bluuebunny/crossref_metadata_2025 huggingface dataset:
Epithelial cell-derived chemokines induce DC recruitment to the gastric epithelium upon H. pylori infection
Authors: Hashimi Marziah , Seberll Thomas A , Sidar Barkan , Wilking James N , Bimczok Diane
Published: January 2023

H. pylori is a non-invasive bacterial pathogen that infects the lumen of the stomach and interacts with epithelial cells. In tissues from H. pylori infected and non-infected patients, DCs are found in close association with the gastric epithelium, which likely promotes DC uptake of luminal H. pylori. We hypothesized that DCs are recruited to the gastric epithelium in a chemokine-dependent manner. The aim of this study was to identify the epithelial chemokines that recruit DCs under steady state conditions and upon H. pylori infection. In chemotaxis assays, supernatants from human gastric organoids microinjected with H. pylori induced a 2-fold higher recruitment of monocyte-derived DCs compared to mock-infected organoids. Microscopic analysis of DC organoid-co-cultures similarly revealed DC recruitment to the epithelium of non-infected organoids and significantly increased recruitment upon H. pylori infection. DCs showed only low chemotactic activity towards H. pylori or bacterial supernatants, pointing to a role for epithelial rather than bacterial factors. DC migration towards the supernatants of both mock-infected and H. pylori-infected organoids was abrogated by pertussis toxin, indicating that recruitment was chemokine dependent. Using a chemokine array, we showed that organoid gastric organoids secreted increased amounts CXCL1, 5, 8 and CCL20 upon H. pylori infection, while CXCL16 and 17 remained constant. Chemotaxis assays confirmed that human DCs show chemotactic responses to CXCL1, 5, 8, 16, 17 and CCL20. In summary, our study indicates that human gastric epithelial cells actively recruit DCs to the mucosal interface by secreting chemokines, with increased recruitment induced upon H. pylori infection.
Exit of human cutaneous resident memory CD4 T cells that enter the circulation and seed distant skin sites
Authors: Morawski Peter A , Klicznik Maria , Hoellbacher Barbara , Varkhande Suraj , Motley Samantha , Duhen Thomas , Gratz Iris , Campbell Daniel J.
Published: January 2023

Tissue-resident memory T cells (TRM) persist locally in non-lymphoid tissues, providing front-line defense against recurring insults. However, strict tissue compartmentalization of memory may pose certain disadvantages for a large barrier organ like the skin, and the long-term migratory behavior of human TRM and their contribution to the memory pool have not been fully elucidated. TRM at barrier surfaces are defined in part by expression of the markers CD103 and/or CD69 which function to retain TRM in epithelial tissues. Here, we found that CD4+CD69+CD103+ TRM in human skin can downregulate CD69, exit the tissue and be identified as a phenotypically unique population in the circulation of healthy individuals. These circulating TRM produce the cytokines IL-22 and IL-13, and express genes consistent with a role in host-defense and tissue-repair responses. RNA- and TCR-sequencing demonstrated that CD103+ TRM in the blood are transcriptionally and clonally related to CD69+CD103+ TRM in the skin. Furthermore, using a skin xenograft model, we confirmed that human cutaneous CD103+ TRM can exit the skin, enter the circulation, and recirculate to secondary human skin sites where they re-assume a TRM phenotype. Thus, although as a population CD4+ TRM in the skin are largely sessile, recirculation of cutaneous CD4+CD103+ TRM does occur in the steady state in humans, and these recirculating (rc)TRM cells can promote the spread of this functionally specialized T cell population throughout the skin.
EBI3 impacts Leishmania braziliensis-induced inflammation in localized cutaneous leishmaniasis
Authors: DE SANTANA ALYNNE KAREN M , Gupta Gaurav , Gomes Ciro , Turatti Aline , Filho Roberto Bueno , Carregaro Vanessa , Roselino Ana , Almeida Roque , Silva João
Published: January 2023

Localized Cutaneous Leishmaniasis (LCL) can ultimately progresses to chronic ulcerated lesion with strong local inflammatory reaction. We hypothesized that regulatory cytokines, such as Interleukin-27 (IL-27) and interleukin-35 (IL-35) can play a role in susceptibility to Leishmania braziliensis infection. IL-27 and IL-35 are members of the interleukin-12 (IL-12) family structurally related by sharing Epstein–Barr virus-induced gene 3 (EBI3), which forms a heterodimer either with the p28 subunit to build IL-27 or with IL-12p35 to form IL-35. We used RT-PCR and Immunohistochemistry analysis to detect high expression of IL-27 and IL-35 subunits in the patients biopsies compared to healthy controls, suggesting that these cytokines can be involved in immunopathology induced by the infection. Moreover, L. braziliensis infection also induced IL-27/35 mRNA expression both in vitro and in vivo. We here analysed the function of EBI3 during infection with the intracellular parasite Leishmania braziliensis. We showed that EBI3 defficient mice displayed bigger lesion and an elevated inflammatory immune response in the ear compared to Wild-type mice with similar parasites levels in the ear and lymph nodes. The lack of EBI3 led to an increased expression of T helper type 1(Th1-), Th2- and Th17-derived cytokines in the infected ears. So far, our results demonstrate that EBI3 acts as a regulator of inflammatory immune responses in experimental cutaneous leishmaniasis, although, it is not required for control of L. braziliensis infection.

Financial Support: CAPES, FAPESP, CRID.
RGC-32 regulates expression of insulin-like growth factor binding proteins in astrocytes
Authors: Tatomir Alexandru , Boodhoo Dallas , Nguyen Vinh , Cudrici Cornelia , Badea Tudor , Rus Violeta , Rus Horea
Published: January 2023

Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects and activation of cell cycle. During experimental autoimmune encephalomyelitis (EAE), RGC-32 knock-out (KO) mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from wild-type (WT) mice had a reactive, hypertrophied phenotype, suggesting a role for RGC-32 in astrocyte differentiation. To gain more insight into the role played by RGC-32 in astrocytes, we investigated the transcriptional profile induced by TGF-β in astrocytes using qPCR arrays. The majority of genes differentially expressed were related to cell growth, extracellular matrix remodeling and signal transduction. Among them, a group of genes belonging to the family of insulin-like growth factor binding proteins (IGFBPs) and insulin-like growth factors (IGFs) were found to be most significantly regulated by RGC-32. The IGFBP category includes IGFBP2, IGFBP3, IGFBP6 and connective tissue growth factor (CTGF). Using Real-time PCR and Western Blotting, we confirmed the differential expression of these genes in RGC-32 KO astrocytes after TGF-β stimulation. When glial fibrillary acidic protein (GFAP) and CTGF levels were compared between WT and KO astrocytes after TGF-β stimulation, significantly lower levels of mRNA and protein expression were found in RGC-32 KO astrocytes. These data suggest that TGF-β stimulation of CTGF is RGC-32 dependent and that RGC-32 plays a role in the regulation of CTGF downstream effects. The differential regulation of IGFBPs and GFAP suggests that they might be responsible for the lack of astrocyte differentiation seen in RGC-32 KO mice during EAE.
Multivariate analysis of genes expressed in the colorectal tumor immune microenvironment driven by CXCL13
Authors: Nunez Sha’Kayla K , Griffen Tiara L , Young Corey , Dill Courtney D. , Adebayo Olayinka , Carey Kaylin , Lillard James W
Published: January 2023

Colorectal cancer (CRC) is a heterogenous disease having numerous molecular phenotypes. Key tumor progression markers such as microsatellite instability, BRAF, and KRAS mutation status have been widely studied. Less is known regarding the influence of chemokines and chemokine receptors in CRC. CXCL13 and CXCR5 have been shown to be associated with poor prognosis of advanced CRC. The central hypothesis tested in this study was CXCR5-CXCL13 signaling mediates gene expressions associated with CRC progression. RNAseq patient control and matched primary tumor samples were acquired from The Cancer Genome Atlas (TCGA). DESeq analysis identified differentially expressed genes between normal tissue and primary tumor groups. Weighted Gene Co-expression Network Analysis (WGCNA) elucidated gene networks that correlated with factors influencing CRC prognosis. Canonical pathway, upstream regulator, and protein-protein interaction analyses were performed using Ingenuity Pathway Analysis (IPA). CXCL13 was identified in the WGCNA-designated green module, which also contained XCL1, TNFRSF17, FCRN1, CCL19, IL-17A and IFNG. These genes are associated with immunoregulatory signals found in lymphoid and non-lymphoid cells, BCR-signaling and poor prognosis of CRC. Genes associated with energy metabolism, apoptosis, hypoxia and angiogenesis, including GNG4, PRKCG, WNT11, and KNG1, were found in the WCGNA-designated yellow module. These factors are involved in Wnt/CTNNNB1 and PI3K/AKT signaling, which have previously shown to be driven by CXCR5-CXCL13 interactions. Taken together, our data suggest a role of the CXCL13-CXCR5 axis in shaping the tumor immune microenvironment of CRC.
The association between the statistical significance of the AUC and measure of stochastic superiority when modeling cytokines: an example from colon cancer
Authors: Qeadan Fares , Stalter Lily , Beswick Ellen
Published: January 2023

Early successes in clinical trials suggest that immunomodulation through cytokine blockade may be an effective approach for developing new colorectal cancer (CRC) immunotherapies. It has been shown that certain cytokines shape a pro-tumorigneic tumor microenvironment and are linked with metastasis. Thus, identifying differentials in the cytokine response with respect to CRC metastatic stage is needed for further exploration to identify potential new therapeutic targets. In this study, we (a) use the area under the receiving operating characteristics curve (AUC) from logistic regression to classify metastasis and non-metastasis cases for CRC patients, (b) use the measure of stochastic superiority (denoted by A using the Wilcoxon-Mann-Whitney test), to give the probability of concordance between predicted and observed outcomes of metastasis and non-metastasis, and (c) examine the association between the statistical significance of the two measures (i.e. p-value for AUC vs. p-value for A) noting that, while it was shown that AUC is the same as A, limited studies were done examining the relationship between their statistical significance.

We obtained the AUC and A measures on a panel of 44 cytokines and chemokines identified in 69 subjects (35 with Metastasis and 34 without Metastasis) with CRC, and revealed that 7 cytokines (GCSF, IL-6, IL-10, MCP-1, IL-8, IL-1a, and IL-17A) were statistically significant in discriminating metastasis and non-metastasis patients according to the p-values of the A measure while only 5 cytokines were significant according to the p-values of the AUC measure (GCSF, IL-6, IL-10, MCP-1, and IL-8). This observation indicates that, while the two measures agree, the AUC test is subject to more type II errors.
Cell-targeted steroid signaling in the thymus determines T cell repertoire strength
Authors: Taves Matthew Donald , Mittelstadt Paul , Presman Diego , Hager Gordon , Ashwell Jonathan D
Published: January 2023

Thymocyte selection is critical for generation of a competent and tolerant T cell repertoire, and glucocorticoids (GCs) antagonize T cell receptor (TCR) signaling and negative selection to strengthen the TCR repertoire. While circulating GCs from the adrenals coordinate systemic responses, the thymus contains all the enzymes needed to independently synthesize GCs. However, the biological relevance, source, and targets of locally-synthesized GCs is unclear. To test the source and importance of local GC synthesis in vivo, we generated mice with targeted deletion of the GC-synthetic enzyme Cyp11b1 in thymic epithelial cells (Cyp11b1foxn1-Cre) or thymocytes (Cyp11b1lck-Cre). We found that expression of GC-responsive genes was reduced in Cyp11b1foxn1-Cre but not Cyp11b1lck-Cre thymocytes, confirming that epithelial cells are a major source of GCs in the mouse thymus. Cyp11b1foxn1-Cre T cells had reduced alloreactivity and antiviral responses, demonstrating a weakened TCR repertoire. To identify targets of epithelial GCs we used chromatin-associated GC receptors as a cell-specific biosensor of endogenous GC exposure. With this approach we detected and quantified targeted GC delivery to activated CD4+8+TCRhi thymocytes undergoing antigen-specific selection. Negative selection, as measured by PD-1 and Bim expression, was specifically increased in CD4+8+TCRhi thymocytes of Cyp11b1foxn1-Cre mice. These findings 1) demonstrate the biological importance of local GC synthesis, 2) show cell-specific targeting of paracrine GCs, and 3) quantitate intra-organ heterogeneity in GC levels within the thymus. GCs may similarly function as paracrine immunoregulatory signals across a range of tissues and contexts.
Advancing threespine stickleback as an outbred immunogenetics model by pinpointing the onset of adaptive immunity
Authors: Niebergall Emily R , Beck Emily A , Bassham Susan , Cresko William A.
Published: January 2023

The development of an outbred immunogenetics model system is needed to understand how genetic variation impacts phenotypic variation of disease states in humans. Threespine stickleback fish (Gasterosteus aculeatus) provide just such a model. Stickleback are genetically tractable laboratory organisms with a well-annotated genome, but with lines drawn from populations inhabiting vastly different habitats. Individuals from different populations show high levels of genetic variation. The onset of the adaptive immune system is currently unknown in stickleback, a significant hindrance in studies of immunodeficiency diseases. To characterize the early development of adaptive immunity, we will analyze the expression of known early indicators of adaptive immunity maturation. These include recombination activating genes, rag1 and rag2, which are essential to the maturation of T and B lymphocytes, and T cell receptor genes, tcr-β and tcr-γ, which are expressed in mature T lymphocytes. To analyze the expression of rag1, rag2, tcr-β, and tcr-γ, we will perform whole mount in situ hybridization throughout a developmental time series to detect when and where the genes are first expressed, with special focus on the head kidney and the thymus, followed by qPCR to quantify the expression of the early adaptive immunity genes. Knowing when adaptive immunity onset occurs in threespine stickleback advances threespine stickleback as an outbred disease model in immunogenetics studies, allowing manipulative studies of immunological disease phenotypes in the context of genetic variation.
mTORC1 signaling mediates T cell lineage choices
Authors: Bastardo Blanco Daniel E. , Yang Kai , Chi Hongbo
Published: January 2023

The thymus supports and guides the generation of a diverse repertoire of mature T cells from precursors derived from the bone marrow. The interaction between extrinsic factors and intrinsic signal strength governs thymocyte development, but the mechanisms linking these two aspects of T cell development remain elusive. Capitalizing on genetic deletion of RAPTOR, here we report that RAPTOR-dependent mTORC1 signaling couples microenvironmental cues with metabolic programs in orchestrating reciprocal development of two fundamentally distinct T cell lineages: αβ and γδ T cells. Loss of RAPTOR impairs αβ but promotes γδ T cell development while disrupting metabolic remodeling of oxidative and glycolytic metabolism. Mechanistically, we identify mTORC1-dependent control of reactive oxygen species (ROS) production as a key metabolic signal that, upon perturbation of redox homeostasis, impinges upon T cell fate decision. Our results establish mTORC1-driven metabolic signaling as a fundamental mechanism underlying thymocyte lineage choices.
Skin microbiome regulates SCF level in Keratinocytes and defines mast cell maturation
Authors: WU CHIA-CHI , Wang Zhenping , Di Nardo Anna
Published: January 2023

Background

Mast cell (MC) progenitors enter the skin and become mature in the surrounding microenvironment during embryogenesis and they fully mature 8 to 15 days after birth (in mouse). We published that skin commensal bacteria work together with skin resident cells (especially keratinocytes), in order to retain and differentiate mast cells into the skin. In the present study, we aim to demonstrate that this effect is due to TLR2 sensing of microbiome.

Methods

we used germ free mice (GF), hairless (hr/hr) mice and hr/hr/Tlr2−/− mice. Analyses were performed by immunohistochemistry, qPCR and FACS. IgE cross-linking and 48/80 have been used to test in vivo MCs degranulation.

Results

we found that GF mice present with less mature skin MCs than pathogen free mice (PFM). MCs immaturity correlated with a low level of stem cell factor (SCF) in keratinocytes, a critical MC differentiation and growth factor. Furthermore, compound 48/80, a known degranulation agent, induced less edema in the paws of GF mice than in those of conventional mice. A smaller population of c-Kit positive MCs in the GF mice normalized after being co-housed with PFM mice for 5 weeks to reconstitute their microbiome. On the other hand, hr/hr mice, have a skin increased number of mature and proliferating MCs that correlates with a more gram positive microbiome than their littermates. When hr/hr were backcrossed into Tlr2−/− (so they cannot sense the gram positive commensals) the number of MCs in skin dramatically decreased.

Conclusions

While baseline keratinocyte SCF expression is essential to recruit MCs into the skin, TLR2 stimulation by gram positive skin microbiome, promotes SCF keratinocyte production, which then enhances the maturation and proliferation of skin MCs.
Silencing of PD-L2/B7-DC by Topical Application of Small Interfering RNA Inhibits Elicitation of Contact Hypersensitivity
Authors: Furusawa Emi , Ohno Tatsukuni , Nagai Shigenori , Noda Taisei , Komiyama Takuya , Kobayashi Katsunori , Hamamoto Hidetoshi , Miyashin Michiyo , Yokozeki Hiroo , Azuma Miyuki
Published: January 2023

PD-L2 is a ligand for the immune checkpoint receptor PD-1, however, its regulatory function is unclear. We previously reported that silencing of CD86 in cutaneous dendritic cells (DCs) by topical siRNA application inhibits the elicitation of contact hypersensitivity (CHS). Here we investigated the effects of topical PD-L2 siRNA application on allergic skin disease. PD-L2 was induced in DCs concurrently with the elevation of MHC class II and CD86 expression. Topical PD-L2 siRNA application inhibited elicitation of CHS by suppressing early proinflammatory cytokine expression and migration of hapten-carrying DCs into lymph nodes. Local injection of neutralizing anti-PD-L2 mAb comparably inhibited CHS. PD-L2 siRNA treatment inhibited the CHS in PD-1/PD-L1 double-knockout mice and in the sensitized T-cell-transferred skin. These results suggest that the effects of PD-L2 silencing are independent of PD-1 but dependent on local memory T cells. Most of the inhibitory effects of PD-L2 and CD86 silencing on CHS were comparable, but PD-L2 siRNA treatment did not inhibit atopic disease-like manifestations and Th2 responses in NC/Nga mice. Our results suggest that PD-L2 in cutaneous DCs acts as a costimulator rather than a regulator. Local PD-L2 silencing by topical siRNA application represents a new therapeutic approach for contact allergy.
The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction
Authors: Kunimura Kazufumi , Fukui Yoshinori
Published: January 2023

Introduction

Mutations of DOCK8 in humans cause a combined immunodeficiency characterized by atopic dermatitis. However, the molecular link between DOCK8 deficiency and atopic skin inflammation is unknown.

Materials and Methods

The skin disease development was compared between Dock8−/− and Dock8+/−mice expressing TCR transgene. After stimulation with the cognate antigen, CD4+T cells were used for cytokine production assay, microarray analysis and ChIP assay. IL-31 promoter activation, EMSA and immunoprecipitation were performed by standard techniques. Nuclear translocation of EPAS1 was examined in mouse embryonic fibroblasts generated from wild-type and Dock8−/−mice.

Results

We found that unlike Dock8+/−mice, Dock8−/−mice spontaneously developed severe atopic skin inflammation, when crossed with transgenic mice expressing TCR with a particular antigen specificity. Upon stimulation, CD4+T cells from Dock8−/−mice produced large amounts of IL-31, a major pruritogen associated with atopic dermatitis. This IL-31 induction critically depended on the transcription factor EPAS1, and its conditional deletion in CD4+ T cells abrogated skin disease development in Dock8−/−mice. Although EPAS1 is known to form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) and control hypoxic responses, EPAS1-mediated Il31 promoter activation was independent of ARNT, but in collaboration with SP1. In addition, we found that DOCK8 acted as an adaptor and negatively regulated nuclear translocation of EPAS1.

Conclusions

EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL31 induction in CD4+T cells.
Oral tolerance to peanut allergy is mediated by CTLA-4-positive regulatory T cells
Authors: Krempski James W , Iijima Koji , Kobayshi Takao , Kita Hirohito
Published: January 2023

Peanut allergies are life-long diseases that cause severe anaphylactic responses to affected individuals. Oral ingestion of peanut products prevents infants from developing peanut allergy. However, little information is currently available regarding the immunologic mechanisms for this protection. Therefore, the goal of this project was to establish an oral tolerance model to peanut allergy in mice and to determine the immunologic mechanisms.

Naïve BALB/c mice that were exposed intranasally (i.n.) to peanut flour (PNf) developed peanut allergy as demonstrated by increased serum levels of IgE antibody as well as acute anaphylactic response when challenged with peanut extract. When mice were fed peanut butter (PNB) prior to i.n PNf exposure, they were protected from developing peanut allergy; these mice showed decreased serum levels of IgE antibody and lacked anaphylactic response. T follicular helper (Tfh) cells and germinal center (GC) B cells were decreased in lung draining lymph nodes of mice fed PNB. Furthermore, scRNSseq revealed 2 distinct CD4+ T cell subpopulations that are increased in those protected mice. CTLA-4, a checkpoint molecule, was highly expressed in one of these subpopulations. Systemic administration of a CTLA-4 blocking antibody to mice fed PNB increased the numbers of Tfh cells and GC B cells and increased serum levels of IgE antibody. Finally, anti-CTLA-4 reversed the clinical efficacy of oral PNB administration, resulting in manifestation of acute anaphylaxis when challenged with peanut extract.

Thus, oral administration of peanut product(s) prevents development of peanut allergy in mice. CTLA4+CD4+ T cells likely play a pivotal role in this process by regulating the development of Tfh cells.
An epigenetic link between the gut microbiome and immune responses in atopy
Authors: Cait Alissa , Hughes Michael R , Bilenky Misha , Cait Jessica , Pepin Deanna , Hirst Martin , Mohn William W. , McNagny Kelly
Published: January 2023

Atopy is the most common childhood disease in developed countries. Dysbiosis of the microbiome is now understood to be a driving force behind the development and severity of allergy/atopy. We previously showed that antibiotic treatment of newborn mice leads to a life-long perturbation of the gut microbiome marked by reduced bacterial production of the short chain fatty acid, butyrate. These mice exhibit a striking pro-inflammatory phenotype, marked by a heightened Th2 response, that was completely reversible with dietary supplementation of butyrate.

Butyrate is known to exert its effects on target cells via inhibition of histone deacetylases that have long term effects on gene expression. Consistent with a role for epigenetic skewing on the hematopoietic compartment of antibiotic treated mice, we found that bone marrow transplantation was sufficient to transfer the pro Th2 inflammatory phenotype into chimeric mice. Strikingly, we found unique regulatory states (H3K27ac) in purified hematopoietic stem and progenitor cells of these Th2 skewed mice. Single cell sequence analyses identified a distinct stem/progenitor cell transcriptomic signature in butyrate depleted mice that was reversed by butyrate supplementation.

We identified a metagenomic gene signature present in fecal microbiome samples of 3-month-old children who subsequently developed atopy. Consistent with our observations in mice, this signature included depletion of the bacterial genes required for breakdown of indigestible carbohydrates in breast milk and the loss of genes required for butyrate fermentation. This work provides novel evidence for a butyrate-driven epigenetic process that links the fetal microbiome to long term sculpting of the immune response.
Effector CD8 T Cell Infiltration into the Skin to Mediate Contact Hypersensitivity Responses Requires TLR4 Signaling on Precursors of Gr-1+CXCR2+Bone Marrow Cells
Authors: Kish Danielle , Fairchild Robert L
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and subsequent challenge. Challenge of dinitrofluorobenzene (DNFB) sensitized mice generates hapten-presenting endothelial cells (EC) that activate hapten-primed CD8 T cells to produce IFN-γ and IL-17. These cytokines stimulate the EC to produce CXCL1 that directs the recruitment and activation of Gr-1+CXCR2+cells required for hapten-reactive CD8 T cell infiltration into the skin parenchymal tissue. This study investigated the role of TLR4 receptor signaling during elicitation of CHS to DNFB. The production of CXCL1, ICAM, IFN-γ, CXCL9/10, and TNF-α and neutrophil infiltration evoked by hapten challenge of sensitized WT mice was markedly decreased following challenge of sensitized TLR4−/−mice. Hapten-primed CD8 T cells stimulated cultured hapten-labeled WT but not TLR4−/− EC to produce CXCL1. DNFB sensitization of WT and TLR4−/−mice induced similar numbers of hapten-reactive CD8 T cells producing IFN-g and IL-17 in the lymph nodes, but the CHS response to challenge in sensitized TLR4−/−mice was decreased > 50% vs. WT mice. Challenge of DNFB sensitized WT, but not TLR4−/−, mice induced the bone marrow (bm)-derived Gr-1+CXCR2+cells needed to direct T cells into the challenge site. Transfer of hapten-primed WT CD8 T cells to naïve WT, but not TLR4−/−, mice induced CHS in response to hapten challenge. However, transfer of WT bm Gr-1+CXCR2+cells restored T cell infiltration and the CHS response to hapten challenge of sensitized TLR4−/−mice. Collectively, these results indicate that TLR4 signaling in bm Gr-1+CXCR2+precursor cells is required to evoke the innate immune response required for subsequent hapten-reactive T cell mediated CHS.
Metabolic shift induced by systemic activation of T cells in PD-1-deficient mice perturbs brain monoamines and emotional behavior
Authors: Miyajima Michio , Zhang Baihao , Guerrini Matteo , Sugiura Yuki , Fagarasan Sidonia
Published: January 2023

Introduction

While change of intracellular metabolism in T cell activation is well studied, whether T cell activation affects systemic metabolome outside cells is largely unknown. In present study, we examined systemic metabolome change in T cell activation and its effect on brain functions like emotional behaviors.

Results

Concentrations of amino acids in sera were decreased in PD-1-deficient mice in a T cell-dependent manner. On the other hands, concentrations of amino acids in lymph node were increased in lymph node of PD-1 deficient mice with increased number of total and activated T cells. Activated T cells increased expression of amino acid transporter and intracellular incorporations of tryptophan and tyrosine, two representative amino acids. Decreased concentrations of serum tryptophan and tyrosine lead to decreased serotonin and dopamine in brain of PD-1-deficient mice. PD-1-deficient mice showed enhanced anxiety-like behaviors and fear response.

Conclusions

Enhanced incorporation of amino acids in activated T cells leads to serum decrease in amino acids, which leads to serotonin and dopamine decrease in brain and enhanced anxiety-like behavior and fear response in PD-1-deficient mice.
CD71+erythroid cells promote Tregs and via ectonucleotides CD39 and CD73 modulate T cell responses
Authors: Elahi Shokrollah , Shahbaz Shima , Koleva Petya
Published: January 2023

CD71+erythroid cells are abundant in newborns with immunomodulatory properties. Recently, we showed a subset of neonatal CD71+erythroid cells express significant levels of V-domain Ig Suppressor of T Cell Activation (VISTA) and via TGF-β play a pivotal role in promotion of naïve CD4+T cells into regulatory T cells (PLOS Biology Dec. 2018). Here, we have demonstrated another novel immunomodulatory mechanism for CD71+erythroid cells. A subset of CD71+erythroid cells express significant levels of ectonucleotides CD39 and CD73, which culminating in the formation of adenosine. Extracellular adenosine has a marked dampening effects on the immune cells, suppressing effector T cells and assisting regulatory T cells. In addition, we found that CD71+erythroid cells express adenosine receptor (A2A) and constitutively produce TGF-β. Interestingly, exogenous or endogenous TGF-β upregulates expression of CD73 on CD71+erythroid cells. Collectively, these studies provide a novel insight into the crosstalk between CD71+ erythroid cells and regulatory T cells. Our results highlight the important biological role of CD71+erythroid cells in immune homeostasis.
Immune subsets in the murine pituitary gland and potential impacts on the HPG axis
Authors: Nicholas Dequina , Lawson Mark A.
Published: January 2023

Tissue-resident immune cells are known to regulate local homeostasis, including but not limited to inflammation and tissue repair. Recent research has implicated the importance of the immune system in reproduction. However, little data exists delineating the role of the immune system in the hypothalamic-pituitary-gonadal (HPG) axis, an important regulator of reproduction. The goal of this study was to apply conventional immunological techniques to assess the tissue-resident population of immune cells in the pituitary. We hypothesized that, like most tissues, a resident population of immune cells exists to promote homeostasis and may be dysregulated in disease. To test this, we prepared single cell suspensions of pituitary tissue from ~8–10 wk old wildtype male C57BL/6 mice and stained for CD45+ leukocytes and GnRHR+ gonadotropes. We found that 5–10% of the pituitary comprises CD45+ leukocytes. We designed an 11-color flow cytometry panel to immunophenotype subsets in the CD45+ population. Surprisingly, B cell and T cell populations, including CD4+ and CD8+ T cells, are underrepresented in the pituitary relative to mouse peripheral blood, spleen, and lymph nodes. B cells were ~9% of CD45+ cells while CD3+ T cells were ~ 15% of CD45+ cells. Conversely, approximately 40% of CD45+ cells in the pituitary are CD11b+ (likely macrophages) which constitute ~3% of all cells in the pituitary. The substantial presence of CD11b+ cells in the pituitary implicates a role for macrophages in pituitary homeostasis and function. Future work will focus on the role of pituitary tissue resident macrophages in the HPG axis.
A loss-of-function single nucleotide polymorphism in cytochrome b5 reductase 3 amplifies the response of bone marrow-derived macrophages to high salt
Authors: Jerome Jacob A , DeVallance Evan R , Pagano Patrick J , Straub Adam C
Published: January 2023

Introduction

High dietary salt intake is a risk factor for hypertension and autoimmune diseases. Recent studies show high salt intake can result in high-concentration interstitial salt accumulation. In macrophages, similar concentrations induce pro-inflammatory gene expression and amplify activation by LPS. African Americans, a notably salt-sensitive population, have an exclusively high frequency (~23%) for a missense single nucleotide polymorphism (SNP) in cytochrome b5 reductase 3 (CYB5R3), an enzyme important for oxidative stress resistance. We have shown this SNP, T117S, decreases enzymatic activity. Given macrophages’ sizable productive capacity for reactive oxygen species (ROS), we hypothesize T117S expression amplifies salt-mediated pro-inflammatory gene expression and ROS production.

Results

Culture of bone marrow-derived macrophages (BMDMs) from WT or T117S-expressing mice in high salt medium (+51mM NaCl) increased expression of pro-inflammatory genes iNOS, IL-6, and IL-1β. However, the increase was significantly greater in T117S BMDMs. High salt also increased hydrogen peroxide (H2O2) production in both groups, but more so in T117S BMDMs. Of note, pretreatment with inhibitors targeting p38, a MAP kinase involved in the inflammatory response, or NOX2, the major macrophage superoxide source, ablated salt-mediated H2O2 production in both groups.

Conclusions

We show presence of loss-of-function variant T117S CYB5R3 in macrophages enhances salt-mediated pro-inflammatory gene expression and ROS production in a p38 and NOX2-dependent manner. This suggests CYB5R3 may be involved in the response to high salt and raises the possibility that T117S carriers may have an exacerbated response to high salt intake.
Identification of TRM subsets with distinct cellular states and memory potential
Authors: Omilusik Kyla , Milner J Justin , Kurd Nadia , Toma Clara , Chang John T. , Goldrath Ananda W
Published: January 2023

The memory CD8+ T cell population consists of distinct subsets that collaborate to provide robust immunological protection. Within the circulation, two broad classes exist – the shorter-lived effector and effector memory (TEM) cells with heightened effector function for immediate protection and the longer-lived central memory (TCM) cells with stem-like qualities and increased proliferation potential. Tissue-resident memory (TRM) cells are the prominent cell type permanently residing in non-lymphoid tissues acting as sentinels against reinfection. Despite the critical importance of the TRM subset, the extent of their phenotypic and functional heterogeneity remains unknown. Using single-cell gene expression analyses, we assessed small intestine intraepithelial TRM cells over the course of acute infection. Distinct subsets were identified and distinguished by reciprocal expression of effector- and memory-associated transcriptional regulators, Blimp1 and Id3, respectively. While the Blimp1hi subset was prominent at early time points, the Id3hi population persisted and was more abundant over time. Importantly, examination of the subset-specific transcriptional profiles revealed considerable enrichment for the effector/effector-memory gene signature within the Blimp1hiTRM subset and the central-memory gene signature within the Id3hi TRM population. Furthermore, Id3hi TRM cells exhibited more stem-like characteristics including increased capacity to proliferate following rechallenge. Taken together, this study reveals that similar to circulating memory, the TRM population is heterogeneous, comprising cells with contrasting differentiation states and memory potential.
NUR77 HAS A CELL INTRINSIC ROLE IN NATURAL KILLER T CELL DEVELOPMENT AND FUNCTION
Authors: Kumar Amrendra , Suryadevara Naveenchandra , Hill Timothy M , Gordy Laura E , Bezbradica Jelena S , Wu Lan , Acharya Pankaj , Hiebert Scott W , Van Kaer Luc , Joyce Sebastian
Published: January 2023

Natural killer T (NKT) cells are innate-like lymphocytes that respond to self and foreign glycolipids under inflammatory conditions. NKT cells have constitutively high levels of Nur77, an orphan nuclear receptor family transcription factor that is induced very early during their ontogeny. Nur77 function in NKT cells has not been explored, however. Nur77 is known to transcriptionally control negative selection of conventional T cells, development of regulatory T cells and differentiation of effector CD8+ T cells. Thus, we hypothesized that Nur77 controls a lineage-specific gene expression program essential for the proper development of functional NKT cells. We found that, akin to conventional T cells, NKT cells poorly developed in transgenic mice that overexpressed wild type Nur77 (wtNur77tg) in T lineage only. Introgression of the rearranged Va14i a-chain gene into wtNur77tg mice (wtNur77tg; Va14tg mice) rescued NKT cell development but was arrested at the precursor stage 0. NKT cells in wtNur77tg; Va14tg mice expressed lower levels of PLZF when compared to NKT cells in non-transgenic mice. So also, NKT cells in wtNur77tg; Va14tg mice do not respond to glycolipid agonists. These findings are consistent with differences in the transcripomes of stage 0 NKT cells isolated from Va14tg versus wtNur77tg; Va14tg mice. Collectively, our data suggest that Nur77 induction initiates a gene expression program in developing NKT cells that are critical for developmental progression and function of NKT cells.
Supporting scientific knowledge communication and translation efforts by engaging appropriately with the Pacific audience
Authors: Sika-Paotonu Dianne , Laing Bridie , Anitelea Toni , Uiese Tina , Puni Ron , Vaipuna Tevita , Fa’atoese Adam
Published: January 2023

It is recognised that Indigenous Māori and Pacific Peoples are affected disproportionately by Acute Rheumatic Fever (ARF) and Rheumatic Heart Disease (RHD). Rates of ARF and RHD in New Zealand and the Pacific Region are of concern. Appropriate community engagement and knowledge translation efforts that support dissemination of research work and findings of relevance to Pacific populations, are of importance for effective dialogue. Efforts to communicate scientific research work and engage appropriately with Pacific communities and audiences have been limited, and require cultural considerations to ensure communication and interactions are culturally appropriate, clear and meaningful. The purpose of this work was to facilitate discussion between researchers and Pacific communities, regarding ARF and RHD Penicillin related research works of direct relevance and importance to Pacific communities in the Wellington area. Led by a Pacific Biomedical Scientist and supported by University Pacific staff and Medical students, a Pacific Fono (gathering) outreach event in the Hutt Valley was held on the 24th July, 2018. Scientific information and research work with immunological components related to ARF and RHD were presented, followed by a question answer session. Members of the Pacific community that attended represented different Pacific population groups with written and verbal feedback indicating the event had been informative and was enjoyed by those in attendance. This scientific knowledge translation and science communication event demonstrated appropriate engagement had been achieved to support dialogue between researchers and the Pacific community, regarding ARF and RHD Penicillin related research efforts.
Cinemimmunology-the use of cinema to learn immunology
Authors: Siddiqui Sana , Olk Sandra , Collier Rashad , Fazal Nadeem
Published: January 2023

Film can be a powerful educational tool known as cinemeducation. We at College of Pharmacy applied this universal learning method to the first year pharmacy class of basic immunology. Two workshops were introduced to the course and the student’s assessment pre- and post-workshop were analyzed. We used two Hollywood blockbuster movies, i.e., The Incredibles 1 and 2 to enhance first year pharmacy students to grasp concepts of basic immunology. The students watched movies. The main plot was that the immune system is like a family with dad, mom, girls, boys and babies. Giving due credit to Brad Bird, the writer and director of The Incredibles. In his words (changed within brackets)-quoted as follows: The characters of “The dad is always expected in the family to be strong, so we made him (T cell) strong. The moms are always pulled in a million different directions, so we made her stretch like taffy (B cell). Teenagers, particularly teenage girls, are insecure and defensive, so we made her turn invisible and turn on shields (Antibody). And ten-year-old boys are hyperactive energy balls. Babies are unrealized potential. So, we made babies as cytokines.” Our results showed a significantly enhanced conceptual learning and long-term retention of immunological concepts. Even students with severe learning difficulties and disabilities who struggle with any kind of academic curriculum can often relate to film. It was also an example of associated learning using common movies with high yield of performance within a short period of time. We recommend the use of such strategies in immunology education. Commonly accepted education method is known as cinemeducation for imparting medical education and when we used it to teach immunology, we called it cinemimmunology.
Contextualisation of Immunology based teaching for undergraduate Medical science students that supports knowledge application
Authors: Sika-Paotonu Dianne , Yang Anne , Vaipuna Kalilea , Buckley Eilish , Bartlett Hannah , Kalyanasundaram Shiva , Faatoese Adam
Published: January 2023

Provision of appropriate clinically relevant Immunology examples to support understanding of the related mechanistic and cellular Immunology teaching content within undergraduate Medical sciences education is important for knowledge application. This work in part involved the incorporation of Immunologically based clinical research examples for undergraduate Medical science students, that were relevant to current health issues associated with Māori and Pacific population groups living in New Zealand (NZ) and the Pacific Region. Immunologically based Cancer and Acute Rheumatic Fever (ARF) related teaching examples were presented in class to 4th year undergraduate Medical science students. Untreated Group A Streptococcal (GAS) infection of the throat (and skin) can trigger ARF which is an autoimmune condition that can lead to development of permanent heart damage known as Rheumatic Heart Disease (RHD) if left untreated. Māori, Pacific and Indigenous Aboriginal population groups are affected disproportionately by ARF and RHD. As the Cancer burden for Pacific population groups in NZ and the Pacific Region remains of public health concern, Immunologically based Cancer research work was also presented. In-depth interest and understanding of the presented material was demonstrated in class and indicated in student feedback. A subset of students became subsequently engaged in Summer Scholarship research placements focused on varying Cancer related aspects. Based on student responses and subsequent research efforts, the contextualisation of the Immunology information was successfully achieved and raised awareness of the heath priorities for Māori and Pacific population groups living in NZ and the Pacific Region.
An online workshop integrated into Immunology course to teach pharmacogenomics to the first-year pharmacy students
Authors: Fazal Nadeem , Joly Rifka , Jassem Nuhad , Edquiban Marianne , Lakhani Baneen , Shaikh Kiran
Published: January 2023

Genetic basis of antibody structure is taught as a part of didactic course of Immunology to first-year pharmacy students. Therefore, we designed a three-hour workshop for P-1 students as a part of didactic Immunology course. Online workshops offer a rich multimedia learning experience, the interactive format includes guided learning videos, engaging discussion, assessment and comprehensive survey. Accreditation Council for Pharmacy Education (ACPE) Standards 2016 include pharmacogenomics, it is the responsibility of pharmacy schools to prepare pharmacists entering clinical practice to effectively use genetic data in the delivery of precision medicine. However, in a 2010 survey the state of pharmacogenomics instruction at most pharmacy schools was reported as “poor” or “not at all adequate”. Pharmacogenomics is defined as the study of how genes affects a person’s response to drugs. We proposed a new method of instruction using online learning resources to drive better learning and awareness of pharmacogenomics at a very early stage in pharmacy education. 64 students were included in this study. Two YouTube videos 45 minutes each, i.e., one describing basic genetics and the other pharmacogenomics were posted on Moodle. A case-study model was used to bring home the basic concepts. A pre- and post-workshop google forum was posted to assess student’s learning. A Moodle-based chat forum was also incorporated to take students questions during the workshop. The data showed that most of the students were unware of the importance and relevance of pharmacogenomics and the workshop improved learning this significantly. Online learning tools like workshops were found to be student-friendly and a flexible format with no scheduled meeting times.
Development of an interactive simulation and app (Pipette Master) to teach undergraduate students how to read, set and use and automatic laboratory pipette
Authors: Costabile Maurizio , Vedova Chris Della
Published: January 2023

An automatic pipette is a key piece of laboratory equipment. The ability to accurately and reproducibly deliver any volume is a critical skill to master for all undergraduate students in the laboratory sciences. Over many years we have noted that students struggle in learning several facets of correct pipette usage. These include, choosing the correct pipette for a given volume, correctly setting the pipette and correctly using the plunger to aspirate and dispense the liquid. Students also struggle with unit conversions between ml and μl and vice versa. Lastly, students fail to retain these skills and information in following semesters of teaching. In an attempt to help students with converting between units (μl and ml) as well as choosing the most appropriate pipette for a given volume, I developed an interactive simulation which covered all these major aspects. Using a Gilson pipette as an example, the simulation covered how to set the pipette for a number of defined volumes. An Android/iOS app of the simulation has also been developed which can be used during laboratory classes. In 2017, we polled first year undergraduate students about their level of experience, knowledge of pipettes and level of comfort in converting between units and we then explained the simulation to them. The feedback on their skills and the software will be discussed. We also tested the impact of the simulation using 2nd year students enrolled in genetics. Using a laboratory quiz, we found significant improvements in student performance when students used the simulation compared to those that did not. We suggest this interactive approach is a simple and effective way to enhance student learning of key aspects of laboratory pipettes. The simulation is freely available.