title: Discovering novel immunoregulatory molecules that can be manipulated for clinical advantage
Creators: name: Wang, Yulin
Publisher: name: Griffith University
Year: 2022
Subjects: subject: malaria
subject: Plasmodium
subject: controlled human malaria infection (CHMI)

description: The successful control of malaria parasites by an infected host requires complex and balanced interactions between pro-inflammatory and regulatory immune responses. An excessive, dysregulated pro-inflammatory response results in tissue damage and the subsequent development of severe pathologies, with the routinely fatal cerebral malaria among them. Conversely, uncontrolled immunoregulatory responses promoted by Plasmodium species serve to inhibit protective host immunity, contributing to the delay, or even failure, of parasite clearance. The most advanced anti-malarial vaccine candidate RTS,S exhibited only 30% efficacy in individuals suffering repeated exposure to malaria parasites from malaria-endemic settings. A major impediment to enhancing vaccine efficacy is malaria-induced immunoregulatory pathways that hamper the establishment of long-term anti-parasitic immunity. Thus, any viable therapeutic intervention must promote anti-parasitic immunity while concurrently minimizing pathology. CD4+ T cells respond to specific immunological and disease settings by differentiating into distinct subsets with corresponding effector functions. Key components in the response to parasitic disease, IFNγ-producing helper T (TH1) cells protect the host via the activation of phagocytic cells and subsequent clearance of intracellular parasites; while IL-21-producing follicular helper T (TfH) cells promote parasite-specific antibody generation via support of germinal centre B cell function. Alongside these protective CD4+ T cell subsets, Plasmodium spp. also induce a population of type I regulatory (TR1) cells that actively impair TH1-mediated protective immunity. To characterise, and thus better understand the development and function of, TH1 and TR1 cells during malaria, these CD4+ T cell subsets were isolated from human volunteers participating in studies of controlled human malaria infection (CHMI) with Plasmodium falciparum. RNA-sequencing was then employed to identify differentially expressed genes (DEGs) between these subsets. Among those identified, we determined that TMEM173, the gene encoding the DNA-sensing receptor stimulator of interferon genes (STING), was more highly expressed and phosphorylated in TR1 than TH1 cells. STING is an intracellular innate signalling receptor that recognises pathogen DNA and RNA. Required for effective host defence, STING is an essential signalling adaptor that functions primarily by initiating the production of type I interferons (IFNs). Extracellular self-DNA, cell-intrinsic mitochondrial DNA, or genomic DNA released by infected cells have all been demonstrated to activate the STING pathway. Emerging evidence further suggests that the STING signalling pathway shapes adaptive, in addition to innate, immune responses. STING activation in antigen-presenting cells (APCs) is associated with augmented inflammatory immune responses. In T cells, STING signalling has been shown to promote apoptosis and inhibit the proliferation. In the context of autoimmunity, gain-of-function mutation of STING corresponded to an auto-inflammatory syndrome, despite featuring T and B cell deficiencies. In malaria, Plasmodium falciparum genomic DNA delivered to monocytes can activate the STING-TBK1 pathway associated with type I IFN production. However, the role of STING in CD4+ T cell responses to Plasmodium infection remains unknown. To study the roles of STING in CD4+ T cells, we employed CRISPR-Cas9 to disrupt TMEM173 transcription in human primary CD4+ T cells. We determined that STING signalling promotes the production of cytokines IL-10, IFNγ, and type I IFN; and the development of TR1 (LAG3+CD49b+) cells in vitro. To further characterize the roles of STING during infection in vivo, we utilised an experimental model of malaria characterised by Plasmodium berghei ANKA (PbA) infection of C57BL/6J mice. Relevant parasite-specific responses were assessed using a T cell receptor (TCR)-transgenic mouse that features CD4+ T cells (PbTII) expressing a TCR specific for an antigen expressed by all rodent malaria parasites. We detected increased frequencies of PbTII∆Sting IL-10+ IFNγ+ TR1 and Tbet+ IFNγ+ TH1 cells compared to corresponding PbTIIWT cell subsets, suggesting that STING promoted the generation of TR1 cells and inhibited TH1 cells in vivo during experimental malaria. To explore the underlying mechanisms, we performed bulk RNA-sequencing on PbTIIWT and PbTII∆Sting cells isolated from C57BL/6J mice infected with PbA. Gene set enrichment analysis revealed downregulation of genes related to type I IFN signalling in PbTII∆Sting cells. Thus, we hypothesized that STING regulates CD4+ T cell subsets through type I IFN signalling. To address this, we treated TMEM173-disrupted CD4+ T cells with exogenous type I IFN (IFNβ1), and determined that STING-dependent regulation of TR1 cells occurred via type I IFN signalling. To investigate the role of type I IFN signalling in vivo during experimental malaria, we generated type I IFNα receptor knockout (Ifnar-/-) crossed with PbTII mice, featuring antigen-specific PbTII∆Ifnar cells deficient in type I IFN signalling. We subsequently performed an adoptive transfer of PbTII∆Ifnar (CD45.2+) combined with PbTIIWT (CD45.1+CD45.2+) cells into recipient wild-type (WT; CD45.1+) mice, and challenged with PbA. STING was found to promote the generation of TR1 cells through type I IFN signalling, and appeared to inhibit TH1 cell effector function through a STING-intrinsic, type I IFN-independent mechanism. To investigate the role of STING in human disease, we isolated PBMCs from volunteers participating in CHMI studies and stimulated them with uninfected red blood cells (uRBC) or parasitised RBCs (pRBCs) in the presence or absence of STING agonist cGAMP. We determined that parasite antigen has the potential to activate STING signalling in human CD4+ T cells within PBMC culture. Furthermore, exposure to Plasmodium antigen enhanced TR1 cell sensitivity to STING agonist, which then, in turn, promoted TR1 generation. Further experimentation revealed that a JAK1/JAK2 inhibitor ruxolitinib (Rux) inhibits the STING-dependent generation of TR1 cells in vitro, highlighting Rux as a promising drug candidate in the improvement of malaria control. Collectively, these results increase our understanding of how malaria parasites manipulate the immunoregulatory networks that shape anti-malarial immunity, and suggest that targeted inhibition of the STING pathway has the potential to improve anti-parasitic responses and subsequent parasite control.