Murine bactericidal/permeability-increasing protein inhibits the endotoxic activity of lipopolysaccharide and gram-negative bacteria

Recognition of LPS by TLR4 initiates inflammatory responses inducing potent antimicrobial immunity. However, uncontrolled inflammatory responses can be detrimental. To prevent the development of septic shock during an infection with Gram-negative bacteria, the immune system has developed mechanisms to neutralize LPS by specialized proteins. In this study, we report the recombinant expression and functional characterization of the mouse homolog of human bactericidal/permeability-increasing protein (BPI). Purified recombinant mouse BPI was able to neutralize LPS-mediated activation of macrophages and to block LPS-dependent maturation of dendritic cells. Recombinant mouse BPI neutralized the capacity of Gram-negative bacteria to activate immune cells, but did not influence the stimulatory properties of Gram-positive bacteria. Unlike human BPI, mouse BPI failed to kill or inhibit the growth of Pseudomonas aeruginosa. Together, these data demonstrate that murine BPI is a potent LPS-neutralizing protein that may limit innate immune responses during Gram-negative infections.     

Positive and negative regulation of the Drosophila immune response

Insects mount a robust innate immune response against a wide array of microbial pathogens. The hallmark of the Drosophila humoral immune response is the rapid production of antimicrobial peptides in the fat body and their release into the circulation. Two recognition and signaling cascades regulate expression of these antimicrobial peptide genes. The Toll pathway is activated by fungal and many Gram-positive bacterial infections, whereas the immune deficiency (IMD) pathway responds to Gram-negative bacteria. Recent work has shown that the intensity and duration of the Drosophila immune response is tightly regulated. As in mammals, hyperactivated immune responses are detrimental, and the proper down-modulation of immunity is critical for protective immunity and health. In order to keep the immune response properly modulated, the Toll and IMD pathways are controlled at multiple levels by a series of negative regulators. In this review, we focus on recent advances identifying and characterizing the negative regulators of these pathways.     

Primed Immune Responses to Gram-negative Peptidoglycans Confer Infection Resistance in Silkworms

A heightened immune response, in which immune responses are primed by repeated exposure to a pathogen, is an important characteristic of vertebrate adaptive immunity. In the present study, we examined whether invertebrate animals also exhibit a primed immune response. The LD₅₀ of Gram-negative enterohemorrhagic Escherichia coli O157:H7 Sakai in silkworms was increased 100-fold by pre-injection of heat-killed Sakai cells. Silkworms pre-injected with heat-killed cells of a Gram-positive bacterium, Staphylococcus aureus, did not have resistance to Sakai. Silkworms preinjected with enterohemorrhagic E. coli peptidoglycans, cell surface components of bacteria, were resistant to Sakai infection. Silkworms preinjected with S. aureus peptidoglycans, however, were not resistant to Sakai. Silkworms preinjected with heat-killed Sakai cells showed persistent resistance to Sakai infection even after pupation. Repeated injection of heat-killed Sakai cells into the silkworms induced earlier and greater production of antimicrobial peptides than a single injection of heat-killed Sakai cells. These findings suggest that silkworm recognition of Gram-negative peptidoglycans leads to a primed immune reaction and increased resistance to a second round of bacterial infection.     

Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila

In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes. The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections. In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin.     

In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and our genetic studies suggest that GNBP1 acts upstream of the Toll ligand Sp?tzle. Altogether, our results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1.     

果蝇蛋白质激酶Pitslre通过调控抗菌肽表达参与天然免疫反应

为鉴定新的参与黑腹果蝇(Drosophila melanogaster)天然免疫信号通路调控的分子及作用机制,应用果蝇的Gal4/UAS系统敲低54个蛋白质激酶编码基因,分别利用革兰氏阳性菌（Enterococcus faecalis, E.faecalis）或革兰氏阴性菌（Erwinia carototovovora carototovovora 15, Ecc15）感染基因敲低果蝇,筛选参与果蝇天然免疫反应的蛋白质激酶。结果显示,全身性敲低蛋白质激酶Pitslre的果蝇感染E.faecalis或Ecc15 后,生存率降低,半致死时间LT50分别降低为对照组的66.7%和28.6%。相应的,Pitslre功能缺失导致革兰氏阳性菌和阴性菌分别感染后,Toll及IMD通路下游抗菌肽Drosomycin和Diptercin表达水平明显下降。在脂肪体和血淋巴细胞中特异性敲低Pitslre基因,导致革兰氏阳性菌及阴性菌感染后的果蝇半致死时间LT50分别缩短75%和90%,细菌载量分别升高约10倍。在果蝇S2细胞中,敲低Pitslre基因,导致细胞的抗菌肽Drosomycin、Attacin和Diptercin表达水平分别降低约50%。此外,通过免疫共沉淀实验检测Pitslre与预测存在相互作用的蛋白质TSC1、Rcd5和pbl之间的相互作用。综上所述,蛋白质激酶Pitslre参与果蝇天然免疫反应,在正向调控果蝇天然免疫Toll和IMD通路中发挥重要作用。     

Diversification of MIF immune regulators in aphids: link with agonistic and antagonistic interactions

Background

The widespread use of genome sequencing provided evidences for the high degree of conservation in innate immunity signalling pathways across animal phyla. However, the functioning and evolutionary history of immune-related genes remains unknown for most invertebrate species. A striking observation coming from the analysis of the pea aphid Acyrthosiphon pisum genome is the absence of important conserved genes known to be involved in the antimicrobial responses of other insects. This reduction in antibacterial immune defences is thought to be related to their long-term association with beneficial symbiotic bacteria and to facilitate symbiont maintenance. An additional possibility to avoid elimination of mutualistic symbionts is a fine-tuning of the host immune response. To explore this hypothesis we investigated the existence and potential involvement of immune regulators in aphid agonistic and antagonistic interactions.

Results

In contrast to the limited antibacterial arsenal, we showed that the pea aphid Acyrthosiphon pisum expresses 5 members of Macrophage Migration Inhibitory Factors (ApMIF), known to be key regulators of the innate immune response. In silico searches for MIF members in insect genomes followed by phylogenetic reconstruction suggest that evolution of MIF genes in hemipteran species has been shaped both by differential losses and serial duplications, raising the question of the functional importance of these genes in aphid immune responses. Expression analyses of ApMIFs revealed reduced expression levels in the presence, or during the establishment of secondary symbionts. By contrast, ApMIFs expression levels significantly increased upon challenge with a parasitoid or a Gram-negative bacteria. This increased expression in the presence of a pathogen/parasitoid was reduced or missing, in the presence of facultative symbiotic bacteria.

Conclusions

This work provides evidence that while aphid’s antibacterial arsenal is reduced, other immune genes widely absent from insect genomes are present, diversified and differentially regulated during antagonistic or agonistic interactions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-762) contains supplementary material, which is available to authorized users.     

Responses of alternative complement expression to challenge with different combinations of Vibrio anguillarum, Escherichia coli and Staphylococcus aureus: evidence for specific immune priming in amphioxus Branchiostoma belcheri

The existence of specific immunological priming in protection upon secondary exposure in invertebrates remains controversial. By exploring the changes in the expression patterns of Bf, C3 and C6, key genes involved in the alternative complement pathway (AP), after challenge with different combinations of three bacteria Vibrio anguillarum, Escherichia coli and Staphylococcus aureus, we show that re-exposure to the same species of bacteria or the different species of the same (Gram-negative or Gram-positive) class of bacteria results in a significant increase in the expression of Bf, C3 and C6, and an earlier occurrence of gene expression peak compared with the first exposure in amphioxus Branchiostoma belcheri; in contrast, re-exposure to the different class of bacteria did not induce such responses. These characteristics appear to bear some analogy to the immunological memory in vertebrates, suggesting that amphioxus B. belcheri possesses the ability to discriminate between classes of microorganisms. Moreover, our results for the first time establish a link between the alternative complement components and the specific immune priming.     

果蝇蛋白质激酶Pitslre通过调控抗菌肽表达参与天然免疫反应

为鉴定新的参与黑腹果蝇(Drosophila melanogaster)天然免疫信号通路调控的分子及作用机制,应用果蝇的Gal4/UAS系统敲低54个蛋白质激酶编码基因,分别利用革兰氏阳性菌（Enterococcus faecalis, E.faecalis）或革兰氏阴性菌（Erwinia carototovovora carototovovora 15, Ecc15）感染基因敲低果蝇,筛选参与果蝇天然免疫反应的蛋白质激酶。结果显示,全身性敲低蛋白质激酶Pitslre的果蝇感染E.faecalis或Ecc15 后,生存率降低,半致死时间LT50分别降低为对照组的66.7%和28.6%。相应的,Pitslre功能缺失导致革兰氏阳性菌和阴性菌分别感染后,Toll及IMD通路下游抗菌肽Drosomycin和Diptercin表达水平明显下降。在脂肪体和血淋巴细胞中特异性敲低Pitslre基因,导致革兰氏阳性菌及阴性菌感染后的果蝇半致死时间LT50分别缩短75%和90%,细菌载量分别升高约10倍。在果蝇S2细胞中,敲低Pitslre基因,导致细胞的抗菌肽Drosomycin、Attacin和Diptercin表达水平分别降低约50%。此外,通过免疫共沉淀实验检测Pitslre与预测存在相互作用的蛋白质TSC1、Rcd5和pbl之间的相互作用。综上所述,蛋白质激酶Pitslre参与果蝇天然免疫反应,在正向调控果蝇天然免疫Toll和IMD通路中发挥重要作用。