Canine polarized macrophages express distinct functional and transcriptomic profiles

Abstract

Macrophage differentiation and function in disease states is highly regulated by the local microenvironment. For example, macrophage exposure to IFN-γ (interferon gamma) initiates the development of inflammatory (M1) macrophages, which acquire anti-tumoral and antimicrobial activity, while exposure to IL-4 (interleukin-4) and IL-13 (interleukin-13) drives an anti-inflammatory (M2) macrophage phenotype, which promotes healing and suppression of inflammatory responses. Previous studies of canine polarized macrophages have identified several surface markers that distinguished GM-CSF (granulocyte macrophage colony stimulating factor), IFN-γ and LPS (lipopolysaccharide) derived M1 macrophages or M2 macrophages; and reported a subset of genes that can be used to differentiate between polarization states. However, the need remains to understand the underlying biological mechanisms governing canine macrophage polarization states. Therefore, in the present study we used transcriptome sequencing, a larger panel of flow cytometry markers, and the addition of antimicrobial functional assays to further characterize canine macrophage polarization. Transcriptome analysis revealed unique, previously unreported signatures and pathways for polarized canine M1 and M2 macrophages. New flow cytometric markers were also identified, along with new characterization of how macrophage polarization impacted antimicrobial functions. Taken together, the findings reported here provide new insights into canine macrophage biology and identify new tools for the evaluation of polarized macrophages in dogs.

Characterization of polarization states of canine monocyte derived macrophages

“…Remarkably, a comparison of Gene Ontology and KEGG-pathways between studies shows that not one or two specific pathways stand out that define M1 or M2 macrophages. Also completely opposite outcomes were observed regarding the phagocytic capacity of M1 compared to M2 macrophages between the current study and Chow et al [19] These differences probably reflect the plasticity of macrophages and perhaps small differences in experimental setup. In addition, genetic differences between donors and their health status may contribute to the observed differences.…”

“…In addition an important goal of these studies was also to determine the robustness and reproducibility of producing canine monocyte derived M1 and M2 macrophages. Therefore our methodology to obtain monocyte derived macrophages was largely similar in setup to earlier studies [18,19] with the exception that our study included CD14 + cells and, compared to Chow et al, used different growth factors for polarization. Nevertheless, overall, obtained M1 and M2 subsets were comparable between all studies with respect to morphology, and upregulation of several M1 and M2 surface markers.…”

“…Tools available for characterization and use of polarized macrophages in dogs are limited. In 2017 and very recently in 2022, the polarization into M1 and M2 macrophages derived from monocytes was described [18,19] including microRNA analyses of genes and pathways related to polarization. Canine histiocytic cells (DH82 cells) and monocytes polarized towards two subsets resembling M1 and M2a macrophages, were described in a similar way, but based on a relatively small set of genes and markers [20].…”

Tumor-associated macrophages: Prognostic and therapeutic targets for cancer in humans and dogs

“…Activated macrophages (both M1 and M2) upregulated MHC class II, a finding supported by Heinrich et al. as well ( 72 , 74 ). Using IF, however, M1 macrophages were shown to significantly upregulate intracellular iNOS, while M2 macrophages upregulated intracellular CD206, transglutaminase 2 and suppressor of cytokine signaling 1 (SOCS1) as compared to M1 macrophages.…”

“…(It is worthwhile to note that although it is widely accepted that M2 macrophages have decreased ability to respond to infectious insults, some conflicting data in the human literature describe M2 macrophages as highly phagocytic ( 75 , 76 )). RNA-sequencing (RNA-seq) was used to identify unique canine gene signatures of polarized macrophages, resulting in 6 distinct clusters of macrophage genes ( 74 ). Macrophage phenotype has also been studied in a variety of canine pathologies, including (non-exhaustively) intestinal disease, spinal cord disease and Leishmania infections ( 77 – 79 ).…”