bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune-neural transmission regulates gut sensation
Authors: Wu Chuan , Chen Zuojia , Luo Jialie , Li Jian , Kim Girak , Chesler Alexander , Urban Joseph , Trinchieri Giorgio , Li Wei
Published: February 2023

The gastrointestinal tract is known as the largest endocrine organ that encounters and integrates various immune stimulations and neuronal responses due to constant environmental challenges. Enterochromaffin (EC) cells, which function as chemosensors on the gut epithelium, are known to translate environmental cues into serotonin (5-HT) production, contributing to intestinal physiology. However, how immune signals participate in gut sensation and neuroendocrine response remains unclear. Interleukin-33 (IL-33) acts as an alarmin cytokine by alerting the system of potential environmental stresses. We here demonstrate that IL-33 induced instantaneous peristaltic movement and facilitated Trichuris muris expulsion. We found that IL-33 could be sensed by EC cells, inducing release of 5-HT. IL-33-mediated 5-HT release activated enteric neurons, subsequently promoting gut motility. Mechanistically, IL-33 triggered calcium influx via a non-canonical signaling pathway specifically in EC cells to induce 5-HT secretion. Our data establish an immune-neuroendocrine axis in calibrating rapid 5-HT release for intestinal homeostasis.
SARS-CoV-2 related cytokines induce barrier function changes in human aortic endothelial cell monolayers: Implications for cytokine storm and vascular dysfunction.
Authors: Campbell Carmen Elaine , Boisvert William
Published: February 2023

In 2019, a new strain of coronavirus, severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China. Nearly 25 million cases have since been reported in the US and almost 500,000 Americans have died. The aggressive inflammatory response of the host to SARS-CoV-2 infection plays a significant role in disease severity. Severe SARS-CoV-2 infection often leads to Acute Respiratory Distress Syndrome (ARDS), Severe Inflammatory Response Syndrome (SIRS), and Cytokine Release Syndrome (CRS). Observed in about a third of fatal COVID-19 patients, CRS is correlated to high mortality. Patients experiencing CRS also suffer from systemic vascular dysfunction. Thus, methods to assess endothelial cell function may prove essential to evaluate effectiveness of therapeutic interventions targeting cytokines. Our objective was to characterize dynamic responses of vascular endothelial cell monolayer barrier resistance (Rb) when treated with COVID-19 related cytokines. We quantified the effect of eight different cytokines to induce Rb changes in human aortic endothelial cell (HAoEC) monolayers using Electric Cell-substrate Impedance Sensing. We found that Rb of HAoEC monolayers was significantly reduced when treated with TNF-α, IL-1β, or IFN-γ compared to untreated controls. In the case of TNF-α, no significant difference in Rb was measured in cell monolayers treated with TNF-α and anti-TNF-α monoclonal antibody. On the other hand, anti-IL-1β antibody did not block the effect of IL-1β which induced a significant reduction in Rb compared to cytokine free controls. A significant reduction in Rb values were also noted in cell monolayers treated with TNF-α and IL-1β combinations suggesting a systemic synergistic effect.
Activation of Lyn kinase and translocation of its PKCδ target regulate phosphorylation in the IL-4-induced alternate pathway for BCR signaling
Authors: Khan Naeem , Hu Yongmei , Lowell Clifford A , Rothstein Thomas L
Published: February 2023

B cell signaling for activation via the BCR occurs as an isolated event only in vitro; in real life, BCR signaling takes place within a complex milieu that involves interactions with agents that trigger additional receptors. Chief among these agents is IL-4. We have shown that BCR signaling is reprogrammed by IL-4 receptor engagement, and that this reprogramming involves creation of a new, signalosome-independent, Lyn-dependent alternate signaling pathway out of pre-existing, otherwise noninteracting, elements. A unique aspect of alternate pathway BCR signaling is PKCδ phosphorylation. In dissecting this pathway, we unexpectedly found that IL-4 alone induces phosphorylation and activation of Lyn, and that Lyn immunoprecipitated from IL-4-treated B cells capably phosphorylates PKCδ in a cell-free system, although PKCδ phosphorylation does not occur in the absence of BCR triggering in vivo. This raised the question of why IL-4 alone failed to produce PKCδ phosphorylation. We considered the possibility that Lyn and PKCδ may be spatially separated. As expected, Lyn is located primarily in the membrane fraction, whereas PKCδ is located mainly in the cytosol fraction. However, when anti-Ig follows IL-4 treatment, PKCδ is found in the membrane fraction and phosphorylated. This translocation of PKCδ to the membrane fraction is not affected by loss of Lyn, although PKCδ is not phosphorylated without Lyn. Thus, PKCδ phosphorylation through the alternate pathway appears to consist of several discrete steps: 1.IL-4 induces Lyn phosphorylation and activation; 2. IL-4 produces a change such that PKCδ is translocated from the cytosol to the membrane after BCR engagement; 3. Activated Lyn in the membrane phosphorylates PKCδ in the membrane.
Perturbations of Marrow Stromal Cell function during Acute Inflammation
Authors: Matkins Victoria , Camacho Virginia , Hoang Ashley , Patel Sweta , Welner Robert S
Published: February 2023

Mesenchymal stromal cells (MSCs) reside in a complex network of blood cells and can differentiate into mature chondrocytes, osteoblasts, and adipocytes. This trilineage differentiation process is tightly regulated by intrinsic and extrinsic signals, leading to transcriptional programming of balanced lineage output. Inflammatory insults to this stromal homeostatic balance occur through the upregulation of activating cytokines. Pro-inflammatory cytokines’ impact on the hematopoietic lineage is well-defined, but similar impacts to stroma was lacking. To define these changes, we studied two well-characterized forms of inflammation: polyinosinic-polycytidylic acid (Poly(I:C)), that activates IFNα, and Lipopolysaccharide (LPS), that through TLR4 promotes IFNγ. Prior studies of inflammatory conditions show bone loss; therefore, we hypothesized that there will be an increase in the MSC pool to compensate for loss of bone maturation. Using flow cytometry and lineage tracing models for stroma, we phenotypically characterized the non-hematopoietic cells’ changes following perturbation. The lineage-specific Cre models used were stroma (Prx1), adipocytes (AdipoQ), and osteoblasts (OCN). Our data shows an increase in MSCs with decreases in osteoblasts and adipocytes with stimulation. Furthermore, we used reverse-phase protein array (RPPA) and cytokine arrays to map the signaling mechanisms and activated pathways. We found an increase in specifically MAPK pathway with an accompanying increase in cytokines linked to this pathways’ activation. Understanding changes in the bone marrow microenvironment during inflammation will allow for therapeutic intervention in mediating hematopoietic diseases such as leukemia.
The subunits of IL-12, originating from two distinct cells, can functionally synergize to protect against pathogen dissemination in vivo
Authors: Gerber Allison N , Singh Nevil J
Published: February 2023

Cytokines are typically single gene products that potently modify immune responses. The IL-12 family is an exception to this paradigm as these cytokines are heterodimers. In the case of IL-12, the subunits, IL-12p40 and IL-12p35 are encoded by two unlinked genes. However, it is canonical that activated myeloid cells co-express both these subunits simultaneously and secrete a fully active heterodimer of IL-12 which then drives IFNγ production by innate and adaptive cells. Using a chimeric mouse model, we identified a novel method of formation for generating functional IL-12 activity in vivo involving IL-12p40 monomers originating from hematopoietic cells which extracellularly associate with IL-12p35 from other non-hematopoietic cells. In the context of a parasitic challenge, we found that this two-cell mechanism of IL-12 formation was sufficient to drive T cell differentiation in local sites, distal to the initial infection, and thereby regulate systemic dissemination of the pathogen. Accordingly, we identify a new collaboration between hematopoietic and non-hematopoietic cells to produce functional IL-12 through the regulated release of individual subunits as a systemic alert system to prepare host tissues for potential pathogen arrival.
Destination active chromatin: RAG2 regions that pave the way to V(D)J recombination
Authors: Simpson Destiny A , Rodgers William , Rodgers Karla K
Published: February 2023

V(D)J recombination is required to establish the diversity of antigen receptors in the adaptive immune system. Recombination activating gene 2 (RAG2), a protein subunit of the nuclear V(D)J recombinase, rearranges gene segments to generate functional genes that encode antigen receptors. Based on our previous results, we hypothesized that a negatively charged region (the acidic hinge) in RAG2 functions to regulate localization of the V(D)J recombinase with chromatin. To test this, we generated the acidic hinge mutant 1 (A1; D/E 370–383 A), acidic hinge mutant 2 (A2; D/E 404–410 A), and D400H (a known mutation that causes immunodeficiency) to determine impact on chromatin association and V(D)J recombination activity. Dynamics of RAG2 mobility with the chromatin were determined with fluorescence recovery after photobleaching (FRAP) experiments with stably expressed GFP-tagged wild type (WT) or mutant RAG2 in RAG2−/− pro-B cells. Changes in V(D)J recombination activity and in RAG2 localization was determined for cells expressing the WT versus mutant RAG2 proteins. FRAP experiments indicated that the acidic hinge mutants had more tightly bound interactions with chromatin, as compared to WT RAG2. A1 resulted in increased V(D)J recombination, whereas, A2 and D400H led to decreased V(D)J recombination. Interestingly, these results correlated with an increased and a decreased nuclear localization of A1 and A2, respectively, relative to WT RAG2. These results indicate that the acidic region has a role in the cellular localization of the V(D)J recombinase, affecting its recombination efficiency. However, D400H localized similarly to WT RAG2, indicating the residue has a more direct role in the nuclear function of the V(D)J recombinase.
Individual zinc finger domains of Ikaros have specific roles in regulating murine myeloid hematopoietic development
Authors: Vestbostad Johanne , Podgorica Mirjam , Strege Matthew , Pinheiro Douglas , Munthe Ludvig , Schjerven Hilde
Published: February 2023

The Ikzf1 gene (encoding Ikaros) is essential for B cell development, but also plays important roles in the development of other hematopoietic lineages. Ikaros is a zinc finger (ZnF) transcription factor, with ZnF1 through ZnF4 involved in DNA binding, and ZnF5-ZnF6 required for dimerization. Previous work in our group has shown that germline deletion of the first or fourth ZnFs (ΔF1 and ΔF4 mice) resulted in different defects in lymphoid development. Here, we present our results from investigation of the roles of ZnF1 and ZnF4 in myeloid cell development.

Flow cytometry analysis of bone marrow (BM), peritoneal cavity, lung and spleen cells show that ΔF1 and ΔF4 mice have significantly altered levels of eosinophils, basophils, monocytes, and mast cells. Most importantly, the observed changes differ between the two mutants.

Interestingly, we observed a systemic increase in eosinophils in all tissues investigated in one of our ZnF-mutant mice. When culturing BM cells ex-vivo, we see that cells from the mutant mice proliferate at a higher rate than that of wt, and respond more actively to cytokine stimulation. Furthermore, ex vivo differentiation assay displayed a ZnF-dependent defect in mast cell/basophil development, and skewing towards eosinophil development, supporting the finding that ZnF1 and ZnF4 of Ikaros differentially regulate granulocyte development.

In addition to studying immune cell subsets and ex-vivo stimulation assays, we aim to sort granulocyte cell populations from the ΔF1 and ΔF4 mice and perform RNA-seq to investigate differential gene regulation. We conclude that Ikzf1 plays a role in the regulation of eosinophils, mast cells and basophils, and will present results to date.
Single-cell trajectory analysis reveals a melanoma-driven distinct hematopoietic response in murine spleen
Authors: Kara Nihan , Samusik Nikolay , Shi Xiaoshan , Lomas Chip , Widmann Stephanie , Tyznik Aaron J
Published: February 2023

Hematopoietic stem and progenitor cells (HSPCs) are a rare population of precursor cells residing in bone marrow that replenish blood cells throughout adult life. Growing evidence suggests that tumor progression can interfere with normal hematopoiesis, skew the host system to undergo myeloid biased changes and cause extramedullary hematopoiesis (EMH) in organs such as the spleen. In this study, we investigated tumor-driven phenotypic and molecular alterations in HSPCs during EMH using single-cell multiomics. We used a B16-F10 melanoma mouse model which showed splenomegaly and alterations in the composition of the HSPCs in the spleen consistent with EMH. We then isolated four HSPC populations from the spleen and bone marrow of melanoma-burdened mice using the BD FACSMelody™ Cell Sorter and used the BD Rhapsody™ Single-Cell Analysis System for the downstream single-cell multiomics analysis. We utilized oligo-conjugated antibodies for NGS-based sample multiplexing (BD® Single-Cell Multiplexing Kit) and protein detection (BD® AbSeq Assay), along with whole transcriptome analysis for a comprehensive analysis of mRNA expression. We observed tumor-driven distinct hematopoietic response and alterations in developmental trajectories of HSPCs during EMH using the X-shift algorithm paired with single-cell force-directed layout visualization. Our findings highlight melanoma-driven perturbations in hematopoiesis by utilizing single-cell multiomics in a murine model.

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Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice
Authors: Costa Thiago Alves da , Peterson Jacob , Lang Julie , Shulman Jeremy , Freed Brian M , Boackle Susan A , Lauzurica Pilar , Torres Raul M , Pelanda Roberta
Published: February 2023

Central B cell tolerance, the process responsible for mitigating the development of autoreactive B cells, has been extensively studied in mice, while little is known of how human B cells are tolerized in the bone marrow due to the inability to phenotypically distinguish autoreactive and non-autoreactive immature B cells and the difficulty in accessing fresh human bone marrow biopsies. Using a human immune system mouse model where all human Igk+ B cells are autoreactive and therefore tolerized in the bone marrow, we show that human autoreactive immature B cells are phenotypically different from non-autoreactive due to the differential expression of CD69, CD81, CXCR4 and other surface glycoproteins, while also exhibiting lower activation of ERK. Upon treatment of human immune system mice and autoreactive mice with a CXCR4 antagonist we show that signaling through this receptor is necessary to prevent both human and mouse autoreactive B cell clones to egress the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.
MiRNA-17-92 cluster is required for the embryonic development of Langerhans cells and dendritic epidermal T cells
Authors: Zhou Li , Landschoot Lauren , Yu Qian , Yao Yi , Mi Qing-Sheng
Published: February 2023

Mouse epidermis contains two major immune cells, antigen presenting Langerhans cells (LCs) and dendritic epidermal T cells (DETCs), which regulate skin immunity and involve in disease pathogenesis. Recent lineage-tracing studies have uncovered that mouse DETCs and LCs are derived from embryonic yolk-sac-derived hematopoietic precursors and self-maintain after birth. However, detailed regulating mechanisms related to their ontogeny and homeostasis remain unclear. MicroRNAs (miRNAs) are important post transcriptional regulators of protein-coding genes. Using a CSF1R-cre-mediated Dicer deletion mouse model, we found the critical involvement of miRNAs in the ontogeny of both LCs and DETCs. To further investigate the role of individual miRNAs in the ontogeny of LCs and DETCs, we evaluated the expression of miRNAs in the precursors of LCs and DETCs at different embryonic developmental stages and identified that miR17-92 cluster was highly expressed and dynamically regulated. We next generated a Csf1rCre-mediated miR17-92 deletion mouse model (miR17-92 KO) and found that conditionally deletion of miR17-92 led to significantly reduced number and interrupted phenotypes of epidermis LC and DETC precursors at E18.5 and P0 of miR17-92 KO embryos. Defective LC precursors were also identified at E14.5 and E16.5 embryonic skin, while diminished number and blocked maturation of Vg3+ DETC precursors were identified in the thymus of the same embryonic stages in miR17-92 KO mice. Overall, our results indicate that miR17-92 serves as a key epigenetic regulator in the ontogeny of LCs and DETCs. Detailed molecular mechanisms underlining the miR17-92 mediated LC and DETC developmental regulation are currently under investigation.
The role of Klf4 in thymic epithelial cells
Authors: Depoërs Lucyle , Brochu Sylvie , Houques Chloé , Dumont-Lagacé Maude , Perreault Claude
Published: February 2023

Thymic epithelial cells (TECs) are essential for the development and the selection of naïve T cells that will be able to discriminate the non-self from self-peptides. The thymus is the organ that starts degenerating the earliest in the organism, leading to a decrease in naïve T cell production. Starting from 1 year old in humans, thymic involution accelerates at puberty, partially caused by an increase in circulating sex steroids (SS). SS induce thymic involution, mediated indirectly by nuclear receptors in TECs. Little is known about how and why the age-related thymic involution is induced. We identified Klf4 as a potential regulator of TEC function and differentiation. Klf4 is a transcription factor that induces differentiation and inhibits proliferation in epithelial cells from various tissues. We showed that Klf4 expression is negatively correlated with TEC proliferation in different contexts. Interestingly, Klf4 is known as a mediator of nuclear receptors response and is a sex hormone target in several tissues. We discovered that Klf4 was more expressed in male TECs than in female TECs. Importantly, TECs are less proliferative in males than in females. Moreover, Klf4 expression was reduced when males were castrated. These results suggest that Klf4 has an important role in TEC homeostasis and is a mediator of SS response. We are currently testing the role of Klf4 in TECs by phenotypic and transcriptomic analysis on transgenic mice in which Klf4 is specifically deleted in TECs. Using testosterone treatment, we are studying the effect of SS on TEC and thymic output with or without Klf4 expression. Finally, we will assess the direct KLF4 target genes in TECs using ChIP-Seq.
Topological and transcriptional control of TCRβ antigen receptor diversification
Authors: Nganga Vincent K , Allyn Brittney , Hayer Katharina , Collins Patrick , Oyeniran Clement , Bassing Craig , Oltz Eugene
Published: February 2023

Antigen receptor (AgR) loci undergo distinct developmentally regulated changes that include a transcriptional burst and locus compaction to facilitate recombination between V and (D)J gene segments during the primary diversification process. In order to understand how this contributes to diverse AgR repertoires, we generated mice lacking T cell receptor beta (TCRβ) cis-regulatory and CTCF binding elements (CBEs) with potential to serve as locus loop anchors. We then systematically compared epigenetics, transcriptomes, and 3D genome topologies of wild-type and mutant thymocytes populations coupled with their TCRβ repertoires. We find that in DN thymocytes, contacts to the recombination center (RC) are limited to other transcriptionally active chromatin comprising of all Vβ segments, while excluding neighboring heterochromatic regions, supporting our prediction that compaction is driven by homotypic chromatin forces. We identify CBEs and cis regulatory elements that either a) serve as boundary elements, b) control select Vβ accessibility and interaction with the RC, or c) control proximal versus distal skewing of the repertoire. Collectively, our results reveal mechanisms by which CBEs cement TCRβ topological structures and allow efficient recombination of the distal Vβs during AgR diversification.
Natural killer cells as key regulators of cutaneous wound healing
Authors: Jordon Alexander R.D , Crane Meredith J , Jamieson Amanda M
Published: February 2023

Annually in the U.S, the cost to treat the more than 6 million sufferers of chronic, non-healing wounds exceeds 50 billion U.S.D. Despite extensive characterization of the functionally diverse cellular and molecular constituents found in the wound bed, wound healing therapies are lacking. The pro- and anti-inflammatory capabilities of the innate immune system make it critical in the wound healing process. Natural killer (NK) cells are innate lymphocytes which exhibit similar pro- and anti-inflammatory capabilities. Despite the breadth of their functions, ranging from anti-microbial to maintaining a healthy pregnancy, there is a paucity of information regarding NK cells in the wound healing process. Using mouse wound models, we have identified 2 distinct populations of NK cells abundantly present in seven-day old sterile, cutaneous wounds. In addition, we have identified NK cells in the wound effluent of patients who underwent corrective surgery for idiopathic scoliosis. Previous work in different models suggests that these 2 NK cell populations exhibit proliferative effector functions related to their potential to facilitate organogenesis and T cell activation. Interestingly, these are both processes implicated in late stage wound healing. Using our mouse model of cutaneous wound healing we have shown that NK cell depletion delays closure. Furthermore, we show that NK cell depleted wounds demonstrate an aberrant growth factor response. With this work we aim to determine the impacts wound natural killer cell phenotype and function have on cutaneous wound healing outcomes and to determine the role of natural killer cells in wound growth factor expression.
Interleukin-22 by altering type O-glycan profile of mucin plays a protective role in animal model of experimental colitis
Authors: Singh Ankita , Beaupre Michael , Lin Xun , Gaudino Stephen , Kumar Pawan
Published: February 2023

IL-22 is known to play both protective and inflammatory role in the intestine. Thus, the effect of IL-22 in intestine is complex, and cell type-specific function of this cytokine requires further investigation.

To identify target cell type of IL-22, we have used entire intestinal epithelial cell (IL-22Ra1ΔIEC), Paneth (IL-22Ra1ΔDefa6) and Lgr5+ intestinal stem cell (IL-22Ra1ΔLgr5)-specific IL-22Ra1 knockout mice models. Experimental colitis was developed by providing dextran sulphate sodium (DSS) in drinking water for first 8 days, and change to normal water for next two days. On examining DSS induced colitis development, significant increase in disease severity as revealed by reduced colon length, greater weight loss and histopathological changes in tissues were observed in IL-22ra1ΔIEC as compared to IL-22ra1fl/fl mice. No difference in disease severity was observed in IL-22ra1ΔDefa6 and IL-22ra1ΔLgr5 mice compared to their respective littermate control IL-22Ra1fl/fl mice. Quantitative PCR data show increased expression of inflammatory cytokines in distal colon of IL-22ra1ΔIEC compared to IL-22ra1fl/fl mice. Alcian blue staining revealed no difference in goblet cell number, however, expression of selective glycosyltransferase, required for mucin type-O glycosylation core extension, was significantly reduced in distal colon of IL-22Ra1ΔIEC compared to IL-22Ra1fl/fl mice on day 10 DSS/water treatment. Mucin type-O glycan analysis by MALDI-TOF revealed an increase in core 1 and decrease in core 4 structure in IL-22Ra1ΔIEC mice compared to IL-22Ra1fl/fl mice at steady state.

Thus, IL-22 through glycosyltransferase activity may maintain intestinal barrier integrity and alleviate severity of experimental colitis.
SerpinB7 deficiency contributes to development of psoriasis via Calcium-mediated keratinocyte differentiation dysfunction
Authors: Zheng Huaping
Published: February 2023

Defective execution of protease and protease inhibitor, which meditates abnormal signaling cascade, is merging as key contributors to autoimmune disease. SerpinB7, is identified as a skin-specific endogenous protease inhibitor, the role and underlying mechanism in psoriasis is poorly understood. Here we found that SerpinB7 is highly expressed in psoriatic keratinocytes of patient and imiquimod-induced psoriatic lesion in mice. SerpinB7−/− mice showed abnormal epidermal barrier integrity and skin architecture in homeostasis, and aggravated psoriatic lesion with inhibiting terminal differentiation and increasing inflammatory cells infiltration compared to WT after IMQ treatment. Mechanistically, SerpinB7 deficiency resulted in excessive proliferation and impaired differentiation, as well as increased chemokines and antimicrobial peptide expression in human epidermal keratinocyte and mouse primary keratinocyte. Transcriptome analysis demonstrate that SerpinB7 has a significant correlation with keratinocyte differentiation, consistent with WGCNA analysis from GTEx. Notably, we demonstrated that SerpinB7 deficiency prevented the increase of intracellular Ca2+ influx and the generation of mitochondria-derived reactive oxygen species (ROS), which were partly eliminated by antioxidant NAC and intracellular Ca2+ chelator BAPTA-AM. More importantly, iTraq and iTraq-tail proteomics demostrated SerpinB7 deficiency attenuated proteolytic networks that control calcium flux in keratinocytes. Our findings first described the critical role of SerpinB7 in the regulation of keratinocytes differentiation and psoriatic microenvironment, implying a new candidate for therapeutic targets in psoriasis.
Microvesicle-associated immunomodulators are critical regulators of immune dysfunction following burn injury
Authors: Willis Micah L , Mahung Cressida , Coleman Leon , Wallet Shannon , Cairns Bruce , Maile Robert
Published: February 2023

~450,000 people in the USA suffer from burn injury each year, with ~3,500 deaths. Burn injury is characterized by early (0–72h post injury) systemic cell death and widespread release of cytokines and chemokines, followed by immunosuppression with increased risk of opportunistic infection. We and others have described DAMP (damage associated molecular patterns)/TLR (Toll-like Receptor) ligation as driving this response. Microvesicles (MV) are extracellular vesicles released during physiological or pathological processes. We have previously reported that following burn injury there is an increased frequency of heterogenous MV, with a population of MV that contained co-localized HMGB1 DAMP and IL-1b. We hypothesize that burn induced MV are an unidentified reservoir of immunomodulators and drive immune dysfunction. To test this, we utilized burn patients from the NC Jaycee Burn Center, and our established model of burn injury in which wildtype C57BL/6 mice were exposed to a cutaneous burn. MV isolated from the plasma of patients 24h after injury were either lysed or left intact. Concentrations of membrane-associated and free cytokines/chemokines were quantified by Luminex assay. We observed that some immunomodulators such as IL-27, IL-10, IL-16 and MCP-1 were exclusively found in MV. We then performed adoptive transfer studies whereby MV from burn or unburned mice isolated 24h after injury were transferred into unburned mice by tail vein injection. We observed that adoptive transfer of burn-induced MV caused significantly increased plasma levels of IL-10, IL-6, IL-8 and MCP-1 24h after transfer compared to MV isolated from uninjured controls. These data indicate that injury-induced MV play a critical role in immune dysfunction.
Changes in peripheral intraepidermal nerve fiber densities in CD137 ligand – mediated neuropathic pain following sciatic nerve crush
Authors: Jordan Ethan E , Thakkar Bianca , Cao Ling
Published: February 2023

Previously, we have shown that the co-stimulatory molecule, CD137L plays a significant role in the development of neuropathic pain-like behaviors following sciatic nerve crush (SNC). Intraepidermal nerve fiber (IENF) density has shown high specificity and reliability in testing for small fiber sensory neuropathy. Here, we investigated the effects of CD137L depletion on the changes of IENFs following SNC. Both wild type (WT) C57BL/6 and B6_CD137LKO mice underwent SNC and sham surgeries. The ipsilateral and contralateral hind paws from individual mice were collected on days 0 (naïve), 3, 7, 14, 28, and 56 following surgery (2 males and 2 females per group per time point). All tissues were processed for fluorescent immunohistochemistry (IHC), which was performed with antibodies against PGP9.5 (pan-neuronal marker for total nerve fibers) and CGRP (peptidergic nociceptive fibers) followed by respective fluorescence-labeled secondary antibodies and fluorescence-labeled IB4 (non-peptidergic nociceptive fibers). The density of fibers was determined based on the respective IENF numbers (PGP9.5+, CGRP+ or IB4+) and the length of corresponding skin tissue. At the baseline, CD137L KO and WT mice had comparable levels of IENF densities for all types of fibers examined. Although surgery (SNC or sham) induced a transient increase in IB4 IENF density in CD137L KO mice at day 28, neither surgery induced significant changes in IENF densities over time. We did not observe significant differences between SNC vs. sham groups in either WT or CD137L KO mice. Therefore CD137L-mediated neuropathic pain behaviors cannot be attributed to reduction of hind paw IENF densities.
Immune and microRNA differentials post-simulated deep spaceflight
Authors: Paul Amber M , Cheng-Campbell Margareth , Blaber Elizabeth A , Anand Sulekha , Bhattacharya Sharmila , Zwart Sara R , Crucian Brian E , Smith Scott M , Meller Robert , Grabham Peter , Beheshti Afshin
Published: February 2023

Spaceflight can cause immune system dysfunction in astronauts with little understanding of immune outcomes in deep space. This study assessed immune responses in mice following simulated deep spaceflight, along with retrospective data from astronauts on the International Space Station (ISS). We simulated microgravity using the hindlimb unloaded mouse model alone or in combination with the newly developed simulated galactic cosmic rays or solar particle events irradiation at NASA’s Space Radiation Laboratory. Immune differentials revealed unique immune diversity following each experimental platform, suggesting consequences for deep spaceflight. Furthermore, circulating plasma microRNA sequencing analysis revealed immune system dysregulation. An ISS astronaut cohort showed elevated inflammation during low-Earth orbit missions, which supports our simulated ground experiments. Herein, distinct circulating immune biomarkers are defined by irradiation type, coupled to simulated microgravity, which can be promising targets for countermeasure developments and programmatic queries.
Targeted gastrointestinal tract homing of regulatory T-cells reduces acute graft-versus-host disease severity by simulating an increase in Treg dose in vivo
Authors: Nicholls Jemma H , Loschi Michael , Jin Sujeong , Wagers Sara Bolivar , Thangavelu Govindarajan , Zaiken Michael C , McDonald-Hyman Cameron , Saha Asim , Aguliar Ethan G , Koehn Brent , Osborn Mark J , MacDonald Kelli P.A. , Kim Sangwon V , Littman Dan R , Blazar Bruce R
Published: February 2023

Damage to the gastrointestinal (GI) tract following hematopoietic stem cell transplant (HSCT) has been shown to drive the development of acute graft-versus-host disease (aGVHD). We have demonstrated clinically that infusing regulatory T-cells (Treg) during HSCT reduces aGVHD risk while supporting immune recovery; however, high Treg doses were necessary. Here we demonstrate that by promoting Treg homing to the GI tract early post-HSCT, aGVHD severity was reduced. Ex vivo expanded murine Treg were retrovirally transduced to overexpress the orphan chemoattractant receptor GPR15 for colon specific T-cell homing. Despite no change in Treg phenotype or in vitro suppressive function when administered to HSCT recipient mice at the time of transplantation, mice that received GPR15+ Treg exhibited significantly reduced aGVHD severity and prolonged survival compared to control Treg mice (p = 0.0035). To confirm that this resulted from an increased Treg accumulation in the GI tissues, we then assessed the frequency and retention of GPR15+ Treg in mice post-HSCT by in vivo and ex vivo bioluminescent imaging as well as flow analysis. From these data, we observed not only superior localization of GPR15+ Treg within GI tissues, but also significantly reduced inflammation and tissue damage in GPR15+ Treg recipient mice early post-HSCT. Our data provides evidence that deliberate targeting of ex vivo expanded Treg to the GI tract during HSCT significantly reduces injury to the GI tract and reduces aGVHD severity by simulating an increase in Treg dose in vivo. These data provide a rational for the future development of clinical Treg products for increased control of GI aGVHD following HSCT.
Integrin α4β1-dependent regulatory B-cell migration into the skin limits cutaneous inflammation
Authors: Diaz Lazaro Emilio Aira , Debes Gudrun Fiona
Published: February 2023

B cells continuously recirculate through the skin. However, their localized functions within the cutaneous environment are still largely unknown. A subset of IL-10+ B regulatory cells (Bregs) migrate into inflamed skin in an α4β1-integrin-dependent manner, thus we hypothesize that an impaired skin-migration of these Bregs could aggravate skin inflammation. Using two types of skin inflammation (IMQ-induced psoriasiform and DNFB-mediated delayed contact hypersensitivity) in a mouse model with a B cell-specific inducible deletion of α4β1-integrin, we found that loss of α4β1-integrin expression in B cells led to a significant decrease in the numbers of IL-10+ B cells in the inflamed skin without affecting their numbers in lymphoid tissues. The reduced homing of Bregs into the inflamed skin was associated with a significant increase in the clinical and histopathological parameters of cutaneous inflammation in both mouse models. Additionally, transfer of IL-10 competent B cells into Bregs-deficient mice lead to a significant improvement in the psoriasiform skin phenotype. Thus, our data show a key role for skin-homing IL-10+ Bregs in the suppression of skin inflammation, supporting the notion that Bregs are critical players in the cutaneous environment during inflammatory skin diseases.
Syndecan-1 modulates plasma cell motility and competition within the bone marrow
Authors: Benet Zachary , Fooksman David R
Published: February 2023

Plasma cells (PC) are terminally differentiated B cells that secrete antibodies. PCs residing in the bone marrow (BM) express high levels of Syndecan-1 (Sdc1), and we have shown expression promotes PC maturation and survival in a cell intrinsic manner. Sdc1 may also mediate integrin activation and contribute to cell motility and adhesion, which may contribute to PC retention and survival. However, as PCs express heterogenous levels of Sdc1, it is unknown whether the amount of Sdc1 surface expression directly contributes to their overall fitness. Therefore, we assessed the fitness of PCs with altered Sdc1 expression within the BM.

Using co-transfers of WT and Sdc1+/− donor B cells, we tracked PC responses following immunization within both WT and Sdc1+/− recipients. Donor-derived WT PCs expressed higher surface expression of Sdc1 than Sdc1+/− PCs, and WT PCs were present at higher frequencies than those derived from Sdc1+/− donors within the BM but similar within spleen. Interestingly, in Sdc1+/− recipients, there were similar frequencies of donor-derived WT and Sdc1+/− PCs suggesting reduced PC competition based on endogenous Sdc1 levels. Sdc1 has been implicated in integrin activation and potentially affects cell motility thus we assessed PC dynamics of WT, Sdc1+/−, and Sdc1−/− using intravital microscopy. PCs were motile in all hosts, but speeds were proportional to Sdc1 genotype. We screened potential ligands of Sdc1-dependent integrin activation and found that WT but not Sdc1−/− PCs bound to and moved on fibronectin. Using in vitro live cell imaging, we found binding was mediated by Sdc1 through integrin recruitment or activation. These data suggest that PC fitness is driven by Sdc1 and correlates with enhanced PC motility within the BM.
Defining the role of Siglec-6-DOCK8 axis in cell adhesion and migration in Chronic Lymphocytic Leukemia
Authors: Nunes Jessica Judith , Ventura Ann , Hu Eileen , Zapolnik Kevan , Mo Xiaokui , Byrd John , Rader Christoph , Muthusamy Natarajan
Published: February 2023

Siglec-6 is a sialic acid-binding immunoglobulin-like lectin that is involved in diverse functions in trophoblasts. We recently identified expression of Siglec-6 on B cells from chronic lymphocytic leukemia (CLL) patients (B-CLL cells) but not normal donors. To determine the role of Siglec-6 in B-CLL cells, we used the CRISPR-Cas9 technique to knock-out (KO) Siglec-6 in a human CLL cell line, MEC1. We then evaluated the migratory role of Siglec-6+MEC1 cells towards sialyl-Tn (sTn), a known Siglec-6 ligand, or sTn+ CLL patient derived bone marrow stromal cells (BMSCs). Compared to Siglec-6+ MEC1 cells, Siglec-6− MEC1 cells exhibited significant reduction in adhesion to and migration towards sTn or sTn+ CLL-BMSCs in cell adhesion and trans-well migration assays. Interestingly, sTn is overexpressed on CLL-BMSCs compared to normal donor derived BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells to the spleen and bone marrow in NSG mice. Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, a downstream target of DOCK8, and actin polymerization which was compromised in Siglec-6 or DOCK8 KO MEC1 cells. In summary our studies have identified a novel role for Siglec-6 and its ligand in CLL through promotion of Siglec-6 and DOCK8 dependent activation of Cdc42, actin polymerization, cell migration, and subsequent attachment of B-CLL cells to CLL-BMSCs. Ongoing studies are focused on molecular mechanisms of Siglec-6 mediated regulation of actin polymerization and CLL-BMSC interactions in cell migration and adhesion.
Modeling relationship of pharmacokinetics, in vitro potency, and α4β7 receptor occupancy with intestinal cell trafficking in a gut-homing mouse model of IBD with MORF-057
Authors: Redhu Naresh S. , Hussain Ali , Srinivasan Sathish , Lee Dooyoung , Bannister Brianna , Blanco Natalia , Bonesteel Raegan , Camblin Adam , Cappellucci Laura , Cohen Rhianna , Harrison Bryce , Hahn Kristopher N , Kim Kwangsoo , Krumpoch Megan , Lin Fu-Yang , McManus Kevin , McShea Molly , Pondish Jessica , Linde Peter , Lugovskoy Alex , Mangada Maloy , Moy Terence I. , Sang Allison , Gelais Sarah St. , Sullivan Andrew , Troast Dawn , Zhong Cheng , Wang Liangsu , Cui Dan , Bursavich Matthew G. , Lippa Blaise , Rogers Bruce N. , Ray Adrian S. , Wong Jamie
Published: February 2023

Objective:

MORF-057 is an orally bioavailable, selective, and potent small molecule inhibitor of α4β7 integrin being developed for inflammatory bowel diseases (IBD) that is currently in phase 1 clinical testing. We have previously presented work that characterized its nonclinical pharmacologic profile. The current study integrates data to generate a pharmacokinetic (PK) and pharmacodynamic (PD) model of MORF-057.

Methods:

To determine in vivo potency, MORF-057 was tested in murine gut homing assays and the PD response was determined relative to the non-protein bound drug in mouse plasma. A cell adhesion assay (CAA) for α4β7 was refined to enable detection of picomolar-level sensitivity. Murine receptor occupancy (RO) assays for α4β7 and α4β1 were established under physiologic conditions, and MORF-057 was evaluated for its potency and selectivity in fresh mouse whole blood. These datasets were used to build and validate predictive models of PD response.

Results:

MORF-057 strongly inhibited the homing of α4β7hi cells to murine gut lymphoid tissues with an IC90 of 7.9 nM. MORF-057 showed high potency in CAA with an IC90 of 8.8 nM. Similarly, RO assays confirmed MORF-057 to be a highly potent inhibitor of α4β7 in mouse whole blood with an IC90 of 20.5 nM and over 1500-fold selectivity vs. α4β1. The predictive models built upon these datasets revealed a strong PK-PD relationship of α4β7 inhibitors in vivo.

Conclusions:

We observed consistently high potency of MORF-057 across multiple assay platforms. Integrated modeling based on these assays, particularly the RO assay, successfully predicted the PD response to MORF-057. These data begin to establish the relationship between PK, target engagement, and PD with MORF-057.
Macrophage polarization in obesity induced inflammation: The effect of HSP70
Authors: Botros Fady , Pytynia Matt , Kohlmeir Ellen , Incrocci Ryan , Swartzendruber Julie A , Evans Christian , Ciancio Mae J
Published: February 2023

Obesity is a chronic inflammatory disease that plagues our nation’s healthcare system. Tissue macrophages and recruited monocytes are implicated in the systemic release of pro-inflammatory cytokines that foster disease progression. Diet-induced obesity (DIO) is prevented in heat shock protein 70 over-expressing (HSP70 transgenic; TG) mice. While there are several mechanisms responsible for this protective response, prevention of macrophage polarization to a pro-inflammatory phenotype (M1) may be one contributory mechanism that warrants further investigation. Using a TG mouse line with selective over-expression of HSP70 in villin-expressing epithelial cells, male TG and non-transgenic (NTG) littermates were fed a low fat (LF; 10 kcal% fat) or high fat (HF; 60 kcal% fat) diet for 13 weeks (Research Diets, Inc). Peritoneal cavity macrophages were harvested from these mice and analyzed by flow cytometry (Beckman Coulter Cytoflex) using the following markers: total (F4/80 APC), M1 (CD38 PE), and M2 (CD301 APC/Fire 750) macrophages. TG mice demonstrated increased F4/80+high macrophages and more F4/80+high M2 cells in comparison to their NTG littermates (p=0.051). These results demonstrate HSP70 over-expression in vivo increases the population of macrophages, particularly anti-inflammatory M2 macrophages, in the peritoneal cavity. Preferential M2 phenotype could be a significant contributory factor to HSP70-mediated protection from DIO.
S100A9 modulates neutrophil extracellular trap formation thereby enhancing macrophage killing of bacterial pathogens.
Authors: Monteith Andrew J , Miller Jeanette M , Maxwell C Noel , Chazin Walter J , Skaar Eric P
Published: February 2023

Professional phagocytes form the backbone of the innate immune response and are equipped with an arsenal of antimicrobial processes. While many of these processes have been explored in isolation, how immune cells cooperatively combat bacterial pathogens at the site of infection is unclear. The proteins forming calprotectin, S100A8 and S100A9, are the most abundant cytosolic proteins in neutrophils and important components of the innate immune response. Surprisingly, S100A9-deficient (A9−/−) mice are protected from Staphylococcus aureus infection with lower bacterial burdens in the heart. We have uncovered an intracellular function for S100A9, where A9−/− neutrophils produce more mitochondrial superoxide, thereby promoting the terminal formation of neutrophil extracellular traps (suicidal NETosis). Increased suicidal NETosis does not heighten neutrophil killing of S. aureus in isolation, but rather augments macrophage (Mϕ) killing. More specifically, NET formation enhances Mϕ antibacterial activity coinciding with Mϕ phagocytosis of S. aureus and transferring neutrophil-specific antimicrobial peptides to Mϕs. Finally, similar results are observed in response to other phylogenetically distinct bacterial pathogens implicating this as an immune defense mechanism that broadly enhances antibacterial activity. Based on these results, we propose that intracellular S100A9 is a critical rheostat for neutrophil function during infection. Further, NET formation acts as a conduit for neutrophils to enhance the antibacterial activity of Mϕs, where accelerated suicidal NETosis by A9−/− neutrophils makes them especially adept at increasing the antibacterial activity of Mϕs against multiple bacterial pathogens.
Essential Role of ZIP8 in myeloid-mediated host defense against pneumococcal pneumonia
Authors: Hall Sannette C , Smith Deandra R , Wyatt Todd A , Knoell Daren L
Published: February 2023

Zinc (Zn) is essential for proper immune function. Dietary deficiency increases susceptibility to bacterial infections, including Streptococcus pneumoniae, a leading cause of community acquired pneumonia. Zn homeostasis is tightly regulated by 24 Zn transporters. ZIP8, an importer, is rapidly upregulated in myeloid cells following bacterial infection and regulates immune function in a Zn-dependent manner. Knowing that myeloid cells (macrophages, neutrophils, and dendritic cells) play critical roles in host defense against S. pneumoniae, we hypothesized that ZIP8 loss would alter myeloid cell function and adversely impact the host response to pneumococcus.

Using a myeloid-specific Zip8-KO mouse model, we observed that intranasal instillation of S. pneumoniae resulted in increased inflammation, lung injury and mortality with 40% of Zip8-KO animals succumbing to infection by day 4. Based on this, the immune response between 24 and 72 hours post infection was further examined. Analysis of BAL fluid revealed a significant increase in macrophages in Zip8-KO animals and lung homogenates revealed a significant increase in total lung leukocytes, macrophages and inflammatory DCs. The increase in myeloid cell populations in Zip8-KO mice was also associated with bacterial extravasation as measured by increased bacterial burden in the spleen. Collectively, these results indicate that ZIP8 loss aggravates the immune response through alteration of the myeloid cell landscape and lung architecture in response to S. pneumoniae. Further studies are warranted to determine how ZIP8 loss drives myeloid dysfunction, tissue damage and pathogen dissemination and whether genetic deficits in ZIP8 function can be overcome by Zn supplementation.