bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-22 receptor signaling in Paneth cells is critical for their maturation and antimicrobial function
Authors: Gaudino Stephen , Beaupre Michael , Lin Xun , Rathi Sonika , McLaughlin Patrick , Mehta Neil , Eskiocak Onur , Yueh Brian , Van der Velden Adrianus , Beyaz Semir , Kumar Pawan
Published: January 2023

Interleukin-22 (IL-22) acts in the intestine to promote critical tissue protective functions. However, since a diverse array of intestinal cell types (absorptive, secretory, and stem cells) express IL-22Ra1, a receptor for IL-22, it has been difficult to determine what cell type(s) specifically respond to IL-22 to mediate mucosal host defense. To address this question, we used entire gut epithelium, intestinal stem cell (ISC)-specific, and Paneth cell-specific IL-22Ra1 knockout mice. Entire epithelium-specific IL-22Ra1 knockout (Il22Ra1fl/fl;Villin-cre) mice displayed defects in Paneth cell function. Using ISC-specific IL-22Ra1 knockout mice (Il22Ra1fl/fl;Lgr5-EGFP-creERT2) and lineage tracing mice, we ruled out the involvement of Lgr5+ ISC-dependent IL-22Ra1 signaling in regulating the lineage commitment of epithelial cells, including Paneth cells. Using novel Paneth cell-specific IL-22Ra1 knockout mice (Il22Ra1fl/fl;Defa6-cre), we show that IL-22Ra1 signaling in Paneth cells is required for small intestinal host defense. We show that Paneth cell maturation, antimicrobial effector functions, gene expression of specific WNTs (Wnt6 and Wnt9b), and enteroid morphogenesis are dependent on cell-intrinsic IL-22Ra1 signaling. Furthermore, IL-22 signaling in Paneth cells regulates the intestinal commensal bacteria and microbiota-dependent IL-17A immune responses. Finally, we show Paneth cell-specific IL-22Ra1 signaling helps provide immunity against Salmonella typhimurium. Collectively, our findings provide a unique and novel role of IL-22 in Paneth cell-mediated small intestinal host defense.
Differential regulatory mechanisms underlie the control of Vγ4 and Vγ6 γδT17 development by HEB transcription factors
Authors: Anderson Michele K , Selvaratnam Johanna , Wong Alison
Published: January 2023

IL-17-producing γδ T-cells (γδT17) are critical components of the innate immune system, but the pathways that control their development are not well understood. We have previously shown that HEB transcription factors operate upstream of Sox4 and Sox13 to initiate the γδT17 program. However, the defects in fetal Vγ4+ γδT17 cells appear to be distinct from those in the Vγ6+ γδT17 cells. In the absence of HEB, fetal Vγ4+ γδT17 cells are unable to develop, whereas Vγ6+ γδT17 cells develop but are not able to produce IL-17. To further understand the molecular mechanisms that underlie these defects, we performed single cell RNA-seq on E18.5 γδTCR+ cells from WT and HEB-deficient mice, and did differential gene expression analysis on clusters expressing Vγ5, Vγ6, or Vγ4 mRNA. Our analysis revealed that in addition to Sox4 and Sox13, an entire suite of genes associated with the development and function of γδT17 cells was downregulated in HEB-deficient cells. Furthermore, Vδ4, which is normally restricted to cells expressing Vγ6 or Vγ5, was expressed at high levels in nearly all clusters, whereas the levels of Vγ4 and Vδ5 mRNA were severely decreased. These results suggest that direct control of Vγ and Vδ chain mRNA expression underlie the defect in Vγ4+ cell development, whereas Vγ6+ cells require a direct input from HEB into the gene program controlling γδT17 differentiation even in the presence of proper γδTCR pairing.
Affinity threshold conversion: the progressive differentiation checkpoint at which the TCRα/CD3δ signaling axis controls thymic positive selection
Authors: Canfield Jenna M , Ferrer Alejandro , Neier Steven C , Wilton Katelynn M , Stiles Robert J , Davis Tessa , Schrum Adam G , Pages Diana Gil
Published: January 2023

Upon positive selection of conventional αβ T cells, CD4+ CD8+ double-positive (DP) cells transition to single-positive (SP) and reduce their responsiveness to weak peptide/MHC ligands (affinity threshold conversion, ATC). We report that ATC actually begins prior to the completion of positive selection in the DP stage, and this checkpoint is where the TCRα/CD3δ signaling axis controls positive selection. We dissected ATC in the OT1 TCR transgenic system in conjunction with several different genes whose null mutations block thymic development at DP stage: β2m, CD3δ, and/or TCRα-Connecting Peptide Motif (αCPM). In the absence of MHC class I (β2m0), CD3δ or αCPM mutants were blocked at pre-selection DP stage and displayed immature, high-sensitivity to weak TCR ligands. In contrast, when MHC class I was present (β2m+), CD3δ or αCPM mutants were blocked at DP stage but underwent partial ATC; they began positive selection but did not complete it. Upon exogenous TCR engagement of partial-ATC thymocytes, the proximal signaling protein interaction network showed overall weakened activity, notably decreasing SOS1 participation linked to the downstream Ras/Raf/ERK selection pathway. Provision of high affinity peptide/MHC in FTOC rescued completion of positive selection for partial-ATC mutant thymocytes. Finally, comparing nontransgenic, polyclonal B6 vs. CD3δ0 DP thymocytes, although most cells showed equal maturation state, a subset of cells in B6 progressed in maturation relative to CD3δ0. We propose the existence of an intermediate checkpoint inside DP stage, in which ATC already begins to reduce responsiveness to selection ligands, yet further TCR signaling is required to complete positive selection.
Novel role(s) for Irf5 in controlling myelopoiesis and erythropoiesis
Authors: Barnes Betsy J , Vagrecha Anshul , LaPan Margaret , Kalfa Theodosia A. , Blanc Lionel
Published: January 2023

Causes of erythropoiesis dysregulation during anemia of inflammation remain largely unknown. Mammalian erythropoiesis can be divided into differentiation of early progenitors and maturation of precursors within anatomic niches called erythroblastic islands (EBI). EBIs consist of a central macrophage surrounded by differentiating erythroblasts and myeloid cells. While recent studies have focused on interactions of the macrophage and surrounding cells, much less is known about the transcription factors that regulate EBI formation and function. Data from scRNAseq revealed that interferon regulatory factor 5 (Irf5) is expressed by the central macrophage within EBIs. At steady state, Irf5−/− mice have decreased numbers of EBIs in the bone marrow (BM) with altered composition - increased CD71 (erythroid marker) and CD11b and Ly6G (myeloid markers). We also found increased numbers of reticulocytes in Irf5−/− BM. We hypothesize that Irf5 has a conserved function in mammalian blood formation by regulating the cellular output of erythroid and myeloid cells. To test this, we induced stress erythropoiesis in wild-type (wt) and Irf5−/− age- and littermate-matched mice by submandibular bleeding. Mice were bled twice (Day 0 and 2) and complete blood counts, erythropoiesis, and EBI formation monitored over recovery (12 days). Using CD44/Ter119 and Forward Scatter as markers of terminal erythropoiesis, we found that erythroid differentiation was significantly delayed in Irf5−/− mice resulting in increased mortality and corresponding to a significant decrease in EBI formation. These data identify Irf5 as a new regulator of the erythroid-myeloid balance within the EBI that results in altered function(s) during anemia of inflammation.
Cell-Intrinsic Regulation of Cytokine Responses by I-mfa Controls Eosinophil Development
Authors: Houser Jeremy S , Onopiuk Marta , Tsiokas Leonidas , Humphrey Mary Beth
Published: January 2023

The response of hematopoietic stem and progenitor cells to cytokines and other signals in the bone marrow (BM) niche is regulated by a complex network of cell-intrinsic factors that is still not well understood. We have identified a small cysteine-rich protein, the Inhibitor of MyoD Family A (I-mfa), as a novel regulator of hematopoiesis and lineage development. I-mfa is known to regulate β-catenin signaling, a pathway that is activated by the IL-3-family of cytokines (IL-3, IL-5, GM-CSF) which are important during myeloid lineage differentiation. We therefore hypothesized that I-mfa would influence the production of myeloid progenitors and mature cells by regulating the response to these cytokines. To test this, we compared the BM and spleens of wild-type (WT) and I-mfa-knockout (KO) mice by flow cytometry. I-mfa KO animals had expanded common myeloid progenitor and granulocyte/macrophage progenitor populations compared to WT, and showed eosinophilia in the spleen. The function of the BM was analyzed by myeloid colony forming unit (CFU-M) assays, showing significantly higher granulocyte colony output from I-mfa KO BM compared to WT. Mechanistically, myeloid progenitor cell lines over-expressing I-mfa were resistant to β-catenin activation in response to cytokine and Wnt ligand stimulation. These data taken together suggest that I-mfa is a novel regulator of eosinophil development through its regulation of β-catenin signaling downstream of cytokine receptors. These results present a new insight into disease states mediated by eosinophilia such as allergic asthma and may mechanistically underscore eosinophilia during the progression of myeloid leukemias.
The effects of macrophage-specific Vhl knockout on steady state and stress erythropoiesis in mice
Authors: Pan Szu-Yu , Tsai Pei-Zhen , Lin Shuei-Liong
Published: January 2023

Objective

Macrophages are known to assist erythropoiesis. However, the effect of macrophage specific Vhl knockout on erythropoiesis is unclear.

Methods

We generated Tg(Csf1r-Cre/Esr1);VhlF/F mice to achieve inducible macrophage-specific Vhl knockout. Tg(Csf1r-Cre/Esr1);ROSA26CAG-tdTomato/+ was also generated to examine the knockout efficiency.

Erythropoiesis was determined by analyses of complete blood count (CBC), spleen weight, and cell surface expression of TER-119 and CD71 in the bone marrow and spleen. Based on FSC-A and surface CD71 expression, we defined three different red blood cell (RBC) maturation stages as erythroblast, reticulocyte, and mature RBC. Stress erythropoiesis was induced by administration of recombinant human erythropoietin (rhEPO) 4U/gBW subcutaneously thrice per week for 6 doses.

Results

Tamoxifen administration in Tg(Csf1r-Cre/Esr1);ROSA26CAG-tdTomato/+ mice resulted in expression of tdTomato in CD11b(+)F4/80(+) cells in the spleen (79%) and peripheral blood mononuclear cell (PBMC) (48%).

In steady state erythropoiesis, tamoxifen administration in Tg(Csf1r-Cre/Esr1);VhlF/F mice did not result in changes of CBC, spleen weight, ratio of TER-119(+) cells in live cells, or RBC maturation stages in the spleen and bone marrow.

In stress erythropoiesis, administration of rhEPO resulted in polycythemia. However, no significant changes in Hct, ratio of TER-119(+) cells in live cells, or RBC maturation stages could be observed between Tg(Csf1r-Cre/Esr1);VhlF/F and littermate control mice after tamoxifen administration.

Conclusion

Macrophage specific knockout of Vhl in mice did not have significant effects on steady state erythropoiesis and rhEPO induced stress erythropoiesis.
B cells sialylate bone marrow progenitors to inhibit granulocyte production
Authors: Irons Eric Edward , Lee-Sundlov Melissa , Zhu Yuqi , Neelamegham Sriram , Hoffmeister Karin , Lau Joseph
Published: January 2023

Effective hematopoiesis is predicated on the utilization of limited resources and space in the bone marrow during homeostasis and stress. During acute inflammation, secreted factors induce the egress of mature B cells from the medullary compartment, reclaiming space for the emergency proliferation of myeloid precursors. However, it remains unclear whether reciprocal mechanisms between B cells and myeloid cells exist during homeostasis. Recent work by our group has demonstrated the ability of extracellular glycosyltransferases to remodel plasma membrane glycans, altering cell sensitivity to growth factors. Here, we report that B cells secrete active ST6GAL1 sialyltransferase, capable of glycosylating cells by a non-cell autonomous, extrinsic mechanism. B cells secreted ST6GAL1 to glycosylate hematopoietic progenitors in co-culture, suppressing G-CSF-driven neutrophil production. In vivo, B cell-derived ST6GAL1 restored circulating enzyme levels and altered the sialylation of diverse hematopoietic populations after transplantation into St6gal1-deficient mice. In the bone marrow, colonizing IgD+ mature B cells colocalized with regions rich in sialylation. Finally, in the bone marrow of multiple myeloma patients, expression of ST6GAL1 correlated negatively with neutrophil abundance. Our findings underscore a novel mechanism of cross-talk between mature lymphocytes and myeloid progenitors involving paracrine extracellular sialylation, with potential implications for human disease.
Altered Gut DNA virome diversity associated HMGB1 release regulates reactive Astrocytes-induced IL6 release preferably via TLR4-NFkB pathway in experimental Gulf War Illness
Authors: Seth Ratanesh K , Bose Dipro , Saha Punnag , Kimono Diana , Mondal Ayan , Lloyd Patricia Janulewicz , Klimas Nancy , Horner Ronnie D , Sullivan Kimberly , Lim Efrem S , Nagarkatti Mitzi , Nagarkatti Prakash S. , Chatterjee Saurabh
Published: January 2023

In a recent study, we showed that the gut DNA bacteriophage dysbiosis in Gulf War Illness (GWI) was strongly associated with compromised intestinal epithelial cell integrity, increased circulatory IL6 and neuroinflammation. The current study further investigates the mechanism of DNA bacteriophage-IL6 axis in neuroinflammation. Advancing the previous findings, we show that viral dysbiosis positively correlated with high mobility group box protein 1 (HMGB1; a damage-associated molecular pattern) expression and release in circulation following GWI induction in mice. The circulatory HMGB1 activated brain Astrocytes via altering brain endothelial tight junction proteins and crossing the blood-brain barrier. Interestingly, both in GWI mice and mouse primary Astrocytes cell culture showed an increased brain IL6 mRNA and subsequent protein expression. However, GWI mice treated with Ribavirin (used for partial gut viral sterility) showed decreased intestinal HMGB1 and brain IL6 expression. Mechanistically, HMGB1 activated innate immune response via IRAKs-IKKα-NFkB instead of either RAGE-MAPK or PI3K-mTOR pathway. Surprisingly, inhibition of RAGE or PI3K pathway in HMGB1 primed mouse Astrocyte cells showed a significant increase in IL6 expression and release suggesting NFkB activation as a preferential pathway in GWI-Astrocyte-induced IL6 release. In summary, GWI-associated gut viral dysbiosis associated intestinal HMGB1 release activates brain Astrocytes and IL6 release via toll-like receptor 4-NFkB dependent pathway, thus causing neuroinflammation in GWI. The above mechanism can form a basis for studying inflammation-associated neurocognitive abnormalities in GWI.
Brain transforms natural killer cells that exacerbate brain edema after intracerebral hemorrhage
Authors: Liu Qiang , Li Zhiguo , Li Minshu , Shi Samuel , Zhu Zilong , Zhang Xiaoan
Published: January 2023

Perihematomal edema (PHE) occurs within hours after intracerebral hemorrhage (ICH), leading to secondary injury manifested by impaired blood-brain-barrier (BBB) integrity and adjacent tissue destruction. To dissect mechanisms underlying PHE formation, we profiled human and mouse perihematomal tissues and identified natural killer (NK) cells as a predominant immune cell subset that outnumbers other infiltrating immune cell types during early stage of ICH. Unbiased clustering of single cell transcriptional profiles revealed two major NK cell subsets in the brain following ICH that possess either high cytotoxicity or robust chemokine production features, which distinguish them from NK cells in the periphery. NK cells exacerbate BBB disruption and brain edema after ICH via cytotoxicity toward cerebral endothelial cells and recruitment of neutrophils that augment focal inflammation. Thus, brain-bounding NK cells acquire specific features that contribute to PHE formation and neurological deterioration following ICH.
Clonally expanded CD8 T cells patrol Alzheimer’s cerebrospinal fluid
Authors: Gate David , Saligrama Naresha , Leventhal Olivia , Davis Mark M , Wyss-Coray Tony
Published: January 2023

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder in which neuroinflammation plays a critical function. Yet, little is known about the contribution of the adaptive immune response in AD. Here we used integrated analyses of multiple cohorts to identify peripheral and central adaptive immune changes in AD. First, we performed mass cytometry of peripheral blood mononuclear cells and discovered an immune signature of AD consisting of increased numbers of CD8+ T effector memory CD45RA+ (TEMRA) cells. In a second cohort, we found that CD8+ TEMRA cells were negatively associated with cognition. Furthermore, single cell RNA sequencing revealed their enhanced T cell receptor (TCR) signaling. Notably, by utilizing multiple single cell TCR sequencing strategies on a third cohort, we discovered clonally expanded CD8+ TEMRA cells in patient cerebrospinal fluid (CSF). Finally, we used machine learning, cloning, and peptide screens to demonstrate specificity of clonally expanded AD CSF TCRs to two separate Epstein-Barr virus antigens. These results reveal a novel blood-CSF adaptive immune response in AD and provide evidence of clonal, antigen-experienced T cells patrolling the intrathecal space of brains affected by age-related neurodegeneration.
LRRK2-mediated microglial activation via NFATc2: a novel mechanism of neurotoxic inflammation in synucleinopathies
Authors: Kwon Somin , Beilina Alexandra , Smith Nathan , Li Yan , Kim Minhyung , Kumaran Ravindran , Kaganovich Alice , Mamais Adamantios , Adam Anthony , Iba Michiyo , Lee Won-Jae , Shin Soo-Jean , Rissman Robert , You Sungyong , Lee Seung-Jae , Singleton Andrew B , Cookson Mark R , Masliah Eliezer , Kim Changyoun
Published: January 2023

Neuroinflammation is a key pathological feature of a set of neurological disorders called synucleinopathies, which are characterized by abnormal a-synuclein (a-syn) dynamics and include Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), and multiple system atrophy (MSA). Microglia, the resident macrophages of the central nervous system, are critical for orchestrating the inflammatory response in these diseases. In particular, microglia can directly bind neuron-released a-syn via receptors such as Toll-like receptor 2 (TLR2) to adopt a reactive phenotype. Here we explored the pathogenic role of Leucine-rich repeat kinase 2 (LRRK2), a PD-associated gene also known to be highly expressed in T cells, in microglial activation. We observed that neuron-released a-syn enhances LRRK2 kinase activity in microglia in a TLR2-dependent manner to induce the release of neurotoxic cytokines, TNFa and IL-6. We further determined that LRRK2 directly modulated nuclear translocation of activated T-cells cytoplasmic 2 (NFATc2) through multi-site phosphorylation. As such, modulating the LRRK2-NFATc2 cascade may be a promising therapeutic strategy for ameliorating neuroinflammation in synucleinopathies.
IL-9 promotes allergic lung inflammation by differentially affecting macrophage populations
Authors: Fu Yongyao , Kaplan Mark H
Published: January 2023

IL-9 promotes to the development of allergic lung disease through several responsive populations including mast cells, lymphocytes and eosinophils. However, it is not known if these represent the entire IL-9-responsive cell repertoire. In this study, we detected high IL-9R expression on macrophages and these cells occupied a large proportion of IL-9R+ cells in allergic lung. Allergic inflammation leads to a decrease in the number of alveolar macrophages (AM) in wild type mice. However, in Il9r−/− mice, the AM population is not diminished as greatly, suggesting that IL-9 represses AM self-renewal. The level of the IL-9R expression on blood monocyte is corelated with CCR2 expression, which is required for blood monocyte migration to the inflammatory site. In the chronic allergic airway disease model, the circulating monocyte population decreased coincident with an increase in the number of interstitial macrophages in the control mice compared to the Il9r−/− mice, suggesting IL-9 promotes monocyte migration and differentiation in the lung. Interestingly, IL-9 also induces a CD11c+ interstitial macrophage population in the allergic lung. Adoptive transfer of CD11c+ wild type macrophages to the Il9r−/− mice increased the inflammatory response. Thus, our study demonstrated that macrophages are important IL-9-responsive populations in allergic lung disease, and that IL-9 inhibits AM numbers as it promotes the development of pro-inflammatory CD11c+ and CD11c− interstitial macrophages. This balance is important in determining the level of allergic inflammation in the lung.
Cellular senescence and SASP in the pathobiology of IFN-γhigh severe asthma
Authors: Kale Sagar Laxman , Gorry Michael , Ray Prabir , Ray Anuradha
Published: January 2023

Background

5–10% of severe asthmatics are resistant to corticosteroids but account for 50% of health care costs spent on asthma care in the US and in Europe. 30–40% of corticosteroid-resistant severe asthmatic patients show elevated IFN-γ levels (Type1 immune response) in BAL cells that is closely associated with expression of CXCL10, a downstream IFN-γ target, and a marker of senescence associated secretory phenotype (SASP).

Objective

The goal of this study was to evaluate the role of SASP in severe asthma associated with an elevated level of corticosteroid-refractory IFN-γ in the airways.

Methods

Wild type BALB/c and IFN-γ−/− mice were sensitized with house dust mite (HDM) in combination with cdi-GMP and then subsequently repeatedly challenged with HDM. Lungs from these and control mice were subjected to either RNA-seq analysis or used for β-gal staining to demonstrate cellular senescence.

Results

Whole lungs from WT mice subjected to the severe asthma (SA) model demonstrated significantly higher expression of several SASP genes which included p16INK4a, CXCL10, MMP12 and IL-6 as compared to that detected in naïve WT mice. p16INK4a is a key regulator of premature cellular senescence that arrests cell cycle in G1 phase by inhibiting CDK4 and CDK6 and has been associated with corticosteroid resistance. Further, the SASP genes p16INK4a, CXCL10 and CCL8 were downregulated in the absence of IFN-γ suggesting a possible role of elevated IFN-γ in SASP induction. The lung sections from mice subjected to the SA model showed increased numbers of b-gal positive cells as compared to that found in control mice.

Conclusion

SASP may underlie persistent inflammation and corticosteroid resistance observed in a subset of severe asthma patients.
Mast cell hyperplasia underlies respiratory syncytial virus infection-induced predisposition for asthma
Authors: St. John Ashley L , Balla Jozef
Published: January 2023

Asthma is a chronic inflammatory airway disease, which affects approximately 300 million people worldwide. A growing number of epidemiological cohort studies supports an association between pediatric allergic asthma susceptibility and prior respiratory syncytial virus (RSV) infection. In this study, we aimed to experimentally test whether RSV infection promotes the subsequent development of allergic asthma. Either wild type mice or mast cell-deficient congenic KitW-sh mice were infected with RSV or sterile vehicle. After infection clearance, the mice were sensitized to Ragweed (A. artemisiifolia) pollen allergens and subsequently challenged intranasally with Ragweed to induce asthma. Disease severity was measured as airway hyperreactivity to inhaled β-methylcholine using a mechanical ventilator. RSV caused an acute and productive infection in mice, which was associated with a rapid cytokine response in the lungs. In our murine allergic asthma model, the wild type animals which had previously cleared the RSV infection developed more severe disease, when compared to mice that have not experienced RSV. We found that there was a sustained increase in the numbers of airway mast cells following the resolution of an acute RSV infection. The induction of allergic asthma disease in our animal model was dependent on mast cells, as it was strongly attenuated in mast cell-deficient KitW-sh mice. Furthermore, there was no potentiation of allergic asthma severity by a previous RSV infection in Sash mice, indicating this process is mast cell-dependent. Thus, RSV induces airway mast cell hyperplasia, which predisposes mice for more severe allergic responses.
TAK1 contributes to IgE-FcɛRI-mediated calcium mobilization and mast cell degranulation
Authors: Watson Colton J. F. , Rouillard Melissa M. , Crozier Robert W. E. , Fajardo Val A. , MacNeil Adam J.
Published: January 2023

Allergic inflammation is an inappropriate immune response, typically triggered by activated mast cells that have been IgE-sensitized to innocuous environmental agents. Allergic inflammatory events can be split into two phases, early and late, initiated by allergen-mediated mast cell activation. The early phase of the allergic response occurs within minutes of an allergen binding and crosslinking IgE-FcɛRI complexes on the surface of a sensitized mast cell. This recognition of a perceived ‘threat’ rapidly induces mast cell activation and subsequent release of pro-inflammatory mediators from preformed granules, a process known as degranulation. We have identified TAK1 as a novel contributor to induced mast cell signaling events and aim to characterize the mechanistic contribution of TAK1 in mast cell degranulation. To this end, we have employed a primary murine model of bone marrow-derived mast cells (BMMC) to examine contributions of TAK1 to IgE-mediated calcium mobilization and degranulation. β-hexosaminidase release assays and Indo-1-based spectrofluorometry demonstrated a significant inhibition (−67% ±1.44 p<0.0001) in degranulation and a reduction in calcium transients (both peak amplitude p<0.01 and area under curve p<0.05) in allergically-activated BMMCs treated with the TAK1 inhibitor, 5Z-7-oxozeaneol (OZ). These results were supported by restoration of normal degranulation with the use of an inactive OZ analog (5Z), whereas an alternative TAK1 inhibitor (AZ) also resulted in degranulation inhibition (−32% ± 2.64 p<0.0001). These results provide novel evidence to support the mechanistic control of mast cell degranulation by TAK1, highlighting its potential as a therapeutic target for allergic pathologies.
SG-SP1 suppresses mast cell-mediated allergic inflammation via inhibition of FcɛRI signaling
Authors: KIM Min-Jong , Baek Moon-Chang , Kim Sang-Hyun
Published: January 2023

As the number of allergic disease cases increases, studies to identify new treatments take on new urgency. In previous study, gallic acid (GA), a part of EGCG, a major component of green tea, has shown anti-allergic inflammatory effect. However, GA has low metabolic stability and structure-activity relationship. Therefore, we designed SG-SP1 based on GA. We aimed to assess the effects of SG-SP1 on mast cell-mediated allergic inflammation using various animal and in vitro models. For in vitro experiments, IgE-mediated various mast cells (RBL-2H3 and primary cultured peritoneal and bone marrow-derived mast cells) were used to determine the role of SG-SP1 (0.1–1 nM). Immunoglobulin (Ig) E-induced passive cutaneous anaphylaxis and Ovalbumin-induced systemic anaphylaxis are standard animal models for immediate-type hypersensitivity. For in vitro, SG-SP1 reduced degranulation of mast cells by down-regulating intracellular calcium levels in a concentration-dependent manner. In addition, SG-SP1 decreased expression and secretion of inflammatory cytokines in activated mast cells. This suppressive effect was associated to inhibition of the phosphorylation of Lyn, Syk and Akt, and the nuclear translocation of nuclear factor-κB. Due to the strong inhibitory effect of SG-SP1 on Lyn, the known upstream signaling of FcɛRI-dependent pathway, we confirmed the direct binding of SG-SP1 to FcɛRI, a high affinity IgE receptor by surface plasmon resonance experiment. We determined that SG-SP1 directly interacts with FcɛRI and propose SG-SP1 as a therapeutic candidate for mast cell-mediated allergic inflammatory disorders via inhibition of FcɛRI signaling.
IgE-induced mast cell function is suppressed by antagonizing isoprenylation or Ras activation
Authors: Dailey Jordan , Ballance William , Sebti Said M , Ryan John J
Published: January 2023

Mast cells are tissue resident innate immune cells activated by IgE and other ligands. Upon activation they release histamine, cytokines, chemokines, proteases, and lipid mediators evoking allergic symptoms. New ways of targeting mast cells could greatly benefit allergic disease therapy. We previously found that statin drugs reduced IgE-mediated inflammation in vitro and in vivo by inhibiting production of the isoprenoid lipid geranylgeranyl pyrophosphate. However, this suppression was dependent on genetic background. To overcome this limitation, we tested a dual farnesyl and geranylgeranyl transferase inhibitor (FGTI 2734). Using mouse mast cells, we show that FGTI 2734 reduced IgE-mediated degranulation and cytokine production similar to statin effects. Unlike statins, FGTI 2734 did not show a significant difference in IC50 based on genetic background. Furthermore, FGTI 2734 suppressed IgE-mediated systemic anaphylaxis in vivo. Ras GTPases are important for mast cell function and among the signaling proteins modified by isoprenylation. We show that Pan-Ras inhibitors can mimic statin and FGTI 2734 effects on cytokine production in mouse mast cells. These data indicate that dual farnesyl and geranylgeranyl transferase inhibition may be effective in suppressing IgE-induced inflammation, possibly due by disrupting Ras-mediated signals.
Developing a nonhuman primate basophil activation assay by flow cytometry
Authors: Chu Haiyan , Lucchini Amanda , Frantz Aric
Published: January 2023

The Human Basophil Activation test (BAT) has been validated for many disease conditions, used for the diagnosis of allergy to drugs and foods, and for monitoring the role of basophils in immunotherapy. While several BAT kits are commercially available and widely used for humans, the BAT assay in nonhuman primate (NHP) is scarcely present in the literature. Given that NHPs are used as a nonclinical model in drug development and safety assessment when other animal species cannot be used, we developed a BAT using peripheral blood of cynomolgus macaques (Cyno). For human, the mAb panels 1) CCR3+SSClow,2) CD203c+SSClow, 3) CRTH2+CD3−, and 4) CD66−CD3−CD123+HLA-DR− have been used to identify total basophils. Following stimulation with allergens or a positive control (eg. anti-human FcɛRIα or IgE), the activated basophils can be gated as CD63+ and/or CD203+. For the Cyno BAT development, first, the anti-human mAbs noted above were tested for their cross-reactivity with Cyno. Second, the anti-human FcɛRIα and IgE mAbs were tested for their ability to crosslink and activate the Cyno basophils. Third, the basophil stimulation buffer (BSB) was formulated with calcium and heparin with or without IL-3 for blood collected in EDTA tubes. The FcɛRIα but not the IgE mAb, consistently activated basophils and upregulated CD63 expression. IL-3 added to BSB resulted in high background CD63 expression on unstimulated basophils and therefore was not used in the finalized method. Our data indicates that the only panel with all mAbs cross-reactive to Cyno was 4) CD66−CD3−CD123+HLA−. This panel identified the Cyno basophils that were SSCMed not SSCLow. In summary, a BAT has been successfully developed for use in exploratory NHP studies.
Neural Dysfunction Following Francisella tularensis Infection
Authors: Tena Anahis , Muniz Mireya G Ramos , Spencer Charles T
Published: January 2023

Francisella tularensis is a gram-negative intracellular coccobacillus that is the causative agent of tularemia. Local, acute inflammation is beneficial since it can clear bacterial infections, remove cellular damage and promote the healing of tissue. However, F. tularensis is capable of bypassing the immune system and causing an overactivation of inflammation, known as a “cytokine storm”. This cytokine storm increases host cell damage, nutrient availability and serves an immune distraction allowing the bacterium to replicate. Other infections that cause cytokine storms include ebola, pandemic influenza, hantavirus, and HIV, all BSL3 pathogens.

The routes of transmission for F. tularensis are many and intertwined. Infection could be the result of inhaling air droplets containing infectious bacteria, coming into direct contact with an infected animal or being bitten by an infected arthropod such as a tick, flea or mosquito. Eating and drinking contaminated meat and water can also lead to infection and different disease manifestations occur depending on the route of transmission.

While extensive research has documented infection of the spleen, liver and lung, there hasn’t been any reports of F. tularensis productively infecting the brain, until now. This work focuses on characterizing the effects of F. tularensis infection of the brain. We hypothesize that damage caused by F. tularensis neural infection will have a negative effect on behavior. This work aims to identify these negative effects based on the anatomical location of the bacteria foci, regions containing numerous bacteria. With the use of brain mapping and behavioral assays, we seek to determine the impact of F. tularensis infection on behavior and cognition.
Essential role of CCL2-CCR2 axis in the pathogenesis of chronic irritant contact dermatitis
Authors: Shibuya Rintaro , Kitoh Akihiko , Hanakawa Sho , Ishida Yoshihiro , Kabashima Kenji
Published: January 2023

Chronic irritant contact dermatitis (ICD) including hand eczema is a common skin disorder caused by the repetitive exposure to irritants such as chemical detergents and organic solvents. Products including sodium dodecyl sulfate (SDS), such as soap or shampoo, are the leading cause of occupational ICD. Previous studies have shown that irritants cause epidermal damage which induce the production of proinflammatory mediators, followed by a dermal influx of inflammatory cells. However, the detailed immunological cascades occurring in the chronic ICD remains unclear.

Repeated application of SDS, onto murine ear skin induced ear swelling, accompanied by epidermal hyperplasia and barrier dysfunction. Dermal infiltration of neutrophils, eosinophils and monocytes were remarkable in the SDS-applied skin. Our observation described above was consistent with the features of human chronic ICD. Here, we found that CCR2 deficiency or blockade of CCL2, a major ligand for CCR2, significantly attenuated SDS-induced ear swelling as well as inflammatory cell accumulation. Among the differentially expressed genes, which were up-regulated by SDS application, genes related to cell interaction at the vascular wall were significantly affected by CCR2 deficiency. Further, we investigated whether CCR2 on radiosensitive cells or radioresistant cells is critical. Lethally irradiated CCR2−/− mice transplanted with CCR2+/+ bone marrow cells (BMC) exhibited impaired SDS-induced ICD, while CCR2+/+ mice transplanted with CCR2−/− did not.

Collectively, our data suggest that CCR2 expression by radioresistant cells plays an essential role in the pathogenesis of chronic ICD.
Cytosolic LPS sensing triggers the release of a lectin DAMP that promotes inflammation
Authors: Russo Ashley Jean , Vasudevan Swathy , Menoret Antoine , Duduskar Shivalee , Fettis Margaret , Li Chuan , Huergo Santiago Mendez , Kumari Puja , Chandran Karthik , Vanaja Sivapriya Kailasan , Bauer Michael , Sponholz Christoph , Rabinovich Gabriel , Hudalla Gregory , Vella Anthony , Deshmukh Sachin , Zhou Beiyan , Rathinam Vijay
Published: January 2023

Activation of inflammatory caspases, such as caspase-11 and caspase-4, by lipopolysaccharide (LPS) from Gram-negative bacteria, is a key mechanism of innate immune defense against infections. This cytosolic LPS sensing pathway results in activation of a pore-forming protein, gasdermin D, which mediates an inflammatory form of cell death called pyroptosis. In addition, caspase-11-mediated cytosolic LPS sensing leads to the activation of caspase-1 and the maturation of IL-1b and IL-18. Importantly, cytosolic LPS-induced pyroptosis is associated with the release of intracellular proteins that act as alarmins or damage-associated molecular patterns (DAMPs) to modulate the inflammatory response and play an important role during infectious diseases and sepsis. However, the identity and function of DAMPs released downstream of caspase-11 activation are poorly characterized. Using a proteomic approach involving ProteomeLab’s PF2D fractionation system followed by mass spectrometry, we have discovered that galectin-1, a b-galactoside-binding lectin, is released as a consequence of cytosolic LPS sensing. By utilizing various mutant cell lines and primary cells we show that galectin-1 secretion is dependent on caspase-11 and gasdermin D, but not NLRP3 or caspase-1. Importantly, in vivo studies using galectin-1-deficient mice showed that galectin-1 acts as a DAMP to promote systemic inflammation and plays a detrimental role during septic shock. Additionally, human septic patients were found to have increased levels of galectin-1 in their serum. Taken together, we have characterized the secretion and function of a novel lectin DAMP released upon lytic cell death and provide new insights into outcomes of inflammasome activation.
Innate immunity and bacterial conflict: the deep origins of cGAS-STING signaling and discovery of uncharacterized animal immunity pathways
Authors: Burroughs A Max , Aravind L
Published: January 2023

Just as animal innate immunity pits endogenous response pathways against invading extragenic threats, microbiomes are governed by myriad pathways mediating inter-genomic resource procurement, collectively known as ‘conflict systems.’ Leveraging the continually-increasing amount of genome data, we have probed such systems and in the process uncovered the evolutionary origins of core components of animal immunity. This includes the discovery that cyclic-nucleotide synthesizing enzymes like human cGAS and OAS1 and their receptors like STING were acquired from comparable conflict-triggered nucleotide signaling systems in bacteria. While our analyses point to previously-unknown diversity animal innate immunity, particularly in marine invertebrates, this diversity pales in contrast to bacterial pathways which contain a wealth of completely uncharacterized nucleotide synthetase enzymes, nucleotide receptors, and response effector domains.

Further, studies from our group point to entirely-uncharacterized components of the animal innate immunity response that have also been acquired from bacterial conflict systems. These include the viral RNA-acting human Leng9 phosphoesterase and the human N4BP2L1 nucleotide kinase. These studies also uncover a connection between immunity-driven second messenger nucleotide generation and a previously-unrecognized receptor domain in the human apoptotic factor MAP3K5.
The Rab11-family interacting protein 2 is an important regulator of inflammatory cell death in human macrophages
Authors: Husebye Harald , Gravastrand Caroline , Skjesol Astrid , Yurchenko Mariia , Jakovljev Aleksandra , Afset Jan Egil , Espevik Terje
Published: January 2023

Evidence that membrane trafficking and anchoring at the trans-Golgi network is important for NLRP3 inflammasome activation is now emerging. The Rab11-family interacting protein 2 (FIP2) is a key regulator of intracellular vesicle trafficking and has been shown to bind to a number of phosphatidylinositol phosphate (PtdIns) species, including PtdIns4P that is prevalent in the membranes of the Golgi apparatus.

Here we report a role of FIP2 in NLRP3 inflammasome activation downstream of TLR4. High resolution confocal microscopy data revealed that FIP2 was resident in the Golgi and the trans-Golgi-network. In LPS primed and Nigericin treated cells, FIP2 was frequently observed on specks containing the inflammasome adaptor ASC. FIP2 positive ASC-specks were observed in mouse macrophages, THP-1 monocytes, primary human monocytes and macrophages.

In addition, FIP2 showed a marked co-localisation with Caspase-1 in Golgi. Also, FIP2 co-localised with NLRP3 in additional cellular compartments. Indeed, co-immunoprecipitation studies in HEK293 cells showed that FIP2 could bind both NLRP3 and Caspase-1.

Moreover, FIP2 silenced THP-1 monocytes showed impaired ASC-speck formation and marked reduction in secretion of cleaved IL1-β and Caspase-1. Similar results were found when FIP2 silenced THP-1 cells were LPS primed and treated with virulent clinical isolates of uropathogenic Escherichia coli (UPEC). Together these data show that FIP2 is a regulator of NLRP3 inflammasome assembly and activation.
DDX39A interacts with LGP2 to inhibit RLR responses
Authors: Stone Amy E.L. , Thompson Ahna B , Jamieson Abby W , Feliciano Shelby , Said Ali , Gale Michael J
Published: January 2023

RIG-I-like receptors (RLRs) are DEAD-box helicases and cytoplasmic pathogen recognition receptors that recognize and bind to nonself/viral RNA and trigger innate antiviral immunity. RLRs are essential for the detection of RNA virus infection and include RIG-I, MDA5, and LGP2. LGP2 has been implicated as RIG-I or MDA5 co-factor to regulate their activity. We conducted a yeast 2-hybrid (Y2H) screen (using LGP2 as bait) of a human hepatic cDNA library to identify protein binding partners of LGP2. Identified proteins were validated as LGP2 interactors through overexpression and co-immunoprecipitation assays of LGP2 binding. DDX39A, a DEAD Box helicase protein with known roles in RNA splicing, was identified as a binding partner. DDX39A overexpression was found to inhibit interferon-beta production from RLR-signaling in a cell-based ligand-free model in a dose-dependent manner. Further, in a cell-based interferon beta-promoter-luciferase assay model of Sendai virus (SenV) infection, DDX39A and LGP2 synergistically inhibited promoter activity. Similar results were seen in a cell-based NFkB-luciferase assay of the same system. Endogenous expression of DDX39A was assayed through the Huh7 human hepatoma cell line system with various stimuli including viral infection with SenV and Interferon-beta treatment showing that DDX39A is constitutively expressed in those cells. Structure-function mutants of DDX39A and LGP2 were used to determine the protein domains and functions required for interaction via co-immunoprecipitation assays. Through these studies, we will define the interactions between LGP2 and DDX39A, and reveal the roles these proteins have as co-factors in RLR signaling.
Complement regulators and mechanisms vary by sex in intestinal ischemia reperfusion injury
Authors: Wu Miaomiao , Rowe Jennifer , Fleming Sherry D
Published: January 2023

Complement activation plays a pivotal role in intestinal ischemia reperfusion (I/R)-induced injury, leads to significant morbidity and mortality. The injury includes significant inflammation including complement activation. Although established primarily using a male models, intestinal I/R-related diseases occur in both sexes. However, multiple animal models of ischemia and complement activation demonstrate sexual. Thus, we hypothesized that complement regulation may differ by sex during intestinal I/R altering the timing, quantity or required molecules. Both sexes of C57Bl/6 mice were subjected to Sham treatment or 30 min intestinal ischemia followed by 15 min, 30 min, 60 min, and 120 min reperfusion. We demonstrate that compared to male mice, females sustain attenuated intestinal I/R-induced tissue damage. In response to I/R, males produce PGE2 regulated by COX-2, while females produce LTB4 by A5-LOX. Complement initiation pathways differed with males tending to use classical pathway while females tend to use MBL pathway. Together these data suggest that local control of complement activation and regulation, eicosanoid production and neutrophil infiltration in response to intestinal I/R varies between sexes suggesting that distinct therapeutic intervention may be needed in clinical ischemic diseases.
Single cell RNA sequencing reveals novel inflammatory mechanisms of human acute respiratory distress syndrome
Authors: Rosborough Brian R , Chen Jie , Jiang Yale , Kitsios Georgios , McVerry Bryan , Lee Janet S , Ray Anuradha , Chen Wei , Ray Prabir
Published: January 2023

The acute respiratory distress syndrome (ARDS) is a common etiology of acute respiratory failure that results from overwhelming pulmonary inflammation. Since prior bulk RNA sequence analyses have provided limited insights into ARDS pathogenesis, we used the approach of single cell RNA sequencing (scRNA-seq) to probe ARDS at a higher resolution. Towards this end, scRNA-seq data of peripheral blood mononuclear cells (PBMCs) of patients with pneumonia and sepsis with early ARDS were compared to that of sepsis patients who did not develop ARDS. We detected a previously unrecognized reduced frequency of NK cells in the PBMCs of ARDS patients, which also showed complete loss of IFNGR1 expression. Monocyte clusters from ARDS patients revealed multiple distinguishing characteristics in comparison to monocytes from patients without ARDS. This included a pro-inflammatory signature with up-regulation of multiple type I IFN-induced genes, especially in CD16+ cells. To generate an ARDS risk score, we identified 29 differentially expressed genes in the monocytes of these patients, 17 of which showed a similar profile in cells of patients in independent cohorts. Genes with increased expression in monocytes that may have relevance in ARDS pathogenesis include RAB11A, known to inhibit neutrophil efferocytosis, ATP2B1, a calcium pump that exports Ca2+ implicated in endothelial barrier disruption, and SPARC, associated with processing of pro-collagen to collagen. Together, our findings identify molecules that are likely involved in ARDS pathogenesis that may inform biomarker and therapeutic development.
TGF-beta-mediated lung fibroblast-to-myofibroblast transition exhibits IFN-γ resistance via SHP2/SOCS3 activation
Authors: Chen Chia-Ling , Chang Chun-Jung , Lin Chiou-Feng
Published: January 2023

Idiopathic pulmonary fibrosis is a lethal disease of unknown etiology. Abnormal activation of TGF-β is a common cause in developing fibrotic lesions, where Src homology-2 domain-containing phosphatase 2 (SHP2) plays a critical regulator in fibroblast activation and fibrosis. We found that a marked expression of phospho-SHP2, phospho-STAT3 and the suppressor of cytokine signaling 3 (SOCS3) were detected in bleomycin-induced rat lung fibrosis, which was highly association with pulmonary parenchymal lesions and collagen deposition. Human pulmonary fibroblasts differentiated into myofibroblasts via TGF-β1 treatment were characterized with the increased bundles of F-actin, enhanced expression of α-smooth muscle actin (α-SMA), fibronectin and collagen, which also exhibited the activation of SHP2, SOCS1/3, PIAS1, STAT3, IL-6 and IL-10. An AKT-regulated glycogen synthase kinase (GSK)-3β/SHP2 axis was induced in ROS-dependent regulation during myofibroblast differentiation. The significant retardation of interferon (IFN)-γ signaling in myofibroblasts was revealed by the decrease expression of phospho-STAT1 and IFN-γ-associated genes along with IFN-γ-inducible protein 10 (IP-10) reduction. Treatment of nintedanib (bibf1120) in myofibroblasts effectively inhibits both SHP2 and SOCS3 activation accompanying by a suppression of fibroblast-to-myofibroblast transition (FMT). Moreover, synergistic treatment of nintedanib and IFN-γ markedly ameliorated TGF-β1-mediated FMT. Accordingly, we speculate that the activation of both ROS/AKT/GSK-3β/SHP2 and IL-6/STAT3/SOCS3 axis signaling causes myofibroblasts hyposensitivity to IFN-γ whereby escaping from IFN-γ immune-surveillance during FMT.