Abstract
Interleukin-10 (IL-10) is a pleiotropic cytokine that acts primarily on immune cells to regulate inflammatory, innate, and adaptive immune responses to maintain tissue homeostasis. Viruses have evolved mechanisms to manipulate immunosurveillance by their hosts. A common strategy is the genetic acquisition and production of host proteins such as IL-10. Many viruses have been reported to encode IL-10, including members of the Herpesviridae and Poxviridae. The viral IL-10 proteins have diversified in sequence and function relative to those of their host species, but generally inhibit immune function and promote infection. A prime example is the IL-10 protein produced by Orf virus (ORFV) from the Parapoxvirus (PPV) genus of Poxviridae, which shares the immune suppressive and immune stimulatory properties of hIL-10. Four additional PPV members, namely Pseudocowpox virus (PCPV), Bovine papular stomatitis virus (BPSV), Red deer parapoxvirus (RDPV) and Grey seal parapoxvirus (GSPV), have been reported to encode variants of IL-10. As prior studies of viral IL-10 proteins provided great insight into this family of proteins, it was hypothesised that investigations into the evolution, structure, and function of these natural IL-10 variants would increase understanding of viral IL-10s as virulence factors, host IL-10s as master regulators of immune function, and recombinant IL-10 proteins as therapeutics for immune-mediated diseases.
To investigate the evolution of the viral IL-10 family, an updated list of viral and host IL-10s was compiled and phylogenetic analysis was performed. The 36 viral IL-10 proteins formed seven distinct clades, with varying evolutionary distances to the IL-10 proteins from their respective hosts. The PPV IL-10s formed one clade in the phylogenetic tree, clustering near each other and the IL-10s from humans and their mammalian hosts. To predict the structure of the PPV IL-10s, their amino acid sequences were aligned, revealing moderate levels of amino acid identity and similarity with human IL-10. Furthermore, structural models indicated the PPV IL-10s would adopt an overall tertiary structure analogous to hIL-10. This structural homology, in turn, suggested they would engage the IL-10 receptor complex in a similar manner to hIL-10. However, some variations in structure, glycosylation and receptor interactions were predicted between the PPV IL-10s and that of hIL-10, implying there could be differences in their downstream signalling and immunoregulatory effects.
To facilitate functional analyses of the PPV IL-10s, recombinant proteins were expressed in a mammalian cell line and then purified using affinity chromatography. Purification of GSPV IL-10 proved particularly challenging, requiring method optimisation. Biochemical analysis of the purified PPV IL-10s revealed some interesting findings. The molecular weights for the ORFV, PCPV, and human IL-10s were higher than predicted and consistent with dimer formation. The RDPV and GSPV IL-10s also exhibited higher molecular weights than predicted, with N-glycosidase digestion confirming the presence of N-linked glycans. These biochemical differences between the human and PPV IL-10s could also influence their molecular interactions and biological effects.
To investigate the immune suppressive and immune stimulatory functions of the PPV IL-10s, the recombinant proteins were applied to human monocytic and murine mast cells, stimulated with lipoteichoic acid (LTA) or IL-3 and IL-4, respectively. All PPV IL-10s retained some degree of immune-suppressive and immune-stimulatory activities analogous to hIL-10, however, the levels of these activities varied. The ORFV and BPSV IL-10s were the most potent and effective agonists of LTA-induced cytokine production by monocytes, and ORFV and RDPV IL-10s were the most potent and effective agonists of IL-3 and IL-4-stimulated mast cell proliferation. By contrast, the PCPV and GSPV IL-10s were partial agonists with reduced potency and efficacy in these cellular assays. These quantitative differences indicate that while the PPV IL-10s interact with the IL-10 receptor complex, they may vary in their molecular interactions and induction of signalling pathways.
To investigate receptor binding by the PPV IL-10s, various methods were trialled with varying success. Bioluminescence resonance energy transfer assays utilising a human cell line expressing various combinations of IL-10 receptor (IL-10R) detected IL-10R1-IL-10R1 homodimerisation in response to excessive amounts of hIL-10 but failed to detect IL-10R2-IL-10R2 homodimerisation, or IL-10R1-IL-10R2 heterodimerisation. Competitive binding assays utilising recombinant IL-10R-1 proteins detected concentration-dependent binding by human, ORFV, BPSV, PCPV and RDPV IL-10s, but not GSPV IL-10. Detection of IL-10R2 binding by hIL-10 required excessive receptor amounts. Co-immunoprecipitation of the human and PPV IL-10s with the recombinant receptors, followed by Western blot detection of each binding partner, supported the findings from the competitive IL-10R1 binding assay. It also revealed variable binding by the human and PPV IL-10s to IL-10R2, the greatest extent by
BPSV IL-10, and the least by GSPV IL-10. However, when both IL-10Rs were included, competition was observed, with the level of each receptor interaction decreasing. Finally, the signalling pathways induced by the PPV IL-10s were examined in LTA-stimulated monocytes. Phosphorylation of STAT3 at tyrosine 705, for the most part, aligned with the receptor binding profiles of the human and PPV IL-10 in Western blot and AlphaLISA analyses, with the PCPV, BPSV and RDPV IL-10s showing the greatest potency, and the GSPV IL-10 being the least potent.
Overall, the combined analyses revealed that the PPV IL-10 proteins share a common origin and structure, but have important differences in their biochemical properties, immunomodulatory functions, receptor interactions, and signalling responses. Taken together, these findings provide crucial insights into the structure-function relationship in the IL-10 family. This knowledge has implications for how viral IL-10s modulate host immunity to enable virus persistence and how host IL-10s control immune homeostasis. This study may lead to the development of the PPV IL-10s, or other modified IL-10s, as new, improved anti-inflammatory therapeutic agents.