bluuebunny/crossref_metadata_2025 huggingface dataset:
mDia1 promotes T cell extravasation and enables induction of Experimental Autoimmune Encephalitis
Authors: Thompson Scott Beckett , Estin Miriam Laquer , Jacobelli Jordan
Published: January 2023

Self-reactive T cells play a central role in Multiple Sclerosis (MS) and invade the central nervous system (CNS) to drive neurological damage. To enter tissues, T cells extravasate through the vascular endothelial cell wall via a process known as transendothelial migration (TEM). While many of the adhesion molecules and chemokine signaling pathways required for TEM are well characterized, little is known about how downstream cytoskeletal effectors mediate the mechanical forces and shape changes needed for T cell TEM. Mouse Diaphanous Homolog 1 (mDia1) is a terminal cytoskeletal effector involved directly in actin network remodeling. It has been associated with the formation of membrane protrusions that likely function in migration and cell-cell interactions and is highly expressed by activated T cells. The goal of our study was to examine the role of mDia1 in T cell extravasation into the CNS and subsequent induction of neuroinflammation. mDia1 knock-out (KO) mice were partially protected from Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of MS. mDia1 deficient ex-vivo activated auto-reactive T cells displayed similar activation kinetics compared to control T cells. However, when these activated mDia1 deficient T cells were transferred into wild-type mice, they were impaired in their ability to induce EAE. In vitro time-lapse microscopy data suggest that mDia1 deficient T cells have a reduced ability to complete TEM across brain endothelium. This defect may explain the reduced ability of mDia1 deficient T cells to induce EAE. Collectively our data suggest that mDia1 may be a promising therapeutic target for regulating T cell trafficking in MS.
Sirt1 modulates immunoglobulin class switch DNA recombination and somatic hypermutation by regulating B lymphocyte Aicda expression through NF-kB deacetylation
Authors: Gan Huoqun , shen Tian , Sanchez Helia N. , Vasudev Anusha , Taylor Julia R. , Zan Hong , Casali Paolo
Published: January 2023

Maturation of the antibody response entails Ig class switch DNA recombination (CSR) and somatic hypermutation (SHM), which perfect the antigen-binding activity and diversify the effector functions of antibodies. By showing that HDAC inhibitors selective for class I/II HDACs dampened CSR and SHM by downregulating AID through upregulation of selected microRNAs that target Aicda, we have outlined an important role of these Zn-dependent HADCs in the modulation of the antibody response. Here we showed that Sirt1, one of the NAD+-dependent Class III HDACs or Sirtuins, also plays an important role in the regulation of B cell CSR and SHM. Sirt1 deletion in AID-expressing B cells of AicdacreSirt1fl/fl mice, whose B cells contain the normal complement of Aicda genes together with a Cre gene under control of an extra Aicda promoter, led to increased class-switched and somatically mutated antibodies in response to NP-CGG, as a result of upregulated AID expression, without alteration of Prdm1 expression or plasma cell differentiation. Sirt1-deficient B cells hyperacetylated p65, thereby activating the canonical NF-kB pathway, which mediates Aicda activation, without changes in DNA methylation or histone acetylation of the Aicda promoter. This was mimicked by increased concentrations of glucose, which reduced the NAD+ Sirt1 co-factor, leading to p65 hyperacetylation and Aicda upregulation in B cells. Conversely, treatment of B cells with NAD+ or SRT1720, a potent Sirt1 activator, dampened Aicda expression. Thus, our findings outline an important B cell-intrinsic role of Sirt1 in the modulation of the antibody response.
Unrelated respiratory infections promote the loss of pre-existing tissue resident influenza-specific memory CD8 T cells in the lung
Authors: Hayward Sarah L , Takamura Shiki , Kohlmeier Jacob E
Published: January 2023

Tissue resident memory CD8 T cells mediate protective immunity at common sites of infection such as the skin, gut, female reproductive tract, and lung. Lung-resident memory T cells (lung TRM) have been found to be important for protective heterosubtypic immunity to influenza, however the efficacy of cellular immunity to influenza viruses wanes over time. Our data suggests this decline in protective immunity is due to the gradual loss of flu-specific lung TRM, which, in contrast to circulating flu-specific memory T cells and TRM in other tissues, steadily decrease in number for several months following primary infection. Notably, the loss of flu-specific lung TRM occurs throughout the TRM pool regardless of expression of the tissue residency markers CD69 and CD103. Furthermore, parabiosis experiments using influenza memory mice show that flu-specific lung TRM are not exiting the tissue, and thus the gradual loss of these cells is likely due to increased cell death compared to the systemic memory T cell pool. As the lung is continually exposed to environmental and biological insults that can result in localized inflammation, we sought to determine whether lung inflammation could promote the loss of pre-existing flu-specific lung TRM. Infection of influenza-immune mice with Sendai virus resulted in a significant decrease of the number of pre-existing flu-specific lung CD8 TRM compared to PBS controls. Importantly, Sendai infection had no impact on the number of systemic flu-specific memory CD8 T cells in the spleen. Together, these data show that lung inflammation induced by unrelated respiratory infections can promote the loss of pre-existing lung TRM and may explain the gradual loss of cellular immunity in the lung.
West Nile virus antagonism of a RIG-I, type I interferon, and STAT5 signaling axis in human dendritic cells
Authors: Bowen James R , McDonald Circe E , Suthar Mehul S
Published: January 2023

West Nile virus (WNV) is a neurotropic flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Murine models have found dendritic cells (DCs) are pivotal for viral control and programming of antiviral immunity in a RIG-I like receptor (RLR)- and type I interferon (IFN)-dependent manner. To better understand human WNV infection, we evaluated infection of monocyte derived DCs (moDCs). To define the antiviral landscape within moDCs, we combined mRNA sequencing with weighted gene co-expression analysis. Using molecular signatures for RLR and IFN signaling as filters, we found WNV partially induced expression of genes related to antiviral immunity. Specifically, WNV induced delayed IFN responses and failed to up-regulate inflammatory (IL-1β, IL-6, IL-12, TNF) or T cell co-stimulatory molecules (CD86, CD40, MHC II). Using cis-regulatory sequence analysis, STAT5 was identified as a regulatory node involved in RLR- and IFN-induced DC responses, but was minimally activated during WNV infection. Infected DCs failed to secrete multiple STAT5 signaling cytokines (GM-CSF, IL-4, IL-15) and STAT5 was not phosphorylated during WNV infection. Mechanistically, WNV blocked STAT5 phosphorylation downstream of RIG-I, IFN, and IL-4 signaling. Zika virus (ZIKV), a closely related flavivirus that also fails to activate human DCs, also blocked STAT5 phosphorylation. Our findings suggest STAT5 antagonism is a conserved mechanism used by pathogenic flaviviruses to subvert host immunity. In summary, we employed a systems biology approach to define the antiviral landscape within human DCs, identifying viral antagonism of a RIG-I, IFN, and STAT5 signaling axis that subverts DC immunogenicity during WNV and ZIKV infection.
Suppression of type-2 mediated allergic diseases by dectin-1
Authors: Gour Naina , Lajoie Stephane , Wills-Karp Marsha
Published: January 2023

Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental proteins. Arthropods, such as mites, cockroaches, and crustaceans, are a major source of allergens, yet the majority of us live with or consume these invertebrates with no ill effects. Recent discoveries have raised the possibility that altered innate immune recognition of allergens, through pattern recognition receptors (PRR), may underlie disease susceptibility. Here, we identify the PRR, dectin-1 (CLEC7A), as a key receptor in mediating protection against dust mite-driven type 2 immune responses through the inhibition of epithelial cell IL-33 production, and the downstream recruitment of IL-13-producing innate lymphoid 2 cells (ILC2). Surprisingly, we observed that this effect was not mediated by the prototypical dectin-1 ligand, β-glucan, within allergen extracts. We discovered a novel protein ligand conserved across the arthropod phyla, sensing of which confers protection against aberrant type 2 responses that drive respiratory allergy and food anaphylaxis in mice. While this homeostatic pathway is functional in respiratory epithelial cells from healthy humans, dectin-1 is repressed in asthmatics and patients with chronic-rhinosinusitis. Importantly, we report a novel SNP in CLEC7A in the SAGE and GALAII GWAS studies, associated with lung function. Collectively, our findings reveal a previously unrecognized negative regulatory pathway in which ligation of dectin-1 results in reduction of type 2 immune responses, while disruption of this critical regulatory circuit is associated with susceptibility to a spectrum of allergic disorders.
Studies on the mechanisms of nickel induced immunosuppression
Authors: Pandolfi Franco , Altamura Simona , Nucera Eleonora , Frosali Simona , Rizzi Angela , Di Rienzo Alessia , Pascolini Lucilla , Buonomo Alessadro , Carusi Valentina , Chini Raffaella , Schiavino Domenico
Published: January 2023

Nickel (Ni) inhibits lymphoproliferative responses. Proliferation was performed on PBMC of 20 controls and 10 patients with Ni allergy. NiSO4 was used at a concentration of 0.1mM. PHA was added at a concentration of 5 μg/ml. Cells were incubated for 5 days and proliferation assessed by CFSE staining. At 0.1mM Ni strongly reduced (>40%) PHA-induced proliferation. The inhibition decreases in a dose-dependent manner and response to PHA is restored at 20 mM Ni. Ni alone induces small (<5%) proliferative response in both allergic and non allergic donors. Nickel inhibition requires the presence of Ni in the supernatant and is abrogated when Ni is incubated with the cells for up to 4 hours and the supernatant is washed.

Strong inhibition was also observed in preliminary experiments with tuberculin purified protein derivative (PPD) stimulated PBMC.

Staining for CD8 on CFSE cultures showed that PHA response was due to both CD8 and CD4 cells. Ni increased % of CD4 cells and decreased the % of CD8 cells.

PPD response was predominantly a CD4 response, with Nickel reducing the % of CD4 cells at higher concentations, while unaffecting the CD8.

Ni inhibits IL-6 production essential to normal PHA response. The addition of 10–100 IU of IL-6 and/or IL-2 to the cultures did not affect Ni inhibition of PHA proliferation. Addition of IL-6 increased CD25+ cells.

Our data suggest that Ni induced inhibition of PHA response is due to steric interference between PHA and its cellular ligand. Further studies will clarify the precise mechanism of Ni mediated inhibition of immune responses and its the role in the pathogenesis of Ni allergy.
Impaired BCR-induced NFAT1 nuclear translocation is implicated in low TACI expression in newborn B lymphocytes
Authors: Sakai Jiro , Coleman Adam S , Akkoyunlu Mustafa
Published: January 2023

Neonates and infants have impaired immune responses to immunization compared to adults, resulting in high susceptibility to microbial infection. B cells have a central role in humoral immunity to immunization and infection, and signals via B cell receptor (BCR) are essential for the development and maintenance of B lymphocytes. T cell-independent type 2 (TI-II) antigens activate B lymphocytes through BCR and transmembrane activator and CAML interactor (TACI). Newborns, however, are unable to generate TI-II responses, partly because TACI expression is severely reduced in newborn B cells. Since BCR signaling regulates TACI expression, we hypothesized that suboptimal BCR activation may be responsible for the reduced TACI expression on newborn B cells. We found that engagement of murine newborn BCR with anti-mouse IgM f(ab′)2 results in ablated Nuclear Factor of Activated T-cells 1 (NFAT1) nuclear translocation as compared to adult BCR. Neither anti-IgM stimulation nor the calcium influx inducer, Ionomycin stimulation elicits a significantly different calcium influx profile between newborn and adult B cells, suggesting that the factors downstream of calcium influx are responsible for the suppression of NFAT1 activation. Diminished NFAT1 translocation is likely responsible for the reduced TACI expression in newborn B lymphocytes because blocking NFAT1 pathway with the selective NFAT inhibitor, FK506 prevents the upregulation of TACI in anti-IgM-stimulated adult B lymphocytes. We are currently investigating the activities of calcium-dependent NFAT1 activators calmodulin and calcineurin in newborn B lymphocytes to assess whether insufficiencies in these molecules are responsible for the ablated translocation of NFAT1.
K+ Channel Activation Promotes Tumor infiltrating Lymphocyte (TIL) Expansion and Enhances Expression of CCR7
Authors: Ritthipichai Krit , Machin Marcus , Simpson-Abelson Michelle , Mosychuk Christopher , Veerapathran Anand , Lotze Michael
Published: January 2023

Only two K+ channels are known to be expressed by T-cells. Activated effector T cells express high levels of Kv1.3; activated naïve and central memory T-cell subsets express high levels of KCa3.1. Overexpression of Kv1.3 in murine T-cells increases K+ efflux, improves effector function (IFNγ production) and promotes anti-tumor activity1. Inhibition of KCa3.1 suppresses murine T-cell proliferation and cytokine production. In human PBMCs and TIL, KCa3.1 expression was relatively low with upregulation observed within 24 h following stimulation with anti-CD3 and anti-CD28. The addition of the KCa3.1 agonist SKA-31 did not alter KCa3.1 expression but enhanced CCR7 expression significantly on TIL and human PBMCs. TILs propagated in the Rapid Expansion Protocol (REP) had a 1.42-fold greater expansion (p=0.002) in the presence of SKA-31 and significant increases in the CD8+CD28+ (p=0.04), CD8+CD27+(p=0.04), and CD8+CD27+CD28+subsets (p=0.002), consistent with a less differentiated phenotype. We thus demonstrate that SKA-31 treatment enhances CCR7 expression associated with memory cells, promotes TIL expansion, and attenuates T-cell differentiation. Targeting the KCa3.1 channel is a novel strategy to expand and sustain less differentiated TILs and may improve the clinical application of adoptive T-cell therapy.

1: Eil R, Vodnala SK, Clever D, Klebanoff CA, Sukumar M, Pan JH, Palmer DC, Gros A, Yamamoto TN, Patel SJ, Guittard GC, Yu Z, Carbonaro V, Okkenhaug K, Schrump DS, Linehan WM, Roychoudhuri R, Restifo NP. Ionic immune suppression within the tumour microenvironment limits T cell effector function. Nature. 2016;537):539–543.
Reprogramming of exhausted T cells following elimination of chronic antigen exposure
Authors: Abdel-Hakeem Mohamed , Khan Omar , Stelekati Erietta , Ali Mohammed-Alkhatim , Wherry E. John
Published: January 2023

T-cell exhaustion is a hallmark of immunological failure to control cancer and chronic infection. Blocking inhibitory receptors such as PD-1 can re-invigorate exhausted T cells (TEX), but clinically most patients fail to achieve durable tumor control. Thus, a deeper understanding of other mechanisms underlying reversal of T-cell exhaustion is needed. It is also unknown whether antigen elimination would allow recovery of TEX into durable functional cells. To address this, we investigated whether TEX become “reprogrammed” into more functional effector or memory T cells following non-immunological antigen elimination. Using the lymphocytic choriomeningitis virus (LCMV) model we adoptively transferred TEX from chronically-infected mice to immune mice, thus eliminating chronic exposure to viral antigens. We aim to determine the cellular profiles, molecular mechanisms, and population dynamics of recovering T cells (TREC). Our data indicate that some markers and capacities of memory T cells may be recovered upon cure of infection, though other aspects of TEX biology do not appear to be corrected simply by eliminating exposure to chronic infection. Ongoing studies are investigating whether these changes are linked to selective recovery of a specific subset of TEX, and whether improvements are accompanied by changes in the molecular pathways of these TREC. We expect these studies to enhance our understanding of the molecular signatures and mechanisms of recovery of TEX, and to identify candidate transcriptional circuits differentially regulated in TREC that could represent novel therapeutic targets for reversal of immune exhaustion.
Myeloid PPAR-γ suppresses acute host diseases and inhibits chronic fibrosis development following influenza infection
Authors: Sun Jie , Huang Su , Zhu Bibo , Jiang Li
Published: January 2023

Influenza virus is the leading cause of respiratory infection. In addition to its induction of acute pulmonary diseases, severe influenza infection can lead to the development of chronic lung conditions including pulmonary fibrosis. Currently, the underlying mechanisms regulating the development of influenza sequelae in the respiratory tract are poorly defined. We found that PPAR-γ expression in macrophages is differentially regulated during low pathogenic (sublethal) or highly pathogenic (lethal) influenza infection. Myeloid deletion of PPAR-γ resulted in increased host mortality and morbidity following influenza infection. In the acute phase, myeloid PPAR-γ deficient mice exhibit enhanced macrophage cytokine production, diminished T cell immunity (day 10), delayed viral clearance and impaired lung repair responses following influenza infection. Strikingly, myeloid PPAR-γ deficiency also triggered the development chronic pulmonary fibrosis following influenza infection. We demonstrated that myeloid PPAR-γ deficiency caused scar formation, collagen deposition and fibrotic gene expression in the lungs at 60 days post infection. Mechanistically, myeloid PPAR-γ expression is required for the regeneration of tissue resident alveolar macrophages following influenza clearance, and the depletion of alveolar macrophages at the recovery stage resulted in impaired lung repair and chronic collagen deposition. Thus, our data has revealed that myeloid PPAR-γ expression is critical for the suppression of both acute and chronic pathogenesis of influenza, and suggested that the promotion of PPAR-γ activation may be employed to treat both acute influenza-associated diseases and to prevent chronic fibrosis development.
NKT cells control tumor associated macrophages and metastatic growth in neuroblastoma
Authors: Courtney Amy N , Tian Gengwen , Marinova Ekaterina , Wei Jie , Guo Linjie , Jin Jingling , Gao Xiuhua , Ghaghada Ketan B. , Heczey Andras , Metelitsa Leonid S
Published: January 2023

Va24-invariant NKT cells (NKTs) control tumor growth via poorly understood interactions with CD1d-positive tumor-associated macrophages (TAMs). TAMs comprise M1- and M2-like subsets, but only CD163high M2-like TAMs are associated with poor outcome in neuroblastoma (NB) patients. Here, we demonstrate that NKTs selectively target M2-like TAMs via contact-dependent and independent mechanisms. Upon direct contact with antigen-pulsed M1 or M2, NKTs selectively kill the latter. Additionally, we found that antigen-activated NKTs could reprogram M2 into functional M1-like macrophages via GM-CSF production. Furthermore, adoptive transfer of human NKTs resulted in M1-like polarization of TAMs in metastatic NB xenografts in humanized NSG mice. To further explore the role of NKT–TAM axis in tumor immune surveillance, we examined the effect of NKT deficiency on tumor progression and TAM accumulation in NB-Tag transgenic model of NB. Mice lacking either type I (Jα18−/−NB-Tag) or all (CD1d−/−NB-Tag) NKTs had shortened survival compared with NB-Tag mice (P < 0.0002). At four month of age we observed an increase of CD11b+Ly6G-Ly6C-F4/80+ TAMs in primary tumors of NKT deficient groups compared with NB-Tag mice. Despite no difference in the size of primary adrenal tumors between groups, the increase of TAM frequency coincided with metastatic spread in NKT deficient groups as detected by CT imaging and confirmed by pathological analysis. By five months, nearly all mice in NKT deficient groups had distant metastases in liver and lungs but none of the NKT replete mice had detectable distant metastases. Thus, our results reveal a novel mechanism of immune regulation, in which NKTs selectively control M2-like TAMs and suppress tumor metastasis.
Dampened inflammation with ruxolitinib treatment leads to CD8+ T cell exhaustion and induction of T regulatory cells in a murine model of HLH
Authors: Albeituni Sabrin H , Verbist Katherine , Tedrick Paige , Tillman Heather , Nichols Kim
Published: January 2023

Hemophagocytic lymphohistiocytoses (HLH) is a heterogeneous group of disorders characterized by severely damaging immune responses. In HLH, activated CD8+ T cells and macrophages overproduce pro-inflammatory cytokines, which contribute greatly to morbidity and mortality. In a murine preclinical model, HLH is recapitulated by infecting perforin knockout mice with lymphocytic choriomeningitis virus (LCMV). The inability of CD8+ T cells to kill infected cells results in a high production of inflammatory cytokines and ultimately death. We recently reported that treatment with the JAK1/2 inhibitor ruxolitinib significantly reduces the manifestations of HLH in this model. In this study, we sought to: 1) compare the effect of ruxolitinib treatment with IFN-γ blockade (currently in clinical trials) in the acute phase and 2) determine the long-term effects of treatment upon discontinuation. On day 9 p.i., ruxolitinib treatment was superior to IFN-γ blockade in reducing splenomegaly, numbers of inflammatory foci in the liver, LCMV-specific hepatic CD8+ T cells, and splenic IFN-γ+TNF-α+ CD8+ T cells. Surprisingly, on day 35 p.i., ruxolitinib treated mice achieved 100% survival, while only 20% of the IFN-γ Ab-treated mice survived. Since ruxolitinib dampens inflammation, we hypothesized that lower inflammation and viral persistence would induce T cell exhaustion. Indeed, CD8+ T cells stimulated with LCMV-specific peptide produced less IFN-γ and TNF-α. In addition, a significant population of CD8+CD44hi T cells co-expressed PD-1 and Tim-3. This was also correlated with enhanced percentages of Foxp3+CD4+ Tregs in treated mice. Overall, this study provides evidence and mechanistic insight for ruxolitinib as a novel treatment for HLH.
Blockade of critical immune regulators results in different roads to pathogenesis in Primary Sjögren’s syndrome
Authors: Voynova Elisaveta Ninova , Braley-Mullen Helen , Ettinger Rachel
Published: January 2023

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease that affects exocrine glands resulting in severe dryness of mucosal surfaces, primarily the mouth and eyes. Female, but not male NOD.H-2h4 mice develop pSS-like manifestations in aged mice. Previously, we have shown that pSS disease that arise in these mice is driven by early peripheral dysregulation that manifests as spontaneous germinal centers (sGC) that are dependent on CD40/CD40L interactions (Mahmoud, T et al., Sci Transl Med 2016). We now examined if other key immune pathways also play a role in pathogenesis of pSS. We found that NOD.H-2h4 mice deficient in either CD28/IFNg or PD1/IFNg developed exacerbated pSS manifestations, compared to the NOD.H-2h4 wild type (WT) mice. As in the WT mice, a single treatment with anti-CD40L mAb in CD28/IFNg−/− mice diminished both sGC and salivary gland tertiary lymphoid structures (TLS). Unexpectedly, however, the aggressive disease noted in PD1/IFNg−/− animals was not similarly inhibited by treatment with anti-CD40L mAb. While anti-CD40L dissolved the sGC, it had no effect on the TLS that develop in the salivary glands of these PD1/IFNg−/− mice. The different results following anti-CD40L mAb treatment in CD28/IFNg−/−vs. PD1/IFNg−/− mice is likely not due to lack of functional Tregs in CD28/IFNg−/− mice, as while transient depletion of Tregs in PD1/IFNg−/− mice resulted increased salivary gland TLS, anti-CD40L still had minimal impact on disease.
A serum-free medium for differentiation of monocytes to macrophages
Authors: Tabatabaei-Zavareh Nooshin , Wang Tinya , Khan Fareeha , Karas Evan A , Le Fevre Tim A , Szilvassy Stephen J , Thomas Terry E , Eaves Allen C , Wognum Albertus W
Published: January 2023

Macrophages play an important role in defense against pathogens and in tissue homeostasis. They are classified into two main groups depending on how they are activated: 1) M1 (activated by IFN-γ and LPS) and 2) M2, which include M2a (activated by IL-4 or IL-13), M2b (immune complexes with IL-1β or LPS) and M2c (IL-10) subsets. It is difficult to control macrophage activation and polarization in cultures containing serum, in which variable amounts of M1- and M2-specific cytokines and other factors may be present. Here we describe a serum-free culture system that selectively supports the differentiation of monocytes into either M1 or M2a macrophages. Monocytes were isolated using EasySep immunomagnetic cell separation and cultured for 4 or 6 days in serum-free ImmunoCult medium with 50 ng/mL M-CSF. The cells were then stimulated by adding 10 ng/mL LPS plus 50 ng/mL IFN-γ for M1 or 10 ng/mL IL-4 for M2a macrophage polarization and cultured for 2 more days. The yields of M1 and M2a macrophage were 51±18% and 58±14% (mean±SD), respectively. M1 macrophages expressed high levels of CD80 and CCR7 (96±3% and 62±15%, n=57), but very low levels of CD206 and CD209. In contrast, M2a macrophages expressed CD206 and CD209 (90±10% and 92±13%, n=59), but were negative or low for CD80 and CCR7. M1 macrophages produced TNF-α and IL-12 (mean±SEM: 2821±396 and 656±86, pg/mL, n=24). M2a macrophages produced small amounts of IL-10 (29±6 pg/mL, n=21). Both M1 and M2a macrophages were functional as demonstrated in a phagocytosis assay using fluorescently-labeled E.coli. By selecting appropriate stimuli this culture method can be easily adapted to generate other macrophage subsets as well and should prove useful for the study of macrophage biology.
The Effects of Childhood, Adolescent and Adult Obesity on Epithelial T Cell Homeostasis in the Intestine
Authors: Limon Natalie M
Published: January 2023

According to the CDC, 30% of adults in the U.S population are obese, while 17% of children and adolescents are obese. Obesity has resulted in a wide array of complications including disruption of barrier permeability as well as problems with tissue repair. The epithelial layer of the intestines contains intraepithelial intestinal lymphocytes (IEL) that are important in maintaining epithelial homeostasis and repairing tissue. To outline the mechanism by which obesity disrupts intestinal epithelial function among different age groups, childhood, adolescent, and adult mice were placed in a high fat diet (HFD) for 7 weeks to observe IEL number and function. In all age groups, mice administered a HFD exhibit a significant decrease in IEL within the epithelial layer of the small intestine. Notably, in the childhood cohort, IEL seeding is disrupted resulting in limited IEL numbers during adolescence. Interestingly, T cells in the epidermis of the skin are not reduced in number after 7 weeks of HFD suggesting that the intestine is more sensitive to early obesity. This study shows the harmful impact of obesity on the immune system in the small intestine whether obesity occurs in youth or in adulthood.
IL-2 producing and non-producing effector CD8 T cells phenotypically and transcriptionally coalesce to form memory subsets with similar protective properties.
Authors: Kahan Shannon M , Bakshi Rakesh K , Luther Rita , Harrington Laurie E , Hendrickson R. Curtis , Lefkowitz Elliot J , Weaver Casey T , Zajac Allan J
Published: January 2023

Interleukin-2 (IL-2) acts as a differentiation factor that drives effector CD8 T cell formation and also programs robust memory recall responses. However, the roles of intrinsic IL-2 production in shaping effector and memory CD8 T cell differentiation remain uncertain. To address this, we utilized novel IL-2 and IFN-γ double cytokine reporter systems to trace the cytokine production profiles and fate decisions of effector and memory virus-specific CD8 T cells. Notably, effector and memory CD8 T cells segregate into populations that co-produce IL-2 and IFN-γ or populations that produce only IFN-γ. IL-2-producing effector CD8 T cells preferentially survive and develop memory traits, including the ability to mount protective recall responses. These cells also display transcriptional profiles more closely associated with memory formation. Additionally, cells that produce IL-2 appear refractory to signaling by IL-2 through their STAT5 pathways, which potentially directs their developmental choices. These data illustrate that the intrinsic ability of effector CD8 T cells to produce IL-2 can dictate their sensitivity to the cytokine milieu, predict their developmental fates, and forecast their ability to mount vigorous secondary responses, resist exhaustion, and protect the host. Despite having distinct properties during the effector phase of the response, IL-2-producing and non-producing CD8 T cells transcriptionally and phenotypically coalesce as memory matures to form subsets which respond similarly to secondary lymphocytic choriomeningitis virus challenge. Thus once memory is established, robust secondary responses are not limited to polyfunctional IL-2-producing virus-specific CD8 T cells.
The human γδ T cell repertoire is shaped by age, tissue compartmentalization and viral exposure
Authors: Sant Sneha Ashok , Dash Pradyot , Wang Zhongfang , Pizolla Angela , Ngyuen Oanh , Koutsakos Marios , Lappas Martha , Crowe Jane , Loudovaris Tom , Mannering Stuart I , Westal Glen P , Wakim Linda , Xu Jianqing , Thomas Paul G , Loh Liyen , Kedzierska Katherine
Published: January 2023

γδ T cells can provide immunity against foreign pathogens and internal tissue malignancies. Despite numerous studies in mice, the potential role of γδ T cells in human viral infections and their TCR profiles are understudied, especially at different tissue locations. We utilised a single-cell multiplex-nested-RT-PCR to dissect the TCRγδ repertoire (n=372 γδ pairs) in healthy individuals across three major age groups (neonates, adults and elderly donors) in peripheral blood and tissues (spleen, lymph node and lungs). Neonatal γδTCRs display diverse TCRγδ repertoires, while adults show an enrichment of γ9δ2 TCRs with diverse CDR3 regions. Strikingly, elderly donors displayed distinct TCRγδ profiles with large clonal CDR3 expansion. Tissue-associated TCRγδ usage differs markedly from γ9δ2 cells that dominate the adult peripheral blood with more restricted CDR3 regions. Using our established in vitro co-culture assay of influenza virus infection in lung epithelial cells, we found that human γδ T cells respond to influenza infection by IFN-γ production and enrichment of γ9δ2 T cells. Our findings further support the role of monocytes and IL-15 in γδ T cell activation after influenza infection. Our study provides novel insights into the composition and diversity of TCRγδ repertoires and demonstrates how human γδ TCRs are shaped by the age, tissue compartmentalization and viruses. Our findings have future implications for immunotherapies involving γδ T cells in patients across different ages.
Non-cytopathic bovine viral diarrhea virus 2 induce autophagy in MDBK cell
Authors: Du-Gyeong Han , Kyoung-Seong Choi
Published: January 2023

Objective

Bovine viral diarrhea virus (BVDV) is classified into two distinct species, BVDV1 and BVDV2. Both species consist of two biotypes, cytopathic (cp) and non-cytopathic (ncp). Autophagy is an essential control process by which cells degrade unnecessary or dysfunctional cellular components or organelles in the cytoplasm. In this study, we explored autophagy activity in ncp BVDV2-infected MDBK cells.

Materials and methods

Ncp BVDV 2 (12F001) strain was cultured in MDBK cell and titration of the 50% tissue culture infective dose (TCID50) was conducted using the Kärber method. Transfection with GFP-LC3 was performed using the LipofectamineTM 2000 reagent. MDBK cells (8′105) were seeded in 6-well plates, infected with ncp BVDV2 or treated with rapamycin or DMSO, and then incubated. Subsequently, cells were used for western blot for LC3B, p62/SQSTM1, ATG14, and β-Actin.

Results

Our results showed that LC3 protein expression levels were increased in ncp BVDV2-infected MDBK cells by western blot and confocal microscopy. This expression was high in rapamycin-treated cells, followed by ncp BVDV2 infection, and DMSO treatment. We determined autophagy activity in ncp BVDV2-infected MDBK cells by assessing the protein levels of SQSTM/p62, LC3 and ATG14 by western blot. The decreased levels of SQSTM/p62 were accompanied by an enhanced expression of LC3 and ATG14 in ncp BVDV2-infected MDBK cells.

Conclusion

These results suggest that ncp BVDV 2 infection activate autophagy in MDBK cells. We conclude that autophagy may be linked to the establishment of persistent infection through virus survival. Thus, further studies should be needed to determine the association between autophagy and virus replication.
Increased generation of regulatory T cells in the presence of reduced T cell receptor signaling capacity correlates with a reduction in positive selection of thymocytes
Authors: Palin Amy , Lee Jan , Oliva Miles , El Khoury Dalal , Love Paul E
Published: January 2023

The T cell antigen receptor (TCR) is the primary signal transducing structure expressed on thymocytes and mature T cells, transmitting signals necessary for maturation and activation. The TCRα and β chains are rearranged from germline gene segments to generate a highly variable repertoire of antigen receptors. Thymocytes undergo a selection process in the thymus to ensure presence of a functional, but not highly autoreactive, T cell receptor on their surface. These processes, and T cell function, depend on the invariant CD3 molecules to transduce intracellular signals from the surface TCRαβ chains. We have created a knock-in mouse with Cre-recombinase-inducible tyrosine (6Y)-to-phenylalanine (6F) mutations in the 6 Immunoreceptor Tyrosine Activation Motif (ITAM) tyrosines of the 2 CD3ζ chains of the TCR/CD3 complex, resulting in a theoretical 60% decrease in TCR signaling potential. We found that pre-selection, but not post-selection, switching of wild-type (6Y) CD3ζ to non-signaling (6F) CD3ζ results in a significant increase in the percentage of thymic and peripheral regulatory CD4+ T cells (Tregs) relative to control germline 6Y CD3ζ mice. Mice with a pre-selection 6Y to 6F CD3ζ switch also have increased CD4+ CD25+ Foxp3− Treg precursors. This increase in Tregs and thymic Treg precursors is cell autonomous, and correlates with a reduction in positive, but not negative, selection of CD4SP thymocytes. Tregs with reduced signaling capacity do not, however, have suppressive capacity equivalent to wild-type Tregs, despite expressing normal levels of transcription factors and surface proteins characteristic of Tregs. Collectively, the data support an instructive, TCR-signal-based model of Treg lineage commitment in the thymus.
Necrotic liver induces pyroptosis through caspase-1 in eosinophils.
Authors: Macapagal Daphne , Connor Jane , Mustelin Tomas Mikael , Ramalingam Thirumalai , Wynn Thomas , Davidson Todd
Published: January 2023

During sterile inflammatory reactions, damage associated molecular patterns (DAMPS) are released from necrotic cells and recruit immune cells to the sites of tissue injury. We found that necrotic liver homogenates, when injected intraperitoneally, can recruit large numbers of immune cells. Among these immune cells were activated eosinophils. While eosinophils are well known to accumulate in the lungs of asthmatics or in response to helminths and parasites, several studies have shown the presence of eosinophils in liver biopsies of drug-induced liver injury, primary biliary cirrhosis, chronic hepatitis C, hepatic allograft rejection, and liver fibrosis. The prevalence of eosinophilic infiltrates in the liver points to a correlation between infiltration and disease outcome. Our results demonstrated eosinophil activation upon exposure to necrotic liver components. Exposure to necrotic liver cells induced eosinophil degranulation as measured by expression of CD107a and eosinophil peroxidase release into the supernatant as measured by ELISA. Further study of eosinophil activation during necrotic liver exposure showed IL-1β and IL-18 release as well as cell death. All of these responses were dependent upon caspase-1 activation. Caspase-1 mediated cell death accompanied by IL-1β and IL-18 release are hallmarks of pyroptosis, an inflammatory cell death pathway. We verified eosinophil pyroptosis in vivo using a liver fibrosis model involving Schistosoma mansoni infected mice. Eosinophils extracted from S. mansoni infected livers showed caspase-1 activation and cytokine release indicating that these cells undergo pyroptosis in vivo in response to hepatocyte injury.
Lyn/Fyn-STAT5 cascade in IgG-mediated inflammation
Authors: Paranjape Anuya R , Ryan John J
Published: January 2023

The Src family kinases Lyn and Fyn provide critical and antagonistic controls over mast cell responses to IgE and IgG mediated activation. Similarly, Fyn activates, while Lyn impedes IgE-induced STAT5 phosphorylation, which is critical for IgE-activated cytokine production. However, the role of STAT5B, the paralog that selectively influences cytokine production downstream of IgE, remains uncovered in the context of IgG-induced responses. Therefore, we examined the importance of STAT5 in IgG signaling, and broadened these studies to test the role for Lyn, Fyn, and Stat5 in IgG-mediated inflammation in vivo. Consistent with IgE, STAT5B KO mast cells produced significantly less cytokines and chemokines after IgG-induced activation in vitro. On the other hand, IgG-induced cytokine production in STAT5B KO macrophages was unaffected, indicating that the non-redundant role of STAT5B might be lineage-restricted. To expand these studies, we employed the K/BxN model of inflammatory arthritis, in which macrophages do play a critical role. In this model, Fyn or STAT5B deficiency did not alter arthritic inflammation. By contrast, Lyn deficiency significantly exacerbated arthritis. These studies indicate clinically relevant, lineage-restricted role for the Lyn/Fyn-STAT5 cascade, showing that inhibitory effects of Lyn kinase are critical in macrophage-driven inflammatory responses.
Induced negative regulation decodes PAMP quality and quantity to generate bacteria-specific inflammatory responses
Authors: Gottschalk Rachel A , Dutta Bhaskar , Krauss Kathleen , Dorrington Michael G , Martins Andrew J , Uderhardt Stefan , Tsang John S , Torabi-Parizi Parizad , Fraser Iain D.C. , Germain Ronald N
Published: January 2023

Toll-like receptors (TLRs) expressed on macrophages are ligated in various combinations by diverse pathogen-associated molecular patterns (PAMPs), but how mixtures of signals through these receptors are integrated to mediate distinct biological responses to different pathogens remains unknown. By stimulating macrophages using a wide matrix of TLR2/TLR4 concentration combinations, we identified conditions resulting in non-linear responses. Transcriptomic and RNAi analyses identified negative regulators induced in a TLR-specific manner that regulated cytokine production dynamics and chemokine milieus across the combinatorial matrix. TLR4 ligand concentration explained much of the variation in production of many chemokines. However, combinations of sub-maximal amounts of either single ligand produced a zone of non-linear responses with sustained production of select inflammatory mediators, due to lack of negative regulation induced only at higher individual ligand concentrations. A similar sustained response was observed for Gram-positive bacteria, as compared to a panel of Gram-negative species, which express high amounts of the TLR4 ligand LPS. The novel TLR4-specific negative regulators we describe thus aid the innate immune system in discriminating Gram-negative from Gram-positive species, supporting qualitatively distinct inflammatory responses to these classes of bacteria.

This research was supported by the Intramural Research Program of the NIH, NIAID
The regulatory actions of retinoic acid on M2a macrophage polarization of porcine and human myeloid-derived cells.
Authors: Dawson Harry D , Chen Celine , Urban Joseph F.
Published: January 2023

Previously we demonstrated that the most bioactive vitamin A metabolite, all-trans retinoic acid (ATRA) increased T helper 2-associated responses induced by Ascaris infection of pigs. We also demonstrated, on a limited basis, that ATRA potentiated the mRNA expression of several IL-4 induced chemokines (chemokine (C-C motif) ligand 11 ((CCL11), CCL17, CCL22 and CCL26) associated with alternative activation (M2a) in porcine macrophages in vitro. Herein, we describe several mechanisms whereby ATRA affects IL-4 signaling and extend these findings to porcine monocytes/macrophages and human macrophages. In human THP-1 cells, IL-4 exposure led to an increase in the mRNA for the M2a macrophage-associated chemokines, CCL11, CCL13, CCL17, CCL18, CCL22 and CCL26 as well as the M2a-associated surface markers, CD206, CD209 and CD274. ATRA synergistically increased IL-4–induced CCL2, CCL13, CCL18, CCL22 and CCL26 mRNA and CCL13, CCL18 and CCL26 protein levels while having no effect or slightly down regulating MRC1, CD209 and CD274 mRNA expression. The effects of ATRA on mRNA levels were greater at 24 compared to 48 hrs; however, greater differences in protein levels were observed at 48 hrs for CCL13, CCL18 and CCL26. In contrast to porcine monocytes and macrophages, ATRA decreased IL-4 induced CCL17 mRNA and protein expression in THP-1 cells. Thus, in porcine monocytes and macrophages and in human macrophages, ATRA selectively increased signaling in response to IL-4. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces.
Common Patterns of Gene Expression in Tissues of Patients with Systemic Lupus Erythematosus Imply Similar Pathways of Molecular Pathogenesis
Authors: Heuer Sarah Elise , Catalina Michelle , Robl Robert , Geraci Nicholas , Lipsky Peter , Grammer Amrie
Published: January 2023

The mechanisms underlying tissue pathology in SLE are not completely understood. To gain additional insight, gene expression profiles from lupus affected skin, synovium, and kidney were obtained and analyzed for commonalities. More than 300 arrays from lupus patients and controls were analyzed to determine differentially expressed (DE) genes (8279 discoid lupus skin, 5465 synovium, 6381 glomerulus, 5587 kidney tubulointerstitum). Notably, the majority of lupus tissue DE genes were detected in more than one tissue and 439 were differentially expressed in all tissues. Curated STRING-based interaction analysis of these 439 DE genes identified a number of functions and pathways that were commonly activated in all four tissues such as p38 MAPK and Ras signaling, T cell signaling, the IFN-signature, and hallmarks of antigen presentation. IPA identified 65 pathways shared among these tissues, including PI3K signaling, B cell receptor signaling, TLR signaling, IL-6 signaling, and T cell signaling. When DE genes in at least 3 of 4 tissues were examined, 1,379 and 1,066 genes that were commonly up-regulated and down-regulated, respectively, were identified. Curated STRING-based interaction analysis of the up-regulated genes in at least three of the four tissues identified several additional cellular and molecular processes, including neutrophil chemotaxis, T cell adhesion, DNA damage response, cell proliferation, and hallmarks of CD8+ T cells, monocytes, and Th2 cells. These results indicate that common cellular and molecular pathways can be identified in all of the affected lupus tissues, implying that related processes might be involved in the pathology of multiple organs in this autoimmune disease.
Development of new immune reagents for swine health, vaccine and disease studies
Authors: Manirarora Jean Nepomuscene , Bailey Michael , Renukaradhya Gourapura , Kenney Scott , LaBresh Joanna , Sang Yongming , Francis Ore , Wooldridge Linda , Lunney Joan
Published: January 2023

Current research efforts require a broad range of immune reagents; those available for pigs are limited. The goal of the US-UK Collaborative Swine Immune Toolkit Initiative is to generate priority immune reagents, based on international input, and pipeline them for marketing. UK researchers focus on mucosal targets, including production of monoclonal antibodies (mAbs) to chemokine receptors and IgE. Plans for mAbs to each 7-transmembrane G-protein coupled receptor (CCR3, CCR9, CCR10, CXCR1, CXCR6, F4/80) are to target peptides from extracellular loop 2 (ECL-2) based on threaded protein structures. These peptides have been designed for CCR3 and CCR9 and mAb plans are underway. US efforts are aimed at expression of soluble proteins and swine CD molecules, and production of panels of mAbs. The team has set up collaborations with commercial partners for protein expression and mAb production, and updated protocols to evaluate reagent specificity. New panels of mAbs reactive with IL-6, IFNβ and IFNγ are currently being evaluated. Immunizations are underway to produce panels of mAbs for CXCL10, CX3CL1 (fractalkine), IL-13, IL-17A, IL-17F, IFNω1 and IFNω5. Plans for mAbs to porcine CD1d using 4 synthetic peptides are in progress using CD1d knockout (KO) mice and screening on CD1d KO pig cells. Plans continue to use reagents produced to develop new assays for swine immune markers, e.g., multiplex assays and intracellular staining to provide the veterinary community with new commercial reagents and techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease and biomedical research efforts. Supported by USDA ARS, NIFA grant #2015-67015-23216 and BBSRC grant BB/M028232/1.