Experimental Trichinellosis in rats: Peritoneal macrophage activity

The influence of Trichinella spiralis infection on macrophage activity in rats during the first 28 days of infection was examined by measuring the production of NO and IL-6, as well as the expression of mannose receptor on the surface of peritoneal macrophages. During the course of a dynamic shift in the 3 life-cycle stages of the parasite, intermittent variations in NO production were observed but ended with increased values that coincided with the highest values for IL-6 release in the final, muscle phase of infection. No change in mannose receptor expression was observed during the course of infection. These results confirm that the Trichinella spiralis infection provokes changes in macrophage activity that could influence not only the course of the parasitic disease but also the overall immune status of the host

Obrazac traka u western blot analizi karakterističan za detekciju specifičnih anti-trichinella spiralis antitela kod različitih vrsta domaćina

Western blot (Wb) is considered to be the gold standard test for Trichinella infection serology, since this method allows specifi c Trichinella antigens to be distinguished from cross-reactive antigens. This is not the case with widely used antibody assay techniques - indirect immunofl uorescence and ELISA - which are sensitive, but subject to crossreactions that make the interpretation of weakly positive results diffi cult. Application of Trichinella spiralis muscle larvae excretory-secretory (ES) antigens for the specifi c antibody detection in ELISA resulted in improved specifi city compared to that of crude worm extract that was previously in use, but since production of ES has not yet been standardized, differences among laboratories occur. For this reason, the Wb profi le of serum samples from different T. spiralis infected host species: human, horse, swine and dog, was investigated in the Serbian National Reference Laboratory for Trichinellosis (NRLT). The common feature of the obtained Wb profi les was the appearance of a triad of bands with molecular masses (Mw) of 45, 49, and 53 kDa. The very same triad was recognized by a monoclonal antibody (mAb) 7C2C5 specifi c for an immunodominant epitope unique to the muscle larvae stage of all species in the genus Trichinella. Inhibition studies confi rmed that mAb and anti-Trichinella antibodies from sera competed for the same parasite epitope. Based on the obtained results, the NRLT introduced the recognition of the above mention triad as the basis for specifi c anti-Trichinella antibodies detection in the sera of infected hosts.Western blot (Wb) metoda se smatra zlatnim standardom u serološkoj dijagnostici trihineloze obzirom da ova metoda omogućava razlikovanje specifičnih Trichinella antigena od onih koji daju ukrštenu reakciju. Ovo nije slučaj sa drugim, u praksi češće korišćenim tehnikama baziranim na detekciji antitela – indirektna imunofl uorescencija i ELISA, koje su senzitivne ali podložne unakrsnoj reaktivnosti što otežava interpretaciju slabo pozitivnih rezultata. Primena ekskretorno-sekretornog (ES) antigena larvi u mišićima, Trichinella spiralis u detekciji specifičnih antitela u ELISA testu, rezultirala je povećanom specifičnošću testa u odnosu na prethodni koji je bio baziran na primeni totalnog solubilnog ekstrakta parazita. Ipak, obzirom da proizvodnja ES antigena još uvek nije standardizovana, postoje izvesne razlike između rezultata dobijenih u različitim laboratorijama. Iz ovog razloga, u Nacionalnoj Referentnoj Laboratoriji Srbije za Trihinelozu (NRLT) ispitivali smo Wb profi l uzoraka seruma različitih vrsta domaćina infi ciranih sa T. spiralis, i to ljudi, konja, svinja i pasa. Zajedničko svojstvo dobijenih Wb profi la bila je pojava tripleta traka koje odgovaraju proteinima sa molekulskim masama (Mw) od 45, 49, 53 kDa. Isti triplet prepoznaju i monoklonska antitela (mAt) 7C2C5 specifična za imunodominantni epitop karakterističan za stadijum mišićnih larvi svih vrsta parazita iz genusa Trichinella. Eksperimentalne studije inhibicije su pokazale da postoji kompeticija između mAt i anti-Trichinella antitela iz seruma za isti epitop parazita. Na osnovu dobijenih rezultata, NRLT uvodi prepoznavanje pomenutog tripleta traka kao osnovu za detekciju specifičnih anti-Trichinella antitela u serumima infi ciranih domaćina

Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity.

The immunomodulatory potential of Trichinella spiralis muscle larvae excretory-secretory products (ES L1) has been well documented in vitro on dendritic cells (DCs) and in animal models of autoimmune diseases. ES L1 products possess the potential to induce tolerogenic DCs and consequently trigger regulatory mechanisms that maintain immune homeostasis. The use of ES L1 as a potential treatment for various inflammatory disorders proved to be beneficial in animal models, although the precise immunomodulatory factors have not yet been identified. This study aimed at the isolation and characterization of ES L1 components that possess galectin family member properties. Galectin-1-like proteins (TsGal-1-like) were isolated from ES L1 based on the assumption of the existence of a lactose-specific carbohydrate-recognition domain and were recognized by anti-galectin-1 antibodies in Western blot. This TsGal-1-like isolate, similar to galectin-1, induced DCs with tolerogenic properties and hence, the capacity to polarize T cell response towards a regulatory type. This was reflected by a significantly increased percentage of CD4+CD25+Foxp3+ regulatory T cells and significantly increased expression of IL-10 and TGF-β within this cell population. Proteomic analysis of TsGal-1-like isolate by mass spectrometry identified nineteen proteins, seven with annotated function after blast analysis against a database for T. spiralis and the UniProt database. To our surprise, none of the identified proteins possesses homology with known galectin family members. Nevertheless, the isolated components of ES L1 possess certain galectin-1 properties, such as specific lactose binding and the potential to elicit a regulatory immune response, so it would be worth further investigating the structure of sugar binding within isolated proteins and its biological significance

Myeloid-derived suppressor cells prevent disruption of the gut barrier, preserve microbiota composition, and potentiate immunoregulatory pathways in a rat model of experimental autoimmune encephalomyelitis

Over-activated myeloid cells and disturbance in gut microbiota composition are critical factors contributing to the pathogenesis of Multiple Sclerosis (MS). Myeloid-derived suppressor cells (MDSCs) emerged as promising regulators of chronic inflammatory diseases, including autoimmune diseases. However, it remained unclear whether MDSCs display any therapeutic potential in MS, and how this therapy modulates gut microbiota composition. Here, we assessed the potential of in vitro generated bone marrow-derived MDSCs to ameliorate experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats and investigated how their application associates with the changes in gut microbiota composition. MDSCs differentiated with prostaglandin (PG)E2 (MDSC-PGE2) and control MDSCs (differentiated without PGE2) displayed strong immunosuppressive properties in vitro, but only MDSC-PGE2 significantly ameliorated EAE symptoms. This effect correlated with a reduced infiltration of Th17 and IFN-gamma-producing NK cells, and an increased proportion of regulatory T cells in the CNS and spleen. Importantly, both MDSCs and MDSC-PGE2 prevented EAE-induced reduction of gut microbiota diversity, but only MDSC-PGE2 prevented the extensive alterations in gut microbiota composition following their early migration into Payer's patches and mesenteric lymph nodes. This phenomenon was related to the significant enrichment of gut microbial taxa with potential immunoregulatory properties, as well as higher levels of butyrate, propionate, and putrescine in feces. This study provides new insights into the host-microbiota interactions in EAE, suggesting that activated MDSCs could be potentially used as an efficient therapy for acute phases of MS. Considering a significant association between the efficacy of MDSC-PGE2 and gut microbiota composition, our findings also provide a rationale for further exploring the specific microbial metabolites in MS therapy

NANOMATERIALS-BASED STRATEGY FOR MYELOID CELLS ACTIVATION RESULTS IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS AMELIORATION AND GUT MICROBIOTA MODULATION

ntroduction: Recent studies implicated overactivated myeloid cells and gut microbiome, along with our work, in multiple sclerosis (MS) pathogenesis. As we have shown before, prostaglandin (PG)E2 promotes suppressive properties of myeloid cells leading to amelioration of symptoms in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Additionally, we investigated how the changes of gut microbiota associate with EAE and the effects of therapy. Materials & Methods: MOG35-55 in Complete Freund Adjuvans was used for EAE induction in C57BL/6 mice. Gold nanoparticles (GNP) conjugated with PGE2 and MOG were applied on the day 1, 3, 5, 7, and 9 post-immunization. We performed extensive immunophenotyping and metagenomic analysis in order to decipher association between gut microbiome and efficacy of GNP-MOG-PGE2 treatment. Results: GNP-MOG-PGE2 treatment alleviates EAE symptoms, decreased levels of pro-inflammatory cytokines in sera, and increased proportion of suppressive MDSCs in CNS-infiltrates. Furthermore, EAE induction significantly affected species richness, while GNP-MOG-PGE2 treatment increased the gut microbiota diversity and preserved the richness of species with immunomodulatory properties. Conclusion: Taken together, our data indicate that targeted activation of myeloid cells by GNP-MOG-PGE2 together with gut microbiota modification is very promising therapeutic strategy for MS.International Society of Microbiota 10th ISM World Congress on Targeting Microbiota October 17-19, 2023 – Venice, Ital

Reduced Expression of Autophagy Markers and Expansion of Myeloid-Derived Suppressor Cells Correlate With Poor T Cell Response in Severe COVID-19 Patients

Widespread coronavirus disease (COVID)-19 is causing pneumonia, respiratory and multiorgan failure in susceptible individuals. Dysregulated immune response marks severe COVID-19, but the immunological mechanisms driving COVID-19 pathogenesis are still largely unknown, which is hampering the development of efficient treatments. Here we analyzed similar to 140 parameters of cellular and humoral immune response in peripheral blood of 41 COVID-19 patients and 16 age/gender-matched healthy donors by flow-cytometry, quantitative PCR, western blot and ELISA, followed by integrated correlation analyses with similar to 30 common clinical and laboratory parameters. We found that lymphocytopenia in severe COVID-19 patients (n=20) strongly affects T, NK and NKT cells, but not B cells and antibody production. Unlike increased activation of ICOS-1+ CD4+ T cells in mild COVID-19 patients (n=21), T cells in severe patients showed impaired activation, low IFN-gamma production and high functional exhaustion, which correlated with significantly down-regulated HLA-DR expression in monocytes, dendritic cells and B cells. The latter phenomenon was followed by lower interferon responsive factor (IRF)-8 and autophagy-related genes expressions, and the expansion of myeloid derived suppressor cells (MDSC). Intriguingly, PD-L1-, ILT-3-, and IDO-1-expressing monocytic MDSC were the dominant producers of IL-6 and IL-10, which correlated with the increased inflammation and accumulation of regulatory B and T cell subsets in severe COVID-19 patients. Overall, down-regulated IRF-8 and autophagy-related genes expression, and the expansion of MDSC subsets could play critical roles in dysregulating T cell response in COVID-19, which could have large implications in diagnostics and design of novel therapeutics for this disease

Myeloid derived suppressor cells-therapy attenuates experimental autoimmune encephalomyelitis and modulates gut microbiota composition

The role of gut microbiota composition in efficacy of various immune-based therapies is increasingly recognized. Thus, the aim of our study was to investigate if the efficacy of myeloid-derived suppressor cells (MDSC)-Prostaglandin E2 (PGE2) therapy for multiple sclerosis (MS) correlates with gut microbiota composition and function. MDSC generated from bone marrow cells in the presence of PGE2 were applied to spinal cord homogenate/CFA-induced experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats, an animal model of MS. MDSC-PGE2 therapy resulted in a significant attenuation of EAE symptoms over 30 days of disease monitoring. These results correlated with lower percentage of proinflammatory interferon- gamma and interleukin-17 producing cells and higher percentage of anti-inflammatory IL-4 producing cells in spinal cord and spleen. Gut microbial composition were studied using amplicon(16S rRNA)-based metagenomic analyses of fecal samples collected prior to the induction of EAE and MDSC-PGE2 therapy application, and at the peak of the disease. The induction of EAE resulted in a decrease of microbiota diversity, whereas the MDSC-PGE2 therapy preserved the diversity in EAE-induced animals. The induction of EAE in control group associated with a higher relative abundance of Peptococcaceae, but the lower levels of Veillonellaceae and different groups of Prevotellaceae, known to produce immunosuppressive short chain fatty acid (SCFA), and Lactobacillus reuteri, known for its anti-inflammatory function. In contrast, there were no changes in levels of these immunoregulatory taxa in EAE-animals treated with MDSC-PGE2 therapy. Also, SCFA producing Ruminococcaceae, and Coriobacteriaceae, known to metabolize phytoestrogens to immunosuppressive metabolites were more abundant in EAE-animals treated with MDSC-PGE2 therapy. Predicted metabolic profiling obtained by PICRUSt2 revealed that pathways involved in biosynthesis of polyamines, metabolites known to contribute to homeostasis of gastrointestinal mucosa, were enriched in MDSC-PGE2 treated animals. Considering these results, the modification of gut microbiota composition and function could further increase efficacy of MDSC-PGE-2 based therapy of autoimmune diseases.Book of Abstracts: Belgrade BioInformatics Conference 202

DOI:10.2298/ABS1001015G EXPERIMENTAL TRICHINELLOSIS IN RATS – PERITONEAL MACROPHAGE ACTIVITY

Abstract – The influence of Trichinella spiralis infection on macrophage activity in rats during the first 28 days of infection was examined by measuring the production of NO and IL-6, as well as the expression of mannose receptor on the surface of peritoneal macrophages. During the course of a dynamic shift in the 3 life-cycle stages of the parasite, intermittent variations in NO production were observed but ended with increased values that coincided with the highest values for IL-6 release in the final, muscle phase of infection. No change in mannose receptor expression was observed during the course of infection. These results confirm that the Trichinella spiralis infection provokes changes in macrophage activity that could influence not only the course of the parasitic disease but also the overall immune status of the host