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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Publications at this Location » Publication #326436
Title: Functional characterization of the turkey macrophage migration inhibitory factor

Author
item PARK, MYEONSEON - Virginia Tech
item KIM, SUNGWON - Roslin Institute
item Fetterer, Raymond
item DALLOUL, RAMI - Virginia Tech

Submitted to: Developmental and Comparative Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2016
Publication Date: 6/1/2016
Citation: Park, M., Kim, S., Fetterer, R.H., Dalloul, R.A. 2016. Functional characterization of the turkey macrophage migration inhibitory factor. Developmental and Comparative Immunology. 61:198-207.

Interpretive Summary: Turkeys are an important commodity in US agriculture. About 214 million birds are produced annually with a farm level value of 5.3 billion dollars. Although important to agriculture, the responses to infectious diseases and general immune responses in turkeys have not been as well characterized as those of chickens and some other avian species. Recently the turkey genome has been sequenced and characterized. This provides an important tool to investigate molecules important to turkey immune responses. MIF, macrophage migration inhibitory factor, is a soluble protein that plays a pivotal role in modulating immune responses and is widely distributed in mammals and lower vertebrates but has not been characterized in turkeys. The aim of the present study was to clone the turkey MIF gene, express the active form and characterize its basic function. The results of the study indicate that the structure of the gene from turkey is nearly identical to that from chicken and similar to MIF from other avian species. In addition, turkey MIF possesses biological properties that regulate immune responses in a manner similar to chicken MIF. The results of this study help us to better understand the biological function of an evolutionary conserved MIF in the turkey's immune system. This knowledge will aid in development of strategies to control infectious diseases in turkeys and other birds.

Technical Abstract: Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characterize its basic function. The full-length TkMIF gene was amplified from total RNA extracted from turkey spleen, followed by cloning into a prokaryotic (pET11a) expression vector. Sequence analysis revealed that TkMIF consists of 115 amino acids with 12.5 kDa molecular weight. Multiple sequence alignment revealed 100%, 65%, 95% and 92% identity with chicken, duck, eagle and zebra finch MIFs, respectively. Recombinant TkMIF (rTkMIF) was expressed in E. coli and purified through HPLC and endotoxin removal. SDS-PAGE analysis revealed an approximately 13.5 kDa of rTkMIF monomer containing T7 tag in soluble form. Western blot analysis showed that anti-chicken MIF (ChMIF) polyclonal antisera detected a monomer form of TkMIF at approximately 13.5 kDa size. Further functional analysis revealed that rTkMIF inhibits migration of both mononuclear cells and splenocytes in a dose-dependent manner, but was abolished by the addition of anti-ChMIF polyclonal antisera. qRT-PCR analysis revealed elevated transcripts of pro-inflammatory cytokines by rTkMIF in LPS-stimulated monocytes. rTkMIF also led to increased levels of IFN-' and IL-17F transcripts in Con A-activated splenocytes, while IL-10 and IL-13 transcripts were decreased. Overall, the sequences of both the turkey and chicken MIF have high similarity and comparable biological functions with respect to migration inhibitory activities of macrophages and enhancement of pro-inflammatory cytokine expression, suggesting that turkey and chicken MIFs would be biologically cross-reactive.