Avian Macrophage Responses to Virulent and Avirulent Clostridium perfringens

The present study evaluated the avian macrophage responses against Clostridium perfringens that varied in their ability to cause necrotic enteritis in chickens. Strains CP5 (avirulent-netB+), CP1 (virulent-netB+), and CP26 (highly virulent-netB+tpeL+) were used to evaluate their effect on macrophages (MQ-NCSU cells) and primary splenic and cecal tonsil mononuclear cells. The bacilli (whole cells) or their secretory products from all three strains induced a significant increase in the macrophage transcription of Toll-like receptor (TLR)21, TLR2, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and CD80 genes as well as their nitric oxide (NO) production and major histocompatibility complex (MHC)-II surface expression compared to an unstimulated control. The CP1 and CP26-induced expression of interferon (IFN)γ, IL-6, CD40 genes, MHC-II upregulation, and NO production was significantly higher than that of CP5 and control groups. Furthermore, splenocytes and cecal tonsillocytes stimulated with bacilli or secretory products from all the strains showed a significant increase in the frequency of macrophages, their surface expression of MHC-II and NO production, while CP26-induced responses were significantly higher for the rest of the groups. In summary, macrophage interaction with C. perfringens can lead to cellular activation and, the ability of this pathogen to induce macrophage responses may depend on its level of virulence.

Caveolin-1 Down-Regulation Reduces VEGF-A Secretion Induced by IGF-1 in ARPE-19 Cells

The insulin-like growth factor 1 (IGF-1) stimulates expression and secretion of vascular endothelial growth factor-A (VEGF-A), the main actor in ocular neovascularization, by RPE cells. Activity of IGF-1 is regulated by interaction between its receptor and Caveolin-1 (Cav-1), the main component of caveolae. The aim of this study was to investigate whether modulation of Cav-1 expression affects synthesis and secretion of VEGF-A. ARPE-19 cells were transfected with small interfering RNA for Cav-1 (si-Cav-1) and with control siRNA (si-CTR) and stimulated with IGF-1. We found that down-regulation of Cav-1 did not affect activation of IGF-1R but regulated in an opposite manner the phosphorylation of Akt and Erk1/2. Moreover, we found that IGF-1 increased mRNA levels of VEGF-A in both si-CTR and in si-Cav-1 ARPE-19 cells and that Cav-1 silencing significantly reduced basal and IGF-1-stimulated VEGF-A release. Then we investigated the response of the microvascular endothelial cell line HMEC-1 to secretory products of ARPE-19 cells by evaluating wound healing closure, finding that conditioned media from si-Cav-1-ARPE-19 cells reduced endothelial cell migration rate. These data demonstrate that Cav-1 regulates secretion of VEGF-A, and that the depletion of Cav-1 reduces IGF-1 induced VEGF-A secretion in ARPE-19 cells and the migratory potential of their secretory products.

Helminths make themselves at home

Drurey et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20211140) show that excretory/secretory products from the parasitic helminth Heligmosomoides polygyrus suppress the host-protective small intestinal epithelial response. These findings establish that helminths directly modulate the tissue in which they live, shining new light on the host–parasite interaction.

Exopolysaccharides from Bifidobacterium animalis Ameliorate Escherichia coli-Induced IPEC-J2 Cell Damage via Inhibiting Apoptosis and Restoring Autophagy

Enteropathogenic Escherichia coli (EPEC) is a common zoonotic pathogen that causes acute infectious diarrhea. Probiotics like Bifidobacterium are known to help prevent pathogen infections. The protective effects of Bifidobacterium are closely associated with its secretory products exopolysaccharides (EPS). We explored the effects of the EPS from Bifidobacterium animalis subsp. lactis (B. lactis) on ameliorating the damage of an intestinal porcine epithelial cell line (IPEC-J2) during EPEC infection. Pretreatment with EPS alleviated EPEC-induced apoptosis through the restoration of cell morphology and the downregulation of protein expressions of cleaved-caspase 8, cleaved-caspase 3, and cleaved-PARP. EPS-mediated remission of apoptosis significantly improved cell viability during EPEC infection. EPEC infection also resulted in impaired autophagy, as demonstrated by decreased expressions of autophagy-related proteins Beclin 1, ATG5, and microtubule-binding protein light chain-3B (LC3B) and the increased expression of p62 through western blot analysis. However, EPS reversed these effects which indicated that EPS promoted autophagosome formation. Furthermore, EPS prevented the lysosome damage induced by EPEC as it enhanced lysosomal acidification and raised lysosome-associated protein levels, thus promoted autophagosome degradation. Our findings suggest that the amelioration of EPEC-induced cell damages by EPS is associated with the limitation of detrimental apoptosis and the promotion of autophagy flux.

Intestinal epithelial tuft cell induction is negated by a murine helminth and its secreted products

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.

Mining Anti-Inflammation Molecules From Nippostrongylus brasiliensis-Derived Products Through the Metabolomics Approach

Hookworm is one type of soil-transmitted helminth, which could exert an anti-inflammatory effect in human or animal host, which provides a beneficial possibility for the discovery of inflammatory-related disease interventions. The identification of hookworm-derived anti-inflammatory molecules is urgently needed for future translational research. The emergence of metabolomics has become a powerful approach to comprehensively characterize metabolic alterations in recent times. Herein, excretory and secretory products (ESPs) were collected from cultured adult worm, while small intestinal contents were obtained from Nippostrongylus brasiliensis (N. brasiliensis, Nb)-infected mice. Through ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) platform, metabolomics analysis was used to explore the identification of anti-inflammatory molecules. Out of 45 differential metabolites that were discovered from ESPs, 10 of them showed potential anti-inflammatory properties, which could be subclassed into amino acids, furanocoumarins, linear diarylheptanoids, gamma butyrolactones, and alpha-keto acids. In terms of intestinal contents that were derived from N. brasiliensis-infected mice, 14 out of 301 differential metabolites were discovered to demonstrate anti-inflammatory effects, with possible subclassification into amino acids, benzylisoquinolines, quaternary ammonium salts, pyrimidines, pregnane steroids, purines, biphenyls, and glycerophosphocholines. Furthermore, nine of the differential metabolites appeared both in ESPs and infected intestinal contents, wherein four were proven to show anti-inflammation properties, namely, L-glutamine, glutamine (Gln), pyruvate, and alanine-Gln (Ala-Gln). In summary, we have provided a method for the identification and analysis of parasite-derived molecules with potential anti-inflammatory properties in the present study. This array of anti-inflammatory metabolites could provide clues for future evaluation and translational study of these anti-inflammatory molecules.

Parasitic nematode fatty acid- and retinol-binding proteins compromise host immunity by interfering with host lipid signaling pathways

Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda.

Identification of Myoferlin, a Potential Serodiagnostic Antigen of Clonorchiasis, via Immunoproteomic Analysis of Sera From Different Infection Periods and Excretory-Secretory Products of Clonorchis sinensis

Clonorchiasis, which is caused by Clonorchis sinensis, is an important foodborne disease worldwide. The excretory-secretory products (ESPs) of C. sinensis play important roles in host-parasite interactions by acting as causative agents. In the present study, the ESPs and sera positive for C. sinensis were collected to identify proteins specific to the sera of C. sinensis (i.e., proteins that do not cross-react with Fasciola hepatica and Schistosoma japonicum) at different infection periods. Briefly, white Japanese rabbits were artificially infected with C. sinensis, and their sera were collected at 7 days post-infection (dpi), 14 dpi, 35 dpi, and 77 dpi. To identify the specific proteins in C. sinensis, a co-immunoprecipitation (Co-IP) assay was conducted using shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) to pull down the sera roots of C. sinensis, F. hepatica, and S. japonicum. For the annotated proteins, 32, 18, 39, and 35 proteins specific to C. sinensis were pulled down by the infected sera at 7, 14, 35, and 77 dpi, respectively. Three proteins, Dynein light chain-1, Dynein light chain-2 and Myoferlin were detected in all infection periods. Of these proteins, myoferlin is known to be overexpressed in several human cancers and could be a promising biomarker and therapeutic target for cancer cases. Accordingly, this protein was selected for further studies. To achieve a better expression, myoferlin was truncated into two parts, Myof1 and Myof2 (1,500 bp and 810 bp), based on the antigenic epitopes provided by bioinformatics. The estimated molecular weight of the recombinant proteins was 57.3 ku (Myof1) and 31.3 ku (Myof2). Further, both Myof1 and Myof2 could be probed by the sera from rabbits infected with C. sinensis. No cross-reaction occurred with the positive sera of S. japonica, F. hepatica, and negative controls. Such findings indicate that myoferlin may be an important diagnostic antigen present in the ESPs. Overall, the present study provides new insights into proteomic changes between ESPs and hosts in different infection periods by LC-MS/MS. Moreover, myoferlin, as a biomarker, may be used to develop an objective method for future diagnosis of clonorchiasis.

Hypoxic and nitrosative stress conditions modulate expression of myoglobin genes in a carcinogenic hepatobiliary trematode, Clonorchis sinensis

Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1—CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity.