bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel self-regulatory mechanism of Th17 cells controls autoimmune uveitis through interleukin-24
Authors: Chong Wai Po , Mattapallil Mary , Raychaudhuri Kumarkrishna , Silver Phyllis B. , Jittayasothorn Yingyos , Chan Chi-Chao , Chen Jun , Horai Reiko , Caspi Rachel R
Published: January 2023

The Th17 response is critical in driving inflammation and is associated with many autoimmune diseases. However, clinical trials targeting the Th17 signature cytokine, IL-17A in some autoimmune diseases, including uveitis, have been disappointing. We investigated the role of IL-17A in determining the pathogenicity of uveitogenic Th17 cells. Unexpectedly, IL-17A deficiency in these cells did not reduce the severity of uveitis in recipient mice. We found that IL-17A deficient Th17 cells produced elevated amounts of other Th17-related cytokines, i.e. IL-17F, GM-CSF and IL-22. RNA-seq analysis and the follow up in-vitro experiments revealed that IL-17A exerts a negative feedback on Th17 cells by inducing them to produce IL-24, which in turn suppresses the production of Th17-related cytokines in an autocrine fashion. In vivo results showed that IL-24 treatment of recipients ameliorated Th17-induced adoptive EAU, and conversely, silencing IL-24 expression in retina-specific Th17 cells increased their pathogenicity, supporting the relevance of this pathway in vivo. Mechanistic studies confirmed that IL-17A activates the NFkB signalling pathway in Th17 cells, whereas site mutagenesis at the NFkB binding sites In IL-24 promoter abrogates the IL-17A-induced IL-24 expression. In vitro experiments implicated SOCS 1 and 3 in the downstream effects of IL-24. Importantly, we also observed a similar inhibitory IL-17A/IL-24 circuit in polarized human Th17 cells. We conclude that IL-17A exerts a negative feedback on Th17 cells by inducing autocrine IL-24, which limits their expression of other Th17-lineage cytokines and dampens their pathogenicity.
LAG3 limits the diabetogenicity of intra-islet CD8 T cells
Authors: Grebinoski Stephanie J , Zhang Qianxia , Cillo Anthony R , Szymczak-Workman Andrea , Workman Creg , Vignali Dario
Published: January 2023

Tight control of effector T cells is necessary to limit autoimmune diabetes (AD). One mechanism by which this is achieved is through inhibitory receptor (IR) expression. Lymphocyte Activating Gene 3 (LAG3) is one critical IR that limits disease progression and incidence of AD. Using a mouse model in which LAG3 deletion is restricted to CD8+ T cells, Lag3L/L-YFPE8iCRE-GFP.NOD, and wild type (WT) controls, E8iCRE-GFP.NOD, we find that CD8+ T cell-restricted deletion of LAG3 is sufficient to accelerate diabetes incidence and insulitis. Interestingly, we see Lag3L/L-YFPE8iCRE-GFP.NOD mice have an increased intra-islet CD8:Treg ratio, and increased tetramer (Nrpv7, IGRP mimotope) staining. These observations drive our hypothesis that LAG3 limits those autoreactive CD8+ T cell. 5′ single cell RNAseq in conjunction with TCRseq reveals that LAG3-deficient (Lag3−/−) intra-islet CD8+ T cells are transcriptionally unique compared to WT controls, exhibiting a more activated phenotype along with lower geneset enrichment for markers of exhaustion. This was validated by flow cytometry in which WT CD8+ T cells possess an expanded PD1Hi, Tox+ population, while Lag3−/− CD8+ T cells have an expanded PD1mid population, express more CD44, KLRG1, BrdU, Ki67, and less aCasp3. This indicates that WT CD8+ T cells are able to appropriately upregulate PD1 and Tox in response to chronic self-antigen stimulation, while Lag3−/− CD8+ T cells maintain a more activated population. Strikingly, LAG3 deletion on CD8+ T cells appears to be sufficient to overcome a normal “exhaustion-like” program in intra-islet CD8+ T cells, resulting in accelerated diabetes incidence. These data highlight an important role for LAG3 and CD8+ T cell exhaustion programs in AD.
Dissection of T-cell tolerance induction by dendritic cell surface glycoproteins
Authors: Dustin Michael , Saadawi Ahmed , Mayya Viveka , Cespedes Pablo , Valvo Salvatore , Chen Lina , Demetriou Philippos
Published: January 2023

Tolerogenic dendritic cells (DC) induce T-cell clonal deletion, anergy, generation and activation of regulatory T cells. In vitro production of TolDC has been accomplished, however the full molecular circuitry is not known. We aim to decipher the chemical signals and physical characteristics leading to T-cell tolerance in an artificial system. We have performed surface proteomic analysis of human peripheral blood cDC2 subset without and with in vitro stimulation with polyIC in vitro. The surface proteomics is based on biotinylation of sialic acids and the signal strength was 55-fold higher in the resting compared to the activated condition, presumably due to decreased sialation of glycoproteins in the activated cDC2. We found that label free quantification (LFQ), corrected for differences in sialation, agreed well with quantitative surface expression by flow cytometry. We combined the two datasets and ranked the top 120 glycoproteins with extracellular domains (ECD) > 60 amino acids by LFQ with approximately one third invariant, one third up-regulated/induced and one one-third down-regulated/lost on activation. The 120 ECD were expressed in HEK293 cells with C-terminal 10His tags and purified by Ni2+ affinity chromatography. Supported lipids bilayers (SLB) representing the tolerogenic and activating DC are being designed with different combinations of the 120 ECDs. We will adjust sialation to match resting and activated states. The SLB compositions will be tested for immunological synapse formation (fluorescent self-pMHC/CD80, ICAM-1 and CD58 for the cSMAC, pSMAC and dSMAC) and tolerance/activation spectrum with identification of relevant molecular combinations by machine learning.
UVB light exposure promotes IgE autoantibody production and skin autoreactivity in mice that express a lupus-associated isoform of Foxp3
Authors: Domeier Phillip P , Chang Sarah E , Spath Sabine , Utz Paul J , Zhou Baohua , Ziegler Steven F
Published: January 2023

Foxp3 activity is required for the maintenance of self-tolerance, but humans encode an additional exon 2-deficient splice variant isoform of FOXP3 with no known function. To determine how exon 2 of FOXP3 contributes to Treg activity, we generated mice that express exon 2-deficient FoxP3 (called B6.FoxP3ΔExon2). B6.FoxP3ΔExon2 mice develop mild autoimmunity (splenomegaly, autoantibodies and kidney nephritis) with significantly elevated titers of skin-reactive IgE. We also observed skin-reactive IgE antibodies in SLE patients with active photosensitivity, so we challenged B6 and B6.FoxP3ΔExon2 mouse skin with ultraviolet B (UVB) light to determine if UVB damage can induce skin-reactive IgE production. When compared to B6 controls, B6.FoxP3ΔExon2 mice developed greater skin inflammation with increased infiltration of Tregs (CD4+Foxp3+), effector T cells (CD4+CD44+Foxp3−) and IgE+ plasma cells (B220lowCD138+Blimp1+) at the site of UVB damage. B6.FoxP3ΔExon2 mice also had higher numbers of IgE+ antibody-forming cells, IL4-producing T cells (CD4+CD44+Foxp3−) and IgE-producing B cells, but lower numbers of CTLA4+ Tregs in draining lymph nodes from UVB-treated skin. Finally, UVB-treated B6.FoxP3ΔExon2 mice also had worsened kidney disease, phenocopying multi-systemic flares that occur in SLE patients with photosensitivity. Collectively, we found that exon 2 of the Foxp3 gene is required to control UVB-induced cutaneous inflammation though the regulation of type 2 immune activation and skin-reactive IgE. Thus, our findings suggest that photosensitivity reactions in SLE patients may be mediated by altered Foxp3 activity and skin-reactive IgE that could be therapeutically targeted to control systemic autoimmune flares.
Characterization of Aire-expressing DCs using a novel Aire-reporter strain
Authors: Matsumoto Mitsuru , Matsumoto Minoru , Miyazawa Ryuichiro , Morimoto Junko , Tsuneyama Koichi , Nishijima Hitoshi
Published: January 2023

Aire is predominantly expressed by medullary thymic epithelial cells (mTECs), and it is essential for maintaining self-tolerance by controlling the expression of tissue-restricted self-antigens (TRAs). Besides mTECs, extra-thymic Aire-expressing cells (eTACs) have been reported. However, the exact nature of eTACs has been difficult to study because of the small numbers together with low expression levels of Aire within the cells. We have recently established a novel Aire reporter strain in which endogenous Aire was replaced by the human AIRE-GFP-Flag tag fusion gene (Aire/hAGF-knockin: J. Immunol. 2015). hAGF-reporter protein was efficiently produced and retained within the mTECs as authentic Aire nuclear dot protein. With this high-sensitivity and high-fidelity Aire-reporter strain, we found that DCs in the spleen and lymph nodes expressed Aire although their expression levels were rather low. Aire-expressing DCs showed high levels of MHC-II and CD80/CD86, and half of which expressed CD8. In the thymus, we found that there were two types of DCs expressing Aire: hAGF-low DCs and hAGF-high DCs. Interestingly, hAGF-low DCs had small hAGF dots within the nuclei, whereas hAGF-high DCs retained larger hAGF dots which were mainly present outside the nuclei, suggesting that hAGF-high DCs had acquired hAGF protein from mTECs. Acquisition of hAGF protein by hAGF-high DCs from mTECs was confirmed by the bone-marrow transfer experiment. Mechanisms underlying the transfer of nuclear protein (i.e., hAGF protein) from mTECs to thymic DCs, and immunological consequence of this event are now under investigation.
Pathogenic role of plasmacytoid dendritic cells in the development and onset of Sjögren’s syndrome-like exocrinopathy in non-obese diabetic mice
Authors: Zhou Jing , Zhang Xiaofeng , Yoo Bora , Yu Qing
Published: January 2023

Plasmacytoid dendritic cells (pDCs), which are the most potent producers of type I IFNs, have been demonstrated to be critically involved in the induction of multiple autoimmune diseases. In Sjögren’s syndrome (SS), the presence of pDCs and activated type I IFN pathways has been shown in the salivary glands of patients, and the disease severity closely correlates with type I IFN activity. However, the particular role of pDCs in the pathogenesis of SS remains unknown. To investigate whether pDCs play a crucial role in the development and onset of SS, we administrated a pDC-depleting anti-BST2 antibody to female non-obese diabetic (NOD) mice, a spontaneous model of SS during the early stage of SS development, from 4 to 7 weeks of age. Mice were analyzed at 10 weeks of age around the clinical onset of the disease. Anti-BST2 treated mice had significantly higher salivary flow rate than IgG-treated control group, indicating the remission of SS-like xerostomia by pDC depletion. Meanwhile, anti-BST2 treatment inhibited leukocyte infiltration of submandibular glands (SMGs), as demonstrated by H&E staining. Consistent with these results, flow cytometric analysis provided evidence that administration of anti-BST2 not only lowered the frequency of total leukocytes, CD4 T cells, CD8 T cells, B cells and pDCs, but also reduced the proportion of CD44+CD4 T cells, CD44+CD8 T cells and CD86+B cells among the total SMG cells of NOD mice. These changes were accompanied by downregulation of CD40, CD40L, Lt-α, Lt-β, TNF-α, IFN-γ, CXCL9, CXCL11 and NOS2, as determined by RT2 Profiler PCR array, suggesting an attenuation of SMG inflammation. Taken together, pDC depletion at the early stage of disease development hinders the pathogenesis of SS-like exocrinopathy.
Helminth-induced modulation of neuroinflammation in a mouse model of multiple sclerosis
Authors: Fettig Naomi M , Horwitz Marc S , Osborne Lisa C
Published: January 2023

Environmental factors play an important role in multiple sclerosis (MS) disease progression, but the exact cause remains unknown. The hygiene hypothesis suggests that a lack of exposure to microbes may alter disease susceptibility. Helminths in particular may play a role in preventing autoimmune disease due to the type 2 immune response they typically induce. Trichinella spiralis (Ts) first forms a transient intestinal infection followed by chronic infection where it exerts type 2 and regulatory immune responses, respectively. To evaluate the role of helminth infection in development and progression of MS, C57BL/6J mice were first chronically infected with Ts, then immunized with MOG35–55 to induce experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Ts-infected EAE mice have an altered disease course compared to uninfected controls, demonstrating delayed onset, reduced incidence, and symptom remission. Despite an overall decrease in lymphocyte recruitment to the central nervous system (CNS) in Ts/EAE mice, elevated numbers of infiltrating Th2 cells are maintained through the EAE disease course. In contrast, regulatory T cell expansion is dependent on the presence or absence of EAE symptoms. Histological analysis of cellular infiltration and demyelination in the spinal cord indicate limited demyelination with reduced infiltration into the parenchyma of Ts/EAE mice compared to EAE controls. Further immunophenotyping in the CNS to evaluate the contributions of infiltrating macrophages will be performed, as well as analyses of the cytokine milieu in the CNS. These results suggest that chronic Ts infection limits neuroinflammation, as shown by reduced clinical, immunological, and pathologic signs of EAE.
Foxp3+ regulatory T cells play an indispensable role during glucocorticoid-induced treatment of autoimmune inflammation
Authors: Kim Dongkyun , Min Booki
Published: January 2023

Glucocorticoid (GC) is a class of steroid hormones with profound immunosuppressive and anti-inflammatory properties that is widely used as the first-line treatment option in chronic inflammatory conditions such as autoimmunity. Despite the broad usage, its working mechanisms remain largely unclear. Herein, we report that Foxp3+ CD4+ regulatory T cells (Tregs) play an irreplaceable role during GC-mediated treatment of experimental autoimmune encephalomyelitis (EAE), a murine autoimmune inflammation model in the central nervous system that resembles multiple sclerosis in human. Dexamethasone (Dex) administered at the disease onset or ongoing disease was unable to suppress EAE without Tregs, while adoptive transfer of Tregs restored Dex effects. Glucocorticoid receptor (GR) expression in Tregs was essential for Dex effects, suggesting that GC directly acts on Tregs. From RNA sequencing analysis we found that Dex treatment induced miR-342 expression only in Tregs but not in effector T cells. We also found that miR-342 targets the Rictor, a subunit of the mTORC2 complex. Inhibiting Dex-induced miR-342 expression in Tregs not only restored Rictor expression but also interfered with Treg-mediated suppression of EAE even with Dex treatment. While Dex-stimulated Tregs displayed robust oxidative phosphorylation rather than glycolysis as the energy source, miR-342 inhibition in Tregs reversed metabolic program to robust glycolysis. Taken together, this is the first report demonstrating that the miR-342-Rictor axis may represent a novel mechanism by which GC controls inflammation by metabolic programming in Tregs.
Peripherally-induced brain tissue resident memory CD8 T cells mediate protection against CNS infections
Authors: Urban Stina Lisa , Jensen Isaac J , Pewe Lecia L , Badovinac Vladimir P , Harty John T
Published: January 2023

The central nervous system (CNS) is classically viewed as an immune privileged site; however, recent advances in the field highlight the importance between the interaction of the peripheral immune system and the CNS in maintaining tissue homeostasis. Tissue resident memory (Trm) CD8 T cells have been described in almost every organ and are shown to provide a first line of defense against secondary invading pathogens. Trm CD8 T cells in the CNS have been described using multiple models of brain infections, but it is unknown whether CNS infection is required for development of brain Trm populations. Here, we show that peripheral infections and immunizations generate robust antigen (Ag)-specific CD8 memory T cells in the brain that adopt a Trm phenotype with a unique brain resident signature not prevalent in other organs. Upon peripheral antibody mediated depletion of circulating memory T cells, this brain signature was enriched without reducing proportion or number of CNS residing memory CD8 T cells. 2-photon microscopy revealed peripherally-induced Trm are highly dynamic in the CNS. CD8 T cells surveyed the tissue with multiple mechanisms including moving along the blood vessels, traveling long distances in apparently random fashion or remaining stationary. Most importantly, peripherally-induced Trm cells in the CNS showed enhanced cytokine production and provided protection against brain infections by reducing both bacterial and viral loads and morbidity and mortality associated with infections. These data increase our understanding of how peripheral immunization strategies can generate brain Trm populations and will guide potential use of T cells as therapeutic strategies against CNS infections and neurological diseases.
DAMP-mediated cardiac dysfunction: cardiomyocytes as actors and target of innate immune response
Authors: Weber Birte , Lackner Ina , Baur Meike , Gebhard Florian , Relja Borna , Marzi Ingo , Kalbitz Miriam
Published: January 2023

Objective

Severe trauma often led to a systemic inflammatory response, accompanied by release of danger associated molecular patterns such as the high mobility group box-1 protein (HMGB-1) and extracellular histones. The aim of this study was to clarify the influence of HMGB-1 and extracellular histones on human cardiomyocytes, in context of posttraumatic cardiac dysfunction.

Material and Methods

Cell viability, mitochondrial respiration and calcium handling of human cardiomyocytes in presence of extracellular histones or HMGB-1 were evaluated. A hemadsorption filter was tested to eliminate extracellular histones in vitro and from blood samples of multiple trauma patients (mean injury severity score >16).

Results

Incubation of human cardiomyocytes with HMGB-1 or histones were associated with impaired calcium handling, mitochondrial respiration and reduction of cell viability. Furthermore, human cardiomyocytes release further cardio-depressive cytokines in the presence of extracellular histones and HMGB-1 such as IL-1b and TNF. Hemadsorption of human plasma after multiple trauma showed a significant reduction of histone levels. Furthermore, in vitro analysis of hemofiltration capacity detected a dose dependant reduction of extracellular histones.

Conclusion

Despite their positive intention as part of the immune reaction, HMGB-1 and extracellular histones are associated with detrimental effects on cardiomyocyte viability, calcium handling and mitochondrial function. Moreover, presence of DAMPs resulted in additional release of cardiodepressive cytokines. Therefore, in the context of tissue trauma cardiomyocytes are target and actor at the same time.
Immunosenescence in Type 2 Diabetic Patients with Chronic Renal Disease
Authors: Shu Kai-Hsiang , Chiu Yen-Ling
Published: January 2023

Background

Immunosenescence is an important challenge for an aging population. It is known that patients with end-stage renal disease suffer from immunosenescence and premature T cell aging but whether such changes occur in diabetic patients with less severe chronic renal disease is unclear.

Methods

832 patients with type 2 diabetes with different levels of renal function were recruited in this study. Immunosenescence was analyzed by staining peripheral blood with two immunophenotyping panels and analyzed by multicolor flow cytometry.

Results

Out of all the 832 participants, there were 171 patients with stage 3 CKD and 46 patients with 4/5 CKD. Compared to patients with eGFR>60 ml/min, patients with more severe CKD showed progressive decreased total CD3+ T cell and CD4+ and CD8+ T cell, but not monocyte numbers. In addition, immunosenescence, as defined by various phenotypic markers, showed significant upregulation in both stage 3 and stage 4/5 patients. However, immunosenescence was not associated with proteinuria level nor worse glucose control. In age and sex adjusted regression models, stage 3 CKD patients already exhibited significant elevated percentages of CD28−, CD127− and CD57+ cells among CD8+ T cells when compared to patients with eGFR>60 ml/min. In addition, stage 3 CKD patients exhibited depressed HLA-DR and CX3CR1 expression in specific monocyte subpopulations.

Conclusion

Level of immunosenescence is not significantly associated with proteinuria nor glucose control in type 2 diabetes patients. Both T cell and monocyte compartment exhibit characteristics of immunosenescence during renal function decline, starting from stage 3 CKD.
The IgM and IgG subclass distribution of anti-Galactose-alpha-1,3-galactose and anti-N-glycolylneuraminic acid antibodies in healthy adults
Authors: Kang Hee Jung
Published: January 2023

Background

The major xenoreactive antibodies (Abs), anti-galactose-alpha-1,3-galactose (Gal) and anti-N-glycolylneuraminic acid (Neu5Gc), exist naturally in humans and can contribute to several immunological pathogenesis in xenotransplantation. In this study, we investigated the serum titers of anti-Gal and anti-Neu5Gc Abs including the IgM, IgG, and IgG subclasses in humans to gather basic population data before launching clinical xenotransplantation.

Methods

Serum titers of anti-Gal and anti-Neu5Gc Abs were measured in 380 healthy adults using an in-house-developed enzyme-linked immunosorbent assay. The detection frequency and distribution of anti-Gal and anti-Neu5Gc IgM, IgG and IgG subclass were evaluated.

Results

The detection frequencies of Abs were as follows: for anti-Gal, IgG, 95.0%; IgM, 99.2%; IgG1, 23.2%; IgG2, 94.5%; IgG3, 12.4%; and IgG4, 3.4%, and for anti-Neu5Gc, IgG, 96.6%; IgM, 87.4%; IgG1, 1.6%; IgG2, 46.3%. The median titers of anti-Gal IgG (1,198 ng/ml, range <401 – 128,758 ng/ml) and IgM (996 ng/ml, range <113 – 24,698 ng/ml) were significantly higher than those of anti-Neu5Gc Abs (IgG, 194 ng/ml, range <30 – 12,415 ng/ml; IgM, 328 ng/ml, range <154 – 6,514 ng/ml; all P<0.001). IgG2 of both anti-Gal and anti-Neu5G Abs correlated better with the IgG class than other IgG subclasses (rs = 0.762, P < 0.001; rs = 0.606, P < 0.001).

Conclusions

Most healthy adults possess anti-Gal and anti-Neu5Gc IgG and IgM in their sera. Overall, the titers of anti-Gal Abs are higher than those of anti-Neu5Gc Abs. IgG2 seems to be the main IgG subclass in both anti-Gal and anti-Neu5Gc natural Abs. Changes in the titers of these Abs would be a clue to understand an immunological pathogenesis in the recipients of xenotransplantation.
Obesity contributes to a dysfunctional regulatory T Cell phenotype within adipose tissue
Authors: Smith Alan J , Liu Joey , Yilmaz Ahmet , Wright Valerie , Bradley David , Hsueh Willa A
Published: January 2023

Adipose tissue (AT) regulatory T Cells (Tregs) play a key role in regulating inflammatory and metabolic responses. Obesity is associated with a decrease in AT Tregs as well as an increase in tissue inflammation and systemic insulin resistance in both humans and mice. We hypothesize that, in addition to the decrease in AT Treg number, there is a dysfunctional Treg phenotype caused by the increased inflammatory state within obese AT. We isolated Tregs from AT and peripheral blood(PB) from lean and obese individuals. Real-time PCR analysis was used to measure the expression of several known markers for T cell exhaustion. Protein expression of each marker was validated using FACS analysis. When compared to PB, there was a significant increase in PD-1 expression on AT Tregs. PD-1 and TIGIT were increased in Tregs isolated from obese vs. lean individuals, while CD127 expression was reduced. To determine if PD-1 expression correlates with Treg dysfunction, isolated AT and PB Tregs were added to CFSE-labeled PBMC. After 4 days culture, PB Tregs significantly inhibited proliferation but there was less suppression by AT Tregs. RNAseq analyses of PD-1 high vs. PD-1 negative AT and PB Tregs revealed a unique expression phenotype within AT Tregs. AT PD-1 high Tregs express altered metabolic and inflammatory pathways promoting loss of Tregs and increased inflammation. These data suggest that Treg exhaustion may explain the decreased Treg abundance in AT of obese individuals, which has important metabolic implications. (Partially supported by Foods for Health, a focus area of the Discovery Themes Initiative at The Ohio State University)
Characterization of DOCK8 as a novel gene associated with hemophagocytic lymphohistiocytosis
Authors: Cron Randy Q , Zhang Mingce , Cron Remy R , Absher Devin , Atkinson Prescott , Chatham Winn
Published: January 2023

Background

Hemophagocytic lymphohistiocytosis (HLH) is life threatening, presenting with fever, pancytopenia, coagulopathy, & multi-organ dysfunction (MOD). Familial HLH (fHLH) results from homozygous defects in perforin mediated cytolytic pathway genes used by NK & CD8 T cells. Up to 40% of secondary HLH (sHLH) patients have heterozygous defects in fHLH genes resulting in decreased cytolytic function, prolonged interaction with antigen presenting cells, & subsequent increased pro-inflammatory cytokines resulting in MOD. As NK cell dysfunction is common in HLH, we screened for novel gene associations.

Methods

By whole genome sequencing, 4 sHLH patients had mutations in DOCK8 (guanine exchange factor) critical to NK cell function. DOCK8 mutations, or wild-type (WT) sequence, were introduced into NK92 cell lines by foamy virus (FV), or by disrupting endogenous NK92 DOCK8 genes (CRISPR). WT & mutant DOCK8 NK92 cells incubated with K562 cells were compared for cytolysis, degranulation (CD107a), & cytokines [interferon-γ (IFNγ), tumor necrosis factor (TNF)].

Results

2 HLH patients had rare heterozygous DOCK8 mutations; 2 had the same DOCK8 polymorphism (D63N). 1 novel mutation was missense (A261V); 1 was a splice acceptor variant (c.54-1G>T, 0.03%). The A261V mutant decreased NK lytic activity by CRISPR or FV (50% vs. WT, p=0.007), & decreased CD107a by >50% (p=0.0129). The A261V DOCK8 NK cells increased expression of IFNγ & TNF by >200% (p=0.0192 & p=0.0027). The D63N NK cells had similar results. The DOCK8 splicing mutant disrupted splicing by exon trapping.

Conclusion

Heterozygous DOCK8 mutations contribute to HLH by dominant-negative or hypomorphic effects yielding decreased cytolysis & increased inflammatory cytokines.
Cell-specific transcriptional signature of human immune cells in GESTALT
Authors: Kaila Mary , Shapiro Benj , Dunn Christopher , Singh Amit , Bektas Arsun , Kim Jaekwan , Piao Yulan , Wood William , Nguyen Cuong , Wallace Tonya , Mckelvey Julia , Zukley Linda , Battle Alexis , De Supriyo , Weng Nan-Ping , Sen Ranjan
Published: January 2023

The Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing (GESTALT) study is designed to understand the basis of immune dysfunction that accompanies human aging. Twelve immune cell subsets were purified from hundred healthy donors of age range 20 – 90 and used for transcriptomic, proteomic and epigenomic analyses. Additionally, the chronic inflammatory components of aging were assessed by analysis of serum cytokines. The results were first used to identify cell-specific signatures of each omic study and determine their inter-relationships. These observations define lineage identity of human immune cells and change that occur with differentiation of adaptive immune cells to memory cells.
A triple “no” to the enrichment of immune genes at X files
Authors: Meester Irene , Manilla-Muñoz Edgar , Solís-Soto Juan Manuel , Paniagua-Frausto Gustavo A. , Carrión-Alvarez Diego , Rodríguez-Rangel Ximena , Ruiz-Rodríguez C. Orelli
Published: January 2023

Objective

As sex bias in immune function has been contributed partly to a preponderance of immune system-related genes (ISRG) on the X-chromosome, we performed a comparative study to verify this viewpoint.

Methods

The comparative study consisted of three complementary strategies:

Results

The X chromosome had less ISRG than the median of the autosomal chromosomes. Among X-linked genes, ISRG ranked 4th after genes dedicated to the reproductive and nervous systems or to development, proliferation and apoptosis (not ranking genes dedicated to basal metabolism nor genes of unknown function). On the Y chromosome, ISRG ranked 2nd, and at the pseudoautosomal region 1st. Various epigenetic controllers, X-linked MSL3 and Y-linked KDM5D and UTY, were preferentially expressed in leukocytes and deserve further attention with respect to a possible role in sex biased expression or its neutralisation.

Conclusions

The X chromosome is not enriched for ISRG. Thus, gene-related causes for sex bias in immune responses cannot be due to the X-linked ISRG number, but may be due to 1) particular sex chromosome-linked genes, 2) sex-biased regulation of the expression of ISRG in leukocytes and/or lymphoid organs. (Funding: UDEM, UIN19592)
A clinical trial comparing aliskiren with enalapril for the treatment of C3 glomerulopathy
Authors: Karpman Diana , Hallander Hilda , Kristoffersson Ann-Charlotte , Bekassy Zivile
Published: January 2023

Introduction

To date there is no established, effective treatment for C3 glomerulopathy, encompassing both Dense Deposit Disease and C3 glomerulonephritis. We have previously reported that renin mediates complement activation by cleaving C3 and found that the direct renin inhibitor aliskiren blocked complement activation by renin and inhibited complement activation systemically and in the kidneys in three patients with Dense Deposit Disease. Theoretically enalapril should increase renin without blocking its effect and therefore we will conduct a clinical trial to compare these two treatments.

Material and methods

We will include patients to be treated with aliskiren or enalapril (15 in each arm). Recruitment will take place in Swedish hospitals. Children 6 years or older with a glomerular filtration rate equal to or above 50 ml/min/1.73 m2 and adults equal to or above 30 ml/min/1.73 m2 will be included. Patients will be randomized for treatment with aliskiren or enalapril, as monotherapy or in combination with immune suppressive medications. After 6 months patients will switch to the other treatment and continue on the second treatment for an additional 2.5 years. Patients will be followed regularly regarding renal function, proteinuria, blood pressure, activation of the renin-angiotensin system and complement activation assays. After 1–3 years, when medically indicated or at the end of the study, a repeat renal biopsy will be performed to validate the effect of treatment on renal morphology and complement deposition.

Conclusions

The results of this study may provide more scientific evidence for, or against, the use of aliskiren in patients with C3 glomerulopathy.
Single cell multiplex mass cytometry reveals differential abundance of specific cytotoxic memory and regulatory T cell populations in human hypertension
Authors: Alexander Matthew , Dale Bethany L , Elijovich Fernando , Wogsland Cara E , Irish Jonathan M , Madhur Meena S
Published: January 2023

Emerging evidence from animal models demonstrates the importance of a variety of innate and adaptive immune cells in the pathogenesis of hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets is altered in human hypertension reflecting disease pathophysiology. We thus performed high dimensional, single cell profiling of peripheral blood mononuclear cells in humans with a panel of 31 cell surface markers using mass cytometry. Unsupervised computational analysis from 11 control and 10 hypertensive individuals matched for age, gender, race, and body mass index revealed an approximately two-fold increase in a novel memory helper CD62L+CCR7−CD161lo T cell subset in hypertensive subjects. In addition, manual gating for major known immune cell populations revealed a 40% decrease in memory cytotoxic T cells and a 29% decrease in regulatory T cells in hypertensive individuals. Unsupervised analysis of the cellular subsets of these populations using Phenograph demonstrated a selective 63% decrease in PD-1+ memory cytotoxic T cells and 49% decrease in CCR10+ regulatory T cells in hypertension. In a validation cohort, circulating PD-1+ memory cytotoxic and CCR10+ regulatory T cells were significantly decreased by 30% and 50%, respectively, without a difference in the memory helper T cell subset. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1+ memory cytotoxic and CCR10+ regulatory T cell populations in human hypertension. These findings may provide new insights into hypertension pathogenesis and potential therapeutic targets.
The expression and function of complement inhibitory protein CD55 in chronic rhinosinusitis with nasal polyp
Authors: Ruan Jianwen , Wang Hai , Liu Zheng
Published: January 2023

Background

The factors that regulating classic complement activation in chronic rhinosinusitis with nasal polyps (CRSwNP) remain largely unknown. CD55 functions as a complement regulatory protein by inhibiting the formation of C5 convertases.

Objectives

To investigate the expression, regulation, and function of CD55 in CRSwNP.

Methods

The expression of CD55 in sinonasal mucosal samples was investigated by quantitative RT-PCR, Western blotting, and immunofluorescence. The levels of C3d and C5b-9 in nasal tissue homogenates were analyzed by ELISA. Primary human nasal epithelial cells were cultured to investigate the regulation and function of CD55.

Results

The mRNA and protein levels of CD55 were reduced in eosinophilic nasal polyps compared with those in noneosinophilic nasal polyps and control tissues. CD55 was prominently expressed by nasal epithelial cells in sinonasal mucosa, which was downregulated by IL-4, IL-13, and Dermatophagoides pteronyssinus group 1. The levels of C3d and C5b-9 were upregulated in eosinophilic nasal polyps and were inversely correlated with CD55 expression levels. The conversion of C3 to C3d by primary human nasal epithelial cells was enhanced in those from eosinophilic nasal polyps in comparison to those from noneosinophilic polyps. The mRNA and protein expression of E-cadherin and ZO-1 were downregulated in eosinophilic nasal polyps and were positively correlated with CD55 expression levels.

Conclusion

The reduced expression of CD55 caused by local type 2 milieu lead to hyperactivation of complement and subsequent C5b-9 formation, which may further disrupt epithelial integrity in patients with eosinophilic CRSwNP.
Understanding Genetic and Immune Cellular-Signaling Defects in Common Variable Immunodeficiency with Granulomatous Lymphocytic Interstitial Lung Disease
Authors: Lui Victor , Baxter Ryan , Routes John Michael , Verbsky James , Hsieh Elena WY
Published: January 2023

Common Variable Immunodeficiency (CVID) is a heterogenous disorder characterized by hypogammaglobulinemia, failure to mount vaccine responses, recurrent sinopulmonary infections. Furthermore, a subset of CVID patients develop various autoimmune and lymphoproliferative complications, highlighting the dysregulated immune mechanisms underlying the disorder. One such autoimmune complication is granulomatous lymphocytic interstitial lung disease (GLILD) which is a major cause of mortality in CVID. The clinical heterogeneity of CVID indicate that it is a collection of disorders with monogenic and polygenic defects. Due to the genetic and phenotypic complexity of CVID, our understanding of the immunopathogenesis of CVID and the development of its complications is limited. Our goal is to elucidate relationships between genetic variants and their downstream phenotypic and signaling defects in CVID patients with GLILD. We hypothesize that specific genetic variants will result in unique immune cellular (and signaling) signatures in CVID with GLILD. To identify these cellular and molecular signatures, we integrate whole exome sequencing and mass cytometry data. We examined cellular subsets affected by perturbed T and B cell receptor signaling pathways via mass cytometry, and then correlate these signatures with specific genetic variants identified through whole exome sequencing. These results will reveal mechanisms of immune dysregulation in CVID with GLILD. In CVID patients with GLILD, we find expansion of CD57+ CD8+ T cells, which may represent a class of senescent cells. We also find enrichment of CD21-low B cells within CVID patients, regardless of GLILD status.
Cholesterol crystals use both extracellular and intracellular complement to increase NLRP3 signalling pathways in atherosclerosis
Authors: Espevik Terje , Niyonzima Nathalie , Bakke Siril Skaret , West Erin , Rahman Jubayer , Aukrust Pål , Kemper Claudia , Mollnes Tom Eirik , Halvorsen Bente
Published: January 2023

During atherosclerosis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall which trigger plaque inflammation. In the present, study we examined the relation between complement, NLRP3 signalling pathways and CC in patients with coronary artery disease and carotid atherosclerosis. We analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with different degrees of atherosclerosis applying ELISAs, multiplex, qPCR, immunohistochemistry, gene profiling and bioinformatics. Transcripts of IL-1β and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome. Immunohistochemistry of carotid plaques showed pronounced accumulation of C1q and terminal C5b-9 complement complex (TCC) around CC-clefts. Transcripts of C5aR1, C5aR2 and C3aR1 were increased in carotid plaques compared to control arteries. Priming of carotid plaques ex vivo with C5a and TNF followed by CC incubation resulted in pronounced upregulation of NLRP3 inflammasome components. Importantly, we found that intracellular “C5 system” is a major contributor of CC induced IL-1β in human macrophages through the action of C5aR1 present on mitochondria. Mechanisms underlaying the role of intracellular C5 in this process will be described. The data suggest that CC formation is an important extracellular and intracellular complement activator which in turn results in IL-1β expression and activation of NLRP3 in atherosclerotic plaques.
Myeloid-derived suppressor cells modulate aortic aneurysm formation via CXCR2 and IL-17 signaling
Authors: Sharma Ashish Kumar , Elder Craig , Su Gang , Lu Guanyi , Qi Xiaoyan , Jiang Zhihua , Moldawer Lyle Leonard , Efron Philip , Upchurch Gilbert R
Published: January 2023

Aortic aneurysm formation is associated with vascular inflammation and remodeling mediated via CD4+ T cell-secreted IL-17A production. Our hypothesis is that innate myeloid-derived suppressor cells (MDSCs), which are a heterogeneous population of myeloid precursor and progenitor cells, exhibit an immunoregulatory role in the progression of aortic inflammation during abdominal aortic aneurysm (AAA) formation. The trafficking and migration of MDSCs to the aortic tissue and role of CXCR2 and programmed death-1 (PD-1) receptor signaling was deciphered using an in vivo elastase-treatment model of AAA as well as in vitro co-culture experiments. We used an established murine AAA model of topical elastase-treatment in the present study. Preliminary data showed that elastase-treatment of C57BL/6 (wild-type; WT) mice resulted in significant increase in infrarenal aortic diameter causing AAA formation on day 14 that was significantly inhibited by a combined treatment with anti-CXCR2 and PD-1 antibodies. A multi-fold increase in MDSCs (CD11b+Gr-1+) in aortic tissue of elastase-treated WT mice on days 7 and 14 occurred compared to controls. Co-culture of MDSCs with aortic endothelial cells in the presence of anti-CXCR2 antibody inhibited the transmigration of MDSCs through the endothelial barrier. Moreover, transient elastase treatment of co-cultures of anti-CD3/CD28 treated mononuclear cells with MDSCs significantly enhanced IL-17A secretion compared to cultures without MDSCs. Our results indicate that MDSCs may play a contributory role in aortic inflammation by trafficking in a CXCR2-dependent manner and promotion of IL-17A secretion, signifying the importance of MDSC-mediated immunoregulation of aortic aneurysms.
A natural mouse model reveals genetic determinants of Systemic Capillary Leak Syndrome (Clarkson disease)
Authors: Xie Zhihui , Raza Abbas , Chan Eunice , Chen Wei-Sheng , Scott Linda M , Eisch A. Robin , Krementsov Dimitry N , Rosenberg Helene F , Parikh Samir M , Blankenhorn Elizabeth P , Teuscher Cory , Druey Kirk M
Published: January 2023

The Systemic Capillary Leak Syndrome (SCLS, Clarkson disease) is a disorder of unknown etiology characterized by recurrent episodes of vascular leakage of proteins and fluids into peripheral tissues, resulting in whole-body edema and hypotensive shock. The pathologic mechanisms and genetic basis for SCLS remain elusive. Here we identify an inbred mouse strain, SJL, which recapitulates cardinal features of SCLS, including susceptibility to histamine- and infection-triggered vascular leak. We named this trait “Histamine hypersensitivity” (Hhs/Hhs) and mapped it to Chromosome 6. Hhs is syntenic to the genomic locus most strongly associated with SCLS in humans (3p25.3), revealing that the predisposition to develop vascular hyperpermeability has a strong genetic component conserved between humans and mice and providing a naturally occurring animal model for SCLS. Genetic analysis of Hhs may reveal orthologous candidate genes that contribute not only to SCLS, but also to normal and dysregulated mechanisms underlying vascular barrier function more generally.
The complex allergen house dust mite (HDM) dramatically increases the abundance of the autophagy cargo adapter SQSTM1/p62 in macrophages and suppresses Torin 1-induced degradative autophagy
Authors: Gao Hongjuan , Qi Xiulan , Jackson William , Keegan Achsah D
Published: January 2023

Autophagy is an essential homeostatic process involving degradation of intracellular components, including soluble proteins, aggregated proteins, organelles, macromolecular complexes and foreign bodies. Several components of the autophagy pathway have been linked to allergic asthma and macrophage phenotypes, however the contribution of autophagy to these processes is unclear. We previously showed that house dust mite (HDM) increased the number of fluorescent foci (autophagosomes) in bone-marrow derived macrophages (BMM) in a manner similar to the autophagy inducer Torin 1. Further, electron microscopy revealed an increase in double membraned vesicles in response to HDM and Torin 1. While Torin induced a specific increase in lipidated LC3, treatment with HDM caused equal increases in both non-lipidated and lipidated LC3. Torin 1 stimulated autophagic degradation as measured by reduction of SQSTM1/p62. In contrast, SQSTM1/p62 levels increased dramatically in response to HDM. The increase was blocked by actinomycin D and cycloheximide indicating that new gene transcription and protein synthesis is required for the HDM-induced effect. HDM-stimulated increase in SQSTM1/p62 was also observed in alveolar macrophages treated in vitro. An increase in SQSTM1/p62 is often interpreted as a block in autophagic degradation. Indeed, we observed that in the presence of HDM, Torin 1 treatment did not lead to the loss of SQSTM1/p62 below baseline; however, the protein did not accumulate to the high level observed in the HDM-alone treatment group. Our results suggest that HDM engages autophagy machinery at multiple points, both inducing expression of SQSTM1/p62 and suppressing its degradation.
Resolvin D2 Promotes the Resolution of Allergen-Induced Lung Inflammation
Authors: Peh Hong Yong , Bruggemann Thayse R , Ho Wanxing Eugene , Cheng Chang , Tan WS Daniel , Wong WS Fred , Levy Bruce D
Published: January 2023

Background

Asthma is a chronic inflammatory disease without complete resolution. The production of specialized pro-resolving mediators (SPMs) is defective in asthma, so they represent a potential therapeutic opportunity. We hypothesize that docosahexaenoic acid derived resolvin D2 (RvD2) can promote the resolution of asthma.

Methods

BALB/c mice were challenged intra-tracheally with house dust mite (HDM). Bronchoalveolar lavage (BAL) fluid was collected at predefined protocol endpoints for peak inflammation and during resolution. To determine impact on resolution, some mice were given RvD2 (100 ng, intra-nasally) for 2 days after the last HDM challenge, and the resolution index for eosinophils and other markers were calculated.

Results

Airway HDM challenge led to marked increases in BAL eosinophils and BAL fluid pro-inflammatory cytokine and chemokine levels. Histology demonstrated changes in airway inflammation and mucous cell metaplasia in HDM-challenged lungs. NF-κB and Nrf2 expression were increased the lungs of HDM allergic mice. Compared to vehicle, treatment with RvD2 significantly decreased BAL inflammatory macrophage and eosinophil counts, and accelerated the resolution of HDM-evoked inflammation with a marked decrease in the resolution index for BAL eosinophils from 5 days to 1 day. Interestingly, RvD2 attenuation of eosinophils specifically targeted the inflammatory eosinophil population, but not the resident eosinophils. The impact of RvD2 on cytokine expression and NF-kB and Nrf2 activity is under current investigation to determine mechanisms for RvD2’s pro-resolving actions.

Conclusion

RvD2 promotes resolution of allergen-induced inflammation in a murine model of allergic asthma.
Evaluation of the skin sensitizing potential of gold nanoparticles and the impact of established dermal sensitivity on the pulmonary immune response to various forms of gold
Authors: Roach Katherine Adair , Anderson Stacey , Stefaniak Aleksandr , Shane Hillary L , Roberts Jenny R
Published: January 2023

Gold compounds are associated with diverse immune effects, including allergy. Whether gold nanoparticles (AuNP) cause similar effects remains unknown. To address this, three in vivo studies were performed. AuNP (29nm)dermal sensitization potential was assessed via Local Lymph Node Assay (LLNA) along with soluble gold salts (AuCl3) and larger particles (Au, 942nm). AuCl3 caused lymph node (LN) expansion (SI 10.9), whereas Au and AuNP did not, indicating minimal potential for dermal sensitization. Next, pulmonary immune effects of AuNP (10μg, 30μg, 90μg) were assessed 1d, 4d, 8d post-aspiration. AuNP had no effect on lung injury or inflammation, but did increase LN CD4+ T- and B-cells by 4d. Finally, mice were dermally sensitized to AuCl3 (d1-3), then aspirated 1× (d10),2× (d10, 14), or 3× (d10, 14, 18) with Au or AuNP in doses normalized for mass or surface area (SA), to assess impact of existing contact sensitivity on lung immune responses. In dermally sensitized groups, increased BAL lymphocytes were seen after two and three aspirations, which correlated to the administered SA. The highest AuNP dose caused the greatest increase in total BAL lymphocytes, as well as increased number, proportion, and activation of BAL/LN CD8+ T-cells. Comparatively, the lower SA-based doses of Au/AuNP resulted in elevated numbers of BAL T-cells(predominantly CD4+ phenotype), exposure-dependent increases in serum IgE, and selective expansion/activation of LN CD4+ T- and B-cells, suggestive of a Th2-polarized state. Overall, these findings suggest that AuNP are not likely to cause allergic sensitization; however, established contact sensitivity to gold may be associated with increased immune reactivity to AuNP following respiratory exposure.