bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nlrp6 limits inflamation in mouse colon by regulating microbiota
Authors: Seregin Sergey S , Golovchenko Natasha , Schaf Bryan , Pudlo Nicholas , Mitchell Jonathan , Chen Jiachen , Schloss Patrick , Martens Eric , Eaton Kathryn , Chen Grace Yi 
Published: January 2023

Abstract
Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that involves chronic inflammation involving the colon. We have previously shown that NLRP6 protects against the development of chemically-induced colitis. Here, we demonstrate that NLRP6 is important for suppressing the development of spontaneous colitis in the IL-10−/− mice model of IBD. IL10−/−Nlrp6−/− mice have significantly increased inflammation based on histologic scoring, spleen weight, fecal lipocalin levels, intestinal permeability, infiltration of immune cells, and upregulation of proinflammatory mediators. To determine whether NLRP6 protects against colitis by modulating the microbiome, we generated and recolonized germ-free (GF) Nlrp6−/− mice with the microbiota from WT donors and compared the composition of the microbiome to that of recolonized GF WT mice. We determined that GF WT and Nlrp6−/− mice acquire distinct microbiota upon re-colonization. Importantly, Nlrp6−/− mice have reduced a-diversity and dramatically increased colonization of Akkermansia muciniphila, which was also observed in IL10−/−Nlrp6−/− mice. To determine whether Akkermansia contributes to increased inflammation in IL-10−/− mice, we monocolonized GF IL-10−/− mice with Akkermansia muciniphila and found that these mice developed increased colitis compared to GF IL-10−/− mice gavaged with a control non-colitogenic Bacteroides strain. Altogether these results demonstrate that NLRP6 regulates the colonization of Akkermansia, which in turn, can precipitate colitis in a genetically susceptible host. Our work furthered the insight into the pathogenesis of UC and will be instrumental in the development of therapeutics that reduce pathobionts colonization to treat IBD.
Intestinal microbiota influence postnatal thymic T cell development
Authors: Jain Nitya , Seed Brian 
Published: January 2023

Abstract
The immune system faces unique challenges in the period immediately following birth. Thrust from a sterile fetal environment, it is abruptly exposed to an immense array of foreign antigens, the major burden of which is in the form of the microbiota newly colonizing the gastrointestinal tract. Adaptive immunity at the post-natal stage is largely immature, and the newborn’s immune system relies initially on innate immune effectors for protection against infections. In the early post-natal period, increasing numbers of innate and adaptive lymphocytes mature in the thymus and migrate to populate peripheral lymphoid organs including intestinal mucosal tissue. Intestinal microbiota regulate the maturation of the immune system at mucosal sites and also influence host physiology and function beyond the mucosal tract such as the brain and the pancreas. However, whether and how intestinal microbial colonization influences immune cell development in the thymus remains unknown. Here we show that the thymus of neonatal mice harbor precursor cells with distinct gene expression profiles compared to adult thymocytes that could potentially favor the development of innate-like lymphocytes. There are discrete patterns of distribution of PLZF and RORgt expressing thymic cell subsets and the prevalence of these cells is influenced by microbial colonization status of the neonatal intestine. Finally, we propose that migratory dendritic cells are one mediator of thymic-intestine crosstalk that transmit microbiota-specific signal to influence development of these thymic subsets. Thus, intestinal microbiota regulate yet another fundamental aspect of immune function- the development of lymphocytes itself in the neonatal thymus.
Cutaneous microbiota drive the accumulation of IL-17A-producing T cells within the lungs
Authors: Constantinides Michael G , Ridaura Vanessa K , Belkaid Yasmine 
Published: January 2023

Abstract
The microbiome consists of a diverse array of commensal microorganisms that reside at barrier sites of the human body and promote local immune homeostasis. While recent work has demonstrated that intestinal commensals can influence immunity throughout the body, the distal effects of other microbial communities have not been fully explored. Here we show that application of distinct human commensals to the skin of mice induces the accumulation of IL-17A-producing T cells in the lungs. The induction of these cells occurs in a manner that is partially dependent on both antigen presentation and IL-23 signaling. Response kinetics and adoptive transfer experiments suggest that the T cells migrate from the skin-draining lymph nodes, while transcriptional analysis has identified specific chemokine receptors that may facilitate trafficking to the lungs. Furthermore, in the context of pulmonary infection, cutaneous application of commensals promotes the production of IL-17A and recruitment of neutrophils to the lungs. This work demonstrates that the skin microbiome can enhance pulmonary immunity, but also has the potential to exacerbate inflammation through the production of IL-17A, thus providing a possible mechanism for why children with atopic dermatitis have an increased propensity to develop asthma, a progression commonly referred to as the “atopic march.”
Macrophage-Dependent Regeneration of Pulmonary Epithelia Requires Trefoil Factor 2 for Wnt Expression
Authors: Hung Li-Yin , Sen Debasish , Oniskey Taylor K. , Nieves Wildaliz , Urisman Anatoly , Krummel Matthew F , Herbert DeBroski R 
Published: January 2023

Abstract
Coordination between epithelial and myeloid cell lineages may facilitate tissue repair, but mechanistic evidence is lacking. Independently of Type 2 cytokines (interleukin-4/13), and T, B or ILC populations, lung macrophages promoted epithelial proliferation following injury caused by Nippostrongylus brasiliensis or bleomycin sulfate. Multiple myeloid populations up-regulated Trefoil factor 2 (TFF2) following lung injury and CD11c-driven Tff2 deletion impaired the proliferative expansion of pro-SpC+ distal lung epithelial progenitors. Direct interactions between macrophages and damaged epithelia resulted Wnt production, which accelerated epithelial proliferation, trans-epithelial resistance, and barrier function in a TFF2-dependent manner. In summary, the current study demonstrates that TFF2 is a regenerative cytokine expressed by macrophages to facilitate repair of infectious or non-infectious lung damage.
Interferon regulatory factor 1 (IRF1) expression patterns by respiratory epithelial cells reveal non-redundancy of type I versus type III interferon.
Authors: Novatt Hilary , Renn Lynnsey , Theisen Terence , Massie Tammy , Massie Tristan , Rabin Ronald L 
Published: January 2023

Abstract
Types I and III interferon (IFN) are co-expressed by respiratory epithelial cells (REC) in response to viral infection. In turn, these IFN stimulate neighboring REC to express a set of interferon-stimulated genes (ISG) through shared signaling pathways. Whether types I and III IFN have non-redundant functions in anti-viral defense of respiratory infections is unknown. Because transcription factors dictate cellular phenotype and function, we hypothesized that ISG that are transcription factors (TF-ISG) mediate non-redundant functions of types I or III IFN. We treated BEAS-2B human REC with increasing doses of IFN-β or IFN-λ1 alone or together, and measured expression of TF-ISG and a set of canonical ISG by qRT-PCR and Western blot. Alone, IFN-β and IFN-λ1 each induced expression of canonical ISG and a subset of TF-ISG. Expression of several ISG differed in response to type I versus type III IFN, either in peak levels or kinetic patterns, or both. Uniquely, IRF1 is induced early in response to IFN-β alone, but is poorly expressed in response to IFN-λ1. Western blots also revealed that while IFN-β alone induced early and transient IRF1 expression, it was lower but sustained (through 24h) after IFN-λ1 alone. In contrast to transcript expression, the two IFN together enhanced expression of IRF1 protein greater than either alone, and IRF1 expression was sustained through 24h. The distinct selective and rapid expression of IRF1 transcript and protein in response to IFN-β suggests that this TF-ISG mediates non-redundant qualitative functional responses of REC to types I and III IFN.
Challenge with Staphylococcus aureus induces a lipid response in newborn piglets
Authors: Alvarez Sandy , Bartlett Jennifer , Pollock Nicolette , Vazquez Jessica , McCray Paul , Porter Edith 
Published: January 2023

Abstract
The innate immune system provides the first line of defense against microbes. Epithelial cells recognize pathogen-associated molecular patterns and detection leads to induction of antimicrobial peptides, proinflammatory cytokines, and chemokines. Recent evidence suggests that antimicrobial lipids, including fatty acids and cholesteryl esters, are also important effectors in the innate mucosal defense of the airways. We have previously shown that cholesteryl linoleate and cholesteryl arachidonate are active against Pseudomonas aeruginosa and other bacteria, and that the expression of SOAT1, a key enzyme for cholesteryl ester biosynthesis, is increased in sinus mucosa of patients with chronic rhinosinusitis. The objective of this study was to further elucidate the regulation of antimicrobial lipid production by assessing the gene expression of enzymes involved in lipid biosynthesis in an in vivo pig infection model. Newborn pigs were challenged intratracheally with aerosolized heat-killed Staphylococcus aureus (n = 7) or saline (n = 5) and RNA was extracted from trachea tissues 4 h later. Gene expression of the enzymes SOAT1, FADS2, FASN and ACSL1 was statistically significantly increased (p &lt; 0.05 in One-Way ANOVA) in infected animals. In a second experiment (n = 3 for each group), formalin fixed tissues were subjected to a lipid preservation protocol prior to processing for histology and staining with the lipophilic dye Oil Red O. We observed in the trachea of animals challenged with S. aureus an increased staining with Oil Red O consistent with increased lipid accumulation. These data provide evidence that infection leads to increased lipid biosynthesis further supporting the concept of lipid-mediated innate mucosal defense.
Distinct tissue-specific functions for TSLP and IL-33 in the atopic march
Authors: Han Hongwei , Roan Florence , Johnston Laura , Bryce Paul J. , Ziegler Steven F 
Published: January 2023

Abstract
Atopic dermatitis (AD) often precedes asthma and food allergy, indicating that epicutaneous sensitization to allergens may be important in the induction of allergic responses at other barrier surfaces. Our study demonstrated a central role for IL-33 in this “atopic march.” We showed that mice exposed to antigen in the skin, in the presence of IL-33, developed severe airway inflammation when later challenged in the lung, and developed allergic diarrhea following oral antigen challenge. In addition, IL-33-driven allergic disease occurred in a thymic stromal lymphopoietin (TSLP)-independent manner. In contrast, IL-33 signaling was required for local inflammation following epicutaneous TSLP/OVA sensitization and during challenge in allergic gastrointestinal disease, but was dispensable during challenge in TSLP-mediated airway disease. These data reveal critical, site-specific roles for IL-33 in the “atopic march” that lead from atopic dermatitis to allergic asthma and gastrointestinal allergy.
Tissue-resident monocytes promote the establishment of lung-resident CD8 T cell memory following influenza infection
Authors: Dunbar Paul , Wein Alexander N , McMaster Sean Ryan , Hayward Sarah Louise , Kohlmeier Jacob E 
Published: January 2023

Abstract
Lung tissue resident memory CD8 T cells (TRM) have been shown to be critical in protecting against heterologous influenza virus challenge by limiting early viral replication and promoting more rapid viral clearance. Unlike other mucosal tissues, we have found that environmental signals are insufficient for the establishment of lung TRM, and that pulmonary antigen encounter is necessary for TRM to differentiate in the lung. This indicates a likely role for pulmonary antigen presenting cells (APC’s) in the process of lung TRM development, and thus we investigated the role of lung APC subsets on the establishment of lung CD8 TRM following influenza infection. Although many lung APC subsets contain flu nucleoprotein (FluNP) over the course of infection, most lung APC’s do not contain FluNP once virus has been cleared by day 10 post-infection. In contrast, lung monocytes continue to have high numbers of FluNP-bearing cells through day 15 post infection, the same time we observe the appearance of flu-specific CD8 T cells with a TRM phenotype. As these data suggest a potential role for monocytes as drivers of TRM differentiation, we tested mice deficient for the chemokine receptor CCR2 (CCR2−/−), which have a significant defect in the recruitment of monocytes to the lung. In our murine influenza model these CCR2−/− mice had significantly less flu-specific lung CD8 TRM than WT controls. Notably, this defect was not observed in the flu-specific CD8 memory T cells in the lung vasculature or mediastinal lymph node (MedLN), indicating this effect is specific for tissue-resident memory cells. Thus, these data show that antigen presentation by CCR2+ monocytes have a likely role in the lung in the establishment of lung CD8 TRM.
Releasable RapidSpheres™ enable immunomagnetic purification of highly viable and functional immune cells from complex tissues in less than 30 minutes
Authors: Clarke Samuel , Kokaji Andy I , Kellerman Drew W , Ewen Catherine , Chambers Martina N , Chan Mandy , Woodside Steven M 
Published: January 2023

Abstract
Recent advances in cellular therapy and diagnostics promise dramatic improvements in disease prevention and treatment. This research is driven by scientists studying how cells of the immune system communicate and relies on the success of cell-based assays. However, it remains challenging and time-consuming to purify large numbers of high-quality cells from complex tissues. EasySep™ Release is a fast and easy cell isolation method which utilizes the Releasable RapidSpheres™. This novel magnetic particle technology improves cell purities by reducing contaminants and allows for gentle particle removal to mitigate potential interference in downstream assays. The 29-minute protocol involves first incubating target cells with antibody complexes followed by the Releasable RapidSpheres™. Next, labelled cells are purified using a hand-held magnet. Particle-free purified cells are obtained by applying a mild dissociation reagent and a final magnetic separation. We validated EasySep™ Release by purifying T, B and NK cells from peripheral blood mononuclear cell samples containing 5 to 800 million cells. Cell purities were 99.7 ± 0.1% (n=17) for CD3+ T cells, 99.4 ± 0.4% (n=17) for CD4+ cells, 98.8 ± 0.5% (n=17) for CD8+ T cells, 98.6 ± 0.7% (n=6) for CD19+ cells and 95.6 ± 1.7% (n=6) for CD56+ cells. Isolated cells were confirmed particle-free, viable and functional. We obtained similar high performance using unprocessed leukapheresis samples of up to 5 billion cells, demonstrating excellent scalability and compatibility with more complex samples. Finally, we show how EasySep™ Release can be paired with commercially available antibodies or sequential separations to isolate almost any cell type, including those with a complex phenotype.
New dyes for highly sensitive analysis of cells in immunofluorescence applications
Authors: Richter Anne , Vahldieck Marco , Jansen Nicole , Schiminsky Esther , Siewert Christiane , Krauthäuser Susanne , Dose Christian 
Published: January 2023

Abstract
The potential of flow cytometry and fluorescence light microscopy in the analysis of complex biological systems is still limited by the range of bright fluorochromes. The sensitivity of fluorescent probes in cell staining is usually limited by several factors including i) the ability of the fluorochrome to absorb energy and convert it into fluorescence light, ii) the extent to which the intrinsic autofluorescence of the system interferes with the fluorochrome emission, and iii) for certain applications, the resistance of the fluorochrome to fixation agents.
Here we present a new set of fluorescent dyes, VB515 and VB667, which are detected in the channels of flow cytometers and fluorescence microscopes. We demonstrate that these fluorescent molecules are superior to commercially available alternatives, e.g., FITC, Alexa Fluor® 488, VioBright™ FITC, BD™ Horizon Brilliant™ Blue 515, or Alexa Fluor 647, and can even exceed the fluorescence signal of Allophycocyanine (APC)- and Phycoerythrin (PE)-conjugated antibodies. While offering higher brightness than APC- and PE, VB515 and VB667-conjugated antibodies also provide higher stability towards fixation agents in comparison to their protein counterparts. They are thus ideally suited for applications requiring high sensitivity for the detection of especially low expressed cell markers.
With these new fluorochromes a multicolor panel and a gating strategy has been developed for the flow analysis of regulatory T (Treg) cells derived from PBMC and lysed blood. Treg cells play a crucial role in the suppression of non-specific or excessive immune responses. Therefore, the detailed characterization of these cells and their function is of great interest for basic and translational research.
Novel tissue clearing method for preservation of morphology, fluorescence and epitope integrity permits quantitative cellular analysis in intact organs
Authors: Gerner Michael Y. , Li Weizhe , Germain Ronald N 
Published: January 2023

Abstract
Spatial organization of cells within tissues is critical for most biological processes, such as cellular differentiation, signal relay, and localized function. Our understanding of such events is historically based on section imaging, which discards 3-dimensional (3D) information and is problematic for understanding structure-function relationships of complex tissues and rare events. Although recent innovations in tissue clearing methods have paved way for whole organ microscopy, they suffer from various technique-specific artifacts, such as fluorophore quenching, poor epitope preservation, or morphological distortions. Here we report a new clearing method, Clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency, preserves cellular morphology and protein fluorescence, and retains epitopes for antibody-based labeling. We use Ce3D to demonstrate whole-tissue microscopy of various cell types in lymphoid and non-lymphoid organs. Further, when coupled with highly multiplexed antibody labeling and Histo-Cytometry, Ce3D permits quantitative analysis of phenotypically complex cells in intact tissues. Collectively, this methodology provides a comprehensive strategy for whole-organ quantitative imaging and analysis, paving the way for improved understanding of cellular organization and physiology.
This research was supported by the Intramural Research Program of the NIH, NIAID.
Direct immunomagnetic depletion of RBC without hypotonic lysis or sedimentation.
Authors: Peters Carrie , Lee Taylor N , Woodside Steven M , Thomas Terry E , Eaves Allen C 
Published: January 2023

Abstract
Isolation of specific cell types for functional studies frequently requires red blood cell (RBC) depletion as a first step. However, hypotonic lysis of samples may be deleterious to certain cell types, and RBC removal by hetastarch sedimentation is inconsistent. Furthermore, RBC depletion with agents such as ammonium chloride (NH4Cl) is often ineffective, leaving samples that still contain many RBC. We have developed an immunomagnetic method to deplete RBC that is rapid, can be used with a variety of sample types, and leaves untouched total nucleated cells (TNC) ready for downstream assays.
RBCs were removed from four different types of samples (whole blood, leukopak, buffy coat and bone marrow) either magnetically using the EasySep™ RBC Depletion Reagent or by NH4Cl lysis. To perform magnetic RBC depletion, samples were diluted 1:1, the reagent added, and the sample placed in an EasySep magnet. Magnetically labeled RBC were held in the magnet and TNC were poured or pipetted off. The reagent addition, magnet incubation and removal steps were repeated a second time, and in some cases the magnet incubation and removal steps were repeated a third time with whole blood and buffy coat samples.
RBC contamination of the TNC fraction was &lt;2% for whole blood (n=8), leukopak (n=2), and bone marrow (n=5) samples treated with EasySep™, and 12 ± 10% for buffy coat (n=4) samples treated with EasySep™; in all cases this was equivalent to or significantly lower than the RBC contamination in the same samples treated with NH4Cl. TNC recovery from samples RBC-depleted by either EasySep™ or NH4Cl lysis was similar. RBCs can be immunomagnetically depleted from samples in 6–15 minutes, depending on the sample type, and TNC are immediately ready for downstream assays.
Targeted inhibition of proinflammatory cytokines produced by myeloid cells
Authors: Efimov Grigory , Rozov Fedor , Mokhonov Vladislav , Drutskaya Marina , Kruglov Andrey , Tillib Sergey , Nedospasov Sergei A. 
Published: January 2023

Abstract
Proinflammatory cytokines, in particular IL-6 and TNF, are crucial factors in the development of autoimmune immune response. Their systemic inhibition has been successfully used as a therapy of multiple autoimmune and inflammatory diseases. However, recent data from murine genetic models suggest that the cellular source of proinflammatory cytokines may significantly affect their biological effects. Cytokines derived from particular cellular sources may be pathogenic while the same cytokine produced by other cell may play a protective role. Thus, systemic cytokine inhibition may be far from the optimal therapeutic approach. To independently confirm these findings and to evaluate potentially more effective therapeutic approaches we are developing bispecific antibodies, which may serve as inhibitors of proinfllammatory cytokines targeted to specific cells. Using myeloid-specific TNF inhibitor we have demonstrated that limiting TNF inhibition to macrophages and monocytes results in superior neutralization activity in the TNF-induced hepatotoxicity model. This approach could lower both the required dose and potentially the incidence of side effects. Currently we are developing the similar approach for IL-6. Our bispecific reagents are based on two different VhH antibodies, thus we immunized the lama with recombinant IL-6 and selected high affinity single-domain antibodies by phage display.
Funding: Russian Scientific Fund grant 14-25-00160 (anti-IL6 antibodies), RFBR grant 14-04-01656 (anti-TNF).