bluuebunny/crossref_metadata_2025 huggingface dataset:
Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use
Authors: Sun Jing , Li Ning , Oh Kyu-Seon , Dutta Bhaskar , Vayttaden Sharat J , Lin Bin , Ebert Thomas S , De Nardo Dominic , Davis Joie , Bagirzadeh Rustam , Lounsbury Nicolas W , Pasare Chandrashekhar , Latz Eicke , Hornung Veit , Fraser Iain
Published: January 2023

Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)–based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor–associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and pro inflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.
Pattern recognition channels in anti-viral host defense in humans and an insect vector
Authors: Carrithers Michael D
Published: January 2023

A novel mechanism of innate immune signaling is proposed. The central hypothesis is that viral-associated molecular patterns directly activate intracellular splice variants of voltage-gated sodium channels to increase transcription of anti-viral genes. Past work revealed that splice variants of human voltage-gated sodium channels are expressed intracellularly on endosomes and contain exon deletions that alter channel kinetics.

Here it is shown that intracellular dsRNA activates the human macrophage SCN5A variant to initiate a signaling cascade that leads to increased transcription of type I interferons and SP100-related genes. This pathway is regulated by sequential downstream activation of a calcium-dependent adenylate cyclase, adcy8, and the cyclic AMP-dependent transcription factor ATF-2. Knockdown of channel expression in human macrophages reduces the host response to HSV-1.

Additional work reveals expression of an evolutionarily related sodium channel gene, AAEL006019, in Aedes aegypti, the yellow fever mosquito. This intracellular channel contains an alternative exon deletion that reduces peak current but does not alter ion selectivity. In contrast to human macrophage SCN5A, the AAEL006019 channel is activated by mimics of virally-derived ssRNA, but not dsRNA. Channel activation increases transcription of mediators of insect innate immunity, RelA and Ago2.

These data suggest that channel variants initiate anti-viral responses independently of other pattern recognition receptor pathways. It is postulated that pattern recognition by channel variants are an evolutionarily conserved mechanism of innate immune signaling that reflect a complex interplay between human host, vector, and virus.
Isoforms of ADAR1 independently control MDA5-driven autoimmunity and multi-organ development
Authors: Pestal Kathleen , Funk Cory C , Snyder Jessica M , Price Nathan D , Treuting Piper M , Stetson Daniel B
Published: January 2023

Mutations in the ADAR gene that encodes the ADAR1 RNA editing enzyme cause Aicardi-Goutières Syndrome (AGS), a severe autoimmune disease associated with an aberrant type I interferon response. How ADAR1 prevents autoimmunity remains incompletely defined. Here, we demonstrate that ADAR1 is a specific and essential negative regulator of the MDA5-MAVS RNA sensing pathway. Moreover, we uncovered a MDA5-MAVS-independent function for ADAR1 in the development of multiple organs. We showed that the p150 isoform of ADAR1 uniquely regulated the MDA5 pathway, whereas both the p150 and p110 isoforms contributed to development. Abrupt deletion of ADAR1 in adult mice revealed that both of these functions were required throughout life. Our findings delineate genetically distinct roles for both ADAR1 isoforms in vivo, with implications for the human diseases caused by ADAR mutations.
Elucidating regulatory mechanisms of RAG2 in response to DNA damage.
Authors: Byrum Jennifer , Rodgers William , Rodgers Karla
Published: January 2023

V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. Live cell imaging reveals export of RAG2 within minutes of DNA damage, demonstrating a rapid response to genotoxic stress. Intriguingly, dose response assessment exhibited RAG2 export following the generation of very few DNA DSBs, indicating that DNA damage-triggered export of RAG2 can occur under normal physiological conditions. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr490. In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulation of cellular responses to DSBs in developing lymphocytes.
Relative contribution of medullary thymic epithelial cells and dendritic cells to thymocyte central tolerance as assessed by multi-photon microscopy
Authors: Ehrlich Lauren Ilyse Richie , Lancaster Jessica N , Hu Zicheng , Tran Andy , Sasiponganan Chayanit
Published: January 2023

Following positive selection, thymocytes migrate into the medulla where they encounter diverse tissue-restricted antigens (TRAs) that induce self-tolerance. Both medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present TRAs to induce negative selection of autoreactive thymocytes, but their relative contribution remains unresolved. Previous studies using genetic models to impair antigen presentation by mTECs or DCs demonstrated both cell types could induce negative selection. However, because interactions between thymocytes and stromal cells maintain a properly differentiated stromal compartment, these studies may not reflect physiologic interactions or relative contributions of mTECs and DCs when both are intact. Thus, we developed a 2-photon microscopy approach to directly observe interactions between thymocytes and DCs or mTECs during negative selection, while monitoring T cell receptor (TCR) activation. Despite earlier studies indicating DCs are required for negative selection to a soluble TRA, we find that mTECs and DCs contribute equivalently to TCR stimulation of CD4 and CD8 single positive (SP) thymocytes responding to membrane-bound or secreted TRAs. Interactions with mTECs and DCs generally accounted for all TCR activation events; however, a significant fraction of CD4SP activation events were not accounted for in response to a soluble TRA, suggesting presentation by other medullary stromal subsets. Initial analysis of polyclonal thymocytes also indicates equal contributions of mTECs and DCs to medullary TCR activation. In summary, mTECs and DCs contribute equivalently to presentation of soluble and membrane-bound mTEC-expressed TRAs on MHC-I and MHC-II during negative selection.
The role of nutrients in B lymphocyte growth and survival responses.
Authors: Heyse Shannon A , Connolly Timothy , Chiles Thomas Crane
Published: January 2023

B cell activation is an energetically demanding process during which B lymphocytes shift from a resting state to a proliferative, metabolically active state. Little is known about the metabolic reprogramming process or the role extracellular nutrients play in the activation response. We demonstrate that there are distinct requirements for the nutrients L-glutamine and glucose during activation. We show that cells activated in glucose-depleted conditions are still able to undergo growth and signaling events. In contrast, we show that extracellular L-glutamine is essential for all but the earliest activation events, and cells cultured in L-glutamine-deprived conditions are unable to enter the cell cycle. Consistently, we show that extracellular supplementation of the cell-permeable derivative of a-ketoglutarate (a-KG) is able to rescue cell activation in the absence of glutamine. We also show the induction of the high affinity amino acid transporter ASCT2 is required for glutamine uptake following BCR crosslinking. Specifically, we found that halting glutamine uptake or processing by inhibiting ASCT2 or the glutaminolytic enzyme glutaminase causes activation defects that parallel those observed in glutamine deprived conditions, indicating a requirement for glutaminolysis during the very early stages of activation. Finally, we found that a-KG does not contribute to epigenetic remodeling, but is necessary for mTORC1 activation. In turn, mTORC1 activity is required for upregulation of the glucose transporter Glut1 during activation, as well as increased glucose uptake. These findings indicate a distinct metabolic profile that begins with glutamine uptake, and acts through mTORC1 signaling to later promote glucose uptake.
The effect of sex on immune cells in healthy aging: elderly women have more robust natural killer (NK) lymphocytes than do elderly men
Authors: Lutz Charles T , Al-Attar Ahmad , Presnell Steven R. , Peterson Charlotte A. , Thomas D. Travis
Published: January 2023

Gender differences in blood leukocytes and immune responses have been reported, but there is limited knowledge of gender differences in elderly humans. We compared monocyte and lymphocyte subsets, along with soluble immune mediators in healthy men and women over the age of 70. We also measured natural killer (NK) lymphocyte cytotoxic granule exocytosis, chemokine synthesis, and cytokine synthesis in response to a variety of stimuli. We found that elderly women had significantly more circulating B cells than men, whereas men had more CD4 central memory T cells. Men also had higher monocyte levels. Women had a higher ratio of immature CD56bright NK cells to mature CD56dim NK cells, indicating a gender difference in NK cell maturation in the elderly. Moreover, compared with NK cells from men, mature NK cells from women had more vigorous cytotoxic granule responses to K562 leukemia cells and IFN-g responses to NKp46 crosslinking. Female mature NK cells were also more likely to produce MIP-1b than male mature NK cells in response to a variety of stimuli. We also measured soluble factors that affect immune cells or are produced by immune cells. Plasma adiponectin levels were higher in women, plasma levels of retinol-binding protein 4 were higher in men, but there was no significant gender difference in C-reactive protein, IL-15, or sphingosine-1-phosphate. These data support the hypothesis that gender influences immunity in general, and NK cell activity in particular, in elderly humans.
Increased antioxidant activity mitigates age-associated dysfunction of thymic stromal cells in aged mice.
Authors: Cepeda Sergio , Xiao Yangming , Venables Thomas , Griffith Ann
Published: January 2023

Age-associated thymic atrophy results in diminished production of new T lymphocytes and a concomitant decrease in responsiveness to new pathogens and vaccines. In addition to loss of thymic size with age, we have shown that critical stromal functions, including tissue-restricted antigen (TRA) expression, are diminished with age. We previously identified deficiency of the hydrogen peroxide quenching enzyme catalase (CAT) in thymic stromal cells as a key cause of thymic atrophy during aging, and established that thymic atrophy can be mitigated by genetic or dietary complementation of catalase antioxidant activity. Here, we find that in addition to maintaining thymic mass with age, long-term increases in catalase activity may mitigate age-associated loss of stromal function. Our preliminary studies indicate that life-long overexpression of catalase in mitochondria of transgenic mice (mCAT Tg) results in increased TRA expression, mitigated acquisition of an aged global gene expression signature, and increased maintenance of cortico-medullary organization in aged mCAT Tg mice relative to non-transgenic littermates.
The role of Indoleamine 2,3-dioxygenase (IDO) in the survival of bone marrow resident long lived Plasma cells
Authors: Maharaj Shivana M , Nair Jayakumar R , Carlson Louise M , Lee Kelvin P
Published: January 2023

Interactions within the bone marrow microenvironment provide critical survival signals to normal resident long lived plasma cells (LLPC). Previous work in our lab has shown that CD28 is required for LLPC survival, and we know that CD28 back signaling in to CD80/86 induces IDO production from DCs within the bone marrow microenvironment. IDO is classically known to contribute to an immunosuppressive environment (specifically with respect to T cell activity) in multiple myeloma, giving these cells the ability to survive within the bone marrow. However, to our great surprise previous results demonstrate that IDO is required for durable specific antibody responses in a normal bone marrow microenvironment. Thus we hypothesize that IDO has an additional role acting as a factor to maintain the bone marrow niche for long lived plasma cell survival, and sustained antibody production. Here we show that human myeloid derived DCs co-cultured with plasma cell lines produce IDO in vitro. In vivo models were also used, including the IDO knockout mouse model to analyze their LLPCs and the bone marrow compartment. A decrease of plasma cells in the bone marrow from wild type mice compared to IDO knock out mice was observed. These findings indicate that plasma cells can interact with dendritic cells within the bone marrow to produce IDO, and that a lack of IDO leads to a decreased number of these cells. Due to IDO deficient mice being unable to maintain long term antibody production, we propose a model wherein CD28 signaling induces IDO production, which plays a role in providing a supportive niche within the bone marrow for long lived plasma cell survival and antibody production.

Supported by: R01CA121044-08
Impaired metabolic reprograming in antigen-activated recent thymic emigrants
Authors: Cunningham Cody Alan , Fink Pamela J
Published: January 2023

Recent thymic emigrants (RTEs) are the youngest peripheral T cells that have completed thymic selection and egress to the lymphoid periphery. However, T cell maturation does not end in the thymus; rather, RTEs require 2 – 3 weeks in the lymphoid periphery to achieve functional maturity and enter the mature but naïve (MN) T cell pool. Following antigen encounter, RTEs are functionally distinct from MN T cells as they exhibit dampened proliferation, altered cytokine production, and increased expression of some anergy-associated genes. RTEs are a clinically important population as these cells contribute significantly to the T cell pool in neonates and adults recovering from HIV infection or chemotherapy. Here we show that, compared to MN T cells, RTEs are impaired in their ability to switch from oxidative phosphorylation to aerobic glycolysis following antigenic stimulation. This impaired metabolic reprograming contributes to the reduced proliferation and cytokine production observed in antigen-activated RTEs. This failure to undergo antigen-induced metabolic reprograming is associated with reduced expression of the transcription factor Myc. Critically, high levels of IL-2 are able to rescue the expression of Myc in RTEs resulting in proper metabolic reprograming that mimics their mature counterparts. These results suggest that an altered metabolic phenotype underlies the functional differences observed between RTEs and MN T cells and highlights a novel point for therapeutic intervention for neonates and individuals recovering from lymphoablation.
The histone lysine methyltransferase G9a maintains the transcriptional profile of mature CD8 T cells
Authors: Verbaro Daniel , Kim Byungil , Chou Chun , Shinkai Yoichi , Egawa Takeshi
Published: January 2023

Lineage identities of developing lymphocytes are established by both activation of lineage specific genes and repression of alternative lineage genes. The latter process depends on active gene repression by continued expression of transcriptional repressors or epigenetic silencing mediated by methylation of cytosine on DNA and lysine residues in histone tails, including Lys9 (K9) of histone H3. Here we show that the histone H3K9 methyltransferase G9a is essential for silencing of helper lineage-related genes in mature CD8+ T cells. Despite the development of normal CD4− CD8+ T cells, a proportion of G9a-deficient CD8+ T cells, when forced to divide with anti-CD3e and anti-CD28 stimulation, depressed surface CD4 in vitro. Furthermore, a substantial fraction of G9a-deficient CD8+ T cells carrying an OT-I T cell receptor derepressed surface CD4 during homeostatic proliferation following an adoptive transfer to lymphopenic host mice. These results suggest that G9a establish stable silencing of alternative lineage genes in dividing mature CD8+ T cells. We are currently conducting gene expression analysis to assess the requirement for G9a in establishment of the lineage identity of CD8+ T cells at a genome-wide level.
A pilot study to determine the prevalence of human rat lungworm (Angiostrongylus cantonensis) exposure on Hawaii Island
Authors: Jarvi Susan Irene , Quarta Stefano , Eamsobhana Praphathip , Howe Kathleen , Jacquier Steven , McHugh Robert , Kramer Kuilei , Meyers McKayla
Published: January 2023

The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that is the cause of human eosinophilic meningitis and ocular angiostrongyliasis. It is considered as a global, emerging infectious disease commonly known as rat lungworm disease (RLWD). Rats are the definitive hosts, mollusks are the obligatory intermediate hosts. Humans can become infected by ingesting intermediate hosts containing infective larvae in contaminated food or water. There are currently no diagnostic tests for RLWD available in Hawaii and prevalence of infection is unknown. To estimate prevalence of exposure to RLW parasites, working with Clinical Labs of Hawaii, LLP and Puna Community Medical Center in Pahoa, HI, we collected serum from 436 human volunteers from east Hawaii where most of the cases of RLWD in the USA originate. The presence of RLW antibodies were evaluated by indirect ELISA using crude antigen extracted from adult A. cantonensis nematodes. Of the 360 samples successfully analyzed, 76 (21%) were strongly positive, 166 (46%) weakly positive and 118 (33%) negative. Based on self-reported data, 92 (26%) believed they have or had RLWD, and 15 (4.2%) were definitively diagnosed. 77% of volunteers reported catchment systems as their main source of household water. More studies would need to be completed to evaluate the potential for water transmission of RLWD. Due to the potential for false positives using crude RLW antigen, results will be verified using dot-blots containing a 31 kDa protein which is expected to provide greater specificity.
IFN-γ produced by brain-resident cells is crucial to control cerebral infection with Toxoplasma gondii
Authors: Sa Qila , Ochiai Eri , Tiwari Ashish , Perkins Sara , Mullins Jeremi , Gehman Marie , Huckle William , Eyestone Willard H. , Saunders Thomas L. , Shelton Brent J. , Suzuki Yasuhiro
Published: January 2023

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations including LPS, a component of the outer membrane of Gram-negative bacteria. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii, an obligate intracellular protozoan parasite, using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b+ cells suggested that IFN-γ production by microglia, which is the only CD11b+ cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ production by brain-resident cells is pivotal for upregulating cerebral expression of CXCL9 and CXCL10 chemokines and recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.
Characterization of immunomodulatory cytokine IL-27 in tuberculosis infection.
Authors: Gideon Hannah Priyadarshini , Flynn JoAnne L
Published: January 2023

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the world’s most successful human bacterial pathogen. Lung granulomas are the pathologic hallmark of TB. The cellular components of the granulomas play an important role in containment and progression of Mtb infection. The balance of pro and anti-inflammatory cytokines within granulomas is associated with reduction in bacterial burden. IL-27 is an immunomodulatory cytokine that modulates T cells. The properties of IL-27 as an enhancer or silencer of inflammation are yet to be evaluated in the context tuberculosis in a non-human primate (NHP) model. We used macaques, a NHP model that recapitulates human TB, to characterize the expression and modulatory effects of IL-27, especially at an individual granuloma level. In this preliminary evaluation, we characterized the cell types producing IL-27 in response to Mtb and in granulomas and correlated IL-27 production with pro and anti-inflammatory cytokines such as IFN-g, IL-2, IL-17 and IL-10. In addition we also correlated IL-27 expression with bacterial burden in granulomas. Overall, these data provide insight into immunomodulation of cytokines in tuberculosis.
Oleo-gum-resin suppresses indomethacin-induced elevation of proinflammatory biomarker and gastric injury in rats
Authors: AlSharari Shakir , Al-Rejaie Salim
Published: January 2023

AIM

To investigate the mucosal protective effect and the mechanisms of action of the anti-ulcer drug oleo-gum-resin in gastric injury induced by indomethacin in rats.

METHODS

Gastric mucosal injury was induced in male Wistar rats by oral administration of indomethacin at a dose of 60 mg/kg. One hour before indomethacin treatment, animals were orally pretreated with oleo-gum-resin at doses of 125 mg/kg, 250 mg/kg or 500 mg/kg. Four hours after indomethacin administration, the animals were sacrificed and their stomachs were rapidly removed and processed for the evaluation of gastric mucosal damage and the determination of the concentrations of TNF-α, IL-1β, IL-6, NO, PGE2 and myeloperoxidase (MPO) in mucosal tissues.

RESULTS

Linear hemorrhagic mucosal lesions were observed primarily in the glandular stomach 4 h after oral administration of indomethacin. Pretreatment with oleo-gum-resin markedly reduced the number and severity of gastric lesions in a dose dependent manner. The mucosal concentrations of TNF-α, IL-1β, IL-8, NO, and MPO, which indicates the degree of mucosal infiltration by neutrophils, increased concomitantly with the occurrence of gastric injury in the indomethacin administered rats while PGE2 levels inhibited in this group. Pretreatment with oleo-gum-resin significantly decreased the levels of these inflammatory factors in gastric tissue elicited by indomethacin and enhanced the PGE2 levels.

CONCLUSION

The mucosal protective effects afforded by oleo-gum-resin on gastric injury induced by indomethacin are mediated by inhibition of mucosal proinflammatory cytokine production and neutrophil infiltration, leading to suppression of mucosal inflammation and subsequent tissue destruction.
MAdCAM signaling through integrin α4β7 modulates surface expression of CCR5 and markers of CD4+ T cell activation.
Authors: Perrone Ian , Cicala Claudia , Nawaz Fatima , Ray Jocelyn , Olowojesiku Ronke , Waliszewski Mia , Jelicic Katija , Wei Danlan , Van Ryk Donald , Ansari Aftab Ahmed , Fauci Anthony S , Arthos James
Published: January 2023

Integrin α4β7 mediates homing of lymphocytes, including CD4+ T cells, to gut-associated lymphoid tissue (GALT) through an interaction with Mucosal Addressin Cell Adhesion Molecule (MAdCAM), expressed on gut endothelial venules. MAdCAM facilitates rolling adhesion of CD4+ T lymphocytes along the gut endothelium prior to extravasation. Previous work also indicates that MAdCAM interactions with α4β7 provide a co-stimulatory signal to T cells. α4β7+/CD4+ T cells are a preferred target of HIV during the acute phase of infection. We hypothesized that signaling through α4β7 via MAdCAM renders cells susceptible to HIV infection and replication. In this study we characterized the response of α4β7+/CD4+ T cells to costimulation via MAdCAM in order to better understand whether the induced phenotype would support an enhanced degree of viral replication. We found that CD4+ T cells upregulated CCR5, an HIV coreceptor, in response to MAdCAM costimulation. We also observed the rapid induction of CD69, a known marker of activation. This induction was restricted to α4β7+ cells, including both the CD45RO+ α4β7+ memory subset and CD45ROneg/α4β7+ naive cell subset. We also observed decreased surface expression of two MAdCAM ligands; integrin β7+ was down-regulated within four hours post MAdCAM costimulation and L-selectin was shed with similar kinetics. This shedding occurred primarily in the CD45ROneg subset. Overall these results indicate that MAdCAM-mediated costimulation through α4β7 activates CD4+ T cells in a unique manner that is likely conducive to viral replication. Of note we found that CCR5, the principal HIV coreceptor, was upregulated. Interestingly, the surface expression of two MAdCAM ligands, L-selectin and α4β7, were both reduced.
Regulator of G Protein Signaling 10 is a novel regulator of intestinal inflammation
Authors: Houser Madelyn C , Tansey Malú G
Published: January 2023

Sustained intestinal inflammation has been implicated in the development and progression of numerous diseases, including neurodegenerative disorders such as Parkinson’s disease (PD). Identifying mechanisms that regulate intestinal immune responses can provide insight into the pathogenesis of these disorders and present new therapeutic targets.

Regulator of G protein Signaling 10 (RGS10) is a GTPase-activating protein that alters proinflammatory activity in microglia, macrophages, and T cells and also regulates immune cell chemotaxis. Furthermore, RGS10 is protective in animal models of PD pathology and potentially deficient in human PD patients. These findings and the known involvement of the intestine in PD prompted an investigation of the function of RGS10 in the gut, a system in which it had not previously been examined.

This study determined that RGS10 is expressed in intestinal epithelial cells and at higher levels in intestinal immune cells where it acts as an anti-inflammatory regulator. RGS10-null mice exhibit greater baseline intestinal inflammation, compromised tight junction integrity, and altered microbiota composition with greater abundances of pathobionts compared to WT mice. These changes are physiologically relevant, as RGS10-deficient animals exhibit more severe pathology in response to intestinal injury and inflammation than do their WT counterparts. Levels of α-synuclein – the protein that aggregates in brains and colons of PD patients – are also higher in the intestine of RGS10-null mice, likely as a result of chronic inflammation. This study identifies RGS10 as a novel regulator of mucosal immune function and suggests that reduced levels of this protein may contribute to pathology in human disease.
Innate immune protein lipocalin-2 and iron mitigate EGCG-mediated inhibition of myeloperoxidase
Authors: Yeoh Beng San , Olvera Rodrigo Aguilera , Singh Vishal , Xiao Xia , Kennett Mary J. , Joe Bina , Lambert Joshua D. , Vijay-Kumar Matam
Published: January 2023

Green tea (Camellia sinensis, Theaceae) contains a plethora of antioxidant polyphenols that may promote health. Amongst these polyphenols, the (−)-epigallocatechin-3-gallate (EGCG) is extensively studied for its potential therapeutic properties in models of inflammatory bowel disease (IBD), although its molecular mechanism is not completely understood. Here, we demonstrate that EGCG can inhibit the activity of myeloperoxidase (MPO; a pro-oxidant neutrophil enzyme associated with IBD flares) in a dose-dependent manner in vitro. By using spectral analysis, we show that EGCG prevents the MPO-catalyzed reaction by reverting the reactive peroxidase heme back to its native inactive ferric state. Oral administration of EGCG to dextran sodium sulfate (DSS)-induced colitic mice significantly reduced the colonic MPO activity and alleviated pro-inflammatory mediators associated with gut inflammation, confirming that our findings extend to in vivo conditions as well. In light of previous reports that EGCG can complex with innate immune protein lipocalin-2 and iron, we next tested whether these factors can affect the bioactivity of EGCG. Intriguingly, the presence of lipocalin-2 or iron significantly mitigate the ability of EGCG to inhibit MPO in vitro. Further, the efficacy of EGCG against DSS-induced gut inflammation is diminished when orally co-administered with iron. These findings indicate that EGCG-mediated inhibition of MPO could be one of the mechanisms by which EGCG exerts its mucoprotective effects. However, counter-regulatory factors such as host lipocalin-2 and dietary iron could explain why EGCG does not ameliorate gut inflammation in some IBD patients.
Toll-Like Receptor abundance in colonic epithelium is selectively altered in HIV disease and partially normalizes with effective Antiretroviral Therapy
Authors: McCausland Marie R , Levine Alan D
Published: January 2023

Background

Since the intestinal mucosa and microbiome are disrupted in HIV disease, we studied the abundance of innate immune sensors, Toll-Like Receptors (TLR), in the epithelia of patients with Viremia, prior to antiretroviral therapy (ART) initiation, Immune Success (IS- >500 CD4 T cells/ul after 2 years of ART; suppressed viremia), and Immune Failure (IF- <350 CD4 T cells/ul after 2 years of ART; suppressed viremia). Viremia and IF show increased systemic immune activation; therefore, modulation of intestinal TLR abundance may offer a mechanism behind persistent inflammation in HIV.

Method

Immunofluorescence for TLR3, TLR4, and TLR9 on sections cut from paraffin embedded colonic biopsies of uninfected, viremic, IS, and IF was imaged with a DeltaVision microscope and quantified with Metamorph software.

Results

Viremic patients have significantly higher viral TLR9 and TLR3, and significantly lower bacterial TLR4, on the surface epithelium when compared to levels seen on uninfected control. Viremic patients have significantly higher TLR9, similar TLR3, and significantly lower TLR4 expression, in the crypts when compared to uninfected control. TLR9 abundance remains elevated in IF, while normalization occurs in IS. TLR3 abundance on surface epithelium and crypt is even more elevated in IF and IS, suggesting an effect of ART. TLR4 abundance on surface epithelium normalizes in IF and IS; in the crypt, in IS TLR4 decreases further.

Conclusion

Epithelial TLR abundance is selectively altered in HIV disease; effective ART partially normalizes some TLRs, and enhances TLR3. Mucosal TLR desensitization or hyper-vigilance, possibly caused by disrupted cytokine levels in the HIV+ colon, may contribute to systemic inflammation.
The role of commensal microbiota on CD8 T cell response to Listeria monocytogenes infection
Authors: Cho Kyungjin , Surh Charles D
Published: January 2023

Commensal microbiota is composed of trillions of microorganisms including fungi, protozoans, viruses and bacteria, which have co-evolved with the host immune system. In particular, the gut is the largest reservoir of microorganisms that are involved in dynamic interaction with the immune system, including maintaining intestinal homeostasis, induction of several metabolic pathways, and protection from pathogenic microbes. To better understand the mechanisms involved microbiota-mediated protection from the pathogenic bacteria, we have studied oral infection of germ-free (GF) mice with a strain of Listeria monocytogenes (L. monocytogenes) that has been engineered to efficiently infect the intestinal epithelial cells of the mouse. We found that GF mice are more susceptible to orally administrated L. monocytogenes than conventional SPF mice, with an increased Listeria burden in the mLN, small intestine and feces. In addition, in the infected GF mice, L. monocytogenes-specific CD8 T cells were vigorously activated and induced to differentiate into memory precursor effector cells (MPECs) and tissue resident memory (CD69+ CD103+) T cells in the intestinal epithelium at a faster rate than in infected SPF mice. L. monocytogenes-specific CD8 T cells in GF mice also secrete more effector cytokines, such as interferon-γ and tumor necrosis factor-α. The mechanisms by how commensal microbiota confers resistance to infection by L. moncoytogenes in SPF mice is currently under investigation.
The APC capture: rapid identification of target CD8+ T cell epitopes by flow cytometry
Authors: Roesler Alexander S , Krishna Sri , Qiu Ji , LaBaer Joshua , Anderson Karen S.
Published: January 2023

Identifying immunoreactive CTLs by current technologies (cytokine secretion, intracellular cytokine, cytolytic release, ELISPOT, and MHC tetramer assays) is often difficult when probing multiple target antigens. We developed a rapid flow cytometry assay to detect targeted IFN-γ secretion by CD8+ T cells using commonly available reagents. PBMCs were obtained from HLA-A2+ healthy donors, and CD8+ and CD8− fractions were purified. FluMI-specific T cells were confirmed by HLA-A2/M1 tetramer, and pooled CEF peptides (a mixture of CMV-, EBV-, and Flu-specific HLA class I epitopes) immunoreactivity was shown by IFN-γ ELISPOT. The CD8+ fraction was rested for 5 days in IL-2 (20 U/ml) and IL-7 (5 ng/ml). In parallel, autologous APCs were generated from the CD4− CD8− fraction using recombinant CD40L (100 ng/ml), IL-4 (5ng/mL), and cyclosporin A (1 μg/ml). After 5 days, the APC culture was >70% CD19+ CD86+ HLA-DR+ activated B cells. The APCs were pulsed with DMSO control or CEF peptide pool (10ug/mL) overnight, labeled with commercial IFN-γ catch (Miltenyi Biotec), washed, and co-cultured for 6–8 hours with purified CD8+ T cells. APC-surface-bound IFN-γ was detected by flow cytometry. CEF-pulsed APCs showed robust presence of surface-captured IFN-γ (ranging from 14%–56.8%) compared to DMSO-pulsed APCs (ranging from 1.6%–4.5% IFN-γ+). APC IFN-γ capture was sensitive even at low effector-target ratios (as low as 1:1) and was applicable to the artificial APC K562/A2. In conclusion, we have demonstrated epitope-reactive CD8+ T cell detection of target cells by capturing IFN-γ onto APCs. We are evaluating capture of other cytokines and cytolytic effectors on target APCs in response to CD8+ recognition.
BioWardrobe: an integrated platform for analysis of epigenomics and transcriptomics data
Authors: Barski Artem , Kartashov Andrey
Published: January 2023

Development of next generation sequencing (NGS) has revolutionized molecular biology by enhancing our ability to perform genome-wide studies. However, due to the need for bioinformatics expertise and the sheer size of resulting datasets, use of these technologies is still beyond the capabilities of many laboratories. Here we present BioWardrobe, a platform that allows storing, visualizing and analyzing epigenomics and transcriptomics NGS data using a biologist friendly web-based graphical user interface without the need for programming expertize. BioWardrobe can be installed on consumer class hardware within an institutional local network. Analysis capabilities include predefined pipelines that allow the user to download data from either institutional core facility or public databases, perform quality control, map reads, and visualize data on a built-in mirror of the UCSC genome browser. Reads Per Kilobase per Million mapped (RPKMs) are calculated for RNA-Seq, and islands of enrichment are identified for ChIP-Seq and similar datasets. Advanced analysis capabilities include differential gene expression and binding analysis, and creation of average tag density profiles and heatmaps. BioWardrobe package and documentation is available at http://biowardrobe.com.
Column-free magnetic enrichment of pDCs yields significant contamination with DNA-containing dead cells that affect the activation status of the isolated cells
Authors: Richter Anne , Morrissey-Wettey Frank Ralf , Angerer Caroline , Kurig Stefanie , Dzionek Andrzej
Published: January 2023

Despite their role in the induction of adaptive immune responses, plasmacytoid dendritic cells (pDC) play also a crucial role in innate immune defense by recognizing pathogen derived molecular patterns. Unmethylated CpG motifs of bacterial DNA bind specifically endosomal and lysosomal TLR9 in pDCs, thereby inducing secretion of type I IFN or proinflammatory cytokines and classical DC maturation, respectively. However, TLR9 also recognizes CpG motifs in vertebrate DNA of damaged cells as danger-associated molecular patterns (DAMPs) that activate pDCs.

For in-vitro research pDCs may be isolated by indirect magnetic cell separation using either Miltenyi Biotec’s column-based or a column-free system from other suppliers. Here we describe the influence of the isolation method on the functionality of the purified pDCs. In addition to the significantly better separation performance, our column-based method enriched 10- and 30-fold less DNA-containing dead cells and debris, respectively. Although both methods yielded pDCs with a comparable phenotype instantly after isolation, after 24h of culture without stimulation pDCs isolated with the column-free system appeared pre-activated as demonstrated by elevated expression of CD80, −83, −86 and HLA-DR and by significantly higher basal IFN-α secretion. Moreover, upon stimulation with CpG-B pDCs isolated with the column-free method induced extraordinary high amounts of IFN-α, whereas pDCs isolated with our system exhibited normal responses to CpG-A and CpG-B. These results clearly demonstrate that unspecific enrichment of dead cells and debris may significantly influence the activation status of the isolated pDCs and therefore affect the results of downstream applications.
VDJPipe: a pre-processing pipeline for immune repertoire sequencing data
Authors: Christley Scott , Levin Mikhail , Fonner John , Monson Nancy , Rounds William H , Rubelt Florian , Scarborough Walter , Scheuermann Richard H , Toby Inimary , Cowell Lindsay G
Published: January 2023

Pre-processing of high-throughput sequencing data for immune repertoire profiling is essential to insure high quality input for downstream analysis. VDJPipe is a flexible, high-performance tool that can perform multiple pre-processing tasks with just a single pass of the data. These tasks include base composition statistics, read quality statistics, numerous quality filters, homopolymer filtering, length and nucleotide filtering, barcode demultiplexing, 5′ and 3′ PCR primer matching, and filtering of duplicate reads. VDJPipe utilizes a “pipeline” approach whereby multiple processing steps are described in a sequential workflow, with the output of each step passed as input to the next step. The workflow is flexible enough to handle the complex barcoding schemes used in many immunosequencing experiments. VDJPipe is an integral part of the VDJServer (http://www.vdjserver.org) web resource for performing immune repertoire analysis and can be accessed via the VDJServer Software page.
Measuring frequency and quality of pathogen-specific T cells
Authors: Anikeeva Nadia , Grosso Dolores , Flomenberg Neal , Sykulev Yuri
Published: January 2023

The frequency and functional activity of pathogen-specific T cells reflect the effectiveness of immune responses. We have developed an assay to measure the frequency and efficacy of antigen-specific CD8 T cells. The assay measures changes of intracellular Ca2+ in real time by fluorescent microscopy in individual CD8 T cells, which are immobilized on a glass surface to form a monolayer enabling the cells to present cognate peptides to each other. The T cells were labeled with Ca2+ dependent fluorophore, and intracellular fluorescence in the cells was analyzed before and after stimulation with specific antigenic peptides. Subtraction of the fluorescent intensity measured prior and after the stimulation revealed responding T cells and the kinetics of intracellular Ca2+ accumulation. The proposed assay permits comparison of the same cell before and after stimulation providing a significant increase in the sensitivity and utility of the assay. We found that as little as 3–5 responding T cells among 1×104 CD8 T cells could be reliably detected and that as much as 100 T cells with different specificities could be identified in a single round of analysis. We utilized the assay to characterize the frequency and quality of human cytomegalovirus-specific CD8 T cells from peripheral blood of healthy donors and patients who underwent haploidentical stem cell transplantation. The data revealed potential diagnostic and prognostic values of the proposed assay.
Diagnostic for assessing NKT cell function in cancer patients
Authors: Derakhshandeh Roshanak , Webb Tonya J
Published: January 2023

Significant efforts have been invested into harnessing the therapeutic potential of the immune system for the treatment of cancer. Natural killer T (NKT) cells play a critical role in anti-tumor immunity. However, NKT cells are approximately 50% lower in cancer patients compared to healthy donors of the same age and gender. Thus, prior to initiating immunotherapy, a specific and highly sensitive assessment of immune cell function should occur; yet, no such assay is currently available. Our lab has developed a novel cell-based molecular diagnostic for quantitation of a patient’s NKT cell activity by monitoring IFN-γ induction utilizing qPCR post activation with CD1d-based artificial antigen presenting cells (aAPCs). In this study we sought to assess NKT cell function in healthy donors and cancer patients in order to investigate the potential of NKT cell-based immunotherapeutic strategies. We assessed NKT cell function in 22 healthy donors (HD), 30 breast cancer patients (BCP), and 50 lymphoma patients (LP). The aAPC-qPCR method was more sensitive than flow cytometry and ELISA. We detected NKT cell function in 81% HD, 66% BCP, and 44% LP. In contrast, total T cell function was observed in 91% HD, 90% BCP, and 74% LP. These data suggest that NKT cell-based immunotherapeutic strategies may be more effective in specific cancer types. Collectively, these data demonstrate the need for assessing baseline immune function prior to the initiation of immunotherapy.
Characterization of a small molecule IRAK4 kinase inhibitor for the treatment of autoimmune and inflammatory diseases
Authors: Lamagna Chrystelle , Chan Meagan , Yi Sothy , Young Chi , Frances Roy , Siu Stacey , Braselmann Sylvia , Payan Donald G , McMurtrie Darren , Kelley Ryan , Heckrodt Thilo , Yen Rose , Chen Yan , Li Hui , Singh Rajinder , Park Gary , Masuda Esteban , Taylor Vanessa
Published: January 2023

IRAK4 is a key protein kinase in immune cells. It is responsible for initiating MyD88-dependent signaling from most Toll-Like Receptors (TLR) and Interleukin-1 Receptors (IL-1R), resulting in downstream production of pro-inflammatory cytokines. Hence, inhibitors of IRAK4 kinase activity represent valuable therapeutic tools to treat cytokine-driven autoimmune and inflammatory diseases. Through cell-based screening, we identified potent small molecule IRAK4 kinase inhibitors that block TLR4- and IL-1R-induced cytokine production with potencies less than 100nM. Our inhibitors exhibit good selectivity against a broad panel of kinases. In vivo, our lead compound decreases serum IL-6 in an acute mouse model of IL-1beta-induced cytokine release, demonstrating excellent pharmacokinetics properties. In addition, our IRAK4 inhibitor blocks monosodium urate crystals-induced peritonitis in mice, reduces paw swelling in the chronic rat model of collagen-induced arthritis, as well as the clinical score and overall inflammatory phenotype of mice in the imiquimod-induced psoriasis model. The in vitro and in vivo characterization of our lead candidate is promising and confirms IRAK4 as an attracting therapeutic target for the management of cytokine-driven diseases.
NALP1 inflammasome activation in adjuvant arthritis rats is a novel therapeutic target of carboxyamidotrizole in the treatment of rheumatoid arthritis
Authors: Zhu Lei , Li Juan , Ye Caiying
Published: January 2023

BACKGROUND AND PURPOSE

Pro-inflammatory cytokines play an important role in rheumatoid arthritis (RA) and their production is mainly regulated by NF-κB and inflammasomes. Carboxyamidotrizole (CAI) has been reported to exhibit potent anti-inflammatory activities by decreasing cytokines. Therefore, we investigated NALP inflammasomes in the synoviums of a rat RA model, and explored the therapeutic effects of CAI on RA model and the involvement of inflammasomes relative pathways in the actions of CAI.

EXPERIMENTAL APPROACH

The anti-arthritic effects of CAI were assessed in rat adjuvant arthritis (AA) model. NALP1 and NALP3 inflammasomes, nuclear factor (NF)-κB pathway, and pro-inflammatory cytokines levels were determined.

RESULTS

CAI administration significantly decreased arthritis index, improved radiological and histological manifestation, and reduced synovial IL-1β, IL-6, IL-18 and TNF-α levels in AA rats. As compared to the normal rats, the level of 70 kDa NALP1 isoform was significantly up-regulated, NALP3 was down-regulated, and the levels of 165kDa NALP1 isoform and the adaptor protein ASC were unchanged in AA synoviums. CAI reduced 70kDa NALP1 isoform and restored NALP3 level in synoviums of AA rats. And CAI inhibited caspase-1 activation in AA synoviums, although it did not directly inhibit the enzymatic activity in vitro. In addition, CAI reduced p65 NF-κB subunit expression and IκBα phosphorylation and degradation in AA rats.

CONCLUSION

NALP1 inflammasome was activated in arthritic synoviums, which might be a novel therapeutic target for RA. CAI may have therapeutic value in RA by inhibiting the activation of NALP1 inflammasome and NF-κB and by decreasing pro-inflammatory cytokines.