bluuebunny/crossref_metadata_2025 huggingface dataset:
Cul4b promotes DNA damage repair during T cell proliferation
Authors: Dar Asif Amin , Dybas Joseph , Sawada Keisuke , Moser Emily K , Oliver Paula
Published: January 2023

Ability of T cells to become activated and clonally expand during pathogen invasion is pivotal for the induction of protective immunity. Understanding how T cell receptor (TCR) signaling, cell cycle, and survival are coordinated could allow development of therapeutics designed to regulate the numbers of antigen-specific T cells. Activating mutations in Cul4b and its orthologue Cul4a are known to drive cell cycle and cancer development. Loss-of-function mutations in Cul4b have been identified in X-linked mental retardation patients. However, how Cul4b controls the function of primary T cell remains unknown.

Using a novel proteomics screen, we identified Cul4b as highly active following TCR stimulation. Cul4b is expressed in naïve T cells but its levels and activity increased on TCR stimulation. We found that Cul4b was more abundant in T cells than Cul4a, and a significant proportion of Cul4b was associated with chromatin. To study the role of Cul4b in T cells we generated mice lacking Cul4b specifically in T cells. Loss of Cul4b impaired maintenance, proliferation and survival of activated T cells. Supporting this, Cul4b-deficient T cells were unable to expand and drive colitis. Cul4b deficient cells showed enrichment of cell cycle and DNA damage related pathways, with concomitant accumulation of pATM, P53, pCHK1, and γ-H2AX. In CD4 T cells, Cul4b preferentially associated with the substrate receptor DCAF1 to form an active CRL4B complex. Additionally, both Cul4b and DCAF1 were found to interact with proteins associated with DNA damage response and repair pathways. These findings demonstrate an essential role for Cul4b in promoting the rapid repair of damaged DNA to allow proliferation, survival and overall expansion of activated T cells.
Inhibins/TGFβRIII modulate T cell activation and Th1/Th17 effector differentiation
Authors: Soldevila Gloria , Ortega-Francisco Sandra A , Morales Alexandra , de Oca Selina Montes , Albarran Adrian , Hernandez-Campos Oscar , Olguin-Alor Roxana
Published: January 2023

Inhibins, ligands of the TGFβ superfamily, and their molecular pair, TGF-β type III receptor (TβRIII), have been shown to regulate the development of T cells within the thymus. Moreover, we recently reported that TβRIII acts as an activation marker after TCR stimulation However, the intrinsic role of Inhibins in T cell activation and differentiation is currently unknown. Here, we describe for the first time that T cells are able to produce Inhibin A after a polyclonal TCR stimulation which raises the possibility of this ligand playing a direct role in T cell responses. Interestingly, Inhα−/− naïve T cells showed lower expression of activation markers, including TβRIII, after TCR stimulation, compared with their wild type counterparts. Moreover, Inhα−/− naïve T cells showed a significant decrease in Th1 differentiation under polarizing conditions in vitro, that correlated with lower levels of activation. Furthermore, when we evaluated the differentiation of Inhα−/− naïve T cells towards Th17, a TGFβ-dependent lineage, we observed an increase in the percentage of IL-17+ T cells compared to wild type. Interestingly, this altered Th differentiation could be rescued by addition of exogenous Inhihin A, leading to a significant decrease in IL-17 production and a trend towards an increase in IFNγ production. These data suggest that Inhibins play an intrinsic role during T cell activation and may be key modulators of T effector cell differentiation during an immune response.
Homeostasis and transitional activation of regulatory T cells require c-Myc
Authors: Saravia Jordy , Zeng Hu , Dhungana Yogesh , Chapman Nicole M , Wang Yanyan , Raynor Jana L , Chi Hongbo
Published: January 2023

Regulatory T (Treg) cell activation and expansion during neonatal life and in response to inflammation are critical for immunosuppression, yet the mechanisms governing these events are incompletely understood. We report that the oncogene and transcriptional regulator c-Myc (Myc) controls immune homeostasis through regulation of Treg cell accumulation and functional activation. Myc activity is enriched in Treg cells generated during neonatal life and responding to inflammation. Myc-deficient Treg cells show cell-intrinsic defects in overall accumulation and ability to transition to an activated state during early life or acute inflammation. Consequently, loss of Myc in Treg cells results in a rapid, early-onset autoimmune disorder accompanied by uncontrolled effector CD4+ and CD8+ T cell responses. We also provide evidence that Myc regulates mitochondrial oxidative metabolism but is dispensable for fatty acid oxidation (FAO). Indeed, Treg cell-specific deletion of Cox10, which is required for oxidative phosphorylation, but not Cpt1a, the rate-limiting enzyme for FAO, results in impaired Treg cell function and maturation. Thus, Myc coordinates Treg cell accumulation, transitional activation and metabolic programming to orchestrate immune homeostasis.
LSECtin interacts with BTN3A1 to inhibit T cell activation
Authors: Wang Jinghua , Manick Brain , Renelt Mark , Gerassenkov Tatsiana , Bi Ming , Kalabokis Vassilios , Person Anthony , Wu Guoping
Published: January 2023

LSECtin is a type II transmembrane protein which belongs to the C-type lectin receptor superfamily. Previous studies revealed that LSECtin is expressed on melanoma cells, acts as a co-inhibitor of human T cell immunity and may hamper anti-tumor T cell immune responses. However, the mechanisms by which LSECtin suppresses T cell immunity remain unknown. In this study, we demonstrate that the LSECtin protein inhibits human T cell activation through the butyrophilin family receptor BTN3A1. LSECtin markedly suppressed human T cell proliferation in the presence of T cell receptor signaling. Furthermore, LSECtin significantly reduced IFN-γ, IL-2, IL-17 and TNF-a cytokine and IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10, and TARC/CCL17 chemokine production by anti-CD3 activated human T cells. Moreover, a functional ELISA binding assay revealed that LSECtin bound to BTN3A1. Anti-BTN3A1 neutralizing antibody attenuated the binding of LSECtin to BTN3A1. BTN3A1 is expressed on both resting and activated human T cells, and pretreatment of immobilized LSECtin with the BTN3A1 protein or an anti-BTN3A1 antibody partially attenuated the ability of LSECtin to inhibit IL-2 secretion from anti-CD3-activated T cells, suggesting that BTN3A1 acts as a receptor for LSECtin to deliver a negative signal to T cells. Thus, we show that LSECtin/BTN3A1 represents a novel negative regulatory pathway on T cells and may serve as a unique target for developing new immunotherapy strategies for the treatment of immune-related diseases, including cancers.
Treatment with natural AhR ligand leads to amelioration of LPS-induced acute lung injury by decreasing microRNA 29b-2-5p expression targeting increased IL-22 production
Authors: Holloman Bryan L , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are severe pulmonary inflammatory diseases that cause destruction and remolding of the lung epithelial barrier, which may lead to restriction of pulmonary functionality and/or death. Indole-3-carbinol (I3C), a naturally occurring Ahr ligand has been shown to ameliorate multiple inflammatory disorders. Our studies are aimed at determining whether treatment with the I3C affects reparative reepithelialization and restores lung functioning through an immune-mediated mechanism. Towards this, 5mg/kg of LPS was intranasally injected in C57BL/6 mice to induce ALI. Three hours following LPS administration, the mice were treated with 40mg/kg I3C. After 48 hours, we examined lung functions using Buxco plethysmography and noticed LPS + I3C treated-mice have similar functionality as naïve mice in multiple parameters including ratio of time peak expiratory flow (Rpef), rejection index (Rinx), and tidal volume (TV) basal pulmonary functions whereas LPS+vehicle treated mice showed alterations. Histopathological analysis revealed fibrotic tissue in lung sections of LPS + Vehicle administered mice, but not in LPS + I3C treated mice. In addition, I3C increased the expression of Th22 (CD3+CD4+IL22+) cells, which are key mediators of epithelial cell layer protection, antimicrobial peptide production, and reepithelialization improvement. I3C increased IL-22 expression in bronchoalveolar lavage fluid, and decreased the expression in miR-29b-2-5p transcript that directly regulates IL-22 expression. In conclusion, our studies suggest that Ahr ligand I3C alleviates lung injury by maintaining lung epithelium integrity which may be mediated by IL-22 secreting T cells.
Induction of autophagy or mitophagy improves the cardiovascular lesions of Kawasaki Disease in a murine model of vasculitis
Authors: Marek-Iannucci Stefanie , Gomez Angela C , Moreira Debbie , Lane Malcolm , Porritt Rebecca , Lee Youngho , Shimada Kenichi , Crother Timothy R , Rivas Magali Noval , Arditi Moshe
Published: January 2023

Kawasaki Disease (KD), an acute febrile illness and systemic vasculitis of unknown etiology, is the leading cause of acquired heart disease among children. NLRP3 activation and IL-1 signaling are required for the development of coronary arteritis and abdominal aorta aneurysms (AAA) in the KD vasculitis murine model. NLRP3 is also activated by mitochondrial (mt) DNA released from impaired mitophagy-autophagy processes. How autophagy and mitophagy participate in KD cardiovascular lesions remain unknown. We investigated the role of autophagy/mitophagy by using the well-accepted Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis. LCWE injection in WT mice results in coronary arteritis, aortitis, myocarditis as well as AAA. p62 and Parkin, were expressed significantly higher in whole lysate and mt fractions of cardiovascular tissues from LCWE-injected mice compared to control, suggesting an impaired autophagic flux and lack of degradation and cell clearance. Parkin−/− mice developed significantly higher LCWE-mediated cardiovascular lesions and AAA, compared to control mice. Conversely, autophagy induction in WT mice by intermittent fasting or by treatment with a Glucagon-like peptide-1 receptor agonist, led to a significant decrease in LCWE-mediated cardiovascular lesions and AAA. This data suggests that induction of autophagy leads to decreased cardiovascular lesions, whereas impaired mitophagy results in exacerbation of LCWE-induced cardiovascular lesions in this murine model of KD vasculitis. These findings enhance our understanding of the pathogenesis of the cardiovascular lesions in KD vasculitis and may provide novel therapeutic targets to prevent or treat these complications.
Regulatory Role of STAT3-PLSCR2 Axis in Antiviral and Inflammatory Response
Authors: Lee Chien-Kuo , Tsai Ming-Hsun
Published: January 2023

Cytokines are key factors for initiating inflammatory response and among them IFN-I is one of the most versatile because of its dual roles in antiviral and inflammatory activities. While STAT1 and STAT2 are critical for triggering antiviral response, the signaling events downstream of IFN-I receptor contributing to inflammation have yet to be defined. Here we demonstrate that STAT3 and one of its interacting partners PLSCR2 negatively regulate IFN-I-mediated antiviral and inflammatory response. Enhanced expression of IFN-I-stimulated genes (ISGs) is found in cells either absence of STAT3 or PLSCR2, leading to increased resistance to viral infection in vitro and in vivo. Moreover, STAT3 can cooperate with PLSCR2 to suppress IFN-I response. Interestingly, GSEA analysis for IFN-I-stimulated cells lacking PLSCR2 versus WT control displays an inflammatory signature with increased expression of inflammatory cytokines and chemokines, their receptors and TLRs. Stimulation of TLR ligands, including LPS, R848 or CpG, also results in enhanced expression of IFN-I and inflammatory cytokines and chemokines in PLSCR2KO cells as opposed to WT control. PLSCR2KO mice are also more susceptible to LPS-induced endotoxic shock in vivo with increased serum TFN. Similarly, PLSCR2KO mice also display increased acute lung injury in response to intratracheal administration of LPS with increased infiltration of macrophages and neutrophils and production of inflammatory cytokines and chemokines. Enhanced TLR response is also found in STAT3KO BM-derived macrophages. Therefore, these results demonstrate that STAT3 coordinates with PLSCR2 to regulate antiviral and inflammatory response and define a novel axis in modulating IFN-I-mediated response.
Damage-induced pyroptotic cell death facilitates regeneration of the thymus
Authors: Kinsella Sinéad , Evandy Cindy A , Hopwo Kayla S , Cooper Kirsten , Iovino Lorenzo , Smith Colton W , Jain Reema , deRoos Paul , Dudakov Jarrod A
Published: January 2023

Endogenous thymus regeneration is a critical process that enables the restoration of immune competence after damage caused by infection, age and common cancer therapies; however, the molecular mechanisms regulating this process are poorly understood. Previously, we identified that depletion of CD4+CD8+ thymocytes (DPs) after injury precedes the induction of regenerative factors such as BMP4 from endothelial cells (ECs), and IL23 from dendritic cells (DCs), and that the presence of homeostatic apoptotic DPs can suppress BMP4 and IL23. Together with our findings that the metabolic needs of key thymus populations alter drastically following injury due to damage-induced metabolic remodeling, we hypothesize that further to the loss of DP-specific suppression after damage, metabolic dysfunction in DPs triggers mitochondrial-induced pyroptotic cell death, which can directly promote the production of regenerative factors. Here we demonstrate a disruption in glycolytic flux, increased mitochondrial biogenesis, an induction of ROS, and increased cleavage of caspase 1 in murine DPs after damage; key processes that can induce inflammasome activation, pyroptosis and the release of cellular DAMPs such as ATP and HMGB1. Interestingly, these metabolic perturbations were not observed in the regeneration-inducing ECs and DCs, suggesting metabolic stability may facilitate their radio-resistance. Secreted HMGB1 levels are increased in the thymus after damage, and extracellular stimulation of ECs in vitro with ATP increased Bmp4 expression. These studies describe a novel regulatory mechanism of metabolic-mediated tissue regeneration, and have the potential to lead to a superior therapeutic strategy to boost thymic function.
Effect of mesenchymal stem cell in adriamycin-induced renal nephropathy
Authors: Park Eun Jae , Kim Hyung Suk , Kang Yu Jeong , Lee Hye Jin , Jun Hye Won , Lee Hong Kyung , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Mesenchymal stem cells (MSCs) are multi-potent progenitor cells able to differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs secrete various soluble factors that are advantageous for tissue repair, anti-apoptosis, anti-fibrosis, and immunomodulation. Recently, MSCs improve the renal inflammation in adriamycin (ADR)-, adenine-, and cisplatin-induced animal model, but the mechanisms underlying their efficacy are unclear. In this study, we examined effect of MSCs in ADR-induced renal nephropathy. MSCs prolonged survival, restored body weight, and decreased serum creatinine and proteinuria levels in ADR-induced animal model. MSCs also inhibited renal fibrosis and podocyte damage by decreasing the expression of fibronectin, α-smooth muscle actin, and collagen 1α1. MSCs inhibited renal inflammation by decreasing the expression of CCL4, CCL7, CCL19, IFN-α/β, TNF-α, TGF-β and chitinase 3-like 1. Taken together, our data suggest that MSCs improve renal injury by decreasing inflammatory factor in ADR-induced nephropathy.
The specific transcriptomic response of porcine intestinal epithelial cells to Escherichia coli and Salmonella enterica Typhimurium infections is modulated by bovine colostrum fractions
Authors: Blais Mylene , Bouchard Michael , Asselin Ckaude , Lessard Martin
Published: January 2023

Enterotoxigenic Escherichiacoli (ETEC) and Salmonella enterica Typhimurium are both enteric pathogens, but the mechanisms by which they infect intestinal epithelium are different. Recently, we observed that bovine colostrum (BC) increases protection against enteric infections in weaned piglets. In this study, we described the transcriptomic response of porcine intestinal epithelial cells IPEC-J2 infected by each pathogens, and we measured the specific effect of defatted BC, as well as bovine serocolostrum and casein fractions (SC and CAS respectively) on IPEC-J2 transcriptomic response to ETEC and Salmonella by microarray analysis. We also measured the impact of BC, SC and CAS on pathogen-induced epithelial integrity loss using transepithelial electrical resistance (TEER), and on NF-kB transcriptional activity by luciferase assays. Results showed that transcriptomic profile of cells challenged with ETEC and Salmonella both induced genes involved in inflammatory response, while ETEC specifically induced the expression of genes involved in morphogenesis and response to lipids, and Salmonella specifically regulated genes involved in oxidative stress, migration and apoptotic process. The ETEC induction of inflammatory genes was decreased by BC and SC, while Salmonella induction of inflammatory genes was specifically reduced by BC and CAS fraction. BC prevented epithelial integrity disruption caused by both pathogens, while SC only prevented the one caused by Salmonella. Finally, BC and SC decreased NF-kB activity induced by each pathogen. Altogether, these results indicated that BC fractions modulate in a specific manner cellular mechanisms involved in intestinal epithelial response to these pathogens.
Lattice light-sheet microscopy Multi-Dimensional Analyses (LaMDA) of T-cell receptor dynamics predict T-cell signaling states
Authors: Huang Jun
Published: January 2023

Cell surface receptors dynamically change in response to cell signaling, but a direct linkage between receptor dynamics and cell state has not been established. Here we report the development of lattice light-sheet microscopy multi-dimensional analyses (LaMDA), a pipeline that combines high spatiotemporal-resolution four-dimensional lattice light-sheet microscopy, machine learning, and diffusion maps to analyze T-cell receptor (TCR) dynamics and predict T-cell signaling states without the need for complex biochemical measurements. LaMDA images thousands of TCR microclusters on the surface of live primary cells to collect high-dimensional dynamic data for machine learning, which extracts key dynamic features to build predictive diffusion maps. LaMDA spatiotemporally reveals global changes of TCRs across the 3D cell surface, accurately differentiates stimulated cells from unstimulated cells, precisely predicts attenuated T-cell signaling after CD4 and CD28 receptor blockades, and reliably discriminates between structurally similar TCR ligands. We anticipate broad usage of this approach for other receptors and cells, as well as for guiding the design and development of future immunotherapies for cancer, infection, and autoimmunity.
IFN-γ control of metabolic stress response during T. gondii infection
Authors: Reyes JS B , Zhao Yanlin , Yap George S
Published: January 2023

IFN-γ is essential for host resistance against Toxoplasma gondii infection; however, overproduction of IFN-γ systemically results in increased host morbidity. During acute infection, mice lose weight due to anorexia and hypermetabolism and exhibit high levels of circulating IFN-γ. We wish to understand the role of IFN-γ in the host stress response during infection. We focused on two peptide stress hormones: GDF15 and FGF21. Growth differentiation factor 15, a member of the TGF-β family, is an anorexic agent while fibroblast growth factor 21 is a hormone that upregulates energy expenditure. Indeed, we observed increased levels of serum GDF15 and FGF21 during acute infection preceding weight loss. To investigate the contribution of IFN-γ in the regulation of GDF15 and FGF21, we transiently neutralized IFN-γ in T. gondii infected mice. We observed a decrease in serum GDF15 and FGF21 levels following IFN-γ blockade. Moreover, IFN-γ neutralization not only prevented further weight loss but also led to weight gain. However, there was no difference between control and treated mice in the tissue levels of GDF15. Altogether, these data suggest that IFN-γ contributes to the sickness response during infection by regulating the systemic levels of GDF15 and FGF21. Ongoing studies are focused on the role of IFN-γ in promoting the maturation and release to the circulation of these metabolism-regulating stress hormones during infection.
KLRG1+ Memory CD8 T cells Combine Properties of Short-lived Effectors and Long-lived Memory
Authors: Huggins Matthew A , Renkema Kristin R , da Silva Henrique Borges , Knutson Todd P , Henzler Christy M , Jameson Stephen C , Hamilton Sara E
Published: January 2023

An effective memory CD8 T cell response must generate cells that respond robustly to antigen re-encounter to produce an effective recall response while also maintaining a stable pool of durable cells in the absence of antigen. During the effector response KLRG1+ CD127low cells are described predominantly as short-lived effectors that will undergo apoptosis during the contraction phase or downregulate KLRG1. However, we have shown that long-lived KLRG1+ CD127low T cells are still present at the memory phase, and that these T cells (termed long-lived effector cells or LLEC) display robust protective function during acute re-challenge with bacteria or viruses. Here, we find that LLEC are derived exclusively from KLRG1+ effector cells, and that this contributes a substantial fraction of the resulting memory pool. Our work challenges the concept that the KLRG1+ CD127low population is dominated by short-lived cells, shows that KLRG1 downregulation is not a prerequisite to become a long-lived protective memory T cell, and highlights the diversity within the memory compartment. Furthermore, using single-cell sequencing we demonstrate that LLEC are not simply a remnant effector population that has failed to undergo initial contraction, but rather a discrete population of T cells with a distinct transcriptional signature that shares characteristics of both early effectors (GzmA, GzmB, Klrg1, Cx3cr1, Zeb2), long-lived memory cells (Bcl2, Il7r, Aqp9, Dmrta1), and a pattern of inhibitory receptors associated with NK cell function (Klrc1, Klre1, Klrd1). Overall, the unique transcriptional profile of LLEC translates to an identity encompassing the hybrid characteristics of effector-like cytotoxicity paired with longevity.
Blimp-1 in adipose resident Tregs controls adipocyte beiging and obesity
Authors: Beppu Lisa , Qu Xiaoyao , Marrero Giovanni , Fooks Allen , Frias Adolfo , Helfrich Katherine , Sipula Ian , Xie Bingxian , Watkins Simon , Poholek Amanda , Jurczak Michael , D’Cruz Louise
Published: January 2023

Visceral adipose tissue regulatory T cells (VAT Tregs) protect against systemic inflammation and metabolic disease by limiting expansion of pro-inflammatory Th1 cells and M1 macrophages, and by preserving insulin sensitivity and glucose tolerance. Although their basic markers and roles have been studied, less is known about the transcriptional machinery regulating their differentiation and function.

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional regulator known to be involved in the development, polarization, and maintenance of various immune cells including CD4+ T cells. Using Blimp-1 reporter mice, we discovered that Blimp-1 is constitutively expressed in a subset of VAT Tregs compared to lymphoid Tregs, and that Blimp-1+ VAT Tregs are phenotypically distinct from their Blimp-1− counterparts. Blimp-1 is not required for VAT Treg development, however Treg-specific deletion of Blimp-1 led to unique changes in VAT Treg markers in lean versus obese adipose tissue. In addition, Blimp-1 knockout mice fed high fat diet had fewer adipose-resident NK cells and increased CD8 T cells. Surprisingly, loss of Blimp-1+ Tregs led to less adipose tissue IL-10, increased expression of thermogenic genes, reduced body fat, decreased weight, and improved insulin sensitivity. Based on these data, we hypothesize that Blimp-1+ Treg dependent IL-10 production suppresses adipocyte beiging, and that loss of these cells results in increased thermogenesis, greater weight loss and improved insulin sensitivity in obese mice.
Using Electric Cell-Substrate Impedance Sensing (ECIS) as a novel method to evaluate bacterial urinary tract infections
Authors: Horseman Tim , Campbell Carmen , Gomes Grace , Fong Keith
Published: January 2023

Urinary tractinfections (UTIs) are commonly acquired bacterial infections that cause significant morbidity, worsened by the rise of antimicrobial resistance among uropathogenic bacteria. Understanding the dynamics of bacterial UTIs is essential to the proper use of antimicrobial therapies in patient treatment. In this study, Electric Cell-Substrate Impedance Sensing (ECIS) technology was used to monitor bacterial-mediated cytotoxicity in-vitro enabling evaluation of antibiotics. Using the ECIS system, real-time changes of barrier function were recorded for human bladder epithelial cells infected with either antibiotic sensitive or resistant bacteria. This study employed four common uropathogens: Pseudomonas aeruginosa, Escherichia coli, Enterococcus species, and Klebsiella pneumoniae. The effect of different antimicrobials, antibiotic titers, and organism concentrations were evaluated. The addition of an antibiotic helped confirm and differentiate sensitive and resistant bacterial strains. Antibiotic concentrations at sensitive and resistant breakpoints were not as effective at high organism concentrations but were completely effective at lower more clinically relevant bacterial loads. The data showed bacterial strains resistant to co-cultured antibiotic took longer on average to degrade the cell monolayer than sensitive strains. Overall, these results indicate that antibiotic resistance is often associated with a fitness cost. Our findings support the use of ECIS to augment routine susceptibility data allowing the direct phenotypic measurement of bacterial virulence and the effects of antibiotic treatment on these strains.
Development and organization of pulmonary lymphoid aggregates after Klebsiella pneumoniae infection of the murine respiratory tract
Authors: Wasbotten Rachel , Smith Catherine Diane Morffy , Rosen David A.
Published: January 2023

Klebsiella pneumoniae is a worrisome nosocomial pathogen given the widespread emergence of antibiotic resistant strains. Little is known about the host immune response to K. pneumoniae; however, it has recently been shown that aggregates of lymphoid cells can be found in murine lungs 4 weeks after surviving K. pneumoniae pulmonary infection. Others have characterized pulmonary collections of lymphoid cells in response to other pathogens and coined them induced bronchus-associated lymphoid tissue (iBALT). iBALT is believed to assist in protection against re-infection. In our nonlethal model of K. pneumoniae, mice that survive the primary infection and exhibit these structures are indeed protected from K. pneumoniae challenge. To further characterize the development and organization of these structures, pulmonary immune cell populations were analyzed post-infection by flow cytometry, histology, and immunofluorescence. As expected, pulmonary neutrophil populations increase over the first 10 days post-infection. T cells appear to rapidly expand between 7 and 10 days post-infection and both CD4+ and γδ T cell subsets make up the majority of this population. Immunofluorescent imaging of lymphoid aggregates demonstrate that CD4+ and γδ T cells are found within these structures at early time-points. Experiments using TCRβδ−/− mice lacking T cells reveals the absence of lymphoid aggregates 28 days post-infection, suggesting that T cells are essential in the initial formation and development of these structures.
Nanobody LAMPoles: Novel tools for dengue virus and Zika virus diagnosis
Authors: Grab Dennis J , Sharma Anuj , Burbulis Ian , Lehrer Axel T , Nerurkar Vivek , Magez Stefan
Published: January 2023

Detection of antibodies by ELISA for verifying exposure to dengue virus (DENV) and Zika virus (ZIKV) has been a challenge. Point-of-care (POC) diagnostics with detection of very low concentrations of viral antigens would improve critical disease staging and surveillance to reduce morbidity and mortality. LAMPoles are protein-DNA hybrids where a ‘DNA barcode’ is covalently linked to a single site on a protein. Recombinant camelid nanobodies (Nb), which are variable region of the heavy chain antigen binding domain (VHH), can be tailored to bind almost any molecular target to maximize assay sensitivity and specificity. LAMPole fusions displaying Nb that bind ZIKV and DENV antigens will improve the sensitivity and specificity of virus antigen measurement at the POC. Llama were vaccinated with recombinant DENV envelope or ZIKV NS1 proteins and VHH phagemid libraries constructed and screened for DENV and ZIKV antigen-specific Nb. For DENV 70 full-length Nb were identified, belonging to 26 different CDR3 groups. For ZIKV 63 full-length anti-ZIKV Nb were identified, belonging to 42 different CDR3 groups. Nb screened by surface plasmon resonance spectroscopy with Koff kinetics in the 10−3 sec range were conjugated to chalcone synthase specific DNA elements using psoralen-based photocross-linkers. In a pilot experiment, LAMP targeting the CHS DNA tail of one Nb-LAMPole detected less than 10 fg of Nb-LAMPole protein. Nb-LAMPole recognized DENV-2 E-protein antigen in sandwich ELISA and in DENV-2 infected cells by immunofluorescence. We assert that LAMPole fusions displaying Nb that bind viral antigens may improve the sensitivity and specificity of DENV and ZIKV antigen detection that will facilitate markedly improved POC diagnosis.
A therapeutic vaccine strategy to prevent Pneumocystis pneumonia in an immunocompromised host in a NHP model HIV and Pneumocystis co-infection
Authors: Rabacal Whitney , Rayens Emily , Jimenez Viviana Cobos , Schweitzer Finja , Kling Heather M , Norris Karen
Published: January 2023

Pneumocystis (Pc) is a ubiquitious fungal pathogen that causes pneumonia (PCP) and Pc-related pulmonary sequelae in HIV-infected individuals and other immunocompromised populations. Deficiencies in CD4 T cell populations and immunologic dysfunction can limit humoral immunity and vaccine efficacy in Pc-susceptible populations. The ultimate goal of the field is to develop an effective vaccine that can overcome immune deficits in at risk populations and induce long-lasting protective immunity to Pc. Toward this goals, our laboratory has established a model of PCP co-infection in simian immunodeficiency virus (SIV)-infected non-human primates (NHP). We have previously identified a recombinant protein sub-unit vaccine, KEX1, that induces robust anti-Pc immunity in immune-competent macaques that is durable and prevents Pc pneumonia following simian immunodeficiency virus (SIV)-induced immunosuppression and subsequent Pc challenge (Kling and Norris, 2016). In the present study, we used the SIV model of HIV infection to address whether therapeutic vaccination with KEX1 can be effective following virus-induced immunosuppression. Type I, or invariant natural killer T (iNKT) cells have the potential to provide CD4-independent B cell help. When activated, iNKT cells are capable of rapidly expressing a wide array of cytokines and co-stimulatory molecules that can enhance humoral responses. The central hypothesis of this study is that a therapeutic vaccination strategy can induce invariant natural killer (iNKT) cell-B cell help to boost anti-KEX1 titers and enhance immunity to Pc during virus-induced immunosuppression.
Effective clearance of Chlamydia from the female reproductive tract in the absence of T-bet+ Th1 cells
Authors: Rixon Jordan , McSorley Stephen J
Published: January 2023

Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted CD4 Th1 responses are thought to be vital for bacterial clearance, due to their capacity to secrete IFN-γ. However, other studies suggest protective or pathogenic roles for additional CD4 T helper subsets. We used a variety of gene-deficient mice to re-examine bacterial clearance following primary genital infection of C57BL/6 mice with Chlamydia muridarum. As seen previously, IFN-γ-, and IFN-γR-deficient mice resolved much of FRT infection, but experienced systemic dissemination and 100% mortality with an average median survival of approximately 4 weeks. Mice lacking the Th1 subset defining transcription factor T-bet or the Th2 transcription factor STAT6 both effectively cleared infection from the reproductive tract, suggesting that neither Th1 nor Th2 cells are essential for local bacterial control. In contrast, RORγt mutant mice selectively deficient in Th17 responses exhibited a week delay in clearance compared to wild-type mice. Th17 subset markers were not observed to be upregulated in T-bet−/− mice and markers of Th1 subsets were minimally upregulated in RORγt mutant mice, indicating that compensation between these subsets does not explain bacterial clearance. In conclusion, timely local clearance of Chlamydia muridarum from the reproductive tract requires Th17 CD4 T cells, but will ultimately clear independent of classical T-bet+ Th1 and RORγt+ Th17 cells individually. Studies are underway to examine which alternative CD4 T cell mechanisms contribute to bacterial clearance within the FRT.
Development and characterization of first ever panels of human helminth-specific IgE monoclonal antibodies (mAb)
Authors: Hadadianpour Azadeh , Daniel Jacob Luke , Smith Scott Alan , Bogdan Olivia
Published: January 2023

According to WHO estimates, more than 1.5 billion people (>24% worldwide) are living with some type of helminth infection and there is no FDA-approved anti-helminth vaccine available. The type 2 immune response is understood to be involved in our defense against helminth infection. In developing countries, elevated serum IgE level is a hallmark of parasitic disease. However, many aspects of the human immune response to helminth infection are still unknown; including, the identity of helminth proteins targeted by IgE antibodies and the size and complexity of the functional IgE repertoire. In this study, we have generated and characterized first ever panels of full-length naturally occurring human helminth specific IgE mAbs. We isolated IgE B cells from individuals with helminth infection and used human hybridoma technology to generate IgE mAbs. ELISA and Western blot techniques were used to test binding of purified IgE mAbs to helminth lysate. The helminth proteins targeted by IgE mAbs were determined using immunoprecipitation, mass spectrometry and de novo peptide sequencing techniques. The information obtained regarding IgE targets and their immune dominance will assist in our basic understanding of the role IgE plays in defending us against helminth infection and will ultimately aid in the rational design of anti-helminth vaccines. Lastly, we analyzed IgE mAb sequences and found remarkable variation in antibody variable region gene usage as well as mutation frequency.
The influence of the lung virome on pulmonary B cells
Authors: Aihara Fumiaki , Feng Feng , Wang Yu Mei , Breen Michael , Elledge Stephen J. , Mizgerd Joseph P. , Fearns Rachel , Kepler Thomas B.
Published: January 2023

The human microbiome is a complex and diverse environment that is implicated in human health and disease. Major locations that host a microbiome include the gut, respiratory system, and the urogenital tracts. While there have been steady advances of the effect of our commensal flora in regions such as the gut, the impact of the pulmonary microbiome, specifically the viral arm of the microbiome (virome), remains under-researched. Studies that have addressed this field provided insight into a complex interplay between viruses and the immune system. On one hand, there are reports of viruses inducing protection from other microorganisms, while on the other, they are correlated with increased risk towards other diseases. Despite this growing body of work on how the lung virome influences human health through the immune system, the immunological mechanisms that underlie this interaction remains unclear.

We hypothesize that constant exposure to pulmonary virus will generate a unique B cell repertoire distinct from peripheral B cells that is adapted specifically to pulmonary viruses. To address this hypothesis, we compared memory B cells from patent-paired lung and blood samples by flow cytometry as well as their secreted antibody affinity towards viral epitopes. Our results show B cell population distributions unique in the lung relative to blood. Secreted antibodies collected from cultured B cells have also shown specificity towards human viral pathogens. We believe our results will provide insight into the influence of a unique and chronic viral presence on pulmonary immunity.
Zinc supplementation modulates T helper 17 cells via its effect on gut microbiome
Authors: Gordon Sarah R , Vaishnava Shipra
Published: January 2023

Zinc is an essential micronutrient involved in many cellular functions in both humans and bacteria. In the gut, the availability of zinc may impact survival and pathogenicity of the gut microbiota. Recent studies show that changes in levels of dietary zinc can alter the composition of the gut microbiota, however how this impacts mucosal immune responses is not known. Here, we show that supplementing mice with zinc sulfate in drinking water for a week resulted in significant changes in gut microbial composition, as well drastic reduction in microbial diversity. More importantly we saw that zinc supplementation attenuated the T helper 17 cell number and activity in murine small intestine. Th17 induction via adherent microbes is mediated by upregulation of Serum Amyloid A (SAA) from epithelial cells. We see a reduction in SAA in zinc-supplemented mice. Furthermore, T regulatory cell numbers and IL-10 cytokine levels, which are known to antagonizes Th17 induction, is unchanged during zinc supplementation. Using 16S rRNA sequencing, we found major shifts in community composition of the mucosal-associated microbiota upon zinc supplementation. To determine if zinc-dependent changes in gut microbiota are sufficient to limit Th17 response, we transplanted microbiome from zinc treated mice into germ-free mice. We saw that germ-free mice that received cecal contents from zinc treated mice have reduced Th17 response thus establishing that dietary zinc changes immune potential of gut microbiome. Th17 cells mediate antimicrobial response in the gut and are involved in the pathogenesis of many autoimmune diseases. Our work thus provides a novel nutritional approach for remodeling microbiota to dampen inflammatory immune response in the gut.
Microbiota-derived lactate control hematopoiesis and erythropoiesis through regulating stem cell factor from leptin receptor+ niche cells in the bone marrow
Authors: Kim Tae-Young , Lee Yong-Soo , Kim Yeji , KIM SEUNGIL , Lee Su-Hyun , Kweon Mi-Na
Published: January 2023

Although the functional interplay between the intestinal microbiota and the distant sites beyond the gut have been identified, it is still unveiled how microbiota-derived metabolites are involved in determining the fate of hematopoietic stem cells (HSC). In this study, we are aiming to investigate a role of microbiota-derived lactate in hematopoiesis using G-protein-coupled receptor (Gpr) 81, a known lactate receptor, deficient (Gpr81−/−) mice. We found that total numbers of hematopoietic cells and numbers of Lin−c-Kit+Sca-1+ HSC were significantly reduced in the bone marrow (BM) from Gpr81−/−mice when compared to heterogenic (Gpr81+/−) mice in steady-state condition. Of note, expression levels of stem cell factor (SCF), which is required for maintenance of HSC and erythropoiesis, were significantly decreased in Leptin-receptor expressing (LepR+) stromal cells around sinusoidal vascular of BM tissue obtained from Gpr81−/−mice when compared to those from Gpr81+/− mice. In addition, Gpr81deletion considerably reduced hematopoietic recovery and SCF expression in the BM damaged by irradiation. Oral administration with probiotics or lactate stimulated SCF secretion from LepR+ BM stromal cells and increased hematopoiesis and erythropoiesis. Furthermore, colonization of lactate-producing bacteria in GF mice also accelerated HSC number and SCF expression. Taken together, these results demonstrate that microbiota-derived lactate stimulates SCF secretion by LepR+ BM stromal cells and subsequently activate hematopoiesis and erythropoiesis in a Gpr81-dependent manner.
Short chain fatty acids regulate T cell metabolism to promote IL-22 production and gut immunity
Authors: Yang Wenjing , Yu Tianming , Huang Xiangsheng , Bilotta Anthony J , Yao Suxia , Cong Yingzi
Published: January 2023

Interleukin-22 (IL-22) is critical in gut immunity. Both innate lymphocyte cells (ILCs) and CD4+ T cells produce IL-22. ILCs provide a rapid source of IL-22 that is essential for early protection of epithelial barrier function upon enteric infection and inflammatory insult, while T cells become the dominant source of IL-22 in the intestinal lamina propria during chronic intestinal inflammation. Microbiota is central in IL-22 production in the intestines, however, the factors that regulate IL-22 production in T cells and ILCs in the intestines, and the mechanisms involved remain unclear. We found that microbiota-derived short-chain fatty acids (SCFAs) promoted IL-22 production in T cells and ILCs through their inhibition of HDAC activity but not GPR43. Additionally, SCFAs upregulated IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor (HIF)1α expression, which were differentially regulated by mTOR and Stat3 pathways. Furthermore, HIF1α directly bind to the il22 promoter, and SCFAs increased HIF1α binding to the il22 promoter through histone modification. SCFAs enhanced oxygen consumption and glycolysis, which mediated SCFA-induced IL-22 production. Hypixic condition enhanced the SCFAs induction of IL-22, and glucose facilitated SCFAs induction of IL-22 production in a dose-dependent manner. SCFA supplementation protected intestines from Citrobacter Rodentium infection and dextran sodium sulfate (DSS) insult, which was mediated by enhanced IL-22. SCFAs promoted T cell IL-22 production from patients with Crohn’s disease through inducing AHR and HIF1α. These findings establish the roles of SCFAs in inducing IL-22 production in T cells and ILCs to maintain the intestinal homeostasis.
Complement C3aR loss drives colorectal cancer by modulating gut microbiota
Authors: Guglietta Silvia , Carloni Sara , Darwich Abbass , Fosso Bruno , Weber Lukas M , Robinson Mark D , Pesole Graziano , Rescigno Maria , Krieg Carsten
Published: January 2023

Several lines of evidence suggest that inflammation plays a pivotal role in the development and progression of colorectal cancer (CRC) and can be unleashed by the loss of innate immunosurveillance. The complement system is a central component of immunity and is essential for intestinal homeostasis. Emerging evidence suggests that complement dysregulation is involved in the development and progression of CRC. Here we show for the first time that methylation of the complement anaphylatoxin C3a receptor (c3aR1) in CRC patients results in decreased overall survival and events-free survival. Based on our clinical findings, we ablated c3ar1 in the spontaneous APCMin mouse model of intestinal tumorigenesis. Loss of C3aR in this model resulted in a shift of tumorigenesis from the small intestine to the colon, therefore more closely mirroring human CRC. By performing microbiota sequencing and fecal microbiota transplantation (FMT) experiments, we found that loss of C3aR resulted in the establishment of a pro-inflammatory microbial flora, which fostered strong Th1/Th17 cell inflammation. Our findings highlight a previously unrecognized oncosuppressive role for C3aR in CRC that could be exploited as a biomarker or for therapeutic intervention.
Steady-state interferon-stimulated gene expression in the intestinal epithelium depends on interferon lambda and the bacterial microbiome
Authors: Van Winkle Jacob , Peterson Stefan , Wheadon Michael , Lee Sanghyun , Baldridge Megan , Nice Timothy
Published: January 2023

Distinct cellular responses have evolved that are specialized for eliminating pathogens in mucosal surfaces, such as the gastrointestinal tract. In particular, intestinal epithelial cells (IECs) are uniquely responsive to interferon lambda (IFN-λ) and depend on this cytokine for antiviral defense to a greater extent than other types of interferon. IFN-λ signaling protects IECs by inducing expression of antiviral IFN-stimulated genes (ISGs), but it is unclear how this signaling pathway interacts with the intestinal microbiome. We find that depletion of the intestinal microbiome with broad spectrum antibiotics significantly reduces steady-state ISGs in the gastrointestinal tract when assessed by whole-tissue RNA sequencing. Steady-state ISG expression is also significantly reduced in mice with conditional knockout of IFN-λ receptor (IFNLR) in IECs. These data suggest that enteric bacteria stimulate expression of IFN-λ and, subsequently, epithelial ISGs at homeostasis. Strikingly, imaging data reveals distinct pockets of IFNLR-dependent ISGs throughout the small intestinal epithelium. These highly localized ISGs are also dependent on the presence of bacteria and IFNLR expression by IECs. Notably, in addition to localized ISG expression along the length of the gastrointestinal tract, ISG expression is concentrated in the most mature IECs at the tips of individual villi. These novel observations indicate that ISGs are robustly expressed in localized regions of the intestinal epithelium at homeostasis. This robust ISG expression depends on the bacterial microbiome and may preemptively activate antiviral defenses in vulnerable IECs to improve host fitness against enteric viruses.