Intestinal TLR4 deletion exacerbates acute pancreatitis through gut microbiota dysbiosis and Paneth cells deficiency

Abstract

Toll-like receptor 4 (TLR4) has been identified as a potentially promising therapeutic target in acute pancreatitis (AP). However, the role of intestinal TLR4 in AP and AP-associated gut injury remains unclear. This study aimed to explore the relationship between intestinal TLR4 and gut microbiota during AP. A mouse AP model was establish by intraperitoneal injection of L-arginine. Pancreatic injury and intestinal barrier function were evaluated in wild-type and intestinal epithelial TLR4 knockout (TLR4ΔIEC) mice. Gut microbiota was analyzed by 16S rRNA sequencing. Quadruple antibiotics were applied to induce microbiota-depleted mice. Differentially expressed genes in gut were detected by RNA sequencing. L. reuteri treatment was carried out in vivo and vitro study. Compared with wild-type mice, AP and AP-associated gut injury were exacerbated in TLR4ΔIEC mice in a gut microbiota-dependent manner. The relative abundance of Lactobacillus and number of Paneth cells remarkably decreased in TLR4ΔIEC mice. The KEGG pathway analysis derived from RNA sequencing suggested that genes affected by intestinal TLR4 deletion were related to the activation of nod-like receptor pathway. Furthermore, L. reuteri treatment could significantly improve the pancreatic and intestinal injury in TLR4ΔIEC mice through promoting Paneth cells in a NOD2-dependent manner. Loss of intestinal epithelial TLR4 exacerbated pancreatic and intestinal damage during AP, which might be attributed to the gut microbiota dysbiosis especially the exhausted Lactobacillus. L. reuteri might maintain intestinal homeostasis and alleviate AP via Paneth cells modulation. Abbreviations: AP Acute pancreatitis, TLR4 Toll-like receptor 4, IL-1β Interleukin-1β, IL-6 Interleukin-6, TNF-α Tumor necrosis factor-α, SIRS Systematic inflammatory response syndrome, LPS Lipopolysaccharides, SPF Specific pathogen-free, ZO-1 Zonula occludens-1, CON Control, H&E Hematoxylin and eosin, FISH Fluorescence in situ hybridization, DAPI 4′,6-diamidino-2-phenylindole, PCoA Principal co-ordinates analysis, SCFA Short chain fatty acid, LEfSe Linear discriminant analysis Effect Size, ANOVA Analysis of variance, F/B Firmicutes/Bacteroidetes, PCA Principal component analysis, NOD2 Nod-like receptor 2, ABX antibiotics, PCNA proliferating cell nuclear antigen

Intestine microbiota and SCFAs response in naturally Cryptosporidium-infected plateau yaks

“…Results showed that Cryptosporidium infection increased the alpha diversity index of Shannon (p<0.01) and Simpson (p<0.01) (Figure 1B), which demonstrated the increased microbiota complexity of infected animals. The current results are in line with our previous results found in Cryptosporidium-infected horses (Wang et al, 2022). Beta diversity analysis through NMDS, PCoA, Qiime 2b, and PCA analysis revealed microbiota differences between healthy and infected yaks (Figure 3), which were confirmed by comparing the dominating gut microbiota in different taxa (Figures 2, 4).…”

“…Then we explored the significantly different bacteria between the H and INF groups through DESeq 2 volcano diagram and LEfSe chart analysis. The results showed that a total of 11 phyla and 58 genera were significantly different (Figure 5), which is in accordance with the previously reported results in a study conducted on infected people and horses (Chappell et al, 2016;Wang et al, 2022). The increased genera in yaks were in line with previous studies that found a higher abundance of Desulfovibrio and Butyrivibrio in colitis patients (Berry and Reinisch, 2013;Gryaznova et al, 2021), Prevotellaceae_Prevotella in diarrheic pigs (Yang et al, 2017), Anaerovorax in slow growth performers in nursery pigs (Zhai et al, 2020), Succiniclasticum in LPS induced dual-flow continuous culture system (Dai et al, 2019), Pseudobutyrivibrio in chronic kidney people (Wu et al, 2020), Anaerorhabdus in pulmonary fibrosis persons (Tong et al, 2019), Selenomonas in gastric cancer patients (Zhang et al, 2021), Anaerostipes in diabetic nephropathy patients (Du et al, 2021), Pyramidobacter in endoscopic sphincterotomy surgery gallstone patients (Shen et al, 2021), and Anaerofustis in Alzheimer people (Hou et al, 2021).…”

“…Intestine microbiota is composed of millions of complex and diverse microorganisms, which contribute greatly to host health, nutrition absorption, host metabolism, and immunological development (Zeineldin et al, 2018). Previous studies demonstrated that this bacteria was related to various diseases like Type 2 diabetes (Martinez-Lopez et al, 2022), acute pancreatitis (Mei et al, 2022), obesity (Salazar et al, 2022), and diarrhea (Han et al, 2017;Zeineldin et al, 2018;Li et al, 2022b). Short-chain fatty acids are metabolic products of microbiota, which contribute to the cellular metabolism of the host (Bachem et al, 2019), regulating immune function and suppressing inflammatory reactions (Abdalkareem Jasim et al, 2022).…”

“…In our previous study, we observed prominent changes in intestinal microbiota in a horse infected with Cryptosporidium spp. (Wang et al, 2022). However, scarce information is available about microbiota and SCFAs changes in plateau yaks infected with Cryptosporidium spp.…”

Chemotherapeutic Drugs Induce Different Gut Microbiota Disorder Pattern and NOD/RIP2/NF-κB Signaling Pathway Activation That Lead to Different Degrees of Intestinal Injury

“…Gut microbiota disorder aggravates intestinal injury. and the two promote each other's deterioration ( 26 , 27 ). Although chemotherapeutic drugs, such as 5-FU, CPT-11 and L-OHP, can induce intestinal injury ( 28 ), studies have yet to compare the differences between the intestinal injury degree and gut microbiota disorder pattern among them, which form the FOLFIRI (folinic acid, 5-FU, and CPT-11) and FOLFOX colorectal cancer chemotherapy regimens ( 29 ).…”