Using In Vitro Granulomas to Identify the Type 1 And Type 3 Interferons In Granuloma Formation and Antimicrobial Activity

Abstract

The interferons (IFNs) include three major categories of cytokine including type 1 (α, β, ε, κ, and ω), type 2 (γ), and type 3 (λ). Type 3 (IFNs) are further classified into IFNλ1, IFNλ2, IFNλ3, and IFNλ4 in humans and non-human primates (NHPs). These cytokines play important roles in immunity against pathogenic microorganisms where in some cases, they confer protection, and in other cases, they promote pathology. In Mycobacterium tuberculosis (Mtb) infection, type 1 IFNs are known well for their pathogenic roles, whereas the role that type 3 IFNs play in TB is less known and has received little attention. To address this knowledge gap, we made granulomas under in vitro conditions by infecting PBMCs isolated from macaques with Mtb. We pre-treated the PBMCs with IFNs and IFN inhibitors to identify their role in early immune responses in TB. We understood that type I IFNs play a crucial role in promoting granuloma formation and maintenance, while IFN λ signaling shows promise to be a regulator. Interestingly, supplementation with exogenous type I IFN led to a modest reduction in granuloma size, while the addition of IFNλ1 or IFNλ4 resulted in a slight increase in granuloma dimensions. This led to our understanding of the contrasting effects of type I IFN supplementation and IFNλ supplementation on granuloma dimensions, suggesting opposing roles in modulating granuloma formation. A lactose dehydrogenase (LDH) assay was performed to determine if IFN inhibition or supplementation had cytopathic effects in each treatment condition and IFN-supplemented conditions were found to be modestly cytoprotective. To determine how cytokine inhibition or supplementation affected immune responses, we measured cytokine and IDO1 expression. A cytokine bead array performed on the supernatants of the samples led to the identification of IL-1β, TNF-α, and IL-6 exhibiting the highest expression after type I IFN neutralization and a downward trend upon IFN supplementation of both type 1 IFN and IFN λ. The data obtained from flow cytometry suggests that myeloid cells didn’t get influenced by type 3 IFNs to exhibit any change in IDO1 expression