Immunohistochemical Characterization of Protease TMPRSS2 Expression in Porcine Tissue

Many human respiratory viruses rely on interactions with host cell proteases for their activation, necessary for their successful replication in host cells. Type II transmembrane protease, serine S1 member 2 (TMPRSS2) competently activates influenza A and is involved in the activation of other viruses including coronaviruses and members of Paramyxoviridae . Research using TMPRSS2 knock out mice showed resistance to multiple human influenza strains. However, little is known regarding TMPRSS2's tissue distribution in animals other than human and mouse. Mice are not naturally susceptible to influenza, respond differently than humans, and data has shown a difference in TMPRSS2 expression in certain tissues. Swine are naturally susceptible to human influenza viruses and vice versa. Consequently, swine are a logical model for further characterization of the role of TMPRSS2 in influenza A activation, potentially providing knowledge that could lead to development of novel antiviral treatments. To analyze the distribution of TMPRSS2, various tissues from the circulatory, respiratory, digestive, immune and reproductive systems of four pigs were collected, fixed in 10% neutral buffered formalin, processed to paraffin using standard techniques and sectioned at 4μm onto positively charged slides. Tissue sections were deparaffinized and rehydrated using xylenes and graded ethanols and heat induced antigen retrieved in sodium citrate pH 6 using a vegetable steamer technique for 20 minutes. Endogenous peroxidase was blocked by incubating slides in 3% hydrogen peroxide for 15 minutes. Anti‐TMPRSS2 monoclonal rabbit antibody (Abcam 92323; Cambridge, MA) was diluted 1:2500 and incubated on the sections for an hour. The primary antibody was detected using VECTASTAIN Elite ABC Kit (PK‐6101 Rabbit IgG; Burlingame, CA), visualized with 3,3′‐Diaminobenzidine (DAB; PK‐4100, VL), counterstained with hematoxylin and mounted with Permount (Electron Microscopy Sciences; Hatfield, PA). Slides were washed in Tris Buffered Saline 1× with 0.01% Tween‐20 between steps. Previous knowledge of porcine TMPRSS2 expression was limited to primary cells derived from the lower respiratory tract. We found expression throughout the entire respiratory tract. In addition, TMPRSS2 protein expression was present in the digestive tract, some lymphoid organs, kidneys, and reproductive system. Using IHC to visualize TMPRSS2 expression showed some interesting cell type localization in these tissues. These results extend the knowledge of TMPRSS2 expression in swine. Confirmation of TMPRSS2 mRNA expression using SYBR RT‐qPCR assays is underway. This research contributes to the advancement of our knowledge regarding this host cell protease involved in activation of multiple medically relevant viruses for which antiviral development is of great interest. Support or Funding Information This project is the result of funding provided by the Science and Technology Directorate of the United States Department of Homeland Security under contract number D15PC00276.TMPRSS2 Expression in Porcine Tissue (A) Widespread labeling of varying intensity dependent on cell type in lung. (B) Representative rabbit IgG isotype reagent control in lung. (C) Differential labeling of kidney tubules with sparing of glomeruli. (D) Labeling of ureter transitional epithelium. Bar 200um. Images extracted from Aperio GL whole slide digital scan.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .