bluuebunny/crossref_metadata_2025 huggingface dataset:
B Cell Activation Results in IKK-Dependent, but Not c-Rel- or RelA-Dependent, Decreases in Transcription of the B Cell Tolerance-Inducing Gene Ets1
Authors: Kearly Alyssa , Ottens Kristina , Battaglia Michael C , Satterthwaite Anne B , Garrett-Sinha Lee Ann
Published: January 2023

Ets1 is a key transcription factor in B cells that is required to prevent premature differentiation into Ab-secreting cells. Previously, we showed that BCR and TLR signaling downregulate Ets1 levels and that the kinases PI3K, Btk, IKK, and JNK are required for this process. PI3K is important in activating Btk by generating the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate, to which Btk binds via its PH domain. Btk in turn is important in activating the IKK kinase pathway, which it does by activating phospholipase Cγ2→protein kinase Cβ signaling. In this study, we have further investigated the pathways regulating Ets1 in mouse B cells. Although IKK is well known for its role in activating the canonical NF-κB pathway, IKK-mediated downregulation of Ets1 does not require either RelA or c-Rel. We also examined the potential roles of two other IKK targets that are not part of the NF-κB signaling pathway, Foxo3a and mTORC2, in regulating Ets1. We find that loss of Foxo3a or inhibition of mTORC2 does not block BCR-induced Ets1 downregulation. Therefore, these two pathways are not key IKK targets, implicating other as yet undefined IKK targets to play a role in this process.
IL-6/STAT3 Signaling Axis Enhances and Prolongs Pdcd1 Expression in Murine CD8 T Cells
Authors: Powell Michael D , Lu Peiyuan , Neeld Dennis K , Kania Anna K , George-Alexander Lou-Ella M M , Bally Alexander P R , Scharer Christopher D , Boss Jeremy M
Published: January 2023

CD8 cytotoxic T cells are a potent line of defense against invading pathogens. To aid in curtailing aberrant immune responses, the activation status of CD8 T cells is highly regulated. One mechanism in which CD8 T cell responses are dampened is via signaling through the immune-inhibitory receptor Programmed Cell Death Protein-1, encoded by Pdcd1. Pdcd1 expression is regulated through engagement of the TCR, as well as by signaling from extracellular cytokines. Understanding such pathways has influenced the development of numerous clinical treatments. In this study, we showed that signals from the cytokine IL-6 enhanced Pdcd1 expression when paired with TCR stimulation in murine CD8 T cells. Mechanistically, signals from IL-6 were propagated through activation of the transcription factor STAT3, resulting in IL-6–dependent binding of STAT3 to Pdcd1 cis-regulatory elements. Intriguingly, IL-6 stimulation overcame B Lymphocyte Maturation Protein 1–mediated epigenetic repression of Pdcd1, which resulted in a transcriptionally permissive landscape marked by heightened histone acetylation. Furthermore, in vivo–activated CD8 T cells derived from lymphocytic choriomeningitis virus infection required STAT3 for optimal Programmed Cell Death Protein-1 surface expression. Importantly, STAT3 was the only member of the STAT family present at Pdcd1 regulatory elements in lymphocytic choriomeningitis virus Ag-specific CD8 T cells. Collectively, these data define mechanisms by which the IL-6/STAT3 signaling axis can enhance and prolong Pdcd1 expression in murine CD8 T cells.
Variability in Severe Acute Respiratory Syndrome Coronavirus 2 IgG Antibody Affinity to Omicron and Delta Variants in Convalescent and Community mRNA-Vaccinated Individuals
Authors: Tu Michael K , Chiang Samantha H , Wong David T W , Strom Charles M
Published: January 2023

The emergence of the omicron and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters, and whether existing mRNA vaccines for the original SARS-CoV-2 strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1-specific salivary Abs to the omicron and delta variants using a cohort of mRNA-vaccinated (n = 109) and convalescent (n = 19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5, 26.7, and 14.7% in measurable signal to the omicron strain and 13.4, 2.4, and −6.4% mean reduction to the delta variant for convalescent, Pfizer-, and Moderna-vaccinated groups, respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves.
Dam–Infant Rhesus Macaque Pairs to Dissect Age-Dependent Responses to SARS-CoV-2 Infection
Authors: Langel Stephanie N , Garrido Carolina , Phan Caroline , Travieso Tatianna , Kirshner Helene , DeMarco Todd , Ma Zhong-Min , Reader J Rachel , Olstad Katherine J , Sammak Rebecca L , Shaan Lakshmanappa Yashavanth , Roh Jamin W , Watanabe Jennifer , Usachenko Jodie , Immareddy Ramya , Pollard Rachel , Iyer Smita S , Permar Sallie , Miller Lisa A , Van Rompay Koen K A , Blasi Maria
Published: January 2023

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for other respiratory viral diseases. To investigate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam–infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analyses of the respiratory tract and evaluated systemic cytokine and Ab responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam–infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum at day 1 postinfection whereas CXCL10 was induced in all animals. Both groups mounted neutralizing Ab responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal airway cells isolated at day 14 postinfection revealed significant upregulation of multiple IFN-stimulated genes in infants compared with adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared with infants. Our study in rhesus macaque monkey dam–infant pairs suggests age-dependent differential airway responses to SARS-CoV-2 infection and describes a model that can be used to investigate SARS-CoV-2 pathogenesis between infants and adults.
Characterization of Circulating Fibrocytes in People Living with HIV on Stable Antiretroviral Therapy
Authors: Dean Logan S , Chow Dominic C , Ndhlovu Lishomwa C , Boisvert William A , Chang Sandra P , Shikuma Cecilia M , Park Juwon
Published: January 2023

Highly effective combination antiretroviral therapy has reduced HIV infection to a manageable chronic disease, shifting the clinical landscape toward management of noninfectious comorbidities in people living with HIV (PLWH). These comorbidities are diverse, generally associated with accelerated aging, and present within multiple organ systems. Mechanistically, immune dysregulation and chronic inflammation, both of which persist in PLWH with well-controlled virally suppressive HIV infection, are suggested to create and exacerbate noninfectious comorbidity development. Persistent inflammation often leads to fibrosis, which is the common end point pathologic feature associated with most comorbidities. Fibrocytes are bone marrow–derived fibroblast-like cells, which emerged as key effector cells in tissue repair and pathologic fibrotic diseases. Despite their relevance to fibrosis, the circulating fibrocyte concentration in PLWH remains poorly characterized, and an understanding of their functional role in chronic HIV is limited. In this study, utilizing PBMCs from a cross-sectional adult HIV cohort study with matched uninfected controls (HIV−), we aimed to identify and compare circulating fibrocytes in blood. Both the percentage and number of fibrocytes and α-smooth muscle actin+ fibrocytes in circulation did not differ between the HIV+ and HIV− groups. However, circulating fibrocyte levels were significantly associated with increasing age in both the HIV+ and HIV− groups (the percentage and number; r = 0.575, p ≤ 0.0001 and r = 0.558, p ≤ 0.0001, respectively). Our study demonstrates that circulating fibrocyte levels and their fibroblast-like phenotype defined as collagen I and α-smooth muscle actin+ expression are comparable between, and strongly associated with, age irrespective of HIV status.
Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic–Epigenetic Cross-Talk
Authors: Greenwood Dalton L , Ramsey Haley E , Nguyen Phuong T T , Patterson Andrew R , Voss Kelsey , Bader Jackie E , Sugiura Ayaka , Bacigalupa Zachary A , Schaefer Samuel , Ye Xiang , Dahunsi Debolanle O , Madden Matthew Z , Wellen Kathryn E , Savona Michael R , Ferrell P Brent , Rathmell Jeffrey C
Published: January 2023

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit–enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor–treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.
Cytotoxic T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection. Breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load.
Authors: Harrer T , Harrer E , Kalams S A , Barbosa P , Trocha A , Johnson R P , Elbeik T , Feinberg M B , Buchbinder S P , Walker B D
Published: January 2023

Although vigorous activated and memory CTL have been associated with HIV-1 infection, data are lacking regarding the breadth of epitopes recognized in a given individual and the relationship to the viral quasispecies present in vivo. In this study we performed a detailed analysis of the HIV-1-specific CTL response in a seropositive person with documented HIV-1 infection of 15 yr duration, stable CD4 counts above 500 cells/ml, and viral load persistently below 500 molecules of RNA/ml of plasma. Epitope mapping studies revealed the presence of HLA class I-restricted CTL responses to six different epitopes in p17, p24, RT, Env, and Nef, which conferred broadly cross-reactive recognition of reported HIV-1 variants. Sequence analysis of autologous viruses revealed the absence of immune escape variants within five of the six epitopes. Despite consistently low viral RNA levels in plasma and viral DNA levels in PBMC, in vivo-activated circulating CTL were detected against three of the epitopes. Five of the six epitopes, including the three dominant epitopes, have been detected in persons with progressive disease, suggesting that nonprogressors may not target unique epitopes. This study demonstrates that HIV-1-specific CTL can be highly activated and broadly directed in the setting of an extremely low viral load, and that neither high viral load nor antigenic diversity is required for the generation of a multispecific CTL response. Although the detection of strong CTL responses, low viral load, and lack of immune escape are consistent with the hypothesis that CTL may contribute to lack of disease progression in this individual, the contribution of these responses to maintenance of the asymptomatic state remains to be determined.
Impaired learning resulting from respiratory syncytial virus (VIR2P.1028)
Authors: Espinoza Janyra , Bohmwald Karen , Céspedes Pablo , Gomez Roberto , Riquelme Sebastían , Cortes Claudia , Valenzuela Javier , Sandoval Rodrigo , Pancetti Floria , Bueno Susan , Riedel Claudia , Kalergis Alexis
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of respiratory distress in infants worldwide. Neurologic alterations, such as seizures and ataxia, have been associated with RSV infection. We demonstrate the presence of RSV proteins and RNA in zones of the brain—such as the hippocampus, ventromedial hypothalamic nucleus, and brainstem—of infected mice. One month after disease resolution, rodents showed behavioral and cognitive impairment in marble burying (MB) and Morris water maze (MWM) tests. Our data indicate that the learning impairment caused by RSV is a result of a deficient induction of long-term potentiation in the hippocampus of infected animals. In addition, immunization with recombinant bacillus Calmette-Guérin (BCG) expressing RSV nucleoprotein prevented behavioral disorders, corroborating the specific effect of RSV infection over the central nervous system. Our findings provide evidence that RSV can spread from the airways to the central nervous system and cause functional alterations to the brain, both of which can be prevented by proper immunization against RSV.
The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRβ and TCRα Genes and Influences Development of αβ T Cell Lineages
Authors: Burn Thomas N , Miot Charline , Gordon Scott M , Culberson Erica J , Diamond Tamir , Kreiger Portia A , Hayer Katharina E , Bhattacharyya Anamika , Jones Jessica M , Bassing Craig H , Behrens Edward M
Published: January 2023

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRβ and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRβ and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αβ T cells, decreased genesis of invariant NK T lineage αβ T cells, and mature CD4+ αβ T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRβ and TCRα gene recombination and influence differentiation of αβ T lineage cells, thereby establishing replete diversity of αβ TCRs and populations of αβ T cells while restraining generation of potentially autoreactive conventional αβ T cells.
Co-administration of recombinant chicken IL-7 or chicken NK-lysin peptide 2 increases efficacy of Eimeria tenella elongation factor-1α in Eimeria maxima infected chicken
Authors: Lee Youngsub , Park In-kyung , Wickramasuriya Samiru S , Lillehoj Hyun S
Published: January 2023

This study was conducted to improve the efficacy of recombinant Eimeria tenella elongation factor-1α (EF-1α) antigen by co-administrating recombinant chicken IL-7 or chicken NK-lysin peptide 2 in Eimeria maxima-challenged chickens. On day 4, birds were administered with following five groups: (CON) control birds without Eimeria infection, (NC) sterile PBS solution plus adjuvant (ISA 78 VG), (COM 1) 100 μg of recombinant EF-1α plus ISA 78 VG, (COM 2) COM 1 component plus 1 μg of recombinant chIL-7, and (COM 3) COM 2 component plus 5 μg of cNK2 peptide. One week after, the boosting immunization were administrated with same concentration of components as the primary immunization. All birds except CON were orally inoculated with E. maxima (1.0 × 104 oocysts/bird) on day 19. Animal experiment demonstrated that the birds immunized with recombinant EF-1α showed greater serum anti EF-1α level, and co-administration of recombinant chIL-7 increased the level of IL-7 in serum. Vaccination of COM 2 mitigated intestinal lesion formation at 6 days post-infection (dpi) and increased body weight at 6 dpi and 9 dpi. Vaccination of COM 3 reduced fecal oocyst shedding compared with NC. E. maxima infection up-regulated expression level of jejunal proinflammatory cytokines (IL-β, IL-17F) and type I cytokines (IFN-γ and IL-10), and those expression levels were decreased by administration of COM 1, COM 2 or COM 3. Down-regulated expression of tight junction (TJ) protein (Jam2 and Occludin) by E. maxima challenge were increased by COM 2or COM 3, respectively. Overall animal experiment results demonstrated a potential effect that co-administration of recombinant IL-7 or cNK-2 peptide can increase the efficacy of recombinant EF-1α in E. maxima-infected chicken.

Funding was partly supported by ARS CRIS 8042-32000-107-00D, NIFA SBIR 2015-33610-23499, and NIFA SBIR 2016-33610-25686.
Direct MHC Class I Antigen Presentation Regulation by Ribosomal Biogenesis Inhibitor CX-5461
Authors: Kazemi Soheila , Dolan Brian P
Published: January 2023

Direct MHC class I antigen presentation relies on on-going protein synthesis by the antigen presenting cell. This is especially true for the presentation of peptides derived from Defective Ribosomal Products (DRiPs) which are inherently defective proteins rapidly degraded following their synthesis. Recently an inhibitor of RNA polymerase I, known as CX-5461, has been described which can inhibit ribosome biogenesis. Here we queried if interruption of ribosome biogenesis could impact direct antigen presentation using a reporter protein whose stability can be controlled post-translationally. We first determined a dose and time of treatment with CX-5461 which was not toxic to cells, but greatly decreased the levels of RNA within EL4 cells. Once an acceptable dose was determined, we examined presentation of a model peptide derived from the Shield-controlled recombinant antigenic protein (SCRAP) which is a fusion protein consisting of a destabilization domain, the SIINFEKL peptide, and a fluorescent reporter protein. SCRAP levels can be controlled through the addition of the small molecule Shield-1, which stabilizes SCRAP and prevents its degradation. After treatment with CX-5461, we found the levels of SCRAP protein partially decreased within the cell, indicating a loss in protein synthesis. Concurrently, presentation of the reporter peptide was diminished from both the DRiP and non-DRiP form of SCRAP. These data indicate that interruption of ribosome biogenesis has a minor impact on direct antigen presentation and protein synthesis.
Profiling MHC-I and MHC-II canonical and out-of-frame epitopes from SARS-CoV-2 and seasonal human coronavirus infected human cells
Authors: Nanaware Padma Pandurang , Becerra-Artiles Aniuska , Cruz John , Calvo-Calle J Mauricio , Khaja Muneeruddin , Shaffer Scott A , Stern Lawrence J
Published: January 2023

Understanding the targets of adaptive immunity to SARS-CoV-2 is essential for vaccine development and interpretation of coronavirus disease 2019 (COVID-19) pathogenesis. SARS-CoV-2 and other human coronaviruses share substantial sequence homology raising the possibility that the previous exposure to seasonal “common cold” human coronaviruses could impact the T cell response upon SARS-CoV-2 infection. Detailed investigation of the antigenic peptides presented in SARS-CoV-2 and other common coronavirus infections would be beneficial to understand the impact of preexisting T cell mediated immunity on SARS-CoV-2 infection. The repertoire of naturally processed and presented viral peptides by MHC II upon SARS-CoV-2 and seasonal human coronaviruses infections remains largely uncharacterized. Here, we report the MHC I and II immunopeptidome of cells infected with SARS-CoV-2 or hCoV-OC43, one of the seasonal coronaviruses. We identified 11 MHC I peptides and 13 MHC II peptides of SARS-CoV-2 infected cells including both canonical and out-of-frame spike epitopes, 27 MHC I and 91 MHC II peptides from membrane, nucleocapsid, spike and hemagglutinin-esterase from OC43 infected cells using mass spectrometry. We validated some peptides using HLA peptide binding assays, and these peptides were shown to recall T cell responses in donors with presumed history of common cold viral infection. Some of these peptides share substantial homology with common cold coronaviruses, indicating possibility of conserved T cell epitopes eliciting the protective immunity in COVID-19. The identification of naturally presented peptides and their striking homology with other coronavirus could aid in the peptide selection for the vaccine development.
Dysregulation of T follicular cells and antibody responses in high fat-diet associated lupus development in MRL/lpr mice
Authors: Zhang Xin , Meng Juan , Shi Xuhua , Patel Ronak , Ray Alicia , Hellmers Linh , Davis William , Zakem Jerald , Keshavamurthy Chandana , You Zongbing , Quinet Robert
Published: January 2023

Background/Purpose

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by persistent inflammation, autoantibodies production, and organ damage. Genetic predisposition and environmental factors such as fat diet/obesity contribute to lupus pathogenesis. Here, we investigated the role of immune cells, especially T follicular helper (Tfh) cells and T regulatory (Treg) cells, in bridging obesity and SLE manifestations using lupus prone mice.

Methods

Fifty MRL/lpr mice were fed and grouped in a regular diet (RD) or high fat diet (HFD). Body weights and skin lesions were recorded weekly. Urine protein, serum IgG, anti-dsDNA antibody (Ab), and anti-nuclear Ab were detected. At week 14, kidney and skin biopsy were collected for H&E and PAS staining for histopathological lesions and quantified as kidney index and skin score. Immune cells in spleen were examined by flow cytometry and confirmed by slides staining.

Results

HFD induced a significant increase in body weight than RD (p<0.01). SLE features, such as skin lesions, splenomegaly, proteinuria, higher kidney index, increase of anti-dsDNA Ab and IgG titer were observed in HFD mice. There were significant increase of germinal center B cells and plasma cells in the spleen of HFD mice. The percentage of Tfh cells and the ratio of Tfh/Treg were significantly increased in HFD group (p<0.05).

Conclusion

HFD induced an exacerbated lupus development with dysregulated Tfh/Treg cells associated with increased of anti-dsDNA Ab in MRL/lpr mice, suggesting the central role of Tfh/Treg cells in linking HFD to autoimmunity in SLE. Intervention of healthy diet or restoring balance of Tfh/Treg cells may improve lupus symptoms and outcomes in genetically predisposed individual.

Supported by None
Nexinhib20 inhibits neutrophil adhesion and β2 integrin activation to prevent myocardial ischemia-reperfusion injury
Authors: Fan Zhichao , Liu Wei , Cronin Chunxia G , Wang Chengliang , Ruan Jianbin , Johnson Jennifer L , Catz Sergio , Sun Hao , Groisman Alex , Chen Yunfeng , Hu Liang , Vella Anthony T , Liang Bruce
Published: January 2023

Primary percutaneous coronary intervention (PPCI)—a therapy intended to restore blood flow after acute myocardial infarction—can also induce myocardial ischemia-reperfusion (I/R) injury, exacerbating cardiomyocyte death. Neutrophil-mediated cardiac tissue damage contributes prominently to the process of myocardial I/R injury. Therefore, limiting neutrophil recruitment and exocytosis may prevent myocardial I/R injury after PPCI. Here, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits both neutrophil adhesion and exocytosis and prevents myocardial I/R injury in a mouse model. Using a microfluidic chamber, we found that Nexinhib20 inhibited interleukin 8 (IL-8)-induced β2 integrin-dependent human neutrophil adhesion under flow. Using dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL-8 stimulation. Western blots of Rac-1-GTP pull-down assay confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. In vitro competing assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454, R41HL156322, R44HL152710, and K99HL153678), a Career Development Award from American Heart Association (18CDA34110426), and a startup fund from UConn Health.
High-fat diet-induced immune dysregulation in Th17/regulatory T cells promotes inflammation through protein kinase A (PKA) and PPAR-γ pathways
Authors: Kiran Sonia , Rakib Ahmed , Boddu Geetha , Singh Udai P
Published: January 2023

Obesity is a hallmark around the globe, which is defined as a complex situation associated with various metabolic abnormalities and cardiovascular diseases. The increasing evidence suggests that type 1 inflammation-associated immune cells are dominant in adipose tissue and exert metabolically deleterious impacts. The precise mechanism of alteration of adipose tissue immune system and its effect on metabolic homeostasis is far from clear. We investigated how a high-fat diet (HFD) alters adipose tissue (AT) immune system and influences the inflammation. HFD consumption amends the metabolic parameters including body weight, glucose, and insulin levels. We noticed that there is increased infiltration of Th17 cells, dendritic cells, and macrophages in AT of mice fed HFD as compared to a normal diet (ND). In mice consuming HFD, we also find a reduction in regulatory T cells as compared to ND. We noticed a higher level of proinflammatory cytokines and chemokines in the HFD group as compared to ND. We also noticed an increase in IL-6 and KLF4 expression in the AT of HFD as compared to ND. Further, an increased level of AT protein kinase A in HFD fed and a decrease in PPAR-γ as compared to ND, suggesting acting as a negative regulator of Th17 cell differentiation. Taken together, these results suggest that HFD induces Th17, macrophage, dendritic cells, inflammatory cytokine and reduces Tregs and PPAR-γ to sustain AT inflammation. This study supports a key role of Th17/Tregs dysregulation and macrophages to induce AT inflammation through PKA and PPAR-γ pathways during obesity.

(Supported by NIH grant R01 AI140405).

Supported by NIH grant R01 AI140405.
Farnesol induces protection against CNS inflammatory demyelination and decreases spinal infiltration of CD4+ T-Cells
Authors: Doyle William Jerome , Sell Lacey B , Ramelow Christina C , Kohl Hannah M , Hoffman Kristina R , Bains Jasleen K , Strawn Kevin D , Hervin Theresa , Kirby Trevor O , Gibson K Michael , Roullet Jean-Babtiste , Ochoa-Repárez Javier
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune disease that causes T-cells to attack and degrade the myelin sheath of neurons in the spinal cord and brain. Farnesol is synthesized by plants and mammals and has anti-inflammatory along with neuroprotective activities. We used the MOG35–55 induced c57BL/6 murine EAE (experimental autoimmune encephalomyelitis) model due to model’s neurodegenerative and inflammatory properties. We predicted that farnesol would protect against EAE and increase autoimmunity markers. We collected spinal cords and spleens for flow cytometry analysis at the end of the study. This study found that farnesol significantly reduced spinal infiltration of CD4+ T cells, and increased infiltration of Tregs compared to untreated mice. Interestingly the proportion of CD25+Foxp3+ was increased compared to untreated mice, and statistically significant compared to vehicle treatment. We did not observe significant changes in CD4+, or CD25+Foxp3+ frequencies in the spleens. FOL treatment showed significant increase in CD11b+F4/80+ monocyte-derived macrophages (MDM) and F4/80int granulocytes/monocytes. FOL also showed significant weight retention and reduction of disease severity compared to untreated. These findings show that farnesol helps mediate the invasion of CD4+ T cells in the EAE model. Future studies should study how farnesol affects T-cell activation and differentiation, along with affects on macrophages and dendritic cells.

This work was supported in part by the National Institutes of Health (grant R15NS107743)
Hapten skin sensitization induces development of TLR4-dependent inflammatory monocytes that are required for elicitation of skin contact hypersensitivity responses αααα
Authors: Kish Danielle , Fairchild Robert L
Published: January 2023

Contact hypersensitivity (CHS) to skin sensitization and challenge with haptens is mediated by hapten-primed CD8 T cell populations producing IFN-γ and IL-17. Following hapten challenge these primed CD8 T cells induce hapten-presenting endothelial cells (EC) to produce CXCL1/CXCL2 directing the recruitment and activation of a population of inflammatory monocytes (IM) expressing Gr-1, Ly6C, CXCR2, CD11c, FasL and perforin that are required for hapten-primed CD8 T cell infiltration into the parenchymal skin tissue to mediate CHS. This study investigated mechanisms promoting development of these IM. The IM were absent in the bone marrow of hapten sensitized mice with defective IL-1, TNFα or TLR4 receptor signaling. Sensitized TLR4−/− mice lacked CHS responses to challenge despite equivalent numbers of hapten primed CD8 T cells producing IFNγ and IL-17 in skin-draining lymph nodes as sensitized wild type (WT) mice. IM infiltration into hapten challenged skin of sensitized TLR4−/− was absent at 6 hrs. post-challenge and was accompanied by absent skin mRNA expression of CXCL1, CXCL2, IFNγ, GCSF, TNFα, and CCL20. Whereas hapten primed CD8 T cells from sensitized TLR4−/− mice transferred CHS responses to challenged naïve WT mice, transfer of hapten-primed WT CD8 T cells to naïve TLR4−/− mice failed to elicit CHS responses. IM isolated from the bone marrow of sensitized WT mice and transferred at hapten challenge to sensitized TLR4−/− mice restored the absent CHS response and challenged skin expression of CXCL2, IFNγ, and G-CSF mRNA. These results indicate that TLR4 signaling is required for the bone marrow development of IM that are critical for hapten-primed CD8 T cell infiltration into challenged skin during elicitation of CHS responses.
Role of Mycoplasma pneumoniae CARDS toxin in inducing mitochondrial damage and inflammation.
Authors: Benavides Andrea Milenia , Liendo Nathaniel Steven , Segovia Jesus A
Published: January 2023

Known for its ability to cause damaging airway inflammation and decreased lung compliance, Mycoplasma pneumoniae does so through the production of the Community-Acquired Respiratory Distress Syndrome Toxin (CARDS toxin.) This toxin plays a key role in the inflammation seen during infection. Once M. pneumoniae is able to establish infection in the lungs and produce CARDS toxin, harmful inflammation followed by cell death and tissue injury follows. Although CARDS toxin is known to have vacuolating and ADP-ribosylating properties, the exact mechanism by which it triggers an inflammatory response is still being elucidated. Experimental evidence from our lab supports that CARDS toxin can localize to the mitochondria in vitro, and this localization likely triggers mitochondrial damage and an increase in cell death. In our current study, recombinant CARDS toxin was administered to lung epithelial cells and macrophages exogenously, as well as endogenously via the expression of the CARDS toxin gene on a mammalian expression plasmid. Endogenously-expressed CARDS toxin was analyzed by western blot. Additionally, markers of mitochondrial damage and cytokine production were measured. The ADP-ribosylation and vacuolation properties of CARDS toxin were also tested for their ability to trigger inflammatory cytokine production. Our results support our hypothesis that CARDS toxin localization to the mitochondria is required to trigger mitochondrial damage and thus promote inflammation. By better understanding the mechanism through which CARDS toxin affects the mitochondria, we will gain more insight into how M. pneumoniae infects the host, and which therapeutic strategies could be most effective for future outbreaks.

Supported by grant from NIH T34GM008073
Epithelial cell-specific role of HMGB1 in murine lungs with muco-obstructive lung disease
Authors: Mao Yun , Choudhary Ishita , Patial Sonika , Saini Yogesh
Published: January 2023

Introduction

Mucoobstructive lung diseases are characterized by defective mucociliary clearance, mucin hypersecretion, and mucoobstruction. High mobility group box 1 protein (HMGB1) plays a key role as an inflammatory mediator. However, the role of HMGB1 in mucoobstructive lung diseases remains unclear. Using Scnn1b-Tg+ mice, a mouse model of cystic fibrosis, we hypothesized that the airway epithelial cell-specific HMGB1 acts as an anti-inflammatory mediator in the Scnn1b-Tg+ lungs.

Methods

Airway epithelial cell-specific HMGB1-deficient (CCSP-iCre+/Hmgb1flox/flox) and control (CCSP-iCre− /Hmgb1flox/flox) mice were generated. The bronchoalveolar lavage fluid (BALF) and lung tissue were collected for endpoints analyses.

Results

Compared with wild-type (WT) mice, HMGB1 expression was significantly increased in BALF and airway epithelial cells of Scnn1b-Tg+ mice. Unlike HMGB1-sufficient Scnn1b-Tg+ mice, HMGB1-deficient Scnn1b-Tg+ mice exhibited enhanced lung inflammation evident from the elevated total protein and dsDNA contents in the BALF. HMGB1 deletion in airway epithelial cells in Scnn1b-Tg+ mice also exaggerated immune cell recruitment, which was accompanied by increased levels of chemokines and cytokines, including KC, G-CSF, MCP-1, MIP-2, MIP-1α, MIP-1β, and IP-10. Furthermore, HMGB1 deletion in airway epithelial cells in Scnn1b-Tg+ mice enhanced the type 2 inflammation manifested by increased levels of Th2 cytokines such as IL-4 and IL-5. Additionally, HMGB1-deficient Scnn1b-Tg+ mice showed increased airway mucus plugging and impaired bacterial clearance.

Conclusion

Epithelial cell-derived HMGB1 may play an anti-inflammatory role in murine lungs with mucoobstructive lung disease.

Supported by NIEHS (RO1, ES030125)
Pulmonary macrophage activation and recruitment in lipopolysaccharide-induced acute lung injury mediates neutrophil infiltration: Role of AhR ligation in intervention
Authors: Holloman Bryan L , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

CCL2-CCR2 signaling plays an essential role in the recruitment of macrophages and neutrophils following tissue injury. During inflammation, CCR2+ macrophage secretion of CCL2 induces an autocrine effect that leads to an intracellular signaling cascade of macrophages that promotes upregulation of CCL2, CXCL2, and CXCL3 expression, which stimulates the chemotaxis of blood circulating CCR2+ monocytes and CXCR2+ neutrophils to the disease site. Interestingly, the aryl hydrocarbon receptor (AhR) ligand has been shown to regulate effector cell recruitment. Therefore, we studied the effects of the AhR ligand, indole-3-carbinol (I3C) on the recruitment of circulating CCR2+ monocytes and CXCR2+ neutrophils during acute lung injury (ALI). To induce ALI in C57BL/6 mice and Ccr2gfp mice (mice deficient in the CCR2 receptor), they were given 5mg/kg of lipopolysaccharide intranasally. Mice were treated with I3C or vehicle following disease induction. Interestingly, I3C downregulated neutrophils expressing CXCR2 (a receptor associated with neutrophil recruitment) and CCR2+ macrophages in lungs of C57BL/6 diseased-mice. Furthermore, to determine if CCR2+ macrophages recruit CXCR2+ neutrophils, we induced ALI in Ccr2gfp mice. Abolishing the expression of CCR2 eliminated the recruitment of CXCR2+ neutrophils to the lungs during ALI. Interestingly, scRNASeq of macrophage/monocyte cells showed that I3C reduced expression of CXCL3. CXCL3 gene translates into the chemokine CXCL3, which binds to CXCR2 and is involved in neutrophil recruitment to the disease site. These findings suggest that CCR2 macrophages are involved in the recruitment of CXCR2+ neutrophils, and the AhR ligand I3C can regulate immune cell trafficking capabilities.

Supported by NIH grants P01AT003961, P20GM103641, R01ES030144, R01AI129788, R01AI123947, R01AI160896 and R01AI123947-S2
T-cell-derived Exosomal Proteins ECP and BPI Induce Cytokines and Tissue Inflammation in Systemic Lupus Erythematosus
Authors: Chuang Huai-Chia , Tan Tse-Hua
Published: January 2023

T cells play critical roles in the pathogenesis of systemic lupus erythematosus (SLE). Serum-derived exosomes are increased in human SLE patients and are correlated with disease severity; however, the property of T-cell-derived exosomes from SLE patients remains unclear. We characterized proteins of SLE T cell-derived exosomes by exosome MACSPlex analysis and proteomics using T-cell supernatants from human SLE patients and healthy controls. We identified two exosoaml proteins, eosinophil cationic protein (ECP, also named human RNase 3) and bactericidal/permeability-increasing protein (BPI), which were overexpressed in SLE T-cell-derived exosomes and T cells. Both T-cell-specific ECP transgenic (Lck-ECP Tg) and BPI transgenic (Lck-BPI Tg) mice displayed multi-tissue inflammation. Lck-ECP and Lck-BPI Tg mice displayed early induction of serum IFN-γ levels and IL-1β levels, respectively. Single-cell RNA sequencing (scRNA-seq) showed the induction of IFN-γ mRNA and inflammatory pathways in ECP Tg T cells. ScRNA-seq also showed the reduction of Treg population in BPI Tg T cells; conversely, Treg differentiation was enhanced by BPI knockout. Remarkably, adoptively transferred ECP-containing or BPI-containing exosomes stimulated serum autoantibodies levels and induced nephritis/nephritis/arthritis in the recipient mice. Collectively, ECP or BPI overexpression in T-cell-derived exosomes or T cells are novel biomarkers and pathogenic factors for human SLE nephritis, hepatitis, and arthritis.
TH9-derived IL-9 promotes CCR2-dependent mast cell accumulation in the allergic lung
Authors: Pajulas Abigail , Kaplan Mark H
Published: January 2023

Allergic asthma is a chronic lung disease characterized by airway hyperresponsiveness and cellular infiltration that is exacerbated by IgE-dependent mast cell activation. Our work in mouse models of allergic airway inflammation identified interleukin-9 (IL-9)-producing T helper 9 (TH9) cells as important effector cells in promoting mast cell accumulation in the lungs. However, the precise mechanism of IL-9-mediated mast cell expansion remains unclear. Here, we demonstrate that intranasal recombinant IL-9 expands, while blockade of IL-9 reduces, mast cell progenitor numbers in the lungs and the bone marrow. These findings suggests that there are systemic effects of local IL-9 production in the allergic lung. Using adoptive transfer models and newly generated mice with an inactivation of the IL-9 gene restricted to T cells generated by CD4-Cre/loxP-mediated targeting of the IL-9 gene, we show that TH9 cells promote mast cell progenitor and mature mast cell recruitment from the bone marrow to the lungs in a chemokine receptor 2-dependent manner. Our research further demonstrates that T cell-derived IL-9 is critical for mast cell-mediated airway hyperresponsiveness. Together, these findings define a contribution of T cell-derived IL-9 in promoting mast cell expansion and function in allergic airway inflammation. Therefore, IL-9 may be a promising therapeutic target for targeting mast cell-specific pathologies.

Supported by grants from NIH (R01 AI129241, T32 AI060519)
Cytotoxic CD4+ tissue-resident memory T cells are associated with asthma severity.
Authors: de la Mata Sara Herrera , Ramírez-Suástegui Ciro , Mistry Heena , Kyyaly Mohammad A , Simon Hayley , Liang Shu , Lau Laurie , Barber Clair , Mondal Monalisa , Arshad Syed Hasan , Kurukulaaratchy Ramesh J , Vijayanand Pandurangan , Seumois Grégory
Published: January 2023

Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling that is refractory to corticosteroid treatment. To determine T cell subsets and effector molecules that drive pathogenesis of severe asthma, we performed single-cell transcriptome analysis of >50,000 airway CD4+ T cells isolated from bronchoalveolar lavage (BAL) samples from 30 patients with mild and severe asthma. We observed striking heterogeneity in the nature of CD4+ T cells present in asthmatics’ airways with tissue-resident memory (TRM) cells making a dominant contribution. Notably, in severe asthmatics a subset of CD4+ TRM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4+ T cells in the airways of male patients with severe asthma when compared to mild asthma (13%). This subset was enriched for transcripts linked to T cell receptor (TCR) activation (HLA-DRB1, HLA-DPA1, CD40LG) and cytotoxicity (GZMB, GZMH), and following stimulation expressed high levels of transcripts encoding for pro-inflammatory non-TH2 cytokines (CCL3, CCL4, CCL5, TNF, LIGHT) that could fuel persistent airway inflammation and remodeling. Our findings indicate the need to look beyond the traditional T2 model of severe asthma to better understand the heterogeneity of this disease.

This work was supported by (i) NIH research grant R01HL114093 (PV) and equipment grants (S10RR027366 - BD FACSAria Fusion, and S10OD025052 - Illumina Novaseq 6000); the William K. Bowes Jr. Foundation (P.V.). The WATCH study is supported by the Southampton NIHR Biomedical Research Centre and the Southampton NIHR Clinical Research Facility which are funded by the NIHR and are a partnership between the University of Southampton and University Hospital Southampton NHS Foundation Trust.
Robust homotypic and heterosubytpic immunity against Influenza A Virus in mice lacking Th1/Tc1 transcriptional machinery
Authors: Dhume Kunal , Gallahan Samatha , McKinstry Karl Kai
Published: January 2023

T cell responses against Influenza A Virus (IAV) are dominated by IFN-γ producing CD4 (Th1) and CD8 (Tc1) cells with little IL-17 production. However, loss of the transcription factors T-bet (Tbx21), the master Th1 regulator, and its paralog, Eomesodermin (Eomes), that influences both Th1 and Tc1 functionality, unleashes a strong Th17/Tc17 response while nearly eradicating IFN-γ production and other Th1/Tc1 hallmarks of antiviral T cells. Although Tbx21−/−Eomes−/− mice infected with LCMV were shown to succumb to Tc17 mediated wasting disease, we find that Tbx21−/−Eomes−/− mice survive primary H1N1 IAV infection and clear the virus with similar kinetics as do wild type (WT) mice. Long-lived IAV-specific IgG titers are also similar between Tbx21−/−Eomes−/− and WT mice, correlating with comparable follicular T helper cell responses. Furthermore, primed Tbx21−/−Eomes−/− mice maintain robust homotypic protection against supralethal H1N1 rechallenge indicating highly functional humoral immunity. Strikingly, H1N1-primed Tbx21−/−Eomes−/− mice also survive supralethal H3N2 IAV challenge, indicating intact heterosubtypic immunity as judged by similar viral control, weight loss kinetics and remarkably, no differences in immunopathology versus WT mice despite an overwhelming Th17/Tc17-biased inflammatory response. This cross-protection is T cell dependent as H1N1-primed Tbx21−/−Eomes−/− mice depleted of CD4 and CD8 T cells do not recover or control the H3N2 challenge virus. Our observations thus support that T-bet- and Eomes-independent effector T cells can provide robust long-term protection, potentially extending the available mechanisms for vaccines to target T cell-mediated immunity against IAV.

Supported by grants from NIH (R21AI146647)
Differential serological immune imprinting following SARS-CoV-2 infection, vaccination, and breakthrough infection
Authors: Voss William , Huang Yimin , Marchioni Jeffrey , Seeger Allison , Paresi Chelsea , Kain Jessica , Townsend Douglas , Munt Jenny , Baric Ralph , Georgiou George , Lavinder Jason , Ippolito Gregory
Published: January 2023

Extensive efforts have been made to study the human immune response following SARS-CoV-2 infection. Recent work by our lab provided insights into the molecular-level composition of the circulating IgG antibody repertoire evoked by SARS-CoV-2 primary infection and revealed key properties of the spike-directed antibodies and the epitopes they target in convalescent plasma (Voss et al., Science 2021). We showed that the antibody response in mild SARS-CoV-2 infection is directed to epitopes outside the RBD and primarily to the S2 domain of the spike glycoprotein.

The continual emergence of globally circulating SARS-CoV-2 variants underscores the need for an ongoing study of the long-term evolution of our adaptive immune response to this virus. Collective knowledge about the molecular composition of human serological IgG memory to SARS-CoV-2 is limited. Our research uses single-cell sequencing and plasma IgG proteomics to better understand the evolving plasma antibody response to infection, vaccination, and breakthrough infection.

In volunteers with a primary infection prior to vaccination, we can identify the serological recall of somatically mutated, pre-existing IgG antibodies targeting the S2 domain of the SARS-CoV-2 spike that cross-react with conserved epitopes expressed by endemic β-coronaviruses, indicating the existence of serological immune imprinting by prior infections. In contrast, in volunteers who received a full vaccine regimen and then had a breakthrough infection, the serological recall is markedly different—as shall be discussed. Collectively, these data reveal that the molecular nature of serological IgG recall appears to depend on the sequence and mode of exposure to SARS-CoV-2 spike antigen.

Supported by NIH NCI COVID-19 SeroNet grant U54-CA260543 (R.S.B.)