bluuebunny/crossref_metadata_2025 huggingface dataset:
A critical epithelial survival axis regulated by MCL-1 maintains thymic function
Authors: Jain Reema , Sheridan Julie M. , Policheni Antonia , Heinlein Melanie , Gandolfo Luke C. , Dewson Grant , Smyth Gordon K. , Sansom Stephen N. , Fu Nai Yang , Visvader Jane E. , Mintern Justine D. , Tan Iris , Holländer Georg , Strasser Andreas , Gray Daniel H.D.
Published: January 2023

T cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function. Age-related thymic involution is recognised as a potentially fatal brake on immune recovery following haematopoietic stem/progenitor cell transplantation and cancer treatments. To uncover new strategies to restore thymic function, we determined the molecular mechanisms that control life and death decisions in TEC. We recently published that while TEC-specific deletion of pro-survival proteins BCL-2 and BCL-XL did not cause gross abnormalities, MCL-1 deficiency impacted on TEC as early as E15.5, resulting in early thymic atrophy, T cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was continually required for the survival of medullary TEC, including autoimmune regulator (AIRE) expressing TECs and the maintenance of overall thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via the MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. However, these findings do not imply that the intrinsic pathway of apoptosis necessarily controls TEC death during homeostasis. Therefore, we removed essential effectors of this pathway, BAX and BAK, specifically in TECs and found that there was a specific increase in a medullary TEC subset leading to an overall increase in TEC numbers both in young and old mice. This signalling axis governing TEC survival, death and thymic function represent a compelling strategy for protecting the thymus during blood cancer treatment to prevent immunodeficiency, without counterproductive survival of haematopoietic lineages.
A requirement for Lysine-specific histone demethylase 1A (Lsd1) for T cell development
Authors: Stamos Daniel , Li Liqi , Rosenfeld Michael , Love Paul E.
Published: January 2023

Lysine-specific demethylase 1A (Lsd1), also known as Kdm1a, is a part of the histone demethylase family of enzymes. Histone demethylases are key epigenetic regulatory proteins of the mammalian genome that participate in chromatin remodeling. It has been shown that Lsd1 mediates H3K4me2/me1 histone demethylation, and this event has been connected with gene repression in hematopoietic stem cells (HSC) and HSC maintenance. While Lsd1’s role in plasma cell differentiation and as a therapeutic target in cancer are well established, its role in T cell development has not been examined. To investigate Lsd1’s role in T cell development, we used the CD2i-Cre system to conditionally delete Lsd1 in immature double negative (DN) thymocytes. Here we show that deletion of Lsd1 causes a severe reduction in total thymocyte numbers attributable to a defect at the CD8+CD4−CD3lo intermediate single positive (ISP) stage. In addition, Lsd1 deficient T cells are reduced in number, exhibit a memory phenotype, and express low surface levels of T cell antigen receptor (TCR) as assessed by TCRβ and CD3 staining. Together, these findings identify an important role for Lsd1 in T cell maturation and indicate that regulation of H3K4 histone demethylation is essential at several critical stages of thymocyte development.
Lck activity regulates NKT subset development.
Authors: Alberola-Ila Jose , Berrett Hannah , Henry Sara
Published: January 2023

Invariant natural killer T cells (iNKT cells) are a conserved population that expresses a canonical TCR α rearrangement, Vα14-Jα18 in mice, and recognizes CD1d+glycolipid antigens. Different subsets of iNKT cells have been reported, including NKT1, NKT2 and NKT17 cells. We have shown that a small alteration in E protein activity during development of iNKT development results in a decrease in the development of NKT1s at the expense of NKT2 and NKT17 cells. E protein activity is regulated by the expression levels of Id proteins, which are induced downstream TCR signals during positive selection. Therefore, we hypothesized that changes in TCR signal strength may influence NKT subset differentiation. To test this we have used a gain-of-function, loss-of-function model, where signal strength is modified at the level of Lck by expression of a constitutive active (dLGF) or catalytically inactive (dLGKR) Lck mutant at low levels. Our results show that in dLGKR mice, which have less Id induction and increased E protein activity during positive selection, the percentage of NKT2 in the thymus is increased. In contrast, in dLGF(Balb/c) mice there is a dramatic shift from the NKT2 and NKT17 subsets in the thymus towards the NKT1 subset. These results suggest that TCR strength may bias NKT subset differentiation, but it is unclear what determines signal strength during NKT positive selection. Differential Vβ usage affects NKT TCR affinity for different ligands, and there is evidence that Vβ usage is biased (i. e. Vβ7 is overrepresented in NKT2). However, despite the major shift in subset representation in dLGF mice, there are only minor changes in the Vβ usage. Therefore, Vβ expression is unlikely to to be the only factor influencing TCR signal strength.
Vβ recombination signal sequences enforce TCRβ allelic exclusion
Authors: Wu Glendon S. , Yang-Iott Katherine , Bassing Craig
Published: January 2023

The assembly of antigen receptor (AgR) genes via RAG1/RAG2 (RAG) mediated recombination of variable (V), diversity (D), and joining (J) gene segments is essential for adaptive immunity. V(D)J recombination of Tcrb, Igh, and Igk loci is regulated such that functional AgR genes are assembled on and expressed from one allele (allelic exclusion) in most cells. It is assumed that allelic exclusion is achieved by mono-allelic initiation and feedback inhibition of the V-to-(D)J recombination step, and intrinsic features of recombination signal sequences (RSSs) have been proposed to mediate mono-allelic V-to-DJ recombination of Tcrb and Igh loci. To investigate the role of Vβ RSSs in enforcing allelic exclusion, we established and analyzed mice with the Trbv2 or Trbv31 RSS replaced by a RSS possessing greater recombination activity. Each of these RSS substitutions causes a profound increase in the development of T cells expressing either Trbv2+ or Trbv31+ TCRβ chains at the expense of cells expressing other TCRβ chains, reflecting that the RSSs flanking Vβ segments are major determinants of Vβ recombination frequency. Each RSS replacement also promotes a modest increase in the fraction of cells expressing Trbv2+ or Trbv31+ TCRβ chains along with another type of TCRβ chain. Strikingly, mice with Trbv2 and Trbv31 RSS replacements on opposite alleles have a 30-fold higher than normal level of bi-allelic Trbv2+ and Trbv31+ expression, demonstrating that the RSSs flanking Vβ segments enforce TCRβ allelic exclusion. Our data suggests that the infrequency of Vb recombination, as determined by weak Vβ RSSs, limits bi-allelic assembly of Tcrb genes within the time window before feedback inhibition permanently cements allelic exclusion.
Infantile-onset primary alveolar proteinosis with hypogammaglobulinemia caused by heterozygous mutations of 2′-5′-oligoadenylate synthase 1
Authors: Okano Tsubasa , Cho Kazutoshi , Kawamura Shunsuke , Onai Nobuyuki , Fujii Wataru , Kakuta Shigeru , Kanai-Azuma Masami , Ohteki Toshiaki , Imai Kohsuke , Kanegane Hirokazu , Otsu Makoto , Ariga Tadashi , Morio Tomohiro
Published: January 2023

2′-5′-oligoadenylate synthase 1 (OAS1) is one of the interferon-stimulated genes (ISG) which plays a critical role in innate anti-viral immunity. Stimulated by viral double strand RNA (dsRNA), OAS1 produces 2′-5′ linked oligomer of adenylates (2′-5′A) and the produced 2′-5′A activates ribonuclease-L (RNase-L) which results in degradation of viral RNA and inhibition of viral replications.

We identified autosomal inherited OAS1 missense mutations in three Japanese pedigrees with infantile-onset primary alveolar proteinosis with hypogammaglobulinemia. Patients’ peripheral blood monocytes and CD11c+ myeloid dendritic cells (mDC) were progressively decreased; CD27+ memory B cells were lacking. The alveolar macrophages showed decreased phagocytosis activity; and the enlarged and foamy form was scarcely observed, which were typical in conventional pulmonary alveolar proteinosis (PAP). Their bone marrow monocyte progenitors were decreased and showed reduced colony forming potentials. One of the patients is currently alive without PAP after hematopoietic stem cell transplantation.

The recombinant mutant OAS1 protein had unaltered dsRNA binding activity. Patient-derived lymphoblastoid cell lines showed normal expression of OAS1 protein and mRNA, and unaltered function in OAS1 - RNase-L pathway. We generated model OAS1 knock-in mice by CRISPR/Cas9 based gene editing and analyzed immune-phenotypes. The mice show normal B and DC numbers and normal serum immunoglobulin levels so far. Detailed functional study on mutant OAS1 protein with the multi-Omics approach is currently underway.
Cell-intrinsic PD-L1 and PD-1 signal effects in bladder cancer
Authors: Zhang Deyi , Sun Xiujie , Gupta Harshita B. , Reyes Ryan M. , Svatek Robert S. , Curiel Tyler J.
Published: January 2023

PD-1/PD-L1 provides a mechanism of immune escape, the blockage of which has reinvigorated interest in the treatment of urothelial cancer. We recently reported on cell-intrinsic PD-L1 effects in melanoma and ovarian cancer, and considered that effects in bladder cancer cells could differ based on distinct mutational landscapes. Using CRISPER/Cas9 methodology, we knockout PD-L1 in the murine bladder cancer cell line MB49 and the human bladder caner cell line RT. We show here that cell-intrinsic MB49 and RT4 cells express PD-L1 and PD-1 that each mediate cell-intrinsic signals. PD-L1 knockout has little effect on cells proliferation in vitro, but significant difference could be found in tumor size when we used MB49 Ctrl or MB49 PD-L1KO cells to challenge C57B16 mice. αPD-L1 and αPD-1 antibodies reduced bladder cancer cell proliferation in vitro demonstrating direct signaling effects. Bladder cancer cell-intrinsic PD-L1 promoted mTORC1 and tumor initiating cell generation similar to melanoma and ovarian cancer cells. By contrast to melanoma and ovarian cancer cells, bladder cancer cell PD-L1 promoted autophagy and had little effect on in vivo immune-independent growth. Base one the functional role, PD-L1 knockout increased proliferation inhibition induced by Rapamycin in both mouse and human bladder cancer cells. Furthermore, we found intrinsic PD-L1 suppress cytokine production, including CXCL10. Corresponding with increased CXCL10 secretion, more CXCR3+ NK cells could be attracted by PD-L1KO cancer cells. Overall, our findings further illustrated the intrinsic role of PD-L1 in bladder cancer development and found PD-1 expression on human bladder cancer cells for the first time.
Herpesvirus co-infection alters immune responses to human influenza virus infection.
Authors: Souquette Aisha , Oshansky Christine , Tang Li , Wong Sook-san , Jeevan Trushar , Webby Richard , DeVincenzo John , Thomas Paul G.
Published: January 2023

Herpesviruses (HVs) cause lifelong infections, and the majority of humans are infected with at least one; whether co-infection with HVs is beneficial or detrimental to the host is a current debate. We sought to determine if co-infection with HVs alters the magnitude or quality of the immune response to influenza infection, and, if so, the extent to which each virus affects illness outcome. We used a longitudinal study of naturally acquired influenza virus infection and linear regression modeling to determine differences in influenza viral load and shedding, cytokine levels, and severity scores, while taking into account the effects of confounding variables. Our results show co-infection with HVs does not significantly affect influenza virus kinetics or clearance, but Cytomegalovirus is associated with decreased symptom severity and increased antibody titers. Furthermore, co-infection alters cytokine levels involved in each step of the immune response. Changes in plasma and nasal wash cytokines were predominately associated with Herpes simplex virus 1/2 and Epstein-Barr virus co-infection, respectively. The cytokines most affected by HV serostatus in the plasma and nasal wash are MIP1b (adj. R2 = 0.3073) and RANTES (adj. R2 = 0.3604), respectively. The strongest effects on cytokine levels are associated with HV interactions and are primarily found in the plasma. In the nasal wash, co-infection mostly results in decreased levels of cytokines, including those implicated in immunopathology during influenza infection (e.g. IL1, MIP1, TNF-α, IL6). Understanding host-virus interactions during co-infection and how that relationship impacts disease severity can ultimately be used for better prognosis and/or prophylactic measures.
A blood immune suppression index (BISI) based on IL10/IL12 and ARG1/NOS2 ratios is predictive of immune dysfunction and respiratory failure after burn injury in patients
Authors: Stepp Wesley H. , Malfitano Madison , Saklayici Irmak , Jones Samuel W. , Ortiz-Pujols Shiara , Maile Robert , Cairns Bruce A.
Published: January 2023

Few molecular markers exist that can detail the extent of an injury or predict outcomes in burn patients. Understanding the immune status of patients could minimize complications following burn injury by reducing non-emergent surgical interventions when infection risks are high and allowing more aggressive interventions in patients at higher risk for pulmonary compromise. Here we detail a novel molecular signature to identify these at-risk patients. PBMCs from healthy volunteers (n=30) or burn trauma patients (n=110) were isolated under UNC IRB 15-1662 and assayed for IL-10, arginase-1 (ARG1), IL-12 and nitric oxide synthase-2 (NOS2) using quantitative, real-time PCR (qRT-PCR) or Nanostring mRNA arrays. A blood immune suppression index (BISI) was calculated using a ratio of anti- to pro-inflammatory gene expression and molecular pathway analysis of Nanostring data was conducted using Ingenuity Pathway Analysis. PBMCs from severe burn patients had increased expression of the anti-inflammatory mediators interleukin-10 (IL-10) and arginase-1 (ARG1) within the first 48 hours of hospital admission. Conversely, the pro-inflammatory cytokines, interleukin-12 (IL-12) and nitric oxide synthase-2 (NOS2), were markedly down-regulated. The BISI for patients who eventually developed acute lung injury or suffered skin graft failures was increased by 2.2-fold and 3.1-fold, respectively. Further, large-scale mRNA profiling of key immune regulatory markers (n=596 genes) using Nanostring mRNA profiling highlighted key pathways of immune dysfunction that confirmed our initial qRT-PCR findings and added additional immunoregulatory markers that are differentially expressed in conditions of immune dysfunction.
Spatial map of human B cell compartmentalization
Authors: Borghesi Lisa A. , Weisel Nadine , Liu Ailing , Shlomchik Mark J.
Published: January 2023

We have performed a comprehensive analysis of human lymphoid tissue from relatively inaccessible sites including SPL, BM and LN from normal donors. Most of what is known about human B cells comes from blood, a tissue that contains only ~2% of the immune cells in the body. Examination of different tissues from 14 independent donors reveals for the first time the global B cell population structure. We performed unbiased computational clustering on samples stained with antibodies to markers indicative of antigen experience, activation status, and tissue retention. We found that the population structure of splenic B cells is remarkably conserved across a large number of donors who vary by age, gender, environment and history of pathogen exposure. By contrast, blood B cell composition is highly individualized with respect to each donor while BM has a relatively uniform population structure with some individual tailoring. Comparison of paired tissues within individuals further demonstrates that SPL and blood contain largely non-overlapping populations, suggesting geographic segregation of B cell subsets within each person. Recently published Ig clonal tracking studies from this donor cohort demonstrates partitioning of B cell clonal sharing into two broad networks, one across blood rich tissues and the other restricted to portions of the GI tract. Knowledge about the mechanisms that direct the trafficking and retention of discrete B cell subsets to specific tissues has direct implication to how infections are controlled throughout the body. Our findings constitute the basis for a geographic understanding of B cell population structure that will inform studies on the human humoral immune response.
Macrophage-associated lipin-1 enzymatic activity contributes to modified low-density lipoprotein-induced pro-inflammatory signaling and atherosclerosis.
Authors: Vozenilek Aimee E. , Navratil Aaron R. , Green Jonette M. , Coleman David , Blackburn Cassidy M.R. , Finney Alexandra C. , Pearson Brenna H. , Chrast Roman , Finck Brian N. , Klein Ronald L. , Orr A Wayne , Woolard Matthew D.
Published: January 2023

Macrophage proinflammatory responses induced by modified low-density lipoproteins (modLDL) contribute to atherosclerotic progression. How modLDL causes macrophages to become proinflammatory is still enigmatic. Macrophage foam cell formation induced by modLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to modLDL-elicited macrophage proinflammatory responses in vitro. The objective of this study was to determine if macrophage-associated lipin-1 contributes to atherogenesis and to assess its role in modLDL-mediated signaling in macrophages. We developed mice lacking lipin-1 in myeloid-derived cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared to control mice despite similar plasma lipid levels. Stimulation of bone marrow-derived macrophages with modLDL activated a persistent PKCα/βII-ERK1/2-cJun signaling cascade that contributed to macrophage proinflammatory responses that was dependent on lipin-1 enzymatic activity. Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a PKCα/βII-ERK1/2-cJun signaling cascade that contributes to foam cell proinflammatory responses. Taken together these results suggest modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation, but rather are both directly influenced by enhanced lipid synthesis.
Granulocyte-derived extracellular vesicles activate monocytes and are associated with mortality in ICU patients
Authors: Norris Philip , Inglis Heather C. , Abdel-Mohsen Mohamed , Deng Xutao , Adelman Avril , Schechtman Kenneth B. , Keating Sheila Marie , Cohen Mitchell J. , Jacobs Evan S. , Pillai Satish , Lacroix Jacques , Spinella Philip C. , Danesh Ali
Published: January 2023

To understand how extracellular vesicle (EV) subtypes differentially activate monocytes, a series of in vitro studies were performed. We found that plasma-EVs biased monocytes toward an M1 profile. Culturing monocytes with granulocyte-, monocyte- and endothelial-EVs induced several pro-inflammatory cytokines. In contrast, platelet-EVs induced TGF-β and GM-CSF, and red blood cell (RBC)-EVs did not activate monocytes in vitro. The scavenger receptor CD36 was important for binding of RBC-EVs to monocytes, while blockade of CD36, CD163, CD206, TLR1, TLR2, and TLR4 did not affect binding of plasma-EVs to monocytes in vitro. To identify mortality risk factors, multiple soluble factors and EV subtypes were measured in patients’ plasma at intensive care unit (ICU) admission. Of 43 coagulation factors and cytokines measured, two were significantly associated with mortality, TPA and cystatin C. Of 14 cellular markers quantified on EVs, 4 were early predictors of mortality, including the granulocyte marker CD66b. In conclusion, granulocyte-EVs have potent pro-inflammatory effects on monocytes in vitro. Furthermore, correlation of early granulocyte EV levels with mortality in critically ill patients provides a potential target for intervention in management of the pro-inflammatory cascade associated with critical illness.
The source of fat in a high fat diet affects systemic inflammatory markers and modulates gene expression in intestinal, hepatic and adipose tissues of growing pigs.
Authors: Blais Mylene , Gonzalez Jose Luis Martinez , Pouliot Yves , Gauthier Sylvie , Boutin Yvan , Roy Denis , Marette Andre , Asselin Claude , Lessard Martin
Published: January 2023

While high fat diets have been associated with increased level of inflammation and metabolic health impairment, new evidences show that the source of fat plays an important role on these outcomes. In order to investigate the specific role of dairy fats on metabolic health, a model of growing pigs was used. A total of 40 pigs aged 6 weeks were fed a low fat diet, or high fat diets (17.5% fat) containing either lard only, a combination of lard and butter (butter: 4.5% fat; lard: 13% fat) or a combination of lard and cheddar cheese (cheddar: 4.5% fat; lard: 13% fat) for 10 weeks. After 5 and 10 weeks, blood samples were collected to determine hyperlipidemia and inflammatory markers level. After 10 weeks, pigs were euthanized to collect intestinal, hepatic and adipose tissues to measure the expression of a selection of genes involved in inflammation, oxidative stress and energy metabolism by qPCR. Blood levels of IL-1β and TNF-α were decreased in pigs fed either butter or cheddar compared to the lard-only high fat diet (P < 0.05), while no changes were observed in the level of blood lipids. To visualize the impact of dairy fats on gene expression, a Heatmap of gene expression was drawn and showed that both butter and cheddar affected gene expression in jejunum, while butter specifically modulated the gene expression in liver and fat tissues, and cheddar specifically changed gene expression in colon. In conclusion, replacement of lard for dairy fats in a high fat diet protects from systemic inflammation and changes gene expression in pig intestinal, hepatic and adipose tissues.
A missense mutation impairs ITK function in a patient with severe Epstein-Barr virus disease.
Authors: Dowdell K. C. , Howe Matthew , Roy Amitava , Niemela Julie , Wilson Wyndham , McElwee Joshua , Cohen Jeffrey
Published: January 2023

T cells are critical in controlling EBV. IL-2-inducible T cell kinase (ITK) is an integral component of T cell signaling and modulates cytotoxic function. ITK activation leads to ITK mediated phosphorylation of phospholipase C (PLC)-γ, resulting in an intracellular calcium flux, activation of ERK, and translocation of NF-κB and NFAT into the nucleus. ITK is expressed in cells critical for controlling EBV infection, including T cells, NK cells, and invariant NKT (iNKT) cells. Mutations in ITK have been previously reported in 9 patients, ranging from 5 to 18 years of age who presented with low numbers of CD4 T and iNKT cells and EBV driven B cell lymphoproliferation. Here we identify a patient with a previously unreported homozygous missense mutation (ITK NM_005546 c.1618G>A p.D540N) in a highly conserved residue in the kinase domain of ITK. This patient presented at age 22 with lung nodules due to lymphomatoid granulomatosis with EBV-positive large tumor cells. She was treated with interferon and cytotoxic chemotherapy and her disease ultimately went into remission. She has since had persistent elevation of EBV DNA in the blood, low CD4 T cells, low NK cells, and nearly absent iNKT cells. Stimulation of her T cells resulted in reduced ITK signaling with diminished PLC-γ and Erk activation, and impaired calcium flux. Western blot analysis showed normal levels of ITK, indicating that the mutation affects the function, but not the stability of ITK. The patient’s CD8 T cells were impaired for degranulation, while her NK cells had reduced expression of the NK cell receptor, NKG2D. Our findings highlight the critical role of ITK to modulate T cell activation and control EBV driven B cell lymphoproliferation.
Cardiac damage after experimental blunt chest trauma and multiple trauma with hemorrhagic shock
Authors: Kalbitz Miriam , Weber Birte , Braun Christian , Lackner Ina , Haffner-Luntzer Melanie , Huber-Lang Markus , Gebhard Florian
Published: January 2023

INTRODUCTION

Heart injury represents an independent predictor for a poor outcome after trauma. However, molecular mechanisms of depressed cardiac function after trauma remains elusive.

METHODS

Multiple trauma (PTHS) included a blast-wave induced blunt chest trauma, traumatic brain injury and closed femoral fracture as well as pressure controlled hemorrhagic shock. In vitro studies were performed using human cardiomyocytes (Cellular Dynamics). Data were analyzed by one-way ANOVA analysis of variance followed by Dunnett’s multiple comparison test.

RESULTS

4h after PTHS in mice plasma concentration of pro-inflammatory cytokines such as MCP-1 and chemokine KC were significantly increased in comparison to sham treated animals. Plasma concentration of cardiac troponin levels were significantly increased early after experimental PTHS. HMGB1 staining provided significant increase in left ventricular tissue after PTHS. Distribution of the gap junction protein connexin 43 (Cx43) was was disintegrated in histological sections.

In presence of TNF reactive oxygen species were significantly increased in human cardiomyocytes, indicating a redox imbalance indicative of an oxidative state. Furthermore, presence of TNF in cultured human cardiomyocytes was associated with increased expression of the NLRP3 inflammasome and increased concentration of troponin in supernatant, which is specific marker for cardiac cell damage.

DISCUSSION

The cellular and subcellular events demonstrated in this report may explain molecular mechanisms associated with cardiac dysfunction after trauma. Better understanding of cardiac damage and molecular mechanisms of cardiac dysfunction after trauma will improve patient care in future.
TLR3 contributes to degeneration of dopamine neurons in an MPTP mouse model of Parkinson’s disease
Authors: Chung Young Cheul , Jeong Jaeyeong , Jin Byung Kwan
Published: January 2023

Toll like receptor 3 (TLR3), a pattern recognition receptor, is highly expressed in the brain. However, its role in neurodegenerative diseases is unknown. The present study investigated the effects of TLR 3 on dopamine (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease (PD). Here we show that TLR3-deficient mice are resistant to MPTP neurotoxicity. TH immunohistochemical analysis revealed that number of nigral TH+ neurons and striatal density of TH+ fibers were significantly maintained in MPTP-treated TLR3−/− mice, compared to MPTP-treated TLR3+/+ control mice. The observed neuroprotection was accompanied by reducing blood brain barrier (BBB) leakage and infiltration of immune cells such as CD-3+ T cells in the substantia nigra (SN). Moreover, genetic deletion of IKK-β (IKK-β−/−) in microglia, the downstream signaling of TLR3 and inhibitor of NF-κB kinase subunit beta (IκB) rescued dopamine neurons in the SN of MPTP-treated mice, compared to MPTP-treated IKK-β+/+ control mice. Similar to TLR3−/− mice, number of CD-3+ T cells were significantly attenuated in MPTP-treated IKK-β−/− mice, compared to MPTP-treated IKK-β+/+ control mice. Collectively, these results suggest that TLR 3 via IKK-β signaling pathway may play a crucial role in degeneration of dopamine neurons in the MPTP mouse model of PD.
Prenatal Maternal Immune Activation Sensitizes the Neuroinflammatory Responses to Neonatal Hypoxic-Ischemic Brain Injury
Authors: Kuan Chia-Yi , Chen Hong-Ru
Published: January 2023

Epidemiological evidence suggests that prenatal maternal immune activation (MIA) is a risk factor for autism spectrum disorders (ASD). Yet, because not all human MIA offspring develop ASD, we hypothesize that “second hits” may be needed to induce severe brain damage and disruption of the neural network. We tested this hypothesis using an established MIA model with Poly I:C (20 mg/kg) intraperitoneally injected at mid pregnancy (E12.5) in C57BL/6 mice. The offspring were challenged by mild hypoxia-ischemia (HI) at P10 as the second hit. We found that MIA alone infrequently caused clusters of amoeboid microglial cells (AMCs) expressing a high level of Complement component 3 (C3) in the offspring brains. Elevated NFkB signaling and a higher basal level of IL-6, IL-17 and MMP9 mRNA were also detected in the P11 MIA offspring brains. When challenged by HI, the MIA offspring showed significantly greater NFkB activity, TUNEL-positive apoptosis, and mRNAs for pro-inflammatory cytokines including IL-6, IL-17, TNFα, IL-23, TSPO and MCP-1 at 24 h post-injury, when compared to those injured by HI alone. At 7 d recovery, the MIA/HI-injured mice showed more AMCs, monocyte infiltration, and greater brain atrophy than singularly MIA- or HI-injured mice. Finally, the MIA/HI-injured mouse brains showed a significant reduction of PSD95 and synaptotagmin/PSD-96 punctates, correlated with greater C3 expression in the hippocampal CA2/3 region than MIA alone or HI-injured counterparts. Together, these results suggest that MIA elevates the inflammatory activity in the offspring brain, and acts as a primer to sensitize the neuroinflammatory responses to a secondary insult, leading to greater brain damage, and potentially ASD-like cognitive deficits.
Absence of CD137L decreases mechanical hypersensitivity and functional recovery time following sciatic nerve crush
Authors: Wakley Alexa A. , Cao Ling
Published: January 2023

CD137 Ligand (CD137L), a tumor necrosis factor superfamily molecule, is involved in immune responses such as migration, proliferation and production of cytokines. To investigate the involvement of CD137L in pain development and nerve regeneration following peripheral nerve injury, we examined various pain-like and functional recovery-related behavioral responses in CD137L knockout (KO) mice and wild type (WT) B6 mice following sciatic nerve crush (SNC). Male and female B6 WT and CD137LKO mice underwent a sham or SNC surgery and were evaluated for hypersensitivity (von Frey, Hargreaves & cold water tail withdrawal tests), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functional recovery. Behaviors were assessed before surgery and on post-surgery days 1–77. On post-SNC Day 1, both KO and WT mice showed increased hypersensitivity and decreased sensory and functional responses. By Day 7, CD137LKO mice showed reduced mechanical hypersensitivity compared to WT and were responding at sham levels by Day 42, whereas WT controls reached sham levels by Days 56–63. Lack of CD137L did not alter warm or cold thermal hypersensitivity. Grip strength improved in both strains beginning on Day 7, but CD137LKO mice reached sham levels faster than WT controls (Day 17 vs. Day 70). No differences were found on toe spread reflex and toe spacing scores. Finally, pin prick scores revealed that sensory recovery in both strains of mice emerged on Day 7, but the CD137LKO mice responded similar to shams by Day 28, one week earlier than WT. Together, these results suggest that CD137L mediates the sensory hypersensitivity and impedes the restoration of sensory and motor functions following peripheral nerve injury.
A novel variant in ATM gene causes ataxia telangiectasia revealed by whole-exome sequencing
Authors: Alonazi Noufa Afri
Published: January 2023

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder caused by variants in ATM gene and characterized by progressive neurologic impairment, cerebellar ataxia, and oculo-cutaneous telangiectasia. Immunodeficiency with a recurrent sinopulmonary infections are observed in patients with A-T. Here, we report a novel stop codon variant, c.5944 C>T (p.Gln1982*), revealed by whole-exome sequencing in a 9- year old boy. He presented with recurrent upper respiratory tract infections, failure to thrive, developmental delay, ataxic gait, and bulbar telangiectasia.
Effects of HMGB1 gene silencing on respiratory syncytial virus-induced inflammatory response
Authors: Kempaiah Kempaiah Rayavara , Kurosky Alexander , Hosakote Yashoda Madaiah
Published: January 2023

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections among infants and young children worldwide. High mobility group box 1 protein (HMGB1) is a redox-sensitive multifunctional protein that serves as both DNA regulatory protein and an extracellular cytokine signaling molecule that mediates the activation of innate and adaptive immune responses to infection, injury and inflammation. The goal of this study is to investigate the effect of HMGB1 gene silencing using short interfering RNA (siRNA) in RSV-induced airway inflammation. Studies were conducted in airway epithelial cells, human lung carcinoma cell line (A549) and normal small alveolar epithelial (SAE) cells. Cells were transfected with siRNA followed by infection with RSV, and NFκB, Nrf2, HDAC1 and catalase expression were determined. HMGB1-siRNA achieved a reduction of ~60–70% in nuclear HMGB1 expression in the airway epithelial cells. The Knockdown of nuclear HMGB1 was found to worsen inflammation in RSV-infected lung epithelial cells, highlighting a role for intracellular HMGB1 in cellular protection. We also found that HMGB1 gene silencing resulted in decreased HMGB1 release with significant downregulation of Nrf2, catalase and HDAC1 and increased NFκB and MAPK expression. These results indicated that downregulation of nuclear HMGB1 by siRNA might not only decrease its proinflammatory extracellular role by reducing its release but also serve to maintain its beneficial intracellular role, thus protecting against RSV -induced airway inflammation. Understanding the role of HMGB1 and associated cellular signaling in RSV-induced lung inflammation may pave the way for the development of novel therapeutic interventions.
Cessation from smoking improves innate host defense and clearance of experimentally inoculated nasal S. aureus
Authors: Cole Alexander M. , Schmidt-Owens Mary , Beavis Ashley C. , Chong Christine , Tarwater Patrick M. , Schaus James , Deichen Michael G. , Cole Amy Liese
Published: January 2023

Staphylococcus aureus (SA) nasal carriage is transient in most humans and usually benign, but dissemination of SA to extranasal sites causes the majority of clinical infections, and SA is a major cause of serious infections in the U.S. A better understanding of innate nasal decolonization mechanisms is urgently needed, as are relevant models for studying SA clearance. Here, we screened a population of healthy smokers for nasal SA carriage, and compared participants’ abilities to clear experimentally applied nasal SA before and after completion of a smoking cessation program. We determined that cigarette smoking increases mean nasal SA load (2.6×104 CFU/swab) compared to healthy non-smokers (1.7×103 CFU/swab) and might increase the rate of SA nasal carriage in otherwise healthy adults: 22 of 99 smokers carried SA at the screening visit, while only 4 of 30 non-smokers screened positive during the same time period. Only 6 of 19 experimental inoculation studies in active smokers resulted in SA clearance within the month of follow-up, while in the cessation group, 6 of 9 subjects cleared nasal SA and carriage duration averaged 21±4 days. Smoking cessation associated with enhanced expression of SA-associated IL-1β and G-CSF in nasal fluids. Participants who failed to clear SA exhibited higher nasal SA load and elevated nasal interleukin-1 receptor antagonist (IL-1RA) expression at the pre-experiment study visits. We conclude that smokers exhibit higher SA load than non-smokers, and that innate immune pathways including G-CSF expression and signaling through the IL-1 axis are important mediators of nasal SA clearance.
Purine nucleotide metabolism regulates expression of human MICA.
Authors: O’Callaghan Chris , McCarthy Michael T. , Moncayo Gerald , Hiron Thomas K. , Jakobsen Niels A. , Valli Alessandro , Soga Tomoyoshi , Adam Julie
Published: January 2023

Human MICA is upregulated in abnormal or stressed cells, including cancer cells, virus-infected cells and rapidly proliferating cells. Recognition of MICA by the activating NKG2D receptor on cytotoxic immune cells provides a mechanism for identification and potential elimination of aberrant cells. Despite the wide range of possible therapeutic applications of MICA manipulation, the factors that control MICA expression are unclear. We have used metabolic interventions and metabolomic analyses to show that the transition from quiescent cellular metabolism to a ‘Warburg’ or biosynthetic metabolic state induces MICA expression. Glucose transport into the cell and active glycolytic metabolism are both necessary to upregulate MICA expression. Active purine synthesis is required to support the effect of glucose on MICA expression and increases in purine nucleotide levels are themselves sufficient to induce MICA expression. This metabolic induction of MICA expression influences NKG2D-dependent cytotoxicity by immune cells. These findings are consistent with a model of MICA regulation whereby the purine metabolic activity of a cell can be indicated at the cell surface by MICA expression and is the subject of surveillance by NKG2D receptor-expressing immune cells.
The engagement of the aryl hydrocarbon receptor by tryptophan derivatives can prevent the development of anti-FVIII antibodies in an experimental model of hemophilia A
Authors: Matino Davide , Gargaro Marco , Scalisi Giulia , Manni Giorgia , De Luca Antonella , Puccetti Paolo , Quintana Francisco Javier , Alfonso Iorio , Fallarino Francesca
Published: January 2023

Patients affected by haemophilia A require treatment with factor VIII (FVIII) protein. The most relevant complication is the development of neutralizing FVIII-specific antibodies or “inihibitors”. We reported that the inhibitor-positive status was associated with reduced activity of the immune-regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1), that promotes regulatory effects via the production of tryptophan catabolites, known as kynurenines. Some of those tryptophan derivatives are endogenous ligands for the Aryl hydrocarbon receptor (AhR). In this study we tested the potential of tryptophan-related AhR ligands for inhibiting the development of anti-FVIII antibodies in hemophilic (F8 KO) mice. To this aim, F8 KO mice were treated with recombinant human FVIII (rhFVIII) alone or in combination with selected AhR ligands once weekly for four weeks. Antibody titers were tested by specific ELISA and Bethesda test. All mice treated with rhFVIII developed high-titer anti-FVIII antibodies after 4 weeks of treatment. Administration of a specific tryptophan metabolite prevented the generation of anti-FVIII antibodies in almost 80% of F8 KO mice. The protective effect of these AhR ligands was negated by co-administration of the AhR antagonist CH-223191 or in AhR KO mice. Similar results were obtained by administration of engineered gold nanoparticles loaded with the same tryptophan metabolite and rhFVIII. These results suggest that the engagement of AhR, by specific tryptophan derivatives, may be a possible new strategy to control the immune response to rhFVIII. Our findings might lead to the development of novel immunomodulatory interventions for preventing or eradicating inhibitors in hemophilia A patients.
Regulation of NKT cell metabolism by PLZF
Authors: Kumar Ajay , Pyaram Kalyani , Yarosz Emily , Giri Shailendra , Chang Cheong-Hee
Published: January 2023

Cellular metabolism and signaling pathways are the key regulators to determine T cell fate, survival and function, particularly during activation. Resting CD4 and CD8 T cells use oxidative phosphorylation as a primary energy source but switches to glycolysis upon activation, which is necessary to produce biomolecules for cell proliferation and function. Failure of this reprogramming is detrimental for T cell mediated immunity. However, little is understood about how NKT cells control their metabolism to survive and function. We found that NKT cells operate distinctly different metabolic programming from CD4 T cells for their survival and function. Supporting evidence includes that, unlike CD4 T cells, oxidative phosphorylation seems to be preferred over glycolysis in NKT cells even after stimulation as revealed by lower level of intracellular lactate produced by NKT cells than CD4 cells after activation. These cells also seem to have high energy as revealed by higher ATP in NKT cells. Although the proper maintenance and functions of stimulated NKT cells need glucose, they are sensitive to the elevated glycolytic potential compared to CD4 T cells resulting in spontaneous cell death accompanied by hyperproliferation. Importantly, PLZF is the essential regulator of NKT cells’ metabolism so that similar changes of glucose metabolism were found in CD4 T cells expressing PLZF. Conversely, NKT cells with haplodeficient PLZF were more tolerant to increased glycolysis, strongly supporting the key role of PLZF in NKT cell metabolism and homeostasis. We are currently investigating the underlying mechanisms by which PLZF controls metabolism in NKT cells using several approaches including metabolomics.
Pseudomonas aeruginosa-induced lung epithelial production of IL-8 contributes to PMN sub-mucosa recruitment but not trans-epithelial migration in vitro.
Authors: Lanter Bernard B. , Eaton Alex D. , Gipson Kevin S. , Yonker Lael M. , Hurley Bryan P.
Published: January 2023

Background

Numerous lung associated diseased states such as acute pneumonia and cystic fibrosis (CF) are associated with increased concentrations of IL8 and infiltration of neutrophils (PMNs) in the airspace. IL8 production by lung epithelial cells (ECs) is stimulated by Pseudomonas aeruginosa infection, an opportunistic pathogen involved in acute pneumonia, and lung infection of CF patients. The exact role IL8 plays in migration of circulating PMNs into the infected lung and airspace is still uncertain.

Objective

Assess the role of IL8 in mediating PMN migration in response to P. aeruginosa infected lung epithelium.

Methods

CRISPR/Cas9 was used to delete the IL8 gene from human lung ECs, which were grown as polarized monolayers in a co-culture transwell model. Basolateral to apical PMN migration to PAO1 infection was assessed. A novel double transwell model was developed to observe PMN submucosa migration in response to PAO1 infection in the presence and absence of IL8 lung EC production.

Results

PAO1 induced PMN trans-epithelial (TE) migration was not impacted in the absence of IL8 expression. When PMNs were placed in a separate transwell suspended 5 mm above the basolateral side of the infected epithelium, the IL8 gradient produced was sufficient to cause relevant migration of PMNs to the basolateral side of the infected epithelium. The level of migrated PMNs in the double transwell system with tissues devoid of the IL8 gene was equivalent to the presence of PMNs which responded to un-infected epithelial tissues.

Conclusion

These findings further support that IL8 is integral in PMN recruitment, and suggests that IL8 is most important towards recruiting PMNs to the submucosa during P. aeruginosa PAO1 infection of lung epithelium.
Ablation of adaptive immune responses facilitates increased bacterial clearance in murine model of defective mucociliary clearance
Authors: Lewis Brandon W. , Patial Sonika , Saini Yogesh
Published: January 2023

Defective mucociliary clearance (MCC) associated with muco-obstructive lung diseases may compromise effective clearance of aspirated bacteria. Mucus hypersecretion is often associated with mucous cell metaplasia (MCM) of airway epithelium, which is a Type 2 immune response. Whether ablation of Type 2 response abolishes MCM/muco-obstruction, and thus, enhances bacterial clearance remains untested. Accordingly, we hypothesized that the ablation of adaptive immune responses will promote bacterial clearance in murine model of defective MCC, i.e., Scnn1b [Sodium channel, non-voltage-gated 1, beta] transgenic (Tg+) mice. The Tg+ model recapitulates hallmarks of muco-obstructive airways, including MCM and bacterial infections. The Type 2 response deficient (Il2rg−/−, Rag1−/− and Il2rg −/−\Rag1−/−) mice strains on Tg+ background were observed at neonatal age (6–7 days) for bacterial clearance response. Our data suggest that while Il2rg−/− mice [lacking innate lymphoid cells (ILC)] had significantly impaired bacterial clearance in control (Tg−) as well as Tg+, the Rag1−/−mice [lacking adaptive immune response] did not exhibit impaired bacterial clearance in Tg− as well as Tg+ neonates. The defective bacterial clearance in Il2rg−/− was not due to reduced phagocyte recruitment. Interestingly, Il2rg −/−\Rag1−/− mice had significantly improved bacterial clearance, an observation that remains a focus of our ongoing investigation. Our analyses revealed absence of IL-10 in Rag1−/−mice was associated with morphological activation of macrophages. Taken together, our current data suggest that while ILCs are essential for bacterial clearance, the adaptive immune responses suppress macrophage’s antibacterial function.
Early, in situ re-activation of lung memory T cells drives chronic lung inflammation
Authors: Turner Damian L. , Tanna Nicole , Barahona Josselyn
Published: January 2023

Acute immune responses within the respiratory tract generate memory T lymphocyte populations that are retained within the lung tissue and contribute to chronic inflammatory responses upon subsequent re-exposures to antigen. The generation and maintenance of antigen-specific memory T cell populations within the lung are therefore processes which, if inhibited, could ameliorate the symptoms of chronic inflammatory lung diseases. We sought to develop a model system that consistently generates lung inflammation and antigen-specific memory T cells within the lung. We show that prolonged respiratory exposure to lipopolysaccharide and a protein antigen generates a long-lived memory T cell population in the lung. These cells are retained in this compartment even after a week-long period of induced lymphopenia. Furthermore, lung memory T cells are re-activated in an antigen-specific manner upon intranasal challenge with antigen. The early in situ reactivation of memory T cells in the lung induced the recruitment of innate immune cells into the lung parenchyma and into the airways. Thus, elucidating the pathways involved in the tissue retention of the lung-retained memory T cell population may lead to therapies that can reduce harmful T cell populations within the lung.