bluuebunny/crossref_metadata_2025 huggingface dataset:
Modified low density lipoprotein (mLDL) uptake by B cells alters B cell phenotype and impacts atherosclerosis.
Authors: Waseem Tayab , Gjurich Breanne , Butcher Matthew , Alekseev Andrei , Nurieva Roza , Galkina Elena
Published: January 2023

Atherosclerosis is a multifaceted chronic inflammatory disease characterized by the accumulation of modified lipoproteins and immune cells in the aortic wall and vascular dysfunction. The uptake of modified low density lipoproteins (mLDL) by MFs plays an important role in atherosclerosis through the modulation of cholesterol metabolism and MF activation. To date, the impact of mLDL on B cell phenotype and functions is unaddressed. We show that B cells can uptake acetylated and oxidized LDL (acLDL and oxLDL, respectively) via a dynamin-dependent pathway in a concentration, time, and temperature-dependent manner. Assessment of the transcriptional profiles of in vivo acLDL+ B cells and acLDL- B cells by RNAseq suggests that acLDL+ B cells assume an anergic like phenotype. Anergy plays an important role in keeping autoreactive B cells in a state of unresponsiveness and the loss of anergy contributes to the development of autoimmune diseases. Currently, nothing is known about the role and regulation of B cell anergy in atherosclerosis. We show here for the first time that GRAIL, an E3 ubiquitin ligase associated with T cell tolerance, is expressed in B cells. To determine whether B cell-specific GRAIL expression plays a role in atherosclerosis, we performed adoptive transfer of GRAIL-deficient or GRAIL-sufficient B cells into B-cell-deficient mice on apolipoprotein-E deficient background (uM−/− Apoe−/−) mice and examined atherogenesis after 8 weeks of western diet feeding. B cell-specific GRAIL-deficiency supported plaque burden and orchestrated inflammation in the aortic wall. These data suggest that there is a specific mechanism by which B cells recognize mLDL and regulate their anergy-dependent response to mLDL in atherosclerosis.
The endogenous regulator IL-27 suppresses Th2 and Th17 responses in the kidney to ameliorate renal fibrosis in a model of obstructive kidney disease
Authors: Muallem Gaia , Aronson Lillian , Hunter Christopher A
Published: January 2023

Chronic kidney disease (CKD) is a major healthcare issue, causing significant morbidity and mortality as well as high healthcare costs. Early detection of CKD and prevention of disease progression are important strategies for management, and accordingly an improved understanding of fibrotic processes is a critical focus of kidney research. Aberrant immune responses promote fibrosis in the kidney, and exploring the endogenous regulators of these pathways has relevant implications for clinical therapies. Consistent with other organ systems, CD4+ T cell production of the cytokines IL-4, IL-13, and IL-17 has been associated with renal fibrosis in preclinical models of disease, and alternatively activated macrophages (AAMs) have been shown to promote fibrosis in the kidney. The cytokine interleukin-27 (IL-27) limits the magnitude of T cell responses, and our data indicate that after unilateral ureteral obstruction (UUO) mice deficient in IL-27 develop increased kidney fibrosis that is associated with the emergence of Th2 and Th17 responses as well as an increase in alternative activation of macrophages. Therefore, IL-27 is an endogenous regulator that limits clinically relevant immune pathways in the kidney and has potential as an important diagnostic and therapeutic tool in the management of renal fibrosis.
T cell responses to exercise stress do not adequately model those from tactical stress events
Authors: Siedlik Jacob A , Deckert Jake A , Bhatta Anuja , Dunbar Amanda J , Gigliotti Nicole M , Chan Marcia A , Benedict Stephen H , Vardiman John P , Gallagher Philip M
Published: January 2023

Military training environments are rigorous, requiring service members to endure not only physical and psychological stress but also sleep deprivation, caloric restriction, and severe thermic challenges. The convention has been to use immunoendocrine responses induced by various exercise regimens to generalize results to the layered stress environments of wartime. The purpose of this study was to determine whether exercise models are accurate representations of the physiologic response to the stress experienced in an operational environment. To achieve this aim we identified changes in T cell proliferative capacity (costimulation through CD3+CD28 or stimulation with PHA) and relevant immunoendocrine interactions following an environment comparable to that experienced in Basic Combat Training. An increased proliferation response was observed post-exercise in T cells isolated from whole blood in the layered stress group with those increases still significant at 6 h post-exercise. In contrast, the moderate exercise group saw no significant changes in proliferative ability immediately after exercise exhibiting what could be expressed as a characteristic exercise response. Analyses of serum stress hormones, immunomodulatory cytokines, and immunoglobulins -G, -M, and -A failed to reveal any correlated variations that could clarify the T cell findings. These data suggest that exposure to a high stress environment leads to increased T cell proliferation that appears to be independent of changes in stress hormone concentrations and immunomodulatory cytokines. We submit that variations in exercise intensity and duration do not necessarily approximate military operational or tactical stress responses.
IL-23 mediated skin inflammation is dependent on the expansion and function of RORγt expressing cells
Authors: Gauld Stephen B , Wang Yibing , Gauvin Donna , Huang Susan , Lays Laura , Lippert Stephanie , Edelmayer Rebecca , Wetter Joseph , Salte Katherine , Olson Lauren , Namovic Marian , Donnelly-Roberts Diana , Su Zhi , Scott Victoria , Honore Prisca , McGaraughty Steven , Kort Michael
Published: January 2023

While playing an important role in host defense against extracellular pathogens, the IL-23/IL-17 axis is inherently linked to autoimmune disease. Biologic therapies have been developed that target the p19 subunit of IL-23 and IL-17A or IL-17R; and show remarkable efficacy against a range of autoimmune conditions including Psoriasis (PsO). Despite this clinical validation, the cellular pathways induced by IL-23 remain poorly defined. In order to better understand the role of RORγt in this pathway, we administered a potent and selective inhibitor of RORγt in a model of IL-23 mediated skin inflammation, which closely models human PsO. Looking specifically at skin, to differentiate from previous studies, we show that the accumulation of RORγt expressing cells is an early event following exogenous IL-23 exposure. Expansion of these RORγt expressing cells paralleled disease progression (inflammation) with both αβ and γδ T cells contributing to this population. Inhibition of RORγt with a novel small molecule inverse agonist significantly attenuated ear inflammation and histological endpoints related to human PsO. Furthermore, use of this compound attenuated both the accumulation of RORγt positive cells and their effector function (IL-17A production). Finally, inhibition of RORγt had profound downstream consequences, leading to significant recovery of keratinocyte dysregulation and normalization of anti-microbial gene expression. Collectively our studies confirm that RORγt expressing cells are an early feature of IL-23 mediated skin inflammation, reside predominantly in the T cell compartment, and play an active role in keratinocyte biology and disease pathology.
Sphingosine kinase 2 deletion in CD4+ T cells protects against kidney fibrosis
Authors: Perry Heather , Bajwa Amandeep , Huang Liping , Okusa Mark D
Published: January 2023

Progressive fibrosis, a hallmark of chronic kidney disease (CKD), can occur following an acute insult such as ischemia-reperfusion injury (IRI). Sphingosine kinase 2 (Sphk2) deletion in CD4+ T cells protects from folic acid-induced fibrosis; an effect mediated IFNγ. Mitochondrial metabolism has emerged to be important in CD4+ T cell function. We hypothesized that Sphk2 regulates mitochondrial function and IFNγ production in CD4+ T cells in protection against IRI induced fibrosis.

CD4+ T cells were isolated from WT, Sphk2−/− or Sphk2−/− Ifng−/− mice and adoptively transferred to WT mice prior to unilateral IRI. After 14 days, plasma creatinine (PCr) was quantified as a measure of kidney function and kidney transcript levels of a fibrosis marker αSMA (Acta2) was quantified by RT-qPCR. Mitochondrial function was assessed by Seahorse and TMRE uptake by flow cytometry.

WT mice that received Sphk2−/− CD4+ T cells had better kidney function indicated by a reduced PCr level (0.68 +/− 0.01, n = 3) compared to vehicle (0.99 +/− 0.07, n = 4), WT (0.86 +/− 0.04, n = 3), and Sphk2−/− Ifng−/− (1.16 +/− 0.06, n = 4) CD4+ T cells. Additionally, Sphk2−/− CD4+ T cell recipients had reduced transcript levels of Acta2. Naïve and effector Sphk2−/− CD4+ T cells had increased basal oxygen consumption rate compared to WT CD4+ T cells. Effector Sphk2−/− CD4+ T cells had increased TMRE fluorescence compared to WT control CD4+ T cells.

Sphk2−/− CD4+ T cells protect against IRI induced decline in kidney function and fibrosis, an effect mediated by production of IFNγ. Additionally, deletion of Sphk2 may promote mitochondrial function and effector CD4+ T cell function. Thus, targeting the Sphk2 pathway in T cell adoptive therapy may be a therapeutic strategy in the treatment of CKD.
A brain-specific chemokine, FAM19A5, inhibits RANKL-induced osteoclast formation
Authors: Park MinYoung , Lee Ha Young , Kim Hyeung Sik , Lee Mingyu , Kim Ye Seon , Seong Jae Young , Bae Yoe-Sik
Published: January 2023

Since osteoclast plays a key role in bone resorption, the identification of novel molecules that regulate osteoclastogenesis has been important issue. A neurokine or brain-specific chemokine, FAM19A5, a member of C-C chemokine family, is mainly expressed in central nervous system. In this study, we found that FAM19A5 strongly stimulated chemotactic migration of mouse bone marrow-derived macrophages (BMDM). FAM19A5 also markedly stimulated the activation of protein kinase B (Akt) and ERK, resulting in phosphorylation of the kinases in BMDM. FAM19A5 also blocks RANKL-induced osteoclastogenesis. It was confirmed by tartrate-resistant acid phosphatase (TRAP) staining and osteoclast-related gene level. Collectively, we suggest that FAM19A5 can act as a new ligand that regulates chemotactic migration of BMDM and osteoclastogenesis.
CD1a on Langerhans cells controls inflammatory skin diseases.
Authors: Kim Ji Hyung , Hu Yu , Winau Florian
Published: January 2023

In contrast to conventional T cells that recognize peptides on MHC proteins, CD1 molecules present lipid antigens to T lymphocytes. The abundant expression of CD1a hallmarks Langerhans cells in the skin, a subtype of dendritic cell (DC) with antigen-presenting functions. CD1a is able to bind and display a broad spectrum of lipid antigens derived from exogenous sources, such as bacteria, or host origin. The complex immune system of the skin is critically involved in responses to extrinsic insults like allergens, as well as in autoimmune diseases, such as psoriasis. However, the in vivo role of CD1a on Langerhans cells remains enigmatic since CD1a is lacking in mice. Using CD1a-transgenic mice, we show that the lipidic substance urushiol from the plant poison ivy induces severe skin inflammation in a CD1a-dependent fashion. The immune response is driven by CD1a-expressing Langerhans cells that elicit the generation of CD4 T cells, producing the inflammatory cytokines IL-17 and IL-22. Among different urushiol congeners, we identified diunsaturated pentadecylcatechol (C15:2) as dominant antigen for CD1a-restricted T cells. We determined the crystal structure of the CD1a-urushiol (C15:2) complex, demonstrating the molecular basis of urushiol interaction with the antigen-binding cleft of CD1a. In a model for psoriasis, CD1a massively amplified inflammation mediated by Th17 cells reactive with self lipid antigens from skin. Strikingly, treatment with blocking antibodies versus CD1a fully abrogated skin inflammation. Patients suffering from psoriasis showed strong inflammatory T cell activation in response to CD1a. Thus, we propose CD1a as a potential target for future therapeutic strategies against inflammatory skin diseases.
A self-glycerophospholipid suppresses immunity and inflammation via recruitment of Ly6C+ myeloid cells
Authors: Singh Ram Raj , Prasad Priti , Tran Cynthia , Halder Ramesh
Published: January 2023

Membrane lipids function as essential components of biological membranes, as signaling molecules, and as energy storage molecules. Phosphatidic acid is a vital membrane lipid that serves as a precursor for the synthesis of all acylglycerol lipids in the cell. Phosphatidic acid participates in a wide range of cellular processes, including cytoskeletal organization, secretion, endocytosis, and cell proliferation. Here, we examined the effect of phosphatidic acid on myeloid cells and its ability to modulate tumor immunity and inflammatory disease. We found that in vivo administration of phosphatidic acid protected against immune mediated liver inflammation induced by concanavalin A, but worsened tumor spread in the B16F10 melanoma lung metastasis model. Phosphatidic acid reduced pro-inflammatory cytokines but increased anti-inflammatory cytokine interleukin-10 in the liver. Phosphatidic acid did so by inducing the mobilization and migration from the bone marrow to peripheral organs of Ly6C+ myeloid cells that upon adoptive transfer protected against autoimmune liver inflammation. Our novel observations indicating immune suppressive functions of phosphatidic acid may have wide implications for a variety of conditions with altered lipid metabolism and inflammation such as atherosclerosis and autoimmune disease and conditions with local or systemic alterations in phosphatidic acid such as osteoarthritis and Alzheimer’s disease.
Neutrophil polarization in the airways of infants with bronchiolitis
Authors: Cavallaro Elena C , Liang Kar-Kate , Forsyth Kevin D , Dixon Dani-Louise
Published: January 2023

Pulmonary neutrophilia is observed in pediatric patients with viral-induced bronchiolitis. Despite an alleviation in clinical symptoms, our findings suggest that airway neutrophil infiltration and activation does not appear to resolve by discharge in hospitalized infants. The recent identification of neutrophil plasticity and polarization into distinct phenotypic pro-inflammatory (N1) and immunosuppressive (N2) subsets provides an unexplored avenue for regulation of the neutrophilic inflammatory response in bronchiolitis. Admission nasopharyngeal aspirate (NPA) samples were assessed from twelve bronchiolitic infants presenting to Flinders Medical Centre, Adelaide, Australia in 2013. An overall strong positive correlation between N2 soluble mediator TGF-β1 and the percentage of neutrophils stained for N2 marker MMP-9 was observed; with no correlation observed between TGF-β1 and neutrophils positive for N1 marker ICAM-1. In 2015 and 2016, eleven bronchiolitic infants provided consecutive NPAs across hospitalization. In both cohorts, concentrations of active TGF-β1 detected were comparable to levels previously identified to significantly suppress neutrophil activity in vitro (>1 pg/mL). Temporal assessment of neutrophil polarization is underway. The detection of pre-established neutrophil polarizing mediators, and the correlation of these mediators with different neutrophilic subsets within NPAs indicates a potential N1/N2 paradigm occurring within the airways of bronchiolitic infants. Further investigation is merited, with analyses over disease trajectory and by disease severity required to assess biological significance.
Majie Pingchuan Cataplasm Regulates Thymic Stromal Lymphopoietin in Rats with Asthma
Authors: Wang Xueqian , Cheng Fafeng , Zhu Wenxiang , Yin Xiangjun , Liu Yuanjun , Li Changxiang , Ma Chongyang , Kong Hui , Wang Qingguo
Published: January 2023

As a new type of external preparations, cataplasm is widely used in clinical practice. In this study, we aimed to explore the efficacy and mechanism of Majie Pingchuan Cataplasm (MJPC) on asthma. By treating the brown-norway asthmatic rats with MJPC, it was found that the incubation period was significantly longer compared with rats injected with dexamethasone (P<0.05); in addition, the airway smooth muscle spasm was relieved compared with the asthmatic rats (P<0.05); it was observed that the number of inflammatory cells (such as eosinophils and neutrophils) was reduced both in the blood and lung tissue (P<0.05). The efficacy of MJPC was also explored in vitro. Components of MJPC had an antagonistic effect on the tracheal smooth muscle contraction of guinea pigs induced with histamine phosphate and acetylcholine chloride. It has been proven that thymic stromal lymphopoietin (TSLP) plays an important role in the pathogenesis of atopic dermatitis and asthma. So we hypothesized that TSLP may be an important regulator of immune system in the skin and lung. In this study, TSLP levels in different tissues were detected to explore the mechanism of MJPC in the treatment of asthma. The results showed that expressions of TSLP in the lung tissue, skin, bronchoalveolar lavage fluid and serum were significant lower in the MJPC group compared with the model group (P<0.05). Our study confirmed that MJPC has a therapeutic effect on asthma in rats, it can not only play a role in blood after percutaneous absorption, but also regulate the skin immune system to achieve the treatment of asthma by affecting the expression of TSLP in local skin.
Serum soluble Siglec-8 in hypereosinophilic syndromes
Authors: Legrand Fanny , Cao Yun , Bochner Bruce S , Klion Amy D
Published: January 2023

Siglec-8, selectively expressed on eosinophils, basophils and mast cells, is the target of novel biologics in clinical development for eosinophil and mast cell-associated disorders. Since soluble Siglec-8 (sSiglec-8) can be detected in serum and could interfere with the efficacy of biologics targeting Siglec-8, the aim of the present study is to assess sSiglec-8 levels in a large cohort of subjects with hypereosinophilic syndrome (HES). sSiglec-8 levels were quantified in serum from normal (n=9) and eosinophilic (n=123) subjects using an in-house sandwich ELISA (limit of detection: 0.5 ng/mL) and correlated with Siglec-8 expression on the surface of eosinophils (measured by flow cytometry on whole blood eosinophils gated as granulocytes CD45+CD16neg) and absolute eosinophil count (AEC). The effects of treatment were also examined.

Serum levels of sSiglec-8 were undetectable in 28 subjects, did not differ between untreated HES subjects and normal donors (GM 1.23 and 1.53 ng/mL), and did not correlate either with AEC (n=123) or with Siglec-8 surface expression on blood eosinophils (n= 35). There was also no clear relationship between the clinical subtype of HES and levels of sSiglec-8. Levels measured before and after varied HES treatments in 7 subjects showed a significant decrease from 1.82 to 0.50 ng/mL (P<0.05). In contrast, serum from 8 HES patients at two different time points on no treatment showed no significant change (GM 2.35 and 2.10 ng/mL respectively). These data suggest that, despite the lack of correlation with AEC, eosinophils are a significant source of sSiglec-8 in serum. Whether serum sSiglec-8 is predominantly from tissue eosinophils or mast cells, or requires eosinophil activation, is currently under investigation.
Adam8 limits emphysema development in smoke-exposed mice: A new player in the pathogenesis of COPD
Authors: Rojas Joselyn Joanna , Polverino Francesca , Gai Xiaoyan , Rout Amit , Wang Xiaoyun , Yambayev Ilyas , Owen Caroline Anne
Published: January 2023

Background

A disintegrin and metalloproteinase-8 (ADAM8) is expressed by lung cells implicated COPD pathogenesis. It’s not known whether ADAM8 contributes to COPD pathogenesis.

Methods

ADAM8 expression was measured in the lungs of COPD patients and controls, and mice exposed to cigarette smoke (CS) versus air by staining lung sections for ADAM8 and markers of different lung cells. Emphysema development and small airway fibrosis were evaluated wild-type (WT), Adam8−/− and Adam8 bone marrow chimeric mice exposed to air or CS for 6 months. Lung inflammation, alveolar septal cell apoptosis, and lung oxidative stress levels were measured in WT vs. Adam8−/− mice exposed acutely to air or CS.

Results

Adam8 expression is reduced in alveolar macrophages and lung epithelial cells in COPD patients and CS-exposed WT mice. CS-exposed Adam8−/− mice had greater airspace enlargement, alveolar septal cell apoptosis, and oxidative stress levels (but similar small airway fibrosis) versus CS-exposed WT mice. Both leukocyte- and lung parenchymal cell-derived Adam8 mediate Adam8’s protective activities in the lung. CS-exposed Adam8−/− mice had greater lung macrophage counts than WT mice. ADAM8 does not degrade mediators of inflammation or regulate monocyte adhesion or migration. Adam8−/− alveolar macrophages were protected from CS-induced activation of the intrinsic apoptosis pathway.

Conclusions

ADAM8 protects against CS-induced emphysema development by reducing lung inflammation by increasing macrophage apoptosis and reducing alveolar septal cell death. However, ADAM8’s expression is reduced in COPD lungs. ADAM8 may be a therapeutic target for COPD.
RELM protein-induced CXCR3 ligands in pulmonary hypertension
Authors: Van Raemdonck Katrien , Lin Qing , Fang Xia , Skinner John , Fan Chunling , Yamaji-Kegan Kazuyo , Johns Roger A.
Published: January 2023

In pulmonary hypertension (PH), inflammation and angiogenesis go hand in hand. Inflammatory mediators exert direct effects on endothelial (EC) and vascular smooth muscle cells (VSMC) and attract leukocytes to affected vascular beds. Resistin-like molecules (RELM) were previously implicated in vascular remodeling in PH. Structural and functional parallels can be drawn between chemokines and RELMs. Resistin (Retn) stimulates SMC proliferation and regulates EC adhesion and apoptosis. The less-studied Relm-β has similar effects. RELMs also modulate chemokine production. Our data suggest that Relm-β contributes to pathogenic upregulation of CXCL10 and CXCL11, both IFNγ-inducible CXCR3 ligands. These are known players in angiogenic disorders and have been associated with PH. Under hypoxia, mice did exhibit increased pulmonary expression of CXCL11 in a pilot study. In vitro chemokine levels indicate that RELMs stimulate CXCL10 and CXCL11 release by activated monocytic THP1 cells. Because chemokines may locally affect VSMC proliferation, cause EC apoptosis, and attract T cells that exacerbate inflammation, we hypothesize that CXCR3-driven vascular remodeling contributes to the pathogenic role of RELMs in PH. While the receptor remains unknown, our preliminary data suggest Relm-β-induced inhibition of PPARγ activity to regulate chemokine release. Our endeavors continue to unravel the receptor and downstream pathways involved. As development of RELM-targeted therapies progresses, understanding the full range of biological activities of, not only Retn, but also Relm-β, remains crucial. CXCR3 ligands have the potential to serve as valuable biomarkers as we evaluate the efficiency of innovative therapies in the field of PH.
Using MHC Binding Assays to Investigate HLA-Dependent Drug Directed Immune Responses
Authors: Kaever Thomas , Metushi Imir G , Pavlos Rebecca , Sidney John , Ostrov David A , Buus Soren , Redwood Alec , Sette Alessandro , Hunt Donald F , Phillips Elizabeth J , Peters Bjoern
Published: January 2023

Immune mediated adverse drug reactions (IM-ADRs) pose a significant risk to human health, and have been shown to be linked to specific human leukocyte antigen (HLA) alleles. Hypersensitivity syndrome induced by abacavir, the strongest such genetic association found to date, is driven by drug-specific activation of cytokine producing, cytotoxic CD8+ T cells exclusively found in individuals expressing the HLA-B*57:01 allele variant. We have previously shown that abacavir interacts with the peptide binding cleft of the HLA-B*57:01 molecule, thus increasing the affinity of certain self-peptides generating an altered self-peptide repertoire. In MHC binding studies a second drug, acyclovir, also revealed a dose dependent increase in affinity for peptides that contain small, hydrophobic residues at the C terminus. Peptide elution studies performed in the presence of acyclovir showed an elevated number of endogenously bound peptides with a C-terminal isoleucine. Accordingly, we concluded that acyclovir acts by the same mechanism as abacavir, albeit at a lesser magnitude. Our data showed a maximum increase of affinity of 2.5-fold for acyclovir, when the effect of abacavir was as high as 1000-fold. Acyclovir is not known to cause IM-ADRs. We concluded that the moderate effect of acyclovir on binding affinity was not sufficient to cause a hypersensitivity reaction. Here we applied the same screening strategies to four compounds that are involved in IM-ADRs with strong known HLA association, to determine if drug specific effects of these molecules could be observed. While no effect as striking as that for abacavir could be detected, several notable changes in repertoire were associated with the presence of these compounds.
High output flow cytometry array profiling facilitates discriminant phenotyping of peripheral blood cells from patients with different forms of autoimmune uveitis
Authors: Nowatzky Johannes , Manasson Julia , Resnick Ezra , Stagnar Cristy , Manches Olivier
Published: January 2023

The information content of multi-parametric flow cytometry-based immune-phenotyping experiments is often underexploited given the paucity of adequate tools and strategies for large-scale unbiased data analysis. Here, we developed and applied a data analysis approach taking into account all mathematically possible combinations of markers in a given flow cytometry panel. We analyzed flow cytometry data generated from peripheral blood samples of healthy humans and patients with autoimmune uveitis in the setting of 2 different autoimmune diseases: Behcet’s disease and sarcoidosis. Original FACS data files were mined from Dryad Digital Repository http://dx.doi.org/10.5061/dryad.v6ste with reference to http://dx.doi.org/10.1371/journal.pcbi.1003215, and gated in bivariate plots. We employed combinatory mathematics to generate a matrix quantifying the representation of all possible cell populations using a given set of markers within the respective starting population and coded the algorithm to create a Java-based platform enabling the computation of large numbers of markers. Resulting metadata were visualized in a heat map approach. Our method enabled clustering of healthy vs diseased subjects using only 4 common markers (CD3, C8, CD197, and CD45), and of sarcoid vs Behcet’s patients with minimal error. Our approach demonstrates that multi-dimensional analysis of flow cytometry data allows meaningful large-scale screening of biologically relevant markers enabling classification and characterization of states of health and autoimmune disease. The approach is unbiased and has the potential to facilitate the discovery of cell populations with relevance as potential biomarkers or biological research targets.
Bacterial Dysbiosis Associates with Functional Intraepithelial Lymphocyte Changes in Inflammatory Bowel Disease and Spondyloarthritis
Authors: Ohri Neha , Gerich Mark E , Fennimore Blair P , Ir Diana , Robertson Charles E , Regener Emilie , Caplan Liron , Wagner Brandie D , Frank Daniel N , Kuhn Kristi
Published: January 2023

Dysbiosis occurs in spondyloarthritis (SpA) and inflammatory bowel disease (IBD), subdivided into Crohn’s Disease (CD) and Ulcerative Colitis (UC). The immunologic consequences of dysbiosis have not been defined. Intraepithelial lymphocytes (IELs) are T cells within the intestinal epithelium that are in close contact with bacteria, and as such, are likely to be modulated by dysbiosis. We correlated IELs with resident bacteria in SpA, IBD, and controls. Subjects with biopsy-proven IBD (N=10 with CD and 7 with UC), SpA fulfilling ASAS criteria (N=5), and healthy controls (N=15) were evaluated for fecal microbiome by 16S rRNA sequencing and IELs from colon biopsies analyzed by flow cytometry and ELSIA. Subjects with SpA had significantly lower numbers of IELs compared to controls (p=0.03). Subjects with CD had significantly increased IL-17A, (p=0.03) and IFN-γ (p<0.01) whereas those with UC had higher IL-1β (p=0.01) compared to controls. Both subjects with CD and SpA had significantly increased secretion of TNF-α (p=0.04). Correlating cytokines to bacteria revealed an association between TNF-α and the Simpson Diversity Index in subjects with UC (Spearman’s r=0.943, p<0.01); in subjects with CD, Fusobacterium had a negative correlation with TNFα+IFNγ+IL-1β (Spearman’s r=−0.786, p=0.02). Sequencing results from subjects with SpA are pending. Our data indicate differences in IEL function among subjects with SpA, CD, and UC compared to healthy controls. We hypothesize that the correlations between dysbiosis and IEL function are relevant to the pathogenesis of SpA and IBD. Future studies will be aimed at further understanding mucosal T cell and microbial interactions and may offer new therapeutic targets in these diseases.
Expression and regulation of chemokine receptor CXCR3 (CD183) on human gastrointestinal stem cells (hGISC) derived primary epithelial cells
Authors: Panja Asit B , Panja Prerona , Bhatt Rachana , Joseph Ansamma
Published: January 2023

Expression of Chemokine receptor CXCR3 (CD183) by cells in inflamed tissues play essential role in the development of chronic conditions by attracting leucocytes to the inflammatory sites. CXCR3 expression in intestinal epithelium has also been implicated in celiac disease by means of its potential role in peptide (gliadin) uptake and triggering of immune responses in the small intestine. Therefore, having an CXCR3 expressing intestinal epithelial cell system in long-term culture would be a valuable tool for discovery and development of new treatments for celiac and/or other gastrointestinal inflammatory diseases. Earlier, we have developed a method for growing human intestinal primary epithelial cells (HIPEC) from isolated human gastrointestinal stem cells (hGICSs) in long-term culture. Now we examined two small intestinal HIPEC lines for CXCR3 expression by immunocytochemical staining and subsequent fluorescence microscopic analysis.

Our results showed that CXCR3 was either undetectable or weakly expressed in normal jejunal HIPECs that had columnar characteristics whereas CXCR3 was detected on HIPECs with enriched MUC2 positive goblet epithelial cells. CXCR3 was also detectable in HIPECs derived from an actively inflamed tissue from a patient with ulcerative colitis (UC). Stimulation of normal HIPECs with proinflammatory signaling agents LPS, TNFα or IFNγ showed a minimum effect on the level of CXCR3 expression when added individually. However combinations of LPS with either TNFa or IFNg or both increased the CXCR3 expression on HIPECs.

These findings may help better understand of the disease mechanisms and development of new treatment for inflammatory conditions such as celiac disease and/or ulcerative colitis.
Skin inflammation induced by lupus serum was inhibited in IL-1R deficient mice
Authors: Deng Guo-Min , Li Xiaoyan , Guo Xuanxuan , Liu Huicheng , Gao Gongmin , Xu Guangqiong , Fei Xibin , Fang Xiang , Qiao Wei
Published: January 2023

Skin lesion is the second common clinical manifestations of systemic lupus erythematosus (SLE), but its pathogenesis is not clear. IL-1 is a proinflammatory cytokine, and its role in SLE skin lesion is still unclear. We used IL-1R deficient mice and other gene deficient mice to study the role of IL-1 in the lupus serum -induced skin inflammation. We found that the severity of skin inflammation induced by lupus serum was significantly reduced in IL-1R deficient mice and caspase-1 deficient mice. IL-1R deficiency did not affect the expression of FcγRI (CD64), FcγRII (CD32) and MHC class II (CD74) induced by lupus serum. IL-1R deficiency also reduced the lipid raft clustering, and decreased expression of MCP-1 and TNFa in monocytes. Skin inflammation and keratinocyte proliferation were significantly decreased in TNFa deficient mice. Our findings indicate that IL-1 plays an important role in skin lesions of SLE. This study suggests that IL-1 is a therapeutic target in skin lesions of SLE.
The chromatin organizer SATB1 governs the epigenetic repression of the co-inhibitory receptor PD-1 in T cells
Authors: Payne Kyle K , Stephen Tom L , Chaurio Ricardo A , Allegrezza Michael J , Zhu Hengrui , Perez-Sanz Jairo , Perales-Puchalt Alfredo , Nguyen Jenny M , Vara-Ailor Ana , Eruslanov Evgeniy B , Borowsky Mark E , Zhang Rugang , Laufer Terri M , Conejo-Garcia Jose R
Published: January 2023

Despite the importance of Programmed Cell Death-1 (PDCD1/PD-1) in inhibiting T-cell effector activity, the mechanisms regulating its expression in anti-tumor lymphocytes remain poorly defined. Here we show that the chromatin organizer Special AT-rich Sequence-Binding Protein-1 (Satb1) is required to restrain activation-induced PD-1 expression. We demonstrate that, mechanistically, Satb1 physically interacts with a nucleosome remodeling deacetylase (NuRD) complex, which it recruits to Pdcd1 regulatory regions. This molecular complex thus drives histone de-acetylation and results in PD-1 repression in T cells. Accordingly, T-cell-specific Satb1 deficiency results in a 40-fold increase in PD-1 expression. Intriguingly, tumor-derived Transforming growth factor (Tgf)-β decreases Satb1 expression in T cells through binding of Smad Family Member (Smad) proteins to the Satb1 promoter, while Smad also competes with Satb1/NuRD for binding to Pdcd1 enhancers, cooperatively unleashing PD-1 expression in a Satb1-dependent manner. Consequently, Satb1-deficient tumor-reactive T cells lose their effector activity more rapidly than wild-type T cells at PD-L1+ tumor beds, but these differences are abrogated by sustained PD-L1 blockade. Therefore, we demonstrate that Satb1 is an epigenetic controller of PD-1 expression, and that Tgf-β signaling contributes to T cell dysfunction within the tumor microenvironment by inhibiting Satb1-mediated repression of PD-1.
Resistance to PD1 blockade in the absence of metalloprotease-mediated LAG3 shedding
Authors: Andrews Lawrence Peter , Moskovitz Jessica , Szymczak-Workman Andrea L , Bruno Tullia C , Workman Creg J , Vignali Dario AA
Published: January 2023

Inhibitory receptors control immune responses preventing exacerbated T cell activation and limiting antitumor immunity. LAG3 co-expression with PD1 phenotypically marks functionally exhausted tumor-specific T cells, however LAG3 expression and function is itself regulated by ADAM metalloproteinase-mediated cell surface cleavage. To investigate the impact of LAG3 shedding on T cells within tumors, a conditional knock-in mouse was generated that results in a non-cleavable form of LAG3 (LAG3.NC). LAG3.NC CD4Cre, LAG3.NC ThPOKCreERT2 and LAG3.NC E8ICre mice (restricting LAG3.NC to all T cells, CD4+ T cells and CD8+ T cells, respectively) exhibit enhanced LAG3 expression on the respective T cell subsets infiltrating MC38 tumors. Upon therapeutic administration of anti-PD1, MC38 tumor-bearing wild-type mice show significant tumor regression and 40% become tumor-free, resulting in long-term survival. LAG3.NC CD4Cre and LAG3.NC ThPOKCreERT2 resist anti-PD1 therapy and succumb to tumor growth. However, this phenotype is not observed in LAG3.NC E8ICre mice, resulting in a similar frequency of tumor-free mice to controls. CD8+ TIL isolated from LAG3.NC CD4Cre mice do not show enhanced IFN-g/TNF-a production or proliferation as observed in controls or LAG3.NC E8ICremice following anti-PD1. Failure to mediate an antitumor immune response by LAG3.NC CD4Cre, but not LAG3.NC E8ICre mice, suggests that LAG3 cleavage on CD4+ T cells, including Tregs, is necessary for CD8+ TIL to elicit an effective immune response with anti-PD1. Selective ADAM10 inhibition in vivo, preventing LAG3 shedding, also reduces cytokine release, proposing that metalloproteinase-mediated LAG3 cleavage limits the efficacy of the antitumor immune response.
Staphylococcus aureus alpha-toxin and Clumping Factor A are pathogenic and immunotherapeutic targets against a hematogenous implant-related biofilm infection
Authors: WANG YU , Cheng Lily , Helfer David R , Ashbaugh Alyssa G , Miller Robert J , Tzomides Alexander J , Thompson John M , Ortines Roger V , Tsai Andrew S , Liu Haiyun , Dillen Carly , Archer Nathan , Cohen Taylor S , Tkaczyk Christine , Sellman Bret R , Miller Lloyd S
Published: January 2023

Infection is a major impediment to the long-term success of implantable medical devices. Treatment of these infections is complicated by bacteria biofilms, which form on the implants and block penetration of immune cells and antibiotics. Hematogenous implant-related infections following a transient bacteremia are particularly problematic because they can occur at any time in a previously stable implant. To evaluate alternative targeted immune-based therapies against these infections, we developed a hematogenous infection model in which an orthopaedic titanium implant was surgically placed in the legs of mice followed by an intravenous injection of Staphylococcus aureus 21 days later. This resulted in a marked propensity for a hematogenous implant-related infection comprised of septic arthritis, osteomyelitis with neutrophil abscess formation in the bone and biofilm formation on the implants in the surgical legs compared with sham surgical legs without implant placement or contralateral nonsurgical normal legs. Prophylaxis with two neutralizing human monoclonal antibodies directed against S. aureus virulence factors, secreted alpha-toxin (AT) and surface expressed clumping factor A (ClfA) inhibited biofilm formation in vitro and the hematogenous implant-related infection in vivo. Our findings suggest that AT and ClfA are important pathogenic factors that could be targeted as a novel immunotherapeutic against S. aureus hematogenous implant-related infections.
Bone marrow T cells populations are differently affected by sepsis than their splenic counterparts in the murine sepsis cecal ligation and puncture (CLP) model
Authors: Skirecki Tomasz , Swacha Patrycja , Hoser Grażyna , Kozłowska Ewa
Published: January 2023

Sepsis is known to induce lymphopenia which correlates with secondary infections and mortality. In sepsis survivors the lymphocyte count restores with time, although the T cell responses are impaired. So far, the mechanisms of T cells recovery are not fully understood and the murine studies were focused on the peripheral lymphatic organs in which the spleen was most common. Our study was designed to elucidate the recovery of bone marrow (BM) T cells which contain population of memory cells, after the CLP murine sepsis. In our model, mild CLP produced peripheral lymphopenia which was lowest at 48h after CLP but after 7 days circulating T cells count was rebounding. 7 days after CLP the BM frequencies of naïve and central memory (CD44+CD62L+) CD4 cells were unchanged while they were reduced in the spleen. Percentage of effector memory (CD44+CD62L−) CD4 and CD8 cells increased in the BM and at the same time they dramatically decreased in the spleen. On the contrary, the effector (CD44−CD62L+) CD4 and CD8 cells expanded at that time point in the spleen while they did not change in the BM. At D7 after CLP, the rates of BrdU+ proliferating CD4+ and CD8+ cells were markedly higher in the BM than spleen. Among the BM CD4+ cells most of the proliferating cells were the effector memory cells, while the effector CD4+ had highest proliferation rate in the spleen. Similarly, CD8 effector memory cells in the BM and CD8 effector cells were most extensively proliferating in the BM and the spleen, respectively. Taken together, our data point to the BM as the place of effector memory T cell proliferation during post-sepsis recovery.
Cold-induced stress modulates the maturation of immune cells of mice during Chlamydia muridarum genital infection
Authors: Belay Tesfaye
Published: January 2023

Chlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease (STD) in the United States. Results in a cold-induced stress mouse model established in our laboratory show that cold-induced stress increases the intensity of chlamydia genital infection. This study was to survey the production of T helper cell type 1 (Th1) and (Th2) cytokines in the stress mouse model. Cells harvested from bone marrow or T cells from spleens of stressed and non-stressed mice during Chlamydia muridarum genital infection were allowed to proliferate in vitro. Cytokine production in culture supernatants was tested by ELISA. Gene expression of GATA 3, T-bet and INF-γ in splenic T cells were determined by QPCR. Production of IL-12 by dendritic cells of stressed mice was 0μg compared to 41.06 μg in non stressed mice. However, C. muridarum infection induced increased IL-12 production (97.27 μg and 123 μg) in stressed and non-stressed mice, respectively. Stress induced a 2-fold decrease in T-bet gene expression in T-cells compared to non-stressed group. In contrast, gene expression of GATA 3 in T cells of stressed mice was significantly increased compared to all other groups tested. INF-γ gene expression in T cells of stressed mice showed a 4-fold decrease compared to T cells of non stressed mice. Stress resulted in decreased IL-12 production in dendritic cells and macrophages, INF-γ production and gene expression of T-bet by Th1 cells. Furthermore, the increased gene expression of GATA 3 only in stressed chlamydia infected T cell suggests that cold-induced stress leads to a skewed immune response toward marked increase in production of Th2 cytokines that favor immunosuppression during chlamydia genital infection.
An alternative mechanism for durable protective immunity to Staphylococcus aureus skin infection
Authors: Dillen Carly A , Farber Orly N , Pinsker Bret L , Liu Haiyun , Archer Nathan K , Lee Da B , Wang Yu , Ortines Roger V , Lee Steven K , Cai Shuting , Ashbaugh Alyssa G , Holland Steven M , Freeman Alexandra F , Milner Joshua D , Miller Lloyd S
Published: January 2023

The immune mechanisms for durable immunity against S. aureus skin infections are unclear as reinfections are common despite specific antibodies and Th1/Th17 cells. Using a CA-MRSA skin reinfection model in mice involving a primary infection (1°) in the lower back (cleared by 14 days) followed by a secondary infection (2°) in the upper back on day 28, we found that 1° and 2° wt mice had similar responses. In contrast, 1° IL-1β−/− mice had increased lesions and bacterial burden with impaired neutrophil recruitment whereas 2° IL-1β−/− mice were protected and resembled wt mice. Transfer of serum containing CA-MRSA specific antibodies did not confer protection to 1° IL-1β−/− mice and depletion of CD4+ T cells did not alter the protection of 2° IL-1β−/− mice, indicating a lack of a role of antibodies or CD4+ T cells. However, 2° IL-1β−/− mice were no longer protected if the egress of lymphocytes from lymph nodes was blocked in 2° IL-1β−/− mice (FTY720 treatment) and the immune impairment in naïve IL-1β−/− mice could be rescued by adoptive transfer of total lymph nodes cells from d28 IL-1β−/− mice. Moreover, transfer of only 50,000 γδ T cells from lymph nodes of day 28 IL-1β−/− mice conferred protection to 1° IL-1β−/− mice. These γδ T cells produced IFNγ/TNF-α, but not IL-17/IL-22, and neutralizing IFNγ/TNF-α activity resulted in loss of protection in 2° IL-1β−/− mice. In humans, similar IFNγ/TNF-α-producing γδ T cells were found in individuals with IRAK4 deficiency whose susceptibility to S. aureus skin infections improves with age but not from individuals with chronic granulomatous disease whose susceptibility is lifelong. These findings define an alternative mechanism to therapeutically target for long-term protection against CA-MRSA skin infections.
CD4+ T cell control of Salmonella infection and persistence is dependent on different macrophage subsets
Authors: Goldberg Michael F , Jenkins Marc K
Published: January 2023

The control of the persistent phagosomal pathogen, Salmonella enterica (Se), by the adaptive immune system is dependent on pathogen-specific CD4+ T-helper 1 (Th1) cells. To explore the hypothesis that Se uses a specific cellular niche to evade Th1 cells in order to persist, we generated a strain of Se that expresses a chromosomally-encoded dTomato protein that displays bright and stable fluorescence in vivo. Following oral infection in 129sv/J mice we found dTomato+ cells in macrophage (MΦ), monocyte, and neutrophil populations. Depletion of neutrophils during infection had no effect on bacterial burden or survival. In contrast, depletion of monocytes and MΦ in CCR2 or LysM-Cre CD115 stop-flox diphtheria toxin (DT) receptor transgenic mice increased bacterial counts and death 6–8 days after DT treatment, indicating an important role for these cells in maintaining Se persistence. Depletion of CD4+ T cells led to increases in bacterial counts and an altered infected cell profile compared to control animals. The frequency of infected CD11bHi MerTKInt infiltrating MΦ was increased, while infected CD11bIntMerTKHi tissue resident MΦ remained relatively constant, suggesting that Th1 cells interact preferentially with infiltrating MΦ. Infected tissue resident MΦ expressed less MHCII, CD86 and more PD-L1 and PD-L2 than uninfected counterparts, and a reciprocal expression pattern was observed on infected infiltrating MΦ. These data suggest that Th1 cells promote killing of intracellular Se in infiltrating MΦ and either ignore or are inhibited by infected tissue resident MΦ providing a reservoir where Se can persist for the lifetime of the infected animal.
Exploring the differences in intestinal dysbiosis induced by CNS inflammatory demyelination and diabetes in non-obese diabetic mice
Authors: Kirby Trevor O , Keever Abby , Seagrave Elizabeth , Turkistani Abdulgader , Lopez Susana , Garcia Daniel , Johnson Cory , McClendon Paige , Ramelow Christina , Harding Christopher , Tigranyan Arman , Rogers Jacqueline , Gonzales Marlene , Magori Krisztian , Ochoa-Repáraz Javier
Published: January 2023

Intestinal dysbiosis is being evaluated as an influencing factor in human diseases. Our laboratory works on the general hypothesis that the association is bidirectional and that disease induction affects the composition of the microbiota. Non-obese diabetic (NOD) mice develop spontaneous diabetes unless active experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis, is induced. It was proposed that the complete Freund’s adjuvant used for active induction of EAE protect against diabetes. Interestingly, although oral antibiotics treatment reduces EAE severity in SJL and C57BL/6 mice, others showed that the same treatment exacerbates diabetes in adult NOD mice. We questioned whether two different diseases affecting one animal strain, the NOD mice, affected the microbiota composition differently. We hypothesized that the gut microbiota differs between the acute inflammatory and chronic progressive stages that characterize the NOD EAE model. Further, we proposed differences in the microbiota/disease axis in EAE and diabetes. We observed significant changes in the microbiota of NOD mice that developed a severe secondary form of EAE when compared with healthy control mice. The genera Coprococcus, Ruminococcus, and Akkermansia were found elevated in EAE mice while undetermined members of the families Lactobacillaceae and Christensenellaceae were significantly reduced. Furthermore, we compared the EAE and diabetes microbiome and evaluated the effects of transplantation stool samples obtained from EAE, diabetes and control mice on diabetes progression. Our findings support the hypothesis that there are reciprocal effects between disease induction and the modification of the microbiome.