bluuebunny/crossref_metadata_2025 huggingface dataset:
Vaccination modulates pulmonary eosinophil subsets upon breakthrough influenza infection.
Authors: Chang Lauren A , Rathnasinghe Raveen , Jangra Sonia , Choi Angela , García-Sastre Adolfo , Schotsaert Michael
Published: January 2023

Eosinophils play a critical role in mucosal tissue homeostasis, anti-helminth responses, and allergy. Lung eosinophilia is associated with aberrant T cell responses to respiratory viral infection and is also a consequence of vaccine-associated enhanced respiratory disease (VAERD). A growing body of literature has demonstrated there are different phenotypic subsets of eosinophils and, depending on subset, can play a protective role and aid in lung recovery following infection. Previous work by Choi et al. showed recruitment of eosinophils to the lung in BALB/c mice vaccinated with alum-adjuvanted trivalent inactivated influenza vaccine (TIV) following a sub-lethal H1N1 (A/New Caledonia/20/1999; NC99) influenza challenge. In this study, flow cytometric analysis of the lung showed an increase in total lung eosinophil numbers in all NC99-challenged mice compared to controls. Furthermore, in TIV-vaccinated then challenged mice, flux of inflammatory eosinophil (iEos) and resident eosinophil (rEos) numbers at 7 days post-infection (DPI) resulted in iEos/rEos ratios similar to that of OVA-sensitized allergic control mice; this effect was independent of alum adjuvant. Treatment groups with increased lung eosinophil numbers did not show signs of enhanced morbidity or inflammatory cytokine signatures compared to unvaccinated mice. Unsupervised clustering using CATALYST confirmed findings from manual gating analysis and revealed differences in eosinophil activation status based on surface expression levels of MHC Class II. Our findings provide a starting point for more sophisticated phenotypic characterization of eosinophil subsets and their distinct functional contributions to vaccine- and infection-induced immunity.

This research was partly funded by NIAID T32 Virus-Host Interactions Training Grant (5T32AI007647-22) and CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C).
Alveolar macrophages act as an early viral sponge inducing long-lived functional enhancement
Authors: Waldstein Kody , van de Wall Stephanie , Anthony Scott , Harty John T , Varga Steven M
Published: January 2023

Alveolar macrophages (AM) are one of the first immune cells to encounter pathogens in the lung. However, the role of AMs early after infection remains to be completely defined. To track virally infected cells, mice were infected with a panel of fluorescent reporter-expressing respiratory viruses. By 6 hr post-infection (p.i.), AM numbers significantly declined. The remaining AMs accounted for the majority of infected cells at early time points, surpassing the epithelium, but did not support significant viral replication. Strikingly, AMs represented over 80% of all respiratory syncytial virus (RSV)-infected cells which was confirmed by intravital microscopy and ImageStream flow cytometry. To track cells that survive infection, we utilized a novel cre recombinase-expressing RSV in a floxed-reporter mouse in which infected cells are indelibly marked. Cre-mediated reporter activation required direct infection, and reporter-positive AM numbers remained constant up to 30 days p.i., long after viral clearance. Reporter-positive and -negative AMs were isolated at 30 days p.i., and their functional capacity was assessed ex vivo. Both reporter-positive and -negative AMs exhibited increased basal levels of pro-inflammatory cytokines as compared to naïve AMs which was further increased upon heterologous infection. Reporter-positive AMs displayed an enhanced inflammatory response as compared to reporter-negative AMs from the same lung, indicating innate immunity can be further increased post-direct infection. Our results confirm that AMs play an essential role in limiting early viral access to the respiratory epithelium by serving as a reservoir for non-productive infection resulting in long-lived functional enhancement.

Supported by grants from NIH (R01AI124093-01A1, T32AI007485) and fellowships from NASA (Iowa Space Grant Consortium) and the University of Iowa Graduate College.
Innate lymphoid cells; essential responders to pulmonary cytomegalovirus infection
Authors: Mundy Miles Alexander , Brossay Laurent
Published: January 2023

Cytomegalovirus (CMV) is a β-herpesvirus that is ubiquitously spread throughout the human population. In healthy individuals, there is minimal impact to the host and the virus remains in a latent state. However, in elderly individuals, neonates and immunocompromised hosts, CMV can subvert immune responses and reactivate leading to significant pathology, including interstitial pneumonia. CMV induced pneumonitis is among the most common manifestations of perinatal human cytomegalovirus (HCMV) infection. Although an abundance of research has been conducted on the immune response to CMV, the true route of initial infection is still in contention. Through research with murine cytomegalovirus (MCMV), it is thought that infection first occur in the upper respiratory tract, specifically the olfactory epithelium, with a subsequent primary seeding of susceptible cells in the lung. While the role of innate lymphoid cells (ILCs) in combatting CMV infection in the periphery has been well established, the importance of these cells in the context of pulmonary CMV remains largely unknown. Utilizing an intranasal infection method in murine models, we have begun to uncover the role of ILCs such as natural killers (NK), ILC1 and ILC2s in pulmonary CMV infection. Our preliminary results have suggested an outsized role of NK cells in response to pulmonary CMV as compared to other ILC compartments as shown by early interferon gamma response by NK cells, as well as their increased abundance as compared to ILC1. Additionally, though ILC2 are the most predominant tissue resident ILC compartment in the lung, our data suggests that their contribution may not be critical to the antiviral response, though they may potentially aid in the resolution of tissue damage.

Supported by NIH Research Grants AI046709 and AI12221 and T32 HL134625 Training Grant from NHLBI
Distinct antibody profiles against Ebola virus track with the development of Post-Ebola Syndrome.
Authors: Velazquez Jalene V , Bond Nell G , Schieffelin John S , Gunn Bronwyn M
Published: January 2023

Survivors of Ebola virus disease (EVD) have reported a wide range of symptoms following recovery from infection. These long-term sequelae, collectively termed post-Ebola syndrome (PES), can significantly impact the daily lives of EVD survivors, yet little is known about the underlying mechanism of PES pathogenesis. Antibodies against Ebola virus provide protection against infection through both neutralization and recruitment of innate immune effector functions via the antibody Fc region, yet persistent antibody-mediated inflammation may contribute to PES manifestations. To investigate the potential role that virus-specific antibodies have in PES, we analyzed the antibody immune profiles in a cohort of EVD survivors and household contacts that had been previously characterized for clinical sequelae. Antibodies isolated from survivors an average of 2.5 years after recovery were measured for induction of Fc-mediated innate effector function against the immunodominant antigen Ebola glycoprotein-coated targets. We found that antibodies in asymptomatic EVD survivors were qualitatively different from survivors experiencing musculoskeletal and gastrointestinal manifestations of PES. Specifically, antibodies from asymptomatic individuals induced higher levels of antibody-dependent complement deposition and monocyte-mediated phagocytosis, but not neutrophil-mediated phagocytosis, and differed in NK cell activation profiles compared with individuals with PES. Together, these data suggest that the development of qualitatively different antibodies may shape susceptibility to/protection from the development of PES and may help identify potential therapeutic targets for EVD survivors suffering from PES.

Supported by Washington State University, College of Veterinary Medicine
Iron chelators and HDAC inhibitors are potent inducers of Epstein-Barr Virus Lytic cycle in Stomach Adenocarcinoma
Authors: Chakravorty Srishti , Wang Luopin , Kazemian Majid
Published: January 2023

Epstein-Barr Virus (EBV) is a complex oncogenic γ-herpesvirus that infects around 90% of the global adult human population. Upon primary infection, EBV typically persists asymptomatically in form of a latent infection. However, under certain circumstances the virus can malignantly transform lymphocytes and epithelial cells leading to cancers such as Diffuse Large B Cell Lymphoma (DLBCL) and Stomach Adenocarcinoma (STAD) respectively. Unfortunately, it is difficult to target latent EBV using the current immuno-therapeutic strategies, specifically due to reduced antigen expression. Cytolytic Virus Activation (CLVA) therapy is an approach that can specifically target and kill tumor cells that harbor EBV in a lytic state. The switch from latent to lytic phase can be mediated by a plethora of chemical compounds or lytic inducers. Recently, in our lab, we have developed an intuitive in-silico drug prediction approach to rapidly screen and identify FDA-approved or clinically available compounds that can be repurposed to induce lytic cycle in different EBV+ tumors. Using this strategy, we identified a range of HDACi and Iron chelators as inducers of lytic cycle in EBV+ epithelial cancers. Interestingly, these drugs also significantly induced the expression of Programmed Death Ligand-1 (PD-L1) protein, a major target of Immune checkpoint blockade (ICB) therapy. This led us to hypothesize that by utilizing such an in-silico drug prediction approach, we can identify cancer specific drugs that are potent inducers of EBV lytic cycle. To better understand the underlying mechanisms, we are now investigating the effect of these cytolytic compounds in vivo using xenograft mouse models.

Supported by SIRG Graduate Research Assistantship Award PCCR-SIRG-FY2022-01 (P30CA023168) from Purdue University
Cleavage of DNA and RNA by PLD3 and PLD4 limits autoinflammatory triggering by multiple sensors, including endolysosomal TLRs and a STING dependent sensing pathway.
Authors: Nemazee David , Peng Linghang , Blane Tanya , Huang Deli , Gavin Amanda
Published: January 2023

We have studied the biochemistry and genetics of Phospholipase D3 (PLD3) and PLD4 because polymorphisms of these proteins have been associated with several important inflammatory and autoimmune diseases, including rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus and Alzheimer’s disease. We found that both PLD3 and PLD4 are 5’ exonucleases able to degrade both ssDNA and ssRNA. Deficiency in PLD3, PLD4 or both results in a variety of inflammatory defects, with the most serious being lethal hemophagocytic lymphohistiocytosis (HLH) in double knockout mice. Pld3−/− Pld4−/− mice represent a unique spontaneous model of HLH apparently independent of microbes, whereas Pld4−/− mice develop a range of phenotypes depending upon genetic background ranging from macrophage activation syndrome to lupus like disease. Evidence will be presented showing that the diseases in these mice result from autorecognition of nucleic acids and can be reversed by mutation of nucleic acid sensing pathways. PLD3 and PLD4 tend to limit TLR9 recognition but have a more complex effect on recognition by TLR7 and TLR8. Unc93b13d/3d Pld3−/− Pld4−/− mice spontaneously signal through STING suggesting that PLD3 and PLD4 also may regulate cytoplasmic nucleic acid sensing.

Supported by NIH grants R01 AI142945 and R56AG070775
Inhibition of the Pentose Phosphate Pathway in Cytotoxic T Lymphocytes Preserves Neuronal Function and Ameliorates Neuroinflammatory Disease
Authors: Grund Ethan , Clarkson Ben D S , Howe Charles L
Published: January 2023

In diseases such as multiple sclerosis and autoimmune encephalitides, activated CD8+ cytotoxic T cells infiltrate the brain and are found in clonally expanded clusters that outnumber CD4+ cells in progressive stages of disease. At present, the pathophysiological role of these cells is poorly understood and therapeutic strategies for preventing or ameliorating autoinflammatory disease progression are limited. T cells exhibit profound metabolic reprogramming that facilitates the energetic and proliferative demands of effector T cells. Key metabolic pathways in this reprogramming include upregulation of glycolysis and the pentose phosphate pathway (PPP). Here we show that 1) pharmacologic inhibition of the PPP suppresses effector phenotype acquisition and proinflammatory cytokine secretion by CD8+ T cells; 2) engagement of the PPP is enhanced by pharmacologic Nrf2 activation, implicating reactive oxygen species-induced activation as a transcriptional regulator of PPP-dependent immune activation; 3) Nrf2-driven PPP flux promotes the acquisition of specific CD8+ effector phenotypes; 4) limiting the metabolic flux of glucose-derived metabolites through the PPP prevents CD8+ T cell-mediated injury of neurons expressing cognate antigen and preserves their electrophysiological function; and 5) pharmacologic inhibition of the PPP in B6 mice at symptom onset is sufficient to delay and ameliorate motor disability in the MOG EAE preclinical model of MS. Overall, these results underscore the potential of immunometabolic therapies to limit T cell mediated injury in autoinflammatory and autoimmune diseases across organ systems including the prevention of neurologic deficits in patients with T cell driven neurologic disease.

This work was supported by the NIH (R01NS115126 to CLH; T32GM065841 to EMG), the Mayo Clinic Graduate School of Biomedical Sciences, the Mayo Clinic Center for MS and Autoimmune Neurology, and generous donations from Eugene and Marcia Applebaum, Donald J. and Frances I. Herdrich, Bob and Loralee Marvin, and Rob and Debbie Mudge.
Characterization of thymic epithelial cell and thymocyte function and development in patients with thymic defects using scRNAseq profiling
Authors: Bosticardo Marita , Pala Francesca , Oguz Cihan , Branco Mackenzie , Zhao Chen , Carr Shamus R , Rajan Arun , D’Udekem Yves , Delaney Meghan , Notarangelo Luigi D
Published: January 2023

The interaction between thymocytes and thymic epithelial cells (TEC) in the thymus is critical for the correct function and maturation of both cell populations and for the development of a diverse repertoire of non-self-reactive T-cell lymphocytes. Limited information is available on lympho-stromal crosstalk in the thymus of patients carrying thymic defects. We have previously shown that thymic tissues from DiGeorge syndrome (DGS) and Down syndrome (DS) patients have alterations in architecture and TEC composition, leading to altered T-cell development. Based on these results, we are performing single cell RNAseq (scRNAseq) to explore the transcriptional landscape of both stromal and hematopoietic components in the human thymus. Thanks to a collaboration with the pediatric cardiothoracic surgery center at the Children’s National Medical Center in Washington D.C., we have access to discarded thymic samples obtained from pediatric patients with partial DGS and DS, and from patients without immune defects. Preliminary results of scRNAseq analysis comparing tissues from normal donors and DS patients revealed a different distribution of cell subsets in both hematopoietic and stromal compartments, with several pathways differentially regulated, such as stress and metabolic processes. Additionally, we established a collaboration with the Thoracic and Gastrointestinal Malignancies Branch at the National Cancer Institute and are evaluating samples obtained from resected thymic epithelial tumors. Integration of data obtained from normal and diseased thymic tissue will provide novel insights into the function and development of the human thymus that could be helpful in the clinical management of patients with thymic defects.

This research was supported by the Intramural Research Programs of the NIAID and the NCI (Center for Cancer Research), NIH
Immune and hematological disorders in Primary Immunodeficiency in children
Authors: Fazal Nadeem , Nasrullayeva Gulnara M , Mammadova Vafa R , Aliyeva G Z , Babayeva B A
Published: January 2023

Primary immunodeficiency (PID) was previously considered an extremely rare pathology in childhood, now it has been established that congenital deficit of quantity and functional activity of the lymphocytes, platelets and neutrophils occurs in 1:500–1:4000 newborns. Severe chronic neutropenia in PID is characterized by a stable decrease or absence of circulating neutrophils (<500/cm) and accompanied with recurrent bacterial skin and lung infections. We identified 135 patients with PID. Patient’s examination included clinical observation, general blood test, immunological tests, X-ray of chest and ultrasound examination. We report that six children with PID had significant hematological disorders. The age of these patients was from 1 month to 13 years. Four of them were born from consanguineous marriages, 3 patients died before the age of 5 years. 2 patients with PID had congenital neutropenia, repeated skin infections, pneumonia, diarrhea, stomatitis. Two brothers with WAS syndrome had repeated bleeding, skin hemorrhages, anemia, lymphadenitis, hepatosplenomegaly. The platelet count was about 30–70/ml. Genetic analysis in these patients revealed a hemizygous c.12_13insGG mutation in exon 1 of the WAS gene. 2 children with SCID were found to be carriers of thalassemia B. All our patients showed a significant decrease in Ig classes, CD3+, CD4+, CD8+ and NBT test. We conclude that patients with suspected PID should be continuously monitored by Immunologists and Hematologists. Timely detection of hematological disorders in patients with PID allows for correct diagnosis and prevents fatal complications. Genetic testing can help in making a definitive diagnosis.

Supported by Azerbaijan Medical University
Analysing the cellular mechanisms underlying activated PI3K δ Syndrome (APDS) reveals differences between APDS1 and APDS2
Authors: Deenick Elissa , Lau Anthony , Nguyen Tina , Bier Julia , Brink Robert , Tangye Stuart
Published: January 2023

APDS is a primary immunodeficiency condition caused by heterozygous gain-of-function mutations in PIK3CD (APDS1), which encodes the leukocyte-restricted p110-δ catalytic subunit of phosphoinositide 3-kinase (PI3K) or heterozygous loss-of-function mutations in PIK3R1 (APDS2), which encodes the regulatory subunit of PI3K. PI3K is activated downstream of many receptors expressed by lymphocytes and plays important roles in their activation and differentiation. The increased PI3K activity in cells from APDS patients leads to multiple immune manifestations including lymphoproliferation, respiratory tract infections, Th2-related pathologies, impaired Ab responses and autoimmunity.

We previously dissected the cellular defects that lead to disease in APDS1 by studying patient cells and our novel mouse model. This revealed changes in lymphocytes including increased memory T cells, defective Tfh function, decreased isotype switching and a break in B cell tolerance and production of autoantibodies. We have now generated a mouse model of APDS2 (Pik3r1E11SpD) and use this together with studies of patients’ cells to determine whether increased PI3K signalling due to loss of regulatory function causes the same cellular dysregulation. While we observed many of the same changes previously observed in APDS1, including altered cytokine production and decreased isotype switching, we also identified multiple differences in the cellular phenotype in APDS2 compared to APDS1.

Together, these studies reveal that although APDS1 and 2 both cause increased PI3K signalling and result in similar clinical phenotypes, there are distinctions between how these two types of mutations affect cellular function.

Supported by grants from the NHMRC and an AAI Careers in Immunology Fellowship
Dysfunction in B cell tolerance and activation in obesity.
Authors: Vanz Carlo , Hägglöf Thomas , Dudley Elizabeth A , Leadbetter Elizabeth
Published: January 2023

Obesity is a complex condition that affects a large part of the global population, making it a major risk factor for premature mortality and medical complications. Obesity profoundly engages the immune system, impacting cancer immunity, infections, and autoimmunity. Additionally, obesity is associated with a low-grade chronic inflammation which exacerbates metabolic dysfunction. B cells play a significant role in obesity-associated inflammation and metabolic dysfunction, but their effect is subset-dependent and can be either protective or pathogenic. Therefore, understanding how different B cell populations contribute to these phenotypes represent a crucial goal.

Our lab has identified a subset of B lymphocytes which is expanded in the adipose tissue of humans and mice during obesity. These B cells co-express the transcription factor T-bet and the integrin CD11c, and they produce IgG2c antibodies. In vivo studies with mice fed a high fat diet revealed that these Tbet+ B cells promote inflammation through the production of proinflammatory antibodies. Additionally, these B cells express high levels of the scavenger receptor CD36, a lipid-transport molecule associated with inflammation and atherosclerosis. As a potential consequence of the CD36 expression, the Tbet+ B cells maintain a higher intracellular lipid content and are capable of increased uptake of oxidized LDL compared to conventional B cells, suggesting a unique lipid metabolism.

Because of their unique nature, T-bet+ B cells represent a potential link between obesity, inflammation, and autoimmunity. Identifying the mechanisms regulating their function will provide new potential targets to reduce inflammation and immunological dysfunction in obese patients.

Supported by R01 AI132798
Diet-induced obesity promotes CD11c+ T-bet+ B cell expansion in liver and adipose tissue
Authors: Enslow Benjamin , Vanz Carlo , Dudley Elizabeth A , Hägglöf Thomas , Leadbetter Elizabeth A
Published: January 2023

Immune cells are increasingly appreciated to play a role in inflammation in adipose and hepatic tissue during obesity and may contribute to the development of non-alcoholic fatty liver disease (NAFLD). Both invariant Natural Killer T (iNKT) cells and B cells are enriched in the liver and adipose tissues of humans and mice, but the degree and nature of their influence in the development of NAFLD remains unclear. iNKT cells interact with B cells in many contexts, including during the chronic inflammation associated with autoimmune disease and infection, so it is likely they interact during obesity. Emerging evidence implicates intrahepatic B cells in the progression of human NAFLD, and proinflammatory B cells increase in the steatotic livers of HFD-fed mice. Our preliminary studies demonstrate an iNKT cell-dependent expansion of inflammatory CD11c+ T-bet+ B cells in the subcutaneous adipose tissue of obese humans and perigonadal adipose tissue of obese mice. We now extend those findings to other iNKT cell-rich fatty depots. Flow cytometry identified increased frequencies of T-bet+ B cells in the livers, but not the omenta or mesenteric adipose tissue, of obese mice. In parallel to the in vivo studies, in vitro co-cultures revealed that iNKT cells are sufficient to mediate expansion of T-bet+ B cells. Ongoing experiments will examine the potential role and mechanism of iNKT cells in mediating hepatic Tbet+ B cell expansion. Characterization of the specific contribution of immune inflammation in the progression NAFLD to NASH will allow for the design of successful therapeutic interventions for this increasingly frequent public health problem.

Funding support from NIH K12GM111726 (BTE), NIH TL1 TR002647 (TS), Swedish Research Council (TH), and NIH R01 AI32798-01A1 (EAL).
A gut feeling about Complement in Early Onset Colorectal Cancer
Authors: Anand Sudarshan , Affleck Arthur , Gardner Ivy , Watson Katherine , Ruhl Rebecca , Tsikitis Liana V
Published: January 2023

Colorectal cancer (CRC) remains the second leading cause of cancer-related death in the United States, with a significant increase in incidence observed in young adults below the screening age. We hypothesized that differences in the immune tumor microenvironment (TME) between early-onset (≤50 years) and late-onset (≥65 years) CRC drives tumor progression and responses to treatment. Our studies in a cohort of 121 patients indicate that complement pathway genes including C7 and Complement Factor D (CFD) and the anti-apoptotic gene Bcl2 are significantly increased in early onset CRC vs late onset disease. Moreover, the immune genes in this signature also correlate with progression free survival in our cohort. Analyses of this signature in an independent TCGA cohort also shows association of these genes with progression free survival. Surprisingly, tumor intrinsic CFD gain of function experiments in preclinical mouse tumor models increased tumor burden and recapitulated a subset of the early onset human immune gene signature. Inhibition of CFD, in combination with a Bcl2 inhibitor, significantly decreased clonogenic survival in human CRC lines in vitro. Our data associates a distinct complement enhanced immune microenvironment and hints at a putative role for the microbiome in shaping this immune microenvironment in younger patients with more aggressive disease. Taken together, our work highlights complement and the innate immune response as a potential contributor to early onset CRC.

Supported by grants from NIH to SA (R01 HL137779 and R01 HL143803)
Circulating extracellular vesicles from patients with severe COVID-19 upregulate Cathepsin B and activate STAT3 in normal human mesangial cells
Authors: Glover Sarah C , Scindia Yogesh , Liu Lauren P , Dhaliwal Neha , Williams Haley , Pride Yilianys , Owings Anna H , Robinson Tanya , Alli Abdel A
Published: January 2023

Severe COVID-19 disease is associated with acute kidney injury in up to 57% of infected patients. The etiology of this is likely multifactorial. Extracellular vesicles (EVs) contain unique molecular cargo that contributes to health and disease in many different organ systems and can contain useful surrogate biomarkers. To gain insight into SARS-CoV2’s impact on the human kidney, we prospectively collected blood and urine from individuals hospitalized with severe COVID-19 infection and isolated EVs. Using these EVs, we investigated the impact of circulating EVs released during COVID-19 infection on healthy human glomerular mesangial cells. Nanoparticle tracking analysis and electron microscopy showed that EVs isolated from the plasma of COVID-19 patients were larger in size compared to those isolated from the plasma of non-COVID-19 patients, suggesting the presence of unique cargo within circulating EVs in the setting of COVID-19. The plasma derived EV’s from COVID-19 patients also increased glomerular cell proliferation, STAT3 activation, and cathepsin B protein expression in normal human glomerular cells when compared to EVs isolated from the plasma of non-COVID-19 patients. These data suggest systemic EVs during COVID-19 infection alter normal human mesangial cell function. and provide insight into the effects of SARS-CoV2 on the kidney.

This work was supported by the University of Florida College of Medicine and the University of Mississippi Medical Center.
Single-Cell Transcriptomics Of CD8 T Cells During CMV Viremia In Kidney Transplant Recipients Identify Transcriptional States Of Advanced Differentiation
Authors: Sen Subha , Pickering Harry C , Sun Yumeng , Parmar Rajesh , Reed Elaine F
Published: January 2023

Acute versus chronic viral infections define the dogma of strictly segregated pathways of generating memory or exhausted T cells. Paradoxically, chronic human cytomegalovirus (HCMV) infection elicits an inflationary T cell response with accumulation of progressively differentiated T cells occupying up to 30% of total CD8 T cells. The mechanisms of HCMV-driven memory T cell inflation remains poorly understood.

HCMV infection is asymptomatic but immunocompromised transplant recipients often experience clinical episodes of CMV viremia correlating with graft injury and mortality. We leveraged a defined clinical cohort of renal transplant recipients and profiled the T cells from 31 CMV PCR+ patients with/without prior CMV exposure and 31 matched PCR− controls at baseline, longterm post-transplantation and at 1wk and 1month post-viremia for PCR+ patients.

We observed contraction of naïve and central memory T cells and reciprocal expansion of terminal effector memory CD8 T cells post-viremia. ScRNASeq of CD8 T cells further defined early (CD28+CCR7+), transitional (CD28+GZMK+) and advanced (CD28-KLRG1+) stages of differentiation. Pseudotime analyses revealed specific transcriptomic modules and transcription factors associated with distinct CD8 T cell differentiation states. We report that HCMV induced progressively differentiated T cells with memory-progenitor features lacking inhibitory receptors but with stemness (kat6a, rnf138) and anti-apoptotic markers (bcl2, braf). Thus HCMV skewed the balance of CD8 T memory cells from pro-exhaustion to a prolonged non-classical memory phenotype. This study revealed important mechanisms underlying memory inflation over exhaustion in the setting of chronic antigen exposure.

This work was funded by NIH grant U19AI128913 to EFR.
Differential CD4+ and CD8+ T cell recognition of mycobacteria antigens in pediatric versus adult TB
Authors: Lancioni Christina , Morrow Erin , Liu Qijia , Swarbrick Gwendolyn , Kiguli Sarah , Nsereko Mary , Toerien Megan , Cansler Meghan , Lewinsohn David Michael , Lewinsohn Deborah
Published: January 2023

T cells from young children may be limited in capacity to mount pro-inflammatory responses to intracellular pathogens, thus contributing to vulnerability to TB disease. We compared CD4+ and CD8+ T cell cytokine production in response to mycobacterial antigens and mitogen SEB, between Ugandan children <5 yr (PedTB) and adults (ATB) with culture-confirmed pulmonary TB. HIV-uninfected children (n=12) and adults (n=41) had blood drawn for PBMC isolation prior to TB treatment. CD4+ and CD8+ T cell production of IFNγ, IL2, and TNFα in response to ESAT6/CFP10 (EC), ESXJ, PE12/13, PE3, PPE15, and PPE51 peptide pools, and SEB, were assessed by intracellular flow cytometry. Proportions and frequencies of CD4+ and CD8+ T cells producing cytokines were compared using Fisher’s Exact and Kruskal-Wallis tests, respectively, with adjusted p-values <0.05 significant. PedTB and ATB had equivalent CD4+ and CD8+ T cell production of IFNγ, IL2, and TNFα, including polyfunctional responses, to EC. T cell cytokine responses to ESXJ and PPE51 were equivalent; significant differences were noted in CD4+ T cell responses to PE3 and PPE15, and CD8+ T cell responses to PE12/13. Frequencies of CD4+ T cells producing IFNγ or IL2 in response to SEB were lower (p<0.0001) in PedTB versus ATB. Young children with PedTB exhibited adult-like capacity to generate Th-1 cytokines in response to EC, ESXJ, and PPE 51. Pediatric responses to PE3, PPE15, and PE12/13 were impaired, suggesting that the breath of antigen recognition differs between infants and adults with TB. Differential T cell recognition of mycobacterial antigens between adults and children should be considered in development of novel T cell based diagnostics and vaccines targeting pediatric TB.

Supporting from grants by NIH: AI157807; HHSN272200900053C
Distinct Subpopulations of Mouse and Human Kidney Resident Macrophages in Acute Kidney Injury
Authors: Cheung Matthew David , Erman Elise , Lever Jeremie , Porrett Paige , Agarwal Anupam , George James
Published: January 2023

Introduction

Kidney resident macrophages (KRMs) are important in renal homeostasis and the response to acute kidney injury. Preliminary data suggests that the KRM population consists of several undescribed subpopulations. Here, we combined single-cell RNA sequencing (scRNAseq) and spatial transcriptomics to identify and localize KRM subpopulations during homeostasis and injury.

Methods

KRMs were isolated from C57BL/6J mice without treatment and after bilateral ischemia-reperfusion injury (BIRI) as well as from a human donor kidney. ScRNAseq was performed using the 10X Genomics Chromium platform and spatial transcriptomics using the 10X Visium platform. Gene expression data were integrated and analyzed using the R package, Seurat 4.0.

Results

UMAP plots of integrated data revealed seven major clusters of mouse KRMs with unique transcriptional profiles associated with distinct functions. Spatial transcriptomics revealed that these clusters reside in distinct cellular compartments within the kidney and appear to associate with specific renal structures. Following BIRI, these subpopulations appear in cellular compartments distinct from those occupied in the controls. Several human KRM clusters were correlated with those of the mouse and localized to specific regions of the kidney.

Conclusion

Transcriptionally distinct subpopulations of mouse KRMs reside within specific kidney microenvironments and change location as a function of injury. Similar subpopulations of KRMs were identified in the human kidney as well. Therefore, further study of the temporal and spatial characteristics and signaling pathways of these subpopulations in the context of homeostasis and injury is warranted.

Supported by grants from NIH (R01-DK-59600, T32-AI007051)
Role of CD73 on peritoneal macrophages in age-associated immune dysfunction and tumor immunity
Authors: Rivera-Escalera Fátima , McGinty Mitchell T , Rutkowski Melanie R , Murphy Patrick S , Elliott Michael R
Published: January 2023

Aging is a major risk factor for cancer and many other life-threatening diseases due in part to a wide range of age-associated immune dysfunctions. In the context of cancer, chronic, low-grade inflammation seen with aging is thought to contribute to cellular transformation and tumorigenesis. Tissue resident macrophages (TRM) are key innate immune regulators of the inflammatory milieu in all tissues, and studies from our laboratory have shown that expression of the 5’-ectonucleotidase CD73 on macrophages suppresses inflammatory responses and promotes the resolution of tissue inflammation in the lung and peritoneum. However, the role of CD73 on TRM in modulating inflammation during aging or how age-associated changes in CD73 on TRM contribute to cancer development are not known. We have found that large peritoneal macrophages from aged mice (18+ months) show significantly increased levels of surface CD73 compared to young mice (<4 months). To test the role of CD73 in regulating inflammation in vivo, we utilized mouse models of ovarian cancer and peritonitis. Our preliminary studies suggest that blocking CD73 in aged mice reduces LPS-mediated production of TNF-a in vivo and ex vivo when compared to young mice. In addition, we found that treatment of ID8-Def29/Vegf-a tumor-bearing mice with anti-CD73 monoclonal antibody significantly prolongs the survival of aged mice compared to young mice. In ongoing experiments, we are utilizing conditional deletion approaches to address the role of CD73 specifically on macrophages in these models. Results from this study will provide insight into the immunoregulatory role of CD73 on TRM during aging and cancer, and will help refine therapeutic approaches targeting CD73 in cancer immunotherapy.

Supported by NIH grants T32 AI007496 and R01 AI114554.
Complement-dependent phagocytosis as a regulator of antibody-dependent cellular cytotoxicity
Authors: Van Wagoner Carly M , Rivera-Escalera Fátima , Comeau Laurey Dea , Bauchle Casey , Chu Charles C , Zent Clive , Elliott Michael Rusty
Published: January 2023

While monoclonal antibodies (mAb), such as αCD20, have significantly improved patient outcomes for B cell malignancies, patient responses vary and drug resistance can occur. One proposed limitation is the reduction of innate immune effector functions such as antibody dependent cellular phagocytosis (ADCP) – the principal cytotoxic mechanism for many mAb-based therapeutics. ADCP is mediated by Fcγ Receptor- and complement-dependent pathways (FcγR-ADCP and cADCP, respectively). However, our lab has shown that the capacity of macrophages (Mϕ) to engulf mAb opsonized targets is finite and after brief period of engorgement, Mϕ enter a phase of negligible phagocytic activity termed hypophagia. We have previously described the mechanisms that negatively regulate FcγR-ADCP, but complement-induced hypophagia is less understood. To begin to characterize complement-induced exhaustion, we used live cell imaging to measure mAb opsonized thymocyte engulfment by primary mouse Mϕ in the presence absence of complement. Preliminary data suggest that cADCP kinetics and cytotoxic capacities are distinct from FcγR-ADCP; however, both lead to a hypophagic state that impinges on future target cell clearance. Furthermore, Mϕ refractory to additional FcγR-ADCP still phagocytose through complement receptors up until the cADCP pathway also becomes exhausted. Published data from our lab also show that FcγR driven hypophagia involves surface receptor downregulation, but interestingly, there is no reduction in complement receptor surface levels following cADCP-induced hypophagia. Therefore, a better understanding of cADCP and the mechanistic overlap with FcγR-ADCP is critical to optimizing mAb therapy by minimizing immune cell exhaustion.

Supported by grants from NIH (T32 AI007496, 26A1)
The effects of fragmented sleep on monocyte and neutrophil distribution within the tissues during atherosclerosis
Authors: Moriarty Alina K , Keeter William Coles , Ma Shelby , Stahr Natalie , Hatler Julian , Wellman Laurie , Sanford Larry , Galkina Elena
Published: January 2023

Atherosclerosis is a chronic inflammatory disease of large and medium size vessels that progresses across a lifespan and has been extensively linked to insufficient sleep. Poor fragmented sleep quality is strongly associated with all-cause mortality. Accumulating evidence demonstrates the independent effects of atherosclerosis and fragmented sleep on chronic inflammation within multiple tissues. However, there is limited data on the combined effects of sleep fragmentation (SF) and atherosclerosis on the inflammatory environments of multiples tissue. To address this question, we used flow cytometry and qPCR to investigate immune alterations within the peripheral blood, bone marrow, spleen, aorta, small intestine, and brains of female Apoe−/− mice following 12 weeks of high fat diet (HFD) feeding and SF. SF was induced by waking up mice at the day time using a moving bar. Importantly, we used an activity control (AC) group of mice that had a moving bar during nighttime. We found that chronic SF accelerated atherogenesis and induced increased myelopoiesis. Particularly, we detected increases in pro-inflammatory monocytes and neutrophils in the bone marrow, blood, spleen, aortas and small intestines of SF vs AC groups of HFD-fed female Apoe−/− mice. Additionally, we detected increase inflammatory markers in the brains of SF vs the control AC mice. Our data suggests that SF accelerates atherosclerosis and alters the immune composition of multiple tissues including the blood, bone marrow, spleen, small intestine, and aorta of atherosclerosis-prone mice. Furthermore, the increased peripheral monocytosis and neutrophilia may serve as a link to the elevated inflammatory markers in the brains of HFD-fed, female SF Apoe−/− mice.

Supported by the American Heart Association Pre-Doctoral Fellowship (20PRE35180156)
Profiling adrenal gland resident macrophage response to acute and chronic stress
Authors: Xu Yingzheng , Gallerand Alexandre , Dolfi Bastien , Zhu Alisha , Schrank Patricia R , Patterson Michael T , Acharya Nisha , Zaitsev Konstantin , Ivanov Stoyan , Williams Jesse W
Published: January 2023

Macrophages are found across all organs in the body and play key roles in health and disease. Systemic stress is a major risk factor for a variety of chronic inflammatory diseases. However, the primary source for stress hormones, the adrenal gland (AGs), have yet to be thoroughly assessed for immune cell diversity. Here we utilized single cell RNA-sequencing (scRNA-seq) to investigate macrophage heterogeneity in nascent AGs, compared against acute stress (cold challenge) or a chronic stress model of cardiovascular disease. In steady state, we observed two major macrophage populations in the AG. Following acute and chronic stress, AG macrophages upregulated expression of inflammatory cytokines (Il1b, TNFa) and key lipid uptake molecules, including Trem2. These observations were replicated using imaging analysis showing hormone uptake by AG macrophages. Using a monocyte-fate-mapping approach, we observed increased recruitment following stressed conditions, which correlated with reduced AG macrophage proliferation. Finally, we observed exacerbated systemic corticosterone levels following AG macrophage depletion and Trem2 deficiency. Overall, these data define AG macrophage heterogeneity and suggest a regulatory function of AG macrophage following exposure to acute and chronic stress.
Glycogen pathway intermediate uridine diphosphate glucose alters dendritic cell mitochondrial respiration in a nitric oxide-dependent manner
Authors: Vagher Bay L , Amiel Eyal
Published: January 2023

Dendritic cells (DCs), the professional antigen presenting cells of the immune system, undergo metabolic reprogramming in response to stimulation through Toll-Like Receptors (TLRs). Our lab and others have shown that metabolic reprogramming in DCs is accompanied by increased reliance on glycolytic metabolism through use of glucose and glycogen as fuel. Both metabolites are required by DCs to perform immune functions associated with activation including secretion of cytokines, upregulation of costimulatory molecules, and initiation of the T cell-mediated immune response. While our lab has published that glycogen is required within 4–8 hours after activation, production of nitric oxide (NO) regulates many metabolic processes during the later stages of activation via NO-dependent suppression of mitochondrial respiration. Recent studies have shown that glycogen pathway intermediate uridine diphosphate glucose (UDPG) is continually elevated in inflammatory macrophages past 24 hours of activation. Additionally, UDPG can be exported extracellularly and act as an autocrine signaling molecule to increase proinflammatory gene expression. We tested the hypothesis, that, like inflammatory macrophages, DCs elevate production of UDPG in response to activation and that this metabolite can bolster NO-mediated processes during DC activation. To investigate the role of UDPG in DCs, we characterized DC mitochondrial respiration in response to activation by LPS and the addition of exogenous UDPG. Additionally, we performed functional assays to assess NO production, cytokine secretion, as well as inflammatory gene expression to begin elucidating an axis by which UDPG and NO work together to regulate DC metabolism and immune function.

Supported by grants from UVM (COBRE VCIID Funding Support and Pilot Project) and NIH (NIAID 1R21AI135385-01A1)
Influenza A virus modulates ACE2 expression and SARS-CoV-2 infectivity in human cardiomyocytes
Authors: Rajaram Murugesan , Wu Qian , Kumar Naresh , Salijoughian Noushin , Zani Ashley , Patton Ashley , Ganesan Latha , Yount Jacob , Lafuse William
Published: January 2023

Influenza A virus (IAV) and SARS-CoV-2 are both acute respiratory viruses currently circulating in the human population. Prior IAV infection enhances SARS-CoV-2 infectivity and lung pathogenesis in mice; however, underlying mechanisms and the extent that co-infection leads to involvement of other organs in disease severity remains unknown. Herein, we investigated the impact of prior IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. IAV infection induces the expression of ACE2 in human lung epithelial cells, lung fibroblasts, macrophages, cardiac fibroblasts (HCFs), and hiPSC-Cardiomyocytes (CMs). Interestingly, we detected poorly glycosylated ACE2 in lung epithelial cells and cardiac fibroblasts. In contrast, expression of a heavily glycosylated form of ACE2 is induced by IAV in CMs. In all cell types, IAV infection enhances SARS-CoV-2 viral entry. However, efficient SARS-CoV-2 replication was uniquely inhibited in CMs. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Angiotension II, induction of eNOS and nitric oxide production, providing a mechanistic underpinning for restricted viral replication in CMs. Our results indicate that IAV-mediated induction of ACE2 is double-edged, providing increased risk for SARS-CoV-2 coinfection in epithelial cells and HCFs, while limiting SARS-CoV-2 replication in CMs. We conclude that differential glycosylation of ACE2 may be a molecular determinant, not of SARS-CoV-2 infection, but of replication.

Funding: NIH grants R01-AI 146252, R21-AI 146690

Funding: NIH grants R01-AI 146252, R21-AI 146690
Defining complex mechanistic interactions and responses by macrophages during Mycobacterium abscessus infection
Authors: Gilliland Haleigh , Olive Andrew
Published: January 2023

Phagocytosis by macrophages is critical to eradicate invading pathogens. Phagocytosis is regulated by parallel networks that control the binding and uptake of particles, subsequent lysosome delivery, and activation of downstream innate immune responses. However, whether distinct host pathways drive unique responses to targeted substrates within the lung environment remains unknown. Mycobacterium abscessus (MAB) is a rapidly growing non-tuberculous mycobacterium that infects patients with chronic lung diseases, like cystic fibrosis. While interactions between MAB and macrophages contribute to its pathogenesis, how macrophages bind, phagocytose, and control MAB remains entirely unclear. We hypothesize that understanding key MAB-macrophage interactions will identify critical host targets that can be inhibited to prevent disease. To test this hypothesis, using a genome-wide knockout library of immortalized bone marrow derived macrophages, we completed a forward genetic screen to identify host pathways that contribute to fluorescent MAB uptake. Our results show glycosaminoglycan synthesis (sGAGs) in macrophages is required to efficiently take up MAB. Both enzymatic and genetic approaches found key sGAG biosynthesis regulators, UGDH, B3GLCT, B4GALT7 and B3GAT3, influence macrophage interactions with MAB and other pathogens like Mycobacterium tuberculosis. Using a recently developed alveolar macrophage model, we are now dissecting the role of sGAGs in lung specific macrophages. Future work will determine how sGAG pathways modulate bacterial replication, cytokine responses and disease progression following MAB infection with the goal of defining key mechanisms driving host-pathogen interactions in the lungs.