bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced immunoglobulin somatic hypermutation in human APOBEC3 transgenic mice
Authors: Jones Sean , Guo Kejun , Barrett Brad , Santiago Mario L.
Published: January 2023

Eliciting a broadly neutralizing antibody (bNAb) response remains a major hurdle in HIV-1 vaccine development, as a majority of these bNAbs are heavily mutated. Immunoglobulin (Ig) somatic hypermutation (SHM) is primarily driven by AID. However, our previous data in Friend retrovirus (FV) infected mice revealed that an evolutionarily-related deaminase, mouse APOBEC3 (mA3), may complement AID in mutating virus-specific Abs (Halemano et al, PNAS 2014). AID mutates primarily in an RC context (R=purine), whereas the A3s mutate in a YC context (Y=pyrimidine). Of note, mice encode only one A3 gene, but humans encode seven. To date, it remains unclear if the human APOBEC3 (hA3) deaminases can directly mutate Ig genes. To study the role of hA3s in Ig SHM, bacterial artificial chromosome (BAC)-transgenic mice expressing the 7 hA3s (hA3-Tg) were crossed onto an mA3 KO background. All hA3s were expressed in hA3-Tg B cells during FV infection. hA3-Tg (n=13) and mA3 KO (n=12) mice were vaccinated with live-attenuated FV and analyzed at 28 d. Surprisingly, the hA3-Tg mice have an impaired plasma NAb response (p=0.0003) despite similar virus-specific binding IgG titers (p>0.1). Illumina sequencing of bone marrow IgG transcripts showed increased deamination mutations (C to T or G to A) in hA3-Tg versus mA3 KO mice (p=0.0043). Interestingly, this increase was accounted for by AID-type (RC), but not hA3-type (YC), mutations. Our findings reveal that hA3 can increase IgG SHM rates following vaccination, but the initial impact on the vaccine NAb response may be detrimental. Studies are ongoing to monitor the development of virus-specific antibodies in hA3-Tg mice, as harmful mutations may be more prominent during early time points post-vaccination.
Investigation of the immunological mechanisms underlying the attenuation of vaccinia virus lacking host-range factor C7
Authors: Deng Liang , Yang Ning , Dai Peihong , Wang Yi , Wang Weiyi , Merghoub Taha , Wolchok Jedd D. , Shuman Stewart
Published: January 2023

Vaccinia virus C7 protein is an important host-range factor for the vaccinia virus life cycle in mammalian cells. Type I IFN plays an important role in host defense of viral infection. However, the role of C7 in immune modulation of the IFN pathway is unclear. Here, we find that C7 functions as a dual inhibitor of IFN production and IFN signaling by interfering with the activation of two transcription factors IRF3 and STAT2. Furthermore, vaccinia virus with deletion of C7 (VACVΔC7L) is non-pathogenic in wild-type C57BL/6J mice in an intranasal infection model. We elucidated the immunological mechanisms underlying the attenuation phenotype of VACVΔC7L, which can be summarized as follows: (i) intranasal infection of VACVΔC7L triggers the production of interferons (IFNs), proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BAL), whereas WT VACV does not; (ii) intranasal infection of VACVΔC7L results in the recruitment of dendritic cells, inflammatory monocytes, neutrophils, CD8+, and CD4+ in the BAL, whereas WT VACV does not; and (iii) infection of primary type II lung alveolar epithelial cells (AECs) with VACVΔC7L induces IFN, proinflammatory cytokine and chemokine gene expression and protein secretion, whereas WT VACV does not. However, VACVΔC7L dramatically gained virulence in STAT2 or IFNAR1-deficient mice, with increased titers in the lungs and systemic dissemination of the viruses to the blood and distant organs. Taken together, our results provide evidence that vaccinia C7 is a key virulence factor that antagonize both IFN production and signaling and IFNAR1 and STAT2 are critical in restricting vaccinia viral replication in the lung alveolar epithelial cells.
Single cell analysis revealed distinct B-cell subpopulations that produce or respond to type I interferon in SLE
Authors: Hamilton Jennie A. , Wu Qi , Yang PingAr , Luo Bao , Liu Shanrun , Li Jun , Hong Huixian , Mattheyseys Alexa , Chatham Winn , Hsu Hui-Chen , Mountz John D.
Published: January 2023

We previously showed that B-cell endogenous expression of interferon beta (IFNβ) is associated with the development of autoreactive B cells in BXD2 autoimmune mice. As among all type I IFNs, IFNβ exhibits the highest affinity to IFNAR1 and IFNAR2 and IFNβ also can stimulate IFNαs, there is a pressing need for a better understanding of the source and responses of IFNβ in systemic lupus erythematosus (SLE). Flow cytometry and super-resolution imaging analysis showed increased expression of IFNβ in B cells obtained from PBMCs of SLE patients compared to healthy controls. The importance of SLE B-cell endogenous IFNβ in regulating TLR7-mediated responses was identified when CL264 (a TLR7 ligand) plus anti-Ig-induced CD69 and survival of purified B cells were significantly and equivalently suppressed by an anti-IFNβ or an anti-IFNAR1 blocking antibody. We next carried out single cell qRT-PCR analysis to determine if B-cell endogenous IFNβ acts in an autocrine or paracrine manner to modulate IFN stimulated genes (ISGs). Hierarchical analysis revealed three prominent and consistent clusters, consisting of an IFNB+ cluster, an ISG+ cluster, and an IFNA+ cluster in naïve B cells isolated from 3 SLE patients. Cells within the IFNB+ cluster expressed higher levels of IFNB, IFNA1, IFNA8, and TLR7. Cells within the ISG+ cluster expressed higher levels of IFNAR1, IFNAR2, BAFFR, MX1, RIG1 and TLR9. Cells within the IFNA+ cluster expressed higher levels of IFNA4, IFNA10, IFNA14, IFNA16, IFNA17, and TLR3. Together, the results reveal (1) B cells as an important source of IFNβ in modulating TLR7 responses in SLE; (2) a well-orchestrated program in modulating IFNβ donor versus responder B cells; and (3) some patients may benefit from specific targeting of IFNβ.
Loss of B cell Ezrin in lupus-prone Lyn-deficient mice downregulates B cell activation and autoimmune pathology
Authors: Gupta Neetu , Pore Debasis , Parameswaran Neetha , Huang Emily , Dejanovic Dina
Published: January 2023

Systemic lupus erythematosus (SLE) is an aggressive autoimmune disorder that afflicts 1.5 million Americans. Human GWAS studies have reported a strong association between the Src family tyrosine kinase Lyn and incidence of SLE, and B cells from SLE patients exhibit decreased Lyn expression. Genetic deletion of Lyn in mice results in loss of peripheral B cell tolerance and development of human SLE-like autoimmunity, characterized by hyperactive B cell antigen receptor (BCR) signaling, autoantibody production and glomerulonephritis. However, the molecular regulators of B cell pathogenesis in Lyn-deficient mice have not been fully characterized. We have previously shown that the membrane-cytoskeleton linker protein ezrin regulates multiple aspects of B cell function through its dynamic switching between phosphorylated and dephosphorylated states. We observed that ezrin is hyperphosphorylated in Lyn-deficient B cells, suggesting that it may impact B cell activation in this context. To test this hypothesis, we generated conditional deletion of ezrin in the B cells of Lyn-deficient mice (hereafter referred to as DKO mice). Compared to Lyn-deficient mice, the DKO mice exhibited a significant decrease in splenomegaly and reduction in autoantibodies against a variety of autoantigens. Glomerulonephritis and deposition of IgG and complement factor C3 in the kidneys of DKO mice were also markedly reduced. The DKO B cells were hyporesponsive to BCR stimulation and displayed a calcium mobilization pattern reminiscent of that observed in anergic B cells. Our data demonstrate that ezrin regulates B cell hyperactivation and associated pathology in the absence of Lyn, and suggest that ezrin may be a novel molecular target in SLE.
Characterization of MMP-28 in autoimmune neurodegenerative diseases
Authors: Tokmina-Roszyk Dorota , Onwuha-Ekpete Lillian C. , Refai Mohammed , Tokmina-Lukaszewska Monika , Bothner Brian , Fields Gregg
Published: January 2023

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix components and are crucial for normal physiological processes; however, their dysregulation has been implicated in numerous pathologies associated with autoimmunity as well as neurological disorders. MMP-28 is the least characterized member of this family and it has been implicated as a regulator of myelination during neural development and repair. Moreover, recent studies have suggested a role for MMP-28 in determining the strength of the immune response. To unravel the complex role of MMP-28 in an autoimmune neurodegenerative disease such as Multiple Sclerosis (MS) we utilized the Experimental Autoimmune Encephalomyelitis (EAE) murine model. We compared disease progression in both WT and MMP-28 null mice and did not observe significant difference between both groups. However, weight loss and therefore disease severity was overall higher in MMP-28 null mice. Subsequently, we subjected brain samples from both WT and MMP-28 null to global protein profiling using a shotgun proteomics approach and corresponding protein profiles were compared. Our findings, provide further evidence that MMP-28 is an important factor in the autoimmune response. Moreover, in addition to the expected impact of MMP-28 on various proteins associated with myelin, we identified mitochondrial protein complexes that were upregulated in samples derived from MMP-28 null mice. These data provide evidence of a previously unknown intracellular role of MMP-28 and suggest a possible novel mechanism of immune regulation. Our data suggest that MMP-28 might be an important antitarget in autoimmune neurodegenerative diseases such as MS.
A detrimental effect of interleukin-22 on salivary gland tissue integrity and function
Authors: Yoo Bora , Zhou Jing , Yu Qing
Published: January 2023

Interleukin-22 (IL-22) has well-documented effects in promoting epithelial wound healing and protecting tissue damage in the intestine and lung. However, in certain inflammatory diseases such as psoriasis, IL-22 can also exert tissue-detrimental effect. Although the up-regulation of IL-22 has been reported in patients with Sjögren’s syndrome (SS), the role of IL-22 in this disease is not well-characterized. Herein, we investigated the effects of IL-22 on SS-like salivary gland disorder using both in vivo and in vitro approaches. To assess the effects of exogenous IL-22 on salivary gland tissues in vivo, IL-22 was directly injected into submandibular glands (SMGs) of 7–12 weeks old female C57BL/6 and the non-obese diabetic (NOD) mice, a model of SS disease. The salivary flow rate was decreased and the caspase-3 level was increased in the SMGs of the IL-22-treated C57BL/6 mice. IL-22 treatment also decreased the salivary flow rate and increased the level of capase-3 in the SMGs in the NOD mice. We determined the effects of endogenous IL-22 using an anti-CD3 antibody-induced acute exocrinopathy model. Anti-CD3 antibody was intraperitoneally injected into 8-week-old female C57BL/6 mice every other day for 3 days, together with a neutralizing anti-IL-22 antibody or its isotype control IgG. Neutralization of endogenous IL-22 improved saliva production and reduced caspase-3 activation in the SMGs in anti-CD3-treated C57BL/6 mice. Moreover, in vitro IL-22 treatment of a human salivary gland epithelial cells induced STAT3 activation and inhibited cell expansion. Taken together, these results provided strong evidence that IL-22 can directly act on salivary gland epithelial cells and promote damage and dysfunction of salivary gland tissues.
Hemopexin deficiency prevents joint injury following collagen antibody-induced arthritis
Authors: Baig Sahar , Du Yong , Ling Qin , Paglicawan Laura , Vanarsa Kamala , Mohan Chandra
Published: January 2023

Background

Rheumatoid arthritis is an autoimmune disorder in which the immune system attacks its own tissues, resulting in swelling and inflammation of the joints. Hemopexin (Hx), an acute-phase protein synthesized by hepatocytes in response to proinflammatory cytokines, binds to heme, and controls heme-iron availability in tissues and T lymphocytes. Its absence has been associated with increased inflammatory properties of high-density lipoproteins and the development of autoimmune disease, such as experimental autoimmune encephalomyelitis (EAE). The aim of this study is to investigate the role of Hx in the development of arthritis using the collagen antibody-induced arthritis (CAIA) mouse model.

Methods

CAIA was induced in Hx-deficient (Hx−/−) mice and wild-type (WT) littermates, via i.p injection of antibodies directed against type II collagen, followed by the administration of lipopolysaccharide (LPS). Paw thickness and disease severity was evaluated daily. Blood samples were collected to measure the serum Hx levels using ELISA.

Results

During CAIA, serum Hx level became elevated and remained high. When CAIA was induced in Hx−/− mice, they exhibited reduced arthritis compared to WT mice, as arthritic Hx−/− mice had lower average paw thickness (1.32 ± 0.02 vs 2.67 ± 0.05, 2-tail t test, P < 0.05 ) and reduced arthritis scores ( 2.0 ± 0.5 vs 7.3 ±1.25, 2-tail t test, P < 0.01) at the end of the experiment compared to WT mice. Pathology studies are in progress.

Conclusion

Hx−/− mice are protected from CAIA, with reduced arthritis, indicating a pro-inflammatory role for Hx in the development of arthritis. Further studies are warranted to unravel the pathogenic mechanisms involved and clinical relevance of this molecule.
Multi-parametric Human Cell-Based Inflammation Assay for Cytokines and Cell Surface Antigens
Authors: Cromwell Evan , Hoxha Ori , Hammer Matthew , Sirenko Oksana
Published: January 2023

Inflammation is a complex event in which cells respond to various endogenous and exogenous stimuli. Factors such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ activate signaling pathways leading to the expression of cytokines and cell-surface antigens. The ability to monitor up-regulation of these molecules provides an important physiological read-out for cell-based models of inflammation. We present results from a multiparametric cell-based assay that uses immunoassays for secreted cytokines and fluorescence read-outs of cell surface markers to evaluate the effect of different mediators on inflammatory response. Primary human umbilical vein endothelial cells (HUVEC) were stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β) for 20 hours. Then, a newly developed microfluidic-based assay system, PuMA, was used to quantify amount of MCP-1, IL-8, and IL-6 in cell supernatants. Expression of adhesion molecules VCAM and HLADR was quantified by measurement of total fluorescence intensity after staining cells with directly conjugated antibodies using an ImageXpress Nano Automated Imaging System. Concentration dependent inhibition of the inflammation responses by the compounds AG126 and MG132 were measured. Clear differences in cytokine expression were seen between the compounds consistent with their reported mechanisms of action. The combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can be used to test the efficacy of anti-inflammatory compounds versus toxicity and also provide significant insight into the mechanism of action by selective inhibition of markers triggered by different signaling pathways.
Aortic intima microenvironment instructs macrophage gene expression signature in steady state and progressing atherosclerosis
Authors: Williams Jesse W. , Zaitsev Konstantin , Kim Kiwook , Kim Kyeongdae , Zinselmeyer Bernd , Choi Jae-Hoon , Artyomov Maxim N. , Randolph Gwendalyn J.
Published: January 2023

Macrophages reside in the vascular intima of the aorta in plaque prone regions. Intima macrophages were found to be ‘tissue resident’, possessing the ability to self-renew independent of circulating monocytes and having a unique dependence on Csf1 and Flt3 pathways for development. Lineage tracing and depletion experiments showed these cells were regulators of early plaque development in models of atherosclerosis. Using bulk RNAseq on intima resident macrophages and single cell sequencing data from total leukocytes from C57Bl/6 mouse aorta, we identified a core gene expression profile associated with intima resident macrophages. Comparing this gene signatures to single-cell gene expression data obtained from total leukocytes from atherosclerotic aortas identified overlapping gene expression with foamy macrophages, despite foamy macrophages developing exclusively from recruited monocytes at this stage of disease. The intima resident gene signature was expressed in foamy cells in addition to cholesterol handling genes, but not in newly recruited monocytes. Importantly, the intima gene signature was only associated with cells lacking expression of inflammatory genes. Together this suggests there is a niche-driven gene expression program within the aortic intima influencing macrophage differentiation and function. Targeting this newly identified gene set may be important for regulation of the inflammatory pathways and foamy macrophage differentiation in atherosclerosis.
Dendritic cell derived IL-12p40 binds extracellular proteins to make heterodimeric cytokines
Authors: Gerber Allison N. , Abdi Kaveh , Singh Nevil J.
Published: January 2023

IL-12 and IL-23 are heterodimeric cytokines which share a common subunit, p40. Binding of p40 to p35 results in IL-12, while binding to p19 generates IL-23. Assembly of these cytokines is thought to occur only when both relevant subunits are expressed inside a dendritic cell (DC) allowing for covalent linkage and secretion. Intriguingly, the p40 subunit is also released from DC as a monomer with unknown function. We previously hypothesized that monomeric p40 can bind p35 (or p19) released by another cell in the extracellular space. Consistent with this, recombinant p40 and p35 were shown to assemble in cell free extracts. To test this hypothesis in a physiological setting, in vitro and in vivo assays were performed by mixing p40−/− and p35−/− cells. In these assays, since no one cell can make both subunits, the only way IL-12 can be present is through extracellular assembly. In vitro bioassays for IL-12 function showed proliferation and IFNγ production in response to mixing. In vivo, mixed bone marrow chimeras infected with Leishmania major were able to induce IFNγ production by T cells. These results suggest that functional IL-12 assembly can occur in the extracellular milieu from component peptides. Importantly, this mechanism may allow even non-hematopoietic tissues to direct T cell differentiation by releasing proteins capable of binding to DC-derived p40. In order to identify novel heterodimeric cytokines capable of fulfilling that paradigm, we have used a panel of proteins that can bind p40, identified from an unbiased mass spectrometry based analysis. Recombinant versions of these proteins expressed as dimers with p40 are being evaluated for functional activity.
Response gene to complement 32 interacts with SMAD3 to promote TGF-β-induced extracellular matrix production in multiple sclerosis
Authors: Tatomir Alexandru , Boodhoo Dallas , Tegla Cosmin , Rus Violeta , Rus Horea
Published: January 2023

We have previously shown that response gene to complement 32 (RGC-32) mediates transforming growth factor-β (TGF-β)-induced astrocyte reactivity and extracellular matrix production. However, the molecular mechanisms underlying RGC-32 expression and function remain to be elucidated. In the present study, we found that TGF-β induced expression and nuclear translocation of both RGC-32 and SMAD3 in astrocytes. Using specific pathways inhibitors, we have found that RGC-32 induction is mediated by SMAD-dependent and independent mechanisms. We investigated whether RGC-32 interacts with SMAD2 or SMAD3 in astrocytes and if this association plays a role in extracellular matrix synthesis. Co-immunoprecipitation showed that SMAD3, but not SMAD2 physically interacts with RGC-32 in astrocytes. SIS3, an inhibitor of SMAD3 phosphorylation, significantly reduced RGC-32 translocation to the nucleus, indicating that SMAD3 phosphorylation is required for RGC-32 nuclear translocation. On the other hand, silencing RGC-32 with siRNA had no effect on SMAD3 nuclear translocation. Y27632, a selective ROCK inhibitor was also able to significantly inhibit RGC-32 nuclear translocation, while the SMAD3 nuclear translocation was unaffected. The inhibition of SMAD3 phosphorylation also blocked the RGC-32-induced collagen type I expression, indicating a synergistic action between RGC-32 and SMAD3 to induce the expression of collagen type I. Taken together, our data demonstrate for the first time that RGC-32 interacts with SMAD3 to mediate the extracellular matrix production in astrocytes. These data suggest that a similar mechanism might be involved in mediation of gliosis seen in chronic multiple sclerosis lesions.
Visual analysis of multidimensional CyTOF data from allergic asthmatics pre and post allergen challenge
Authors: Wang Jing M. , Bhakta Nirav , Ansel Mark , Antalek Bobby , Nguyen Christine P. , Woodruff Prescott G.
Published: January 2023

Allergen-induced airway inflammation is a central feature of asthma. The ability to deeply phenotype cells with mass cytometry (CyTOF) can lead to new insights into the immune response after allergen challenge (AC). We have developed a 33 marker CyTOF-based panel and applied it to human bronchoalveolar lavage (BAL) and whole blood cells from 10 subjects with atopic asthma in a segmental AC study. Because the data generated by CyTOF are high dimensional, traditional bivariate analysis is complicated and limited to traditionally-defined cell types. Therefore, we applied the unbiased visualization method, viSNE. We report data from our initial subject before (baseline) and after diluent (DIL) or AC. Our results show that viSNE captures marker expression differences, cell grouping differences by sample type and rare cell populations. BAL cells showed more macrophages (CD206+) at baseline and more macrophages and neutrophils (CD16++CD15++CD24+) after DIL versus AC. In contrast, AC led to a tremendous influx of eosinophils (CD24++CCR3+) with some basophils (HLA-DR−CD123+FcɛRI+). Interestingly, eosinophils in AC were CD69+ in the BAL but not in the blood, implying that eosinophils were activated after recruitment into the airways. There were no changes in T cell (CD3+CD4+ or CD3+CD8+), B cell (CD19+ or CD20+) or dendritic cell (CD1c+CD123−HLA-DR+ or CD1c−CD123+HLA-DR+) proportions between baseline, DIL and AC BAL cells; additional analyses from this clinical study will focus on subsets of these cells. In conclusion, visual analysis of high-dimensional data in allergic airway inflammation identifies differences and potentially meaningful but as yet undefined inflammatory cell populations.
IRF4 expression by lung dendritic cells promotes allergic Th2 responses by controlling OX40L, IL-10, and IL-33 expression during sensitization
Authors: Camacho Daniel F. , Howard Chanie L. , Darnell Eli P. , Wang Esther , Hrusch Cara L. , Sperling Anne I.
Published: January 2023

The specific mechanisms by which DCs initiate Th2 responses are not completely understood. We previously found that mice lacking the transcription factor IRF4 in DCs do not develop type 2 lung inflammation in response to house dust mite extract (HDM). Further, IRF4-deficient DCs had a reduced capacity for restimulating T cells towards a Th2 phenotype ex vivo. We hypothesized that IRF4 is necessary for DCs to perform one or more of the functions required to successfully initiate Th2 responses in vivo. Here we demonstrate that during in vivo HDM sensitization, IRF4 in DCs is required neither for CD11b+ CD24+ cDCs to take up allergen in the lungs, nor for the lung DCs to process antigens. Instead, IRF4-deficient lung DCs display lower levels of the Th2-associated costimulatory molecule OX40L and express less of the Th2-promoting cytokines IL-33 and IL-10 during sensitization. While HDM-bearing DCs lacking IRF4 arrive in the lung-draining lymph nodes at proportions comparable to WT DCs and are capable of processing antigens, reduced numbers migrate to the lung-draining lymph nodes in the absence of IRF4. Following HDM sensitization, mice with IRF4-deficient DCs display impaired CD4+ effector/memory T cell recruitment to the lung parenchyma and reduced T cell expression of CD69. Thus, IRF4 controls a pro-Th2 program in DCs including expression of OX40L, IL-10, and IL-33 as well as migration to the draining lymph nodes, culminating in Th2 priming in response to HDM.
The crosstalk between developing sympathetic nerves and immune cells contributes to early-life Th2 bias in the lung
Authors: Wang Wei , Cohen Jonathan , Trieu Kenneth , Bai Yan , Ai Xingbin
Published: January 2023

The adaptive immunity in early life is polarized towards Th2 response, which contributes to increased susceptibility to asthma in children. However, the molecular mechanism of age-related Th2 bias remains poorly understood. Building upon critical roles of nerves in the modulation of immune responses, we investigated the crosstalk between the developing nervous system and the immune cells in postnatal lungs using murine models and human lung tissues/cells. Notably, like human, mice exhibit similar early-life Th2 bias, as allergen exposure elicits more robust inflammation in neonates than in adult mice. In addition, our previous studies revealed a similar profile of neurogenic factors between mouse and human lungs. Therefore, the neonatal mouse model of asthma provides an invaluable tool to identify potential modulatory roles of nerves in early life Th2 immunity bias. In this study, we identified dopamine as a potent inducer of Th2 phenotypes in a screen for the role of neurotransmitters in the differentiation of CD4+ T cells, which is consistent with a previous finding. Dopamine is selectively expressed by the sympathetic nerves in developing lungs. Besides, we discovered that sympathetic nerves transited from dopamine-producing in early life to norepinephrine-producing in adult life. We proved that the dopamine-rich sympathetic nerve played a role in allergic Th2 inflammation in neonatal mice lungs. However, with age, the level of dopamine decreased and therefore, this immune modulation reduced. Taken together, our findings reveal the crosstalk between the developing nerves and Th2 prone immunity in early life. Our study may provide the foundation for potential therapy strategies for kids with asthma.
Role of TNFα Antagonists in Susceptibility to Mycobacterial Infection in Association with Genetic Polymorphisms in Crohn’s Disease
Authors: Qasem Ahmad , Naser Saleh
Published: January 2023

Crohn’s disease (CD) is a relapsing inflammatory bowel disease that has been associated with bacterial triggers such as Mycobacterium avium subspecies paratuberculosis (MAP). Overreactive immune response in CD patients involves a significant elevation in Tumor Necrosis Factor alpha (TNFα), which causes upregulation in multiple cytokine levels, leading to formation and maintenance of granuloma. Standard CD treatment includes immunomodulators and biologics such as Anti-TNFα. We hypothesize that blocking TNFα promotes MAP survival which worsens the disease condition. Therefore, CD treatment plan should include MAP-specific antibiotics and novel cytokine targets based on personalized medicinal approach. RHB-104, a triple antibiotics formulation ((63.3 %) clarithromycin, (6.7 %) clofazimine, and (30 %) rifabutin), recombinant TNFα, adalimumab (Humira®), and certolizumab pegol (Cimzia®) were evaluated in vitro for effect on MAP culture and macrophages infected with MAP. As expected, RHB-104 was detrmintal to MAP (MIC< 2ug/mL), while anti-TNFα therapeutics had no direct bactericidal effects at concentrations as high as 4x Cmax. Recombinant TNFα reduced MAP survival in infected macrophages by 2.63 logs. On the contrary, both anti-TNFα therapeutics promoted MAP survival by 1.54 logs. Additionally, we have identified 2 significant single nucleotide polymorphisms (rs767455 and rs3397) related to TNFR superfamily 1(A/B) in 54 CD patients compared to 50 healthy subjects by using TaqManTM genotyping assay. This study should provide i) data about the safety of Anti-TNFα biologics in CD patients-associated with MAP infection and ii) predictions toward response to treatment plan based on patient’s pharmacogenomics.
Hypoxia boosts Th17-cell responses in inflammatory bowel disease through increased efflux of ligands reactive with the aryl-hydrocarbon-receptor
Authors: Longhi Maria Serena , Xie Anyan , Robles Rene’ J. , Zhang Haohai , Csizmadia Eva , Wu Yan , Moss Alan C. , Robson Simon C.
Published: January 2023

Despite beneficial influence of hypoxia-inducible-factor (HIF) stabilization in animal models of mucosal inflammation and experimental colitis, hypoxia can be deleterious during protracted inflammation by inhibiting T-regulatory-1 and favoring differentiation of T-helper (Th)-17 cells.

We report here that Th17 cells from Crohn’s disease patients display heightened levels of HIF-1α and become refractory to the immunoregulatory effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl hydrocarbon receptor (AhR), when exposed to hypoxia in vitro. Molecular blockade of HIF-1α reconstitutes Th17 cell ability to respond to UCB and upregulates CD39, an ectonucleotidase that catalyzes ATP and ADP into immunosuppressive adenosine. Inability of Th17 cells to respond to AhR derives from induction of ATP transporters multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4) by HIF-1α; this results in increased efflux of AhR ligands like UCB out of Th17 cells. Ritonavir, a drug with inhibitory effects over HIF-1α, MDR1 and MRP4, restores UCB regulation over Th17 cells in Crohn’s and enhances UCB salutary effects in dextran-sulfate-sodium-induced experimental colitis.

We conclude that inhibitory effects of hypoxia in limiting Th17 cell response to AhR in Crohn’s disease are mediated through upregulation of ATP transporters that decrease AhR substrate availability. Concurrent treatment with HIF-1α inhibitors and AhR ligands provide effective therapeutic options to overcome Th17 functional alterations in inflammatory bowel disease.
Novel UNC13D intronic variant disrupting a NF κB enhancer in a patient with recurrent macrophage activation syndrome and systemic juvenile idiopathic arthritis
Authors: Cron Randy Q. , Schulert Grant S. , Zhang Mingce , Husami Ammar , Fall Ndate , Brunner Hermine , Zhang Kejian , Grom Alexei A.
Published: January 2023

Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (SJIA), and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH-3 (FHL3), but the role of non-coding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS. A custom enrichment library was constructed to sequence approximately 1 MB flanking UNC13D in 24 patients with SJIA, recurrent MAS, and negative prior genetic (exon/coding) testing. Functional consequences of intronic variants were assessed using quantitative PCR on patient derived peripheral blood mononuclear cells (PBMC), electrophoretic mobility shift assay (EMSA), in vitro transcriptional enhancer assays, and in vitro NK cell degranulation assays. We report a patient with SJIA and recurrent episodes of MAS, found to have a novel functional intronic variant in UNC13D, c.117+143A>G. This variant occurs in a proposed regulatory region that drives an alternative, lymphocyte specific UNC13D transcript, and is associated with reduced transcript levels in patient PBMC. In support of this, the patient’s variant disrupts NFκB binding to a functional transcriptional enhancer, leading to reduced enhancer activity and NK cell degranulation in vitro. Another patient was identified with a previously described UNC13D intronic variant, for a total non-coding variant hit rate of 8.3% (2/24). These findings highlight that intronic variants in key regulatory regions may be associated with MAS in patients with SJIA, and support deep sequencing approaches when causative coding variants are not identified.
Blood-based untargeted metabolomics in Relapsing-Remitting Multiple Sclerosis revealed testable therapeutic target
Authors: Giri Shailendra , poisson Laila , Singh Jaspreet , Datta Indrani , Suhail Hamid , Mangalam Ashutosh K. , Cerghet Mirela , Elias stanton B. , Rattan Ramandeep
Published: January 2023

Relapsing-remitting (RRMS), a most common form of MS, is characterized by acute attacks alternated by partial or complete recovery periods. The major focus of our research is to identify the therapeutic target using metabolomics. Metabolomics is a fast emerging field which can provide a direct “functional readout of the physiological state” of an organism. Identification of blood-based metabolic pathway(s) in relapsing-remitting form of MS (RRMS) which could be used for therapy. Using untargeted ultra-performance liquid and gas mass spectrometry, we measured serum metabolites from 33 RRMS patients, and 14 healthy subjects (HS). A total of 621 known metabolites were detected and 60 metabolites were significantly altered in the serum of RRMS compared to HS. Bioinformatics analysis revealed four metabolic pathways altered in RRMS including glycerophospholipid, citrate cycle, sphingolipids, and pyruvate metabolism. PBMCs isolated from RRMS patients exhibited higher glycolysis suggesting altered metabolic state of immune cells. EAE mice treated with glycolytic inhibitor 2-deoxyglucose (2-DG; once daily), resulted in a significantly delayed (P<0.001) the disease progression. 2DG inhibited (P<0.01) interleukin 17 production by reducing glycolysis (P<0.01) in monocytes of treated EAE group. Using untargeted metabolomic and Seahorse bioanalyzer approaches, we document that RRMS patients showed altered metabolic state “metabotype”. Targeting glycolysis, upstream of metabolic pathways altered in RRMS, using pharmacological inhibitor ameliorated the disease progression and pathology in a preclinical model of MS.
Single-cell RNA sequencing of skin and kidney cells in lupus nephritis provides insights into pathogenesis and indicates novel potential biomarkers
Authors: Der Evan B. , Suryawanshi Hemant , Ranabothu Saritha , Goilav Beatrice , Belmont H. Michael , Izmirly Peter , Bornkamp Nicole , Jordan Nicole , Wang Tao , Wu Ming , James Judith A. , Guthridge Joel M. , Raychaudhuri Soumya , Buyon Jill , Tuschl Thomas , Putterman Chaim
Published: January 2023

Classification and treatment decisions in lupus nephritis (LN) are largely based on renal histology. Single-cell RNAseq (scRNAseq) analysis may accurately differentiate types of renal involvement at the transcriptomic level, and better inform treatment decisions and prognosis. scRNAseq was performed on kidney and non-lesional skin tissue collected from clinically indicated renal biopsies, and skin biopsies obtained at the time of renal biopsies, in 20 systemic lupus erythematosus (SLE) patients. Cell-types were determined using principal component analysis and tSNE plotting, resulting in the definitive identification of keratinocytes, tubular cells, mesangial cells, fibroblasts, endothelial cells, and leukocytes. Tubular cells in patients with proliferative nephritis demonstrated upregulated TNF signaling compared with membranous nephritis. Interestingly, keratinocytes of patients with proliferative nephritis also demonstrated upregulated TNF signaling. Furthermore, tubular cells of patients who did not respond to standard immunosuppressive treatment showed upregulation of extracellular matrix proteins and fibrotic markers at the time of biopsy. Using logistic regression analysis, a 4-gene tubular fibrosis score was created which predicted response to treatment, with an area under curve of 0.9. We conclude that scRNAseq using small amounts of renal biopsy tissue in SLE can differentiate between the different classes of LN, and provide important insights into potential pathogenic mechanisms. Further, changes in the skin of LN patients can provide a useful source of biomarkers and may reflect important information concerning concurrent kidney pathological events.
Formation of an Argonaut-independent miRNA-mRNA Complex with DDX6 Orchestrates MTOR- dependent Regulation of T-cell differentiation.
Authors: Zhang Nannan , Hu Guowu , Pena Hanna , Qiu Jin , Park Yoon-Dong , Uzel Gulbu , Datta Sandip K. , Steiner Joseph P. , Kelsall Brian L. , Williamson Peter R.
Published: January 2023

The mechanistic target of rapamycin(MTOR) is an important global regulator of growth, metabolism and immunity; however, how MTOR integrates global and sequence-specific signals remains poorly understood. Herein we utilize the MTOR signaling pathway involved CD4+FOXP3+ regulatory T cells (Tregs) and identify sixty-three genes regulated during differentiation including a key transcription factor, Forkhead box 3 (FOXP3). MTOR-dependent DCP2 phosphorylation potentiated decapping/degradation and was facilitated by MIR1246 miRNA binding to the 3′-UTR of FOXP3 mRNA. The physiological relevance of DDX6-dependent FOXP3 mRNA degradation was established in experimental autoimmune encephalitis and fungal mening oencephalitis using Ddx6flox/floxX CD4cre mice, as well as in patient cells having mutations incytotoxic T-lymphocyte associated protein 4 (CTLA4) associated with autoimmunity. These data provide a novel mechanism of miRNA-dependent mRNA regulation of MTOR-dependent targets highlighted by regulation during T cell polarization and suggests new targets for immunotherapy.
Understanding protective plasma levels of penicillin and monitoring antibody responses for the prevention of GAS infection in a paediatric population in New Zealand
Authors: Sika-Paotonu Dianne , Tiatia Ramona , Thornley Craig , Betty Bryan , Wineera-Parai Ranei , Eddie Barbara , Baker Michael , Salman Sam , Marsh Julie , Manning Laurens , Joseph John , Hand Rob , Carapetis Jonathan
Published: January 2023

Acute Rheumatic Fever (ARF) is an autoimmune condition resulting from untreated Group A Streptococcal (GAS) infection of the upper respiratory tract and possibly skin. Repeated untreated and severe episodes of ARF can cause permanent cardiac damage known as Rheumatic Heart Disease (RHD). Rates of ARF in New Zealand and Australia are among the highest in the world, particularly amongst Māori and Pacific and Indigenous Aboriginal children. Monthly injections of Benzathine Penicillin G (BPG) are given intramuscularly to prevent GAS infections that can lead to ARF and cause RHD. Early studies exploring the Pharmacokinetics (PK) properties of BPG were conducted in healthy, fit, young military recruits without ARF/RHD. BPG bioavailability, metabolism may differ in paediatric populations groups with ARF who have comorbidities and vary in Body Mass Index. This project seeks to determine the PK characteristics in a paediatric population currently receiving monthly injections of BPG while at the same time monitoring antibody immune responses to possible GAS infections. This is a prospective cohort study in a paediatric population of Wellington based children and teens (aged 5–21 years of predominantly Pasifika and Māori heritage) with a previous diagnosis of ARF and are receiving monthly BPG injections via community services. Participants will contribute finger prick blood samples over time for Dried Blood Spot assays designed to quantify penicillin G levels in the blood and measure streptococcal serology. Routine throat swab samples will also be collected with ASOT antibody levels monitored for GAS breakthrough infections. This is the first population PK study exploring use of BPG in Māori and Pacific children previously diagnosed with ARF.
A Novel Mendelian Disease of Autoimmunity caused by Mutations in a small GTPase
Authors: Leney-Greene Michael Alexander , Park Ann , Chao Brittany , Vilarinho Silvia , Su Helen , Ozen Ahmet , Lenardo Michael J.
Published: January 2023

In this study, we harness Whole Exome Sequencing (WES) to interrogate the underlying genetic cause in a patient cohort suffering from a Mendelian disease of immune dysregulation characterized by severe lymphopenia, splenomegaly, anemia, thrombocytopenia and liver failure. Bioinformatic analysis revealed novel mutations in a small GTPase that led to a near complete loss of mature protein in patient cells. Animal models lacking these genes develop a disease remarkably similar to that observed in the human patients, however, the molecular role of this GTPase in the immune system remains a fundamental gap in our knowledge. Due to a remarkable similarity in phenotypes between mice deficient for this small GTPase and T cell specific deletions of regulators of mTORC1, we hypothesized that this gene may be involved in the mTORC1 signaling pathway. We observe a robust association between this GTPase and members of the ragulator complex which is required for recruitment of mTORC1 to the lysosome and subsequent signalling. Furthermore, treatment with inhibitors of mTORC1 led to remarkable clinical improvements in our mouse model and also one of our human patients. Overall, we have described a novel, T cell specific member of the ragulator complex and it’s role in maintaining immune homeostasis in humans.

This research was supported by the Intramural Research Program of NIAID, NIH.
Hypomethylation of STAT1 and HLA-DRB1 in CD8+ T cells is associated with type-I interferon dependent activation of CD4+ T cells in systemic lupus erythematosus
Authors: Sawalha Amr , Miller Shaylynn , Coit Patrick , Gensterblum-Miller Elizabeth , Renauer Paul , Kilian Nathan , Schonfeld Mark
Published: January 2023

Background

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by autoantibody and type I interferon (IFN) production. The goal of this study was to explore possible pathogenic roles of CD8+ T cells in lupus through characterizing DNA methylation changes.

Methods

Genome-wide DNA methylation of SLE and matched healthy control CD8+ T cells was measured using Infinium Human Methylation 450k arrays. Cell surface expression of HLA-DRB1 on CD8+ T cells with and without IFNα was measured by flow cytometry. Co-incubation of IFNα-treated CD8+ T cells with autologous naïve CD4+ T cells was performed.

Results

SLE CD8+ T cells had 188 hypomethylated CpG sites compared to healthy controls. Among the most demethylated were sites associated with HLA-DRB1 (Δβ = −0.33) and STAT1 (Δβ = −0.15). The proportion of CD8+ T cells expressing HLA-DRB1 was significantly higher in SLE compared to controls. Treatment with IFNα upregulated cell surface expression of HLA-DRB1 on CD8+ T cells of SLE patients but not healthy controls, as measured by median fluorescence intensity. Co-incubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to increased expression of the stimulation marker CD69 on CD4+ T cells in SLE patients, but not in healthy controls. There was a significant increase in STAT1 mRNA levels in both SLE patients and controls with IFNα treatment.

Conclusion

HLA-DRB1 and STAT1 loci are hypomethylated and epigenetically poised for overexpression in SLE CD8+ T cells in the presence of type-I interferon. IFNα-treated lupus CD8+ T cells stimulate autologous CD4+ T cells in vitro. These data suggest a possible pathogenic role for CD8+ T cells that is dependent upon a high type-I interferon environment in SLE patients.
Liver-specific programming of myeloid cells promotes intrahepatic immunosuppression
Authors: Guha Prajna , Gardell Jillian , Lopes Mikayla , Espat N. Joseph , Katz Steven C.
Published: January 2023

Liver is a tolerogenic organ and has variety of immune cells resulting in a profoundly immunosuppressive space. We recently reported that GM-CSF/JAK2/STAT3 axis drives liver myeloid suppressor cell (L-MDSC) proliferation and STAT3 inhibition causes activation of apoptosis signaling via Bax up-regulation. Herein, we explore liver specific programming events that promote L-MDSC suppressive conditioning. Bone marrow derived MDSC (BM-MDSC) were expanded in CD45.1+ mice in response to intraperitoneal MC38 tumors. CD45.1+ BM-MDSC were adoptively transferred into CD45.2+ recipient mice via tail vein (TV) or portal vein (PV). CD11b+ cells were harvested 48 hrs later from recipient liver and lungs. Liver from PV and lung from TV CD45.1+ MDSC recipients were compared. There was increased expansion of CD45.1+ MDSC (CD11b+Gr1+) in PV-liver as compared to TV-lung group (Liver-PV 45±3% vs. Lung-TV 15±2% vs. Tumor 43±3, p<0.001, n=5) with increased numbers of the more immunosuppressive monocytic MDSC (M-MDSC) subtype in CD45.1+ transferred cells in liver as compared to lung (Liver-PV 61±4%, Lung-TV 40±3%, Tumor 58±3 p<0.005, n=5). Enhanced pSTAT3 expression (mediator of MDSC expansion) in CD45.1+CD11b+Gr1+ cells in liver was observed as compared to lung (pSTAT3: Liver-PV 54±4, Lung-TV 30±5, p<0.05 n=5). Quantitative PCR of MDSCs isolated from Liver-PV showed significantly decreased levels (8.5 fold, p<0.05, n=4) of pro-apoptotic Bax protein as compared to Lung-TV indicating MDSC apoptosis resistance following conditioning in the liver. These data indicate that L-MDSCs are directed towards suppressive programming and blocking STAT3 may have clinical application for enhancing the efficacy of immunotherapy for liver tumors.
Spanning tree progression analysis of density normalized events (SPADE) identification of novel myeloid derived suppressor cells (MDSC) subsets
Authors: Talmadge James E. , Warkentin Phyllis , Briitton Holly , Klassen Lynell W. , Cole Kathryn
Published: January 2023

MDSC’s are a heterogeneous population of myeloid cells at various differentiation stages. The human MDSC phenotypes, are controversial and subdivided into macrophage (M-), granulocytic (G-) and immature (i-) MDSC’s. Our studies used a single staining tube with antibodies to linage markers (CD3, CD19 and CD56), HLA-DR, CD11b, CD14, CD15, CD16, CD33, CD45, PD-L1 and LOX-1. Mobilized lymphoma patient apharesis (CA) products provided a product with a high frequency of MDSCs compared to normal donor blood samples (PB). SSCA and FSA identified lymphocytes, granulocytes and monocyte populations. A total population (CD45+), MDSCs (Lin-HLA-DR-CD11b+CD14+, Lin-HLA-DR-CD11b+CD14-CD33+ and Lin-HLA-DR−CD11b+ CD15+) and non MDSC myeloid populations (Lin-HLA-DR+CD14+, Lin-HLA-DR+CD33+ and Lin-HLA-DR+ CD15+Lin-HLA-DR-11b-) were exported from FlowJo for Spade analysis. This identified 11 nodes with 4 MDSC sub-populations. Impressively, cells from the non-MDSC populations, including monocytes and granulocytes did not occur within these nodes. SPADE identified an M-MDSC, G-MDSC and two iMDSC subsets with an increased frequency in the CA samples. Increased expression of LOX-1 and CD16 occurred in CA MDSCs vs. PB. Intracellular staining for expression of NOS-2 and Arg-1 were consistent with the M and G-MDSC subsets. Node 7 mainly expressed M-MDSC’s with a low frequency of CD15 dull G-MDSC’s. The cell frequency for G-MDSC’s in node 7 was 1,334 for CA and 0 for PB with M-MDSC’s 1,079 and 43 respectively. Node 8 had a frequency (3,319) of G-MDSC’s for CA and 2,286 for PB with M-MDSC’s 188 and 15 respectively. In summary SPADE analysis can be used to cleanly identify MDSCs from monocytes and granulocytes and provide insight into novel MDSC subsets.