Genhong Cheng - US grants

Tumor necrosis factor (TNF) has been considered as an anti-cancer agent since its discovery two decades ago. Members of the TNF receptor (TNFR) superfamily can send both survival and death signals to cells, and play important roles in a wide range of biological effects that include acute phase responses and lymphocyte activation. CD40, as a member of this receptor family, activates multiple signaling pathways, induces expression of dozens of genes, and is essential for many important events in T-cell-dependent humoral responses. Our goal is to find connections that can link the CD40 receptor to multiple signal transduction pathways, and that link each signaling pathway to its downstream effector genes and to the CD40-mediated biological functions. The recent discovery of several early signaling mediators, including the TNF receptor-associated factor (TRAF) family proteins, the TRAF- associated NF-kappaB activator (TANK) and the NF-kappaB-inducing kinase (NIK), has provided an opportunity to dissect multiple CD40-mediated signal transduction pathways. This proposal will focus on the early events of CD40 receptor-initiated signaling. First, we will determine the specificities of multiple TRAF proteins for receiving signals from CD40 and for sending out downstream signals to activate both the NF-kappaB and stress-activating protein kinase (SAPK) signal transduction pathways. Second, we will determine the molecular mechanisms of TRAF and TANK cooperation. We will also test the possible role of TANK as a switching molecule in controlling the threshold of CD40-induced NF-KB and SAPK activation. Our work will: 1) provide new insights into the molecular mechanisms by which a single receptor interacting with its ligand can generate multiple signal transduction pathways and control multiple biological events; and 2) identify new therapeutic targets in the multiple CD40 and TNF signaling pathways for treatment of cancers and immune diseases.

DESCRIPTION (Provided by the Applicant): CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is expressed on the surface of B-cells, activated macrophages, epithelial cells, dendritic cells and a variety of tumor cells. CD40 is activated as a trimer after interaction with its partner, the CD40 ligand (CD40L), which is expressed on the surface of activated CD4+ helper T-cells. Mutations in the CD40L gene are responsible for X-linked hyper-IgM syndrome, a severe inherited human immunodeficiency disease. CD40-CD40L interaction plays an essential role in most of the events of the T-cell-dependent humoral response, including immunoglobulin heavy-chain class switching, antibody affinity maturation, generation and maintenance of memory B-cells, and T-cell activation. However, the molecular mechanisms of CD40 that are involved in these fundamental events in the basic immune response remain to be elucidated. In addition, antibodies specifically blocking the CD40L-CD40 interaction in mouse models can prevent organ rejection after transplantation and can be used to prevent autoimmune diseases such as rheumatoid arthritis, lupus nephritis and encephalomyelitis, as well as to reduce the risk of atherosclerosis. However, these blocking antibodies would also inhibit physiological functions of CD40 in normal immune responses. Our long-term goal is to use CD40 signaling pathways as molecular tools to dissect individual events in the T-cell-dependent humoral response, and to find a way to specifically inhibit CD40-associated diseases without affecting normal immune responses. In this proposal, we will take a genome-wide approach to systematically search for CD40-regulated genes using newly developed DNA microarray technology. We will: 1) investigate signaling and functional specificities between CD40L, LPS and TNF-a; 2) elucidate the molecular mechanisms by which multiple signal transduction pathways control a single CD40-mediated biological event; 3) identify NF-kB subunit specific genes responsible for CD40-mediated cell proliferation and isotype switching.

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to gain insight into the molecular mechanisms of cell apoptosis in response to microtubule disruption during cancer treatments. Microtubule disruption agents such as vinblastine, vincristine, and paclitaxel represent a growing family of anti-cancer drugs. They can effectively inhibit growth and induce apoptosis in a large variety of cancer cells, regardless of their p53 status. Little is known, however, about their mechanisms in inducing cell apoptosis. We have recently demonstrated, using a somatic knockout approach in DT40 cells, that MEKK1 is essential for JNK activation and cell apoptosis in response to microtubule or actin disruption. Our studies also indicated that this apoptotic process is independent of cell cycle arrest, but requires new protein synthesis. Through recent microarray analysis, we identified caspase 3 as a potential MEKK1-dependent apoptotic gene that is upregulated during microtubule disruption. In this proposal, we will test our hypothesis that cell apoptosis in response to microtubule or actin disruption proceeds through the MEKK1/JNK signal transduction pathway to upregulate proapoptotic genes such as caspase 3. Specific Aim 1 is focused on the molecular basis for the activation of the MEKK1. We will take structural and functional approaches, using MEKK1 -/- DT40 cells, to determine domains of MEKK1 required for JNK activation and apoptosis in response to microtubule and/or actin disruption, and the signaling specificity between MEKK1 and other MEKK family members. In Specific Aim 2, we will define the MEKK1-dependent apoptotic pathway by studying the role of SEK/JNK pathway and the upregulation of caspase 3 in microtubule disruption-induced apoptosis. In Specific Aim 3, we wilI determine the role of the MEKK1-dependent signaling pathway in the treatment of human cancer cells. We will measure the correlation between the drug sensitivity and the MEKK1 kinase activity, and determine whether it is possible to sensitize otherwise resistant human cancer cells to the treatment of microtubule disruption agents by increasing the activity of the MEKK1-dependent apoptotic pathway. We believe that our study will not only elucidate the molecular mechanisms of a large family of microtubule and actin disruption agents used in chemotherapy, but also will provide critical information for future design of more effective and specific anti-cancer drugs.

DESCRIPTION (provided by applicant): The long term goal of this application is to further elucidate the Toll-Like Receptor (TLR)-mediated signaling and gene expression network (gene program) in regulating Th17 cell differentiation and its associated autoimmune diseases. Upon recognizing pathogen-associated molecular patterns or host danger signals, host pattern recognition receptors such as the TLRs can initiate a series of signal transduction and gene expression cascades for host defense against invading pathogens and other stresses. The signaling specificity of individual TLRs, initiated through differential recruitment of adaptor molecules such as MyD88 and TRIF, can be further amplified by differential induction of cytokines, which are not only important for innate immune responses but also play critical roles in instructing the directions of adaptive immune responses, including different types of T-cells. Th17 cells are a subtype of CD4+T helper cells believed to be important for host defense against pathogen infections, whereas elevated Th17 responses can lead to many autoimmune and inflammatory diseases such as multiple sclerosis in human and its similar disease in mouse, Experimental Autoimmune Encephalomyelitis (EAE). Recent studies have identified IL-1, IL-6, TGF2, and IL-23 as the promoters and IL-27 and IL-10 as the suppressors for the differentiation of Th17 cells. However, the mechanisms responsible for regulating these cytokines during in vivo immune responses are still not fully understood. Our recent studies have shown that while MyD88-deficient mice failed to develop EAE, mice lacking either TRIF or type I interferon (IFN) receptor (IFNAR) have elevated Th17 cells in the central nervous system and develop much more severe EAE than wild type mice. We hypothesize that TRIF-mediated type I IFN induction plays critical roles in suppressing Th17-associated autoimmune and inflammatory diseases such as EAE. Based on our preliminary studies, we further hypothesize that the IFN2-mediated inhibition of Th17 cell differentiation through IL-27 induction as the mechanism by which IFN2 can successfully treat human multiple sclerosis. The goal of this proposal is to understand the mechanisms by which TLR-mediated gene programs regulate Th17 cell differentiation and modulate Th17-associated autoimmune and inflammatory diseases through activation of the type I interferon pathway. We will first define which TLR mediates MyD88- dependent pathways in promoting EAE and which TLR mediates TRIF-dependent pathways in suppressing EAE. We will also determine the contributions and the mechanisms of IL-27 induction in type I IFN-mediated negative regulation of Th17 differentiation. Finally, we will explore the possibilities of modulating this TRIF and IFN1/2-dependent gene program to treat Th17-mediated inflammatory diseases. Our proposed studies will not only elucidate the physiological role of endogenous type I IFNs in inhibiting the development of autoimmune disease, but will also provide insight to understand how IFN2 works in the treatment of Multiple Sclerosis and to help design additional strategies to treat autoimmune and inflammatory disease.

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to gain insight into the mechanisms of action of the receptor interacting protein 2 (RIP2) in host biodefense against Listeria monocytogenes infection. RIP2 is a member of the RIP family of serine/threonine (Ser/Thr) kinases. It has been implicated in the signal transduction pathways activated by the Nod receptor family proteins, potential receptors for intracellular pathogens. We recently created knockout mice lacking the RIP2 gene, and found that RIP2-/- mice are severely impaired in their ability to defend against infection with L. monocytogenes. Our preliminary results also indicated that RIP2-/- macrophages have lost their ability to respond to muramyl dipeptide (MDP), the minimal immunostimulatory subunit of peptidoglycan from gram positive bacteria. In addition, RIP2-/- T helper 1 (Thl) and natural killer (NK) cells have reduced interferon gamma (IFN-gamma) production upon IL-12 stimulation. We hypothesize that RIP2 may be involved in multiple signaling and cellular events to coordinate innate and adaptive immune responses in host biodefense against pathogen infection. We propose experiments to investigate RIP2- mediated signal transduction pathways and to determine the in vivo role of RIP2 in immune responses during pathogen infections. First, we hypothesize that RIP2 is involved in MDP-induced activation of innate immune responses. We will determine the role and the mechanism of RIP2 in mediating signal transduction and cytokine production by macrophages in response to MDP stimulation. Second, we hypothesize that RIP2 is involved in Thl differentiation by modulating the activity of IL-12-induced STAT4 activation and interferon gamma (IFN-gamma) production. We will first confirm the intrinsic defects of RIP2-/- Thl cells, and then explore potential signaling events where RIP2 might be involved in IL-12-induced STAT4 activation and interferon IFN-(, production. Third, we hypothesize that RIP2 is involved in host defense against microbial infections by affecting both innate and adaptive immune responses. We will determine the susceptibility of RIP2-/- mice to gram-positive and gram-negative extracellular and intracellular bacteria to understand the role of RIP2 in determining the pathogen specificity. We will also use L. monocytogenes infection of RIP2-/- mice as a model to determine the contribution of RIP2 in innate and adaptive immune responses against microbial infections. We believe that the insights obtained from these studies will provide new knowledge about pathogen recognition and coordination between innate and adaptive immune systems, and suggest new avenues of immunologic intervention to prevent and treat many human infectious diseases.

[unreadable] DESCRIPTION (provided by applicant): Since its discovery in 1957, type I interferon has been recognized as the major antiviral cytokine in vertebrates. To combat viral infections, most nucleated vertebrate cells are able to both produce and respond to type I interferon. The antiviral function of type I interferon is carried out through its binding to the type I interferon receptor (IFNAR1), activation of the JAK/STAT pathway and subsequent induction of a large set of target genes important in antiviral responses On the other hand, as a result of co-evolution, different viruses have developed various strategies to inhibit the ability of host cells to either produce or respond to type I interferons. To understand this paradox of host and pathogen interactions, we seek to determine how host cells can recognize different viruses and how this recognition event results in the production of type I interferons. We, together with other groups, have previously demonstrated that toll-like receptors (TLRs) can mediate type I interferon production and antiviral responses through activation of interferon regulatory factors, IRF3 and IRF7. While TLRs are able to recognize certain viral infections in innate immune cells such as macrophages and plasmacytoid dendritic cells (pDCs), additional intracellular receptors such as PKR and RIG-I are involved in type I interferon production in response to viral infections in non-immune cells. Our preliminary studies indicated that TNF receptor associated factor 3 (TRAF3) might be a critical modulator of type I interferon induction in both immune and non-immune cells during viral infection. The goal of this application is to further develop our functional as well as mechanistic understanding of the role of TRAF3 in host biodefense against infection by different types of viruses. In this research proposal, we will take advantage of TRAFS-deficient cells and mice to determine the antiviral functions of TRAF3, use biochemical approaches to dissect TRAFS-mediated signal transduction pathways, and also explore the contributions of the TRAFS-mediated pathways to interferon induction in response to infections with different types of viruses such as Influenza, Yellow Fever, Herpes Simplex, Ectromelia and Vesicular Stomatitis virus. We believe that our studies will provide significant insights into the mechanisms of type I interferon induction and potential novel therapeutic targets for improving our immune response against different types of viral infections. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to gain insight into the role and the mechanisms of action of the tumor necrosis factor (TNF) receptor-associated factors 2 and 3 (TRAF2 and TRAF3) and NF-KB inducing kinase (NIK) in the regulation of NF-KB activation and in suppression of inflammatory and autoimmune diseases. The NF-kB transcription factors, including p50, p52, p65, c-Rel and Rel-B, are critical regulators of inflammation, proliferation and apoptosis. While defective NF-KB activity can lead to cell apoptosis and immunodeficiency, overactivated NF-KB has been associated with numerous diseases such as cancers and a variety of inflammatory syndromes. Recently, the NF-KB signaling pathways have been categorized into the type 1 (classical or canonical) pathway, which activates the degradation of I:B1 and the release of active NF- :B complexes containing p50, and the type 2 (alternative or noncanonical) pathway, which involves the induced processing of p100 to p52 and the formation of NF-KB complexes containing p52. Our genetic studies have demonstrated that loss of TRAF3 results in constitutive type 2 NF-KB activity. The early post-natal lethality observed in TRAF3-deficient mice is rescued by compound loss of the type 2 NF-KB p100 gene. In addition, our preliminary studies indicate that TRAF3 is also a potent inhibitor of type 1 NF-KB activation in response to TNF1 and IL-12 stimulation. Lack of inhibition of both NF-KB pathways in TRAF3-/- cells correlated with constitutive stimulus-independent survival of B cells ex vivo and profound super-induction of inflammatory cytokines in vitro and in vivo. Recent studies indicated that another TRAF family member, TRAF2, is also a negative regulator of the type 2 NF-KB pathway. However, the molecular mechanisms responsible for TRAF2- and TRAF3-mediated negative regulation of type 2 NF-KB activation remain to be elucidated. Our recent studies showed that NIK is expressed at very low basal levels in unstimulated cells but is highly induced with a slow kinetics (8-12 hours to reach its maximum) after activation of receptors such as CD40, B-cell activating factor (BAFF) receptor (BAFF-R) and lymphotoxin 2 receptor (LT2R). Interestingly, bothTRAF2-/- and TRAF3-/- cells have high basal levels of NIK (equivalent to receptor activated wild type cells), which correlate with constitutively activated type 2 NF-KB. We hypothesize that TRAF2 and TRAF3 negatively regulate NF-KB by recruiting a NIK ubiquination complex to constantly degrade NIK and to keep basal levels of NF-KB activation low. We also hypothesize that receptor activation induces dissociation of TRAF2 and TRAF3 from the NIK ubiquination complex allowing NIK accumulation and subsequent type 2 NF-kB activation. In this grant proposal, we will determine: 1) how TRAF2 and TRAF3 recruit a NIK ubiquination complex to control ubiquitination and degradation of NIK at basal levels;2) how receptor activation leads to dissociation of such a ubiquination complex to allow NIK accumulation;3) how NIK kinase activity is regulated in order to induce type 2 NF-kB activation;and 4) how type 2 NF-kB can crosstalk with type 1 NF-kB and involve in inflammation. Project Narrative: Many diseases such as cancers and numerous inflammatory diseases are associated with overactivation of NF-kB transcription factors. The proposed studies will focus a critical negative regulator of NF-KB by examining how it functions as a gate keeper to prevent overactivation of NF-kB and how loss of such a gate keeper would lead to inflammatory responses and lethal phenotypes in animal models. Elucidating the molecular mechanisms responsible for inhibiting NF-kB activity will undoubtedly help in designing novel strategies to treat human diseases associated overactivation of NF-kB.

DESCRIPTION (provided by applicant): The long term goal of this application is to elucidate the mechanisms and biological significance of crosstalk between the innate immune response and nuclear hormone receptors in host defense against infections and in pathogen-associated metabolic diseases. Preliminary data in our lab has identified a novel Interferon Regulatory Factor 3(IRF3)-dependent but type I interferon independent pathway induced during innate immune response to viral product stimulation or viral infection, leading to strong transcriptional repression of Retinoid X Receptor 1 (RXR1). As RXR1 is the major heterodimer partner for most of the nuclear hormone receptors involved in numerous cellular metabolic processes, we hypothesize that repression of RXR1 and its regulated genes during viral infections can have strong impacts on host metabolisms especially drug metabolisms. We further suggest this can contribute to the pathogenesis of viral associated metabolic diseases such as Reye's Syndrome, a hepatotoxicity disease that occurs when children are given aspirin in the context of a viral infection, or acetaminophen (APAP)-induced hepatotoxicity, which accounts for about half of the acute liver failures in US. Interestingly, our preliminary studies indicate that innate immune responses to viral products can potentiate aspirin-induced but protect APAP-induced hepatotoxicity, which may explain why during viral infections patients are safer to use Tylenol than Aspirin. These results further suggest that, depending upon the nature of the drugs, viral infections may differentially affect the accumulation of the drug's intermediate metabolites, which could lead to opposite toxic effects on host tissues. In this application, we will first develop mouse models that mimic Reye's Syndrome and acetaminophen (APAP)-induced hepatotoxicity. We will then use these two opposite drug metabolism models as examples to determine how innate immune responses to viral infection could differentially alter stability or toxicity of different drugs. Furthermore, additional preliminary data in our lab showed that nuclear hormone receptor agonists can suppress the induction of antiviral genes and promote viral replications. We therefore also hypothesize that repression of nuclear hormone receptors by the innate immune response may contribute to an effective anti-viral response. We will further analyze the effects of nuclear hormone receptors and their agonists on anti-viral innate immune responses to determine if the repression of nuclear hormone receptors by innate immune response is necessary for the proper host defense against viral infections. We believe our investigation of the crosstalk between the innate immune response and nuclear hormone receptor-mediated metabolism will not only help us to understand the mechanisms responsible for virally induced metabolic diseases and drug induced immuno-suppressions but will also provide novel strategies to prevent or treat patients with viral infections and their associated metabolic diseases. PUBLIC HEALTH RELEVANCE: Numerous diseases including metabolic diseases are associated with virus infections. On the other hand, many metabolic products and drugs can also affect our body's ability to defend against invading viruses. However, the molecular mechanisms responsible for such associations are not clear. We recently found a novel crosstalk between host immune response and metabolisms, which could explain not only viral associated liver diseases such as Reye's Syndrome but also how metabolic drugs can inhibit host response against viral infections. Further investigation on such crosstalk will provide novel strategies to prevent or treat patients with viral infections and their associated metabolic diseases such as acetaminophen (APAP)-induced hepatotoxicity, which accounted for about half of all acute liver failures in America.

The increasing global energy demands and ensuing threat, be it accidental or intentional, of the release of nuclear material require a greater understanding of how to treat and mitigate radiation injury. While many studies describe the nature ofthe host response to radiation injury, established models provide very compelling evidence for a role for innate immunity in the process. Pathogen associated molecular patterns (PAMPs) released following gut injury stimulate tissue repair and host immune pathways. A recently described class of endogenous ligands released by injured cells, the damage associated molecular patterns (DAMPs), stimulate a similar group of innate immune receptors and so instigate a related program of tissue repair. These observations emphasize the point that very similar gene programs are involved in radiation repair and host immunity. The UCLA-CMCR has identified a number of different compounds and small molecules that are successful mitigators of radiation damage;the majority of which activate similar pathways as pathogens. From this comes the emerging understanding that a successful radiation mitigator suppresses excessive inflammation and supports robust regenerative gene programs leading to tissue repair. The optimal balance is exampled by lead compounds such as MIS416, an immune adjuvant that can successfully mediate crosstalk between Innate stimulation and radiation repair by regulating a number of signaling pathways. The same is true for other lead mitigators, such as IL-12, anti-inflammatory small molecules, or Tilorone, a type I interferon inducer. However, the molecular mechanisms responsible for mitigation remain unclear. In this application, we propose to discover which ofthe innate system pattern recognition receptors and which signal transduction pathways are required to support the mitigating activity of MIS416 and other UCLA-CMCR lead molecules. Furthermore, we will determine which genetic programs or cytokines contribute to the mitigating mechanism by inducing regeneration of hematopoietic stem cells. Thirdly, utilizing this increased understanding of the interaction between lead mitigators and innate immune regulatory systems, we shall develop a nanovesicle platform for radiation mitigation. Finally, we will examine a live vaccine model to explore the crosstalk between tissue repair mechanisms utilized in response to radiation injury and infection. Our proposed studies, by dissecting the receptors and pathways that mitigate radiation injury, will provide novel targets for therapeutic intervention and lead molecule verification. Our improved understanding ofthe similarities between responses activated by radiation and infection will drive design of additional novel strategies for intervention.

DESCRIPTION (provided by applicant): Dimeric NF-:B transcription factors play critical roles in a wide variety of immune processes. Cytosolically sequestered in resting cells, NF-:B dimers are released to initiate gene transcription through the action of two basic signaling pathways known as the canonical and non-canonical NF-:B signaling pathways. These two pathways lead to release of NF-:B dimers on vastly different time scales and are regulated distinctly. While canonical NF-:B signaling is activated within minutes by a large number of receptors on a wide variety of cell types, non-canonical NF-:B signaling is activated over hours by a select group of receptors such as BAFF, LT2R, CD40 and RANK on a limited number of cell types such as B-cells, fibroblasts, and macrophages. However, both of these pathways play critical, non-redundant roles in the generation and survival of B-cells, osteoclasts, and secondary lymphoid tissues. It is now appreciated that hyperactivity of both canonical and non-canonical NF-:B signaling can lead to a variety autoimmune and proliferative diseases including Systemic Lupus Erythematosus, Multiple Myeloma, and Diffuse B-cell Lymphoma. While multiple negative feedback mechanisms have been described in the case of canonical NF-:B signaling which serve to terminate signaling and prevent pathological hyperactivation, no negative feedback mechanisms have been described for the non- canonical NF-:B signaling pathway. Our preliminary studies have demonstrated that IKK1, activated by NIK, can induce not only p100 phosphorylation and processing but also phosphorylation and destabilization of NIK. We have further mapped the IKK1 phosphorylation sites in NIK and shown that disruption of IKK1-dependent NIK phosphorylation can significantly increase NIK levels after receptor activation. Based on these preliminary results, we hypothesize that while previously reported TRAF-cIAP complex is responsible for NIK degradation in unstimulated cells, this novel IKK1-dependent NIK phosphorylation and destabilization mechanism plays an important negative feedback role in regulating non-canonical NF-:B activity after receptor activation. The goal of this R21 grant is to unravel the molecular components and mechanisms by which this feedback occurs. We propose to first identify the additional molecular components functioning within NIK-IKK1 feedback complex, then determine how these components assemble within the stimulus-induced NIK degradative complex, and finally determine the role of this feedback mechanism in regulation of both canonical and non-canonical NF-:B signaling. Together, we believe these studies will significantly enhance our understanding of non-canonical NF- :B regulation. Given the pathological potential of this pathway's hyperactivity in causing autoimmune diseases and cancers, further understanding of the molecular factors, biochemical relationships, and functional roles of negative feedback within the pathway will assist in future attempts to pharmacologically intervene in the pathway's activity. PUBLIC HEALTH RELEVANCE: It is now appreciated that hyperactivity of the non-canonical NF-:B pathway can lead to a variety autoimmune diseases such as Systemic Lupus Erythematosus and cancers including Multiple Myeloma and Diffuse B-cell Lymphoma. The goal of this R21 grant is to unravel the molecular components and mechanisms responsible for a novel feedback control pathway within non-canonical NF-kB signaling which we have identified based on our recent exciting preliminary results. We believe further understanding of the molecular factors, biochemical relationships, and functional roles of negative feedback within the non-canonical NF- :B pathway will assist in future attempts to pharmacologically intervene in treating autoimmune diseases and cancers.

The goal of this research project is to understand the molecular mechanisms responsible for the opposing functions of Type I versus Type II interferon (IFN) in controlling the replication of and the pathogenesis associated with the intracellular pathogen Mycobacterium leprae (mLEP) in skin. The disease associated with mLEP varies in different patients with a large spectrum from self-limited, tuberculoid (T-lep) patients with expression of the Th1 cytokine IFN-gamma (Type II) in lesions to disseminated lepromatous (L-lep) patients with expression of immunosuppressive cytokines such as IL-10 in lesions. We were among the first groups made the initial discovery that, while Type II IFN is required for, Type I IFN (mainly IFN-alpha-s and IFN-Beta) plays a detrimental role in host defense against bacterial infections. However, the molecular mechanisms responsible for the effects of Type I and Type II IFNs on mLEP infection and disease development remain to be elucidated. Through close collaboration with Dr. Robert Modlin's group over the past ten years, it became clear that Type I IFN inducible genes are preferentially upregulated in L-lep lesions whereas Type II IFN inducible genes are preferentially upregulated in T-lep lesions. More importantly, we have identified a subset of Type II IFN inducible genes in T-lep lesions that have potential antimicrobial activities and a subset of Type I IFN inducible genes in L-lep lesions that may not only suppress immune responses but also promote pathogenesis of leprosy. We hypothesize that Type ll and Type I IFNs play opposite roles in controlling the mLEP infection and associated diseases by differentially inducing antimicrobial and immunosuppressive gene programs, respectively. We propose to : 1) identify the Type I vs. Type II IFN gene programs in leprosy lesions; 2) determine the anti-mLEP activities of type II interferon inducible genes upregulated in T-lep; 3) define type I IFN inducible gene programs in suppressing host immunity against mLEP and promoting pathogenesis. We believe our proposed studies will not only help us understand the molecular mechanisms responsible for the opposing functions of Type I and II IFN in controlling mLEP but also provide insight for novel therapeutic tools for skin and systemic inflammatory associated diseases.

The increasing global energy demands and ensuing threat, be it accidental or intentional, of the release of nuclear material require a greater understanding of how to treat and mitigate radiation injury. While many studies describe the nature ofthe host response to radiation injury, established models provide very compelling evidence for a role for innate immunity in the process. Pathogen associated molecular patterns (PAMPs) released following gut injury stimulate tissue repair and host immune pathways. A recently described class of endogenous ligands released by injured cells, the damage associated molecular patterns (DAMPs), stimulate a similar group of innate immune receptors and so instigate a related program of tissue repair. These observations emphasize the point that very similar gene programs are involved in radiation repair and host immunity. The UCLA-CMCR has identified a number of different compounds and small molecules that are successful mitigators of radiation damage; the majority of which activate similar pathways as pathogens. From this comes the emerging understanding that a successful radiation mitigator suppresses excessive inflammation and supports robust regenerative gene programs leading to tissue repair. The optimal balance is exampled by lead compounds such as MIS416, an immune adjuvant that can successfully mediate crosstalk between Innate stimulation and radiation repair by regulating a number of signaling pathways. The same is true for other lead mitigators, such as IL-12, anti-inflammatory small molecules, or Tilorone, a type I interferon inducer. However, the molecular mechanisms responsible for mitigation remain unclear. In this application, we propose to discover which of the innate system pattern recognition receptors and which signal transduction pathways are required to support the mitigating activity of MIS416 and other UCLA-CMCR lead molecules. Furthermore, we will determine which genetic programs or cytokines contribute to the mitigating mechanism by inducing regeneration of hematopoietic stem cells. Thirdly, utilizing this increased understanding of the interaction between lead mitigators and innate immune regulatory systems, we shall develop a nanovesicle platform for radiation mitigation. Finally, we will examine a live vaccine model to explore the crosstalk between tissue repair mechanisms utilized in response to radiation injury and infection. Our proposed studies, by dissecting the receptors and pathways that mitigate radiation injury, will provide novel targets for therapeutic intervention and lead molecule verification. Our improved understanding of the similarities between responses activated by radiation and infection will drive design of additional novel strategies for intervention.

PROJECT 1: Acute and Long Term Immune Responses to Radiation and Mitigation PROJECT SUMMARY The long-term goal of this application is to understand the role of immune responses in and the mechanisms responsible for the short and long term diseases caused by exposure to sources of ionizing radiation. One important aspect of radiation injury is the release of both endogenous damage associated molecular patterns (DAMPs) from the damaged tissues and pathogen associated molecular patterns (PAMPs) from the gastrointestinal system. These DAMPs and PAMPs released after irradiation interact with common pattern recognition receptors such as Toll-Like Receptors (TLR) and activate overlapping gene programs to regulate innate and adaptive immune responses as well as tissue damage and repair. In our previous studies, we found that MIS416 ? a particle based on the cell wall components of P. acnes decorated with a single stranded TLR9 ligand CpG-A (Vironyx Corp.) ? which likely contains multiple PAMPs, can function as a mitigator to rescue lethally irradiated mice. We have further evidence that different TLR agonists induce different profiles of growth factors, cytokines and chemokines, suggesting that multiple innate immune pathways might be required to mitigate radiation damage. Based on our results from multiple tissue damage models, we have recently hypothesized that over reactive innate immune responses to DAMPs and PAMPs released after irradiation can further trigger secondary tissue damages by proinflammatory cytokines and autoantibodies. We have further developed a widely available and easily deliverable DAMP blocking compound, glycyrrhizic acid (GA), as a potent radiation mitigator. In addition, while GCSF has been used as a standard mitigator, we have developed a bivalent GCSF (Bi-GCSF) as a more potent and stable radiation mitigator. Through close collaboration with Dr. William McBride's group, we have found that although many mitigators can effectively rescue lethally irradiated mice during acute radiation syndrome (ARS), many rescued mice often develop delayed effects of acute radiation exposure (DEARE) exhibiting chronic diseases such as heart and kidney failures around a year after WBI. Our preliminary studies indicate that these chronic diseases are associated with proinflammatory responses. Based on these studies, we hypothesize that balanced immune and inflammatory responses are critical not only for rescuing acute phase tissue damages but also for preventing chronic diseases caused by radiation. In this application, we will evaluate the effects of individual mitigators on the short and long term innate and adaptive immune systems and further develop a combination of mitigators that can effectively treat both acute and chronic diseases after radiation.

Abstract/Summary Our group of investigators has been collaborating over the last two years to describe the clinical aspects and pathogenesis of in utero transmission of ZIKA virus (ZIKV) infection. We have embarked in a strong collaborative effort to document and understand the pathogenesis of ZIKV. We have available to us a unique prospective cohort of well characterized ZIKV-infected mother-infant pairs who have been followed since the antenatal period, with infants now between 12 to 24 months of age and specimens collected over time. We propose to characterize ZIKV humoral immune responses over time in our cohort of mother-infant pairs who became infected during the Rio de Janeiro 2015/16 epidemic, in order to determine the timing of development of immune responses to ZIKV in perinatally infected infants, and possible correlation with intermittent viral shedding. We plan to evaluate whether infant immune responses are associated with the breadth and potency of maternal immunologic responses to ZIKV over time. To do so we will measure neutralizing antibody activity in 100 mother-infant pairs over 3 years and explore potential associations with annual infant neurodevelopmental assessments and timing of infection in gestation. We will also investigate genetic viral evolution in their ZIKV isolates and are presently sequencing whole ZIKV genome from mother-infant pairs from the same cohort who had adverse pregnancy outcomes, normal pregnancy outcomes, recurrent or relapsing infections, while also checking whether there is variability in viral sequences by compartment (CSF, blood, urine, placenta). We are also evaluating specific ZIKV-immune responses at the cellular and molecular levels and determining mechanisms by which the virus evades host immune responses during pregnancy. Our findings will answer important questions pertaining to the mechanisms of immune pathogenesis and intrinsic virologic factors associated with ZIKV mother-to-child transmission which may be predictive of longer term infant outcomes. Our well characterized population of mother-infant pairs with detailed clinical follow-up and specimens collected over time allows us to perform state of the art translational studies to elucidate mechanisms of viral pathogenesis.

ABSTRACT The goals of this proposal are to determine the type I interferon (IFN-I)-mediated antiviral gene program against SARS-CoV-2 infection and to develop novel broad-spectrum antiviral agents (BSAAs) for the treatment of COVID-19 and other emerging infectious diseases. There are no effective therapeutic agents currently available in the fight against the global COVID-19 pandemic, in which SARS-CoV-2 has infected millions of people in confirmed cases and caused hundreds of thousands of fatalities. Drugs that target a single virus, like the inhibitors of HIV reverse transcriptase and influenza neuraminidase, require a comprehensive understanding of the lifecycle and disease mechanisms of the virus, which makes development of these drugs necessarily time-consuming. Outbreaks of infection caused by novel emerging highly pathogenic viruses, including avian influenza, SARS, Ebola, Zika virus (ZIKV) and SARS-CoV-2, have become a major concern in the past two decades. We cannot rely on the traditional virus-specific drugs to treat diseases caused by these unpredictable emerging viruses. Therefore, it is extremely important to develop BSAAs effective against a range of viruses. My laboratory has been studying anti-viral innate immune responses, particularly the IFN-I signaling pathway and its downstream gene program, for the last 20 years. While the field has previously focused only on interferon-stimulated genes (ISGs), we have demonstrated that ISGs like cholesterol 25- hydroxylase (CH25H) and IFN-I downregulated genes like fatty acid synthase (FASN) both play important roles in limiting viral infection and replication. We have also identified multiple small molecules for use as BSAAs, including 25-hydroxycholersterol (25HC), the metabolic product of CH25H, and the FASN inhibitor C75. In addition, we have extensively studied host innate immune responses to coronaviral infection: we have published multiple papers that explain how the host IFN-I signal transduction pathway is activated in response to infection by coronaviruses like murine hepatitis virus (MHV) and how coronaviruses can suppress their host?s innate immune responses through the viral papain-like protease (PLpro). Most importantly, in our preliminary studies we found that 25HC and C75 both have strong inhibitory effects against SARS-CoV-2 infection. We hypothesize that the IFN-I-mediated antiviral gene program involves not only upregulation of antiviral ISGs but also downregulation of the host genes required for viral infection and replication. We further hypothesize that by identifying the IFN-I-mediated antiviral gene program against SARS-CoV-2, we will be able to develop novel antiviral agents to combat COVID-19 and other emerging threats. In this proposal, we will first determine the IFN-I gene program in innate immune response to SARS-CoV-2. We will also develop 25HC, 25HC analogs and FASN inhibitors as novel antiviral agents against SARS-CoV-2. We believe our studies will not only determine the IFN-I-mediated antiviral gene program in host innate immune response to SARS-CoV-2 infection but also develop BSAAs to treat COVID-19 and other emerging infectious diseases.