bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric Oxide (NO) regulation of Dendritic cells (DC) metabolism and immune effector functions
Authors: Gullickson Soyeon , Snyder Julia , del Rio-Guerra Roxana , Sweezy Andrea , Vagher Bay L , Hogan Tyler , Lahue Karolyn , Reisz Julie , D’Alessandro Angelo , Krementsov Dimitry , Amiel Eyal
Published: August 2023

Dendritic cell (DC) activation is characterized by a sustained commitment to glycolysis, a requirement for survival in DC subsets expressing inducible nitric oxide synthase (iNOS, encoded by Nos2 gene). Nitric oxide (NO) can mediate both host-protective and host-cytotoxic effects by interfering with pathogen replication and/or by inhibiting the host mitochondrial respiration, resulting in metabolic rewiring in DCs. These phenomena primarily have been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6), where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced post-activation survival in LPS-stimulated PWD DCs due to attenuated NO production. To genetically map PWD’s NO phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2; 11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. The 11.2 DCs show comparable iNOS expression with lower NO production levels than B6 DCs. We demonstrate that activated 11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production and suggest a useful model to investigate the contribution/regulation of NO in DC metabolism and immune function.

This work was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (P30GM118228, 1R21AI135385-01A, T32AI055402-15, and R21 AI145306), the NIH, National Institute of Neurological Disorders and Stroke (R01 NS097596), and the National Multiple Sclerosis Society (RG-1901-33309).
Human respiratory syncytial virus activates beta-catenin pathway in lung epithelial cells to regulate pro-inflammatory response
Authors: Mohanty Indira , Pokharel Swechha , Maddison Lisette , Bose Santanu
Published: August 2023

Respiratory Syncytial Virus (RSV) is a non-segmented negative-sense RNA virus belonging to the paramyxovirus family. RSV infects respiratory tract to cause pneumonia and bronchiolitis in infants, elderly, and immunocompromised patients. Effective clinical therapeutic options and vaccine to combat RSV infection are still lacking. Therefore, to develop effective therapeutic interventions, it is imperative to understand virus-host interactions during RSV infection. Canonical Wnt/beta-catenin pathway is a well-established signaling cascade regulating expression of Wnt target genes involved in various biological and physiological functions. Our study shows RSV infection of human lung epithelial A549 cells triggering stabilization of beta-catenin protein and subsequent activation of beta-catenin mediated transcriptional activity. Further studies with beta-catenin inhibitors and beta-catenin lacking cells revealed a role of beta-catenin in positively regulating pro-inflammatory response in RSV infected A549 cells. Activation of beta-catenin during RSV infection was mediated by cell surface receptor complex comprising of frizzled receptors and low-density lipoprotein receptor-related protein 5 and 6 (LRP5 and LRP6). Thus, our studies have revealed an involvement of beta-catenin pathway in inducing pro-inflammatory response during RSV infection. The mechanism(s) and host derived factors regulating RSV-mediated beta-catenin activation will be discussed.
SOCS3 Regulates Dectin-2-Induced Inflammation in PBMCs of Diabetic Patients
Authors: Alrashed Fatema , Haider Mohammed , Albaqsumi Zahraa , Al-Mulla Fahd , Ahmad Rasheed
Published: August 2023

The C-type lectin receptors (CLRs) Dectin-1 and Dectin-2 are involved in several innate immune responses and are expressed mainly in dendritic cells, monocytes, and macrophages. This study aims to evaluate the expression and function of Dectin-2 in peripheral blood mononuclear cells (PBMCs) isolated from diabetic patients and non-diabetic controls. Our results show that Dectin-2 expression was the highest in monocytes compared with other leukocyte subpopulations. The expression of Dectin-2 was also found to be significantly higher in T2D patients monocytes compared with non-diabetic controls. The expression of Dectin-2 was found to be positively correlated with markers of glucose homeostasis, including HOMA-IR and HbA1c. Interestingly, SOCS3, a negative regulator of inflammation, was expressed significantly lower in the PBMCs of T2D patients. Moreover, SOCS3 expression was negatively correlated with Dectin-2 expression level. Further analysis of inflammatory signaling pathways showed a persistent activation of the Dectin-2-Syk-NFkB pathway that was instigated by the diminished expression of SOCS3. Dectin-2 activation failed to induce SOCS3 expression and suppress subsequent inflammatory responses in the PBMCs of diabetic patients. siRNA-mediated knockdown of SOCS3 in PBMCs displayed a similar inflammatory phenotype to diabetic PBMCs when exposed to Dectin-2 ligands. Altogether, our findings suggest that elevated Dectin-2 and its relationship with SOCS3 could be involved in the abnormal immune response observed in T2D patients.

This work was supported and funded by the Kuwait University Research Grant No. [SL01/21] and by the Kuwait Foundation for the Advancement of Sciences (KFAS) grant No. (RA MoH-2022-002).
Impact of Ezh2 on macrophage responses against lipopolysaccharide (LPS), experiments on conditional Ezh2 deletion mice using LysM-Cre system
Authors: Hirankarn Nattiya , Kunanopparat Areerat , Leelahavanichkul Asada , Visitchanakuna Peerapat , Kueanjindaa Patipak , Phuengmaung Pornpimol , Sae-khow Kritsanawan , Boonmee Atsadang , Benjaskulluecha Salisa , Palaga Tanapat
Published: August 2023

Because the macrophage responses to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis (severe infection), impacts of the enhancer of zeste homologue 2 (Ezh2; a histone lysine methyltransferase of epigenetic regulation) were explored. The transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2 silencing, using small interfering RNA (siRNA), in a macrophage cell line (RAW264.7) indicated the non-different LPS responses between Ezh2-reducing cells and the control, higher supernatant TNF-α in Ezh2-reducing cells were demonstrated after twice LPS stimulations (LPS tolerance). With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/−) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre−/−), perhaps due to an upregulation of Socs3 (suppressor of cytokine signaling 3; another inhibitor controlled by Ezh2 gene). In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control supporting loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than Ezh2 control mice after an LPS injection (less severe LPS-induced hyper-inflammation) with similar serum cytokines after LPS tolerance (no reduction of serum cytokines after the 2nd dose of LPS indicating the less severe LPS tolerance). In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation with the less severe LPS tolerance (higher cytokine production), partly, through an upregulated Socs3.

Supported by grants from NSRF via the Program Management Unit for Human Resources and Institutional Development, Research and Innovation (B16F640175 and B05F640144). A. K. was supported by the Second Century Fund (C2F) for Postdoctoral Fellowship, Chulalongkorn University.
Characterization of gene alterations during Macrophage Activation Syndrome associated with central nervous system disorders
Authors: Kulangara Varsha Kontham , Nagarkatti Prakash , Nagarkatti Mitzi
Published: August 2023

Macrophage activation syndrome (MAS) is the immense proinflammatory condition that occurs most frequently in patients with systemic juvenile idiopathic arthritis (SJIA) and systemic lupus erythematosus (SLE). An important feature of MAS is central nervous system dysfunction as evident in the form of seizures as well as altered mental status. In current study, MAS was induced in C57Bl/6 female mice by repeated injections of CpG. We performed transcriptome analysis of spleen from MAS and vehicle-treated control mice. Differential expression analysis revealed significantly altered expression of 3873 genes in mice harboring MAS. Pathway analysis indicated upregulation of heme biosynthesis while downregulation of pathways involved in 14-3-3 mediated signaling, oxytocin signaling and dopamine receptor signaling. Decreased expression of genes involved in these pathways was associated with incidents of depression, schizophrenia and Parkinsonism. The major downregulated genes were dopamine receptor D5 (DRD5), glutamate decarboxylase 1 (GAD1) and protein kinase C (PRKC) family of genes. Decreased activity of these genes are reported in depression, schizophrenia, epilepsy and Parkinson’s. At the same time, significant increase in the expression of glutamate ionotropic receptor NMDA type subunit associated protein 1 (GRINA), synuclein alpha (SNCA), glucocerebrosidase (GBA) and tubulin (TUB) genes were observed in MAS induced mice. In this study, we unravel the novel pathways as well as differential expression of genes associated with central nervous system dysfunction during episodes of MAS.

(Supported by NIH grants P01AT003961, P20GM103641, R01ES030144, R01AI129788, R01AI123947 and R01AI160896 to PN and MN).
Ruminococcus gnavus is a pathobiont expanded by genetic susceptibility and dietary tryptophan in a lupus-prone mouse model
Authors: Ma Longhuan , Brown Josephine , Choi Seung-Chul , Elshikha Ahmed , Kanda Nathalie , Terrell Morgan , Ge Yong , Mohamadzadeh Mansour , Morel Laurence
Published: August 2023

Gut microbial dysbiosis has been associated with the development of systemic lupus erythematosus (SLE). Dietary tryptophan modifies the composition of gut microbiota as well as disease activity in a lupus-prone mouse (TC). To directly test the modulatory function of tryptophan on the gut microbiota, we transplanted the gut microbiota from TC and B6 control mice into germ-free or antibiotic treated B6 recipients fed with high or low dietary tryptophan. Interestingly, a putative human lupus pathobiont Ruminococcus gnavus (Rg) was only enriched in the recipients of TC microbiota fed with high dietary tryptophan, which produced anti-dsDNA IgA and presented T cell infiltration in the kidney. A higher level of Rg was detected in untouched TC mice as compared to B6 controls, in correlation with the levels of lupus signature cytokines. Depletion of this bacterium from the gut of TC mice decreased autoantibody production and increased the frequency of regulatory T cells. Conversely, monocolonization of antibiotic-treated TC mice with Rg induced the production of autoantibody as well as increased the frequency of IFN-γ+ T cells, IL-10+ non-T regulatory cells and GC B cells as compared to age-matched TC mice. These results indicate that monocolonization with Rg is sufficient to induce the autoimmune activation in lupus mouse model and that this bacterium is expanded by the combination of lupus genetic susceptibility and tryptophan.

This project is supported by grant RO1 AI143313 from the NIH to LMo.
Discovery and characterization of novel inhibitors of CTP synthase 1 (CTPS1) for the treatment of autoimmune and inflammatory disease
Authors: McElwee Joshua J , Kaila Neelu , Carreiro Samantha , Kumar Sheetal , Toms Angela V , Phadke Avinash , Jacques Suzanne , Caravella Justin A , Kreutter Kevin , Campos Sebastien , Verras Andreas , Albanese Steven K , Demarco Kevin R , DiMattia Michael A , Lynch Eric , Kollman Justin , Tummino Peter
Published: August 2023

The de novo pyrimidine biosynthetic pathway is an inducible cellular program which allows the rapid synthesis of pyrimidine nucleotides in proliferating cells, and the final rate-limiting step is catalyzed by cytidine triphosphate synthase (CTPS1 or CTPS2). A CTPS1 loss-of-function mutation in humans has illustrated the essential and non-redundant role of CTPS1 in activated lymphocyte proliferation. Therefore, selective CTPS1 inhibition is an attractive approach to specifically target pathological immune activity in autoimmune and inflammatory disease.

Using cryo-EM and structure-based drug design, we have identified a series of potent, orally bioavailable, CTPS1-specific inhibitors, exemplified by the compound NTX-871. In-vitro, NTX-871 significantly inhibits the proliferation of T-cell derived lines and activated primary T-cells. These inhibitory effects are reversed by the addition of exogenous cytidine, illustrating the specificity of NTX-871 for CTPS1 and the de novo pyrimidine synthesis program.

NTX-871 was also tested in several mouse in-vivo PD and autoimmune disease models. Oral dosing of NTX-871 significantly reduced T-cell proliferation in an ovalbumin-induced T-cell proliferation model, and QD or BID dosing of NTX-871 significantly improved disease phenotypes in models of delayed-type hypersensitivity, collagen-induced arthritis, and experimental autoimmune encephalomyelitis.
Novel NFkB Variant V213E: Phenotypic Description & Molecular Modeling
Authors: Chaudhri Eman N , Weber Caleb A , Coban Mathew A , Savytskyi Oleksandr V , Squire Jacqueline D , Wierenga Klaas J , Caulfield Thomas R
Published: August 2023

BACKGROUND

Mutations of NFkB1 are a known cause of inborn errors of immunity resulting in immunodeficiency due to the role of NFkB1 as a transcriptional regulator of immunomodulating proteins. We present a patient with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Our proband, a 22-year-old male, presents with a clinical picture of immunodeficiency, and has a maternal cousin with a similar gene mutation but differing phenotype. Next generation panel sequencing (NGPS) was utilized to discover a novel, single point mutation variant in NFkB1, V213E, that is present in our proband.

METHODS

The novel single-point mutation V213E in the NFkB gene was modeled in-silico using the I-TASSER homology modeling software. Molecular dynamics simulations of both the wild type and variant mutant form fitted to accommodate the novel mutation were conducted and analyzed to assess for possible pathogenicity of the novel mutation.

RESULTS

This variant replaces Valine with Glutamic Acid at position 213 in the transcription regulator’s sequence. We perform molecular modeling to analyze the proteomic impact and difference in molecular dynamics (MDS) which depicted suppressed dynamics in the ankyrin region and death domain of the protein complex. Analysis of the proband’s family showed no significant family history except for the maternal cousin with a mutation in NFkB1.

CONCLUSION

This case both highlights the heterogeneity in phenotype of NFkB1 pathogenic variants as shown in this patient, full penetrance of the variant mutation, and suggests the pathogenicity of a new variant through protein modeling techniques and molecular dynamic simulations.
Tumor Progression Locus 2 Promotes Mouse Plasmacytoid Dendritic Cell Cytokine Secretion and Signaling
Authors: Watford Wendy Tharpe , Shirke Neeti , Choudhury Farhatul
Published: August 2023

The autoimmune disorder systemic lupus erythematosus is thought to be caused by the inefficient clearance of dying cells. This releases nucleic acid debris that stimulates type I interferon (T1 IFN) secretion. Plasmacytoid dendritic cells (pDCs) are the major source of T1 IFNs, which are causally linked to lupus pathogenesis. Both T1 IFNs and pDCs are thus the focus of intense studies for immunotherapies. The serine-threonine kinase, tumor progression locus 2 (TPL2), negatively regulates T1 IFN secretion by conventional dendritic cells (cDCs) and macrophages in response to Toll-like receptor 9 (TLR9) stimulation in vitro. In contrast, we and others have shown that TPL2 is required for T1 IFN production in TLR9-stimulated pDCs. The goal of this study is to validate TPL2-dependent regulation of pDC function in vivo and to delineate the mechanisms responsible. Both cDCs and pDCs develop normally in Tpl2−/− mice, but Tpl2−/− mice show defective secretion of T1 IFNs and proinflammatory cytokines in response to administration of CpG (TLR9) or R848 (TLR7) in vivo. The same defect in T1 IFN and proinflammatory cytokine production was observed in bone marrow-derived pDCs and cDCs in vitro. A modest defect in TLR-induced expression of maturation markers CD80, CD86 and MHC class II was seen in Tpl2−/− pDCs, suggesting impaired NFkB activation. Flow cytometric measurement of phosphoproteins demonstrated that TPL2 is also required for TLR-induced activation of MAPK and ribosomal S6 kinase pathways in pDCs. Ongoing studies are examining the role of TPL2 in TLR trafficking and lupus pathogenesis. These data establish TPL2-dependent regulation of pDC functions, which may be exploited for therapeutic intervention in lupus or other IFN-related diseases.

Supported by grants from NIH/NIAMS (R21 AR075317) titled “Tpl2 regulation of pDC function and SLE pathogenesis”
Fusion partners matter: Factor VIII light chain binding enables CD16-mediated recombinant Factor VIII-Fc fusion protein natural killer cell activation
Authors: Ou Jiayi , Lagasse H A Daniel , Sauna Zuben , Golding Basil
Published: August 2023

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with multiple Fc gamma receptors (FcγR) resulting in the activation or inhibition of a wide variety of FcγR expressing immune cells. We demonstrated that rFVIIIFc activates natural killer (NK) cells via Fc mediated interactions with FcγRIIIA or CD16. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, we assessed cellular activation by measuring inflammatory cytokine secretion (IFNγ by ELISA) or cellular degranulation (flow cytometry-based surface LAMP1/CD107a). In some cases, we introduced specific blocking molecules (anti-FVIII, anti-Fc, or anti-CD16) into our assays to act as inhibitors of NK cell activation. We identified the FVIII light chain as playing a key role in the activation of CD16+NK cells by rFVIIIFc. Using FVIII domain-specific inhibitors, we demonstrate that blocking C1 or C2 domain-mediated membrane binding potently inhibits rFVIIIFc-mediated CD16+NK cell activation, while targeting FVIII heavy chain domains does not. Our results suggest FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface and allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. This working model may help explain our previous results where we observed recombinant Factor IX-Fc fusion protein and rFVIIIFc exhibited different CD16-signaling properties despite having identical IgG1 Fc domains.
Psychological stress triggers the accumulation of myeloid-derived suppressor cells in the liver through adrenergic receptor signaling
Authors: Ninkov Marina , Rudak Patrick Thomas , Wang Nicole Ilayda , Rashu Rasheduzzaman , Inoue Wataru , Haeryfar S M Mansour
Published: August 2023

Chronic physiological stress affects tightly regulated crosstalk between the nervous and the immune system. However, the exact mechanism(s) of stress-induced immunomodulation is far from clear. Immature myeloid cells such as myeloid-derived suppressor cells (MDSCs) impair immune responses, which may in turn increase susceptibility to infections and cancer or their complications. In a mouse model of chronic psychological stress through physical restraint, we found mononuclear-MDSC (Mo-MDSC) cells, defined as CD11b+Ly6ChighLy6Ghigh cells, to accumulate in the liver. These cells expressed high levels of Mo-MDSC markers such as CD14, TLR4, IL-4Rα as well as markers indicative of anti-apoptotic and immunosuppressive functions (HIF-1α, G-CSFR, Bcl2, Arg2, iNOS). In addition, FACS sorted MDSCs suppressed production of IFN-ɣ by T cells in vitro. Increased levels of proinflammatory mediators were detectable in serum samples of stressed mice, with circulating levels of IL-6 and G-CSF being the most pronounced mediators. The observed intrahepatic accumulation of Mo-MDSC-like cells was mediated by IL-6 and G-CSF and could be reversed by treatment with corresponding receptor-blocking monoclonal antibodies. In addition, this phenomenon was driven by norepinephrine (NE), the main neurotransmitter released from sympathetic neurons, and could be recapitulated by in vivo administration of NE to non-stressed mice. Our work reveals a novel mechanism of immunosuppression that is governed by adrenergic and inflammatory cues and alters the cellular landscape of the liver with implications for immune surveillance against microbes and cancer.

This work was funded by NSERC
Restraint of IFN-γ expression through a distal silencer CNS–28 for tissue homeostasis
Authors: Cui Kairong , Chen Zuojia , Cao Yaqiang , Liu Shuai , Hu Gangqing , Fang Difeng , Wei Danping , Liu Chengyu , Zhu Jinfang (Jeff) , Wu Chuan , Zhao Keji
Published: August 2023

Interferon-g (IFN-g) is a key cytokine in response to viral or intracellular bacterial infection in mammals. While a number of enhancers are described to promote IFN-g responses, no silencers for the Ifng gene have been identified. By examining H3K4me1 histone modification in naïve CD4+ T cells within Ifng locus, we identified an unrecognized silencer (CNS–28) that is responsible for restraining Ifng expression. Mechanistic study further demonstrates that CNS–28 maintains Ifng silence by diminishing enhancer-promoter interactions within Ifng locus in a T-bet independent manner. Functionally, CNS–28 restrains Ifng transcription in Th1, Tc1, and NK cells during both innate and adaptive immune responses. Moreover, CNS–28 deficiency resulted in repressed type 2 responses due to elevated IFN-g expression, shifting Th1 and Th2 paradigm. Thus, CNS–28 activity ensures immune cell quiescence by cooperating with other regulatory cis elements within the Ifng gene locus to minimize autoimmunity.

This research was supported by the Intramural Research Programs of National Heart, Lung and Blood Institute, National Cancer Institute, and National Institute of Allergy and Infectious Diseases of National Institutes of Health.
Modeling the regulation of the Il12b locus by NF-κB dimers
Authors: Oh Yeuran , Md Toufiqur Rahman Shah , Aqdas Mohammad , Sung Myong-Hee
Published: August 2023

The transcription factor NF-κB is composed of a family, which regulates the expression of various genes in immune and inflammatory responses. The NF-κB family consists of five members: RelA/p65, RelB, c-Rel, p50/p105, and p52/p100, which control the transcription of target genes by binding to regulatory κB sites in homo-or heterodimer forms. Unlike the widely known RelA, the mechanisms related to the regulation of target genes by c-Rel is less understood. Interleukin 12b(Il12b), which encodes the p40 subunit of IL12 and IL23, is one example of gene that depend on c-Rel for proper activation in macrophages. The differences in intrinsic binding properties of c-Rel- and RelA-containing dimers at various regulatory sites around Il12b can be a key to understand the mechanism of c-Rel-containing dimers’ role in Il12b regulation. Using c-Rel and Il12b reporter mice with fluorescence live-cell imaging, we observe the single-cell dynamics of nuclear c-Rel and Il12b gene expression in bone marrow-derived macrophages induced by NF-κB activating ligands. We analyze the live-cell imaging data and extract quantitative features of nuclear c-Rel and Il12b expression from the time-series data. For additional mechanistic insights of the Il12b gene regulation by NF-κB dimers, we propose a mathematical model of dynamic gene regulation which includes actions of RelA- and c-Rel-containing dimers at κB sites proximal and distal to the Il12b locus. Our combined experimental and mathematical modeling approach will reveal quantitative insights that may provide a more comprehensive understanding of regulatory mechanisms underlying Il12b gene expression employed by NF-κB dimers.
Analysis of STAT Protein Phosphorylation in T Cells, NK Cells, and B Cells Using High Sensitivity Flow Cytometry
Authors: Pugsley Haley Renee , Sutton Alexandra G
Published: August 2023

In the immune system, cytokine-induced protein phosphorylation is a key cell signaling event that modulates diverse functions, including proliferation, differentiation, apoptosis, and migration. The degree of protein phosphorylation is determined by both the cytokine concentration and the number of cytokine-specific receptors on the cell surface, which are differentially expressed across immune cell populations. Signal transducer and activator of transcription (STAT) proteins are transcription factors that translocate to the nucleus upon activation by tyrosine phosphorylation and alter gene expression. Cytokines such as interleukins (IL) initiate the signaling cascade that activates STAT proteins. In addition, phosphorylated STAT3 and STAT6 have been associated with prognosis in cancer and other malignancies.

Flow cytometry is a powerful method to study the orchestrated response of immune cell subsets to stimulation by combining immunophenotyping markers with fluorescent antibodies specific to phosphorylated signaling molecules. However, because many phosphorylation events occur on weakly expressed proteins and with low stoichiometry, flow cytometers must be highly sensitive to track the subtle changes in signaling magnitude. Flow cytometers with sensitive detection, such as the Amnis® systems, can be used to study phosphorylation events that are key to understanding the processes required for vaccine and immunotherapy development. In this study, we used the Amnis® CellStream® Flow Cytometer to monitor the phosphorylation of STAT3 and STAT6 in CD4+ T cells, NK cells, and B cells after stimulation with IL-4 and IL-6.
Mesenchymal Stem Cells (MSCs) Treatment Alleviates Smoke Inhalation and Burn-Induced Acute Respiratory Distress Syndrome (ARDS) by Inhibiting HMGB1-TLR4 Signal
Authors: Yang Zhangsheng , Thorpe Catherine R , Cancio Tomas S , Willis Robert P , Snook John K , Necsoiu Corina , Meyer Andrew D , Cap Andrew P , Cancio Leopoldo C , Batchinsky Andriy I , Salinas Jose , Young Matthew D
Published: August 2023

ARDS is one of the leading causes of high morbidity and mortality in trauma patients. Previously our data demonstrated autologous MSCs treatment improved survival in a swine model of smoke inhalation and burn injury. However, the pathophysiological mechanisms are still largely unknown.

Twenty anesthetized female swine underwent smoke inhalation injury and 40% TBSA burns, were then randomly assigned to either mock treatment (IC, n=10), or autologous MSCs treatment (MSCs, n=10), followed by ICU care up to 72 hours except in case of early death. Three doses of MSCs collected by bone marrow aspiration and concentrated using a bedside cell concentrator device were applied at 2, 24, and 48 hours of post-injury (PI). Blood and tissue samples were collected for ELISA and IHC analyses.

In IC group, 10/10 injured pigs developed ARDS, but only 6/10 pigs in MSCs group developed ARDS. Serum analysis revealed that the HMGB1 level gradually increased after injury and reached a peak at 48h PI (5.7-fold increase vs. baseline). The SDC-1 and C3a levels also increased after injury but reached a peak at 24h (2.9-fold) and 72h PI (2.2-fold), respectively. The MSCs treatment significantly reduced the HMGB1 level in the serum, especially at 6h and 12h PI compared to the IC group but failed to inhibit the SDC-1 and C3a increases. IHC analyses showed that injury triggered a significantly higher expressions of HMGB1 and TLR4, as well as co-localization of HMGB1 and TLR4 in the lung; while MSCs treatment was able to disrupt the HMGB1-TLR4 interaction and significantly reduce their expressions.

Our data indicate that the way MSCs treatment mitigates ARDS might be by reducing HMGB1 release and inhibiting the HMGB1-TLR4 signal pathway activation in pigs after smoke and burn injury.

This work was supported by the US Army Medical Research and Development Command (USAMRDC) under Grant No. W81XWH-13-2-0005.
The nuclear receptor Nurr1 regulates the expression and activity of PPARγ in human pro-inflammatory macrophages
Authors: Solís Miguel Ángel , Santana Eduardo , Segovia Norma Cristina , Sánchez-Torres Carmen
Published: August 2023

Nurr1 is a member of the orphan nuclear receptor family NR4A that modulates inflammation in several cell lineages through inhibition of the NF-κB signaling pathway. Nurr1 was found to be able to interact with the peroxisome proliferator-activated receptor (PPAR)γ, as well as to be recruited to the PPARG promoter in LPS-activated microglial cells; however, the functional relationship between Nurr1 and PPARγ is not clearly established. Here, we aimed to investigate the role of Nurr1 on PPARγ activity in human macrophages. Blood monocytes were cultured with GM-CSF or M-CSF to generate pro- (GM-MDMs) and anti-inflammatory (M-MDMs) macrophages, respectively. GM-MDMs showed enhanced expression of both Nurr1 and PPARγ compared to M-MDMs, and they were preferentially located at the cytoplasmic (Nurr1) and nuclear (PPARγ) compartments. PPARγ activation in GM-MDMs with the agonist rosiglitazone did not modify Nurr1 expression. Conversely, Nurr1 activation with the agonist C-DIM12 increased PPARγ expression. This was concomitant with enhanced expression of two PPARγ target genes, CD36 and PLIN2, while silencing Nurr1 expression led to a decrease in the amounts of CD36 transcripts. Co-treatment of GM-MDMs with C-DIM12 and rosiglitazone synergistically enhanced the transcriptional activity of PPARγ, whereas C-DIM12 alone had no effect. PPARγ and Nurr1 agonists alone exerted anti-inflammatory effects on GM-MDMs, and also showed synergistic activity in combined form. In sum, our data suggests that Nurr1 positively regulates PPARγ activity at least partially through induction of PPARγ expression, which may represent a novel mechanism of Nurr1 targeting metabolic pathways and inflammation.

Supported by grants from the Secretaría de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)
Age-Related Changes in B1a Derived IgM Repertoire Affect their Atheroprotective function
Authors: Bontha Sai Vineela , Srikakulapu Prasad , Deroissart Justine , Binder Christoph J , Rothstein Thomas , Holodick Nichol , McNamara Coleen
Published: August 2023

Perivascular adipose tissue (PVAT) harbors immune cells and locally regulates atherosclerosis. The B1a cells present in PVAT secrete IgM in young mice. This IgM has been shown to confer atheroprotection by binding to ox-LDL and other oxidation specific epitopes (OSEs). The impact of aging on B1a-B cells in PVAT, the PVAT B-1a cell IgM repertoire and how aged B-1a cells affect atherosclerosis has not been explored. We sequenced the V-D-J gene segments of IgM in aged B-1a cells from hyperlipemic mice with atherosclerosis. Results demonstrated greater N-additions as well as changes in the usage of the VDJ gene regions in PVAT B1a cells when compared to the B1a cells residing in peritoneal cavity: the primary niche of B1a cells where they self-renew. Further, our previous results have shown similar changes in IgM repertoire in secondary antibody producing sites like spleen and bone marrow. ELISA demonstrated that these changes in IgM repertoire during aging reflect in the systemic IgM pool. The ratio of atheroprotective IgM (IgM to OSEs) to total IgM was significantly decreased with age when 10-, 50- and 100-week-old ApoE−/− mice plasma IgM levels were compared. Further, when B1a cells from young and aged CD45.2 Apoe−/− mice were adoptively transferred to PCSK9 injected CD45.1 STEM mice, those that received B1a from young mice had lower atherosclerosis compared to those that received B1a cells from aged mice. With these results we conclude that with age B1a derived IgM repertoire substantially changes to be less atheroprotective.

“Supported by grants from NIH (RO1 HL107490 and RO1 HL136098 (CM); Ro1AI154539 (NH)); and AHA (award number 18CDA34110392 (PS)).”
The prognostic implications of macrophage polarization in advanced and therapy-resistant prostate cancer
Authors: Martinez Shannalee R , Muriel Nicole Torres , Barrientos Risso Carla M , Wall Nathan R , Dutil Julie , Diaz Osterman Carlos J
Published: August 2023

Tumor-associated macrophages (TAMs) are components of the spatial microenvironment of many solid neoplasms. In prostate cancer (PCa), TAMs are linked to a poor prognosis. PCa is characterized by enhanced tumor lipid production and TAM lipid accumulation. However, the interplay between lipid-producing tumor cells and TAM lipid uptake has yet to be determined. Human prostate tumor tissue microarrays were evaluated for TAM (CD68+) and M2/M1 (CD206+/CD86+) levels correlated with tumor aggressiveness by multiplex immunohistochemistry. Standard PCa cells 22rv1, PC3 and DU145 were used to develop chemoresistant sublines which were validated using dose-response viability assays and immunoblotting for resistance-associated markers and lipid-producing enzymes. Cancer cell-derived extracellular vesicles (EVs) were isolated from cell culture supernatants by size exclusion chromatography and were incubated with U937-derived macrophages, followed by flow cytometric evaluation of M1 and M2 markers. Proteomic evaluation of human prostate tumors indicates that CD68+ TAMs correlate with aggressive, metastatic PCa. Furthermore, a higher M2-to-M1 ratio also correlates with metastatic disease compared to normal or localized tumors. Chemoresistant PCa cells exhibited an enhanced lipogenic phenotype compared to naive cells and the over-production of lipids was linked to EV-mediated reprogramming of macrophages toward an M2-like phenotype. These findings indicate that alternative activation of macrophages is linked to aggressive, therapy resistant PCa. Moreover, our data suggest that tumor lipid metabolism may influence tumor-associated macrophage phenotype and function through EV-mediated intercellular signaling.

Puerto Rico Science Research and Technology Trust
Essential role of IL-17A in activating Treg cells in autoimmune uveitis
Authors: Chong Wai Po , Zhong Yajie , Zhang Xuan , Deng Yujie , Mak Kingston King-Lun
Published: August 2023

The balance between Th17 cells and regulatory T (Treg) cells is critical for host homeostasis. The signature cytokine of Th17 cells, IL-17A, is a pro-inflammatory cytokine associated with various autoimmune diseases in patients and in animal models. Our previous study demonstrated that IL-17A limits pathogenicity of Th17 cells by inducing IL-24. In this study, we investigate the role of IL-17A in regulating Treg cells. In-vitro studies showed that IL-17A promotes both the differentiation and suppressive functions of Treg cells. Adoptive transfer of IL-17A-treated Treg cells is more potent to ameliorate experimental autoimmune uveitis (EAU) in mice when compared to the control Treg cells. We found that the induction of Treg cells is impaired in Il17ra−/− mice after EAU immunization and Il17ra−/− naïve T cells are less effective in their differentiation into Treg cells in EAU compared to wild-type cells. Mechanistic studies revealed that IL-17A activates NF-kB signaling and inhibits TAZ, a Foxp3 suppressor, during Treg cell differentiation. In conclusion, our data suggest that IL-17A is involved in Tregs induction and/or maintenance, and that this has a role in controlling the autoimmune response.

Natural Science Foundation of Guangdong Province (2019A1515010956, 2021A1515010569, 2022A1515012450) Fundamental Research Fund of the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center.
Spatial phenotyping of cytokine signatures reflecting the immunotherapy responses in head and neck cancer
Authors: Ji HaYeun , Jhaveri Niyati , Pratapa Aditya , Ma Ning , Ben Cheikh Bassem , Monkman James , O’Byrne Ken , Hughes Brett , Kulasinghe Arutha , Braubach Oliver
Published: August 2023

Immune checkpoint inhibitor (ICI) therapy has drastically improved the treatment strategies for mucosal head and neck squamous cell cancer (HNSCC). To increase treatment response rates for highly targeted ICI therapies, predictive and prognostic biomarkers are being actively explored. Specifically, the cytokine expression patterns in the tumor microenvironment (TME) are now recognized as key to understanding immune responsive and resistant phenotypes. Cytokines play an essential role in the regulation of the TME, specifically in modulating the proliferation and differentiation of immune cells. Here, we have studied spatial signatures of various cytokines within the TME of metastatic/recurrent HNSCC tumors treated with Pembrolizumab/Nivolumab. We utilized the PhenoCycler-Fusion to perform whole-slide, single cell resolution spatial phenotyping of the TME of HNSCC tumors from a cohort of n=40 patients. The discovery cohort consisted of patients who had complete vs. partial vs. stable vs. progressive responses to ICI therapy. Transcriptomic profiling of more than 60 RNA targets for various subfamily of chemokines, interleukins, and immune cell lineages was achieved using Akoya’s novel high plex RNA detection technology. Our study identified distinct spatial signatures that implicate certain cytokines in either tumor progression or regression. Specifically, we have identified areas of high and low CXCL9 and CXCL10 expression in several tumor regions that reflect immune-cell landscapes associated with resistance and sensitivity to immunotherapy. Our study demonstrates the power of unbiased spatial phenotyping with whole-slide imaging to identify biomarkers associated with response to ICI therapy in HNSCC.
CD244 loss on monocytes promotes differentiation into M1 macrophages and potentiates PD-L1 blockade in melanoma
Authors: Kim Jeongsoo , Kim Tae-Jin , Chae Sehyun , Ha Hyojeong , Park Yejin , Lee Hyemin , Hwang Youngsoo , Hwang Daehee , Lee Kyung-Mi
Published: August 2023

Accumulating data have highlighted the role of monocytes/macrophages in immune escape by generating immunologically “cold” tumors that do not respond to immunotherapy. CD244 (SLAMF4, 2B4), a member of the signaling lymphocyte activation molecule family, is expressed on myeloid cells, but its precise role has not been elucidated. Using monocyte lineage-specific CD244-deficient mice challenged with B16F10 melanoma, we report for the first time that CD244 negatively regulates tumor immunity by inhibiting the differentiation and functional maturation of monocytes into macrophages within the tumor microenvironment. CD244-deficient macrophages more effectively activated antigen-specific T cell responses compared to WT macrophages, thus delaying tumor growth in the B16F10 melanoma model. Moreover, combinatorial intervention of anti-PD-L1 antibodies with CD244-KO BMDM markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with WT BMDM. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset, revealed DEGs of CD244− monocytes and macrophages were associated with phagocytosis, antigen presentation, and autophagy. Additionally, cell type deconvolution analysis within melanoma patients bulk RNA-seq datasets from TCGA database, revealed presence of CD244-monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Hence, we proposed that CD244 serve as a critical immunoregulatory receptor on macrophages, and CD244-deficient macrophages may represent a novel therapeutic modality, which can function synergistically with checkpoint blockade therapies.

Supported by grants from National Research Foundation of Korea (NRF-2020R1A2C2103061, NRF-2018M3A9D3079288, NRF-2016M3A9B6948342)
ADAM9 drives immunosuppressive environment for lung tumor progression
Authors: Sher Yuh-Pyng , Liu Jing-Pei , Liu Shih-Jen
Published: August 2023

Cancer cells develop the immune suppressive tumor environment, but the underlying molecular mechanism for it is still elusive. Here, we demonstrated that ADAM9 contributed to lung tumor progression and promoted IFN-related gene expression in lung cancer cells that participated in immune regulation in the tumor environment. ADAM9 knockout (KO) lung cancer cells reduced lung cancer metastasis with a decrease in neutrophil infiltration and an increase in CD8+ T cell infiltration. By performing the genome-wide approach to explore ADAM9-regulated genes in lung cancer cells, we revealed that ADAM9 regulated cytokine production and lymphocyte activation. It enhanced the master transcription factor of IFN signaling, STAT1 activation in lung cancer cells. Moreover, ADAM9 promoted IL6/IL6 receptor-STAT3 axis signaling that lead to suppression of IL12p40 secretion and MHC class II expression in dendritic cells and macrophages. Notably, we found that type I IFN related-proteins and steroid biosynthesis-related proteins were related to ADAM9-mediated STAT signaling activation by LC-MS/MS analysis. We demonstrated that ADAM9 created an immune suppressive environment by regulating IFN signaling for tumor progression.

Supported by grants from NHRI (NHRI-EX112-11219BI)
CAR Tregs protect alloimmune islets against autoimmune destruction
Authors: Wardell Christine M , Fung Vivian , Sanchez Isaac Rosado , Mojibian Majid , Levings Megan K
Published: August 2023

Regulatory T cell (Treg) therapy is a promising approach to induce tolerance of transplanted tissues and/or self antigens that drive autoimmunity. Early clinical trials with polyclonal Tregs have proven feasible, safe and tolerable. The next wave of Treg therapies is genetically enhanced with antigen specificity. We have previously shown that Tregs engineered to express a chimeric antigen receptor specific for HLA-A2 (A2-CAR) delay rejection of HLA-A2+ grafts; however, it was unknown whether they would limit T cell responses targeting antigens other than HLA-A2 via bystander suppression. To investigate this possibility, we used a model of HLA-A2-mismatched islet transplantation and asked if A2-CAR Tregs could inhibit rejection mediated by diabetogenic T cells. HLA-A2+ islets (150–200) isolated from NOD-HLA-A2+ mice were transplanted into immunodeficient NSG mice. After 3.5 weeks, diabetogenic BDC2.5 CD4+ T cells were adoptively transferred with or without polyclonal or A2-CAR Tregs (1 CD4+:5 Tregs). Mice that received BDC2.5 CD4+ T cells without Tregs or polyclonal Tregs rapidly developed hyperglycemia. In contrast, mice that received A2-CAR Tregs were completely protected from hyperglycemia. Longitudinal monitoring of injected Tregs showed that A2-CAR Tregs were more numerous and activated relative to polyclonal Tregs. Removal of the HLA-A2+ islet graft from a protected A2-CAR Treg-treated mouse did not induce hyperglycemia, suggesting that the ability of BDC2.5 CD4+ T cells to mediate autoimmune attack of the endogenous (HLA-A2-) pancreas was suppressed. These data suggest that A2-CAR Tregs mediate bystander suppression in vivo and may have the ability to induce long-term tolerance to both allo- and autoantigens.

Supported by a CIHR Canada Graduate Scholarship doctoral research award and a CIHR foundation grant (FDN-154304).
Inflammasome activation occurs in CD4+ and CD8+ T cells during graft-versus-host disease
Authors: Talley Sarah , Campbell Edward
Published: August 2023

A severe complication of hematopoietic stem cell transplantation is graft-versus-host disease (GvHD), a reaction that occurs following the transfer of donor immune cells (the graft) into an allogeneic host. Transplanted cells recognize host alloantigens as foreign, resulting in the activation of donor T cells and migration of these pathological cells into host tissues. In this study, we found that caspase-1 is active in alloreactive murine and human CD4+ and CD8+ T cells early during acute GvHD (aGvHD) development. The presence of inflammasome-bound, active caspase-1 (p33) and ASC speck formation confirmed inflammasome activation in these cells. Isolated T cells with high levels of active caspase-1 had a strong inflammatory transcriptional signature and a metabolic phenotype similar to inflammatory myeloid cells, including the upregulation of proinflammatory cytokines and metabolic switch from oxidative phosphorylation to aerobic glycolysis. We also observed oxidative stress and mitochondrial dysfunction, in alloreactive T cells. For the first time, this study characterizes caspase-1 activation in transplanted T cells during aGvHD, using mouse and human models, adding to a body of literature supporting inflammasome function in cells of the adaptive immune system.

NIH
CD45.1/CD45.2 congenic markers induce a selective bias for CD8+ T cells during adoptive lymphocyte proliferation in lymphocytopenia mice
Authors: Kandy Rakhee Rathnam Kalari , Fan Xiaoxuan , Cao Xuefang
Published: August 2023

CD45.1/CD45.2 congenic markers have been frequently used to track hematopoietic lineage differentiation following hematopoietic stem and progenitor cell (HPSC) transplantation. However, several studies suggest that a bias exists in CD45.1 versus CD45.2 hematopoietic cell reconstitution from the transplanted HPSCs. Meanwhile, no definitive comparison has been reported for mature immune cells as to whether the CD45.1/CD45.2 disparity can skew immune cell response. In this study using lymphocytopenia Rag1−/− CD45.2 mice as hosts, we assessed the proliferative potential of CD45.1 versus CD45.2 lymphocytes following adoptive transfer of mature T and B cells harvested from the spleens of CD45.1 and CD45.2 mice. We have found that a selective bias for CD8+ T cells in that CD45.1 CD8+ T cells showed significantly higher proliferation and higher expression of PD-1 compared CD45.2 CD8+ T cells in the Rag1−/− CD45.2 hosts. This bias is likely due to MHC-I restricted allogeneic stimulation as CD45.1 versus CD45.2 CD4+ T cells and CD19+ B cells contained the same graft as CD8+ T cells showed equivalent proliferation. These results suggest that CD45.1/CD45.2 markers induce an alloreactive response specific to CD8+ T cells, and therefore call for caution for using them as congenic markers in immunologic models.

This study was supported by funds through the National Cancer Institute (R01CA184728), the Maryland Department of Health’s Cigarette Restitution Fund (CRF) Program and used shared core facilities supported by University of Maryland Greenebaum Comprehensive Cancer Center (UMGCC) Support Grant (P30CA134274).