bluuebunny/crossref_metadata_2025 huggingface dataset:
AhR expression on both Rorc-specific immune cells and Vil1-expressing colonic epithelial cells are essential for I3C-mediated protection against colitis
Authors: Mitchell Chandani , Staley Shanieka , Wilson Keisha , Rutkovsky Alex , Wisniewski Pj , Dopkins Nicholas , Nagarkatti Mitzi , Nagarkatti Prakash , Busbee Philip B
Published: February 2023

Colitis is an inflammatory bowel disease (IBD) characterized by dysregulation of the immune system. In our previous reports, we showed indole-3-carbinol (I3C), an aryl hydrocarbon receptor (AhR) ligand, was able to reduce colitis-induced disease severity in an interleukin-22 (IL-22) dependent manner, mainly though innate lymphoid type 3 (ILC3) cells. In the current study, we generated cell-specific conditional knockout (KO) mice that had AhR deficiency in either Rorc-expressing immune cells (ILC3s), or Vil1-expressing colonic epithelial cells (CECs). We induced colitis using the dextran sodium sulfate (DSS) model in control mice and conditional KO strains, those deficient of AhR in Rorc (AR mice) or Vil1 (AV mice) cells. Results showed that compared to control mice, both AR and AV mice lost the expected efficacy effects of I3C treatment during colitis, with higher disease score and inflammation in the colon. Interestingly, immune cell profiling by single-cell RNAseq and flow cytometry showed only AR mice lost the ability to increase IL-22 secretion by ILC3s after I3C treatment during colitis. Since AV mice were still able to increase IL-22 via ILC3s, this suggests AhR deficiency in the CECs leads to alterations in other I3C-mediated mechanisms used to prevent colitis development, independent of the IL22-ILC3 axis. Collectively these data show AhR expression in both specific immune cells and CECs play a pivotal role in I3C-mediated prevention of colitis, though their mechanisms differ.
Altered levels of immune checkpoint molecules in colon biopsies and sera from microscopic colitis and ulcerative colitis patients compared to controls
Authors: Lushnikova Alexandra , Sjöberg Klas , Münch Andreas , Lange Anna , Bohr Johan , Hultgren Olof , Hörnquist Elisabeth Hultgren
Published: February 2023

Background:

Microscopic colitis (MC), comprising lymphocytic colitis (LC) and collagenous colitis (CC), is an inflammatory bowel disorder. MC patients have a lower risk of developing colorectal cancer (CRC) than ulcerative colitis (UC) patients. We hypothesize that the immune response in MC is geared more towards immune surveillance of tumor cells than that of UC, which instead contributes to inflammation-associated CRC.

Methods:

Using Luminex, protein levels of 14 immune checkpoints (TIM-3, CD28, CD137, CD27, CD152, HVEM, IDO, LAG-3, BTLA, GITR, CD80, PD-1, PD-L1, PD-L2) in protein lysates from colon biopsies (controls, n = 9; diarrhea controls, n = 7; LC, n = 14; CC, n = 15; UC, n = 17) were analyzed. Soluble checkpoints were analyzed in serum (23 controls, 17 LC, 36 CC and 2 UC).

Results:

In patients with active LC and CC, CD137, IDO, and CD80 levels were increased compared with one or both control groups. CD152 and PD-1 levels were increased in patients with active CC compared with both control groups. In patients with active UC, levels of CD137, CD152, BTLA, PD-1, and PD-L2 were increased compared with both control groups, IDO levels were increased compared with controls, and CD80 levels were raised compared with diarrhea controls.

In sera, CD27, IDO, CD80, PD-1, and PD-L2 levels were decreased in LC patients compared to controls.

Conclusions:

Increased levels of immune checkpoint molecules in colon biopsies from UC and MC patients are likely a sign of inflammation and may indicate what kind of homeostatic feed-back mechanisms are active to balance inflammation. Lowered concentrations of soluble immune checkpoint molecules in sera from patients with LC indicate a different level of homeostatic balance systemically in LC patients versus controls.
Fibroblastic reticular cells sustain innate lymphoid cell niches in the intestinal lamina propria
Authors: Cheng Hung-Wei , Mörbe Urs Michael , Lütge Mechthild , Engetschwiler Celine , Onder Lucas , Novkovic Mario , Gil-Cruz Cristina , Perez-Shibayama Christian , Rülicke Thomas , Hehlgans Thomas , Scandella Elke , Ludewig Burkhard
Published: February 2023

Innate lymphoid cells (ILC) in the small intestine govern immune homeostasis and protect the host against gut pathogens. While distinct cell-intrinsic signals have been identified that determine ILC development and differentiation, it has remained unclear which cell population regulates ILC sustenance. Using unbiased transcriptomic analysis of intestinal fibroblasts, we have identified a specialized Ccl19-expressing fibroblastic reticular cell (FRC) population that underpins solitary intestinal lymphoid tissue (SILT) structures including cryptopatches and isolated lymphoid follicles. Conditional ablation of lymphotoxin-β receptor (LTβR) signalling in SILT FRC impeded the maturation of isolated lymphoid follicles and blocked ILC maintenance resulting in the elevated susceptibility to bacterial infection. Moreover, specific Ltbr ablation in FRC during adulthood revealed that sustained LTβR-dependent FRC-ILC interaction is required to maintain SILT structures and ILC populations. Taken together, our study unveils a critical intestinal FRC niche that secures protective gut immunity.
Regulatory T Cell Transfer Increases Trem2+ Uterine Macrophages and Decreases Fetal Loss
Authors: Lewis Emma L , Porrett Paige M , Elovitz Michal A
Published: February 2023

Intrauterine fetal demise (IUFD) – fetal loss after 20 weeks – affects 6 pregnancies per 1,000 live births in the United States, and the majority of IUFDs are of unknown etiology. We investigated immunological mechanisms of IUFD using the CBA mouse model, which has mid-late gestation fetal loss. Regulatory T cells (Tregs) are critical to establishing maternal-fetal tolerance, thus we hypothesized they may be protective from fetal loss in this model. Adoptive transfer of 2×105 Tregs decreased fetal loss 2.7-fold (p=0.04) and increased pup viability by 69% (p=0.003) (N=7–9/group). To examine how systemic Treg transfer influenced local uterine immune cells, we used single-cell RNA-sequencing of uterine CD11b+ cells from pregnant dams that had received Tregs vs. PBS (N=3/group). Sequencing identified 15 uterine CD11b+ populations, with Trem2+ macrophages increased (p=2.2e–12) and CCL3+ neutrophils decreased (p=3.6e-12) by Treg transfer. Compared to other uterine macrophages, this Trem2+ population was characterized by low expression of MHC-II genes and high expression of Trem2, Apoe, Hexb, cathepsin genes, and C1q genes. The uterine Trem2+ macrophages are enriched for gene sets from Trem2+ macrophages found in other tissues, including those in atherosclerotic plaques and skin. We identified this Trem2+, MHC-II-low macrophage population in the uterus of normal and CBA pregnant mice by flow cytometry (N=6–8/group); and found that CBA mice had 2-fold fewer of this macrophage subtype than normal mice (p=0.0004). These data identify a novel uterine macrophage population that may be critical for healthy pregnancy and demonstrate systemic Treg transfer altering tissue immune populations, which has broad implications for immunotherapy.
Type I interferon enhances γδ intraepithelial lymphocyte migratory behavior via CD47 upregulation
Authors: Fischer Matthew , Jia Luo , Edelblum Karen L
Published: February 2023

γδ intraepithelial lymphocytes (IEL) migrate along the basement membrane and into the lateral intracellular space (LIS) between adjacent intestinal epithelial cells (IEC) and is critical to limit microbial translocation across the barrier. Although activated γδ IELs produce interferon (IFN)-α, whether IFNα directly influences γδ IEL migratory behavior remains unknown. To test this, intravital microscopy was performed on GFP γδ T cell reporter mice treated with PBS or IFNα (1 μg, i.v., 3h). We found that γδ IEL track speed was increased 30% and migration into the LIS was enhanced by 75% in IFNα-treated mice compared to controls. To identify candidate genes involved in regulating γδ IEL motility, we performed RNAseq on γδ IELs isolated from control and IFNα-treated mice. As expected, IFNα induced IFN-stimulated gene expression including CD47 (3.5-fold increase), a transmembrane protein involved in neutrophil transepithelial migration. Stimulation of ex vivo cultured γδ IELs with IFNα resulted in a 3-fold increase in CD47 expression compared to unstimulated controls. Next, to investigate whether IFNα enhances γδ IEL motility in vitro, GFP γδ IELs were co-cultured with enteroids expressing membrane tdTomato and treated with 10ng/ml IFNα for 3h. IFNα increased γδ IEL speed (4.5±0.2 vs 3.4±0.2 μm/min; p<0.05) and displacement (47±3.0 μm vs 34±3.1, p<0.05) relative to untreated controls. Addition of anti-CD47 blocking antibody (MIAP301) abrogated the IFNα-mediated increase in γδ IEL migratory behavior. Taken together, these data demonstrate that IFNα enhances γδ IEL motility via a CD47-mediated mechanism, and suggests that CD47 may represent a conserved mechanism of leukocyte migration within the epithelial barrier.
TCDD exposure differentially alters antigen-specific IgA levels when given before or after immunization
Authors: King Aspen E , Ortiz Linda F , Schoenherr Elléanour M , DeKrey Gregory K
Published: February 2023

In mice, activation of the aryl hydrocarbon receptor (AhR) prior to primary antigen challenge can suppress antigen-specific serum IgM and IgG responses. Secondary antibody responses have been reported to be refractory to AhR-mediated suppression. Using ELISAs and female C57Bl/6 mice pretreated with the prototypic AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 40 μg/Kg in peanut oil) one day prior to oral cholera toxin (CT) immunization (10 μg, repeated weekly), we demonstrated significant suppression of CT-specific IgA levels in the feces after two weeks (by 64.4%); however, fecal IgA levels recovered to control levels by week four. Unlike fecal IgA levels, CT-specific serum IgA levels remained significantly suppressed (by 37.1%) through week four. At that time-point, when compared to control mice, the spleens of TCDD-treated mice contained 40.4% fewer leukocytes, and contained 66.4% fewer IgA+ plasma cells (analyzed by ELISpot) (all significantly different). When TCDD exposure was delayed for three weeks after beginning CT immunization, in comparison to control mice, serum CT-specific IgA levels were significantly suppressed by 33.2%, but fecal CT-specific IgA levels were not different, as in TCDD pre-treated mice. Because enhanced migration of some T cells into the gut has been reported after AhR activation, one explanation for the differential effect of TCDD exposure on CT-specific IgA levels when given either before and after immunization could be enhanced migration of plasma cells away from reticuloendothelial tissues and into mucosal tissues.
MicroRNA-146a limits tumorigenic IL-17 signaling in colorectal cancer
Authors: Gopal Murugaiyan , Garo Lucien P. , Ajay Amrendra K. , Fujiwara Mai , Gabriely Galina , Raheja Radhika , Kadowaki-Saga Ryoko
Published: February 2023

Interleukin-17 (IL-17) is a major inflammatory cytokine implicated in colorectal cancer (CRC) development. However, the mechanisms that control tumorigenic IL-17 signaling are poorly understood. Recently, expression changes and polymorphisms in the small non-coding RNA, microRNA-146a (miR-146a), have been associated with clinical outcomes in inflammatory bowel disease and CRC patients. Here, we identified a novel role for miR-146a as a major negative regulator of colonic inflammation and tumorigenesis via modulation of IL-17 responses. MiR-146a-deficient mice are susceptible to both colitis-associated and sporadic CRC, and present with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, an intermediate in NOD2 signaling, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17 levels. Accordingly, myeloid cell-specific deletion of miR-146a leads to CRC susceptibility. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an intermediate in IL-17R signaling, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, an enzyme for PGE2 synthesis. IEC-specific deletion of miR-146a confers marked CRC susceptibility. Importantly, preclinical administration of miR-146a mimic or direct inhibition of miR-146a targets, TRAF6/RIPK2 can ameliorate CRC. In conclusion, miR-146a prevents CRC by two interlinked mechanisms: 1) by limiting myeloid cell-mediated IL-17 production; and 2) by inhibiting tumorigenic IL-17R signaling in IECs. Overexpression of miR-146a may be a promising therapeutic approach for CRC to limit multiple pathways converging on tumorigenic IL-17 signaling.
NF-κB signaling in Cx3cr1+ cells is required for neonatal intestinal homeostasis and for NEC-induced monocyte differentiation
Authors: Managlia Elizabeth , Yan Xiaocai , De Plaen Isabelle
Published: February 2023

Necrotizing enterocolitis (NEC) is a life-threatening intestinal disease affecting premature infants. Intestinal macrophages (Mφ) express Cx3cr1 and have been reported to mediate the gut immune response at this age. At birth in mice, embryonic (e) Mφ (Ly6C−CD64+MHCII+Tim4+) are the primary intestinal myeloid cell with few Ly6C+ monocytes. At 3 weeks, differentiated monocyte-derived-Mφ replace intestinal eMφ. In a NEC model, we showed decreased intestinal Ly6C+ cells due to monocyte activation, recruitment, and differentiation. NF-κB inhibition in monocytes decreased their recruitment and differentiation, improved NEC, but did not reduce IL1β. Whether eMφ contribute to inflammation in the neonatal intestine remains unknown. When IKKβ was deleted in Cx3cr1+ cells, we unexpectedly found intestinal IL1β mRNA to be increased in pups exposed to experimental NEC (8.2 ± 0.8; p≤0.05) but also in dam fed (DF) pups (12.6 ± 2.5; p≤0.01). When small intestinal lamina propria Cx3cr1+ cells were traced (using Cre-reporter mice) by flow cytometry, both eMφ (CD11b+Ly6C−CD64+Tim4+) and differentiating monocytes (CD11b+Ly6C+CD64+) expressed Cx3cr1. In mice lacking IKKβ in Cx3cr1+ cells (Cx3cr1cre+/−IKKβf/f but not in Cre- IKKβf/f controls, experimental NEC caused a 2.4 (±0.4)-fold increase in Ly6C+ cells compared to DF, and 30.4% (±8.3) of the Ly6C+ cells expressed CD64 compared to 6.7 (±0.26) in the DF (p≤0.05). CD64+ cells had a 2.4 (± 0.64; p≤0.05) and 8.3 (± 2.9; p≤0.05)-fold decrease in Tim4 and MHCII MFI respectively. Therefore NF-κB signaling in eMφ is required for neonatal intestinal homeostasis and for completing monocyte Ly6C down-regulation and MHCII up-regulation upon gaining CD64 during NEC-induced monocyte differentiation.
Early life stress in mice promotes chronicity of experimental colitis
Authors: Muir Rachel Quinn , Klocke Barbara J. , Foote Jeremy B. , Molina Patrick A. , Pollock Jennifer S. , Maynard Craig L.
Published: February 2023

Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract with peak onset during adolescence. The etiology of IBD remains poorly understood, but is believed to involve genetic susceptibility, improper immune regulation, and environmental factors. Psychological stress is one environmental trigger that has been associated with an increased risk of inflammatory diseases. Specifically, previous studies have linked early life stress (ELS) with hypothalamic–pituitary–adrenal (HPA) axis dysfunction leading to adverse health outcomes later in life. Our goal was to determine whether ELS in mice affects susceptibility to, and severity of, colitis – induced subsequent to stress exposure. We used an established mouse model of ELS, maternal separation with early weaning (MSEW), in which newborn mice are separated from their mothers for 4–8 hours daily from day of life (DOL) 2 until early weaning on DOL 17. Then, starting on DOL 28, colitis was induced in MSEW mice and their normally-reared (NR) counterparts by transient blockade of the interleukin-10 receptor (IL-10R). Prior to induction of colitis, MSEW mice showed systemic and colon-specific depletion of corticosterone relative to NR mice. In addition, MSEW mice with active colitis harbored significantly reduced numbers of colonic Foxp3+ T cells compared to colitic NR mice. Finally, whereas NR mice were mostly in remission by 15 days following cessation of IL-10R blockade, MSEW mice showed consistently elevated Tnf in the proximal colon and sustained epithelial damage. Our results suggest that ELS-mediated HPA-axis dysfunction causes hypo-corticosteronism, which may subsequently promote prolonged colonic inflammation in susceptible hosts.
Induction with MHC variant peptide MOG 45D generates MOG-reactive T cells but no clinical disease
Authors: Faust Michael A , Kolawole Elizabeth Motunrayo , Evavold Brian D
Published: February 2023

Demyelinating disease can be driven by CD4+ T cells specific for myelin oligodendrocyte glycoprotein (MOG). We have previously decreased the affinity of MOG for Class II MHC by mutating the P6 anchor residue from an S to a D amino acid, creating the 45D MHC variant peptide. 45D is incapable of inducing experimental autoimmune encephalomyelitis (EAE) despite its CD4 epitope bearing the same T cell receptor contact residues as the wildtype MOG epitope (wtMOG). Although the wtMOG and 45D peptides generate CD4 T cells specific for MOG in the periphery and CNS, there is an overall decrease in the number of high-affinity CD4 T cells in response to 45D priming. Analysis of the responding T cell populations to 45D also found increases in MOG-reactive Tregs and decreases in MOG-specific CD8 T cells. Thus, the variant peptide 45D alters the MOG-specific T cell response in favor of peripheral tolerance mechanisms that ultimately result in the absence of clinical disease.
Innate immunity contributes to pathogenesis following Mannheimia haemolytica infections.
Authors: Slate Jamison Ray , Briggs Robert , McGill Jodi L
Published: February 2023

Mannheimia haemolytica (MH) is a common commensal bacteria found in the nasopharynx of healthy cattle, and its colonization is generally well controlled by innate and adaptive immune defenses. Following immunological stressors, MH can migrate into the lungs and develop into lower respiratory tract infections or pneumonia. These infections are often exacerbated by infiltrating cytotoxic leukocytes and excessive inflammation. IL-17A is thought to play an integral part in MH pathogenesis by driving inflammatory pathways, invoking neutrophilia, and modulating innate immune defenses. Therefore, to better understand the immune-induced tissue damage that follows MH infection, this study employed the use of an IL-17A inhibitor, ursolic acid (UA), and determined its impact on immune responses and disease severity. Two independent experiments were performed using 4 week old Holstein calves; each of these studies challenged a non-treated group (UA−/MH+; n = 8) and a prophylactic UA treated group (UA+/MH+; n=8). Serum samples, whole blood, and nasal swabs were collected throughout the course of both studies, with bronchoalveolar lavage fluid and lung tissue sections collected at necropsy. UA treated calves had reduced lung pathology and bacterial loads in the lungs compared to control calves. Subsequent analysis of lung tissue confirmed UA treatment modulated host immune responses, including decreased expression of the inflammatory mediators IL-6, IL-17, and STAT3; furthermore, expression of innate defense molecules, such as antimicrobial peptides, mucins, and metalloproteases, was altered. Taken together, these results outline a mechanistic role for IL-17A-mediated immunity to contribute to the pathogenesis of MH infection.
Identification of porcine ileal innate lymphoid cells using single-cell RNA sequencing
Authors: Wiarda Jayne E , Sivasankaran Sathesh K , Loving Crystal L
Published: February 2023

Innate lymphoid cells (ILCs) are integral in maintaining intestinal defense and homeostasis, but intestinal ILCs have not been identified in pigs. Pigs are an important global food source and human biomedical model, thus identifying and characterizing porcine intestinal ILCs can have broad implications. Single-cell RNA sequencing of porcine ileal lymphocytes yielded 31,983 cells distributed across 54 clusters. Five clusters were closely related to T cell clusters yet lacked expression of the pan-T cell marker, CD3E. One cluster had high expression of genes related to type 3 immunity, including IL22, RORC, and CXCL8, indicating identification of putative ILC3s. The other four clusters expressed genes encoding for NK receptors (KLRK1, KLRC1, KLRD1) and effector molecules (CCL5, PRF1), yet they lacked expression of genes traditionally expressed by porcine NK cells (CD8A, KLRB1, HCST). Thus, we putatively identified the four clusters as ILC1s. Profiles of putative porcine ILC clusters were further compared to transcriptomic signatures of murine intestinal ILC subsets. Results indicated a relative enrichment of murine ILC3 gene signatures in the porcine ILC3 cluster, while relative enrichment of murine ILC1 gene signatures was observed for the other four clusters of porcine ILC1s. We further utilized single-cell gene expression profiles to develop a gating strategy to identify porcine intestinal ILCs as CD45+CD2+CD3ɛ−CD79α− lymphocytes at the protein level by flow cytometry. Altogether, the data allowed us to create the first transcriptomic descriptions of porcine intestinal ILCs and develop approaches to identify ILCs at the protein level, which will be applicable for future research.
The pandemic matrix gene is associated with increased pro-inflammatory immune responses during influenza virus infection
Authors: Griffin Emily Fate , Curran Shelly , Tompkins Mark
Published: February 2023

The 2009 H1N1 influenza pandemic (pdmH1N1) arose from multiple genetic reassortment events between three swine influenza A virus (swIAV) lineages and became established as a seasonal H1N1 human influenza virus. Through reverse-zoonotic events, the pdmH1N1 strain contributed gene segments to endemic swIAVs and the pandemic matrix gene (pdmM) completely replaced the classical swine matrix gene (swM), suggesting a fitness benefit. We assessed a panel of swIAVs in the BALB/c mouse model and demonstrated that swIAVs containing the pdmM gene caused increased morbidity, lung pathology, and mortality compared to swIAVs containing the swM gene. We hypothesized that swIAVs containing the pdmM gene induce dysregulation of the innate response, causing increased disease. To test this, mice were infected with swIAVs containing either the pdmM or the swM gene segment, and immune cell infiltrates, cytokine production, and histopathology were assessed. We found that pdmM viruses elicited increased cellular infiltration, NK cell activation and increased IFNγ production in bronchial alveolar lavage (BAL) fluid compared to mice infected with swM viruses. We also detected increased numbers of infiltrating macrophages, neutrophils, DCs, and eosinophils, as well as increased proinflammatory cytokine production. Additionally, elevated myeloperoxidase content was detected in BAL fluid, suggesting increased neutrophil activation. Finally, infection with pdmM viruses resulted in greater histologic lesion scores compared to swM viruses. We conclude the pdmM gene elicits increased innate immune responses, causing greater disease in a mouse model of infection. Future studies will identify determinants of increased disease severity.
Unique immune signatures predict potential cardiometabolic risk in severe COVID-19 patients and COVID-19 recovered individuals
Authors: Liyanage Namal , Gunasena Manuja , Wijewantha Yasasvi , Bowman Emily , Gabriel Janelle , Kumar Amrendra , Kettelhut Aaren , Ruwanpathirana Anushka , Weragalaarachchi Krishanthi , Kasturiratna Dhanuja , Vilgelm Anna , Pannu Sonal , Bednash Joseph , Demberg Thorsten , Funderburg Nicholas
Published: February 2023

Covid-19, the disease caused by SARS-CoV-2 infection, has resulted in millions of deaths and led to a global public health emergency. SARS-CoV-2 infected patients exhibit a wide variety of phasic clinical manifestations ranging from asymptomatic to severe complications and death. SARS-CoV-2 infection can lead to excessive immune activation, inflammation and multi-organ damage. Clinical data showed that COVID-19 may promote the development of cardiovascular disorders (CVDs). Immune activation, thrombosis, cytokine storm, and altered adhesion molecule expression on leukocyte populations have been proposed as possible mechanisms that trigger COVID-19 associated CVDs. A lack of systematic studies on how SARS-CoV-2 infection triggered immune responses that may lead to CVDs, hinder early risk identification and therapeutic interventions. In this study, by using deep immune profiling and extensive cytokine and chemokine profiling, we explore potential mechanisms of developing CVDs in severe COVID-19 patients (ICU) (n=20) as well as patients recovered from COVID-19 (RD) (n=30). We identify core immune signatures in ICU patients and RD compared to healthy controls (n=17) that may predict potential cardiovascular risk. We found that significantly elevated eosinophils and neutrophils and increased circulating levels of tissue factor, fatty acid binding protein 4 and, LPS binding protein in ICU patients suggested increased immune activation and thrombotic risk. Interestingly, we found significant elevation of several immune parameters (TIMP-1, TIMP-2, Monocytes) that were associated with cardiometabolic risk, in RD group. Thus, our data suggest a possible mechanistic link between severe COVID-19 and cardiometabolic risk.
Using single cell spatial and transcriptional analysis to understand early host response to RNA viral infections.
Authors: Arakkal Leanne , Sturdevant Gail , Robertson Shelly , Best Sonja M , Germain Ronald N , Speranza Emily
Published: February 2023

The innate immune system serves as the first line of defense against pathogens. One innate defense mechanism is the RIG-I like receptor pathway (RLR) which signals through the mitochondrial anti-viral signaling protein (MAVS) which plays a role in initiating the interferon (IFN) response to RNA viruses such as vesicular stomatitis virus (VSV). To investigate the mechanisms the MAVS-dependent pathway has on early viral control following VSV infection in vivo, we used a combination of two approaches: single cell RNA sequencing and multiplex imaging. These two techniques allow us to assess the individual cellular response to VSV in whole lymph node tissue. After intradermal inoculation, skin draining lymph nodes were assessed using single cell sequencing. Macrophages and plasmacytoid dendritic cells (pDCs) showed significantly elevated IFN stimulated gene (ISG) detection in C57Bl/6J (B6) mice compared to the MAVS−/− animals at 8 hours post infection (hpi), yet there was no detectable viral reads. This early initiation of ISG expression played a role in detection of viral RNA at 24 hpi which saw significantly elevated viral RNA in the MAVS−/− animals compared to the B6 animals. Interestingly, the virus replication in both conditions was contained to the macrophage populations suggesting that without the MAVS pathway active, the virus was still contained. Combining this information along with the observed spatial organization through multiplex tissue imaging, we aim to develop a model illustrating the role of the MAVS-dependent pathway in controlling early virus replication.

Animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional ACUC. This research was supported by the IRP of NIH/NIAID.
IFN-lambda orchestrates an IL-10 immunoregulatory program in dendritic cells critical for T cell-mediated protection against influenza virus infection
Authors: Hemann Emily A , Green Richard
Published: February 2023

Influenza virus (IAV) infection remains a major global health burden that requires development therapeutic and vaccination strategies to limit infection and drive robust and durable protective immune responses. Type III interferon (IFN), IFN-lambda, restricts IAV infection in vivo compared to type I IFN. However, the contribution of IFN-lambda to generating adaptive immune responses against IAV remains unknown. Utilizing a murine model of IAV infection in mice lacking the IFN-lambda receptor (Ifnlr1−/−), we determined that IFN-lambda signaling is required for full induction of effector and memory CD8 T cell responses Ifnlr1−/− are significantly more susceptible to mortality following heterosubtypic IAV re-infection. This defect in the CD8 T cell response was not intrinsic to the T cells, but rather due to aberrant dendritic cell (DC) function in the absence of IFN-lambda signaling. Transcriptional analysis of conventional DCs (cDC) sorted from draining lymph nodes of IAV-infected mice revealed specific modulation of an immunoregulatory program in cDC from Ifnlr1−/−, but not WT mice. Indeed, we found IL-10 to be secreted only by Ifnlr1−/−, and not WT, BMDC following IAV infection. Addition of IL-10 to WT BMDC only partially recapitulated defects in DC maturation observed in Ifnlr1−/−, suggesting more additional pathways of regulation of DC by IFN-lambda. Our analysis further revealed shared and unique Ifnar1- and Ifnlr1-dependent transcriptional responses in DCs, highlighting the distinct role IFN-lambda plays in regulating DC function during IAV infection. Overall, these studies reveal a unique role for IFN-lambda programming of DCs to optimally activate protective CD8 T cell responses during influenza virus infection.
Anti-insulin B cell receptors display skewed immunoglobulin gene usage and limited mutation in pre-symptomatic type 1 diabetes donors
Authors: Scaglione Steven R , Joosse Bryan A , Westlake Camille , Kim Eunice , Santhin Austin B , Forchetti Mason V , Cisneros Alberto , Moore Daniel J , Smith Scott A , Bonami Rachel H
Published: February 2023

Antibodies targeting insulin and other islet antigens predict type 1 diabetes (T1D) onset. Insulin autoantibody (IAA)-positive individuals progress to diabetes more rapidly, yet little is known about how IAA-precursor anti-insulin B cells (AIBCs) recognize insulin. Complementarity-determining regions (CDRs) of the B cell receptor (BCR) are structurally diverse loops that constitute the antigen-binding site, and amino acid variability in BCRs increases the breadth of repertoire antigen-specificities. To investigate anti-insulin BCRs during the earliest detectable stages of T1D, we enrolled eight insulin therapy-naïve Type 1 Diabetes TrialNet Pathway to Prevention participants who were positive for ≥2 islet autoantibodies and thus at high risk for diabetes. We stimulated donor peripheral blood mononuclear cells to drive BCR secretion as antibody, screened for AIBC-containing wells by ELISA, and immortalized these cells as hybridomas. We validated 25 monoclonal anti-insulin hybridoma lines and sequenced 16 heavy chain (IgH) and 14 light chain (IgL) immunoglobulin genes. 25% of VH and 50% of VL sequences contained at least one mutation within a CDR compared to germline, with 13% of VH and 29% of VL sequences having ≥6 mutations within the V-region. We surveyed previously and currently reported AIBC VH (n=25 BCRs), which revealed skewed VH and JH gene use compared to the total polyclonal IgH repertoire (n= 8790 BCRs, p < 0.001 and p < 0.05, respectively, chi-squared test). These data expose unique features of insulin recognition among AIBCs isolated from pre-symptomatic T1D donors, including V and J gene usage bias and potential for germline IgH insulin recognition, providing genetic and molecular insight into AIBC origins.
Intra-islet CD8+ T cells are restrained by an exhaustion program that can be reversed by LAG3 deletion
Authors: Grebinoski Stephanie J , Zhang Qianxia , Cillo Anthony R , Das Jishnu , Workman Creg , Vignali Dario
Published: February 2023

CD8+ T cell exhaustion or dysfunction is a differentiation state in which T cells become unresponsive to stimulation and lose effector function1. Impaired chronic viral and tumor clearance has been attributed to CD8+ T cell exhaustion and can be partially reversed upon blockade of inhibitory receptors (IR) such as programmed cell death protein 1 (PD1). Despite detailed analysis of CD8+T cell in tumors and chronic viral infection, the role of the exhaustion program and the transcriptional networks that control CD8+T cell function and fate in autoimmunity are still unknown. Here we show that intra-islet CD8+T cells phenotypically and transcriptionally possess features of exhausted T cells, yet maintain key differences, such as an expanded repertoire of effector mechanisms, that contribute to their persistent pathogenicity in autoimmune diabetes. This dual identity, which we refer to as a ‘divergent’ exhaustion program, can be perturbed substantively by CD8+ T cell-restricted deletion of the IR Lymphocyte Activating Gene 3 (LAG3), causing enhanced effector-like function and pathogenicity. Understanding this divergent exhaustion program and its key mediators will guide future immunotherapeutic interventions by characterizing ways to promote exhaustion in autoimmunity and reversing exhaustion programs in chronic viral infections and tumors.
Impact of B cell-depletion on plasma cells in a pre-clinical model of meningeal inflammation and subpial demyelination
Authors: Wang Angela , Ward Lesley , Ramaglia Valeria , Urich Eduard , Gommerman Jennifer L
Published: February 2023

Anti-CD20 B cell depletion therapy has provided evidence for the pathogenic roles of B cells in multiple sclerosis (MS). Terminally differentiated plasma cells (PCs) are spared by anti-CD20, and our lab has previously shown that during experimental autoimmune encephalomyelitis (EAE), IgA+ PCs mediate protection against neuroinflammation. One hypothesis is that in addition to depleting pro-inflammatory B cells, anti-CD20 also removes the major consumers of BAFF (i.e. B cells) thus promoting a favourable niche for regulatory PCs and may represent an additional mechanism by which anti-CD20 therapy confers protection.

Adoptive transfer EAE of PLP-primed Th17 cells into SJL/J mice leads to ascending paralysis and meningeal inflammation which parallels brain pathology seen in human MS. Mice were prophylactically treated with anti-CD20 (10mg/kg) or isotype control antibodies which we re-administered throughout the course of the experiment and monitored daily for clinical symptoms. We find that anti-CD20 treatment effectively depleted B cells (but not T cells) in the periphery; within the central nervous system, meningeal immune clusters formed in both anti-CD20 and isotype control treated mice, with the former being devoid of B220+ B cells. Compared to control treated mice, anti-CD20 treated mice showed limited subpial demyelination and pathology adjacent to B cell-less meningeal immune clusters, and is accompanied by a reduction in clinical score. In the serum, anti-CD20 treatment elevated levels of BAFF and IgA, which inversely correlated with disease severity. We thus propose that anti-CD20 exerts its protective effects by providing a favourable niche for suppressive IgA+ PCs.
Evaluation of differential autoantibody profiles in gut and brain tissues in children with Autism Spectrum Disorder (ASD).
Authors: Celis Nora Alexandra Ramirez , Van de Water Judy
Published: February 2023

Autoimmunity is a comorbidity often associated with autism spectrum disorder (ASD), and a better understanding of immune dysbiosis and autism is crucial to identify differential ASD phenotypes and develop better biomarkers. We aimed to study autoantibody (aAb) reactivity to gut and brain tissues to find aAb profiles in children with autism that could serve to identify ASD subpopulations with biomarker potential.

Methods:

We used plasma from typically developing children (TD; n=149) and children diagnosed with ASD (n=208). To assess aAb reactivity, we probed plasma brain and gut tissues by Western blot.

Results:

While we found reactivity against brain (28% ASD and 32% TD), and gut (15% ASD and 11% TD) in both groups, we observed reactivity to antigens in defined molecular weight ranges in the gut tissue recognized by aAbs from the ASD subjects and not the TD samples. Further, some individuals had aAb reactivity to both brain and gut (8.6% ASD and 8% TD). These data are consistent with our previous studies against cerebellum tissue, where we reported aAb reactivity in both groups (ASD and TD), but some autoantibodies were differentially reactive in the ASD population.

Conclusion:

We identified aAbs reactive to brain and gut antigens in both TD and ASD children, with differential recognition of plasma from children with ASD to proteins in the gut extract. We are currently working on identification of the differentially recognized proteins in the gut tissue. Further, we can link our findings to the clinical data of these children, including reported GI issues. Such aAbs have the potential to serve as predictors of a subgroup of ASD individuals or facilitate a better understanding of the different sub-phenotypes of ASD based on differential aAb profiles.
Interleukin-38 promotes skin tumorigenesis via regulation of epidermal cell hyperplasia and pro-tumorigenic microenvironment in an IL-1Rrp2-dependent manner.
Authors: Zhou Hong , Zhao Qi-xiang , Yu Jia-dong , Yue Cheng-cheng , Hao Yan , Zheng Hua-ping , Hu Jing , Wang Zhen , Li Jiong
Published: February 2023

As the newest member of the IL-1 family, interleukin-38 (IL-38) has strong associations with chronic inflammatory diseases. However, its roles in tumorigenesis and the underlying mechanism remain poorly understood. Here, we demonstrated that IL-38, which was highly expressed in skin, downregulated in human cutaneous squamous cell carcinomas (CSCC) and 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoyl phorbol-13-acetate (TPA) induced mouse skin tumorigenesis. IL-38 keratinocyte-specific knockout mice (IL-38 cKO) dramatically promoted skin tumor formation and their malignant progression. Mechanistically, IL-38 is dispensable for epidermal mutagenesis. IL-38 keratinocyte-specific deletion reduced proliferative gene expression along with epidermal cell proliferation and hyperplasia. Additionally, keratinocyte-specific deletion of IL-38 was associated with reduced secretion of inflammatory cytokines leading to a decreased infiltration of myeloid cells into the local tumor microenvironment, these inflammation-driven tumorigenesis are essential for carcinogenesis process. Furthermore, we first demonstrated that IL-38 activated the c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) signal transduction pathway to promote proliferation and migration of tumor cells and expression of cancer-related inflammatory cytokines in an IL-1 receptor (IL-1R)-related protein 2 (IL-1Rrp2)-dependent manner. Together, our findings highlight the role of IL-38 in the regulation of epidermal cell hyperplasia and pro-tumorigenic microenvironment through IL-1Rrp2 /JNK and establish a new defined factor IL-38/ IL-1Rrp2 as therapeutic targets in skin cancer.
Generation of mice with a conditional Il9 allele
Authors: Pajulas Abigail , Kaplan Mark H
Published: February 2023

Interleukin (IL)-9 is a pleotropic cytokine that induces cell-specific functions associated with asthma, with target cells including T cells, mast cells, eosinophils, and epithelial cells. Previous findings have demonstrated that IL-9 promotes mast cell growth and differentiation, suggesting that IL-9 contributes to mast cell-driven allergic diseases. Although studies have demonstrated the impact of IL-9 in allergic asthma and other allergic diseases, the cellular source of IL-9 in each setting remains unclear. IL-9 can be produced by several cell types including natural killer T cells, innate lymphoid cells, mast cells, and CD4+ T helper cells (Th). We speculate that T helper 9 (Th9) cells are a major source of IL-9 and that Th9-dervied IL-9 promotes mast cell expansion, activity, and function. Our hypothesis is supported by findings in patients with asthma in which IL-9 expression and Th9 cell numbers are significantly elevated. Here, we address the role of T cell–derived IL-9 in mice with an inactivation of the IL-9 gene restricted to T cells generated by CD4-Cre/loxP-mediated targeting of the IL-9 gene. Preliminary results demonstrate efficient deletion of Il9 in T cells. Il9 fl/fl CD4-Cre Th9 cultures have minimal production of IL-9. Ongoing studies will directly examine the contribution and function of TH9-derived IL-9 during models of allergic airway inflammation and future studies will define the requirement for IL-9 in other cell populations during allergic immune responses.
AKST4290, a CCR3 Inhibitor, Reduces Age-Related Inflammation and Immune Cell Infiltration
Authors: Teichert Arnaud , Masumi Juliet , Rege Sanket , Braithwaite Steven , Minami Sakura
Published: February 2023

Eotaxin, a potent pro-inflammatory mediator, is a chemokine whose levels in plasma increase with age. CCR3, the primary receptor for eotaxin, is expressed on immune cells such as eosinophils and subsets of T lymphocytes, making it a potential therapeutic target for age-related inflammation. Using the CCR3 selective antagonist AKST4290 we aimed to assess the effects of modulating eotaxin/CCR3 signaling in mouse models of aging, inflammation and peripheral immune cell infiltration.

In an aging model, using 18-month-old C57BL/6 mice, AKST4290 treatment reverted the increased blood levels of immune cells to those seen in young mice, with no effects on immune cell levels in young mice.

In inflammation models, using either 8-week-old SKH1-Elite mice challenged with Oxazolone or 8-week-old C57BL/6 mice challenged with LPS, AKST4290 reverted the increased blood levels of immune cells to baseline levels and did not affect cell types unchanged by the stressors. In contrast, Dexamethasone produced a broad, non-specific, decrease of total white blood cells and critical subpopulations.

In an immune cell infiltration model, using 8-week-old C57BL/6 mice challenged with experimental autoimmune encephalomyelitis (EAE), AKST4290 significantly decreased EAE-induced T cell infiltration and microgliosis in the brain.

These findings suggest that AKST4290 is a potent immunomodulator that normalizes immune cell populations related to inflammation to healthy levels, both in the context of disease and aging. These immune modulatory and anti-inflammatory effects have functional consequences in aging models, indicating its therapeutic potential in multiple age-related disorders.
Detection of Cardiotrophin-like Cytokine Factor 1 (CLCF1) by flow cytometry
Authors: Laplante Véronique , Nadeau Ulysse , Rousseau Marine , Lesage Sylvie , Gauchat Jean-François , Pasquin Sarah
Published: February 2023

Cardiotrophin-like Cytokine Factor 1 (CLCF1) belongs to the IL6 family of cytokines and possesses pro-neurotrophic and immuno-modulating functions. Coding mRNA for CLCF1 has been detected in primary and secondary lymphoid organs (i.e. lymph nodes, spleen and bone marrow), as well as in the lungs and feminine reproductive organs. Modulation of CLCF1’s mRNA levels has been associated with the Th17 polarization in CD4+ T cells. However, little information is available regarding CLCF1 protein levels in these tissues or the nature of the immune cells responsible for its production. This can be explained by a lack of in situ detection options for CLCF1. We have therefore developed a methodology for the detection of human and murine CLCF1 by flow cytometry in permeabilized cells. This technique has been validated using derivatives of the Ba/F3 cell line in which cDNAs coding for human and murine CLCF1 were introduced by transduction with recombinant retroviruses. We are currently using this approach to study CLCF1 production by human and murine immune cells. Preliminary results indicate a production of CLCF1 by Th17 T cells. The development of a method to detect CLCF1 by flow cytometry will be beneficial for the study of CLCF1’s functions in the regulation of the immune response.
Functional characterization of gamma-delta (γδ) T cells in allergy
Authors: Yu Esther Dawen , Wang Eric , Sutherland Aaron , Westernberg Luise , Frazier April , Peters Bjoern , Sette Alessandro , da Silva Antunes Ricardo
Published: February 2023

Despite a growing consensus on the involvement of γδ T cells in allergy and other human immunological disorders, the detailed mechanisms remain hypothetical due to lack of investigative tools. Herein, we sought to develop functional assays to study the role of γδ T cells in allergy. Using an Activation-Induced Marker (AIM) assay, based on the upregulation of 4-1BB (CD137) and CD69, we were able to detect ex vivo allergen-specific responses from γδ T cells to multiple allergen extracts in human PBMCs, including mouse, cockroach (CR), house dust mite (HDM) and timothy grass (TG) allergens, which were reproducible and observed in multiple allergic cohorts. The magnitude of allergen-reactive γδ T cells differed between allergic and non-allergic cohorts with HDM and TG allergy, but not mouse or CR allergy. Further characterization showed that the γδ T cells reactive to mouse allergen were mostly the Vδ2 subset and from effector memory (Tem and Temra) compartments. Interestingly, in mouse-specific allergic donors we found differential polarized responses for γδ and ab T cells, with a Th1 and Th2 pattern, respectively. Using a complementary AIM assay for CD40L (CD154) combined with intracellular staining (ICS), we observed that Th1 polarization (INFg and TNFa) in responses to mouse extract was also common in non-allergic donors. Conversely, In the case of CR responses, Th1 polarization of Vδ2 T cells was only observed in the allergic cohort. Overall, these results suggest that allergen-specific Vδ2 T cells are potential to skew dysregulated cytokine responses in immunotherapy. In conclusion, we developed new functional assays that serve as tools for in-depth characterization and deciphering the roles of γδ T cells in allergy.
Human and mouse mast cells express surface PrP that is shed upon activation and affects degranulation
Authors: Willows Steven D , Kulka Marianna
Published: February 2023

Misfolded prion protein (PrP) is the causative infectious agent in prion diseases in both humans and animals. Despite this, the physiological role of PrP in its native conformation (PrPC) is still poorly understood. While best known for its presence on neurons, PrPC is also present on various immune cells. We determined whether mast cells also expressed surface PrPC and whether PrPC expression was linked to changes in mast cell function. Mast cells are tissue-resident immune cells that modulate many physiologic responses such as allergic inflammation, tissue remodeling, response to infection and neuroplasticity. When activated, mast cells degranulate rapidly, releasing several pre-formed signaling molecules and enzymes. We hypothesized that mast cells expressed PrP and that its expression was influenced by mast cell activation. Human (HMC-1 and LAD2) and rodent (BMMC) mast cell lines expressed surface PrPC with the highest expression in BMMC. Activation of mouse bone marrow derived mast cells (BMMC) with A23187 caused a 12 fold decrease in surface PrP expression after 30 min. Surprisingly, levels of PrP recovered to almost half of untreated levels 3 hrs post-stimulation, and reached similar levels to untreated by 48 hrs. BMMCs derived from PrP knockout mice activated by IgE/antigen released 15–25% less granule contents compared to wild type BMMC, suggesting that PrP is somehow involved in degranulation. There were no significant differences in cell surface expression of the IgE receptor FcɛRI in wild type and knockout BMMC. Our results indicate that PrP is highly expressed on human and rodent mast cells and that it may play an important role in degranulation and activation of mast cells, independent of FcɛRI expression.