Helminthiasis may ameliorate inflammatory diseases, such as inflammatory bowel disease and asthma. Information about immunomodulators from Ascaris lumbricoides is scarce, but could be important considering the co-evolutionary relationships between helminths and humans. We evaluated the immunomodulatory effects of a recombinant cystatin from A. lumbricoides on an acute model of dextran sodium sulphate (DSS)-induced colitis in mice. From an A. lumbricoides cDNA library, we obtained a recombinant cystatin (rAl-CPI). Protease activity inhibition was demonstrated on cathepsin B and papain. Immunomodulatory effects were evaluated at two intraperitoneal doses (0.5 and 0.25 μg/G) on mice with DSS-induced colitis. Body weight, colon length, Disease Activity Index (DAI), histological inflammation score, myeloperoxidase (MPO) activity, gene expression of cytokines and cytokines levels in colon tissue were analysed. Treatment with rAl-CPI significantly reduced DAI, MPO activity and inflammation score without toxic effects. Also, IL-10 and TGF-B gene overexpression was observed in rAl-CPI-treated group compared to DSS-exposed control and healthy mice. Furthermore, a reduction in IL-6 and TNF-A expression was found, and this was confirmed by the levels of these cytokines in colonic tissue. In conclusion, rAl-CPI reduces inflammation in a mouse model of DSS-induced colitis, probably by increasing the expression of anti-inflammatory cytokines and reducing pro-inflammatory ones.

Severe helminth infections are negatively associated to allergic diseases like asthma; therefore, the immunomodulatory properties of parasite-derived components have been analyzed, raising the possibility of their use as anti-inflammatory molecules. We evaluated the immunomodulatory properties of Ascaris lumbricoides recombinant cysteine protease inhibitor (rAl-CPI) in a mouse model of allergic airway inflammation induced by the house dust mite (HDM) Blomia tropicalis and its effects on human monocyte-derived dendritic cells (HmoDCs). The B. tropicalis sensitized/challenged mice developed extensive cellular airway inflammatory response, which was significantly reduced upon treatment with rAl-CPI prior to B. tropicalis sensitization, affecting particularly the perivascular/peribronchial infiltrate cells, eosinophils/neutrophils, and goblet cells. A significant decrease of Th2 cytokines, total, and specific IgE antibodies was observed in rAl-CPI treated mice. The antibody response was biased to IgG, mainly IgG2a. Administration of rAl-CPI-alone and rAl-CPI before mite sensitization were associated with a significant increase of regulatory T cells (Tregs) in spleen and elevated IL-10 levels in BAL and splenocytes culture supernatants, which was partially affected by anti-IL10 receptor use. In vitro, rAl-CPI showed a modulatory effect on HmoDCs, lowering the expression of HLA-DR, CD83, and CD86, while inducing IL-10 and IL-6 production. This suggests an inhibition of HmoDC maturation and a possible link with the inhibition of the allergic response observed in the murine model.

BackgroundChronic obstructive pulmonary disease (COPD) is associated with increased risk of severe COVID-19, but the mechanisms are unclear. Besides, patients with severe COVID-19 have been reported to have increased levels of several immune mediators.MethodsNinety-two proteins were quantified in 315 plasma samples from 118 asthmatics, 99 COPD patients and 98 healthy controls (age 40-90 years), who were recruited in Colombia before the COVID-19 pandemic. Protein levels were compared between each disease group and healthy controls. Significant proteins were compared to the gene signatures of SARS-CoV-2 infection reported in the “COVID-19 Drug and Gene Set Library” and with experimentally tested protein biomarkers of severe COVID-19.ResultsForty-one plasma proteins showed differences between patients and controls. Asthmatic patients have increased levels in IL-6 while COPD patients have a broader systemic inflammatory dysregulation driven by HGF, OPG, and several chemokines (CXCL9, CXCL10, CXCL11, CX3CL1, CXCL1, MCP-3, MCP-4, CCL3, CCL4 and CCL11). These proteins are involved in chemokine signaling pathways related with response to viral infections and some, were found up-regulated upon SARS-CoV-2 experimental infection of Calu-3 cells as reported in the COVID-19 Related Gene Sets database. An increase of HPG, CXCL9, CXCL10, IL-6, MCP-3, TNF and EN-RAGE has also been experimentally detected in patients with severe COVID-19.ConclusionsCOPD patients have altered levels of plasma proteins that have been reported increased in patients with severe COVID-19. Our study suggests that COPD patients have a systemic dysregulation in chemokine networks (including HGF and CXCL9) that could make them more susceptible to severe COVID-19. Also, that IL-6 levels are increased in some asthmatic patients (especially in females) and this may influence their response to COVID-19. The findings in this study depict a novel panel of inflammatory plasma proteins in COPD patients that may potentially associate with increased susceptibility to severe COVID-19 and might be useful as a biomarker signature after future experimental validation.

To analyze the impact of Ascaris lumbricoides infection on the pathogenesis and diagnosis of allergic diseases, new allergens should be identified. We report the identification of a new Ascaris lumbricoides allergen, Asc l 5. The aim of this study was to evaluate the physicochemical and immunological features of the Asc l 5 allergen. We constructed an A. lumbricoides cDNA library and Asc l 5 was identified by immunoscreening. After purification, rAsc l 5 was physicochemically characterized. Evaluation of its allergenic activity included determination of Immunoglobulin E (IgE) binding frequency (in two populations: 254 children and 298 all-age subjects), CD203c based-basophil activation tests (BAT) and a passive cutaneous anaphylaxis (PCA) mouse model. We found by amino acid sequence analysis that Asc l 5 belongs to the SXP/RAL-2 protein family of nematodes. rAsc l 5 is a monomeric protein with an alpha-helical folding. IgE sensitization to rAsc l 5 was around 52% in general population; positive BAT rate was 60%. rAsc l 5 induced specific IgE production in mice and a positive PCA reaction. These results show that Asc l 5 has structural and immunological characteristics to be considered as a new allergen from A. lumbricoides.

BackgroundAsthma is not well investigated in equatorial Africa and little is known about the disease-associated allergen molecules recognized by IgE from patients in this area. The aim was to study the molecular IgE sensitization profile of asthmatic children and young adults in a semi-rural area (Lambaréné) of an equatorial African country (Gabon), to identify the most important allergen molecules associated with allergic asthma in equatorial Africa.MethodsFifty-nine asthmatic patients, mainly children and few young adults, were studied by skin prick testing to Dermatophagoides pteronyssinus (Der p), D. farinae (Der f), cat, dog, cockroach, grass, Alternaria and peanut. Sera were obtained from a subset of 35 patients, 32 with positive and 3 with negative skin reaction to Der p and tested for IgE reactivity to 176 allergen molecules from different allergen sources by ImmunoCAP ISAC microarray technology and to seven recombinant Blomia tropicalis (Blo t) allergens by IgE dot blot assay.ResultsThirty-three of the 59 patients (56%) were sensitized to Der p and 23 of them (39%) were also sensitized to other allergen sources, whereas 9 patients (15%) were only sensitized to allergen sources other than Der p. IgE serology analyses (n=35) showed high IgE-binding frequencies to the Blo t allergens Blo t 5 (43%), Blo t 21 (43%) and Blo t 2 (40%), whereas the Der p allergens rDer p 2, rDer p 21 and rDer p 5 (34%, 29% and 26%) were less frequently recognized. Only few patients showed IgE reactivity to allergens from other allergen sources, except to allergens containing carbohydrate determinants (CCDs) or to wasp venom allergens (i.e., antigen 5).ConclusionOur results thus demonstrate that IgE sensitization to mite allergens is very prevalent in asthmatics in Equatorial Africa with B. tropicalis allergen molecules representing the most important ones associated with allergic asthma.

RATIONALE: Flagellin which is abundant in gram-negative bacteria including pseudomonas is reported to be related with development of asthma. However, its effect on airway epithelial cells regarding asthma pathogenesis is not elucidated yet. We investigated the effect of flagellin on the production of cytokines, chemokines and metalloproteinase related with airway inflammation and remodeling in the primary human epithelial cells. METHODS: Primary human bronchial epithelial cells were grown and differentiated in air-liquid interface culture for 14-16 days. The cells were treated with flagellin in vitro at 10 and 100ng/ml for 3, 24, and 48hours. The conditioned media and cells were harvested to measure cytokines, chemokines and metalloproteinase related with asthma and their mRNA expressions by ELISA or western blot and quantitative PCR, respectively. RNA-sequencing was performed to screen the up-regulated and downregulated transcriptomes by flagellin. RESULTS: Flagellin upregulated mRNA expressions of GM-CSF at 3hrs of treatment and increased GM-CSF secretion significantly at 24hr in conditioned media. Flagellin enhanced mRNA expressions and secretions of CCL5, CXCL10, CXCL11 chemokines, which were detected in the RNA-sequencing. In addition, mRNA expressions and protein productions of MMP9 and MMP13 were also enhanced by flagellin. TGF-b1 and TGF-b2 augmented the mRNA expressions of GM-CSF, MMP9 and MMP13 induced by flagellin. CONCLUSIONS: Flagellin is an inducer of cytokines, chemokines and metalloproteinase contributing to airway inflammation and remodeling in bronchial epithelial cells. Further studies are needed to clarify their interaction with other inflammatory and structural cells and clinical implications in terms of asthma.Abstracts AB187 SUNDAY

BackgroundRespiratory diseases such as chronic obstructive pulmonary disease (COPD) have been associated with increased risk of severe SARS-CoV-2 infection, but the mechanisms and putative immune pathways are unclear. Besides, increased levels of several immune mediators in patients with severe coronavirus disease 19 (COVID-19) have been reported.ObjectiveTo perform an immunoproteomic profiling of dysregulated plasma proteins in patients with asthma and COPD and to evaluate their relationship with biomarkers of severe COVID-19.Methods92 protein biomarkers were quantified in 315 plasma samples from adult subjects (age 40-90 years) including 118 asthmatics, 99 COPD patients and 98 healthy controls, that have been recruited in two reference pneumology clinics in Colombia before the beginning of the COVID-19 pandemic.ResultsForty-one plasma proteins showed differences between patients and controls. Asthmatic patients have increased levels in IL-6 and CCL3 while COPD patients have a broader systemic inflammatory dysregulation driven by HGF, OPG, and several chemokines (CXCL9, CXCL10, CXCL11, CX3CL1, CXCL1, MCP-3, MCP-4, CCL3, CCL4 and CCL11). Functional annotation revealed their enrichment in chemokine signaling pathways related with response to viral infections. Some of these proteins were found up-regulated upon SARS-Cov-2 infection of Calu-3 cells. Also, HPG, CXCL9, CXCL10, IL-6, MCP-3, TNF and EN-RAGE have been found increased in patients with severe COVID-19. HGF, the most significant protein in this study as well as its correlated proteins could be associated with the increased risk of severe COVID-19 in COPD patients.ConclusionsAsthma and COPD patients have altered plasma levels of proteins that have been found associated with increased risk of severe COVID-19. The reasons for sharing these profiles with this infection are unknown, but our study suggest that adult patients with asthma and COPD have a systemic dysregulation in chemokine networks that could make them more susceptible to severe COVID-19.

Only few allergens derived from house dust mite (HDM) species have been evaluated in terms of their potential to induce allergic inflammation. In this study, we aimed to evaluate different aspects of the allergenicity and allergenic activity of Blo t 2, a Blomia tropicalis allergen. Blo t 2 was produced as a recombinant protein in Escherichia coli. Its allergenic activity was tested in humans by skin prick test and basophil activation assays, and in mice, by passive cutaneous anaphylaxis and a model of allergic airway inflammation. Sensitization rate to Blo t 2 (54.3%) was similar to that found to Blo t 21 (57.2%) and higher than to Der p 2 (37.5%). Most Blo t 2-sensitized patients showed a low intensity response (99.5%). Blo t 2 elicited CD203c upregulation and allergen induced skin inflammation. Additionally, immunized animals produced anti-Blo t 2 IgE antibodies and passive transfer of their serum to non-immunized animals induced skin inflammation after allergen exposure. Immunized animals developed bronchial hyperreactivity and a strong inflammatory lung reaction (eosinophils and neutrophils). These results confirm the allergenic activity of Blo t 2 and supports its clinical relevance.