Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

Human TLR8 Senses RNA From Plasmodium falciparum-Infected Red Blood Cells Which Is Uniquely Required for the IFN-γ Response in NK Cells.

During blood-stage malaria, the innate immune system initiates the production of pro-inflammatory cytokines, including IFN-γ, that are critical to host defense and responsible for severe disease. Nonetheless, the innate immune pathways activated during this process in human malaria remain poorly understood. Here, we identify TLR8 as an essential sensor of Plasmodium falciparum-infected red blood cells (iRBC). In human immune cells, iRBC and RNA purified from iRBC were detected by TLR8 but not TLR7 leading to IFN-γ induction in NK cells. While TLR7 and 9 have been shown to lead to IFN-γ in mice, our data demonstrate that TLR8 was the only TLR capable of inducing IFN-γ release in human immune cells. This unique capacity was mediated by the release of IL-12p70 and bioactive IL-18 from monocytes, the latter via a hitherto undescribed pathway. Altogether, our data are the first reported activation of TLR8 by protozoan RNA and demonstrate both the critical role of TLR8 in human blood-stage malaria and its unique functionality in the human immune system. Moreover, our study offers important evidence that mouse models alone may not be sufficient to describe the human innate immune response to malaria.

Toll-Like Receptor 3-TRIF Pathway Activation by Neospora caninum RNA Enhances Infection Control in Mice.

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as one of the main transmissible causes of reproductive failure in cattle and consequent economic losses to the sector. In that sense, this study aimed to evaluate the role of Toll-like receptor 3 (TLR3)-TRIF-dependent resistance against N. caninum infection in mice. We observed that TLR3-/- and TRIF-/- mice presented higher parasite burdens, increased inflammatory lesions, and reduced production of interleukin 12p40 (IL-12p40), tumor necrosis factor (TNF), gamma interferon (IFN-γ), and nitric oxide (NO). Unlike those of T. gondii, N. caninum tachyzoites and RNA recruited TLR3 to the parasitophorous vacuole (PV) and translocated interferon response factor 3 (IRF3) to the nucleus. We also observed that N. caninum upregulated the expression of TRIF in murine macrophages, which in turn upregulated IFN-α and IFN-ß in the presence of the parasite. Furthermore, TRIF-/- infected macrophages produced lower levels of IL-12p40, while exogenous IFN-α replacement was able to completely restore the production of this key cytokine. Our results show that the TLR3-TRIF signaling pathway enhances resistance against N. caninum infection in mice, since it improves Th1 immune responses that result in controlled parasitism and reduced tissue inflammation, which are hallmarks of the disease.

Neutralization of the Plasmodium-encoded MIF ortholog confers protective immunity against malaria infection.

Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory factor, PMIF, which modulates the host inflammatory response to malaria. Using a novel RNA replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host response and observed improved control of liver and blood-stage Plasmodium infection, and complete protection from re-infection. Vaccination against PMIF delayed blood-stage patency after sporozoite infection, reduced the expression of the Th1-associated inflammatory markers TNF-α, IL-12, and IFN-γ during blood-stage infection, augmented Tfh cell and germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells. Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to promote immunity to Plasmodium and potentially other parasite genera that produce MIF orthologous proteins.

Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors.

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.

Influence of Leishmania RNA Virus 1 on Proinflammatory Biomarker Expression in a Human Macrophage Model of American Tegumentary Leishmaniasis.

Backgound: Species of the Leishmania Viannia (L. V.) subgenus harbor the double-stranded Leishmania RNA virus 1 (LRV-1), previously identified in isolates from Brazil and Peru. Higher levels of LRV-1 in metastasizing strains of L. V. guyanensis have been documented in both human and murine models, and correlated to disease severity. Methods: Expression of proinflammatory biomarkers, including interleukin (IL) 1ß, tumor necrosis factor alpha (TNF-α), CXCL10, CCL5, IL-6, and superoxide dismutase, in human macrophages infected with 3 ATCC and 5 clinical isolates of L. V. braziliensis, L. V. guyanensis, and L. V. panamensis for 24 and 48 hours were measured by commercial enzyme immunoassay. Analyses were performed at 24 and 48 hours, stratified by LRV-1 status and species. Results: LRV-1-positive L. V. braziliensis demonstrated significantly lower expression levels of TNF-α (P = .01), IL-1ß (P = .0015), IL-6 (P = .001), and CXCL10 (P = .0004) compared with LRV-1-negative L. V. braziliensis. No differences were observed in strains of L. V. panamensis by LRV-1 status. Conclusions: Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific.

Innate sensing of malaria parasites.

Innate immune receptors have a key role in immune surveillance by sensing microorganisms and initiating protective immune responses. However, the innate immune system is a classic 'double-edged sword' that can overreact to pathogens, which can have deleterious effects and lead to clinical manifestations. Recent studies have unveiled the complexity of innate immune receptors that function as sensors of Plasmodium spp. in the vertebrate host. This Review highlights the cellular and molecular mechanisms by which Plasmodium infection is sensed by different families of innate immune receptors. We also discuss how these events mediate both host resistance to infection and the pathogenesis of malaria.

Recruitment of PfSET2 by RNA polymerase II to variant antigen encoding loci contributes to antigenic variation in P. falciparum.

Histone modifications are important regulators of gene expression in all eukaryotes. In Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var) are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD) of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2) has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.

Combined action of nucleic acid-sensing Toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice.

"Triple-defective" (3d) mice carrying a mutation in UNC93B1, a chaperone for the endosomal nucleic acid-sensing (NAS) Toll-like receptors TLR3, TLR7, and TLR9, are highly susceptible to Toxoplasma gondii infection. However, none of the single or even the triple NAS-TLR-deficient animals recapitulated the 3d susceptible phenotype to experimental toxoplasmosis. Investigating this further, we found that while parasite RNA and DNA activate innate immune responses via TLR7 and TLR9, TLR11 and TLR12 working as heterodimers are required for sensing and responding to Toxoplasma profilin. Consequently, the triple TLR7/TLR9/TLR11-deficient mice are highly susceptible to T. gondii infection, recapitulating the phenotype of 3d mice. Humans lack functional TLR11 and TLR12 genes. Consistently, human cells produce high levels of proinflammatory cytokines in response to parasite-derived RNA and DNA, but not to Toxoplasma profilin, supporting a more critical role for NAS-TLRs in human toxoplasmosis.

Induction of protective immunity against toxoplasmosis in mice by immunization with Toxoplasma gondii RNA.

Toxoplasma gondii enters the mucosal surfaces of the host, and so immunity at these sites is of major interest. Due to the compartmentalization of the immune response, systemic immunization does not induce high levels of immunity at mucosal surfaces. Intranasal immunization has been shown to be very effective in inducing both systemic and mucosal immune responses. Immunization with mRNA can induce both humoral and cell-mediated immune responses, both of which are important in conferring immunity to T. gondii. The efficacy of RNA vaccination by the nasal route with T. gondii RNA was evaluated. We assessed the percentage of cumulative survival after an oral challenge with a lethal dose of T. gondii cysts (40 cysts), and the number of brain cysts following a challenge with a sublethal dose of T. gondii 76 K cysts (15 cysts). Vaccinated mice were found to be significantly better protected than non-immunized mice after a challenge with a lethal dose of cysts; and a challenge with a sublethal dose also resulted in fewer brain cysts than in non-immunized mice. Sera and intestinal secretions of immunized mice recognized T. gondii antigens, suggesting that a specific humoral immune response may occur. Moreover, a specific lymphoproliferative response observed in cervical lymph nodes may confer protection. These preliminary findings suggest that RNA vaccination by a mucosal route could be feasible.

Cutting edge: innate immune system discriminates between RNA containing bacterial versus eukaryotic structural features that prime for high-level IL-12 secretion by dendritic cells.

RNA derived from bacterial but not eukaryotic sources, when transfected into human monocyte-derived dendritic cell precursors, induces high-level IL-12 secretion in conjunction with dendritic cell maturation stimuli. In vitro-transcribed mRNA that mimics the structure of bacterial mRNA in the lack of a long 3'-poly(A) tail likewise induces IL-12 secretion, but this property is lost upon efficient enzymatic 3'-polyadenylation. Among other tested RNAs, only polyuridylic acid induced IL-12 p70. This RNA response phenomenon appears biologically distinct from the classically defined response to dsRNA. RNA-transfected APC also polarize T cells in an IL-12-dependent manner toward the IFN-gamma(high)IL-5 (low) Th1 phenotype, suggesting a link between the detection of appropriately structured RNA and the skewing of immune responses toward those best suited for controlling intracellular microbes. RNA structured to emulate bacterial patterns constitutes a novel vaccine strategy to engender polarized Th1-type immune responses.

Mouse antibody response to a microsporidian parasite following inoculation with a gene coding for parasite ribosomal RNA.

This study found that a plasmid construct encoding the small-subunit ribosomal RNA (SSUrRNA) of the microsporidian Microgemma caulleryi generates a humoral response upon intramuscular inoculation in mice. The plasmid used was pCMV, following preliminary trials indicating efficient beta-galactosidase gene expression in mouse muscle cells transfected with pCMV/beta-Gal. The antibodies produced after inoculation with pCMV/SSUDNA recognized parasite spore antigens and reached maximum levels at 30 days postinoculation, subsequently remaining stable for at least 120 days. Due to the highly conserved sequence of the SSUrDNA in different microsporidian species, these results open up interesting prospects for broad-spectrum vaccination.

Mammalian cell expression of malaria merozoite surface proteins and experimental DNA and RNA immunisation.

The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.