bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidating the role of HLA-DO/H2-O in modulating the diversity of the MHC Class II self peptide repertoire
Authors: Nanaware Padma Pandurang , Jurewicz Mollie M. , Leszyk John , Shaffer Scott , Stern Lawrence J.
Published: January 2023

The classical MHC Class II molecules HLA-DP, DQ and DR proteins bind antigenic peptides in the endosomes of APCs and present them to CD4+ T cells whereas HLA-DM and DO are the non-classical proteins. DM catalyzes the exchange of CLIP peptides with other endosomal peptides whereas DO acts as an inhibitor of DM activity. The clear biochemical effect of DO in inhibiting DM is reported but its effect on the peptide repertoire is still not clear. We used Quadrupole Orbitrap mass spectrometry for characterizing peptides presented by the MHC Class II protein DR1 from CRISPR/Cas9 deleted LG2 cells. We identified 6139 peptides in wild type cells (WT) but only 5227 peptides from DO−/− cells (DO-KO). The label free quantitation analysis of WT Vs DO-KO showed DM sensitive CLIP peptides were present in lower abundance in DO-KO, consistent with cell surface staining experiments. We analyzed peptide diversity in the eluted peptidome using diversity measures like Shannon’s Entropy, Simpson’s diversity and Berger-Parker Index. In each case the diversity was higher for the WT peptidome as compared to DO-KO. We screened several peptides for binding affinity and DM susceptibility by analyzing their relative abundances in WT and DO-KO. Peptides from group WT > DO-KO, WT only showed lower binding affinity and high DM sensitivity as compared to peptides in DO > WT, DO only. The same phenomenon was observed for peptides eluted from I-Ab purified from B cells and thymus of BL/6 as compared to H2-O−/− mice. These results indicate that DO/H2-O broadens the peptide repertoire whereas its absence restricts the repertoire resulting in presentation of fewer different epitopes, and highlights the role of DO in regulating the MHC-II peptide repertoire by modulating DM peptide editing.
SMAD4-deficient Dendritic Cells in the Murine Lung shows an Activated Phenotype
Authors: Root Sierra Hope , Menoret Antoine , Cauley Linda S.
Published: January 2023

SMAD4 is a signaling intermediate that acts downstream of the TGFb receptor. This signaling pathway is known to regulate expression of the Itgae gene (CD103) in activated CD8 T cells. Whether SMAD4 plays a role in induction or maintenance of CD103+ dendritic cells (DCs) has not been investigated. To address this question, we crossed SMAD4 floxed mice with Cre under the control of the CD11c promoter. DCs in the lung, spleen and bone marrow of naïve CD11c-SMAD4 mice were analyzed by multi-parameter flow cytometry. The lungs of Smad4-deficient mice contained smaller numbers of CD11c+ cells than control animals, and the remaining cells expressed costimulatory molecules and CD11b at high levels. Lung suspensions were metal barcoded for time of flight cytometry (cyTOF) and viSNE maps were contracted using 25 parameters. Under these homeostatic conditions, Smad4-abalation caused a dramatic decrease in CD11c+ DCs with activation of pSTAT5, when compared to DCs from littermate controls. Activation of pSTAT5 in KO was specific to CD11c+ DCs as no difference was seen in other cell types including T cells. Our data indicate that the lungs of SMAD4-CD11c KO mice were depleted of migratory DCs, which migrated to the draining lymph nodes after spontaneous maturation. We currently are exploring whether DC-depletion undermines cell mediated immunity to respiratory virus infection.
Structure of a TAPBPR/nanobody complex reveals dynamic plasticity of unbound regions of TAPBPR
Authors: Jiang Jiansheng , Natarajan Kannan , Ingram Jessica , Margulies David H.
Published: January 2023

Recently determined structures of the tapasin homolog, TAPBPR, complexed with peptide-receptive MHC-I molecules, revealed interaction of TAPBPR with the a2-1 helix of MHC-I, and displacement of the C-terminal region of the peptide binding groove, resulting in a conformation amenable to peptide loading. These structures were based on X-ray data at ~3.4 Å resolution and a number of regions of the electron density map were ill-defined. In an effort to obtain a higher resolution structure of TAPBPR, we isolated TAPBPR-specific nanobodies (camelid-derived single domain antibody fragments), from phage display libraries generated from peripheral blood lymphocyte mRNA of an alpaca immunized with TAPBPR. E. coli-expressed and purified nanobodies were complexed with TAPBPR. Crystal structures of a TAPBPR/nanobody complex and of nanobody alone were determined at 2.8 Å and 1.55 Å resolution respectively. In the structure of the complex the nanobody binds to the C-terminal Ig domain of TAPBPR, rigidifying the region of contact and thus permitting modeling of TAPBPR residues that were not visible in the reported TAPBPR/MHC-I structures. Unliganded surfaces of TAPBPR were more disordered than in the MHC-I liganded form. These data are consistent with the view that the luminal domains of TAPBPR, in solution, are structurally dynamic, and rigidify on ligand (i.e. MHC-I) interaction. Such structural plasticity may explain the ability of TAPBPR to interact with a variety of MHC-I allelomorphs.
Systematic profiling of HLA-C epitopes
Authors: Keskin Derin B. , Sarkizova Siranush , Klaeger Suzan Klaeger , Li Letitia , Le Phuong M. , Keshishian Hasmik , Hartigan Christina R. R. , Zhang Guang Lan , Clauser Karl , Carr Steven A. , Hacohen Nir , Wu Catherine J.
Published: January 2023

Accurate predictive algorithms have required elucidation of HLA-peptide binding motifs from extensive HLA binding peptide ligand databases. Historically, however, little attention has been paid to HLA-C because of its perceived lower surface expression, even as newer studies have identified important HLA-C restricted viral epitopes. Robust predictive algorithms for HLA-C are thus lacking and yet, if improved, could markedly expand the spectrum of immune targets contributing to protective immune responses. We generated 21 distinct single HLA-C allele-expressing cell lines and subjected them to immunoaffinity purification and MS-based sequencing. From 33851 peptides identified from the 21 HLA-C expressing cell lines, we discovered novel anchor motifs. These data were used to train machine-learning algorithms to generate an accurate and novel HLA-C epitope prediction tool. Finally, we used our novel algorithm to predict HLA-C restricted neo-epitopes out of tumor biopsies.
DO:DM tunes antigen presentation in a BCR affinity-dependent manner
Authors: Jiang Wei , Adler Lital N. , Macmillan Henriette , Mellins Elizabeth
Published: January 2023

HLA-DO, a non-classical class II major histocompatibility complex (MHCII), competitively inhibits HLA-DM in B cells. B cells lower DO levels during antigen-mediated activation in germinal centers, implying a regulatory function of DO in resting B cells. We transfected T2 (T/B hybrid) DR4.DM cells with DO and used index FACS to measure single cell variation in DO:DM ratios. High DO:DM ratios allowed groove occupancy by class II-associated Ii peptides (CLIP), and low DO:DM ratios enabled CLIP removal. 3D structured illumination microscopy revealed DMDO concentrated in LAMP1+ endosomes. Both DM+DO+ and DM−DO− cells express CLIP/DR complexes, but a larger fraction of CLIP/DR localizes to LAMP1+ endosomes in DM+DO+ cells. Using memory and naïve human tonsillar B cells, we saw significant downregulation of DO after cell activation. CLIP/MHCII density relative to free DM (estimated by 1-DO/DM) was generally higher in resting vs. activated B cells, especially in memory cells. This memory B cell phenotype primarily reflected higher intracellular CLIP/MHCII; upon BCR-mediated stimulation, total CLIP/MHCII fell to levels in activated naïve cells. We hypothesized that higher CLIP/MHCII and DO in resting memory B cells sets a threshold for the activation needed to efficiently recruit Tfh and selects for cells with higher affinity BCR. Using antigen stimulation of 2 clonal B cell lines expressing BCRs with different affinities for GAD65, we confirmed that, in this system, BCR affinity correlates with the amount of reduction in total CLIP/MHCII after activation. A positive correlation between BCR affinity and the extent of peptide exchange on MHCII is likely mediated by the effects of BCR affinity on signaling and antigen delivery.
Investigating a role for the MHC class II accessory molecule HLA-DO in autoimmune disease
Authors: Welsh Robin A. , Song Nianbin , Boronina Tatiana , Cole Robert , Foss Catherine , Sadegh-Nasseri Scheherazade
Published: January 2023

HLA-DO (DO), H2-O in mice, is a non-classical, non-polymorphic MHC Class II accessory molecule. Expressed primarily in the thymic medulla and B cells little is known about its physiological role in vivo. To gain insight into what role DO might have in CD4 T cell selection, our lab previously used in vitro biochemical assays to show that DO itself enhances the binding of DM insensitive peptides to the HLA-DR1 molecule, while inhibiting the binding of DM sensitive peptides. This led us to hypothesize that in vivo, DO might be important for ensuring proper presentation of immunodominant epitopes in the thymic medulla; thereby ensuring proper negative selection occurs. Lack of DO then could lead to a larger precursor pool of autoreactive CD4+ T cells in the periphery due to faulty negative selection. To test this, we utilized H2-O knock-out (KO) mice and the autoimmune model experimental autoimmune encephalomyelitis (EAE). With this model, we have found that KO mice have a faster onset of disease when compared to C57BL/6 wild-type mice. This increase in onset correlated with the recovery of more MOG specific cells from the CNS of KO mice at all stages of disease. Moreover, naïve mouse data also supports our hypothesis with naïve KO mice having a larger number of MOG specific CD4+ T cells. More recently, we found that KO mice also harbor increased percentages of regulatory T cells (Tregs) in both the naïve and diseased states. Correlating this finding with disease onset, we saw that KO Tregs appear to have impaired upregulating of the inhibitory molecule CTLA-4 in early disease. As a whole, our data suggests that KO mice not only have an increased precursor pool of autoreactive CD4 T cells but might also have impaired Treg function; all leading to faster disease onset.
One size doesn’t fit all: SLAM-Associated Protein is dispensable for type 1 diabetes but required for autoantibody-mediated arthritis
Authors: Bonami Rachel , Nyhoff Lindsay , Hulbert Chrys , Felton Jamie , Kendall Peggy L. , Thomas James W
Published: January 2023

Signaling lymphocytic activation molecule-associated protein (SAP), a critical intracellular signaling molecule for T-B interactions, drives Tfh development in germinal centers (GC) in the setting of protective immune responses. SAP promotes long-lived T cell-B cell interactions necessary to sustain germinal center reactions that lead to antibody production. High-affinity islet autoantibodies predict type 1 diabetes (T1D) but do not cause beta cell destruction. This paradox intimates T follicular helper cells (Tfh) as key pathologic effectors. To understand Tfh contribution to autoimmune processes, we investigated the role of SAP in T1D and autoantibody-mediated arthritis. Whereas spontaneous autoimmune arthritis depended on SAP, organized insulitis and diabetes onset were unabated, despite a blocked anti-insulin vaccine response. SAP-deficient T cell function was therefore investigated in the non-obese diabetic (NOD) T1D model. B lymphocyte antigen presentation drove SAP-deficient NOD T cell proliferation. Although germinal center B cell formation was markedly reduced, GC-Tfh were found at sites of autoimmune attack in SAP-deficient NOD mice. Thus, Tfh induced in germinal center reactions are dispensable for T1D and suggest that the autoimmune process in NOD retains pathogenic Tfh in the absence of prolonged B cell interactions. These findings demonstrate that SAP is essential for Tfh formation and autoimmunity when autoantibodies play a central role in autoimmune arthritis but not in cell-mediated T1D. SAP inhibition is thus a compelling strategy for autoantibody-mediated disease treatment, but additional cognate cellular interaction molecules must be targeted to treat cell-mediated autoimmune diseases.
Tryptamine treatment results in the amelioration of EAE by inducing an anti-inflammatory shift in the Th17/Treg balance mediated by regulation of miRNA expression profile
Authors: Dopkins Nicholas , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

In this study we focus on understanding the novel immunotherapeutic potential of tryptamine, a bioactive indole compound derived from microorganisms and various botanicals, in the context of ameliorating symptoms of Experimental Autoimmune Encephalomyelitis (EAE). EAE is used in the laboratory setting to replicate the etiology as well as pathology of Multiple Sclerosis (MS) in murine organisms for research purposes. Globally MS, an incurable autoimmune disorder, is experiencing increases in incidence and treatment costs which places emphasis on the discovery of novel immunosuppressents with limited toxicity. In this study we show that tryptamine poses potent immunosuppressive activity and ameliorates the clinical parameters of EAE by shifting the balance of CD4+ T cells from a pro-inflammatory T helper 17 cell (Th17) towards an anti-inflammatory T regulatory cell (Treg) dominated landscape in chronic progressive EAE. Based on preliminary miRNA array data, we have found that the observed shift in the Th17/Treg balance following tryptamine treatment is due to an anti-inflammatory miRNA expression profile within the lymphocytes of treated mice. MiR-3473g and miR-140-3p, putative targets of the enzyme SIRT1, were found to be upregulated within the lymphocytes of tryptamine treated mice. SIRT1 in the context of inflammatory disorders works as a marker of pro-inflammatory responses in lymphocytes due to SIRT1’s activation of the master transcription factor of Th17 cells known as RORγt. This epigenetic regulation of gene expression provides a clearly linkable mechanism explaining the shift in the Th17/Treg balance as well as the amelioration of symptoms observed following tryptamine treatment in EAE mice.
Lymphocytes populations in murine models of Systemic Lupus Erythematosus exhibit different responses to treatment with metabolic modulators
Authors: Cornaby Caleb , Zeumer-Spataro Leilani , Choi Seung-Chul , Li Wei , Morel Laurence
Published: January 2023

An often debilitating autoimmune disease involving multiple immune cell types and tissues, Systemic Lupus Erythematosus (SLE) afflicts nearly 3 in a 1000 people, an estimated 9.7 million in the United States. Murine models of lupus are essential for the study of disease progression, prevention, and treatment of SLE, with B6.Sle1.Sle2.Sle3 triple congenic (TC) and NZB X NZW F1 hybrid (NZB/W) being two murine models commonly used for these purposes. While these murine models show similar disease symptomology, they exhibit immune cell subsets that differ metabolically. CD4+ T cells from TC mice show increased glycolysis and phosphorylation (OXPHOS) function, however CD4+ T cells from NZB/W mice demonstrate similar activity of OXPHOS and glycolysis compared to control B6 mice. B cells from both strains present similar elevated metabolism. Further, CD4+ T cell and B cell populations from these two strains display differential metabolic modulation during treatment with the same metabolic inhibitors. This contrasts with a similar therapeutic efficiency of a treatment inhibiting glycolysis with 2DG and mitochondrial respiration with metformin in both murine models of lupus. This implies metabolic inhibitors may have therapeutic efficiency across cell-specific differences in metabolic defects.
Evaluating cooperative roles for STAT3 and Aiolos in lymphocyte mimicry associated with metastatic disease
Authors: Sandhu Simran , Read Kaitlin , Jones Devin , Dickerson Sam , Oestreich Kenneth J
Published: January 2023

Inappropriate expression and function of cytokine and chemokine receptors by both hematopoietic and non-hematopoietic cell populations have been linked to human disease. One example of this is the expression of lymphocytic genes by metastatic cancers, termed lymphocyte mimicry. Specifically, the aberrant expression of chemokine receptors by cancer cells allows for inappropriate responses to immune signals, migration, and increased ability to survive in circulation. The mechanisms that regulate lymphocyte mimicry are currently incompletely understood. Our laboratory recently identified a novel transcriptional complex composed of the Ikaros Zinc Finger transcription factor Aiolos and STAT3 that regulates gene expression in CD4+ T cells, including that of chemokine receptors. Interestingly, Aiolos and STAT3 have been independently implicated in multiple cancers, including metastatic breast cancer. We hypothesized that Aiolos and STAT3 may cooperate to induce chemokine receptor expression in metastatic breast cancer cells. Our study observed increased Aiolos expression and STAT3 activation in metastatic breast cancer cells as compared to non-tumorgenic and non-metastatic controls. This correlated with increased expression of the chemokine receptor CXCR4, which has been previously linked to metastatic cancer. Importantly, shRNA knockdown of Aiolos in metastatic cells resulted in decreased expression of CXCR4. Collectively, these data support a role for Aiolos, possibly in collaboration with STAT3, in the induction of a metastatic gene signature in human breast cancer.
Neuroinflammation functionally regulates CNS lymphatic vasculature and drainage
Authors: Hsu Martin , Rayasam Aditya , Kijak Julie A. , Choi Yun Hwa , Harding Jeffrey S. , Marcus Sarah A. , Karpus William J , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

There are no conventional lymphatic vessels within the CNS parenchyma, although it has been hypothesized that lymphatics near the cribriform plate or dura maintain fluid homeostasis and immune surveillance during steady-state conditions. However, the role of these lymphatic vessels during neuroinflammation is not well understood. We report that lymphatic vessels near the cribriform plate undergo lymphangiogenesis in a VEGFC – VEGFR3 dependent manner during experimental autoimmune encephalomyelitis (EAE) and drain both CSF and cells that were once in the CNS parenchyma. Lymphangiogenesis also contributes to the drainage of CNS derived antigens that leads to antigen specific T cell proliferation in the draining lymph nodes during EAE. In contrast, meningeal lymphatics do not undergo lymphangiogenesis during EAE, suggesting heterogeneity in CNS lymphatics. We conclude that increased lymphangiogenesis near the cribriform plate can contribute to the management of neuroinflammation-induced fluid accumulation and immune surveillance.
Analysis of leukocyte populations and nerves in developing murine corneas
Authors: Abou-Slaybi Abdo , Jamali Arsia , Harris Deshea L , Seyed-Razavi Yashar , Hamrah Pedram
Published: January 2023

The avascular cornea is the most densely innervated tissue in the body and is endowed with resident bone marrow-derived cells. We have identified a novel subset of resident corneal plasmacytoid dendritic cells (pDCs). We have shown pDCs support corneal nerves through release of nerve growth factor (NGF).

This study was to examine the presence of pDCs in the developing cornea. We examined corneas from E17, P2, and 8 week adult C57BL/6 mice. Corneas were stained for the pan-leukocyte marker CD45, the pDC marker plasmacytoid dendritic cell antigen-1 (PDCA-1), the pan-neuronal marker bIII-tubulin. Corneal CD45+ and PDCA-1+ cells and nerves were quantified. Significance was assigned by ANOVA and post-hoc Tukey test. All data is reported as mean ± standard deviation.

Corneal CD45+ leukocyte density increased from E17 (65.4±14 cells/mm2) to P2 pups (301.9±19 cells/mm2, p<0.05). Both E17 and P2 corneas had lower CD45+ density when compared to adults (345±34 cells/mm2, p<0.05). We observed pDCs in the anterior stroma proximal to corneal nerves in E17, P2, and adult corneas. In E17, few pDCs were present in the peripheral cornea (1.5±2.6 cells/mm2), with none in the central cornea. In P2, pDCs were found in the periphery (68.19±8.99 cells/mm2), but not central corneas. The density of pDCs in adult peripheral (82.2±5.1 cells/mm2) and central (51.6±4.9 cells/mm2) corneas was greater than E17 (p<0.001) and P2 (p<0.05) corneas. Nerve density increased from E17 (45.43±6.75 mm/mm2) when compared to P2 (83.15±.21.6 mm/mm2 p<0.05) and to adult (151.6±3.25 mm/mm2 p<0.05) corneas.

Our data show pDC density increased from embryonic, to neonatal, and adult corneas. Concurrently corneal nerve density increased during development from embryonic to adult mice.
Systemic Lupus Erythematosus (SLE) patients’ Peripheral Blood Mononuclear Cells (PBMCs) display an altered transcript isoform repertoire
Authors: Singh Prashant , Veiga Diogo Troggian , Rossi Robert , Marches Radu , Belaid Djamel Nehar , Pascual Virginia , Banchereau Jacques
Published: January 2023

Alternative splicing (AS) is a key mechanism of biological diversity in eukaryotes allowing a single gene to generate multiple mRNA transcript isoforms, yielding unique proteins that may have distinct or opposing functions. Alterations in AS have been associated with numerous diseases. SLE patients display autoantibodies to components of the spliceosome, chiefly anti-Sm/RNP, anti-Ro/La, and anti-dsDNA. We performed Long-Read sequencing using Pacific Biosciences equipment (LR-Seq), as well as short read RNA-Seq, from adolescent SLE patients’ and healthy controls’ PBMCs, to discover and quantitate splice junctions. A computational pipeline was built to differentially quantify splice junctions in the LR-Seq sequenced isoforms, both known and novel using rMATS (Multivariate Analysis of Transcript Splicing). Compared to Gencode, LR-Seq revealed ~9000 novel transcript isoforms. 55% of all transcripts were expressed in 2 or more samples. A majority of these transcripts belonged to genes associated with cell cycle, metabolism, and inflammation. Additionally, we interrogated our SLE LR-Seq generated transcriptome with publicly available RNA-Seq data from adult SLE and healthy donors. We observed 250 differential expression of exon skipping (ES) splice junctions in transcript isoforms of genes, a majority of which did not exhibit any significant change in gene expression. Of the 250 ES transcript isoforms, 160 exon inclusion events were observed in SLE PBMCs. Fifty percent of the exon inclusion events were found to be in novel transcript isoforms. Thus LR-Seq, besides revealing novel transcripts, allows us to study changes in transcript isoform expression.
The impact of the TRPM2 ion channel on inflammation and macrophage metabolism in gastrointestinal models
Authors: Algood Holly Marie Scott , Dixon Beverly R.E.A. , Olivares-Villagomez Danyvid , Serezani Carlos Henrique , Washington M. Kay , Rathmell Jeffrey C. , Coburn Lori A.
Published: January 2023

Macrophages can play a vital role in regulating pro-inflammatory pathways that drive chronic inflammation and impact carcinogenesis. We previously published that TRPM2 regulates ROS production and the pro-inflammatory cytokine profile of macrophages in vivo and in vitro in H. pylori infection models. Since macrophage function and activation profiles can be regulated by metabolism, the impact of the TRPM2 channel on macrophage metabolism was assessed using bone marrow derived macrophages in Seahorse Extracellular Flux Assays and Mitotracker assays. The data demonstrated an unfavorable shift in mitochondrial content in Trpm2−/− macrophages after M1 activation. Consistent with this shift, the oxygen consumption rate was reduced in Trpm2−/− macrophages compared to WT macrophages, and the extracellular acidification rate at baseline and glycolytic reserve were higher in Trpm2−/− macrophages following classical activation. To investigate the role of TRPM2 in controlling other gastrointestinal pathologies, including acute colitis and carcinogenesis, the dextran sulfate sodium (DSS) colitis model and the azoxymethane (AOM)/DSS colitis-associated cancer (CAC) model were used. Trpm2−/− mice were not protected against acute DSS-induced colitis. Our endpoint analysis in the CAC model demonstrated that Trpm2−/− mice developed more numerous but smaller tumors than WT mice and immunohistological analysis suggests a shift in numbers of innate cells in the Trpm2−/− tumors compared to WT tumors. In the non-tumor areas, the histological injury score was reduced in the Trpm2−/− mice compared to WT mice. These data suggest that TRPM2 regulates Mϕ metabolism and impacts inflammation in the gastrointestinal tract.
Comparing docosahexaenoic acid as a prophylactic treatment for acute and chronic particle-exposed Balb/c mice
Authors: Fletcher Paige , Hamilton Ray F , Buford Mary , Postma Britten , Pestka James J , Holian Andrij
Published: January 2023

Rationale

There is a rising trend in the use of nanomaterials in various consumer products; increasing the risk of human exposures. Inflammation is a vital contributing factor to pulmonary diseases which can be triggered by exposure to airborne toxicants such as nanomaterials. However there are no effective treatments. One potential treatment shown to have anti-inflammatory effects is the dietary supplement docosahexaenoic acid (DHA); an omega-3 polyunsaturated fatty acid found in fish oil. This study compares DHA as a prophylactic treatment for acute and chronic particle-induced inflammation.

Method

Balb/c mice were fed a control or 1% DHA-containing diet for 5 weeks (acute) or 18 weeks (chronic). At week 4 (acute) or weeks 2, 3, 4, and 5 (chronic) the mice were anesthetized and oropharyngeal instilled with dispersion media (50 μl) or a highly inflammatory multi-walled carbon nanotube (50 μg). One week (acute) or 13 weeks (chronic) after instillation, macrophage NLRP3 inflammasome activation and lung lavage fluid (LLF) levels of lactate dehydrogenase (LDH) and protein levels were evaluated. Inflammatory cytokines were evaluated in LLF and plasma of the mice. DHA’s impact on macrophage phenotype was also examined.

Result for both exposures

Macrophage production of IL-1β and LLF levels of LDH, protein, and pro-inflammatory cytokines, such as IL-33 and TNF-α, decreased in mice fed the DHA diet. DHA appears to be altering macrophage phenotype within the lung and spleen tissues; specifically the M2 phenotype.

Conclusion

DHA could potentially be used as a prophylactic treatment for acute and chronic particle exposures since it was demonstrated to have a number of anti-inflammatory effects on Balb/c mice exposed to a common nanomaterial.
Sex-differences in the response to inhaled nanoparticles
Authors: Ray Jessica , Holian Andrij
Published: January 2023

Multi-walled carbon nanotubes (MWCNTs) are a versatile engineered nanomaterial of which the production and integration into consumer products is rapidly expanding. Unfortunately, the risk of occupational and environmental exposures continues to increase, yet long-term consequences and potentially sensitive populations remain undefined. Notably, the respiratory response to MWCNTs closely resembles that seen in asthma, including airway hyper-responsiveness (AHR), Th2-type cytokine production, and eosinophil recruitment to the airways. Based on the similarities in disease pathology and the evidence that asthma occurs most commonly in women of reproductive age, I hypothesized that female animals would be more sensitive to MWCNTs than males. Male and female C57BL/6 mice were exposed to a single dose of MWCNTs or exposed once per week for 4 weeks and harvested 8-weeks after the last exposure. Lung lavage fluid was analyzed for cytokine expression and immune cell recruitment; histology was performed on lung sections. Comparable to the results seen in asthma, female animals mounted a greater Th2-type immune response and subsequent lung pathology. At all time points airway eosinophil recruitment and IL-33 expression, key mediators in both allergic asthma and MWCNT-induced inflammation, were significantly elevated in females compared to males. Lung pathology in the repeated, long-term exposure study reflected the differences seen in the acute response and was more severe in female animals than in males. The next step in this project is to investigate the modification of alveolar macrophage phenotype through steroid hormone signaling as an explanation for the sex-differences observed. Work supported by R01 ES023209.
EGF-like repeats and discoidin I-like domain 3 (EDIL3) deficiency improves post-myocardial infarction healing via neutrophil extracellular traps (NETs) mediated pro-inflammatory macrophage polarization
Authors: Wei Xiaoqiong , Zhang Chengxin , Xie Zhonghui , Teng Xiu , Wang Zhen , Zheng Huaping , Zou Song , Huang Nongyu , Cui Kaijun , Li Jiong
Published: January 2023

After myocardial infarction (MI), injured cardiomyocytes recruit neutrophils, leading to extravasated monocytes polarized to inflammatory and reparative macrophages sequentially, which contribute to the healing and regenerative process. As an endogenous leukocyte-endothelial adhesion inhibitor, the role of EDIL3 in MI remains obscure. We found that serum EDIL3 level of MI patients was negatively associated with MI biomarkers, including CK-MB, cTnI and MGB. EDIL3−/− mice showed improved infarct healing with lower mortality, better cardiac function and decreased infarcted size compared with wild type (WT). Enhanced neutrophil infiltration resulted in the increase of Ly6Chi MHClo Mertk− macrophages, which we identified to be pro-inflammatory, protecting EDIL3−/− mice hearts from excessive fibrosis. A neutrophil infiltration antagonist not only eliminated the difference in cardiac function between WT and EDIL3−/− mice, but also reduced Ly6Chi MHClo Mertk− macrophage polarization. These data implied that improved cardiac healing was associated with increased infiltration of neutrophil and it mediated macrophage polarization. Notably, we confirmed that this polarization depended on NETs, which were more abundant in hearts and serum in EDIL3−/− mice than in WT. Indeed, self-DNA released from NETs primed the pro-inflammatory polarization of macrophages. The degradation of NETs by DNase I reduced this polarization and thus affected post-MI outcome, indicating the pivotal role of NETs in MI healing. Meanwhile, the pro-inflammatory macrophages induced apoptosis of neutrophils and restrained excessive inflammation. Our findings reveal a novel neutrophil–macrophage crosstalk essential to cardiac healing after MI.
Histone Deacetylase 3 regulates the ontogeny and maintenance of tissue-resident macrophages
Authors: Yao Yi , Liu Queping , Geissmann Frederic , Zhou Li , Mi Qing-Sheng
Published: January 2023

Tissue-resident macrophages (TRMs) are essential for tissue homeostasis, immune-surveillance, and resolution of inflammation, and contribute to the pathogenesis of cancer, autoimmune, and inflammatory diseases. Most adult TRMs are derived from embryonic yolk sac erythro-myeloid progenitors and self-maintain independently from adult hematopoiesis. A few genes have been recently identified to be required for TRM tissue-specific development. However, the master regulator that centrally controls TRM ontogeny remains a mystery. Using a fate-mapping strategy, here we report that conditional deletion of histone deacetylase 3 (HDAC3) in mice essentially abrogates epidermal Langerhans cells and TRMs in dermis, lung, kidney, and heart at embryonic stage and after birth. In contrast, HDAC3 deletion promotes liver TRM ontogeny, while without significant effects on brain TRM microglia. Using lung TRM alveolar macrophage (AM) as a model, we further indicate that HDAC3 controls metabolism and cell death, at least by directly targeting PPAR-γ during AM embryonic development. Moreover, single-cell RNA sequencing identifies four distinct AM sub-clusters and lack of HDAC3 dysregulates AM functions in a cluster-specific manner. Overall, this is the first study to demonstrate that HDAC3 serves as a key epigenetic regulator of embryonic TRM ontogeny and maintenance in a spatial- and tissue-specific manner. Not only do these findings provide a new conceptual framework for our understanding of epigenetic regulation in TRM ontogeny and homeostasis, they also shed light on HDAC3 as a potential therapeutic target for TRM-based intervention in cancer and autoimmune diseases.
IL-27 Receptor Signaling potentiates Angiotensin II induced myelopoiesis and promotes Abdominal Aortic Aneurysm
Authors: Aghayev Turan , Peshkova Iuliia , Fatkhullina Aliia , Makhov Petr , Eguchi Satoru , Tan Yin Fei , Kossenkov Andrew , Sykes Stephen , Koltsova Ekaterina
Published: January 2023

Abdominal Aortic Aneurysm (AAA) is a vascular disease, where aortic wall degradation is in part mediated by the accumulation of immune cells leading to aortic wall rupture and bleeding which is often fatal for the patient. Smoking, age, male gender, hypertension and atherosclerosis are major risk factors, however the mechanism of AAA development is still elusive. Though cytokines regulate the inflammatory milieu within the aortic wall, their possible role in systemic regulation of this disease, particularly in the control of hematopoietic stem cell proliferation, myeloid cell differentiation and their subsequent accumulation in AAA remains poorly defined.

Here we report an unexpected pathogenic role of IL-27R signaling in the development of AAA. We found that in an animal model of AAA, prolonged infusion of Angiotensin (Ang) II robustly induces AAA formation in hyperlipidemic Apoe−/− and Apoe−/−Il27ra+/− mice but not in IL-27R deficient Apoe−/− mice. This mitigation of AAA formation in Apoe−/−Il27ra−/− mice is associated with a blunted accumulation of myeloid cells in suprarenal aortas due to a reduction of Ang II-induced hematopoietic stem and progenitor cell (HSPCs) expansion. We found that the absence of IL-27R signaling engages transcriptional programs that promote HSCs self-renewal program and suppress myeloid lineage differentiation, resulting in decreased mature myeloid cell production and thus a concomitant lack of Ang II-induced myeloid cell accumulation in the suprarenal aortas.

Collectively, these data demonstrate that IL-27R signaling influences AAA development by potentiating Ang II induced myelopoiesis and may represent a molecular point of therapeutic intervention in AAA.
CCCTC-binding factor regulates the development and function of dendritic cells
Authors: Yang Bobae , Kim Tae-Gyun , Kim Sueun , Kim Hyoung-Pyo
Published: January 2023

Dendritic cells (DCs) are professional antigen presenting cells, which present antigen to cognate T cells. DC activation through diverse toll-like receptors is prerequisite for triggering efficient immune responses to foreign antigens. CCCTC-binding factor (CTCF) is a DNA binding protein that regulates 3D genome structure which is believed to be important to control of gene expression. Here we described that CTCF is required for development and function in DCs. CTCF is critically required for the FLT3L-dependent CD11c+ DCs (FL-DCs) development, unlike for those in the GM-CSF-dependent CD11c+ DC (GM-DCs) development. However, the resultant CTCF-deficient DC number was decreased by 50% in both FLT3L- and GM-CSF-supplemented culture conditions. Although CTCF was required for bone marrow (BM) progenitor proliferation in both FLT3L-and GM-CSF-supplemented cultures, CTCF-deficient GM-DCs showed normal cellular apoptosis rate and higher CD11c+ DC differentiation. Upon TLR4 ligation, CTCF-deficient DCs showed higher expression levels of surface MHCII and co-stimulatory molecules compared to those WT DCs. Thus, our data indicate that CTCF plays a critical role in the overall differentiation, survival, and maturation of DCs.
The DNA-binding protein ARID3a is Required for Human Erythrocyte Lineage Differentiation using the K562 Model Cell Line
Authors: Garton Joshua W , Chapman Brittany , Shankar Malini , Rose Kira , Webb Carol
Published: January 2023

Our previous studies demonstrated that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein associated with increased immunoglobulin heavy chain transcription and chromatin accessibility, is required for normal hematopoiesis in mice and in human cells. ARID3a knockout mice exhibited low numbers of erythrocytes and died due to failed erythropoiesis between days 9 and 12 of gestation. The role of ARID3a in human erythropoiesis has not been studied. Stimulation of the human K562 cell line with hemin induces fetal globin synthesis and erythrocyte differentiation. K562 cells constitutively express ARID3a, so we used this cell line to determine if ARID3a is important for human erythrocyte differentiation. Knockdown of ARID3a with shRNA in hemin-stimulated K562 cells resulted in >75% inhibition of globin. To identify genes affected by ARID3a loss, we performed RNA-seq experiments at various times after hemin stimulation with and without ARID3a. ARID3a shRNA-inhibited cells confirmed inhibition of globin gene products, including HBA1, HBA2, and HBZ. Erythroid specific transcription factors were also inhibited in samples treated with ARID3a shRNA while myeloid surface markers were induced. Several critical upstream regulators of ARID3a were also identified in these analyses. These data suggest that ARID3a is important for cell fate decisions in erythropoiesis in human cells and identifies potential important targets of ARID3a in this process.
An Nfil3-Zeb2-Id2 pathway imposes Irf8 enhancer switching during cDC1 development
Authors: Bagadia Prachi
Published: January 2023

Classical type 1 dendritic cells (cDC1) are required for anti-viral and anti-tumor immunity, motivating a need to understand their development. We recently discovered that the development of the cDC1 progenitor requires an E protein-dependent +41 kb Irf8 enhancer, but its maturation instead requires a BATF3-dependent +32 kb Irf8 enhancer. To identify the genetic circuitry underlying this switch, we performed single-cell RNA-sequencing of the multipotent common dendritic cell progenitor (CDP). We found a cluster of cells within the CDP that already expressed transcription factors influencing cDC1 development, including Nfil3, Id2, and Zeb2. Genetic epistasis between these factors revealed a hierarchy of action in which Nfil3 expression is required for a transition from Zeb2hi and Id2lo CDPs into Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E protein activity to exclude pDC potential within this progenitor and also explains the switch in Irf8 enhancer usage during cDC1 development.
Sargassum horneri ethanol extract down-regulates the Th2 polarization via STAT5 in ovalbumin sensitized murine splenocytes
Authors: Mihindukulasooriya Suyama Prasansali , Herath Kalahe Hewage Iresha Nadeeka Madushani , Kim Areum , Kim Hyo Jin , Ko Mi-Ok , Jeon You-Jin , Youngheun Jee
Published: January 2023

Sargassum horneri, a brown marine algae distributed across East Asian countries, is known to exhibit several health promoting effects such as anti-virus, anti-inflammation, and anti-oxidation. In this study, we aimed to clarify the anti-allergic effect of S. horneri ethanol extract (SHE). BALB/c mice were first sensitized with intraperitoneal injection of ovalbumin (OVA; 20 μg) and alum (Days 0 and 14; 2 mg). After the final administration, splenocytes were obtained and challenged with OVA (100 μg/mL) in the presence or absence of SHE. SHE was not cytotoxic on murine splenocytes at any of the concentrations tested. Furthermore, SHE exhibited suppressive effect on the proliferation of murine splenocytes. SHE treatment decreased the population of CD3+CD4+ helper T cells in OVA-sensitized and OVA-challenged splenocytes compared with the SHE non-treated controls. We also discovered that the increase in the population of CD45+Gr-1+ granulocytes due to OVA challenge was subdued significantly through SHE treatment. Importantly, SHE simultaneously attenuated Th1 and Th2 responses: it reduced the production of both Th1 (INF-γ, TNF-α) and Th2 (IL-4, IL-5, IL-13) cytokines induced through the OVA challenge. Also, SHE mitigated OVA induced activation of STAT5. The down-modulation of IL-13 and STAT5 activation was abrogated when a STAT5 inhibitor (CAS 285986-31-4) was applied. Our results suggest that SHE can mitigate OVA induced allergic immune responses through the attenuation of OVA induced TH2 polarization probably via STAT5 pathway and be therapeutically useful for alleviating symptoms in allergen mediated immune diseases. This research is a part of the project (No. 20150306) funded by Ministry of Oceans and Fisheries, Korea.
Bacillus subtilis-mediated suppression of house dust mite allergic airway inflammation
Authors: Swartzendruber Julie A , Negris Olivia , Incrocci Ryan , Knight Katherine L
Published: January 2023

The incidence of allergic diseases is increasing, with house dust mite allergy, afflicting up to 20% of the population in developed countries. House dust mites (HDM) are a common allergen that can be difficult to avoid and can trigger allergic reactions, including wheezing and shortness of breath. We recently showed that two pretreatments with Bacillus subtilis, a gram-positive bacterium, can prevent murine HDM-induced allergic lung inflammation. We assessed infiltration of eosinophils in bronchial alveolar lavage fluid and found significantly reduced eosinophils following B. subtilis treatment. Our current work addresses the capacity of B. subtilis to provide long-term protection from HDM challenge. Following pretreatment with B. subtilis, we found protection from HDM challenge, determined by quantifying infiltrating eosinophils for two weeks, but lost protection after three weeks. These data suggest a transient protection elicited by B. subtilis and support the need for frequent dosing to maintain suppression of allergic airway inflammation.
Clearance of Apoptotic cells by Lung Macrophages Prevents Development of House Dust Mite-induced Asthmatic Lung Inflammation
Authors: Miki Haruka , Gracias Donald Tom , Croft Michael
Published: January 2023

Allergen inhalation can cause epithelial damage and induce cell death while driving asthmatic lung inflammation. It has been suggested that patients with severe asthma may have a defect in clearance of apoptotic cells, of significance as apoptotic cell phagocytosis is thought important to maintain tissue homeostasis. Previously we have reported that lung resident macrophages in a non-inflamed lung generate regulatory T cells (Tregs) and can promote airway tolerance, and hypothesized that this ability was related to uptake of apoptotic cells. We investigated this by infusing apoptotic thymocytes into the lungs to determine their impact on macrophage function and allergic asthma. We found that lung macrophages were the primary cell to engulf apoptotic cells and this was not modified with intranasal exposure to House Dust Mite (HDM) allergen challenge. The uptake of apoptotic cells by alveolar macrophages in vivo or in vitro resulted in increased expression of RALDH1 and RALDH2, enzymes that drive retinoic acid production. When apoptotic cells were transferred into the lung during sensitization with HDM, eosinophilic airway inflammation, and Th2 cytokine production in the BALF and lung, were significantly reduced compared to mice given viable cells. This was accompanied by an increase in the frequency of antigen-specific Treg in the lung draining lymph nodes. These results indicate that apoptotic cell clearance by lung macrophages is important to maintain airway tolerance and prevent induction of allergic asthma.
Ly-6C+CD11b+conventional dendritic cells accumulate in inflamed lung and differentiate into type 2 dendritic cells
Authors: Izumi Gentaro , Nakano Keiko , Thomas Seddon Y , Whitehead Gregory , Grimm Sara A , Nakano Hideki , Cook Donald N
Published: January 2023

Allergic asthma is a complex inflammatory disease characterized by airway obstruction and airway hyperresponsiveness. Pulmonary dendritic cells are critical for allergic sensitization as they take up inhaled allergens and direct differentiation of allergen-specific helper T cell lineages in lung-draining lymph nodes. In this study, we used mass cytometry to study lung DC subsets that increase in frequency after allergen inhalation. This approach revealed a novel cell population displaying Ly-6C, in addition to surface markers routinely used to identify type 2 conventional DCs (cDC2), including CD11b and CD26. These Ly-6C+ cells had dendrites, lacked monocyte/macrophage markers such as F4/80 and CD115, and were absent in the mice lacking FMS-like tyrosine kinase 3ligand (FLT3L), a growth factor essential for cDC differentiation. Based on these observations, we conclude that this novel population represents bona fide cDCs. Their numbers in the lung were low at steady state, but increased dramatically after allergic sensitization. Cultured DC precursors (preDCs) displayed Ly-6C, but lost that marker by time. Ly-6C+CD11b+cDCs freshly isolated from mouse lung also lost Ly-6C expression after ex vivo culture. Together, these results suggest that Ly-6C+CD11b+cDCs are the precursor of cDC2. Given that the number of these DCs increase dramatically in the lung after allergen inhalation, they might play important roles in allergic sensitization and the induction of allergic asthma.
Critical role for IL-33 in orchestrating group 2 innate lymphoid cell and natural killer cell function in the lungs
Authors: Ferrini Maria M , Jaffar Zeina , Roberts Kevan
Published: January 2023

The alarmin, IL-33, acting via ST2 receptors initiates innate immunity to inhaled allergens and environmental particulates. IL-33 belongs to the IL-1 family and is largely derived from epithelial cells, endothelial cells and fibroblasts. The cytokine plays a critical role in underpinning type 2 immunity at mucosal sites and the IL-33/ST2 axis has been implicated in bronchial asthma and virus-induced exacerbations of allergic airway disease. In this study, we found that IL-33 instillations into the airways of C57BL/6 mice, for 3 days over a 7 day period, induced a marked increase in the number of group 2 innate lymphoid cells (ILC2s) and their production of type 2 cytokines IL-13 and IL-5 and this was associated with a pronounced pulmonary eosinophilic inflammation and airway mucus production. Interestingly, IL-33 administration also resulted in an increase in the number of CD11b+CD11C+MHCII+ dendritic cells and evoked a marked expansion of Th2 cells in the lung mucosa. Concomitant with this response, there was a significant reduction in IFN-γ production by pulmonary CD3−CD19−DX5+NK1.1+ NK cells. Flow cytometry revealed that ST2 expression was predominantly expressed by pulmonary ILC2s rather than NK cells, raising the possibility that ILC2s, either directly or indirectly, regulate the NK cell response. These findings reveal that IL-33 plays critical roles in both initiating pulmonary innate and adaptive immune responses, thus providing an essential axis for rapid immune responses and tissue homeostasis in the lung as well as influencing the development of chronic airway inflammation and remodeling.