bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoantigen specific T-cell receptor induces organ-specific autoimmunity by escaping T cell negative selection
Authors: Yin Mianmian , Smith Jennifer A , Chou Marissa , Chan Jackie , Hu Yongmei , Lowell Clifford A , Gould Douglas B , Anderson Mark S , DeFranco Anthony L
Published: January 2023

Previously we reported a new model of spontaneous autoimmune uveitis in which mice with a hypomorphic mutation of Aire (AireGW/+) are combined with deletion of Lyn. The key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+ Lyn−/− IRBP−/− mice failed to develop uveitis. We performed single-cell RNA-seq with TCR sequencing for IRBP 271–290 (P2)-specific CD4+ T cells from eye-draining lymph nodes (LN) of these mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCRs was expressed in thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR. In addition, we generated TCR transgenic mice with this TCR clonotype. The TCR transgenic mice spontaneously developed uveitis with expansion of P2-specific CD4+ T cells. Combined with AireGW/+ or IRBP−/−, there were more P2-specific CD4+ T cells in the thymus of TCR transgenic mice. Thus, suboptimal Aire function in AireGW/+ Lyn−/− mice results in a partial defect in negative selection of T cells recognizing IRBP in the thymus, promoting development of uveitis. This disease is accelerated in mice expressing a TCR transgene recognizing IRBP.

Supported by AI138479
Type III interferon enhances thymic B cell licensing
Authors: Martinez Ryan J , Breed Elise , Worota Yosan , Ashby Katherine , Qian Lily , Hogquist Kristin Ann
Published: January 2023

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate central immune tolerance. The processes leading to thymic B cell licensing are still not fully understood. By comparing thymic B cells to activated Peyer’s Patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by immunoglobulin class switch recombination (CSR) without germinal center B cell phenotypes or structures. Transcriptional profiling supported the finding of extrafollicular activation of thymic B cells, but also demonstrated a strong interferon signature distinct from peripheral B cell activation. Removal of type I, II and III IFN receptors revealed thymic B cell activation and CSR were partially dependent on type III IFN signaling, while type I and II IFN signaling showed minimal contribution. Loss of type III IFN signaling in thymic B cells resulted in reduced licensing and alterations in self-antigen presentation. Together, these findings reveal the importance of type III IFN in generating tolerogenic thymic B cells and suggests inflammation in the thymus has a significant impact on the generation of central immune tolerance to B cell self-antigens.
The impact of pulmonary fibrosis on neutrophil aging and trafficking
Authors: Warheit-Niemi Helen I , Moore Bethany B
Published: January 2023

Neutrophil aging is the process through which neutrophils released from the bone marrow into circulation acquire specific phenotypic and functional changes over time. After neutrophils age in circulation, they return to the bone marrow or other organs for clearance. Aged neutrophils are highly proinflammatory and can potentially contribute to tissue injury if their return to and clearance within the bone marrow is dysregulated. We have found that the development of pulmonary fibrosis in mice is associated with decreased numbers of aged neutrophils within the bone marrow compared to non-fibrotic mice 21 days after induction of fibrosis, indicating that neutrophils are not returning to the bone marrow for clearance after aging in circulation. Indeed, we observed a corresponding increase in the numbers of aged neutrophils in peripheral blood and bronchioalveolar fluid (BALF) of fibrotic mice compared to non-fibrotic mice, supporting the idea that pulmonary fibrosis prevents neutrophil migration back to the bone marrow after aging. Pulmonary fibrosis is a disease characterized by aberrant wound healing and lung and vascular injury. In accordance with this, we have observed increased levels of extracellular DNA and neutrophil elastase in plasma and BALF of mice 21 days after induction of fibrosis. These products are released in high levels by aged neutrophils during the formation of neutrophil extracellular traps, suggesting that aberrant aged neutrophil behavior may contribute to lung and vascular injury in pulmonary fibrosis. Our study seeks to understand the role of neutrophils in fibrotic disease progression by evaluating how fibrosis may alter the trafficking of aged, highly proinflammatory neutrophils.

Supported by grants from NIH (R35 HL144481, F31 HL152509) and the Scleroderma Foundation (Arnold Postlethwaite, M.D. Memorial Pre-Doctoral Summer Fellowship Award)
CCR2 identifies two subtypes of pathogenic human type 17 Th cells and has a non-redundant role in their transendothelial migration
Authors: Parween Farhat , Singh Satya P , Kathuria Noshin , Zhang Hongwei , Farber Joshua M
Published: January 2023

We have shown previously that CCR2 is expressed on highly differentiated human Th memory cells as well as mucosal associated invariant T (MAIT) cells. CCR2+ T cells typically co-express a combination of other inflammation-associated chemokine receptors, including CCR6, which is found on all type 17 T cells. We found that the CCR6+ CCR2+ human Th cell population is enriched in cells with a pathogenic cytokine and cytotoxic profile. Single-cell analysis revealed two subtypes of such CCR6+ CCR2+ cells, either biased to produce GM-CSF or IFNg. Pathogenic activity requires migration into inflamed tissue, and to understand the roles for CCR2, CCR6 and other chemokine receptors expressed on these cells in extravasation we used flow chambers containing HUVEC activated with TNFa and/or IFNg. We found that although CCR6, CCR5 and CXCR3 (but not CCR2) were capable of mediating T cell arrest, only CCR2 was active in transendothelial migration. CCR2’s activity was a function of the localization of its principal ligand, CCL2, in endothelial cells. Native CCL2 was found intracellularly but not on the cell surface. Immediate pre-treatment of cells with CCL2 or using HUVEC transduced to express a CCL2-CXCL9 chimeric chemokine that bound to surface glycosaminoglycans enabled CCR2 to mediate T cell arrest. These studies reveal the integration of transendothelial trafficking and a pathogenic effector profile in type 17 human Th cells and demonstrate redundant and non-redundant roles for the multiple chemokine receptors on these cells due to chemokine positioning. Our findings could inform more effective combinatorial inhibition or expression of chemokine receptors/ligands to regulate the migration of highly inflammatory T cells.

Supported by NIH
Role of paracrine mTOR signaling in regulating thymus size and function.
Authors: Wedemeyer Sarah A , Jones Nicholas , Emtage Simon , Griffith Ann
Published: January 2023

The thymus is the primary site of T cell generation, and the unique thymic stromal microenvironment directs T cell differentiation, self-tolerance and self-restriction. However, the size of the thymus declines precipitously relatively early in life, resulting in declining production of naïve T cells. Homeostatic mechanisms drive a shift toward an oligoclonal T cell memory, leaving the elderly less responsive to vaccines and new infections, especially viral infections. Preventing or reversing age-associated thymic atrophy therefore holds potential for extending the healthspan. Mechanisms governing thymic atrophy have been difficult to identify because the primary targets of atrophy, cortical thymic stromal cells, are rare and difficult to isolate. Using an informatic approach to characterize the transcriptional response of thymic stromal cells during age-related atrophy or experimental regeneration, we previously showed that cortical thymic epithelial cells (cTECs) display a unique morphology characterized by extensive looping projections. Atrophy is associated with contraction of these projections, which are renewed during induced regeneration. Further informatic analysis suggested that paracrine mTOR signaling in cTECs promoted by ligands expressed in medullary TECs (mTECs) may be required to maintain the cellular architecture of the cortical stroma and preserve thymus size. To test these hypotheses, we generated two new tissue-specific transgenic mouse lines overexpressing mTOR-activating ligands (IGF1 and FGF21) in most mTECs. Ongoing experiments aim to characterize the impact of increased paracrine mTOR signaling on age-associated thymic dysfunction. This work was supported by N.I.H. R21AI154109.

Supported by grant from NIH (R21 AI154109)
Risky business blockade: RAG2 basic region blocks V(D)J recombinase function upon genotoxic stress in DNA damage response
Authors: Simpson Destiny A , Rodgers Karla K
Published: January 2023

The V(D)J recombinase, composed of the recombination activating gene (RAG) proteins, RAG1 and RAG2, generate functional antigen receptor genes through the repeated generation of DNA double strand breaks and DNA repair. However, this process can lead to increased genomic instability particularly in the presence of excess DNA breaks. Previously, we showed in pre-B cells that full length RAG2, a nuclear protein, will export to the cytoplasm with DNA damage in an ATM-dependent manner with eventual recovery back into the nucleus following DNA repair. We propose that re-localization of RAG2 prevents RAG-mediated DNA cleavage in the presence of excess DNA ends. Here, by using fluorescently tagged RAG proteins, we show that DNA damage-induced export of the V(D)J recombinase can be recapitulated in non-lymphoid cells. Further, we determined that mutations in the intrinsically-disordered region of RAG2 have distinct effects on this process. Specifically, a basic region mutant of RAG2 (K/R 518–524 A) alters the localization pattern of the V(D)J recombinase, and yet its pre-DNA damage localization pattern is re-established upon DNA repair. Conversely, a T490A mutant blocks relocalization of RAG2 following genotoxic stress for both the single T490A mutant and the combined T490A/basic region mutant. Together, these results demonstrate the role of the intrinsically-disordered region in RAG2 in regulating the mobility and subcellular localization of the V(D)J recombinase upon cellular conditions that may be deemed too risky for continued DNA rearrangements.

Supported by NIA T32AG052363, NIH AI128137, OCAST
Sex-Dependent Differences in Serum Autoantibody Levels in the 3×Tg-AD Model of Alzheimer’s Disease
Authors: Ma Donglai , Song Wei , Michalski Bernadeta , Sakic Boris , Fahnestock Margaret
Published: January 2023

Sex-dependent discrepancies in disease prevalence and serum autoantibody levels are observed in patients and animal models of Alzheimer’s disease (AD). The present study examines whether gonadal hormones play a role in sex differences in serum autoantibody levels in the 3×Tg-AD mouse model of AD. 3×Tg-AD and wild-type (WT) mice were gonadectomised or sham-operated at 3 months of age. After behavioural phenotyping at 6 months of age, the animals were assessed for serum autoantibodies by indirect immunofluorescence for antinuclear antibodies (ANA) and by line-immunoblot assay for an additional 16 monospecific autoantibodies including anti-nucleosome antibodies. There were significant differences between the strains in ANA levels, with the major target antigens confirmed as nucleosomes. The results of ANA and anti-nucleosome assays were combined for further analysis. Further analysis revealed: 1) the level of serum autoantibodies in male 3×Tg-AD mice was higher than in female 3×Tg-AD animals, and this was not altered by orchiectomy. 2) sham-operated 3×Tg-AD female mice displayed a significantly lower level of serum autoantibodies than sham-operated WT females. 3) ovariectomy further reduced the level of serum autoantibodies in female 3×Tg-AD mice. The results suggest that dissimilar levels of serum autoantibodies in 3xTg-AD mice are a sex-dependent phenomenon and that female hormones play a role in regulation of their synthesis.

Funded by grant #SVB-158618 from the Canadian Institutes of Health Research to MF.
Interleukin-22 plays a protective role in animal model of experimental colitis by signaling on Math1+ cell.
Authors: Singh Ankita , Beaupre Michael , Lin Xun , Gaudino Stephen , Kumar Pawan
Published: January 2023

Interleukin-22 plays both beneficial and deleterious role in intestinal inflammation. Further investigation is required to understand cell target and mechanism of action of IL-22 in large intestine.

By using various intestinal cell specific IL-22Ra1 knockout mice models, such as entire intestinal epithelial cell (IL-22Ra1ΔIEC), Paneth (IL-22Ra1ΔDefa6), tamoxifen inducible Lgr5+ intestinal stem cell (IL-22Ra1ΔLgr5) and RU486 inducible secretory cell (IL-22Ra1ΔMath1-PGR). We tried to identify specific cell target of IL-22 in intestine. To induce experimental colitis, 8 days dextran sulphate sodium (DSS, 2%) was provided in drinking water followed by two days of normal water. On assessing colitis severity, more weight loss, histopathological changes and reduced colon length was observed in IL-22ra1ΔIEC mice. Increased expression of inflammatory cytokine genes and Lgr5+ stem cell related Olfm4 transcript were observed in distal colon of IL-22ra1ΔIEC as compared to IL-22ra1fl/fl mice. IL-22Ra1 signaling in IL-22Ra1ΔLgr5 and IL-22Ra1ΔDefa6 mice were dispensable in DSS induced colitis. Although goblet cell number was unaltered, expression of selective glycosyltransferases (causing O-glycosylation of mucin) were significantly reduced in colon of DSS treated IL-22Ra1ΔIEC as compared to IL-22Ra1fl/fl mice. Type-O glycan profile analysis of colon mucin using MALDI-TOF reveal premature termination of core 1 and 2 structure in IL-22Ra1ΔIEC mice as compared to IL-22Ra1fl/fl mice at steady state. Our preliminary data show that IL-22Ra1ΔMath1-PGR mice are more susceptible to DSS induced colitis.

IL-22 may act by inducing expression of glycosyltransferases in Math1+ cell to abrogate development of experimental colitis.
Arthroplasty surgery alters the local immune composition of the knee joint by causing increases in specific lymphocyte populations.
Authors: Cichos Kyle H , Raman Chander , Ghanem Elie S
Published: January 2023

Objective

We aimed to determine how the innate and adaptive immune cell composition and cytokine profiles of the knee joint microenvironment are altered in replaced knees compared to native knees.

Methods

Upon IRB approval, we obtained peripheral blood (PBMC) and synovial fluid (SF) from 14 native knees with osteoarthritis (OA) and 12 replaced knees at least 6 months after arthroplasty surgery, comprising 26 total patients. All PBMC and SF samples were analyzed by Flow Cytometry, and utilizing multidimensional reduction analysis via TriMap and clustering analysis by FlowSOM, cell clusters were phenotyped for identification of unique populations of cells. Additionally all samples were analyzed for cytokine/chemokine profiles using 65-Plex ProcartaPlex assays.

Results

Overall, there was no significant different in any of the innate cell populations studied, including neutrophils, monocytes (including G-MDSCs), macrophages (M1 of M2 phenotypes), and dendritic cells. B cells, CD8 effector T cells, CD4 T cells, Naive CD4 T cells, Th17 cells, and CD4 T cells with Th1-phenotypes s were all significantly increased in replaced knees compared to native knees (p < 0.02 in each case). The replaced knee group showed elevated levels of CXCL5, HGF, IFN-gamma, IL-6, IL-16, CXCL10, CCL2 (MCP-1), CCL8, MDC, TNF-alpha, and VEGF-A compared to the native knee group (p < 0.01 for all) and a decrease in CX3CL1, CXCL11, IL-3, IL-5, CXCL9, TRAIL (p < 0.02 in all cases).

Conclusions

Arthroplasty surgery results in a lymphocytic shift in immune cell populations and a generally pro-inflammatory cytokine/chemokine profile. Overall, this lymphocytic shift may have implications for development of periprosthetic infections and warrants further study.
Interrogating mechanisms of CD8 T cell dysfunction in obese asthma
Authors: Hay Ceire , Sayed Samir , Conrey Peyton , Campos Jose , Pastore Christopher F , Sengupta Shaon , Herbert De’Broski , Henrickson Sarah E
Published: January 2023

Pediatric obese atopic asthma (OA) is a complex and poorly understood disease at the intersection of two of the most common chronic inflammatory diseases of childhood. Pediatric OA patients often experience severe asthma exacerbations caused by respiratory viral infections. However, the immunological mechanisms by which obesity modifies immune function in asthma remain poorly understood. Using high dimensional immune profiling, we have demonstrated CD8 T cells from pediatric OA patients have an exhausted-like immunophenotype compared to non-obese asthmatics. To more deeply characterize the role of CD8 T cell dysfunction in OA, we developed a mouse model of OA using high fat diet (HFD) to induce obesity and house dust mite (HDM) to induce asthma. We found that non-naïve CD8 T cells isolated from the lungs of OA mice demonstrate increased expression of multiple activation and inhibitory receptors (e.g., PD-1, CD39 and Tim3) as well as TOX, all consistent with T cell exhaustion, compared to healthy controls. We hypothesize the exhausted-like state of CD8 T cells in OA impairs anti-viral responses, thereby increasing severity of asthma exacerbation during respiratory viral infections. Ongoing work is testing antigen specific responses to influenza infection. Overall, this reveals a previously unrecognized role for exhausted-like CD8 T cell responses in mediating the immunopathogenesis of pediatric obese asthma.

This work was supported by the Translational Research Training Program in Environmental Health Sciences Pre-Doctoral Fellowship to C.A.H. and K08-AI135091 to S.E.H.
The NLRP3 Inflammasome Inhibitor OLT1177® Ameliorates Experimental Allergic Asthma in Mice
Authors: Lunding Lars P , Skouras Damaris B , Vock Christina , Dinarello Charles A , Wegmann Michael
Published: January 2023

Background

The symptoms of asthma arise on the basis of a chronic inflammation of the airways. The NLRP3 inflammasome plays a central role in regulating inflammation e.g. by activating interleukin (IL) 1 cytokines, which in turn promote allergic inflammation. This study aims to evaluate the therapeutic potential of the specific NLRP3 inhibitor OLT1177® on experimental allergic asthma (EAA) in mice.

Methods

The impact of OLT1177® on NLRP3 expression, caspase-1 activation, and IL-1β release were assessed in vivo and in allergen-restimulated mononuclear cells (MNC) from ovalbumin (OVA)-sensitized mice. The effect of intra-peritoneal OLT1177®-treatment on OVA- and house dust mite (HDM) EAA and on poly(I:C)-triggered exacerbation of EAA were assessed. Furthermore, the effect of oropharyngeal aspiration of OLT1177® and oral OLT1177® via an enriched diet were also evaluated in mice with OVA-induced EAA.

Results

OLT1177® reduced NLRP3 expression, caspase-1 activation, and levels of activated IL-1β in vitro and in mice with OVA-induced EAA. Intra-peritoneal treatment with OLT1177® significantly diminished allergic airway inflammation, mucus hyperproduction, and airway hyperresponsiveness (AHR) in mice with OVA- and HDM-induced EAA as well as in mice with poly(I:C)-triggered exacerbation of EAA. While oropharyngeal aspiration of OLT1177® had no effect on EAA, oral OLT1177® via feeding an enriched diet markedly reduced all pathophysiologic hallmarks of EAA in mice.

Conclusion

Intra-peritoneal and oral delivery of OLT1177® is effective to inhibit the NLRP3 inflammasome in vivo and to treat EAA in mice.

This study was mainly supported by the German Center for Lung Research (DZL). The study was also supported by Olatec Therapeutics LLC and the Interleukin Foundation.
GPR43 signaling in lung eosinophils suppresses neutrophilic airway inflammation in asthma
Authors: Yu Jihyun , Kim Seongryong , Kim You-Me
Published: January 2023

Asthma is caused by chronic inflammation in airways. It typically manifests type 2 immune activation and eosinophilia, while more severe, steroid-resistant asthma involves additional activation of Th17 cells and neutrophils. Even though pathogenic roles of eosinophils in asthma have been extensively studied, regulatory mechanisms curving down their overactivation are not well known. We found that eosinophils in asthmatic lung highly upregulate expression of GPR43, a receptor for short-chain fatty acid mediating various anti-inflammatory responses. In eosinophil-specific GPR43-deficient (GPR43ΔEOS) mice, asthma induction by Ova/alum or house dust mite extracts resulted in increased activation of Th17 cells and neutrophils in the lung compared to wild type mice. Consequently, GPR43ΔEOS mice showed more severe lung pathology and disease phenotype. Moreover, we identified a distinct Siglec-F+ neutrophil subset is enriched in GPR43ΔEOS mice by scRNA-seq analysis of the asthmatic lungs. Siglec-F+ neutrophils were recently shown to mediate Th17 cell activation and aggravation of asthma in vivo. We also found that Siglec-F+ neutrophils promote in vitro Th17 differentiation. Co-culture experiments of lung eosinophils and bone marrow neutrophils revealed that GPR43-deficient eosinophils directly mediate conversion of Siglec-F-neutrophils into Siglec-F+ neutrophils via GM-CSF secretion. Combined, our results demonstrate that GPR43 signaling in lung eosinophils prevents generation of Siglec-F+ neutrophils and Th17 cell activation, thereby suppressing severe asthma pathology.
Effect of HIV status on Th17/IL-17 immune function in subjects undergoing tuberculosis treatment in Kenya
Authors: Ongaya Asiko , Files Megan , Martinez-Martinez Yazmin Berenice , Chacha Robi , Kyalo Jeremiah , Mwangi Paul , Huante Matthew B , Navqi Kubra , Amukoye Evans , Endsley Janice J
Published: January 2023

Mycobacterium tuberculosis and HIV remain top global public health concern. Complexities associated with Mtb/HIV coinfections, including in those virally suppressed with anti-retroviral therapy (ART), present significant challenges to efforts to reduce both infections. The pattern of cytokines produced by T helper (Th) lymphocytes plays a central role in susceptibility, relapse and reinfection to TB. To understand how HIV and ART status affects recovery of Th cell effector function, we conducted a prospective study of HIV+ or HIV- subjects from Nairobi, Kenya. Blood samples were taken at start of TB treatment and 5 months after. Cytokine bead array was used to assess human Th cell-derived cytokines isolated from plasma from Nil, TB1 and Mitogen QuantiFERON tubes, including; IL-4, IL-9, IL-10, IL-17A, IL-17F, IL-22, IFN-γ and TNF-α. Intracellular cytokine staining (ICS) of cryopreserved peripheral blood mononuclear cells was also performed to determine changes in baseline numbers of Th1, Th2, Th17, Th22, IL-10+ Th cells, as well as HIV+ Th cells, across treatment groups. Results show reduced plasma IL-9, IFNg and IL-22 activation by mitogen in TB+HIV+ subjects that is independent of ART status, while IL-17A is moderately reduced in TB+HIV+ART- subjects. Cytokine recall to TB1 antigen was observed to be similar across all treatment groups, with the exception that a moderate reduction of IFNg and IL-17A was observed in the TB-HIV+ group. In subjects compliant for ART, ISC analysis revealed greater numbers of HIV+ cells in TB+HIV+, compared to TB-HIV+, groups. These preliminary results identify defects in effector cytokines produced by Th cells that may persist after ART and contribute to poorer TB treatment outcomes in subjects with HIV.

Supported by NIH, NIAID, 1R01AI147948
CD8+ T cell impairment drives severe human metapneumovirus respiratory disease in aged mice.
Authors: Parks Olivia Bartlett , Lan Jie , Zhang Yu , Sojati Jorna , Eddens Taylor John , Byersdorfer Craig A , Williams John V
Published: January 2023

Objective

Human metapneumovirus (HMPV) is a leading cause of severe respiratory disease in children <5, adults >65, and the immunocompromised. CD8+ T lymphocytes are the primary cell responsible for clearing HMPV infection in the lung. We tested the hypothesis that CD8+ T cell impairment in the aged host drives severe respiratory infection and delayed viral clearance.

Methods

B6 aged 72–73wk mice and young 6–7wk mice were infected with 2×106 PFU HMPV. Seven days post infection, mice were euthanized and lung, bronchoalveolar lavage (BAL), spleen, and draining lymph nodes were collected for flow cytometry. Lung and BAL supernatant was collected for viral titer, histology, and cytokine measurement.

Results

Aged mice lost more weight, had delayed viral clearance, and increased clinical scores and lung histopathology. Aged mice produced fewer HMPV-tetramer+ cells in lung and BAL. Aged CD8+ T lymphocytes in BAL co-expressed inhibitory receptors PD-1, TIM-3, LAG-3, and 2B4. Aged CD8+ T lymphocytes also upregulated senescence markers, T-bet and EOMES. Upon ex vivo peptide stimulation, aged CD8+ T cells produced significantly less granzyme B compared to young CD8+ T cells. Adoptive transfer of young T cells into aged mice rescued this severe disease phenotype. Young CD8+ cells in the aged host produced more tetramer+ cells and granzyme B and had less senescent marker expression.

Conclusion

Aged mice exhibit more severe respiratory disease when infected with HMPV. Aged CD8+ T cells appear to be impaired and upregulate senescence markers that contribute to severe HMPV infection in the aged host.

Supported by: NIH/NIAID 5R01 AI085062-09, John V. Williams, MD NIH T32GM008208 Medical Scientist Training Program, University of Pittsburgh School of Medicine
Helminth TGF-β mimic, TGM, increases leukocyte migration and activation while also enhancing cutaneous wound healing and tissue regeneration.
Authors: Lothstein Katherine E , Smyth Danielle J , Maizels Rick M , Gause William C
Published: January 2023

Intestinal parasites express excretory/secretory (ES) molecules, which modulate the type-2 immune response including anti-inflammatory and tissue repair mechanisms. TGF-β mimic (TGM), an ES molecule secreted by Heligmosomoides polygyrus (Hp), binds TGF-β receptors yet lacks structural homology to TGF-β and distinct receptor interactions. We now demonstrate that TGM enhanced wound healing using an in vivo wound biopsy model in which TGM, in a 1.5% carboxymethylcellulose solution, was topically administered beneath a Tegaderm layer. Through histological analysis, greater restoration of normal tissue structure in the wound beds of TGM treated mice was observed during mid to late stage wound healing. These observations included accelerated re-epithelialization as well as hair follicle regeneration, in the absence of increased scarring. Using flow cytometric, histological and gene expression analyses, differential expansion of myeloid populations at different stages of wound healing was observed. This included enhanced early accumulation and persistence of migratory Ly6C+ macrophages in TGM-treated wounds during the early inflammatory phase. Additionally, at mid-stage wound healing, we observed increased CD301b+ myeloid populations, a subset that is critical to efficient tissue restoration. In summary, TGM can enhance skin wound healing and associated recruitment of specific macrophage subsets and elevation of type 2 cytokines. This study indicates a role for TGM as a potential novel therapeutic option for enhanced wound healing.
Delineate the effects of cannabidiol on type 1 IFN mediated inflammation during HIV infection
Authors: Tomer Shallu , Mu Wenli , Ng Hwee , Wang Li , Wennerberg Wally , Razek Valerie , Martin Heather , Zhen Anjie , Kitchen Scott
Published: January 2023

Methods

THP-1 and HMC3 cells were exposed to CBD/vehicle with and without stimulation with 2’3’-cGAMP (cGAMP), LPS, or PMA/ionomycin. qPCR was carried out to investigate the expression of interferon stimulatory genes (ISGs). RNA-Seq was performed to investigate gene expression in cGAMP-stimulated THP1 cells treated with vehicle/CBD. Western blot was performed with THP1 cells treated with vehicle/CBD then cGAMP to investigate the change of expression of phosphorylated STING (pSTING) and autophagy markers (LC3B, p62). Lastly, humanized mice (Hu mice) were constructed, infected with HIV-1 for 12 wks then treated with vehicle/CBD for 2 wks. Flow cytometry and qPCR were performed to examine changes in T cell activation and IFN-1 signaling in peripheral blood before and after CBD treatment.

Results

We observed significant down regulation of ISGs in stimulated, CBD-treated THP-1 and HMC3 cells. RNA-seq and qPCR confirmatory analysis showed that after stimulation IFN-1 genes were down regulated in CBD group compared to vehicle. We observed significantly higher LC3B/p62 and lower pSTING levels in CBD treated, stimulated cells as compared to vehicle treated, stimulated cells. Lastly, we observed a reduction in PD-1, Tim-3 and ISG MX1 expression in HIV-1 infected Hu mice treated with CBD.

Conclusion

We observed down regulation of ISGs in stimulated CBD treated cells. CBD treatment leads to induction of cellular autophagy, responsible for regulating/degrading pSTING after cGAS-STING pathway activation and lower ISGs as observed in our study. Lastly, we observed reduced ISGs, and decreased activation/exhaustion marker expression on T cells in HIV infected Hu mice after CBD treatment, suggesting the potential of CBD as an immunomodulator.

Supported by grants from NIH/NIDA RFA-DA-20-022
Cholesterol, LXR activation, and myelin uptake in the macrophage response to diverse signals
Authors: DeLong Jonathan Howard , Velazquez Sofia Evelyn , Sun Leonardo Shiuchi , Landreneau Margaret J , Sansing Lauren Hachmann
Published: January 2023

Introduction

Monocyte-derived macrophages contribute to neurotoxic inflammation during hemorrhagic stroke. Cholesterol is an immunosuppressive molecule present at high levels in the brain, primarily as a component of myelin. Uptake of myelin and activation of the transcription factor LXR downstream of myelin and cholesterol uptake limits the macrophage response to LPS. However, the roles of these molecules in macrophage responses to the endogenous signals present during sterile inflammation are poorly understood. The present study seeks to determine what role myelin, cholesterol, and LXR activation play in modifying macrophage responses to diverse signals.

Methods and Results

Murine bone marrow-derived macrophages were treated with myelin, cholesterol, or the LXR agonist T0901317 18h before stimulation with S100A9, IFN-g, TNF-a, IL-4, IL-10, TGF-b, p(I:C), Pam3CSK4, or LPS. Production of TNF-a, IL-6, CCL2, and IL-10 were measured by multiplex ELISA. Transcriptome-wide effects were measured by RNA sequencing. Cholesterol limited inflammatory cytokine production in response to LPS but not to other stimuli. The LXR agonist T0901317 did not suppress cytokine production. Principal component analysis revealed that cholesterol had strong effects on the transcriptomic responses to LPS but not to the other stimulations. Myelin affected the transcriptomic response to all inflammatory stimulations.

Conclusions

Cholesterol limits the macrophage response to LPS, partially through activation of LXR, but does not broadly limit macrophage activation. Uptake of myelin has broader effects than cholesterol, suggesting that non-cholesterol components of myelin modulate the macrophage response.

Supported by grants from NIH (R01 NS097728, T32 HL007950) and AHA (Postdoctoral Fellowship 830877)
Vagus nerve stimulation leads to contraction of the spleen and dampened IFNγ production in response to TLR stimulation
Authors: Nash William T , Tanaka Shinji , Okusa Mark D
Published: January 2023

Vagus nerve stimulation (VNS) generates an anti-inflammatory state and limits the pathology of inflammatory diseases, such as sepsis and acute kidney injury. Interestingly, splenectomy prior to stimulation results in a loss of this protection. Thus, the spleen is a central organ for converting VNS signals into protective anti-inflammation. To better understand the interplay between VNS and the spleen and gain insight into the mechanisms of anti-inflammation, we performed a broad query of the spleen post-VNS. Mice received either electrical stimulation of the left-cervical vagus nerve or sham surgery. Spleens were harvested and assessed for mass, immune cell populations, and splenocyte in vitro cytokine production in response to TLR4, 3, and 9 agonists (LPS, poly I:C, and CpG, respectively). We discovered that by 48 hours post-VNS, spleen mass was reduced and the number of CD45+ cells was significantly decreased compared to sham mice. Analysis of CD45+ cells revealed that many individual immune cell populations were diminished. B cells, T cells, NK cells, dendritic cells, and basophils were all significantly reduced and eosinophils and monocytes showed strong trends towards reduction, nearing significance. In response to TLR agonists, our Luminex analysis revealed a strong impact on TLR4 stimulation, with 12 of the 32 measured cytokines showing a significant reduction in supernatants from VNS samples. TLR3 and TLR9 stimulation also exhibited significant reductions in a subset of the measured cytokines, but only IFNγ exhibited strong signs of reduced production in all TLR stimulation conditions. Thus, VNS has profound impacts in the spleen and some of the observed protection could be due to tighter control of immune cell IFNγ secretion.

Supported by grants from the NIH (R01 DK123248)
The DEAH-box helicase RHAU regulates immunoglobulin class switch recombination
Authors: Thavornwatanayong Thongthai , Zheng Simin , Guillaume Sabine Jean , Vuong Bao Q
Published: January 2023

During a humoral immune response, B cells produce alternative immunoglobulin isotypes (IgG, IgA, IgE) through class switch recombination (CSR). Initiation of CSR requires AID-mediated deamination of switch (S) regions in the immunoglobulin heavy chain (IgH) locus. Although G-quadruplex(G4)-forming S region RNAs bind AID and localize AID to S region DNA sequences, the molecular mechanism that regulates the transition of AID binding from RNA to DNA remains uncharacterized. Highly stable G4 structures necessitate helicases to unwind the G-G hydrogen bonds, which in turn may permit the base-pairing of the S transcript to the complementary DNA sequence in the IgH locus during CSR. An S region RNA pull-down assay identified the RNA Helicase associated with AU-rich element (RHAU), a DEAH-box RNA helicase, as an S transcript binding protein. To examine the role of RHAU in CSR, we genetically deleted a floxed RHAU allele in B cells of mice using CD23-Cre. Conditional deletion of RHAU in B cells reduced CSR in vivo and in vitro to approximately 50% of wild-type controls. This data suggests that RHAU plays an important role in CSR. We hypothesize that RHAU unwinds G4 in S transcripts to facilitate the handoff of AID from G4-RNA to S region DNA during CSR.

Supported by The National Cancer Institute (2U54CA132378) and The National Institute of General Medical Sciences (1SC1GM132035-01)
The plasma cell proteome initiates early in B cell activation in advance of Blimp-1.
Authors: Gaudette Brian T , Allman David
Published: January 2023

Plasma cell (PC) differentiation is a highly regulated process that radically changes the morphology and function of a B lymphocyte to that of a professional protein secretor. It is becoming clear that this process precedes increase in immunoglobulin synthesis rather than reacting to it. We have shown previously that large segments of the PC transcriptome are initiated in an mTORC1-dependent manner prior to both Blimp-1 and Xbp1s expression. Still, the kinetics of induction of the PC proteome remain largely unknown. We performed a whole proteome kinetic analysis of activated Blimp-1-reporter follicular B cells in PC-inductive conditions every 12 hours for three days and observed distinct patterns in induction of the PC proteome. To elucidate transcriptional networks, we performed single cell RNA-seq with the same conditions and correlated transcription factor expression with genes coding for these proteome modules within single cells. We found distinct subgroups within proteome modules corresponding to transcriptional patterns each with a highly correlated transcription factor set. These included robust, transient, Myc-associated factors required for translation initiation; rapid, sustained expression of protein synthesis machinery; and gradual, sustained increases in proteins associated with ER function and secretion. Each of these preceded the induction of Blimp-1 and increase in immunoglobulin protein. Finally, using inducible deletion of Blimp-1 in conjunction with the reporter allele we show that these unique transcriptional and proteomic programs are initiated independently of Blimp-1 expression. Thus, activated B cells prepare for protein secretion by initiating the PC proteome prior to antibody secretion.

Supported by grants from the NIH: R01 AI113545, AI139123, AI154932, R21 AI133998 (PI:Allman) and BTG was supported by training grant T32 CA009140 (PI:Pear)
Function of ATM and MSH2 during nonhomologous end joining in DNA repair
Authors: Sible Emily , Attaway Mary , Fiorica Giuseppe , Choi Jee Eun , Chwat-Edelstein Tzippora , Haughton Lyric , Bedasee Donna , Vuong Bao Q
Published: January 2023

Class switch recombination (CSR) produces secondary immunoglobulin isotypes and requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain gene locus. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with APE1, a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also play a role in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, ATM−/− mice were bred to mice deficient for the MMR gene MSH2. Surprisingly, the predicted Mendelian frequencies of ATM−/− MSH2−/− adult mice were not observed. To obtain ATM- and MSH2-deficient B cells, ATM was conditionally deleted on an MSH2−/− background using a floxed ATM allele [ATMF] and B cell-specific Cre recombinase expression (CD23-cre) to generate a deleted ATM allele (ATMD). As compared to the ATMD/D and MSH2−/− mice and B cells, the ATMD/D MSH2−/− mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from ATMD/D MSH2−/− B cells that were induced to switch to IgG1 in vitro revealed a significant loss of blunt end joins and an increase in insertions as compared to wildtype, ATMD/D, or MSH2−/− B cells. This data suggests that the absence of both ATM and MSH2 blocks NHEJ and leads to inefficient end joining and the reduced CSR. We identify complementary roles for ATM and MSH2 in NHEJ and A-EJ during CSR and propose a model whereby ATM and MSH2 function cooperatively to regulate end-joining during CSR through pS38-AID.

This work was supported by The National Institute on Minority Health and Health Disparities (5G12MD007603), The National Cancer Institute (2U54CA132378), and The National Institute of General Medical Sciences (1SC1GM132035-01).
Defining the cis- and trans-regulatory architecture for human PD-1 in T Follicular Helper Cells
Authors: Powell Michael Duane , Rose James R , Neeld Dennis , Rahmberg Andrew R , Scharer Christopher D , Boss Jeremy M
Published: January 2023

The surface immune checkpoint inhibitor Programed Death-1 (PD-1), encoded by PDCD1, is expressed throughout the immune system. Notably, PD-1 expression is amongst its highest in a specialized subset of CD4+ T follicular helper (TFH) cells. TFH cells primarily function by engaging with cognate B cells, facilitating the generation of high affinity vaccine- and pathogen-specific antibodies. Despite the proven clinical benefit of therapies targeting the PD-1 pathway, very little is known regarding the mechanisms by which human PDCD1 is transcriptionally regulated in humans. Here, we seek to elucidate both the cis- and trans-regulatory mechanisms that govern PDCD1 expression in TFH cells. Human TFH cells were sourced from discarded tonsil tissue, circulating blood, or through an ex vivo differentiation model to generate ‘TFH-like’ cells. RNA-seq analysis revealed that ex vivo-derived ‘TFH-like’ cells resembled bona-fide in vivo TFH cells, including induction of PDCD1. Furthermore, ATAC-sequencing experiments identified 8 conserved regions in the PDCD1 locus that exhibited altered accessibility in TFH cells. These cis-regulatory elements were determined to have distinct functions in controlling PDCD1 expression. Additionally, bioinformatic analysis revealed a core TFH gene program, implicating the transcription factors BCL-6, ASCL2, TOX, TOX2, and AIOLOS as regulators of PDCD1 and TFH fate. Taken together, these findings uncover the molecular mechanisms that control PDCD1 expression, informing the design of future therapies aimed at manipulating PD-1.

Supported by National Institute of Health grants: RO1 AI113021 to J.M.B, F32 AI161857 to M.D.P
A temporal switch in T follicular helper cells controls the output of the GC response to influenza
Authors: Arroyo Nicole M , Bachus Holly , Papillion Amber , Rosenberg Alexander F , Bradley John Edwin , Leon-Ruiz Beatriz , Ballesteros-Tato Andre
Published: January 2023

T follicular helper (Tfh) cells are a specific subset of CD4 T cells that localize in the B cell follicle and are essential for the Germinal Center (GC) response. Under polarizing environments, Tfh cells produce effector cytokines, including IFN-γ and IL-4. Beyond promoting class switching, the role played by the different subsets of cytokine-producing Tfh cells remains largely unexplored. Using an influenza infection model, we show here that Tfh cells switch from producing IFN-γ early after infection to mainly producing IL-4 at later time points. Importantly, IFN-γ and IL-4-producing Tfh cells differently control the GC B cell response to influenza. Whereas IL-4-producing Tfh cells help GC maintenance late after infection, interaction with IFN-γ producing Tfh cells skews the GC B cell response towards the memory differentiation pathway early after infection. Consequently, lung-memory B cells fail to differentiate in the absence of IFN-γ producing Tfh cells. Collectively, our results support a model in which temporary changes in cytokine production by Tfh cells dynamically control the outcome of the GC response. Hence, our data provide new insights into GC fate decisions following influenza infection.

Supported by grants from NIH (R56 AI162698, R01 AI110480)
Human Circulating Plasmablasts Are Not Blasting: A Probable Misnomer
Authors: Nguyen Doan C , Saney Celia , Woodruff Matthew C , Sanz Ignacio , Lee F Eun-Hyung
Published: January 2023

Following infection or vaccination, antibody-secreting cells (ASC), also called plasmablasts, appear in circulation transiently; a small fraction of them then migrate to the spleen or bone marrow (BM) to mature into long-lived plasma cells (LLPC). While LLPC, by definition, are quiescent or non-dividing, early-minted blood ASC were mostly thought to be “blasting” or proliferative. In this study, we show <3% of LLPC are Ki-67+ with none taking up BrdU. In contrast, nearly all (>95%) nascent blood ASC express Ki-67 but only 10–15% actually incorporate BrdU. Due to the limitations of traditional flow cytometry, we integrated an optofluidic technology capable of single-cell visualization into a unique ASC survival system to directly evaluate both cell division status and ongoing antibody secretion. In agreement with the literature, LLPC displayed no proliferative potential. Surprisingly, only 11–13% of the blood ASC displaying ongoing antibody secretion had divided by day 10–14. Thus, while newly-generated ASC in the blood have recently undergone proliferation, the majority have exited the mitotic cell cycle and are not actively “blasting”. As they further mature into terminal LLPC, they undergo complete cell cycle arrest. Therefore, the term “plasmablast” may actually be a misnomer.

Supported by NIH/NIAID 1R01AI121252, 1P01AI125180, U01AI141993, U54CA260563, U19AI110483, T32AI070081 and the Bill & Melinda Gates Foundation Grant INV-002351.
Modulating Immunoglobulin Secretion of a Single Human Plasma Cell by Glycolysis Inhibition
Authors: Nguyen Doan C , Woodruff Matthew C , Sanz Ignacio , Lee F Eun-Hyung
Published: January 2023

Early-minted antibody-secreting cells (ASC) undergo further maturation to become long-lived plasma cells (LLPC) in the bone marrow (BM) microniche to afford life-long humoral protection. As terminally-differentiated cells, ASC are thought to be antibody factories that secrete immunoglobulins (Ig) constitutively. This model suggests that Ig secretion is linked with survival and loss of Ig secretion is associated with apoptosis. Here, we show that the Ig secretory function of human ASC is inhibited after treatment with an GAPDH inhibitor as measured by IgG Elispot assays and by direct visualization of IgG secretion from a single ASC. Although Ig secretion ceases quickly with the initial treatment, ASC promptly recover secretory function upon removal of the inhibitor. The modulation of Ig secretory function was directly visualized in both human early-minted blood ASC and BM LLPC by single-cell analysis using an optofluidic technology. Thus, we conclude that ASC Ig secretion is not constitutive but a dynamic process which can be modulated by metabolic processes such as glycolysis.

Supported by NIH/NIAID 1R01AI121252, 1P01AI125180, U01AI141993, U54CA260563, U19AI110483, T32AI070081 and the Bill & Melinda Gates Foundation Grant INV-002351.
B-cell-intrinsic MHCII signaling limits bacteremia and systemic invasion by Citrobacter rodentium
Authors: Roland Mary Melissa , Hall Nia , Peacock Tori , Mohammed Ahmed , Jolly Amy , Kubinak Jason L
Published: January 2023

B-cell-intrinsic major histocompatibility complex class II (MHCII) antigen presentation is central to the development of T-cell-dependent antibody responses. However, the physiological significance of B-cell-intrinsic MHCII signaling during homeostatic conditions and infection are unknown. Here, using a B-cell-intrinsic MHCII knockout mouse model, we demonstrate that B-cell-intrinsic MHCII signaling is important for the development of systemic IgG1 and mucosal, high-affinity IgA responses. We also show that the conditional ablation of MHCII signaling on B cells results in a change in the microbiota composition, exhibiting a significant increase in the mucin-degrading bacteria Akkermansia muciniphila. This change in the microbiome is also associated with elevated bacterial dissemination from the gut into the systemic compartment under homeostatic conditions. Additionally, using a Citrobacter rodentium model of acute gastrointestinal infection, we also demonstrate the relevance of B-cell-intrinsic MHCII signaling in limiting invasion of the systemic compartment by an opportunistic enteric pathogen. Results from these experiments demonstrate that B-cell-intrinsic MHCII signaling is an important factor minimizing the physiological cost of microbial colonization of the gut.

Supported by NIH R21AI142409, R01AI155887