bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polysaccharide capsule allows Group B Streptococcus to avoid killing in a newborn pneumonia model
Authors: Lund Sean J. , Patras Kathryn A. , Kimmey Jacqueline M. , Yamamura Asami , Hernandez Gilberto , Lakhdari Omar , McCoy Alyssa M. , Nizet Victor , Prince Lawrence S. 
Published: January 2023

Abstract
Group B Streptococcus (Streptococcus agalactiae, GBS) causes severe infections in neonates. While not a common adult pathogen, GBS is the leading cause of congenital pneumonia. To determine the molecular mechanisms of neonatal susceptibility, we developed a murine GBS pneumonia model. Neonatal, juvenile, and adult C57BL/6 mice were inoculated intranasally with 2800 CFU/g of GBS (COH1). After 7 d, survival was 100 % in adults and juveniles, with 10 % of neonates dying within 3 d after infection. Histopathological assessments showed severe lung injury scores 24 h after infection, with adult and juvenile mice demonstrating resolution of injury at 3 d post infection and normal histology after 7 d. In contrast, neonatal lungs had persistent lung injury up to 7 d post infection. By FACS, GBS induced rapid neutrophil recruitment into adult lungs and increased CD11b expression on adult lung macrophages. In neonatal lungs, GBS neither increased neutrophil numbers nor altered macrophage marker expression. Confocal imaging showed GBS in adult lungs almost entirely localized to alveolar macrophage phagolysosomes. However in neonatal lungs, GBS was only rarely phagocytosed by macrophages. These data were consistent with GBS killing data, which showed complete GBS killing within 24 h in adult and juvenile mice, but persistent GBS viability within the lung for up to 3 d in neonates. Defective GBS killing in neonates appeared to require the GBS capsule. Neonatal mice were much more efficient in killing the ΔcpsD capsule mutant compared to wild type GBS. Adult mice showed equal killing of ΔcpsD or wild type GBS. Our data suggest that the GBS capsule prevents killing by the newborn lung innate immune system, leading to neonatal disease susceptibility.
Recruited macrophages fail to restrict intracellular growth of Mycobacterium tuberculosis
Authors: Jang Jessica , Ernst Joel D. 
Published: January 2023

Abstract
Mycobacterium tuberculosis (Mtb), a bacterium that predominantly afflicts the lungs, infects over 10 million people per year and causes 1.7 million deaths per year. Immediately following infection, subsets of pulmonary myeloid cells will become infected with Mtb, starting with alveolar macrophages, and eventually spreading to neutrophils, recruited macrophages, monocytes and dendritic cells. These cells differ in their ability to restrict intracellular growth of Mtb. Using reporter Mtb that constitutively express mCherry and express GFP under the tetracycline promoter, we can differentiate between live Mtb (mCherry+ GFP+) and dead Mtb (mCherry+) by flow cytometry. We show that a larger percentage of CD11b+ recruited macrophages and Ly6C+ monocytes in the lungs contain live Mtb and fewer dead Mtb compared to other myeloid populations. This has been validated by sorting infected myeloid cells by flow cytometry and plating colony forming units (CFU). Recruited macrophages averaged 5,000–8,000 live bacilli per 1,000 sorted cells, while alveolar macrophages, neutrophils and dendritic cells harbored 1,000–2,000 live bacteria/1,000 sorted cells. Furthermore, genome-wide transcriptional profiling of these sorted myeloid populations reveal substantial differences in anti-bacterial mechanisms, metabolism and chemokine production between recruited macrophages and other myeloid populations. These include changes in production of interferons, lysosomal genes and tumor necrosis factor superfamily. Altogether, these data reveal that recruited macrophages are unable to restrict the intracellular growth and create a favorable environment for Mtb growth through their deficiency in anti-bacterial cytokines.
T cell IFNγ production is restricted within pulmonary tuberculosis granulomas
Authors: Gern Benjamin Henry , Plumlee Courtney R. , Gerner Michael Y. , Urdahl Kevin B. 
Published: January 2023

Abstract
The direct interaction between CD4 T cells and Mycobacterium tuberculosis (Mtb) infected cells presenting their cognate antigen on MHCII is crucial for optimal bacterial control. Using quantitative imaging (QIM) and a physiologic ultra-low dose (ULD) Mtb infection in mice to visualize this interaction, we have found that while T cell receptor (TCR) signaling in CD4 T cells occurs throughout Mtb-infected lungs, IFNγ production is impaired near infected cells. To examine the antigen-specificity of this observation, we co-transferred Th1-polarized Mtb ESAT-6-specific CD4 cells (C7) and OVA-specific (OTII) CD4 T cells into mice 35 days post ULD Mtb infection. The next day, lungs were harvested for phenotypic and spatial analysis of TCR signaling (using IRF4) and IFNγ production. QIM revealed TCR signaling and IFNγ production in Mtb-specific T cells, but minimal TCR signaling and IFNγ production in control T cells with irrelevant specificity for OVA. Positioning data showed that TCR recognition of Mtb ESAT-6 occurred throughout the lung, including within the center of the granuloma, where most Mtb bacilli are observed. Despite this, only 1–2% of ESAT-6-specific T cells within the granuloma produced IFNγ, whereas 10–15% produced IFNγ outside the granuloma, where Mtb bacilli are sparse. Thus, T cell production of IFNγ is restricted at the site of Mtb infection within the granuloma, despite ongoing TCR signaling at this location. These findings may help explain why IFNγ-producing T cells may be more efficient at preventing extrapulmonary dissemination than in controlling Mtb within the lung. Understanding the mechanisms regulating this local immunosuppression may yield new strategies for vaccine and immunotherapeutic development.
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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 &gt; ctrl. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly &gt; 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 &gt; ctrl, and reduced anti-PD-L1 efficacy against PD-L1KO&gt; 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

Abstract
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

Abstract
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.