bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of the Innate Immune System in Doxorubicin-Induced Cardiotoxicity
Authors: Bhagat Anchit , Kleinerman Eugenie S
Published: January 2023

Doxorubicin (Dox) is one of the most effective agents for the treatment of pediatric cancers. However, survivors treated with Dox are at high risk for developing cardiovascular disease and succumbing to a cardiac event. Indeed, cardiac failure is the most common cause of non-cancer related deaths. Understanding the mechanisms involved in Dox-induced cardiac failure are necessary to reduce this late effect. Because of the link between inflammation, innate immune cells and other types of cardiomyopathies, we hypothesized that migration & activation of innate immune cells mediated by damaged cardiac fibroblasts may contribute to Dox-induced heart damage, cardiac fibrosis and failure. Mice treated with Dox for 2 weeks showed decreased ejection fraction, increased circulating levels of ROS, and increased infiltration of neutrophils in the heart tissue. Incubation of cardiac fibroblasts with Dox in vitro resulted in the upregulation of inflammatory cytokines (IL-1b, TNFa) and chemokines (MCP-1, CXCL1). Taken together, these data support a role for neutrophils and an innate immune response in Dox-induced cardiotoxicity. Identifying agents that can inhibit or decrease this have the potential to improve cardiac health, QOL and decrease cardiac-related deaths in childhood cancer survivors.
IL-1R signaling enhances neutrophil extracellular traps (NETs) formation and leads to increase lung permeability and hypoxemia in LPS/mechanical ventilation-induced acute lung injury mouse model
Authors: Nosaka Nobuyuki , Martinon Daisy , Crother Timothy R. , Arditi Moshe , Shimada Kenichi
Published: January 2023

Although neutrophils extracellular traps (NETs) play a role in host defense against invading microorganisms, NETs alone play a role in pathogenesis of human acute respiratory distress syndrome (ARDS) either with or without infection. However, the mechanism of NET formation and its functional implications in alveolar space during acute lung injury are not less understood. We created a two-hit lung injury mouse model using intratracheal LPS (0.2 mg/kg), followed by 2.5 hours high-volume mechanical ventilation (30 ml/kg). We observed significant hypoxemia along with increased alveolar leakage, neutrophil infiltration, and NET formation using this model. Neutrophil depletion using (Ly6G[1A8]) monoclonal antibody inhibited these effects. Intriguingly, Il1r1−/− mice were significantly protected from hypoxemia and alveolar leakage despite similar alveolar neutrophil infiltration. We also found that Il1r1−/− mice had reduced levels of cell-free DNA and MPO-DNA complexes in bronchoalveolar lavage fluid (BALF), suggesting that NETs were involved in inducing lung vascular permeability and that IL-1R signaling was involved in inducing NET formation in alveolar space. Inhibition of NET formation via Cl-amidine or removal of NET DNA via DNase I led to reduced hypoxemia and alveolar leakage despite unaltered neutrophil infiltration. Additionally we found that recombinant IL-1β treatment of mouse neutrophils induces NETs, and this was inhibited by IL-1R antagonist (Anakinra). Taken together, we have found that IL-1R signaling-induced NETs play a critical role in development of lung injury, and may offer a new target for therapeutics.
Neuropilin-2 Isoforms Regulate Distinct Functions of Tumor-associated Macrophages in Breast Cancer
Authors: Soloff Adam C , Stanton Rebecca A. , Radio Nicholas M. , Freilino-Glunt Maria , Nasarre Cecile , Dimou Anastasios , Nasarre Patrick , Denlinger Chadrick E. , Oesterreich Steffi , Lee Adrian V. , Lotze Michael T. , Dhupar Rajeev , Gemmill Robert
Published: January 2023

Introduction

Neuropilins are neural guidance molecules which contribute to tissue development. We have shown that the two isoforms of neuropilin-2 endow opposing functionality to tumor cells due to distinct signaling pathways, with Nrp2b promoting metastatic behavior. Due to the role of macrophages (Mθ) in organogenesis and metastasis, we examine the role of Nrp2 isoforms in these cells.

Methods

Stable shRNA knockdown of Nrp2a or Nrp2b in Raw264.7 Mθ were generated. Phagocytosis, cytokine production, and migration were assessed in knockdowns in response to stimuli (TGFβ, HGF, VEGF, IL-10, IFNγ, LPS, β-glucan). Nrp2 isoforms in Mθ from mouse mammary tissue or EO771-induced mammary tumors were measured by FACS and RT-PCR. Mθ were phenotyped via FACS for wound-healing or inflammatory markers. Single-cell (sc)qPCR for a 96 gene panel examining components of signaling pathways, autophagy, metabolism, and pro/anti-tumor responses was performed on 576 CD11b+F4/80+ TAMs FACS-sorted from EO771 tumors.

Results

Nrp2b expression was significantly upregulated in TAMs compared to Mθ of the blood, spleen, or mammary tissues. 56% of the scqPCR transcripts analyzed were significantly altered in Nrp2bHigh vs. Nrp2bLow TAMs, and computation analysis (PCA/tSNE) revealed two distinct TAM subsets enriched for Nrp2b. Nrp2b+ Raw264.7 cells showed decreased ability to phagocytose tumor cells, but increased rates of division and migration in response to growth factors compared to Nrp2a+ counterparts.

Conclusions

We demonstrate, for the first time, that the principle neuropilin-2 isoforms are present in Mθ, regulate unique functionality, and that Nrp2b+ TAMs are both upregulated in mammary tumors and represent a phenotypically unique subtype.
miR-222 stimulates M2b macrophage polarization in severely burned mice through the degradation of long noncoding RNA GAS5
Authors: Ito Ichiaki , Finnerty Celeste C , Herndon David N , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

M2bMφ have been characterized as cells responsible for the increased susceptibility of severely burned mice to various opportunistic infections. We have previously demonstrated that a long noncoding RNA GAS5 is not expressed by M2bMφ, significantly. This RNA is involved in CCL1 gene silencing, and CCL1 is essential for the prolongation of M2bMφ. In this study, the mechanism involved in the reduction of a GAS5 level in M2bMφ was investigated. Mφ were isolated from the mesenteric lymph nodes of mice 1–15 days after severe burn injury (3rd degree, 25% TBSA burn). The level of phosphorylated UPF1 (pUPF1), a marker of the nonsense-mediated RNA decay (NMD) pathway, was determined in various Mφ preparations by Western blotting. In some experiments, these Mφ were transfected with miR-222 mimic, and tested for the GAS5 RNA and CCL1 mRNA levels. miR-222 has been shown to directly binds to GAS5. In the results, the levels of pUPF1 and miR-222 were increased in Mφ from mice 10–15 days after burn injury. These Mφ did not express GAS5 RNA and expressed CCL1 mRNA. The GAS5 reduction was also demonstrated in normal mouse Mφ transfected with miR-222, but not in the same Mφ treated with an inhibitor of the NMD pathway. M2bMφ were not demonstrated in severely burned mice injected with an miR-222 inhibitor. These results suggest that, through the reduction of long noncoding RNA GAS5, miR-222 appeared in Mφ of severely burned mice stimulates the M2bMφ polarization.
Functional characterization of a novel myeloid cell-specific lncRNA U90926
Authors: Sabikunnahar Sabikunnahar , Varnum Stella , Bivona J.J. , Cooper Rachel , Krementsov Dimitry
Published: January 2023

Long non-coding RNAs (lncRNAs) are emerging as novel positive and/or negative regulators of different innate immune cell functions. We previously identified a novel mouse lncRNA of unknown function called U90926, whose expression in activated macrophages was highly dependent on p38 MAP kinase (MAPK). Here, we functionally characterized this gene in myeloid cells. Analysis of U90926 expression revealed no expression across multiple tissues, including unstimulated macrophages. However, toll-like receptor (TLR) stimulation of macrophages rapidly and transiently induced high levels of U90926 expression, which were dependent on p38 MAPK. To study the function of U90926, we generated knockout mice using CRISPR-Cas9, as well as developed RAW 264.7 macrophages knockdown and overexpression using lentivirus-based vectors. We investigated the functional role of U90926 lncRNA using ex vivo activated bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs). Deletion of U90926 downregulated production of the anti-inflammatory cytokine IL-10 by BMDCs, a cytokine that we and others have shown to be highly dependent on p38 MAPK. In contrast, U90926-deficient BMDC displayed reduced activation-induced expression of MHC class II and CD40, while U90926-deficient BMDM exhibited reduced expression of CD80 and CD86. Taken together, these results identify the lncRNA U90926 as a novel regulator of myeloid cell function and suggest that U90926 may be a positive regulator of IL-10 induction by p38 MAPK, and a negative regulator of the antigen-presentation pathway. Ongoing studies are aimed at identifying the underlying molecular mechanisms and in vivo functions of this gene.
Protective effect of a brown algae, Sargassum horneri on particulate matter-induced oxidative stress and inflammation in MLE-12 cells
Authors: Kim H.J. , Herath K.H.I.N.M. , Kim A. , Mihindukulasooriya S.P. , Ko M. , Jeon Y. , Jee Y. , Kim H.J.
Published: January 2023

The constant exposure to fine particulate matter (PM) induces oxidative stress and proinflammatory cytokine production. The ROS formed by oxidative stress is related to activate NF-κB signal pathway for inflammation. Sargassum horneri, a brown algae found in East Asia, is known to be an excellent source for bioactive components. In this study, the antioxidant and anti-inflammation effects of Sargassum horneri ethanol extract (SHE) on the PM-induced oxidative stress in MLE-12, a type II alveolar epithelial cell line were investigated. As the exposure concentration of PM increased to 1,000 μg/mL, the cell viability was reduced; however, it was increased when treated with SHE. The ROS generation and lipid peroxidation of MLE-12 cells were increased from the PM concentration of 125 μg/mL and they were reduced after treatment of SHE at 62.5 and 125 μg/mL. The expressions of antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD2), in MLE-12 cells exposed to PM were lower than those exposed to PM with SHE. The PM was proved to increase the expression of 8-OHdG, one of DNA oxidative damage markers, and OGG1, the repair enzyme of 8-OHdG, while SHE protected the DNA damage of cell. The expression of NF-κB signaling was reduced by SHE. These results suggest that SHE can adjust the expression of antioxidant enzymes, help to suppress the oxidative stress induced by PM, and attenuate its oxidative damage and NF-κB pathway to lung epithelial cells by eliminating over-produced ROS.

This research is part of a project titled ‘Development of functional food products with natural materials derived from marine resources’ funded by the Ministry of Oceans and Fisheries, Republic of Korea in 2017 (Project NO. 20172085).
MALAT1 is essential for A-NHEJ pathway choice during B cell immunoglobulin class switch recombination
Authors: Zhao Jianjun , Hu Yi , Lin Jianhong
Published: January 2023

Metastasis-associated lung adenocarcinoma transcript 1(MALAT1) has been found high expressed in multiple cancer type including B cell lymphoma and multiple myeloma. In human cancer murine models, deficiency of MALAT1 reduce the tumor burden and malignancy phynotype. We have identified MALAT1 in alternative-non-homozygous end joining(A-NHEJ) pathway by binding to PARP1 and LIG3 in multiple myeloma cells, two key components of the A-NHEJ protein complex. To investigate requirments for MALAT1 in A-NHEJ pathway, by using MALAT1 deficient mice, we found that the contribution of MALAT1 in the context of AID induced DNA breaks in CSR of B cells in mice. MALAT1 is required for A-NHEJ DNA repair pathway, but not the NHEJ pathway, which is the major DNA repair pathway for CSR. To further prove the hypothesis that MALAT1 promote A-NHEJ pathway in B cells, we have engineered mice that conditionally overexpress MALAT1 in B lymphocytes confers a strong A-NHEJ activity in CSR. More importantly, we also found IgH/c-myc translocations are promoted in MALAT1 B cell specific transgenic mice. Thus, we identified MALAT1 as a critical noncoding RNA in regulating A-NHEJ pathway during CSR.
IL-21-dependent Tbet+ B cells require Bcl6 and receive delayed help from non-Tfh T cells
Authors: Levack Russell C , Popescu Maria , Winslow Gary
Published: January 2023

Tbet+ B cells are a novel lymphocyte population that have been implicated in a multitude of diseases and infections, including hepatitis, AIDs, malaria, SLE, and age-related autoimmunity. Our laboratory has characterized a population of Tbet+ CD11c+ memory B cells that arise during Ehrlichia muris infection in mice, and we have used this infection model to study the ontogeny of these cells. We have previously demonstrated that the generation of Tbet+ memory B cells requires CD4 T cells and IL-21 signaling, implicating a role for Tfh cells in Tbet+ B cell development. However, Tfh cells were not required for the generation of Tbet+ memory B cells. Rather, these data suggest that Tbet+ B cells received help from a population of non-Tfh CD4 T cells capable of secreting IL-21, likely Th1 cells. As expected, the helper CD4 T cells provided CD40:CD40L signals to the Tbet+ B cells; however, these interactions occurred 16 days or later post-infection. While the helper CD4 T cells did not require Bcl6, the same was not true for the Tbet+ B cells, as Tbet+ memory B cells were not generated in the absence of B cell-intrinsic Bcl6. These data suggest that T-bet B cells require a novel transcriptional program whereby Tbet and Bcl6 function cooperatively, perhaps transiently. Collectively, these data support a model whereby a subset of non-Tfh T cells (likely Th1 cells) provide CD40 and IL-21 signals that drive the formation of Bcl6-dependent Tbet+ B cells.
Long-lived effector CD8+ T cells are transcriptionally and functionally distinct from effector memory cells
Authors: Nguyen Hong , Toma Clara , Omilusik Kyla , Goldrath Ananda W. , Milner J. Justin
Published: January 2023

In response to an infection or vaccination, antigen-specific CD8+ T cells have the capacity to differentiate into long-lived memory T cells that ultimately confer critical protection against re-infection. The memory CD8+ T cell population is heterogeneous and comprised of circulating effector memory (Tem) and central memory (Tcm) cells as well as non-circulating memory cells or tissue-resident memory cells (Trm). Each memory T cell subset is characterized by distinct phenotypic and functional attributes as well as localization patterns. It has previously been demonstrated that circulating memory subsets can be further subdivided based on effector function or migration patterns. Here, we contribute to the clarification of this heterogeneity by characterizing the gene expression, functional activity and differentiation requirements of long-lived CD127loCD62Llo effector cells (LLE) that can persist at least one year after infection in mice. LLE were found to be phenotypically and functionally distinct from CD127hiCD62Llo Tem and CD127hiCD62LhiTcm subsets. LLE exhibited a unique transcriptional profile, with genes expressed in common with both CD127loKLRG1hi short-lived effector cells and Tem. Further, we refine roles for key fate-specifying transcription factors in controlling the differentiation of LLE versus Tem subsets. These studies further highlight the extent of memory T cell heterogeneity and may inform novel vaccination strategies.
Distinct lymph node entry efficiencies for CD8+ and CD4+ T cells are eliminated during malnourishment
Authors: Gubbels Bupp Melanie R , Goulmamine Syreen , Gibson David , Little Amie , Murphy Sarah , Patrick Kristin , Thoner Tim
Published: January 2023

Previous work has suggested that glucocorticoids upregulate expression of the IL-7 receptor component, CD127 on peripheral T cells, even though the total number of peripheral T cells declines dramatically during malnourishment. We have proposed that CD127 up-regulation contributes to peripheral T cell diminishment by increasing the scavenge rate of IL-7 and thereby providing a mechanism to rapidly adjust the total number of T cells during malnutrition. As such, each malnourished T cell would receive a higher dose of IL-7 than control T cells. We next wondered if increased exposure to IL-7 during malnourishment might confer additional energy-saving behaviors. Thus, we compared lymph node entry rates of adoptively-transferred malnourished and control T cells in malnourished and control recipients. As expected, control CD4+ T cells were more efficient than control CD8+ T cells at entering the lymph nodes. Interestingly, regardless of recipient diet, malnourished CD4+ and CD8+T cells entered the lymph nodes at equivalent rates. The molecular events that enable faster CD4+ T cell entry into lymph nodes in control mice is currently unknown. An improved understanding of T cell-intrinsic changes that occur during malnourishment should enhance our knowledge of CD4+ and CD8+ T cell migration, as well as uncover strategies to improve vaccination responses in malnourished children.
PRMT5-mediated arginine methylation controls the strength of signaling via γc-family cytokines required for T cell maintenance
Authors: Inoue Maia , Okamoto Kazuo , Takayanagi Hiroshi
Published: January 2023

Protein arginine methylation is a post-translational modification which is involved in a wide range of biological processes such as transcriptional control and pre-mRNA splicing. Various cytokines, including IL-2 and IL-7, play crucial roles in T cell development and activation. These cytokines utilize the common cytokine receptor γ-chain (γc, encoded by Il2rg) and its associated kinase JAK3 for signal transduction, but the regulatory mechanism underlying γc and JAK3 expression remains to be elucidated. Here we show that protein arginine methyltransferase 5 (PRMT5) has a pivotal role in the maintenance of invariant NKT (iNKT), CD4+T and CD8+T cells. T-cell specific deletion of the Prmt5 gene resulted in a severe loss of iNKT cells as well as a decreased number of CD4+T and CD8+T cells. PRMT5-mediated arginine methylation is essential for the expression of γc and JAK3 required for iNKT cell development and the proliferation and survival of peripheral T cells. PRMT5 stimulates the symmetric dimethylation of the Sm proteins that facilitates the splicing of the Il2rg and Jak3 premRNA in T cells. Taken together, our findings show that PRMT5-mediated arginine methylation is a crucial determinant of the strength of signaling via γc-family cytokines and T cell maintenance.
NFAT1 and NFAT2 differentially regulate CTL differentiation upon acute viral infection
Authors: Xu Tianhao , Keller Ashleigh , Martinez Gustavo J
Published: January 2023

CD8+ T cell differentiation orchestrated by transcription regulators is critical for balancing pathogen eradication and long-term immunity by effector and memory CTLs respectively. The transcription factor Nuclear Factor of Activated T cells (NFAT) family members are known for their roles in T cell development and activation but still largely undetermined in CD8+ T cell differentiation in vivo. Here, we interrogated the role of two NFAT family members, NFAT1 and NFAT2, in the effector and memory phase of CD8+ T cell differentiation using LCMVArm acute infection model. We found that NFAT1 is critical for effector population generation whereas NFAT2 is required for promoting memory CTLs in a cell-intrinsic manner. Moreover, we found that mice lacking both NFAT1 and NFAT2 in T cells display a significant increase in KLRG1hi and CD127hi population and are unable to clear an acute viral infection. NFAT-deficient CTLs showed different degrees of impaired IFN-g and TNF-a expression with NFAT1 being mainly responsible for IFN-g production as well as for antigen-specific cytotoxicity. To further comprehend the molecular mechanisms behind this differential CTL commitment upon deficiency of NFAT members, we performed RNA-seq analysis. Our results have identified genes uniquely regulated by NFAT1 or NFAT2, and we are in the process of further understanding how this differential transcriptome translates into the observed distinct CTL differentiation. Overall, our results suggest that NFAT1 and NFAT2 have distinct roles in mediating CD8+ T cell differentiation and function.
Dynamics of antigen re-exposure to CD8+ T cells during Toxoplasma gondii infection
Authors: Shallberg Lindsey , Phan Anthony T , Christian David A , Hunter Christopher A
Published: January 2023

The role of T cell Receptor (TCR) stimulation in activation, expansion, and eventual exhaustion of CD8+ T cells has been well characterized in multiple mouse models of disease. What has not been well characterized is the dynamics of antigen re-exposure to CD8+ T cells in non-lymphoid tissues throughout disease progression, and what proportion of CD8+ T cells have been re-stimulated by antigen at any given time. To answer this question, Nur77-GFP reporter OT-I mice were utilized, which provides a sensitive fluorescent reporter for recent TCR stimulation. In mice infected with a transgenic version of the parasite Toxoplasma gondii expressing OVA, the response of Nur77-GFP OT-I to this stimulus was assessed during the acute and chronic phases of infection. Data from these experiments showed that TCR stimulation varies between infected tissues, but largely correlated with infectious burden. For example, during acute infection, up to 50% of the OT-I in the brain had recently re-encountered antigen whereas during the chronic phase approximately 15% of the OT-I expressed Nur77-GFP. Furthermore, characterization of Nur77GFP+ versus Nur77-GFP− OT-I revealed a role of TCR stimulation in co-expression of the inhibitory receptors Tim3 and PD-1. This model system provides a better understanding of how frequently pathogen specific T cells re-encounter cognate antigen and future studies will focus on the role antigen re-exposure plays in CD8+ T cell effector function and memory formation.
CBLB Constrains Inactivated Vaccine-Induced CD8+ T Cell Responses and Immunity against Lethal Fungal Pneumonia
Authors: Nanjappa Somashekarappa Gowda , Mudalagiriyappa Srinivasu , Fites J. Scott , Suresh M. , Klein Bruce S
Published: January 2023

Fungal infections in CD4+ T cell immunocompromised patients have risen sharply in recent years. Although vaccines offer a rational avenue to prevent infections, there are no licensed fungal vaccines available. Inactivated vaccines are safer but less efficacious and require adjuvants that may undesirably bias toward poor protective immune responses. We hypothesized that reducing the TCR signaling threshold could potentiate antifungal CD8+ T cell responses and immunity to inactivated vaccine in the absence of CD4+ T cells. In this study, we show that CBLB, a negative regulator of TCR signaling, suppresses CD8+ T cells in response to inactivated fungal vaccination in a mouse model of CD4+ T cell lymphopenia. Conversely, Cblb deficiency enhanced both the type 1 (e.g., IFN-γ) and type 17 (IL-17A) CD8+ T cell responses to inactivated fungal vaccines and augmented vaccine immunity to lethal fungal pneumonia. Furthermore, we show that immunization with live or inactivated vaccine yeast did not cause detectable pathologic condition in Cblb−/− mice. Augmented CD8+ T cell responses in the absence of CBLB also did not lead to terminal differentiation or adversely affect the expression of transcription factors T-bet, Eomes, and RORγt. Additionally, our adoptive transfer experiments showed that CBLB impedes the effector CD8+ T cell responses in a cell-intrinsic manner. Finally, we showed that ablation of Cblb overcomes the requirement of HIF-1α for expansion of CD8+ T cells upon vaccination. Thus, adjuvants that target CBLB may augment inactivated vaccines and immunity against systemic fungal infections in vulnerable patients.
Upregulation of Neuronal VEGF in Experimental Cerebral Malaria Coincides with Disease Severity Biomarkers in Humans: A Novel Mechanism of Pathophysiology
Authors: Fain Cori E. , Rana Sabhya , Huggins Matthew A. , Sieck Gary C. , Johnson Aaron J.
Published: January 2023

Cerebral malaria (CM) is a deadly neurological complication of Plasmodium falciparum infection resulting in high mortality or severe cognitive deficit. Current therapeutics are highly ineffective, namely due to drug resistance and lack of mechanistic insight into pathophysiology. CM is characterized by VEGF upregulation, disruption of blood-brain barrier (BBB), vascular permeability, and severe edema detectable by MRI. Using small animal MRI and the established animal model of CM Plasmodium berghei ANKA, we recapitulate these features of human disease. Here we show that regions of brain permeability during CM as identified by T1-weighted gadolinium-enhanced and T2-weighted MRI coincide with VEGF mRNA upregulation. Regions of analysis include granular and glomerular cell layers of main olfactory bulb and superior medullary brainstem. We determined through RNA in situ hybridization that neurons are the major cell type responsible for the increased VEGF production during CM. Interestingly, we identified another cell type found to minimally upregulate VEGF in close proximity to these neurons. These VEGF expressing cells may play a role in support or induction of the neuronal response. Intriguingly, astrocytes show no VEGF upregulation during this pathology. Overall, these findings suggest a novel role for neuronal-VEGF in promoting CNS pathology, and a putative mechanism of BBB disruption and vascular permeability during CM.
Enterohepatic Helicobacter (EHH) species differ in their effects on colonic inflammation and colitis-associated colon tumorigenesis in mice
Authors: Alkarkoushi Rasha Raheem , Chatzistamou Ioulia , Hui Yvonne , Nagarkatti Mitzi , Nagarkatti Prakash , Testerman Traci L.
Published: January 2023

Enterohepatic Helicobacter (EHH) species are gram-negative bacteria that colonize the colons and sometimes the biliary tracts of humans, primates, rodents, and other mammals. Though ~20% of the human population may be infected, EHH species are poorly studied in humans. H. hepaticus is known for causing colitis in mice with severe immunologic abnormalities; however, virtually nothing is known about the influence of EHH species on colitis in wild-type mice, which are more relevant to humans. Dextran sulfate sodium (DSS) treatment and DSS plus azoxymethane (AOM ) are widely used as mouse models of ulcerative colitis and colitis-associated colon cancer. We hypothesized that specific EHH species would alter the course of DSS-induced colitis and colitis-associated colon tumorigenesis in mice. We infected C57BL/6 mice with human- and rodent-associated EHH species +/− DSS, or +/− DSS/AOM and measured the effects on pathology and inflammation markers. We found that H. cinaedi, H. pullorum, H. fennelliae exacerbate DSS-induced colitis and delay recovery, whereas H. hepaticus reduced the effect of DSS-induced colitis severity. H. hepaticus also prevented tumor development in the AOM/DSS colon cancer model. We measured the proinflammatory and the anti-inflammatory cytokines in serum and colon homogenates and found that cytokine induction varied among the EHH species. Therefore, EHH species alter susceptibility to DSS-induced colitis and DSS-AOM colon cancer. These results suggest that EHH species could increase or decrease the risk of inflammatory bowel disease or colorectal cancer in humans, making it important to identify the infecting species.
Protective role of secreted aspartyl proteinase in murine systemic candidiasis caused by non-albicans Candida species
Authors: Shukla Manisha , Alam Shahid , Rohatgi Soma
Published: January 2023

Non-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. The Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis. Groups of Balb/c mice were immunized with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Mice immunized with rSap2 cloned from C. parapsilosis showed highest increase in survival time (p=0.02) and maximum reduction in organ fungal burden (spleen, kidneys, lungs, brain) (p<0.05); compared to sham immunized controls. Immunization with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ~60% homology across species. Mice immunized with rSap2 cloned from C. parapsilosis also exhibited significantly higher levels of IFN gamma and IL-4 cytokine levels just before infection, which correlated with protection. The anti-Sap2 specific antibody titres were measured. No correlation was observed between level of Sap2-specific IgG titres and survival outcome. The IgG response was primarily Th2, belonging to IgG1 subtype. Further cellular and histological studies are in progress to characterize the murine rSap2-parapsilosis specific protective immune response in detail.
Deciphering multicellular microenvironment of tuberculosis lung granulomas associated with bacterial control using high-throughput single-cell mRNA sequencing
Authors: Gideon Hannah Priyadarshini , Hughes Travis K , Wadesworth Marc H , Fortune Sarah M , Shalek Alex K , Flynn JoAnne L
Published: January 2023

Infection with Mycobacterium tuberculosis results in the formation of tuberculosis (TB) lung granulomas, which are complex structures with a network of multiple immune cells closely intertwined and function together. Within a given host, granulomas have unique trajectories and outcomes. Some granulomas are able to control bacterial replication, while others fail resulting in persistent bacterial replication. The molecular features associated with these outcomes are not well understood. Using high-throughput single-cell mRNA sequencing on individual TB lung granulomas and computational algorithms, we are generating models to understand infection dynamics that incorporate immune cell composition, its function and interaction; bacterial burden and trajectories of the granulomas to identify robust drivers of bacterial control. We evaluated 28 granulomas from 4 cynomolgus macaques, that were infected for 10 weeks with the Erdman strain of M. tuberculosis. We identified over 30 functional clusters of cells spanning multiple immune and non-immune cells types including T cells, B cells, plasma cells, dendritic cells, mast cells, macrophages, neutrophils, type 2 pneumocytes, fibroblasts and endothelial cells. We observe differential cellular composition and host-cell intercellular communication constituting granulomas that are associated with bacterial burden, that may inform systems level properties across granulomas and animals. By understanding features of the cellular microenvironment that limit bacterial replication, we hope to identify novel targets for host-directed immune therapies and prophylactics in M. tuberculosis infection and other granulomatous disease.
Production of IL-17A by different subsets of γδ T cells in response to the commensal Corynebacterium mastitidis has distinct requirements for IL-1R and TCR recognition
Authors: xu xiaoyan , St. Leger Anthony , Zhu Wenjie , Bing So Jin , Horai Reiko , Mattapallil Mary , Caspi Rachel R
Published: January 2023

The ocular surface is a mucosal site that interacts with the environment and protects the eye. We previously identified an ocular surface commensal, Corynebacterium mastitidis (C. mast.), which induced IL-17A production from γδ T cells in the conjunctiva, affording protection from infection. In the current study we dissect the requirement for IL-17A production in response to C. mast by Vγ4+and Vγ4−γδ T cell subsets. We found that colonization of C. mast-negative mice with C. mast. increased the frequency of IL-17A+ cells in eye-draining lymph nodes by several fold in both subsets. However, whereas Vγ4−γδ T cells upregulated their expression of IL-1R, Vγ4+γδ cells downregulated their CD3 expression, possibly secondary to TCR engagement. To examine this further, we performed in vitro co-cultures of dendritic cells and γδ T cells. With C. mast as the sole stimulant, only Vγ4+ cells, but not Vγ4− cells, produced IL-17A. Addition of exogenous IL1 to the co-cultures elicited production of IL-17A also from the Vγ4− subset, whereas inhibition of downstream TCR signaling events by FK-506 or CsA abrogated IL-17A production in the Vγ4+ subset. Furthermore, C. mast produced a positive Nur77-GFP signal only in the Vγ4+ subset. IL-1R blocking antibody abrogated IL-17A production in both subsets.

We propose that Vγ4+and Vγ4− subsets may sense and respond to C. mast by IL-17A production using distinct pathways. Although an IL-1R signal is needed by both subsets, their level of dependence on this signal differs. IL-1R signaling is dominant in Vγ4−γδ T cells, whose TCR does not appear to respond to C. mast. In contrast, a much lower level of IL-1 appears to be sufficient for triggering IL-17A production from Vγ4+ γδ T cells, which recognize C. mast via their TCR.
Mycobacterium tuberculosis enhances macrophage P-glycoprotein (MDR-1) expression and activity to promote intracellular survival
Authors: Rajaram Murugesan , Wu Qian , Hossfeld Austin , Gerberick Abigail , Saljoughian Noushin , Tiwari Charu , Ganesan Latha Prabha , Wozniak Daniel
Published: January 2023

Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis (M.tb), a serious problem worldwide with over one third of the global population being infected; resulting in 1.3 million deaths. TB treatment includes a combination of 3 or more anti-TB drugs over a prolonged period of time. In the last few decades, multi drug resistant (MDR-TB) and extensively drug resistant TB (XDR-TB) strains have emerged due to the mismanaged use of anti-TB drugs. In this study, we discovered that M.tb infection increases the expression of macrophage drug efflux pump P-Glycoprotein (MDR-1) to extrude various chemical substances including the anti-TB drugs to benefit the M.tb survival. M.tb infection in macrophages suppresses the heat shock factor-1 (HSF1), a transcriptional regulator of MDR-1 through the up regulation of miR-431. Notably, the non pathogenic mycobacterium M. smegmatis or BCG did not increase the MDR-1 expression in human macrophages. Additionally, the inhibition of M.tb phoPR secretion system by pharmacological inhibitor reverses the M.tb mediated up regulation of MDR-1 in macrophages. Finally, inhibition of MDR-1 in macrophages enhances the anti TB drug mediated killing of M.tb. In conclusion, we report a novel finding that M.tb up regulates the MDR-1 in macrophages which limits the exposure of M.tb to sub-lethal concentrations of drugs that promotes M.tb survival and emergence of resistant variants.
Characterization of a novel Plasmodium Virulence Factor
Authors: Akkaya Munir , Bansal Abhisheka , Sheehan Patrick , Miller Louis , Pierce Susan K
Published: January 2023

Plasmodium is a deadly parasite that claims lives of nearly half a million individuals each year. Identification of the mechanisms the parasite uses to evade the immune response is a critical goal to develop the much needed vaccine against this pathogen. However, we know very little about how individual parasite genes function. Here, by using CRISPR/Cas9 strategy, we altered the DNA binding domain of a Transcription Factor (TF) (PBANKA_011210) that is essential for the completion of Plasmodium Berghei life cycle. In mice, compared to the WT parasite, the mutant one showed differential expression of 46 genes, most of which are thought to be involved in generating parasite surface antigen diversity. Furthermore, unlike WT parasite which successfully hides from immune system, mutant parasite triggered high levels of IFN-g which translated into formation of prolonged germinal centers and development of higher titers of parasite specific IgG2b and IgG2c type antibodies. At low inoculum levels this resulted in successful clearance of infection and development of protective immunological memory in mutant parasite infected animals in contrast to a deadly outcome in WT parasite infected group. The differential development of the immune response was dependent on the host’s ability to produce IFNg as the phenotype was lost in IFNg KO mice. We therefore conclude that (PBANKA_011210) TF is a novel virulence factor acts as an immune modulator and dampens IFNg mediated immune response against parasite
Mosaic response of T cells to commensal bacteria derived polysaccharide
Authors: Alvarez Carlos A , Jones Mark B , Cobb Brian A
Published: January 2023

Polysaccharide A (PSA) is a component the commensal gut bacteria Bacteroides fragilis capsule. Over the last 40 years, PSA has been shown to possess potent inflammatory traits while also serving as a robust stimulator of immune suppression. This duality was shown in rodents in which PSA induces abscess and adhesion formation in sepsis models, but can also inhibit asthma, IBD, and EAE through the induction of regulatory T cells. However, despite decades of study, there remains an inability to explain how PSA is able to balance both potent pro- and anti-inflammatory responses. Here, we utilized a systems immunology approach, consisting of RNAseq transcriptomics, high-throughput flow cytometry and Luminex analyses, to characterize the full impact of PSA-mediated stimulation of both MHCII+ antigen presenting cells and CD4+ T cells. The goal was to better understand the molecular foundations of microbiota-mediated immune homeostasis. We found upregulation of TNF superfamily genes and cell surface markers associated with activated T cells and maintenance of the immune response such as OX40, OX40L and GITR. Immune checkpoints such as CTLA4 and BTLA are downregulated while important cell motility and chemotactic markers such as CD11c and ICAM1 are upregulated in response to PSA. Thus, PSA led to the production of activated and experienced T cells that should be able to navigate towards sources of inflammation, but these cells also showed increases in immune checkpoint markers such as Ly-6A/E, CD73 and PDL1. Taken together, PSA responding cells display a curious mixture of pro and anti-inflammatory markers, consistent with known in vivo function, potentially revealing novel pathways to restore homeostasis in patients with inflammatory disease.
Infection of the spleen by Chlamydia muridarum following per-vaginal inoculation causes splenomegaly and is essential for its dissemination into the gastro-intestinal tract
Authors: Shillova Nita , Howe Savannah E , Konjufca Vjollca H
Published: January 2023

Chlamydia trachomatis is a leading cause of preventable blindness and bacterial sexually transmitted disease (STD) worldwide. C. trachomatis infections in women can result in pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting the female reproductive tract (FRT), Chlamydia spp. colonize the gastro-intestinal (GI) tracts of animals and humans and can be a reservoir for reinfection of the FRT. In mice, C. muridarum disseminates from the FRT to the GI tract via an internal, non-oral, non-rectal route. Here we show that C. muridarum disseminates from the FRT to the GI tract in a step-wise manner, by first infecting the FRT-draining iliac lymph nodes (ILNs), then the spleen, inducing extramedullary erythropoiesis and splenomegaly, which peaks at 7 days post per-vaginal (PV) infection, thus splenomegaly is transient. Infection of and proliferation of C. muridarum in the spleen is essential for its dissemination to the GI tract, as colonization of the GI tract by C. muridarum is significantly reduced or completely inhibited in splenectomized mice following per-vaginal inoculation. While intra-venously infected mice clear C. muridarum from ILNs by day 9 of infection, PV-infected mice do not, indicating that infection of ILNs from FRT is the first step of C. muridarum systemic dissemination. Furthermore, inhibition of cell egress with FTY720 significantly inhibits Chlamydia dissemination from ILNs to the spleen and GI tract, indicating a cell-mediated transport of this pathogen. Further elucidation of the mechanisms of C. muridarum systemic dissemination and ensuing immune responses will be essential for developing therapies and vaccination approaches to target this pathogen.
Moringa oleifera Treatment Induces T Cell Activation in Plasmodium chabaudi Infection
Authors: Pilotos Jennifer , Mowa Nathan C , Opata Michael M
Published: January 2023

Malaria is a chronic infection that induces the generation of effector T cells and pro-inflammatory cytokines but never results in sterile immunity. Anti-malaria drugs can alleviate the life-threatening symptoms associated with primary infection, but many of these life-saving drugs are becoming obsolete due to growing resistance. This calls for the development of new strategies to control the disease, and the herbal plant Moringa oleifera is a promising candidate. Studies have shown that Moringa oleifera possesses potent suppressive and curative antimalarial properties, but these studies have mostly been restricted to assessment of parasitemia. Therefore, we sought to investigate the effect of Moringa on malaria immunity in vivo using the rodent parasite strain P. chabaudi. We observed that Moringa treatment slightly reduced parasite burden compared to the untreated controls. Looking at the immune response, mice treated with high dose Moringa (60 mg/mouse) for a short time (7 days) or low dose Moringa (30 mg/mouse) for a longer time (3 weeks), exhibited increased numbers of Teff cells accompanied by an increase in Tbet expression. To further investigate this, we treated mice with Moringa after infection (curatively) or before infection (prophylactically), and observed that mice that were treated with Moringa after infection exhibited increased Teff numbers, IFNγ and TNFα secretion. Interestingly, the mice that were treated before infection had significantly higher Tbet expression. These data suggest that Moringa treatment leads to CD4 T cell activation and production of pro-inflammatory cytokines after malaria infection. Future studies will investigate the mechanism by which Moringa treatment modulates CD4 T cell functions.
Mycobacterium tuberculosis extracellular vesicles induce the formation of granulomalike structures in an in vitro model
Authors: Wong-Baeza Isabel , Paredes Víctor Gabriel García , Silva-Miranda Mayra , Ruiz-Sánchez Bibiana Patricia , Castañeda-Casimiro Jessica , Hernández-Solis Alejandro , Cicero-Sabido Raúl , Serafín-López Jeanet , Estrada-Parra Sergio , Estrada-García Iris
Published: January 2023

Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis. Only 10% of the infected individuals develop an active disease, while 90% of the infected individuals contain the mycobacteria inside structures known as granulomas, in a latent, asymptomatic state. The lung granuloma is formed by a core of infected macrophages, foam cells and epithelioid macrophages, surrounded by several lymphocyte subsets (CD4 T cells, CD8 T cells, Tγδ cells, NK cells, and B cells). M. tuberculosis releases extracellular vesicles, both spontaneously and under stress conditions; these vesicles carry components, derived from the mycobacterial cell wall, that are ligands for human Toll-like receptors (TLR) 2/6 and 4; as a result, these vesicles activate the innate immune system. To determine if M. tuberculosis extracellular vesicles induce the formation of a granuloma-like structure, we used an in vitro granuloma model, with peripheral blood mononuclear cells from individuals with latent TB (positive for the Quantiferon test). We found that M. tuberculosis extracellular vesicles induced the formation of granuloma-like structures that were similar in numbers, diameters and cellular composition (percentage of monocytes, T cells, Tγδ cells, NK cells and ILCs) to the granuloma-like structures formed in response to live M. tuberculosis H37Rv. These results suggest that M. tuberculosis extracellular vesicles participate in the cellular activation and migration processes that lead to granuloma formation in the lungs of TB patients.

Supported by a grant 20190106 from Secretaría de Investigación y Posgrado, Instituto Politécnico Nacional.
Microbiota metabolites SCFA stimulate epithelial migration to promote wound healing through MFGE8 and PAK1
Authors: Bilotta Anthony James , Ma Chunyan , Huang Xiangsheng , Yang Wenjing , Yao Suxia , Cong Yingzi
Published: January 2023

How microbiota regulate intestinal homeostasis is still not completely understood. Emerging evidence indicates that gut microbiota metabolites play crucial roles in maintenance of intestinal homeostasis and regulation of Inflammatory Bowel Disease (IBD). Many patients with IBD are deficient in the microbial metabolites of dietary fibers, short chain fatty acids (SCFAs). To date, the evidence for use of SCFAs as a treatment for IBD has been inconsistent, highlighting the need for further investigation. In this study, we demonstrate that SCFAs promote intestinal epithelial cell (IEC) wound healing. Treatment of mouse and human IEC in vitro with the SCFAs acetate, propionate, and butyrate enhanced wound closure by promoting cell migration, which was independent of cell proliferation. Further analysis of random cell migration revealed that SCFAs increased cell speed, cell persistence, and consequently mean squared displacement. To investigate the mechanism of enhanced migration, we performed microarray of mouse-derived enteroids, which identified p21 activated kinase 1 (PAK1) and milk fat globule-EGF factor 8 (MFGE8), two genes implicated in tissue repair and motility, to be highly upregulated. We confirmed upregulation of MFGE8 and PAK1 via qRT-PCR and western blot. Inhibition of PAK1 and CRISPR knockout of MFGE8 abrogated the effects of SCFAs on migration. Lastly, the above effects were dependent upon SCFAs inhibition of histone deacetylases (HDAC), as treatment of IECs with the HDAC inhibitor, trichostatin A, fully recapitulated the effects of SCFAs on migration and expression of MFGE8 and PAK1. These findings elucidate a pathway in which SCFAs inhibition of histone deacetylases may enhance wound healing in IBD.