bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual specificity phosphatase 12 deficiency reveals its role in maintaining T cell survival
Authors: Chen Min , Yang Chia-Yu
Published: November 2024

Dual specificity phosphatases (DUSPs) are protein phosphatases that regulate cellular signaling pathways by dephosphorylating threonine/serine and tyrosine residues on the substrates. DUSP12 mediates the regulation of Toll-like receptor signaling, cell cycle, hepatocyte metabolism, cardiac hypertrophy, and fibrosis. Moreover, a nonsynonymous mutation of DUSP12 has been identified in patients with T-cell-mediated autoimmune diseases. However, the regulatory roles and mechanisms of DUSP12 in T cells remain unclear. In this study, we investigated the functional regulation and molecular mechanism of DUSP12 in T cells using T cell-specific DUSP12 conditional knockout mice. We found that DUSP12 cKO mice had severe T-cell lymphopenia. More interestingly, DUSP12 cKO mice spontaneously developed systemic inflammation including splenomegaly and intestinal inflammation. Furthermore, our results showed that DUSP12 localized to mitochondria and was essential for mitochondria integrity and cellular oxidative stress in T cells. Using mass spectrometry analysis, western blotting, immunofluorescent staining, and proximity ligation assay, we identified a novel DUSP12-interacted protein in T cells. The functional regulation of DUSP12 on this novel protein in T cells was also explored. Our current results made a significant breakthrough in DUSP12 functions in T cells. Also, DUSP12 may serve as a potential biomarker and therapeutic target for inflammatory disease.
Dependence of lymphopoiesis on efficient β-catenin degradation
Authors: Cosgun Kadriye , Jumaa Huda , Robinson Mark , Klemm Lars , Oulghazi Salim , Fonseca-Arce David , Xu Liang , Xiao Gang , Khanduja Dhruv , Chan Lai , Lee Jaewoong , Kume Kohei , Song Joo , Chan Wing-Chung , Chen Jianjun , Taketo Mark , Kothari Shalin
Published: November 2024

Wnt/β-catenin pathway is tightly controlled by β-catenin destruction complex to regulate lineage specification and differentiation. Compared to epithelial lineage, lymphoid cells display higher rates of β-catenin degradation to achieve constitutively low levels of β-catenin. Emphasizing the reliance of lymphoid cells on β-catenin degradation, expression of degradation resistant form of β-catenin or deletion of APC or CK1 suppressed lymphoid development. Similarly, small molecules inhibiting GSK3β mediated phosphorylation or proteasomal degradation of β-catenin induced higher toxicity selectively in lymphoid cells compared to other lineages.

Our interactome and CHIP-seq analysis highlighted that the unique dependency of lymphoid cells on efficient degradation of β-catenin is mediated by Ikaros zinc finger (IKZF) proteins and nucleosome remodeling and deacetylase (NuRD) complexes. Unlike activating TCF/β-catenin complexes in epithelial cells, recruitment of repressive β-catenin/IKZF/NuRD complexes to MYC superenhancer (SE) regions lead to suppression of Myc expression and induced cell death. Deletion of both IKZF1 and IKZF3 or mutation of Ikaros binding motifs within the Myc-SE subverted the β-catenin mediated toxicity. Upon loss of Ikaros factors, β-catenin interaction with TCF-factors resulted in de novo activation of epithelial specific genes. Our findings suggest that β-catenin degradation is an evolutionary requirement for Ikaros mediated protection of lymphoid identity.
Spatial Characterization of the Immune Microenvironment within HIV+ Lymph Nodes
Authors: Furler O'Brien Robert , Dunn Daniel , Iñiguez Luis Pedro , Del Rio Estrada Perla , Avila-Rios Santiago , Nixon Douglas
Published: November 2024

Background: Persistent HIV replication is hypothesized to induce chronic inflammation in ART-suppressed people living with HIV (PLWH), however defining this chronic inflammation within tissues is limited. Molecular and spatial characterization of chronic inflammation within human lymph nodes from PLWH provide valuable information on the immunological microenvironment that limits viral eradication.

Methods: We spatially characterized the transcriptomic and proteomic profiles of the lymph node microenvironment from 6 human lymph nodes: three from ART-suppressed PLWH, two from chronically infected PLWH, and one from a male HIV- control donor. For spatial transcriptomics analysis, lymph nodes were either from fresh-frozen or FFPE tissue. Lymph node sections (10μm) were placed onto the 10X Visium slides, stained with H&E, and imaged prior to RNA-Seq library prep. Following sequencing cluster identification was done with single cell RNA-seq datasets and downstream bioinformatics analyses were performed.

Results: Following the bioinformatic integration of spatial transcriptomic datasets, cluster analysis identified pro-inflammatory T cells within the HIV+ tissues compared to the HIV- tissue within the T cell zone. Additionally, two sets of proinflammatory myeloid cells were identified that were spatially located within the afferent and efferent lymphatics in the HIV+ tissues. These proinflammatory clusters further characterize chronic inflammation that occurs in PLWH.
Evaluating the Functional Capability of Circulating Natural Killer Cells in Human Studies
Authors: Siksou Léa , Kong Weimin , Cohen Gadi , Kumar Gaurav , Dotan Or , Schnir Victoria , Gutman Dikla , Tamir Ayala , Rigby Mark , Graves Andrew , Orange Jordan
Published: November 2024

Introduction:

Interleukin-15 (IL-15) appears to be involved in T cell and natural killer (NK) cell homeostasis and immune-mediated diseases. In a first-in-human study of a monoclonal antibody against IL-15 (TEV-53408), a reduction of circulating NK cells was observed in healthy volunteers. In order to determine if there was also a qualitative effect on NK cells, in-vitro functional assessments were performed on the remaining NK cells.

Methods:

PBMCs were obtained from 81 healthy participants(administered TEV-53408 or placebo) in a single-dose (SD) and a multiple dose (MD) study. Samples were stimulated with phorbol myristate acetate (PMA)/ionomycin to induce degranulation, stained with an NK cell-extracellular panel and CD107a. After fixation, permeabilization, and intracellular staining for perforin, cells were run through a flow-cytometry assay.

Results:

The collected data demonstrated a standard CD56 bright:dim ratio at all time points. Perforin load (MFI) and % of CD107a expression with or without stimulation suggest that TEV-53408 had no effect on the degranulation ability of NK cells.

Discussion:

These assessments provide an effective method to assess the phenotype and functionality of remaining NK cells following anti-IL-15 treatment. The results suggest that treatment with TEV-53408 does not affect the functionality of the remaining NK cells during the studied timeframe, despite the reduction of circulating NK cells.
CD38 modulates CD8 TRM responses after respiratory viral infection
Authors: de Almeida Santos Gislane , Wu Yue , Sen Chaudhuri Arka , Tang Jinyi , Cheon In Su , Narasimhan Harish , Sun Jie
Published: November 2024

Aged hosts have increased susceptibility to develop persistent pathology and sustained inflammation after exposure to respiratory viral infection. Our group previously demonstrated that this condition is associated with an exuberant CD8 tissue resident memory (TRM) cell response. However, the underlying mechanisms that contribute to exuberant TRM accumulation and pathological activity in this context are still unknown. We found that CD8 TRM cells isolated from the lungs of influenza-infected young and aged mice express high levels of CD38. Interestingly, aging further increases the expression of this molecule on CD8 TRMs. CD38 is a molecule with NAD-depleting activity, which can affect a broad diversity of cellular processes. To address the physiological consequence of high expression of CD38 by CD8 TRMs in the lung during influenza infection, young CD38KO mice and Wild type (WT) mice were infected with influenza. We observed reduced numbers of antigen specific CD8 TRMs in the lung of infected CD38KO mice compared with infected WT mice. Mixed bone marrow chimera (1:1 ratio) experiments showed decreased frequency of antigen specific CD8 T cells in the lungs following CD38 deficiency, demonstrating an intrinsic role for CD38 in CD8 TRM responses. Similar results were observed in CD8 T cell-specific CD38-deficient mice (CD38fl/flCD8cre). Together these results show an important role for CD38 in modulating CD8 TRM responses in the lung during influenza infection.
2-Deoxy-D-glucose as a potential therapeutic agent for reducing neuroinflammation following spinal cord injury
Authors: Vinit Stephane , Michel-Flutot Pauline , Mansart Arnaud
Published: November 2024

High spinal cord injuries (SCIs) lead to persistent diaphragm paralysis and related respiratory dysfunctions. The absence of functional recovery is due to limited neuroplasticity processes, mainly caused by chronic neuroinflammation occurring in the injured spinal cord. In this study, we aimed at reducing glucose metabolism that supports neuroinflammatory processes in an acute preclinical model of C2 spinal cord lateral hemi-section in rats. We administered 2-Deoxy-D-glucose (2-DG; 200 mg/kg/day s.c. for 7 days) and evaluated the effect on respiratory function and chondroitin sulfate proteoglycans (CSPGs) production around spinal phrenic motoneurons. Contrary to our initial hypothesis, our 2-DG treatment did not have any effect on diaphragm activity and CSPGs production in injured rats, even if a slight increase in tidal volume can be observed. Unexpectedly, it led to deleterious effects in uninjured (sham) animals, characterized by an increased ventilation and CSPGs production. Globally, our results seem to indicate that this 2-DG treatment paradigm could create a neuroinflammatory state in healthy animals, without altering or modulating the already installed spinal inflammation in injured rats. Given the established beneficial effects of 2-DG observed in other studies on neuronal activity and inflammation, adapted2-DG doses and/or longer treatment duration should be explored for reducing deleterious inflammatory processes occurring after SCI.
Role of the cGAS DNA sensing pathway in acute intestinal ischemia-reperfusion injury
Authors: Jayakumar Preethi , Hu Jiannan , Huang Hai , Deng Meihong
Published: November 2024

Acute intestinal ischemia-reperfusion injury (AIRI) often occurs during trauma, shock, and surgical procedures associated with high mortality. Cyclic GMP-AMP synthase (cGAS) is a DNA sensor sensing double-stranded DNA in the cytoplasm to initiate immune responses. As circulating cell-free DNA levels are known to increase during AIRI, we hypothesized that the cGAS DNA sensing pathway is involved in the pathogenesis of AIRI. To test our hypothesis, we subjected C57BL/6 (WT) and cGAS-/- mice to 60 min intestinal ischemia via superior mesenteric artery occlusion followed by 4h reperfusion to induce AIRI. Intestinal and pulmonary injury were assessed by histology, cytokine levels, and neutrophil infiltration. The jejunum was severely injured after AIRI, evidenced by damaged intestinal villi, increased IL-6 levels, and neutrophil infiltration. The cGAS signaling was activated in the jejunum after AIRI, evidenced by increased levels of cGAS and the downstream mediator pIRF3. Notably, knockout of cGAS alleviated AIRI by attenuating the damage in intestinal villi and decreasing levels of IL-6 in the jejunum compared with WT mice. The levels of IL-6 and the numbers of neutrophils in the lung were lower in the cGAS-/- mice compared with WT mice after AIRI, suggesting that the cGAS signaling was involved in AIRI-induced lung injury. Our results demonstrate that the cGAS signaling plays critical roles in the pathogenesis of AIRI. The cGAS pathway maybe targeted for treating AIRI.
Highly efficient IL-2-dependent gene activation by a single wave of IL-2R signaling in human regulatory T cells
Authors: Vujanac Milos , Yu Aixin , Malek Thomas
Published: November 2024

Low-dose IL-2 is being advanced as a therapy for autoimmunity based on selectivity toward Tregs. Regarding human CD4+ Treg and effector memory (TEM) T cells, the IL-2 sensitivity of Tregs is 10- and 100-fold greater for proximal signaling and down-stream gene activation, respectively. To further investigate the efficiency of IL-2R signaling in Tregs, we monitored the persistence of a single wave of pSTAT5 and assessed if this supported IL-2-dependent transcription. When Treg and TEM cells were pulsed with a high dose of IL-2 to normalize initial STAT5 activation, pSTAT5 persisted for >2 hr only in Tregs. Lower amounts of pulsed-IL-2 also led to persistent pSTAT5 in Tregs. The short persistence of pSTAT5 in TEM cells is not cell intrinsic because pulsing TEM cells with IL-7 also led to lasting pSTAT5. Thus, the high expression of CD25 and CD127 in Treg and TEM cells likely contributed to persistent pSTAT5. This conclusion implies that a component of proximal STAT5-dependent signaling is limiting in comparison to the numbers of bound receptors. RNA Prime Flow demonstrated that transient pulse of IL-2 or IL-7 in Treg and TEM cells caused upregulation of CISH mRNA. These results indicate that a limited exposure of T cells expressing high amounts of the IL-2R and IL-7R are linked to a mechanism to sustain proximal signaling to support gene transcription. This mechanism may fine tune gene expression enabling cells to maximize utilization of limiting cytokines in vivo.
Profiling the interactions of epigenetic regulators and their control of T cell states
Authors: Inge Melissa , Siggers Trevor
Published: November 2024

Characterizing the gene regulatory mechanisms that define distinct T cell states is essential to understanding T cell biology in health and disease. Despite advances in identifying transcriptional and epigenetic differences across T cell states, the regulators of these changes remain poorly defined. We performed a systems-level proteomic and genomic study to identify transcription factor (TF) and chromatin modifying cofactor (COF) complexes that might regulate cell state in resting, activated, and exhausted T cells. We used a high-throughput, protein-binding microarray-based approach called CoRec, to profile numerous COFs and identified unique TF-COF recruitment networks for each T cell state. In our study, some COFs are recruited specifically to TFs known to drive T cell state changes, including TFs implicated in T cell exhaustion. Exhaustion-specific recruitment of COFs suggests that these TFs may drive exhaustion through targeted alteration of epigenetic marks by these COFs. This alters expression of exhaustion-related genes and can create a functional change in T cell state. Additionally, by integrating our CoRec data with other methods, we examine how these TF-COF networks relate to chromatin accessibility and genome-wide chromatin marks. The CoRec method and its integration with other methods provides an exciting new approach for profiling cell-specific TF-COF complexes and allows us to better understand and identify the epigenetic regulators of T cell states.
Cytokine production during neonatal Escherichia coli sepsis drives disease severity through pathogen-specific pathways
Authors: Muske Josey , Knoop Kathryn
Published: November 2024

Late-onset neonatal sepsis (LOS) – often resulting from the dissemination of E. coli from the gut into the bloodstream – has an inverse correlation between severity and gestational age. In our previous data, we observed multiple E. coli strains - both pathogenic and commensal - were able to translocate from the gut and circulate systemically, resulting in variations of disease outcomes. It is unknown how the translocated E. coli strains can cause cytokine storm, lethality, and LOS, while similar E. coli is unable to cause such reaction. Additionally, we sought to understand how the neonatal immune response to sepsis is unique from that of older pups. We infected pups on either postnatal day 5-7 or 17-20 with a single-organism infection of pathogenic or commensal E. coli. During pathogenic E. coli infection, neonatal mice had increased cytokine levels, bacterial burden, and mortality, with contributions from IL-6 and TLR4 signaling, compared to periweaning mice. During co-culture with pathogenic E. coli, RAW-Blue cells produced more IL-6, similar TNFα, and less IL-1β compared to the commensal strain. The distinct difference in cytokine production suggests differential signaling by a pathogenic-specific pathway to cause lethality, cytokine production, and bacterial burden, specifically in neonates.
Discovery of clinical candidate GS8-164, a highly potent oral NLRP3 inflammasome inhibitor for inflammation disorders
Authors: Lv Yue , He Weiming , Zhai Wenqiang , Xia Yuanfeng , Yang Fanglong
Published: November 2024

The NLRP3 inflammasome is a component of the innate immune system that plays a crucial role in the pathophysiology of multiple metabolic diseases. But so far, no marketed drugs specifically targeting NLRP3 have been approved. Here, we report the characterization of GS8-164, a potent and selective NLRP3 inflammasome inhibitor with a new phthalazine skeleton. In vitro, GS8-164 displayed potent inhibition of IL-1b production in THP-1, human PBMC and human whole blood assays without inhibiting either IL-6 or TNF-α. In vivo, GS8-164 strongly inhibited production of IL-1β in an acute mouse peritonitis model, and attenuated pathological progress and production of IL-1β in a rat acute pericarditis model. GS8-164 has a good ADME and toxicology profile. In 14-day rat and dog toxicology studies, GS8-164 displayed good tolerance, excellent PK profile and a good safety window (>50 fold). GS8-164 is under IND-enabling study and the clinical development is planned towards early 2025.
Infection-suppressed surfactant protein C alters alveolar type 2 cell lineage
Authors: Jain Krishan , Ji Hong Long , Zhao Runzhen
Published: November 2024

Suppressed surfactant protein C is a hallmark of respiratory infections. Acute Respiratory Distress Syndrome (ARDS) is a critical condition in pneumonia, COVID-19, and sepsis and involves damaged epithelial lining, cytokine storms, and lung edema. Alveolar type 2 (AT2) epithelial cells are stem cells of the distal lung and are vital for lung repair. AT2 cells secrete surfactant protein C which protects alveoli from collapse and infections. Whether downregulated SP-C affects the re-alveolarization via regulating AT2 fate remains unclear. We used alveolar organoids to compare AT2 lineages in wild-type (wt), sftpc gene mutated L184Q mice, and disrupted sftpc gene (KO) mice. H1N1 infection reduced AT2 cells in KO mice. Sftpc deficiency led to fewer AT2 cells and reduced organoid counts and organoid colony-forming efficiency in KO mice compared to wt controls. Confocal images and western blotting indicated increased AT1 differentiation in sftpc-disrupted organoids. Similarly, L184Q mice also exhibited reduced AT2 yield, organoid count, and organoid formation efficiency. Further, scRNAseq identified differentially expressed genes in sftpc-deficient AT2 cells. This study demonstrated that sftpc could be essential to regulate alveolar regeneration by modulating AT2 lineage. Suppressed surfactant protein C in infected lungs could be a potential druggable target for ARDS and COVID-19.
Weight cycling alters the immune composition of reproductive tissues, worsening infertility in female mice
Authors: Kalev-Altman Rotem , Hamilton Riley , Martinez-Navarrete Wenceslao , Guha Ray Aleepta , Wiseman Destini , Weinstock Ada
Published: November 2024

Obesity increases the risk of several comorbidities, such as infertility. While weight loss is highly recommended, maintaining a normal weight is typically unsuccessful, leading to weight regain (WR), which exacerbates inflammation and worsens comorbidities, compared to maintaining stable obesity, with reasons unknown. Hence, understanding the deleterious effects of WR holds great potential for decreasing the burden of metabolic diseases as well as infertility. Here, we hypothesized that WR has an adverse effect on female fertility compared to stable obesity. Females were fed high-fat diet for 14wks to establish obesity, then subjected to weight loss by reducing food intake by 30% for 2wks, followed by 3wks of free access to food to induce WR. Controls were age-matched mice that were freely fed high-fat diet without weight cycling. Females were assessed for immune composition of reproductive tissues or outcomes of mating with lean males. WR females showed lower pregnancy rate, smaller litter size and higher dystocia prevalence, resulting in in-uterus death of the fetuses, as compared to non-cycling females. Moreover, the uterus myometrium showed lipid accumulation only in WR females, suggesting a mechanism by which the fatty myometrium cannot contract properly leading to dystocia. In conclusion, weight cycling exacerbates obesity-related infertility. Further histology and flow cytometry analysis will determine whether and how immune cells in reproductive tissues are involved.
Adenosine analogs have immunomodulatory antiviral properties through the Adenosine A2A Receptor pathway
Authors: King Brionna , Huang Zhi , Hewins Peter , Cook Thomasina , Mirzalieva Oygul , Larter Kristina , Wyczechowska Dorota , Crabtree Judy , Garai Jones , Li Li , Zabaleta Jovanny , Barbier Mallory , Del Valle Luis , Jurado Kellie , Miele Lucio , Monticone Giulia
Published: November 2024

The COVID-19 pandemic has unveiled an urgent need for novel antivirals to control emerging infectious diseases. Classic antivirals are often insufficient to clear infections in the absence of an effective immune response. Immunotherapy could complement the use of antivirals; however, its application to infectious diseases remains largely unexplored. Adenosine analogs (AAs) are antiviral drugs which resemble the structure of adenosine, a metabolite which suppresses the immune response through activation of the Adenosine A2A Receptor (A2AR) in immune cells. We hypothesized that selected AAs have previously unrecognized immunomodulatory properties by acting as A2AR antagonists. In this study, we used molecular docking modeling to screen AAs for their capacity to bind to A2AR and test AAs for immunomodulatory properties in cell-based and in vivo assays. We found that selected AAs restored T-cell functions from adenosine-mediated immunosuppression in T-cells, suggesting that they are endowed with immunomodulatory properties through A2AR antagonism, in addition to their intrinsic antiviral properties. These immunomodulatory functions contribute to the antiviral activity of the AAs as we found that the analogs could restore antiviral T-cell responses in COVID-19 models. In conclusion, selected AAs could be used as novel dual – immunomodulatory and intrinsic – antiviral drugs and could represent effective therapies against emerging viral diseases and future pandemics.
Dysregulation of fetal-derived cochlear resident tissue macrophage mediate CMV-associated sensorineural hearing loss
Authors: Otsuka Kelly , Trujillo Emilio , Stout Jordan , Kfoury Peter , Park Albert , Beaudin Anna
Published: November 2024

Cytomegalovirus (CMV) is the most common congenital viral infection in the developed world and leading cause of non-genetic sensorineural hearing loss (SNHL) in children. Here we investigated the role of fetal-derived resident tissue macrophages (RTMs) in normal cochlear development and their role in CMV-associated SNHL.

Using genetic fate mapping, we uncovered dual sources of fetal-derived RTMs from yolk sac (YS) and fetal liver (FL) hematopoiesis. YS-derived RTMs naturally recede after cochlea maturity while FL-derived RTMs expand, revealing a temporal layering of RTMs. Additionally YS-derived and FL-derived RTMs demonstrate spatial specificity, seeding distinct cochlear tissues. Both anti-CSF1R depletion of fetal-RTMs and embryonic genetic deletion of YS-derived RTMs by CSF1R-flox lead to SNHL and impaired cochlear tissue development, revealing the requirement for fetal RTMs in normal cochlear development.

During CMV infection, scRNAseq and fate-mapping analysis revealed an infiltration of bone marrow-derived macrophages at the expense of yolk-sac derived RTMs, indicating that CMV infection disrupted the normal temporal layering of fetal-derived RTMs in the cochlea. Fetal-derived RTMs also upregulated inflammation-associated pathways and downregulated pathways critical for inner ear development, such as FGF and TGFb signaling.

Together these data reveal perturbation of developing RTMs that regulate normal cochlear development as a mechanism underlying CMV-mediated SNHL.
Oral tolerance inhibits DTH in the sensitization phase by Treg-mediated suppression of DC functions in skin dLNs
Authors: Akagi Arisa , Shibuya Rintaro , Hanakawa Sho , Kitoh Akihiko , Kabashima Kenji
Published: November 2024

Antigen (Ag)-specific skin immune responses such as delayed-type hypersensitivity (DTH) are suppressed by prior oral administration of Ag, depending on the induction of regulatory T cells (Tregs) in gut-draining lymph nodes (dLNs). Here, we aimed to clarify the exact phase and location where Tregs induced in gut-dLNs suppress DTH.

Prefeeding ovalbumin (OVA) attenuated DTH against OVA, consistent with previous studies. This attenuation was abrogated by in vivo depletion of Tregs. DTH responses elicited in OVA-fed and unfed mice, subsequent to the transfer of cells from dLNs of OVA-immunized skin, were comparable. In contrast, DTH responses were significantly attenuated in mice transferred with cells from dLNs of OVA-immunized skin in OVA-fed mice, suggesting that OVA-feeding inhibited the responses in the sensitization phase of DTH. Next, we assessed the capacity of DCs isolated from dLNs of OVA-immunized skin to prime OT-II T cells. OVA feeding did not affect T-cell priming capacity of DCs at 24 h post-immunization (d1), but impaired it at 48 h (d2). In vivo DC migration assay revealed OVA feeding did not affect the migration of Ag-bearing (Ag+) DCs to the dLNs. Expression levels of CD80 and CD86 on Ag+ DCs in tolerized mice were comparable to non-tolerized mice at d1 but lower at d2.

In conclusion, our data suggest that DTH responses in orally tolerized mice are inhibited in the sensitization phase via Treg-mediated suppression of DC functions in the skin-dLNs.
Expanded GATA3+ T regulatory cells fail to control germinal center responses during pulmonary fibrosis in humans and mice
Authors: Velez Tania , Yoon Young me , Hollinger Maile , Blaine Kelly , Lee Cathryn , Strek Mary , Guzy Robert , Sperling Anne
Published: November 2024

Interstitial lung disease (ILD) describes heterogenous conditions of progressive lung fibrosis. Interestingly, lung lymph nodes (LLN) of different types of ILD patients are enlarged and predicts survival outcomes. Our recent studies demonstrate that there are active germinal centers (GC) in the LLN of ILD patients, suggesting on-going antigen-specific responses. To determine whether these responses are dysregulated, we investigated T regulatory cells (Tregs). Our data shows that Tregs are expanded in the LLN of all ILD patients compared to controls. Intriguingly, there was a predominance of GATA3+ Tregs that co-expressed ICOS and CD137, indicating recent activation. GATA3+ Tregs can have detrimental effects in asthma models, but their role in pulmonary fibrosis is unknown. Thus, we hypothesize that GATA3+ Tregs in ILD patients may be failing to control GC responses and potentially facilitating lung fibrosis. To test this hypothesis, we utilized a mouse model of bleomycin (bleo)-induced pulmonary fibrosis. We find that GATA3+ Tregs expand in the lung and mediastinal LN (mdLN) of bleo-treated mice, and there is evidence of GC responses. Depletion of all Tregs is detrimental and leads to enlargement of mdLN. In contrast, specific depletion of GATA3 in Tregs is protective and does not lead to mdLN enlargement. In summary, these data show that GATA3+ Tregs may have roles in promoting pathogenic mechanisms during pulmonary fibrosis in humans and mice.
Neurophysiological phenotypes are uncoupled from toll-like receptor (TLR)-mediated systemic disease in a model of Systemic Lupus Erythematosus (SLE)
Authors: Stumpf Cecilia , Rodriguez Vanessa , Cuda Carla
Published: November 2024

SLE is a multisystemic autoimmune disease with diverse clinical presentations, including neuropsychiatric symptoms (NPSLE). Prior work implicates uncontrolled MyD88-dependent TLR activation, increased interferon regulatory factor 5 (IRF5) activity and elevated type I interferon (IFN) levels in disease. Immune complexes containing self-nucleic acids can activate endosomal TLRs 7/8/9, which require signaling adaptor MyD88 to modulate downstream gene expression through transcription factor 1) IRF7 to upregulate IFN, which can subsequently act on the IFN-α/β receptor (IFNAR) or 2) IRF5 to upregulate proinflammatory genes. SLE- and NPSLE-prone mice with conditional deletion of signaling mediators MyD88, IRF5 and IFNAR in dendritic cells and tissue resident-macrophages were generated. MyD88 and IRF5 deletion ameliorated systemic disease phenotypes including splenomegaly, lymphadenopathy and glomerulonephritis, but NPSLE-associated phenotypes in the brain, including expansion of total and disease-associated microglia, macrophages and extravascular T cells, persisted. In contrast, IFNAR deletion worsened systemic disease, yet reversed several NPSLE-associated phenotypes. These discoveries suggest an uncoupling of systemic and neurophysiological phenotypes in SLE. Namely, neurophysiological phenotypes manifest independently from the TLR-dependent pathway that drives systemic disease and are instead in part linked to aberrant signaling through IFNAR via mechanisms yet unexplored.
Characterizing inflammasome activation by bacterial amyloid curli complexes from biofilms in dendritic cells and its role in SLE
Authors: Mintri Shrutika , Grando Kaitlyn , Tükel Çagla , Fitzgerald Katherine A , Gallucci Stefania
Published: November 2024

Bacterial infections are a crucial environmental trigger for flares in Systemic lupus erythematosus (SLE). Although these infections are common in lupus patients, a precise mechanism of action remains unknown. Bacteria protect themselves from stresses by embedding in an extracellular matrix to form biofilms. Biofilms from bacterial species such as E. coli and Salmonella contain an amyloid protein called curli. Previous work from our labs has shown that curli/DNA complexes can accelerate lupus-like disease in mice, and increased levels of anti-curli antibodies are associated with flares in SLE patients, making these complexes important in autoimmunity. We had previously found that curli/DNA is a potent trigger of immune activation in dendritic cells (DCs) and macrophages. In macrophages, curli/DNA was also reported to induce IL-1b production through activation of the NLRP3 inflammasome. Here we expand these studies to delineate how curli/DNA complexes regulate IL-1b in murine and human DCs. Using curli/DNA alone or in combination with inflammasome stimuli, we found that curli/DNA stimulate pro-IL1b expression and inflammasome assembly, leading to caspase 1 activation, cleavage and release of mature IL-1B by DCs. However, we observed no cell death post inflammasome activation, suggesting a hyperactivated state in stimulated DCs. A deeper knowledge of this molecular mechanism might shed light on the role of inflammasome activation by bacterial infections in escalating SLE.
Deficiency in CD103DC function leading to decreased pTregs in female lupus-prone mice is due to defects in intestinal lamina propria stromal cell function that can be restored by male microbiota.
Authors: Ma Jing , Alard Pascale , Kosiewicz Michele
Published: November 2024

Active lupus in humans is associated with low Treg percentages, but not function. We have found that lupus-prone female BWF1 have decreased peripherally-derived Tregs (pTregs), but not function vs lupus-resistant male BWF1 mice. Gut CD103+ DC (CD103DC), the major pTreg inducer, function is decreased in female mice due to defects in the retinoic acid (RA) synthesis pathway, i.e., lower RALDH2 (critical enzyme) and higher Zfp36 expression (degrades RALDH2 RNA). Both normal CD103DC development/function and RALDH2 expression require exogenous RA. Treating female CD103DC with RA restored function and increased RALDH2 and decreased Zfp36 expression indicating CD103DC are not defective themselves. Lamina propria stromal cells (LPSC) are required for CD103DC development via RA and GM-CSF production. Female BWF1 LPSC had decreased RALDH2 expression/activity and GM-CSF suggesting lower CD103DC function is due to decreased LPSC function. We have found differences in female and male gut microbiota in BWF1 mice, and transfer of male microbiota to female mice protects against disease and increases survival, increases percentages/numbers of total Tregs/pTregs, and restores CD103DC and LPSC function that appears to be mediated by metabolites, phytol/phytanic acid, produced by male microbiota. Lower pTregs in female BWF1 mice may be due to defects in LPSC support of CD103DC development and can be restored by metabolites produced by male microbiota that contribute to protection from disease.
Exogenous IL-27 prevents the development of alopecia areata
Authors: Connell Samuel , Kahl Payton , Zhu Zhaowen , Lensing Maddison , Crotts Sydney , Bai Xue-Feng , Jabbari Ali
Published: November 2024

Alopecia areata (AA) is an autoimmune disease with a 2% lifetime incidence. Commonly presenting as defined patches of hair loss on the scalp, AA is characterized by the presence of T cells surrounding the hair bulb and is thought to result as a consequence of the breakdown of immune privilege of the hair follicle. IL-27 is a cytokine with context-dependent pro- and anti-inflammatory properties that has been used as a potential therapy in models of autoimmune diseases and cancer. The objective of this study was to determine if IL-27 has therapeutic potential in AA. To address this, we used an adeno-associated virus that drives the overexpression of IL-27 (AAV-IL27) in our skin-graft induction model of AA. We found that mice that received AAV-IL27 were fully protected from disease development and had minimal CD8 T cell infiltration into the hair follicles. Further, we saw an increase in IL-10 producing CD4 T cells, supporting a potential mechanism by which IL-27 prevents disease development. Interestingly, we found that mice treated with AAV-IL27 generated a robust population of NKG2D expressing CD8 T cells, which has previously been associated with the presence of disease. These results suggest that IL-27 may act in an immunoregulatory manner in AA, by promoting immunosuppressive T cells in the SDLNs. Further studies are needed to further define the specific mechanisms by which IL-27 contributes to the prevention of AA and its utility as a treatment for AA.
Skewing of UV-Induced Cell Death Pathways using TLR4 Ligands
Authors: Chaudhry Sumra , Williford Zachary , Downes Shae
Published: November 2024

Ultraviolet (UV) radiation induces DNA damage resulting in epidermal cell mutations and immunosuppression contributing to the formation of nonmelanoma skin cancer. UV-induced cellular damage is commonly associated with apoptosis characterized by a non-inflammatory cell death through the cleavage and activation of caspase-3 which requires Toll-like receptor 4 (TLR4) signaling to activate the apoptotic signaling cascade. TLR4 is a cell associated innate immune receptor and has been shown to contribute to UV-induced carcinogenesis. We hypothesize that skewing cell death from apoptosis to necroptosis using lipid A mimetics to reduce TLR4 expression through tolerization can reduce apoptosis and lead to inflammatory cell death. Hexa-acylated and tetra-acylated lipid A memetics from Yersinia pestis (Y. pestis) are used to tolerize TLR4 in thioglycolate-induced macrophages (ThioMΦ). This will likely lead to the ThioMΦ cells being less susceptible to UV-induced carcinogenesis. To analyze the role of TLR4 in UV-induced carcinogenesis, cells that are TLR4-proficient, TLR4-deficient, and TLR4 tolerized with lipid A were exposed to multiple doses of UVB radiation. UV-irradiated ThioMΦ cells were surveyed for markers of DNA repair and cell death mechanism. Our data indicates that exposure to lipid A attenuates TLR4-mediated apoptotic cell death and, likely, downstream immune suppression. TLR4 ligands may be useful in prophylactic treatment approaches against UV-induced carcinogenesis.
Complement receptor 2 as a novel therapeutic target to suppress germinal center-derived autoantibody production in a SLE mouse model
Authors: Rattan Ajitanuj , Zhu Danni Y , Holers V Michael , Huber Brigitte T , Carroll Michael C
Published: November 2024

Presence of spontaneous germinal center (GCs) in SLE patients correlates with the production of pathogenic, isotype-switched antibodies (Abs) and epitope spreading. In lupus mice, spontaneous GCs are a major site for self-reactive B cells to undergo clonal selection to self-antigens. Limited efficacy of current B cell-based lupus therapies highlights the need to explore alternative targets for inhibiting auto-Ab secreting B cells (ASCs) generation. We propose that complement receptor 2 (CD21) is a novel therapeutic target to block expansion of autoimmunity and pathogenic Ab production. To test this, we set-up mix bone marrow chimeras and adoptive transfer models with 564Igi lupus mice and human CD21-transgenic mice, which allowed us to selectively block CD21 function of either B cells or FDC, or both. CD21 blockade on B cells not only restricted recently activated B cells but also memory B cells (MBC) to participate in GC-derived ASC generation. CD21 blockade on FDCs intriguingly biased MBC fate towards ASC generation, though impeded recently activated B cell capacity to participate in GC response. Dual blockade of CD21 on B cells and FDC exhibited strongest inhibitory effect on GC-derived ASC generation and auto-Ab production. As expected, blockade of ASC generation by donor B cells further correlated with reduced Ab deposition and ameliorated kidney pathology. In brief, CD21 presents itself as a promising therapeutic target to suppress pathogenic auto-Ab generation in SLE.
Selective depletion of anti-dsDNA long-lived plasma cells in SLE
Authors: Deokar Aditi , Huang Yina
Published: November 2024

Systemic lupus erythematosus (SLE) is an autoimmune disease driven by secretion of autoantibodies including those specific for double stranded-DNA (dsDNA) by short- or long-lived plasma cells (PCs). Most SLE treatments are generally immunosuppressive and indirectly decrease short-lived PCs. New therapies are needed to selectively eliminate SLE-causing long-lived PCs while preserving immunity and avoiding side effects. Prior research fusing the model antigen ovalbumin (OVA) with an antibody specific for a PC-specific protein, CD138, showed selective depletion of anti-OVA long-lived PCs. We hypothesize that a similar fusion protein comprising anti-CD138 antibody and a dsDNA peptide mimic can selectively deplete long-lived dsDNA-specific PCs. We tested the ability of two previously reported peptide mimics, ALW and DWEYS, to competitively inhibit detection of anti-dsDNA autoantibodies from NZB/NZW mice. We found DWEYS but not ALW inhibited detection. We also validated a method to indirectly measure anti-dsDNA PCs by measuring levels of secreted antibody by ELISA. We then confirmed efficient in vivo CD138 depletion. The next step is to fuse DWEYS with anti-CD138 antibody and test for selective depletion in vitro and in vivo. Successful selective depletion of anti-dsDNA PCs using a DWEYS/anti-CD138 fusion protein may support its future development as an improved treatment for SLE patients not benefited by current treatments.
Protective Effects of Delta-8-THC Against EAE-Induced Enteric Neuropathy and Neuroinflammation through Regulation of miRNA-Mediated Signaling Networks
Authors: kakar khadija , Halder urmi , nagarkatti Mitzi , nagarkatti Prakash
Published: November 2024

Multiple sclerosis has long been associated with neurological symptoms, yet the connection between neuroinflammation and enteric neuropathy remains understudied. To test the complex brain-gut connection, we administered Δ8-THC (10mg/kg) i.p to mice with Experimental autoimmune encephalomyelitis (EAE), animal model of MS. We observed that treatment significantly reduced the clinical score and reversed the weight loss in treated group by suppression of proinflammatory Th1 phenotypes such as Tbet and IFN-g and increase anti-inflammatory phenotype such as Tregs and IL-10.miRNAseq analysis of brain infiltrating CD4+ cells revealed that decrease in neuroinflammation following treatment was associated with downregulation of miRNAs, involved in Pyroptosis, Th1 and Th2 pathway regulation. The EAE mice exhibit persistent gastrointestinal symptoms. To investigate bowel dysfunction miRNAseq analysis was performed for colonic and ileal tissues and contents. A highly dysregulated miRNA6538 found in all four miRNAseq analyses. miRNA6538 downregulation in colonic and ileal tissues, along with ileal content in treated EAE mice, suggests a protective role against EAE-induced bowel symptoms while its upregulation in colonic content could be due to the shedding of cells as a part of homeostasis during cellular turnover.In summary, Δ8-THC's suppression of EAE involves a dual impact on the brain and gut, showing its potential as a therapeutic strategy for mitigating neurodegenerative disorders.
Multi-omic studies in Cutaneous T Cell Lymphoma (CTCL) reveal progression-associated alterations in epigenetic and lymphocyte signaling pathways
Authors: Payton Jacqueline , Dorando Hannah , Andrews Jared , Borcherding Nicholas , Quinn Chaz , Schmidt Jennifer , Aluri Jahnavi , Cooper Megan , Musiek Amy , Mehta-Shah Neha
Published: November 2024

Patients with advanced stage CTCL have a 20% 5-year survival rate and molecular factors that drive disease progression are undefined. To address this, we collected 52 longitudinal tumor and normal samples from 10 CTCL patients and subjected them to exome + whole genome (eWGS), single-cell (sc)RNA and TCR sequencing. Clustering and cell-type inference of scRNAseq (UMAP/Seurat/singleR) from 92496 total cells showed canonical CTCL marker genes and high proportions of patient-specific TCR clonotypes. Somatic genome copy number alterations (CNA) and deleterious/damaging single nucleotide variants (SNV) (cnvkit, GATK/Mutect2, copykat, cb_sniffer) were identified. We defined molecular changes associated with progression and/or resistance to therapy (APR) as CNA and SNV detected in samples collected at later timepoints with progression and/or lack of therapy response in >30% of patients. This novel approach identified pathways in chromatin modification, particularly DNA and histone methylation, lymphocyte signaling and transcriptional regulation. A novel-to-CTCL SNV in STAT3 had gain-of-function activity and aberrant genomic binding (CUT&RUNseq). CNA and mutant allele levels correlate with disease progression and therapy response in scRNAseq. Our findings represent new insights into CTCL progression, reveal new markers for response assessment, and identify new targets for therapeutic development. Based on these results, we have initiated two clinical trials at WUSM.
Molecular Mimicry in Pathogen Immune Evasion
Authors: Santambrogio Laura , Clement Cristina , Osan Jaspreet , Becerra Aniuska , Mota Ines , Nanaware Padma , Franco Alessandra , Stern Lawrence
Published: November 2024

Molecular mimicry is the immunological mechanism by which pathogen-derived peptides can modulate immune responses due to sequence similarity with self-peptides. Herein, we report molecular mimicry associated with pathogen immune evasion. A quantitative analysis of the immunopeptidome presented by in vivo mycobacteria tuberculosis (MTB)-matured dendritic cells in HLA-DR0101 (DR1) transgenic mice identified an MTB-processed peptide which presented immunosuppressive capability and shared a DR1 core sequence with a human and mouse self-protein. The TB peptide could expand nTreg with generation of immunosuppressive cytokines in DR1 mice and DR1 subjects diagnosed with active or latent TB. In TB-infected patients the tolerogenic TB peptide reduced the inflammatory response generated to a series of immunogenic TB peptides as quantified by the QuantiFERON test. Additionally, the tolerogenic TB peptide presented an MHC-binding core sequence shared by a multitude of pathogens. Our analysis characterizes a pathogen-derived peptide which, through molecular mimicry facilitate immune evasion by activating/inducing nTreg.
Detection of lipid nanoparticles phagocytosed by antigen presenting cells using imaging flow cytometry
Authors: D'Cruz Louise , Phan Anthony , Shi Xiaoshan , Widmann Stephanie , Alameh Mohamad-Gabriel , Hunter Christopher , Tyznik Aaron
Published: November 2024

mRNA vaccines were critical for the prevention of severe disease during the COVID-19 pandemic and efficacy of their delivery was achieved through nucleic acid-encapsulated lipid nanoparticles (LNPs). Despite the effectivness of mRNA vaccines, the mechanisms by which antigen-presenting cells (APCs) recognize and phagocytose the LNP cargo are not fully understood. Here, we used the BD CellView™ Image Technology on the BD FACSDiscover™ S8 Cell Sorter to visualize LNPs phagocytosed by host cells. LNPs formulated with green fluorescent (DiO)-labeled lipids were injected intramuscularly into mice. With the imaging capabilities of the BD FACSDiscover™ S8 Cell Sorter, we visualized LNPs in different APCs and observed different morphology of LNPs in dendritic cell subsets and monocytes. A time course experiment uncovered the kinetics of antigen recognition upon LNP-mediated cargo delivery. Analysis using the instrument imaging parameters revealed unique cell clusters associated with distinct morphology of LNP phagocytosis. We then sorted the LNP+ cells and evaluated their transcriptomic signature by real-time PCR. This work provides a workflow to visualize LNP phagocytosis followed by image-based cell sorting for downstream characterization enabled by the BD FACSDiscover™ S8 Cell Sorter. With increased adoption of LNP-mediated nucleic acid vaccines, the image-based cell sorter provides a powerful tool to understand the host immune response upon vaccination.
Reversal of dysfunction of dendritic cells induced by Treg cells for enhancing cancer immunotherapy
Authors: Liu Xia , Zhang Yuanqin , Tao Yan , Zhao Lei , Peng Guangyong
Published: November 2024

Different types of Regulatory T (Treg) cells create immunosuppressive microenvironments in cancer patients, which are a major obstacle to effective antitumor immunity. A precise understanding of the molecular mechanisms utilized by Treg cells for immune suppression on different types of immune cells is critical for the development of effective strategies for cancer immunotherapy. We investigated the interaction between breast cancer tumor-derived gamma-delta Treg cells with dendritic cells (DCs) and determined the phenotypic and functional changes of DCs. We showed that tumor-derived gamma-delta Treg cells promote the development of senescent DCs with tolerogenic functions in breast cancer. In addition, we demonstrated that PD-L1 and STAT3 signaling pathways are involved in senescence induction in DCs mediated by gamma-delta Treg cells. Importantly, our complementary in vivo studies further demonstrated that blockages of PD-L1 and STAT3 signaling can prevent gamma-delta Treg-induced senescence and reverse tolerogenic functions in DCs, as well as enhance anti-tumor immunotherapy in the human breast cancer models. These studies not only dissect the suppressive mechanism mediated by tumor-derived gamma-delta Treg cells but also provide novel strategies to enhance anti-tumor efficacy mediated by tumor-specific T cells for cancer immunotherapy.