bluuebunny/crossref_metadata_2025 huggingface dataset:
Oscillatoria sp. LPS activates human monocytes in a manner that is distinct from E. coli LPS
Authors: Swanson-Mungerson Michelle Anne , Incrocci Ryan , Subramaniam Vijay , Radowska Kinga , Hall Mary , Williams Philip , Mayer Alejandro
Published: January 2023

Within the gut-associated lymphoid tissue (GALT) reside monocyte/macrophages that sample products from normal flora and pathogens. After consumption of the pathogen cyanobacteria, toxins and lipopopolysaccharide (LPS) from this bacterium can induce fever, gastro-intestinal illness, and even death. The toxicity of one species of cyanobacteria, Oscillatoria sp. (Osc-sp.), is not well understood. Previous studies from our laboratories demonstrate that Osc-sp. LPS activates murine B cells, but to a lesser extent than E. coli LPS, due to a decrease in NF-kB and a loss of IRF-3 activation. Since ingestion of Osc-sp. exposes resident GALT monocyte/macrophages to the LPS of Osc-sp., we tested the effects of this cyanobacteria’s LPS on human monocytes. Due to our previous findings we hypothesized that Osc-sp. LPS would activate human monocytes, but may change the predominant functions and cytokines produced in comparison to E. coli LPS. Therefore, we purified monocytes from purchased human buffy coats and activated the cells in the absence or presence of Osc-sp. LPS or E. coli LPS. Our data indicate that Osc-sp. LPS activates monocytes to proliferate and upregulate markers important for T cell activation to a lesser extent that E. coli LPS. In contrast, Osc-sp. LPS was able to induce greater phagocytosis of FITC-Dextran and produce a different cytokine environment when compared to E. coli LPS. These findings indicate that Osc-sp. may be more effective in activating certain functions of human monocytes than E. coli LPS, in contrast to its effects on murine B cells. As cyanobacteria, such as Osc-sp., continue to contaminate our fresh water sources, our understanding of the toxic effects of these bacteria on humans and animals will be critical.
A functional androgen-regulated subset lacking KLRG1 contributes to the sex bias in ILC2s
Authors: Kadel Sapana , Ainsua-Enrich Erola , Hatipoglu Ibrahim , Turner Sean , Singh Simar , Khan Sohaib , Kovats Susan
Published: January 2023

Humans show significant sex differences in the incidence and severity of respiratory diseases including asthma and virus infection. Sex hormones contribute to the female sex bias in type 2 inflammation associated with respiratory diseases, including greater numbers of group 2 innate lymphoid cells (ILC2s). We determined if sex hormone levels govern sex differences in the numbers, phenotype and function of ILC2s in the murine lungs and bone marrow (BM). Our data show that lungs of female mice harbor significantly greater ILC2 numbers in homeostasis, in part due to a major subset of ILC2s lacking killer-cell lectin like receptor G1 (KLRG1), a population largely absent in male lungs. The KLRG1− ILC2s were capable of type 2 cytokine production and increased with age after sexual maturity, suggesting that a unique functional subset exists in females. Experiments with gonadectomized mice or mice bearing either global or lymphocyte restricted estrogen receptor alpha deficiency showed that androgens rather than estrogens regulated these ILC2 phenotypes in the lungs. While BM ILC2 numbers were similar in both sexes, male levels of androgens led to a significant population of functional KLRG1+ ILC2s. However, mice with abnormally elevated androgens showed a reduced frequency of BM ILC2s with decreased expression of the transcription factor GATA-3 required for the development of ILC2s. Furthermore, the numbers of BM PLZF+ILC precursors (ILCPs) were higher in males and increased by excess androgens, suggesting that androgens act to inhibit the transition of ILCPs to ILC2s. Taken together, these data show that a functional subset of KLRG1− ILC2s in females contributes to the sex bias in lung ILC2s that is observed after reproductive age.
Centrifuge-less immunostaining of suspension cells for flow cytometry analysis by DA-Cell washer and plate for superior data and workflow
Authors: Kim Namyong , Lye Melvin
Published: January 2023

We describe the DA-Cell™ system, a novel wall-less plate and laminar-flow cell washer that enables the automated washing of suspension cells and retains more than 95% cells at a fraction of the time and with higher viability of cells. The wall-less DropArray (DA) plate consists of an array of 96 hydrophilic spots separated and surrounded by a hydrophobic surface, which functions as a virtual wall. In a typical immunostaining assay, a 50 μl drop, containing cells and antibody mix, is dispensed on each spot of the DA plate. During incubation, cells settle on the surface of the spots. The plate then undergoes a laminar-flow washing process in DA-Cell washer by repeated cycles of aspiration and dispensing of buffer through two sets of nozzles. The controlled buffer flow minimizes turbulence and cell loss. The cell washing process only requires 3–4 minutes by eliminating the need of a centrifuge, which also reduces stress on the cells and possible cross-contamination of antibodies on cell membranes, leading to better segregation of cell populations in flow cytometry. Additional incubation time of 10–20 minutes improves cell retention to more than 99%. Most importantly, cell incubation and washing on a DA-Cell system minimizes operator variability as mixing and washing steps are mechanically controlled and mostly automated, significantly improving reproducibility and consistency of flow cytometry analysis.

Here, we show a series of immunostaining assays comparing the DA-Cell system with a conventional centrifugation. Based on the staining index, absolute cell counts and additional data, we demonstrate that the DA-Cell system produces superior data while simplifying and expediting cell preparation for flow cytometry analysis.
Biologically Inspired, iterative engineering of a Human Lymphoid Follicle Chip
Authors: Goyal Girija , Long Jaclyn , Levy Oren , Ingber Donald E.
Published: January 2023

Organs Chips are microengineered, three-dimensional (3D) in vitro models that simulate organ-level pathophysiology and therapeutic responses with high fidelity. Here, we report the development of an in vitro model of lymphoid follicles seen in germinal centers and sites of chronic inflammation. The Human Lymphoid Follicle Chip was designed to mimic lymph flow through the lymph node as well as its cellular and matrix composition. A subcapsular sinus like channel allows the media to flow around the follicles with a fraction of it flowing through the parenchyma. We provide the first known evidence that 3D organization, cellular density and physical forces such as interstitial flow play a role in formation of tertiary lymphoid organs and germinal centers. Like tertiary lymphoid organs, CXCL3 production by lymphocytes is observed in the follicle chip concomitant with the aggregation of T and B cells. The follicles formed in vitro recapitulate the gross expansion and production of antibodies and cytokines seen in vivo in response to bacterial stimulation. While trying to incorporate vasculature into the follicle on chip, we found that our in vitro cultures mimic the killing of allogeneic endothelial cells, a clinically important phenotype in graft rejection that previously couldn’t be modeled without humanized mice.

Mice and conventional two-dimensional (2D) cultures of circulating human immune cells, even together, can fail to replicate human biology, leading to low efficacy and unpredicted, sometimes severe toxicity in clinical trials. In future experiments, the Follicle Chip will enable the assessment of candidate therapeutics, and specifically immunotherapies, in a patient-specific manner in vitro.
STAT3 regulation of effector Th17 cells and its implications for treatment of autoimmunity
Authors: Raphael Itay , McGeachy Mandy J.
Published: January 2023

Th17 cells are implicated in the pathogenesis of many autoimmune and inflammatory diseases, including multiple sclerosis (MS) and rheumatoid arthritis. Early Th17 cell development relies on key cytokines that signal through STAT3, including IL-6, IL-21 and IL-23. These signals are critical for gain of effector Th17 cell functions, including the expression of the inflammatory cytokines IL-17, IFN-γ and GM-CSF. Thus, development of drugs that target STAT3 signaling are a major research focus to alleviate disease. However, the functions of STAT3 signaling post Th17 cell development in late effector cells remain elusive. Here, we developed a model to study STAT3 deficiency effects specifically in late effector Th17 cells, termed Th17ΔSTAT3. We show that Th17ΔSTAT3 mice are resistant to development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To determine mechanisms by which STAT3 regulates Th17 cell pathogenicity we performed bioinformatics analysis of effector Th17 cell transcriptome. Corresponding to changes in gene expression, we found that Th17ΔSTAT3 cells were reduced in numbers in lymph nodes and CNS during EAE. We demonstrated that STAT3 is critical to drive G1 to S phase transition of the cell cycle by regulating key cell cycle genes. Moreover, we found that STAT3 maintains Th17 cell mitochondrial integrity. Unexpectedly, our data show that lineage specific gene expression, including cytokines, is not altered in Th17ΔSTAT3 cells. However, Th17ΔSTAT3 cells have reduced IL-17 expression, but not IFN-γ or GM-CSF upon antigen restimulation. Our data reveal hitherto unknown mechanisms for STAT3 in regulating effector Th17 cells, which have major implications for use of Th17 cell-targeted therapies.
Cdc42: A New therapeutic target in drug-resistant multiple myeloma
Authors: Nguyen Thi Thuy Phuong , Chakrabart Jayati , Guo Fukun
Published: January 2023

Multiple myeloma (MM) is an incurable hematologic malignancy essentially due to acquired drug resistance, highlighting the need for alternative therapeutic strategies. Here we report that CASIN, a Cdc42 GTPase selective inhibitor, significantly inhibited cell proliferation and survival of both melphalan/bortezomib-sensitive and melphalan/bortezomib-resistant MM cell lines. Intriguingly, while CASIN was less efficient than melphalan/bortezomib in killing melphalan/bortezomib-sensitive cells, it was much more potent than melphalan/bortezomib in killing melphalan/bortezomib-resistant cells. Combined CASIN and melphalan/bortezomib treatment revealed that CASIN sensitized melphalan/bortezomib-resistant cells to melphalan/bortezomib. Mechanistically, Cdc42 activity was significantly higher in melphalan-resistant MM cells than that in melphalan-sensitive counterparts, and CASIN inhibited melphalan-induced ubiquitination of FANCD2 of the Fanconi anemia DNA repair pathway in melphalan-resistant MM cells, but not in melphalan-sensitive cells, sensitizing melphalan-resistant MM cells to melphalan-induced DNA damage. On the other hand, CASIN inhibited EGFR/STAT3/ERK pathway in bortezomib-resistant cells, but not in bortezomib-sensitive cells. An ERK inhibitor acted similarly to CASIN. ShRNA Cdc42-mediated Cdc42 knockdown mimicked CASIN effect, suggesting that CASIN didn’t cause off-target effect. CASIN had minimum effect on normal B cells and peripheral blood mononuclear cells, suggesting that its side effect is negligible. Taken together, our data have demonstrated a proof of concept that targeting Cdc42 represents a promising approach in overcoming MM drug resistance.
Cell-intrinsic and spatially divergent tumor programmed death ligand 1 (PD-L1) signals modify local and systemic anti-tumor immunity through novel chemokine effects
Authors: Curiel Tyler J. , Gupta Harshita B. , Padron Alvaro , Pandeswara Srilakshmi , Sareddy Gangadhara , Yuan Bin , Reyes Ryan M. , Turk Mary Jo , Hurez Vincent , Li Rong , Vadlamudi Ratna , Clark Curtis A.
Published: January 2023

PD-L1, expressed on many tumors, inhibits anti-tumor T cells by programmed death (PD)-1 engagement. Tumor PD-L1 predicts anti-PD-L1 treatment outcomes through incompletely understood mechanisms. We used naturally PD-L1+ murine B16 melanoma (ctrl) and made PD-L1KO B16 by CRISPR/Cas9. PD-L1KO B16 grew faster when ctrl B16 was on the opposite flank (trans). By contrast, ctrl B16 grew slower when trans to PD-L1KO B16. Anti-PD-L1 slowed ctrl but not PD-L1KO B16 tumors as expected. However, when PD-L1KO was trans to ctrl αPD-L1 now also slowed PD-L1KO growth. Anti-PD-L1 increased CD3+ T cell infiltration into ctrl and PD-L1KO tumors similarly in vivo, but increased natural killer (NK) cell numbers and functions (e.g., CD107a, IFN-g) in PD-L1KO tumors > ctrl. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl, but NK cells migrated 2-fold more to PD-L1KO vs ctrl. PD-L1KO tumors produced more CCL2 than ctrl in vitro and in vivo, and this CCL2 induced CCR2+ NK chemotaxis in vitro. Both NK cells and T cells were required for optimal trans responses to anti-PD-L1 treatment in vivo. In wild type mice, anti-CCL2 reduced growth of trans PD-L1KO tumors > ctrl, and reduced anti-PD-L1 efficacy against PD-L1KO> ctrl tumors. In CCR2KO mice, anti-PD-L1 efficacy was retained against trans ctrl B16, but abolished against PD-L1KO trans tumors. PD-L1 also regulated other chemokines recruiting receptor+ cells, with similar effects seen in MB49 bladder cancer and 4T1 breast cancer cells. Thus, tumor PD-L1 alters immune infiltrates and anti-PD-L1 efficacy through novel chemokine effects. Our models help understand anti-PD-L1 (and likely anti-PD-1) responses based on tumor PD-L1 expression and can define ways to improve immunotherapy for PD-L1 null tumors.
Enhanced killing of triple-negative breast cancer cells by blocking LLT1-CD161 inhibitory signal to NK cells
Authors: Marrufo Armando Magallanes , Mathew Stephen O. , Chaudhary Pankaj , Mathew Porunelloor Abraham
Published: January 2023

Triple-negative breast cancer (TNBC) is known to be the most invasive form of breast cancer due to the lack of expression of estrogen, progesterone, and human epidermal growth factor-2 receptors, which makes it difficult to eradicate tumor growth with conventional chemotherapeutic and hormonal treatments. TNBC accounts for approximately 20 percent of all breast cancer cases and is associated with poorer prognosis and higher incidences of relapse. Hence, natural killer cell-mediated immunotherapy is a promising option for treatment of TNBC. Natural killer cells (NK) are innate lymphoid cells that magnifies its role within the immune system to recognize and lyse infected and tumor cells. NK cells regulate its own function through its receptors interacting with activating and inhibitory ligands on target cells. Lectin-like Transcript-1 (LLT1, CLEC2D, OCIL) is a counter-receptor that interacts with CD161 (NKRP1A) and suppresses NK cell activation. We have identified the expression and function of LLT1 on TNBC cell lines MDA-MB-231 and MDA-MB-436 by flow cytometry, western blot, immunofluorescent microscopy, and chromium-release assay. Blocking LLT1 on TNBCs with antibodies disrupts interaction with CD161 and enhanced NK cell-mediated lysis of TNBCs. We have also shown that gene knockdown of LLT1 decreased cell surface expression of LLT1 and increased lysis of TNBCs. Our results demonstrate that expression of LLT1 on TNBCs function in evasion from immunosurveillance by NK cells. Blocking interaction of LLT1-CD161 on TNBCs activates NK-mediated lysis and will potentially open a new immunotherapeutic strategy for patients diagnosed with TNBC.
Metastasis-specific, NK cell-mediated, immune surveillance of lung cancer
Authors: Chockley Peter , Keshamouni Venkateshwar
Published: January 2023

Natural Killer (NK) Cells are the first line of defense against virally transformed and malignant cells. Failure of immune surveillance and clearance allows for cancer initiation and progression.(Ribatti, 2017; Swann and Smyth, 2007) Solid tumor progression to a metastatic stage is associated with end stage disease and worsening patient prognosis. Metastasis is a coordinated and complex process that is initiated by epithelial-mesenchymal transition (EMT).(Lambert et al.) EMT allows epithelial cancer cells to trans-differentiate into highly-motile, invasive, mesenchymal-like cells giving rise to disseminating tumor cells. Only few of these disseminated cells successfully metastasize. Immune cells and inflammation in the tumor microenvironment was shown to drive EMT, but few studies investigated the consequences of EMT on tumor immune surveillance.(Chockley and Keshamouni, 2016)

In addition to initiating metastasis, we demonstrate that EMT confers increased susceptibility to NK cells and contributes, in part, to the inefficiency of the metastatic process. Depletion of NK cells allowed spontaneous metastasis without effecting primary tumor growth. EMT-induced modulation of E-cadherin and cell adhesion molecule 1 (CADM1) mediated increased susceptibility to NK cytotoxicity. Higher CADM1 expression correlates with improved patient survival in two lung and one breast adenocarcinoma patient cohorts and decreased metastasis. Our observations reveal a novel NK-mediated, metastasis-specific, immune surveillance in lung cancer and presents a window of opportunity for the prevention of metastasis by boosting NK cell activity.
Type II interferon signaling plays a critical role in inducing T helper type 1 immune responses and adaptive immunity after influenza vaccination
Authors: Kim Ki-Hye , Lee Young-Tae , Hwang Hye suk , Ko Eun-Ju , Lee Youri , Jung Yu-Jin , Kang Sang-Moo
Published: January 2023

The roles of interferons (IFN) remain poorly understood in generating long-term protective immune responses to vaccination despite their well-known anti-viral innate immunity. We investigated the roles of IFN receptor (IFNR) signaling in inducing adaptive immune responses and protective immunity to influenza virus-like particle (VLP) vaccination by using A129 and AG129 mouse models. AG129 mice showed a significant defect in raising IgG2a Th1 antibody responses despite of the induction of comparable IgG1 Th2 type and hemagglutination inhibiting antibodies compared to those in A129 mice. After lethal challenge infection, the AG129 vaccinated mice were not protected as evidenced by 20% severe weight loss, high airway hyperresponsiveness, histopathology, excessive infiltration of neutrophils, inflammatory chemokine and cytokines, and 80 folds higher lung viral titers compared to the A129 vaccinated mice that were 100% protected without displaying weight loss. AG129 mice were not able to induce rapid recall of plasma cells, germinal center B cells, and IFN-r+ CD4 and CD8 T cells. Depletion of T cells in A129 mice after vaccination resulted in lower efficacy of protection. The role of type I IFNR were determined in AB6 mice by comparing with B6 mice after vaccination. The AB6 mice did not show significant defects in developing IgG1 and IgG2c antibodies and in conferring protection after vaccination and challenge, despite of inability to recruit monocytes, compared to those in B6 mice. These results suggest that type II IFNR signaling plays a more critical role in developing adaptive immunity after vaccination than type I IFNR signaling, and that both type I and II IFNR signaling is required for effective control of influenza virus infection.
IL-1 cytokines promote ADAMTS-mediated remodeling of the lung extracellular matrix during severe influenza A infection in mice
Authors: Boyd David F. , Guo Xizhi , Thomas Paul G.
Published: January 2023

The lung extracellular matrix (ECM) is extensively remodeled following respiratory viral infection. This remodeling occurs during inflammation when immune cells migrate to the site of infection and during repair as the ECM is modified to regenerate damaged tissue. Matrix proteases, including the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) gene family, play a critical role in these remodeling processes. The immune mechanisms that regulate matrix protease activity and their effect on host responses to lung infection have not been well characterized. Previously, we found that ADAMTS-4 knockout mice exhibited improved survival following lethal influenza A (IAV) challenge and signs of reduced immunopathology, including fewer numbers of inflammatory T cells. Here, by flow cytometric sorting of immune and stromal cell populations, we identified fibroblasts as the main producers of ADAMTS-4 in the lung during severe IAV infection, with ~50-fold higher levels of mRNA compared to other lung cell populations. Stimulation of primary lung fibroblasts in vitro with a wide range of inflammatory cytokines demonstrated that only IL-1β and IL-1α induced expression of ADAMTS-4. Co-stimulation of lung fibroblasts with IL-1 cytokines and TNF-α resulted in a synergistic effect and an increase of ADAMTS-4 expression to greater than 6-fold above baseline levels. IL-1 treatment of lung fibroblasts also induced expression of the lung ECM proteoglycan versican (VCAN), a major enzymatic target of ADAMTS-4. These results suggest a role for IL-1 cytokines in regulating lung ECM remodeling, and highlight the potential of modulating the IL-1/ADAMTS-4 pathway to reduce immunopathology following severe respiratory infection.
Metformin alleviates liver injury through mTOR-mediated T cell regulation in viral hepatitis in mice
Authors: Sun Jiaren , Liang Yuejin , Xu Lanman
Published: January 2023

Metformin is not only the first-line medication for the treatment of type 2 diabetes, but also has other effects, such as anti-inflammatory, anti-oxidative, and anti-tumor activities, in animal models. However, the therapeutic potential of metformin during viral hepatitis is uncertain. In this study, C57BL/6 mice were treated with oral administration of metformin (250 mg/kg) for 7 days daily, infected with different doses of adenovirus (i.v. inoculation), and then received metformin for another 7 days. All mice were sacrificed at 7 days post-infection. We found that metformin treatment ameliorated the liver injury induced by different doses of adenovirus infection, as judged by the significant decrease in liver alanine transferase levels, liver histopathology scores, as well as intrahepatic lymphocyte (IHL) and splenocyte counts. Compared to the PBS-treated groups, metformin-treated mice had significantly decreased numbers of activated T cells in the liver, as well as expression of IL-2, IFN-γ, and TNF-α in CD4+ and CD8+ T cells, especially among CD8+ T cells. Metformin treatment also significantly reduced phosphorylated S6 kinase beta 1 (S6K1)-positive CD4+ and CD8+ T cells in the liver, indicative of decreased mTORC1 activity in these cells. These data indicate that metformin can alleviate adenovirus-induced acute liver injury in mice, and that the beneficial effects of metformin may be mediated in part through mTORC1 pathway in T lymphocytes.
Bacterial amyloids from biofilms induce type I interferon response in plasmacytoid dendritic cells
Authors: Qiu Connie Claire , Gallucci Stefania
Published: January 2023

An increased expression of type I interferon (IFN) regulated genes is a hallmark of systemic lupus erythematosus, a complex autoimmune inflammatory disease. Although the exact etiology of lupus is unknown, infections can be an important environmental trigger for flares, and are a major cause of morbidity and mortality. Medically, bacterial biofilms are important for the establishment of chronic, persistent infections. Bacteria within these aggregates are embedded into their biofilms, allowing them to dramatically increase their ability to withstand physical insults and tolerate host defenses. To strengthen the extracellular matrix of their biofilms, bacteria produce amyloids, complex proteins with a conserved beta sheet structure. We have reported that Salmonella and E. coli biofilms contain the functional amyloid protein curli, which complexes with eukaryotic and bacterial DNA. Systemic administration of curli-DNA complexes accelerated autoimmunity in lupus-prone mice. Infections with curli-expressing Salmonella or E. coli gave the same results, implicating curli as a pathogenic factor in how biofilm-producing infections may contribute to lupus pathogenesis. We present here for the first time that bacterial amyloid curli activates plasmacytoid dendritic cells (pDCs), the primary producer of type I IFNs, in vitro. Additionally, we show that curli induces a robust production of IFNα and expression of interferon-stimulated genes, including CXCL10. Furthermore, curli induces in vivo activation of DCs in lupus-prone and wild-type mice. Our results identify bacterial amyloid curli as a novel activator of pDCs, and suggest that curli may accelerate lupus by directly activating pDCs and their powerful type I IFN activity.
A protective MHC class II molecule prevents autoimmune diabetes by selecting for specific intestinal microbes early in ontogeny
Authors: Silverman michael A. , Denu Lidiya , Lubin Jean-Bernard
Published: January 2023

Type 1 diabetes (T1D) affects millions of people worldwide. The incidence has risen significantly over the past few decades. Both genetic and environmental factors contribute to T1D. Certain HLA haplotypes such as DRB1*1501-DQB1*0602 dominantly protect against T1D yet the mechanism of this remarkably protection remains unknown. We recently demonstrated that the protective major histocompatibility complex (MHC) E molecule prevents autoimmune diabetes in NOD mice by shaping the intestinal microbiome early in ontogeny. This finding presents a novel paradigm in which genetic protection from immune system dysregulation is mediated via commensal microbiota. An important gap in knowledge is which intestinal microbes prevent autoimmunity by modulating the immune system. We hypothesize that those microbes that differentially stimulate humoral immune responses in the previously reported MHCII E-expressing NOD (Eα16/NOD) mice may also modulate the immune system to prevent autoimmunity. We address this question by leveraging the exquisite sensitivity and specificity of the humoral immune system to identify a subset of microbes via flow cytometry that stimulate mucosal and/or systemic immune responses to prevent autoimmunity.
Bacterial amyloids from biofilms break tolerance in lupus by simultaneous BCR/TLR signaling
Authors: Lee Michael Hweemoon , Tursi Sarah A. , Nicastro Lauren K. , Caricchio Roberto , Tükel Çagla , Gallucci Stefania
Published: January 2023

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease, in which infections are considered to play a pathogenic role, but the cellular and molecular culprits remain unknown. Bacterial biofilms are multicellular bacterial communities important in the establishment of chronic infection by pathogens. We have previously shown that curli fibers, functional bacterial amyloids present in Salmonella and E. coli biofilms, can complex with DNA. Systemic administration of these curli/DNA complexes accelerated autoimmunity in lupus-prone mice and induced autoantibody production in wild-type mice, suggesting curli/DNA complexes as novel players in SLE pathogenesis. This affect was diminished in TLR2 or TLR9 deficient mice. We present here evidence that curli/DNA complexes polyclonally activate B cells in vivo in wildtype mice, lupus-prone mice and 3H9 mice, the latter expressing an anti-DNA Ig heavy chain and whose B cells are normally tolerized. Interestingly, curli/DNA complexes can activate B cells in the absence of T cell help. Indeed, curli/DNA complexes induce polyclonal proliferation and activation in B cells purified from wild-type and lupus prone mice in vitro. Interestingly, curli/DNA complexes also induce isotype switching and aicda, the master regulator of class switch recombination, in the absence of T cells help in vitro. The induction of non-canonical NFkB in the absence of T cells suggests that the fibrillar structure of curli/DNA complexes can cross-link BCR, some recognizing DNA. Our results suggest that curli/DNA complexes may induce anti-DNA antibody production by simultaneous BCR/TLR signaling, which leads to B cell antibody production in the absence of T cell help.
Systemic recognition of immunogenic insulin peptides promotes T cell diabetic autoimmunity in multiple lymphoid tissues
Authors: Wan Xiaoxiao , Zinselmeyer Bernd , Zakharov Pavel , Vomund Anthony , Hu Hao , Lichti Cheryl , Unanue Emil R.
Published: January 2023

Recognition of insulin is essential for initiating autoimmune diabetes in the non-obese diabetic (NOD) mice; however, how this autoantigen is presented to pathogenic T cells remains unclear. In patients with type 1 diabetes, insulin autoantibodies (IAAs) are the earliest sign of disease. In NOD mice IAAs are detected before immune infiltration in islets, suggesting that insulin autoreactivity originates from peripheral lymphoid sites. This is confirmed by our previous studies showing formation of insulin-specific germinal centers in multiple lymph nodes accompanied by differentiation of cognate T follicular helper cells, indicating widespread insulin recognition. In this study, we used a two-photon imaging system that robustly and accurately detected presentation of the limited amounts of insulin peptide-MHC complexes. We demonstrate that insulin recognition by CD4 T cells takes place concomitantly in various lymph nodes, a process precisely guided by the specific epitope but not the autoimmune environment. Importantly, a unique set of insulin-reactive T cells specifically recognized free insulin peptides but not the conformational protein. During chronic antigen recognition, these T cells exhibited an effector but not exhaustion phenotype, manifested by transcriptional activation, metabolic reprogramming and enhanced pathogenicity. These findings reveal a unique pattern of insulin recognition that may account for its prime role in triggering autoimmune diabetes.
CX43 is essential for optimal cGAS function during cytosolic DNA-sensing
Authors: Nagarajan Uma , Zhang Yugen , Bodnar Wanda , Kiatthanapaiboon Amy , Hagan Robert , O’Connell Catherine , Tripathy Manoj Kumar
Published: January 2023

During intracellular Chlamydia infection, cytosolic DNA is sensed by cyclic GMP-AMP synthase (cGAS) that catalyzes the synthesis of cGAMP from ATP and GTP. cGAMP is a key mediator of IFN beta expression during DNA sensing and can also transfer to adjacent uninfected cells through intercellular connexin gap-junctions to induce intrinsic immunity. In this study, we investigated the role of gap junction protein connexin 43 (CX43) during Chlamydia infection. Using RNA-in situ hybridization, we discovered a distinct intracellular contribution of CX43 in IFN beta expression in the infected cells and cells transfected with DNA, a function distinct from its intercellular gap-junction role affecting IFN beta expression in adjacent uninfected cell. CX43 is essential for IFN beta expression during DNA-sensing upstream of STING activation, but not required during RNA-sensing. CX43 co-localized with transfected DNA and on the chlamydial inclusion membrane with cGAS. CX43-depleted cells showed significantly reduced cGAMP production during DNA-sensing. CX43 forms hemichannels on intracellular membrane, which open and close to allow nucleotide transfer. Blocking this transport across CX43 hemichannel reduced IFN beta expression during infection and DNA-sensing, without altering CX43 localization. These results uncover a novel role of CX43 in cGAMP synthesis by cGAS during cytosolic DNA-sensing.
The scaffolding protein IQGAP1 interacts with Nucleotide Binding Domain Leucine Rich Repeat CARD containing protein (NLRC3) and inhibits type I interferon production
Authors: Davis Beckley K. , Tocker Aaron M. , Durocher Emily , Talento Suzanna M. , Trieschman Kate , Rechnitzer Alma A. , Jacob Kimberly , Roberts David M.
Published: January 2023

Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I interferon. One of several upstream receptor cGAS (cyclic-GMP-AMP synthase) binds to cytosolic DNA and generates di-cyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to Stimulator of Interferon Genes (STING). STING recruits TANK binding kinase 1 (TBK1) which acts as a critical node that allows for efficient activation of interferon regulatory factors (IRFs) to drive the anti-viral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine rich repeat containing protein (NLR) that negatively regulates the type I interferon pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I interferons. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two hybrid screening. Here we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3:STING interaction in the cytosol of human epithelial cells. Furthermore, knock down of IQGAP1 in THP1 and HeLa cells causes significantly more interferon-b production in response to cytosolic nucleic acids. This result phenocopies NLRC3 deficient macrophages and fibroblasts and shRNA knock down of NLRC3 in THP1 cells. Our findings suggest IQGAP1 is a novel regulator of type I interferon production possibly via interacting with NLRC3 in human monocytic and epithelial cells.
Myeloperoxidase Deficiency Attenuates Dietary and Systemic Iron-induced Toxicity
Authors: Xiao Xia , Saha Piu , Yeoh Beng San , Kumar Matam Vijay
Published: January 2023

Background

Iron is highly reactive and could induce oxidative stress via augmenting the activity of heme enzymes, i.e., NADPH oxidase and myeloperoxidase (MPO). Unlike the membrane-restricted NADPH oxidase, MPO remains active upon its release into circulation and thus may potentially exacerbate iron-induced oxidative damage.

Objective

To investigate the extent to which MPO is involved in iron-induced toxicity.

Methods

Acute toxicity was induced by intraperitoneal injection of FeSO4 to MPO deficient (MPOKO) mice and their WT littermates. In some studies, WT mice were pretreated with 4-aminobenzoic acid hydrazide (a MPO inhibitor). Chronic toxicity was induced by feeding 2% carbonyl iron-diet to WT and MPOKO mice. Iron status, MPO levels, inflammatory (SAA, lipocalin 2, KC, IL-6 and IL-1β), organ damage (AST, ALT) and oxidative stress (MDA) markers were assessed.

Results

Acute iron administration upregulated circulating MPO in WT mice, which may be due to increased neutrophil degranulation or oxidative burst. However, the loss of MPO activity in mice, either via inhibition or genetic deficiency, markedly reduced iron-induced organ damage and systemic inflammation. In contrast, long-term iron feeding to WT mice reduced circulating MPO, but promoted its accumulation in liver. While both MPOKO and WT mice showed similar levels of diet-induced hyperferremia, MPOKO mice displayed significantly reduced inflammatory response and oxidative stress.

Conclusion

Genetic deficiency or pharmacologic inhibition of MPO substantially attenuated acute and chronic iron-induced toxicity. Our results suggest that targeting MPO during iron overload may be a promising approach to reduce iron-induced toxicity/side effects.
An IL-2/STAT5/Eos regulatory axis promotes Th1 differentiation.
Authors: Sreekumar Bharath Krishnan Nair , Read Kaitlin A. , Powell Michael Duane , Baker Chandra E. , Zafar Jawad , Martin Emily , Allen Irving C. , Oestreich Kenneth J.
Published: January 2023

Members of the Ikaros Zinc Finger Transcription Factor (IkZF) family are known regulators of hematopoiesis, including the development of CD4+ T helper cell populations. Our recent work established that the IkZF family member Aiolos cooperates with STAT3 to positively regulate Bcl-6 expression in T follicular helper (Tfh) cells. Given the conservation between members of the STAT and IkZF families, and their roles in T helper cell development, we hypothesized that additional IkZF/STAT interactions may regulate the differentiation of other T helper cell subsets. Here, we find that IL-2 signaling induces expression of the IkZF factor Eos in Th1 cells. Knockdown of Eos results in a significant reduction in the expression of signature Th1 genes including Prdm1, Il2ra, and Ifng. Mechanistically, we find that Eos and STAT5 physically interact and form a transcriptional complex that is capable of inducing Prdm1 expression. In support of these data, we also find that Eos and STAT5 are co-enriched at the regulatory regions of Th1 genes including Prdm1 and Il2ra. Taken together, these findings are suggestive of a novel, cooperative role for the IkZF family member Eos and STAT5 in regulating the Th1 differentiation program.
Differing effects of TGFβ family members in the generation of IL-9-secreting T cells
Authors: Ulrich Benjamin Joseph , Kaplan Mark H.
Published: January 2023

The development of Th subsets results from cellular and cytokine cues that are present in the inflammatory environment. The developing T cell is able to integrate multiple signals from the environment, and those signals sculpt the cytokine-producing capacity of the effector T cell. Even when cytokines activate similar intracellular signaling pathways, the T cell can discriminate the cytokine signal to generate distinct outcomes. IL-9-secreting Th9 cells regulate multiple immune responses, including immunity to pathogens and tumors, allergic inflammation, and autoimmunity. In combination with IL-4, both TGFβ and Activin A promote IL-9 production, yet it is not clear if both cytokines generate Th9 cells with identical phenotype and function. We observed that in contrast to TGFβ that efficiently represses Th2 cytokines in Th9 cultures, Activin A produced a multi-cytokine phenotype with secretion of IL-4, IL-5, IL-13, IL-10, in addition to IL-9. Ongoing experiments will define the function of these populations of cells in vivo. These observations suggest that although TGFβ and IL-4 were identified as cytokines that stimulate optimal IL-9 production, they might not be the cytokines that generate optimal function from Th9 cells in immunity and disease.
Egr2 is required for the activation of the TH17 pathogenetic program
Authors: Gao Yuanyuan , Wang Yan , Bouladoux Nicolas Jean , Belkaid Yasmine , Lazarevic Vanja
Published: January 2023

CD4+ TH17 lineage are a heterogenous population of CD4+ effector cells; some of which have been guardians of tissue homeostasis while others have been linked to tissue inflammation and autoimmunity. TH17 cells can alter their differentiation program ultimately giving rise to either protective non-pathogenic or pro-inflammatory pathogenic cells. Molecular switches that control the development of “pathogenic” versus “non-pathogenic” TH17 cells remain largely unknown. In this study, we identified the transcription factor Egr2 as a critical regulator of the TH17 pathogenic program. We found Egr2 was transiently expressed in the early-intermediate stage (24–48 hours) of TH17 differentiation. When ectopically expressed under TH17-polarizing conditions, Egr2 significantly enhanced the expression of TH17 signature genes in a RORγt-dependent manner. Although Egr2 was dispensable for TH17 lineage commitment, its expression was required for the generation of pathogenetic TH17 cells. Mice with T cell-specific deletion of Egr2 were less susceptible to experimental autoimmune encephalomyelitis (EAE) than WT mice. Myelin-specific Egr2-deficeint TH17 cells failed to induce a chronic inflammatory response in the CNS. Transcriptional analysis revealed that Egr2 promoted the pathogenicity of TH17 cells by regulating the expression of pathogenicity-associated genes. Interestingly, Egr2 was not required for the effector function of protective gut-resident TH17 cells in response to Citrobacter rodentium challenge. These findings indicate that Egr2 represents an attractive candidate for the therapeutic targeting of pathogenic TH17 cells while preserving tissue-protective functions of TH17 cells at barrier sites.
The role of tumor-derived exosomes in the formation of a pre-metastatic niche in cancer
Authors: Morrissey Samantha M. , Kong Xiangyu , Noe Jordan , Yan Jun
Published: January 2023

Purpose

Determine if tumor derived exosomes (TDE) can drive cancer progression through polarization of immunosuppressive cell phenotypes capable of restricting αβ T cell functioning. Preliminary data shows that exosome stimulation differentially upregulates PD-L1 (programmed-death-ligand-1) expression on peritoneal macrophages and lung resident γδ T cells while also upregulating PD-1 expression on αβ T cells. The ultimate goal is to assess whether elimination of TDE can synergize with current PD-1/PD-L1 treatments by removing antagonizing factors and improving clinical outcomes.

Methods

TDE were isolated using serial ultracentrifugation from Lewis Lung Carcinoma cells. Exosome stimulated peritoneal macrophages and γδ T cells were co-cultured with ova-transgenic –I (CD8+) T-cells to assay for suppressive function. Anti-PD-1 antibody was added to the culture to confirm that the PD-1/PDL-1 axis mediates the immune suppression. The effect of TDE on γδ T cell metabolism was also investigated. Lastly, human peripheral blood samples from lung cancer and anti-PD-1 treated lung cancer patients were compared via flow cytometry for γδ/PD-L1 expression profiling.

Results

The results suggest that TDE play a role in tumor metastasis by polarizing γδ T cells and macrophages to an immunosuppressive PD-L1+ phenotype capable of restricting αβ T cell effector functioning.

Discussion/Conclusion

TDE could be directly antagonizing the effects of immune checkpoint therapies. Therefore, future experiments will focus on eliminating exosome secretion by using siRNARab27 inhibition and the small drug compound, cambinol, concurrently with anti-PD-1 or anti-PD-L1 monoclonal antibody in efforts to increase progression free survival.
Immunogenicity of diffuse intrinsic pontine glioma (DIPG)
Authors: Marquez-Manriquez Juan P. , Fernandez Lorena Montijo , BRISENO JOSE ANTONIO MATUTE , MONTANO GEOBANNI TORRES , VERBURG MARK L. , CAMACHO-HERNANDEZ ALEJANDRO , LUCERO-DIAZ PEDRO-ALEJANDRO
Published: January 2023

Background

Little information exists about the potential immunogenicity and immunotargets for DIPG. Most of the pediatric neurotumors are mainly unexplored immunologically. We studied twenty repository samples of DIPG tumors. We selected two relevant immune markers to demonstrate immune infiltration such as CD8 and Tbet and eight biologically and clinically relevant proteins found in aggressive neoplasias including DIPG. According with this preliminary data DIPG is immunogenic and six of eight relevant proteins were overexpressed. This data will open the possibility to treat this lethal tumor with active antigen-specific immunotherapy either alone and in combination.

Methods

N=20 repository DIPG pediatric tumors were analyzed by immunohistochemistry (IHC). Moreover we analyzed by granzyme B and IFN-gamma ELISPOT the naturally occurring immune response against predicted Th1 and CD8 epitopes from the eight proteins and we performed univariate and multivariate analysis using SAS/SAT and prism.

Results

six of eight bad prognosis proteins were overexpressed as following RCAS1 (80%), Fascin-1 (90%), EGFR (55%), VCP (85%), Ape-1 (95%) and Sox2 (70 %). We found 35% of expression of CD8 cells and 40% of Tbet, which is an interesting finding in terms of immune infiltration in DIPG. CD8 antigen specific by Granzyme ELISPOT against Ape-1, RCAS1 and Fascin-1 were significant as well (P=0.0001), (p=0.005) and (9=0.0001), respectively.

Discussion

DIPG is immunogenic according with our preliminary data and we will expand the number of samples in order to validate a large repository and move to the clinic. We predicted immunogenic peptides for each protein using repository PMBC’s and most of them were recognized by autologous PBMCS’s.
MR image-guided focused ultrasound immune modulation for glioma therapy
Authors: Sheybani Natasha Diba , Witter Alexandra R. , Bullock Timothy N. , Price Richard J.
Published: January 2023

Glioblastoma (GB) is the most common and malignant brain tumor. Despite standard treatment with surgery, radiation and chemotherapy, its diffuse nature and proclivity for recurrence renders it largely intractable. Immunotherapy (ITx) approaches (e.g. anti-PD1) may hold promise for treating GB; however, the blood-brain (BBB) and blood-tumor (BTB) barriers hinder delivery of systemically administered ITx drugs. A potential approach to enhancing ITx delivery is MRI-guided focused ultrasound (FUS), a non-invasive technique that, when combined with concomitant systemic injection of microbubbles (MB), can transiently disrupt the BBB/BTB and mechanically perturb the tumor microenvironment. Here, we investigate whether localized BBB/BTB disruption with FUS+MB enhances anti-tumor immune responses and inhibits tumor growth, as a prelude to eventual combination with immune checkpoint blockade. One week after FUS+MB (peak negative acoustic pressure=0.6 MPa) treatment of a murine glioma model stably transfected with luciferase (GL261-luc2), CD86 mean fluorescence intensity on dendritic cells (DC) increased ~3-fold in deep cervical lymph nodes, intratumoral CD4+ T cells doubled, and intratumoral CD8+ T cells increased by ~17% (by flow cytometry). Serial bioluminescence imaging of tumors revealed significant reduction in total photon flux as early as 6 days following FUS+MB (p=0.0106), indicating tumor growth inhibition. We conclude that FUS+MB can promote DC maturity and potentially mediate adaptive immunity against glioma, independent of drug delivery. Ongoing studies entail combining FUS+MB with anti-PD-1 delivery to evaluate whether an allied treatment approach can promote an even more robust anti-glioma response.
A modest change in regulation of NOD1 expression has a major impact on inflammation and gastric cancer
Authors: Subramanian Naeha , Rommereim Leah M. , Dutta Bhaskar , Akhade Ajay Suresh , Hutcheon Carolyn , Rostomily Clifford , Lounsbury Nicolas , Savan Ram , Fraser Iain D.C. , Germain Ronald N.
Published: January 2023

Altered protein dosage by defects in single genes leads to haploinsufficiencies and monogenic disorders, but the impact of small changes in gene expression on multifactorial disease is unknown. Here we show that a persistent small (~1.5 fold) increase in expression of NOD1 (Nucleotide-binding oligomerization domain-containing protein 1), a key innate sensor of bacterial infection, precipitates a large physiological effect with dramatically altered cellular function associated with carcinogenesis. Inhibition of miR-15b and miR-16 microRNA function leads to a ~1.2–1.4 fold increase in NOD1 protein concentration, with even slightly greater increases leading to ligand-independent, switch-like NOD1 activation. miRNA regulation of NOD1 plays a crucial role in limiting the development of an inflammatory state is impaired in gastric cancer with a small increase in NOD1 being associated with greater early patient mortality. Overall, our data show that tight control of NOD1 expression by miRNA prevents this sensor from exceeding a physiological switching checkpoint that promotes persistent inflammation and lethal cancer progression, and reveal the impact of a single and modest cellular alteration on cancer. These findings also have broader implications for understanding how small expression changes caused by genetic variation impact development of complex diseases like autoimmunity and cancer.