Precise and dynamic alterations in gene expression are critical determinants of the regulation of host immunity to microbial pathogens. Pathogenic rickettsiae in the spotted fever group cause some of the most severe infectious diseases in humans, characterized by microvascular inflammation and dysfunction attributed to disseminated infection of endothelial cells and increased vascular permeability resulting in pulmonary/cerebral edema. Long non-coding (lnc) RNAs of ? 200 nucleotides regulate a panoply of biological responses through an array of mechanisms and changes in their expression levels are now intricately linked to the determination of innate as well as cell-mediated immune responses. As an important subset of lncRNAs, enhancer lncRNAs implement their regulatory roles by enhancing protein coding genes (PCGs) in a cis- or trans-acting manner. We performed RNA-sequencing on the lungs as one of the predominantly affected target organs of susceptible mice infected with R. conorii to identify up-regulation of 179 lncRNAs. Via follow-up analysis to differentiate enhancer (elnc) from promoter-associated (plnc) RNAs based on the ratio of single- versus tri-methylation of histone 3 at lysine 4 (H3K4Me1:H3K4Me3) and other active enhancer signatures based on POLR2A, p300, DNase I hypersensitivity sites, CTCF, and Hi-3C ChIP-Seq datasets, we further determined significantly higher expression of an active elncRNA013718 and its target PCG Inhibitor of DNA binding 2 (ID2) in the mouse lungs, spleen, and CD8+ T-cells during Rickettsia conorii infection. Our preliminary findings further suggest that elncRNA013718 positively regulates the expression of ID2, a protein antagonist of E protein transcription factors and a regulator of T cells in the immune system. Accordingly, we refer to elncRNA013718 as elncRNA- ID2 and hypothesize novel contributory roles for elncRNA-ID2:Id2 interplay in the regulation of protective host immunity during rickettsial infections. We propose to test this hypothesis via two independent yet thematically interlinked specific aims.
Aim 1 will distinguish cell type-specific expression and functional roles of elncRNA- ID2 and ID2 in the host lungs and spleen in experimental murine models of R. conorii and R. australis infection.
In Aim 2, we will determine the modulatory effects of both global and cell-specific interference with elncRNA- ID2 on host immune responses and disease progression/outcome. Given the complexity of cellular immune responses, we will employ two independent and established in vivo models of infection closely mimicking the pathophysiology of human rickettsioses and cutting-edge approaches of cellular microbiology and immunology to determine the regulatory potential of elncRNA-ID2 as a novel elncRNA in the molecular circuitry underlying regulation of host immunity. The acquired insights will enhance our knowledge of context-specific physiological roles of elncRNA-ID2 in the determination of host responses to pathogenic rickettsiae and reveal potentially unique entry points for novel strategies to enhance host immunity against intracellular microbial infections.
Long non-coding (lnc) RNAs are emerging as important regulators of a range of host responses to infectious insults, including differentiation, maturation, migration, activation, and functions of a variety of immune cells. This application addresses the functional implications of elncRNA-ID2, a novel enhancer lncRNA element, in the context of mechanisms of pathogenesis and host immunity using murine models of tick-borne spotted fever rickettsioses. As a fundamentally important area of scientific enquiry at the crossroads of Cellular Microbiology, Immunology, and RNA Biology, definition of regulatory functions of novel lncRNAs will exert a sustained and potentially cross-disciplinary impact on their roles in inflammation and immunity during human diseases.
