bluuebunny/crossref_metadata_2025 huggingface dataset:
Intratumoral CD56bright Natural Killer cells are associated with improved survival in bladder cancer
Authors: Mukherjee Neelam , Ji Niannian , Tomasini Maggie E. , Hurez Vincent , Curiel Tyler J. , Montgomery Maureen , Braun Andrew J. , Nicolas Marlo , Aguilera Marcela , Liu Qianqian , Ruan Jianhua , Svatek Robert S.
Published: January 2023

Antitumor immune responses are largely mediated by cytotoxic lymphocytes that recognize tumor-associated antigens presented in the context of a major histocompatibility complex class I molecule (MHC I). However, many tumors downregulate MHC I expression as a means of immune escape. Under these conditions, additional effector lymphocytes, such as natural killer cells (NK cells) which mediate non-MHC restricted cytotoxicity, provide antitumor defenses. Using flow cytometric analysis, we discovered that NK cells predominate among lymphocytes infiltrating human and carcinogen-induced mouse bladder cancer (BC). Using transcriptomics and clinical outcomes from the TCGA dataset we found that the presence of intratumoral NK cells is associated with increased survival after cystectomy, especially in low MHC class I expressing tumors. We validated this finding using a local BC cohort and determined that functional NK cells decline with increasing patient age. Using in vitro assays, we found that intratumoral NK cells from bladder tumors exhibit MHC class I dependent cytotoxicity against BC cells. Importantly, intratumoral CD56dim NK cells increase with higher pathologic stage and are less functional (cytotoxicity and cytokine secretion) than CD56bright counterparts. The presence of intratumoral CD56brightNK cells predicts improved patient survival independent of tumor stage. We conclude that intratumoral NK cells are functional and prognostically relevant in BC, but their presence and function is diminished with aging or tumor MHC class I. Intratumoral CD56bright NK cells are associated with better clinical outcomes whereas CD56dim NK cells are dysfunctional and associated with advanced BC.
Nanoemulsion adjuvant augments retinaldehyde dehydrogenase activity in dendritic cells and regulates expression of gut homing receptors on T cells
Authors: Farazuddin Mohammad , Goel Rishi R. , Landers Jeffery J. , Kline Nicholas J. , Toufaily Nour , Uppula Pradyumna , O’Konek Jessica J. , Baker James R.
Published: January 2023

Mucosal surfaces are the primary point of entry for most infectious agents. Therefore, induction of mucosal immune responses serves as the primary defense by preventing and clearing pathogens. In order to mount an effective immune response, secretory IgA and T cell homing towards mucosal surfaces are two very important factors. Conventional vaccines induce strong systemic immunity, but often fail to produce mucosal immune responses. Our group has developed an oil-in-water nanoemulsion which has been shown to provide protection against mucosal pathogens when administered as an intranasal vaccine adjuvant. In the present study, we demonstrate that intranasal immunization with nanoemulsion indirectly activates the retinaldehyde dehydrogenase in dendritic cells, leading to an increase in secretory IgA production as well as expression of α4β7 and CCR9 gut homing receptors on the mesenteric lymph node cells. Ex vivo stimulation of splenocytes from NE immunized animals established increase in Th1/Th17 cytokines while suppressing Th2, suggesting RALDH increase does not suppress Th1/Th17 response. Our nanoemulsion based adjuvant activates retinaldehyde dehydrogenase without possessing any active ligand and imprints T cells for gut homing without altering systemic immunity. These results provide new insights into the mechanism of our nanoemulsion adjuvant and have significant implications for the design of effective mucosal vaccines.
HIV vaccine mimicking the RV144 regimen tightly controls and ablates SHIV BaL.P4 infection in rhesus macaques
Authors: Hessell Ann Jones , Pandey Shilpi , Sutton William F. , Liu Lily , Kong Xiang-Peng , Haigwood Nancy Logan , Gorny Miroslaw K.
Published: January 2023

The RV144 human HIV-1 vaccine trial provided the first and only immunologic signal to define a correlate of reduced risk. This sole independent correlate of reduced risk of HIV infection among the >150 variables studied, was the level of antibodies (Abs) reactive with the V1V2 region of HIV-1 Envelope protein. We designed vaccine/challenge experiments designed to mimic the RV144 vaccine trial to study the protective effects of vaccine-elicited immune responses in rhesus macaques (RM). Nine RM were co-immunized at weeks 0, 4, 12 and 20 intradermally with gp160 92TH023 and SIV gag DNA via gene gun together with gp120 A244 and MN proteins formulated in Adjuplex. Two weeks after the final immunization, all RM and naïve controls were challenged intrarectally with SHIVBaL.P4. Blood and mucosal secretions were collected weekly. Plasma viral load (PVL) and PBMC-associated VL were significantly different between controls and immunized RM (p=0.0091, p=0.0045, respectively). Time to infection was greater in immunized NHP (p=0.0235, Kaplan Meier). PVL in 5 RM were either undetectable or transient at 102–103 copies/ml. Midpoint titers of anti-V2 plasma binding antibodies (Abs) were ~1 Log10 lower compared to titers against cognate and heterologous gp140 and gp120. The vaccine regimen induced strong Ab responses with neutralizing activity that inversely correlated with VL to tightly control or ablate SHIV infection in 5 of the 9 immunized macaques. At necropsy no virus was detectable in lymphoid tissues. Efficacy based on this outcome is 55%, representing a milestone in pre-clinical vaccine protection. This study provides a strong basis to investigate mechanisms associated with tight viremia control, including antibody Fc-mediated activities.
Poxvirus subversion of B7-mediated T cell co-stimulation
Authors: Elliott Jabari I. , Wang Xiaoli , Piersma Sytse J. , Nelson Christopher A. , Yokoyama Wayne M. , Fremont Daved H.
Published: January 2023

Poxviruses encode a broad array of proteins that serve to undermine host immune defenses. Our group has been investigating a large sequence-diverse family of secreted poxvirus proteins that appear to share a conserved beta-sandwich fold, but differ in their binding specificities. Host proteins discovered to interact with this viral protein family include: chemokines, GM-CSF, IL-2, MHC class I, and GAGs. We have termed members of this superfamily; Poxvirus Immune Evasion (PIE) proteins, and there appears to be at least 20 distinct subfamilies. As it turns out, cowpox virus (CPXV BR) contains 10 PIE proteins, one of which is secreted from virally infected cells and specifically binds B7.1 (CD80) and B7.2 (CD86), two membrane proteins expressed on professional antigen presenting cells and activated T cells. We have termed this viral protein B7 Response Modifier (BRM) and demonstrated that it competes with the CD28 as well as CTLA4 receptors expressed on T-cells for binding to B7 proteins. However, at low concentration, BRM competes primarily with CD28 for binding to B7.2, consistent with the idea that cowpox uses BRM to undermine T cell activation but not checkpoint control. Quantitative interaction analysis reveals that BRM binds the ectodomain of B7 proteins with higher affinity than that reported for CD28 or CTLA-4. Functionally, BRM can potently disrupt T-cell proliferation and IL2 production co-stimulated by B7 proteins. Furthermore, supernatants of wild-type but not BRM deleted CPXV are capable of suppressing B7.2-mediated T-cell activation. In sum, our findings define a novel mechanism of viral immune evasion that highlights the role of CD28 co-stimulation in host defense of poxvirus infections.
Immunoprotective γδ T cell subsets in the cornea during early HSV-1 infection.
Authors: Fitzpatrick Steffani E. , Lausch Robert , Barrington Robert A.
Published: January 2023

Herpes Simplex Virus-1 (HSV-1) is the leading cause of infectious blindness in the U.S.A., with an annual incidence of new ocular infections of 11.8 per 100,000. Both mice and humans develop similar pathology after intracorneal (i.c) HSV-1 infection, making mice a useful model to understand underlying mechanisms of disease. Mice lacking γδ T cells exhibit reduced Interleukin 17A (IL-17A), increased virus titers and develop lethal encephalitis after i.c. infection. Therefore γδ T cells are required for immunoprotection against i.c. HSV-1. To understand the diversity of the γδ T cell response to corneal infection, we examined corneal γδ T cell subpopulations based on C-C chemokine receptor 6 (CCR6) levels, IL-17A production, and the presence of activation markers, CD44 and CD62L. Both CCR6+ and CCR6neg γδ T cells were observed in the cornea. The number of CCR6+ γδ T cells increased within the first 48 hours post infection (p.i.). In addition, the CCR6+ γδ T cell subset were the predominant producers of IL-17A and possessed an activated cell phenotype (CD44hiCD62Lhi), consistent with central memory T cells.

To test whether the CCR6+ γδ T cell population is responsible for immunoprotection in the cornea, we measured corneal pathogenesis in HSV-1 infected CCR6 deficient (CCR6−/−) mice. CCR6−/− mice had significantly higher corneal pathology scores at days 3 and 7 p.i. compared to wild-type mice after HSV-1 i.c. These data suggest, that CCR6+ γδ T cells produce IL-17A and limit early corneal pathology.
Structural and Antigenic Features of Powassan Virus Envelope Protein
Authors: Errico John M. , VanBlargan Laura A. , Nelson Christopher A. , Diamond Michael S. , Fremont Daved H.
Published: January 2023

Flaviviruses, such as West-Nile virus and Dengue virus, are insect-transmitted positive-sense RNA viruses that cause substantial morbidity and mortality worldwide. Powassan virus is an emerging encephalitic tick-borne flavivirus endemic to the northern United States, and is currently the only tick-transmitted flavivirus known to infect humans in North America. In cases of severe neurological disease, up to 10% of patients die of Powassan encephalitis, while many survivors are left with long-term neurological sequelae. Despite this, no vaccines or therapeutics are currently available to treat Powassan virus infection. Flaviviral vaccination efforts are commonly foiled by an inability to generate potent neutralizing responses, and by genesis of cross-reactive antibodies that paradoxically enhance infection by heterologous flaviviruses. These neutralizing antibodies, and their cross-reactive counterparts, commonly target structurally homologous epitopes present on many flaviviruses. Thus, understanding these epitopes has important implications for vaccine design. Little is known about the structure of Powassan virus envelope protein or host antibodies that target it. We have recombinantly expressed Powassan virus envelope protein and are currently working to determine its structure. As well, a panel of Powassan-virus specific monoclonal antibodies has been developed. Utilizing our recombinant protein, we are characterizing the epitopes these antibodies utilize as well as their biochemical and functional properties. These findings will aid vaccine and therapeutic design for Powassan virus and expand our understanding of the quintessential antigenic and structural features of flaviviral envelope proteins.
An in vitro/ex vivo human lung infection model for studying the macrophage innate immune response to Ebola virus Kikwit
Authors: Josleyn Nicole M. , Kuehne Ana , O’Brien Cecilia , Brannan Jennifer , Edwards Darin , Parkhill Robert , Dye John M.
Published: January 2023

Ebola virus causes a severe and often fatal hemorrhagic fever in people, for which no approved vaccines or therapeutics are available. Disease pathogenesis and countermeasure studies are typically performed using animal models; however, ex vivo models recapitulating human immune responses may provide valuable information. We optimized engineered lung tissue constructs comprised of human lung epithelial cells and human endothelial cells that mimic the vascularized lower airway alveolar tissue micro-environment to study the human innate immune response to Ebola virus Kikwit (EBOV/Kik). Human lung tissue macrophages are difficult to obtain due to practical and ethical considerations, so macrophages were chemically derived from human monocytes. We inoculated the constructs, in the presence and absence of monocyte derived macrophages (MDM), and quantified cytokines/chemokines secreted on days 0, 3, 6, 9, and 15 post-infection using 29-plex and 1-plex human cytokine/chemokine kits. Prior to infection, MDM were secreting IL-13, MCP-1, MIP-1a, MIP-1b; however, these levels rapidly declined by day 3 post-infection (pi). The MDM secreted peak levels of IL-4, IFN-a2, Eotaxin, IL-12P40, and IFNg at day 6pi, and IL-1ra peaked at day 9 pi. In response to EBOV/Kik, the human lung epithelial cells in the engineered lung tissue construct secreted G-CSF, IL-1ra, IL-8, IP-10, and PDGF-AA/BB. G-CSF and IL-8, cytokines known to mobilize hematopoietic progenitors from bone marrow to blood and recruit neutrophils to sites of infection, were abundantly secreted by both endothelial cells and macrophages in response to infection. This model system may be a valuable resource to the field to study human immune responses during acute infections.
Antibodies that bind to an EBV derived peptide mimic of dsDNA, have pathogenic potential
Authors: Spatz Linda A. , Sarbaugh Dylan , Singh Divya , Kryvda Olga , Oudit Omar
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of antibodies to double stranded DNA (dsDNA) that can deposit in the kidney leading to nephritis. Our laboratory has previously been studying the association of SLE with the Epstein Barr Virus (EBV). We generated two monoclonal antibodies designated 3D4 and 16D2, that bind to a peptide (PFM-15) in the carboxyl region of a major nuclear protein of EBV (EBNA-1) and cross-react with dsDNA. In the present study, we demonstrate that both of these monoclonal antibodies can bind to fixed preparations of rat glomeruli suggesting pathogenic potential. Immunostaining is dramatically reduced when glomeruli are pre-treated with DNase indicating that these antibodies recognize chromatin associated structures. In addition, the PFM-15 peptide inhibits 3D4 and 16D2 from binding to glomeruli and inhibits both antibodies from binding to dsDNA coated plates by ELISA. This suggests that PFM-15 can act as a molecular mimic of dsDNA. Our results demonstrate that antibodies that recognize a viral peptide and cross-react with dsDNA can bind to glomeruli and therefore have pathogenic potential. This study further supports an association between EBV and SLE and supports the role of molecular mimicry in autoimmune disease.
Exposure to the Epstein-Barr viral antigen induces affinity-maturated anti-myelin antibodies.
Authors: Lomakin Yakov Anatol’evich , Belogurov Alexey
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune chronic inflammatory disease of central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered as one of the possible mechanisms of MS triggering. Previously we showed that monoclonal myelin basic protein (MBP)-specific IgGs are cross-reactive with Epstein-Barr virus (EBV) protein LMP-1. Here we report evidence that exposure of mice to LMP1 may result in induction of myelin-reactive autoantibodies in vivo. Only part of such anti-MBP antibodies are cross-reactive towards LMP1 but majority is occurred as a result of epitope spreading. We suggest that chronic contact with viral antigen rather than multiple rapid exposures to it is more sufficient in inducing switch of B cells from viral to myelin antigen. Moreover, even in inbred animals being almost identical in terms of genome, such switch was observed only in 20% of animals, indicating that this evidence is occurred by a chance rather than systematically. Our findings provide novel insights into still enigmatic link between EBV infection and MS development, determining several criteria that are beneficial for induction of self-reacting antibodies on the background of viral infection.

Current study was supported by Russian Science Foundation grant #17-74-30019.
Plasticity and functionality of Th17 subsets in EAE
Authors: Butcher Matthew J. , Zhu Jinfang (Jeff)
Published: January 2023

Several studies have suggested that IFNγ+IL-17A+ (or Tbet+RORγt+) Th17-like cells may be the key players in Experimental Autoimmune Encephalomyelitis (EAE). However, direct evidence demonstrating the importance and functionality of Tbet+RORγt+cells is lacking. Therefore, we examined the generation and stability of Th17 subsets during EAE, and assessed the contributions of RORγt and T-bet to the functionality of these cells. Using Tbx21ZsGreenRorcE2CrimsonFoxp3Rfp/y reporter mice, we were able to isolate T-bet and RORγt single and dual expressors after EAE induction. In a passive EAE model, we observed that donor RORγt-expressing Th17 cells elicited EAE passively and either maintained their phenotype, expressed T-bet, or became non-canonical Th1 cells (expressing T-bet only) in the recipients’ CNS. By contrast, T-bet and RORγt dual expressors largely assumed a non-cannonical Th1 cell fate; while donor Tbet+RORγt− cells maintained their phenotype and failed to elicit EAE. To assess if sustained RORγt expression in Tbet+ Th17 cells is required for the pathogenesis of EAE, we immunized Tbx21CreRorcFl/Fl and RorcFl/Fl mice. Interestingly, Tbet-dependent Rorc knockout mice did not develop EAE symptoms, and Tbet+ Th17 cells were absent in the CNS and dLNs. To examine the importance of RORgt downregulation by T-bet, we generated T-bet and Runx motif mutations within the Rorc promoter via Crispr/Cas9. RorcΔTbet/Runx mice similarly failed to develop EAE symptoms. Together these data suggest that Th17 cell subsets are functionally plastic and that the balance between T-bet and RORγt is required for the functionality of Th17/Th1 cells in EAE.
Interleukin 27 signaling in T cells is essential for the development of type 1 diabetes
Authors: Ciecko Ashley E. , Foda Bardees M. , Mueller Kevin W. , Geurts Aron M. , Chen Yi-Guang
Published: January 2023

Human genome wide association studies have identified a 0.73 Mb region on chromosome 16 significantly linked to the development of type 1 diabetes (T1D) and IL27 is proposed to be the causal gene. IL27 encodes the p28 subunit of the heterodimeric cytokine interleukin 27 (IL-27). Human eQTL studies suggest a disease promoting role for IL-27 in the pathogenesis of T1D. However, the function of IL-27 in T1D has not been completely defined. The focus of this study was to test the role of IL-27 in the progression of T1D using the NOD mouse model. We generated NOD mice deficient in IL-27 or IL-27 receptor (IL-27Rα). In sharp contrast to wildtype NOD mice, both NOD.Il27−/− and NOD.Il27ra−/− strains were completely protected from T1D. Additionally, histological examination of pancreatic sections revealed that both NOD.Il27−/− and NOD.Il27ra−/− mice had significantly less insulitis compared to the wildtype NOD control. Furthermore, IL-27 production by myeloid antigen presenting cells was sufficient to drive T1D progression. Adoptive transfer of IL-27 deficient T cells into NOD.Rag1−/− recipients resulted in diabetes development indicating that IL-27 deficient mice still harbor diabetogenic T cells. However, NOD.Rag1−/− recipients of IL-27Rα deficient T cells did not develop diabetes demonstrating that direct IL-27 signaling is essential for their diabetogenic activity. We further demonstrated that direct IL-27 signaling promoted the accumulation of CD8 T cells and altered CD4 T cell subsets (reducing Foxp3+ but increasing T-BET+ frequency) in pancreatic islets. Our observations reveal that IL-27 signaling in T cells is required for diabetes development in the NOD mouse and provide additional evidence to support its potential role in human T1D.
Understanding the role of STAT4 in experimental autoimmune encephalomyelitis
Authors: Anderson Ashlyn E. , Shin Boyoung , McWilliams Ian , Harrington Laurie E.
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune disease that affects over two million people worldwide. MS is characterized by the demyelination of axons in the central nervous system (CNS), leading to vision problems, muscle weakness and poor coordination. Among the various immune cells that contribute to the disease, a subset of CD4 T cells, Th17 cells has been associated with MS pathogenesis. Importantly, IL-23 signaling in Th17 cells is essential for the pathogenicity of these cells. A transcription factor downstream of IL-23 signaling, STAT4, is dispensable for in vivo and in vitro Th17 development, however STAT4 is crucial for the development of CNS inflammation and polymorphisms in STAT4 are linked to MS susceptibility. As STAT4 deficiency does not prevent Th17 differentiation, but still limits inflammation, we hypothesize that STAT4 is necessary for the pathogenic properties of Th17 cells in MS. Using the adoptive transfer model of experimental autoimmune encephalomyelitis (EAE), we find that STAT4 expression by Th17 cells is necessary to induce CNS inflammation. Furthermore, global gene expression analysis indicates that STAT4 regulates the recently described pathogenic and nonpathogenic Th17 gene signatures. In the absence of STAT4, the levels of pathogenic Th17 genes including Tbx21, Il22 and Cxcl3 are significantly reduced, while the expression of nonpathogenic genes including Il10 and Ahr is increased. Interestingly, we find that STAT4 influences Th17 gene expression regardless of the differentiation conditions. Together, these data reveal a novel role of STAT4 in controlling Th17 pathogenicity, which may provide a promising therapeutic target for MS patients.
Non-coding RNA regulation of immunity
Authors: Schaefer Jeremy , Schnupp Trevor
Published: January 2023

Non-coding RNAs (ncRNAs) comprise a diverse range of biologically functional RNA species that include long ncRNAs (lncRNA) and short ncRNAs such as piwi-interacting RNAs (piRNA) and microRNAs (miRNA). These ncRNAs are capable of regulating gene expression via distinct mechanisms depending on their structure---miRNAs bind specific messenger RNAs (mRNA) to block translation while lncRNAs tend to bind chromatin to modify chromatin states to interfere with gene expression. NcRNAs have been identified in numerous diseases as having altered expression. To further elucidate the role of ncRNAs in immunity, we conducted a screen of lncRNA expression in the interleukin-10 knockout mouse (IL-10−/−) model of intestinal inflammation. This screen identified several candidate lncRNAs with dysregulated expression. Importantly, our data demonstrate that Malat1 and Hoxa11as expression was significantly decreased in mice with severe intestinal pathology. These results suggest that an association exists between these lncRNAs and the progression of disease in the IL-10−/− mouse model of colitis.
IL-1α facilitates the entry of immune cells into the brain during chronic Toxoplasma gondii infection
Authors: Batista Samantha , O’Brien Carleigh , Harris Tajie H.
Published: January 2023

Toxoplasma gondii is a parasite that causes chronic infections in the brain. Infection is asymptomatic in healthy hosts, but immunocompromised hosts experience severe disease in which fast-replicating parasites destroy brain tissue. Constant immune pressure is required to maintain control of the parasite, leading to the localized recruitment of immune cells. During chronic T. gondii infection, cell death occurs in focal areas of inflammation in the brain, suggesting the possibility of inflammatory molecule release. The alarmin IL-1α is expressed in the brain, and is upregulated by T. gondii infection. IL-1α is expressed by brain-resident microglia and infiltrating macrophages, implicating these cell types as sources of this cytokine. Mice lacking IL-1α or its receptor (IL-1R1), but not IL-1β, have defects in macrophage infiltration into the brain during chronic infection, as well as increased parasite burden in the brain. IL-1α has been the subject of less investigation than IL-1β, but may still rely on inflammatory cell death for its release. Indeed, this IL-1α-dependent immune response appears to be dependent on pyroptosis, as caspase-1/11−/− mice phenocopy IL-1R1−/− mice displaying decreased recruitment of myeloid cells into the brain and increased parasite burden. IL-1R1 is expressed on endothelial and other stromal cells in the brain vasculature, and IL-1R1−/− mice display decreased expression of the adhesion molecules ICAM-1 and VCAM-1 in the brain. These data suggest that during chronic T. gondii infection, IL-1α released from a macrophage population in the brain through pyroptosis acts on brain vasculature to modulate expression of adhesion molecules, facilitating the infiltration of monocytes to sites of infection.
Cox10-deficient NK cells have increased apoptosis during MCMV infection, leading to susceptibility
Authors: Mah Annelise Y. , Keppel Molly P. , Saucier Nermina , Sexl Veronika , French Anthony R. , Cooper Megan A.
Published: January 2023

Studies in diverse cell types have rediscovered the importance of basic cellular metabolism in regulating immune responses. For example, glycolytic and oxidative metabolism are counter-regulated in T cells to develop inflammatory effectors vs. memory and regulatory cells, respectively. NK cells, which are first responders to viral infection and malignant transformation, may be similarly regulated. We recently showed that NK cell cytotoxic activity during infection with murine cytomegalovirus (MCMV) required glucose metabolism, whereas other NK cell responses such as cytokine production did not. We hypothesized that oxidative metabolism would be required for some of these glycolysis-independent responses. To test this, we examined responses to MCMV in mice with an NK-specific deletion of Cox10, which is required for assembly of electron transport chain complex IV. These “Ncr1-Cox10” mice had normal NK cell development and numbers, and stimulated Cox10-deficient NK cells survived and proliferated normally in vitro. However, Ncr1-Cox10 mice were more susceptible to MCMV infection, with impaired expansion of MCMV-specific Ly49H+ NK cells and increased apoptosis of splenic NK cells. Thus, we find that Cox10 is required by NK cells to expand and survive in response to MCMV infection. These deficiencies were not found in vitro, emphasizing the importance of using NK-specific genetic models to investigate metabolism in physiological contexts.
γδ T cells orchestrate the induction of protective Type 2 immunity in neonatal influenza infection
Authors: Guo Xizhi , Thomas Paul G.
Published: January 2023

Influenza is a cause of hospitalization and mortality in infants. γδ T cells are the first T cells to develop during embryogenesis and are a significant component of the neonatal immune system. In the neonatal period, conventional αβ T cell responses are functionally less prominent. γδ T cells play critical regulatory and effector roles, but their relevance in neonatal influenza remains to be investigated. In our neonatal infection model, we found γδ T cells were protective against mortality associated with neonatal influenza infection. Infection induced the accumulation of γδ T cells, which transiently expressed IL-17a, rather than IFN-γ. Subsequently, this led to stronger Type 2 immune responses than the TCRδ-deficient animals, particularly in IL-33 production and amphiregulin-mediated tissue repair. Loss of γδ T cells did not alter viral clearance or conventional anti-viral responses. We also observed an association between IL-17a levels and clinical outcomes. Thus, our results identify a specific requirement for γδ T cells in influenza-infected neonates for initiating Type 2 immune responses, mediating tissue homeostasis, and promoting lung integrity, which may provide a new therapeutic target for pediatric influenza treatment.
NOD2-mediated downregulation of the TLR pathways contributes to protection against colitis and colorectal tumorigenesis
Authors: Nashir Udden S. M. , Peng Lan , Zaki Hasan
Published: January 2023

NOD-like receptor family member NOD2, which activates the NF-kB and MAPK signaling pathways in response to the bacterial cell wall component MDP, is the first identified inflammatory bowel disease (IBD) susceptibility gene. IBD is a risk factor for colorectal tumorigenesis. However, the role of NOD2 in colorectal tumorigenesis has not been studied adequately and the precise mechanism of NOD2-mediated protection against IBD is elusive. We studied the role of NOD2 in colorectal tumorigenesis using azoxymethane plus dextran sulfate sodium-induced colorectal tumorigenesis in mice. We found that Nod2-deficient mice are highly susceptible to colorectal tumorigenesis with increased tumor burden as compared to wild-type mice. Interestingly, increased tumorigenesis in Nod2−/− mice was independent of gut microbial composition. Increased tumorigenesis in Nod2-deficient mice was associated with higher inflammatory responses and activation of the inflammatory signaling pathways, such as NF-kB, ERK, and STAT3. Consistently, in vitro studies demonstrate that while NOD2 activates NF-kB and ERK in response to MDP, it inhibits TLR-mediated activation of NF-kB and ERK, and production of inflammatory mediators. Notably, NOD2-mediated downregulation of TLR signaling pathways is independent of MDP-mediated activation of NOD2. We further explored the underlying mechanism of NOD2-mediated downregulation of TLR signaling and revealed that NOD2 induces IRF4 which negatively regulates TLR-NF-kB signaling axis. Taken together, this study therefore shed light on a novel function of NOD2 which may be implicated in genetic association of NOD2 mutations with IBD and colorectal cancer.
Nucleic acid sensing in the cytoplasm of C2C12 cells directly after plasmid DNA electroporation
Authors: Semenova Nina P. , Znidar Katarina , Bosnjak Masa , Cemazar Maja , Heller Loree C.
Published: January 2023

Mounting evidence stresses the importance of nucleic acid sensing in the cytoplasm of living cells in a variety of physiological and pathological processes, including anti-pathogen defense, autoimmunity and development of malignant tumors. A subset of pattern-recognition receptors (PRRs) are nucleic acid sensors widely expressed in a variety of cells, but the detailed mechanisms of nucleic acid sensing have not been clarified. Our previous studies confirmed the cell type-specific upregulation of PRRs on both the mRNA and protein levels after electroporation of plasmid DNA (pDNA) into the cells. Type I interferon was found to be upregulated as well, which implicates innate immunity pathway activation in response to pDNA entry into the cytoplasm. Still, the sequence of events after pDNA electroporation remains unclear. Using molecular and proteomic techniques, we identified DAI/ZBP1 protein as the initial responding PRR to pDNA entering the cytoplasm after electroporation and estimated the participation of other PRRs in this process in mouse myoblast C2C12 cells. Better understanding of molecular determinants of nucleic acid sensing by PRRs may allow us to manipulate this process, developing new and improving existing therapies for immunity- and tumor-related diseases.
Defining how mitochondrial DNA stress regulates innate immune responses
Authors: Martinez Camila Guerra , Lei Yuanjiu , West Phillip
Published: January 2023

Polymerase gamma (pol γ) is nuclear-encoded protein responsible for mtDNA replication. POLG mutations are recognized as a significant cause of primary mitochondrial diseases and are implicated in a range of disorders. Several mouse models of POLG-related disease have been reported, the most well studied is the POLG-mutator mouse. Animals homozygous for the mutant alleles exhibit elevated mtDNA instability, and POLG-mutator mice exhibit pathology that mirrors various aspects of human POLG-related diseases. The literature has few descriptions about the immune system of POLG-mutator mice or patients with POLG-related disorders, despite the fact that aberrant immune responses have been implicated in the pathology of diseases with similar phenotypes. We have found that mitochondrial dysfunction, resulting from POLG mutation, alters innate immune responses at baseline and after challenge, which we hypothesize may exacerbate pathology in POLG-related mitochondrial disorders. To explore this hypothesis, we have stimulated macrophages from WT and POLG-mutator mice with various innate immune stimuli, and have uncovered that POLG-mutator macrophages display alterations in type I interferon responses, but strikingly, exhibit profound decreases in inflammasome-dependent interleukin-1 beta (IL-1b) secretion. Ongoing work will determine how mitochondrial dysfunction impairs inflammasome activation and IL-1b secretion in order to appreciate how aberrant innate immune responses influence POLG-related disease progression.
LRRK2 is required for macrophage homeostasis and the control of type I IFN
Authors: Weindel Chi Gidley , Bell Samantha L. , Vail Krystal , West A. Phillip , Patrick Kristin L. , Watson Robert O.
Published: January 2023

The use of genetic association studies has helped us appreciate that inflammation and immune dysfunction play an integral role in the pathology of multiple chronic disorders including cancer, type II diabetes, and neurodegenerative disease. We became interested in Leucine-rich repeat kinase 2 (LRRK2), a multifunctional kinase associated with Parkinson’s disease (PD), when it was revealed that LRRK2 polymorphisms are also associated with bacterial susceptibility to Mycobacterium leprae, a close relative of Mycobacterium tuberculosis, both of whose pathology relies heavily upon type I interferon (IFN), an innate immune response that activates effector cells. The regulation of type I IFN is critical to prevent a systemic inflammatory state and for the maintenance of healthy neurons. We therefore hypothesized that LRRK2 has a role in regulating innate immunity, contributing to both PD pathogenesis and bacterial infection. We found that in the absence of LRRK2 (Lrrk2−/−), macrophages failed to produce type I IFN in response to multiple cellular insults including, bacterial infection, mitochondrial damage, and DNA/RNA transfection. Interestingly the defect in type I IFN was a result of an imbalance at the level of the mitochondria, which altered macrophage homeostasis. These data suggest that LRRK2 is critical component of the innate immune system through its role maintaining mitochondrial health. The fact that LRRK2-deficient cells cannot induce type I IFN has major implications on both the maintenance of a proper cytokine milieu in the brain and management of the body’s immune response to infection.
Targeting of Diffuse Large B-cell Lymphomas using MyD88 small molecule inhibitors
Authors: Snyder Greg A. , Brown Lindsey J. , Taylor Savannah , Faupel Ciara E. , Sherman Matthew , Montague Justin J. , Saikh Kamal , Wang Yajing
Published: January 2023

A mutation associated with nearly 1/3 of human diffuse large B cell lymphomas (DLBCLs) has been identified within MyD88. This mutation correlates with tumor cell proliferation and survival involving spontaneous and sustained activation of NF-κB signaling. MyD88 is a central signaling adapter for the Interleukin-1 (IL-1R) and Toll-like receptors (TLRs). In normal healthy cells, MyD88 is thought to be held in an auto-inhibitory state with its own death and TIR domains fused together in negative self-regulation until activated by appropriate receptor mediated ligand engagement.

Observation

CADD derived small molecule compounds inhibit MyD88 dimer formation and protect against Staphylococcal enterotoxin B (SEB) induced death in animal models.

Hypothesis

Based on this observation we hypothesize that MyD88 specific small molecule inhibitors may be useful in treating DLBCLs bearing MyD88L265P.

Approach

Using in vitro and in vivo studies we characterize MyD88 specific SMIs for the ability to inhibit tumor cell proliferation and signaling in cancer cells bearing the oncogenic mutation MyD88L265P.

Results

1) MyD88 SMIs are able to inhibit cell proliferation of DLBCLs bearing the MyD88L265P as measured by MTS cell proliferation assay, 2) recombinant MyD88 and SMIs exhibit unique binding chromatograms in comparison to DMSO controls as measured by thermal shift assay and 3) MyD88 SMIs are partially able to inhibit LPS activated TLR4 cell NF-kB signaling in comparison to the TLR4 specific inhibitor TAK242. Future studies defining the molecular mechanism of this mutation with additional human patient tumor isolates will inform and propel development of novel therapeutics to counteract both inflammation as well as tumor formation.
CARD19: a mitochondrial caspase recruitment domain protein with a role in regulating pro-inflammatory innate immune responses
Authors: Rios Kariana Elizabeth , Liem Ayesha , Kashyap Anuj , Maynard Sean , Paul Suman , Schaefer Brian C.
Published: January 2023

CARD19 is a ubiquitously expressed protein of unknown function. This protein was originally named BinCARD, and early data suggested that it interacts with Bcl10 and inhibits NF-κB activation in T cells. However, these early data were based on overexpression studies, and we are aware of no in vivo data that support this proposed function. Additionally, the cellular localization of CARD19 remains uncharacterized. We sought to address these gaps in knowledge by examining macrophages, thymocytes, and splenocytes from Card19 +/+ and Card19 −/− mice using superresolution microscopy and flow cytometry; we also compared secretion of TNF-alpha, IL-6, and MCP-1 in Card19 +/+ and Card19 −/− mice injected with LPS. Superresolution microscopy revealed that CARD19 is localized to the outer mitochondrial membrane, with no evidence of co-localization with BCL10. Deletion of the predicted transmembrane domain resulted in nuclear and cytosolic distribution, while a partial deletion of the C-terminus resulted in localization in the endoplasmic reticulum. Data to date do not indicate significant differences between Card19 +/+ and Card19 −/− thymocytes and splenocytes in development, activation, or effector function. Interestingly, Card19 −/− mice displayed higher plasma concentrations of TNF-alpha, IL-6, and MCP-1 relative to Card19 +/+ mice in response to LPS. We conclude that CARD19 does not measurably impact BCL10 function in vivo, and we suggest that CARD19 is likely involved in regulation of a mitochondrial function that affects pro-inflammatory innate immune responses via a mechanism independent of BCL10.
Maternal immune activation induced by lipopolysaccharide triggers response inflammatory, oxidative damage and damage to blood brain barrier integrity and placental barrier in Wistar rats pregnant and fetus
Authors: Simões Lutiana Roque , Generoso Jaqueline S. , Sangiogo Gustavo , Faller Cristiano Julio , Réus Gislaine Zilli , Zugno Alexandra Ioppi , Petronilho Fabricia , Scaini Giselli , Giridharan Vijayasree Vayalanellore , Barichello Tatiana
Published: January 2023

Evidence suggest that prenatal immune system disturbance contributes largely to the pathophysiology of neuropsychiatric disorders. A bacterial infection animal model widely accepted is administration of lipopolysaccharide (LPS). We investigated if maternal immune activation (MIA) could induce inflammatory alterations, oxidative damage and damage to blood brain barrier integrity (BBB) and placental barrier (PB) in fetal brain and pregnant Wistar rats. On gestational day 15, Wistar pregnant rats received LPS (0.25 mg/kg) to induce MIA. After 6, 12 and 24 h, fetus brain, placenta, and amniotic fluid were collected to evaluate early effects of LPS. Other group of rats from postnatal day (PND) 54 received injection of ketamine at the doses of 5, 15, and 25 mg/kg. On PND 60 rats were subjected to the spontaneous locomotor activity and pre-pulse inhibition test (PPI). MIA increased oxidative stress in the amniotic fluid and fetal brain. The BBB integrity in the hippocampus and cerebral cortex as well integrity of PB in the placenta and fetus brain were dysregulated after LPS induction. We also found elevated levels of cytokines such as interleukin (IL)-1β, IL-2, IL-4, IL-5, interferon (IFN)-γ, macrophage colony stimulating factor (M-CSF), IL-6, IL-10, IL-18, macrophage inflammatory protein (MIP)-3a, tumor necrosis factor (TNF)-α-, IL-7, MIP-1a and erythropoietin. Rats that receive MIA plus ketamine had locomotor activity impairment and a deficit in the PPI. In the present study, it was concluded that MIA increased the permeability of BBB and PB, the circulation of inflammatory mediators in fetal brains. This immune response in the prenatal period may be responsible for the behavioral changes in the adult life of offspring induced by MIA.
RNA editing is required for innate immune homeostasis through inhibiting cytosolic RNA receptor MDA-5 activated by cellular endogenous RNAs
Authors: Wang Qingde , Li Xiaoni , Qi Ruofan , Zhang Wenshuo , Zheng Pengyuan , Billiar Timothy R.
Published: January 2023

Cytosolic RNA receptors, RLRs, play an essential role in antiviral infections that specifically detect the invaded viral RNAs leading to IFN and ISG expression and antiviral reactions through RNA sensing signaling pathways. This viral RNA sensing mechanism tolerate cellular “self” RNA in normal cells. However, how RLRs distinguish viral and self RNA molecules remains ambiguous. Our recent study showed that insufficient RNA editing by editing enzyme ADAR1 enables the cellular endogenous RNAs to stimulate the RLRs and activate the innate immune signaling pathway that lead to type I IFN and ISGs expression. Specific deletion of ADAR1 in the hepatocytes in a mouse model causes profound inflammation in the liver. As a result, the mortality rate is dramatically increased in the young animals. However, deletion of MDA-5 in these ADAR1 KO mice completely rescues the mice, and all the inflammatory phenotype was reversed. This finding indicated that RNA editing is necessary for the innate immune homeostasis, and MDA-5 plays a critical role in sensing endogenous self RNAs. Studies on this endogenous RNA sensing signaling pathway, its regulation and potential involvement in autoimmune and inflammatory diseases is warranted.
Differential signaling through TLR7 or TLR8 determines the phenotype of human monocytes during RNA virus infection
Authors: Dominguez-Villar Margarita , de Marcken Marine , Dhaliwal Khushwant
Published: January 2023

Despite being a major cell population in blood, and one of the major cellular targets of many RNA virus infections in peripheral blood, the molecular consequences of interactions between human monocytes and RNA viruses, and the signaling pathways responsible for the activation of these cells during infection are not well understood. Toll-like receptors (TLR) are a major family of pattern recognition sensors that trigger specific activation pathways in cells from both the innate and adaptive arms of the immune system. There are at least 10 TLRs in humans, from which TLR7 and TLR8 recognize single-stranded RNA. Despite both recognizing the same general ligand, we and others have demonstrated different phenotypic outcomes on cells stimulated through either TLR7 or TLR8. Our laboratory has recently observed fundamental differences in the phenotype and function of monocytes stimulated via either TLR7 or TLR8, in the context of RNA virus infections, and specifically, in terms of type I IFN responses and effector cytokines they produce, as well as differences in cell surface markers. We have defined the molecular mediators that are responsible for these differences in phenotype, performing ex vivo experiments with human monocytes isolated from blood and we have shown the relevance of these data in common RNA virus infections, demonstrating that TLR7 and/or TLR8 stimulation by RNA viruses in human monocytes accounts for much of the phenotype the cells acquire upon virus interaction.
Stingray venom activates IL-33 producing cardiomyocytes, but not mast cell, to promote acute neutrophil-mediated injury
Authors: Silva Carla Lima , dos Santos Janaina Cardoso , Grund Lidiane Zito , Seibert Carla Simone , Marques Elineide Eugenio , Soares Anderson Brito , Quesniaux Valerie F. , Ryffel Bernhard , Lopes-Ferreira Monica
Published: January 2023

One of the hallmarks of acute inflammation is neutrophil infiltration of tissues. We investigated molecular mechanisms implicated in acute neutrophilic inflammation induced by the venom of a freshwater stingray (Potamotrygon cf. henlei) in mice. Ray venom induced early mobilization of neutrophil in the microvasculature of cremaster mice and infiltration of the peritoneal cavity 2 hours after injury, in a dose-response manner. IL-1b, IL-6, TNF-a, and KC were produced. The neutrophilic infiltration did not occur in mice with ST2 receptor and MyD88 adapters neutralized, or in those with PI3K and p38 MAPK signaling blocked. Drastic reduction of neutrophil infiltration to peritoneal cavities was observed in ST2−/−, TLR2/TLR4−/−, MyD88−/−, TRIF−/− and IL-17A−/− mice, and a partial reduction was observed in IL-18R−/− mice. Mast cell Kit W(sh)/W(sh)-, AHR-, NLRP3-, ICE-, IL-1b-, P2RX7-, CD39-, IL-17RA-, and TBX21 KO mice retain the ability to induce neutrophilia in peritoneal cavity after ray venom injection. IL-6 and TNF-a alone were insufficient for promote neutrophilia in the absence of ST2 signaling. Finally, abundant production of IL-33 by cardiomyocytes was observed. These results refine our understanding of the importance of the IL-33/ST2 axis and IL-33-producing cardiomyocytes in the early acute neutrophilia induced by freshwater stingray venoms.
C-reactive protein is an atheroprotective molecule
Authors: Pathak Asmita , Singh Sanjay K. , Agrawal Alok
Published: January 2023

The co-localization of plasma C-reactive protein (CRP) and atherogenic low-density lipoprotein (LDL) at the atherosclerotic lesions raises the possibility of a role of CRP in the disease process. In vitro, in its native pentameric structural form, CRP does not bind to oxidized LDL. However, in its non-native pentameric structural form, CRP is capable of binding to oxidized LDL. It has been proposed that CRP changes its structure at sites of inflammation to gain the oxidized LDL-binding activity. In vivo, native CRP is neither pro-atherosclerotic nor atheroprotective in animal models of atherosclerosis. We assumed that native CRP shows no effect because inflammatory microenvironment in the arterial wall in animal models of atherosclerosis is not appropriate and, therefore, the structure of CRP remains unchanged. Accordingly, we hypothesized that a CRP mutant, generated by site-directed mutagenesis, capable of binding to oxidized LDL without the requirement of any further structural change should show an effect on the disease. In the current study, we evaluated the impact of such a CRP mutant on the development of atherosclerosis employing LDL receptor knockout mouse model of atherosclerosis. We found that there was a two-week delay in the development of atherosclerotic lesions in the aortic root of mice treated with mutant CRP compared to mice that did not receive CRP. The development of atherosclerotic lesions in the whole aorta was also reduced; there was 30% reduction in the size of lesions in mice treated with mutant CRP. Overall, the data indicate that CRP is indeed an atheroprotective molecule.