Gene signatures for intestinal and peripheral innate lymphoid cells in pigs reveal tissue-specific imprinting and similarities to human cells via single-cell RNA sequencing in pigs

Authors: WIARDA, JAYNE - Oak Ridge Institute For Science And Education (ORISE); Trachsel, Julian; SIVASANKARAN, SATHESH - Iowa State University; TUGGLE, CHRISTOPHER - Iowa State University; Loving, Crystal

Submitted to: American Association for Immunology
Publication Type: Abstract Only
Publication Acceptance Date: 3/7/2022
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Intestinal innate lymphoid cells (iILCs) impact intestinal health outcomes, but study of iILCs in humans is limited. Human peripheral ILCs (pILCs) are easily obtained but may vary substantially from iILCs, thus requiring comparison of iILCs and pILCs to determine applicability of pILCs as surrogates to study iILC function. Pigs have anatomic, physiological, nutritional, and immune similarities to humans that are lacking in rodent models, making pigs a relevant biomedical model; however, ILCs are poorly defined in pigs. Single-cell RNA sequencing was performed to compare porcine iILCs to pILCs and determine potential similarities of porcine to human ILCs. Porcine pILCs matched porcine NK cell descriptions, while iILCs were annotated as group 1 and group 3 ILCs. Gene modules obtained independent of cell annotations were distilled to core signatures of genes most highly specific to pILCs, iILCs, group 1 iILCs, and group 3 iILCs. The pILC signature included conventional NK genes, while the group 3 iILC signature included genes associated with type 3 immunity. Pan iILC and group 1 iILC signatures included genes associated with tissue residency, cell activation, and metabolism, indicating tissue-specific, activation-associated imprinting. Gene profiles were used to develop in situ detection methods for iILCs, establishing group 1 iILCs were intraepithelial, while group 3 iILCs resided in lamina propria/gut associated lymphoid tissue. Findings indicated iILCs were ILC subsets distinct from pILCs, thus opposing pILCs as surrogates to study iILC dynamics. Moreover, gene profiles and in situ locations of iILCs drew close parallels to human iILCs functions, supporting pigs as a biomedical model for iILC research. Four-wk-old pigs were inoculated by subcutaneous (SQ), intramuscular (IM), or IV route with BCG or saline (noBCG – control pigs). A sample size of 8-10 pigs/treatment for each study was maintained. To assess adaptive immune responses, at 5-8 weeks post-inoculation (wpi), ex vivo peripheral blood mononuclear cell (PBMC) IFN responses to PPD bovine (PPDb) were measured by ELISA. Innate memory was assessed at 2-8 wpi by culturing monocytes with LPS and measuring TNF production. At 6 wpi, inoculation site (SQ group) and liver (SQ and IV groups) samples were collected for BCG culture. PBMC IFN production following ex vivo PPDb stimulation was increased in SQ and IM groups (5.1 and 3.6-fold, respectively), but not in IV inoculated pigs. Conversely, TNF production from monocytes isolated from IV inoculated pigs and stimulated with LPS was heightened over SQ and IM groups (3.9 fold). However, BCG was recovered from the site of SQ inoculation and liver of IV but not SQ pigs. The lack of peripheral adaptive immune responsiveness following IV but not SQ or IM inoculation along with the converse enhancement of innate immune responses suggests that how or where an antigen is presented to the immune system plays a critical role in the type of immune response generated. In addition, the recovery of BCG from organs of animals with enhanced innate responses calls into question whether constant exposure to BCG or true cellular changes are associated with innate memory.