bluuebunny/crossref_metadata_2025 huggingface dataset:
Environmental pollutant and lipid peroxidation product, acrolein, inhibits interferon‐alpha mediated antiviral signaling in human hepatocytes: relevance for HCV therapy
Authors: Joshi‐Barve Swati , Amancherla Kiranmayi , Patil Madhuvanti , Bhatnagar Aruni , Srivastava Sanjay , Gobejishvili Leila , McClain Craig , Barve Shirish
Published: June 2021

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease in the US and is a precursor to cirrhosis, end‐stage liver disease, hepatocellular carcinoma and death. The FDA approved standard of care for chronic hepatitis C, is pegylated interferon‐alpha (IFNα) combined with ribavirin which is effective only in about 50% of patients. Studies have identified potential cofactors that contribute to the failure of anti‐HCV therapy, including obesity, elevated lipid peroxidation, oxidative stress and environmental pollutants. Acrolein (ACR), a highly reactive aldehyde, is formed endogenously through cellular lipid peroxidation and is also a common, ubiquitous environmental pollutant. Exposure to ACR induces considerable oxidative stress and disrupts cellular signaling. This study examines the effect of ACR on IFNα mediated anti‐viral signaling in hepatic cells. Our data demonstrate that relevant concentrations of ACR significantly inhibit interferon‐alpha mediated JAK/Stat signaling, leading to reduced expression of anti‐viral proteins. ACR dramatically inhibits IFN‐induced tyrosine phosphorylation of both STAT1 and STAT2, and their upstream kinases, JAK1 and Tyk2. A corresponding decrease is observed in the nuclear translocation of phospho‐STAT1 and 2. Acrolein down regulated IFNα. inducible expression of anti‐HCV proteins such as PKR, OAS, ISG15 and ISG54, primarily by decreasing ISRE‐controlled gene transcription. This study identifies acrolein, a derivative of lipid peroxidation and a common environmental pollutant, as a potential cofactor that contributes to poor treatment outcomes and failure of IFNα therapy.
CD44 and Posterior Capsular Opacification (PCO)
Authors: Desai Vivek Dhaval , Duncan Melinda , Wang Yan
Published: June 2021

PCO arises from epithelial‐mesenchymal transition (EMT) of lens epithelial cells remaining behind following cataract surgery. In cancer systems, CD44 mediates changes that eventually result in EMT, although CD44 function in EMT has not been previously studied in the lens. In the normal adult mouse lens, we found CD44 protein and mRNA only in the lens fiber cells. CD44 null mice did not exhibit any qualitative differences in the timing or extent of expression of the EMT marker, α‐SMA, as compared to WT following cataract surgery suggesting that CD44 is not essential for EMT. But CD44 expression is highly up‐regulated in the remaining lens epithelial cells 12 hours following the surgery. This suggests that it is the earliest molecular marker ever described for lens response to injury and may serve as a good model to understand the early events in the lens epithelial response to injury and the sensitization of these cells to later TGFβ mediated EMT. CD44 expression is known to be up‐regulated in response to hepatocyte growth factor (HGF) mediated signaling and HGF has been reported to be a potent mitogen for lens epithelial cells. Future work will investigate the hypothesis that HGF mediated signaling up‐regulates in the lens in response to injury leading to up‐regulation of CD44 expression and sensitization of lens cells to EMT. Funded by the Beckman Foundation, Barry M. Goldwater Scholarship, HHMI, and National Eye Institute.
Polypodium leucotomos extract prevents from solar‐induced DNA damage in human peripheral blood mononuclear cells
Authors: Reyes Eduardo , Guerrero Antonio , Brieva Aurora , Valdes‐Hevia Marta D , Gonzalez Salvador , Alvarez‐Mon Melchor
Published: June 2021

Exposure of human cells to solar radiation (SSR) leads to deleterious effects such as immune suppression and cancer. The mechanism of cell damage is complex and can be simplified in necrosis and apoptosis of cells that caused inflammation. Polypodium leucotomos extract (PPL‐240) has photoimmunoprotective properties. To analyse the effect of PPL‐240 on the cellular damage of PBMC induced by SSR. PBMC were incubated during 2 h in presence or absence of PPL‐240. Then, PBMC were exposured to SSR (HB406, Phillips). Finally, PBMC, before and after 24 h culture in presence of polyclonal mitogen, were labeled with annexin V (AV) to identify apoptotic cells, followed by a incubation with 7‐Amino Actinomycin D (7AAD). The cell populations discriminated are viable cells (AV−7AAD−), early apoptotic cells (AV+7AAD−), late apoptotic cells (AV+7AAD+), and early necrotic cells (AV−7AAD+). SSR increases spontaneous and mitogen‐induced apoptosis (annexin V+ cells) in PMNC (from 12.8±4.6 to 78.3±1.2% and from 51.8±5.1 to 85.2±0.6% respectively). The treatment with PPL‐240 reduces the percentage of AV+ cells (18.8±8.8 and 29.6±7.2% respectively). These apoptosis is mainly due to a reduction of early apoptotic cells (44.4±3.4 and 50.2±11.5%, for spontaneous and mitogens‐induced respectively). We conclude that PPL‐240 reduces apoptosis in PBMC stimulated with SSR. This reduction is attributable to AV+7AAD− cells.
Mutiplex Detection of Apoptotic Protein Biomarkers By Luminex‐based Suspension Array
Authors: Hwang Joseph Bumsuh , Wiese Rick
Published: June 2021

The vast number of intracellular signaling pathways controlled by changes in the activation/inactivation of intracellular proteins has created demand for more sensitive and efficient analysis techniques. Traditional methods to detect apoptosis and related cell signaling pathways include flow cytometry, Western blotting, and ELISA. We have developed a multiplex suspension array based on Luminex® xMAP® technology. This technology allows for the simultaneous detection of multiple signaling proteins in cell lysates in a 96‐well plate format. Unlike traditional methods, this assay simultaneously measures changes in multiple targets in a single well of sample, with increased sensitivity, significantly smaller samples volumes, and less preparation time. The assay procedure is a simple fluorescent bead‐based sandwich immunoassay consisting of uniquely color‐coated bead‐sets, which are covalently coupled to capture antibodies specific for each target molecule. A second reporter antibody labeled with biotin is used for the detection of specific phosphorylation/cleavage/epitope sites on the target proteins. The level of total protein or site specific phosphorylation/cleavage for each bead set is then quantified using the Luminex® 100™ IS system. Using the Apoptosis Multiplex Panel, increased levels of cleaved caspase‐3, cleaved PARP, and phosphorylation of histone H2A.X were detected in stimulated cells when compared to their unstimulated counterparts. Multiplex suspension array results were more quantitative, sensitive, and required much less labor compared to Western blotting when used to analyze apoptotic protein biomarkers in cellular lysates.
Neuroanatomical and neurochemical specificity of pathological changes in the posterior inferior temporal cortex in AD
Authors: Thangavel Ramasamy , Van Hoesen Gary W. , Zaheer Asgar
Published: June 2021

The posterior inferior temporal cortex or Brodmann's area 37 (BA37) has been investigated in Alzheimer's disease (AD). Previous studies suggested a relationship between severity of AD and the degree of neurofibrillary tangles (NFTs) clustering pattern in different areas of the cortex in brains. The cellular architecture and connectivity of BA37 is unclear. We studied the laminar changes in the distribution and topography of NFT pathology of BA37 in AD subjects using thionin staining for Nissl substance and thioflavin‐S staining for NFTs. We used immunohistochemical methods to reveal the neurochemical alterations of BA37 in AD. Neurons with neurofibrillary processes and degeneration were detected in layers II–VI of this cortex. NFTs were predominantly expressed in bilaminar pattern in entire regions of BA37. To compare the corresponding cortical region in the monkey brain, we studied the modular and columnar organizations of inferior temporal cortex by using the retrograde tracer fast blue (FB). Clustering of FB labeled neurons was densely distributed in layers III, V and VI. We also observed the vertical columnar cluster pattern through layers II–VI. Based on clinical history, neuroanatomical, neurochemical and neuropathological features in the BA37, we hypothesize that the disruption of feed forward, feedback and efferent cortical projections is responsible for the development of NFT formation in AD.
Tools to enhance biochemistry undergraduate laboratory exercises using Escherichia coli beta‐galactosidase
Authors: Markwell John , Boanca Gina , Madhavan Soundararajan , Bailey Cheryl
Published: June 2021

Our undergraduate biochemistry program has developed a comprehensive one‐semester laboratory course based on the E. coli β‐galactosidase enzyme. The rationale for this central theme is that students perceive more connections between different techniques or instruments with concepts that build upon foundational material in undergraduate biochemistry and microbiology courses. Two bio‐materials have proven critical to these exercises. The first is use of the E. coli ML308 (ATCC 15224) which is constitutive for β‐galactosidase expression. This strain is used to provide students with consistently high levels of expression for enzyme kinetics and inhibition experiments. The second biomaterial is a plasmid (Novagen Cat. No. 69257‐0.2ml) that contains the β‐galactosidase coding sequence inserted into pET15b with a 6X His‐tag on the N‐terminus. Experiments with this plasmid allow students to explore the kinetics of induction with lactose or IPTG, and also exposes them to affinity chromatographic procedure through a one step purification of the enzyme using affinity resin binding to the 6x His‐tag.
B7‐1 and B7‐2 contribute unequally to peripheral homeostasis of regulatory T cells (Tregs)
Authors: Zeng Menghua , Guinet Elisabeth , Nouri‐Shirazi Mahyar
Published: June 2021

Tregs maintain immunological self tolerance, prevent autoimmunity and control immune responses to transplants. Observations of decreased numbers of Treg cells in CD28‐ or B7‐1/B7‐2‐deficient non‐obese diabetic (NOD) mice suggest that the CD28/B7 interaction is essential for the development and peripheral homeostasis of the Tregs. Although targeting the CD28/B7 interaction remains among the most promising approaches to treat autoimmune diseases and transplant rejection, little information exists regarding the relative role of B7‐1 and B7‐2, the only two known ligands of CD28, on the generation and maintenance of Tregs. To clarify whether B7‐1 and B7‐2 have identical or overlapping effects on Tregs development and homeostasis, in this study we measured the frequencies of CD4+CD25+Foxp3+ Tregs in the blood, thymus and peripheral lymphatic tissues of mice deficient in B7‐1, B7‐2 or both B7‐1/B7‐2 and compared with that in wild type mice. Our results suggest that the absence of both B7‐1 and B7‐2 results in a dramatic reduction in the frequencies of Tregs. Interestingly, we observed a significant but similar reduction in the frequency of Tregs in thymus of both mice deficient for either ligands, however, with more impact in Treg peripheral homeostasis seen in mice deficient in B7‐2 rather B7‐1. Consistent with these results we found that B7‐2 deficient DCs were less efficient in promoting the expansion of Tregs in vitro. Our data provides the first direct comparison of B7‐1 and B7‐2 roles in thymic development and peripheral homeostasis of Tregs. TAMUSHSC‐OVPRGS Award.
Endogenous galectin‐1 enforces class I‐restricted TCR functional fate decisions in thymocytes
Authors: Liu Scot Daren , Whiting Chan C , Tomassian Tamar , Pang Mabel , Bissel Stephanie J , Baum Linda G , Mossine Valeri V , Poirier Francoise , Miceli M Carrie
Published: June 2021

During thymocyte development, the TCR can discriminate MHC/peptide ligands over a narrow range of affinities and translate subtle differences into functional fate decisions. How small differences in TCR input are translated into absolute differences in functional output is unclear. We examined the effects of galectin‐1 ablation in the context of class‐I‐restricted thymocyte development. Galectin‐1 expression opposed partial‐agonist‐driven positive selection, but promoted agonist‐driven negative selection of conventional CD8+ T cells. Further, galectin‐1 promoted TCR agonist‐driven CD8αα IEL development. Mechanistically, galectin‐1 opposed ERK activity in thymocytes undergoing positive selection and promoted the ERK negative‐selection‐signaling signature in thymocytes undergoing negative selection. We propose that galectin‐1 aids in discriminating TCR fate decisions by enforcing agonist and opposing partial‐agonist TCR signals, widening the distinction between TCR‐directed functional fate cues. This work was supported by grant NIH RO1A1056155 to M. C. Miceli. S. D. Liu was supported by the Microbial Pathogenesis Training Grant T32 AI07323‐15, Clinical and Fundamental Immunology Training Grant AI07126‐30, and Warsaw Fellowship.
HEB and E2A enforce TCR checkpoint in T lymphocyte development
Authors: Zhuang Yuan , Jones Mary Elizaberth
Published: June 2021

The T cell receptor (TCR) is required for positive selection and the subsequent transition from CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) stage of αβ T cell development. The molecular mechanism which maintains DP fate prior to the acquisition of a functional TCR is not clear. We will present genetic data to show that the structurally and functionally related transcription factors HEB and E2A work together to maintain DP fate and to control the DP to SP transition. We demonstrate that simultaneous deletion of HEB and E2A in DP thymocytes is sufficient for DP to SP transition independent of TCR. Loss of HEB and E2A allows DP cells to bypass the requirement for TCR‐mediated positive selection, down‐regulate DP associated genes, and up‐regulate SP specific genes. Our work identifies HEB and E2A as the gatekeepers that maintain cells at the DP stage of development until a functional αβ TCR is produced.
A Conserved Salt Bridge in the G‐Loop of Src Family Protein Tyrosine Kinases is Essential for Lyn Function in Mice
Authors: Sauer Karsten , Herman Ann , Lee Christian , Che Jianwei , Watson James , Velentza Anastasia , Kim Sunjun , Ziaee Niusha , Miller Andrew , Jackson Carie , Young Mike , Batalov Serge , Liu Yi , Warmuth Markus , Wiltshire Tim , Cooke Mike , Barouch‐Bentov Rina
Published: June 2021

The glycine‐rich G‐loop is important for ATP binding and phosphate transfer in protein kinases. Here we show that the function of Src family protein tyrosine kinase G‐loops requires an electrostatic interaction between a conserved N‐terminal basic and a conserved C‐terminal acidic amino acid. By limiting G‐loop flexibility, this salt bridge is required for high affinity ATP binding and catalysis. In WeeB mice, mutation of the conserved acidic amino acid results in expression of a mutant Lyn protein with strongly reduced catalytic activity. This leads to moderate perturbation of B cell receptor signaling, which is strongly impaired in Lyn−/− mice. Surprisingly, WeeB mice still show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis, key aspects of the Lyn−/− phenotype. This demonstrates the physiological importance of the conserved G‐loop salt bridge and in addition unveils an extraordinary sensitivity of B cell function to the Lyn kinase activity.
Disruption of cyclin D3‐cdk4 complexes in the human diffuse large B‐cell lymphoma OCI‐LY18 results in caspase‐3‐dependent apoptosis
Authors: Gumina Maria R. , Chiles Thomas C.
Published: June 2021

Normal human B lymphocytes express cyclins D1 and D3. By contrast, we have found that the human diffuse large B‐cell lymphoma (DLBCL), OCI‐LY18 (LY18) uniquely expresses cyclin D3. This non‐redundancy of D‐type cyclin expression suggests that inhibition of cyclin D3‐cdk4 complexes may represent a potential therapeutic target. To investigate the contribution of cyclin D3‐cdk4 complexes in LY18 cells, we treated cells with a chemical cdk4 inhibitor (Cdk4i) in order to selectively disrupt cyclin D3‐cdk4 complexes. We report herein that Cdk4i treatment of LY18 cells leads to G0/G1 phase arrest and apoptosis. Consistent with these findings, transduction of p16(INK4a) peptidyl mimetics (TAT‐p16) into LY18 cells led to disruption of cyclin D3‐cdk4 complexes and apoptosis. Depletion of cyclin D3‐cdk4 complexes resulted in decreased retinoblastoma protein phosphorylation on cdk4‐targeted S807/S811. In addition, we found that apoptosis was dependent on caspase‐3 activation. These findings suggest that cyclin D3‐cdk4 holoenzymes are required for LY18 cell survival. The results also suggest that targeting cyclin D3‐cdk4 complexes in human DLBCLs may prove to be an effective therapeutic strategy.

This work was supported by USPHS grant AI60896 and AI49994.
B Cell Clonal Contraction Following BCR‐triggered, Innate Immune System‐driven Activation Involves a p53‐regulated Apoptotic Process
Authors: Mongini Patricia K. A.
Published: June 2021

Activation‐induced apoptosis (AIA) contributes to the clonal contraction of human B2 cells stimulated by surrogate C3dg‐bound Ag (a‐IgM:a‐CD21:dextran) and IL‐4. Albeit only partially effective, BAFF hinders AIA through a BAFF → COX‐2 → PGE2 pathway that sustains Mcl‐1 levels (J. Immunol. 175:6143 [2005] and J. Immunol. 176:6736[2006]). Presently, the mechanism responsible for AIA within human B cells stimulated by BCR and innate immune system cytokines was studied. AIA involves an intrinsic apoptotic pathway, but unlikely an extrinsic pathway mediated by TRAIL:DR or Fas:FasL. The former was evidenced by (a) elevated levels of Bim, and p53‐regulated Bax, Bad, and Bid at d3 prior to AIA; (b) sustained expression of Bax and Bad at d5; and (c) detection of active caspase 3 within d5 apoptotic progeny. Dramatically reduced levels of intact Bid from d3 to d5 suggests that AIA may involve Bid cleavage to pro‐apoptotic tBid. A role for p53 was confirmed by evidence of elevated p53 within activated blasts and evidence that p53 siRNA treatment of pre‐activated B2 cells reduces subsequent clonal contraction. Taken together, it appears that p53 upregulation via cell stress and/or DNA damage during activation is, at least in part, responsible for AIA. This process can be circumvented when progeny, expressing elevated levels of p53‐regulated pro‐apoptotic molecules, receive pro‐viability signals via a COX‐2 → PGE2 → Mcl‐1 pathway.
The role of the ERK MAP kinase pathway in CD4 T cell proliferation and differentiation
Authors: Chang Chiung‐Fang , D'souza Warren N. , Talukdar Saswata , Hedrick Stephen M.
Published: June 2021

The ERK (Extracellular signal‐regulated protein kinase) signaling pathway is thought to be critical for T cell survival, death, proliferation and differentiation. We used germline ERK1 knockout mice and tamoxifen‐regulated conditional ERK2 knockout mice to identify the role of these two major ERK isoforms within mature CD4 T cells. Our in vitro studies revealed that ERK1 was dispensable for most aspects of CD4 T cell activation, while ERK2 performed several non‐redundant functions. We observed that ERK2 supported early CD4 T cell proliferation, as well as the differentiation of Th1, but not Th2 or Th17 cells. In addition, virus‐specific Th1 responses in the absence of ERK2 were greatly impaired, demonstrating that ERK2 also plays an important role in CD4 T cell proliferation and differentiation in vivo.
A role for TRAF5 in CD8+ T cell responses.
Authors: Kraus Zachary James , Haring Jodie , Harty John , Bishop Gail
Published: June 2021

Effective clearance of and immunity to intracellular pathogens depends on activation of T lymphocytes and generation of effective antigen specific memory. The quality and magnitude of T cell memory is modulated by costimulation. TNFR family members CD27, OX40 and GITR are integral to T cell function and are expressed at different stages in T cell activation. These molecules interact with TRAF5, an adaptor molecule that translates signals from these receptors into modulation of gene transcription and kinase activation. However, the significance of TRAF5 in T cell immunity in vivo remains unclear. We hypothesize that TRAF5 is a key mediator of signaling through these receptors and the absence of this molecule will result in defects in T cell expansion due to decreased survival. Infection of mice deficient in TRAF5 with L. monocytogenes resulted in significantly fewer antigen‐specific CD8+ T cells during the primary and secondary response. Memory levels of CD8+ T cells were also reduced. Our results show that this defect is not due to defects in APC function and is T cell intrinsic in nature. Preliminary results suggest that CD8+ T cells from TRAF5 deficient mice are more susceptible to apoptosis following activation.
Vasoactive intestinal peptide (VIP) induces differentiation of unique Th17 cells independently of IL‐6 and IL‐21
Authors: Yadav Mahesh , Rosenbaum Jennifer , Goetzl Edward
Published: June 2021

Vasoactive intestinal peptide (VIP) effects on many immune functions are transduced by VIP G‐protein‐coupled receptors type 1 (VPAC1) and type 2 (VPAC2) on immune cells. The Th17 subset of CD4 T helper cells that develops under the control of transforming growth factor‐beta (TGFβ) and interleukin (IL)‐6 mediates local tissue and systemic inflammation through IL‐17, IL‐6, IL‐21 and IL‐22. We now show that VIP in the presence of TGFβ stimulates development of a distinctive type of Th17 cell that generates IL‐17, but not IL‐6 or IL‐21. That the level of VIP‐induced Th17 cells was higher in VPAC2 knockout mice, whose CD4 T cells express only VPAC1, suggests VPAC1 is the principal transducer of VIP effects. Th17 cells elicited by VIP and by IL‐6 were similar in their capacity to secrete IL‐17 and IL‐22, but only the IL‐6‐elicited Th17 cells secreted IL‐21. Suppression of VIP‐induced Th17 cell differentiation by protein kinase A inhibitors and the induction of similar Th17 cells by phosphodiesterase inhibition supports a critical signaling role for increases in 3′‐5′‐cyclic adenosine monophosphate (cAMP). The novel ability of VIP‐VPAC1 axis signals to evoke IL‐6‐independent development of a distinct type of Th17 cells demonstrates the unique specificity of neuroregulatory mechanisms in the immunological environment
CCR3 chemokine expression is increased by tumor necrosis factor‐alpha in neuroblastoma cells
Authors: Buels Kalmia S , Jacoby David B , Fryer Allison D
Published: June 2021

Eosinophils cluster around parasympathetic nerves in asthmatic lungs and in antigen challenged animals where they mediate airway hyperreactivity. CCR3 receptors are found on eosinophils and blocking them with antagonists prevents airway hyperreactivity in antigen challenged guinea pigs. To determine what CCR3 chemokines are made by nerves during an inflammatory response, we treated human neuroblastoma cells (SK‐N‐SH) with a cytokine that is increased in asthma, tumor necrosis factor‐alpha (TNFγ; 3 to 3000 U/mL), for 24 h. We screened mRNA (by real‐time PCR) and soluble proteins (by ELISA) for known CCR3 chemokines including CCL5 (RANTES), CCL7 (MCP‐3), CCL8 (MCP‐2), CCL11 (eotaxin‐1), CCL13 (MCP‐4), CCL15 (MIP‐1d), CCL24 (eotaxin‐2), CCL26 (eotaxin‐3), and CCL28 (MEC). We found that SK‐N‐SH cells expressed mRNA for CCL5, CCL7, CCL8, and CCL11. TNFα increased mRNA expression for these same chemokines in a concentration dependent manner. TNFα also increased protein expression for three of these chemokines (CCL5, CCL7, and CCL11). Thus, neuroblastoma cells express a subset of CCR3 chemokines that can be increased by TNFα. These data suggest a mechanism whereby neurons can actively recruit eosinophils that can subsequently mediate neuronal hyperreactivity in asthma.

Funded by: NIH: GM071338‐02, HL54659, HL55543, ES014601
CD4+ CD25+ Foxp3+ regulatory T cells induce cytokine deprivation apoptosis of the effector CD4+ T cells
Authors: Pandiyan Pushpa , Lenardo Michael
Published: June 2021

A key question in mammalian immunology is how CD4+CD25+Foxp3+ regulatory T cells (Tregs) suppress immune responses. Here we show that Tregs induce apoptosis of effector CD4+ T cells in vitro and in a mouse inflammatory bowel disease (IBD) model without affecting their early activation or proliferation. They also induced Foxp3 expression in Tresps that resisted death in vitro. Common gamma chain cytokines reversed Treg‐induced apoptosis. Death required Bcl‐2 interacting mediator of cell death (BIM) and Akt1/Protein Kinase‐B induced dephosphorylation of the bcl‐2 antagonist of cell death (BAD) protein. Thus, cytokine deprivation induced apoptosis is a prominent event in Treg‐inhibition of T cell responses.

This work was supported by the intramural research program of NIAID, NIH. P.P is supported by a National Academy of Sciences/National Research Council fellowship.
Interleukin 15 signal negatively regulates Dendritic Cell Activity in contact hypersensitivity
Authors: He Donggou , Li Hui , Wu Lizhi , A Craig
Published: June 2021

Interleukin‐15 (IL‐15) plays important roles in the regulation of immune responses. In IL‐15 knockout (IL‐15−/−) mice, the activation and function of NK and CD8+ T cells are impaired whereas CD4+ T cells are less affected. Evidence indicates that dendritic cells (DC) is able to produce IL‐15 and that DC derived IL‐15 is required for the stimulation of NK and T cells. However, the effect of IL‐15 on DC cells is less clear. We show here that IL‐15−/− DC are more active than wild type DC in the activation of CD4+ T cells and induction of contact hypersensitivity responses (CHS). IL‐15−/− or IL‐15 receptor knockout (IL‐15R−/−) DC produced a lower level of IL‐12p70 and IFN‐g than wild type DC, as reported in previous studies. However, we found that they also produced a higher level of IL‐12 p40 and IL‐23 and a lower level of IL‐10 than wild type DC. Addition of exogenous recombinant IL‐15 in cultures reduced the production of IL‐12p40 and IL‐23 but increased IL‐12p70, IFN‐g and IL‐10 by IL‐15−/− but not IL‐15R−/− DC. Adoptive transfer of hapten labeled IL‐15−/− or IL‐15R−/− DC into naïve wild type mice induced a higher level of CHS than wild type DC. IL‐15−/− DC that were treated with IL‐15 induced a similar level of CHS as wild type DC. Similarly, IL‐15−/− DC stimulated higher levels of IFN‐g and IL‐17 production by hapten primed T cells and OT‐II cell proliferation in cultures than wild type DC. This effect was abrogated by pre‐treatment of IL‐15−/− DC with IL‐15. The data provide insights into a novel mechanism for IL‐15 mediated regulation of immune responses. It implicate that IL‐15 increases the production of immune suppressive cytokines IL‐10 and inhibits the function of DC in the stimulation of T cells and induction of immune responses.
POLYMORPHISM OF THE HUMAN TNF PROMOTER REGION IN BRAZILIAN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS.
Authors: Pancoto João Alexandre Trés , Mendes Junior Celso T. , Lucisano‐Valim Yara M. , Dias‐Baruffi Marcelo , Donadi Eduardo A.
Published: June 2021

INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by multisystemic organ involvement, polyclonal B‐cell activation and the production of autoantibodies. Several family studies have shown convincing evidences of multiple genetic loci associated with SLE development. TNF is a potent proinflammatory cytokine that plays an important role in the pathogenesis of SLE, and certain alleles of the tumor necrosis factor (TNF) gene have been consistently associated with the disease, particularly the ‐308‐A/G functional promoter polymorphism. In the present study we analyzed the allelic distribution of TNF‐308A/G in Brazilian SLE patients.

METHODS AND RESULTS: A total of 148 SLE patients and 130 matched healthy controls were typed for the TNF‐308A/G polymorphism by polymerase chain reaction using allele‐specific oligonucleotide primers. A significant increase of the TNF G/A 308 genotype was observed in SLE patients when compared with healthy controls (p < 0.0001; OR = 7.156; 95% I. C.: 3.462–14.792).

CONCLUSIONS: Since the Brazilian population is highly genetically diverse, the results reported here are in agreement with those reported for other populations, and corroborate the role of TNF in disease pathogenesis and the TNF‐308A/G genotype, which has been associated with high production of TNF, as conferring susceptibility to the disease.

RESEARCH SUPPORT: CAPES
Spinal glial modulation of neuropathic pain using a Spared Nerve Injury model in the mouse
Authors: Nguyen Thao , Vallejo Ricardo , Benyamin Ramsin , Kramer Jeffery , Vogel Laura A.
Published: June 2021

Neuropathic pain presents a clinical challenge because there are few optimal therapeutic treatments available. The mechanisms of neuropathic pain must be elucidated to provide a foundation for improved pain therapy. Recent evidence implicates spinal cord microglia as a potential cellular modulator of neuropathic pain. We hypothesized that inhibiting microglial activation would decrease pro‐inflammatory cytokine production and alter the neuropathic pain symptom of allodynia in animals with peripheral nerve injury. Animals were treated with propentofylline (PPF; 10 mg/kg/day intraperitoneal injection), an inhibitor of microglial activation, or saline for 14 days starting on day 0. On day 7, animals underwent a sham surgery or Spared Nerve Injury (SNI). Spontaneous pain and cold allodynia behaviors were measured on multiple days post‐surgery. On day 14, lumbar spinal cord tissue was harvested for molecular analysis. RT‐PCR was performed to measure mRNA levels of pro‐inflammatory cytokines TNF‐α and IL‐6 and the microglial activation marker ITGAM. Expression levels of ITGAM, TNF‐α and IL‐6 were decreased in animals administered with PPF. Likewise, spontaneous pain and cold allodynia were attenuated for PPF‐treated animals. The effect of propentofylline on neuropathic pain appears to be a potential tool to examine the mechanism for neuropathic pain. Funded by the Millennium Pain Center.
IL13 Gene Variants Influence Early Life Weight Gain in Breastfed Male Infants
Authors: Lohman Carla , Stern Debra , Klimecki Walt , Vercelli Donata , Wright Ann , Halonen Marilyn
Published: June 2021

Rationale: Although IL‐13 has been described mainly as an immune factor involved in Th‐2 related events, there are a few studies suggesting that this cytokine may have roles beyond this context. In the context of studies suggesting an involvement in mammary gland development, we hypothesized that IL13 gene variants might differentially affect growth of breastfed infants.

Methods: Children were enrolled in the population based Tucson Infant Immune Study. Weight and type of infant feeding assessed prospectively from birth. Genotypes were determined with high‐throughput Taqman® assays for IL13 SNPs +2044 G/A (rs20541). Analyses were limited to breastfed non‐Hispanic White children. Non‐parametric tests were used to assess differences between groups.

Results: Male breastfed infants homozygous for GG at +2044 were heavier than those with other alleles (homozygous or heterozygous for the A allele) in the first month of life (p=0.010). This IL13 gene variant did not influence birth weight. Calculating both weight gain and rate of weight gain also revealed significant increases for +2044 GG homozygote males (p=0.007 and 0.010, respectively). Female infants did not show these weight‐IL13 gene relations.

Conclusion: IL13 genotypes appear to influence growth in early infancy among breastfed male but not female infants.

Funded by: NIH grants AI 42268 AI 61811, HL66391
Gene Sequence Homology of Chemokines Across Species
Authors: LaBresh Joanna W. , Steffens Carrie S. , Olson Erik , Baldwin Cynthia , Lunney Joan
Published: June 2021

The abundance of expressed gene and protein sequences available in the biological information databases facilitates comparison of protein homologies. A high degree of sequence similarity typically implies homology regarding structure and function and may provide clues to antibody cross‐reactivities among species. We compared the predicted soluble portions of chemokine sequences across eight species (human, mouse, rat, canine, feline, bovine, swine, and equine) in order to aid in protein expression and hybridoma production. The chemokine family has approximately 50 small (8–10 kDa) secreted pro‐inflammatory activation‐inducible cytokines involved in a variety of immune responses, acting primarily as long distance chemoattractants and activators of specific types of leukocytes. The chemokine family is further subdivided into four subfamilies: C; CC; CXC; and CX3C based on the position of conserved cysteine residues involved in intramolecular cysteine bonds. Chemokines were assessed because of their importance in inflammation, disease and stress responses.
Macrophage Activation by TNF alpha in Nasal Mucus of Patients with Chronic Rhinitis
Authors: Schmidt Loren Janes , Velicu Irina , Henkin Robert I.
Published: June 2021

The role of TNF alpha in inflammation has not been well defined. To evaluate this phenomenon we investigated relationships between various cytokines and infection by comparing levels of TNF alpha, IL‐1 alpha and beta, and IL‐2R in blood, urine, saliva and nasal mucus of patients with and without chronic nasal infection. Chronic nasal infection was observed in the infected group by the presence of frank nasal pus. In patients with chronic nasal infection, nasal mucus TNF alpha was eight times higher and IL‐2R was six times higher than these levels of infected patients while IL‐1 alpha and beta levels were not significantly different between the two groups. Correlation between the cytokines showed an antagonistic relationship in nasal mucus in chronically infected patients between TNF alpha and T‐cell activation as measured by IL‐2R (R = −.77) but no such relationship existed in uninfected patients. This relationship was apparent only in nasal mucus since IL‐2R levels in both plasma and urine were strongly positively correlated with nasal mucus TNF alpha (R = .92 and R = .91 respectively). These data suggest that during chronic nasal infection macrophage activation is highly up‐regulated leading to T‐cell activation and then migration out of the site of infection as activated macrophages combat the infection and promote T‐cell activation.
Combination of multiple genes from lupus and diabetes prone mice leads to a novel mouse model for lupus
Authors: Sim Davis Lok , Bagavant Harini , Ge Yan , Gaskin Felicia , Fu ShuMan , Deshmukh Umesh S
Published: June 2021

The multigenic nature of systemic lupus erythematosus (SLE) is responsible for the varied serological and clinical manifestations observed in patients. Depending on the genotype, spontaneous mouse models of lupus only recapitulate certain features of the disease. This study was undertaken to test the hypothesis, that a mouse with a combination of most autoimmune genes would develop lupus similar to that seen in patients. For this purpose we crossed the lupus‐prone NZM2328 mice with the diabetes‐prone NOD mice and investigated the female F1 mice for autoimmunity.

While the female F1 mice did not develop any diabetes, they developed severe proteinuria with >80% incidence by 10 months of age. Kidneys showed glomerular IgG and C3 deposits, with onset of acute proliferative GN between 3–5 months and chronic GN by 7 months of age. All F1 females developed ANA and anti‐dsDNA antibody. Also, sera from 70% of F1 mice immunoprecipitated the snRNP particle compared to only 20% NOD mice and 0% NZM2328 mice. The F1 mice showed elevated expression of type 1 interferon (IFN) inducible genes, in comparison to the NOD mice.

Our study demonstrates that combination of multiple autoimmune genes from the NZM2328 and NOD mice results in a novel spontaneous mouse model for lupus nephritis. The disease phenotype in this model represents a large fraction of the human SLE patients.
Bone marrow cell intrinsic defect drives autoimmunity in New Zealand Black chromosome 13 congenic mice
Authors: Loh Christina M , Pau Evelyn , Chang Nan‐Hua , Wither Joan E
Published: June 2021

Introgression of a New Zealand Black (NZB) chromosome 13 interval onto a lupus‐resistant B6 background (denoted, c13) is sufficient to produce many of the hallmarks of lupus, including, high titre anti‐chromatin antibody (Ab) production, abnormal T cell activation, and renal disease. These phenotypes occurred in the presence of a dendritic cell (DC) expansion. In this follow‐up study, we sought to localize and characterize the immune defects leading to these pathogenic abnormalities; this was achieved by generating subcongenic mice and bone marrow (BM) reconstituted mice, respectively. Subcongenic mouse strains, c13(47–89 Mb), c13(73–114 Mb), and c13(81–114 Mb), all demonstrated low titre anti‐chromatin Ab production, abnormal T cell activation, and DC expansion. Lethally irradiated B6.Thy1aIgHa mice were reconstituted with BM cells from B6.Thy1aIgHa, c13, or a mixture of both. Only mice reconstituted with c13 BM cells developed the aforementioned phenotypes. Moreover, in mixed BM chimeras, these abnormalities were seen in both B6.Thy1aIgHa‐ and c13‐derived cells. Thus, this study shows that a BM cell‐intrinsic defect that is not cell‐autonomous is driving the c13 mouse phenotype. Furthermore, this defect can be localized within the overlapping 81–89 Mb subcongenic chromosome 13 interval. Source of research support: The Arthritis Society and Canadian Institutes of Health Research.
Development of Collagen II (CII)‐induced Arthritis Was Associated with High AID and IL‐17 Expression in BXD2 Mice
Authors: Zang Song , Hsu Hui‐Chen , Yang PingAr , Wu Qi , Job Godwin , Guentert Tanja , Nandakumar Kutty Selva , Holmdahl Rikard , Mountz John D.
Published: June 2021

BXD2 mice develop spontaneous arthritis associated with increased activation‐induced cytidine deaminase (AID), development of pathogenic autoantibodies, and increased TH‐17 T cells. CII arthritis (CIA) was induced to determine the relative contribution of autoantibodies and IL‐17 to the development of arthritis. BXD2 mice with a dominant negative AID transgene (BXD2 AID‐DN Tg) were produced. Autoantibodies for CII and specific triple helical CII epitopes (U1, J1, C1) were determined by isotype‐specific ELISA. Expression of Il17 and Il17r mRNA was determined by RT‐PCR and immunohistochemical staining. Despite the heritage of a CIA‐resistant H2b MHC haplotype, BXD2 mice developed accelerated and more severe CIA compared to DBA/1 mice. Both BXD2 and DBA/1 mice developed significantly higher levels of antibodies to C1, U1, and CII compared to B6 mice (p<0.01). BXD2 mice had an increased T‐ and B‐cell activation after CII injection, and T cells from BXD2 mice exhibited a significantly elevated IL‐17 production, compared to DBA/1 and B6 mice. Anti‐CII antibodies were associated with increased TH‐17 in the spleens and increased IL‐17R on GC B cells in BXD2 mice. The presence of AID‐DN Tg significantly suppressed the development of these autoantibodies. Our results suggest that IL‐17 acting on IL‐17R+ B cells in the spleens may be a novel mechanism for induction of AID leading to the development of CII‐specific antibodies and acceleration of CII arthritis. This work is supported by an NIH T32 Award, VAMC Merit Review Award, American College of Rheumatology, Arthritis Foundation, and Daiichi‐Sankyo Co., Ltd.
Activation of Autoreactive B cells by Interaction with CpG‐stimulated Plasmacytoid Dendritic Cells
Authors: Ding Chuanlin , Yan Jun
Published: June 2021

Plasmacytoid dendritic cells (pDCs), specialized type I interferon (IFN) producers, play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies have demonstrated that pDCs could induce B cells to differentiate into plasma cells via secretion of type I‐IFN and IL‐6. In this study, we aimed to investigate whether pDCs could directly or indirectly modulate autoreactive anti‐small nuclear ribonucleoprotein particle (snRNP) B cell responses. In vitro studies demonstrated that pDCs synergistically up‐regulated anti‐snRNP B cell proliferation, cytokine secretion such as IFN‐γ and IL‐6, and autoAb production upon TLR‐9 agonist CpG stimulation. Neutralization of IFN‐α and IL‐6 with mAbs partially abrogated the enhanced autoreactive B cell proliferation and autoAb production mediated by pDCs. Strikingly, in vitro transwell study indicated that these effects required a direct cell‐to‐cell contact between pDCs and autoreactive B cells. The enhanced B cell proliferation and autoAb production mediated by pDCs were also dependent on the activation status of pDCs since freshly isolated pDCs did not have such function. Further studies demonstrated that CD70, but not CD40L, appeared to participate to some extent in this interaction. Thus, pDC‐mediated enhanced autoreactive B cell activation is dependent both on cell‐to‐cell contact and on the release of soluble cytokines.
Hierarchical requirement for CpG Motifs in dendritic cell activation induced by DNA‐containing immune‐complexes
Authors: Yasuda Kei , Uccellini Melissa B. , Richez Christophe , Viglianti Gregory A , Akira Shizuo , Marshak‐Rothstein Ann , Rifkin Ian R.
Published: June 2021

In systemic lupus erythematosus (SLE), immune complexes (ICs) containing mammalian DNA activate dendritic cells (DC) at least in part through Toll‐like receptor 9 (TLR9). However, the DNA sequence required for this TLR9 activation is unclear because, unlike bacterial or viral DNA, mammalian DNA contains very few of the classical unmethylated CpG motifs believed to be important for TLR9 activation. To address this question, we treated mouse DC with immune complexes containing DNA fragments of defined sequence, including sequences obtained from CpG islands within the mammalian genome, and measured cytokine production and co‐stimulatory molecule upregulation. We found that although the strongest activation was elicited by classical unmethylated CpG motifs, effective activation was also elicited by methylated classical CpG motifs and by unmethylated non‐classical CpG motifs. The activation was entirely TLR9‐dependent. In contrast, no activation was elicited by DNA lacking CG sequences. Overall, the data indicate that IC containing DNA sequences found in mammalian DNA are able to activate DC, providing a possible explanation for how DNA‐containing IC might cause immune system activation in patients with SLE.

This research is supported by a NIH (NIAMS) program project grant.
Monocyte STAT5 Dysfunction in Human Autoimmune Diabetes
Authors: Garrigan Erin , Shi Jingda , Wasserfall Clive , Atkinson Mark , Clare‐Salzler Michael , Litherland Sally A
Published: June 2021

Myeloid antigen presenting cells fail to maintain T cell tolerance and regulate inflammatory responses in autoimmune type 1 diabetes (T1D). Cytokine signaling during myeloid cell differentiation and in inflammatory activation of mature monocytes and macrophages via Signal Transducer and Activator of Transcription (STAT) 5 is dysfunctional in T1D, leading to persistent STAT5 phosphorylation and DNA binding, abnormal expression of prostaglandin synthase 2 (PGS2/COX2), and overproduction of GM‐CSF. NOD mouse sequence analysis revealed polymorphisms in epigenetic regulatory regions controlling expression of the genes encoding GM‐CSF (Csf2, Idd4.3) and PGS2/COX2 (Ptgs2, Idd5). Congenic ChIP analyses confirmed that NOD STAT5 binding at both regions is enhanced, and disrupts auto‐feedback regulation of GM‐CSF and GM‐CSF‐induced Ptgs2 expression. Human monocyte analysis shows a 3‐fold difference in mean STAT5 phosphorylation in T1D subjects compared with controls (p=0.0066, controls 3.689±4.074, T1D 12.69±11.82). Age and gender appear to affect this biomarker, with younger female subjects exhibiting the highest unactivated monocyte STAT5 phosphorylation. Monocyte STAT5 phosphorylation, alone or in concert with GM‐CSF and PGS2/COX2 expression and/or genetic polymorphisms in Csf2/Ptgs2 regulatory regions, may be useful as a biomarker for TID risk.
Type I IFN‐mediated Inhibition of Inflammatory Th cell Responses by a Subset of SLE Patient Sera
Authors: Mangini Alyson J. , Nagai Taro , Ozonoff Al , Fritzler Marvin J. , Lafyatis Robert A. , van Seventer Jean Maguire
Published: June 2021

Inflammatory, IFN‐γ‐secreting Th1 cells are implicated in mediating serious forms of systemic lupus erythematosus (SLE), including nephritis and CNS lupus. In SLE, triggering of plasmacytoid DC (pDC) Toll‐like receptors (TLRs) by circulating anti‐nucleic acid‐containing autoimmune complexes stimulates pDC secretion of high levels of type I interferon (IFN) (IFN‐α/β). Study of both human and murine lupus disease strongly implicates these IFNs as key disease effectors. However, the role of pDC‐derived type I IFN in regulating the function of inflammatory Th cells in SLE is unknown. We and others have shown that, although classically considered to promote Th1 cell mediated inflammation, type I IFN can also function as a potent inhibitor of both inflammatory Th1 and Th17 responses. We employed the pan‐type I IFN neutralizing reagent, B18R, to investigate how type I IFN regulate Th cell responses in SLE. Recent observations indicate a subset of SLE patient sera that inhibits IFN‐γ secretion by superantigen‐stimulated healthy donor PBMC in a type I IFN‐dependent manner. This effect positively correlates with PBMC secretion of the Th cell inflammatory cytokines, LT and IL‐17, while negatively correlating with IL‐10 secretion. Remarkably, the ability of SLE patient serum to inhibit IFN‐γ secretion in a type I IFN dependent manner correlates with positive serum Sm and RNP titers. Our findings suggest that in SLE patients with significant serum type I IFN activity, type I IFN blockade may exacerbate Th cell‐mediated autoimmune inflammation.
Intravenous immunoglobulins (IVIg) inhibit antigen presentation in vitro via an FcgammaR‐independent mechanism.
Authors: Aubin Eric , Paquin‐Proulx Dominic , Trepanier Patrick , Roberge Chantal , Lemieux Real , Bazin Renee
Published: June 2021

Intravenous immunoglobulins (IVIg) are used to treat a variety of autoimmune diseases, but their mechanism of therapeutic action remains unclear. It has been observed that IVIg could reduce autoantibody titers in treated patients, suggesting that IVIg may inhibit the ongoing and future production of pathogenic autoantibodies. Several studies also suggested that the anti‐inflammatory effects of IVIg were mediated by either inhibitory or activating Fc gamma receptors (FcγRs). In the present work, we have studied the possibility that IVIg interfere with the process of antigen presentation by APC to T cells which is essential for antibody production in a sustained immune response, and whether this effect could be FcγRs‐dependent. Using an in vitro antigen presentation assay with different types of murine APC, we observed that IVIg inhibited OVA presentation by more than 50%. Surprisingly, IVIg still significantly inhibited OVA presentation by dendritic cells isolated from mice deficient in either inhibitory FcγRIIb or activating FcγRIII (gamma chain deficient), suggesting that IVIg inhibit antigen presentation by an FcγR‐independent mechanism. In addition, we showed that this inhibition may not be dependent on a modulation of expression of co‐stimulatory molecules such as CD80, CD86 or MHC‐II expression. We are currently investigating other mechanisms by which IVIg could interfere with antigen presentation.