Synthesis Of Antimicrobial Peptides Research Articles

Manipulation of the silkworm immune system by a metalloprotease from the pathogenic bacterium Pseudomonas aeruginosa

Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 ΔlasB mutant and found that PO activity in hemolymph of the PAO1 ΔlasB-infected larvae was significantly higher than that in the wild type-infected. The mutant-inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 ΔlasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the ΔlasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection.

Evolutionary ecology of parasitic fungi and their host insects

Abstract The coevolution of parasites and their hosts is accompanied by rapid reciprocal adaptations. Recent studies focusing on the evolutionary ecology of parasitic fungi and their host insects have revealed an unexpectedly complex molecular arms race. Insects recognize the presence of invading fungi when molecular patterns associated with the fungal cell wall bind to host receptors and respond by inducing the synthesis of proteinase inhibitors and antifungal peptides such as metchnikowin. Parasitic fungi can in turn sense the presence of these host-derived defense molecules and respond by synthesizing chymotrypsin and metalloproteases to digest them. As a counter-response, insects have evolved the ability to perceive the presence of microbial metalloproteinases, inducing the synthesis of further antimicrobial peptides and the insect metalloproteinase inhibitor to regulate these virulence-associated fungal proteinases. These specific responses and counter-responses indicate that coevolution has resulted in communication between fungal parasites and their insect hosts, which was recently shown to involve transcriptional reprogramming controlled by histone acetylation/deacetylation and DNA methylation. Consequently, the reciprocal adaptations associated with host–parasite coevolution can be expanded beyond the genetic level to encompass epigenetic mechanisms.

A clip domain serine protease stimulates melanization activation and expression of antimicrobial peptides in the Chinese oak silkworm, Antheraea pernyi

Abstract Clip domain serine proteases (Clips) mediate various biological processes, especially in insect immune responses such as prophenoloxidase (proPO) activation and Toll pathway initiation. In the present study, we cloned and studied the functions of a clip domain serine protease from Antheraea pernyi (ApSnake). ApSnake contains a typical clip domain and a trypsin-like serine protease domain. It shares high similarity with serine protease snake in Bombyx mori, and groups into the CLIPC family. ApSnake mRNA expressions were detected in all tested tissues, particularly high in the hemocytes. Challenged with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus or Nuclear polyhedrosis virus), the expressions of ApSnake mRNA were induced significantly compared with control, particularly challenged by B. bassiana and M. luteus. Recombinant ApSnake protein stimulated melanization reaction and prophenoloxidase activation in A. pernyi hemolymph, but had no effect on phenoloxidase activity. Injection of recombinant ApSnake into A. pernyi larvae increased the transcript levels of proPO and antimicrobial peptides (AMPs) in hemocytes significantly. Our results suggested that ApSnake might be a modulating protein that both stimulate the melanization activation process and AMPs synthesis, and plays an important role in the innate immunity of A. pernyi.

Identification and characterization of serpin genes in Manduca sexta

Insects adopt a variety of immune mechanisms to defend against invading pathogens, such as phagocytosis, encapsulation, melanization and antimicrobial peptide synthesis. Serine protease cascades play an important role in some of these immune responses, and serpins (serine protease inhibitors) regulate the proteases after zymogen activation. Serpins are a superfamily of proteins, which share a conserved tertiary structure composed of three β‐sheets, 8–9 α‐helices, and an exposed reactive center loop (RCL). Once encountering its specific serpin, the protease cleaves the serpin RCL at the scissile bond, resulting in the insertion of RCL into β‐sheet A as a new strand and the formation of an irreversible inhibitory complex. Our group uses Manduca sexta a model insect to study the insect immune system. By searching the whole genome of M. sexta, 32 serpin genes were identified. MsSerpin1, 15 and 28 are capable of encoding more than one polypeptide with different RCL sequences via alternative splicing. Besides 12 isoforms of MsSerpin1 previously known, two additional isoforms were identified, increasing the total number of splicing isoforms of MsSerpin1 to 14. Phylogenetic analysis of serpins in M. sexta and the silk worm Bombyx mori indicate evolutionary conservation of some serpins, with 17 orthologous pairs identified. These include 7 characterized M. sexta serpins that are involved in protease cascades activating the prophenoloxidase or Toll immune pathways. The majority of M. sexta and B. mori serpin genes contain 6–10 exons except for a clade including 10 M. sexta serpin genes and 7 B. mori serpin genes with only 1–3 exons. The analysis of RCL regions indicates that about three quarters of M. sexta serpin proteins are likely to be inhibitory. Transcription level of MsSerpin‐1–7, 11–12, 14–15, 17–18 and 22 displayed an up‐regulation in fat body or hemocytes upon immune challenge. Transcript profiles give hints to the temporal and spatial expression patterns of each serpin, which lead to suggestions for their functional study in the future.Support or Funding InformationNIH grants GM041247, GM58634 and AI11266This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Toll immune signal activates cellular immune response via eicosanoids

Upon immune challenge, insects recognize nonself. The recognition signal will propagate to nearby immune effectors. It is well-known that Toll signal pathway induces antimicrobial peptide (AMP) gene expression. Eicosanoids play crucial roles in mediating the recognition signal to immune effectors by enhancing humoral immune response through activation of AMP synthesis as well as cellular immune responses, suggesting a functional cross-talk between Toll and eicosanoid signals. This study tested a cross-talk between these two signals. Two signal transducing factors (MyD88 and Pelle) of Toll immune pathway were identified in Spodoptera exigua. RNA interference (RNAi) of either SeMyD88 or SePelle expression interfered with the expression of AMP genes under Toll signal pathway. Bacterial challenge induced PLA2 enzyme activity. However, RNAi of these two immune factors significantly suppressed the induction of PLA2 enzyme activity. Furthermore, RNAi treatment prevented gene expression of cellular PLA2. Inhibition of PLA2 activity reduced phenoloxidase activity and subsequent suppression in cellular immune response measured by hemocyte nodule formation. However, immunosuppression induced by RNAi of Toll signal molecules was significantly reversed by addition of arachidonic acid (AA), a catalytic product of PLA2. The addition also significantly reduced the enhanced fungal susceptibility of S. exigua treated by RNAi against two Toll signal molecules. These results indicate that there is a cross-talk between Toll and eicosanoid signals in insect immunity.

Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees

Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21 days post inoculation (21 days pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host’s whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6 days pi. At 7 days pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14 days pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14 days pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse.

The GCN2-ATF4 Signaling Pathway Induces 4E-BP to Bias Translation and Boost Antimicrobial Peptide Synthesis in Response to Bacterial Infection

Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. Here, we use a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). We found that 4E-BP is induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5' UTRs of AMPs score positive in cap-independent translation assays, andthis cap-independent activity is enhanced by 4E-BP. These results are corroborated invivo using transgenic 5' UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis.

Synthesis and biological evaluation of novel aliphatic acid-conjugated antimicrobial peptides as potential agents with anti-tumor, multidrug resistance-reversing activity and enhanced stability.

Compared with traditional anti-tumor drugs, antimicrobial peptides as novel anti-tumor agents have prominent advantages of higher specificity and circumvention of multi-drug resistance. BP100 is a multifunctional membrane-active peptide with high antimicrobial activity. Taking BP100 as a lead peptide, we designed and synthesized a series of aliphatic chain-conjugated peptides through solid-phase synthesis. Biological evaluation revealed that these peptides exhibited better anti-cancer activity than BP100. Further investigations revealed that these peptides could disrupt the cell membrane and trigger the cytochrome C release into cytoplasm, which ultimately resulted in apoptosis. Meanwhile, these peptides also exhibited effective anti-tumor activity against multidrug resistant cells and had multidrug resistance-reversing effect. Additionally, conjugation of aliphatic acid to those peptides could enhance their stability in plasma. In conclusion, aliphatic acid-modified peptides might be promising anti-tumor agents for cancer therapy.

The oyster immunity

Oysters, the common name for a number of different bivalve molluscs, are the worldwide aquaculture species and also play vital roles in the function of ecosystem. As invertebrate, oysters have evolved an integrated, highly complex innate immune system to recognize and eliminate various invaders via an array of orchestrated immune reactions, such as immune recognition, signal transduction, synthesis of antimicrobial peptides, as well as encapsulation and phagocytosis of the circulating haemocytes. The hematopoietic tissue, hematopoiesis, and the circulating haemocytes have been preliminary characterized, and the detailed annotation of the Pacific oyster Crassostrea gigas genome has revealed massive expansion and functional divergence of innate immune genes in this animal. Moreover, immune priming and maternal immune transfer are reported in oysters, suggesting the adaptability of invertebrate immunity. Apoptosis and autophagy are proved to be important immune mechanisms in oysters. This review will summarize the research progresses of immune system and the immunomodulation mechanisms of the primitive catecholaminergic, cholinergic, neuropeptides, GABAergic and nitric oxidase system, which possibly make oysters ideal model for studying the origin and evolution of immune system and the neuroendocrine-immune regulatory network in lower invertebrates.

A chymotrypsin-like serine protease from Portunus trituberculatus involved in pathogen recognition and AMP synthesis but not required for prophenoloxidase activation

Clip domain serine proteases (clip-SPs) play critical roles in various immune responses in arthropods, such as hemolymph coagulation, antimicrobial peptide (AMP) synthesis, cell adhesion and melanization. In the present study, we report the molecular and functional characterization of a clip domain serine protease (PtcSP2) from the swimming crab Portunus trituberculatus. The N-terminal clip domain and the C-terminal SP-like domain of PtcSP2 were expressed in Escherichia coli system, and assayed for their activities. Sequence similarity and phylogenetic analysis revealed that PtcSP2 may belong to the chymotrypsin family, which was confirmed by protease activity assay of the recombinant SP-like domain. The clip domain of PtcSP2 exhibited strong antibacterial activity and microbial-binding activity, suggesting the potential role in immune defense and recognition. Knockdown of PtcSP2 by RNA interference could significantly reduce PtcSP2 transcript levels, but neither decrease the total phenoloxidase (PO) activity in crab nor significantly alter the expression levels of serine protease inhibitors PtPLC and PtSerpin. These results indicate that PtcSP2 is not involved in the proPO system. However, suppression of PtcSP2 led to a significant change in the expression of AMP genes PtALFs and PtCrustin but not PtALF5. All these findings suggest that PtcSP2 is a multifunctional chymotrypsin-like serine protease and may participate in crab innate immunity by its antibacterial activity, immune recognition or regulation of AMP expression.

Shrimp humoral responses against pathogens: antimicrobial peptides and melanization

Diseases have caused tremendous economic losses and become the major problem threatening the sustainable development of shrimp aquaculture. The knowledge of host defense mechanisms against invading pathogens is essential for the implementation of efficient strategies to prevent disease outbreaks. Like other invertebrates, shrimp rely on the innate immune system to defend themselves against a range of microbes by recognizing and destroying them through cellular and humoral immune responses. Detection of microbial pathogens triggers the signal transduction pathways including the NF-κB signaling, Toll and Imd pathways, resulting in the activation of genes involved in host defense responses. In this review, we update the discovery of components of the Toll and Imd pathways in shrimp and their participation in the regulation of shrimp antimicrobial peptide (AMP) synthesis. We also focus on a recent progress on the two most powerful and the best-studied shrimp humoral responses: AMPs and melanization. Shrimp AMPs are mainly cationic peptides with sequence diversity which endues them the broad range of activities against microorganisms. Melanization, regulated by the prophenoloxidase activating cascade, also plays a crucial role in killing and sequestration of invading pathogens. The progress and emerging research on mechanisms and functional characterization of components of these two indispensable humoral responses in shrimp immunity are summarized and discussed. Interestingly, the pattern recognition protein (PRP) crosstalk is evidenced between the proPO activating cascade and the AMP synthesis pathways in shrimp, which enables the innate immune system to build up efficient immune responses.

Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus.

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole.

Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism, mediates immune responses

Tissue damage or pathogen invasion triggers the auto-proteolysis of an initiating serine protease (SP), rapidly leading to sequential cleavage activation of other cascade members to set off innate immune responses in insects. Recently, we presented evidence that Manduca sexta hemolymph protease-1 zymogen (proHP1) is a member of the SP system in this species, and may activate proHP6. HP6 stimulates melanization and induces antimicrobial peptide synthesis. Here we report that proHP1 adopts an active conformation (*) to carry out its function, without a requirement for proteolytic activation. Affinity chromatography using HP1 antibodies isolated from induced hemolymph the 48 kDa proHP1 and also a 90 kDa band (detected by SDS-PAGE under reducing conditions) containing proHP1 and several serpins, as revealed by mass spectrometric analysis. Identification of tryptic peptides from these 90 kDa complexes included peptides from the amino-terminal regulatory part of proHP1, indicating that proHP1* was not cleaved, and that it had formed a complex with the serpins. As suicide inhibitors, serpins form SDS-stable, acyl-complexes when they are attacked by active proteases, indicating that proHP1* was catalytically active. Detection of M. sexta serpin-1, 4, 9, 13 and smaller amounts of serpin-3, 5, 6 in the complexes suggests that it is regulated by multiple serpins in hemolymph. We produced site-directed mutants of proHP1b for cleavage by bovine blood coagulation factor Xa at the designed proteolytic activation site, to generate a form of proHP1b that could be activated by Factor Xa. However, proHP1b cut by Factor Xa failed to activate proHP6 and, via HP6, proHP8 or proPAP1. This negative result is consistent with the suggestion that proHP1* is a physiological mediator of immune responses. Further research is needed to investigate the conformational change that results in conversion of proHP1 to active proHP1*.

Microwave-Assisted Synthesis of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are emerging as one of the unsurpassed therapeutic tools to treat various devastating diseases that are affecting millions of lives. Conventional synthesis of peptides requires longer times, and hence automated microwave technology could be regarded as an alternative implement which offers advantages like less reaction times and higher yields. In this sense, we herein describe a methodology to prepare AMPs through solid-phase peptide synthesis under microwave conditions. We have used LL37 as an example to discuss the synthetic protocol including the difficulties involved in the preparation of so-called long and difficult peptides and also remedial procedures to overcome these obstacles.

Are Th17 Cells Playing a Role in Immunity to Dermatophytosis?

Despite their superficial localization in the skin, pathogenic dermatophytes can induce a complex but still misunderstood immune response in their hosts. The cell-mediated immunity (CMI) is correlated with both clinical recovery and protection against reinfection, and CD4+ T lymphocytes have been recognized as a crucial component of the immune defense against dermatophytes. Before the discovery of the Th17 pathway, CMI was considered to be only dependent of Th1 cells, and thus most studies on the immunology of dermatophytosis have focused on the Th1 pathway. Nevertheless, the fine comparative analysis of available scientific data on immunology of dermatophytosis in one hand and on the Th17 pathway mechanisms involved in opportunistic mucosal fungal infections in the other hand reveals that some key elements of the Th17 pathway can be activated by dermatophytes. Stimulation of the Th17 pathway could occur through the activation of some C-type lectin-like receptors and inflammasome in antigen-presenting cells. The Th17 cells could go back to the affected skin and by the production of signature cytokines could induce the effector mechanisms like the recruitment of polymorphonuclear neutrophils and the synthesis of antimicrobial peptides. In conclusion, besides the Th1 pathway, which is important to the immune response against dermatophytes, there are also growing evidences for the involvement of the Th17 pathway.

Complete genome sequence of Bacillus thuringiensis serovar alesti BGSC 4C1, a typical strain with toxicity to Lepidoptera insects

Bacillus thuringiensis serovar alesti was used to control caterpillars from 1970s. Here we reported the complete genome of BGSC 4C1, the type strain of this serovar. It has a circular chromosome and six plasmids. The largest plasmid pBMB267 contains five insecticidal crystal protein genes (two copies of cry1Ae, cry1Gb, cry2Ab, and a novel cry1M-type gene) and three vegetative insecticidal protein genes (a novel binary toxin gene operon vip1-vip2 and vip3Aa). Besides, the strain also has many genes encodeing virulence factors, and some secondary metabolite biosynthetic gene clusters involved in synthesis of antimicrobial peptides and bacteriocins. In addition, there is a poly γ-glutamate synthesis gene cluster, whose product is a candidate to control inflammasome-mediated disorders and potential in many other fields.

Fat body and hemocyte contribution to the antimicrobial peptide synthesis in Calliphora vicina R.-D. (Diptera: Calliphoridae) larvae.

Antimicrobial peptides accumulated in the hemolymph in response to infection are a key element of insect innate immunity. The involvement of the fat body and hemocytes in the antimicrobial peptide synthesis is widely acknowledged, although release of the peptides present in the hemolymph from the immune cells was not directly verified so far. Here, we studied the presence of antimicrobial peptides in the culture medium of fat body cells and hemocytes isolated from the blue blowfly Calliphora vicina using complex of liquid chromatography, mass spectrometry, and antimicrobial activity assays. Both fat body and hemocytes are shown to synthesize and release to culture medium defensin, cecropin, diptericins, and proline-rich peptides. The spectra of peptide antibiotics released by the fat body and hemocytes partially overlap. Thus, the results suggest that insect fat body and blood cells are capable of releasing mature antimicrobial peptides to the hemolymph. It is notable that the data obtained demonstrate dramatic difference in the functioning of insect antimicrobial peptides and their mammalian counterparts localized into blood cells' phagosomes where they exert their antibacterial activity.

Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag

An important aspect related to infectious pathogens is their exceptional adaptability in developing resistance, which leads to a perpetual challenge in the discovery of antimicrobial drugs with novel mechanisms of action. Among them, antimicrobial peptides (AMPs) stand out as promising anti-infective molecules. In order to overcome the high costs associated with isolation from natural sources or chemical synthesis of AMPs we propose the expression of Pa-MAP 2, a polyalanine AMP. Pa-MAP 2 was fused to an ELP-intein tag where the ELP (Elastin-like polypeptide) was used to promote aggregation and fast and cost-effective isolation after expression, and the intein was used to stimulate a controlled AMP release. For these, the vector pET21a was used to produce Pa-MAP 2 fused to the N-termini region of a modified Mxe GyrA intein followed by 60 repetitions of ELP. Purified Pa-MAP 2 showed a MIC of 25μM against E. coli ATCC 8739. Batch fermentation demonstrated that Pa-MAP-2 can be produced in both rich and defined media at yields 50-fold higher than reported for other AMPs produced by the ELP-intein system, and in comparable yields to expression systems with protease or chemical cleavage.

Regulation of antimicrobial peptide synthesis in larvae of Calliphora vicina (Diptera, Calliphoridae): a dose-dependent effect of ecdysteroids

Ecdysteroids are multifunctional hormones regulating virtually all morphogenetic processes in insects. Their role in stress and immune response regulation is less known. Here we studied 20-hydroxyecdysone effect on synthesis of the antimicrobial peptides in larvae of Calliphora vicina. An inverse correlation was found between 20-hydroxyecdysone titer and the concentration of antimicrobial peptides in the hemolymph of unaffected and bacteria-immunized insects. High and low doses of 20-hydroxyecdysone, injected simultaneously with bacterial cells, had an opposite effect on antimicrobial peptide synthesis in the diapausing larvae. Morphogenetically effective doses of 20-hydroxyecdysone demonstrated immuno-suppressive activity. Low doses of 20-hydroxyecdysone, on the contrary, moderately stimulated synthesis of the antimicrobial peptides. These data suggest that ecdysteroids are directly involved in regulation of the immune system activity and the final effect is dose-dependent.

Rumen-derived lipopolysaccharide enhances the expression of lingual antimicrobial peptide in mammary glands of dairy cows fed a high-concentrate diet

BackgroundLong-term high-concentrate diet (HCD) feeding can cause subacute ruminal acidosis in cows and subsequently trigger systemic inflammatory and immune responses. Therefore, we conducted the present study in which twelve lactating cows installed with ruminal fistula were randomly assigned to the HCD group (forage:concentrate = 4:6, n = 6) or the low-concentrate diet (LCD) group (forage:concentrate = 6:4, n = 6) and were fed for 20 weeks. Ruminal fluid, plasma and mammary gland tissue samples were collected at week 20 for analysing lipopolysaccharide (LPS), pro-inflammatory cytokines, and immune relevant gene expression. The aim of this study was to investigate the effect of rumen-derived LPS on lingual antimicrobial peptide (LAP) synthesis and immune responses in mammary glands of lactating cows fed a HCD.ResultsCompared with the LCD group, the ruminal pH was lower in the HCD group, while LPS concentrations in the rumen, lacteal artery and vein were higher. The expression of LAP, BNBD5, IL-1β, IL-6, IL-8, and TNF–α was enhanced in the HCD group. LAP protein expression was higher in the HCD group than that in the LCD group. The expression of nuclear factor kappa B (NF-кB) did not change, but was activated, as the amounts of phosphorylated NF-kB and phosphorylated inhibitory kBα increased in the HCD group compared with that in the LCD group.ConclusionsAfter long-term HCD feeding, rumen-derived LPS translocated to the blood stream, triggered inflammatory and immune responses and enhanced LAP synthesis via the NF-kB signalling pathway in mammary glands of lactating cows.