bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered cellular metabolism in Mucosal-associated invariant T cells drives dysregulated effector functions in obesity
Authors: Hogan Andrew E. , O’Brien Aisling , Finlay David K. , Lynch Lydia , O’Shea Donal 
Published: January 2023

Abstract
Obesity is a global epidemic and responsible for the development of numerous chronic diseases including type II diabetes. It is well established that obesity negatively alters circulating and tissue resident immune cell frequencies and functions. Mucosal associated invariant T (MAIT) cells are a population of innate T cells, which upon activation can rapidly produce cytokines including IFN-y, TNF-a and IL-17. We have previously reported that MAIT cells are dysregulated in obesity, with altered circulating and adipose tissue frequencies and a reduction in IFN-y production. The mechanisms driving their defective cytokine production are currently unknown. We performed RNA sequencing of MAIT cells isolated from lean and obese adults and reveal extensive alterations in metabolic pathways. Upon activation, immune cells undergo metabolic reprogramming, this allows for the increased demand for energy and biological intermediates required for the formation of effector proteins such as cytokines. Metabolic reprogramming can also determine the type of immune response elicited by specific immune cells. The metabolic pathways utilized by MAIT cells are currently unknown. In this report, we demonstrate that both cellular glycolysis and oxidative phosphorylation are important for MAIT cell cytokine production. We show that obesity induces defects in glucose consumption, mTOR activation and glycolytic metabolism, which explains the loss of IFN-y production previously reported in obesity. Collectively our data show for the first time the intrinsic metabolic pathways controlling MAIT cell cytokine production and highlight a mechanism for the altered MAIT cell responses reported in obesity.
Characterization of Macrophage Subtypes during the Acute Inflammatory Phase of Fracture Healing
Authors: Cottrell Jessica 
Published: January 2023

Abstract
Inflammation has been associated with immunological dysfunctions and chronic inflammatory diseases but is important for normal repair processes like bone healing. Macrophages (mØ) are important for bone growth, maintenance, and regeneration. MØ are distinct from other bone cells and play an important role in the inflammatory stage of bone healing. Previous data has shown that ablation of mØs during the inflammatory stage can severely impair bone healing and exacerbate bone loss in osteoporotic models. However, little research has focused on characterizing the mØ subtypes found during the inflammatory stage. We hypothesized that different mØ subtypes are activated during inflammation and release factors to regulate bone repair. Therefore, bone marrow was collected from mice femurs at days 0, 1, 2, 4, and 7 after fracture and mØ were isolated using established methods. MØ subtypes were identified using anti-F4/80, anti-CD80, and anti-CD86 antibodies via flow cytometry and cytokine expression was quantified using Luminex. When compared to unfractured controls, a 40–50% increase in MHC class II+/CD80+ double positive mØs and MHC class II+/CD86+ double positive mØs were found on day 2 post-fracture, which remained elevated through day 4 or 7, respectively. No differences were found in mØ populations between femurs in naïve (unfractured) mice. mØs of the fractured limbs expressed higher levels of cytokines overtime. Our results suggest that different subtypes of mØs are present during the inflammatory stage and may support diverse functions such as effertocytosis, chemotaxis, and tissue anabolism or catabolism, which provides insight into their contribution in normal or uncontrolled inflammatory related processes and conditions.
Chronic Sleep Fragmentation Plays A Pro-Atherogenic Role In Atherogenesis
Authors: Moriarty Alina K. , Waseem Tayab C. , Butcher Matthew J. , Heaton Julia L. , Wellman Laurie , Sanford Larry D. , Galkina Elena V. 
Published: January 2023

Abstract
Atherosclerosis is a chronic, inflammatory disease characterized by the hardening and narrowing of the small and medium sized arteries caused by the accumulation of fatty plaque lesions. In the USA, approximately 30% of the population reports insufficient sleep, and nearly 70 million people have a diagnosed sleep disorder. Chronic sleep fragmentation (SF) has been shown to have a connection with the development of many common diseases such as, type 2 diabetes, obesity, and cardiovascular disease; however the exact relationship between SF and the progression of atherosclerosis is unknown. Importantly, SF is related to stress conditions, but also has additional unique components that may affect atherogenesis. To examine the effects of SF on atherosclerosis, Apoe−/− mice were placed on a high fat diet (HFD) and sleep was fragmented using a motorized slow moving mechanical sweeper every 2 minutes during the light period each day. After 9 weeks on HFD and SF we found that chronic SF led to increases in plaque formation in HFD fed Apoe−/− mice. The development of atherosclerotic lesions is characterized by the recruitment of leukocytes and myeloid cells into atherosclerotic arteries. To determine if SF alters the immune composition within atherosclerotic aortas, we examined aortic infiltrating leukocyte subpopulations by FACS. We found that the relative proportion of CD45+ cells from SF Apoe−/− aortas was elevated in comparison with control Apoe−/− aortas. We also detected an increased percentage of CD11b+CD115+ macrophages in SF Apoe−/− aortas. Together our data suggests that chronic SF is pro-atherogenic, alters the immune composition of atherosclerosis-prone aortas and facilitates the accumulation of macrophages within the aortic wall.
Gut microbiota enhances neutrophil resolution in immunocompromised hosts to improve response to pneumococcal pneumonia.
Authors: Felix Krysta M. , Jaimez Ivan , Nguyen Thuy-Vi , Ma Heqing , Raslan Walid , Klinger Christina , Doyle Kristian , Wu Hsin-Jung Joyce 
Published: January 2023

Abstract
Streptococcus pneumoniae is a major cause of disease and death worldwide, particularly in neonatal, elderly, and immunodeficient patients. We examined the impact of the gut microbiota on lung disease (known as the gut-lung axis) using traditional immunocompetent as well as novel immunocompromised mouse models of S. pneumoniae lung infection. Adaptive immunity against S. pneumoniae depends largely on T-independent II (TI-II) antibodies against the bacterial polysaccharide capsule. We found that colonization with the gut commensal Segmented Filamentous Bacteria (SFB) increased anti-S. pneumoniae TI-II antibodies, but did not further enhance protection against S. pneumoniae in wild type mice. Indeed, basal B cell activity in the absence of SFB colonization was sufficient to provide essential protection against S. pneumoniae. However, in adaptive immune-compromised mice, we demonstrated a gut-lung axis of communication, as SFB colonization modulated the innate immune response to enhance protection against S. pneumoniae lung infection. Neutrophils are important first responders during pneumonia, but proper neutrophil resolution is essential to prevent excessive tissue damage. We found that SFB colonization led to more efficient clearance of lung neutrophils at 3 days after infection, which correlated with a decrease in CD47, a transmembrane glycoprotein that inhibits clearance of apoptotic cells, on lung neutrophils in SFB+ immunocompromised mice. This accompanied a shift in lung neutrophils from CD18hiCD62L−/lo inflammatory neutrophils to CD18−/loCD62Lhi pro-resolution neutrophils. These data demonstrate that a gut commensal modulates the innate immune response to enhance protection against a lung infection.
Alveolar and monocyte-derived macrophages differentially engage antibacterial programs during adaptive immunity to Mycobacterium tuberculosis.
Authors: Delahaye Jared , Plumlee Courtney R. , Plaisier Christopher , Baliga Nitin , Sherman David R. , Urdahl Kevin B. 
Published: January 2023

Abstract
Cognate interactions between antigen-specific CD4 T cells and Mycobacterium tuberculosis (Mtb)-infected cells are critical for mucosal immunity to tuberculosis (TB). However, Mtb resides intracellularly within a variety of myeloid cell populations, and the relative impact of CD4 T cells on distinct infected cell types remains unknown. Here we report that intratracheally-transferred, Mtb-specific T cells are unable to reduce the bacterial burden in the first week after aerosol infection, when Mtb resides almost exclusively within alveolar macrophages (AMs). However, transferred T cells do mediate protection by day 14, at which point Mtb has disseminated to monocyte-derived macrophages (MDMs). Using an Mtb “replication clock” strain, which loses plasmid at a fixed rate with bacterial division, we observe that Mtb continues to replicate in AMs even after the onset of adaptive immunity, whereas replication in MDMs is curtailed. We also find that MDMs are less reliant on oxidative metabolism and express more NOS2 than AMs, which may explain their superior antibacterial activity. Finally, RNA-Seq analysis of these two macrophage populations reveals enriched expression of proinflammatory pathways (i.e. NFkB signaling) in Mtb-infected MDMs compared to AMs. Together, our results indicate that AMs may serve as a privileged niche for Mtb, despite the presence of Mtb-specific T cells. As many current TB vaccine candidates seek to induce lung-resident T cells that can recognize and control Mtb early after aerosol infection, the relative resistance of Mtb-infected AMs to T cell-mediated immunity could represent a previously unappreciated barrier to this strategy.
Measuring total IFNα at fg/mL concentrations in human blood
Authors: Lambert Jeremy , Ferrell Evan , Wickman Jason , Chang Lei , Hanlon David , Johnson Joe , Kiesel Gregory 
Published: January 2023

Abstract
Total IFNα has been challenging to quantify in normal human plasma and serum due to its low abundance and variety of subtypes. Here we describe the development of a digital immunoassay capable of measuring all 12 subtypes of human IFNα in plasma and serum using Quanterix’s Simoa technology. The Simoa digital ELISA technology counts signal generated from single immunocomplexes formed on superparamagnetic beads confined in arrays of femtoliter-sized wells in which fluorescent molecules are highly concentrated. The Simoa IFNα multi-subtype assay has a dynamic range of approximately 3.5 orders of magnitude and a sensitivity below 100 fg/mL. The median LOD (2.5 SD) over 12 runs was approximately 30 fg/mL and the LOQ was approximately 80 fg/mL. This represents a significant improvement over commercial ELISAs, which claim IFNα sensitivities in the pg/mL range. In repeated screens of 30 normal samples, up to 60% measured above LOD and testing of serum from Lupus patients and stimulated PBMCs resulted in concentrations within the dynamic range of the assay. Depletion experiments conducted by incubating unlabeled capture or detection antibody with these same samples demonstrated that the assay is specific for IFNα. IFNα has been useful in several applications including as a therapeutic agent, as a biomarker for monitoring response to immunotherapy, and as a marker of disease progression in human clinical and preclinical studies. In each example, increased sensitivity for detection of all subtypes of IFNα provides researchers and clinicians with the ability to measure the biomarker in both normal and acute samples with robustness required to advance precision medicine programs.
Centrifuge-less red blood cell lysis and immunostaining of whole blood for flow cytometry using DA-Cell washer and plate
Authors: Kim Namyong , Lye Melvin 
Published: January 2023

Abstract
Blood cells are prime indicators of immuno-surveillance, and the ease of blood sampling makes blood analysis a key interest for clinical and research applications. While current flow cytometry methods are high-throughput and provide fine resolution in the segregation of white blood cell (WBC) populations, WBC enrichment involving red blood cell (RBC) lysis are laborious and typically performed manually, contributing to experimental variability especially as blood cells are sensitive to physical and chemical stress.
We describe RBC lysis and leukocyte immunostaining on a centrifuge-less platform DA-Cell™, using a novel wall-less plate and laminar flow washer. The DropArray (DA) plate consists of an array of 96 hydrophilic spots surrounded by hydrophobic surface, which functions as a virtual wall that separates each spot. The maximum volume of each spot can be increased to 300μL with an insert. During lysis, WBC settle to the surface of the spot, allowing the spent lysis buffer to be removed. After removal of the insert, the plate goes through a gentle 3–4min laminar-flow washing process in the DA-Cell washer, decreasing cell washing time by at least 50% while eliminating centrifugation that stresses cells and disrupts antibody binding.
In studies comparing mouse whole blood lysis (1X RBC Lysis buffer, eBioscience) and antibody staining by conventional tube centrifuge and DA-Cell, DA-Cell achieved dramatically higher staining index and improved resolution of cell cluster by flow cytometry. CD45+ leukocyte recovery and viability was uncompromised compared to conventional tube centrifuge. In summary, DA-Cell system provides gentle, fast and convenient blood lysis, while improving data quality with superior antibody staining.
Real-time detection of LPS induced cytokine-storm using Vium Smart Housing
Authors: Ichim Christine V. , Mechanic Jordan , Shah Puja , Maraganore Colin , Hutto David , Schaevitz Laura 
Published: January 2023

Abstract
The objective of this study was to use Vium Smart Housing to determine if in-life digital readouts (e.g. changes in motion and breathing rate) can correlate with cytokine response profiles in an LPS-induced mouse model of endotoxemia. Real-time detection of cytokine release has the potential to reduce or eliminate the need for expensive blood sampling and biochemical analyses. Eighty Balb/c mice were injected with 5 mg/kg of UltraPure LPS (E. coli 0111:B4, Invivogen) or vehicle control. Subjects were housed in Vium’s Digital Vivarium, where sensors and a HD camera coupled with computer vision, data algorithms, and cloud capabilities allow for continuous monitoring of animals and collection of automated metrics such as motion, % time running on wheel and breathing rate. All study data is available in real-time. As early as 1 hr post-LPS administration, breathing rate increased from baseline, peaked 7 hours post-induction (8.7%±3.5% vehicle vs 39.5±3.1% LPS treated) and remained elevated relative to vehicle for over 24 hrs (p&lt;0.05). Breathing rate changes were accompanied by decreased motion as early as 1 hr post-LPS administration, peaked at 2 hours post-induction (28.73%±53.2% vehicle vs –82.69%±1.72% LPS treated), and remained decreased for over 24 hrs (p&lt;0.05). Increased breathing positively correlated, while decreased motion negatively correlated with plasma levels of TNFα, and IL-10 at 1.5 hrs, and IL-6, IL-1B, and IFN-γ levels at 6 hrs post-LPS (p&lt;0.05). We found that Vium’s automated metrics was able to detect onset of LPS-induced endotoxemia in real-time. This could be used to 1) screen compounds that prevent immune responses or alternatively 2) to identify cytokine release syndrome when screening immuno-oncology candidates.
PLK1 as a potential therapeutic target of lupus
Authors: Wu Tianfu , Li Yaxi , Mohan Chandra 
Published: January 2023

Abstract
System lupus erythematosus (SLE) is a complex autoimmune disease with high mortality and morbidity. A targeted therapy is desirable for personalized medication of SLE. Recently we used a novel Activity-based Kinome Screening and identified several novel signaling molecules which are up- or downregulated in murine lupus. Totally we screened 196 kinases in spleens from two lupus mouse models, B6.Sle1.Sle3 and MRL.lpr. Compared to the B6 control, PLK1 and its upstream regulator Aurora A exhibited significantly increased activities. We further found the activation of both Aurora-A and PLK1 were significantly upregulated in splenic B220+ B cells of lupus mice MRL.lpr. We found a PLK1 specific inhibitor NMS-P064 ameliorated splenomegaly (P &lt; 0.05), anti-dsDNA antibody production (P &lt; 0.001), proteinuria and BUN, and renal pathology GN score in MRL.lpr. In addition, we found IL-6, IL-17 and kidney injury molecule 1 (KIM-1) were significantly decreased in the serum of MRL.lpr mice after PLK1 inhibition. In vitro, after 24 h stimulation of splenocytes with CD40 ligand, IL-6 secretion was significantly reduced upon PLK1 blockade (P &lt; 0.05). Whereas IL-10 secretion was significantly increased in anti-IgM Fab’ fragment stimulated splenocytes upon PLK1 blockade. Given the phosphorylation of PLK1 was significantly increased on Thr 210 in murine lupus, it is possible that this could be the consequence of the direct phosphorylation of PLK1 by Aurora A. Taken together, PLK1 may contribute to the pathogenesis of lupus via Aurora-A/PLK1 signaling cascade and might be a potential therapeutic target of lupus.
ALCAM and VCAM-1 as potential urinary biomarkers in Caucasian lupus patients
Authors: Habazi Deena , Vanarsa Kamala , Stanley Samantha , Gokaraju Sirisha , Ding Huihua , Parodis Ioannis , Gunnarsson Iva , Mohan Chandra 
Published: January 2023

Abstract

Background
Lupus affects millions of people worldwide and is difficult to diagnose early in patients. By utilizing enzyme-linked immunosorbent assays (ELISA), this study seeks to identify urinary biomarkers that may detect lupus in its earlier stages, allowing for better, earlier treatment regimens.

Methods
197 urine samples from a Swedish SLE cohort were divided into four groups, each matched for demographics: 23 active non-renal SLE samples (SLEDAI&gt;0, rSLEDAI=0), 29 active renal SLE samples (SLEDAI&gt;0, rSLEDAI&gt;0), 46 inactive SLE samples (SLEDAI=0), and 99 healthy controls. These samples were screened for 2 human proteins using ELISA, selected because they exhibited biomarker potential in our earlier screens.

Results
VCAM was noted to be significantly elevated (2-tailed t-test, p&lt;0.0001) in SLE patients compared to healthy controls. More specifically, VCAM was elevated in active renal samples when compared to healthy samples (p&lt;0.0001). An ROC curve shows that VCAM can be used to differentiate between active renal samples and control samples (AUC=0.8889, p-value=0.0250). ALCAM was also elevated (p&lt;0.0001) in SLE patients compared to healthy patients. However, ALCAM was not as highly elevated in active renal samples compared to healthy samples (p&lt;0.001). Additionally, an ROC curve does not show ALCAM strongly differentiating between active renal samples and control samples. Based on these results, VCAM is the more promising biomarker of the two.

Conclusions
Urinary VCAM and ALCAM are both potentially viable biomarkers for lupus that warrant further investigation. Studies are in progress to compare the performance of these urinary markers to conventional yardsticks and their relationship to other clinical parameters.
Macrophage heterogeneity correlates with autophagy-dependent anti-mycobacterial activity and ability to present antigen in human macrophages
Authors: Singh Vipul Kumar , Khan Arshad , Papanna Ramesha , Mann Lovepreet , Wan Jon Hak , Canaday David , David Sunil A , Jagannath Chinnaswamy 
Published: January 2023

Abstract
Mycobacterium tuberculosis (Mtb) kills around 1.5 million people each year. Albeit, only 10% of humans develop active disease after aerosol infection, the basis for differential susceptibility of humans remains unclear. To determine if human macrophages are heterogeneous in their ability to kill Mtb, cord blood derived macrophages were treated with either IFN-γ or IL-4 to prepare M1 and M2 phenotype respectively in vitro. Macrophages were infected with a 1:1 ratio of Mtb and days 3 and 5 post infection, macrophage lysates were plated for CFU counts. In addition, intracellular autophagy was measured in gfpMtb infected macrophages using cytoID stains to detect autophagosomes and LC3 lipidation through western blot of macrophage lysates. Mtb infected macrophages were overlaid using CD4 T cells specific for the Antigen-85B epitope to measure antigen presentation. M1 macrophages showed a bacteriostatic ability and restricted the growth of Mtb while IL-4 treated M2 macrophages showed nearly 1 log increase in the growth of Mtb. Thus M2 macrophages were permissive for growth of Mtb. Consistent with this observation, gfpMtb co-localized with autophagosomes more frequently in M1 macrophages suggesting a role for autophagy during growth inhibition. M1 macrophages also presented Ag85B to CD4 T cells better than M2 macrophages. Enhanced antigen presentation by M1 macrophages was also found to be dependent on IFITM3, Atg5 and Atg 16 genes as siRNA knock down of these specific genes resulted in reduced antigen presentation by M1 macrophages. These observations suggest that autophagy pathways within macrophage may govern their heterogeneity into M1 or M2 hence their innate ability to kill intracellular Mtb and trigger immune activation.
Alterations of Specific Lymphocytic Subsets with Ageing and with Aged-Related Metabolic and Cardiovascular Diseases
Authors: Liu Chao Lien , Tram Van Thi Ngoc , Lin Chung Hao , Liao Kuo Chen , Chen Ying Jen 
Published: January 2023

Abstract
Ageing of the immune system (immunosenescence (ISC)), is a complex subject characterized by a decrease in cell-mediated immunity, particularly with T-cell function. We conducted a clinical study to analyze peripheral blood (PB) for alterations in T-cell subsets among young healthy (YH) controls, elderly healthy (EH) controls, age-matched subjects with metabolic diseases (MDs; E-MDs), and cardiovascular diseases (CVDs; E-CVDs). The frequencies of expressions of CD3T, CD8T, and invariant natural killer T (NKTs; iNKT) cells were reduced in the EH, E-MD, and E-CVD cohorts; in contrast, CD4T and regulatory T (Treg) cell frequencies tended to increase with ageing, whereas they were lower in subjects with aged-related MDs and CVDs. Moreover, subsets including naïve, effector memory T (TEM) cells, effector memory T cells re-expressing CD45RA (TEMRA), as well as the CD28−CD57+phenotype in the CD4T and CD8T subpopulations consistently showed accumulation of senescent T-cell subsets in ageing, and in patients with E-MDs and E-CVDs compared to YH volunteers. Notably, serum cytokines, including Interferon (IFN)-γ, Transforming growth factor (TGF)-β, and Growth differentiation factor (GDF)-15 levels were consistently reflection of the alterations in T-cell subsets for within-group comparisons. Ageing-, E-MD-, and E-CVD-associated changes in PB T-cell subsets were paralleled by alterations in expressions of cytokines involved in lymphopoiesis, which might play important roles in ageing, and aged-related MD- and CVD-mediated dysregulation of immune functions. Furthermore, identifying these altered T-cell subsets can potentially serve as diagnostic markers for preventing ageing problems and treating aged-related MDs and CVDs.