bluuebunny/crossref_metadata_2025 huggingface dataset:
Using Rexinoids to Program Effector T cells for Homing to Mucosal Sites of Virus Transmission
Authors: Manhas Kavita , Blattman Joseph N. , Marshall Pamela A. , Wagner Carl E. , Jurutka Peter W.
Published: January 2023

Vaccines are the most effective defense against infectious diseases. To be most successful, vaccines must target the induced immune response to chief sites of pathogen entry: epithelial and mucosal surfaces. We have previously shown that the biologically active form of Vitamin A, all-trans retinoic acid (ATRA), can directly modulate the expression of gut mucosal-homing receptors on T cells as binding of ATRA to the retinoic acid receptor (RAR), which partners with the retinoid X receptor (RXR), promotes transcription of specific genes. Rexinoids, synthetic ligands for the RXR, have potential to mimic or improve the immune effects of vitamin A by enhancing RAR-mediated transcription. Additionally, rexinoids have a greater potential for clinical use due to their improved solubility and stability, making them candidates to incorporate into vaccines. Here, we explored the possibility of different rexinoids to function as adjuvants during vaccination to program effector T cell expression of the gut-homing receptors CCR9, α4β7, and CD103. CD8 T cells obtained from transgenic mice were co-cultured with peptide and set concentration of each rexinoid, followed by flow cytometric analysis of receptor expression. We observed increased expression of gut-homing receptors during activation in the presence of certain rexinoids, indicating that they can mimic the effects of ATRA. Furthermore, we saw a cooperative relationship between other rexinoids and ATRA, as co-culturing with sub-optimal doses of each resulted in enhanced expression. Our findings lend support for the prospective use of these rexinoids as adjuvants during vaccination and in therapeutic approaches against diseases transmitted across mucosal surfaces.
Utility of recombinant Hedgehog proteins and RANKL to generate bone cell models in vitro
Authors: Soto Hortensia , Moyron-Quiroz Juan , Kim Daniel , Chattopadhyay Mohar , Tam Miguel , Yang Xifeng , Nguyen Dzung , Zhou Jie
Published: January 2023

Bone remodeling is a lifelong process that begins during the embryonic stage and continues after birth. The two main bone cells involved in bone remodeling are osteoblast (bone formation) and osteoclast (bone resorption). The strict balance between bone formation and bone resorption is maintained by several cytokines and growth factors, including RANKL, the Hedgehog family of proteins (SHH, DHH, and IHH), and M-CSF secreted by bone cells, as well as systemic hormones. Any imbalance in bone remodeling might result in bone related diseases. Osteoblast differentiation was first studied using mouse mesenchymal cell line C3H10T1/2 cells. Recombinant SHH, DHH, and IHH were able to induce osteoblast differentiation of C3H10T1/2 cells. Bone morphogenetic protein 9 (BMP-9) enhanced the differentiation of C3H10T1/2 cells induced by Hedgehogs. Osteoclasts are multinuclear cells that are derived from hematopoietic precursors of the monocyte/macrophage lineage and their differentiation depends on osteoblast-produced RANKL and M-CSF. In order to study osteoclast differentiation, and the role of different cytokines in bone homeostasis; murine bone marrow derived macrophages (BMDM) were obtained and treated with mouse GM-CSF for 7 and 14 days. FACS analysis of cellular markers and functional assays confirmed the identity of macrophages. Subsequently, BMDM were differentiated to osteoclast with mouse M-CSF and RANKL. The multinucleated cells were identified by cell morphology and functional TRAP detection. Similar results were obtained with RAW264.7 cells, a mouse macrophage cell line. Our results confirm that BMDM and osteogenic cells lines are good models to study cytokines involved in cell differentiation and bone remodeling.
Bone marrow lympho-myeloid malfunction in obesity requires precursor cell-autonomous TLR4
Authors: Borghesi Lisa A. , Liu Ailing , Chen Minhui , Stefanovic-Racic Maja , Kumar Rashmi , O’Doherty Robert , Ding Ying , Jahnen-Dechent Willi
Published: January 2023

Obesity, a prevalent condition in adults and children, impairs bone marrow (BM) function. However, the underlying mechanisms are unclear. Here we show that obese mice exhibit poor emergency immune responses in a toll-like receptor 4 (TLR4)-dependent manner. Canonical myeloid genes (c-fms, pu.1, runx) are enhanced, and lymphoid genes (flt3, e47, ebf) are reduced. Using adoptive transfer and mixed BM chimera approaches we demonstrate that myeloid>lymphoid bias arises after only six weeks of high fat diet and depends on precursor cell-autonomous TLR4. Further, lean mice exposed to the TLR4 ligand lipopolysaccharide (LPS) at doses similar to that detectable in obese serum recapitulates BM lympho-myeloid alterations. Together, these results establish a mechanistic contribution of BM cell-intrinsic TLR4 in obesity-driven BM malfunction and demonstrate the importance of LPS. Our findings raise important questions about the impact of maternal obesity and endotoxemia to fetal hematopoiesis since fetal immune precursors are also sensitive to TLR4 signals.
Common lymphoid progenitor derivation of a conserved dendritic cell subtype is mediated by Bcl11a
Authors: Dekker Joseph D. , Rhee Catherine , Hu Zicheng , Lee Bum-Kyu , Lee Jiwon , Iyer Viswanath R. , Ehrlich Lauren I. , Georgiou George , Ippolito Gregory C. , Tucker Haley O.
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are a distinct subset of dendritic cell that specializes in the production of type I interferons (IFNs) upon engagement of pattern recognition receptors to promote antiviral immune responses. They have been implicated in the pathogenesis of autoimmune diseases that are characterized by a type I IFN signature, yet, pDC also can induce tolerogenic immune responses. Whereas an understanding of pDC physiology, immune function, and clinical significance has increased considerably, their development has not been unequivocally traced to either lymphoid or myeloid lineages nor has its transcriptional regulatory network been fully clarified. We observed that mb1-Cre mediated deletion of the Bcl11a transcription factor in common lymphoid progenitors (CLP) resulted in a ~15% reduction in pDC cellularity. Adoptive transfer of bone marrow from Bcl11aF/Fmb1-Cre mice recapitulated partial pDC loss. Mb1-Cre:YFP+ reporter mice contained ~85% YFP− and ~15% YFP+ pDCs, indicating a myeloid–lymphoid dichotomy of pDC generation. As compared with myeloid pDC, CLP-derived pDC (termed B-pDC) display a B cell-like gene expression profile, including hallmark targets directly transactivated by Bcl11a in B cells. B-pDCs demonstrate preferential homing to secondary lymphoid organs, are inherently activated compared to myeloid pDC, expand more rapidly upon TLR9 engagement, and are genetically and phenotypically homologous to a DC subtype recently discovered in human blood. We conclude that the pDC compartment comprises two cell types with overlapping but distinct functions that are conserved between mouse and man.
Genetic analysis of Bphse and Shs, novel loci controlling hypersensitivity to histamine in mice
Authors: Raza Abbas , Diehl Sean A. , Finstad Kristiaan H. , Case Laure K. , Krementsov Dimitry N. , Blankenhorn Elizabeth P. , Teuscher Cory
Published: January 2023

Histamine (HA) is a biogenic amine produced by mast cells and basophils that regulates many physiological processes including acute and chronic inflammation, immune regulation, and immediate hypersensitivity reactions. Previously, we mapped Bphs, the autosomal dominant locus in mice controlling hypersensitivity to HA following exposure to either Bordetella pertussis or pertussis toxin (PTX) to chromosome 6 (Chr6). Positional candidate gene cloning identified Bphs as HA H1 receptor (Hrh1/H1R). The Bphs-susceptible H1R allele possesses the Pro263, Val312 and Pro330 haplotype while the Bphs-resistant H1R allele have the Leu263, Met312 and Ser330 haplotype. Unexpectedly, when we tested a panel of inbred wild-derived strains for Bphs, we discovered strains e.g. MOLF/EiJ, that are phenotypically Bphs-susceptible despite carrying the resistant H1R allele. Phylogenetically, these strains are confined to a unique branch of wild-derived strains. This supports the existence of an evolutionary selected mechanism capable of complementing Hrh1resistance. Linkage analysis mapped the locus to Chr6, in linkage disequilibrium (LD) with Hrh1. We have designated this locus Bphs-enhancer (Bphse). Additionally, we identified a second Hrh1-linked gene in aged and CFA immunized SJL/J mice controlling HA hypersensitivity in the absence of Bordetella pertussis/PTX-sensitization. We have designated this spontaneous histamine sensitivity (Shs/Shs). Taken together, these data support the existence of a LD domain harboring multiple genes that control hypersensitivity to HA. The identification of the genes underlying both Bphse and Shs has the potential to significantly extend our understanding of the genetic control of HA signaling.
Saturated fat-induced food allergy: A paradigm for unfolded protein response (UPR)-mediated allergic inflammation
Authors: Samavedam Unni Krishna SRL. , Morris Suzanne C. , Khodoun Marat , Wu David , Potter Crystal , Hogan Simon P. , Finkelman Fred D.
Published: January 2023

Murine food allergy induction and maintenance is promoted by increased epithelial permeability and requires the pro-Th2 cytokines: IL-25, IL-33, and TSLP, but how allergens increase permeability and induce pro-Th2 cytokines is unknown. Saturated fats increase intestinal permeability, promote intestinal epithelial pro-Th2 cytokine expression and induce the UPR in several cell types. Two common food allergens, milk and eggs, contain considerable saturated fat. These observations suggested that saturated fats in food might promote allergy by inducing the UPR, which in turn, induces the pro-Th2 cytokines. To test this, quantitative PCR was used to evaluate the ability of heavy cream and egg yolk plasma (EYP, the fat-containing fraction of egg) to induce UPR-associated and pro-Th2 cytokine gene expression in transformed human intestinal epithelial CACO2 cells. Metformin, 4-PBA and TUDCA were used to block the UPR. EYP and cream, but not protein-rich, lipid-poor egg white, increased CACO2 cell UPR-associated gene expression, followed by increased pro-Th2 cytokine expression. This was blocked by the UPR antagonists and by a lipase antagonist that prevents fat absorption. EYP and cream, but not egg white, also increased epithelial permeability; this was blocked by metformin. In BALB/c mice, food allergy (detected as hypothermia and diarrhea) was induced by skin sensitization with egg white + EYP, followed by oral gavage with egg white + EYP. Metformin and TUDCA each prevented food allergy development and strongly suppressed established food allergy. These observations support our hypothesis and suggest that the saturated fat content of egg and milk contributes to their allergenicity.
A disintegrin and metalloproteinase 17 is required for IL-33 induced ILC2 expansion and cytokine production.
Authors: Lownik Joseph Cornelius , Martin Rebecca , Conrad Daniel
Published: January 2023

Type 2 innate lymphoid cells (ILC2s) rapidly respond to the alarmins IL-25 and IL-33, resulting in the initiation of downstream Th2 responses, particularly in parasitic helminth infections. We show that A disintegrin and metalloprotease (ADAM) 17 is required for IL-33 mediated ILC2 activation. In vivo, mice that lack ADAM17 specifically on ILC2s (ADAM17ILC2−/−) exhibit decreased ILC2 expansion in response to intranasal IL-33 as well as Nippostrongylus brasiliensis (Nb) infection. In both of these models, the few ILC2s present in ADAM17ILC2−/− produce less IL-13 than WT ILC2s. Loss of ILC2 expansion in an Nb infection resulted in decreased numbers of IL-13 producing T cells as well as increased worm burden. However, ADAM17ILC2−/− mice have normal ILC2 numbers in a naïve state, suggesting this defect in ILC2 function is limited to cell activation. In vitro, IL-33 treatment of purified ILC2s in the presence of ADAM17 inhibitors show increased apoptosis and decreased IL-13 production compared to control (no ADAM17 inhibitor) ILC2s. The defect in cytokine production following ADAM17 inhibition was not observed in response to IL-25 stimulation, suggesting this defect is limited to IL-33 stimulation Mechanistically, ADAM17 inhibition in ILC2s specifically causes a defect in IL-33 mediated ERK activation, potentially explaining the reduced survival and IL-13 production following ADAM17 inhibition in these cells. Overall, these studies provide a targetable protease to target for the treatment of ILC2 mediated diseases.
Sphingosine-1-phospate receptor 4 regulates passive systemic anaphylaxis in mice but is dispensable for canonical IgE-mediated responses in mast cells
Authors: Kulinski Joseph M. , Proia Richard L. , Tuymetova Galina , Metcalfe Dean D. , Olivera Ana
Published: January 2023

Mast cells are key players in the development of inflammatory allergic reactions. Cross-linking of the high-affinity receptor for IgE (FcɛRI) on mast cells leads to the generation and secretion of the sphingolipid mediator, sphingosine-1-phosphate (S1P) which can in turn activate its G-protein coupled receptors on mast cells. Previous reports have identified the expression of two of the five receptors for S1P on mast cells, S1P1 and S1P2, with functions in IgE-receptor-mediated chemotaxis and degranulation, respectively. Here, we show that cultured human and mouse mast cells also express abundant message for the S1P4 receptor and demonstrate its presence at the plasma membrane in the human mast cell line LAD2. Genetic deletion of S1pr4 resulted in exacerbation of passive systemic anaphylaxis to IgE/anti-IgE in mice. However, exhaustive characterization of IgE-mediated responses in S1P4-deficient bone marrow and peritoneal mouse mast cells indicated that this receptor is dispensable for normal in vitro mast cell growth and responses to IgE-mediated activation including degranulation, cytokine/chemokine release and chemotaxis. Thus, we provide evidence that S1P4 modulates mast cell-mediated anaphylaxis in vivo in an unexpected manner that does not involve regulation of mast cell responsiveness to IgE stimulation.
RNA Binding Protein HuR is critical for the regulation of IL-2 Expression in CD4+ T Cells
Authors: Taylor Tiffany , Ellis Jason S. , Ridenhour Suzanne , Atasoy Ulus
Published: January 2023

The RBP HuR regulates T helper cell differentiation and gene expression by binding to AU-rich elements in the 3′ UTR of its target mRNA and modulating localization and stability. We hypothesized that HuR plays an important role in the regulation of the IL-2 signaling during T cell differentiation and activation. To test this hypothesis, we used the conditional dLck-Cre ROSA HuRfl/fl knock-out mouse to delete HuR late in T cell development but prior to T cell activation. The OVA challenge model of airway inflammation was used to ascertain the effects of HuR KO in vivo. While only 50% of CD4+ T cells in HuR KO mice lack HuR, in vivo and in vitro Th2 responses were essentially nonexistent and there were only background levels of lung inflammation using an OVA challenge model of airway inflammation. The few lung infiltrating CD4+ T cells present had reductions in IL-2 and CD25 expression. HuR KO T cells also had profound antigen-specific proliferation defects in vitro compared to OVA immunized controls and non-specific CD3/CD28 stimulation only partially restored proliferation defects. Furthermore, there is a decrease in IL-2Rα (CD25) which results in decreased levels of phosphorylated (active) STAT5 protein and Blimp1 mRNA and protein levels. To further assess HuR role in regulating IL-2, we developed the pSTAT5+ dLck-Cre HuRfl/fl mouse model, containing a transgene producing constitutively active STAT5. In these mice, there is significant pSTAT5 protein in activated CD4+ T cells, which should circumvent IL-2 downstream signaling, and correct the observed phenotypes. pSTAT5+ HuR KO T cells also had alterations in their expression of multiple CD3 subunits. We conclude that HuR is essential for regulating IL-2 homeostasis and T cell differentiation.
Manipulation of CXCR4 signaling by human cytomegalovirus (HCMV) interleukin-10
Authors: Spencer Juliet , Tu Carolyn
Published: January 2023

Herpesviruses have developed numerous strategies for manipulating host immune responses to avoid clearance and establish lifelong latent infection. Human cytomegalovirus (HCMV) encodes cmvIL-10, an ortholog of human interleukin-10 that binds to the cellular IL-10 receptor (IL-10R) with high affinity to elicit potent immunosuppressive effects, including inhibition of inflammatory cytokine production. In direct contrast, we found that cmvIL-10 promotes heightened chemokine signaling. CXCR4 is an essential cellular chemokine receptor that guides cell movement toward its natural ligand, CXCL12. We found that monocytes and epithelial cells exhibited significantly increased calcium mobilization and migration to CXCL12 in the presence of cmvIL-10. Treatment with a Stat3 inhibitor or siRNA blocked these effects, indicating that Stat3 activation is required for potentiation of CXCR4 signaling activity by cmvIL-10. Using immunofluorescence microscopy, we found evidence of association between IL-10R and CXCR4 in cell membranes and observed that cmvIL-10 treatment led to dramatic changes in the cellular distribution of Stat3. Since intracellular calcium release is a rapid response, Stat3 is likely acting here via a non-canonical pathway, rather than in its classic role as a transcription factor. These findings demonstrate that cmvIL-10 triggers a complex series of events that augment the signaling activity of a cellular chemokine receptor. Given that monocytes are a primary reservoir of latent virus, manipulation of CXCR4 signaling may enable HCMV to modify cell trafficking patterns to promote virus dissemination.
GILT in thymic epithelial cells preferentially facilitates MHC class II-restricted presentation in generating central T cell tolerance to a self and melanoma antigen
Authors: Meador Lydia , Rausch Matthew P. , Metzger Todd C. , Anderson Mark S. , Hastings Karen Taraszka
Published: January 2023

Central tolerance prevents autoimmunity, but limits T cell responses to tumor antigens that are self antigens. In peripheral APCs, gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance is unknown. We found that GILT enhanced the clonal deletion of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells. TRP1 expression was restricted to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for deletion of TRP1-specific thymocytes. Chimeras with GILT expression in TECs also had an increased frequency of TRP1-specific regulatory T (Treg) cells. These data demonstrate an advantage of TEC-derived GILT for antigen processing and subsequent T cell tolerance. Only chimeras that lacked GILT in both TECs and hematopoietic cells were protected from melanoma and had a high effector T cell:Treg cell ratio. Thus, GILT diminishes T cell-mediated protection from melanoma through promoting thymic deletion and Treg cells.
The use of alternative promoters in T cell development
Authors: Chiang Jeffrey , Ku Wai Lim , Cui Kairong , Zhao Keji , Hodes Richard J.
Published: January 2023

Many mammalian genes, including a number involved in immune development and function, use multiple promoters encoding expression of the same protein product. To understand the function of alternative promotors during T cell development, we carried out genome-wide RNA-Seq analysis of DN, DP, CD4 and CD8 thymocytes. 26% (2469/9595) of genes expressed by thymocytes used two or more alternative promotors at some point in T cell development. However, only 3.2% (80/2469) of the genes which utilized two or more promoters showed the difference of promotor activity among different T cell developmental stages which were validated by selected representative genes with qPCR assay. To analyze mechanisms of alternative promoter regulation, DNase hypersensitivity sites were mapped, and ChIP-Seq with anti-H3K4me3 antibodies was performed in the same thymic subpopulations. To determine the in vivo functional role of alternative promotors for T cell development, several genes were selected for study by deletion of individual promoters by CRISPR technology. We have initially generated mice with deletion of distal or proximal promotor of the lck gene and demonstrated distinct functional roles of the two promoters: the proximal lck promoter is critical for early stages of thymic T cell development while the distal promoter was necessary for normal T cell activation. Ongoing studies will characterize more broadly the functional role of alternative promoter expression in thymocyte development, and the mechanisms mediating selective promoter activity.
Control of T cell development and function by Protein Arginine Methyltransferase 5
Authors: Hagman James R. , Fleenor Courtney J. , Arends Tessa , Pandey Ahwan , Abraham Christopher G. , Dege Carissa L. , Straign Desiree , Danhorn Thomas , Reinhardt R. Lee , Espinosa Joaquin M. , O’Connor Brian P.
Published: January 2023

Temporally and spatially regulated gene expression is essential for the development of thymocytes and functional T cells. Gene transcription is controlled, in part, by reversible post-translational modifications (PTM) of histones by epigenetic regulators. In turn, histone PTMs are bound by reader proteins, which mediate transcriptional activation and repression. Together, PTMs and readers enhance or suppress the accessibility of DNA to sequence-specific DNA-binding proteins. Epigenetic mechanisms are often dysregulated in human diseases and are promising targets for cancer treatments and immunotherapy. One such candidate is Protein Arginine Methyltransferase 5 (PRMT5), a type II arginine methyltransferase. PRMT5 catalyzes symmetric dimethylation (Rme2s) of arginines on histones H2A, H3, and H4. Rme2s strongly correlates with transcriptional repression. However, the roles of PRMT5-dependent histone arginine methylation in thymocyte development and T cell activation are poorly understood. Dynamic expression of Prmt5 transcripts suggests that the methyltransferase modulates transcription at select stages in T lymphopoiesis. Previous studies employing hematopoietic system-wide depletion of Prmt5 detected reduced cellularity of T cell progenitor populations, but effects on thymocyte transcriptomes were not determined. Here, we describe consequences of the loss of PRMT5 on T cell progenitor stage-specific transcription. PRMT5 suppresses activities of key transcription factors by regulating DNA accessibility, which in turn prevents inappropriate transcription in thymocytes in vitro and in vivo. Together, our data support a role of PRMT5 as an essential ‘gatekeeper’ of T cell development and function.
Elevation of choline metabolism contributes to the prolonged B cell survival and expanded B cell compartment in the spleen of B cell-specific TRAF3-deficient mice
Authors: Gokhale Samantha , Zhu Sining , Lu Wenyun , Xie Ping
Published: January 2023

Specific deletion of the tumor suppressor gene TRAF3 from B lymphocytes (B-TRAF3−/−) in mice leads to prolonged survival of mature B cells and expanded B cell compartment in secondary lymphoid organs. To understand the metabolic basis of the prolonged survival of TRAF3−/− B cells, we performed LC-MS-based metabolome and lipidome screening with resting splenic B cells of young adult B-TRAF3−/− and littermate control (LMC) mice. Our metabolome screening data showed that phosphocholine (P-Cho) and phosphoethanolamine, two small metabolites and precursors of phospholipids, were significantly increased in TRAF3−/− B cells. Consistently, our lipidome screening revealed markedly elevated levels of multiple species of phosphatidylcholine (PC) and phosphatidylethanolamine in TRAF3−/− B cells. Interestingly, our transcriptome profiling identified Chkα, the rate-limiting enzyme of choline metabolism, as a significantly up-regulated gene in TRAF3−/− B cells. To investigate the functional importance of elevated choline metabolism, we examined the effects of two Chkα inhibitors, MN58B and RSM932A. We found that both MN58B and RSM932A potently induced cell apoptosis in TRAF3−/− mouse B lymphoma and human multiple myeloma cell lines in vitro. Furthermore, in vivo administration of MN58B and RSM932A partially decreased the spleen size and B cell compartment in B-TRAF3−/− mice. Together, our results indicate that TRAF3 specifically re-engineers the P-Cho-PC-PE metabolic pathways to regulate mature B cell survival. Our findings also suggest that the P-Cho-PC-PE metabolic pathways have diagnostic and therapeutic value for B cell malignancies with TRAF3 deletion or relevant mutations.
Cell Death Markers in Sepsis
Authors: Madiraju Charitha , Mallarpu Chandra Shekar , Prasad Sudhir , Singarapu Maneendra , Kim Jean H. , Haririparsa Neda , Bratic Nemanja , Brar Harvinder , Cashman John R. , Chelluri Lakshmi Kiran
Published: January 2023

Cell death mechanisms play an important role in the pathogenesis of sepsis. The goal of our study was to understand cell death regulatory mechanisms such as pyroptosis, necroptosis and apoptosis in sepsis. Our preliminary study design included patients diagnosed with sepsis that were treated in clinical settings. Patients diagnosed with sepsis (test) were compared with healthy individuals (control). Cell death-regulating markers were assessed in plasma, peripheral blood mononuclear cells (PBMCs) and PBMC cell lysate samples derived from patients (N =8) and healthy controls (N=8). Samples were analyzed by ELISA, flow cytometry and immunoblotting methods. Preliminary flow cytometry evaluation of PBMC samples from patients showed elevated levels of cell death markers when compared to samples from healthy individuals. Results from ELISA experiments showed both plasma and PBMC cell lysate samples from septic patients had an increase in a marker for necroptosis, in comparison with control samples (P < 0.05 and P < 0.1, respectively). Interestingly, test samples showed modulation in a marker for apoptosis and a marker for pyroptosis in comparison with controls (P < 0.05). Preliminary results suggest that there exists distinct cell death mechanisms with respect to the clinical outcomes observed in septic patients.
Epigenetic Regulation of the COX-2/PGE2 Pathway in Macrophages Results in Impaired Wound Healing in Diabetes
Authors: Davis Frank M. , Joshi Amrita , Kimball Andrew , den Dekker Aaron D. , Schaller Matthew , Wilke Carol A. , Moore Bethany B. , Gallagher Katherine
Published: January 2023

Macrophage (Mϕ) plasticity, allowing for transition of Mϕs from an inflammatory to a reparative phenotype, is critical for normal wound healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. There is increasing evidence that epigenetic-based mechanisms, including DNA hypomethylation, control Mϕ function. The cyclooxygenase (COX)-2/Prostaglandin E2 (PGE2) axis as well as upstream pathways including cytosolic phospholipase (c(PL)A2), involved in the release arachidonic acid, have been associated with chronic inflammation, however they have not been examined in diabetic wounds. The purpose of this study was to investigate if epigenetic modifications alter gene expression of the COX-2/PGE2 and c(PL)A2 pathway leading to derangements in macrophage inflammation in diabetes. Using the murine diet induce obesity (DIO) model, we evaluated diabetic wound healing compared with control mice. We demonstrate that TGFβ is elevated in DIO wound Mϕs in comparison to control wound Mϕs. Further, the increase in TGFβ stimulates miR-29B production in wound Mϕs resulting in hypomethylation and overexpression of the COX-2 gene. This results in diabetic wound Mϕs displaying increased levels of COX-2 which shunts amino acid metabolism towards the COX-2 pathway increasing PGE2 while limiting leukotriene synthesis via 5-lipoxygenase (5-LO) pathways. Lastly, c(PL)A2 appears to be upregulated in diabetic wound Mϕs and human diabetic monocytes. In summary, these results demonstrate that the COX-2/PGE2 pathway is increased in diabetic wound Mϕs by elevated TGFβ/miR-29b and results in elevated PGE2 that drives Mϕ-mediated inflammation preventing wound repair.
Molecular mimicry and signaling by human monoclonal autoantibody derived from human myocarditis and heart failure may contribute to fibrosis and remodeling in cardiomyopathy
Authors: Myers Jennifer M. , Alvarez Kathy M. , Reim Sean , Bentley Rebecca , Garman Lori , Chen Allshine , Wiley Graham , Bebak Melissa , Montgomery Courtney G. , Gaffney Patrick , Simpson Kathleen E. , Stavrakis Stavros , Cooper Leslie T. , Cunningham Madeleine W.
Published: January 2023

Previous studies in animal models of myocarditis suggest that immune responses against cardiac myosin may lead to remodeling in the heart and dilated cardiomyopathy. In human myocarditis and dilated cardiomyopathy, anti-cardiac myosin autoantibodies significantly associated with myocarditis in cohorts of adults and children and reacted significantly with both human cardiac myosin (HCM) and the β-adrenergic receptors (βAR). We produced a human monoclonal antibody (mAb) derived from human myocarditis which reacted with HCM and specific peptides from pathogenic regions of HCM. Autoantibodies against HCM and βAR from myocarditis and dilated cardiomyopathy including the human myocarditis-derived mAb targeted heart cells by signaling protein kinase A (PKA) in a primary heart cell line. To further define potential pathogenic mechanisms of the human mAb and serum autoantibodies from myocarditis and cardiomyopathy and to test our hypothesis that autoantibodies may alter gene expression related to extracellular matrix, remodeling and fibrosis, we investigated genes differentially expressed by RNA sequence analysis of a primary heart cell line after treatment with the human mAb and sera from both children and adults with myocarditis. The overall heatmap of differentially expressed genes in heart cells demonstrated a significant difference between the control sera group and both human mAb and myocarditis sera treatment groups. We identified upregulated genes related to fibrosis, apoptosis, inflammation and cardiomyopathy, suggesting that the myocarditis- and cardiomyopathy-derived autoantibodies may induce signaling in primary heart cells and potentially contribute to fibrosis and remodeling in the heart.
Altered cellular metabolism in Mucosal-associated invariant T cells drives dysregulated effector functions in obesity
Authors: Hogan Andrew E. , O’Brien Aisling , Finlay David K. , Lynch Lydia , O’Shea Donal
Published: January 2023

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

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

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

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

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

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

Blood cells are prime indicators of immuno-surveillance, and the ease of blood sampling makes blood analysis a key interest for clinical and research applications. While current flow cytometry methods are high-throughput and provide fine resolution in the segregation of white blood cell (WBC) populations, WBC enrichment involving red blood cell (RBC) lysis are laborious and typically performed manually, contributing to experimental variability especially as blood cells are sensitive to physical and chemical stress.

We describe RBC lysis and leukocyte immunostaining on a centrifuge-less platform DA-Cell™, using a novel wall-less plate and laminar flow washer. The DropArray (DA) plate consists of an array of 96 hydrophilic spots surrounded by hydrophobic surface, which functions as a virtual wall that separates each spot. The maximum volume of each spot can be increased to 300μL with an insert. During lysis, WBC settle to the surface of the spot, allowing the spent lysis buffer to be removed. After removal of the insert, the plate goes through a gentle 3–4min laminar-flow washing process in the DA-Cell washer, decreasing cell washing time by at least 50% while eliminating centrifugation that stresses cells and disrupts antibody binding.

In studies comparing mouse whole blood lysis (1X RBC Lysis buffer, eBioscience) and antibody staining by conventional tube centrifuge and DA-Cell, DA-Cell achieved dramatically higher staining index and improved resolution of cell cluster by flow cytometry. CD45+ leukocyte recovery and viability was uncompromised compared to conventional tube centrifuge. In summary, DA-Cell system provides gentle, fast and convenient blood lysis, while improving data quality with superior antibody staining.
Real-time detection of LPS induced cytokine-storm using Vium Smart Housing
Authors: Ichim Christine V. , Mechanic Jordan , Shah Puja , Maraganore Colin , Hutto David , Schaevitz Laura
Published: January 2023

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

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