While Wild-type Research Articles

RIP1 inhibition protects retinal ganglion cells in glaucoma models of ocular injury

AbstractReceptor-interacting protein 1 (RIP1, RIPK1) is a critical mediator of multiple signaling pathways that promote inflammatory responses and cell death. The kinase activity of RIP1 contributes to the pathogenesis of a number of inflammatory and neurodegenerative diseases. However, the role of RIP1 in retinopathies remains unclear. This study demonstrates that RIP1 inhibition protects retinal ganglion cells (RGCs) in preclinical glaucoma models. Genetic inactivation of RIP1 improves RGC survival and preserves retinal function in the preclinical glaucoma models of optic nerve crush (ONC) and ischemia–reperfusion injury (IRI). In addition, the involvement of necroptosis in ONC and IRI glaucoma models was examined by utilizing RIP1 kinase-dead (RIP1-KD), RIP3 knockout (RIP3-KO), and MLKL knockout (MLKL-KO) mice. The number of RGCs, retinal thickness, and visual acuity were rescued in RIP1-kinase-dead (RIP1-KD) mice in both models, while wild-type (WT) mice experienced significant retinal thinning, RGC loss, and vision impairment. RIP3-KO and MLKL-KO mice showed moderate protective effects in the IRI model and limited in the ONC model. Furthermore, we confirmed that a glaucoma causative mutation in optineurin, OPTN-E50K, sensitizes cells to RIP1-mediated inflammatory cell death. RIP1 inhibition reduces RGC death and axonal degeneration following IRI in mice expressing OPTN-WT and OPTN-E50K variant mice. We demonstrate that RIP1 inactivation suppressed microglial infiltration in the RGC layer following glaucomatous damage. Finally, this study highlights that human glaucomatous retinas exhibit elevated levels of TNF and RIP3 mRNA and microglia infiltration, thus demonstrating the role of neuroinflammation in glaucoma pathogenesis. Altogether, these data indicate that RIP1 plays an important role in modulating neuroinflammation and that inhibiting RIP1 activity may provide a neuroprotective therapy for glaucoma.

The presence of circulating human apolipoprotein J reduces the occurrence of cerebral microbleeds in a transgenic mouse model with cerebral amyloid angiopathy

BackgroundCerebral amyloid angiopathy (CAA) is characterized by amyloid-β (Aβ) deposition in cerebral vessels, leading to lobar cerebral microbleeds (CMB) and intracerebral hemorrhages (ICH). Apolipoprotein J (ApoJ) is a multifunctional chaperone related to Aβ aggregation and clearance. Our study investigated the vascular impact of chronic recombinant human Apolipoprotein J (rhApoJ) treatment in a transgenic mouse model of β-amyloidosis with prominent CAA.MethodsTwenty-month-old APP23 C57BL/6 mice received 25 doses of rhApoJ (1 mg/kg) (n = 9) or saline (n = 8) intraperitoneally for 13 weeks, while Wild-type (WT) mice received saline (n = 13). Postmortem brains underwent T2*-weighted magnetic resonance imaging (MRI) to detect hemorrhagic lesions. Aβ levels and distribution, cerebral fibrinogen leakage, brain smooth muscle actin (sma), and plasma matrix metalloproteinases and inflammatory markers were analyzed after treatments. Additionally, plasma samples from 22 patients with lobar ICH were examined to determine the clinical relevance of the preclinical findings.ResultsrhApoJ-treated APP23 presented fewer cortical CMBs (50–300 μm diameter) (p = 0.012) and cortical larger hemorrhages (> 300 μm) (p = 0.002) than saline-treated mice, independently of Aβ brain levels. MRI-detected hemorrhagic lesions correlated with fibrinogen cerebral extravasation (p = 0.011). Additionally, rhApoJ-treated mice presented higher number of sma-positive vessels than saline-treated mice (p = 0.038). In rhApoJ-treated mice, human ApoJ was detected in plasma and in occasional leptomeningeal vessels, but not in the parenchyma, suggesting that its mechanism of action operates through the periphery. The administration of rhApoJ induced an increase in plasma Groα (p = 0.035) and MIP-1α (p = 0.035) levels, while lower MMP-12 (p = 0.046) levels, compared to the saline-treated group. In acute lobar ICH patients, MMP-12 plasma levels correlated with larger hemorrhage volume (p = 0.040) and irregular ICH shape (p = 0.036).ConclusionsChronic rhApoJ treatment in aged APP23 mice ameliorated CAA-related neurovascular damage by reducing the occurrence of CMB. We propose that rhApoJ may prevent blood-brain barrier (BBB) leakage and CMB appearance partly through circulating MMP-12 modulation.

Distinct roles for interferon-α and interferon-β in recovery from Sindbis virus encephalomyelitis

Abstract Alphaviruses are viruses that can cause encephalomyelitis, which is inflammation of the brain and spinal cord due to the immune response to viral infection. Neurons are the primary target cells of Sindbis virus (SINV), the prototypic alphavirus. Interferon (IFN) regulatory factor (IRF) 7 is a transcription factor important for amplification of type I IFN and is required for survival from SINV infection. Irf7-/- mice do not produce IFNα and rapidly succumb to infection – despite local production of IFNβ – while wild type (WT) mice recover. Treatment of Irf7-/- mice with IFNα increases survival and promotes viral clearance, suggesting that IFNα is required for recovery, and further, myeloid-specific expression of IRF7 is necessary for survival. Additionally, Ifnb-/- mice do not phenocopy Irf7-/- mice, further suggesting that IFNα is critical for survival while IFNβ is dispensable despite usage of the same receptor. Interestingly, Ifnb-/- mice have similar peak viral titers to Irf7-/- mice but clear virus with kinetics similar to WT mice, implying that the protective effects of IFNα go beyond restriction of viral replication. Irf7-/-Ifnb-/- double knock-out mice have disease similar to Irf7-/- mice but with earlier onset, suggesting an immunomodulatory role for IFNβ in delaying clinical disease. These findings show subtype-specific differences in protection by type I IFN and can inform treatment options for more severe forms of alphavirus-induced encephalomyelitis.

Mice lacking cyclophilin B, but not cyclophilin A, are protected from the development of NASH in a diet and chemical-induced model.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development.

Interferon Regulatory Factor 7-mediated induction of interferon-α is required for survival from alphavirus encephalomyelitis

Abstract Alphaviruses are a group of arthropod-borne viruses that have the capacity to cause encephalomyelitis, which is inflammation of the brain and spinal cord due to the immune response to viral infection, and can result in fever, headache, and neurologic disease such as cognitive impairment, seizures, ataxia, and paralysis. Neurons are the primary target cells of Sindbis virus (SINV), the prototypic alphavirus; however, due to the indispensable nature of neurons, mechanisms of viral clearance from the central nervous system (CNS) during recovery cannot depend on immune-mediated killing of infected neurons and is therefore specialized. We have previously shown that interferon (IFN) regulatory factor (IRF) 7, a transcription factor important for amplification of type I IFN (mainly IFNα), is required for survival from infection with SINV. Mice deficient in IRF7 succumb to infection – despite local production of IFNβ – while wild type (WT) mice recover. Treatment of Irf7−/−mice with IFNα rescues the WT phenotype, suggesting that IRF7-mediated induction of IFNα and subsequent downstream signaling is required for survival from SINV infection. Additionally, Ifnb−/−mice do not phenocopy Irf7−/−mice, further suggesting that IFNα is required for survival while IFNβ is dispensable despite usage of the same receptor. Interestingly, Ifnb−/−mice have similar viral titers to Irf7−/−mice, implying that the protective effects of IFNα are independent from restriction of viral replication. These findings show subtype-specific differences in protection by type I IFN and can inform treatment options for alphavirus-induced encephalomyelitis. These works have been funded by the U.S. National Institutes of Health (R01 NS087539 and T32 AI007417).

Sensitization of Tumor Cells to IFNy-Mediated Cell Death

Abstract There is increasing evidence that immunotherapies make tumor cells more vulnerable to the host’s own immune system that can lead to long-term and durable remission. To date, only a small percentage of cancer patients have benefited from this approach and advances in this field will likely come from a greater understanding of the interaction between immune cells and their tumor targets. T cells are critical to a successful anti-tumor response due to their ability to directly lyse tumor cells by releasing cytolytic granules, and to produce inflammatory cytokines, such as interferon gamma (IFNγ). IFNγ has several functions in an anti-tumor response. On the immune cell side, IFNγ is a major driver of Th1 differentiation that leads to the activation of T cells and NK cells. On the tumor side, IFNγ can enhance MHC class I presentation but also inhibitory receptors such as PD-1. However, it is not as clear whether IFNγ is directly cytotoxic for tumor cells. If that is the case, it is also likely that due to selection pressure exerted by T cells, tumor cells may have acquired mechanisms to protect themselves against this possibility. Thus, I hypothesized that tumor cells are normally resistant to killing by IFNγ but can be sensitized if the death protection mechanisms are disabled. In preliminary studies using the B16F1 murine melanoma model, we found that while wild-type (WT) melanoma cells were completely resistant, tumor cells with knockouts of two kinases (DKO) became highly sensitized to IFNγ-mediated apoptotic death. Furthermore, the DKO tumors were also more easily killed by T cells in an IFNγ-dependent manner. These findings will provide insight into potential targets for therapeutic intervention to enhance immune-mediated destruction of tumors.

PDGF gene expression and p53 alterations contribute to the biology of diffuse astrocytic gliomas

Diffuse, histologically lower grade astrocytomas of adults (LGAs) are classified based on the mutational status of the isocitrate dehydrogenase (IDH) genes. While wild-type (WT) LGAs often evolve quickly to glioblastoma (GBM), mutant tumors typically follow an indolent course. To find possible effectors of these different behaviors, we compared their respective transcriptomes. Unlike mutant LGAs, platelet-derived growth factor (PDGF) signaling was significantly enriched in WT tumors, and PDGFA was the top overexpressed gene in the pathway. Moreover, methylation of the PDGFA and PDGFD promoters emerged as a possible mechanism for their low expression in mutant tumors. Copy number gain of chromosome 7 co-occurred with high expression of PDGFA in WT cases, and high expression of PDGFA was associated with aneuploidy, extracellular matrix (ECM)-related immunosuppressive features and poor prognosis. We also noted that high PDGFA expression in WT cases occurred irrespective of tumor grade and that multiple mechanisms of p53 pathway inactivation accompanied progression to GBM in PDGFA-overexpressing tumors. Conversely, TP53 point mutations were an early and constant feature of mutant LGAs. Our results suggest that members of the PDGF gene family, in concert with different p53 pathway alterations, underlie LGA behaviors.

The COPD-Associated Polymorphism Impairs the CFTR Function to Suppress Excessive IL-8 Production upon Environmental Pathogen Exposure.

COPD is a lifestyle-related disease resulting from irreversible damage to respiratory tissues mostly due to chronic exposure to environmental pollutants, including cigarette smoke. Environmental pathogens and pollutants induce the acquired dysfunction of the CFTR Cl- channel, which is invoked in COPD. Despite the increased incidence of CFTR polymorphism R75Q or M470V in COPD patients, the mechanism of how the CFTR variant affects COPD pathogenesis remains unclear. Here, we investigated the impact of CFTR polymorphisms (R75Q, M470V) on the CFTR function in airway epithelial cell models. While wild-type (WT) CFTR suppressed the proinflammatory cytokine production induced by COPD-related pathogens including pyocyanin (PYO), R75Q- or M470V-CFTR failed. Mechanistically, the R75Q- or M470V-CFTR fractional PM activity (FPMA) was significantly lower than WT-CFTR in the presence of PYO. Notably, the CF drug Trikafta corrected the PM expression of R75Q- or M470V-CFTR even upon PYO exposure and consequently suppressed the excessive IL-8 production. These results suggest that R75Q or M470V polymorphism impairs the CFTR function to suppress the excessive proinflammatory response to environmental pathogens associated with COPD. Moreover, Trikafta may be useful to prevent the COPD pathogenesis associated with acquired CFTR dysfunction.

Depletion of chop suppresses procedural apoptosis and enhances innate immunity in loach Misgurnus anguillicaudatus under ammonia nitrogen stress.

Ammonia nitrogen is highly toxic to fish, and it can easily cause fish poisoning or even high mortality. So far, many studies have been conducted on the damages to fish under ammonia nitrogen stress. However, there are few studies of ammonia tolerance improvement in fish. In this study, the effects of ammonia nitrogen exposure on apoptosis, endoplasmic reticulum (ER) stress, and immune cells in loach Misgurnus anguillicaudatus were investigated. Loaches (60 d post fertilization) were exposed to different concentrations of NH4Cl, and their survival rates were examined every 6 h. The results showed that high-concentration and long-time NH4Cl exposure (20 mM + 18 h; 15 mM + 36 h) induced apoptosis and gill tissue damages, finally causing a decline in survival. chop plays an important role in ER stress-induced apoptosis, and thus we constructed a model of chop-depleted loach by using CRISPR/Cas9 technology to investigate its response to ammonia nitrogen stress. The results showed that ammonia nitrogen stress down-regulated the expressions of apoptosis-related genes in chop+/- loach gills, while wildtype (WT) exhibited an opposite gene expression regulation pattern, suggesting that the depletion of chop suppressed apoptosis level. In addition, chop+/- loach showed a larger number of immunity-related cells and higher survival rate than WT under the NH4Cl exposure, indicating that the inhibition of chop function strengthened the innate immune barrier in general, thus increasing survival. Our findings provide the theoretical basis for developing high ammonia nitrogen-tolerant germplasm with aquaculture potential.

Editing of the rice importin gene IMPα1b results in sequestration of TAL effectors from plant cell nuclei

Transcription activator-like effectors (TALEs) produced by plant pathogenic bacteria mainly belonging to the genus Xanthomonas cause plant diseases through activation of host susceptibility genes in plant cell nuclei. How TALEs enter plant cell nuclei was not clear until recent studies about PthXo1 and TALI, two TALEs produced by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), the rice (Oryza sativa) pathogens that cause bacterial blight and bacterial leaf streak, respectively. Here, we report that rice importin IMPα1b serves as a nuclear transport receptor in rice plants to facilitate the nuclear import of PthXo1 and TALI from Xoo and Xoc, respectively. While wild-type (WT) rice plants support the nuclear import of PthXo1 and TALI, nuclear trafficking is defective in OsIMPα1b loss-of-function mutants generated by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated 9 (Cas9)-based gene editing. In the edited plants infected by Xoo, OsIMPα1b sequesters PthXo1 from the nucleus, the PthXo1-targeted rice susceptibility gene is no longer activated, and bacterial virulence and blight disease are alleviated as a result. In the edited plants infected by Xoc, OsIMPα1b sequesters TALI from the nucleus, the role of TALI in suppressing rice defense responses is nullified, and rice defense responses are in turn activated to inhibit bacterial virulence and alleviate bacterial leaf streak severity.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy

While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice

Abstract Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and anti-viral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathology in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost only 10% of their body weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice. Supported by grants from the NIH (R01 AI130110, R21 AI151230, R01 HL154001, R21 AI142256, and U54 CA260582)

Th17-driven immunity to oral candidiasis is dependent on the microbiome and can be triggered by mono-colonization with segmented filamentous bacteria.

Abstract The opportunistic infection oropharyngeal candidiasis (OPC; oral thrush) is a common oral infection among the immunocompromised. The oral cavity is a major entry portal to the body and oral health impacts several aspects of host immunity. While much is now known about microbiome dependent Th17 induction and protection against intestinal candidiasis and other Th17 driven responses, our understanding of the role of the microbiome in oral Th17 induction and oral antifungal immunity is rather rudimentary. We demonstrate a direct role for the microbiome in triggering protection against OPC. While wildtype (WT) mice raised under specific pathogen free (SPF) conditions are resistant to OPC, WT germ free (GF) mice succumbed to infection. Reconstitution of WT-GF mice with segmented filamentous bacteria (SFB) was sufficient to restore protection against OPC with associated increase in Th17 cells, neutrophils, and antimicrobial responses in the oral mucosa. Thus, SFB mono-colonization in WT-GF mice triggers oral mucosal Th17 induction to protect against oral thrush and reveals a role of the microbiome in promoting oral mucosal immunity. Supported by R37-DE022550

Mechanisms of Arrhythmogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant I79N.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T—TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (k off). To investigate further, a heterozygous I79N+/− TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/− hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (V m) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/− TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/− vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Contribution of STAT1 to innate and adaptive immunity during type I interferon-mediated lethal virus infection.

Signal transducers and activators of transcription (STAT) 1 is critical for cellular responses to type I interferons (IFN-Is), with the capacity to determine the outcome of viral infection. We previously showed that while wildtype (WT) mice develop mild disease and survive infection with lymphocytic choriomeningitis virus (LCMV), LCMV infection of STAT1-deficient mice results in a lethal wasting disease that is dependent on IFN-I and CD4+ cells. IFN-Is are considered to act as a bridge between innate and adaptive immunity. Here, we determined the relative contribution of STAT1 on innate and adaptive immunity during LCMV infection. We show that STAT1 deficiency results in a biphasic disease following LCMV infection. The initial, innate immunity-driven phase of disease was characterized by rapid weight loss, thrombocytopenia, systemic cytokine and chemokine responses and leukocyte infiltration of infected organs. In the absence of an adaptive immune response, this first phase of disease largely resolved resulting in survival of the infected host. However, in the presence of adaptive immunity, the disease progressed into a second phase with continued cytokine and chemokine production, persistent leukocyte extravasation into infected tissues and ultimately, host death. Overall, our findings demonstrate the key contribution of STAT1 in modulating innate and adaptive immunity during type I interferon-mediated lethal virus infection.

Overexpression of a TIFY family gene, GsJAZ2, exhibits enhanced tolerance to alkaline stress in soybean

Previous studies have indicated the potential role of a TIFY family gene, GsJAZ2, for its response to alkaline stress in Arabidopsis. Here, we transformed GsJAZ2 into an elite soybean (Glycine max) using particle bombardment-based genetic transformation system to assess whether overexpression of GsJAZ2 in soybean improved alkaline tolerance. Results revealed that transgenic soybean overexpressing GsJAZ2 grew well under alkaline conditions, while wild-type (WT) plants exhibited chlorosis, stunted growth, or even death. Higher chlorophyll concentration and lower of Malondialdehyde (MDA) content were detected in the transgenic plants compared to WT under alkaline stress. Moreover, the transcription levels of two alkaline-responsive genes were up-regulated in the GsJAZ2 overexpression at the early or later stage of alkaline stress, respectively, indicating that GsJAZ2 overexpression enhanced tolerance to alkaline stress in soybean, which might modulate intracellular pH homeostasis and osmotic balance through regulating the expression of alkaline-responsive genes. Additionally, GsJAZ2 is a member of the JAZ subfamily that belongs to the TIFY family. Overexpression of GsJAZ2 in soybean conferred JA insensitivity, including JA-mediated root growth inhibition. The transcripts of some JA-responsive genes were examined and altered under alkaline stress, implying that GsJAZ2 overexpression may involve in JA signal pathway for accumulating anti-osmotic substances in defense to alkaline stress. Collectively, the findings of this study facilitate our understanding of GsJAZ2 in response to alkaline stress as well as enrich the function of TIFY genes in crop.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB.

SummaryThe family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.

628-P: Insulin's Regulation of CXCL1 Expression Is Critical for Neutrophil Recruitments and the Development of Periodontitis and Poor Wound Healing in Diabetes

A major cause of poor wound healing and periodontitis in diabetes is defective immune response to infections. Insulin signaling via the PI3K/Akt pathway is selectively inhibited in the gingival and wound fibroblasts of diabetic rodents and patients. To determine how loss of insulin actions in gingival fibroblasts can affect periodontitis development, myofibroblast insulin receptors (IR) were deleted by crossbreeding IRβ-flox/flox mice with SM22-Cre C57BL/6 (SMIRKO) mice, which were fed with regular diet (RD), while wild type (WT) littermates were fed with high-fat diet (HFD) for 10 weeks. To induce periodontitis, 7-0 silk ligatures were tied around maxillary molars for 4, 7 or 14 days. When mice were sacrificed to assess alveolar bone loss, immune cell populations and inflammatory cytokine expression in the gingiva was assessed. Gingival IRβ expression in SMIRKO and HFD-fed mice was decreased by 70% and 50%, respectively, with comparable reductions in insulin-induced (100nM) phosphorylation of Akt (pAkt). Ligature-induced alveolar bone loss in SMIRKO and HFD-fed mice was greater by 70% and 40%, respectively, as compared to RD-fed WT mice. Post-ligature time course analysis showed that while neutrophil recruitment was delayed significantly, macrophages and lymphocyte numbers were elevated in both SMIRKO and HFD-fed mice. Similarly, neutrophil-recruiting factors CXCL1 and 2 expression were decreased in the gingiva, whereas TNFα and IL-1β expression were elevated. Gingival myofibroblasts from SMIRKO and HFD-fed mice showed dramatic reductions of IRβ expression and insulin-induced pAkt with parallel lowering of E.coli LPS-induced CXCL1 expression via downregulation of TLR2, compared to RD-fed mice. These data provided the first evidence that insulin action is critical for neutrophil recruitment during infection by enabling CXCL1 expression in gingival myofibroblasts, which is inhibited in diabetes. Disclosure T. Shinjo: None. Q. Li: None. A. Ishikado: Employee; Self; SunStar Inc. Employee; Spouse/Partner; SunStar Inc. K. Park: None. R. St-Louis: None. H. Yokomizo: None. J. Fu: None. M. Yu: None. H. Wang: None. H. Hasturk: None. T.E. Van Dyke: None. G.L. King: Research Support; Self; Sanofi. Funding Sunstar Foundation; Iacocca Family Foundation

Haploinsufficiency of CALR Confers Hematopoietic Stem Cells (HSCs) with a Clonal Advantage over Wild-Type Cells, and, in Setting of Myeloproliferative Neoplasms, Compensates for the Functions of HSCs Impaired By the Calr Mutation

CALR exon9 frameshift mutations function as driver mutations in essential thrombocythemia (ET) and primary myelofibrosis patients with non-mutated JAK2 or MPL. The mutations augment signal transducer and activator of transcription activity in the presence of MPL, induce increased cell proliferation and growth factor independence in cell lines, and cause ET-like myeloproliferative neoplasms (MPN) in mice.In tumor cells bearing the CALR mutation, mutant CALR protein expression occurs while wild-type (WT) CALR expression is decreased by half. Although the biological activity of mutant CALR has been elucidated in detail, the significance of CALR haploinsufficiency is unclear. The purpose of this study was therefore to clarify the influence of CALR haploinsufficiency on hematopoiesis in normal and CALR-mutated MPN in mice.First, we analyzed the effect of CALR haploinsufficiency on hematopoiesis using CALR heterozygous knockout (CALR-hKO) mice (Tokuhiro et al. Sci Rep. 2015). Blood cell counts, liver and spleen weight, histology, cell fraction in bone marrow (BM) and spleen, and survival of CALR-hKO mice were all equivalent to that observed in WT mice. In the analysis of progenitor cells by fluorescence-activated cell sorting and colony formation assays, no difference was observed in the amount of progenitor cells and in colony replating capacity between WT mice and CALR-hKO mice. Interestingly, in competitive serial transplantation experiments using whole BM cells in primary and secondary transplanted mice, CALR-hKO cells showed higher levels of chimerism than WT cells.Next, the effect of CALR haploinsufficiency on hematopoiesis in mutant CALR-del52 overexpressing mice was analyzed. We compared three groups of mice, WT mice, CALR-del52 transgenic (TG) mice (Shide et al. Leukemia 2017) and CALR-del52 TG/CALR-hKO double-mutant (TG-hKO) mice. Both TG mice and TG-hKO mice developed ET-like MPN. Compared to WT mice, increases in megakaryocytes, platelets and hematopoietic progenitor cells (including HSCs) were observed in these mice. However, no differences were observed between TG mice and TG-hKO mice.Finally, 4000 LSK cells sorted from WT mice, TG mice, and TG-hKO mice (B6-Ly5.2) and B6-Ly5.1 competitor cells (1 × 106 WT BM cells) were mixed and injected into lethally irradiated B6-Ly5.1 recipient mice, and the percent chimerism of donor cells was followed for 1 year after transplantation. From 12 weeks after transplantation, the chimerism of TG cells was significantly lower than that of control WT cells, suggesting that the CALR mutation has a negative influence on clonal expansion of HSCs. The recipient mice transplanted with TG LSK cells showed very mild thrombocythemia. On the other hand, chimerism in TG-hKO cells was significantly higher than that in control WT cells up to 12 weeks after transplantation. Chimerism at 20 weeks after transplantation was equivalent to that in control WT cells, and the recipient mice transplanted with TG-hKO LSK cells showed severe thrombocythemia. These findings show that CALR haploinsufficiency compensates for HSCs functioning, which has been impaired by the CALR mutation, enhancing the ability of HSCs to develop ET.Mice experiments have showed that HSCs with the JAK2V617F mutation are fragile, and it is considered that mutations in DNMT3A or TET2 often occurred prior to mutations in JAK2 to compensate for the defect in self renewal capacity. Conversely, since the CALR mutation is not typically preceded by any mutations, it is considered that the CALR mutation may not require any preceding mutations. Our results showed that, like JAK2 V617F mutants, overexpression of the CALR-del52 mutant impairs HSC functioning. Furthermore, we found that CALR haploinsufficiency restores the functions of impaired HSCs to the same extent as that found in WT HSCs. When an HSC acquires the CALR mutation, “defect” and “recovery” states thus occur simultaneously in the cell. This finding may explain why mutant CALR clones expand without needing to undergo any preceding mutation. DisclosuresNo relevant conflicts of interest to declare.