bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of SAM in Plasma Cell Differentiation and Function
Authors: Sievert Evelyn P , Franke Marissa , Sciammas Roger
Published: January 2023

Plasma cells (PCs) are terminally differentiated B cells which secrete high levels of immunoglobulin (Ig). While Ig is essential for neutralizing and clearing pathogens, an excess of Ig is detrimental in autoimmune conditions. The transcription factor Blimp-1 is necessary and sufficient for this transition to PCs and is known to regulate Ig secretion. PC differentiation is reliant on multiple levels of regulation - epigenetic, post-transcriptional, and translational, which we hypothesize are dependent on methyltransferase activity for dynamic methylation.

PCs also upregulate high levels of CD98, which functions as a membrane transporter for amino acids such as methionine, and thus initialization of the methionine cycle. In this cycle, methionine adenosyltransferase (MAT) coordinates the production of the methyl donor S-adenosylmethionine (SAM) from methionine. We are interested in determining whether this strong expression of CD98 is dependent on Blimp-1 or regulated by PCs, and to understand the role of SAM is this differentiation event.

Utilizing a Blimp-1-YFP reporter mouse line, we are able to detect the transition to PCs in-vitro and study the requirement for SAM during this transition. Depletion of the precursor to SAM, methionine, resulted in a loss of PC number and secretory capacity. To specifically target the role of SAM for PCs, we treated in-vitro cultured PCs with inhibitors of MAT2A. Preliminary data provides evidence that inhibition of SAM reduces PC phenotype, function, and viability. We aim to determine the role of SAM in Ig secretion through transcriptional analysis and metabolomics, and hypothesize that Ig post-transcriptional regulation in PCs is controlled by methyltransferase activity.
H3K27me3 demethylases are important epigenetic regulators of B cell differentiation
Authors: Kania Anna , Price Madeline , Scharer Christopher D. , Boss Jeremy M
Published: January 2023

The humoral immune response relies on robust differentiation of naïve B cells into antibody secreting plasma cells (PC) in response to foreign, but not self-antigens. To ensure proper immune responses, B cell differentiation is regulated through processes that remodel the epigenome. The histone modification H3 lysine 27 trimethylation (H3K27me3) is associated with a repressive chromatin state and gene silencing. This histone modification is deposited by EZH2, which has been shown to regulate all stages of B cell differentiation leading to PC. However, a role for the two demethylases UTX and JMJD3 that remove H3K27me3 in B cells is still to be elucidated. To determine how these enzymes modulate B cell differentiation, we crossed Utxfl/flJmjd3fl/flmice onto the Cd19Cre/+background (dKO mice). Stimulation of Cd19Cre/+(CreCtrl) and dKO mice with the T cell independent antigen LPS led to a significant increase in PC in dKO mice. This phenotype was attributed to enhanced differentiation of marginal zone (MZ), but not follicular B cells (FoB). UTX- and JMJD3-deficient PC unregulated genes associated with oxidative phosphorylation metabolism and resulted in a significantly higher spare respiratory capacity. In response to infection with influenza virus, we observed a significant increase in GC B cells in UTX- and JMJD3-deficient mice. Taken together, our data place H3K27me3 demethylases as critical epigenetic regulators that restrict B cell responses to T cell dependent and independent stimuli.
Interferon regulatory factor 5 expression in myeloid cells and T cells negatively impacts the outcome of visceral leishmaniasis
Authors: Mai Linh Thuy , Fabié Aymeric , Smans Mélina , Hammami Akil , Stäger Simona
Published: January 2023

Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani and/or L. infantum. VL is characterized by hepatosplenomegaly, immune suppression, and chronic inflammation. We have previously reported that the transcription factor IRF-5 largely contributes to the induction of inflammation and the development of splenomegaly in mice infected with L. donovani. Moreover, Irf5−/− mice only develop limited IFNγ+ CD4 T responses and are more susceptible to infection. However, the cellular source of IRF-5 responsible for these effects is yet unknown. IRF-5 is constitutively expressed in several cells, such as macrophages, B cells, and dendritic cells. We have recently reported that CD4 T cells also express IRF-5 during chronic VL. Here, we investigate IRF-5 function in myeloid and CD4 T cells during VL, using cell-specific knockout mice. First, we show that IRF-5 expression in CD11c+ cells is required for the generation of splenomegaly and leads to increased disease susceptibility. We also demonstrate that IRF-5 expression in CD11c+ cells is not crucial for the development of Th1 responses. Moreover, we demonstrate that IRF-5 activation in Th1 cells results in cell death during chronic L. donovani infection. Triggering of TLR7 by apoptotic cell material promotes IRF-5 activation in Th1 cells. This results in the upregulation of death receptor 5 and caspase 8, making IFNγ+ CD4 T cells more prone to cell death. Because chronic inflammation and tissue disruption are common characteristics of persistent infections, the TLR7- IRF-5 pathway in CD4 T cells may represent a novel mechanism aimed at protecting tissues against sustained inflammation.
Expression of virulence factors and pro-inflammatory cytokines by Candida species associated with Vulvovaginal Candidiasis in Eastern Nigeria
Authors: Emele Felix Emele , Anieto Ezinne Chinelo
Published: January 2023

Introduction

Candida albicans (CA) is a major cause of vulvovaginal candidiasis (VVC). Adherence to vaginal epithelial cells (VECs) is considered the first step in Candida colonization and eventual invasion of the vaginal mucosa; extracellular hydrolytic enzymes play important role in the invasion of the host. Transition from colonizing yeast cells to tissue-invading hyphae results in secretion of pro-inflammatory mediators, such as IFN-γ, IL-1β, and IL-17. Our earlier reports suggested increasing involvement of non-albicans Candida species (NAC) in VVC, in our locality. We, therefore, saw the need to compare local isolates of CA and NAC for virulence potentials and production of key inflammatory cytokines.

Materials and Methods

Isolates of CA and NAC were investigated for proteinase, phospolipase, and haemolytic activity, as well as ability to adhere to vaginal epithelial cells (VECs) of women of Child-bearing age. The isolates were also inoculated (intra-vaginally) into mice, and assayed for IFN-γ, IL-1β, and IL-17.

Results

Production of phospholipase, proteinase, and haemolysis, by the isolates, were as follows: (CA = 57%, 91%, and 100%; NAC = 10%, 83%, and 78%, respectively). Mean adherence to VECs was: NAC (2.90+3.40); CA (2.81+2.0). Comparable levels of cytokines were generated by CA and NAC in vaginal wash and serum, although NAC produced slightly more IL-17 than CA in day 14.

Conclusion

Based on the results, it could be concluded that the increasing prevalence of NAC in VVC could possibly be as a result of increasing immuno-compromising states of women in our locality, rather than etiological factors.
Immune reactivity to tuberculosis and candida antigens in subjects with latent tuberculosis infection and unexposed subjects: A validation cohort study
Authors: Escalante Patricio , Pennington Kelly M. , Van Keulen Virginia P. , Erskine Courtney L. , Peikert Tobias
Published: January 2023

Rationale

Candida albicans (CA) is ubiquitous and individuals are often immunized against it. However, skin testing reactivity with CA-antigens (CAAg) have been variable during latent tuberculosis infection (LTBI) screening. We hypothesized that the use of a CAAg in an ELISPOT assay would provide a positive control to study tuberculosis (TB)-antigen specific immune responses.

Objective

To test an ELISPOT with TB-antigens and control conditions, including a purified protein derivative of CA, CAAg, that differentiates subjects with LTBI from unexposed subjects.

Methods

An ELISPOT that detects interferon-γ from PBMCs (2×105 cells) after 2-day incubation with TB antigens, controls, including CAAg, and co-stimulatory antibodies. We studied two independent cohorts of subjects with LTBI diagnosis based on TST and IGRA results per clinical guidelines.

Results

One-hundred thirty-nine subjects were studied in 2010–12, including 27 patients with LTBI and 21 IGRA(−) unexposed subjects. ELISPOT with TB-antigens but also CAAg - nil (78 [21 – 182] vs. 9 [1 – 50] cfu) differentiated LTBI subjects from unexposed ones (P<0.005). A second cohort of 76 subjects were studied in 2015–17, including 14 patients with LTBI and 24 unexposed controls, which showed similar statistical results. Positive control (SEAB/antiCD3) did not differentiate these study groups. ELISPOT results with CAAg correlated with results of ELISPOT with TB antigens in all subjects (P<0.001), and in the LTBI and unexposed groups independently.

Conclusion

An ELISPOT assay that includes CAAg revealed immune reactivity that is prevalent in LTBI. The immune subsets that react to both CA and mycobacterial antigens are unclear, but may be relevant for both TB and candida immunity.
Investigating Staphylococcus aureus tolerance to innate immunity
Authors: Kluthe Kennedy Elizabeth , Daubert Trevor , Page Alexis , Carlson Kim , Nuxoll Austin
Published: January 2023

S. aureus accounts for approximately 80,000 infections every year, resulting in 11,000 deaths. While antibiotic resistance has been researched heavily, antibiotic tolerance has not. Persister cells are dormant like cells that are tolerant to antibiotic treatment. Persister cells could be the cause of many chronic and relapsing infections. Recent experiments have shown central metabolism is a critical component of persister cell formation. Results suggest that interruptions in the tricarboxylic acid (TCA) cycle increase persister cell formation. While many studies have been performed concerning the mechanism of formation of persister cells, few have been done to analyze how persister cells react in in vivo models or against aspects of the innate immune system. Antimicrobial peptides (AMPs) are a key component of the innate immune system. Challenging wild type S. aureus with AMPs, LL-37 and hBD-3, showed several logs of killing. Interruption of the TCA cycle (fumC) resulted in 100-fold more surviving cells compared to wild type. Following infection in Drosophila melanogaster, the fumC knockout exhibited increased survival. Furthermore, D. melanogaster that were infected with fumC had reduced survival. Interruption of the TCA cycle also led to increased survival within a macrophage suggesting persister cells represent a multifaceted problem for the immune system. These data suggest that persisters not only present a challenge during antimicrobial therapy but also for the innate immune system.
Polymicrobial sepsis impairs antigen-specific memory CD4 T cell-mediated immunity
Authors: Sjaastad Frances V , Kucaba Tamara A , Dileepan Thamotharampillai , Swanson Whitney , Dail Cody S , Cabrera-Perez Javier , Murphy Katherine A , Badovinac Vladimir P , Griffith Thomas S
Published: January 2023

Sepsis strikes 750,000 Americans annually. Patients who survive sepsis display prolonged immune dysfunction and heightened risk of secondary infection. CD4 T cells support a variety of cells required for protective immune responses, and perturbations to the CD4 T cell compartment can decrease overall immune system fitness. Using the cecal ligation and puncture (CLP) mouse model of sepsis, we investigated the impact of sepsis on endogenous Ag-specific memory CD4 T cells generated in C57Bl/6 (B6) mice infected with attenuated Listeria monocytogenes (LM) expressing the I-Ab-restricted 2W1S epitope (LM-2W). Sepsis transiently reduced the number of 2W1S-specific memory CD4 T cells on day 2, but numbers recovered by day 14. In contrast to the transient numerical change, there was prolonged impairment in the functional capacity of the 2W1S-specific memory CD4 T cells after sepsis, evidenced by a reduced recall response (proliferation and effector cytokine production) after restimulation with cognate Ag. We then assessed 2W1S-specific memory CD4 T cell-mediated protection to a secondary bacterial challenge after sepsis induction. B6 mice were infected with attenuated Salmonella-2W 30 days before sham or CLP surgery, and then challenged with virulent Salmonella-2W after surgery. Pathogen burden was significantly higher in the CLP-treated mice compared to shams. Similar reductions in functional capacity were noted for the endogenous OVA323–339-specific memory CD4 T cell population in septic mice. Our data collectively show CLP-induced sepsis alters the number and function of Ag-specific memory CD4 T cells, which contributes (in part) to the characteristic long-lasting immunoparalysis seen after sepsis.
Activation of skin-resident group 2 innate lymphoid cells by cold-sensing TRPM8+ neuron-derived signals in thermo-homeostatic regulation and tissue inflammation
Authors: Xu Ming
Published: January 2023

Innate lymphoid cells (ILCs) in the skin are a heterogeneous population of innate lymphocytes that function in local homeostatic regulation but could also cause inflammation. How homeostatic versus inflammatory functions of skin ILCs are regulated is poorly understood. To explore this question, we analyzed gene expression profiles of skin ILCs of wild-type and Rag1−/− mice and discovered that the chemokine receptor CCR10+ skin ILCs dominant in the health skin were of homeostatic properties while CCR10− skin ILCs are activated effector cells with elevated expression of various stimulatory cytokine receptors and signaling molecules. In stearoyl-CoA desaturase 1-knockout (KO) mice whose skin was defective in thermohomeostatic maintenance due to impaired fatty acid synthesis and lipid barrier formation, there was predominant activation of CCR10−CD81+ST2+ group 2 ILCs (ILC2s) while raising these mice at a thermo-neutral room temperature or feeding them with high-fat diet reduced the ILC2 activation, suggesting that skin ILC2s are activated by cold-induced signals to help thermo-homeostatic maintenance. Consistent with this, topical application of menthol, an agonist ligand for the cold-sensing receptor TRPM8, led to activation of CCR10−CD81+ST2+ skin ILC2s and increased dermal adipocyte maturation and uncoupling protein 1 production. Furthermore, compared to wild-type controls, menthol-treated TRPM8-KO and IL-18-KO mice had reduced CCR10−CD81+ activated skin ILC2s, suggesting that IL-18 mediated the TRPM8 activation of skin ILC2s. Our study reveals a neuron-derived cold-sensing pathway in activation of skin ILC2s that helps thermo-regulation but could also cause tissue inflammation if chronically activated.
Migratory memory CD4 T cells surveilling the lower reproductive tract direct rapid clearance of Chlamydia muridarum following vaginal challenge
Authors: Kohlmeier Alison , Li Tiger , Lobby Jenna , Kost Kirsten , Dunbar Paul , Lowen Anice , Zheng HaoQiang , Kohlmeier Jacob E
Published: January 2023

Despite their importance for immunity against sexually transmitted infections, the regulation and function of CD4 T cells in the female reproductive tract (FRT) remains poorly defined. Here we show that across human, non-human primate, and mouse species the FRT memory CD4 T cell pool consists of distinct subsets corresponding to migratory (TMM) and resident (TRM) memory T cells. TMM in the FRT exhibit broad expression of trafficking molecules, restricted distribution to discreet areas of the FRT, and differential responses to local infection. To investigate the contribution of TMM to protective immunity in the FRT, we constructed an influenza virus (HK/x-31) expressing an immunodominant CD4 T cell epitope from the Chlamydia muridarum probable outer membrane protein (PmpG-1303–311). C57Bl/6 mice were infected intranasally with x31-PmpG1 to generate PmpG1-specific CD4 TMM and investigate their ability to surveil distant mucosal tissues. PmpG1-specific CD4 T cells were found restricted into the lower, but not upper, FRT and expressed chemokine receptors and adhesion molecules consistent with TMM. Vaginal challenge of x31-PmpG1 memory mice with C. muridarum showed a significant decrease in bacterial burden at 14 days post-infection (dpi), when bacterial clearance was first detectable in naïve control mice. Notably, x31-PmpG1 memory mice treated with FTY720 during the initial stages of C. muridarum challenge failed to limit bacterial growth, suggesting this effect was due in part to TMM localization to the FRT. These data show that vaccinating at distant mucosal sites can generate surveilling CD4 TMM cells that are distinct from classical central, effector and resident memory CD4 T cells, and can protect against pathogens in the FRT.
The role of CCL27 in the establishment, maintenance, and homeostasis of skin-resident CCR10+ lymphocytes
Authors: Davila Micha L , Xiong Na
Published: January 2023

The skin hosts an array of immune cell populations that are important for maintaining local tissue integrity; dysregulation of these cells can lead to inflammatory diseases such as psoriasis and atopic dermatitis. Understanding how skin-resident immune cells are regulated is important in the effort to treat skin diseases. The majority of skin-homing and skin-resident lymphocytes express the chemokine receptor CCR10. Our lab has found that CCR10 is important for the homeostatic establishment of skin-resident lymphocytes. The skin-specific ligand of CCR10 is CCL27, a chemokine primarily expressed by keratinocytes. While the regulation and functions of CCL27 are not well understood we have found evidence to suggest that CCL27 has a homeostatic role in the skin. We report that CCL27 expression in keratinocytes and within regions of cycling hair follicles is developmentally regulated, independent of commensal bacterial stimulation. Further, CCL27 expression correlates with the localization of CCR10+ lymphocytes to specific regions within the skin. Newly generated CCL27-knockout mice show that the loss of CCL27 results in the diminished migration and localization of CCR10+ lymphocytes into the skin, particularly within regulatory and CD8+ T cell populations. Preliminary data also indicates CCL27-knockout mice had more severe skin inflammation than wild-type mice in a model of psoriasis. These findings reveal that the regulation of CCL27 expression is important for directing the localization of CCR10+ lymphocytes to specific niches of the skin, helping to maintain local homeostasis under steady-state conditions and hair follicle cycling, and that the loss of CCL27 may contribute to inflammatory diseases.
IL17 and Candida albicans colonization in the vaginal mucosa
Authors: Coleman Bianca M , Peters Brian M , Willems Hubertine M.E. , Barker Katherine S. , Aggor Felix E.Y. , Cipolla Ellyse , Verma Akash H , Bishu Srinivas , Huppler Anna H , Bruno Vincent M , Childs Erin E , Conti Heather , Gaffen Sarah L
Published: January 2023

Candida albicans is a ubiquitous commensal fungus that colonizes human mucosal tissues. C. albicans can become pathogenic, clinically manifesting most commonly as oropharyngeal, dermal or vulvovaginal candidiasis (VVC). Studies in mice and humans convincingly show that Th17/IL-17-driven immunity is essential to controlling oral and dermal candidiasis. In contrast, the role of the IL-17 pathway during VVC remains controversial, with conflicting reports from human data and mouse models. It has been observed that a strong IL-17-related gene signature is induced in the vagina during estrogen (E2)-dependent murine VVC. IL-17R deficient mice are resistant to VVC in this standard model of E2-induced disease. Since estrogen increases susceptibility to vaginal colonization and subsequent immunopathology, first we asked whether its use in the standard VVC model masks a role for the Th17/IL-17 axis. Here, we demonstrate that mice lacking IL-17 receptor signaling showed no evidence for altered VVC susceptibility or immunopathology, regardless of estrogen administration, supporting the emerging consensus that Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of VVC. We further considered the epithelial dynamics during murine VVC. C. albicans colonizes mucosal epithelial tissues, particularly those expressing Keratin 13 (K13). When estrogen levels are high, this epithelium becomes increasingly keratinized, characterized by a thick layer of thin, flat pieces of tissue. In order to visualize the expression patterns in the vaginal epithelium, we developed a reporter mouse where LACZ is expressed in all tissues containing K13. We present data using these mice to assess epithelial dynamics during VVC.
Cigarette Smoke Induces Intestinal Inflammation via a Th17 Cell-Neutrophil Axis
Authors: Kim Myunghoo
Published: January 2023

Epidemiological evidence finds cigarette smoking is a common risk factor for a number of diseases, not only in the lung but also in other tissues, such as the gastrointestinal tract. While it is well-documented that smoking directly drives lung inflammatory disease, how it promotes disease in peripheral tissues is incompletely understood. In this study, we utilized a mouse model of short-term smoke exposure and found increased Th17 cells and neutrophilia in the lung as well as in the circulation. Following intestinal inflammatory challenge, smoke exposed mice showed increased pathology which corresponds to enhanced intestinal Th17 cells, ILC3 and neutrophils within intestinal tissue. Using cellular depletion and genetic deficiencies, we define a cellular loop by which IL-17A and downstream neutrophils drive cigarette smoke-enhanced intestinal inflammation. Collectively, cigarette smoke induced local lung Th17 responses lead to increased systemic susceptibility to inflammatory insult through enhanced circulating neutrophils. These data demonstrate a cellular pathway by which inflammatory challenge in the lung can sensitize the intestine to enhanced pathological innate and adaptive immune responses
Revisiting the critical role of Th17 cell plasticity in the Peyer’s patch for gut IgA responses using single cell analysis unexpectedly demonstrated unperturbed responses to oral immunization in their complete absence
Authors: Lycke Nils , Gribonika Inta , Bemark Mats
Published: January 2023

Gut Th17 cells are present at the Peyer’s patch inductive site as well as at the lamina propria effector site of the intestinal immune system. These cells are responsive to pathogens as well as to the commensal microbiota and they appear to be critical for gut homeostasis. While they have been associated with immune protection against pathogens they are also known to drive autoimmune inflammation in the gut. Hence, plasticity is a hallmark of gut Th17 cells and pro- as well as anti-inflammatory functions can be expected. This places Th17 cells at center stage for immune regulation of the gut immune system. However, their role for gut IgA responses to oral immunization awaits to be better defined, although earlier studies suggested that Th17 cells play a central role as their plasticity can convert them into follicular helper T (Tfh) cells that stimulate IgA class-switching and drive gut IgA responses. Here we used an IL-17 fate-reporter mouse and a cholera toxin B-subunit (CTB)-specific tetramer to isolate CTB-specific CD4 T cell subsets following oral immunization with CT. We found no evidence of CTB-specific Th17 development in the PP, rather a clear dominance of Tfh cells was observed. Single-cell RNAseq analysis revealed a massive clonotypic dominance of Tfh cells, but no Th17 or regulatory T cells (Tregs) were found among the CTB-tetramer-specific CD4 T cells. While 3 clusters of Tfh cells were found only one cluster hosted the CTB-specific CD4 T cells. An adoptive transfer of rorgt-deficient CD4 T cells into nu/nu recipient mice exhibited unperturbed gut IgA responses to oral CT immunization, clearly demonstrating that Th17 cells are, indeed, dispensible for gut IgA responses following oral immunization.
The role of glutamate-NMDA-receptor in mice alpha beta T cells from intestinal mucosa
Authors: De Oliveira Marília Garcia , Andreoni Rafaela , Brandao Wesley , Peixinho Beatriz , Polonio Carolina Manganeli , Zanluqui Nagela Ghabdan , Oliveira Lilian , Peron Jean Pierre S
Published: January 2023

The intestinal mucosa consists of a simple layer of epithelial tissue and loose connective tissue called the lamina propria that is formed by several immune cells mainly T cells that play important roles in local tolerance to microorganisms of the microbiota and food antigens. In addition to mucosa the small and large intestines also present the submucosal and the muscular layers that present ganglionic enteric plexuses formed by networks of interconnection between autonomic neurons of the enteric nervous system. These neurons secrete various neurotransmitters including glutamate and their innervations reach the lamina propria. It is known that T cells have receptors for neurotransmitters even the ionotropic glutamate receptor NMDAR. Thus, the present study intends to evaluate the role of NMDAR in αβ T cells from intestinal mucosa. C57BL/6 Grin1f/f and CD4crexGrin1flox mice of both sexes were used at 8 weeks of age. CD4+ T cells, CD8+ T cells, γδT cells, dendritic cells and macrophages present in mesenteric lymph nodes and at intraepithelial compartment of small intestine were evaluated. We found that CD8+ T cells were decreased and γδT cells were increased in mesenteric lymph nodes of CD4crexGrin1flox mice in comparison to the control group. Thus, intraepithelial CD4+ T cells and macrophages were increased, but intraepithelial CD8+ T cells were decreased in small intestine of CD4crexGrin1flox mice compared to the control group. Our preliminary results shows that the absence of NMDAR exclusively in αβ T cells alters the percentage of immune cells present both in mesenteric lymph nodes and intraepithelial compartment of small intestine suggesting a modulation by glutamate. New evaluations are still in progress to better understand that.
Optimizing bacteriotherapy to prevent or treat food allergy
Authors: Hesser Lauren A. , Hubbell Jeffrey , Nagler Cathryn
Published: January 2023

Depletion of protective gut microbes strongly correlates with the increasing prevalence of non-communicable diseases, including food allergies. In previous work we transferred feces from healthy or cow’s milk allergic (CMA) infants to germ free mice and showed that healthy infants harbor intestinal bacteria that are causally protective against food allergy in this model. Monocolonization with a single bacterial species, Anaerostipes caccae, was sufficient to mimic the protective effect of the healthy microbiota. These studies have provided the conceptual basis for the use of bacteriotherapy to prevent food allergy. We are taking a synbiotic approach in which we co-deliver a new sub-strain (A. caccae_lahuc), isolated from the feces of infants in our study, with prebiotic substrates that enhance the ability of A. caccae_lahuc to grow in mice with a replete microbiota and produce butyrate, a short-chain fatty acid with immunoregulatory properties. In vitro studies of A. caccae_lahuc cultured with fecal bacteria from a CMA infant identified potato starch or the combination of lactate and acetate, the intermediate products of fiber breakdown, as prebiotics which maximize butyrate output. We are optimizing delivery of these prebiotics with A. caccae_lahuc to determine whether they stimulate butyrate production in vivo. We found that A. caccae_lahuc bacteriotherapy without prebiotics leads to its stable colonization in mice already colonized with the CMA microbiota without measurable increases in fecal or cecal butyrate. By decoupling growth from butyrate release, we are obtaining mechanistic insights into the components necessary to optimize a synbiotic formulation efficacious for the prevention or treatment of food allergy.
Physiologic expression of AhR regulates CX3CR1+ resident intestinal macrophages and promotes diabetes in NOD mice
Authors: Ehrlich Allison , Kahalehili Heather , Shan Rachel
Published: January 2023

Susceptibility to type 1 diabetes (T1D) is influenced by both genetic and environmental factors. The environmental sensor, the aryl hydrocarbon receptor (AhR), is expressed in mucosal immune cells and can be activated by metabolites derived from the diet and microbiome. We hypothesized that these metabolites may influence the autoimmune response and subsequent susceptibility to T1D. To test this hypothesis, we generated NOD AhR knockout mice and examined the intestinal immune response and development of T1D. Female AhR knockout mice had reduced insulitis at 12 weeks of age, and a decreased incidence and onset of hyperglycemia in comparison to their wild type littermates. To determine whether physiologic expression of AhR was altering the immune response, we analyzed CD4+ T cell subsets and identified an increase in IL-10R+Foxp3+ Tregs in the lamina propria (LP) of AhR knockout mice. We also discovered a population of resident-like macrophages (CD11b+CX3CR1+) in the small intestine LP that were negatively regulated by AhR. These cells have a very strong positive correlation to LP Tregs, and correlate with protection against insulitis in NOD mice. To identify the potential source of AhR ligands that regulate these resident-like macrophages, mice were fed an AhR ligand-deficient diet. These cells were not further regulated by dietary ligands, suggesting that AhR activation originates from the intestinal microbiome. Current studies are aimed at determining how AhR regulates the expression of LP resident-like macrophages and how these cells impose their immunoregulatory functions during T1D pathogenesis.
Roles for Leukemia-inhibitory factor receptor signaling in intestinal immunity
Authors: Kahn Laura Maria , Li Haiyan S. , Chrisikos Taylor T. , Zhou Yifan , Medik Yusra B. , Patel Bhakti , Babcock Rachel Lauren , Newton Kathryn , Watowich Stephanie S
Published: January 2023

Intestinal infections trigger immune responses that in some cases become dysregulated and lead to acute or chronic disease; understanding immune regulatory mechanisms in infection will help advance new therapies for intestinal disorders. Citrobacter rodentium infection in mice mimics enteropathogenic Escherichia coli infections in humans. We found that Leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is upregulated in colon and serum following C. rodentium infection; however, the source, targets and effects of this cytokine during intestinal disease remain unclear. To investigate the role of LIF during infection, we generated mice with LIF receptor (LIFR) deficiency in endothelial and hematopoietic cells (Tie2cre Lifrf/f) or CD11c+ cells (CD11c cre Lifrf/f). Upon C. rodentium infection, Tie2cre Lifrf/fandCD11c cre Lifrf/f mice had reduced colon length, increased bacterial dissemination to spleen and liver, and greater colon pathology scores relative to infected controls. Comparison of cytokine and chemokine expression in the colon revealed upregulation of various factors in infected LIFR-deficient mice, with the pro-inflammatory cytokine IL-23 significantly increased in CD11c cre Lifrf/f mice. Our preliminary data from LIFR-deficient, bone marrow-derived myeloid cells suggests LIFR signaling inhibits inflammatory cytokine production in vitro. Collectively, our results suggest LIFR signaling in CD11c+ cells inhibits colonic IL-23 production and protects from immune dysregulation during C. rodentium infection. Our current studies are investigating LIF-producing and -responding populations in colon, to elucidate protective immune mechanisms during infection.
Curated, multi-omic, ML-driven single-cell atlas for characterizing the human immune system across disease states
Authors: Kiner Evgeny , Sharim Hila , Montalbano Antonino , Raveh-Sadka Tali , Yanover Uri , Wells Daniel K. , Satpathy Ansuman
Published: January 2023

The immune system plays a key role in various diseases such as cancer, autoimmune disorders and infections. It has been recently shown that cell surface protein markers on peripheral blood mononuclear cells (PBMCs) can be predictive of response to therapies1, but measurements of such markers is technically limited in the number of cells and markers that can be assessed. Single-cell RNA technology allows the measurement of the entire transcriptome in tens of thousands of cells, and therefore has the potential to survey the immune system in more depth and reveal rare cell states. However, no large single-cell datasets for immune cell states exist. Moreover, attempts to combine smaller distinct datasets are often hindered by batch effects stemming from differences in sample handling, single-cell technologies and computational analyses. We created a large curated multi-omic single-cell human PBMC atlas with clinical annotations from dozens of patients with several conditions across hundreds of thousands of cells. Our standardized end-to-end protocols and quality control processes provide a platform that allows production of large datasets with minimal batch effects. By combining single-cell RNA-seq (scRNA-seq) with surface marker identification by CITE-seq, we generate curated immune gene signatures and train a classifier to robustly identify many cell types and states across patients and different diseases, including rare cell populations. This curated, multi-omic clinically-annotated atlas is particularly suited for use by machine learning algorithms, and we are confident that as this data accumulates, it will be instrumental for inferring disease states and predict responses to various therapies.
CRISPR Cas9 ribonucleoprotein-mediated genomic editing in primary innate immune cells
Authors: Hildreth Andrew D , Riggan Luke , Rolot Marion , Wong Yung-Yu , Satyadi Will , Sun Ryan , Huerta Chris , O’Sullivan Timothy E.
Published: January 2023

CRISPR (clustered, regularly interspaced, short palindromic repeats) genome engineering has become a powerful tool to functionally investigate complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity for therapeutic purposes, current approaches are often inefficient or nonspecific, limiting their use in primary innate immune cells. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naïve NK cells and bone marrow-derived dendritic cell precursors. Using these methods, we demonstrate that XCR1+ conventional dendritic cells (cDC1) are sufficient and necessary for host protection to mouse cytomegalovirus (MCMV) in a MyD88-intrinsic manner. This scalable method will enhance target gene discovery and offer a specific and simplified approach to gene editing in the innate immune system.
Biomarker Trends: Luminex & Olink, Dried Blood Spots & Plasma
Authors: Rosenberg-Hasson Yael , Ho Tiffany C. , Simon Tamara D. , Liang Jing , Chang Serena , Herschmann Iris , Gotlib Ian H. , Maecker Holden T.
Published: January 2023

Biomarker discovery is at the forefront of understanding disease etiology, diagnosis and emerging treatments. One of the hurdles is to compare data generated by different platforms and technologies. Measurements of common biomarkers between vendors is another challenge which increases as we multiplex and generate more complex data. We address both these aspects concentrating on two major platforms: Luminex and Olink, in serum and cerebrospinal fluid samples. We find that among 30 overlapping cytokines and chemokines, about 20 display similar trends across these two platforms with comparable sensitivity. A few cytokines had differential and reduced sensitivity in one of these platforms emphasizing the need for improved biomarker detection. Our analysis offers a simple method to compare the data from the two platforms. In addition, we demonstrate the use of Dried Blood Spots as a reliable source for generating multiplex data. We compare plasma and DBS samples and observe similar trends for four out of the six cytokines analyzed, specifically: IL-1b, IFN-g, IL-6 and TNF-a. We discuss limitations of this approach and the need to analyze additional biomarkers in both matrices.
Global targeting of functional (phospho)tyrosines using sulfur-triazole exchange chemistry
Authors: Hsu Ku-Lung , Borne Adam L , Brulet Jeffrey W , Hahm Heung S , Toroitich Emmanuel K , Libby Adam H , Yuan Kun , Ware Timothy B , McCloud Rebecca L , Ciancone Anthony M
Published: January 2023

Chemoproteomic probes serve as valuable tools to study and perturb protein function on a global scale. Despite advances in methodologies for cysteine and lysine profiling, a large fraction of the human proteome remains inaccessible with current activity-based probes. Here, we describe development and application of sulfur-triazole exchange (SuTEx) chemistry for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can produce sulfonyl probes with high chemoselectivity for tyrosines over other nucleophilic amino acids to investigate thousands of tyrosine sites in lysates and live cells. We discover hyper-reactive tyrosines are enriched in enzymatic, protein–protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Our findings illustrate the broad potential for deploying SuTEx to globally investigate tyrosine reactivity, function and post-translational modification state in complex biological systems.
Single B cell isolation and cloning from rabbits to generate recombinant antibodies
Authors: Alves Bryce , Jelinek Mary Anne , Lu Yanan , Ritland Melissa , Velasco Patricia , Zhao Xi , Adams Eddie , Fernandez Joseph
Published: January 2023

For decades the method for generating antibodies with high avidity and affinity relied upon monoclonal antibody generation from immunized mice or polyclonal antibodies from rabbits. Both methods have their shortcomings. The murine immune system—specifically the high degree of immunological tolerance—prevents robust humoral responses to antigens that closely reasonable self-antigens in the mouse. Additionally, the cost and time necessary to generate a monoclonal antibody from mice can be prohibitively expensive, particularly if multiple attempts at hybridoma generation are required. A work around is to generate polyclonal antibodies in rabbits. Rabbits are further evolutionarily removed from humans than mice and mount a stronger immunological response to immunized human proteins or antigens. Unfortunately, the source of polyclonal antibodies from each rabbit is finite. To combat this problem, here we describe the development of a method to isolate single rabbit plasma B cells after immunization. Once isolated the VH and VL regions of the antibody are sequenced and the sequences are cloned into a recombinant rabbit IgG and IgK backbone that enables the recombinant expression of the single cell-derived, cloned rabbit antibodies. In doing so we have harnessed the immunological response of the rabbit host while eliminating the supply problem of finite reactive serum volumes and the cost of traditional murine monoclonal antibody generation.
Complete multi-chain adaptome amplification from whole blood and PBMC via novel dam-PCR technology
Authors: Depinet Mollye M , Pan Wenjing , Hou Xiaohong , Brown Brittany E , Eisenhower Mary , Weber Daniel , Byrne-Steele Miranda , Han Jian
Published: January 2023

Profiling the immune repertoire through NGS allows detailed, sequence-specific insight into the adaptive immune response. One of the key challenges during immune receptor amplification is the formation of dimers, which can compete with the immune amplicons of interest during library preparation. Therefore, we developed a novel PCR technique, dimer-avoided multiplex PCR (dam-PCR), that effectively avoids primer dimer formation and allows for the all-in-one qualitative amplification of all BCR and TCR receptors in a single, quantitative reaction. Here, we present this technology’s capabilities of bias-free immune repertoire amplification, even for low frequency CDR3s. First, we demonstrate the ability of this method to accurately detect a Jurkat cell line that was spiked into pre-screened sorted donor CD4+ cells at 1%, 0.1%, 0.01%, 0.001%, and 0.0001%. RNA from each spike-in percentage was extracted and amplified in duplicate using our standard amplification protocol to assess repeatability of CDR3 discovery between replicates, as well as establish the limits of detection and quantitation. Then, we demonstrate the method’s ability to amplify a synthetic repertoire, created by spiking RNA from two individuals of known repertoire into each other at 75%, 50%, and 25%. The mixed RNA, as well as pure RNA from each individual, was then amplified to assess correlation between the predicted repertoire and amplified repertoire at each spike-in percentage. For both the Jurkat cell level spike-in test, as well as the RNA level spike-in test, the amplified repertoire was successfully predicted at an R2 value of > 0.99. This single reaction method will allow for a cost effective, all-inclusive, and quantitative analysis of immune repertoires.
Development of an in vivo model to assess immune and angiogenic responses to metallic implants using Humanized TLR-4 mice
Authors: Wood Steven C , Gotipatti Anuhya
Published: January 2023

Metal on metal(MOM) hips often fail due to inappropriate inflammation and angiogenesis which may be due to localized concentrations of metal ions and/or MOM wear particles in the joint. Critically, human, but not mouse TLR-4 receptors, are responsive to nickel, cobalt and MOM particles. Therefore, we are using humanized, hTLR-4 mice in these studies. MOM hips are predominately cobalt, so our initial experiments were focused upon this ion. Spleen cells of hTLR -4 mice were exposed to 3 concentrations of CoCl2 (2.5μM, 25μM, 250μM) for 3 and 6 days. The cultures were analyzed for endothelial cell markers, (MADCAM and CD31)and T cell markers, (CD4 and CD8). A dose dependent induction of endothelial markers was seen on day 3 with a dose dependent reduction on day 6, with no changes in CD4 or CD8 expression. Next, spleen cells of TLR-4 mice were exposed to 3 different ratios of cell to MOM particles (1:5, 1:50, 1:500 per cell) for 3 and 6 days. Endothelial markers were induced, but it was not dose dependent, however the endothelial cell numbers increased between 3 and 6 days. Again, T cell markers were not altered. To assess the in-vivo responses to MOM particles, nude mice were implanted with angioreactors containing MOM particles (2.5×105 and 2.5 × 106). On day 12, the angioreactors were removed and the was a dose dependent increase in endothelial cells. Currently, we are extending the MOM angiogenesis studies in hTLR-4 mice. In sum, both CoCl2 and MOM particles induced endothelial cells and angiogenesis. The hTLR-4 mouse model will greatly aid in the evaluating the angiogenic and immunotoxicological profile of ions and MOM wear debris, particularly during menopause. Funded by FDA Office of Women’s Health.
A robust and interpretable, end-to-end deep learning model for cytometry data
Authors: Hu Zicheng , Tang Alice , Singh Jaiveer , Bhattacharya Sanchita , Butte Atul
Published: January 2023

Cytometry technologies are essential tools for immunology research, providing high-throughput measurements of the immune cells at the single-cell level. Traditional approaches in interpreting and using cytometry measurements include manual or automated gating to identify cell subsets from the cytometry data, providing highly intuitive results but may lead to significant information loss, in that additional details in measured or correlated cell signals might be missed. In this study, we propose and test a deep convolutional neural network for analyzing cytometry data in an end-to-end fashion, allowing direct association between raw cytometry data and the clinical outcome of interest. Using nine large CyTOF studies from the open access ImmPort database, we demonstrated that the deep convolutional neural network model can accurately diagnose the latent cytomegalovirus (CMV) in healthy individuals, even in the presence of heavy batch effects between studies. In addition, we adopted three approaches for interpreting the deep convolutional neural network model and identified potentially novel immune cell subsets significantly associated with latent CMV infection.
HMGB1 A Box prevents neuronal injury from PD via restricting activation of microglia and inhibiting differentiation and infiltration of Th17 cells
Authors: Su Zhaoliang , Tian Yu , Xia Lin , Chen Rong , Xu Huaxi
Published: January 2023

Parkinson’s disease is a complexly neuronal injury and accompanied by many clinical challenges, for example, lacking of early diagnostic strategies and effective treatment. Here, we demonstrate that in the subacute Parkinson’s disease mice model, injection of HMGB1 competitive inhibitor protein HMGB1 A Box restored the number of dopaminergic neurons and TH+ fibers in the substantia nigra striatum. Our data also showed that A Box inhibited the activation of microglia mediated by neuronal injury and the production of TNF α and IL-1β by up-regulating microglia CD200-CD200R signal axis in vitro and in vivo. Microglia overexpressing CD200R produced fewer inflammatory mediators and restored TH+ neurons in vitro. Intravenous injection of A Box also reduced the levels of RANTES and CXCL12 in substantia nigra and significantly inhibited Th17 cells infiltration. The ability of activated microglia to promote naïve CD4+ T cells differentiation into Th17 in MPP+ model in vitro was also inhibited by A Box. Elevated levels of these two chemokines were also detected in the serum of the patients. In conclusion, our studies show that HMGB1 can induce neuronal damage by activating microglia to promote the production of inflammatory mediators. HMGB1 itself does not directly cause neuronal death. Moreover, the differentiation of T cells into Th17 and migration of T cells secondary to microglia activation induced by HMGB1 further aggravates neuronal damage. The significant effect of HMGB1 A Box indicates the possibility that this recombinant protein might become a potent inhibitor of neuroinflammation in Parkinson’s disease.