Amylase, lipase, D-lactate, soluble tumor necrosis factor-α receptor (sTNF-αR), and secretory immunoglobulin A (sIgA) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). The chromogenic limulus amebocyte lysate kit was purchased from Bioendo Technology Co., Ltd (Xiamen, China). Rat interleukin-1beta (IL-1β) and transforming growth factor-β (TGF-β) ELISA kits were purchased from ExCell Biotech Co., Ltd (Shanghai, China). Rat TNF-α and IL-10 ELISA kits were purchased from Cusabio Biotech Co., Ltd (Wuhan, China). Rat IL-6 and IL-18 ELISA kits were purchased from Elabscience Biotech Co., Ltd (Wuhan, China). The rat IL-8 ELISA kit was purchased from Neobioscience Technology Co., Ltd (Shenzhen, China). The rat IL-4 ELISA kit was purchased from Invitrogen (Carlsbad, CA, USA). The specific antibodies against FITC anti-rat CD4, Alexa fluor^®647, anti-rat IFN-γ, PE anti-rat IL-4, and Alexa fluor^® 647 anti-rat Foxp3 were purchased from BioLegend (San Diego, CA, USA). Rabbit anti-rat CD68 and rabbit anti-rat CD103 were purchased from Abcam (Cambridge, MA, UK). PE anti-rat CD25 was purchased from BD Biosciences (San Jose, CA, USA). The sources of other reagents were indicated in the specified methods.

All animal experiments were conducted strictly in accordance with the guidelines and regulations of Southwest Medical University and preapproved by the Animal Ethics Committee of Southwest Medical University (approval numbers: 20180309063, Luzhou, China). The specific pathogen-free grade Sprague–Dawley rats (male, 6 weeks old, weight 200 ± 220 g) were purchased from the Dossy Experimental Animals Co., Ltd (permit number: SCXK 2013-24, Chengdu, China). The rats were housed in a specific pathogen-free environment (23 ± 1°C, 40%–70% relative humidity, and 12 h/12 h light–dark cycle), with free access to water and standard rodent diet.

The rats were randomly assigned to three groups: Normal group, Sham group, and SAP group. The Sham group and the SAP group were allocated into 11 subgroups according to time points of 1, 3, 6, 12, 24, 36, 48, 72, 120, 168, and 336 h after surgery. Rats were fasted for 12 h and anesthetized with 2.0% sodium pentobarbital (Sinopharm Chemical Reagent Co., Ltd., China). The animal model was established according to existing studies (17, 18). In brief, an abdominal incision was made along the midline to expose the biliopancreatic duct, and the liver hilum was temporarily closed with a microvascular clamp. Then, the biliopancreatic duct was cannulated using an intravenous indwelling trocar (Fenglin Medical Devices Co., Ltd., Jiangxi, China) with a catheter through the duodenum. The SAP rat model was established by a retrograde injection of 3.5% sodium taurocholate (Sigma-Aldrich, St. Louis, MO, USA) into the biliopancreatic duct using a micro-infusion pump. The microvascular clamp and intravenous indwelling trocar were removed 3 min later, and the abdominal incision was sutured. Postoperatively, 5 ml of normal saline was subcutaneously injected to compensate for fluid loss. The Sham group was injected with equal doses of normal saline, and the Normal group was not treated. Rats in each group were sacrificed at the above indicated time points. For survival analysis, the survival rate was recorded for 14 days after surgery.

The blood samples were obtained through the abdominal aorta, kept at room temperature for 10 min, and centrifuged at 2,500 rpm for 15 min to collect serum, which was then stored at −80°C until further analysis. The pancreas and small intestines were harvested, washed in ice-cold normal saline, and stored at −80°C for further analysis. A small piece of pancreas and small intestines were fixed in 4% paraformaldehyde for pathological analysis. The mesenteric lymph nodes (MLNs) were harvested and gently ground with the inner core of a 5-ml syringe to make it pass through the 40-μm cell strainer to prepare a single-cell suspension of MLNs. Centrifugation was conducted at 1,000 rpm for 10 min, and the supernatant was discarded. Cell pellets of MLNs were resuspended in complete medium, and the concentration was adjusted to 10⁶/ml by cell counting plate for flow cytometry. An experiment flowchart is shown in Figure 1A.

Pancreas and small intestines were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Slices (5 µm) of the paraffin-embedded tissue were stained with hematoxylin and eosin. All the slices were graded by two experienced pathologists who were blind to the experimental protocol under a light microscope (Olympus Corporation, Tokyo, Japan). Inflammation, edema, hemorrhage, and acinar cell necrosis of pancreas were scored according to Schmidt’s standard (19). The severity of the small intestines was assessed as previously described by Chiu (20).

The activities of serum amylase and pancreatic lipase were determined with the corresponding assay kits according to the manufacturer’s instructions.

The levels of serum D-lactate and endotoxin were assayed using the commercially available detection kits, and the levels of pro- and anti-inflammatory cytokines including IL-1β, TNF-α, IL-6, IL-8, IL-18, TGF-β, IL-10, IL-4, and sTNF-αR in the small intestines were quantified using the relevant ELISA kits according to the manufacturer’s instructions. Absorption at 450 nm was examined using an Epoch 2 microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Immunohistochemical staining was performed to determine the expressions of intestinal CD68 and CD103 to observe the changes in intestinal macrophages and dendritic cells. The paraffin-embedded slices were dewaxed, rehydrated, and treated for antigen retrieval according to standard procedures. Endogenous peroxidase was blocked by 3% hydrogen peroxide. After blocking the nonspecific antigens, the slices were incubated overnight at 4°C with anti-rat CD68 antibody or anti-rat CD103 antibody, followed by incubation with polymer adjuvant and horseradish peroxidase-labeled anti-rabbit IgG polymer (Bioss Biotech Co., Ltd., Beijing, China). Finally, the slices were stained with DAB (Beyotime Biotech Co., Ltd., Shanghai, China), and nuclei were counterstained with hematoxylin (Solarbio Technology Co., Ltd., Beijing, China). The samples were observed, and pictured under a Leica DMi8 inverted microscope (Leica Microsystem Ltd., Wetzlar, Germany). Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

MLN cells were placed in a sterile 6-well plate with complete medium, and cultured with cell activation cocktail (BioLegend, San Diego, CA, USA) for 5 h in an incubator (Thermo Scientific, Waltham, MA, USA) with 5% CO₂, at 37°C. After culturing for 2 h with brefeldin A solution (BioLegend), the cells were collected, centrifuged at 1,000 rpm for 5 min, and resuspended in PBS. Next, the cells were incubated with FITC anti-rat CD4 antibody for 15 min at room temperature in darkness, and then washed twice with PBS. Stained cells were fixed with fixation buffer (BioLegend) for 20 min at room temperature and washed twice with PBS. For intracellular staining of IFN-γ and IL-4, fixed cells were permeabilized using a permeabilization buffer (BioLegend) for 20 min at room temperature and incubated with Alexa fluor 647^® anti-rat IFN-γ antibody or PE anti-rat IL-4 antibody for 15 min at room temperature in darkness. After washing twice with cell staining buffer (BioLegend), the cells were suspended in cell staining buffer and the ratio of Th1/Th2 was determined by a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA) as previously described (21).

The expression of Tregs from the MLNs was detected by a flow cytometer after staining with anti-rat-specific Abs conjugated with FITC, PE, and Alexa fluor 647^®. To detect the proportion of CD4⁺ T cells in MLN cells and CD25 expression on the surface of CD4⁺ T cells, the cells were stained with FITC anti-rat CD4 antibody and PE anti-rat CD25 antibody for 30 min at room temperature. Simultaneously, for the measurement of intranuclear Foxp3, stained cells were washed with PBS and reacted with true-nuclear™ 1× fix working solution (BioLegend) for 60 min at room temperature. After washing twice with true-nuclear™ 1× perm buffer (BioLegend), the cells were stained with Alexa fluor 647^® anti-rat Foxp3 antibody for 30 min. After washing, the cells were suspended in cell staining buffer (BioLegend) and analyzed by a FACSCalibur flow cytometer (BD Biosciences) as previously described (22).

Rats were randomly assigned to four groups: Sham group, Sham + PA group, SAP group, and SAP + PA group. The Sham + PA group and the SAP + PA group were injected with 2.0×10⁸ CFU/kg of PA via the tail vein at 0, 12, 24, 36, 48, and 72 h after Sham or SAP construction, and the other groups were injected with equal doses of normal saline. Rats were sacrificed at 2 h after injection, and small intestines were collected. The levels of intestinal inflammatory cytokines were measured according to the manufacturer’s instructions. Meanwhile, the numbers of macrophages and dendritic cells were analyzed as previously described.

Data were analyzed by GraphPrism 8.0 software (San Diego, CA, USA) and expressed as mean ± standard deviation (SD). The comparison was conducted by Student’s t-test between two groups, and the comparison between multiple groups (including two factors with different time and model processing) was conducted using two-way analysis of variance (two-ANOVA). Counting data were tested by the chi-square test. The survival curves were analyzed using the Log-rank test. p < 0.05 was considered statistically significant.

There was no dead rat in the Sham group and Normal group, while the rats in the SAP group developed drowsiness, abdominal pain, and distension, and started to die at 12 h after model construction. A total of 11 rats died at 12–24 h and 5 rats died at 48–72 h. The 14-day cumulative survival rate of SAP rats was 65.72% (Supplemental Table 1; Figure 1B). The mortality rule of SAP rats was basically consistent with previous reports (23, 24). The results suggested that 12–24 h and 48–72 h were the two death peaks of SAP rats.

To further clarify whether the SAP rat model was successfully established, we observed the pathological changes in pancreas, and measured the activities of serum amylase and pancreatic lipase.

The results showed that the pancreas exhibited intact pancreatic characteristics with normal structures in the Sham and Normal groups. However, the pancreas in the SAP group showed a small amount of inflammation infiltration, edema, hemorrhage, and acinar cell necrosis (black arrow) at 1–3 h, and these pathological changes further deteriorated at 6–48 h. The pancreas of SAP rats were in a self-healing state with massive fibrous hyperplasia and granulation tissue formation (red arrow) at 72–336 h (Figure 2A). The pancreatic pathological score in the SAP group was markedly greater than that in the Sham group at different time points (Figure 2B). Compared with the Sham group, the activities of serum amylase and pancreatic lipase in the SAP group were evidently elevated at 1–48 h and at 3–24 h, respectively (Figures 2C, D). Overall, these results illustrated that the SAP rat presented self-limiting pathological changes from the early injury aggravation (1–48 h) to later continuous repair (72–336 h), indicating that the SAP rat model was successfully established.

The intestinal mucosal barrier function helps maintain intestinal homeostasis (25). As an intestinal metabolite, the serum D-lactate level can reflect the changes in intestinal mucosal permeability (7, 17). The changes in intestinal mucosal barrier function of SAP rats can be determined by observing pathological changes in the small intestines and detecting the serum D-lactate level.

The results showed that the small intestines were structurally intact in the Sham and Normal groups. However, the small intestine in the SAP group showed obvious changes in morphology and structure, such as intestinal dilatation, epithelial cell shedding, and inflammatory infiltration (black arrow) at 6 h, and the tissue damages were further aggravated at 12–48 h, but gradually ameliorated at 48–336 h (Figure 3A). The intestinal pathological score in the SAP group was meaningfully higher than that in the Sham group after 12 h, which increased at 6 h, basically kept a higher level at 12–48 h, but gradually decreased at 48–336 h (Figure 3B). The serum D-lactate level in the SAP group gradually increased at 12 h, and slightly decreased after maintaining a higher level at 48–120 h (Figure 3C). The aforementioned results suggested that the intestinal mucosal barrier function was partially impaired, and the gut permeability was markedly increased in the early stage after SAP model construction.

Given that inflammation is the main initiator of intestinal injury caused by SAP, we sought to explore the dynamic changes in intestinal pro- and anti-inflammatory cytokine levels to reflect changes in intestinal mucosal immune function during SAP.

The detection of intestinal pro-inflammatory cytokines found that, compared with the Sham group, the level of IL-1β in the SAP group increased at 12 h, peaked at 24 h, and hereafter gradually decreased until it dropped to normal levels at 120 h (Figure 4A). Compared with the Sham group, the level of TNF-α in the SAP group was slowly elevated at 1–12 h, then quickly increased at 12 h, and kept at a higher level at 24–48 h, which rapidly declined to normal levels at 120 h (Figure 4B). Compared with the Sham group, the levels of IL-6, IL-8, and IL-18 in the SAP group considerably increased at 6 h and peaked at 24 h. Then, the levels of IL-6 and IL-8 gradually decreased at 24 h, and the former fell to normal levels at 168 h, while the latter dropped to normal levels at 72 h (Figures 4C, D). The level of IL-18 in the SAP group after maintaining a higher level at 24–48 h gradually decreased at 48–336 h, which was still higher than that in the Sham group (Figure 4E). Compared with the Sham group, the level of serum endotoxin in the SAP group increased at 12 h, peaked at 24 h, remained at a higher level at 24–48 h, and gradually decreased to normal levels at 72 h (Figure 4F).

The results of intestinal anti-inflammatory cytokines showed that, compared with the Sham group, the level of intestinal TGF-β in the SAP group slowly increased at 1 h, remained basically unchanged at 1–48 h, and then slowly declined at 48–168 h until it eventually decreased to normal levels at 336 h (Figure 4G). Interestingly, the level of intestinal IL-10 in the SAP group showed the double peaks, which gradually increased at 1–36 h, greatly decreased at 36–72 h, and then gradually increased again at 72–336 h (Figure 4H). The levels of intestinal IL-4 and sTNF-αR in the SAP group gradually decreased at 3 h, remained low at 6–36 h, then gradually increased at 36 h until they finally returned to normal levels at 48 h (Figures 4I, J).

Taken together, these data demonstrated that the inflammatory response of SAP rats was developed at 1–12 h, during which the pro-inflammatory and anti-inflammatory cytokines gradually increased. Subsequently, the inflammatory cytokine storm was observed at 12–24 h, during which the pro-inflammatory response became dominant. Then, both pro-inflammatory and anti-inflammatory cytokines reached their peak levels at 24–48 h and were counterbalanced by each other. Eventually, a hypo-inflammatory response was dominant after 48 h. As a result, the counterbalance of pro- and anti-inflammation occurred at 24–48 h after SAP model construction.

Innate immune cells in the small intestines activate inflammasome signaling pathways via pattern recognition receptors, rapidly initiating inflammatory responses (26, 27). Macrophages and dendritic cells in the small intestines are the main inflammatory cells involved in intestinal innate immune response (28, 29). CD68 and CD103 are regarded as specific markers of macrophages and dendritic cells, respectively (30, 31). An immunohistochemical staining method was used to analyze the dynamic changes in intestinal macrophages and dendritic cells in each group.

The results showed that the expression of intestinal CD68 in the SAP group gradually increased at 1–6 h, greatly decreased at 12–72 h, and gradually increased again at 120–336 h compared with the Sham group (Figures 5A, C). Compared with the Sham group, the expression of intestinal CD103 in the SAP group gradually increased at 1–3 h, slowly reduced at 6–72 h, and gradually rose again at 120–336 h (Figures 5B, D). The above results verified that the number of intestinal innate immune cells increased and the inflammatory response was enhanced at 1–3 h. At 6–72 h, the number of intestinal innate immune cells decreased, resulting in declined cellular immune function and intestinal immune barrier dysfunction. After 120 h, the number of intestinal innate immune cells returned to normal levels.

Th cells, Tregs, and sIgA are indispensable parts in the adaptive immune response (32). Thus, we further evaluated their changes in SAP rats. We firstly investigated the changes in intestinal Th cells in SAP rats. Th cells are the central cells of the body’s adaptive immune response (32). The expressions of CD4⁺/IFN-γ⁺ and CD4⁺/IL-4⁺ serve to quantify the activations of Th1 cells and Th2 cells, respectively (13). The results revealed that the expression of Th1 cells in the SAP group was significantly higher than that in the Sham group at 6–72 h, which did not significantly change at 1–3 h, gradually increased at 6–24 h, peaked at 24 h, and gradually decreased until it dropped to normal levels at 120 h (Figures 6A, D). The expression of Th2 cells in the SAP group was significantly lower than that in the Sham group at 48–336 h. Th2 cells did not obviously change at 1–36 h, but slowly declined at 48–336 h (Figures 6B, E). The ratio of Th1/Th2 in the SAP group was significantly higher than that in the Sham group at 12–336 h, which did not significantly change at 1–3 h, gradually elevated at 6–24 h, peaked at 24 h, and then gradually downregulated at 24–336 h (Figure 6F). As a result, severely imbalanced Th1/Th2 was observed in SAP rats.

Next, we studied the changes in Tregs in SAP rats. Tregs, which are regarded as suppressive T cells, play a crucial role in maintaining the balance between immune activation and tolerance (13). The expression of CD4⁺/CD25⁺/FOXP3⁺ is identified as the most specific marker of Tregs (13). The results showed that Tregs in the SAP group were obviously higher than those in the Sham group at 24–120 h, which greatly increased at 12 h, slowly declined at 24–48 h after peaking at 24 h, gradually decreased at 48–120 h, until it finally returned to normal levels at 168 h (Figures 6C, G).

Finally, we also detected the changes in intestinal sIgA, which is the first line of intestinal mucosa defense and exerts a key role in humeral immunity (33). The results showed that the level of intestinal sIgA in the SAP group was evidently lower than that in the Sham group at 3–36 h, which gradually decreased at 1 h, remained at a lower level at 3–36 h, gradually upregulated at 36 h, and finally returned to normal levels at 48 h (Figure 6H). All the above results indicated that intestinal innate and adaptive immune switch occurred at 12–48 h after SAP model construction.

Obviously, there were still some differences in the immunoswitching points of changes in various intestinal inflammatory cytokine levels and immune cell numbers. Hence, in further experiments, PA was selected as a second hit to further confirm the immunoswitching point from excessive inflammatory response to immunosuppression in SAP rats.

The rats were injected with a sublethal dosage of PA (2.0×10⁸ CFU/kg; Supplemental Table 2) via the tail vein at 0–72 h after Sham or SAP model construction, followed by sacrifice at 2 h post injection (Supplemental Figures 1A, B). The detection of pro- and anti-inflammatory cytokines of Sham or SAP rats challenged with or without PA is shown in Figure 7. The results showed that the levels of IL-1β, TNF-α, IL-6, IL-8, IL-18, and serum endotoxin significantly increased in Sham + PA, SAP, and SAP + PA groups compared with the Sham group at 0–72 h. The same trend was also found in TGF-β level, while the level of IL-4 presented a trend of first gradually decreasing (0–36 h) and then gradually increasing (48–72 h). Compared with the SAP group, most of the cytokines in the SAP + PA group showed a different degree of increase at 0–36 h, but turned around at 48 h, during which even if rats were challenged with PA, their levels did not increase (Figures 7A–H).

We also analyzed the changes in macrophages and dendritic cells. The results found that after challenge with PA, the number of macrophages in the Sham + PA group and the SAP-0 h + PA group significantly increased. Then, the number of macrophages in the SAP + PA group gradually decreased, which was significantly higher than that in the SAP group at 0–24 h, but with a reversal at 48 h. The changing pattern of dendritic cells was obviously different. Except for the SAP-0 h + PA group, there was no significant increase in other groups. Compared with the Sham group, the number of dendritic cells in the SAP group and the SAP + PA group greatly decreased from 48 h (Figures 8A–D). These data confirmed that the rats after challenge with PA showed excessive activation of immune cells and further secretion of cytokines, but the SAP rats changed their immune status into an immunosuppressive one at 48 h, where the immune cells were exhausted, showing a hypo-inflammatory response.