bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-canonical unfolded protein response transcription factor CREB3L2 regulates survival of long-lived plasma cells
Authors: Lau Louis C , Manakkat Vijay Godhev K , Hall David Reed , Singh Harinder
Published: August 2023

Differentiation of activated B cells into antibody-secreting cells (ASCs) involves extensive endoplasmic reticulum (ER) & Golgi biogenesis and remodeling that enable increased antibody synthesis and secretion. These processes are mediated by the unfolded protein response (UPR). Canonical UPR pathway genes are regulated by the transcription factors (TFs) XBP-1 and ATFs, which are activated upon ER or Golgi stress. Using scRNA-seq to analyze the generation of splenic and bone marrow (BM) plasma cells (PCs), we discovered that the Creb3l2 gene, encoding a non-canonical UPR TF, is specifically expressed in PCs, with highest levels in long-lived PCs (LLPCs). CREB3L2 has been shown to regulate transcription of genes encoding components critical to secretory cells of the pancreas and pituitary gland. To analyze the function of CREB3L2 in ASCs, we pursued a two-pronged strategy (i) using CRISPR/Cas9-RNP editing in the murine CH12 B lymphoma cell line to generate out-of-frame deletions in exon 2 of the Creb3l2 gene (Creb3l2−/−) and (ii) generating Creb3l2 B-cell conditional KO (cKO) mice carrying a floxed allele of the Creb3l2 gene by inserting loxp sites flanking exon 7 with a CD19-cre driver (Creb3l2fl/fl CD19cre/+). RNA-seq of Creb3l2−/− CH12 cells shows CREB3L2 positively regulated genes including Cd93 and Cd28 & its signaling adapter SLP-76 (Lcp2), that are involved in plasma cell survival in the BM niche. Expression of CD28/SLP-76 and CD93 proteins were strongly diminished in Creb3l2−/− CH12 cells. Consistent with these findings, Creb3l2 cKO mice had reduced NP-specific LLPCs (90 d.p.i) after NP-KLH immunization. Thus, CREB3L2 appears to selectively regulate the survival of LLPCs by controlling the expression of CD28/SLP-76 and CD93.

Supported by grants from NIAID (U01 AI141990, R01 AI145064).
Phenotypic Analysis of Fibrogenic Monocyte/Macrophage Subsets in Acute Inflammatory Exacerbations of Lung Disease Driven by Mutant Surfactant Protein-C Expression
Authors: Venosa Alessandro , Musci Teresa , Moos Philip
Published: August 2023

Acute inflammatory exacerbations (AIE) represent precipitous deteriorations of a number of chronic lung conditions, including pulmonary fibrosis (PF), COPD, and asthma. AIEs are marked by diffuse and persistent polycellular alveolitis that profoundly accelerate lung function decline and mortality. Excess monocyte mobilization during AIE, and their persistence in the lung are linked to poor disease outcome. Guided by clinical evidence, we have developed an inducible model of pulmonary fibrosis leveraging the PF-linked missense isoleucine to threonine mutation at position 73 [I73T] in the alveolar type-2 cell-restricted Surfactant Protein-C [SP-C] gene [SFTPC]. We have previously shown that ablation of CCR2+ monocytes was protective with respect to lung histopathology, mouse survival, and overall inflammation following SP-C mutant induction. Here we identify and phenotypically characterize resident and recruited monocytes/macrophages populations intervening during the initiation and progression of AIE-PF. Single cell sequencing shows pro-inflammatory and pro-fibrotic signatures originating from immature recruited clusters (itgax-itgam+ ccr2+ cx3cr1+) as well as activated macrophages (cd68+ itgax+ itgam+ arg1+) 14 d after SP-CI73T induction. Using CellChat Explorer database, these cells were predicted to coordinate intense tgfb1, fn1/fibronectin and spp1/osteopontin signaling. Together, these results provide a clear picture of the fibrogenic role of specific activated monocyte/macrophage subsets responding to alveolar epithelial stress.

NIEHS R01ES032553 (AV); ALSAM Foundation for Research Initiatives Grant (AV)
Randomized Clinical Trial of Intermittent Calorie Restriction in People with Multiple Sclerosis: Effects on Immunometabolic and Cognitive Measures
Authors: Tolentino Miguel , Ghezzi Laura , Tosti Valeria , Cantoni Claudia , Salter Amber , Lancia Samantha , Zhou Yanjiao , Obert Kathleen , Mikesell Robert , Sen Monokesh Kumer , Cross Anne , Naismith Robert , Piccio Laura
Published: August 2023

BACKGROUND

Intermittent calorie restriction (iCR) reduces inflammation in preclinical models and in people with Multiple Sclerosis (pwMS). Mechanisms could be related to changes in levels of adipose tissue-derived adipokines with reduced systemic inflammation and gut microbiota composition.

AIMS

Evaluate the effects of iCR on adipokines levels, metabolic and immune/inflammatory biomarkers compared to a control unrestricted diet (Ctr) in pwMS.

METHODS

PwMS (relapsing Remitting MS) were randomly assigned to the iCR or Ctr groups for 12 weeks. Blood was collected at baseline, 6 and 12 weeks. Leptin and adiponectin levels, peripheral blood metabolic and immunologic profiling, anthropometric and total body fat measures, and clinical and cognitive measures were evaluated. Differences between iCR and Ctr were examined using a linear, repeated measures mixed model and adjusted for baseline levels, age, sex and DMTs.

RESULTS

42 pwMS were randomized (iCR=22 and Ctr=20). 34 pwMS completed the study (iCR=17 and Ctr=17). PwMS on iCR showed a significant reduction of anthropometric (weight and BMI) and body adiposity measures (waist circumference and fat mass) over the 12 weeks. Leptin levels decreased and were significantly lower in the iCR group at 6 weeks and approaching significance at 12 weeks. Adiponectin increased from baseline in the iCR group at 6 and 12 weeks. The iCR group showed a significant decrease in Th1 cells and an increase in CD45RO+ regulatory T cells after 6 weeks. Cognitive testing using symbol digit modality test showed significantly greater improvement in the iCR group versus Ctr at 12 weeks.

CONCLUSIONS

Short term iCR is safe and feasible in pwMS, and it can ameliorate metabolic, immunologic, and cognitive profiles.

National Multiple Sclerosis Society # RG-1607-25158
Resistant starch supplementation alters the gut microbial consortium and attenuates kidney inflammation in an experimental model of autoimmune vasculitis
Authors: O’Sullivan Kim Maree , Snelson Matthew , Nguyen Jenny , Le Anne Cao , Coughlan Melinda
Published: August 2023

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is a rare autoimmune disease with suboptimal treatment options which contribute to morbidity and mortality. The characteristic pathological feature is severe inflammation induced by neutrophils which causes life threatening damage to the small blood vessels of the kidney. Neutrophil activity is influenced by microbial metabolites, including short chain fatty acids (SCFAs), produced from fermentation of non-digestible carbohydrates by gut microbiota. We utilised a well-established mouse model of AAV. Mice were randomised to receive either a control diet or a diet supplemented with 15% resistant starch (RS) (n=8 per group). At day 20, mice were culled and kidneys removed to assess injury. Glomerular neutrophil and macrophage infiltration were assessed by immunohistochemistry. Cecal contents were collected, DNA extracted and the V1-V2 hypervariable region amplified using 27F and 338R universal primers to assess the gut microbiota. The resistant starch intervention significantly reduced segmental necrosis in glomeruli (77.63 ± 7.2 % vs 25.59± 4.5 p/gcs, p < 0.0001) and reduced neutrophil (1.3 ± 0.37per glomerular cross section (p/gcs) vs 0.4 ± 0.3p/gcs, p < 0.0001) and macrophage (1.6± 0.3 p/gcs vs 0.4 ± 0.08 p/gcs, p < 0.0001) infiltration into glomeruli. The resistant starch diet significantly altered the gut microbiota consortium and was associated with a substantial expansion of Bacteroidaceae and Muribaculaceae both SCFA producing bacteria. This is the first report of the therapeutic efficacy of a resistant starch supplemented diet in experimental AAV. This data represents a promising therapeutic avenue to limit autoimmune kidney injury in AAV.
Treatment with linsitinib, an IGF-1 receptor inhibitor, attenuates disease development and progression in an experimental model of Graves’ Orbitopathy
Authors: Gulbins Anne Renate Liselotte , Horstmann Mareike , Oeverhaus Michael , Banga J Paul , Floegel Ulrich , Daser Anke , Krause Gerd , Spencer Andrew , Zeidan Ryan , Hammer Gary Douglas , Bechrakis Nikolaos E , Philipp Svenja , Eckstein Anja , Goertz Gina-Eva
Published: August 2023

Study objective

The activity of linsitinib, a small molecule inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), was assessed in a mouse model of Graves’ orbitopathy (GO). GO is the most common extra-thyroidal manifestation of Graves’ disease (GD), which is caused by autoantibodies against the thyroid stimulating hormone receptor (TSHR).

Methods

GD was induced in mice by immunization with a human TSHR A-subunit encoding plasmid. Linsitinib was administered orally for four weeks either in parallel to or after disease induction. Endocrine orbitopathy and inflammation were determined by histology and MRI.

Results

Immunization with the TSHR A-subunit induced hyperthyroidism, as well as CD3+ T-cell infiltration and macrophage infiltration into orbital muscle/adipose tissue. Linsitinib reduced these effects by up to 80% whether given prophylactically or after disease induction. In addition, linsitinib reduced the formation of brown adipose tissue in the orbit and displayed an additional effect on the immune system. An MRI using F19 imaging confirmed marked orbital inflammation, significant muscle edema and formation of brown fat in experimental GO, events that were abrogated upon application of linsitinib. Linsitinib’s pharmacologic effects were observed at a dose that did not impact the concentration of plasma glucose.

Conclusion

Linsitinib improved GO-related endpoints in a TSHR autoantibody dependent mouse model of Graves’ disease. Both prophylactic and therapeutic intervention with linsitinib improved clinically relevant endpoints in the orbita and thyroid gland. Our results are consistent with the therapeutic hypothesis being evaluated in an ongoing Phase 2b clinical study (NCT05276063).

This work was funded by Sling Therapeutics, Inc.
Profiling the gut microbiome unraveled signature bacterial groups in autoimmune diabetes which remain unperturbed regardless of the ionic strength of drinking water
Authors: Jayaraman Sundararajan , Babu Meghana , Aung Saw , Jayaraman Arathi
Published: August 2023

Type 1 diabetes is an autoimmune disease that occurs spontaneously in non-obese diabetic mice, and the gut microbiota has been implicated in disease manifestation. The simple dietary variable, acidified drinking water, exaggerated or reduced diabetes incidence in some studies but not in our investigation. To elucidate the underlying basis of these discrepancies, we profiled the gut microbiome of diabetic mice that were transitioned from acid to neutral water. Quantitative polymerase reactions using primers specific for 16S rRNA genes validated the relative abundance of bacterial taxa longitudinally from 6 weeks of age to adults that overtly manifested diabetes. Among the 15 representative bacterial groups examined, only Lactobacillus sp. remained low throughout the life of the mouse. The frequencies of the Firmicutes Ruminococcaceae and the Bactereoidetes phylum members Anaerophaga sp. and Paludibacter sp. increased during maturation. The level of mucin-degrading Akkermansia muciniphila, implicated in diabetes protection remained low throughout the life of the mouse. The segmented filamentous bacteria could be detected in the gut of young mice and remained abundant in adult mice that developed full blown diabetes. The drinking water’s pH neither impacted the relative abundance of these bacterial taxa investigated herein nor diabetes incidence. Thus, the combination of microbiome profiling and quantitative real-time polymerase chain reactions facilitated validation of the signature gut bacterial taxa in autoimmune diabetes, which remain unperturbed by the pH of the drinking water.

Supported by the University of Illinois at Chicago, Chicago.
Recombinant immune complexes (rICs) as a therapeutic for Multiple Sclerosis (MS): effect on myeloid cell activation
Authors: Wang Jin , Brown Kellie Nicole , Margaret Danehy Caroline , Rice Samuel Worcester , Thomas Fridtjof , Torgbe Kwame , Block David , Olsen Henrik , Strome Scott Eric , Fitzpatrick Elizabeth A
Published: August 2023

Monocyte recruitment into the central nervous system (CNS) is critical for driving pathology in neuroinflammatory diseases such as MS. The monocytic influx as well as activation of resident myeloid cells contribute to recruitment and subsequent re-activation of autoreactive T cells, ultimately leading to demyelinating lesions, axonal damage, and progressive paralysis. Using the murine model for MS, experimental autoimmune encephalitis (EAE) we tested a rIC, M019, to determine its efficacy in reducing myeloid cell activation and subsequently reducing disease severity. Four days following induction of EAE with MOG peptide, daily treatment with M019 inhibited clinical symptoms including weight loss and demyelination in a dose-dependent manner. M019 also delayed or prevented disease in the SJL model of relapsing remitting EAE demonstrating its effectiveness against several forms of EAE. Analysis of the immune cell infiltrate in the spinal cords of M019 and vehicle treated mice revealed that M019 significantly inhibits the infiltration of myeloid and lymphoid cells into the CNS. The myeloid cells that were present in the CNS of M019 treated mice had reduced levels of MHC II suggesting they are inefficient at antigen presentation to autoreactive T cells. Microglia in the M019 treated EAE mice did not upregulate MHC II expression compared to the vehicle treated mice suggesting M019 inhibits their activation. M019 binds directly to monocytes in the periphery via CD16 (FcγRIII) and CD32 (FcγRII) and we detected increased chemokines in the sera of M019 treated EAE mice compared to vehicle treated EAE mice. These results suggest that M019 blocks immune cell migration into the CNS, potentially via phenotypic and functional changes to monocytes.
Chimeric cytokine receptor provides IL-2 signaling for engineered regulatory T cell therapy
Authors: Cortez Jessica T , Ly Han , Trotta Eleonora , Matthaei James F , Kegler Peter , Phong Brian , Fiebiger Benjamin M , Torres Josue , Chase Jack , Mileur Trevor , Beilke Joshua N , Bluestone Jeffrey A , Ramsdell Fred , McKenzie Brent S , Van Gool Frédéric
Published: August 2023

Regulatory T cells (Tregs) are potent immune suppressors and critical to the maintenance of immunological tolerance. Interleukin 2 (IL-2) is essential for Treg survival, expansion, phenotypic stability, and suppressive function. However, Tregs do not produce IL-2 and are dependent on exogenous sources of IL-2. This poses a significant challenge for Treg cell therapies in tissues with no or limited IL-2 availability in the microenvironment. Therefore, we have developed engineered Treg cells containing a chimeric cytokine receptor that provides an IL-2 signal. The chimeric cytokine receptor, termed IL-9 tethered switch receptor (IL-9TSR), consists of interleukin 9 (IL-9) tethered to the extracellular domain of IL9RA paired with the intracellular domain of IL2RB. We have shown that Tregs expressing IL-9TSR can survive and expand in the absence of exogenous IL-2 in vitro and in vivo but require TCR stimulation for prolonged persistency. IL-9TSR Tregs have a stable FOXP3+HELIOS+ phenotype and superior suppressive function when compared to control Tregs. In conclusion, we have generated Treg cells engineered to survive and function in a low IL-2 environment. Treg cell therapies equipped with IL-2 signaling enable increased persistency and allow for potential use in more clinical indications than previously possible.
Donepezil is emerging as a potential therapeutic treatment in improving endothelial barrier function to prevent and treat coronary artery disease in Diabetes Mellitus
Authors: Wilson Kiara D , Kasim Omodolapo , Aliyeva Leyla , Graefen Bahar , Najafova Nurana , Tauseef Mohammad , Fazal Nadeem
Published: August 2023

Current treatments have shown limited success in reversing DM induced vascular endothelial barrier dysfunction. Vascular endothelium forms the inner most lining of the blood vessels, regulates variety of biological processes such as angiogenesis, wound healing and host defense mechanisms. During inflammatory conditions, such as diabetes mellitus and myocardial infarction endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins, such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications. Therefore, we hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. To investigate the signaling mechanism involved in the protection offered by Donepezil, we found that Donepezil prevented loss of VE-cadherin in endothelial junctions, and abrogated stress fiber formation in endothelial cell exposed to high-glucose solution. In conclusion, our study shows that Donepezil maintained endothelial barrier function and improved wound healing process in endothelial cells exposed to high glucose.

Chicago State University CTRE grant to Professor Nadeem Fazal, MD, PhD
A noncanonical NF-kB - β-catenin signaling axis in dendritic cells instructs gut inflammation
Authors: Deka Alvina , Kumar Naveen , Madan Upasna , Bhawna  , Chawla Meenakshi , Awasthi Amit , Basak Soumen
Published: August 2023

Dendritic cells (DCs) balance intestinal homeostasis against inflammatory insults. In particular, the noncanonical NF-kB pathway, which activates RelB:p52 heterodimers, has been implicated in DC differentiation. However, if DC-intrinsic noncanonical signaling modulates DC function in the inflamed gut remains unclear. Here we report that genetic ablation of the noncanonical signaling in DCs accumulated regulatory T cells in the lamina propria and associated lymph nodes, restraining inflammation in the colitogenic gut. Our mechanistic investigation revealed that an impaired RelB:p52 gene function, rather unexpectedly, augmented the abundance of active β-catenin via impairment of the β-catenin destruction complex. In turn, β-catenin transcriptionally upregulated Raldh2, imparting a retinoic acid gene signature and tolerogenic functions to noncanonical NF-kB-deficient DCs. Indeed, mice possessing DC-intrinsic noncanonical signaling defects exhibited an increased frequency of β-catenin+ DCs in the gut and resilience to chemical induced experimental colitis. Taken together, we chart a novel crosstalk between noncanonical NF-kB signaling and the β-catenin pathway that instructs tolerogenic DC functions and modulates inflammation in the gut.

National Institute of Immunology Core funding and BT/PR36631/BRB/10/1862/2020, Department of Biotechnology (DBT).
Deep Characterization of Human γδ T Cell Subsets Defines Lineage-Specific Traits
Authors: Mann Brendan T , Sanz Marta , Ryan Paul , Bosque Alberto , Pennington Daniel , Hackstein Holger , Soriano-Sarabia Natalia
Published: August 2023

Human γδ T cells represent a small fraction of T cells in peripheral blood (1–10%). They constitute a unique subset of T lymphocytes that recognize non-peptide antigens through MHC-independent presentation. Major γδ T cell subsets, Vδ1 and Vδ2 possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that Vδ1 and Vδ2 T cells possess differences at the gene, phenotypic, and functional level that distinguish them as distinct lineages. Comparisons between each subset have been limited by challenges in isolating sufficient cell numbers for characterization. Through multiparametric flow cytometry we identified phenotypic signatures that defined memory and tissue homing properties specific for each subset. The immune checkpoint marker TIGIT marked a Vδ1 T cell population enriched for higher cytotoxic marker expression. Using a stringent FACS-based isolation method, we compared cytotoxicity and gene expression profiles of highly purified human Vδ1 and Vδ2 cells. Cytotoxicity assays revealed similar killing capacity of Daudi cells possibly through dissimilar mechanisms. Microarray analysis identified shared and subset-specific genetic programs including lower expression of CD3ζ in Vδ1 T cells. Analysis of transcription factors associated with subset-specific gene profiles implicated NFAT in Vδ1 and AhR in Vδ2 T cells as key regulators of ex vivo gene expression. In confirmatory assays, using TCR-dependent and independent stimuli we show higher sensitivity to calcium-mediated NFAT activity in Vδ1 T cells while Vδ2 T cells may require additional activating stimuli. These findings will facilitate further studies into the specific contributions of Vδ1 and Vδ2 T cells to the immune system.

NIH NIAID R01 AI125097
miR-19a-3p and miR-19b-3p promote microglia activation associated with neuroinflammation following spinal cord injury
Authors: Sahebdel Faezeh , Morse Leslie , Battaglino Ricardo , Olson Julie K
Published: August 2023

Spinal cord injury (SCI)-induced neuropathic pain represents a significant clinical challenge that affects 10 to 30% of injured persons. Following SCI, microglia activation promotes neuroinflammation through the production of pro-inflammatory cytokines which has been suggested to play a role in neuropathic pain. MicroRNAs (miRs) can be molecular mediators of neuroinflammation. Our group has shown elevated levels of circulating miR-19a and miR-19b in persons living with chronic pain following SCI. We wanted to determine whether miR-19a and miR-19b are involved in neuroinflammation mediated by activated microglia after SCI. Microglia were incubated with inflammatory stimuli in the presence of miR-19a or miR-19b mimics and analyzed for activation. Microglia activation was enhanced in the presence of miR-19a or miR-19b mimics with increased expression of pro-inflammatory cytokines and decreased expression of suppressor of cytokine signaling, SOCS1 and SOCS3. Further, our results show enhanced signaling via NF-kB and Jak pathways. Overall, our results showed that treatment of microglia with miR-19a or miR-19b inhibited SOCS1 and SOCS3 expression and promoted expression of pro-inflammatory cytokines resulting in enhanced microglia activation. These results suggest that increased expression of miR-19a and miR-19b after SCI may be promoting neuroinflammation through activation of the microglia which contributes to neuropathic pain. These results may be useful in developing therapies targeting miRNAs to control neuroinflammation and neuropathic pain in persons living with SCI.

This study was supported by the Department of Defense (W81XWH-10-1-1043), National Institutes of Health (R01AR064793) and The National Institute on Disability, Independent Living, and Rehabilitation Research (NIDILRR, 90SI5015-01-00)
Human neutrophils are a key target of glucocorticoids
Authors: Gadkari Manasi , Tran Thai , Cao Qilin , Howe Katherine N , Randazzo Davide , Kaplan Mariana J , Franco Luis M
Published: August 2023

Neutrophils play central roles in immunity and inflammation. Glucocorticoids (GCs) are the cornerstone of anti-inflammatory and immunosuppressive therapy. While GCs trigger an acute rise in circulating neutrophils, little is known about the mechanisms by which GCs modulate neutrophil biology. We studied the response of 9 primary human cells to in vitro treatment with GCs. Surprisingly, neutrophils were transcriptionally the most responsive cell type, and they express proportionally the most glucocorticoid receptor (GR) transcripts. Long-read RNA-seq revealed 42 full-length GR transcript isoforms in neutrophils, twice as many as had been reported previously in all other cell types. At the protein level, neutrophils also have a unique and diverse set of GR isoforms. In contrast to the widely accepted model of GR signaling, in which GR binds GC molecules in the cytoplasm and then translocates to the nucleus, GR localization in neutrophils is almost exclusively nuclear, even in the absence of GC treatment. To validate our findings in vivo, we then performed a study of healthy volunteers, with blood collected before and serially after GC infusion. We generated a pathway-level map of the human neutrophil transcriptome at baseline and in response to GCs. The in vivo transcriptional response of human neutrophils to GCs was as strong as that observed in vitro, with a predominance of upregulation. Among the most strongly induced genes are several that are either not expressed, or expressed at very low levels, in human neutrophils at baseline. Some of the proteins encoded by these genes may have putative effects on neutrophil function, including SAMSN1 and ZBTB16. These findings indicate that human neutrophils are a key target for GC action.
Cigarette smoke-initiated oxidative stress causes NF-κB activation and lung inflammation via E3 ligase Itch-mediated Txnip degradation
Authors: Chen Chia-Ling , Wu Ying-Jung , Lin Pei-Yun , Shih Fu-Chia , Lin Chiou-Feng
Published: August 2023

Chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD) are characterized by pulmonary structural changes, narrowing of the small airways, and destruction of the lung parenchyma caused by the prolonged inflammation. Sustained inflammation mediated by macrophages is deliberated the critical role in COPD pathogenesis, while inductive mechanisms of the persistent inflammation remain unclear. In this study, we revealed that thioredoxin-interacting protein (Txnip) participated in cigarette smoke-incited NF-κB activation that potentially conducted pulmonary inflammation. Cigarette smoke extract markedly inhibited Txnip expression via an MAPKs-dependent regulation, which accompanied by iNOS/NO and COX-2 induction in RAW264.7 cells. The decreased Txnip was also detected in lung tissues and macrophages obtained from smoking mice, while a higher NF-κB activation and lung inflammation had arisen simultaneously. In addition, cigarette smoke-associated oxidative stress initiated the proteasomal degradation of Txnip followed by the MAPKs-regulated NF-κB activation concurrently. The expression of E3 ligase Itch was elevated in smoking mouse lungs and in hydrogen peroxide-stimulated cells, whereas specific silencing Itch significantly attenuated Txnip degradation as well as NF-κB activation. Moreover, Txnip was distinctly suppressed in lung tissues, bronchoalveolar lavage fluid cells and peripheral blood mononuclear cells obtained from patients with COPD. Accordingly, cigarette smoke-aroused oxidative stress induced Itch-mediated Txnip degradation and further NF-κB inflammation thereby enabling the COPD pathogenesis potentially.

MOST 111-2314-B-038-120-MY3 and

MOST 111-2320-B-038-012
Connexin-43: More than just a gap junction protein. Connexin-43 controls CD4+ T cell activation and clonal expansion
Authors: Bennett Beth E , Bilokon Thomas , Kraj Piotr , Kuczma Michal
Published: August 2023

Activation and proliferation of antigen specific T cells depends on the interaction of multiple signaling molecules with the TCR/CD3 complex. We report that expression of connexin 43 (Cx43), a gap junction protein, is critical for the expansion of antigen-specific or self-reactive effector CD4+ T cells. While gap junction mediated intercellular communication has been the main focus of connexin research, the cytoplasmic C-terminus of Cx43 has multiple functions independent of membrane channel forming properties and may act as a docking molecule for assembling signaling complexes including the immunological synapse. We also report that antigen-specific Cx43-deficient CD4+ T cells transferred into recipient mice immunized with cognate antigenic peptide and adjuvant expanded poorly compared to wild type cells. This protected mice from experimental autoimmune encephalomyelitis (EAE). Decreased proliferation and expansion also protects lymphopenic mice repopulated with naive, Cx43 deficient CD4+ T cells, from inflammatory bowel disease. We have found that Cx43-deficient CD4+ T cells have a signaling defect affecting Erk, p38 and Akt signaling pathways. Transcriptome RNA-seq analysis comparing gene expression profiles of activated Cx43 sufficient and deficient CD4+ T cells shows that differentially expressed genes are enriched in genes involved in G protein and Ca2+ signaling, ATP metabolism, genes regulating cytoskeleton reorganization and T cell homing. In summary, our data demonstrate that Cx43 has a major impact on the generation of effector CD4+ T cells, their ability to respond to antigenic stimulation, or to precipitate autoimmune disease.
The pro-inflammatory phenotype of human macrophages is restricted by the activity of the orphan nuclear receptor Nurr1
Authors: Solís Miguel Ángel , Santana Eduardo , Muñoz José Ramón , Segovia Norma Cristina , Patiño Eduardo , Sánchez-Torres Carmen
Published: August 2023

The nuclear receptor related 1 protein (Nurr1) is a member of the orphan nuclear hormone receptor family whose expression has been associated with several inflammatory diseases. Macrophages are key regulators of the inflammatory processes, yet information about the role of Nurr1 in human macrophages is limited. In the present study we examined the expression and activity of Nurr1 in steady state and activated human monocyte-derived macrophages. Pro- and anti-inflammatory macrophages were derived in vitro by culture of blood monocytes with the hematopoietic cytokines GM-CSF and M-CSF, respectively. Nurr1 expression was predominant in macrophages with a pro-inflammatory phenotype. Accordingly, its expression was induced by a variety of inflammatory stimuli such as TLR-2, -3 and -4 ligands, TNF, IFN-β, and IL-4. Pro-inflammatory macrophages exposed to the Nurr1 agonist C-DIM12 decreased their production of IL-1β, IL-6, and reactive oxygen species. Conversely, Nurr1 deficient macrophages produced enhanced levels of IL-6. Mechanistically, Nurr1 agonists partially abrogated the activation of the NF-κB signaling pathway in pro-inflammatory macrophages exposed to LPS, rendering cells with a lower rate of NF-κB p65 nuclear translocation. These results suggest that Nurr1 expression is linked with the pro-inflammatory phenotype of human macrophages where it may constitute a brake to attenuate the synthesis of inflammatory mediators.

Supported by grants from Secretaria de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)
β-catenin inhibition in tumor microenvironment macrophages promotes immunogenicity
Authors: Pundkar Chetan Y , Kang Xuejia , Babu R Jayachandra , Chen Pengyu , Suryawanshi Amol
Published: August 2023

Melanoma cells activate Wnt/β-catenin signaling in the tumor microenvironment (TME) to promote tumor progression and suppress dendritic cell-mediated anti-tumor immunity. However, the role of β-catenin in macrophages and its effect on anti-tumor immunity is largely undefined. In this study, using XAV939, a tankyrase inhibitor encapsulated in nanoparticles, we explored whether targeting β-catenin in macrophages can promote immunogenicity in TME. Our data show that the co-culture of macrophages with melanoma cell supernatants or melanoma cells in the presence of XAV939 nanoparticles significantly promotes the expression of CD80 and CD86, and suppresses the expression of PD-L1 and CD206 compared to macrophages treated with control nanoparticles. Similarly, XAV939 nanoparticle treatment of macrophages in co-culture conditions resulted in significantly increased TNF-α and IL-6 and reduced IL-10 production by macrophages compared to control groups. Our data suggest that targeted inhibition of β-catenin in tumor associated macrophages could overcome tolerance and promote anti-tumor immunity.
CD4 T cells from mice with alopecia areata express an effector like phenotype and can transfer disease
Authors: Connell Samuel J , Crotts Sydney , Kahl Payton , Lensing Maddison , Dix Cristina , Jabbari Ali
Published: August 2023

Alopecia areata (AA) is an autoimmune disease with a 2% lifetime incidence. AA presents as hair loss and is characterized histologically by the presence of CD4 and CD8 T cells surrounding the hair bulb. Although CD4 T cells comprise a majority of the cellular infiltrate, little is known about the contribution of this population to disease pathogenesis. An emerging animal model of AA involves adoptive transfer of in vitro activated and expanded bulk skin draining lymph node (SDLN) cells from AA mice, resulting in the induction of disease in recipient mice. The objective of this study was to investigate the mechanisms by which those cells transfer disease. Expanded bulk SDLN cells overwhelmingly consisted of CD3+ T cells, including both CD8 and CD4 T cells. To address whether these T cell populations could separately induce AA, we first sorted CD8 T cells from the SDLNs of AA mice and found these cells potently induced disease in recipient mice. Surprisingly, isolated CD4 T cells from AA mice also had a robust capacity to induce disease, and they did so more efficiently than CD4 T cells from unaffected (UA) mice. High dimensional flow cytometric analysis of the SDLNs indicated that an enhanced number of CD4 T cells in AA mice exhibited an effector-like phenotype. Development of AA following transfer of expanded CD4 T cells relied on the presence of endogenous CD8 T cells and host responsiveness to IFN-γ, but, in contrast, loss of IL-17 signaling appeared to have no effect. Together, these data suggest that activated CD4 T-helper type 1 effector cells contribute to the activation of CD8 T cells and the subsequent attack of the hair follicle. Further studies are needed to further define the specific mechanisms by which CD4 T cells contribute to the development of AA.

Dept of VA (I01BX004907), NIH/NIAMS (R01AR077194, K08AR069111, T32AI007485)
MOG-specific T cells skew high affinity during severe demyelinating disease after recombinant protein induction
Authors: Faust Michael A , Kolawole Elizabeth , Liu Tao , Cordero Bethanie , Zhou Zemin , Evavold Brian D
Published: August 2023

Demyelinating diseases can be driven by CD4 T cells specific for self-antigen derived from myelin in the central nervous system (CNS). Mouse models utilizing peptide induction protocols may restrict the breadth of T cell specificity. Here, we compare mice induced with a peptide fragment of myelin oligodendrocyte glycoprotein (MOG), recombinant extracellular domain (ECD), and recombinant full-length MOG protein, which includes the transmembrane domain. We sought to understand why mice induced with recombinant MOG suffer a more severe demyelinating disease than those induced with peptide by focusing on MOG-specific CD4 T cells in the CNS. Fluorescently-labeled peptide-MHC tetramers revealed that mice induced with ECD and protein have a greater frequency of tetramer-positive T cells than peptide induction. To more precisely define the MOG-specific T cell response, we employed the two-dimensional micropipette adhesion frequency assay (2D-MP), which, unlike tetramer, identifies low-affinity antigen-specific T cells. We confirm that 2D-MP identifies more MOG-specific T cells in all three induction protocols; however, mice induced with protein had a lower frequency of MOG-specific T cells than those induced with ECD and peptide, likely due to the epitope within the transmembrane region. 2D-MP also unveiled that T cells from mice primed with protein skew high-affinity as compared to mice primed with peptide. Ultimately, our data indicate that disease after ECD and protein induction is driven by a greater frequency of high-affinity T cells than those from mice induced with peptide. We posit that this is due to a change in antigen presentation after recombinant protein priming.

5R01NS071518-09 5R01AI110113-05
miRNAs targeting TGFβ-signaling disrupt Treg development and function in CNS autoimmunity
Authors: Rau Christina N , Severin Mary E , Lovett-Racke Amy E
Published: August 2023

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. Regulatory T cells (Tregs) exhibit impaired function in MS, and microRNAs (miRNAs) are known to regulate the development and activity of these cells.

Our group recently performed a large miRNA profiling study on naïve CD4 T cells in untreated MS patients. These data revealed that large number of TGFβ-signaling pathway targeting miRNAs are upregulated in MS patients compared to healthy controls. This dysregulation limits the in vitro generation of human regulatory T cells (Tregs). Administration of TGFβ-associated miRNAs in utero or after birth resulted in reduced Treg frequencies compared to controls. miRNA-treated mice of various strains were more susceptible to CNS autoimmunity modelled by induction of experimental autoimmune encephalomyelitis (EAE) at 8–10 weeks of age. Tregs isolated from miRNA-treated mice also showed a loss of T cell receptor (TCR) repertoire diversity after induction of EAE as TCR Vβ deep sequencing revealed.

Our findings indicate that increased levels of TGFβ-associated miRNAs lead to impaired Treg development and function contributing to an increased susceptibility to CNS autoimmunity. This may provide further understanding of Treg dysfunction in MS patients.
Activated memory T cells cross-talk to naïve T cells: A mechanism for generation of autoreactive T cells
Authors: Al Moussawy Mouhamad , Abdelsamed Hossam
Published: August 2023

Cytotoxic CD8 T cells (CTLs) can be auto-reactive under certain environmental and genetic conditions resulting in a wide range of autoimmune diseases. Although, these cells are detected at a very low frequency in healthy subjects, the mechanism(s) responsible for their maintenance/generation is not completely understood. Previously, we demonstrated that activated/memory CD8 T cells influence the fate of autologous naïve CD8 T cells acquiring activated/memory phenotype, suggesting that these cells might be auto-reactive. Since both CD4 and CD8 T cells play a pivotal role in the pathogenesis of autoimmune diseases, we examined whether their interaction could contribute to generation of auto-reactive T cells. Here, we showed that CD8 naive cells acquired activated-memory phenotype (CCR7neg CD45RO+) in the presence of activated memory CD4 T cells. Further using Type I diabetes (T1D) tetramers, we observed ~4% of these cells are autoreactive. These findings revealed a possible mechanism where activated memory CD4 T cells are capable to communicate with CD8 naive cells and generate auto-reactive T cells, which may have implications of autoimmunity following vaccination and transplantation.
Effect of mechanical cues on T cell killing of cancer cells
Authors: Dada Hannah , Do Kun , Cartagena-Rivera Alexander , Altan-Bonnet Grégoire
Published: August 2023

Cancer immunotherapies have focused on strategies to strengthen immune cell activation and effector function. These treatment strategies enhance the effector function of immune cells, particularly cytotoxic T cells (CTL), driving tumor eradication. Methods to improve the killing mechanisms of CTLs are not well studied and largely focused on assessing/strengthening the antigenic responses of T cells. We hypothesize that the mechanical landscape of CTL-tumor interactions can also drastically impact CTL cytotoxicity. By altering the physical context for T cell recognition of tumors, we measured cytotoxicity changes and subsequently used these changes in killing capacities to train and generate a quantitative parametrization of T cell killing. CTLs utilize cytokine expression to kill and shape the inflammatory milieu. Taking advantage of a robotic platform that enables the automatic collection of cytokines, we assembled datasets of T cell attacks of tumor cells grown on substrates with varying stiffness levels. The stiffness levels were altered using polyacrylamide (PAA) gels and ranged from 0.2kPa to approximately 113 kPa. Preliminary results from two different models (i.e., mouse and human TCR-transgenic T cells) suggest that there exists an optimal stiffness value for maximal response by T cells. These results suggest that CTLs utilize biophysical cues in addition to molecular cues (antigen) to properly elicit an immune response—modeling and fine-tuning these mechanical cues can be a novel and effective approach to improving cancer immunotherapies.
Elevated cholesterol uptake by T cells is a biomarker for the efficacy of immune checkpoint inhibitors in mouse models for triple negative breast cancer
Authors: Ciavattone Nicholas G , Guan Jenny , Farfel Alex , Desmond Tim , Stauff Jenelle , Brooks Allen , Luker Gary
Published: August 2023

Predicting the outcome of cancer immunotherapy can help avoid the cost and side effects of ineffective treatment and help develop effective treatment strategies. T cells take up cholesterol during activation, which is necessary for their proliferation. However, there is no literature linking the uptake of cholesterol as a biomarker for T cell activation and less so as a biomarker for immune checkpoint inhibitor therapy (ICI). Our research group has developed a novel radioactive cholesterol analog called 18F-FNP-59, which can be used to measure the uptake of cholesterol in live cells and within living organisms. We hypothesize that elevated cholesterol influx in T cells of the tumor microenvironment is linked to T cell activation and predicts the success of cancer immunotherapy in triple negative breast cancer (TNBC) models. We found that the elevated uptake of exogenous labeled cholesterol analogs functions as a biomarker for T cell activation. When comparing ICI-responsive E0771 tumors to non-responsive AT3 tumors, we found that the uptake of a fluorescent cholesterol analog was significantly higher in the T cells of the ICI-responsive tumors both in vitro and in vivo. Using the 18F-FNP-59 compound, we found that the uptake of cholesterol by T cells was further enhanced in ICI-responding tumors that received anti-PD-1 treatment. These results suggest that the uptake of exogenous cholesterol analogs by tumor-infiltrating T cells may be able to predict T cell activation and the success of ICI therapy. In future studies, we will expand the tumor models and types of cancers that we observe. Additionally we will examine the relationship between T cell cholesterol trafficking and invasion of T cells into the tumor using imaging techniques.
Investigating the Role of HDAC8 in CD4+ T Cells
Authors: Wang Jing , Yang Weiqin , Zhou Jingying , Chen Zhiwei , Cheng Alfred Sze-Lok
Published: August 2023

T cell exclusion restricts immune checkpoint blockade (ICB) efficacy for some hepatocellular carcinoma (HCC) patients. Epigenetic therapy provides new opportunities to rewire transcriptional programs. We have recently elucidated that pharmacological inhibition of histone deacetylase 8 (HDAC8) resulted in increased CD8+ T cell infiltration and enhanced ICB therapy efficacy in HCC. Moreover, this combined immunotherapy induced durable survival benefits even after tumor rechallenge accompanied by elevated memory T cells (Sci Transl Med, 2021). In this study, we found that HDAC8 inhibition increased memory T cells in HCC tumor-bearing mice. Interestingly, HDAC8 knockdown in tumor cells did not influence the effect of pharmacological HDAC8 inhibition in T cell activation and cytotoxic function, suggesting an immune cell-intrinsic role of HDAC8. HDAC8 was highly expressed in T cells instead of other immune cells. Notably, HDAC8 inhibition upregulated memory-related gene expression in CD4+ T cells, but not in CD8+ T cells. Besides, HDAC8 inhibition increased acetylation of structural maintenance of chromosome 3 (SMC3ac), a ring-shaped cohesin component in topologically entrapping enhancers and promoters which may modulate gene expression. Moreover, inhibiting HDAC8 increased SMC3ac binding onto the targeting gene promoter region and induced mRNA expression in CD4-expressing Jurkat cells. These data suggest that HDAC8 inhibition exerts memory T cell formation through SMC3ac-mediated epigenome reprogramming in CD4+ T cells, thus providing insights into developing effective cancer immunotherapy.

Acknowledgment: This work is supported by the RGC Collaborative Research Fund (C4045-18W), Li Ka Shing Foundation, TRS T11-706/18-N and EpiHK.
Sex-specific T cell exhaustion drives differential immune responses in glioblastoma
Authors: Lee Juyeun , Nicosia Michael , Silver Daniel , Lathia Justin
Published: August 2023

Sex differences in glioblastoma (GBM) incidence and outcome are well recognized; males show higher incidence and worse prognosis than females. Emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood. Using orthotopic GBM animal models, we recapitulated these sex differences in immune-competent B6 mice but not in immune-deficient mouse strains (NSG and RAG1KO). By depleting CD8+ T cells, we further confirm that CD8+ T cells play a critical role in driving sex differences in survival. Flow cytometry analysis revealed that male CD8+ T cells from tumor expressed higher levels of exhaustion markers such as PD-1, CTLA-4 and Tox, with higher frequency of progenitor exhausted T cell subset whereas female tumor contained more effector-like CD8+ T cells expressing elevated cytokine production. Anti-PD-1 antibody treatment exclusively extended the survival of male mice via inducing decreased exhausted CD8+ T cell subsets and increased effector function and proliferation. Survival analysis on bone marrow chimera model and adoptive transfer model indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, as the sex of donor cells was a critical contributing factor. Finally, we confirmed these observations in GBM patient tumor samples with male tumors having a higher frequency of progenitor exhausted T cells with increased TOX expression. Taken together, these findings demonstrate sex-specific pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy responses.

Supported by grants from NIH (R35 NS127083, P01 CA245705).