bluuebunny/crossref_metadata_2025_split huggingface dataset:
TSLP acts on Tregs to limit effector Th2 cell generation during type 2 immune responses
Authors: Gurram Rama Krishna , Oh Jangsuk , Leonard Warren J 
Published: August 2023

Abstract
Thymic stromal lymphopoietin (TSLP) is a type 2 alarmin secreted by epithelial cells to promote type 2 inflammation. After antigen recognition, naïve CD4 T cells differentiate into effector T (Teff) cells, whereas regulatory T (Tregs) cells suppress the immune response to prevent adverse effects caused by inflammation. Therefore, a delicate balance between Teff and Treg activity is required for the homeostasis of immune responses. Here we generated a TSLPR conditional KO mice (Crlf2fl/fl) to investigate the functional role(s) of TSLP on Teff and Tregs. Conditional deletion of TSLPR on T cells (Cd4CreCrlf2fl/fl) resulted in lower numbers of eosinophils and Th2 cells with OVA-induced airway inflammation. In contrast, conditional deletion of TSLPR on Tregs (Foxp3CreCrlf2fl/fl) resulted in elevated numbers of eosinophils and increased IL-5- and IL-13-secreting effector Th2 cells. Furthermore, phenotypic analysis of Foxp3+ Tregs during type 2 immune responses shows that Tregs from Foxp3CreCrlf2fl/fl mice have upregulated levels of GATA3 and secrete IL-13. Together, these data indicate that TSLP not only promotes suppressive activity by Tregs in the generation of Th2 cell-mediated responses but that it also restrains GATA3 production and IL-13 secretion by the Treg cells, indicating a key role for TSLP in shaping an immunosuppressive environment during type 2 immune responses.
This research was supported by the Intramural Research Programs of NHLBI, NIH.
Rational design of TGFbRII dominant negative armored GPC3 CAR-T cells for the treatment of hepatocellular carcinoma
Authors: Merlino Ashley , Tu Eric , Overstreet Michael , Gilbreth Ryan , Clarke Lori , Cobbold Mark , Moody Gordon , Giardino Letizia , Chu Nina 
Published: August 2023

Abstract
Chimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity is a concern hampering its broader use. In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we employed a strategy to manage the toxicity profile by lead selecting a single-chain variable fragment with reduced affinity characterized by a high dissociation rate. Compared to a high-affinity construct, the low-affinity CAR maintained cytotoxic function but showed reduced toxicity in a mouse model of hepatocellular carcinoma (HCC). The HCC microenvironment is rich of the immunosuppressive cytokine TGFβ, prompting us to armor the low affinity GPC3 CAR-T cells with a dominant-negative TGFβRII (TGFβRIIDN). In vitro, expression of TGFβRIIDN nearly abolished TGFβ-induced SMAD2/3 phosphorylation and reduced suppression of IL-2 and IFNg production. In a xenograft model of a human TGFβhigh HCC, armored CAR-T cells had 10/10 complete responders (CR) while the unarmored CAR-T had 4/10 CRs. In four TGFβhigh patient-derived xenograft models armored CAR-T cells achieved 60–90% tumor growth inhibition (TGI) while the unarmored CAR-T achieved no significant TGI. The armored CAR-T cells infiltrated TGFβhigh HCC tumors more abundantly than their unarmored counterparts and expressed lower levels of immune checkpoint markers PD1 and LAG3. In line with these observations, we detected significantly more IFNg at peak response, confirming in vivo functional superiority of TGFβRIIDN armored CAR-T therapy. These data support the clinical development of AZD5851, GPC3 CAR-T armored with TGFβRIIDN.
Single cell transcriptomic analysis of the ovarian immune microenvironment reveals hormone-dependent changes
Authors: Brodeur Tia Y , Krementsov Dimitry 
Published: August 2023

Abstract
The ovary is a dynamic organ, where, driven by the estrous cycle, cells undergo continuous waves of apoptosis, proliferation, and differentiation. These changes parallel fluctuations in ovarian hormones such as estradiol (E2), however, the immune microenvironment during high-E2 and low-E2 states has not been described. We induced a high-E2 state by superovulation with i.p. injection of gonadotropins (GN) into 10 week old C57BL/6 mice. Flow cytometry and single-cell RNA sequencing revealed abundant NK cells, B cells, CD8+ T cells, CD4+ T cells, CD3+TCRβ+CD4−CD8− “double negative” T cells, and several distinct myeloid subsets. The frequency of NK cells in the CD45+ compartment was 14% in GN-treated mice and 7% in controls, suggesting increased proliferation or recruitment of NK cells in response to E2. Conversely, mature B cells represented 8% of CD45+ cells in GN-treated mice and 18% in controls. To determine whether CD45+ cells were recruited from circulation or ovarian resident cells, we injected fluorescently-labelled α-CD45 i.v. into live mice and performed flow cytometry on ovaries, spleen, and blood. The majority (88–96%) of ovarian CD45+ cells were unlabeled, suggesting that they were ovary-resident. To assess population dynamics during the naturally occurring low-E2 state of reproductive aging, we performed flow cytometry on ovaries of 40 week old mice. Interestingly, we identified significant increases in the number of macrophages and double-negative T cells in aged ovaries. Our findings reveal a diverse resident immune landscape in the ovary that responds robustly to hormonal changes. These findings may have implications for immune-mediated causes of infertility such as primary ovarian insufficiency and endometriosis.
Support by a grant from the NIH 3P30GM118228-05S3
Maternal high fat diet exposure results in MyD88 dependent alteration in placental and offspring intestinal fatty acid transporters
Authors: Jaleel Sana , Niu Xinying , Lu Dongmei , Mirpuri Julie 
Published: August 2023

Abstract

Introduction
We know that offspring born to parents with obesity are more likely to be obese. We hypothesized that the maternal high fat diet (mHFD) exposure may increase placental and offspring small intestinal transport of fatty acids. We hypothesized both these processes would be dependent on the presence of MyD88.

Methods
Wild-type (WT) and MyD88 deficient mice on either 60% mHFD or regular diet (RD) were profiled. The baseline expression of fatty acid transport proteins FATP1 and FATP4, fatty acid binding proteins FABP1, FABP4, and CD36 were profiled by qRT-PCR from placenta after C-section at E20, and offspring small intestine 2-weeks after birth. Serum from 0-day old RD and HFD WT mice were collected and analyzed using high-throughput, non-targeted mass spectroscopy based metabolic analysis.

Results
In WT mice, we found FATP1 was increased 32-fold and FABP was increased 7-fold in the mHFD placenta. FATP2 was decreased 2-fold and FATP4 was decreased 2.5-fold in the mHFD offspring small intestine. In MyD88 KO mice, we found no difference in any of the above fatty acid transporters. Serum of neonatal pups showed unique clustering of metabolites by PCA analysis in HFD and RD mice, with increased butanoate and propanoate metabolites in mHFD offspring.

Conclusion
Fatty acid transport regulators in the placenta and offspring small intestine were modified by exposure mHFD. In both the placenta and small intestine, this outcome was dependent on the presence of MyD88. Fatty acid metabolites were increased in the serum of neonatal pups exposed to mHFD. These findings suggest that mHFD exposure can modulate fetal metabolism and may be an opportunity for future development of therapeutics that target placental and intestinal transporters.
Regulation of myeloid cells and inflammation marks increased susceptibility in a genetic model of acute viral infection-induced tissue damage
Authors: Annis Jessica L , Crittenden Rowena B , Brown Michael G 
Published: August 2023

Abstract
M.H2k/b mice are identical to MA/My parent strain aside from a C57L-derived region within the MHC, which causes these mice to develop significant splenic tissue damage during acute murine cytomegalovirus (MCMV) infection. To understand how MCMV infection leads to the induction of tissue damage, we probed immunological differences between the M.H2k/b and MA/My mice over time. Using tissue imaging and flow cytometry, we observed increased infiltration of neutrophils to the marginal zones of the M.H2k/b mice aligning with the appearance of necrosis at 2 days post infection (dpi). While TUNEL staining identified increased cell death within these clusters, apoptosis was reduced, suggesting an inflammatory death pathway in the M.H2k/b neutrophils. In line with this hypothesis, multiplex ELISA identified increased IL-6 and TGF-β in the spleens of M.H2k/b mice at this time. Intriguingly, depletion of neutrophils prior to infection did not alleviate M.H2k/b histopathology or inflammation. Seeing the differential regulation of neutrophils, we assessed the state of other myeloid populations in the spleen during acute MCMV infection. We observed significant loss of marginal zone and red pulp macrophages (MZMs, RPMs) beginning at 2dpi and worsening over time in the M.H2k/b mice. Further exploration into how the loss of these populations may contribute to tissue damage identified buildup of toxic lipid peroxidation byproduct 4HNE early in the MZMs and progressing through the RPMs alongside the development of histopathology. In conclusion, we have identified increased inflammation, oxidative stress, and macrophage loss in M.H2k/b spleens during acute MCMV infection that tracks with localization and temporal appearance of histopathology.
NIH NIAID R01AI50072, Department of Medicine/Nephrology, Center for Immunity Inflammation and Regenerative Medicine, Beirne B Carter Center for Immunology Research.
Lupus-like phenotypes emerge from loss of macrophage derived Dnase1L3
Authors: Infante Angelica M , Engavale Minal B , Keyel Peter A 
Published: August 2023

Abstract
One leading cause of death in women is autoimmunity. One prominent autoimmune disease is Systemic Lupus Erythematosus (lupus). During lupus, inflammation is driven by autoantibodies that target cell-free DNA. Cell-free DNA is degraded by two serum endonucleases, Dnase1 and Dnase1L3. The loss of Dnase1L3 causes lupus in humans and mice, but the amount of Dnase1L3 necessary to prevent lupus onset is unknown. Since DnaselL3 is secreted by macrophages and dendritic cells, we hypothesized that loss of macrophage-derived DnaselL3 is sufficient to cause lupus-like phenotypes in mice. We generated conditional knockout (cKO) mice lacking DnaselL3 expression in macrophages. Sera was collected weekly from these mice until 50 weeks of age. To measure autoantibody induction, total IgG, total IgM, anti-dsDNA and anti-nuclear antibody levels were analyzed at 4, 20, 30 and 50 weeks. Sandwich ELISA was used to quantitate antibody levels in control and cKO mice. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgG, anti-ds DNA antibody levels increased in cKO mice. We conclude that SLE-like phenotypes arise in mice due to the loss of macrophage Dnase1L3. This suggest that macrophage derived DnaselL3 is critical to controlling lupus.
Plains Bridges to the Baccalaureate Program (PBB) (NIH 2R25GM83730-B)
Syk-Dependent Responses and Innate Cytosolic Defenses in Orientia tsutsugamushi-infected Macrophages
Authors: Soong Lynn , Fisher James R , Gonzales Casey , Liang Yuejin 
Published: August 2023

Abstract
Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. Human studies and animal models of scrub typhus have shown robust type 1-skewed proinflammatory responses during severe infection. Macrophages play a critical role in initiating such responses, yet mechanisms of innate recognition for O. tsutsugamushi remain unclear. In this study, we investigated whether Syk-dependent C-type lectin receptors (CLRs) contribute to innate immune recognition and the generation of proinflammatory responses. To validate a role of CLRs in scrub typhus, we infected murine primary macrophages with O. tsutsugamushi in the presence of selective Syk inhibitors and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that Mincle/Clec4a and Clec5a transcription was significantly abrogated upon Syk inhibition at 6 hours of infection. The effect of Syk inhibition on Mincle protein expression was validated via western blot. Syk-inhibited MF had diminished expression of type 1 cytokines and chemokines (Il12p40, Tnf, Il27p28, Cxcl1) during infection. Additionally, expression of innate immune cytosolic sensors (Mx1 and Oas 1–3) was highly induced in the brain of lethally infected mice. We established that Mx1 and Oas1 expression was reduced in Syk-inhibited MF, while Oas2, Oas3, and MerTK were not sensitive to Syk inhibition. This study reveals that Syk-dependent CLRs contribute to inflammatory responses to O. tsutsugamushi. It also provides the first evidence for Syk-dependent activation of intracellular defenses during infection, suggesting a role of pattern recognition receptor crosstalk in orchestrating macrophage-mediated responses to this poorly studied bacterium.
Supported by the NIAID grants (R21 AI156536, R01 AI132674, and T32 AI007526). JF was the recipient of a NIAID Emerging and Tropical Infectious Diseases T32 fellowship.
Neutrophils use a novel NLRP3 inflammasome-independent mechanism to release bioactive IL-1β in response to lysosomal disruption
Authors: Miller Brandon A , Pearlman Eric , Dubyak George R 
Published: August 2023

Abstract
IL-1β is an inflammatory cytokine mainly secreted by myeloid cells in response to infection or sterile tissue damage. Non-canonical secretion of IL-1β from macrophages downstream of activated NLRP3/caspase-1 inflammasomes is the best-characterized model; this is mediated by caspase-1 cleavage of GSDMD allowing N-GSDMD to form pores in the plasma membrane that act as conduits for IL-1β release and inducers of pyroptosis as a lytic cell death. The NLRP3 initiator acts as a sensor of perturbed cellular homeostasis including decreased cytosolic [K+]. In macrophages, this K+ efflux mediated NLRP3 activation is also triggered by agents that disrupt lysosomal integrity. While neutrophils also assemble competent NLRP3 inflammasomes and release bioactive IL-1β via GSDMD-dependent mechanisms, they resist the formation of plasma membrane N-GSDMD pores and progression to pyroptosis. We tested whether lysosome disrupting stimuli in neutrophils would phenocopy macrophage mechanisms to secrete IL-1β in an NLRP3 and GSDMD-dependent manner. Surprisingly, our data indicate that lysosome-disrupting stimuli induce neutrophils to release mature IL-1β and undergo rapid lytic cell death independently of NLRP3 inflammasome assembly and caspase-1 activity with only partial dependence on GSDMD. Ongoing studies testing effects of serine protease inhibitors support a mechanism in which cytosolic accumulation of the multiple serine proteases stored in the lysosome-like azurophilic granules coordinately disrupt canonical NLRP3 inflammasome assembly and directly cleave proIL-1β and GSDMD as part of a novel neutrophil-specific signaling mechanism for lysosomal disruption-induced processing and export of IL-1β.
P01-AI141350 R01-EY014362 R21-EY032662
Myeloid Plasticity of Neutrophil Precursors Contributes to Eosinophil Emergency Hematopoiesis
Authors: Haruna Nana-Fatima D , Politanska Yuliya , Abdala-Valencia Hiam , Berdnikovs Sergejs 
Published: August 2023

Abstract
Eosinophils and neutrophils are heterogenous cells that play diverse roles in homeostasis and inflammation. Tissue eosinophil origins are poorly understood, posing challenges in the functional interpretation of heterogeneous phenotypes. New evidence from our lab shows unexpected hematopoietic plasticity of Ly6G(+)IL-5Rα(+) band-stage neutrophils with potential to contribute to eosinophil lineage. Using flow cytometry and multi-omics approaches in mouse and human tissue samples derived from healthy and allergic conditions, we tested the hypothesis that neutrophil plasticity contributes to eosinophil diversity in inflammation. We found at least two distinct populations of neutrophils at a healthy baseline, one of which expressed classical neutrophils markers (S100A8/9) while the other expressed both neutrophil (MPO, ELANE) and eosinophil-specific (EPX, PRG2) granule proteins, indicating an intermediate hematopoietic state. In human and mouse neutrophils treated with IL-5/G-CSF in vitro, a subset with mixed characteristics acquired eosinophil-specific granular proteins and eosinophil cell surface markers (Siglec8, CCR3). In a mouse asthma model, using scRNA-seq, we found overlap in neutrophils and eosinophils with tissue remodeling functions. We also identified activated neutrophils and eosinophils playing unique roles in inflammation. Our findings implicate eosinophil emergency hematopoiesis driven by neutrophil plasticity as an overlooked process driving persistence of tissue eosinophils in inflammation.
Supported by grants from GSK ISS and NIH R01
Epithelial TLR9 upregulation by respiratory viruses impairs pulmonary antibacterial immunity
Authors: Rich Helen Eva Adams , Bhutia Simran , Martínez-Colón Giovanny , Warheit-Niemi Helen , Bame Monica , Moore Bethany 
Published: August 2023

Abstract
While much is known about signaling downstream of the dsDNA sensor TLR9, regulation of its expression is underexplored and has significant consequences for innate immune responses to pathogens. Mice lacking TLR9 surprisingly fare better upon coinfection with influenza and Staphylococcus aureus, and other groups have shown protection from S. aureus alone as well. These TLR9KO mice display lower inflammatory cytokine production, reduced cellular infiltration into airspaces, and increased survival. While macrophages taken from influenza-infected TLR9KO mice display increased bacterial phagocytosis and killing, inhibition of TLR9 on wild-type murine macrophages ex vivo surprisingly decreased their ability to phagocytize and kill S. aureus. Bone marrow chimera experiments revealed that lack of TLR9 in non-hematopoietic cells was responsible for the protection from bacterial super-infection seen in whole-body TLR9KO mice. To assess which structural cell types were important for this protection, we bred epithelial- and endothelial-specific TLR9KO mice, which revealed a role for epithelial cells but not endothelium. Interestingly, we also found that influenza infection caused upregulation of TLR9 in epithelial cells. Moreover, infection with two other respiratory viruses also caused epithelial cells to increase TLR9 expression, as did treatment with the antiviral mediator interferon beta (IFNB). IFNB has long been known to regulate susceptibility to bacterial coinfection during influenza, and these results suggest that it does so in part by upregulating TLR9. Together, these data show a role for epithelial TLR9 upregulation, likely driven by IFNB, in the pathogenesis of respiratory viral/bacterial coinfection.
Funding for this research was made possible in part by the Michigan Postdoctoral Pioneer Program at the University of Michigan Medical School, and by NIH grants NHLBI T32HL007517 (Helen Rich) and NHLBI R35HL144481 (Beth Moore).
Defining how Antibiotic Treatments shape Host Immunity
Authors: Gross Julia , Basu Rahul , Bradfield Clinton , Sun Jing , Moyer Tom , Smelkinson Margery , Antonioli-Schmit Sophia , Fischer Elizabeth , Dekker John , Weiss David , Fraser Iain D C 
Published: August 2023

Abstract
Although antibiotics are clinically crucial, little is known about how they impact a patient’s immune response to infection. We address that gap by investigating how macrophages respond to bacteria treated with different classes of antibiotics. We treated a broadly susceptible E. coli strain with 8 separate antibiotics, infected macrophages, and compared how much TNF the various antibiotic-treated bacteria elicited from macrophages. We found that bacteria treated with cidal antibiotics (that kill bacteria) elicited ~2–3 fold more TNF than macrophages infected with equivalent amounts of bacteria treated with bacteriostatic (growth limiting) antibiotics. A panel of 12 clinical isolates from patients (4 each of E.coli, Enterobacter and Klebsiella) showed a similar pattern of antibiotic class-dependent inflammatory cytokine output. We then evaluated an in vivo IP injection model with antibiotic pre-treated bacteria, heat-killed bacteria, or vehicle alone. At two hr post infection, we saw ~35–200% increases in serum levels of pro-inflammatory cytokines induced by cidal-treated bacteria compared to static-treated bacteria. Consistent with prior data, both the vehicle and the killed bacteria elicited no inflammatory cytokines. Preliminary imaging data suggest that treatment with the cidal drugs causes most of the dead bacteria to form large aggregates, while treatment with the static drugs does not. We now aim to clarify the role of these aggregates in driving this inflammatory outcome. This may identify additional patterns of immune responses to antibiotic treated bacteria that could guide the development of improved antimicrobial treatment strategies. This work is supported by the Intramural Research Program of NIAID, NIH.
Supported by Intramural Research Program of NIAID, NIH.
Deep phenotypic and functional characterization of NK cells during NK-mediated cytotoxicity
Authors: Nickle Rebecca , Chou Lisa , Bhattacharya Sam , Tam Miguel , Wiethe Carsen 
Published: August 2023

Abstract
Natural Killer (NK) cell-mediated cytotoxicity is a critical mechanism of the immune system to respond against harmful stimuli. The development of emerging cell therapies, such as CAR-NK cells, requires a deeper characterization of cultured NK cells. Here we show a phenotyping of ex vivo and in vitro NK cells, and demonstrate how high quality and reliable reagents from BioLegend ensure a more in depth cytotoxic analysis. NK cells were isolated from fresh human PBMCs, using our MojoSort™ Human NK Cell Isolation Kit, and incubated at different ratios with Tag-it Violet™ stained K562 target cells in IMDM + BioLegend’s Cell-Vive™ T-NK Xeno-Free Serum Substitute, in the presence of CD107a-PE. Cells were then harvested for surface phenotyping and the supernatants were collected for cytokine analysis using LEGENDplex™ Human CD8/NK Panel. Tag-it Violet™ and Apotracker™ Green staining of K562 cells demonstrate significant decrease in proliferation and significant increase in apoptosis, respectively. Surface staining of CD107a reveals increased degranulation of both CD56bright CD16− and CD5 + CD16+ NK cells that correlates with increased apoptosis of K562 cells. Supernatant cytokine analysis revealed significant increases in Granzyme A and B, Perforin, and Granulysin, among others. Taken together, these results show a more complete picture of the phenotypical and functional state of NK cells by measuring target cell killing as well as characterizing NK surface markers and secreted molecules involved in NK-mediated cytotoxicity. BioLegend reagents are designed to address ever increasing needs for deeper understanding of immunological processes and support the demand to develop sophisticated and consistent cell-based therapies.