HIV virus can persist latently in CD4+ T-cells and tissue-resident macrophages. This necessitates lifelong antiretroviral therapy (ART) for people living with HIV (PLWH). Reactivation of latent HIV results in killing of infected cells by immune cells. Studies with latency reactivating agents have shown that reactivation alone is not sufficient for the elimination of the HIV reservoir. HIV latency is associated with histone methylation mediated chromatin condensation at viral integration sites. Innate immune memory induced in macrophages by muramyl dipeptide, ß-glucan and Syk inhibition has been shown to relieve chromosome condensation through epigenetic rewiring at immune effector gene loci, thereby increasing phagocytosis, antigen presentation, and lymphoid cell activity. We trained PBMCs of ART-suppressed PLWH in the presence of matched autologous sera to test whether this could lead to specific killing of the HIV infected cells. qPCR analysis with the IPDA HIV reservoir assay showed that training with autologous sera could not only enhance the reactivation of viral RNA transcription, but could also lead to HIV reservoir reduction, potentially through NK cell and CTL activation, enhanced antigen presentation, and phagocytosis by trained macrophages and dendritic cells. Our data highlight a new therapeutic approach for PLWH where training of myeloid cells could both reactivate latent HIV transcription and reduce the viral reservoir through CTL and NK cell dependent killing. This work was supported by the Intramural Research Program of NIAID, NIH and the Office of AIDS Research at NIH. Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Interferon induced protein with tetratricopeptide repeats 1 (IFIT1) is an interferon stimulated gene with antiviral properties mediated through the translational inhibition of viral mRNAs that lack a 2’-O methyl group on their 5’ cap. IFIT1 expression is induced by activation of pattern recognition receptors, e.g. Toll-like receptors. Previously, we showed that IFIT1 differentially modulates the expression of various immune-related genes. IFIT1 positively regulates the type I interferon gene program in response to LPS stimulation-an effect mediated through modulation of an HDAC2 transcriptional regulatory complex-while also negatively regulating pro-inflammatory cytokine expression. We now show that IFIT1 negatively regulates miR-22 expression, an miRNA that inhibits translation of IL-b. Depletion of IFIT1 led to the upregulation of miR-22 and a consequent downregulation of intracellular IL1-b protein levels, along with strongly reduced IL1-b secretion in response to inflammasome activation. In IFIT1 depleted THP1 macrophages, reduced IL-1 levels led to increased infection by the OC43 coronavirus. We also found that RSV induced IL1-b expression was significantly diminished in Ifit1 knockout immortalized mouse macrophages (iMMs), leading to a significant increase in RSV infection levels. Our studies demonstrate a novel function for IFIT1 in supporting IL-1 dependent host defense against viral infection, through a miR-22-regulated circuit controlling IL1-b protein levels. This work was supported by the Intramural Research Program of NIAID, NIH and the Office of the AIDS Research at NIH Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

The induction of innate immune memory and consequent trained immunity has several potential applications within the biomedical field. For instance, training with established stimuli such as BCG vaccination and β-glucan has previously shown to reduce microbial infections and cancer growth; although, some cases presented side effects, such as autoimmune disease. By identifying new trained immunity stimuli with less side effects, this approach could have broad applications, such as the reactivation and killing of latent HIV. Using a high throughput screening assay, we found that the metabolites taurine and α-ketoglutarate (AKG) both induced innate immune memory in macrophages. Our studies further showed that taurine and AKG enhanced the training effect of previously identified stimuli such as Syk kinase inhibitors (SYKi IV or R406), β-glucan, and Muramyl dipeptide (MDP), the active component of the BCG vaccine. Chromatin immunoprecipitation analysis of taurine and AKG treated macrophages showed enrichment of H3K4Me3, a transcriptional activation and innate immune memory marker, at the TNF and IL6 promoters. Our studies also showed that combined application of AKG or taurine with either MDP, β-glucan or SYKi IV, can reverse HIV latency in cell line models more efficiently than training with MDP, β-glucan or SYKi IV alone. We also show that combined application of taurine or AKG with MDP, β-glucan, or SYKi IV can efficiently reactivate HIV from PBMCs of people living with HIV. This work was supported by the Intramural Research Program of NIAID, NIH and the Office of the AIDS Research at NIH. Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

We used established innate immune memory-inducing stimuli to study the reactivation of HIV from latently infected cells. Inducing innate immune training activated transcription of HIV-1 genes from THP89GFP monocytic latent HIV reporter cells. Four out of 17 patients had detectable counts of HIV from PBMC-derived macrophages and demonstrated reactivation with all three training stimuli. We also found that trained macrophages in co-culture can directly induce reactivation of HIV from 2D10 cells, a T cell reporter line for latent HIV, suggesting an innate immune memory-induced crosstalk between macrophages and T cells. To further study this, we performed scRNA-seq and scATAC-seq in PBMCs of ART-suppressed PLWH. We identified enhanced cell-to-cell ligand-receptor cross talk specifically between trained myeloid cells and lymphoid cells. Training of myeloid cells in PBMCs of PLWH also led to enhanced expression of many genes among the known pathways of CD4/CD8 T cell, NK cell and B cell activation. This suggests that innate training of the PBMC myeloid compartment may also trigger an enhanced CD8 T cell or NK cell-mediated elimination of the HIV reservoir. Our studies suggest that induction of innate immune memory could be a viable approach to the reactivation of latently infected HIV-1 and could be developed as a novel therapeutic approach to facilitate the elimination of latent HIV reservoirs. This work was supported by the Intramural Research Program of NIAID, NIH and the Office of the AIDS Research at NIH. Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

AbstractThe immunologic consequences of using bactericidal versus bacteriostatic antibiotic treatments are unclear. We observed a bacteriostatic (growth halting) treatment was more protective than a bactericidal (bacteria killing) treatment in a murine peritonitis model. To understand this unexpected difference, we compared macrophage responses to bactericidal treated bacteria or bacteriostatic treated bacteria. We found that Gram-negative bacteria treated with bactericidal drugs induced more proinflammatory cytokines than those treated with bacteriostatic agents. Bacterial DNA – released only by bactericidal treatments – exacerbated inflammatory signaling through TLR9. Without TLR9 signaling, the in vivo efficacy of bactericidal drug treatment was rescued. This demonstrates that antibiotics can act in important ways distinct from bacterial inhibition: like causing treatment failure by releasing DNA that induces excessive inflammation. These data establish a novel link between how an antibiotic affects bacterial physiology and subsequent immune system engagement, which may be relevant for optimizing treatments to simultaneously clear bacteria and modulate inflammation.

HIV virus can persist in a latent but activatable chronic state in resting CD4 +T-cells and macrophages that are more persistent, leading to the requirement of lifelong combination antiretroviral therapy (cART). The latency of HIV-1 in macrophage cells is associated with increased chromatin condensation induced by histone methylation at HIV-1 integration sites. Innate immune training of macrophages has been shown to relieve chromosome condensation through epigenetic rewiring. We hypothesized that training of macrophages could enhance the reactivation of HIV-1 by opening the chromatin to facilitate transcription in response to Latency Reversing Agents (LRAs). We used beta-glucan, Syk inhibitor and Rutaecarpine to train the THP89GFP cell line, which is an experimental model for latently infected HIV-1 monocytes. We found that these trained THP89GFP cells have higher transcription of HIV-1 specific genes in response to PMA stimulation. To check the epigenetic changes associated with training in the proviral HIV-1 DNA of THP89GFP, we performed a chromatin immunoprecipitation for the histone mark, H3K27Ac. Consistent with the higher induction of transcription of HIV-1 genes, we observed that multiple regions of the HIV-1 LTR had higher deposition of H3K27Ac in the trained macrophages. Our studies thus show that induction of trained innate immunity may be a viable approach to the reactivation of latently infected HIV-1. This finding supports the hypothesis that innate immune training stimuli could be developed as novel candidates for enhancing reactivation of latently infected HIV-1, which may facilitate elimination of macrophage reservoirs. This work was supported by the Intramural Research Program of NIAID, NIH This work was supported by the Intramural Research Program of NIAID, NIH and Office of AIDS research.

Kinase signaling in the tiered activation of inflammasomes and associated pyroptosis is a prime therapeutic target for inflammatory diseases. While MAPKs subsume pivotal roles during inflammasome priming, specifically the MAP3K7/JNK1/NLRP3 licensing axis, their involvement in successive steps of inflammasome activation is poorly defined. Using live-cell MAPK biosensors to focus on the inflammasome triggering event allowed us to identify a subsequent process of biphasic JNK activation. We find that this biphasic post-trigger JNK signaling initially facilitates the mitochondrial reactive oxygen species generation needed to support core inflammasome formation, then supports the gasdermin-mediated cell permeation required for release of active IL-1β from human macrophages. We further identify and characterize a xanthine oxidase-ROS activated MAP3K5/JNK2 substrate licensing complex as a novel regulator of the GSDMD mobilization which precedes pyroptosis. We show that inhibitors targeting this MAP3K5 cascade alleviate morbidity in mouse models of colitis and dampen both augmented IL-1β release and cell permeation in monocytes derived from patients with gain-of-function inflammasomopathies.

Innate immune training molecules have several potential applications in the biomedical field. We developed an assay to screen for new training molecules by generating HiBiT tagged TNF cells in the THP1 monocytic cell line through CRISPR knock-in. The HiBiT tag, an 11 amino acid long peptide, was fused to the TNF C-terminus to produce luminescence upon addition of the LgBiT substrate. To optimize the assay, we first titrated different concentrations of multiple TLR ligands including pIC, p(dAdT), PGN, FLGN, LPS, R848, and P3C. LPS, PGN, and P3C induced the strongest signal to noise (S/N) ratio in the pool of TNF-HiBiT cells. From the TNF-HiBiT pool, we then single cell cloned and characterized them for optimum TNF response. The clones D3 and D6 had significantly higher S/N ratio compared to the pools, especially in response to P3C and PGN. To optimize the TNF-HiBiT cells for a training screen, we used previously known training molecules such as Syk kinase inhibitor, beta-Glucan and Rutaecarpine, to train multiple TNF-HiBiT cell clones. We found that the clones that induced a lower TNF signal upon acute TLR activation exhibited a more robust training phenotype compared to the clones that induced a more robust acute TNF signal. We found therefore that the optimal protocol to assess trained immunity is to use lower concentrations of activating TLR ligands (0.1, 0.1, and 0.0001 ng/mL of P3C, LPS and PGN) in the trained cells. This may be due to saturation of the TNF-HiBit reporter signal when cells are challenged with higher TLR ligand concentrations (10, 100, LPS, and 10 ng/mL of P3C, LPS and PGN). In brief, we have developed and optimized a robust assay for screening novel training molecules. This work was supported by the Intramural Research Program of NIAID, NIH Supported by grants from Intramural Research Program of NIAID, NIH and the Office of AIDS Research at NIH.

We conducted a genome-wide siRNA screen for regulators of LPS-driven TNF expression in macrophages. We used a splicing-independent reporter for the screen, and while we identified very few core spliceosome components among the strongest screen hits, we observed a clear enrichment for peripheral splicing regulators. Among these, a novel putative splicing regulator, UBL5, gave a strong screen phenotype, and a UBL5 shRNA-expressing cell line showed a selective defect in LPS-driven gene expression. While many genes were induced normally by LPS in the UBL5 deficient cells, a subset of genes, including core components of the LPS-activated TLR4 signaling pathway, were substantially diminished in UBL5-deficient cells. We conducted both Illumina and Pacbio long-read sequencing, and we observed marked defects in the LPS-induced splicing of numerous genes, however the primary impact on known components of the TLR4 pathway was observed at the level of overall gene expression rather than alternative splicing. Our data suggests that the putative splicing regulator UBL5 also has key roles in the maintenance and induction of gene expression for several core components of the TLR4 signaling pathway. Key role of UBL5 in the induction of TLR4 signaling pathway was further evidenced by the defective NLRP3 inflammasome activation and protection of OC43 coronavirus infection in UBL5-deficient cells. This work was supported by the Intramural Research Program of NIAID, NIH Intramural Research Program of NIAID, NIH

Biologically active small molecules can impart modulatory effects, in some cases providing extended long-term memory. In a screen of biologically active small molecules for regulators of tumor necrosis factor (TNF) induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing lipopolysaccharide (LPS)-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits β-glucan-induced H3K4Me3 marks at the promoters of several pro-inflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for β-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and OC43 coronavirus infection, highlighting a potential application of this molecule and other SYKis as prophylactic treatments for viral susceptibility.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi's sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman's disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60's role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV+/EBV- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics.

Activation of the TLR4 signaling pathway by lipopolysaccharide (LPS) leads to induction of both inflammatory and interferon-stimulated genes, but the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remain poorly understood. In a genome-wide small interfering RNA (siRNA) screen of the LPS-induced tumor necrosis factor α (TNF-α) response in macrophages, we identify the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identifies a positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced gene classes. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3A-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate a function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program.

The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.

Macrophages play a critical role in the innate immune response to pathogen infection, but few tools exist for systematic dissection of these responses using modern genome-wide perturbation methods. To develop an assay platform for high-throughput analysis of macrophage activation by pathogenic stimuli, we generated reporter systems in human and mouse macrophages with dynamic readouts for NF-κB and/or TNF-α responses. These reporter cells show responsiveness to a broad range of TLR ligands and to gram-negative bacterial infection. There are significant challenges to the use of RNAi in innate immune cells, including efficient small RNA delivery and non-specific immune responses to dsRNA. To permit the interrogation of the macrophage pathogen response pathways with RNAi, we employed the stably expressed reporter genes to develop efficient siRNA delivery protocols for maximal target gene silencing with minimal activation of the innate macrophage response to nucleic acids. We demonstrate the utility of these macrophage cell systems for siRNA screening of pathogen responses by targeting components of the human and mouse TLR pathways and observe species-specific perturbation of signaling and cytokine responses. Our approach to reporter cell development and siRNA delivery optimization provides an experimental paradigm with significant potential for developing genetic screening platforms in mammalian cells.

RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene's phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1-host cell interactions.

The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon's protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections.