Native and Engineered Probiotics: Promising Agents against Related Systemic and Intestinal Diseases.

Intestinal homeostasis is a dynamic balance involving the interaction between the host intestinal mucosa, immune barrier, intestinal microecology, nutrients, and metabolites. Once homeostasis is out of balance, it will increase the risk of intestinal diseases and is also closely associated with some systemic diseases. Probiotics (Escherichia coli Nissle 1917, Akkermansia muciniphila, Clostridium butyricum, lactic acid bacteria and Bifidobacterium spp.), maintaining the gut homeostasis through direct interaction with the intestine, can also exist as a specific agent to prevent, alleviate, or cure intestinal-related diseases. With genetic engineering technology advancing, probiotics can also show targeted therapeutic properties. The aims of this review are to summarize the roles of potential native and engineered probiotics in oncology, inflammatory bowel disease, and obesity, discussing the therapeutic applications of these probiotics.

LACpG10-HL Functions Effectively in Antibiotic-Free and Healthy Husbandry by Improving the Innate Immunity.

Antibiotics are broadly restricted in modern husbandry farming, necessitating the need for efficient and low-cost immunomodulatory preparations in antibiotic-free and healthful farming. As is known to all, CpG oligonucleotides (CpG-ODNs, an effective innate immunostimulatory agent) recognized by TLR9 in mammals (while TLR21 in avians) could collaborate with some united agent to induce stronger immune responses, but the cost is prohibitively expensive for farmers. Here, considering the coordination between TLR2 and TLR9/TLR21, we firstly proposed the idea that the well-fermented Lactococcus lactis could be utilized as a CpG-plasmid carrier (LACpG10) to enhance the host's innate immunity against pathogenic invasion. In the present study, after obtaining LACpG10-HL from homogenized and lyophilized recombinant strain LACpG10, we treated primary chicken lymphocytes, two cell lines (HD11 and IPEC-J2), and chickens with LACpG10-HL, CpG plasmids (pNZ8148-CpG10), and other stimulants, and respectively confirmed the effects by conducting qRT-PCR, bacterial infection assays, and a zoological experiment. Our data showed that LACpG10-HL could induce excellent innate immunity by regulating autophagy reactions, cytokine expression, and motivating PRRs. Interestingly, despite having no direct antiseptic effect, LACpG10-HL improved the antibacterial capacities of lymphocytes and enterocytes at the first line of defense. Most importantly, water-supplied LACpG10-HL treatment reduced the average adverse event rates, demonstrating that LACpG10-HL maintained its excellent immunostimulatory and protective properties under farming conditions. Our research not only contributes to revealing the satisfactory effects of LACpG10-HL but also sheds new light on a cost-effective solution with optimal immune effects in green, antibiotic-free, and healthful husbandry farming.

YBX has functional roles in CpG-ODN against cold stress and bacterial infection of Misgurnus anguillicaudatus.

Misgurnus anguillicaudatus (M. anguillicaudatus) is a widely cultivated fish. However, in M. anguillicaudatus breeding, the frequent cold stress during daily breeding could induce immune suppression and increase the risk of infection, causing serious economic loss. Based on existing findings, CpG Oligonucleotides (CpG-ODNs) may be an ideal protective agent for low temperature fish breeding, performing anti-infective when faced with cold stress with cold shock proteins Y box binding proteins (YBX). Although YBX has pleiotropic functions, its roles in CpG-ODNs-mediated immunity (especially under cold situations) remain largely unexplored. To clarify the relationship among them, we identified the YBX1/YBX2 in M. anguillicaudatus and analyzed using a series of bioinformatics methods. After that, we immunized the fish with 3 types of CpG-ODNs and challenged with Aeromonas hydrophila (A. hydrophila). Here we showed that the best anti-bacterial effect of CpG-B was accompanied by the significant upregulation of YBX1. And the detection of the YBX1 downstream effectors confirmed that CpG-B induced the YBX1-mediated Th1 oriented responses to A. hydrophila by regulation of the NLRP3 (Caspase-A/-B), IL-1ß, IL-12 and IFN-γ. Afterwards, we found that under cold stress, CpG-B can activate the NLRP3 and NF-κB pathways through YBX1, a key mediator of anti-A. hydrophila in CpG-B immunization. In this study, we demonstrated CpG-B protection against infection in low temperature, and its interaction with YBX1, expanded the research of CpG-ODN under cold stress, and provided a new CpG-ODN application for low temperature fish farming.

Overexpression of pEGF improved the gut protective function of Clostridium butyricum partly through STAT3 signal pathway.

Clostridium butyricum (C. butyricum) is a probiotic that could promote animal growth and protect gut health. So far, current studies mainly keep up with the basic biological functions of C. butyricum, missing the effective strategy to further improve its protective efficiency. A recent report about C. butyricum alleviating intestinal injury through epidermal growth factor receptor (EGFR) inspired us to bridge this gap by porcine epidermal growth factor (EGF) overexpression. Lacking a secretory overexpression system, we constructed the recombinant strains overexpressing pEGF in C. butyricum for the first time and obtained 4 recombinant strains for highly efficient secretion of pEGF (BC/pPD1, BC/pSPP, BC/pGHF, and BC/pDBD). Compared to the wild-type strain, we confirmed that the expression level ranges of the intestinal development-related genes (Claudin-1, GLUT-2, SUC, GLP2R, and EGFR) and anti-inflammation-related gene (IL-10) in IPECs were upregulated under recombinant strain stimulation, and the growth of Staphylococcus aureus and Salmonella typhimurium was significantly inhibited as well. Furthermore, a particular inhibitor (stattic) was used to block STAT3 tyrosine phosphorylation, resulting in the downregulation on antibacterial effect of recombinant strains. This study demonstrated that the secretory overexpression of pEGF in C. butyricum could upregulate the expression level of EGFR, consequently improving the intestinal protective functions of C. butyricum partly following STAT3 signal activation in IPECs and making it a positive loop. These findings on the overexpression strains pointed out a new direction for further development and utilization of C. butyricum. KEY POINTS: • By 12 signal peptide screening in silico, 4 pEGF overexpression strains of C. butyricum/pMTL82151-pEGF for highly efficient secretion of pEGF were generated for the first time. • The secretory overexpression of pEGF promoted the intestinal development, antimicrobial action, and anti-inflammatory function of C. butyricum. • The overexpressed pEGF upregulated the expression level of EGFR and further magnified the gut protective function of recombinant strains which in turn partly depended on STAT3 signal pathway in IPECs.

Molecular cloning, characterization and expression profiles of Annexin family (ANXA1~A6) in yellow catfish (Pelteobagrus fulvidraco) and ANX regulation by CpG ODN responding to bacterial infection.

Up to now, many previous reports have emphasized that Annexins (ANX) family played an important role in immune responses. Aeromonas hydrophila (A. hydrophila), the most common zoonotic pathogenic bacteria of yellow catfish (Pelteobagrus fulvidraco), can cause serious economic loss, especially to yellow catfish with high economic value. In our previous work, we demonstrated that synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) owned powerful immunostimulatory activity. However, the relationship among Pelteobagrus fulvidraco Annexins (Pf_ANX), CpG ODN and A. hydrophila is unknown. Therefore, we cloned Pf_ANX1-6 genes and analyzed its sequences, structures, genetic evolution, post-translation modifications (PTMs), Ca2+ ion binding sites and tissue distribution to reveal the relevance. In addition, we investigated the responses of ANXA1-6 and cytokines in intestine and spleen as well as morbidity/survival rate of fish post CpG ODN immunization and/or A. hydrophila infection. The results showed that compared with challenge alone (challenge-CK) group, the CpG immunization following challenge (CpG-challenge) group displayed relatively flat IL-1ß level throughout in both organs. Meanwhile, the expression of IFN-γ and morbidity/survival rate of fish in CpG-challenge group showed a great improvement compared with the challenge-CK group. Our results indicated that CpG ODN could improve morbidity/survival by up-regulating Pf_ANXA 1, 2 and 5 in the intestine and spleen to ameliorate inflammatory responses and promote anti-infective responses. Our findings offer some important insights into ANX related to the immunity of fish infection and lay a theoretical basis for the prevention and treatment of fish infections.

Protective effects of Clostridium butyricum against oxidative stress induced by food processing and lipid-derived aldehydes in Caco-2 cells.

The human body is almost always facing the oxidative stress caused by foodborne aldehydes such as glyoxal (GO) and methylglyoxal (MGO), 4-hydroxyhexenal (HHE), and 4-hydroxynonenal (HNE). When these aldehydes build up, they can cause a range of harm. However, a probiotic, Clostridium butyricum, can increase nuclear factor erythroid-2 related factor 2 (Nrf2) and may have the potential to relieve oxidative stress. If C. butyricum is indeed resistant to aldehydes, the advantages (accessibility, convenience, and safety) will be of great significance compared with drugs. Unfortunately, whether C. butyricum can play a role in alleviating toxic effects of foodborne aldehydes in the intestine (the first line of defense against food-derived toxin) was unclear. To investigate these, we measured the viability, ROS, autophagy, and inflammatory cytokine expression of Caco-2 which were co-cultured with C. butyricum and stimulated by the four aldehydes via Nrf2 pathway (Staphylococcus aureus and Enterococcus faecium as controls). Then, we explored the link among C. butyricum, NLRP6, and Nrf2 signaling pathways when facing the stimuli. In the present study, we demonstrated that Clostridium butyricum relieved the oxidative stress induced by the aldehydes in Caco-2. Most interestingly, we found a "complementary" relationship between NLRP6 and Nrf2 in C. butyricum treatment under aldehyde stress. Our research not only makes a contribution to the popularization of C. butyricum as a probiotic-rich food instead of medicines but also sheds new light on the application of subsequent microecological formulation of C. butyricum. KEY POINTS: • The adverse effects are caused in a dose-dependent manner by foodborne aldehydes. • Clostridium butyricum can significantly ameliorate oxidative stress. • There is a "complementary" relationship between the NLRP6 and Nrf2 signaling pathways. • Using Clostridium butyricum foods to alleviate oxidative stress shows great prospects.

Intestinal guard: Human CXCL17 modulates protective response against mycotoxins and CXCL17-mimetic peptides development.

Mycotoxin contamination is an ongoing and growing issue that can create health risks and even cause death. Unfortunately, there is currently a lack of specific therapy against mycotoxins with few side effects. On the other hand, the strategic expression of CXCL17 in mucosal tissues suggests that it may be involved in immune response when exposed to mycotoxins, but the exact role of CXCL17 remains largely unknown. Using Caco-2 as a cell model of the intestinal epithelial barrier (the first line of defense against mycotoxins), we showed that a strong production of ROS-dependent CXCL17 was triggered by mycotoxins via p38 and JNK pathways. Under the mycotoxins stress, CXCL17 modulated enhanced immuno-protective response with a remission of inflammation and apoptosis through PI3K/AKT/mTOR. Based on our observed feedback of CXCL17 to the mycotoxins, we developed the CXCL17-mimetic peptides in silico (CX1 and CX2) that possessed the safety and the capability to ameliorate mycotoxins-inducible inflammation and apoptosis. In this study, the identification of detoxifying feature of CXCL17 is a prominent addition to the chemokine field, pointing out a new direction for curing the mycotoxins-caused damage.