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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #436673

Research Project: Antigen Presenting Neutrophils in Cattle

Location: Animal Parasitic Diseases Laboratory

Project Number: 8042-32000-116-002-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Aug 1, 2019
End Date: Jul 31, 2022

Preliminary results indicate that expression of interleukin-10, CD25 or major histocompatibility complex class II by bovine neutrophils is potently regulated by antigens from Ostertagia ostertagi, an important bovine abomasum-dwelling nematode parasite. The neutrophils with dual functional phenotypes can be critical in conditioning or biasing the subsequent acquired immune responses mediated by T and B lymphocytes. The resulting immunity can be beneficial or damaging, depending on the types (protective or non-protective) of immunity induced. The hypothesis is that Ostertagia ostertagi antigens can induce immunosuppressive neutrophils, which in turn induce active T and B lymphocyte-mediated immunosuppression, allowing continued survival of the parasite in the host. The objectives are 1) to define the phenotypes of bovine neutrophils isolated from Ostertagia ostertagi-infected cattle, 2) to investigate the ability of bovine neutrophils to present antigens and regulate down-stream T and B lymphocyte responses in vitro, ex vivo and in vivo, and 3) to elucidate mechanisms by which Ostertagia ostertagi infection induces antigen-presenting regulatory neutrophils in cattle experimentally infected with Ostertagia ostertagi. The ultimate goal is to identify and characterize immunosuppressive neutrophils induced by Ostertagia-derived molecules which may be used as vaccine candidates.

1. Phenotyping of bovine neutrophils: Neutrophils will be analyzed with flow cytometry by morphology (polymorphonuclear) and antibodies to cell surface markers and cytosolic cytokines, including CD25, major histocompatibility complex class II and interleukin-10. Cells with major histocompatibility complex class II will possess the ability to present antigens to T lymphocytes, and Cells with CD25 and/or interleukin-10 will have regulatory functions, suppressing T and B lymphocyte responses. T and B lymphocyte responses conditioned by antigen-presenting neutrophils will be analyzed for the ability to produce cytokines which define the type of immunity and respond to antigens by mediating cellular activities. 2. Modulation of neutrophils: Neutrophils expressing CD25, interleukin-10, and/or major histocompatibility complex class II in stomach mucosa, draining lymph nodes, spleen, and peripheral blood of Ostertagia-infected and uninfected cattle will be determined by flow cytometry and quantitative polymerase chain reaction. Neutrophils with defined phenotypes (interleukin-10 positive or negative) will be enriched by sorting with flow cytometry and tested in vitro for the ability to interact with and regulate T and B lymphocytes in co-culture systems. T and B lymphocyte responses will be analyzed by flow cytometry, RNAseq, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. 3. Functional mechanisms of antigen-presenting neutrophils: Neutrophils with defined phenotypes will be sorted to a purity of >99% and studied by high-throughput gene and protein profiling technologies.