bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in solid tumors
Authors: Anderson Kristin G , Oda Shannon K , Bates Breanna M , Chiu Edison Y , Burnett Madison G , Suarez Gutierrez Magdalia L , Garcia Nicolas M , Daman Andrew W , Greenberg Philip D
Published: January 2023

Over half of patients diagnosed with high grade serous ovarian cancer will die within five years, highlighting the need for therapy innovation. Engineering T cells to target proteins uniquely overexpressed in tumors has the potential to limit tumor growth without toxicity—mesothelin (Msln) is a rational immunotherapy target as it contributes to the malignant/invasive phenotype and has limited expression in healthy cells. T cells were engineered to express a high-affinity Msln-specific T cell receptor (TCRMsln) and adoptively transferred into a disseminated ID8VEGF murine model, which recapitulates many features of human ovarian cancer. TCRMsln+ T cells preferentially accumulated within established tumors, delayed tumor growth, and significantly prolonged survival. However, T cell persistence and anti-cancer activity were limited by elements in the tumor microenvironment (TME), including Fas Ligand (FasL) that can induce apoptosis in infiltrating lymphocytes expressing Fas receptor (Fas).

To overcome this potential T cell evasion mechanism, we generated a panel of immunomodulatory fusion proteins (IFPs) containing the Fas extracellular binding domain fused to a co-stimulatory domain, replacing the natural death domain. Relative to T cells modified with only TCRMsln, T cells that expressed both TCRMsln and a Fas IFP preferentially infiltrated tumors and expanded/persisted in the TME of tumor-bearing mice. Moreover, adoptive immunotherapy with TCRMsln+IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCRMsln+ only T cells. As many solid tumors overexpress FasL, IFPs may provide an opportunity to enhance engineered adoptive T cell therapy against many malignancies.
Enhanced Fc-mediated complement activity with broadly neutralizing antibody 10E8v4 does not improve viral outcomes following SHIV challenge in rhesus macaques
Authors: Spencer David A , Goldberg Ben , Dufloo Jérémy , Pandey Shilpi , Cheever Tracy , Bruel Timothée , Schwartz Olivier , Ackerman Margie , Hessell Ann J
Published: January 2023

Fc modified broadly neutralizing antibodies (bNAbs) are being developed for both prophylactic and therapeutic treatment of HIV with some success in extending half-life, but in vivo benefits of enhanced FcγR interaction remain unclear. Here we sought to improve bNAb efficacy by increasing Fc-mediated complement activity. A panel of Fc variants was screened for complement enhancement of 10 bNAbs. Modified 10E8v4, which weakly neutralizes SHIVSF162p3 (IC80 30 mg/ml) and does not mediate ADCC, was selected for macaque studies. Compared to wildtype, the EFTAE modification resulted in greater affinity for multiple FcγRs, >2-fold enhanced complement deposition, viral lysis, and antibody-mediated complement dependent cytotoxicity, but reduced serum half-life. Subcutaneous infusion with a sub-protective 5 mg/kg dose of 10E8v4 followed by a single high dose intrarectal challenge with SHIVSF162p3 showed unmodified 10E8v4 treated animals displayed lower plasma viremia compared to the complement/FcgR dual knockout (LALA) and controls. Unexpectedly, the EFTAE group displayed higher plasma viremia than controls, and viral DNA was significantly enhanced in tissues at necropsy (9 wpi, p <0.0001). Viremia in EFTAE treated animals inversely correlated with plasma neutralization titer at time of challenge; however, elevated viremia was not seen in animals treated with 10 and 20 mg/kg Ab, suggesting that complement mediated enhanced infection was mitigated by greater neutralizing activity. These are the first in vivo results substantiating in vitro reports of complement dependent enhanced infection mediated by non-neutralizing HIV Abs and indicate that future studies modulating complement induction by therapeutic bNAbs are warranted.
Development of a Multiplex Bead Serological Assay for Use in Global Laboratories to Assess Population Immunity to Measles and Rubella
Authors: Coughlin Melissa M , Matson Zachary , Sowers Sun B , Priest Jeffery , Smits Gaby , van der Klis Fiona , Hickman Carole , Scobie Heather , Rota Paul , Bankamp Bettina
Published: January 2023

Serosurveys are an important tool for the evaluation of population immunity and guidance of immunization activities. The multiplex bead assay (MBA) offers multiple benefits over standard serological assays and this study sought to compare the use of MBA to measure humoral immunity to measles and rubella compared to the enzyme immunoassay (EIA) for both viruses and the plaque reduction neutralization (PRN) for measles. Multiplex beads coupled with laboratory-produced purified measles whole virus antigen (WVA) correlated better to EIA and PRN than baculovirus-expressed measles nucleoprotein (N); therefore, a commercial antigen source of measles WVA was evaluated. The commercial measles WVA demonstrated significant correlation using Pearson’s correlation coefficient to the laboratory-produced WVA MBA (R=0.995, p=<0.0001) and demonstrated a strong linear relationship between the two WVA MBA assays (R2= 0.926). The commercial measles WVA MBA correlated strongly to PRN (R=0.9530, p=<0.0001) with a linear relationship comparable to the laboratory-produced WVA and PRN (R2=0.716 and R2=0.768, respectively). Receiver operator curve (ROC) analysis resulted in a seroprotection cutoff of 153 mIU/ml, similar to the established correlate of protection of 120 mIU/ml, with a sensitivity of 98% and a specificity of 84%. Comparison of the rubella MBA to EIA demonstrated a strong linear correlation of the two assays (R=0.959 and R2=0.919). ROC analysis yielded a cutoff of 9.36 IU/ml, similar to the accepted cutoff of 10 IU/ml for seroprotection with a sensitivity of 99% and a specificity of 100%. The results presented here support the use of the MBA as a tool to conduct multiplexed serosurveys to assess population immunity to measles and rubella.
Impact of pre-existing specific influenza antibodies as a determinant of disease severity on influenza virus infected patients: a multicenter observational cohort study
Authors: Aydillo GOmez Teresa A , Merino Alba Escalera , Aslam Sadaf , Carratala Jordi , Krammer Florian , Garcia-Sastre Adolfo , Matia Elisa Cordero
Published: January 2023

It is unclear to what extent pre-existing antibody-mediated immunity shapes influenza virus infection in humans. Similarly, the influence of previous history of influenza vaccination on disease severity and clinical outcome is not well understood. In here, we aimed to determine the balance of pre-existing antibodies against the hemagglutinin (HA) and neuraminidase (NA) protein of influenza virus that correlates with protection in humans. We measured hemagglutination inhibition (HI) titers and anti-HA (stalk and full-length) and anti-NA antibodies at the onset of the influenza infection on a cohort of H1N1/Cal09 influenza virus-infected patients (solid organ transplant recipients, SOTRs). Results demonstrated that the antecedent of TIV vaccination was the only controllable factor associated with lower risk of severity of disease in influenza virus-infected SOTRs. The presence of lower respiratory symptoms (LRS) significantly determined disease outcome at hospital admission (severe vs. mild, 9, 75% vs. 19, 32·1%, OR 95% 6·3, p=0·01). Moreover, a predicted probability curve for both anti-HA full-length and stalk (OR 0·13, p=0·02 and OR 0·06, p=0·03, respectively) demonstrated that the probability of developing LRS decreased with increasing assay log-values. However, multivariate logit model adjusted for HI, anti-HA (full-length and stalk) and anti-NA titers demonstrated that only anti-HA stalk antibodies were independently associated with influenza severity. Our study demonstrated that when HI antibodies fail to confer protection from symptomatic influenza infection, pre-existing HA stalk antibodies protected against severe disease in SOTRs.
Structural basis of neutralization of Powassan virus by monoclonal antibodies
Authors: Errico John M , VanBlargan Laura A. , Nelson Christopher A. , Diamond Michael S. , Fremont Daved H.
Published: January 2023

Powassan virus (POWV) is an emerging tick-borne flavivirus endemic to the northern and northeastern United States. Severe human POWV infection is characterized by fatal encephalitis in 10% of patients and long-term neurological sequelae in 50% of survivors. Little is known about humoral immunity to POWV, and there are currently no licensed vaccines or therapeutics available for treatment. We have generated a panel of 23 murine antibodies against POWV that separate into six epitope groups based on competitive binding. Two of the groups target domain III of the viral envelope protein; mapping by alanine scanning mutagenesis places their epitopes on the A-strand and C-C′ loop of domain III, respectively. These DIII-specific mAbs display potent neutralization in vitro and robust protection in a mouse model of POWV infection. X-ray crystallographic analysis reveals that POWV-80, a C-C′ loop antibody, adopts a binding orientation similar to a class of potently neutralizing flavivirus antibodies that target the lateral ridge epitope on domain III, while POWV-61 binds primarily to the A-strand and B-C loop. Our results indicate that antibodies to POWV can target epitope regions similar to those observed on mosquito-transmitted flaviviruses by employing unique binding modes that may facilitate enhanced neutralizing and protective efficacy. Current efforts are focused on defining the determinants of antibody potency and understanding antibody binding in the context of the virion.
Severe fever with thrombocytopenia syndrome (SFTS) induce innate immunity disorder particularly in monocyte subsets
Authors: Zhang Li , Fu Yuxuan , Wu Allen
Published: January 2023

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with a high mortality rate in humans, which is caused by SFTS virus (SFTSV), a novel phlebovirus in the Bunyaviridae family, is tick borne and is endemic in Eastern Asia. Previous study found that SFTSV can infect and replicate in macrophages in vivo and in vitro. However, the role of macrophages in virus replication and the potential pathogenic mechanisms of SFTSV in macrophage remain unclear. In this study, we provided evidence that the SFTSV infection drove macrophage differentiation skewed to M2 phenotype, facilitated virus shedding and resulted in viral spread. We showed evidence that miR-146a and b were significantly upregulated in macrophages during the SFTSV infection, driving the differentiation of macrophages into M2 cells by targeting STAT1. Further analysis revealed that the elevated miR-146b but not miR-146a was responsible for IL-10 stimulation. We also found that SFTSV increased endogenous miR-146b-induced differentiation of macrophages into M2 cells mediated by viral non-structural protein (NSs). The M2 skewed differentiation of macrophages may have important implication to the pathogenesis of SFTS.

Myeloid cell populations are influenced in patients’ peripheral blood during acute SFTS virus infection. SFTSV specifically targets human CD14+ blood monocytes, inducing phenotypic shift. Our studies imply that monocytes/macrophages may support persistent SFTSV viral infection. However, the evidence that monocytes/macrophages are directly involved in the development of SFTS in patients, particularly those in the acute phase.
HIV-infected apoptotic hepatocytes program macrophages for inflammasome development: role of alcohol
Authors: Osna Natalia A , New-Aaron Moses , Ganesan Murali , Dagur Ragubendra S , Poluektova Larisa Y
Published: January 2023

Liver injury is a second cause of non-AIDS mortality in HIV-infection, and the progression to end-stage liver disease is more rapid in alcohol abusers. Hepatocytes comprise 80% of liver cells and are strong ethanol metabolizers. To elucidate whether hepatocytes are HIV-infected, we measured the kinetics of HIVgag RNA (RT-PCR) and p24 (Western blot) expression in Huh7.5-CYP (hepatocyte-like cells) exposed to HIV-1ADA in the presence or absence of acetaldehyde-generation system (AGS). The measurements were done at days 1, 3 and 5 post-infection. We found that the highest levels of HIV markers were observed at day 1, with further decrease by day 3 and 5. AGS treatment substantially increased these levels. The accumulation of HIV in hepatocytes was accompanied by activation of oxidative stress, judged by increasing reactive oxygen species (ROS) production at day 3 and 5 post-infection and enhanced expression of 4-hydroxynonenal (4HNE) protein adducts, which increased caspase 3 cleavage. To mimic proapoptotic effect of acetaldehyde and study the involvement of apoptotic hepatocytes in liver inflammation, we incubated apoptotic HIV-infected or uninfected Huh7.5-CYP cells with monocyte-derived human macrophages (MDMs) and then measured inflammasome activation based on NLRP3, caspase 1, IL-1β and IL-18 mRNAs. Activation of inflammasome in MDMs was more prominent when HIV-infected apoptotic cells were engulfed by MDM compared with uninfected cell engulfment. We conclude that acetaldehyde synergies with HIV to induce oxidative stress and apoptosis in hepatocytes. The engulfment of apoptotic HIV+ hepatocytes leads to inflammasome activation in macrophages, thereby promoting progression to liver injury.
Glutamine metabolism is required for alveolar macrophage proliferation
Authors: Wang Min , Zhu Bibo , Sun Jie
Published: January 2023

Alveolar macrophages (AMs) are the most abundant immune cells in the lung during homeostasis. AMs are critical for normal lung development, surfactant metabolism and host defense against various respiratory pathogens. Despite being terminally differentiated cells, AMs are able to proliferate and self-renew to maintain their compartment without the input of the hematopoietic system in the adulthood during homeostasis. However, the molecular and metabolic mechanisms modulating AM proliferative responses are still incompletely understood. Here we have investigated the metabolic regulation of AM proliferation and self-renewal. We found that mitochondrial metabolism is critical for AM proliferation in vitro and in vivo. Interestingly, inhibition of glucose uptake or fatty acid oxidation did not significantly impact AM proliferation. Rather, inhibition of the glutamine metabolic process abolished AM proliferation in vitro and in vivo following their partial depletion during influenza infection. We further demonstrated that glutamine metabolism was required for AM mitochondrial respiration. Our data indicate that glutamine is a critical substrate fueling mitochondrial metabolism required for AM proliferation. Our study is expected to shed light on the metabolic regulation of AM maintenance and repopulation during homeostasis and following acute respiratory viral infection.
New insights into the role of N-glycosylation involved in molecular immune checkpoint regulation by human cytomegalovirus glycoproteins with the implication in cancer therapy
Authors: Nemcovicova Ivana , Benko Mario , Lenhartova Simona , Kempova Viera , Nemcovic Marek
Published: January 2023

The regulation of immune checkpoint molecules, such as BTLA or TIGIT, on immune cells occurs during persistent viral infections. These pathways are important for preventing immune-driven pathology but can also limit immune-mediated clearance of the infection. The recent success of immune checkpoint blockade in cancer therapy suggests that targeting these pathways would also be effective for preventing and treating a range of infectious diseases. Here, we report our current study of immune checkpoint pathways in the pathogenesis of human cytomegalovirus and discuss the potential for therapeutically targeting these pathways in this setting.

Human cytomegalovirus (HCMV) belongs to the beta herpes virus family that has co-evolved with the host immune system. UL144 and UL141 are found exclusively in clinical HCMV strains; while UL144 encodes a structural homologue of the herpesvirus entry mediator HVEM (target of BTLA), UL141 mimics TIGIT immunoreceptor. UL144 plays a role in virus-mediated immune evasion by transmitting inhibitory signals to downregulate T-cell responses and UL141 play significant role in NK cell evasion mechanism. Their sequences show considerable number of N-linked glycosylation sites located mostly in extracellular part. Many of them are not present in other viral species that suggest this glycosylation is important in human setting and may play a role in ligand-binding recognition. Here, we present characterization of recombinant HCMV glycoproteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and intact protein analysis. The individual glycan profiles followed protein-binding studies showed specific function of glycosylation in immune recognition.
Role of Tonsillar Innate and Adaptive immune cells during SIV infection in Rhesus macaques
Authors: Liyanage Namal , Shukla Rajni Kant , Reinhold-Larsson Nicole , Duncan Michael , Hatharasinghe Amila
Published: January 2023

In recent years, the incidence of HIV has decreased, however it continues to spread globally. 70-90% of individuals infected with HIV develop oral mucosal infections. Interestingly, HIV infected patients are at higher risk of developing Epstein–Barr virus (EBV)-associated B cell malignancies in tonsils. However, the potential role of oral immunity in pathogenesis of oral lesions is unknown. Tonsils are considered oral-pharyngeal mucosal associated lymphoid tissues and play an important role in oral mucosal immunity. However, detailed study of human tonsillar immune cells during HIV infection is restricted due to limited sample availability. In this study, we investigated the role of innate and adaptive immune cells in macaque tonsils by high dimensional flow cytometry analysis. Our data revealed a novel innate B cell subset that express NK cell receptors (NKB) significantly increased during SIV chronic infection in tonsils. We also found loss of ploy functional memory CD8 T cells during SIV infections. Tonsillar IL17 producing Cytotoxic T cells (Tc17+) were significantly increased during infection despite a loss in Th17 cells. Consistent with our previous studies in gut mucosal tissues, we found a significant loss of NCR+ILC3 in the tonsils during SIV infection. However, in contrast to the gut, we observed an expansion of oral CD56+ NK cells in tonsils during chronic SIV infection. These data suggest that the SIV associated immune responses are significantly distinct in the tonsils compared to other mucosal tissues. These data extend our understanding of the oral innate immune system during SIV infection and could aid future studies in evaluating the role of tonsillar immune cells during HIV associated oral mucosal infections.
Oligoadenylate Synthetase 1 enhances DNA sensor cGAS translation to mediate WNV antiviral activity
Authors: Perez Joseph , Harioudh Munesh K. , So Lomon , Nair Sharmila , McCormick Kevin , Ghosh Arundhati , Shao Lulu , Srivastava Rashmi , Ebert Thomas , Atianand Maninjay , Hornung Veit , Diamond Michael S. , Savan Ram , Sarkar Saumendra N.
Published: January 2023

Interferons inhibit virus replication through the expression of interferon stimulated genes (ISGs). We have found that a specific isoform of one such ISG, Oligoadenylate Synthetase 1 (OAS1) limits host susceptibility to West Nile Virus (WNV) infection through a non-canonical mechanism. This OAS1 isoform (OAS1 P46) in humans is generated due to an alternative splice acceptor site at the C-terminus of OAS1 gene. The SNP rs10774671 at this site has been associated with disease severity to WNV. We show that human OAS1-KO cells have lower basal levels of cGAS protein and can be rescued by OAS1 P46 independent of its enzyme activity. Additionally, through RNA-seq, SILAC, polysome profiling and radiolabeling experiments, we show that OAS1 does not regulate mRNA transcription but instead enhances protein translation of a select set of mRNAs, thereby increasing the steady state and induced levels of specific proteins with antiviral properties. Inducible expression of OAS1 P46 in cGAS-KO cells does not suppress WNV replication, suggesting that the antiviral activity of OAS1 is mediated through cGAS. We also have established functional equivalence between OAS1 P46 and a mouse ortholog, Oas1b (no enzyme activity), which similarly affects WNV susceptibility. Oas1b inhibits WNV infection and pathogenesis in vivo and inhibits WNV infection in vitro in cGAS-dependent manner. Through RNA-protein crosslinking experiments we have identified target mRNAs that bind to OAS1 and have demonstrated increased sensitivity of WNV in Oas1b RNA binding mutants. Our findings suggest a novel mechanism of OAS1 in which it binds to target mRNAs, enhances the translation of these RNAs and limits virus infection.
Characterization of a novel dengue virus strain and its implication for vaccine development
Authors: Chen Rita E , Errico John M , Gordon David , Dowd Kimberly A. , Cardosa M. Jane , Sariol Carlos A. , Kallas Esper G. , Sekaly Rafick-Pierre , Vasilakis Nikos , Pierson Ted , Fremont Daved H. , Diamond Michael S.
Published: January 2023

Dengue virus (DENV) causes up to 390 million infections yearly worldwide. Currently, four serotypes, DENV 1–4, circulate between Aedes mosquitoes and humans and can cause severe dengue, which has been associated with secondary DENV infection by a different serotype. DENV strain DKE-121 was recently isolated from Malaysia and is 12–38% different in its envelope protein from DENV 1–4. While previous studies of DENV genetic variation have described up to 3% amino acid divergence within a serotype, this virus differs by up to 12% from DENV4, suggesting its possible classification as a new serotype. The potential of a new DENV serotype emerging into circulation raises concerns of increased risk of severe dengue and uncertainty of how protective current tetravalent vaccine efforts would be. We tested the ability of serum from mice, non-human primates (NHPs), and humans that were infected or vaccinated with DENV4 and DKE-121 to neutralize infection of DENV4 and DKE-121. NHPs and humans immunized with DENV4 had 2 and 5-fold higher neutralizing titers (EC50 values), respectively, against the homologous DENV4 than DKE-121. However, mice boosted with DENV4 had similar neutralizing titers against DENV4 and DKE-121. In comparison, DKE-121 infection in NHPs and mice elicited 3-fold and up to 21-fold, respectively, higher titers against DKE-121 than DENV4. In addition, DKE-121 was poorly neutralized by type-specific anti-DENV4 monoclonal antibodies (mAbs), and reciprocally neutralizing mAbs against DKE-121 did not inhibit DENV4 infection. Using polyclonal sera and mAbs, DKE-121 and DENV4 show substantive differences in antigenicity. Ongoing studies are aimed at determining how these differences affect antibody-mediated protection in vivo.
Immune responses in Mayaro infected mice and its relation with viral replication and muscle damage
Authors: Neris Romulo , Figueiredo Camila , Leopoldino Daniel , da Silva Mariana , Almeida Juliana , dos-Santos Julio , Figueiredo Claudia , Bellio Maria , Bozza Marcelo , Miranda Iranaia
Published: January 2023

Mayaro virus (MAYV) is a tropical arbovirus first described in forest regions of the South America, and recently associated with urban circulation. Mayaro infections leads to a disease with high rate of persistent arthralgia and myalgia in humans. Despite this, pathogenesis and host immune response of MAYV infections are still limited. The aim of this work was to evaluate the immune response and cell profile during MAYV infections. We developed a disease model infecting SV129 wild-type (WT), SVA129 Type I Interferon receptor deficient (IFNAR−/−), C57BL/6 WT and C57BL/6 recombination activation gene-1 deficient (RAG−/−) mice, and accessing replication, tissue damage, and inflammation through time. While adult WT mice are resistant to infection, IFNAR−/− mice exhibited increasing viral load up to 6 days after infection, footpad swelling, lethargy and weight loss. Histological analysis showed extensive necrosis sites in infected IFNAR−/− mice and also presence of mononucleated cell infiltrate. We detected elevated expression of inflammatory cytokines like TNF, IL-6, KC, IL-1, MCP-1 and RANTES, and decreased TGF-beta in infected muscle. In RAG−/− mice, MAYV infection lead to a persistent replication being detected at blood and tissues up 40 days post infection. However, infected RAG−/− mice did not develop any signs of muscle damage or infiltrate during early and late infection and neither the expression inflammatory cytokines. So far, our data demonstrates that innate and adaptive immunity are critical to restrict MAYV replication. Adaptive immunity is also involved in MAYV-induced tissue damage. These results contribute in underlying MAYV pathogenesis and further experiments must to be made to elucidate the mechanisms involved.
Gp120 plays a role in the hypo-functionality of the cholinergic anti-inflammatory pathway in HIV infection
Authors: Samarani Suzanne , Aldabah Zainab , Ahmad Ali
Published: January 2023

We have investigated the functionality of the cholinergic anti-inflammatory pathway (CAP) in HIV-1 infected individuals. The pathway plays an important role in regulating inflammatory process in the body. Acetylcholine (ACh), a neurotransmitter, is the main effector molecule of the CAP. It is produced by CD4+ T cells, intestinal epithelial cells as well as by neurons in the body, and exerts its effects via nicotinic (n) and muscarinic (m) Acetylcholine receptors (AChR). Through these receptors, ACh modulates differentiation, proliferation and effector functions of immune cells in the body. Little is known about the regulation of the non-neuronal CAP in HIV infections. To investigate it, we measured concentrations of ACh and the Secreted Ly6/Urokinase type plasminogen activator Receptor-related Peptide-1 (SLURP-1, an allosteric ligand for the α-7nAChR) in the circulation of HIV-infected individuals. The concentrations of both ACh and SLURP-1 were increased in treatment-naïve HIV-infected individuals. In contrast to an increase in the concentrations of ACh and SLURP-1, the expression of the α-7nAChR was significantly decreased on immune cells in the virus-infected individuals. We investigated the effects of gp-120 on the expression of this receptor. We found that the treatment of the human PBMC with recombinant gp120 reduces the expression of α-7nAChR on immune cells. Interestingly, the treatment increases the binding of α-bungarotoxin, a ligand for nAChR. Collectively, these results suggest that the non-neuronal CAP is functionally compromised in HIV-infected individuals and gp120 plays a role in the hypo-functionality by down-regulating the expression of α-7nAChR on the peripheral blood immune cells.
Dynamic decay of viral reservoirs in systemic and lymphoid compartments of SIV-infected rhesus macaques on long-term combined antiretroviral therapy
Authors: Wang Xiaolei , Ziani Widade , Shao Jiasheng , Veazey Ronald S. , XU HUANBIN
Published: January 2023

Material & Method

By quantitative real-time/digital PCR, we here longitudinally examined dynamic decay of viral parameters (cell-associated unspliced and multiply spliced viral RNA, episomal 2-LTR circular viral DNA and integrated proviral DNA) in systemic and lymphoid tissues in SIV-infected rhesus macaques on long-term combined antiretroviral therapy (cART, up to 20 months), and evaluated reservoir size and timing of viral rebound after treatment interruption. SIV RNA or DNA copy numbers were normalized and expressed as per 106 cell equivalents. Frequencies and absolute numbers of peripheral CD4+ T cells were analyzed before and after suppressive antiretroviral therapy.

Results

Our results showed that suppressive ART significantly reduced levels of cell-associated unspliced and multiply spliced SIV RNA/2-LTR DNA to undetectable, yet levels of integrated proviral DNA alone were stably maintained in systemic and lymphoid compartments throughout cART, which correlated with levels of viral replication prior to initiating cART. Following ATI, rapid clonal expansion of cells with integrated provirus were detected, as indicated by undetectable spliced tat/rev RNA and 2-LTR but increased levels of total proviral DNA, which may have contributed to subsequent viral rebound. Cell-associated SIV RNA and DNA levels completely recovered to pre-treatment levels in blood, spleen, lymph node, tonsil and gut-associated lymphoid tissues by 3 months after ATI.

Conclusions

These findings suggest that discriminating integrated HIV proviral DNA from non-integrated or defective proviruses in tissues are useful biomarkers for monitoring HIV persistence and latency for treatment and cure strategies.
Fibroblasts with trisomy 21 are more susceptible to damage from Herpes Simplex Virus-1 infection compared to typical fibroblasts
Authors: Singh Gurkirat , Bustillos-Saucedo Amalia , Royzman Kristina , Keefe Caitlin , Whitehead Tyler , Theard Curtis , James Stephanie
Published: January 2023

Down syndrome (trisomy 21; T21) is characterized as the presence of an extra copy of chromosome 21, resulting in cognitive and physical disorders. Individuals with T21 have a higher risk of mortality due to infection compared to typical individuals, despite having an extra interferon-alpha receptor (IFNAR), as this gene is located on q21. Herpes Simplex Virus 1 (HSV) causes the common cold sore, and can also result in serious disease such as encephalitis and periodontitis. There has been little research done with HSV in relationship to T21 although one study showed higher HSV burden in gum lesions in individuals with T21 compared to typical adults. The objective of this research was to measure differences in viral replication\protein expression and cellular responses to HSV infection in fibroblasts with and without T21. Viral replication was measured using qRT-PCR and protein expression by Western blot in cells infected with HSV, cellular gene expression was measured using mRNAseq. Viral replication and protein expression between the two groups was not significantly different although disomic cells had a slight increased rate of viral replication compared to cells with T21. However, it was observed that cells with T21 had increased cytopathic effects after 24 hours compared to typical cells. RNA sequencing data showed significantly different gene expression patterns between the two groups, notably in the EIF2 signaling pathway. Despite similar viral replication and protein expression, data presented here suggests that the fibroblasts with T21 are more susceptible to damage by viral infection. This may be due abnormalities in protein translation mechanisms yet to be delineated.
Demyelination and interferon signaling in axons cause retrograde upregulation of neuronal ISGylation pathway genes
Authors: Clarkson Ben D.S. , Johnson Renee , Howe Charles L
Published: January 2023

Cognitive impairment in MS is associated with diffuse gray matter injury, which remains poorly understood. Given that axons span both white and gray matter regions, it’s possible that signals within these axons contribute to the spread of diffuse injury in the brain. We show that retrograde IFNg signaling in axons (in vitro human and murine) and demyelination (murine in vivo) cause transcriptional / translational changes in neuronal cell bodies. Chief among the candidate signaling pathways identified is ISGylation, a process whereby a multitude of cellular proteins are modified by the attachment of one or more ISG15 molecules. Using custom adeno-associated viral vectors we tested how altering ISGylation in neurons affects neuronal synaptic function and how it affects neuronal responsiveness to specific inflammatory factors in cell cultures. We also measured ISGylation in MS brain tissues to see if neuronal ISGylation correlates with gray matter injury in MS. We report early evidence that increased neuron ISGylation alters the composition of neuron-derived extracellular vesicles. Microglia treated with extracellular vesicles from neurons overexpressing ISGylation pathway genes exhibited morphology consistent with increased phagocytic activity and increased mRNA expression of inflammatory cytokines including: CCL2, IL1b, TNFa, iNOS, CXCL10, CCL5, and IL6. At this point, it is still unclear how these changes are related to gray matter pathology. However, it is possible that microglial activation by these vesicles is involved with driving synapse loss as activated microglia are known to be capable of stripping neuronal synapses.
Chemokine CCL28 treats oropharyngeal candidiasis through rapid membrane disruption
Authors: He Jie , Thomas Monica A , Van Why Emma , Grayson Mitchell H , Volkman Brian F , Huppler Anna R
Published: January 2023

Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of these infections is limited by the paucity of anti-fungal drug classes. Antimicrobial peptides are a component of the immune response to fungi and promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the spectrum of in vitro antimicrobial activity and the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis (OPC), as well as the mechanism of anti-Candida activity. CCL28 is effective against multiple clinical strains of C. albicans, but less effective against C. glabrata. In the mouse model of OPC, application of wild type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to clinical anti-fungal agents. Polyamine protein transporters are not required for CCL28 anti-Candida activity. Application of wild type CCL28 to C. albicans results in membrane disruption as measured by solute movement and enzyme leakage. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results indicate CCL28 functions in the immune response to disrupt fungal membranes and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.
Transcription factor Fli-1 impacts IL-17 expression and affects immune cell infiltration into the kidney in lupus-prone mice
Authors: Sato Shuzo , Zhang Xian , Matsumoto Haruki , Temmoku Jumpei , Fujita Yuya , Matsuoka Naoki , Furuya Makiko , Asano Tomoyuki , Kobayashi Hiroko , Watanabe Hiroshi , Migita Kiyoshi
Published: January 2023

Objectives

Transcription factor Fli-1 affects lupus nephritis development through regulating several cytokines and chemokines. Th17 immune response is important to maintain inflammation in lupus nephritis. The purpose of this study is to elucidate whether Fli-1 affects renal IL-17 expression and influence immune cell infiltration into the kidney.

Methods

Sera were collected from Fli-1-heterozygous (Fli-1+/−) MRL/lpr mice and wild-type (WT) littermates, and the concentration of IL-17 was measured by enzyme-linked immunosorbent assay. Expression of IL-17 and related molecules were measured by real-time polymerase chain reaction (rt-PCR) analysis. Kidneys from Fli-1+/− and WT MRL/lpr mice were stained and evaluated the grade of inflammation. IL-17 expression was detected using anti-mouse IL-17 antibodies. Immunofluorescence staining was performed using anti-CD3, CD4, and anti-IL-17 antibodies to detect T cells related to Th17 responses. In addition, to detect CCL20 expression as a chemoattractant of Th17 cells, anti-CCL20 antibodies and anti-CD11b antibodies were used to detect monocytes expressed CCL20.

Results

Fli-1+/− MRL/lpr mice had tendency of decreased serum IL-17 levels. Expression of IL-17 was significantly decreased in Fli-1+/− MR/lpr mice kidney interstitium, accompanied by decreased IL-6, STAT3, and IL-1β expression by rt-PCR. CD3 or CD4/IL-17 double-positive immune cells were significantly decreased in Fli-1+/− MLR/lpr mice; similarly, CCL20 positive monocytes were significantly decreased.

Conclusion

Fli-1 impacts IL-17 expression into the kidney of MRL/lpr mice by reducing CD3 and CD4 positive lymphocytes expressing IL-17 as well as CD11b positive monocytes expressing CCL20.
Understanding the role of STAT4 in CD4 T cell mediated neuroinflammation
Authors: Anderson Ashlyn E , Shin Boyoung , McWilliams Ian , Bryars Morgan , Harrington Laurie E
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune disease that affects more than one million people in the United States and is characterized by the demyelination of axons in the central nervous system (CNS). While the etiology of the MS is unknown, CD4 T cells are central to disease pathogenesis. The transcription factor STAT4 is critical for Th1 cell development, is associated with risk for MS, and is required for experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Interestingly, we and others find that many Th1 associated molecules such as IL-12, IFNγ and Tbet are dispensable for EAE. Hence, we hypothesize that STAT4 functions in effector CD4 T cells to promote neuroinflammation independent of the classical Th1 pathway. To test the potential role of STAT4 in Th17 cells, we employed the adoptive transfer model EAE. While we note that STAT4 expression does not impact the development of Th17 cells, it is required for the migration of Th17 cells into the CNS and for Th17 mediated demyelination. Moreover, RNA sequencing analysis shows that STAT4 regulates the expression of >200 genes in the Th17 cells, many of which skew the balance between regulatory and inflammatory Th17 cell function such as Il10 and Tbx21, respectively. Together, these data reveal a novel role of STAT4 in controlling the functional properties of Th17 cells, and this may provide a promising therapeutic target for patients affected by Th17 driven autoimmune diseases.
Rapid and sensitive determination of cytokine release from cells without the need for sample transfer
Authors: Lazar Dan F , Kupcho Kevin R , Sondgeroth Casey A , Hsiao Kevin , Thompson David V , O’Brien Martha A , Gilden Julia K , Cali James J
Published: January 2023

We have utilized NanoLuc® Binary Technology (NanoBiT®) to develop a homogeneous and rapid assay method (≤ 70 min completion time) to measure cytokines released from cells in culture without the need for sample transfer and requiring only a standard, plate-reading luminometer for signal acquisition. Separate antibodies to a specific cytokine are labeled with the small, 11-amino acid subunit of NanoBiT luciferase (SmBiT) or its 17.6 kDa complementary subunit (LgBiT). When SmBiT- and LgBiT-labeled antibodies converge on the target cytokine, the resultant proximity of SmBiT and LgBiT reconstitutes a bright luciferase that produces light proportional to analyte levels. In this manner, immunoassays have been developed for several cytokines, including IL-1β, IL-2, IL-6 and IFN-γ. In general, these assays share LLODs < 10 pg/ml and linearity over three logs of analyte concentration, mitigating the need for sample dilution. Following 24 h treatment of human PBMCs in 96-well plates with vehicle, LPS, R848, or a combination of PMA and ionomycin, cytokine detection reagents were added directly to the culture wells containing cells and medium. Depending on stimulus, maximal signal to background ratios (S/B) achieved for the cytokines were 347-, 450-, 580- and 655-fold for IL-1β, IL-2, IL-6 and IFN-γ, respectively. In a cell model comprised of activated T cells and target Raji B cells induced for 20 h with increasing bispecific T-cell engager Blincyto®, release of IL-2 and IFN-γ were observed with an EC50 of ~0.2 ng/ml and maximal S/B for IL-2 and IFN-γ of 82- and 168-fold, respectively. The implementation of this novel detection chemistry will enable rapid “add-and-read” assays for cytokine detection for both low- and high-throughput applications.
Serum Cytokines are regulated in African Americans with High HbA1c
Authors: Kimbro K Sean , Williams Ariel
Published: January 2023

Background

Low grade inflammation is associated with T2D resulting from a cadre of serum cytokines. IL3, IL4, IL7, IL-17A, GM-CSF, and GM-CSF play a coordinated pathogenic role in many inflammatory diseases. It has been previously reported high circulating HbA1c influences serum cytokine levels and cholesterol metabolism.

Methods

A total of 120 serum samples were collected from African Americans: 68 women and 52 men. This group consists of 45 normal HbA1c and 71 high HbA1c participants. Clinical metabolic parameters were measured. Cytokines were measured using Luminex X-MAP® technology. A series of unpaired t-test were computed to determine if there were significant differences between participants with and without elevated HbA1c. Pairwise Pearson Correlation (r) test was computed for each variable.

Results

In women, IL-3 expression was upregulated 1.7-fold in the presence of high circulating glucose. In men, IL-3 expression was downregulated 1.5-fold. Individuals with high HbA1c exhibit a negative correlation between IL-3 and BMI in men and women. Male serum IL-7 levels decrease in presence of circulating high glucose while female serum IL-7 levels increase. Normal HbA1c men have a 2.7-fold higher serum IL-7 level than women with normal HbA1c. Women participants have a positive correlation with serum IL-7 and HbA1c, as well as BMI. These correlations are lost in men. In the presence of high HbA1c, there is an increase in serum levels of IL-17A, G-CSF, and GM-CSF in women with high HbA1c.

Conclusion

An increase in HbA1c increases the expression of inflammatory cytokines and the risk of metabolic disease. Our data suggests that, in a sex specific manner, several cytokines may regulate cholesterol and lipid management in T2D.
Roles of the RNA binding protein Arid5a in IL-17-mediated diseases
Authors: Taylor Tiffany , Amatya Nilesh , Li Yang , Coleman Bianca , Zhou Chunsheng , Gingras Sebastien , Gaffen Sarah L
Published: January 2023

Interleukin 17 is a proinflammatory cytokine required for host defense against extracellular microorganisms but also contributes to the pathogenesis of certain autoimmune diseases. Candida albicans is a commensal fungus that colonizes mucosal tissues. During immunosuppression, C. albicans can cause superficial infections, such as oropharyngeal candidiasis (OPC). Defects in IL-17 signaling increase susceptibility to OPC in both humans and mice. IL-17 receptor signaling involves RNA binding proteins (RBP) that regulate translation and stability of target mRNA transcripts. During OPC, IL-17 induces several RBPs including Arid5a. This RBP interacts with AU-rich elements located in the 3′ UTR of many inflammatory mRNAs. In T cells, loss of Arid5a reduces expression of IL-6 and STAT3 and leads to decreased frequency of Th17 cells. Consequently, Arid5a−/− mice are resistant to experimental autoimmune encephalomyelitis (EAE), an IL-17-driven model of multiple sclerosis. Although made by T cells, IL-17 signals on non-hematopoietic cell types. We reported an in vitro role for Arid5a in the downstream IL-17 receptor pathway. Specifically, Arid5a regulates stability and/or translation of several IL-17-dependent mRNAs including Il6. Accordingly, we postulated that Arid5a plays a critical role in protection against OPC by stabilizing and increasing expression of IL-17-responsive genes. To test this hypothesis, CRISPR/Cas9 was used to generate an Arid5a knockout mouse. We confirmed that Arid5a−/− mice are resistant to EAE. To ascertain the effects of Arid5a in OPC, we infected mice orally with C. albicans and assessed fungal loads and weight loss. Surprisingly, Arid5a−/− mice were resistant to OPC, in contrast to Il17ra−/− mice.
Neutrophilic granule protein is a novel murine LPS antagonist
Authors: Hong Jaewoo , Qu Peng , Wuest Todd , Lin P Charles
Published: January 2023

Neutrophilic Granule Protein (NGP) was previously reported as a granular protein of neutrophils in mouse, but the function has not been known clearly. We found the presence of the possible signal peptide in NGP and validated this protein is circulating in the bloodstream. In our findings, NGP is being modified post-translationally in Golgi apparatus and endoplasmic reticulum, which is a universal character of secretory molecules with a signal peptide. The secreted NGP protein could be detected both in vitro and in vivo. NGP has sequence similarity with an antimicrobial protein cathelicidin, and we observed the aspect of inflammation of NGP. Interestingly, NGP interacts with the complex of LPS and LBP. This interaction blocks the binding of the complex of LPS and LBP to Toll-like receptor 4 (TLR4) and the downstream inflammatory signals. Furthermore, the inhibitory function of NGP against the inflammatory effect of LPS could be observed in both in vitro and in vivo. With these findings, we report NGP is a novel secretory protein to mask LPS and inhibit its function.
Type 1 interferon expression and signaling occur in distinct regions in granulomas during Mycobacterium tuberculosis infection
Authors: Jespersen Alyssa M , Junecko Beth A , Talukdar Priyanka , Mattila Joshua T
Published: January 2023

Type 1 interferon (IFN1s) expression is associated with progression of tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb). This relationship has been assessed in murine models of TB and in human peripheral blood and airway cells, but IFN1 expression in human granulomas, the lesions associated with TB, and disease has not been addressed. To determine if IFN1s are expressed in granulomas, which cells express them, and which cells undergo IFN1-regulated signaling, we investigated these factors in granulomas from macaques, an animal model that recapitulates human TB. We found that IFN1s and interferon stimulated genes (ISGs) were expressed at higher levels in granulomas than uninfected lung. In granulomas, the strongest expression was observed within epithelioid macrophages, a subset of cells that are in close proximity to Mtb, but other myeloid cells including some neutrophils and alveolar macrophages also expressed IFN1s. The lymphocyte cuff showed greater expression of the IFN1 receptor, IFNAR1, and also expressed more of the ISG MX1 than the epithelioid macrophage region. These data indicate that IFN1 is expressed by epithelioid macrophages and IFN1-related signaling occurs within the lymphocyte cuff. These results suggest that IFN1 expression may be due to the presence of Mtb or Mtb antigens and that IFN1-regulated signaling and responses are spatially distinct from the regions and cells where IFN1 expression occurs.