bluuebunny/crossref_metadata_2025 huggingface dataset:
Disturbance of Renal Circadian Rhythm in Lupus Nephritis
Authors: Mishra Rakesh K , Bethunaickan Ramalingam , Zhang Weijia , Davidson Anne
Published: January 2023

Background

Circadian rhythm is a universal phenomenon that allows organisms to anticipate and respond to changes in their environment by regulating various biological processes in the number of tissues, including the kidney. We found a disturbance of expression of key clock transcriptional regulators in the kidneys of mice with SLE nephritis. The purpose of these experiments was to determine how this abnormality affected transcriptional regulation of renally expressed genes and immune cell homeostasis.

Methods

We studied NZB/W mice at the age of 12 weeks (without proteinuria) and 30 weeks (with proteinuria). Kidneys and blood were harvested at 4 hour intervals. Kidneys were perfused with PBS and used to generate RNA, protein lysates and single cells for flow cytometry analysis.

Results

Young NZB/W mice have normal circadian oscillation of peripheral WBCs. There was a marked increase of circulating renal macrophages in the nephritic mice but circadian rhythm of Ly6Chi vs Ly6Clo cells followed a normal pattern. Kidneys of young NZB/W mice displayed a normal circadian pattern of expression of the master transcriptional regulators of circadian rhythm including Bmal1, Clock, Per and Cry genes compared from the nephritic mice had attenuated rhythm of these genes with inversion of the normal pattern. We also examined levels of core clock proteins (BMAL1 & PER2) in the kidney of mice from young and nephritic group at different ZT times. As expected, there was significant reduction in BMAL-1 protein expression at various zeitgeber times in nephritic mice compared to young mice.

Conclusion

Circadian dysfunction in the setting of lupus nephritis may have important implications for the delivery and metabolism of therapeutic intervention.
TACI deficiency delays the onset of systemic lupus erythematosus symptoms and improves the survival of MRL/Lpr mouse
Authors: Liu Lunhua , Allman Windy R , Coleman Adam S , Takeda Kazuto , Akkoyunlu Mustafa
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies and immune-complex deposition in organs such as skin and kidney. Ablation of BAFF with anti-BAFF antibodies alleviates SLE symptoms but the receptor responsible for the generation of pathogenic antibodies has been controversial. Since TACI is critical in the development of antibody secreting plasma cells, we evaluated the contribution of TACI in the formation of autoantibodies and the development of SLE in TACI deficient “lupus mouse”, generated by backcrossing the lupus prone Mrl/Lpr mouse with TACI−/− mouse. We found that TACI deficiency (Lpr-TACI−/−) increased the survival of lupus mouse as compared to the littermates, LPR-TACI +/+ and LPR-TACI +/− mice. The survival of Lpr-TACI−/− mice correlated with a delay in the development of anti-dsDNA and anti-SAM/RNP antibody titers. The prolonged survival of LPR-TACI −/− mice is likely a result of protection from kidney damage since proteinuria levels and IgG accumulation in the renal glomeruli were significantly less in LPR-TACI −/− mice as compared to the age-matched control mice with disease. Coinciding with the preserved kidney function, four month old LPR-TACI −/− mice kidney macrophages manifested M2-skewed phenotype, while macrophages harvested from kidneys of the age-matched control littermates were M1-skewed. Further supporting the protective role of M2-skewed macrophages, adoptively transferred LPR-TACI−/− macrophages improved the proteinuria and glomerulonephritis in control animals with kidney pathology. Collectively, LPR-TACI −/− mice experiments suggest that blocking TACI function may be a novel approach for the treatment of SLE disease.
Deciphering Interleukin 4-induced gene-1 as novel immune regulatory pathway in dendritic cell subsets
Authors: Fallarino Francesca , Gargaro Marco , Briseno Carlos , Murray Peter , Scalisi Giulia , Turco Antonella , Matino Davide , Puccetti Paolo , Murphy Theresa L , Murphy Kenneth M
Published: January 2023

Interleukin 4-induced gene-1 (IL4i1) is as L-phenylalanine oxidase initially described as an early IL-4-inducible gene in B cells [1]. Herein, we analyzed IL4I1 expression in different DC subsets and investigated the possible role of IL4I1 in T-cell regulation. By using a novel and highly specific antibody, reactive to mouse IL4I1, developed in our laboratory, we found that IL4i1 could be induced by IL-4 or CpG olognucleotides (CpGODN) only in classical DCs (cDC). IL4i1 induction, by IL-4 was prevented in cDCs isolated from AhR−/− mice. Moreover, IL-4–treated cDCs cultured with CD4+T cells favored the expansion of FoxP3+ CD4+ T cells (Treg) compared to untreated cDCs. This effect was abrogated in the presence of a small interfering RNA (siRNA) targeting IL4i1 but not by a control siRNA. Notably, IL-4-induced IL4i1 expression in cDCs required aryl hydrocarbon receptor (AhR) in these cells. IL4I1-mediated oxidative deamination of phenylalanine produces H2O2 and phenylpyruvate (PP). Recently we found that PP is a novel ligand of AhR. Phenylpyruvate administration in vivo significantly reduced disease severity in a murine model of multiple sclerosis, such effect was prevented in AhR−/− mice. Overall, these results point IL4i1 as a key enzyme in the regulation of immune responses.
Gestational hypothyroxinemia increases the immune response of the progeny that suffers experimental autoimmune encephalomyelitis.
Authors: Opazo Maria Cecilia , Bugueño Katherinne , Jara Evelyn , Haensgen Henny , Albornoz Eduardo , Gomez Felipe , Binzberger Rebecca , Toledo Paola , Blanco Noelia , Simon Felipe , Elorza Alvaro , Kalergis Alexis , Bueno Susan , Riedel Claudia
Published: January 2023

There are more evidences supporting the relevance of a healthy pregnancy over offspring imprinting. This is the case for the maternal endocrine status and the immune system. Hypothyroxinemia (Hpx) is a highly frequent thyroid hormone deficiency condition in pregnant women characterized by low levels of thyroxin (T4) with normal levels of triiodothyronine (T3) and thyroid stimulating hormone (TSH) in the blood. It is known that this condition is asymptomatic for the mother, however it impairs cognition in the offspring. In this work we have shown that the effects of gestational Hpx go beyond the central nervous system by also affecting the immune response. We observed that gestational Hpx increases the immune response in the offspring that suffers experimental autoimmune encephalomyelitis (EAE). EAE is an experimental model used to study multiple sclerosis (MS). We show here that mice C57BL/6 gestated in Hpx induced with EAE suffer earlier and more intense EAE symptoms compared to C57BL/6 mice gestated in euthyroidism. Even though, the population of T regulatory (Treg) cells in the spleen was similar in mice gestated in Hpx and mice gestated in euthyroidism, the Treg cells from mice gestated in Hpx have less capacity to suppress T effector cells and to secret IL-10. Mice gestated in Hpx have high basal levels of TNF-α and IL-17 and present increased brain blood barrier permeability. In this work we analyzed the population of Th-17 and capacity to secret IL-17 from intestine, meninges and spleen in mice gestated in Hpx that suffer EAE. These results are very novel and impact our knowledge of how the immune system of the offspring could be affected by the deficiency of maternal thyroid hormones during gestation.
Interleukin-15 Enables Septic Shock by Maintaining Natural Killer Cell Integrity and Function
Authors: Guo Yin , Luan Liming , Wang Jingbin , Patil Naeem , Bohannon Julia , Rabacal Whitney , Fensterheim Benjamin , Hernandez Antonio , Sherwood Edward
Published: January 2023

Interleukin-15 (IL-15) is essential for development, differentiation and function of natural killer (NK) and memory (m) CD8+T cells. Our lab previously showed that NK and CD8+T cells facilitate systemic inflammation and physiological dysfunction during septic shock. However, factors that regulate NK and CD8+T cell function during sepsis are not well characterized. We hypothesize that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+T cell numbers and function. To test our hypothesis, the response of IL-15-deficient (IL-15 KO) mice to sepsis was assessed. IL-15 KO mice showed improved survival, attenuated hypothermia, and less pro-inflammatory cytokine production during septic shock caused by cecal ligation and puncture (CLP) or lipopolysaccharide (LPS). Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15 neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion and conferred protection if given 4 days prior to septic challenge. IL-15 SA treatment amplified septic shock, which was prevented by NK cell or IFNγ depletion. IL-15 SA treatment also exacerbated septic shock caused by CLP if given after the onset of sepsis. In conclusion, endogenous IL-15 doesn’t directly augment the pathogenesis of sepsis but enables septic shock by maintaining NK cell numbers and function. Exogenous IL-15 SA exacerbates severity of sepsis by activating NK cells and facilitating IFNγ production.
Monocyte trans-differentiation in healthy and coronary artery disease patients is dependent on the affinity state of CD11c/CD18.
Authors: Hernandez Alfredo Alexander , Simon Scott I , Foster Greg , Fernandez Andrea , Singh Gagan
Published: January 2023

Mon2 CD14++CD16+subset of monocytes are central to the progression of coronary artery disease (CAD) by increasing their numbers in the circulation and upregulation of CD11c, which binds to endothelial VCAM-1 leading to recruitment to the nascent sites of plaque formation in arteries. In contrast, Mon1 CD14++CD16− monocytes are enriched within atherosclerotic plaques. We hypothesized that the number and affinity state of CD11c, which influences the capture efficiency and adhesion to inflamed endothelium, can regulate monocyte fate. Using a lab-on-a-chip assay to capture monocytes from whole blood samples under shear flow we examined the effect of antibodies that stabilize a high (HA) versus low affinity (LA) state of CD11c. In healthy subjects, we found that Mon2 have the capacity to differentiate into Mon3 CD14dimCD16++ (mean: .1 vs 6.79%, P<.001) when stabilized with CD11cHA, or to a Mon1 monocyte (mean: 6.13 vs 36.82%, P<.001) stabilized with CD11cLA. Healthy Mon1 spontaneously upregulate CD16, thereby taking on a Mon2 phenotype (~80% expansion to CD16+) in contrast to CAD Mon2 which progressively lose CD16 (50–60% reduction) and assume a Mon1 phenotype consistent with their fate in atherosclerotic plaques. Stabilizing CD11cHA maintained a Mon2 phenotype antagonizing the shift to Mon1 (mean: 62.37 vs 2%, P<.01) in CAD monocytes. The loss of CD16 on adherent Mon2 monocytes and the conversion to Mon1 is consistent with the phenotype within atherosclerotic plaques. However, Mon2 gain of CD16 and progression to Mon3 in healthy patients suggests divergent pathway for homeostatic function. Taken together these data indicate a role for regulation of monocyte phenotype via outside-in signaling of CD11c on monocytes captured on VCAM-1.
Diversity of Human Monocyte Subsets Revealed by CyTOF Mass Cytometry
Authors: Hamers Anouk AJ , Thomas Graham D , Kim Cheryl , Hedrick Catherine C
Published: January 2023

Background

To date, 3 distinct human monocyte subsets have been identified based primarily on their expression of the surface markers CD14 and CD16. With the emerging knowledge of myeloid-derived suppressor cells and other myeloid subsets, we hypothesized that these monocytes were likely more heterogeneous in composition. Therefore, we set out to use the high dimensionality of mass cytometry to accurately identify and define monocyte subsets in blood of healthy humans.

Methods

Heparinized blood from 12 healthy humans was obtained and analyzed by CyTOF mass cytometry. We employed the Phenograph algorithm to cluster and identify all monocyte subsets based on their phenotypes using a 40-marker mass cytometry panel.

Results

Phenograph identified a total of 15 monocyte clusters based on cell phenotype. By performing hierarchical clustering, we were able to group the 15 clusters into 6 larger meta-clusters and found that most of these meta-clusters fall within the CD14hi classical monocyte population, illustrating significant heterogeneity among the classical CD14hi monocyte population in humans. We also identified two subsets of nonclassical monocytes of which one was high for both CD61 and CD9 expression, pointing to a possible role in cell adhesion/migration and platelet activation.

Conclusion

Monocytes are highly diverse with the conventional classical subset showing the most diversity. Studies of these new subsets should lead to greater understanding of functions of monocyte subsets in diseases.
Neonatal MDSCs compromise macrophage control of encapsulated bacteria
Authors: Robinson Cory , Gleave-Parson Madeline , Grimmett Juanita , Lyda Logan , Labuda Christopher
Published: January 2023

Limitations in innate and adaptive immunity have been described early in life that contribute to susceptibility to microbial infection. Interleukin (IL)-27 is a heterodimeric cytokine produced primarily by antigen presenting cells and suppressive toward a variety of immune cell types. We have shown in humans and mice that neonatal macrophages express IL-27 at an elevated level as compared with adults. In mice this was shown to be maintained through infancy. Using the mouse as a model system, we have recently shown that myeloid-derived suppressor cells (MDSCs) are more abundant in the spleens of neonatal and infant mice and contribute to elevated IL-27 levels in young mice. Functional studies demonstrate that these MDSCs express active nitric oxide synthase, phagocytic oxidase complex, and arginase-1. Few studies have investigated the influence of MDSCs on innate immune function during bacterial infection. Streptococcus agalactiae and Escherichia coli are significant concerns for newborns as common causes of sepsis. We investigated the influence of neonatal and infant MDSCs cultured with infected macrophages and show impaired macrophage control of S. agalactiae and E. coli. Furthermore, the suppressive effect of MDSCs on macrophage function requires IL-27. Inclusion of a soluble receptor to neutralize IL-27 promotes increased control of bacterial growth. Our data suggests that the effect of MDSCs and IL-27 involves a direct influence on macrophage effector functions since inflammatory cytokines such as TNF and IL-6 are not significantly altered by the presence of MDSCs. Taken together these results suggest that the increased abundance of MDSCs and IL-27 production may contribute to some of the limitations in early life immunity.
Using the Circulating Proteome to Assess Type I Interferon Activity in Systemic Lupus Erythematosus
Authors: Smith Michael Alexander , Chiang Chia-Chien , Sinibaldi Dominic , Zerrouki Kamelia , Manna Zerai , White Wendy I. , Kaplan Mariana J. , Hasni Sarfaraz , Siegel Richard M. , Sanjuan Miguel A. , Casey Kerry A.
Published: January 2023

Type I interferon (IFN) signaling drives pathology in systemic lupus erythematosus (SLE) and can be tracked via IFN-inducible transcripts present in whole blood as described by several IFN gene signatures. As SLE is a complex disease affecting diverse organ systems, we examined whether measurement of circulating proteins, which can infiltrate the bloodstream from afflicted tissues, might also offer insight into global IFN activity. The presence of anti-DNA autoantibodies in patient serum has prevented effective use of SOMAmers for the evaluation of circulating proteins in SLE. Here, we adapted protocols to mitigate for those autoantibodies and report high reproducibility and accuracy with 100% QC pass rate and improved correlation with previously validated multi-analyte platform results. Using SOMAmers together with the IFN 21-gene signature1 (IFNGS), we derived an IFN protein signature that can approximate the IFNGS score. In a cohort of 82 SLE patients and 48 healthy donors, the protein signature was found elevated above healthy donors for most of IFNGS-high patients (49/55, 89%) and also for a subgroup of IFNGS-low patients (7/27, 26%). The protein signature correlated with global disease activity (median SLEDAI score of 4 for the cohort) in both lymphopenic and non-lymphopenic patients. Significant associations with skin involvement, low complement, anti DNA auto-antibodies, and thrombocytopenia were also observed. In sum, our results suggest blood derived protein measurements may complement validated gene signatures to monitor IFN activity.
A combined genome-wide DNA methylation and mRNA expression analysis identified aberrant gene regulatory pathways in inflammatory myositis
Authors: Li Quanzhen , Luo Hui , Zhu Honglin , Zhu Chengsong , Wang Li , Zhang Huali , Zuo Xiaoxia
Published: January 2023

Inflammatory myositis (IM) is an immune-mediated inflammatory process primarily involving skeletal muscle and the etiology is still poorly understood. Clinical and molecular evidence indicated that the epigenetic regulation on mRNA transcription of immune cells may drive the pathogenic processing. In this study, we performed genome-scale DNA methylation and mRNA transcription profiling using Illumina HumanMethy450 and HumanHT-12 Beadchips on PBMC of 24 IM patients and matched normal controls (NC). We identified 617 genes showing altered DNA methylation between IM and NC. By integrating DNA methylation and mRNA expression data, we found 108 hypomethylated genes showing concurrent up-regulation and 43 hypermethylated genes showing decreased expression. Integrated pathway analysis indicated that these genes are involved in Th1/Th2 cell activation, arginine degradation, chemotaxis of neutrophils, granulocyte adhesion, cytotoxic T cell-mediated apoptosis, etc. Comparison of DNA methylation profile between dermatomyositis (DM) and polymyositis (PM) identified 66 altered genes which were shared in both DM and PM, 51 genes altered only in PM and 32 genes only DM. The genes which showed most significant hypomethylation and up-regulated are DYSF, IL1R2, OLFM4, ARG1, PDE6H, PPARG, MYB, and the most hypermethylated and down-regulated genes include CXCR6, ABLIM1, LAX1, PRKCH, CD3G and SLFN5. DYSF is a gene encoding dysferlin protein which may involve in the pathogenesis of myositis by interfering membrane repair in muscular dystrophy. Our analysis lays the groundwork for further molecular studies of IM by identifying novel epigenetically dysregulated genes potentially involved in the immune activation and muscular dystrophy.
Our Own Worst Enemy: Psychosomatic effects of perceived stress on IgA levels
Authors: Redmond Sarah , Biggs Erick , Blevins Kasey , Brimer Kelsey , Brown Cameron , Bryan Sandra , Burger Haley , George Anna , Hunt Mckenzie , Jannise Caitlin , Kesterson Melissa , Kwan Anthony , Marshall Jacob , Miller Kenzie , Mohamed Attia , Rajashekara Arpitha Mysore , Pagan Rachael , Peters Mary , Saenz Allison , Steeves Gregory , Stowers Samuel , Welch Nate , Williams Alexis , Williams Devonn , Wingfield Kyle , Zimmermann Krista
Published: January 2023

Our study is to determine and compare human IgA levels to self reported stress levels of college students and faculty at the beginning of the college semester, when stress levels are expected to be low, as compared to a later point in the semester, when stress levels are expected to be higher. This study is significant because the data will provide us with information regarding whether or not people are as biologically stressed as they report at different periods of the semester. Individuals are expected to be at a lower stress level at the beginning of the semester than at the end of the semester. Biological stressors are indications that the psychological and physical aspects of an organism are attempting to maintain an equilibrium in order to function properly. We are interested in quantifying biopsychological stressors between mental and physical stress responses and the effect of psychosomatic influence. To analyze samples for IgA molecules levels in comparison to proclaim stress levels to determine if students are as physically stressed as they are mentally feeling stressed. Stress was reported on a zero to ten scale and these reports will be compared to actual levels of human IgA attained from saliva samples. Geospatial coordinates have been taken to relate the data to location across campus. After a regression test, showing a P value of 0.206, it was clear that there was no significant correlation in the data. This tells us that, overall, the levels of IgA, and therefore stress, did not relate to the reported level of stress from the participants.
Macrophage Migration Inhibitory Factor Serves As an Upstream Regulator of NLRP3 Expression and Subsequent IL-1beta Production in Human Monocytes in Response to Lupus U1-snRNP Immune Complex
Authors: Shin Min Sun , Kang Youna , Leng Lin , Bucala Richard , Kang Insoo
Published: January 2023

The pathologic hallmarks of systemic lupus erythematosus (SLE or lupus) are altered immune responses to nuclear autoantigens with autoantibody production and subsequent tissue injury. TLR7/8 recognize the ssRNA of the self-antigen U1-small nuclear ribonucleorprotein (U1-snRNP) that is targeted by anti-U1-snRNP antibodies (Abs) in lupus. Recently, we showed IL-1β production from human monocytes (MO) exposed to a combination of U1-snRNP and anti-U1-snRNP Abs (snRNP immune complexes) via the NLRP3 inflammasome. Macrophage migration inhibitory factor (MIF), primarily produced from MO and macrophages, promotes the production of IL-1β, IL-6 and TNF-α, which are increased in lupus patients. Data showed an association between serum MIF levels and MIF gene polymorphisms with lupus-related organ damage and decreased glomerulonephritis in lupus-prone mice treated with MIF antagonists. Here we investigated the possible mechanisms underlying the functional link between MIF and the NLRP3 inflammasome. The snRNP immune complexes (ICs) induced high levels of MIF and IL-1β from freshly purified human MO. The small molecule MIF inhibitor MIF098, which interferes the binding of MIF to its receptor complex, decreased the production of IL-1β from MO in response to the snRNP ICs. The inhibitor substantially reduced the expression of NLRP3 and caspase-1 activation. This phenomenon is likely mediated in part by the suppression of NF-kB activation. Plus, exogenous MIF increased IL-1β production by the MO. Our results first show the production of MIF from MO by the lupus snRNP IC and the regulation of the NLRP3 inflammasome by MIF in human, providing a scientific rationale for translating animal studies targeting MIF into human lupus.
The Benzathine Penicillin G (BPG) reformulation preferences study - towards a new Pencillin for Rheumatic Fever and Rheumatic Heart Disease
Authors: Sika-Paotonu Dianne , Tiatia Ramona , Sung Yun K , Lander Lynette , Haira Tia , Thornley Craig , Betty Bryan , Wineera-Parai Ranei , Eddie Barbara , Baker Michael , Chrzan Keith , Maloney Margaret , Felix Manuel , Spector Jonathan , Carapetis Jonathan
Published: January 2023

Acute rheumatic fever (ARF) is an autoimmune condition caused by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause cardiac damage known as rheumatic heart disease (RHD). The most effective recommended treatment of ARF requires monthly intramuscular injections of Benzathine Penicillin G (BPG) known as secondary prophylaxis. The goal of secondary prophylaxis is to prevent GAS infections that may lead to the recurrence of ARF. Rates of adherence to secondary prophylaxis schedules are usually low due to the frequency and duration of injection, pain and access to proper and timely healthcare services. A less painful and longer acting BPG formulation would ideally help prevent ARF recurrence and improve compliance rates to this schedule. The purpose of this work is to explore the BPG reformulation preferences of children and teens currently receiving monthly BPG intramuscular injections, in addition to their families and healthcare providers. A software application has been developed that will explore the experiences of the groups who will then choose their ideal BPG formulation from a range of plausible formulations and associated dosing regimens. The software application has been optimized for use to ensure age and cultural appropriateness and also efficient data collection. Pretesting of the software has been undertaken which has received positive responses with the piloting stage initiated. This is the first time a software application has been successfully developed to collect qualitative and quantitative data on individual preferences for BPG formulations and dosing regimens.
A Fast, Flexible and Low Cost Process for Neo-Epitope Based Immune Monitoring
Authors: Castro Aaron , Derhovanessian Evelyna , Luxemburger Ulrich , Beck Michaela , Gehring Franziska , Wenschuh Holger , Zerweck Johannes , Kern Florian , Reimer Ulf , Sahin Ugur
Published: January 2023

Advances in genomic technologies have paved the way to developing personalized cancer vaccines targeting neo-antigens. Patient-specific vaccines, targeting several neo-antigens in one go have already entered the clinic. Such personalized, multi-target approach poses a challenge for immune monitoring of vaccine-specific T-cell responses in a fast, flexible and cost-effective fashion.

Most immune monitoring protocols use 9–15-mer synthetic peptides originating from the vaccine target antigen. In contrast to detection of shared antigen-specific T-cells, for which the same peptide batch can be used for several patients, immune monitoring of individual neo-antigen-specific T-cell responses requires small amounts of large numbers of peptides (e.g. 40 × 15-mer peptides for 10 neo-epitopes of 27 amino-acids), which can only be used for one single patient. Therefore, standard peptide synthesis approaches applying commercial peptide synthesizers not only lack required throughput and speed but also generate peptides at prohibitive costs. Here, we present data demonstrating the flexible application of our high-throughput, low cost FastTrack peptide synthesis approach in comparison with different specifications of standard peptides in ex-vivo ELISPOT to monitor neo-antigen specific immune responses in patients participating in the IVAC MUTANOME Phase I clinical trial (NCT02035956). Application of the new peptide synthesis method enables the assembly of up to 1200 peptides in less than 3 weeks at appr. 20% of standard synthesis costs.
Development of a powerful next-generation immunoassay platform for low level detection of serum based analytes
Authors: Bourdon David M , Meng Lihao , Alohali Madhawi , Freeman Alisha , Le Lucas , Messina Jessica
Published: January 2023

Early detection of protein biomarkers is critical to the study of human disease (e.g. cancer, inflammation/autoimmunity, neurodegeneration). Basic research scientists as well as translational investigators require powerful protein quantitation tools that provide sensitive low-level detection of disease relevant protein analytes.

ELISAs first developed in the 1970s and slightly improved upon over the years are still considered the gold standard, yet large sample consumption, limited sensitivity, and laborious workflows, leave room for improvement and innovation.

Thermo Fisher Scientific, the worldwide leader in life science tools for researchers, is unveiling an affordable new platform for the next generation of high-sensitivity, ready-to-use immunoassays. Featuring serum compatible, TaqMan™ based Proximity Ligation Assay (PLA) and SiteClick™ antibody labeling technologies, this new assay combines the analyte specificity of high affinity antibody-antigen binding with the signal detection and amplification of real-time PCR.

Key benefits of this platform include: - Greater sensitivity and dynamic range than traditional methods- Smaller sample consumption than traditional methods: 1–2 uL per sample- A fast, simplified workflow - 2 hr. sample-to-answer with no wash steps- The inclusion of an intuitive, robust multi-OS data analysis software- Workflow compatibility with any real-time PCR instrument

Here, we highlight the immunoassay performance including sensitivity, dynamic range, spike-recovery, parallelism, linearity of dilution, and benchmarking to ELISA, all analyzed using a new software available through the Thermo Fisher Cloud.
Enhancement of antigen-specific Natural Killer cell ‘memory’ by removal of a viral innate immune signalling inhibitor
Authors: Ren Hongwei
Published: January 2023

Previous analysis of the vaccinia virus (VACV) K7 protein showed that this protein has a Bcl-2 structure, inhibits the pathways leading to activation of IRF3 and NF-κB and contributes to virus virulence. Here we show that infection with a VACV lacking the K7 protein (vΔK7) induced weaker VACV-neutralising antibody responses 1 month post infection, but still provided better protection against intranasal challenge with VACV. This implied a role for cellular immunity. By transfer of NK cells or CD8+ T cells to recipient mice and subsequent challenge with VACV, it was demonstrated that the NK cells from mice infected with vΔK7 induced better protection against challenge, compared to NK cells derived from wild type immunised mice. In contrast, adoptive transfer of CD8+ T cells from mice immunised with either virus conferred equal protection. In recipient mice, the NK cells transferred from vΔK7-infected mice were more responsive to homologous virus challenge and showed enhanced migration to the site of infection. By staining donor cells with CFSE prior to adoptive transfer, it was seen that donor NK cells proliferated greater in response to VACV than influenza challenge, and NK cells from vΔK7-immunised mice enhanced this effect compared to NK cells from wild type immunised mice.

This study shows that VACV-specific NK memory cells can confer protection against VACV challenge and that the production of these memory cells is enhanced by deletion of the VACV K7 protein.
Development of functionally distinct Treg subsets in the thymus
Authors: Owen David L. , Mahmud Shawn A , Williams Jason B , Jeschke Jonathan C , Aggarwal Praful , Broeckel Ulrich , Alegre Marisa , Zheng Ye , Williams Calvin B , Farrar Michael A
Published: January 2023

Mature thymic regulatory T cells (Tregs) are defined by dual expression of Foxp3 and CD25 on CD4SP thymocytes. However, the cellular pathways by which CD4 thymocytes differentiate into Tregs remain incompletely understood. Previous reports demonstrated that Treg development occurred via a two-step process. In step one, strong TCR signaling induces expression of CD25 leading to a CD25+ Foxp3− Treg progenitor (TRP). In step two, exposure of this TRP to IL2 results in conversion to a CD25+Foxp3+ Treg. Work from the Singer lab has defined a CD25−Foxp3lo TRP that can also be converted by IL2 into CD25+Foxp3+ Tregs. A key question is why two distinct cellular pathways involving distinct TRP populations are needed to reach the same endpoint. Using a combination of maturation marker analysis and RAG2-GFP reporter mice we observed that CD25+Foxp3− TRP arise more rapidly than the CD25−Foxp3lo TRP following positive selection. Analysis of Nur77-GFP reporter mice revealed that these two progenitor populations also display distinct affinity spectrums for self-peptide. Further, TCR sequencing has provided evidence that these 2 progenitor populations contain largely distinct TCR repertoires. Further, we have observed differential dependence on NFκB signaling and the Foxp3 regulatory element Cns3 between the 2 progenitor populations. Our preliminary data suggests that these TRPs mediate tolerance in distinct autoimmune contexts. Our results suggest a model whereby cells are driven by high affinity or high avidity interactions with self-peptide through two discrete pathways to form a comprehensive Treg repertoire capable of protecting against sterile and musical autoimmunity.
Inhibition of properdin is more effective than other inhibitors of the complement system at preventing cell lysis in an in vitro model of atypical hemolytic uremic syndrome
Authors: Galwankar Neeti , Emch Heather N , Lad Apurva C , Cortes Claudio , Ferreira Viviana P
Published: January 2023

The alternative pathway (AP) of the complement system can be activated excessively in several inflammatory diseases, particularly when there is a defect in regulatory components of the complement system. For instance, defects of complement regulatory proteins such as factor H (FH) and lack of CD55 and CD59 are associated with atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH), respectively. Both diseases lead to severe prothrombotic pathologies in which the damage is mediated mainly by the AP. Thus, strategies to protect host cells by effectively inhibiting AP complement-mediated attack are necessary. Here we have developed and characterized monoclonal antibodies against properdin (clones 6E11A4, 3A3E1, 6E9E6, and 1G6D2), the only known positive regulator of complement, to study their effects in preventing lysis in an in-vitro model of aHUS. We determined that our antibodies recognize both human and rabbit properdin, and the non-inhibitory antibodies recognize distinct epitopes as determined in a binding competition assay. More importantly, the data indicate that the inhibitory anti-properdin monoclonal antibodies had lower IC50 values than all other complement inhibitors tested, including Soliris, which is currently used to treat patients with this disease. We have also standardized a highly sensitive sandwich ELISA, using monoclonal antibody pairs, with a lower properdin detection limit than commercially available kits (20 pg/ml). Further studies aimed at determining the therapeutic value of inhibiting properdin in human inflammatory diseases are warranted.
Functional characterization of Irakm
rΔ9–11 splice variant in mice
Authors: Rothschild Daniel E , Zhang Yao , Diao Na , Lee Christina K , Chen Keqiang , Caswell Clayton C , Slade Daniel J , Helm Richard F , LeRoith Tanya , Li Liwu , Allen Irving C
Published: January 2023

Ulcerative colitis (UC) and Crohn’s disease (CD) are two pathologies collectively referred to as Inflammatory Bowel Disease (IBD). While a definitive etiology is currently unknown, a complex interplay of environmental factors and genetic predispositions has been linked to IBD development. The Toll-like receptors (TLRs) are a family of pattern recognition receptors (PPRs) involved in the signaling pathways that regulate the inflammatory response, and as such, represent essential targets for therapeutic intervention of UC and CD. Interleukin receptor associated kinase M (IRAKM) is an important negative regulator of TLR signaling acting to attenuate an overzealous innate immune response. Metadata analysis of human patient samples afflicted with IBD revealed increased expression of IRAKM compared to both healthy controls and patients with inactive forms of IBD. Furthermore, our findings in murine studies have identified that commercially available Irakm−/− mice contain a spliced mRNA variant of the Irakm gene as a result of the knockout strategy; defined here to be IrakmrΔ9–11. We have found that the truncated IrakmrΔ9–11 protein robustly activates a NF-κB dependent luciferase reporter when overexpressed in mammalian cells. These results, together with data supporting the mechanistic role of this truncated protein, suggest an alternative explanation for the function of IRAK-M in TLR signaling and in modulating gastrointestinal immune homeostasis with potential therapeutic implications for IBD patients.
Deficiency in Toll-Interacting Protein (Tollip) Skews Inflamed yet Incompetent Innate Leukocytes in vivo during DSS-Induced Septic Colitis
Authors: Zhang Yao , Diao Na , Li Liwu
Published: January 2023

The Toll-interacting protein (Tollip) is a critical regulator of Toll-like receptor-mediated innate immune responses. However, the role and mechanism of Tollip in the defense to bacterial infection remains poorly understood. In this study, we found that Tollip deficient mice were more susceptible to DSS induced acute colon injury. Tollip deficient mice displayed higher clinical scores with higher inflammatory cytokines in the blood. Further studies showed that Tollip deficient neutrophils had decreased migratory capacity, compromised bacterial killing activity, as well as reduced potential to generate neutrophil extra-cellular trap. On the other hand, Tollip deficient neutrophils had elevated levels of CCR5, responsible for their homing to sterile inflamed tissues. Together, our data reveal a novel mechanism in Tollip alteration that underlies the inflamed and incompetent polarization of neutrophils leading to severe outcomes of colitis.
Inhibition of radiation-induced lung inflammation and NF-kB activation by soy isoflavones.
Authors: Fountain Matthew Donovan , Abernathy Lisa Marie , Cannon Alexa Christine , Joiner Michael C , Hillman Gilda Gali
Published: January 2023

Background

Lung cancer patients treated with radiotherapy (RT) develop late respiratory distress as a result of inflammatory responses and lung fibrosis. We previously demonstrated that soy isoflavones (SIF) inhibited RT-induced inflammation, pneumonitis and fibrosis. NF-kB is a major transcription factor responsible for driving inflammatory responses. We have now investigated the effects of SIF on histopathology and status of NF-kB in lungs and BEAS-2B normal lung epithelial line treated with RT.

Methods

C57BL/6 and BALB/c mice were treated with 10Gy thoracic RT and oral SIF given daily for up to 18 weeks. Lung tissues were processed for histology and western blot protein analysis. In-vitro BEAS-2B cells were treated with SIF and 3Gy RT. NF-kB-p65 was assessed by western blot of cytoplasmic and nuclear protein fractions.

Results

H&E and Masson’s trichrome staining for fibrosis showed SIF reduced RT-induced inflammation and collagen fiber accumulation in lung parenchyma. SIF inhibited the infiltration and activation of macrophages and neutrophils in irradiated lungs. Analysis of NF-kB-p65 subunit, a marker of NF-kB activation, showed that RT induced expression of p65 was reduced by SIF in the lungs at 1–12 weeks after RT. In-vitro studies showed that while RT caused an increase in nuclear p65 at 3 and 24 hours post-RT, treatment with SIF and RT showed reduced p65 translocation to the nucleus.

Conclusions

SIF treatment mitigated radiation-induced histopathological alterations and p65 activation in-vivo and in-vitro. These findings suggest a trend for SIF to regulated NF-kB activation and the inflammatory response after RT. Future studies will decipher the signaling pathways involved in SIF-mediated radioprotection.
Intracellular HMGB1 mediating neutrophil extracellular trap formation during liver sterile inflammation
Authors: Huang Hai , Yazdani Hamza , loughran Patricia , Tsung Allan
Published: January 2023

Neutrophil extracellular traps (NETs) has been recently found exacerbate sterile inflammatory injury during liver ischemia/reperfusion (I/R). High mobility group box-1 (HMGB1) released from damaged cells, mediates liver damage and inflammatory responses after I/R. However, the intracellular role of HMGB1 in NET formation remains unknown. We sought to determine the role of intracellular HMGB1 in NET formation. Myloid cells HMGB1 knockout (LysM-HMGB1 KO) and HMGB1-flox mice were subjected to a non-lethal warm liver I/R. Less NETs was identified in ischemic liver lobes of LysM-HMGB1 KO mice compared with flox mice by confocal immunofluorescence (IF) imaging. This was associated with significantly less NET markers in LysM-HMGB1 KO mice, serum level of myeloperoxidase (MPO)-DNA complexes, and tissue level of citrullinated histone H3. Absence of HMGB1 in myloid cells protected the live from I/R injury indicated by significantly decreased serum transaminases (sALT), less necrosis and inflammatory cytokines production compared to flox mice. In vitro, no NET formation was observed in HMGB1 KO neutrophils under stimulation of PMA (positive control) or extracellular HMGB1 or histones confirmed by IF imaging. Chromatin in HMGB1 KO neutrophils failed to de-condensate in response to PMA which is the key step of NET formation. Additionally, gene deletion of HMGB1 in neutrophils completely blocked citrullination of histone H3 as a key step of NET formation, compared with control neutrophils in response to various stimulations. Our study demonstrates the dominant role of intracellular HMGB1 in NET formation. Lack of HMGB1 in neutrophils leads to diminished NET formation protect the liver from organ damage and cells death after liver I/R.
Foxo3 Transcription Factor Drives Pathogenic T Helper 1 Differentiation by Inducing the Expression of Eomes
Authors: MICHIELETTO MICHAEL , STIENNE CAROLINE , BENAMAR MEHDI , CARRIE NADEGE , CADENAT ISABELLE , SAOUDI ABDEL , LIBLAU ROLAND , HEDRICK STEPHEN M , DEJEAN ANNE S
Published: January 2023

CD4 T cell differentiation is a process finely controlled by specific transcriptional programs leading to the acquisition of specific T helper effector functions. Up to now, the functions of Foxo3 in CD4 T cells have not been investigate. Here, we described for the first time the critical role of the transcription factors Foxo3 in Eomes expression and their involvement in pathogenic T Helper 1 differentiation.

First, we showed that TCR triggering resulted in a dose-dependent upregulation of Foxo3 in CD4 T cells with an increased expression over time. We next addressed Foxo3 functions in CD4 T cells and we observed a decreased production of the Th1 related cytokines IFN-g and GM-CSF by Foxo3 KO CD4 T cells, with no impact survival, proliferation or other lineages differentiation. We then compared the transcriptome of WT and Foxo3 KO CD4 T cells and found that Foxo3 deficiency resulted in a decreased expression of genes related to the IFN- g/IFN-g response pathway and that the most downregulated gene in this pathway is Eomes. We showed, using ChIP and luciferase experiments, that Foxo3 binds and induces the expression of Eomes through direct DNA binding within the eomes locus. Using lentivirus experiments we showed overexpression of Eomes in Foxo3 KO CD4 T cell restored the proportion of IFN-g and GM-CSF producing cells, thus comforting Eomes role’s in the pathogenicity of CD4 T cells. In addition to this defect of differentiation, Foxo3-deficient mice developed a less severe Experimental Autoimmune Encephalomyelitis (EAE) as compared to WT mice, thus emphasizing the role of Foxo3 and Eomes in the development of autoimmunity. Altogether, we described here a new pathway whereby Foxo3 and Eomes drive CD4 T cells pathogenicity and neuro-inflammation.
Regulation of gene expression in Th17 cells by cyclic AMP Response Element Binding protein
Authors: Hernandez Jeniffer B , Cave Si
Published: January 2023

The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of the pro-inflammatory T cell, Th17. Although Th17 cells protect us against specific pathogens, Th17 cells have been found to cause destruction of tissue in patients with autoimmune diseases. Using RNAseq analysis, we have identified several genes that may be regulated by CREB in Th17 cells. Of these potential CREB targets, the G protein coupled receptor 65 is uniquely expressed in Th17 cells and mice deficient in GPR65 are resistant to autoimmunity. Blockade of GPR65 may lead to inhibition of pathogenic Th17 cells while simultaneously leaving non-pathogenic Th17 cells untouched.
Lumican Inhibits the pathogenicity of murine encephalitic TH17 cells
Authors: Yang Xuexian , Castillo Eliseo F , Zheng Handong , Cabanlong Christian , Dong Fei , Luo Yan , Yang Yi , Liu Meilian , Kao Winston
Published: January 2023

TH17 cells play an essential role in the development of both human multiple sclerosis and animal experimental autoimmune encephalomyelitis (EAE). Nevertheless, it is not well understood how the pathogenicity of TH17 cells is controlled in the autoimmune neuro-inflammation. In vitro, we found Lumican (Lum), an extracellular matrix (ECM) protein, is selectively expressed by TH17 cells among tested murine TH subsets. Lum-deficiency leads to earlier onset and enhanced severity of experimental autoimmune encephalomyelitis. This enhanced disease in Lum-deficient mice is associated with increased production of IL-17 and IL-21 and decreased TH17 cell apoptosis. Dysregulation in cytokine production appears to be specific to TH17 cells as TH1 and TH2 cell polarization and/or cytokine production were unaltered. Furthermore, adoptive transfer of MOG-specific TH17 cells derived from Lum-deficient mice led to earlier onset and increased severity of disease compared to controls highlighting a TH17 cell-intrinsic effect of Lum. Taken together, our results suggest that Lum negatively regulates encephalitic TH17 cells, implicating a potential therapeutic pathway in TH17 cell-mediated autoimmune and inflammatory diseases.
BAMBI, a new molecular target in autoimmunity
Authors: Alvarez Pilar , Augustín Juan Jesús , Tamayo Esther , Iglesias Marcos , Merino Jesús , Merino Ramón
Published: January 2023

We have previous demonstrated that BAMBI (BMP and Activin Membrane Bound Inhibitor) negatively regulates the functional differentiation of CD4 T cells into tolerogenic regulatory T-cells (Tregs) and positively the differentiation into pro-inflammatory TH17 cells. Accordingly, mice deficient in BAMBI are resistant against the development of collagen type II-induced arthritis (CIA). To analyze the therapeutic effects of BAMBI inhibition in normal mice in the development of autoimmunity, we developed new inhibitory mouse anti-mouse BAMBI mAbs. Treatment of B10RIII wild type (WT) mice with a high dose of a mouse IgG1 anti-mouse BAMBI mAb (clone B101.37; 2 mg/mice/week), but not with a low dose of B101.37 (0.3 mg/mice/week) or a high dose of an irrelevant IgG1 mAb (2 mg/mice/week), inhibited the development of CIA as efficiently as observed in B10RIII.BAMBI-KO mice.

We further explore the therapeutic potential of B101.37 in experimental models of psoriasis (Ps) and psoriasis arthritis (PsA). A single ip injection of mannan from Saccharomyces cerevisiae induces an acute inflammation resembling human Ps and PsA in B10RIII.WT but with a reduced severity in B10RIII.BAMBI-KO mice. Again, treatment of WT mice with B101.37, but not with an irrelevant IgG1 antibody, protects the animals against mannan-induced PsA and Ps by a Treg- and TGFβ-dependent mechanism. B101.37 also exhibits a therapeutic activity against mannan-induced PsA and Ps once the disease has been established. Similar results have been observed in a model of Ps induced after the topic administration of the TLR-7 agonist imiquimod. Altogether, our results demonstrate the beneficial effects of BAMBI inhibitors in the treatment of autoimmune skin and joint diseases.