Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells.

The maintenance of immune homeostasis requires regulatory T cells (Treg cells). Here we found that Treg cell­specific ablation of Ubc13, a Lys63 (K63)-specific ubiquitin-conjugating enzyme, caused aberrant T cell activation and autoimmunity. Although Ubc13 deficiency did not affect the survival of Treg cells or expression of the transcription factor Foxp3, it impaired the in vivo suppressive function of Treg cells and rendered them sensitive to the acquisition of T helper type 1 (TH1) cell­ and interleukin 17 (IL-17)-producing helper T (TH17) cell­like effector phenotypes. This function of Ubc13 involved its downstream target, the kinase IKK. The Ubc13-IKK signaling axis controlled the expression of specific Treg cell effector molecules, including IL-10 and SOCS1. Collectively, our findings suggest that the Ubc13-IKK signaling axis regulates the molecular program that maintains Treg cell function and prevents Treg cells from acquiring inflammatory phenotypes.

Suppressor of cytokine signaling 3-derived peptide as a therapeutic for inflammatory and oxidative stress-induced damage to the retina.

Purpose: Inflammation and oxidative stress contribute to age-related macular degeneration (AMD) and other retinal diseases. We tested a cell-penetrating peptide from the kinase inhibitory region of an intracellular checkpoint inhibitor suppressor of cytokine signaling 3 (R9-SOCS3-KIR) peptide for its ability to blunt the inflammatory or oxidative pathways leading to AMD. Methods: We used anaphylatoxin C5a to mimic the effect of activated complement, lipopolysaccharide (LPS), and tumor necrosis factor alpha (TNFα) to stimulate inflammation and paraquat to induce mitochondrial oxidative stress. We used a human retinal pigment epithelium (RPE) cell line (ARPE-19) as proliferating cells and a mouse macrophage cell line (J774A.1) to follow cell propagation using microscopy or cell titer assays. We evaluated inflammatory pathways by monitoring the nuclear translocation of NF-κB p65 and mitogen-activated protein kinase p38. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the induction of inflammatory markers. In differentiated ARPE-19 monolayers, we evaluated the integrity of tight junction proteins through microscopy and the measurement of transepithelial electrical resistance (TEER). We used intraperitoneal injection of sodium iodate in mice to test the ability of R9-SOC3-KIR to prevent RPE and retinal injury, as assessed by fundoscopy, optical coherence tomography, and histology. Results: R9-SOCS3-KIR treatment suppressed C5a-induced nuclear translocation of the NF-kB activation domain p65 in undifferentiated ARPE-19 cells. TNF-mediated damage to tight junction proteins in RPE, and the loss of TEER was prevented in the presence of R9-SOCS3-KIR. Treatment with the R9-SOCS3-KIR peptide blocked the C5a-induced expression of inflammatory genes. The R9-SOCS3-KIR treatment also blocked the LPS-induced expression of interleukin-6, MCP1, cyclooxygenase 2, and interleukin-1 beta. R9-SOCS3-KIR prevented paraquat-mediated cell death and enhanced the levels of antioxidant effectors. Daily eye drop treatment with R9-SOCS3-KIR protected against retinal injury caused by i.p. administration of sodium iodate. Conclusions: R9-SOCS3-KIR blocks the induction of inflammatory signaling in cell culture and reduces retinal damage in a widely used RPE/retinal oxidative injury model. As this peptide can be administered through corneal instillation, this treatment may offer a convenient way to slow down the progression of ocular diseases arising from inflammation and chronic oxidative stress.

Temporal trends in acute kidney injury across health care settings in the Irish health system: a cohort study.

BACKGROUND: Complete ascertainment of the true rates of acute kidney injury (AKI) and emerging trends are essential for planning of preventive strategies within health systems. METHODS: We conducted a retrospective cohort study from 2005 to 2014 using data from regional laboratory information systems to determine incidence rates of AKI and severity Stages 1-3 in the Irish health system. Multivariable models were developed to explore annual trends and the contributions of demographic factors, clinical measures, geographic factors and location of medical supervision expressed as adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: From 2005 to 2014, incidence rates of AKI increased from 6.1% (5.8-6.3) to 13.2% (12.7-13.8) per 100 patient-years in men and from 5.0% (4.8-5.2) to 11.5% (11.0-12.0) in women, P < 0.001. Stage 1 AKI accounted for the greatest growth in incidence, from 4.4% (95% CI 4.3-4.6) in 2005 to 10.1% (95% CI 9.8-10.5) in 2014 (P < 0.001 for trend). Compared with 2005, patients in 2014 were more likely to experience AKI [OR 4.53 (95% CI 4.02-5.1) for Stage 1, OR 5.22 (4.16-6.55) for Stage 2 and OR 4.11 (3.05-5.54) for Stage 3], adjusting for changing demographic and clinical profiles. Incidence rates of AKI increased in all locations of medical supervision during the period of observation, but were greatest for inpatient [OR 19.11 (95% CI 17.69-20.64)] and emergency room settings [OR 5.97 (95% CI 5.56-6.42)] compared with a general practice setting (referent). CONCLUSION: Incidence rates of AKI have increased substantially in the Irish health system, which were not accounted for by changing demographic patterns, clinical profiles or location of medical supervision.

Noncanonical IFN Signaling, Steroids, and STATs: A Probable Role of V-ATPase.

A small group of only seven transcription factors known as STATs (signal transducer and activator of transcription) are considered to be canonical determinants of specific gene activation for a plethora of ligand/receptor systems. The activation of STATs involves a family of four tyrosine kinases called JAK kinases. JAK1 and JAK2 activate STAT1 in the cytoplasm at the heterodimeric gamma interferon (IFNγ) receptor, while JAK1 and TYK2 activate STAT1 and STAT2 at the type I IFN heterodimeric receptor. The same STATs and JAKs are also involved in signaling by functionally different cytokines, growth factors, and hormones. Related to this, IFNγ-activated STAT1 binds to the IFNγ-activated sequence (GAS) element, but so do other STATs that are not involved in IFNγ signaling. Activated JAKs such as JAK2 and TYK2 are also involved in the epigenetics of nucleosome unwrapping for exposure of DNA to transcription. Furthermore, activated JAKs and STATs appear to function coordinately for specific gene activation. These complex events have not been addressed in canonical STAT signaling. Additionally, the function of noncoding enhancer RNAs, including their role in enhancer/promoter interaction is not addressed in the canonical STAT signaling model. In this perspective, we show that JAK/STAT signaling, involving membrane receptors, is essentially a variation of cytoplasmic nuclear receptor signaling. Focusing on IFN signaling, we showed that ligand, IFN receptor, the JAKs, and the STATs all undergo endocytosis and ATP-dependent nuclear translocation to promoters of genes specifically activated by IFNs. We argue here that the vacuolar ATPase (V-ATPase) proton pump probably plays a key role in endosomal membrane crossing by IFNs for receptor cytoplasmic binding. Signaling of nuclear receptors such as those of estrogen and dihydrotestosterone provides templates for making sense of the specificity of gene activation by closely related cytokines, which has implications for lymphocyte phenotypes.

A cell penetrating peptide from SOCS-1 prevents ocular damage in experimental autoimmune uveitis.

We describe an immunosuppressive peptide corresponding to the kinase inhibitory region (KIR) of the intracellular checkpoint protein suppressor of cytokine signaling 1 (SOCS-1) that binds to the phospho-tyrosine containing regions of the tyrosine kinases JAK2 and TYK2 and the adaptor protein MAL, and thereby inhibits signaling downstream from these signaling mediators. The peptide, SOCS1-KIR, is thus capable of downregulating overactive JAK/STAT or NF-kB signaling in somatic cells, including those in many compartments of the eye. Attachment of poly-arginine to this peptide (R9-SOCS1-KIR) allows it to penetrate the plasma membrane in aqueous media. R9-SOCS1-KIR was tested in ARPE-19 cells and was found to attenuate mediators of inflammation by blocking the inflammatory effects of IFNγ, TNFα, or IL-17A. R9-SOCS1-KIR and also protected against TNFα or IL-17A mediated damage to the barrier properties of ARPE-19 cells, as evidenced by immunostaining with the tight junction protein, zona occludin 1 (ZO-1), and measurement of transepithelial electrical resistance (TEER). Experimental autoimmune uveitis (EAU) was generated in B10. RIII mice using a peptide of interphotoreceptor retinal binding protein (IRBP161-180) as immunogen. Topical administration of R9-SOCS1-KIR, 2 days before (prophylactic), or 7 days after immunization (therapeutic) protected ocular structure and function as seen by fundoscopy, optical coherence tomography (OCT), and electroretinography (ERG). The ability R9-SOCS1-KIR to suppress ocular inflammation and preserve barrier properties of retinal pigment epithelium makes it a potential candidate for treatment of autoimmune uveitis.

Causes and outcomes of emergency presentation of rectal cancer.

Emergency presentation of rectal cancer carries a relatively poor prognosis, but the roles and interactions of causative factors remain unclear. We describe an innovative statistical approach which distinguishes between direct and indirect effects of a number of contextual, patient and tumour factors on emergency presentation and outcome of rectal cancer. All patients diagnosed with rectal cancer in Ireland 2004-2008 were included. Registry information, linked to hospital discharge data, provided data on patient demographics, comorbidity and health insurance; population density and deprivation of area of residence; tumour type, site, grade and stage; treatment type and optimality; and emergency presentation and hospital caseload. Data were modelled using a structural equation model with a discrete-time survival outcome, allowing us to estimate direct and mediated effects of the above factors on hazard, and their inter-relationships. Two thousand seven hundred and fifty patients were included in the analysis. Around 12% had emergency presentations, which increased hazard by 80%. Affluence, private patient status and being married reduced hazard indirectly by reducing emergency presentation. Older patients had more emergency presentations, while married patients, private patients or those living in less deprived areas had fewer than expected. Patients presenting as an emergency were less likely to receive optimal treatment or to have this in a high caseload hospital. Apart from stage, emergency admission was the strongest determinant of poor survival. The factors contributing to emergency admission in this study are similar to those associated with diagnostic delay. The socio-economic gradient found suggests that patient education and earlier access to endoscopic investigation for public patients could reduce emergency presentation.

Mechanisms and mediation in survival analysis: towards an integrated analytical framework.

BACKGROUND: A wide-ranging debate has taken place in recent years on mediation analysis and causal modelling, raising profound theoretical, philosophical and methodological questions. The authors build on the results of these discussions to work towards an integrated approach to the analysis of research questions that situate survival outcomes in relation to complex causal pathways with multiple mediators. The background to this contribution is the increasingly urgent need for policy-relevant research on the nature of inequalities in health and healthcare. METHODS: The authors begin by summarising debates on causal inference, mediated effects and statistical models, showing that these three strands of research have powerful synergies. They review a range of approaches which seek to extend existing survival models to obtain valid estimates of mediation effects. They then argue for an alternative strategy, which involves integrating survival outcomes within Structural Equation Models via the discrete-time survival model. This approach can provide an integrated framework for studying mediation effects in relation to survival outcomes, an issue of great relevance in applied health research. The authors provide an example of how these techniques can be used to explore whether the social class position of patients has a significant indirect effect on the hazard of death from colon cancer. RESULTS: The results suggest that the indirect effects of social class on survival are substantial and negative (-0.23 overall). In addition to the substantial direct effect of this variable (-0.60), its indirect effects account for more than one quarter of the total effect. The two main pathways for this indirect effect, via emergency admission (-0.12), on the one hand, and hospital caseload, on the other, (-0.10) are of similar size. CONCLUSIONS: The discrete-time survival model provides an attractive way of integrating time-to-event data within the field of Structural Equation Modelling. The authors demonstrate the efficacy of this approach in identifying complex causal pathways that mediate the effects of a socio-economic baseline covariate on the hazard of death from colon cancer. The results show that this approach has the potential to shed light on a class of research questions which is of particular relevance in health research.

Noncanonical IFN Signaling: Mechanistic Linkage of Genetic and Epigenetic Events.

The canonical model of cytokine signaling via the JAK/STAT pathway dominates our view of signal transduction but provides no insight into the significance of the simultaneous presence of activated JAKs and STATs in the nucleus of cells treated with cytokines. Such a mechanistic shortcoming challenges the usefulness of the model in its present form. Focusing on the interferon (IFN) cytokines, we have developed a noncanonical model of IFN signaling that naturally connects activated JAKs and STATs at or near response elements of genes that are activated by the IFNs. Specifically, cells treated with IFNγ showed association of activated STAT1α and JAK2 at the GAS element of genes activated by IFNγ. For IFNα treated cells, the association involved activated STAT1α and TYK2 JAK kinase at the ISRE promoter. The power of the noncanonical model is that it provides mechanistic insight into specific gene activation at the level of the associated epigenetics, akin to that of steroid/steroid receptor signaling.

Risk-adjusted antibiotic consumption in 34 public acute hospitals in Ireland, 2006 to 2014.

As antibiotic consumption rates between hospitals can vary depending on the characteristics of the patients treated, risk-adjustment that compensates for the patient-based variation is required to assess the impact of any stewardship measures. The aim of this study was to investigate the usefulness of patient-based administrative data variables for adjusting aggregate hospital antibiotic consumption rates. Data on total inpatient antibiotics and six broad subclasses were sourced from 34 acute hospitals from 2006 to 2014. Aggregate annual patient administration data were divided into explanatory variables, including major diagnostic categories, for each hospital. Multivariable regression models were used to identify factors affecting antibiotic consumption. Coefficient of variation of the root mean squared errors (CV-RMSE) for the total antibiotic usage model was very good (11%), however, the value for two of the models was poor (> 30%). The overall inpatient antibiotic consumption increased from 82.5 defined daily doses (DDD)/100 bed-days used in 2006 to 89.2 DDD/100 bed-days used in 2014; the increase was not significant after risk-adjustment. During the same period, consumption of carbapenems increased significantly, while usage of fluoroquinolones decreased. In conclusion, patient-based administrative data variables are useful for adjusting hospital antibiotic consumption rates, although additional variables should also be employed.

Influence of socioeconomic status on allograft and patient survival following kidney transplantation.

AIM: Whether socioeconomic status confers worse outcomes after kidney transplantation is unknown. Its influence on allograft and patient survival following kidney transplantation in Ireland was examined. METHODS: A retrospective, observational cohort study of adult deceased-donor first kidney transplant recipients from 1990 to 2009 was performed. Those with a valid Irish postal address were assigned a socioeconomic status score based on the Pobal Hasse-Pratschke deprivation index and compared in quartiles. Cox proportional hazards models and Kaplan-Meier survival analysis were used to investigate any significant association of socioeconomic status with patient and allograft outcomes. RESULTS: A total of 1944 eligible kidney transplant recipients were identified. The median follow-up time was 8.2 years (interquartile range 4.4-13.3 years). Socioeconomic status was not associated with uncensored or death-censored allograft survival (hazard ratio (HR) 1.0, 95% confidence interval (CI) 0.99-1.00, P = 0.33 and HR 1.0, 95% CI 0.99-1.00, P = 0.37, respectively). Patient survival was not associated with socioeconomic status quartile (HR 1.0, 95% CI 0.93-1.08, P = 0.88). There was no significant difference among quartiles for uncensored or death-censored allograft survival at 5 and 10 years. CONCLUSION: There was no socioeconomic disparity in allograft or patient outcomes following kidney transplantation, which may be partly attributable to the Irish healthcare model. This may give further impetus to calls in other jurisdictions for universal healthcare and medication coverage for kidney transplant recipients.

Prevalence and variation of Chronic Kidney Disease in the Irish health system: initial findings from the National Kidney Disease Surveillance Programme.

BACKGROUND: Chronic Kidney Disease (CKD) is a major non-communicable chronic disease that is associated with adverse clinical and economic outcomes. Passive surveillance systems are likely to improve efforts for prevention of chronic kidney disease (CKD) and inform national service planning. This study was conducted to determine the overall prevalence of CKD in the Irish health system, assess period trends and explore patterns of variation as part of a novel surveillance initiative. METHODS: We identified 207, 336 adult patients, age 18 and over, with serum creatinine measurements recorded from a provincial database between 2005-2011 in the Northwest of Ireland. Estimated glomerular filtration rates (eGFR) were determined using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation from standardized creatinine measurements and the presence of CKD was defined as eGFR<60 ml/min per 1.73 m2. Age and sex-specific prevalence estimates were determined for each group while generalized estimating equations (GEE) and multivariable logistic regression were used to explore associations using adjusted odds ratios (AOR) and 95% confidence intervals (95% CI). RESULTS: The prevalence of CKD in the health system was 11.8% (95% CI 11.8-12.1); 10.9% in men (10.7-11.1) and 12.6% in women (12.4-12.8). This corresponded to a detection rate of 4.5% (5.1% in women and 3.9% in men). The prevalence of CKD was significantly higher in women than in men (12.6% versus 10.9%, P<0.001), older age groups, and among patients with a history of Acute Kidney Injury (AKI) than without (45.2% versus 10.7%, P<0.0001). Multivariable analysis identified advancing age, female gender, location of medical supervision, county of residence, and AKI as significant determinants of prevalence. CONCLUSION: The prevalence of CKD in the Irish health system is 11.8% corresponding to a detection rate of 4.5% in the general population. Demographic, geographic factors and acute kidney injury episodes are important determinants of disease burden. Passive surveillance of CKD is both feasible and desirable within the Irish health system, and offers huge opportunities for targeted prevention programmes and improved clinical outcomes.

Inhibition of SOCS1-/- lethal autoinflammatory disease correlated to enhanced peripheral Foxp3+ regulatory T cell homeostasis.

Suppressor of cytokine signaling 1-deficient (SOCS1(-/-)) mice, which are lymphopenic, die <3 wk after birth of a T cell-mediated autoimmune inflammatory disease characterized by leukocyte infiltration and destruction of vital organs. Notably, Foxp3(+) regulatory T cells (Tregs) have been shown to be particularly potent in inhibiting inflammation-associated autoimmune diseases. We observed that SOCS1(-/-) mice were deficient in peripheral Tregs despite enhanced thymic development. The adoptive transfer of SOCS1-sufficient Tregs, CD4(+) T lymphocytes, or administration of SOCS1 kinase inhibitory region (KIR), a peptide that partially restores SOCS1 function, mediated a statistically significant but short-term survival of SOCS1(-/-) mice. However, the adoptive transfer of SOCS1-sufficient CD4(+) T lymphocytes, combined with the administration of SOCS1-KIR, resulted in a significant increase in the survival of SOCS1(-/-) mice both short and long term, where 100% death occurred by day 18 in the absence of treatment. Moreover, the CD4(+)/SOCS1-KIR combined therapy resulted in decreased leukocytic organ infiltration, reduction of serum IFN-γ, and enhanced peripheral accumulation of Foxp3(+) Tregs in treated mice. These data show that CD4(+)/SOCS1-KIR combined treatment can synergistically promote the long-term survival of perinatal lethal SOCS1(-/-) mice. In addition, these results strongly suggest that SOCS1 contributes to the stability of the Foxp3(+) Treg peripheral population under conditions of strong proinflammatory environments.

Steroid-like signalling by interferons: making sense of specific gene activation by cytokines.

Many cytokines, hormones and growth factors use the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway for cell signalling and specific gene activation. In the classical model, ligand is said to interact solely with the receptor extracellular domain, which triggers JAK activation of STATs at the receptor cytoplasmic domain. Activated STATs are then said to carry out nuclear events of specific gene activation. Given the limited number of STATs (seven) and the activation of the same STATs by cytokines with different functions, the mechanism of the specificity of their signalling is not obvious. Focusing on IFNγ (interferon γ), we have shown that ligand, receptor and activated JAKs are involved in nuclear events that are associated with specific gene activation, where the receptor subunit IFNGR1 (IFNγ receptor 1) functions as a transcription/co-transcription factor and the JAKs are involved in key epigenetic events. RTKs (receptor tyrosine kinases) such as EGFR [EGF (epidermal growth factor) receptor] and FGFR [FGF (fibroblast growth factor) receptor] also undergo nuclear translocation in association with their respective ligands. EGFR and FGFR, like IFNGR1, have been shown to function as transcription/co-transcription factors. The RTKs also regulate other kinases that have epigenetic effects. Our IFNγ model, as well as the RTKs EGFR and FGFR, have similarities to that of steroid receptor signalling. These systems consist of ligand-receptor-co-activator complexes at the genes that they activate. The co-activators consist of transcription factors and kinases, of which the latter play an important role in the associated epigenetics. It is our view that signalling by cytokines such as IFNγ is but a variation of specific gene activation by steroid hormones.

Disparate viral pandemics from COVID19 to monkeypox and beyond: a simple, effective and universal therapeutic approach hiding in plain sight.

The field of antiviral therapeutics is fixated on COVID19 and rightly so as the fatalities at the height of the pandemic in the United States were almost 1,000,000 in a twelve month period spanning parts of 2020/2021. A coronavirus called SARS-CoV2 is the causative virus. Development of a vaccine through molecular biology approaches with mRNA as the inducer of virus spike protein has played a major role in driving down mortality and morbidity. Antivirals have been of marginal value in established infections at the level of hospitalization. Thus, the current focus is on early symptomatic infection of about the first five days. The Pfizer drug paxlovid which is composed of nirmatrelvir, a peptidomimetic protease inhibitor of SARS-CoV2 Mpro enzyme, and ritonavir to retard degradation of nirmatrelvir, is the current FDA recommended treatment of early COVID19. There is no evidence of broad antiviral activity of paxlovid against other diverse viruses such as the influenza virus, poxviruses, as well as a host of respiratory viruses. Although type I interferons (IFNs) are effective against SARS-CoV2 in cell cultures and in early COVID19 infections, they have not been broadly recommended as therapeutics for COVID19. We have developed stable peptidomimetics of both types I and II IFNs based on our noncanonical model of IFN signaling involving the C-terminus of the IFNs. We have also identified two members of intracellular checkpoint inhibitors called suppressors of cytokine signaling (SOCS), SOCS1 and SOCS3 (SOCS1/3), and shown that they are virus induced intrinsic virulence proteins with activity against IFN signaling enzymes JAK2 and TYK2. We developed a peptidomimetic antagonist, based on JAK2 activation loop, against SOCS1/3 and showed that it synergizes with the IFN mimetics for potent broad spectrum antiviral activity without the toxicity of intact IFN molecules. IFN mimetics and the SOCS1/3 antagonist should have an advantage over currently used antivirals in terms of safety and potency against a broad spectrum of viruses.

COVID-19 incidence and outcome by affluence/deprivation across three pandemic waves in Ireland: A retrospective cohort study using routinely collected data.

BACKGROUND: Since the pandemic onset, deprivation has been seen as a significant determinant of COVID-19 incidence and mortality. This study explores outcomes of COVID-19 in the context of material deprivation across three pandemic waves in Ireland. METHODS: Between 1st March 2020 and 13th May 2021, 252,637 PCR-confirmed COVID-19 cases were notified in Ireland. Cases were notified to the national Computerised Infectious Disease Reporting (CIDR) system. Each case was geo-referenced and assigned a deprivation category according to the Haase-Pratschke (HP) Deprivation Index. Regression modelling examined three outcomes: admission to hospital; admission to an intensive care unit (ICU) and death. RESULTS: Deprivation increased the likelihood of contracting COVID-19 in all age groups and across all pandemic waves, except for the 20-39 age group. Deprivation, age, comorbidity and male gender carried increased risk of hospital admission. Deprivation was not a factor in predicting ICU admission or death, and diagnosis in wave 2 was associated with the lowest risk of all three outcomes. CONCLUSIONS: Our study suggests that COVID-19 spreads easily through all strata of society and particularly in the more deprived population; however this was not a consistent finding. Ireland is ethnically more homogenous than other countries reporting a larger deprivation gradient, and in such societies, structural racial differences may contribute more to poor COVID outcomes than elements of deprivation.

Enhancement of antiviral immunity by small molecule antagonist of suppressor of cytokine signaling.

Suppressors of cytokine signaling (SOCSs) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as type I and type II IFNs. We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and encephalomyocarditis virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and encephalomyocarditis virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFN-beta, which may play a role in the antagonist antiviral effect at the cellular level. Ab neutralization suggests that constitutive IFN-beta may act intracellularly, consistent with recent findings on IFN-gamma intracellular signaling. pJAK2(1001-1013) also synergizes with IFNs as per IFN-gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the kinase inhibitory region of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the IFN-gamma activation site promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on humoral and cellular immunity as well as an enhancement of polyinosinic-polycytidylic acid activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.

Individual and Synergistic Anti-Coronavirus Activities of SOCS1/3 Antagonist and Interferon α1 Peptides.

Suppressors of Cytokine Signaling (SOCS) are intracellular proteins that negatively regulate the induction of cytokines. Amongst these, SOCS1 and SOCS3 are particularly involved in inhibition of various interferons. Several viruses have hijacked this regulatory pathway: by inducing SOCS1and 3 early in infection, they suppress the host immune response. Within the cell, SOCS1/3 binds and inhibits tyrosine kinases, such as JAK2 and TYK2. We have developed a cell penetrating peptide from the activation loop of the tyrosine kinase, JAK2 (residues 1001-1013), denoted as pJAK2 that acts as a decoy and suppresses SOCS1 and 3 activity. This peptide thereby protects against several viruses in cell culture and mouse models. Herein, we show that treatment with pJAK2 inhibited the replication and release of the beta coronavirus HuCoV-OC43 and reduced production of the viral RNA, as measured by RT-qPCR, Western blot and by immunohistochemistry. We confirmed induction of SOCS1 and 3 in rhabdomyosarcoma (RD) cells, and this induction was suppressed by pJAK2 peptide. A peptide derived from the C-terminus of IFNα (IFNα-C) also inhibited replication of OC43. Furthermore, IFNα-C plus pJAK2 provided more potent inhibition than either peptide alone. To extend this study to a pandemic beta-coronavirus, we determined that treatment of cells with pJAK2 inhibited replication and release of SARS-CoV-2 in Calu-3 cells. We propose that these peptides offer a new approach to therapy against the rapidly evolving strains of beta-coronaviruses.

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ.

We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The ß-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation.

HSV-1-induced SOCS-1 expression in keratinocytes: use of a SOCS-1 antagonist to block a novel mechanism of viral immune evasion.

Keratinocytes are important for the acute phase of HSV-1 infection and subsequent persistence in sensory nervous tissue. In this study, we showed that keratinocytes (HEL-30) were refractory to IFN-gamma induction of an antiviral state to HSV-1 infection, while IFN-gamma did induce an antiviral state in fibroblasts (L929). This led us to examine the possible role of suppressor of cytokine signaling-1 (SOCS-1) in this refractiveness. RT-PCR analysis of SOCS-1 mRNA expression in HSV-1-infected cells showed a 4-fold increase for keratinocytes while having a negligible effect on fibroblasts. A similar pattern was observed at the level of SOCS-1 protein induction. Activation of STAT1alpha in keratinocytes was inhibited by HSV-1 infection. A direct effect of HSV-1 on the SOCS-1 promoter was shown in a luciferase reporter gene assay. We have developed a small peptide antagonist of SOCS-1, pJAK2(1001-1013), that had both an antiviral effect in keratinocytes against HSV-1 as well as a synergistic effect on IFN-gamma induction of an antiviral state. HSV-1 ICP0 mutant was inhibited by IFN-gamma in HEL-30 cells and was less effective than wild-type virus in induction of SOCS-1 promoter. We conclude that SOCS-1 plays an important role in the inhibition of the antiviral effect of IFN-gamma in keratinocytes infected with HSV-1. The use of SOCS-1 antagonist to abrogate this refractiveness could have a transformational effect on therapy against viral infections.