Crocodilians are regarded as possessing a powerful immune system. However, the composition and action of the crocodilian immune system have remained unclear until now. Cathelicidins, the principal family of host defense peptides, play pivotal roles in vertebrate immune defense against microbial invasions. However, cathelicidins from crocodilians have not been extensively studied to date. In the present study, six novel cathelicidins (As-CATH1-6) were identified and characterized from the endangered Chinese alligator (). As-CATH1-6 exhibit no sequence similarity with any of the known cathelicidins. Structure analysis indicated that As-CATH1-3 adopt a random coil secondary conformation, whereas As-CATH4-6 were predicted to mainly adopt an amphipathic α-helix conformation. Among them, As-CATH4-6 exhibited potent, broad-spectrum and rapid antimicrobial activity by inducing the disruption of cell membrane integrity. They also exhibited strong ability to prevent the formation of bacterial biofilms and eradicate preformed biofilms. Furthermore, As-CATH4-6 exhibited potent anti-inflammatory activity by inhibiting the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and pro-inflammatory cytokines in mouse peritoneal macrophages. They directly neutralized LPS toxicity and therefore inhibited the binding of LPS to the TLR4 receptor and the subsequent activation of inflammatory response pathways. In a peritonitis mice model, As-CATH2-6 provided effective protection against bacterial infection through enhanced immune cell recruitment. In the host Chinese alligator, As-CATH1-6 are mainly expressed in immune organs and epithelial tissues. Bacterial infection significantly enhances their expression, which implies an important role in host anti-infective response. Taken together, the diversity and multiple functions of As-CATH1-6 partially reveal the powerful immune system of the Chinese alligator.

Multidrug-resistant Staphylococcus aureus, including MRSA (methicillin-resistant) and VRSA (vancomycin-resistant), causes serious healthcare-associated infections, even sepsis and death. Here, we identified six novel cathelicidins (CATHPb1-6) from Python bivittatu, and CATHPb1 displayed the best in vitro pharmacological and toxicological profile. We further show that CATHPb1 exhibited evident protection in mice MRSA/VRSA infection models, given either 24 h before or 4 h after infection. The protection was all effective through different administration routes, but was blocked by in vivo depletion of monocyte/macrophages or neutrophils. CATHPb1 can rapidly and massively modulate macrophages/monocytes and neutrophils trafficking to the infection site, and potentiate their bactericidal functions. Meanwhile, CATHPb1 remarkably augmented neutrophil-mediated bacteria killing by facilitating neutrophil extracellular traps (NETs) formation and preventing its degradation. Acting through MAPKs and NF-κB pathways, CATHPb1 selectively enhanced the levels of chemokines while reducing the production of pro-inflammatory cytokines without undesirable toxicities. The much improved serum half-life and stabilities confer CATHPb1 an excellent prospect to become a novel therapeutic agent against multidrug-resistant staphylococcal infections.

To protect themselves against the invasion of microorganisms, amphibians, especially the Rana frogs, are possibly equipped with complex combinations of antimicrobial peptides (AMPs). The two major AMP families, ranid skin secretion AMPs and cathelicidins might together constitute the host innate immune system of amphibians. Cathelicidins are a group of cationic peptides found in leukocytes and epithelial cells, and they play a central role in the early innate immune defense found in virtually all species of mammals. However, they have rarely been reported from amphibians. Here, we report the identification and discovery of polymorphism cathelicidins in Limnonectes fragilis. The expression profile indicated high cathelicidin transcript levels in frog spleen, liver and kidney, but lower levels in lung, skin and stomach. According to the amphibian's unique proteolytic pattern, R125 and L121 of the prepropeptides are predicted to be the processing positions for protease to generate the mature peptides, Lf‐CATH1 and ‐2, respectively. Both consist of 30 amino acid residues, of which two were cysteines positionally conserved among a few known amphibian cathelicidins. Homology modeling analysis revealed that Lf‐CATH1 and ‐2 adopt a tertiary structure with a mostly α helix that is representative of small cationic cathelicidin family peptides. Recombinant Lf‐CATH1 (rLf‐CATH1) was produced in Escherichia coli. Synthetic Lf‐CATH1 and ‐2 displayed potent antimicrobial activities in vitro against a broad spectrum of microorganisms, including standard and clinically isolated drug‐resistant strains, while showing neglectable hemolysis and cytotoxicities.

Cathelicidins are short cationic host defense peptides and play a central role in host innate immune system. Here we identified two novel cathelicidins, Cl-CATH2 and 3, from Columba livia. Evolutionary analysis of avian cathelicidins via phylogenetic tree and Ka/Ks calculations supported the positive selection that prompted evolution of CATH2 to CATH1 and 3, which originate from common ancestor and could belong to one superfamily. Cl-CATH2 and 3 both adopt amphipathic α-helical comformations identified by circular dichroism and the 3D structures built by Rosetta. Cl-CATH2 of CATH2 family with the most expression abundance in bird, exhibited relatively weak antimicrobial activity, but acted instead on the innate immune response without showing undesirable toxicities. In macrophages primed by LPS, Cl-CATH2 significantly down-regulated the gene and protein expressions of inducible nitric oxide synthase and pro-inflammatory cytokines while enhancing the anti-inflammatory cytokine, acting through MAPK and NF-κB signaling pathways. Molecular docking shows for the first time that cathelicidin binds to the opening region of LPS-binding pocket on myeloid differentiation factor 2 (MD-2) of toll-like receptor (TLR)4-MD-2 complex, which in turn inhibits the TLR4 pathway. Our results, therefore, provide new insight into the mechanism underlying the blockade of TLR4 signaling by cathelicidins.

Alternative splicing (AS) is a complex coordinated transcriptional regulatory mechanism. It affects nearly 95% of all protein-coding genes and occurs in nearly all human organs. Aberrant alternative splicing can lead to various neurological diseases and cancers and is responsible for aging, infection, inflammation, immune and metabolic disorders, and so on. Though aberrant alternative splicing events and their regulatory mechanisms are widely recognized, the association between autoimmune disease and alternative splicing has not been extensively examined. Autoimmune diseases are characterized by the loss of tolerance of the immune system towards self-antigens and organ-specific or systemic inflammation and subsequent tissue damage. In the present review, we summarized the most recent reports on splicing events that occur in the immunopathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and attempted to clarify the role that splicing events play in regulating autoimmune disease progression. We also identified the changes that occur in splicing factor expression. The foregoing information might improve our understanding of autoimmune diseases and help develop new diagnostic and therapeutic tools for them.

Cutaneous hyperpigmentation from excess melanogenesis causes serious pigmentary disorders and even melasma. Short peptides (SPs) are garnering attention lately owing to their therapeutic potential in dermatological diseases and low systemic side effects. Here, we show an octapeptide, ansin2, designed de novo from antioxidant SPs we previously reported, significantly inhibiting melanogenesis in B16 cells by decreasing tyrosinase production via regulating the MITF pathway. Ansin2 could also inhibit tyrosinase function by covering its catalytic pocket, which was simulated in docking and LIGPLOT studies. Topical application of ansin2 exhibited evident protection in UVB-induced pigmentation in guinea pig models both in terms of prophylaxis and treatment. Interestingly, unlike other hydrophilic and peptidic drugs that need delivery systems, ansin2 can be efficiently delivered topically to the epidermis and dermis per se without an affiliated moiety. Given that ansin2 lacks unwanted toxicities and immunogenicity, it holds great potential in treating hyperpigmentation in the cosmetics and pharmaceutical industries.

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Previous studies have proved that a novel antioxidant system composed of various antioxidant peptides (AOPs) exists in the skin of ranid frogs, keeping the redox homeostasis. However, only a small number of AOPs have been identified so far. Here, a total of 47 cDNA sequences encoding 21 different AOPs belonging to 11 families were cloned from the skin cDNA library of Limnonectes fragilis. Among them, fragilin-A1 (VKRRGQDCIHGFCSD) and fragilin-B1 (GQFNDKRWIPFG) were also purified from skin secretions. They were selected with odorranain-Q-Lf (APIRMWYMYRKLTDMEPKPVA), the newest sequence among all 21 AOPs, to evaluate the antioxidant activities by direct free radical scavenging and lipid peroxidation inhibition assay. Results demonstrated that all peptides possessed strong DPPH and ABTS(.+) scavenging activities, and effectively inhibited lipid peroxidation in linoleic acid emulsion system during a 7- day test. No cytotoxic and hemolytic activity against human erythrocytes was observed for the three AOPs. The homology modeling analysis revealed that they all adopt tertiary structures ideally suited for the key residues to come into contact with the radicals. Current results reveal the existence of antioxidant system constituted of AOPs in the skin of the L. fragilis, and furthermore provide excellent templates for the development of novel antioxidant agents.