bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA-PKcs Regulates Transcriptional Networks in Activated Macrophages
Authors: Morales Abigail , Morales Rosalie , Dominguez Zuley A , Dilgen Teague C , Flores Melissa , Rodriguez Ricardo A , Winkler Michelle
Published: August 2023

The maintenance of genome integrity is critical for organismal survival; however, eukaryotic cells perpetually sustain various types of DNA damage. DNA double-strand breaks (DSBs) are the most insidious of these lesions, as they can lead to chromosomal translocations if they persist unrepaired. A substantial body of work has established, however, that DNA DSBs are also critical signaling intermediates that impact a variety of cell-type-specific processes. Recently published work established that upon infection with the intracellular bacterium Listeria monocytogenes, activated macrophages sustain damage in their genomic DNA. This initiates a DNA damage response (DDR) that depends on both ATM and DNA-PKcs kinases. Though ATM has been implicated in the regulation of transcriptional networks downstream of DNA damage in many different cellular contexts, few studies have directly addressed a potential role for DNA-PKcs in regulating gene expression. Thus, we asked whether DNA-PKcs kinase activity is important for regulating transcriptional responses in macrophages in settings of inflammation. In particular, we asked whether DNA-PKcs kinase activity is required for optimal activation of NF-κB. Our preliminary data suggest reduced translocation of NF-κB to the nucleus in the absence of DNA-PKcs kinase activity with a corresponding diminished expression in key NF-κB-regulated pro-inflammatory genes. As NF-κB is also activated in response to DNA damage, we suggest that in macrophages, pathogen-induced TLR signaling and DNA damage signaling synergize to drive NF-κB-mediated transcriptional responses, thereby regulating the cell’s function in the innate immune response to infection.
A Role for Stimulator of Interferon Genes in Toll-Like Receptor 8 Signaling
Authors: García-Martínez Karla Yvonne , Chen Jingyi , Leifer Cynthia A
Published: August 2023

The innate immune system is equipped with multiple receptors to detect microbial nucleic acids and induce type I interferon (IFN) to restrict viral replication. When dysregulated these receptor pathways induce inflammation in response to host nucleic acids and promote development and persistence of autoimmune diseases like Systemic Lupus Erythematosus (SLE). IFN production is regulated by the Interferon Regulatory Factor (IRF) transcription factor family downstream of several innate immune receptors such as Toll-like receptors (TLRs) and Stimulator of Interferon Genes (STING). Both TLRs and STING activate TANK-binding kinase I (TBKI) and IkappaB kinase-ɛ (IKKɛ), which phosphorylate and activate IRFs. The pathway by which TLRs and STING activate IFN response are considered independent.

Here we show that STING plays a previously undescribed role in human TLR8 signaling. Stimulation with TL8-506 (TL8), TLR8 ligand, induced IFN secretion in primary human monocytes and THP-1 cells and was reduced by inhibition of STING. Using THP-1 cells encoding reporters for IRF and NFkB activity, two major signaling pathways downstream of TLRs, we found that TL8 stimulation increased IRF and NFkB activity. When STING was inhibited, IRF, but not NFkB, activity was reduced. TL8-induced IRF activity was dependent on IKKɛ, not TBKI. Bulk RNA transcriptomic analysis show that TLR8 induces gene signatures associated with SLE which are downregulated by inhibition of STING. These data demonstrate that STING is required for full TLR8-IRF signaling.

These studies build a new framework for crosstalk between cytosolic and endosomal innate immune receptors, which could be used to treat IFN driven autoimmune diseases.
Rosiglitazone improves alveolar macrophage mitochondrial derangements induced by chronic alcohol consumption and infection with Klebsiella pneumoniae
Authors: Yeligar Samantha M
Published: August 2023

Objective

People with alcohol use disorders (AUDs) are 2–4 times more likely to develop lung infections, sepsis, and acute respiratory distress syndrome. Chronic alcohol misuse blunts alveolar macrophage (AM) immunity, in part, due to decreased peroxisome proliferator-activated receptor (PPAR)γ which increases cellular oxidative stress. Mitochondrial (MT) oxidative stress may impair AM phagocytosis; thus, we posited that chronic alcohol plus acute Klebsiella pneumoniae (Kp) dysregulates MT and treatment with rosiglitazone (RSG), a PPARγ ligand, reverses these effects to improve AM phagocytosis of Kp.

Methods

AMs were isolated from 8–10-month-old male C57BL/6J mice fed ± ethanol (EtOH, 20% wt/vol in drinking water) for 12 wks ± intratracheal 2 × 104 CFU of Kp in saline vehicle for the last 24 h of water or EtOH ± RSG (10 mg/kg/d in methylcellulose vehicle) via oral gavage 4 h prior to sacrifice (n=5/group). MT O2•− and H2O2 were assessed by fluorescence microscopy. mRNA and protein levels of PPARγ and MT dynamics mediators, transcription factor A mitochondrial (TFAM), dynamin-related protein 1 (DRP1), and mitofusin-2 (MFN2) were measured by qRT-PCR and cytoimmunostaining. Kp clearance by AMs was determined by plating lysed AMs on MacConkey agar.

Results

EtOH feeding to mice increased MT oxidative stress; decreased PPARγ, TFAM, and MFN2 levels; and increased DRP1 levels in AMs. Kp exacerbated EtOH’s MT derangements by ~51–63% and impaired Kp clearance in AMs by 57.3 ± 11.2%. RSG treatment in vivo reversed EtOH- and Kp-induced MT derangements in AMs and restored AM clearance of Kp.

Conclusion

RSG may provide a novel therapeutic strategy for mitigating AM MT derangements and immune dysfunction in AUD individuals infected with Kp.

NIAAA R01-AA026086
LRRK2 G2019S promotes the development of colon cancer
Authors: Wang Yuhang , Gao Joyce , Xiong Yiqin , Kang Zizhen
Published: August 2023

LRRK2-G2019S is the most prevalent variant associated with Parkinson’s disease (PD), found in 1–3% of sporadic and 4–8% of familial PD cases. Intriguingly, emerging clinical studies have suggested that LRRK2 G2019S mutation carriers have an overall increased risk of cancer, including colon cancer. However, it remains to be experimentally tested whether and how this mutant directly affects cancer development. Here, by using the AOM/DSS-induced colitis-associated colon cancer model combined with LRRK2 G2019S knock-in (KI) mice, we found that LRRK2 G2019S promoted the pathogenesis of colon cancer, characterized by increased tumor loads and tumor-promoting cytokines compared with those in wild-type controls. Immunoblotting and immunofluorescence indicated that expression levels of Cyclin-D1, Ki-67, β-catenin and p-STAT3 were dramatically increased in the colon of KI mice, and these proteins are well known to promote the pathogenesis of colon cancer. As inflammation is a major factor contributing to the progression of colon cancer, we thus further tested if LRRK2 G2019S promotes colitis in the initiation of the disease. Indeed, we found KI mice were more susceptible to colitis induction, evident by rapid body weight loss and elevated clinical histology score. Mechanistically, LRRK2 G2019S promoted gut inflammasome activation and enhanced the activation of the NF-ƙB and MAPK pathways upon Dectin-1 stimulation. Together, our data for the first time provide direct evidence that gain-of-function of kinase activity in LRRK2 promotes colon cancer development, implicating LRRK2 as a potential target in colon cancer, especially in those LRRK2 G2019S carrier patients and cancer with hyper LRRK2 expression.

Startup fund from the Department of pathology at the University of IOWA, NIH R21AG076895, R01 NS104164
An animal model for autoinflammation with infantile enterocolitis
Authors: Wang Yuhang , Gao Joyce Ziyi , Xiong Yiqin , Kang Zizhen
Published: August 2023

Autoinflammation with infantile enterocolitis (AIFEC) is one of the emerging life-threatening diseases caused by inborn errors of NLRC4 inflammasome. The pathogenicity of AIFEC remains largely unknown and standard therapy is lacking due to rare patient cases and high mortality. To fill the gap, it is urgent to set up an effective animal model for this rare disease. We have recently generated a loxP-flanked NLRC4 V341A conditional knock-in (KI) mouse line where human V341A (GTG to GCG) mutation was introduced into the corresponding mouse Nlrc4 coding sequence. When this line is bred with distinct Cre transgenic mice, the KI allele can be expressed globally or cell-type specifically. We bred the KI line with E2a-Cre transgenic mice and got globally expressed V341A KI mice, and the KI mice developed autoinflammation with infantile enterocolitis that recapitulated the symptoms of AIFEC patients. In brief, immunoblot demonstrated KI mice had hyperactivation of NLRC4 inflammasome in gut epithelium and macrophages. The neonatal KI pups had significant intestinal inflammation, accompanied with diarrheal, impaired barrier integrity and dysbiosis. Furthermore, the KI pups had dramatically elevated IL-1β, IL-6 and IL-18 in sera and supernatant of colon explant cultures. Meanwhile, the KI pups exhibited cytopenia, hemophagocytosis, and macrophage hyperactivation. Biochemical assays suggested multi-organ damage in KI mice, including the liver, kidney, and heart, accompanied with hyper serum ferritin level. Without any intervention, the pups died before day 10 postnatally. Taken together, we have established a novel animal model for AIFEC, thus providing a platform to elucidate the pathogenesis of AIFEC and test therapeutic strategies.

Startup fund from the Department of pathology at the University of IOWA, NIH R21AG076895, R01 NS104164
Regulation of Inflammatory NF-kB Target Gene Activation by Jak-STAT Signaling
Authors: Mishra Bikash , Ivashkiv Lionel
Published: August 2023

Macrophages are key innate immune cells that are important for host defense but their aberrant activation by IFN-γ has been implicated in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (RA). IFN-γ, signals through protein tyrosine kinases JAK1/2 to activate transcription factor STAT1 and drive the expression of interferon-stimulated genes (ISGs). IFN-γ primed macrophage induced rapid and enhanced expression of both canonical and non-canonical ISGs when challenged with toll-like receptor 4 (TLR4) ligand (LPS). However, IFN-γ alone was not able to elicit the expression of the noncanonical ISGs that are considered proinflammatory NF-kB target genes. Based on this observation, we hypothesized that any inflammatory stimuli can synergistically activate NF-kB target genes in IFN-γ primed macrophages. Like LPS, TLR2 ligand (PAM3) synergistically induced NF-kB target genes expression but inflammatory cytokine TNF-α failed to do so in IFN-γ primed macrophages. The primed macrophage is similar to RA macrophage where JAK signaling is ongoing and STAT1 is activated in tandem with inflammatory NF-kB signaling. Our model suggests that targeted inhibition of JAK1/2 could be a potential strategy to inhibit the production of inflammatory cytokines by these macrophages. To address this, we used an FDA-approved JAK1/2 inhibitor for RA. JAK inhibition effectively suppressed the expression of a subset of IFN-γ-induced genes, whereas LPS induced genes remained partially resistant to inhibition. This suggests that IFN-γ priming induces long term epigenetic memory which persist even after inhibition of IFN-γ signaling.
GATA2-TGF-b1 axis regulates human NK cell development
Authors: Wang Dandan , Myers Kasiani C , Bresnick Emery H , Verbsky James , Thakar Monica , Malarkannan Subramaniam
Published: August 2023

Natural killer (NK) cells, a subset of innate lymphocytes, produce proinflammatory cytokines and mediate anti-tumor cytotoxicity. GATA2 is a master transcription factor that is essential for the development of erythroid, myeloid, B, and NK cells. Patients with GATA2 haploinsufficiency lack CD56bright NK cells (immature), with or without a reduction in the number of CD56dim NK cells (mature). However, how GATA2 functions to establish and maintain genetic networks during the development and function of human NK cells is unknown. Here, we identify a novel GATA2-TGF-b1 axis that regulates NK cell development. We performed single-cell RNA-seq with NK cells from three GATA2T354M patients and found significantly reduced expression of immediate early genes, which indicated that they are functionally-defective. We discovered a reduction in TGF-b1 transcripts and defective expression of TGF-b1 target genes in NK cells from GATA2T354M patients. Using a reporter assay, we determined that GATA2 occupy to TGFB1 promoter, and this association was decreased in GATA2T354M NK cells. ATAC-seq of GATA2T354M NK cells indicates significantly altered chromatin accessibility. GATA2 CUT&Tag-seq validated that it occupies the TGFB1 promoter region, which correlated with activation of the TGFB1 locus in human NK cells. In summary, we define a mechanism by which GATA2 controls TGF-b1 production in human NK cells and thereby regulating their development. These findings provide vital clues for developmental and functional defects of NK cells in GATA2T354M patients.

ASH Graduate Hematology Award (D.W.); GIRT Award from MCW-C4I (D.W.); NIH R01 AI102893 and NCI R01 CA179363 (S.M.); HRHM Program of MACC Fund (S.M.), Nicholas Family Foundation (S.M.); Gardetto Family (S.M.); MCW-Cancer Center-Large Seed Grant (S.M. and M.S.T.); MACC Fund (S.M.); Ann’s Hope Mela- noma Foundation (S.M.); and Advancing Healthier Wisconsin (S.M.)
Pellino1 promotes immunity in the skin during infection with herpes simplex virus (HSV)
Authors: Jensen Liselotte E , Cai Kathy Q
Published: August 2023

Pellino1 (Peli1) is a ubiquitin ligase involved in IL-1 and Toll-like receptor signaling; however, the role of Pellino1 in skin immunity against HSV is unknown. Here, using the mouse flank HSV-1 skin infection model, we demonstrate that Pellino1 has several critical functions during active viral replication. Analyses of single cell genome wide gene expression data (RNA-seq) revealed expression of Peli1 mRNA throughout the skin including in epithelial, fibroblast and immune cells. Both histology and RNA-seq demonstrated normal development of the epidermis and dermis in Pellino1 knockout (KO) mice. The median survival of Pellino1 KO mice was 12 days compared to greater than 16 days in wild type mice. Pellino1 deficient mice also developed larger zosteriform skin lesions along affected dermatomes. During infection HSV-1 did not replicate in the dermis. Immunohistochemistry revealed delayed recruitment of myeloid and T cells to early infected epidermis in Pellino1 deficient mice. This correlated with lower expression of the inflammatory IL-1 and IL-36 cytokines and the poorly understood chemokine Grp15l. In Pellino1 KO mice, the virus spread extensively through the epidermis, follicular infundibulum into sebaceous glands where sebocytes were found positive for the virus. Histological analyses for HSV-1 and neuronal axons revealed nerve endings throughout the epidermis and axons wrapped around the hair follicles. Dissemination of HSV-1 into the sebaceous glands did not appear to involve a shift in how the virus migrated through these nerve axons as early lesions were only found in the interfollicular epidermis. In conclusion, Pellino1 plays important roles in restricting viral dissemination.

Supported by grant from NIH (R01 AI125111)
Butyrate modulation of inflammatory responses to lipopolysaccharide in porcine monocytes
Authors: Becker Sage R , Loving Crystal
Published: August 2023

Butyrate is a short-chain fatty acid produced through microbial fermentation of dietary fibers in the lower intestinal tract. Butyrate production is associated with enhanced mucosal immunity, increased barrier integrity, and modulation of immune cells. Butyrate driven immunomodulation occurs through a variety of mechanisms, including histone deacetylase (HDAC) inhibitor activity and inhibition of NF-kB signaling. Butyrate impacts lipopolysaccharide (LPS)-induced cytokine production and gene expression by porcine monocytes in a dose-dependent manner. To investigate the mechanisms driving butyrate modulation of porcine monocyte responses to LPS, peripheral monocytes were cultured with LPS +/− butyrate. After incubation, cells were processed for collection of cytoplasmic and nuclear fractions. HDAC activity in nuclear fractions and NF-kB signaling state (IkBa, p-IkBa, p65, p-p65) in cytoplasmic fractions was assessed. Butyrate did not inhibit HDAC activity after 1 hour (h) but at 24 h, butyrate exposure resulted in inhibition of HDAC activity regardless of LPS exposure, in a dose-dependent manner. Neither the low nor high concentration of butyrate altered IkBa, p-IkBa, p65, or p-p65 status, though p-IkBa and p-p65 status was dependent on LPS exposure. Overall, the impact of butyrate on HDAC activity was time and dose-dependent, and butyrate had little to no effect on NF-kB signaling induced by LPS exposure. Continued investigation is warranted to understand the potential mechanisms driving butyrate modulation of monocyte responses to LPS to enhance abilities to modulate immune status.

USDA-ARS CRIS 005
Investigating SARS-CoV-2 ORF3a binding to human TRAF2 and TRAF3
Authors: Busscher Brianna M , Befekadu Henock , Liu Zhonghua , Wang Chuanping , Xiao Tsan Sam
Published: August 2023

Severe COVID-19, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is characterized by widespread inflammation and cytokine storm. A number of viral proteins are capable of inducing inflammatory responses, one of which is open reading frame 3a (ORF3a). ORF3a from SARS-CoV shares 73% homology with SARS-CoV-2 ORF3a and was shown to stimulate NF-κB signaling and inflammasome activation in a manner dependent on TNF receptor-associated factor 3 (TRAF3). TRAF family proteins regulate NF-κB activation and thereby influence inflammatory responses. Both ORF3a homologs contain a TRAF-binding consensus sequence, and SARS-CoV ORF3a has been shown to bind to human TRAFs 2, 3, and 6. We therefore investigated the binding of SARS-CoV-2 ORF3a to TRAF proteins as a potential mechanism through which SARS-CoV-2 may activate inflammatory signaling.

TRAF-C domains from TRAFs 2 and 3 were purified and co-crystallized with a peptide from ORF3a containing the TRAF-binding sequence (PIQAS). Fluorescence polarization (FP) was used to assess binding affinity between TRAF-C domains and ORF3a peptide, and dual-luciferase assays were used to measure NF-κB activation in cells transfected with wildtype ORF3a or ORF3a in which the TRAF-binding sequence was mutated to alanines. X-ray crystallography revealed that the ORF3a peptide is able to bind to the TRAF-C domain of TRAFs 2 and 3, and FP indicated that the binding affinity for these interactions is low. When we compared NF-κB activation in HEK293T cells expressing WT vs mutant ORF3a, there was no significant difference. Therefore, while ORF3a has the potential to bind to TRAFs, this interaction does not significantly contribute to inflammatory signaling through NF-κB in our assay.

This work is supported by NIH grants R01GM127609 and P01AI141350.
CD68+ macrophages are associated with adipogenesis after murine muscle injury and fibrotic material implantation
Authors: Josyula Aditya , Ngo Tran B , Destefano Sabrina , Lokwani Ravi , Fertil Daphna , Faust Mondreakest T , Sadtler Kaitlyn
Published: August 2023

Murphy roths large (MRL) mice are known to regenerate skin and skeletal muscle wounds without scar formation but are also susceptible to metabolic dysfunction and obesity. How biomaterials influence immune cell recruitment and immune-adipocyte interaction after muscle injury in MRL mice is yet to be delineated. To investigate this, we performed volumetric muscle loss surgeries (VML) in C57BL/6 (B6) and MRL mice and evaluated immune responses to injury, regenerative (decellularized extracellular matrix, ECM) and fibrotic (polyethylene, PE) material implants using multiparametric flow cytometry, RNA sequencing, and histopathology. In MRL but not B6 mice, we observed white fat deposition at the site of VML. This was correlated with enrichment of adipogenesis gene sets including Adipoq, Cebpa, Pparg and Acsl1. In addition, F4/80-CD68+ macrophages were significantly more abundant in MRL mice at 3 weeks post-injury in PE and saline groups. The same treatments also highly potentiated expression of the lipid associated macrophage gene signature comprising Trem2, Ly6c2, Cd9, Cd63 and Lyz2. Ly6G+ neutrophils were the second most abundant cell type (~ 25% of CD45+ cells) which was corroborated by increased abundance of Vav1, Ccl2, Adam8 and Rac2. In contrast, ECM implantation produced a largely SiglecF+ eosinophilic response (~ 60% of CD45+ cells) and inhibited CD68+ macrophage prevalence and also potentiated expression of the pro-regenerative Arg1, Chil3, Retnla and Gata3 in both B6 and MRL. Taken together, our results suggest a mechanistic role for CD68+ macrophages in promoting adipogenesis after VML. Moving forward, we will analyze adaptive immune responses to injury and material implants using abovementioned methods.

Intramural Research Program, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health
Injury-induced hair growth response is promoted by tissue macrophages via insulin-like growth factor 1
Authors: Ayush Otgonzaya , Goal Shubham , Viosin Benjamin , Nadella Vinod , Kim Do Young , Sakamoto Keiko , Nagao Keisuke
Published: August 2023

Hair follicles (HFs) produce hair to mediate the first line of defense in mammals. HFs undergo cyclical production of hair (anagen), and while this process is unsynchronized during adulthood, external cues, including shaving, induce synchronized anagen and bolster the surface barrier. Herein, we show that macrophages were crucial in promoting anagen. Shaving triggered the accumulation of macrophages to the secondary hair germs (SHG), which underwent proliferation as early as Day3 post-induction. Constitutive ablation of macrophages markedly delayed anagen, and targeted ablation of macrophages at Day 3, but not at Day 0 or Day 6 post-shaving, impaired SHG activation with delayed anagen. Anagen entry was dependent on Folr2+ tissue macrophages, but not on CCR2+ monocyte-derived macrophages. Single-cell RNA-sequencing and cell-cell communication analyses revealed that Folr2+ macrophages communicated with the SHGs via Igf1 (encoding insulin-like growth factor 1 or IGF1). Ablation of macrophages and deletion of IGF1 from macrophages decreased AKT signaling in SHG and led to delayed anagen, both which were rescued via injection of recombinant IGF1 in vivo. Single-cell ATAC-sequencing revealed that the lack of IGF1-from macrophages resulted in alteration of transcription factor motif enrichment and decreased chromatin accessibility in various genes in SHGs, demonstrating that macrophages primed the SHGs to enter anagen by promoting IGF1R signaling. These results highlight the importance of immune-tissue crosstalk in facilitating hair growth and provide impetus for strategies to promote hair growth.
A novel Fth1-mScarlet reporter mouse supports quantification of monocyte-derived macrophage inflammatory state in vivo
Authors: Michalides Brandon Alan , Shoger Karsen E , Cheemalavagu Neha , Gottschalk Rachel A
Published: August 2023

Monocyte-derived macrophages (mo-macs) acquire a spectrum of pro- and anti-inflammatory functions during infection-associated inflammation and resolution. Quantification of mo-mac functional state is critical to elucidate mechanisms leading to dysregulation of inflammatory control and how these processes can be manipulated therapeutically. Given that binary M1/M2 cell markers often fail to explain macrophage function in vivo, we sought to identify genes predictive of inflammatory gene signatures. We analyzed published single cell RNAseq of macrophages during lung inflammation, using Lasso (L1-penalized linear regression) to weight genes according to their ability to predict all other genes, and identified Fth1 (Ferritin Heavy Chain 1) as the most predictive gene. We generated Fth1-mScarlet reporter mice using CRISPR/Cas9 technology, inserting mScarlet in-frame of Fth1, linked through a T2A self-cleaving peptide. Using live cell imaging in vitro, we found monocyte mScarlet expression peaked 24 hours after stimulation with S. aureus or TLR ligands. We are currently using monocyte adoptive transfer to track mo-mac fate during acute S. aureus lung infection. In this intra-nasal S. aureus model (strain FDA309), pro-inflammatory genes and infiltration of neutrophils and monocytes peak 1–2 days post infection. We transferred Fth1mScarlet bone marrow monocytes 6 hours after infection, analyzed their recruitment into the lungs, and quantified mScarlet expression over time. Increased mScarlet expression was associated with the transition of donor cells from Ly6C+CD11c-monocytes to Ly6C-CD11c+ mo-macs. This model will allow us to quantify the plasticity and persistence of mo-macs during inflammation and resolution.

R35 GM146896 (PI - Gottschalk)
GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation
Authors: Hwang Daniel , Ishikawa Larissa Lumi Watanabe , Seyedsadr Maryam S , Mari Elisabeth , Kasimoglu Ezgi , Sahin Ziver , Boehm Alexandra , Jang Soohwa , Rasouli Javad , Vaccaro Courtney , Gonzalez Michael , Hakonarson Hakon , Rostami Abdolmohamad , Zhang Guang-Xian , Ciric Bogoljub
Published: August 2023

GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.

This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (T32 training grant T32AI134646).
Accessory cells precondition naive T cells and regulatory T cells for cytokine-mediated proliferation
Authors: Sato Noriko , Bamford Richard N , Bryant Bonita R , Tagaya Yutaka , Waldmann Thomas A
Published: August 2023

Naïve T cells and regulatory T cells, when purified, do not proliferate to the common cytokine receptor γ-chain family cytokines interleukin (IL)-2, IL-7 or IL-15, despite their expression of cognate cytokine receptors. Cell-to-cell contact with dendritic cells (DCs) enabled proliferation of the T cell to these cytokines, independent of antigen recognition or T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in mice depleted of DCs. We propose calling this a “preconditioning effect”. Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2-target genes. Preconditioning was necessary to activate these two pathways, and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation and prolonged S6 phosphorylation occurred, and T cells underwent proliferation. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells.
Treatment of pulmonary fibrosis through normalization of alveolar macrophages
Authors: Baek Jiyeon , Kim Minji , Kwon Byungsuk
Published: August 2023

Idiopathic pulmonary fibrosis (IPF) is a chronically advanced interstitial lung disease with unclear etiology and poor prognosis. Despite numerous studies, the pathogenesis of IPF has not been completely understood. In this study, we searched for a drug that can decrease chronic lung inflammation without deteriorating lung fibrosis. We found that fenofibrate, a PPARα agonist that is clinically used to control dyslipidemia, had therapeutic effects on pulmonary fibrosis through normalization of alveolar macrophages. We first showed that 6 days of oral administration of fenofibrate blocked the progression of bleomycin-induced pulmonary fibrosis and resolved inflammation. In addition, fenofibrate suppressed the LPS-induced pro-inflammatory mediators in bone marrow-derived macrophages in a PPARα-independent manner; however, it promoted the expression of tissue repair and anti-inflammatory genes. Finally, we examined the expression of the pro-inflammatory, tissue-repair, and anti-inflammatory genes in alveolar macrophages at days 2 or 6 after oral administration of fenofibrate in mice with advancing pulmonary fibrosis. Administration of fenofibrate resulted in decreased expression of proinflammatory cytokines and increased expression of tissue repair and anti-inflammatory genes at day 2. However, the gene expression pattern of alveolar macrophages of fenofibrate-treated mice at day 6 became similar to that of naïve alveolar macrophages. Taken together, this study suggests that fenofibrate can rapidly reprogram inflammatory macrophage polarization toward normal state through a transient resolving and anti-inflammatory macrophage polarization stage, which is linked to amelioration of pulmonary fibrosis.
CCL3-deficient HLA-DP2 Transgenic (Tg) mice are protected against Be-induced inflammation in the lungs
Authors: Atif Shaikh M , Falta Michael T , Martin Allison , Tucker Rebecca , Mack Douglas G , Palmer Brent E
Published: August 2023

Chronic beryllium disease (CBD) is an inflammatory lung disorder caused by the recruitment and expansion of pathogenic CD4 T cells. In a murine model of Be-disease, we reported the accumulation of CCL3/Be and CCL4/Be-specific CD4 T cells in the lungs of BeO-exposed HLA-DP2 Tg mice (WT). However, the addition of a single dose of LPS on day 14 (d14) in BeO-exposed WT mice, resulted in a significant increase in the release of chemokines (CCL3 and CCL4), increased accumulation of chemokine/Be-specific CD4 T cells and formation of cellular aggregates in the lungs. In the present study, we explored the loss of chemokine(s) CCL3/4 on the pathogenesis of CBD using HLA-DP2 Tg-CCL3-deficient mice (CCL3-DP2 Tg), which were also found to be deficient in CCL4. CCL3-DP2-Tg mice exposed to BeO+LPS (d14) caused a significant reduction in the accumulation of CCL3/4/Be-specific CD4 T cells in the lungs and the formation of cellular aggregates compared to the WT mice. Also, the ELISPOT assay revealed a significant reduction in the IFNγ and IL-17-producing CD4 T cells in the lungs of BeO-exposed CCL3-DP2-Tg mice. Next, we examined the source of CCL3 in the lungs of BeO-exposed WT mice. CCL3 is majorly contributed by CD11c+ dendritic cells compared to the other cells of the hematopoietic compartment. No or little staining of CCL3 was observed in the stromal compartment. Bone marrow transfer from WT to CCL3-DP2 Tg mice showed the role of the hematopoietic compartment in both the accumulation of CCL3/4-specific CD4 T cells and the formation of cellular aggregates in the lungs after Be-exposure. Overall, we showed that hematopoietic cell-derived CCL3 or CCL4 plays an important role in the pathogenesis of CBD, and the lack of CCL3 and CCL4 protects against Be-induced lung damage.

NIH RO1 HL152756
Post-thymic Thpok-deficiency in CD4 T cells compromises control of Toxoplasma gondii infection
Authors: Vacchio Melanie S , Clark Alex , Bouladoux Nicolas , Belkaid Yasmine , Bosselut Remy
Published: August 2023

Toxoplasma gondii, an intracellular parasite, is estimated to chronically infect >25% of the world-wide human population. While generally kept in check by the host immune system, reactivation of infection can occur when individuals become immunocompromised, particularly with T cell deficiencies. Both CD4+ and CD8+ T cells play significant roles in controlling this parasite. The transcription factor Thpok, is critical for commitment of MHC Class II-restricted thymocytes to the CD4 lineage during thymic differentiation, but also plays a significant post-thymic role in shaping the effector function of mature CD4+ T cells. Based on these findings, we interrogated how mice with post-thymic deletion of Thpok manage a chronic infection using Toxoplasma gondii (“Toxo” hereafter). During acute infection, Thpok-deficient mice have equivalent expansion of Toxo-specific T cells compared to WT littermate controls, but these CD4 T cells have undergone “cytotoxic diversion”, upregulation of a CD8 transcriptional program and loss of CD4 helper function. During the chronic phase of infection Thpok-deficient mice die (~3 weeks p.i. vs >12 weeks for WT) and exhibit increased Toxo cysts in the brain compared to WT littermates, suggesting deficient control of the parasite. This death in the early chronic phase is not simply due to lack of CD4+ T cell help, as Class II-deficient mice survive significantly longer into the chronic phase of Toxo infection (~6 weeks, p.i.). This data indicates that Thpok-deficient CD4+ T cells are far worse at controlling Toxo than complete absence of CD4+ T cells. Studies are currently in progress to address the mechanism by which Thpok-deficiency dysregulates parasite control by CD4+ T cells.

Intramural National Institutes of Health
A pathogenic role for regulatory T cells in chronic liver disease
Authors: Arpaia Nicholas , Savage Thomas M , Fortson Katherine T , de los Santos Alexis Kenia , Alsina Angelica Oliveras , Rouanne Mathieu , Gamarra Jennifer R , Cavero Renzo , Li Fangda , Haeusler Rebecca A , Adam Julien , Schwabe Robert F
Published: August 2023

Current dogma holds that regulatory T (Treg) cells preserve tissue function in settings of inflammation and damage through their anti-inflammatory activity and production of epithelial growth factors – the latter of which is independent from immunosuppression. Consistent with this, we observe that Treg cells – in particular those producing the epidermal growth factor receptor (EGFR)-ligand amphiregulin (Areg) – are enriched in the livers of mice and humans with non-alcoholic steatohepatitis (NASH). Mouse and human liver Treg cells undergo substantial transcriptional changes in chronic liver damage, reflecting their increased activation; however, rather than playing a protective role, we find that Treg cell–derived Areg promotes NASH-induced liver fibrosis through the direct activation of EGFR on hepatic stellate cells. Clinically, NASH is closely linked to insulin resistance, but the mechanistic contributions of NASH-induced disease processes to insulin resistance remain unclear. We further observe that Treg cell–derived Areg promotes glucose intolerance in a NASH-dependent manner – through the induction of hepatic gluconeogenesis – a process also mediated through EGFR signaling on hepatic stellate cells. Mechanistically, Treg cell–derived Areg promotes hepatocyte gluconeogenesis through hepatocyte detection of soluble mediators produced by Areg-activated hepatic stellate cells – offering new insight into how chronic liver disease promotes insulin resistance. Taken together, we provide the first evidence that Treg cells mediate a maladaptive role in tissue injury, finding that their production of a growth factor plays a central role in liver disease and promotes liver fibrosis and glucose intolerance in NASH.

This research was supported by grants from NIH (K22 AI127847, R01 HL148718, R01 CA259634, T32 GM007367, P30 CA013696, P30 DK132710) and the Searle Scholars Program (SSP-2017-2179).
IL-4 acts through aryl hydrocarbon receptor to antagonize TLR7 induced double negative 2 B cells in lupus
Authors: Mountz John D , Lu Changming , Barnes Stephen , Wilson Landon , Gao Min , Chatham Winn W , Hsu Hui-Chen
Published: August 2023

We recently showed that in systemic lupus erythematosus (SLE), IL-4R signaling is a powerful antagonist that can effectively suppress the development of activated naïve (aNAV) and CD11c+T-bet+ IgD−CD27− double negative 2 (DN2) B cells promoted by both type I and type II IFNs. In the present study, we used the BXD2 mouse model of lupus to determine the mechanism of IL-4 in suppressing the development of DN2 B cells in vivo. Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentages of CD11c+T-bet+ IgD−B cells. Feature-barcoding single cell RNA-sequencing analysis showed that IL-4 modulated B cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23+CD21− follicular B cells. IL-4 induced the gene encoding interleukin-4-induced1 (IL4i1), an enzyme that metabolizes aromatic amino acids and this was associated with the upregulation of aryl hydrocarbon receptor (AhR) and downstream genes. Metabolomics analysis revealed IL-4 induced AhR agonistic metabolites in B cells including kynurenine (Kyn), indole-3-acetic acid, and indole-3-lactic acid. In the absence of IL-4, Kyn and a potent AhR agonist, formylindolo[3,2-b]carbazole (FICZ), significantly suppressed TLR7 plus IFNβ-induced DN2 B-cell development in vitro. Our results suggest that IL-4 acts through the IL4i1-AhR pathway to inhibit B-cell regulatory response to TLR7 and type I IFN. Identification of small molecular metabolites that act directly in B cells to induce homeostasis may lead to development of orally dosed metabolome modulating therapeutics efficacious in the treatment of SLE.

This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311.
Bacterial catabolism of tryptophan to indole is required to induce collagen induced arthritis via the IL23/Th17 axis
Authors: Seymour Brenda , Allen Brendan , Trent Brandon , Berlinberg Adam , Jubair Widian , Colgan Sean , Kuhn Kristi
Published: August 2023

While intestinal dysbiosis is linked to the pathophysiology of inflammatory arthritides like rheumatoid arthritis (RA) and spondyloarthritis (SpA), the mechanisms by which this occurs are unknown. Therefore, we used the collagen-induced arthritis (CIA) model to connect the microbiome with bacteria-produced metabolites in the development of disease. After initially correlating CIA severity with bacteria-dependent alterations in dietary tryptophan metabolism, and specifically indole, we demonstrate that both bacteria and dietary tryptophan are required for disease, and that indole supplementation fulfills these requirements. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β and Th17 cells. These findings were associated with increased anti-CII IgG2b, decreased anti-CII antibody sialyation, and increased complement fixation, suggesting that indole-mediated Th17 immunity may enhance autoantibody pathogenicity. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in the IL-17 signaling pathway in B and T cells. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of Th17 immunity. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.

Supported by grants from NIH: NIAMS R01AR075933 (KAK), NIAMS T32AR007534 (WKJ, BT, AJB), NIAID T32AI007405 (BJS), NIAID F30AI174817 (BJS); and the Rheumatology Research Foundation: Future Physician Scientist Award (BJS) and Scientist Development Award (AJB).
Targeting glutamine metabolism mitigates Foxp3-deficiency mediated disease
Authors: Zhong Yuelin , Hill Charlotte Nicole , Charbonnier Louis-Marie
Published: August 2023

Regulatory T (Treg) cells play a key role in enforcing peripheral immunological tolerance to self-antigens. Foxp3 has been widely described as a master regulator for Treg differentiation and maintenance of their suppressive functions. Upon Foxp3 loss of function mutations, Foxp3-deficient Tregs (ΔTreg) develop in the thymus, however, these cells present impaired suppressive capacity and an effector T cell (Teff)-like phenotype. Among the Teff-like characteristics is the shift in their metabolism towards glycolysis and enhanced mitochondrial respiration. Metabolomic analysis revealed an increase in glutamine consumption. Here, we used 6-diazo-5-oxo-norleucine (DON), a broad inhibitor of glutamine-utilizing enzymes to treat Foxp3 deficient mice (Foxp3ΔeGFPiCre), and observed a significant decrease in Teff cell activation, and IFNγ production from both ΔTreg and Teff cells together with a significant increase in survival. This effect was enhanced when we combined DON treatment with a ΔTreg specific Rictor depletion (Foxp3ΔeGFPiCre RictorΔ/Δ). Given that glutamine is a key amino acid for T cell activation, we evaluated if this was a pan-T cell effect by inducing Treg depletion in Foxp3DTR mice with concomitant DON treatment. Surprisingly, in the absence of Tregs, DON was not able to prevent autoimmune disease. These results suggest that DON mitigates Foxp3-deficiency mediated disease in a ΔTreg dependent manner, by improving their suppressive capacities. We are currently performing metabolomic studies to better understand the role of glutamine usage in ΔTreg, more specifically if glutamine is directed to glutaminolysis, nucleotide or glutathione synthesis and how this contributes to Treg cell loss of function.

NIH grant 1R01AI153174
Nanotechnology-based drug delivery for targeted therapy of autoimmune arthritis improves the therapeutic profile of anti-arthritis drugs
Authors: Moudgil Kamal D , Nanjaiah Hemalatha , Venkatesha Shivaprasad H , Meka Rakeshchandra R
Published: August 2023

Rheumatoid arthritis (RA) is among the major human autoimmune diseases, and it affects over 1 percent of the population. Although many anti-arthritis drugs are available for RA therapy, their long-term use may lead to severe adverse effects. This, combined with high cost of many newer drugs, poses a hurdle for the effective control of RA as well as patient compliance with therapeutic regimens. Therefore, novel treatment modalities are required to overcome these limitations. In this context, we have developed a nanotechnology-based drug delivery system for targeted therapy of arthritic inflammation. Liposomal entrapment of drugs increases their shelf-life in vivo, whereas the display of a joint-homing peptide ligand on liposomal surface aids in guiding them to the site of inflammation. One such peptide (denoted as ART-1) was previously identified by us by phage peptide-display library screening of arthritic Lewis rats. When administered subcutaneously or intravenously, fluorescence-labeled ART-1 shows preferential homing to arthritic joints compared to normal (control) joints. We exploited this drug delivery system for the treatment of experimental arthritis with dexamethasone (Dex) using the rat adjuvant arthritis (AA) model. At the time of onset of AA, rats were treated with liposomal Dex, unpackaged (free) Dex, or the vehicle on alternate days. All rats were observed and graded regularly for arthritis severity. Sera obtained at the terminal step were tested for a panel of enzymes indicating tissue/organ toxicity. Liposomal Dex showed improved therapeutic profile (efficacy/toxicity ratio) over that of free Dex. We propose that this peptide ligand-targeted drug delivery approach can be adapted for effective control of human RA.

“Supported by grants from NIH (R01 AT004321), Veterans Affairs (5 I01 BX002424) and Silo Pharma”
Efficacy and safety of AK111, in patients with active ankylosing spondylitis: results from a randomized, double-blind, placebo-controlled, phase II clinical study
Authors: Li Zhanguo , Xia Yu , Wang Max , Li Baiyong , Ni Xiang , Wang Guoqin , Zhang Li
Published: August 2023

Background

As an inflammatory cytokine that can induce the repair of barrier tissue, IL-17 not only participates in the anti-infection immune response, but also plays a key role in some chronic inflammatory diseases, including Ankylosing spondylitis (AS). Inhibition of IL-17A can reduce the disease activity of AS and reduce joint damage. AK111 is a humanized IgG1 monoclonal antibody against IL-17A.

Methods

A total of 120 patients (male and female) with age ranging from 18 to 75 years were enrolled. There were 4 cohorts (AK111: 75mg, 150mg, 300mg and placebo) in this study. Subjects were randomized in a 1:1:1:1 ratio to receive one dose of AK111 or matching placebo.

Results

The Assessment Of SpondyloArthritis International Society Criteria For 20% Improvement (ASAS20) response rates of AK111 treatment groups at week 16 were 80.6% (25/31), 71.9% (23/32) and 66.7% (20/30) respectively, which were significantly higher than placebo group 37.5% (12/32) (as same as the ASAS 40 response rates of those groups). All dose groups of AK111 can improve ASAS20/ASAS40 response rate at week 16.

125 subjects enrolled in the study. 99(79.2%) subjects experienced at least one treatment-emergent adverse event (TEAE). 63(50.4%) subjects had TEAE related to study drug (TRAE), including 49(52.7%) subjects in AK111 groups and 14(43.8%) subjects in placebo group. The most of TEAE/TRAE were mild (80.6% and 52.7%) and recovered without any intervention. 2(1.6%) subjects with serious adverse event (SAE). Investigator assessed the SAEs were non-related and possible-related to study drug. No death was reported.

Conclusion

AK111 was generally safe and well tolerated, also able to improve ASAS20/ASAS40 response in Chinese subjects with active ankylosing spondylitis.