bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-cell atlas of the liver myeloid compartment with cure of chronic viral hepatitis
Authors: Alatrakchi Nadia , Cui Ang , Li Bo , Gonnye Anna , Wallace Michael S , Patel Hailey , Oetheimer Christopher , Tonnerre Pierre , Holmes Jacinta A , Lieb David , Ma Aileen , Damasio Marcos , Brown Joelle , Sade-Feldman Moshe , Aneja Jasneet , Gustafson Jenna , Butler Michael Dillon , Epstein Eliana T , Thabet Ashraf , Kim Arthur , Lauer Georg M , Hacohen Nir , Chung Raymond T
Published: August 2023

Background/Aims

Chronic viral infections present serious public health challenges. Direct-acting antivirals (DAAs) are now able to cure nearly all patients infected with the hepatitis C virus (HCV), representing the only cure of a human chronic viral infection to date. DAAs provide a valuable opportunity to study immune pathways in the reversal of chronic immune failures in humans.

Methods

To leverage this opportunity, we used plate-based single-cell RNA-seq (scRNA-seq) to deeply profile myeloid cells from liver fine needle aspirates in HCV patients before and after DAA treatment. We characterized liver neutrophils, eosinophils, mast cells, conventional dendritic cells (cDCs), plasmacytoid DCs, classical/non-classical monocytes, and macrophages, and defined fine-grained subpopulations of several cell types.

Results

We discovered cell-type-specific changes post-cure, including an increase in MCM7+STMN1+ proliferating CD1C+ cDCs, which may support restoration from chronic exhaustion. We observed an unexpected inverse relationship between pre-treatment viral load and post-cure ISG expression in each cell type, revealing a link between viral loads and sustained modifications of the host’s immune system. We found an upregulation of PD-L1 expression in ISG-high neutrophils, pinpointing cell subpopulations crucial for immune regulation. We identified three recurring gene programs shared by multiple cell types, distilling core functions of the myeloid compartment.

Conclusion

This comprehensive scRNA-seq atlas of human liver myeloid cells in response to a cure of chronic viral infections reveals principles of liver immunity and provides immunotherapeutic insights.

The single-cell sorting was done at the MGH Flow and Mass Cytometry Research Core, which was supported by NIH Shared Instrumentation program grants 1S10OD012027-01A1, 1S10OD016372-01, 1S10RR020936-01, and 1S10RR023440-01A1. Additional sorting was done at the Immunology Core Facility of the Ragon Institute, which is part of the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354).
Pivotal role for the non-receptor tyrosine kinase SRMS in IRF3-dependent innate antiviral immune response in murine cells
Authors: Wang Ruixue , Zhang Yunzhi , Li Nan L , Yang Darong , Wang Dang , Zhou Zhi , Potts Malia B , Li Kui
Published: August 2023

Upon viral infections, RIG-I-like receptors (RLRs) sense cytosolic viral RNAs and initiate signaling pathways culminating in the activation of IRF3 and subsequent type I interferon (IFN) antiviral response. This intrinsic protective mechanism is orchestrated by a host of positive and negative regulators, which collectively govern the magnitude and duration of antiviral signaling. SRMS is an understudied non-receptor tyrosine kinase frequently overexpressed in breast cancers. SRMS restrains autophagy and promotes tumor growth, although its physiological function is unclear. We report here that SRMS is critical for IRF3-dependent innate antiviral response in murine cells. Depletion of SRMS impaired virus- or dsRNA-induced expression of type I and III IFNs and ISGs in mouse embryonic fibroblasts (MEFs), mouse hepatoma hepa1–6 cells, and primary mouse lung fibroblasts, concomitant with heightened viral replication. Mechanistically, SRMS loss led to diminished phosphorylations of TBK1 and IRF3, IRF3 dimerization and nuclear translocation. The kinase activity of SRMS, however, was dispensable. Reconstituting the expression of SRMS of mouse or human origin, be it wild-type or kinase-dead protein, in SRMS-null MEFs restored antiviral signaling. Autophagy regulation was not involved, either. Interestingly, antiviral gene expression via the RLRs in human U2OS and MDA-MB-231 cells was not impacted by SRMS deletion, suggesting signaling redundancy in human cells. Altogether, our data uncover a novel role of SRMS in controlling IRF3-dependent innate antiviral responses and describe a non-canonical, kinase- and autophagy-independent regulatory mechanism that operates differentially between mouse and human species.

R21 AI142044
Differential role of TLR7 signaling in SARS-CoV-2-induced antiviral and inflammatory responses
Authors: Ghimire Roshan , Patton Titus , Shrestha Rakshya , Chanda Debarati , More Sunil , Channappanavar Rudragouda
Published: August 2023

Since its emergence in 2019, SARS-CoV-2 has caused significant morbidity and mortality in humans. SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses drive acute lung injury and severe pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and pathogenic inflammatory responses are not well defined. Since CoVs are single-stranded RNA (ssRNA) viruses that make double-stranded RNA (dsRNA) intermediates during replication, we assessed the antiviral and pro-inflammatory roles of ssRNA and dsRNA mimics in mouse macrophages. We show that R837-TLR7 signaling induced excessive inflammation, while PolyIC-TLR3/MDA activation caused robust IFN production. Since R837-TLR7 induced high levels of inflammatory cytokines, using a novel mouse-adapted SARS-COV-2, we examined the role of TLR7 signaling in COVID-19 pathogenesis. Our results show that TLR7 KO mice were more susceptible to SARS-CoV-2 infection as compared to WT mice. Further investigation of SARS-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral interferons (IFNα/β/λ), ISGs (ISG15 and CXCL10), and several pro-inflammatory cytokine/chemokines in TLR7 KO lungs compared to WT. Interestingly, immunolabelling of cells isolated from infected WT and TLR7 KO lungs showed a high neutrophil count in TLR7 KO mice compared to WT mice. Furthermore, reduced lung IFN levels and increased mortality correlated with high lung viral titer in TLR7 KO mice. In summary, our findings conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite TLR7-mediated robust inflammatory cytokine production, TLR7-induced IFN and ISG responses play a key role in host protection.
Role of Caspase-11 in house dust mites-mediated lung inflammation
Authors: Khweek Arwa Abu , Joldrichsen Marisa R R , Boyaka Prosper N , Amer Amal O
Published: August 2023

Asthma is a long-term recurring inflammatory lung disorder characterized by mucus hypersecretion, cellular infiltration, chronic airway inflammation and bronchial hyper-responsiveness. House dust mites (HDM), such as Dermatophagoides pteronyssinus, are an unsurpassed cause of atopic sensitization and the major causes of allergic asthma worldwide. The multiprotein complexness of the canonical and noncanonical inflammasomes assemble in response to pathogen or danger-associated molecular patterns (PAMPs or DAMPs). We have shown that caspase-11 expression is induced in vitro in response to HDM. Treatment with TH2 cytokines such as IL-4 and IL-13 mediates caspase-11 expression. Additionally, caspase-11−/− macrophages show reduced release of IL-6, IL-12, and KC, and express lower levels of costimulatory molecules (e.g., CD40, CD86 and MHCII) in response to HDM stimulation. Notably, similar level of IgE responses and similar degree of hypothermia in response to nasal HDM challenge were seen in WT and caspase-11−/− mice following HDM sensitization. However, altered inflammatory responses and reduced neutrophilia in bronchiolar alveolar lavage fluid (BALF) and in representative histopathological lung tissues were seen in the caspase-11−/− mice. Therefore, caspase-11 is a regulator of airway inflammation in response to HDM exposure.
PTEN regulation of AcetyCoA/HAT activity regulates macrophage activation during Staphylococcus aureus infection
Authors: Shili Cedrick Ndhumba , Salina Ana , Faneuf Eden , Eddie Alex , Serezani Henrique
Published: August 2023

Sepsis is a life-threatening condition characterized by systemic inflammation, multiple organ failure, and death. Macrophage metabolism and actions play a critical role in sepsis pathogenesis. Fatty acid metabolism was identified as one of the most promising metabolic predictors for survival in sepsis. The phosphatase and tensin homolog (PTEN) is a critical regulator of cell growth and metabolism and influences different arms of the innate immune response. Here, we seek to investigate whether PTEN is a bridge between immunometabolism and gene expression in macrophages during S. aureus infection, WT (PTENfl/fl) and KO (PTENfl/fl_LysMCre) were infected i.v. with methicillin-resistant Staphylococcus aureus (MRSA) and kidney and blood were collected on day three. PTEN deficiency decreased MRSA kidney bacterial loads, as evidenced by IVIS. Bulk RNAseq analysis of kidney macrophages from infected WT and PTEN KO mice showed reduced expression of genes involved in fat acids synthesis, such as (FASN), which correlated with decreased levels of cellular acetyl-coenzyme A in MRSA-infected macrophages. Acetyl-CoA is a substrate for the epigenetic enzyme histone acetyltransferase (HAT) and favors gene expression. Infected PTEN KO macrophages showed lower nuclear p300/CBP HAT activity than cells from WT mice and decreased H3K9 and H3K18 acetylation (involved in open chromatin) in infected macrophages. Studies underpinning the role of the PTEN/HAT axis in MRSA infection are underway. These data suggest that PTEN is required for gene expression of genes involved in fatty acid metabolism, which might reduce the cytokine storm seen during sepsis.
Maternal Antibiotic Treatment Dysregulates Goblet Cell Associated Antigen Passages in Neonatal Mice
Authors: McDonald Keely G , Schill Ellen M , Joyce Elisabeth , Floyd Alexandria N , Newberry Rodney D
Published: August 2023

Maternal antibiotic treatment (MAT) disrupts the development of the neonatal microbiome, intestinal immune development and increases susceptibility to sepsis in mouse models. Mechanisms by which MAT disrupts neonatal intestinal immune function will facilitate the development of novel strategies to reduce neonatal sepsis risk. Intestinal goblet cell associated antigen passages (GAPs) are portals for physiologic exposure of the immune system to commensal gut bacteria and luminal antigens. GAPs are inhibited by epidermal growth factor receptor (EGFR) signaling, colonic GAPs are closed prior to postnatal day (P) 10 in mice when luminal EGF levels from breast milk are high, then open after P10 when breast milk EGF levels decline. When colonic GAPs are opened inappropriately due to low levels of EGF prior to P10 or dysbiosis of the gut microbiota after weaning, they can facilitate pathological bacterial translocation. Pups of dams treated with ampicillin and neomycin in their drinking water from pup P1–8 had significantly more open GAPs in the small intestine (SI) compared to controls. These data suggest that MAT creates a permissive environment for SI GAPs in the neonatal period which could allow pathologic bacterial translocation. MAT pups weighed significantly less and were significantly hypoglycemic compared to pups from control dams, after weaning MAT pups recover to a normal weight. We hypothesize that MAT pups have reduced intestinal EGFR signaling, leading to stunted growth and irregular opening of GAPs in the SI and colon. These data suggest maternal antibiotic treatment may be a risk factor for sepsis and growth failure in premature neonates.
Acquired resistance against skin penetration by Strongylodies ratti is dependent on IL-33 expressing APCs
Authors: Jean E Evonne , Herbert De’Broski
Published: August 2023

Interleukin 33 (IL-33) is an alarmin cytokine released from damaged epithelia that is essential for protection against gastrointestinal (GI) helminth (worm) infection, but whether IL-33 serves any role in cutaneous immunity against parasites remains unclear. While epithelial cells are considered the predominant source of IL-33, our recent work demonstrates that CD11c+ myeloid antigen presenting cells can express IL-33 to shape Type 2 immunity. We developed a percutaneous infection model using Strongylodies ratti, a rodent-specific GI helminth, to better understand mechanisms of host protection and the potential role for IL-33. Data show that C57BL/6 mice develop resistance to percutaneous infection upon secondary challenge, but IL-33 deficient mice lack this capacity. Surprisingly, mice with a selective IL-33 deficiency only in myeloid APC (CD11cCre) also failed to develop secondary resistance to S. ratti, suggesting myeloid-derived IL-33 is essential for cutaneous immunity. Mechanistically, we find that loss of myeloid IL-33 impairs recruitment of gd T cells that express the IL-33 receptor ST2 and gene deficient mice that lack all gd T cells show defective protective immunity against S. ratti. Moreover, we propose that IL-33 drives a noncanonical Type 2 immunity, because mice lacking the Type 2 transcription factor STAT6 (Signal transducer and activator of transcription 6), have no defects in primary or secondary cutaneous immunity. Our work implies that myeloid APCs are a necessary source of IL-33 that drives acquired immunity against helminths, potentially through regulating gd T cell effector function(s). Ongoing studies seek to understand how myeloid APC derived IL-33 controls gd T cells to promote cutaneous immunity.

NIH (R01 AI095289 and R01 GM083204) NSF GFRP
Mathematical Modelling of Neutrophil Responses to Aspergillus
Authors: Wheeler Matthew , Yang Ning , Whitten Spencer , Goddard Yana , Vieira Luis Sordo , Scindia Yogesh , Laubenbacher Reinhard , Mehrad Borna
Published: August 2023

Invasive aspergillosis is a dangerous infection commonly affecting immunocompromised patients. The spores of the fungal pathogen, Aspergillus, are found ubiquitously in the environment and are inhaled daily by all humans. These spores are cleared quickly and effectively healthy hosts without developing infection. On the other hand, patients with impaired immunity, can develop an invasive pneumonia with poor outcomes. Neutrophils are essential to the host defense against aspergillosis, both in direct fungal killing and as modulators of immunity against the pathogen. To better understand this role, we conducted a transcriptomic time series comparing the responses of human neutrophils to Aspergillus. Differential gene expression analysis as well as gene enrichment analysis indicated an up regulation of number of transcriptional markers and pathways including NFkB, NR4A, TNF, as well as neutrophil degranulation, cytokine-mediated signaling, chemokine signaling, and TREM1 signaling among others. Combining this data with known mechanisms and pathways from the literature, we built a mathematical model describing the intracellular pathways which determine the neutrophil’s response after detecting Aspergillus. Very few mathematical models have been built modeling intracellular mechanisms of a neutrophil and none that we know of which relates to the interaction between neutrophils and this organism. We intend to use our model to make predictions on the effects of altering different pathways within the cell. Moreover we intend to use this model in a larger systems model describing the immune system response to invasive pulmonary aspergillosis.

Supported by: R01AI135128, U01EB024501, Keck Foundation Medical Research Grant 994413.
Vaccine Immunology in the context of COVID-19
Authors: Sika-Paotonu Dianne
Published: August 2023

Evidence at the start of the pandemic indicated that Māori and Pacific peoples were vulnerable to being disproportionately impacted and affected by COVID-19, indicating prioritisation with respect to COVID-19 vaccination and prevention efforts would be needed. Significant health inequities already existed for Māori and Pacific peoples prior to the COVID-19 outbreak, with the pandemic exacerbating pre-existing health inequities for vulnerable groups including Māori and Pacific peoples. An elimination strategy was pursued at the start of the COVID-19 pandemic in Aotearoa/New Zealand, a suppression strategy was then followed later. The initial lockdown periods helped keep the SARS-CoV-2 virus away while trying to get people vaccinated once the COVID-19 vaccines became available in Aotearoa/New Zealand. Efforts to spread mis/disinformation about the SARS-CoV-2 virus and COVID-19 vaccines were initiated early during the COVID-19 outbreak. The purpose of this work was to focus upon the immunological areas pertaining to the COVID-19 vaccines, to ensure appropriate scientific communication approaches that would support the COVID-19 vaccination roll-out. This work involved the utilisation of an online videoconferencing tool to support urgent communication efforts for the general public and lay audiences that would also support the countering of mis/disinformation about the COVID-19 vaccines in particular. Those who participated responded positively and indicating this approach helped demystify COVID-19 vaccines, explained how they worked, and encouraged vaccination. This work showed how appropriately presented COVID-19 vaccine information in the online setting could help counter mis/disinformation efforts.

Wellington School of Medicine & Health Sciences, University of Otago.
Cross-reactive seasonal coronavirus antibody responses contribute to antibody-mediated effector functions against SARS-CoV-2 in individuals in Kenya
Authors: Ulusan Sinem , Bai Shuangyi , Murrell Amelie , Osoro Eric , Dawa Jeanette , Ngere Isaac , Langat Robert , Robinson James , Norton Elizabeth , Njenga M Kariuki , Gunn Bronwyn
Published: August 2023

Much of sub-Saharan Africa has fared the SARS-CoV-2 pandemic with reduced disease and death even with low uptake of vaccines, little to no infection mitigation strategies, and clear serologic evidence of infection. While warmer weather, a youthful population, and microbiome have been posited to explain the differences in Africa compared to Americas, we aimed to determine if there was a unique humoral immune response to SARS-CoV-2. To do this, we analyzed SARS-CoV-2 seropositive and seronegative samples from Nairobi, Kenya and compared to a cohort collected in the US in late 2020. We measured the levels of antibodies (Abs) against SARS-CoV-2 Spike [S], receptor binding domain [RBD], and nucleocapsid [N] and hCoVs (229E, NL63, OC43, HKU1) S using a multiplexed Luminex-based assay, and we measured induction of antiviral Ab functions including Ab-dependent cellular phagocytosis (ADCP), neutrophil phagocytosis (ADNP), neutralization, NK cell activation (ADNKA), and complement deposition (ADCD). Correlation network analysis revealed that Ab effector functions were strongly associated with SARS-CoV-2 S, RBD and N-specific Abs in both Kenyan- and US-based cohorts. Surprisingly, we observed that hCoV-specific Abs were correlated with the effector functions against SARS-CoV-2 in the Kenyan samples, but not in the US samples. A subset of Kenyan seronegative subjects displayed ADCP and ADNP against SARS-CoV-2, which was reduced when HKU1-specific IgG was depleted from samples, suggesting cross-reactive Ab responses from hCoVs may augment SARS-CoV-2 immunity. Thus, together these data suggests that unique Ab-mediated functions induced by hCoVs in Africa may help explain the disparity in COVID-19 severity observed across the globe.
Robust T cell responses to the Pfizer/BioNTech vaccine compared to infection and evidence of attenuated CD8+ T cell responses due to COVID-19
Authors: Gao Fei , Mallajoysula Vamsee , Altman John D , Wang Taia T , Nadeau Kari C , Boyd Scott D , Pulendran Bali , Davis Mark M
Published: August 2023

The COVID-19 pandemic has resulted in rapid development of several novel vaccine platforms, including the mRNA-based Pfizer/BioNTech vaccine. As of October 2022, more than 3.8 billion Pfizer/BioNTech vaccines were administrated to people in 180 countries. Meanwhile, the total number of COVID-19 cases has reached 627 million globally.

We recently developed a new antigen-specific T cell staining tool— ”spheromer” that is able to label T cells more efficiently and capture a broader swath of the repertoire than any other available platform. Using spheromers loaded with SARS-CoV-2 peptides, we applied a panel covers 49 epitopes spanning the entire genome to characterize the magnitude and durability of antigen-specific T cell responses at single-epitope resolution.

Using very sensitive “spheromer” peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in very robust peripheral blood responses to predicted SARS-CoV-2 spike peptides bound to either class-I or class-II MHC alleles. These peaked at 9.8% and 7.5% for the dominant CD4+ (HLA-DRB1*15:01/S191) and CD8+ (HLA-A*02/S691) T cell epitopes. Interestingly, the antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring one week post the second vaccination (boost), whereas CD8+ T cells didn’t peak until two weeks later. These responses were much higher than in COVID-19 patients. We also found that prior SARS-CoV-2 infection decreased the CD8+ T cell response post vaccination by 2.4 to 41.4-fold at peak, and that the remaining cells were less responsive, suggesting that the virus can profoundly damage an individuals’ CD8+ T cell response.

1. Howard Hughes Medical Institute and NIAID grant AI057229 to Mark.M.Davis; 2. Bill and Melinda Gates Foundation grant OPP1113682, Center for Human Systems Immunology, to Mark.M.Davis; 3. U01 AI140498 to to Mark.M.Davis.
Sensing of SARS-CoV-2 by pDCs and their subsequent production of IFN-I contribute to macrophage-induced cytokine storm during COVID-19
Authors: Laurent Paoline , Yang Chao , Rendeiro Andre L , Nilsson-Payant Benjamin , Carrau Lucia , Chandar Vasuretha , Bram Yaron , tenOever Benjamin , Elemento Olivier , Ivashkiv Lionel , Schwartz Robert , Barrat Franck J
Published: August 2023

Lung-infiltrating macrophages create a marked inflammatory milieu in a subset of patients with COVID-19 by producing a cytokine storm, which correlates with increased lethality. However, these macrophages are largely not infected by SARS-CoV-2, so the mechanism underlying their activation in the lung is unclear. Type I interferons (IFN-I) contribute to protecting the host against SARS-CoV-2 but may also have some deleterious effect, and the source of IFN-I in the lungs of infected patients is not well defined. Plasmacytoid dendritic cells (pDCs), a key cell type involved in antiviral responses, can produce IFN-I in response to SARS-CoV-2. We observed the infiltration of pDCs in the lungs of SARS-CoV-2–infected patients, which correlated with strong IFN-I signaling in lung macrophages. In patients with severe COVID-19, lung macrophages expressed a robust inflammatory signature, which correlated with persistent IFN-I signaling at the single-cell level. Hence, we observed the uncoupling in the kinetics of the infiltration of pDCs in the lungs and the associated IFN-I signature, with the cytokine storm in macrophages. We observed that pDCs were the dominant IFN-α–producing cells in response to the virus in the blood, whereas macrophages produced IFN-α only when in physical contact with infected epithelial cells. We also showed that IFN-α produced by pDCs, after the sensing of SARS-CoV-2 by TLR7, mediated changes in macrophages at both transcriptional and epigenetic levels, which favored their hyperactivation by environmental stimuli. Together, these data indicate that the priming of macrophages can result from the response by pDCs to SARS-CoV-2, leading to macrophage activation in patients with severe COVID-19.

Supported by NCI grant T32CA203702. Supported by NIH grant 1R01AI132447, the Scleroderma Research Foundation, the Scleroderma Foundation, and a grant from the HSS Research Institute to study the role of pDCs in SARS-CoV-2 pathogenesis. Supported by NIH grants AI046712 and AR050401. Supported by NIH grants UL1TR002384, R01CA194547, and P01CA214274; LLS SCOR grants 180078-01 and 7021-20; and Starr Cancer Consortium grant I11-0027. Supported by NIDDK R01DK121072, NIDDK R01DK119667, NCI R01CA234614, and NIAID R01AI107301. Supported as an Irma Hirschl Trust Research Award Scholar.
Prior infection exposure alters the immune response at homeostasis and following SARS-CoV-2 vaccination and experimental infection in laboratory mice
Authors: Baxter Victoria Kate , Knight Audrey C , Dillard Jacob A , Anderson Elizabeth J , Pressey Katia D , Hampton Brea K , Taft-Benz Sharon A , Martinez Sabian A , Kamat Kalika D , Cruz Cisneros Marta C , Madden Emily A , Heise Mark T , Ferris Martin T
Published: August 2023

Variable translatability represents a major criticism of the use of mouse models for preclinical research. Lack of exposure to most endogenous murine pathogens results in natural immune responses of specific pathogen free (SPF) lab mice to more closely resemble that of newborns rather than adult humans. Recently, the effects of prior infection exposure (PIE) on immune maturation and responses have been increasingly explored in mice. Here, we employed a PIE model where BALB/c mice underwent sequential subclinical infection with four pathogens at two-week intervals: 1) MHV-68, a gammaherpesvirus, 2) MCMV, a betaherpesvirus, 3) influenza virus, and 4) Heligmosomoides polygyrus, a murine intestinal nematode. Four-to-12 weeks following H. polygyrus infection, mice were sacrificed, spleens were collected for flow cytometric analysis, and lungs were collected for histological evaluation. Compared to SPF mice, PIE spleens demonstrated increased proportions of CD4+ and CD8+ T effector memory cells. In the lungs, PIE induced the formation of bronchus-associated lymphoid tissue and increased infiltration of CD4+ and CD8+ T cells. Following vaccination against SARS-CoV-2 with either a protein subunit-based (S2P) or inactivated (iCoV2) vaccine, iCoV2-, but not S2P-, vaccinated PIE mice demonstrated significantly reduced serum neutralizing antibodies compared to SPF mice. Following infection with SARS-CoV-2-MA10, PIE mice showed a mild reduction in mortality, weight loss, and lung titers, and a significant increase in lung T cell infiltration compared to SPF mice. These data indicate that PIE results in adaptive immune maturation but has a variable effect on SARS-CoV-2-specific vaccine responses and clinical outcomes in mice.

This work was supported by the NIH (K01OD026529, T32AI007151, U19AI100625, U19AI09680), an AAI Careers in Immunology Fellowship Award, an UNC School of Medicine Junior Investigator Development Award, and a NCTraCS and Emerging Challenges in Biomedical Research COVID Pilot Award.
Alteration of metabolite landscape in hypoxia replicates immune cell programs indicative of dysfunctional immunity in severe COVID-19
Authors: Thompson Elizabeth A , Stoddart Laila , Ngecu Wabathi , Cascino Katherine , Karaba Andrew H , Cox Andrea L
Published: August 2023

Our prior work has demonstrated that altered metabolic programs in T cells define dysregulated immunity in acute SARS-CoV-2 infection. Apoptotic T cells were defined by high expression of the mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1). These cells were markedly increased in elderly patients and correlated with lymphopenia. We devised in vitro systems in healthy T cells to investigate the mechanism driving this unique phenotype. While SARS-CoV-2 did not directly induce this T cell phenotype, factors that enhanced the development of VDAC1+ T cells, including hypoxia, advanced age of healthy T cell donor, and COVID-19 patient-derived plasma. Analysis of patient-derived plasma revealed robust differences in cytokine and metabolomic profiles between people who did or did not exhibit apoptotic T cells. Increased flux through the tryptophan:kynurenin pathway has been suggested as a biomarker of severe COVID-19 and was further enhanced in individuals with apoptotic T cells. Kynurenine directly induced VDAC1+ T cells in a dose dependent manner in vitro. Together, our work indicates metabolic alterations combined with cellular age and hypoxia may induce apoptosis of T cells following SARS-CoV-2 infection. This work highlights mechanisms leading to cellular alterations following infection and highlights key aspects of the host environment that could be targeted therapeutically.

U54CA260492
Elucidating the role played by the NKG2 family of receptors in natural killer cell responses to JC polyomavirus
Authors: Lee Esther , Ahn Jenny , Chen Sarah , Gikundiro Kayitare Eunice , Craemer Melissa , Gressens Simon , Yoder Taylor , Lifton Michelle , Tan C Sabrina , Jost Stephanie
Published: August 2023

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression due to monoclonal antibody treatments for cancer, autoimmune diseases and transplantation, or due to uncontrolled HIV infection. There is currently no effective treatment against PML and novel immunotherapies are urgently needed. Natural killer (NK) cells play critical roles in defense against viral infections, yet NK cell responses to JCPyV remain largely unexplored. Here, we first compared NK and T cell responses against JCPyV VP1 peptide pools by intracellular cytokine staining. In about 40% of healthy donors, we detected robust CD107a upregulation and IFNγ production by NK cells, extending beyond T cell responses. Next, using a novel flow cytometry-based killing assay, we showed that co-culture of NK cells and JCPyV-infected SVG-A cells led to a 65% reduction in infection, on average. To identify receptor-ligand interactions modulating NK cell-mediated responses to JCPyV, we performed co-cultures in the presence of select blocking antibodies. We observed enhanced expression of ligands for the activating NKG2D on infected cells and accordingly, NKG2D blockade resulted in decreased NK cell degranulation. In contrast, blockade of inhibitory NKG2A enhanced NK cell response to JCPyV-infected targets. Moreover, we identified JCPyV-derived peptides that elicit dominant NK cell responses and can stabilize HLA-E, the ligand for NKG2A and the activating NKG2C. Altogether, these findings suggest that modulation of NKG2D and NKG2A/C signaling may play key roles in NK cell-mediated control of JCPyV and may be promising immunotherapeutic targets to boost NK cell anti-JCPyV activity.

Supported by grant from NIH (R01 NS116278)
Neuroinvasive EV-D68 infection in cerebral organoids mimics human pathology
Authors: Vazquez Christine , Han Beatrice , Bannerman Carl , Negatu Seble G , Bongiovanni Angelina , Lanoce Emma , Ming Guo-Li , Jurado Kellie Ann
Published: August 2023

Emerging viruses with the capacity to cause neurological disease remain a global health threat. One such virus, Enterovirus-D68 (EV-D68), has recently received attention due to an associative rise in cases of a polio-like paralytic disease called acute flaccid myelitis (AFM) in some children. Although EV-D68 was identified in the 1960s (prototypic strain Fermon), circulating neuroinvasive EV-D68 strains have evolved to infect cells of the central nervous system. AFM remains a rare infection outcome, suggesting that host immune responses may play a role in limiting disease. Studies of such immune responses as well as tools to examine these responses remain limited. To this end, we seek to understand how neuroinvasive and non-neuroinvasive EV-D68 differentially modulate immune responses using both neuronal cultures and cerebral organoids.

We infected neuroblastoma cells (SH-SY5Y cells) with Fermon and neuroinvasive EV-D68 and found that both viruses were able to replicate in these cells, which is different from what is observed during human disease. Thus, to better dissect strain differences and model human disease, we generated cerebral organoids and infected with our EV-D68 strains. We now found that cerebral organoids were more permissive to neuroinvasive EV-D68 infection compared to Fermon. Further, we found that neuroinvasive EV-D68 infection led to a global increase in the interferon-stimulated gene IFIT1 activation, whereas Fermon infection led to modest IFIT1 activation in bystander cells. These data suggest that immune responses can better control Fermon infection compared to neuroinvasive EV-D68, and also highlight the utility of using cerebral organoids to examine neurotropic virus regulation of immune responses.

Supported by grants from the Burroughs Wellcome Fund and the Office of the University of the Provost
Immune-history based correlates of protection for respiratory infections
Authors: Hertz Tomer , Levy Shlomia , Oppenheimer Hanna , Zismanov Shosh , Friedman Lilach M , Nesher Lior
Published: August 2023

There is growing evidence that immune-history biases responses to vaccination and natural infections. We hypothesized that immune-history antibody profiles can be used to predict risk of infection from respiratory viruses. To study the predictive power of immune-history, we utilized samples from influenza and SARS-CoV-2 clinical trials, in which symptomatic infections were identified using PCR. We used antigen microarrays to generate baseline antibody profiles to (1) a panel of influenza HA and NA proteins spanning the antigenic evolution of A/H1, A/H3 and B human influenza viruses; and (2) to a panel of SARS-CoV-2 variants of concern spike. While there was extensive heterogeneity in baseline immune history of different individuals, we identified IgG and IgA baseline markers that were correlates of risk using logistic regression models adjusted for baseline covariates. Since IgA and IgG profiles were weakly correlated, we then considered combinations of IgG and IgA markers as correlates of risk. We found that combinations yielded improved correlates of protection.

Most importantly, we identified a subset of individuals with low baseline antibody levels that responded with a significant boost in neutralizing antibody titers after vaccination, but were still at significantly increased susceptibility to infection when compared to those who had pre-existing high levels of binding antibodies. Thus, we identify a highly susceptible population that remains susceptible despite apparent responsiveness to vaccines. Our data demonstrate that baseline binding antibody markers are significantly associated with infection risk and may be utilized to identify individuals most at risk from future exposures.

Supported by grants from NIH (75N93021C00016) and the Israeli Science Foundation (882/17 and 2683/21)
Macrophage scavenger receptor 1, a suppressor of HIV infection, is down-regulated in transgender females
Authors: Chang Theresa Li-Yun , Wang Annie , Herbst Alisa C , Hernandez Cyril , Vaishnav Jeet , Lemenze Alexander , Swaminathan Shobha , Piazza Michelle Dalla , Finkel Diana , Bentsianov Sari , Roche Natalie
Published: August 2023

Transgender people are at greater risk than cisgender people of acquiring HIV and other sexually transmitted infections. Globally, the risk of acquiring HIV infection is nearly 49 times higher in transgender women than in other populations. Although behavioral and psychosocial factors contribute to the higher HIV risk in transgender people, gender-affirming hormone (GAH) (i.e., testosterone for transgender individuals assigned female at birth or estrogen/antiandrogen for transgender individuals assigned male at birth) may alter immune cell functions, resulting in increased HIV transmission. We have profiled gene expression of PBMCs from transgender females and males and have identified four specific genes (MTND1P23, IGSF10, MSR1, and DUXAP9) that were differentially expressed in transgender females. Among these genes, macrophage scavenger receptor 1 was down-regulated 0.5- or 0.4-fold compared to cis females and cis males, respectively. Transient expression of MSR1 suppressed in vitro HIV infection, suggesting that MSR1 protects cells against HIV. The data indicate that down-regulation of MSR1 in transgender women may play a role in their increased HIV risk. Future studies on the mechanisms by which MRS1 inhibits HIV infection may offer new strategies for HIV prevention in transgender women.

NIH R21AI55322
Dendritic cell intrinsic role of Ptpn22 and its pro-autoimmune allele enhance anti-viral immunity
Authors: Orozco Robin C , Shaikh Anam , Nasiri Saeideh , Barnes Jenna , Howard Andrew , Orozco Robin
Published: August 2023

The tyrosine phosphatase PTPN22 encodes the enzyme Lyp (PEP in mice) in all immune cells. 5–10% of the North American population expresses the PTPN22 autoimmunity risk allele 1858C>T (rs2476601) causing amino acid substitution R620W (R619W in mice). This mutation alters lymphocyte activation, toll-like receptor signaling, and cytokine production. Ptpn22 knockout (PEP-null) and alternative allele-expressing (PEP-R619W) mice clear persistent virus LCMV-cl13, have enhanced anti-viral T cell function, and a more immunostimulatory dendritic cell (DC) phenotype compared to wildtype (PEP-WT). Adoptive transfer studies suggest a T cell extrinsic mechanism driving enhanced T cell function in PEP-null and -R619W animals. However, the relative contribution of PEP-differing non-T cells during virus infection is not known. We hypothesize that the DC, as the sole PEP-differing cell, is the critical non-T cell driving increased T cell function and enhanced anti-viral immunity. To interrogate this, we employ CD11c Ptpn22 conditional knock out (cKO) mice and bone marrow derived DC (BMDC) cultures from PEP-WT, -null, and -R619W mice. During LCMV-cl13 infection, CD11c Ptpn22 cKO mice have improved disease outcome compared to WT mice. In a T cell: DC co-culture assay, both PEP-null and PEP-R619W DCs lead to more CD4 T cell function over WT DCs. Also, PEP-null and PEP-R619W BMDCs have changed response to virus infection. Taken together, these data suggest Ptpn22 and its allele have DC intrinsic mechanisms that alter anti-viral immunity. Results of this study lead to a more complete understanding of the cellular and molecular mechanisms, particularly in the DC, this common allele alters the immune response during infection.

Supported by grants from the University of Kansas (KU), KU CBID CoBRE Research Award, and KU Center for Genomics.
PI3Kδ shapes Th2 lineage restriction through coordination of IL-2 signaling, Foxo1 inactivation and epigenetic remodeling
Authors: Golec Dominic P , Gazzinelli-Guimaraes Pedro , Chauss Daniel , Hill Tom , Nivelo Luis , Joshi Ilin , Pereira Nicolas , Druey Kirk M , Lack Justin , Afzali Behdad , Villarino Alejandro , Nutman Thomas B , Schwartzberg Pamela L
Published: August 2023

Activated PI3K-delta syndrome (APDS) is a primary immunodeficiency caused by heterozygous activating mutations in PI3Kδ, resulting in recurrent infections as well as allergic diseases. Given that APDS patients show an inability to clear respiratory pathogens (Th1) and development of allergic diseases (Th2), we hypothesized that hyperactivated PI3Kδ (Pik3cdE1020K) may alter CD4 T cell differentiation. Using in vitro polarization of murine naïve CD4 T cells, we observed increased production of IFNγ from Pik3cdE1020K mice under Th1 inducing conditions and marked increases in IL-4 and IL-13 production under Th2 conditions. However, Pik3cdE1020K Th2 cells also expressed high levels of Tbet and aberrantly produced IFNγ. Strikingly, this dysregulation was also observed in vivo; Pik3cdE1020K CD4 cells show pronounced alterations following house dust mite induced airway inflammation, with impaired Th2 and inappropriate polarization to Th1 responses as visualized by scRNAseq. We linked this unstable Th1 and Th2 differentiation in Pik3cdE1020K CD4 T cells in vitro to hyper-responsiveness to IL-2, associated with increased phosphorylation and inactivation of transcription factor Foxo1. CRISPR-mediated targeting of Foxo1 in naïve WT CD4 cells recapitulated the Pik3cdE1020K phenotype. Transcriptomic analysis of Pik3cdE1020K CD4 T cells revealed differential expression of genes involved in histone methylation. Accordingly, Pik3cdE1020K CD4 exhibit altered chromatin modifications, including H3K27 and H3K4 trimethylation. Together these data suggest that balanced regulation of PI3Kδ plays a critical role in enforcing Th1 and Th2 lineage identity.

This research was supported by the Intramural Research Program of NIAID, NIH
BRWD1 establishes germinal center B cell epigenetic states to prevent apoptosis and inflammation
Authors: Wright Nathaniel E , Kennedy Domenick , Veselits Margaret , Ai Junting , Gurbuxani Sandeep , Maienschein-Cline Mark , Mandal Malay , Clark Marcus R
Published: August 2023

Germinal centers (GCs) drive adaptive humoral immunity by selecting for B cells with high affinity antibodies and producing memory B cells and plasma cells. We previously defined three GC B cell subpopulations that are undergoing either selection, proliferation, or somatic hypermutation with distinct transcriptional and chromatin accessibility profiles. We hypothesized that the epigenetic reader BRWD1, which regulates chromatin accessibility during B cell lymphopoiesis, also regulates the chromatin accessibility and differentiation of GC B cells. To study the role of BRWD1 in GC B cells, we generated Aicdacre × Brwd1fl/fl mice and performed ATAC-seq to measure chromatin accessibility. Loss of Brwd1 caused an epigenetic collapse whereby differential chromatin accessibility between GC B cell subpopulations was lost. This was accompanied by SPIB and PU.1 binding motifs becoming more accessible among GC B cell subpopulations. Functionally, loss of Brwd1 promoted proliferation, and GC structures were larger and more frequent. However, the rate of somatic hypermutation was unaltered. Interestingly, Brwd1-deficient GCs had massive apoptotic debris by histology, which was consistent with strong inflammatory and apoptosis transcriptional signatures measured by RNA-seq. Finally, Brwd1 deletion caused a decrease in pre-memory B cell differentiation, and resultant plasma cells had decreased immunoglobulin mutations. We conclude that BRWD1 regulates chromatin accessibility to compartmentalize the molecular functions of each GC B cell subpopulation and to prevent apoptosis-induced inflammation.

This research was funded by grants R01 AI143778 and F30 AI174324 from the NIH/NIAID.
IL-12 drives the differentiation of human T follicular regulatory cells
Authors: Locci Michela , Castano Diana , Wang Sidney , Sharpe Hannah , Bustamante Jacinta , Yang Emily , Feng Allan , Le Coz Carole , Romberg Neil D , Utz P J , Henrickson Sarah E , Casanova Jean-Laurent
Published: August 2023

T follicular regulatory (Tfr) cells can counteract the B cell-helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cues initiating the differentiation of T regulatory (Treg) cells into Tfr cells, which is instrumental for the therapeutic manipulation of diseases associated with a Tfr cell imbalance, is still missing. Despite their opposed roles, Tfr and Tfh cell differentiation appears to be controlled by partially overlapping signals, including TCR stimulation and costimulatory molecules. However, it is still unknown if cytokines that have been shown to control the biology of human Tfh cells, including IL-12 and activin A, can influence the differentiation of Tfr cells. To address this question, we evaluated the impact of these two cytokines on the in vitro differentiation of Tfr cells. Herein, we report a role for IL-12 in driving, on activated Treg cells, the induction of molecules that belong to the Tfr cell program, including CXCR5, PD-1, BCL6 and ICOS meanwhile preserving Tfr regulatory function. Conversely, activin A presented a largely negligible effect on the in vitro differentiation of Tfr-like cells and only modestly strengthened the IL-12-driven differentiation of Tfr-like cells. Importantly, patients with inborn errors of immunity in IL12RB1 gene presented with a severe decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides a novel approach for the in vitro generation of human Tfr-like cells.

AI123738
Cellular cholesterol homeostasis supports visceral adipose tissue (VAT) regulatory T cell (Treg) accumulation and promotes metabolic health
Authors: Elkins C , Sivasami Pulavendran , Bae Jennifer , Li Chaoran
Published: August 2023

A unique population of regulatory T cells (Tregs), characterized by a clonally expanded TCR repertoire and distinct transcriptional profile, is highly enriched in the visceral adipose tissue (VAT) at steady state but is lost during obesity, which exacerbates VAT inflammation and promotes metabolic disease. Therefore, understanding the factors that control the accumulation of VAT Tregs, which might be dysregulated during obesity, is important for developing novel therapies for obesity-associated metabolic diseases. Here we show that cellular cholesterol homeostasis is particularly important for VAT Treg accumulation and function. First, bulk RNA sequencing analysis of VAT Tregs from mice fed long-term high fat diet (HFD) showed a reduction in gene expression involved in cholesterol homeostasis (CH), corresponding to loss of VAT Tregs in vivo. Second, we found that VAT Tregs showed increased cholesterol-associated gene expression, cholesterol levels, and cholesterol uptake compared to lymphoid Tregs at steady state, suggesting this pathway is particularly important for VAT Tregs. Third, CRISPR-Cas9-mediated ablation of Srebf2, the master regulator of cholesterol homeostasis, followed by adoptive Treg transfer or Treg-specific germline deletion of Srebf2 (Foxp3cre Srebf2-flox) reduced Tregs in the VAT, but not those in other tissues. Deletion of Srebf2 in Tregs also increased VAT inflammatory cytokine expression, inflammatory cell infiltration, and insulin resistance in mice following short-term HFD feeding. These data highlight an importance of cellular CH for VAT Treg accumulation and function and implicate this pathway as a potential therapeutic target for treatment of obesity-associated metabolic diseases.

NIH R01 (5R01DK128061-02)
miR-21 promotes B cell autoreactivity, inflammatory signaling, and metabolism in TLR7-driven systemic autoimmunity
Authors: Bricker Kristen N , Fike Adam J , Luckenbill Sara A , Weber Julia L , Jackson Jasmine S , Olsen Nancy J , Rahman Ziaur
Published: August 2023

MicroRNA-21 (miR-21) is upregulated in SLE patients and promotes disease in multiple autoimmune mouse models; however, mechanisms by which miR-21 promote autoimmunity are unknown. Here we show that TLR7 overexpressing SLE-prone B6.Sle1bYaa mice deficient in miR-21 (Sle1bYaamiR-21−/−) resisted autoimmunity development through reduced germinal center (GC) B cell, T follicular helper cell, and plasma cell responses. Sle1bYaamiR-21−/− mice had reduced serum autoantibody titers and numbers of autoantibody-producing antibody forming cells (AFCs) in the spleen and bone marrow. Downstream of TLR7 signaling, PELI1, a negative regulator of the NF-κB family member c-Rel and a direct target of miR-21, was increased in Sle1bYaamiR-21−/− B cells. Consequently, Sle1bYaamiR-21−/− B cells had reduced c-Rel expression and proinflammatory cytokines IL-6 and IL-1β. Additionally, Sle1bYaamiR-21−/− B cells had reduced oxidative phosphorylation, glycolysis, and mitochondrial membrane potential compared to Sle1bYaa B cells. To define the cell-intrinsic mechanisms, we generated B cell-, T cell- and DC-specific miR-21 conditional knockout (cKO) mice on the Sle1bYaa background. Reduced B cell autoreactivity, inflammatory signaling, and metabolism in Sle1bYaamiR-21−/− mice were recapitulated in B cell-specific miR-21 cKO mice. T cell-specific miR-21 cKO mice had a moderate reduction in cellular responses due to reduced co-stimulatory molecules and glycolysis; however, autoimmune responses remained intact. miR-21 deficiency in DCs had no deficit in cellular or autoimmune responses. Our data substantiate a B cell-intrinsic role for miR-21 in promoting inflammatory signaling and metabolic activity in B cells, leading to autoimmunity.