bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Epidermal γδ T Cell Functionality by Obesity due to Acute versus Chronic TNF-α Reception
Authors: Jameson Julie M , Ahmed Jasmine , Rhoiney Mikaela , Matthews Carolyn
Published: January 2023

Epidermal γδ T cells exhibit functional plasticity with roles varying from wound healing to tumor cell lysis. Activation of epidermal γδ T cells occurs via the γδ TCR along with costimulation, suggesting that costimulatory ligand expression regulates functional outcome. Data from our lab indicates that the cytokine milieu adds an additional pressure on epidermal γδ T cells to mediate or inhibit a particular functionality. Previous work from our lab shows that obesity impairs epidermal γδ T cell wound repair function in a TNF-α dependent manner. Here we identify TNF-α as a modulator of epidermal γδ T cell function in wound repair or cellular lysis depending on reception of acute or chronic signals. TNF-α signals through two receptors, tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2), thus the signaling dynamics are complex and induce varied responses including apoptosis, survival and inflammation. We show that epidermal γδ T cells exhibit low TNFR1 and TNFR2 expression prior to TCR activation. Even with low TNFR expression epidermal γδ T cells produce cytokines and chemokines involved in inflammation and macrophage recruitment in response to acute TNF-α signals without TCR activation. During TCR activation or IL-2 stimulation epidermal γδ T cells to upregulate TNFR2 indicating a reliance on TNFR2 signaling during activation. Alternatively, chronic TNF-α stimulation, similar to levels in obese patients, induces a reduction in epidermal γδ T cell cytotoxicity, proliferation and viability. Normal function is restored with IL-2 addition suggesting the inhibition is not permanent, but instead can be modulated. These studies provide potential targets for regulating epidermal γδ T cell functions such as wound repair and tumor lysis.
Selenoprotein I is important for phosphatidylethanolamine synthesis and autophagy during T cell activation
Authors: Avery Joseph , Ma Chi , Hoffmann FuKun , Hoffmann Peter
Published: January 2023

Selenoprotein I (SELENOI) is a type I CDP-alcohol phosphatidyltransferase that synthesizes phosphatidylethanolamine (PE) in the final reaction of the Kennedy pathway. We have found that SELENOI deletion in the whole animal is embryonic lethal, which emphasizes the key role PE synthesis plays in cellular division and development. One important role for PE in cells is the role it plays as a ligand for the conversion of LC3I to the lipidated LC3II molecule, which in turn allows the progression of early autophagosomes to proceed to later stage autophagosomes. Using a T cell-specific knockout (KO) model of SELENOI, we investigated SELENOI loss-of-function studies in these immune cells. In particular, SELENOI KO T cells were stimulated with STING-dependent and STING-independent agonists to test the importance of this selenoenzyme in autophagy and downstream signaling events that depend on efficient autophagy. Our results found that PE levels were lower in TCR-activated T cells lacking SELENOI compared to wild-type (WT) controls. LC3I to LC3II conversion was impaired in SELENOI KO T compared to controls, and STING-dependent autophagosome formation was particularly decreased in the absence of SELENOI. Using bone marrow-derived macrophages (BMDMs) from a doxycycline-inducible transgenic mouse that expresses a short hairpin RNA (shRNA) that lowers SELENOI <50%, we found impaired LC3I to LC3II conversion as well as reduced autophagosome formation but not to the extent of that found with the KO T cell model. Our data suggest that SELENOI is an important enzyme for proper lipidation of LC3-I and autophagosome formation within immune cells.
Cytoskeletal control of the developing and mature B cell antigen receptor repertoire
Authors: Gupta Neetu , Labib Christina , Giudicelli Veronique , Aouinti Safa , Lefranc Marie-Paul , Kossida Sofia
Published: January 2023

The existence of diverse B cell antigen receptor (BCR) specificities is essential for development of protective antibody responses against a wide variety of pathogens. In developing B cells, the BCR repertoire is crafted through sequential rearrangements of heavy and light chain genes, and involves both combinatorial and junctional diversification catalyzed by recombinases and nucleases. Mature B cells undergo further diversification in peripheral lymphoid organs through isotype switching and somatic hypermutations in response to foreign antigens; although these events are not known to alter BCR antigen specificity. Cytoskeletal proteins modulate BCR signal strength and thereby antibody responses, but it is not known if they play a role in the generation of BCR diversity. Using next generation sequencing, and iRepertoire, Inc and ImMunoGeneTics (IMGT) software platforms we examined BCR repertoires in developing and mature B cells that lack the expression of Ezrin, a prominent cytoskeletal regulator of BCR organization, signaling and activation. We show that Ezrin deficiency impacts the generation of BCR repertoire diversity during B cell development, as well as the mature BCR repertoire. Our analysis provides a window into the major processes involved in diversifying the B cell repertoire in control and Ezrin-deficient B cells, including V gene usage, number, frequency and length of unique CDR3s, nucleotide additions and trimming, and shared and unique clonotypes. Our data suggest that the Ezrin cytoskeleton in B cells impacts humoral immunity not only through modulation of B cell activation upon foreign antigen encounter, but also through qualitative and quantitative modulation of the available BCR repertoire.
Co-expression network analysis identified cell cycle signaling hub genes associated with progression and prognosis of multiple myeloma
Authors: Adebayo Olayinka Omoyitola , Dammer Eric , Singh Rajesh , Sanjay Jain , Boise Lawrence , Lillard James W
Published: January 2023

There is a gap in understanding the molecular mechanisms behind early multiple myeloma (MM) progression in the newly diagnosed patient on standard treatment, i.e., lenalidomide, bortezomib, and dexamethasone (RVD). As a result, the relatively high percentage of patients that progress after early treatment with RVD is significant. Therefore, identification of clinically relevant clusters of co-expressed genes or related biomarkers for MM progression, while on RVD, would help identify new molecular mechanisms and drug targets. The objective of this study is to use weighted gene co-expression network analysis (WGCNA) to identify gene-signaling networks associated with early MM progression. Therefore, we performed a novel WGCNA to determine module-trait correlations. We next examined the overrepresentation of upregulators and signal pathway networks from MM patients in the MMRF CoMMpass dataset (n = 175). WGCNA constructed 45 modules based on the correlations between patients on RVD and death using 30,598 gene transcripts. We are using death as a biomarker for MM progression and two years as cut off for treatment. One of the highest modules identified; Pink module has HDAC8, HSPG2, LILRB4, CDC and FOXM1 genes and other 595 genes that are co-expressed together, that are significantly and positively correlated (p < 3.6 × 10-8, R2 =0.38) with overall survival of < 2 years following initial treatment with RVD. These genes are associated with cell cycle signaling, and Jak-Stat signaling pathways and their biological process is largely significant for proliferation and tumorigenesis. In future, connectivity mapping will be used to identify drug signatures that can target any of the hub genes. This study is expected to improve MM’s relapse.
Human B cells respond more strongly to membrane-associated antigen than soluble antigen with the help of mechanosensors
Authors: Kwak Kihyuck , Karell Paul , Yom Jina , Sohn Haewon , Manzella-Lapeira Javier , Pierce Susan K
Published: January 2023

Unlike T cells that recognize peptide-MHC complexes, B cells recognize native antigen in both soluble and membrane-associated forms. It is well established that foreign antigen can be presented in membrane-bound form and induce B cell activation but it remains unclear how mildly autoreactive B cells retained within the repertoire after central tolerance are prevented from inducing humoral autoimmunity despite the presence of their cognate antigen in serum. Here, we demonstrate that membrane-associated antigen provides superior affinity discrimination than soluble antigen. We also show that B cells express various mechanosensors, PIEZO-1, PIEZO-2, TRPA1, and TRPV5, which play an important role in robust B cell activation and in induction of the key B cell regulator gene, Myc, when B cells encounter membrane-associated antigen. We speculate that the mechanisms at play in conferring distinct sensitivity to different forms of antigen helps to explain B cell tolerance and activation in response to auto- and foreign antigens, respectively.
Mechanisms of Activation-Induced Chromatin Decondensation in the Lymphoid Lineage
Authors: Rawlings Jason S , Hurst Madison , Rahn Cassidy , Stuart Sara , Johnson Jamila
Published: January 2023

During an immune response, αβ T cells undergo a dramatic change in chromatin configuration, from a very condensed organization seen in naïve cells to a decondensed state in activated cells. It has previously been demonstrated that this change in chromatin architecture is required for αβ T cell proliferation. Since B cells and γδ T cells share the same lineage as αβ T cells, originating from a common lymphoid progenitor, we hypothesized that chromatin status could regulate their proliferation as well. To test this hypothesis, we analyzed chromatin status as a function of activation and found that in their basal (naïve) state, both B cells and Thy1.2hi (naïve) γδ T cells possess condensed chromatin, similar to αβ T cells, whereas Thy1.2lo (activated/memory) γδ T cells have a more open chromatin configuration. Upon exposure to activation stimuli, all three cell types decondense chromatin, but to different extents that correlate with their known ability to proliferate in response to activation stimuli. To investigate the signaling mechanisms controlling chromatin decondensation in these cells, we selectively activated the IP3 and DAG pathways, demonstrating that activation of either pathway can induce the decondensation of chromatin. Investigating the IP3 pathway further, we show that in B cells, the flux of intracellular calcium is required for the proper decondensation of chromatin, similar to previously published data on αβ T cells. Future studies will investigate if intracellular calcium is sufficient for chromatin decondensation in γδ T cells, establishing a conserved mechanism to control proliferation of cells of the lymphoid lineage during an immune response.
Activation-dependent post-transcriptional regulation of CD40L mediates B cell development and survival in germinal centers
Authors: De Maio Diego Javier Prado , Narayanan Bitha , La Porta James , Xie Ping , Covey Lori R
Published: January 2023

We have previously described a post-transcriptional pathway of induced mRNA stability that occurs at late times of T cell activation and is engaged by the binding of polypyrimidine tract-binding protein (PTBP1) complex to the 3′ UTR of the CD40L transcript. We recently explored the in vivo role of this pathway by generating a genetically modified mouse (CD40LΔ5) bearing a deletion of the PTBP1 stability element. We found that the CD40LΔ5 mice display a dysfunctional germinal center (GC) response including significantly decreased levels of isotype switched antibodies, antibody-secreting cells, memory B cells and GL7+ GC B cells. To extend our earlier findings we measured affinity maturation of antibodies in response to the hapten NP and found a loss of high affinity binding that corresponded to reduced numbers of targeted VH mutations known to be associated with high affinity binding. However, there were no discernible differences in the overall number of mutations across the VH segment, suggesting that the CD40LΔ5 pathway may have a greater effect on the selection and/or expansion of high-affinity B cells rather than on the somatic hypermutation (SHM) in general.

To identify how the CD40L mRNA stability pathway impacted B cells during an immune response, CD19+ B cells from immunized mice were transcriptionally profiled using RNAseq. Expression of genes associated with cell survival and proliferation were found to be down-regulated and apoptotic genes up-regulated in CD40LΔ5 B cells compared to WT cells. Together these finding are consistent with the Δ5 stability element playing a critical role in the proliferation and cell survival of high affinity GC B cells through a pathway of enhanced expression of CD40L in CD4 T cells.
Expression of microglia-specific transcriptional signatures correlates with the severity of behavioral deficits in systemic lupus erythematosus
Authors: Makinde Hadijat M , MIke Elise V , Putterman Chaim , Winter Deborah R , Cuda Carla M
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting multiple organs, including the brain. Though 50% of patients may experience neuropsychiatric symptoms (NPSLE), disease mechanisms remain largely unknown. Microglia are the resident innate immune cell of the brain and suggested to act as drivers of neurological conditions ranging from neurodevelopmental (autism) to neurodegenerative (Alzheimer’s disease) disorders. The majority of investigations into microglia, particularly the recently discovered disease-associated microglia (DAM) subset, have occurred in models of neurodegenerative disease. However, few studies have examined microglia, particularly DAM, in NPSLE. We performed RNA-seq on microglia isolated from two NPSLE-prone mouse strains to correlate gene signatures with behavioral deficits. Similar to patients, both NPSLE models exhibit cognitive impairment. Of the significantly upregulated genes found in NPSLE microglia compared to their respective controls, a common 18-gene ‘NPSLE signature’ is shared and enriched for genes associated with lipid metabolism, scavenger receptor activity and downregulating inflammatory responses and cell chemotaxis processes. NPSLE microglia are also enriched for DAM-associated genes and expression of ‘NPSLE’ and ‘DAM’ signatures significantly correlates with the severity of behavioral deficits. The discovery of our novel ‘NPSLE signature’ and enrichment of the ‘DAM signature’ represents the first to connect microglia-specific transcriptional signatures with clinical outcomes. In future studies, we will assess the penetrance of these signatures to further interrogate how defective microglial function may incite NPSLE.
Protein tyrosine phosphatase SHP-1 modulates microtubule organization in later stages of mast cell activation
Authors: DRABER Pavel , Klebanovych Anastasiya , Sladkova Vladimira , Sulimenko Tetyana , Vosecka Vera , Rubikova Zuzana , Draberova Eduarda , Sulimenko Vadym
Published: January 2023

Antigen-mediated activation of mast cells initiates signaling events leading to degranulation and release of inflammatory mediators. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Important role in microtubule nucleation from centrosomes play γ-tubulin complexes. Here we report on the regulation of microtubule organization in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1). Reciprocal immunoprecipitation experiments revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Pull-down experiments with truncated forms of γ-tubulin and SHP-1 unveiled corresponding interaction domains. When compared to controls, activated BMMCs without SHP-1 had more protrusions containing microtubules, higher level of tyrosine phosphorylation, enhanced activity of Syk kinase, as well as increased expression of cytokines and prostaglandins. Intracellular calcium mobilization and degranulation also increased. Microtubule regrowth experiments in cells lacking SHP-1 revealed stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes of γ-tubulin accumulation in centrosomes. Our data suggest a novel SHP-1 dependent mechanism for the suppression of microtubule formation in later stages of mast cell activation.
Electrophilic properties of itaconate regulating macrophage activation
Authors: Bambouskova Monika , Gorvel Laurent , Lampropoulou Vicky , Sergushichev Alexey , Loginicheva Ekaterina , Jovanovic Marko , Klechevsky Eynav , Stewart Kelly M. , Randolph Gwendalyn J. , Artyomov Maxim N.
Published: January 2023

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite. Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines, including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.
Immune Tolerance Induction by the Exopolysaccharide from the Probiotic Bacillus subtilis
Authors: Zamora-Pineda Jesus Bernabe , Knight Katherine L.
Published: January 2023

Intestinal microbiota can lessen the severity of inflammatory diseases, although the mechanisms by which this occurs are poorly understood. We showed that exopolysaccharide (EPS) from the probiotic Bacillus subtilis induces anti-inflammatory macrophages and protects mice from enteric infection and sepsis. Here, we examined the molecular mechanisms by which EPS induces an anti-inflammatory response. In co-cultures of OVA-loaded bone marrow-derived dendritic cells (BMDC) and OT-II T cells, EPS inhibits T cell activation in a TLR4-dependent manner. We hypothesize that EPS engages TLR4 and activates the noncanonical NF-κB signaling pathway, which up-regulates the expression of indoleamine 2,3-dioxygenase (IDO). IDO degrades the essential amino acid tryptophan into kynurenine. The subsequent tryptophan depletion leads to suppression of T-cell activation and induction of tolerance. Western blot analysis of BMDC treated with EPS showed nuclear translocation of the transcriptional factor RelB, which is central to the noncanonical pathway. Current studies are testing if IDO expression is also induced by EPS treatment of BMDC, and if this is the mechanism by which EPS induces anti-inflammatory responses.
Distinct Changes in the Chromatin Landscape Regulate Maturation of the Alveolar Macrophage Immune Response In Vivo
Authors: Yamamura Asami , Hoeksema Marten A. , Sakai Mashito , Lund Sean J. , Butcher Lindsay D. , Lakhdari Omar , Oppong-Nonterah Gertrude O. , Sajti Eniko , Glass Christopher K. , Prince Lawrence S.
Published: January 2023

Alveolar macrophages (AMs) perform specialized functions in the lung including protection against inhaled microbes and repair following injury. Neonates are particularly susceptible to lung infection and injury, suggesting immaturity of AM function. In mice, AMs differentiate from fetal myeloid precursors within the first week following birth. To measure the molecular and functional differences between neonatal and adult AMs, we performed RNA-seq and ATAC-seq on sorted AMs. Based on RNA-seq data, adult AMs expressed higher levels of genes associated with fatty acid metabolism (Fabp1 and Abcg1), while neonatal AMs expressed higher levels of proinflammatory genes (including Il1b, Il6, Tnf, and S100a8). Consistent with higher proinflammatory gene expression in neonatal AMs, ATAC-seq revealed enrichment of accessible AP1, NF-kB, and IRF binding motifs. In contrast, adult AMs contained open chromatin enriched in motifs for KLF, AR, and STAT. To test if these differences contributed to differential innate immune function, we treated both neonatal and adult mice in vivo with inhaled LPS. LPS-exposed AMs demonstrated robust innate immune responses in both neonatal and adult mice. Interestingly, many of the LPS-induced genes in neonatal AMs had higher basal levels of expression, suggesting constitutive innate immune activation in the neonatal lung. These differences appeared to be driven by the lung microenvironment, as isolating and culturing AMs resulted in rapid loss of differences in gene expression between neonatal and adult cells. Our data therefore suggest the developing environment of the neonatal lung drives unique molecular and functional differences in AM mediated innate immunity.
Antigen Receptor Specificity and Cell Location Influence the Diversification and Selection of the B-1a Cell Pool with Age
Authors: Holodick Nichol E , Tsuji Naomi , Rothstein Thomas L.
Published: January 2023

B-1a cells provide immediate and essential protection from infection through production of natural immunoglobulin, which is germline-like due to minimal insertion of N-region additions. We have previously demonstrated peritoneal B-1a cell derived phosphorylcholine (PC)-specific and total IgM moves away from germline (as evidenced by an increase in N-additions) with age as a result of selection. In young mice, anti-phosphatidylcholine (PtC) antibodies, like anti-PC antibodies, contain few N-additions, and have been shown to be essential in protection from bacterial sepsis. Herein, we demonstrate the germline-like status of PtC specific peritoneal B-1a cell IgM does not change with age. In direct contrast, the splenic PtC-binding B-1a cell population does not preserve its IgM germline status in the aged. Furthermore, splenic PtC-binding B-1a cells displayed more diverse VH use in both the young and aged as compared to peritoneal PtC-binding B-1a cells. While both peritoneal and splenic PtC-binding populations increase VH12 use with age, differential use of VH11 and VH2 is observed between these populations with age. These results suggest disparate selection pressures occur with age upon B-1a cells expressing different specificities in distinct locations. Overall, these results illuminate the need to further elucidate how B-1a cells are influenced over time in terms of production and selection, both of which contribute to the actual and available natural IgM repertoire with increasing age. Such studies would aid in the development of more effective vaccination and therapeutic strategies in the aged population.
T lymphocytes characterization and systemic inflammation after hypoxic ischemia in naturel-resuscitation newborn rats
Authors: Shen Chao-Bin , Gu Yan-Ni , Yu Lan , Wang Li-Min
Published: January 2023

Background

Perinatal hypoxia-ischemia (HI) remains the primary cause of acute neonatal brain injury, leading to a high mortality rate and long-term neurological deficits, such as cerebral palsy of children. In this study we performed investigations of systemic inflammatory cytokines from peripheral blood and T lymphocytes and CD11b+ cells from spleen tissue at acute stage after self-recovery neonatal HI brain injury in rats.

Methods

HI was induced in 4-day old rat and specimen was collected up to 14 days natural resuscitation to investigate expression of immune associated with cell-markers, CD3, CD4, CD8 and CD11b, and multiple inflammation cytokines (IL-1β, IL-6, TNF-α, L-8 and IL-10). We assessed quantity changes of T cell populations and CD11b from the spleen by flow cytometry. We measured cytokines concentration from peripheral blood by ELISA after recovery HI rats.

Results

At 14d after recovery HI rats, FACS analysis showed a marked decrease in the number of CD3, CD4 and CD8 (p<0.001), CD11b+ cells was no relative increase of (p = 0.14). IL-1β, IL-6 and TNF-α was higher than control group in peripheral blood (p=0.0012, 0.001 and 0.003 respectively) and IL-8 and IL-10 were lower than control group (p=0.0042 and 0.018 respectively).

CONCLUSIONS/SIGNIFICANCE

Our findings demonstrate inhibition of systemic immune responses is changes in self-recovery stage after hypoxic ischemic neonatal. The short term immune inhibition observed is of general importance for future studies of the immune response after hypoxic-ischemia as most previous studies have focused on the first few days after damage, while the effects of the late immunocompromise may be remain existed.
Homeostatic maintenance of human T cells in bioengineered secondary lymphoid organoids – a model for studying age-related immune decline
Authors: Gustafson Claire Ellen , Lambert Simon , Weyand Cornelia M , Goronzy Jorg J
Published: January 2023

During aging, homeostatic maintenance of naïve T cells in secondary lymphoid tissues (SLT) is lost, leading to reduced cell numbers, a loss of stem-like properties and partial differentiation. The underlying mechanisms that drive these changes are unclear. Moreover, mechanistic studies in humans are currently limited by poor T cell homeostasis in standard in vitro culture and in humanized mice. Thus, we sought to develop a robust culture system mimicking SLT that maintains human T cell survival and homeostasis. In our system, we encapsulate human T cells into a bioengineered matrix containing primary fibroblastic reticular cells (FRC), which are stromal cells that support T cell homeostasis within SLT in vivo. Within these SLT-like organoids, resting naïve T cells from young donors exhibited significantly higher viability and dramatically longer survival times (> 3 weeks) compared with two-dimensional (2D) T cell-FRC cultures. Survival was not driven by T cell receptor activation or proliferation. Moreover, naïve T cells maintained a quiescent-like phenotype (CD45RAhighCD27high), which is a phenotype preferentially lost by naïve T cells in 2D culture as well as during aging. Using small molecule screening, we found that inhibition of adenosine receptor signaling caused naïve T cells to break quiescence and acquire a partially differentiated phenotype (CD45RAlow) similar to that of naïve T cells from older individuals. As old naïve T cells expressed similar levels of adenosine receptors as young, these data suggest that the loss of adenosine within SLT may contribute to immune aging. Thus, we have developed a new, malleable model system that allows for mechanistic investigation into human T cell homeostasis and the effects of aging.
The role of ERK2 in regulating germinal center B cell fate decisions
Authors: Negron Austin Joseph , Jeffreys Sean , Abdul-Baki Nawal , Perez Rebecca , Perez Christina , Forsthuber Thomas G
Published: January 2023

The regulation of B cell activation, differentiation, and survival is a crucial control point for the maintenance of B cell tolerance in the germinal center (GC) and involves the precise coordination of several complex intracellular signaling pathways. Dysregulation of these pathways can lead to the generation and persistence of autoreactive memory B cells and antibody-secreting cells (ASCs), resulting in autoimmune disease. The mitogen-activated protein kinase (MAPK) pathway is central to signals required for GC B cell selection and differentiation, such as those received through the B cell receptor (BCR) and C-X-C chemokine receptor type 4 (CXCR4), respectively. Extracellular signal-regulated kinases 1 and 2 (ERK1/2), important effectors of this pathway, play a major role in B cell activation and differentiation. Though ERK2 specifically regulates the pro-apoptotic protein, Bmf, its role in regulating GC B cell fate is unknown. Our lab has developed a mouse model in which the deletion of Erk2 is accompanied by the expression of the fluorescent reporter protein, eYFP, allowing us to identify and isolate viable Erk2Δ B cells using flow cytometry. Preliminary data using this model has shown that peripheral Erk2Δ B cells express DEC205, which is associated with follicular T helper cell interactions and CD80, which is involved in co-stimulation and immunoglobulin class switching. Thus, we hypothesize that ERK2 regulates several aspects of GC B cell selection. Our results will help elucidate the molecular mechanisms underlying B cell differentiation, potentially offering new approaches to treating B cell-driven autoimmune diseases.
Utilization of Stable Isotope Tail-Vein Infusion to Assess Glutamine Utilization by Physiologically Activated CD8+ T cells in vivo
Authors: Ma Eric H , Verway Mark J , Roy Dominic G , Steadman Mya , Griss Takla , Sambroska Bozena , Chubukov Victor , Roddy Thomas P , Jones Russell G
Published: January 2023

Metabolic reprogramming is an essential part of the T cell activation program. Upon activation, T cells undergo dramatic rewiring of their metabolic pathways to promote ATP production and biosynthesis sufficient to support the rapid exponential growth of antigen-specific T cells. The metabolic profile of T cells is shaped by both cell-intrinsic factors, such as genetics and receptor-mediated signaling, and environmental conditions, such as nutrient availability. However, our understanding of T cell metabolism has largely been shaped by studies in vitro, where oxygen and nutrients are in excess. Here, we combined bioenergetic profiling and 13C-glutamine infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. Similar to in vitro-activated T cells, glutamine is a prominent source of fuel for the TCA cycle during early expansion (3 dpi). However, at later timepoints of infection (6 dpi), the use of glutamine is substantially decreased by Teff cells. Similar to the observation of decreased glucose usage by T cells over the course of infection (Ma et al Immunity 2019). Our data suggests a dynamic flexibly in nutrient usage by T cells in vivo to fuel the different phases of T cell functions, from the early T cell response (3 dpi) to the late (6 dpi). Our work highlights the differences in T cell metabolism in vivo compared to in vitro cultures and a new method to study T cell nutrient utilization in vivo.
Integrative characterization of the coding and non-coding transcriptome in human memory B cells
Authors: Moroney Justin B , Vasudev Anusha , Pertsemlidis Alexander , Zan Hong , Casali Paolo
Published: January 2023

An effective response against a previously experienced pathogen is contingent upon the generation of memory B cells. These display intrinsic differences over naïve B cells, including extended lifespan and rapid response to antigen, with production of high-affinity class-switched antibodies. However, the transcriptional and regulatory programs that underpin the unique features of memory B cells remain unexplored. Here, we have developed an integrated analysis of differentially expressed (DE) mRNAs, miRNAs, lncRNAs and chromatin profiles to determine how gene expression intersects with epigenetic regulatory elements to shape the identity and functions of human memory B cells. DE analysis revealed an exclusive gene signature shared by CD27+IgG+ and CD27+IgA+ B cells that segregates these memory B cells from CD27−IgD+ naïve B cells – “unswitched” CD27+IgD+memory B cells displayed a transcriptional signature of transition between naïve and switched memory B cells. While changes in DNA accessibility typically predicted changes in mRNA expression, some genes of the “memory” signature deviated from this, suggesting a layer of post-transcriptional regulation mediated by ncRNAs. Indeed, probing the miRNAome revealed downregulation of miR-181 and concomitant upregulation of five target genes (RASSF6, TOX, TRERF1, TRPV3, RORA) in switched memory B cells. In these B cells, select DE lncRNAs clustered proximal to the IgH gene locus on chr 14, suggesting a role in their regulation of BCR expression. Thus, we have identified a set of genes characteristic of memory B cells and have provided evidence these genes are subject to integrated regulation by chromatin remodeling, cis-regulatory elements and distinct sets of miRNAs and lncRNAs.
Bnip3/Nix-mediated mitophagy protects memory B cells by restricting de novo fatty acid synthesis
Authors: Kodali Srikanth
Published: January 2023

Immunological memory preserved by long-lived memory cells forms the basis for long-term protection by the adaptive immune system against repeat infections. Autophagy is crucial for the longevity of memory cells, though the molecular mechanisms for this phenomenon remain unclear. Bnip3 and its homolog Nix are Bcl-2 family proteins that localize to the outer mitochondrial membrane. Here, we show that autophagic clearance of mitochondria mediated by Bnip3 and Nix is critical to maintain mitochondrial homeostasis and prolong memory B cell survival. In mice with deletion of Nix and Bnip3 in B cells, memory B cells formed normally, but declined in numbers thereafter. Nix- and Bnip3-deficient memory B cells had impaired mitophagy and accumulated both total and functional mitochondria. Interestingly, Nix- and Bnip3-deficient memory B cells also had increased lipid droplets and elevated expression of genes required for de novo fatty acid synthesis. Inhibition of the fatty acid synthesis enzyme acetyl-CoA carboxylase 1 (ACC1) prevented lipid accumulation and rescued Nix- and Bnip3-deficient memory B cell numbers in vivo. Our results suggest that Nix and Bnip3 mediate the autophagy of excess functional mitochondria in memory B cells to limit de novo fatty acid synthesis and protect against steatosis-related cell death.
Candida tropicalis infection dramatically affects the gut microbiome in DSS-induced colitis
Authors: Di Martino Luca , Buttò Ludovica , Ghannoum Mahmoud , Hager Cristopher , Osme Abdullah , Buela Kristine-Ann , De Salvo Carlo , Pizarro Theresa , Cominelli Fabio
Published: January 2023

Candidiasis caused by Candida (C.) tropicalis has dramatically increased worldwide. The role of C. tropicalis in the pathogenesis of intestinal inflammation in Crohn’s disease patients has not been fully elucidated. We have previously reported that C57BL/6 mice infected with C. tropicalis show significantly increased severity of intestinal inflammation following induction of colitis by administration of dextran sodium sulfate (DSS) in drinking water. These effects were associated with increased gene expression of Tnfa and Ifng, and decreased expression of Th2-associated genes, such as Il4 and Il13. In the present study, we investigated the effects of C. tropicalis infection on the composition and function of the gut microbiome in this model. Analysis of 16s rRNA in fecal samples of infected mice showed that C. tropicalis significantly altered the gut microbiome composition, affecting, in particular, the abundance of mucin-degrading bacteria, such as Akkermansia Muciniphila and Ruminococcus Gnavus (β diversity analysis: P<.05). Alcian Blue staining showed a significant decrease of mucus-producing goblet cells in infected mice before and after DSS-induced colitis (number of goblet cells per power field: 50.3±9.1 vs. 21.4±5.4; P<.05). Flow cytometry analysis showed that in lymph nodes of infected mice, there was a higher percentage of group 2 innate lymphoid cells (% ILC2: 10.28±2.28 vs. 2.08±0.34, P<.05). Our data demonstrate that C. tropicalis may play a pro-inflammatory role in intestinal injury by altering the gut microbiome community and affecting the abundance of specific mucin-degrading bacteria. Therefore, the resulting dysbiosis may affect the susceptibility of the host to develop inflammatory bowel disease.
Proteomic analysis of exosomes released from Leishmania infected macrophages
Authors: Keller M Murphy , Kelly Patrick H , Pope R Marshall , Thery Fabien , Wilson Mary E
Published: January 2023

Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani or L. infantum. Parasites reside quiescently in macrophages (Mϕ) but dramatically affect systemic immune responses. These Mϕs, like most eukaryotic cells, release small, protein- and RNA-laden extracellular vesicles called exosomes, which facilitate intercellular communication. We hypothesized exosomes released from infected Mϕs mediate some systemic immune changes in VL. If correct, exoproteomes of infected Mϕs must differ from uninfected Mϕs.

Monocyte derived Mϕs (MDMs) from 4 healthy human donors were infected with L. infantum and extracellular parasites removed. Exosomes were collected and exoproteomes were isotope labeled and identified by LC-MS/MS. Data were exported in MaxQuant 1.6.3.3, and analyzed with Perseus 1.6.2.3.

After filtering for quality and for proteins found in all 5 technical replicates of at least one group, 244 proteins were quantified. Pairwise comparison revealed significant differences between donors (FDR 0.05, S0=1), and between infected and uninfected states. 2-way ANOVA (p<0.05) revealed 100 proteins differed significantly by infection status, including histone proteins, HSP-70, Prosaposin, Lipoprotein lipase, CD36, Galectin-9 and several histocompatibility regions and antibody constant chains. The data are reflective of dysregulated lipid metabolism known to occur in visceral leishmaniasis.

These findings highlight a need to investigate Heat Shock proteins, lipid metabolic pathway proteins, histocompatibility antigens, and histones in extracellular vesicles as potential contributors to the immune dysregulation observed during leishmaniasis.
Mice infected with the hypervirulent Mycobacterium tuberculosis HN878 strain develop lung lesions resembling human tubercle granulomas
Authors: Bobba Suhas , Choreño-Parra José Alberto , Rangel-Moreno Javier , Ahmed Mushtaq , Mehra Smriti , Rosa Bruce , Martin John , Mitreva Makedonka , Kaushal Deepak , Zúñiga Joaquin , Khader Shabaana A
Published: January 2023

The aerosol infection of mice with lab-adapted Mycobacterium tuberculosis (Mtb) strains in not an ideal model to study the immune parameters crucial for structural organization of protective human tuberculosis (TB) granulomas. Since experimentation in larger animals that mimic human lung granulomatous responses is expensive, a cost-effective animal model recapitulating morphological aspects of human granulomas is needed. Here, we addressed whether the use of hypervirulent Mtb strains could be a better strategy to improve the mouse model of TB. Hence, we infected C57BL/6 mice with a low dose of aerosolized Mtb HN878 and histologically analyzed infected lung tissues at different time points after infection. We found that granulomas that developed early after Mtb HN878 infection resemble human and NHPs granulomas, except for the lack of multinucleated giant cells. These structures displayed a central core of macrophages surrounded by a lymphocyte cuff. Immunofluorescence analysis showed that the presence of Mtb within mouse human-like granulomas was restricted to the central core area where some macrophages also expressed iNOS. Furthermore, increased formation of B cell lymphoid follicles expressing CXCL13 and germinal center markers was observed at the peripheral lymphocyte cuffs of human-like granulomas. As B cell follicles are indicators of protective immunity in humans, we addressed whether their formation was crucial for Mtb control. In Ighm−/− B cell deficient mice, we found an increased susceptibility to Mtb HN878 infection and enhanced lung inflammation at 50 days post-infection as compared to wild-type mice. Thus, our data supports the use of Mtb HN878 infection to model human TB granuloma formation in mice.
Host resistance to pulmonary cryptococcal infection requires CX3CR1 expression
Authors: Strickland Ashley , Chen Yanli , Shi Meiqing
Published: January 2023

Cryptococcus neoformans is an opportunistic fungal pathogen, and a leading cause of death among HIV-infected individuals. These encapsulated yeast fungi are believed to enter the body through the respiratory tract and initially cause pulmonary infection. If the host immune system fails to contain the fungal cells within the lung, the organism will disseminate to the brain via the bloodstream, consequently resulting in fatal meningoencephalitis. As such, the early interactions between immune cells and C. neoformans in the lung are critical for determining the progression of the disease. CX3CR1 is a chemokine receptor commonly expressed by most myeloid lineage cells. Despite its prevalence though, the role of CX3CR1 during cryptococcal infection has not been fully examined. Here we report that pulmonary infection with C. neoformans enhanced CX3CR1 expression in the lung. Following infection, mice lacking CX3CR1 had significantly higher pulmonary fungal burdens, as well as decreased survival times compared to wild type mice. These infected CX3CR1 knockout mice also displayed higher expression of pro-inflammatory cytokines including MIP-2, MCP-1 and CCL7, but lower expression of anti-inflammatory cytokines such as IL-10. In addition, CX3CR1 deficiency resulted in mice having dramatically enhanced neutrophil accumulation in the lungs following infection. Interestingly, depletion of neutrophils drastically improved lung CFU in infected knockout mice, indicating that excessive inflammation drove fungal growth. Taken together, these data indicate that CX3CR1 expression is essential for host resistance to pulmonary cryptococcal infection by inhibiting excessive lung inflammation.
Subchronic inhalation of Aspergillus versicolor conidia leads to sustained innate immune responses and is associated with irreversible remodeling of pulmonary arterial tissue following recovery
Authors: Barnes Mark A , Croston Tara L. , Lemons Angela R. , Rush Rachael E. , Beezhold Donald H , Green Brett J.
Published: January 2023

Aspergillus versicolor is an environmentally ubiquitous fungal species that is particularly abundant in environments with water infiltration. Respiration of fungal products, including conidia, hyphal fragments, mycotoxins, and other secondary metabolites may cause adverse pulmonary health effects including asthma and allergy. In the present study, pulmonary immune responses in B6C3F1 mice were examined following 1, 2, 4, 8, and 13 weeks of repeated respiratory challenge to 3×105A. versicolor conidia. Increased alveolar macrophages, inflammatory monocytes, neutrophils, and eosinophils, which peaked between 4 and 8 weeks, were detected in the lungs of mice exposed to viable conidia. Type 2 innate lymphoid cells (ILC2s) peaked between 8 and 13 weeks compared to total T cells which increased between 2 and 8 weeks and decreased at 13 weeks. Protein concentrations of the Th2 cytokines IL4, IL5, and IL13 were highest in bronchoaveolar lavage fluid of exposed mice following 4 weeks and exhibited a time-dependent reduction at 8 and 13 weeks. Inhalation of conidia for 13 weeks led to goblet cell hyperplasia, vascular wall thickening, and perivascular inflammation. Finally, mixed perivascular inflammation and smooth muscle hypertrophy were sustained at 4 weeks post-exposure. These data indicate that exposure to A. versicolor elicits a Th2-biased immune response that is driven by innate cell populations and is associated with the induction of remodeling of the pulmonary vasculature.
Neonatal Tregs Suppress the Development of a Stromal Niche for Th2 Cells in Skin
Authors: Boothby Ian C , Habrylo Ireneusz , Boda Devi P , Rosenblum Michael D
Published: January 2023

Many allergic diseases originate during early life and cause chronic inflammation that persists into adulthood. Immune dysregulation during the “critical window” of postnatal development has been hypothesized to establish lifelong susceptibility to inflammation; however, mechanistic evidence is lacking. Regulatory T cells (Tregs) are critical suppressors of inflammation and autoimmunity that are functionally unique during early life. We have found that neonatal Tregs (neoTregs) suppress profound dysfunction of skin immunity and stromal architecture in neonates but not adults. Transient depletion of neoTregs results in the aberrant outgrowth of fibrous bands in the skin subcutis. Single cell RNA sequencing and subsequent validation studies revealed that these fibrous bands are composed of a novel “type 2” fibroblast celltype that localizes to the subcutis and expresses the alarmins IL-18 and IL-33. Concurrently, Th2 cells accumulate in neoTreg-depleted skin and persist through adulthood. These Th2 cells reside near type 2 fibroblasts in the subcutis, and genetic ablation of IL-18 or IL-33 reduces their accumulation. Collectively, these data suggest that neonatal Tregs suppress the formation of a sustained stromal niche that supports pathogenic skin-resident Th2 cells and may predispose to disease later in life. These findings advance our understanding of how pathogenic immune cells arrive and persist in peripheral tissues and may be relevant to human allergic disease.
Antigen-Specific CD4 T cells Protect Against Reproductive Pathology by Subverting Chlamydia-specific CD8 T cell Response
Authors: Li Weidang , Warda Katerina , Moy Lindsay N , Musunuri Tarakarama , Bui Nhi , Wright-McAfee Erika , Murthy Ashlesh K.
Published: January 2023

We have demonstrated previously that CD4 T cells are protective whereas CD8 T cells cause reproductive pathology following genital chlamydial infection. In this study, we explored the interactions of these cell types in context of vaccine-mediated protection. We evaluated three immunization regimens: live-chlamydial elementary body intranasal (EB-i.n.), live-EB i.m. (EB-i.m.), and irrelevant antigen bovine serum albumin i.n. (BSA-i.n.) that induced near-total (90%), partial (50%), and no (0%) protection, respectively against oviduct pathology following i.vag. C. muridarum challenge in C57BL6/J animals. We found no differences in Chlamydia-specific IFN-g, TNF-α, or IL-17 response from purified splenic CD8 T cells on days 7, 14, and 21 after immunization. We further evaluated Chlamydia-specific splenic CD8 T cell responses on days 3, 6, 9, and 12 following i.vag. chlamydial challenge. All groups of challenged animals displayed comparable induction of Ag-specific CD8 T cell cytokine responses on day 6 after challenge. As early as day 9 after challenge, Ag-specific IFN-g and TNF-α production from CD8 T cells was significantly reduced in EB-i.n.-immunized mice, and on day 12, both EB-i.n.- and EB-i.m.-immunized animals displayed minimal Ag-specific TNF-a production, compared to enhanced TNF-a production from CD8 T cells in BSA-i.n. immunized animals. Furthermore, adoptive transfer of Chlamydia-specific CD4 T cells at the time of genital infection induced significant reduction in Chlamydia-specific splenic CD8 T cell response on day 12 after challenge. In summary, these results suggest that Ag-specific CD4 T cells subvert a pathogenic Chlamydia-specific CD8 T cell response to protect against reproductive pathology.