During inflammation, adenosine triphosphate (ATP) is released extracellularly by various stimuli. In general, through the activation of P2X7R, extracellular ATP (eATP) can stimulate inflammation, while the degradation of ATP to adenosine usually exerts anti-inflammation roles. Consequently, ATP/P2X7R signaling can stimulate immune cells to produce various biological activities. Currently, mouse models of colitis have clarified the role of P2X7R in inflammation, which was supported by the increase in extracellular purines in the process of inflammation and the deregulated expression of purine receptor genes and proteins.[1,2] In addition, nuclear factor kappa B (NF-κB) is a pleiotropic transcription factor, which can drive the expression of pro-survival genes in intestinal epithelial cells and coordinate the expression of pro-inflammatory genes in innate and adaptive immune system cells. NF-κB has been proven to be a key regulator in the progression of IBD.
It is well known that the tight junctions at the top of intestinal epithelial cells play a significant role in regulating epithelial barrier permeability. Both IBD patients and modeled animals had increased epithelial barrier permeability, even before the onset of the disease. In addition, submucosal inflammation of IBD may damage the integrity of intestinal mucosal barrier structure, leading to increased permeability of intestinal mucosa, hence allowing paracellular permeation of luminal antigens and aggravation of intestinal inflammation. Based on the findings above, we hypothesized that P2X7R may be over-activated in macrophages of IBD and promote the production of inflammatory cytokines through inactivating the NF-κB pathway, which may further induce dysfunction of the intestinal mucosal barrier and thus exacerbate the progression of colitis.
The experimental animals were 40 C57BL/6 wild-type 7-8-week-old male mice weighing 21 ± 2 g (procured from the Animal Center of Central South University). Drinking water was administrated in mice in the control group while 2.5% Dextran Sulfate Sodium (DSS) in drinking water for the other four groups, namely DSS,3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate(BzATP, Sigma, B6396-5mg, America), brilliant blue G (BBG, Santa Cruz Biotechnology, sc-203733, America), and apyrase, for 8 consecutive days. Additionally, BzATP (5 mg/kg, a P2X7R agonist), BBG (40 mg/kg, the P2X7R antagonist), and apyrase (Sigma, 40 μg/kg, an ATP scavenger), were injected intraperitoneally into mice in the BzATP, BBG, and apyrase groups, respectively, while the same volume of normal saline was injected intraperitoneally in the control and DSS groups. All mice were euthanized on the eighth day after DSS treatment. After weighing and measuring the length of the colon, the whole colon was fixed in 10% formalin to prepare paraffin-embedded sections for hematoxylin & eosin (H&E) staining. A TCS-SP5 AOBS confocal laser scanning microscope (Leica, Heidelberg, Germany) was used to visualize protein expression and localization of P2X7R and F4/80 FITC, with the selection of representative images for further use.
Blood samples were collected from 33 Crohn's disease (CD) patients and 34 healthy subjects from the Department of Gastroenterology, Xiangya Hospital, Central South University from January 2020 and December 2020. Venous blood samples in the EDTA anticoagulant tubes were subject to centrifugation. PBMCs were separated with a lymphocyte isolation agent by ficoll-hypaque density gradient centrifugation. PBMCs washed twice in PBS were suspended at 5 × 105 cells/mL in RPMI-1640 (sigma, R0883-100mL, America) medium and cultured in six-well plates. Macrophages were obtained when the suspended cells were removed after overnight incubation at 37°C containing 5% CO2. The obtained macrophages were cultured continuously in RPMI-1640 medium containing 10% FBS based on different protocols: (1) without any additives for 5 h; (2) lipopolysaccharide (LPS) for 5 h; (3) LPS for 4 h + BzATP for 1 h; (4) LPS for 4 h + BBG for 1 h; (5) LPS for 4 h + apyrase for 1 h; (6) LPS for 4 h + JSH-23 (NF-κB inhibitor; Santa Cruz Biotechnology) for 1 h; and (7) LPS for 4 h + BBG + JSH-23 for 1 h. Among them, the concentrations of LPS, BzATP, BBG, apyrase, and JSH-23 were 1 μg/mL, 300 μmol/L, 1 μmol/L, 4 U/mL, and 20 μmol/L, respectively. The β-catenin mRNA levels were assessed via quantitative polymerase chain reaction. The total and nuclear β-catenin expressions were assessed via western blotting. IL-1β and IL-6 concentrations (picograms per meter) in the supernatants of cultured PBMCs were measured using enzyme-linked immunosorbent assay (ELISA) using corresponding kits (CUSABIO, CSB-E08054M and CSB-E04639M, America) in duplicate.
The weight of mice decreased significantly in the DSS and BzATP groups from the fourth day of DSS administration compared with that in the control group (all P <0.01), with a more serious weight loss in the BzATP than in the DSS group (all P <0.01). However, compared with the DSS group, there was a significant alleviation in weight loss of the mice in both the BBG and apyrase groups (P <0.01) [Supplementary Figure A,https://links.lww.com/CM9/B565]. Furthermore, DAI scores were increased in the DSS and BzATP groups, which, however, were partially prevented in the BBG and apyrase groups (all P <0.01) [Supplementary Figure A,https://links.lww.com/CM9/B565]. In addition, H&E staining [Supplementary Figure B,https://links.lww.com/CM9/B565] revealed that DSS treatment induced massive destruction of colonic epithelial structures, loss of goblet cells, diffuse loss of crypts, and robust inflammatory cell infiltration in the mucosa. Significantly, the BBG and apyrase groups showed partial improvement in the pathological changes of the colonic sections in mice. Previous studies have shown that P2X7R was widely expressed in the epithelium and the crypt bottom of the colon from DSS-induced mice. To determine major immune cells that co-expressed P2X7R, anti-P2X7R was incubated with F4/80 in tissue sections. As presented in Supplementary Figure C, https://links.lww.com/CM9/B565 concerning the co-localization of P2X7R (green) with macrophages (F4/80) (red), in the epithelium of DSS and BzATP treated mice, P2X7R was co-localized with macrophages. However, the expression of P2X7R decreased obviously in the DSS + BBG group (P <0.01) and the DSS + apyrase group (P <0.01) in comparison with that in the DSS group [Supplementary Figure C,D, https://links.lww.com/CM9/B565].
The role of NF-κB in IBD has been recognized and the p65 protein is the representative member, and phosphorylation is the starting point in its activation. In our experiment, NF-κB p65-Ser536 phosphorylation level was elevated in the DSS group compared with that in the control group (P <0.001). In contrast, the DSS + BBG and DSS + apyrase groups showed a decreased NF-κB p65-Ser536 phosphorylation level when compared with the DSS group [Supplementary Figure E,https://links.lww.com/CM9/B565]; similar changes of NF-κB p65-Ser536 were also observed in the western bloting and quantitative polymerase chain reaction [Supplementary Figure F,https://links.lww.com/CM9/B565]. To understand the underlying role of ATP/P2X7R signaling on macrophages, we determine the effect of the NF-kB (P65) pathway on IL-1β and IL-6 levels in an in vitro experimental model. Consequently, the LPS-stimulated IL-1β release from macrophages in CD patients was significantly higher compared with macrophages in CD patients cultured in RPMI alone (P <0.05; Supplementary Figure G,https://links.lww.com/CM9/B565). These changes could also be found in cultured macrophages from healthy controls. However, the LPS-stimulated effect in cultured macrophages from healthy controls was less obvious than that from CD patients. However, the production of IL-1β in the LPS + BBG and LPS + apyrase groups was significantly decreased compared with that in the CD + LPS group (both P <0.05). In addition, similar changes were also observed in IL-6 levels in the macrophages' culture medium [Supplementary Figure G,https://links.lww.com/CM9/B565]. The additional JSH-23 treatment also decreased IL-1β and IL-6 levels in the CD + LPS group, while no difference was found in their release between the LPS + BBG and LPS + JSH-23 groups. Interestingly, there was no synergistic effect when BBG and JSH-23 were used to intervene in macrophages simultaneously.
In general, ATP is derived from symbiotic bacteria harbored in the intestine or from damaged cells in the inflammatory tissue. It can continuously activate P2X7R to increase cell permeability and induce cell death.[4,5] In addition, DSS and BzATP treatment resulted in an elevated count of apoptotic cells in the crypts. The abnormally increased apoptosis of intestinal epithelial cells could further damage the epithelial barrier, lead to overexposure to lumen bacteria, and ultimately destroy the intestinal epithelium's integrity and intestinal homeostasis. Based on these findings, our study established a connection of P2X7R-regulated ATP levels with intestinal inflammation. Interestingly, apyrase can significantly attenuate DSS-induced colitis following the downregulation of ATP levels via promoting ATP hydrolysis. It suggests that the dynamic changes of ATP are a key factor in DSS-induced colitis. Collectively, the findings in our study may provide complementary information to demonstrate that ATP/P2X7R signaling was a potentially important pathway involved in affecting epithelial cell proliferation, differentiation, and cell death.
Our study discovered that macrophages played a pivotal role in mediating the severity of colitis by interacting with ATP and P2X7R. These interactions could both induce macrophage-mediated inflammation and aggravate the progression by promoting macrophage infiltration. Collectively, our study indicates that macrophages have important roles in DSS-induced colitis and human intestinal inflammation via mediating the release of various inflammatory cytokines and mediators.
This work was supported by the National Natural Science Foundation of China (Nos. 82230019, 81770584, and 82000502), China Postdoctoral Science Foundation (No. 2021M693572), Natural Science Foundation of Hunan Province (Nos. 2020SK2068 and 2020JJ5941), Free exploration and innovation project of Central South University (No. 506021711), and Fundamental Research Funds for the Central Universities of Central South University (No. 2020zzts268).
Conflicts of interest
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