High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare

Chitin Modulates Innate Immune Responses of Keratinocytes

Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR) TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined

Immunoblotting detects MsVmp1 in the midgut of feeding, starving and molting larvae.

<p>Crude protein extracts from the anterior, median and posterior midgut were separated by SDS-PAGE, blotted onto nitrocellulose and stained with anti-VMP1 antibodies. The given molecular mass was estimated using standard proteins of known molecular masses. </p

ClustalW alignment of valine-rich midgut proteins from <i>M. sexta</i> (MsVmps).

<p>Highly conserved or identical amino acids are highlighted with light grey, grey or black shadings. The consensus sequence is given below. The accession numbers are as follows: MsVmp1 (Msex012254-PA), MsVmp2 (central part of Msex012257-PA), MsVmp3 (C-terminal part of Msex012257-PA), MsVmp4 (N-terminal part of Msex012261-PA), MsVmp5 (N-terminal part of Msex012257-PA), MsVmp6 (Msex011100-PA), MsVmp7 (C-terminal part of Msex012261-PA), MsVmp8 (Msex012260-PA), and MsVmp (Msex012262-PA).</p

Tissue specific expression of <i>MsVMP</i> genes in fifth instar larvae of <i>M. sexta</i>.

<p>Total RNA was prepared from various tissues and cDNAs were synthesized. RT-PCR was carried out with primers specific to the indicated genes. PCR products of indicated sizes were separated by agarose gel electrophoresis and stained with ethidium bromide. Products for the ribosomal protein MsRpS3 were used as a loading control. Expression was found exclusively in the midgut. </p

Immunodetection of MsVmp1 in the posterior midgut of <i>M. sexta</i> larvae at different physiological conditions.

<p>Cryosections of posterior midguts were stained with CFW and immune-labeled with anti-VMP1 antibodies. Primary antibodies were detected with ALEXA 488-conjugated anti-guinea pig IgGs. Brightfield and fluorescence images (for anti-Vmp1 and CFW), and overlays of fluorescence images are shown. Cryosections were obtained from feeding 2^nd instar larvae (top), starving 2^nd instar larvae (middle) and larvae molting from the 2^nd to the 3^rd instar. C, cuticle; EC, ectoperitrophic space; EN, endoperitrophic space; PM, peritrophic matrix. Size bar, 100 µm.</p

MsVmp1 is extensively <i>O</i>-glycosylated.

<p>(A) <i>MsVMP1</i> was expressed in insect Sf21 cells and purified by Ni-NTA chromatography. After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and stained with Ponceau Red (left) and subsequently with different lectins (right lanes). GNA, <i>Galanthus nivalis</i> agglutinin; PNA, peanut agglutinin; SNA, <i>Sambucus nigra</i> agglutinin; DSA, <i>Datura stramonium</i> agglutinin; MAA, <i>Maackia amurensis</i> agglutinin. (B) For deglycosylation, MsVmp1 (expressed in insect cells) was treated with PNGase F or <i>O</i>-glycosidase in the presence of protease inhibitors. The reaction products were separated by SDS PAGE and stained with Coomassie Blue. <i>Left </i><i>lane</i>, MsVmp1 input without addition of glycosidase; Middle lane, PNGase F treatment; Right lane, <i>O</i>-glycosidase treatment. Std, standard proteins with indicated molecular masses in kDa. Arrows point to reaction intermediates and the terminal deglycosylation product with indicated molecular masses.</p

Phylogenetic tree of lepidopteran VMPs.

<p>The un-rooted maximum likelihood tree was calculated on the basis of a ClustalW alignment of VMPs from different lepidopteran species. Bootstrap values are given in percentages at the internodes; Accession numbers refer to the entries of the dbEST database of Butterflybase. MS, <i>Manduca sexta</i>; BM, <i>Bombyx mori</i>; TN, <i>Trichopulsia </i><i>ni</i>; HA, <i>Helicoverpa armigera</i>; SF, <i>Spodoptera frugiperda</i>; PI, <i>Plodia interpunctella</i>.</p