bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment with TLR4 agonists protect against infection after severe burn injury.
Authors: Bohannon Julia K , Luan Liming , Hernandez Antonio , Fensterheim Benjamin A , Patil Naeem , Guo Yin , Sherwood Edward
Published: January 2023

Infection is the leading cause of death in severely burned patients. Prophylactic treatment with the TLR4 agonist monophosphoryl lipid A (MPLA) induces resistance to subsequent bacterial challenge. Treatment of mice with MPLA enhances bacterial clearance, leading to improved survival in a model of P. aeruginosa burn wound infection. The current study was aimed to define the mechanisms responsible for improved bacterial clearance and survival in MPLA-treated burn-infected mice, and to determine if MPLA could protect against clinically relevant Gram positive and fungal pathogens. Mice underwent severe burn injury, followed by systemic treatment with MPLA or vehicle control for 2 days. Mice were then inoculated with P. aeruginosa topically or intraperitoneally, and responding neutrophils were measured in bone marrow, blood, burn wound and peritoneal cavity. In later experiments, burned mice were challenged systemically with S. aureus or C. albicans. MPLA treatment induced G-CSF production, decreased bone marrow neutrophil numbers and increased neutrophil numbers in the blood, peritoneal cavity and burn wound site. G-CSF was essential for MPLA-mediated survival, bacterial clearance and neutrophil trafficking. MPLA also improved survival against S. aureus and C. albicans systemic infections after burn. This suggests that G-CSF facilitates MPLA-induced trafficking of neutrophils, allowing for a more rapid response to sites of infection. The ability of MPLA to enhance antimicrobial responses and its ability to protect against a variety of clinically relevant pathogens make it an attractive therapeutic candidate for use in burn patients for the prevention of post-burn infections.
Type 2 inflammatory response protects against Staphylococcus aureus-induced sepsis
Authors: Krishack Paulette , Bubeck-Wardenburg Juliane , Sperling Anne I , Verhoef Philip
Published: January 2023

Sepsis is defined as new organ dysfunction due to acute infection and has a mortality rate >20%. Recent studies indicate that type 2 cytokines, best known for their role in allergies, may be beneficial to the host during sepsis. We hypothesized that these responses protect against mortality during Staphylococcus aureus (S. aureus) induced sepsis.

We tested this hypothesis in 2 different mouse models, using either house dust-mite (HDM) sensitization and challenge to activate an adaptive type 2 response, or pre-treatment with IL-33 via intratracheal instillation to induce an innate type 2 response. This was followed by intravenous infection with S. aureus to induce sepsis. Pre-treatment with either HDM or IL-33 protected against S. aureus-induced mortality, indicating that activation of type 2 responses is beneficial during acute infection. Analysis of lung immune cell populations by flow cytometry revealed increased levels of type 2 innate lymphoid cells (ILC2) in mice pre-treated with HDM or IL-33 compared to untreated controls. Further, we found a relative decrease in lung neutrophils and an increase in lung eosinophils in type 2-stimulated compared to untreated mice, suggesting that type 2 responses may suppress an overwhelming neutrophilic response. However, systemic depletion of neutrophils prior to infection resulted in accelerated mortality compared to non-depleted mice, indicating that neutrophils are necessary to protect against S. aureus infection, and that augmented eosinophilia may confer protection against mortality. We conclude that the type 2 response is protective during sepsis, possibly due to the activity of ILC2s and a delicate balance of eosinophils and neutrophils in the inflamed lung.
mTOR deficiency reveals an immunological trade-off in innate resistance to mycobacterial infection in vivo
Authors: Pagan Antonio J , Levitte Steven , Berg Russell D , Hernandez Laura , Zimmerman Joseph , Tobin David M , Ramakrishnan Lalita
Published: January 2023

The Mechanistic Target of Rapamycin (mTOR) has been implicated in myeloid cell development and survival. As adequate myelopoiesis has been recently shown to be a resistance factor in tuberculosis, mTOR deficiency would be expected to increase mycobacterial growth and thus host susceptibility to this disease. In contrast, mTOR inhibition decreases Mycobacterium tuberculosis infection burdens in cultured macrophages, an effect attributed to the triggering of autophagy. Which of these contradictory effects of mTOR deficiency influences infection outcome in vivo is unknown. We have examined the effects of mTOR on mycobacterial infection using the optically transparent and genetically tractable Mycobacterium marinum-zebrafish model of tuberculosis. A forward genetic screen in zebrafish initially identified an mtor mutant as being hypersusceptible to M. marinum, a phenotype that mapped to mTOR complex 1 and was reproduced with rapamycin. However, using very low inoculums typical of human tuberculosis (1–3 mycobacteria) revealed mTOR’s dichotomous role: mTOR-deficient animals were more likely to clear infection early, but those that did not clear the infection progressed rapidly to more severe disease characterized by the death of infected macrophages and subsequent release of mycobacteria into the more growth-permissive extracellular space. Thus, mTOR supports macrophage homeostasis at the expense of a transient enhancement in microbicidal capacity that could reduce the likelihood of mycobacterial colonization but would inevitably thwart long-term immunity.
Immature myeloid/myeloid-suppressor cells and myeloid cell necroptosis mediate lethal pulmonary tularemia
Authors: Periasamy Sivakumar , Avram Dorina , MacNamara Katherine C , Sellati Timothy J , Harton Jonathan A
Published: January 2023

Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that an overt innate cellular response to Ft, not bacterial burden per se, drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature, but subsequently die leading to necrotic lung damage, loss of pulmonary function, and host death. Ensuing tissue damage and host mortality is Necrostatin sensitive and depletion of dying immature Ly6G+ cells is beneficial, implicating a contribution of necroptotic cell death in tularemia pathogenesis. Accelerated recruitment of immature myeloid cells and/or myeloid cell death may account for the rapid mortality seen with a virulent strain of Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the innate myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection
ER stress-induced IRE1α activation obliterates endotoxin tolerance through modulation of cytokine production via GSK-3βand XBP-1
Authors: Chung Hun Taeg , Joe Yeonsoo , Kim Sena , Kim Hyo Jeong , Park Se-ung , Chen Yingqing , Park Jeongmin , Park Hyeok-Jun
Published: January 2023

Endotoxin tolerance occurs in the late phase of sepsis in order to protect cells from the early hyper-inflammatory response. However, as it induces immunosuppressive environment, septic patients in their late phase are suffering from secondary infections, particularly the bacterial pneumonia. Here we show that induction of ER stress leads to activation of GSK-3β and XBP-1 in an IRE1α-mediated manner, which in turn obliterates endotoxin tolerance. Consequently, from the lung of septic host infected with P. aeruginosa, symptoms of pneumonia have been improved and the bacteria cleared. Thus, for septic patients, determination of immune status would be helpful in guiding the selective usage of appropriate immune modulation, in that ER stress can be a novel therapeutic maneuver, restoring the immune response in patients with endotoxin tolerance.
Insights into the development and function of a dendritic cell-like neutrophil in a model of fungal pneumonia
Authors: Fites J Scott , Klein Bruce
Published: January 2023

Phenotypic plasticity in neutrophils has been frequently observed under inflammatory scenarios in both humans and mice. One of the phenotypically converted neutrophil populations has properties of both neutrophils and dendritic cells previously defined as a neutrophil-DC hybrid (PMN-DC). While a modicum of evidence suggests how this morphological conversion occurs, the role that PMN-DCs play in natural settings of infection and inflammation remains speculative. I observe the emergence of PMN-DCs in a fungal pneumonia model of Blastomyces dermatidis infection, which drives strong recruitment of neutrophils to the lung. Although PMN-DCs, as previously described, comprise a small proportion of neutrophils, the pyogranulomatous nature of Blastomyces infection promotes the accumulation of a large number of PMN-DCs allowing interrogation of this poorly understood cell type in the context of an infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) drives neutrophil-to-DC conversion in vitro but has an undefined role in PMN-DC development in vivo. Inactivation and neutralization of GM-CSF during fungal challenge had no effect on the development of neutrophil-DCs suggesting a non-requisite role for GM-CSF. A functional role for PMN-DCs during primary infection was also observed during Blastomyces infection. PMN-DCs not only kill yeast in vivo, but they associate with and kill yeast far out of proportion to their population size. These studies in the Blastomyces infection model indicate that GM-CSF plays a non-requisite role in PMN-DCs emergence and that PMN-DCs play an important role in microbial clearance.
‘Hypoxia reprograms human macrophages towards a proinflammatory direction’
Authors: Varesio Luigi , Raggi Federica , Pelassa Simone , Pierobon Daniele , Cangelosi Davide , Giovarelli Mirella , Bosco Maria Carla
Published: January 2023

Mononuclear phagocytes are recruited from the circulation as primary monocytes to sites of infection, inflammation, and tumor growth, where they undergo terminal differentiation into macrophages. Macrophages can be polarized into classically activated macrophages (M1) or alternatively activated macrophages (M2) which are characterized by a proinflammatory or an anti-inflammatory phenotype, respectively. M1 and M2 polarization is regulated by microenvironment factors. A common feature of pathologic situations is represented by hypoxia. Little is known about the impact of hypoxia on M1/M2 polarization. To address this issue, M1 (CD80+) and M2 (CD206+) macrophages were generated by culturing human monocytes with LPS or IL4 for 24h under normoxia (20%O2) or hypoxia (1%O2). We show that hypoxia amplifies the proinflammatory state of M1 macrophages and reprograms M2 macrophages towards a proinflammatory direction by increasing the production of inflammatory and proangiogenic cytokines/chemokines. The hypoxic pathologic microenvironment can tune the expression of immunoregulatory signaling receptors, whose deregulated expression may result in amplification of inflammation or establishment of immune escape situations. We demonstrate that hypoxia strongly upregulates the expression of one of such receptors, TREM -1, in M1 and M2 macrophages. Engagement of TREM-1 by agonist Ab triggers further production of M1-type cytokines/chemokines in both populations. These results suggest the role of the hypoxic environment present at pathologic sites in skewing macrophages towards a M1-like proinflammatory phenotype by inducing TREM-1, highlighting the potential of targeting TREM-1 in inflammatory disorders and in tumors.
Myeloid cell-specific Sirt1 knockout impairs innate immune responses against intracellular bacteria infection
Authors: Lee Sang-Myeong , Seo Dong-Won , Kang Kyung Won
Published: January 2023

It has been well documented that SIRT1, a member of Class III histone deacetylase directly regulates various functions of macrophages. However, most of studies had focused on its’ role in metabolic diseases although the primary role of macrophages is to remove pathogens as a major component of innate immune responses. Especially, the role of Sirt1 in intracellular bacterial infection is widely unknown. In the present study, we investigated the innate immune response against Listeria monocytogenes (LM) infection in wild type (WT) or SIRT knockout (KO) mice. In the absence of Sirt1, bacterial clearance and secretion of pro-inflammatory cytokines including TNF-α and IL-6 were decreased after LM infection in bone marrow derived macrophages (BMDM). When mice were infected with LM, Sirt1 KO mice were significantly more susceptible to LM infection and showed 100% mortality while less than 40% of WT mice were died. Similarly to in vitro experiments, bacteria clearance and cytokine production were remarkably lower in Sirt1 KO mice compared to WT mice infected with LM. In conclusion, the present study found out that deletion of SIRT1 gene in myeloid cells impairs innate immune system, leading to increased susceptibility due to bacteria accumulation and reduced pro-inflammatory cytokines. These results suggest that SIRT might be a key regulator for innate immune response against intracellular bacterial infection.
Lipopolysaccharide downregulates FcγRIIb via MARCH3-mediated ubiquitination
Authors: Butchar Jonathan P , Fatehchand Kavin , Ren Li , Elavazhagan Saranya , Fang Huiqing , Mo Xiaokui , Vasilakos John P , Dietsch Gregory N , Hershberg Robert M , Tridandapani Susheela
Published: January 2023

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Cellular responses to antibody-coated targets are largely mediated by Fcγ receptors (FcγR), which cluster and become activated upon binding immune complexes. FcγRIIb is an inhibitory FcγR that negatively regulates these responses, so deletion or down-regulation of this receptor may substantially enhance the outcome of antibody therapy.

Toll-like receptor (TLR) agonists are being used and tested as immune-stimulatory agents for cancer. We screened a panel of TLR agonists to test their effect on FcγRIIb expression and found significant receptor downregulation with TLR4 and TLR8 agonists. We selected the TLR4 agonist lipopolysaccharide (LPS) for further examination and found that it led to the ubiquitination of FcγRIIb protein. In a search of our microarray database of monocytes treated with a TLR7/8 agonist (in which FcγRIIb was down-regulated), we found that membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase, was significantly upregulated. We next tested whether this same E3 ligase was also upregulated with LPS treatment and whether it was required for LPS-mediated FcγRIIb downregulation. Results showed that LPS significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the LPS-mediated decrease in FcγRIIb. These data suggest that LPS effects the downregulation of FcγRIIb through ubiquitination and that this is mediated by the E3 ligase MARCH3.
microRNA-142 is critical for peripheral NK cell homeostasis and function.
Authors: Berrien-Elliott Melissa M , Sun Yaping , Sullivan Ryan , Ireland Aaron , Trissal Maria , Wong Terrance , Leong Jeffery , Wagner Julia , Jewell Brea , Schappe Timothy , Abdel-Latif Sara , Link Daniel , Reddy Pavin , Fehniger Todd A.
Published: January 2023

MicroRNAs (miRNA) are small, non-coding RNAs that post-transcriptionally regulate the translation of their targeted mRNAs. Next-generation small RNA sequencing has identified a number of mature miRNAs that are highly expressed in murine NK cells, including miR-142. We hypothesized that miR-142-5p/3p regulated NK cell development, survival, or functionality, and investigated this concept by examining the NK cell compartment in miR-142−/− mice. Despite having normal NK precursor and NK cell frequencies and absolute numbers in the bone marrow (BM), miR-142−/− mice exhibited a marked reduction of NK cells in the spleen (5-fold), blood (5-fold), lymph node (10-fold), and liver (2-fold) compared to controls. The NK deficiency observed in miR-142−/− mice was not due to blocked maturation, as the frequencies of CD27/CD11b stage II, III, and IV NK cells were similar between wild-type and miR-142−/− mice. However, the absolute numbers of each stage were reduced in miR-142−/−, confirming that miR-142−/− mice display a global loss of NK cells in the periphery that is independent of NK cell maturation. Mixed BM chimera studies support a cell-intrinsic role for miR-142 in NK cell peripheral homeostasis. Furthermore, miR-142−/−NK cells do not signal through their IL-12, IL-15, and IL-18 cytokine receptors and thus fail to produce IFN-γ in response to cytokine stimulation thereby limiting their effector functions. miR-142−/− NK cells have a dramatically altered integrin expression profile compared to wild-type, which we hypothesize leads a failure of miR-142−/− NK cells to progress properly through the BM niche, resulting in the loss of NK cells in the periphery. Thus, miR-142 is required for normal NK cell peripheral homeostasis and function.
Dysregulation of immune response to enteric bacteria triggers the development of spontaneous experimental autoimmune encephalomyelitis
Authors: Yadav Sudhir Kumar , Ito Naoko , Mindur John E , Mathay Martin , Dhib-Jalbut Suhayl , Ito Kouichi
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease against the central nervous system (CNS). Although the etiology of MS is still unknown, breakage of peripheral immune tolerance to CNS antigens is believed to be an initial event of CNS autoimmune diseases. It has been indicated that gut microbiota is an important environmental risk factor in MS/EAE pathogenesis; however, the exact mechanism for pathogenesis has not been elucidated.

To examine how the immune tolerance to myelin basic protein (MBP) is broken, we created a humanized transgenic (Tg) mice that express MS-associated HLA-DR2a and MBP-specific TCR (3A6 TCR) genes isolated from an MS patient. The 3A6/DR2a Tg mice develop EAE spontaneously, and antibiotic treatment, which can reduce the population of commensal bacteria, was found to abolish the development of spontaneous EAE. Since fecal IgM is a marker of expansion of enteric bacteria, we examined the association of fecal IgM with spontaneous EAE development. Interestingly, raised levels of fecal IgM and expansion of enteric bacteria were associated with the development of spontaneous EAE, and a massive infiltration of MBP-specific Tg T cells and neutrophils, along with complement deposits, were detected in the colon of spontaneous EAE mice. Furthermore, MBP-specific GM-CSF+ Th1 and Th17 cells were expanding in the colon but not in the small intestine, and CNS-homing integrin, α4β1, was highly up-regulated in the MBP-specific T cells in the mesenteric lymph nodes in the spontaneous EAE mice.

These data suggest that enteric bacteria-mediated dysregulation of gut immunity may be involved in the initiation and progression of CNS-autoimmune diseases.
Identifying T cell antigens in sarcoidosis
Authors: Mitchell Angela M , Falta Michael T , Tinega Alex N , Grunewald Johan , Pinilla Clemencia , Fontenot Andrew P
Published: January 2023

Sarcoidosis is a multisystem disorder which most commonly affects the lungs and involves the accumulation of CD4+ T cells at the sites of disease. The etiology of the disease is unknown, and the lack of a known antigen has hindered the study of disease pathogenesis. However, there is considerable evidence that CD4+ T cells are involved in the initiation and perpetuation of sarcoidosis. Previous work by our lab and others has demonstrated that particular T cell clones within the bronchoalveolar lavage (BAL) have been preferentially expanded in patients with sarcoidosis. These oligoclonal T cell populations accumulate within the lung and disappear with disease resolution. Furthermore, correlations have been observed between the expansions of CD4+T cells expressing the T cell receptor (TCR) α-chain variable (V) region Vα2.3 and the presence of the HLA-DRB1*0301 (DR3) allele in certain patient populations. Preliminary data suggest that cells within the BAL expressing TCRs utilizing Vα2.3, Vβ5.1, and Vβ5.3 are preferentially expanded before and after ex-vivo stimulation with IL-2. We hypothesize that sarcoidosis-specific CD4+ T cells accumulate and expand in the lungs of sarcoidosis patients in response to a specific antigen. To determine whether these expanded CD4+T cell clones play a pathogenic role in disease progression, as well as to address their antigen specificity, the TCRs of these clones have been characterized, and a combinatorial peptide library has been screened to identify candidate antigens that stimulate these clones. These unbiased data will provide clues to the disease-initiating antigens that drive sarcoidosis in a subset of patients and will lead to an extension of our findings to a larger disease cohort.
Mitochondrial bioenergetics of immune cells in Type 1 diabetes
Authors: McDowell Ruth E , Tse Hubert
Published: January 2023

Mitochondrial (mito) bioenergetics of circulating cells change as a function of disease in Alzheimer’s, fibromyalgia, cancer, and Type 2 diabetes. We are exploring the use of mito-bioenergetics as a biomarker of Type 1 diabetes (T1D) progression and immune cell activation. T1D is a polygenic autoimmune disease in which an inflammatory leukocytic infiltrate into pancreatic islets results in destruction of insulin-producing β-cells. Autoantibody synthesis is currently used as a biomarker, but is a poor predictor of T1D since the majority of β-cells are destroyed prior to autoantibody seroconversion. We hypothesize that T1D is a chronic inflammatory disease in which conditions of oxidative stress will diminish the mitochondrial reserve capacity of immune cells and provide a surrogate marker for T1D. We compared mito-bioenergetics of murine immune cells derived from the spleen, pancreatic lymph node and bone marrow between diabetic and pre-diabetic Non-Obese Diabetic (NOD) mice. We show that mitochondrial reserve capacity is reduced in circulating CD4+ T cells from diabetic mice, but not in pre-diabetic mice during T1D progression. To explore the role of mitochondrial oxidative stress on diabetogenic CD4+ T cell effector responses, we used mito-TEMPO to scavenge mitochondrial reactive oxygen species (mROS). Mito-TEMPO reduced IFNγ synthesis in a primary recall assay by 0.24-fold and the combination of mito and cytosolic TEMPO increased Treg-associated IL-2 by 2.5-fold, suggesting that mROS have a causal role in modulating T cell effector responses critical for T1D pathology. We plan to perform human translational studies on mito-bioenergetics of PBMCs from recent onset T1D patients and further dissect the role of mROS on T1D.
Trogocytosis-Mediated Signaling Drives GATA-3 and IL-4 Expression in CD4+ T cells
Authors: Wetzel Scott A , Reed Jim
Published: January 2023

Membrane and membrane-associated proteins are transferred directly from APCs to recipient T cells at the immunological synapse in a process termed trogocytosis. Trogocytosis regularly occurs during CD4 T cell activation, however, the biological significance of these APC-derived molecules on the T cell remains unclear. We previously have shown that trogocytosed molecules drive sustained intracellular TCR signaling (elevated phospho-ZAP-70, phospho-Erk 1/2 and total phospho-tyrosine) after removal of APC leading to preferential survival in culture. Using murine fibroblasts expressing GFP-tagged I-Ek with covalently attached antigenic moth cytochrome c (88–103) along with surface biotinyated APCs, we expanded upon these data to assess whether this sustained signaling influences the effector functions of trogocytosis positive (trog+) CD4 T cells. Using non-polarized in vitro primed CD4+ T cell we observed that the trog+CD4 T cells have significantly elevated GATA3 and IL-4 production and decreased IFNγ, consistent with a TH2-like phenotype. The increase in GATA3 and IL-4 is observable 24 hours post trogocytosis and their expression levels continue to increase during a three day in vitro culture. These findings suggest that the sustained signaling associated with trogocytosed molecules in trog+ CD4 T cells may lead to effector phenotype differentiation and to sustained cytokine production, which has the potential to greatly influence adaptive immune responses.
Costimulatory pathways mediating T-dependent germinal center responses: distinct cellular requirements for CD40 and B7 costimulation
Authors: Watanabe Masashi , Fujihara Chiharu , Jeffrey Chiang Y , Bhatia Sumeena , Hodes Richard J
Published: January 2023

T cell-dependent (TD) germinal center (GC) and antibody responses require coordinated interactions of T cells with B cells and antigen-presenting dendritic cells (DCs). Although B7 and CD40 costimulatory pathways are critical for these responses, a comprehensive analysis of cell type-specific pathways has been hindered by the absence of models for conditional expression of B7 or CD40. Here we report generation of conditional knockout mice for both B7 and CD40, and the use of these lines together with conditional MHC class II (MHCII) knockouts and bone marrow chimeric strategies, to analyze pathways involved in TD GC and antibody responses. Our results indicate that MHCII-restricted recognition of both DCs and B cells is necessary for generation of antigen-specific TFH cells, GC B cells, and antibody response. Notably, the requirements for expression of B7 and CD40 were distinct, with B7 expression required on DCs but not on B cells, while CD40 was required on B cells but not DCs for generation of TFH cells, antigen-specific GC B cells, and antigen-specific class-switched antibody responses. These findings identify requirements for cell and costimulatory interactions in TD GC and antibody responses and support a unique model in which major costimulatory pathways function through interaction of T cells with distinct cell populations.
Helper T cells are required for the development of IgM memory cells, but not IgM bone marrow plasmablasts
Authors: Papillion Amber M , Kenderes Kevin , Winslow Gary
Published: January 2023

The processes and factors that regulate B cell fate decisions during IgM memory cell development and differentiation are unknown, and may differ from those required by canonical memory cells. In our experimental model of Ehrlichia muris infection, we have characterized two long-lived B cell populations: IgM memory cells, and IgM bone marrow (BM) antibody secreting cells (ASCs). The IgM+ memory B cells are required for recall IgG responses, while the BM ASCs produce antigen-specific IgM responsible for maintaining long-term immunity. Both populations are derived from an early AID-expressing CD4 T cell-independent splenic CD11c+ plasmablast population. Because abundant T follicular helper cells are generated in the spleen during early infection, we investigated whether T cell help regulates B cell fate. Infection of MHC class II-deficient mice revealed that the IgM+ memory B cells required CD4+ T cell help, while the IgM+ BM ASCs did not. Moreover, IgM memory cell transfer studies indicated that T cell help was required for the generation of IgM+ memory cells, but not for the generation of BM ASCs. On the basis of these observations, we propose that T cells drive B cell fate: developing spleen plasmablasts that receive T cell signals mature to become IgM memory cells, while cells that do not receive these signals follow a default pathway to become long-lived IgM BM ASCs.
Exploring the role of YAP signaling in CD4+ T cell biology
Authors: Meng Kevin , Butte Manish J
Published: January 2023

The adaptive immune system’s ability to defend against infections and destroy cancerous cells is highly dependent on T cell proliferation. A long-standing observation through in vitro and in vivo studies is that T cells cluster together during activation and subsequent proliferation. In contrast, the vast majority of other cell types turn off division machinery upon cell-cell contact. The highly conserved Hippo signaling pathway controls for cell size and number through proliferation and apoptosis, thereby making it essential for regulating organ size. At the core of the Hippo cascade is the transcription factor YAP. Upon activation, YAP translocates into the nucleus and binds with transcription enhancer factors (TEADs) to drive transcription of proliferation-related genes. While YAP signaling has been extensively studied in epithelial cells, its role in CD4+ T cell effector function and proliferation remains unexplored. Our data indicates that overall YAP protein levels rises during T cell activation. Interestingly, although YAP activation increases proliferation of epithelial cells, our data suggests that YAP is a negative regulator of cellular proliferation in CD4+ T cells. Upon YAP depletion and then restimulating T cells in vitro, we discovered that the T cells knocked down by YAP exhibited greater proliferation and produced more IL-2 than T cells transduced with control shRNA. In support of this surprising finding, expressing a constitutively active YAP with all five phosphorylation sites mutated (YAP 5SA) in T cells led to decreased proliferation of CD4+ T cells. These results hint at potentially novel functions of YAP and warrants further analysis into how YAP signaling affects T cell proliferation.
Bystander-activated memory T lymphocytes cluster around infected cells early after infection
Authors: Maurice Nicholas Joseph , Roepke Sarah , Prlic Martin
Published: January 2023

Innate and adaptive immune responses are typically considered to occur sequentially following an infection. The innate immune system responds initially to an infection and the antigen-specific T and B cell response develops subsequently. Although the peak of the antigen-specific T cell response lags days behind the innate response, inflammatory signals lead to rapid expression of Granzyme B and Interferon-γ in memory CD8+ T cells. This occurs in a T cell receptor (TCR) independent manner and is referred to as “bystander activation.” We and others have demonstrated that these bystander-activated cytotoxic T lymphocytes (BA-CTL) can kill target cells in an NKG2D-dependent, TCR-independent fashion early after infection. This suggests that T cells use innate-like mechanisms for rapid TCR-independent responses to infection. Here we report that during WT Listeria monocytogenes (LM) infection, antigen non-specific BA-CTL rapidly migrate to cluster around infected cells, surrounding foci of LM in splenic white pulp at 24h post infection. These clusters at early time-points closely resemble antigen-specific effector T cell clustering on day 7 post-infection. We describe the mechanisms driving this early activation and define distinct stages of this innate-like effector program. We propose that the purpose of driving bystander-activated T cells to sites of infection and eliciting these innate-like functional capacities is to limit early pathogen replication.
T cell activation requires force generation and is mediated by
Authors: Hu Kenneth H , Butte Manish J
Published: January 2023

Triggering of the T-cell receptor (TCR) has been shown to involve both binding kinetics and mechanical forces. The source of these mechanical forces is likely cytoskeletal activity by the T cell. To understand the contribution of the T-cell cytoskeleton to these forces, we developed an atomic force microscopy (AFM) based method for presenting a stimulatory signal to the T cell and monitoring the force exerted on the cantilever while simultaneously imaging with optical microscopy. T cells respond forcefully, exhibiting pushing and pulling forces that seem temporally correlated with calcium flux. If we perturbed F-actin polymerization through Latrunculin A treatment, we observed abrogation of both force generation and calcium flux. We were then able to rescue calcium flux by applying an oscillatory force through the stimulatory cantilever. Spacing out these forces resulted in a weaker response, suggesting a characteristic timescale for signal integration. We also investigated what components of the cytoskeleton could contribute to the observed force profile through pharmacological inhibition. The pull phase appears to be mediated by myosin contractility. These studies suggest a mechanical-chemical feedback loop whereby initial TCR triggering results in generation of forceful contacts with APCs to amplify signaling.
Macroautophagy regulates T helper cell activation and tolerance
Authors: Dowling Samuel Dae , Mocholi Enric , Gruber Ross , Shafit-Zagardo Bridget , Macian Fernando
Published: January 2023

Macroautophagy is a catabolic process whereby cell components, which range from soluble proteins to whole organelles, are sequestered in de novo formed double-membrane autophagosomes that fuse with lysosomes to degrade their cargo. Effector T helper cells induce macroautophagy during activation to maintain cell proliferation and cytokine production. T cells that are unable to upregulate macroautophagy activity show a reduction in ATP generation, suggesting that macroautophagy is necessary to maintain the energy metabolism required to meet the demands of T cell activation. It is presently unknown whether macroautophagy regulates the activation state and functional capacity of CD4+T cells. We explored the instructive role of macroautophagy in effector T helper cell activation and tolerance. Inhibition of macroautophagy during activation of CD4+T helper type 1 (TH1) cells induces a long-lasting state of hyporesponsiveness, with a molecular signature that resembles that of anergic T cells. Moreover, inhibition of macroautophagy prevented activated TH1 cells from upregulating glycolysis and mitochondrial respiration, supporting that macroautophagy may regulate metabolic homeostasis during activation of effector T helper cells. Consequently, inhibition of macroautophagy in vivo prevents CD4+ T cell activation in response to immunization and results in decreased severity of experimental autoimmune encephalomyelitis. Our data support that macroautophagy, likely by regulating metabolism, is necessary for the activation of effector T helper cells and that its absence induces a tolerant state.
3D biodistribution of pre-treated MSC in mouse model of liver disease using CryoViz™
Authors: Gargesha Madhu , de Witte Samantha , Newsome Philip , Hirschfield Gideon , Clissmann Ciaran , Macarthur Victoria , Elliman Stephen , Watt Suzanne , Hollyman Daniel , Baan Carla , Hoogduijn Martin , Viola Antonella , Roy Debashish
Published: January 2023

‘MERLIN’ (Mesenchymal stem cells to reduce liver inflammation) is an EU FP7-funded project in which BioInVision, Inc. (Cleveland, OH, USA) is partnering with EU establishments that are developing MSC cellular therapy for primary sclerosing cholangitis (PSC), a type of liver disease. MSC have low immunogenicity and possess promising immune modulatory and regulatory capabilities, making them suitable candidates for treating PSC. In MERLIN project, BioInVision’s CryoViz™ cryo-imaging system is being used to study MSC survival under various pre-treatments in a CCL4 liver injury whole mouse model. CryoViz™ consists of a fully automated system for sectioning and tiled microscopic imaging of tissues, providing anatomical brightfield and molecular fluorescence with single cell sensitivity. Umbilical cord-derived MSC (UCMSC) were pre-treated for three days in the presence of pro-/anti-inflammatory cytokines, vitamins and serum-deprivation after which assays were performed to examine their immunogenicity, phenotype, and susceptibility to NK lysis. Subsequently, whole mice intravenously injected with the different pre-treated MSC [no treatment, IFNγ, IFNβ, TGFβ, Starvation, VitB6, (Starvation+VitB6), RA] were frozen and cryo-imaged. Global biodistribution results from CryoViz™ indicate that pretreatment had an effect on cell survival. Localized biodistribution showed preferential distribution in some organs such as the lung and the liver. The cryo-imaging study has revealed that MSC could be optimized to become less immunogenic and more immunomodulatory, which is an important step towards further development of MSC for cellular therapy.
PI3K-AKT-mTOR pathway mediates trafficking of diabetogenic T cells by modulating CXCR3 via T-bet
Authors: Chen Weirong , Ukah Tobechukwu Kenneth , Miller Mindy M , Zaghouani Habib , Wan Xiaoxiao
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta-cells are selectively destroyed by self-reactive T cells. In prior studies, we incorporated mimotope p79 into an Ig molecule and the resulting Ig-p79 chimera was able to prevent adoptive transfer of T1D by diabetogenic BDC2.5 T cells. Further analysis has shown that these T cells are retained in the spleen and cannot migrate to the pancreas due to diminished expression of the transcription factor T-bet and downregulation of the chemokine receptor CXCR3. Investigation of the molecular mechanisms underlying this form of T cell tolerance indicates that expression of mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase, is inhibited and its function is impaired. In contrast, overexpression of mTOR or its positive regulator, AKT, in BDC2.5 T cells restores the expression of T-bet and CXCR3, reestablishes the pancreatic infiltration of those T cells and consequently, nullifies antigen-induced tolerance. These findings suggest that PI3K-AKT-mTOR pathway, via modulating CXCR3, determines the trafficking ability of self-reactive T cells. Thus, this study not only delineates the molecular mechanisms underlying migration of diabetogenic T cell, but also provides a useful target for modulation of pathogenic T cells and treatment of type 1 diabetes.
Class-biased regulation of humoral immunity and mTORC1 inhibition by physiological hypoxia
Authors: Cho Sung Hoon , Ariel Raybuck , Wei Mei , Haase Volker , Boothby Mark
Published: January 2023

B lymphocytes activated by cognate antigen (Ag) proliferate, are selected for affinity maturation, undergo antibody (Ab) class switching dependent on the cytosine deaminase AID, and yield major forms of memory via germinal center (GC) reactions. Although metabolic programming has emerged as a means of mediating or initiating changes in immunity, nothing is known as to the metabolic landscape in lymphoid organs. Here we show that B lymphocytes in GC are hypoxic, and that low oxygen alters B cell Ab response as well as metabolic programming. In addition to increased B cell death, restricted oxygen concentrations impaired Ab class switching by reducing expression of AID and suppressing the pro-inflammatory IgG2c Ab isotype. Prolyl hydroxyl dioxygenases (PHD) and von Hippel-Lindau (VHL) are central to regulation of hypoxia-induced factors (HIF) in hypoxic responses. B cell-intrinsic deficiency of VHL attenuated Ag-specific IgG2c, and decreased levels of Ag-specific GC, early memory B cells, and Ab responses. Hypoxia and VHL deficiency each also inhibited activity of mTORC1 in a manner dependent on HIF proteins. Genetic limitation of mTORC1 in B cells reduced their clonal expansion, AID expression, and IgG2c Ab responses. Collectively, these results provide evidence that GC reactions are sites of physiologic hypoxia that in turn regulates vital functions of B cells through interruption of program that maintains mTORC1 activity. We propose that this restriction of oxygen regulates the balance of fate choices made by GC B cells, and that pathophysiological states that alter oxygen homeostasis may moderate the pro-inflammatory component of humoral immunity.
Glycerol Monolaurate (GML) inhibits human T cell signaling, metabolism, and function by disrupting lipid dynamics
Authors: Zhang Michael S. , Sandouk Aline , Houtman Jon C.D
Published: January 2023

Glycerol Monolaurate (GML) is a naturally occurring fatty acid widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a range of bacteria, fungi, and enveloped viruses. Interestingly, GML suppresses mitogen induced lymphocyte proliferation and inositol triphosphate production, suggesting that GML has immunomodulatory functions. In this study, we have mechanistically examined if GML affects the signaling, metabolism, and functional output of human primary T cells. We found that GML potently altered lipid order and disorder dynamics in the plasma membrane that resulted in reduced membrane localized clustering of the proteins LAT, PLC-γ, and AKT, events integral for proper T cell receptor (TCR) signal propagation. Altered membrane signaling events induced selective inhibition of TCR-induced phosphorylation of SLP-76, regulatory P85 subunit of PI3K, and AKT as well as abrogated calcium influx. In addition to signaling defects, GML treated cells have profoundly altered metabolism profiles characterized by suppressed oxidative phosphorylation and increased glycolysis. Functionally, GML treatment potently reduced TCR-induced production of the cytokines IL-2, IFN-γ, TNF-α, and IL-10. Our data reveal that the widely used anti-microbial agent GML alters the lipid dynamics of human T cells, leading to their defective signaling, metabolism, and function.
PIK3IP1- A novel negative regulator of PI3K
Authors: Uche Uzodinma N , Kane Lawrence P
Published: January 2023

Phosphatidylinositide-3-Kinases (PI3Ks) are a family of lipid kinases that play important intracellular signaling roles in cellular functions such as cell proliferation, motility, growth, intracellular trafficking, differentiation and survival. PI3K produces PIP3 which further facilitates the activation of downstream effectors such as Akt and PDK1. These effectors facilitate the cellular processes associated with PI3K activity. Conversely, because of the nature of PI3Ks roles, dysregulation of PI3K is implicated in many cancers. PTEN, SHIP and INPP4B are negative regulators of PI3K activity that function downstream of PI3K and have been shown to act as tumor suppressors. Recently, PI3K Interacting Protein 1 (PIK3IP1), a novel negative regulator that functions upstream and proximal to PI3K, has been identified. PIK3IP1 is a transmembrane protein that down-regulates PI3K activity, leading to reduced Akt phosphorylation. Our lab recently showed that PIK3IP1 can inhibit TCR signaling and TCR activation. To better understand the role of PIK3IP1 in immune cells, we have begun to carefully define the roles of the various domains that comprise PIK3IP1. In addition, we have produced a PIK3IP1 ecto-domain Fc fusion protein, which we have used to develop monoclonal antibodies against PIK3IP1. This ecto-domain Fc fusion protein is also being used to define possible ligands for PIK3IP1. Finally, we have obtained knockout mice lacking expression of PIK3IP1 in T cell and mast cell compartments, to better define the requirements for PIK3IP1 in primary T and mast cells.
SerpinB1 Deficiency Ameliorates Experimental Autoimmune Encephalomyelitis
Authors: Hou Lifei , Winau Florian , Remold-O’Donnell Eileen
Published: January 2023

Increasing evidence shows that, in autoimmune settings, Th17 cells are converted to pathogenic Th17 cells (patho-Th17), which produce GM-CSF and IFNγ and are pivotal for pathogenesis. We recently discovered that serpinB1, a protease inhibitor, is the signature gene of Th17 cells and forms a regulatory module with cathepsin L that controls Th17 cell generation: Th17 differentiation is restricted by serpinB1 and counter-regulated by cathepsin L.

In the current study, we investigated serpinB1 regulation of Th17 cell pathogenicity in experimental autoimmune encephalomyelitis (EAE). As anticipated, expression of serpinB1 and other Th17 genes were significantly increased in effector CD4 cells in the EAE mouse. Surprisingly, serpinB1 deficient (sb1−/−) mice immunized with MOG35–55 are resistant to EAE, manifested by dramatically decreased disease incidence and severity. Adoptive transfer experiments showed that the protective effect of serpinB1 deficiency is CD4 cell specific. Cytokine profiling showed that, despite producing more Th17 cells, serpinB1 deficiency selectively diminished patho-Th17 cells. Furthermore, Ki-67 and Annexin V staining together with BrdU pulse/chase strategy confirmed that proliferation is not defective in sb1−/− Th17 and patho-Th17 cells. In contrast, sb1−/− patho-Th17 cells showed significantly shortened life span and were eliminated upon antigen-recall. Finally, in vivo administration of E64D, a broad-acting cysteine protease inhibitor, partially reversed the effects of serpinB1 deficiency. Thus, our study identified a protease-protease inhibitor regulatory module acting on Th17 cell differentiation and separately on pathogenicity with potential for targeting in autoimmune diseases.
Autocrine NLPR3 inflammasome activity is critical to normal adaptive immunity via regulation of IFN-γ in CD4+ T cells
Authors: West Erin E , Arbore Giuseppina , Robertson Avril A.B. , Klos Andreas , Woodruff Trent M , O’Neill Luke A. , Coll Rebecca C. , Sher Alan , Leonard Warren J , Köhl Jörg , Monk Pete , Cooper Matthew A. , Afzali Behdad , Lachmann Helen J. , Cope Andrew P. , Mayer-Barber Katrin , Kemper Claudia
Published: January 2023

The NLRP3 inflammasome controls IL-1β maturation in antigen presenting cells but a direct role in human adaptive immune cells has not been described. Here we show that the NLRP3 inflammasome assembles in human CD4+ T cells and initiates caspase-1-dependent IL-1b secretion, thereby promoting IFN-γ production and Th1 differentiation in an autocrine fashion. Importantly, NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which drive reactive oxygen species (ROS) production. The alternative cell surface expressed C5a receptor 2 (C5aR2) negatively regulates this process. Dysregulation of NLRP3 activity in T cells affects inflammatory responses in autoimmune disease or infection. Firstly, CD4+ T cells from patients with cryopyrin-associated periodic syndromes (CAPS), who have constitutively-active NLRP3, exhibit overactive Th1 responses that are normalized by NLRP3 inhibitor treatment. Secondly, IFN-γ production is impaired in T cells from Nlpr3−/− or Il1a/Il1b−/− mice upon viral infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to ‘innate immune cells’ but is an integral component of normal adaptive Th1 responses.
yidC is required for virulent Francisella tularensis to modulate IRF1 and resist killing by host cells
Authors: Drumheller L Brock , Wehrly Tara , Di Russo Case Elizabeth , Celli Jean , Bosio Catharine M
Published: January 2023

Francisella tularensis is a gram negative, intracellular pathogen that causes the lethal disease known as tularemia. One virulence mechanism used by F. tularensis is the inhibition of the activation of Interferon Regulatory Transcription Factor 1 (IRF1). IRF1 is an important transcription factor for Type I and II Interferons and other proteins involved in host defense. The genes encoded by Francisella responsible for modulation of IRF1 activation have not been defined. To identify which genes are involved in the process of IRF1 inhibition, we developed a high throughput assay of IRF1 activation and screened a HIMAR transposon library of Francisella mutants. We identified yidC, which encodes a membrane protein insertase, as a required bacterial gene for inhibition of IRF1. Consistent with its inability to negatively modulate IRF1, yidC mutants were more susceptible to IFN-gamma mediated killing compared to wild type controls. Together, our data points toward a novel role for yidC, and by extension membrane proteins, in the modulation of IRF1 and resistance to host cell antimicrobial responses.