bluuebunny/crossref_metadata_2025 huggingface dataset:
Minipigs as neonatal animal model for tuberculosis vaccine efficacy testing
Authors: Juarrero Mercedes Gonzalez , Ramos Laylaa , Obregon-Henao Andres , Henao-Tamayo Marcela , Bowen Richard , Izzo Angelo A. , Lunney Joan
Published: January 2023

Many vaccines against childhood diseases are administered early after birth. Despite the fact that neonatal-infant immunity differs from adult immunity, vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. A good example of this scenario is Bacillus Calmette-Guerin (BCG). BCG is the only vaccine available against tuberculosis (TB). The vaccine lacks efficacy against child and adulthood pulmonary disease or reactivation of latent TB but prevents disseminated TB in children and is thus widely used in countries with endemic TB as part of the neonatal vaccine regimen. There are several new vaccines that have shown efficacy against TB in adult animal models yet fail to protect infants from TB disease in clinical trials. In this novel approach, we used the minipig as a neonatal animal model for evaluation of immune responses to BCG vaccination. The longitudinal immune responses of neonatal mini-piglets vaccinated with BCG were followed until adolescence, with the same monitoring applied to a group of unvaccinated mini-piglets. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Further, we challenged both vaccinated and non-vaccinated animals via aerosol route with a low dose aerosol of Mycobacterium tuberculosis strain HN878 and we characterized changes between the two groups in the course of immune responses following challenge. Based on comparison of immune responses to BCG in minipigs with similar studies in infants, our findings suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.
Birth weight affects immune response of piglets during the peri-weaning period
Authors: Verso Luca Lo , Matte Jacques , Talbot Guylaine , Lapointe Jérôme , Bissonnette Nathalie , Guay Frédéric , Ouattara Bazoumana , Luna Uanderson Verissimo , Lessard Martin
Published: January 2023

Relationships between birth weight and a range of immune markers were studied in piglets during the peri-weaning period. A total of 47 sows and their respective litters adjusted to 12 piglets were used. Piglets were weighed after birth and within each litter 2 low weight (LW) and 2 high weight (HW) animals were identified: the average weight of the 2 groups was 1.12 and 1.81 ± 0.17 kg, respectively. Within litter, one couple of LW and HW piglets was sacrificed at 16 and 23 d of age, two days after weaning at day 21, to characterise different leukocyte subsets in mesenteric lymph nodes (MLN) and blood by flow cytometry. Jejunal mucosal and MLN samples were also collected to measure the expression of genes involved in immune response by qPCR. The percentage of blood γδT cells was higher in HW piglets before and after weaning (P=0.01 and P<0.001, respectively). At 16 d, LW animals had higher amounts of CD4+CD8+ T cells and dendritic cells in the blood (P=0.01 and P=0.04, respectively) and in MLN (P=0.02 and P=0.009 respectively), and lower count of blood cytotoxic T cells (P=0.01) than HW. Moreover, jejunal expression of TNFα was reduced in LW piglets (P=0.02), while MLN expression of CCL23, BMP2 and SPP1 was increased (P=0.003, P=0.002 and P=0.02, respectively). Two days after weaning (d 23), HW piglets had higher percentages of T cells (P=0.001) and lower amount of non-T cells (P=0.03) and natural killer cells (P=0.04) in blood compared to LW animals. These data revealed that piglet birth weight affects the proportion of leukocyte subsets in different tissues and the expression of genes involved in the immunity. Globally, our results suggest that the development of the immune system after birth differed between LW and HW piglets.
β-glucan induced training and tolerance: alterations to primary monocytes
Authors: Byrne Kristen A. , Beiki Hamid , Tuggle Christopher K. , Loving Crystal L.
Published: January 2023

In mice and humans, exposure to β-glucan can induce epigenetic reprograming in monocytes resulting in enhanced responses to heterologous agonists. Epigenetic reprograming is the basis for innate memory, which includes both decreased immune responsiveness (innate tolerance) and increased responsiveness (innate training). As a valuable food animal and medically relevant species, we sought to understand the phenotypic and mechanistic alterations induced by β-glucan on swine cells to develop methods to enhance health while limiting antibiotic usage. Thus, primary porcine monocytes were stimulated with β-glucan from S. cerevisiae or C. albicans, rested for 5 d, and then restimulated with lipopolysaccharide (LPS; TLR 4 agonist) or Pam3CSK4 (synthetic triacylated lipopeptide; TLR 2 agonist) to determine trained or tolerant phenotype (increase or decrease in cytokine production relative to unstimulated controls). Zymosan (β-glucan from S. cerevisiae) primed monocytes exhibited a tolerant phenotype (decreased IL-1β and TNF-α production compared to controls) when restimulated with LPS or Pam3CSK4. However, β-glucan from C. albicans (the primary β-glucan used in mouse and human studies) primed porcine monocytes for increased cytokine production after LPS and Pam3CSK4 stimulation, an indication of trained immunity. Epigenetic analysis of the accessibility of the genome to transposase (ATAC-sequencing) showed changes in promoter peaks with treatment, providing insight into mechanisms of tolerance versus training. These data indicate that β-glucan can induce both training and tolerance in porcine monocytes, but the source and purity of β-glucan likely impacts innate memory.
Mucosal cellular immune responses following Escherichia coli O157:H7 bacterin vaccination associated with reduced fecal shedding
Authors: Schaut Robert G. , Boggiatto Paola M. , Palmer Mitchell , Loving Crystal L. , Sharma Vijay
Published: January 2023

Parenterally-delivered, bacterin-based vaccines can significantly limit shedding of Shiga-toxin producing Escherichia coli (STEC) O157:H7 from cattle, providing a potential intervention strategy to limit foodborne contamination. While the peripheral antibody response to STEC bacterin has been characterized, induction of STEC-specific cellular immunity, particularly at the site of colonization, remains poorly described. As STEC-specific antibodies likely provide some protection, cellular immune responses likely contribute to limiting colonization also. Following vaccination and challenge, lymphocytes were detected in the rectoanal junction (RAJ), a preferred colonization site for E. coli O157:H7. T cells present in the RAJ were positive for interferon (IFN)-γ mRNA, including a subset of γδ+ CD8+ T cells. Specifically within the RAJ, γδ+ T cells expressing IFN-γ mRNA were greater in number compared to classical αβ+ T cells. Also, distinct lymphoid follicles were present in greater number at the RAJ in vaccinated/challenged animals and fecal STEC-specific IgA was detected. These vaccine -primed responses likely support localized immune responses upon challenge. Local T cell-produced IFN-γ may impact epithelial cells, subsequently limiting E. coli adherence. Thus, distinct immune changes were induced at the mucosa of vaccinated and challenged animals, providing insight on mechanisms associated with limiting shedding. Enhancing mucosal immunity may be critical in the further development of efficacious vaccines for controlling STEC O157:H7 in ruminants, thus limiting human infection.
Somatic hypermutation of TCRα contributes to thymic positive selection in sharks
Authors: Ott Jeannine A. , Castro Caitlin , Deiss Thaddeus , Ota Yuko , Flajnik Martin F. , Criscitiello Michael F.
Published: January 2023

In mammals and probably all vertebrates, receptor editing of TCR alpha genes enhances immature T cell positive selection over a three-day interval in the thymic cortex. Surprisingly we found extensive somatic hypermutation (SHM) operating at the TCRα locus in the nurse shark thymus, implying that SHM contributes to receptor modifications that enhance positive selection. We analyzed mutation in TCRα families of clones with the same VJ rearrangement. Additionally, in situ hybridization showed the strongest activation-induced cytidine deaminase (AID) expression in the central thymic cortex and bordering the corticomedullary junction. The frequency of mutation at TCRα was as high as that seen at B cell receptor (BCR) loci in sharks and mammals. Complementarity determining regions (CDRs) accumulated significantly more mutations than framework regions (FWs), and significantly more mutations in CDRs resulted in amino acid replacement than in silent mutation. We saw a preference for transition mutations as well as a strong bias toward G:C substitutions within AID hotspots, especially within CDR regions. We suggest that TCRα utilizes SHM to boost positive selection and perhaps to broaden diversification of the αβ T cell repertoire in sharks, the first reported use of this process in thymic diversification in vertebrates.
Plasma surfactant proteins and lipid mediators in the airway associate with different stages of viral infection and resolution in a sub-lethal mouse Influenza A model
Authors: Nguyen Allen , Wong Kent , Chakrabarti Ari , Jiang Jenny , Ulufatu Sheila , Liu Shannon , Valle Nicole , Park Summer , Xu Min , Rosenberger Carrie , Anania Veronica , McBride Jacqueline
Published: January 2023

Viral quantification serves as a valid biomarker for detecting Influenza A infection however viral burden is not closely associated with disease severity or clinical outcomes. Biomarkers reflecting the status of the host immune response are needed to monitor and/or predict disease outcomes in a hospitalized setting.

Here, we utilized a sub-lethal mouse model of Influenza A infection to explore plasma and airway cytokines, plasma surfactant proteins (SP-D, SP-A and CC16), and airway bioactive lipid mediators as potential biomarkers of lung leakage and host response, respectively. This study aimed to assess the pharmacodynamic response of these biomarkers after treatment with an efficacious dose of an anti-HA stalk-binding antibody and to assess association of these biomarkers with disease severity metrics.

Plasma SP-D and IP-10 correlated with lung viral titers while SP-A and CC16 tracked more closely with changes in bronchial albumin levels and body weight. Bioactive lipid precursors Arachidonic Acid (AA), Docosahexaenoicacid (DHA) and Eicosapentaenoic acid (EPA), reported to have both pro- and anti-inflammatory properties, increase in the lung with infection. 17-HDHA, a DHA metabolite reported to be pro-resolving, was induced later during disease resolution. With anti-HA antibody treatment, all plasma surfactant proteins and precursor lipids in the airway were reduced and 17-HDHA production was accelerated.

In summary, plasma SP-D may serve as an early biomarker of host response and/or potentially indicate a decline in lung epithelium integrity. Plasma SP-A, CC16, and pro-inflammatory lipid mediators such as AA may reflect the ongoing host response and may therefore be more closely associated with disease outcomes.
Cytokines levels in nasal lavage in pediatric patients infected with respiratory viruses
Authors: Rosas-Taraco Adrian Geovanni , Leon-Icaza Stephen A. , Gonzalez-Chapa Jorge A. , Silva-Ramirez Beatriz , Martinez-Castilla Azalia M. , Treviño-Garza Consuelo , De la O-Cavazos Manuel E. , Montes-Tapia Fernando F. , Salinas-Carmona Mario C.
Published: January 2023

Respiratory viruses are responsible of acute respiratory infections that affect worldwide population causing morbi-mortality and children younger than 5 years are the most affected population. Cytokines can modulate the immune response but little is known about their levels in nasal mucosa and less if these would correlate with symptoms severity. One hundred twenty-one pediatric patients with clinically diagnosed acute respiratory infections were sequentially enrolled in the Department of Pediatrics of the University Hospital. Throat swabs were performed and nucleic acid was isolated to viral detection by Luminex xTAG RVP kit and qRT-PCR. Nasal lavage were performed in each patients to evaluate cytokines levels by multiplex immunoassay. Disease severity was evaluated according to clinical characteristics on the day of enrollment. The most frequently found viral respiratory infections were hRV, RSV, and hMPV. hMPV load correlated with disease severity (P<0.05). Pro-inflammatory, Th1, Th2 and Th17 cytokines were detected in nasal lavage in those patients, however only pro-inflammatory cytokines were found higher in severe disease. Pro-inflammatory, Th1/Th2/Th17 cytokines correlated with symptoms, mainly with fever (P<0.05). Our findings show that disease severity correlates with viral load and cytokines in nasal lavage may regulate local immune response against respiratory virus.
Exchange Proteins Directly Activated by cAMP and the Role of these Proteins in Respiratory Syncytial Virus Infection
Authors: Wu Wenzhe , Choi Eunjin , Ren Yuping , Chen Yu , Liu Shengxuan , Ren Junping , Garofalo Roberto P. , Zhou Jia , Bao Xiaoyong
Published: January 2023

Respiratory syncytial virus (RSV) is a single-stranded, negative-strand RNA virus belonging to Paramyxoviridae family, and is in the genus Pneumovirus. It was discovered in chimpanzees in 1955 and subsequently confirmed to be a human pathogen shortly after that. RSV is one of the most common viruses to infect young children and high-risk adults worldwide, and the leading cause of inflammation of the lungs (pneumonia) or the lungs’ airways (bronchiolitis) in infants. Due in part to the lack of long-term immunity after infection, making reinfection frequent. There is a single antiviral treatment for RSV currently approved, but its use is limited by questionable efficacy, side effects, and cost, and it is recommended that it be used only for patients at-risk for severe disease, on a case-by-case basis. In this study, we discovered an exchange protein directly activated by cyclic AMP (EPAC) that can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, the treatment of EPAC inhibitor (ESI-09), but not PKA inhibitor (H89), significantly inhibits the RSV replication and pro-inflammatory cytokine/chemokine induction. In addition, RSV-activated transcriptional factors belonging to the NF-κB and IRF families are also suppressed by ESI-09. Through isoform-specific gene knock down, we found that EPAC2, but not EPAC1, plays a dominant role in controlling RSV replication and viral-induced host responses. Experiments using both EPAC2 knock out and EPAC2 specific inhibitor support such roles of EPAC2. Therefore, EPAC2 could be a promising therapeutic target to regulate RSV replication and associated inflammation.
Detrimental role of type I interferon signaling in respiratory syncytial virus infection
Authors: Ansar Maria , Komaravelli Narayana , Ivanciuc Teodora , Casola Antonella , Garofalo Roberto P.
Published: January 2023

Respiratory Syncytial Virus (RSV), the primary cause of lower respiratory tract infections in young children, is a poor inducer of type I interferons (IFN) and is resistant to its canonical antiviral activity. Paradoxically, increased type I IFN has been observed in some studies to correlate with more severe disease in RSV-infected children. Previously, we have shown that a pathogenic reactive oxygen species (ROS) production in the course of RSV infection is caused by a progressive reduction in nuclear and cellular levels of the transcription factor NF-E2-related factor 2 (NRF2), the primary regulator of antioxidant enzyme (AOE) gene expression. Thus, the goal of this study was to determine the contribution of type I IFN to lung innate/inflammatory responses, modulation of AOE expression, and airway disease in experimental RSV infection. Using either IFN alpha/beta receptor knockout mice or IFNAR neutralizing antibody, we found that IFN signaling blockade attenuated RSV-mediated NRF2 degradation and rescued AOE levels (i.e. SOD 1 and Catalase) in the lung, resulting in improved airway function and clinical disease. We also showed that NRF2/AOE rescue in mice lacking IFN signaling is due to the reduced expression of the promyelocytic leukemia protein (PML), a key component of PML nuclear bodies. In addition, levels of RSV-induced pro-inflammatory cytokines including IL-1α/β, IL-6, IL-12(p40), TNF-α, and the chemokines CXCL1, CCL2, 3, 4, and 5 were also reduced in IFN deficient mice. In conclusion, this data shows for the first time that IFN type I signaling plays a critical role in enhancing RSV-mediated airway disease through an innate immune pathways that involve the PML-NRF2-AOE axis.
Role of mucin 19 in the respiratory tract.
Authors: McBride Kaitlin E. , Cheemarla Nagarjuna R. , Guerrero-Plata Maria Antonieta
Published: January 2023

Human metapneumovirus (HMPV) is a negative sense, single stranded RNA virus that belongs to the Pneumovirdae family, and represents an important pathogen that causes severe bronchiolitis and pneumonia worldwide. There is currently no vaccine against this virus, so it is important to study the aspects of the immune response induced by HMPV in order to understand its mechanism of infection. Because infiltration of mucus is a hallmark of HMPV infection, it is warranted to study the role of mucus in the disease process. Mucin proteins make up the major component of mucus and can be found within the airway and lungs. Using an in vitro model of HMPV infection and human bronchial epithelial cells, we found that HMPV induced predominantly a mucin 19 expression. Those data were confirmed in mice, where HMPV induced predominant levels of muc19 protein. Mucin 19 is a secreted gel-forming mucin, whose function is largely understudied, particularly in the respiratory tract. Our findings led us to hypothesize that mucin 19 plays a role in HMPV infection. To test that, we used a muc19 knockout mouse model. Lung samples were collected at different time points. Sample analysis showed that the absence of muc19 led to an increased pulmonary inflammation and body weight loss. Interestingly, flow cytometry data indicate that the lack of Muc19 resulted in a decreased CD4+ T cell count in the lung, and reduced numbers of CD4+ and CD8+ T cells in the lymph nodes after HMPV infection. These novel findings yield relevant information regarding the contribution of the mucins, specifically Muc19, on the immune response induced by respiratory viral infections in the lung.
Genome-wide screens identify novel components of the MHC I pathway.
Authors: Kriegsman Barry A. , Cruz Freidrich M. , Merino-Rodriguez Elena , BelleIsle Janice , Rock Kenneth L.
Published: January 2023

Antigen presentation is the process by which cells generate and display peptides on major histocompatibility complex (MHC) molecules for T cell recognition. Although it is well known that antigen presentation is critical for effective adaptive immunity, not all of the machinery driving antigen presentation has been identified. Because there is significant potential to exploit antigen presentation to develop therapeutics against cancers, microbes, and autoimmune diseases, it is important that we advance our understanding of these pathways. To this end, our lab has conducted two independent genome-wide screens to identify novel genes important for MHC I presentation. In one screen, we silenced mouse dendritic cells (DC3.2 cell line) with siRNA and tested the ability of the silenced dendritic cells to cross-present (i.e., present peptides derived from exogenous antigen on MHC I). In another screen, we used a lentiviral sgRNA library to knock out human genes in HeLa-Cas9 cells and identify genes that positively regulate surface MHC I levels. We are currently validating several hits from these screens and characterizing the mechanisms by which they affect MHC I presentation.
A Treg-specific deletion of Helios causes autoimmune lipodystrophy and metabolic syndrome
Authors: Thornton Angela M , Gavrilova Oksana F , Penna Vinay R , Korty Patricia E , Shevach Ethan M
Published: January 2023

Regulatory T (Treg) cells suppress immune activation in a dominant manner and play a critical role in the maintenance of self-tolerance. A subpopulation (60–75%) of Foxp3+T regulatory (Treg) cells express the transcription factor Helios. To examine the function of Helios in Treg cells, we have generated Treg-specific Helios deficient mice (cKO, Heliosflxflx Foxp3cre). Although both Treg development in the cKO mice and their in vitro suppressor function are normal, the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation with a Th1 phenotype, hypergammaglobulinemia, and enhanced germinal center formation. Initially, we observed significant lymphocytic infiltrates only in the salivary gland and not in any other organs typically affected by Treg dysregulation. Strikingly, the mice developed lipodystrophy, hepatic steotosis and insulin resistance. Further analysis revealed a significant lymphocytic infiltrate in both the inguinal and perigonadal adipose tissue, indicating autoimmune mediated destruction of the white adipose tissue (WAT). We have further shown that the lymphocytic infiltrate specific to WAT can be transferred from mice with lipodystrophy to immunodeficient mice, confirming the autoimmune nature of the lipodystrophy. Thus, Helios deficiency in Treg disrupts immune homeostasis in the adipose tissue, leading to the destruction of WAT and redirection of lipids to the liver, and ultimately causes metabolic dysfunction.

Supported by the Intramural Research Program of the NIAID, NIH.
The absence of CD137 ligand upregulates the expression of the immunosuppressive molecule soluble CD137
Authors: Foda Bardees M. , Forsberg Matthew H , Ciecko Ashley E , Mueller Kevin W , Geurts Aron , Chen Yi-Guang
Published: January 2023

The gene encoding the co-stimulatory molecule CD137 is located within the Idd9.3 T1D susceptibility locus and contributes to diabetes progression in NOD mice. We have previously shown that CD137 expression in T cells has dual functions: CD4+CD137+ T cells inhibited T1D development while CD8+CD137+ T cells showed potent diabetogenecity. The protective function of CD137 in CD4+T cells is likely due to the significant amounts of soluble CD137 (sCD137) produced by Foxp3+ Tregs. sCD137 is an alternatively spliced form of CD137 lacking the transmembrane encoding exon. Treating NOD mice with recombinant sCD137 prevents T1D development. We aim to study the impact of CD137 ligand on the expression of sCD137. We used CRISPR/Cas9 to generate a strain of NOD depleted of CD137L (NOD.Tnfsf9−/−). Development of T1D was repressed in the newly generated strain. Deficiency of CD137L was associated with increased levels of serum sCD137. CD137-deficient-T cells contributed significantly to the elevated serum levels of sCD137. Ligand-deficient-Treg cells produced higher levels of sCD137 proteins compared to wild-type Tregs. Moreover, we detected increased levels of sCD137 transcripts in Tregs lacking the expression of CD137L. We direct our efforts to uncover the molecular mechanisms involved in the regulatory role of CD137L influencing the expression of sCD137.
Characterizing a “critical window” of immune development: how perturbation of developmental hematopoiesis shapes immune function and susceptibility to disease in offspring
Authors: Beaudin Anna E , Lopez Diego A , Apostol April , Hernandez Gloria E , Forsberg Camilla
Published: January 2023

During development, fetal hematopoietic stem cells (HSCs) are responsible for the production of “unconventional” innate-like immune cells that persist across the lifespan and contribute to adult immunity. Dysregulation of fetal-derived immune cells contributes to pathogenesis in a variety of immune tolerance disorders, including allergy and asthma; however, the cellular and molecular mechanisms driving pathogenesis are unknown. Using a fate-mapping approach, we have previously identified a developmentally-restricted HSC (drHSC) that is lymphoidbiased and specifically gives rise to innate-like lymphocytes. The drHSC exists during perinatal development but disappears postnatally and does not persist into adulthood under homeostatic conditions. Our discovery of a transient cell-of-origin for a specialized component of adult immunity defines a “critical window” of immune development, during which phenotype can be shaped by extrinsic inputs in early life. We have validated the existence of this critical window of immune development by demonstrating that maternal immune perturbation with a single low-dose injection of poly(I:C) at mid-gestation induces lasting changes to the both hematopoiesis and immunity in offspring. Specifically, maternal immune perturbation causes expansion of the drHSC population in offspring and its inappropriate persistence into adulthood. Furthermore, inappropriate expansion and persistence of the drHSC population consequently results in parallel expansion of innate-like lymphocytes, thereby altering immune landscape and function in offspring. Ongoing work investigates the consequences of developmental perturbation on susceptibility to immune dysfunction, including asthma.
Plasma cells are obligate enhancers of age-associated myeloid skewing
Authors: Pioli Peter , Casero David , Montecino-Rodriguez Encarnacion , Morrison Sherie L , Dorshkind Kenneth A
Published: January 2023

In humans and mice, aging results in significantly elevated myelopoiesis and the increased production of inflammatory cytokines with age is thought to underlie this phenomenon. The observation that treatment of young mice with factors such as interleukin (IL)-1 can skew hematopoiesis towards myeloid development is consistent with this hypothesis. However, the cellular source(s) of inflammatory cytokines in the aging bone marrow has not been fully defined. We observed that plasma cells (PCs) accumulate in the bone marrow with age and acquire an inflammatory gene signature. Furthermore, we observed that their depletion in vivo using anti-CD138 antibodies resulted in a significant decline in myelopoiesis as demonstrated by a reduction in the number of myeloid biased hematopoietic stem cells, myeloid progenitors and mature myeloid cells. We also found that anti-CD138 treated mice exhibited reduced expression of genes encoding inflammatory cytokines in bone marrow stromal cells, indicating that plasma cells can regulate inflammatory cytokine production by other microenvironmental components. We tested whether this inflammatory network was necessary to drive age-associated myeloid skewing by treating old mice with Anakinra or Enbrel to abrogate IL-1 and tumor necrosis factor alpha (TNF-α) signaling, respectively. Concurrent blockade of both signaling pathways in old mice resulted in the significant reduction of bone marrow myelopoiesis, which was not observed upon inhibition of either pathway alone. These data demonstrate a previously unappreciated ability of inflammatory PCs to stimulate myelopoiesis and implicate them at the center of an inflammatory regulatory network in the aging bone marrow.
Merocytic dendritic cell: a new subset of conventional dendritic cells
Authors: Audiger Cindy , Lesage Sylvie
Published: January 2023

Conventional dendritic cells (cDC) are potent antigen-presenting cells that induce the activation of naïve T cells in response to pathogens. cDC activity is mediated primarily by two cDC subsets, namely cDC1 and cDC2, each bearing unique properties. Recently, another DC subset, termed merocytic dendritic cells (mcDC), was defined. In contrast to both cDC1 and cDC2, mcDC are able to reverse T cell anergy, even in non-inflammatory conditions, properties that could be exploited to potentiate cancer treatments. Here, we further characterize mcDC to determine their relationship to cDCs. First, we demonstrate that mcDC express key cDC traits, namely they express the cDC-restricted transcription factor, Zbtb46, and are very potent inducers of mixed lymphocyte reactions. Second, transcriptomic studies reveal that mcDC are more closely related to cDC1 than to cDC2. In contrast, similar to cDC2, mcDC are dependent on IRF4, but not IRF8 and BATF3, two major transcription factors required for cDC1 differentiation. Third, investigating mcDC population dynamics in reconstitution kinetics studies and in parabiotic mice, we demonstrate that, as for cDC1 and cDC2, mcDCs are terminally differentiated cells. Altogether, these data demonstrate that mcDC compose novel cDC subset. Defining the properties of mcDC in mice may help identify a functionally equivalent subset in humans leading to the development of novel cancer immunotherapies.
Isthmin 1 identifies a subset of lung hematopoietic stem cells and it is associated with systemic inflammation
Authors: Rivera-Torruco Guadalupe , Mendiola Carolina Abigail , Floriano Tania Angeles , Franco Rafael , Parra-Ortega Israel , Vilchis Armando , Maravillas-Montero José Luis , García-Contreras Rodolfo , Contreras Oscar Medina , Nava Porfirio , Ortiz-Navarrete Vianney , Licona-Limon Paula , Valle-Rios Ricardo
Published: January 2023

Hematopoiesis is a highly regulated process and it’s widely described in bone marrow, the classical niche for hematopoietic stem cells (HSCs). The lungs are a novel niche of HSCs, as recently described by Lefrançais in 2017, however, little is known about the nature of these cells. Isthmin 1 (ISM1) is a secreted molecule important in developmental hematopoiesis in zebrafish; moreover, it is known that murine lungs express high levels of ISM1 and its secretion is altered after LPS challenge. In this work we performed multiparametric flow cytometry identifying a subpopulation of lung-resident HSCs expressing ISM1. Then we wanted to assess if HSCs ISM1+ cells are affected under inflammatory insults. Acute infection in mice with Pseudomonas aeruginosa showed that lung-derived HSCs ISM1+ are increased, indicating that the lung-HSCs are perturbed. Due to our model produced systemic inflammation we sought to investigate whether circulating ISM1+ cells were affected, we found that the percentage and absolute numbers of ISM1+ cells in blood were slightly diminished. Since ISM1 is a secreted molecule we evaluated its plasmatic levels by ELISA. We discovered a reduction in ISM1 plasmatic levels during murine sepsis with Pseudomonas aeruginosa. Then, we sought to analyze ISM1 levels in human inflammation; we retrieved samples from pediatric individuals with sepsis and adult patients with 2th and 3th grade burn injuries, and in both cases, we detected reduced levels of plasmatic ISM1. Our data suggest that ISM1 is likely a regulator of hematopoiesis in the lung but also may have a function in the regulation of systemic inflammation in mice and humans. We suggest that ISM1 is a novel biomarker of systemic inflammation.
Identification of splenic progenitors for CD11b high myeloid cells in the long-term culture with GM-CSF
Authors: Ryu Seul Hye , Na Hye Young , Sohn Moah , Choi Wanho , In Hyunju , Shin Hyun soo , Park Ji Soo , Park Chae Gyu
Published: January 2023

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a key hematopoietic cytokine for the development of immune cells from progenitors in bone marrow (BM). However, the role of GM-CSF in hematopoiesis outside of BM has been less well understood. In a recent report, we discovered that significant proliferation of CD11bhi myeloid cells, including CD11chi dendritic cells (DCs) and CD11clo macrophages, were detected in the culture of splenocytes with GM-CSF between 1 and 3 weeks. To further characterize this slow myelopoiesis of CD11bhi cells in vitro, we set out to find their progenitors in spleen. We have identified and isolated the distinct splenic populations of myeloid progenitors based on the established cell-surface markers of hematopoietic progenitors in BM. When those isolated splenic progenitors were cultured with GM-CSF, lineage− CD117+CD115−CD16/32+ splenic cells were effective to produce CD11bhi myeloid cells. Our current study indicates that splenic progenitors comparable to BM granulocyte-macrophage progenitor (GMP) are responsible for generating CD11bhi DCs and macrophages in the long-term culture with GM-CSF.
Acute and chronic waves of myeloid cell maturation and myelopoiesis with high fat diet
Authors: Bowers Emily , Abrishami Simin , Singer Durga
Published: January 2023

Diet-induced obesity is associated with an infiltration of monocytes into the adipose tissue in male mouse models. These monocytes differentiate into inflammatory macrophages that drive insulin resistance. This increase in circulating monocytes originates in the bone marrow (BM) where hematopoietic stem cells (HSC) and myeloid-restricted progenitors (GMPs and PreGMs) expand. HSCs isolated from BM of male mice on high-fat diet (HFD) for 16 weeks show myeloid lineage skewing that lasts through successive bone marrow transplants (BMT). This suggests that HFD induces changes to the HSC niche - a multicellular structure that produces regulatory signals that are critical for maintaining the HSC pool. While BM adipocytes expand with HFD exposure, the niche cells promoting myelopoiesis with HFD are unknown. We chose to investigate two cell populations that have been implicated in driving myelopoiesis in other conditions; the BM endothelial cell (BMEC), a critical HSC niche cell, and neutrophils. We hypothesize that HFD induces HSC expansion and reprogramming through neutrophil-BMEC communication. Using 60% HFD in C57Bl/6J mice we evaluated circulating and BM neutrophils and Ly6C monocytes; HSCs and progenitors, and BMECs during short- and long-term HFD exposure. We found that as early as 24 hours after mice went on HFD there is a significant spike in circulating mature neutrophils that return to normal levels by 72 hours only to expand again by 16 weeks HFD. Ly6Chi monocytes rose only after 1 month of HFD exposure. BMECs do not appear to be altered in number with HFD in short- or long-term assays. Further qualitative analysis of BMECs, MAT, and neutrophils are necessary to determine which cells are critical for regulating the HSC niche in HFD.
OPN receptor expression on immune effector cells in RSV infection is regulated by NKT cells
Authors: Qureshi Mahboob Hossain , Qureshi Omar , Durre Kainath
Published: January 2023

RSV infection in early life predisposes to asthma later in life. Role of aberrant immune responses to childhood viral infections is well documented. Involvement of myeloid (mDC) and plasmacytoid (pDC) Dendritic Cells (DC) and Natural Killer T-cells (NKT) has been reported. Osteopontin (OPN) modulates the immune responses to viral infections. The current studies looked into the role of NKT cells in regulating the OPN receptor expression in RSV infection on DC, T and B cells, which will elucidate the role of OPN in preventing RSV-induced hyper-reactive airway and asthma. Five-day-old BALB/C and NKT−/−(CD1d−/−) mice were infected intranasally with RSV; and expression of OPN receptors (CD44 and CD51/61) were examined at designated post-infection days on lung mDC (CD11blo/−CD11chiCD45Rlo/−), pDC (CD11blo/−CD11clo/−CD45Rhi), T (CD3+) and B (CD19+) cells by flow cytometry. Expression of CD44 and CD51/61 were decreased on mDC, pDC, T and B cells in RSV infected wild pups compared to the control pups. On the other hand, OPN receptor expression was enhanced in the RSV-infected NKT−/− pups compared to the infected wild pups and uninfected NKT−/− pups suggesting a regulatory role of NKT cells on expression of OPN receptors, as RSV-infected NKT−/− pups had significantly increased CD44 and CD51/CD61 expression on DC and lymphocytes. Thus, decreased OPN receptor expression during RSV infection very likely diminishes the action of endogenous OPN allowing skewed polarization of DC and subsequent T cell responses. Therefore, abolishing the action of NKT cells to augment the action of endogenous OPN; and/or administration of exogenous OPN warrant further investigation as a new therapeutic strategy against RSV-associated asthma.
The Ikaros zinc finger transcription factor Eos regulates CD4+ TH1 and CD8+ TC1 differentiation programs
Authors: Sreekumar Bharath Krishnan Nair , Powell Micheal Duane , Read Kaitlin , Zafar Jawad , Jones Devin , Baker Chandra , Allen Irving C , Oestreich Kenneth J.
Published: January 2023

CD4+ T helper 1 (TH1) cells are essential mediators of immune responses directed against viruses and intracellular bacteria. As such, elucidation of the regulatory mechanisms underlying their development and function is necessary to direct the function of these cells in immunotherapeutic approaches to treat human disease. Recently, we demonstrated that the Ikaros zinc finger (IkZF) factor Aiolos cooperates with STAT3 to induce Bcl-6 expression in CD4+ T follicular helper (TFH) cells. Given conservation between individual members of the IkZF and STAT families, we hypothesized that additional IkZF/STAT complexes may regulate the development of other T cell subsets. Indeed, we find that the expression of the IkZF factor Eos is elevated in TH1 cells compared to TFH and naive CD4+ T cell subsets. We further find that induction of Eos expression is dependent on IL-2 signaling and is mediated, in part, by STAT5. Furthermore, expression of TH1 markers, including Blimp-1, IL-2rα, and IL-2rβ, as well as the production of the TH1-associated cytokine, IFN-γ were significantly diminished in Eos-deficient TH1 cells. Mechanistically, we found that Eos physically interacts with STAT5 and that these factors are co-enriched at the Prdm1 and Il2ra gene loci in TH1 cells. Intriguingly, we also found that Eos-deficient CD8+ cells differentiated under TC1-polarizing conditions displayed reduced expression of the TC1 genes encoding T-bet, Blimp-1, IFN-γ, and Granzyme B. Collectively, our data support a conserved role for a regulatory axis involving IL-2, STAT5, and Eos that drives the differentiation and function of CD4+ TH1 and CD8+ TC1 cells.
Adventitial stromal cells define group 2 innate lymphoid cell tissue niches
Authors: Molofsky Ari B , Dahlgren Madelene W , Jones Stephen W , Dubinin Alexandra , Farhat Sepideh , Ortiz-Carpena Jorge F , Yu Kevin S , Lee Katharine , Wang Chaoqun , Molofsky Anna Victoria R , Tward Aaron D , Krummel Matthew F , Peng Tien
Published: January 2023

Mobilization of type 2 immune responses is critical to both physiologic tissue remodeling and allergic pathology such as allergic asthma. However, whether there are physical tissue niches with unique cellular constituents that mediate type 2 immunity is unknown. We used quantitative 3D-imaging to define tissue niches of group 2 innate lymphoid cells (ILC2), critical instigators of type 2 immunity. We identified a dominant adventitial ‘cuff’ niche around vessels, airways, and ducts, present in the lung and multiple other tissues. ILC2s, as well as subsets of other tissue-resident hematopoietic cells, localized to these sites. However, ILC2s were most intimately associated with fibroblast-like adventitial stromal cells (ASCs) that were functionally distinct and sufficient to support ILC2 survival and activation. Here we discuss data supporting a model where ILC2s engage in a conversation with micro-anatomically and functionally distinct adventitial mesenchymal cells and other niche components, creating dynamic type 2-biased tissue foci that regulate tissue allergic immunity.
Noncanonical NF-κB controls stem cell signatures in the colonic mucosa and affects susceptibility to inflammation-induced carcinogenesis
Authors: Allen Irving C , Eden Kristin , Morrison Holly , Rothschild Daniel , Brown Stephan , Holl Eda
Published: January 2023

NF-κB signaling is divided into two distinct pathways, defined as either canonical or noncanonical. Canonical NF-κB signaling is well described and characterized. However, significantly less is known regarding mechanisms regulated by the noncanonical NF-κB pathway, especially outside of the lymphoid system. Recently, interest in the role of noncanonical NF-κB in the mucosal immune system has been increasing, with emphasis on NF-κB inducing kinase (NIK). NIK is an essential kinase in this cascade. Our data shows that lack of noncanonical signaling through the complete knockout of Nik in mice results in an aberrant large intestine phenotype characterized by reduced stem cell marker expression, altered regeneration and differentiation capacity under stress, changes in the microbiome, and predisposition to inflammation-associated tumorigenesis. Subsequent mechanistic studies utilizing novel mice with cell type specific deletions of either RelA/p65 or Nik revealed separate and distinct rolls for canonical and noncanonical NF-κB signaling in attenuating inflammation and tumor progression. RelA/p65 attenuates disease progression through the myeloid compartment in the gut, whereas Nik functions through the intestinal epithelial cell compartment. Dysregulation of noncanonical signaling is also observed in human colorectal cancer patients, which have marked suppression of this pathway in colonic biopsy samples. Together, these findings indicate that noncanonical NF-κB signaling is a critical regulator of immune system homeostasis in the gastrointestinal tract and plays a vital protective role against colorectal cancer.
Intestinal Goblet cells and Goblet Cell Associated Antigen Passages regulate the balance between Tregs and Th17
Authors: Kulkarni Devesha Hrishikesh , Gustafsson Jenny , Davis Jazmyne , McDonald Keely , Hsieh Chyi , Newberry Rodney D
Published: January 2023

Tolerance towards dietary antigens and luminal microbes is essential to maintain homeostasis, the breakdown of which results in development of inflammatory bowel disease (IBD). T-helper 17 (Th17) and T-regulatory (Treg) cells are abundant in the intestinal mucosa, where they function to protect the host from pathogens and to restrain excessive effector T-cell responses, respectively. How the balance and switch between Treg and Th17 responses is maintained in the steady state to promote tolerance and how this balance becomes altered to favor Th17 responses in the setting of IBD is not known. We have identified that intestinal goblet cell (GC) associated antigen passages (GAPs), are a major pathway for steady-state luminal antigen transfer to lamina propria - dendritic cells (LP-DCs). In addition, GAPs are necessary for the imprinting of LP-DCs with tolerogenic properties, for the de novo induction of Tregs specific for dietary antigens, and the maintenance of pre-existing LP Tregs. Moreover, GAP inhibition results in the expansion of Th17 cells in the small intestine LP. Consistent with this, single cell profiling of intestinal lymphocytes revealed enhanced Th17 pathway gene signatures in mice lacking GCs. These observations strongly support that GCs/GAPs have a previously unappreciated role in maintaining the functional balance between Treg and Th17 cell.
Dissecting the tissue-specific mechanisms that support the maintenance of intestinal regulatory T cells
Authors: Morales Elisa Cruz , Hart Andrew P. , Laufer Terri M.
Published: January 2023

Intestinal regulatory T cells (Tregs) are important to maintain tolerance toward food antigens (Ag) and commensals. Understanding the mechanisms for localization and maintenance of intestinal Tregs will help to identify novel immunotherapies for inflammatory intestinal diseases.

Previously we used K14 transgenic mice lacking peripheral TCR-MHCII interactions to show that the small intestine lamina propria (siLP) is a niche for Tregs that can be filled and maintained independently of Ag-specific signals. Now, we have begun to dissect the interactions that sustain these MHCII-independent siLP Tregs.

Tregs can be phenotypically and functionally divided into central and effector Tregs. Central Tregs preferentially migrate from thymus toward secondary lymphoid tissues. Effector Tregs reside mainly in non-lymphoid tissues and acquire a tissue-specific phenotype. Although it has been proposed that Ag-specific signals mediate effector Treg differentiation, we find that siLP Tregs acquire an effector phenotype independently of local MHCII. Instead, CTLA4/CD28—but not ICOS—engagement is important to sustain proliferation of MHCII-independent siLP Tregs. These results suggest that, in the absence of MHCII-TCR stimulation (Signal 1), costimulatory pathways (Signal 2) are still important to maintain Tregs in the intestine.

Finally, we find that siLP Tregs localize in two distinct anatomical compartments: randomly within villi and aggregated in organized lymphoid structures known as isolated lymphoid follicles.

Our results show that the intestinal niche can sustain a population of effector Tregs independently of Ag-specific signals but via costimulatory signals like CTLA4/CD28.
Indiscriminate nature of lung resident CD8+ TRM cells reactivation and their varied reactivation profiles
Authors: LOW JUN SIONG , Amezcua Caro , Sefik Esen , Harman Christian C.D. , Jackson Ruaidhri , Jiang Xiaodong , Kelly Joseph Barrack , Cauley Linda S , Flavell Richard A , Kaech Susan
Published: January 2023

CD8+ tissue resident memory (TRM) cells are poised at the portals of infection and confer immediate protection upon reinfection. Despite the critical role of CD8+ TRM cells in long-term protective immunity, the precise mechanics governing their reactivation in-situ are unclear. Seminal work by Leo Lefrançois' group showed that circulating (TCIRC) memory CD8+ T cell depends on CD11c+ antigen-presenting cells (APCs) for reactivation, however, TRM cells are reactivated within peripheral tissues, raising the question of whether the same principle is conserved. We examined this question, focusing on influenza infection, because APCs in the lung are well-defined and lung-resident TRM cells are critical for heterologous immunity to influenza. Using a TCR transgenic Nur77 reporter to delineate TCR-specific from bystander activation in CD8+ TRM cells, we characterized the superior reactivation kinetics in CD8+ TRM cell compared to CD8+ TCIRC. We also identified unique TCR-specific signatures and bystander-specific signatures in the kinetics of CD8+ TRM cells and TCIRC cells reactivation respectively. While we observed the strict requirement for CD11c+APCs in TCR-driven CD8+ TCIRC reactivation, CD8+ TRM cells exhibit promiscuity in their interactions with antigen-presenting partners in that they can be reactivated by both hematopoetic and non-hematopoetic cells. This study explains how the anatomical location of CD8+ TRM cells can confer unique advantage by demonstrating their superior reactivation kinetics and the indiscriminate nature of CD8+ TRM cell reactivation as well as showcasing the transcription profiles of TRM and TCIRC cells following different modes of activation.
Generation of a novel GATA3 reporter and inducible deletion system to study GATA3hi Th2 and ILC2 cells in vivo
Authors: Gurram Rama Krishna , Liu Chengyu , Zhu Jinfang (Jeff)
Published: January 2023

GATA3 is an important transcription factor for the development of CD4 T cells and innate lymphoid cells (ILCs); it is also essential for the differentiation and function of Th2 cells and ILC2s. To understand the physiological role of GATA3 in these cells particularly in vivo, we have generated a novel GATA3 reporter mouse strain by inserting the ZsGreen-T2A cassette in front of the translation initiation site of the Gata3 allele already carrying two loxp sites which allow inducible Gata3 deletion when it is crossed to the CreERT2 transgenic mouse strain. Our initial experiments with this mouse line showed that the reporter expression faithfully reflects the GATA3 protein expression in both in vitro differentiated Th subsets and in vivo isolated T cells and ILCs. Furthermore, the reporter expression was still observed even when the Gata3 exon 4 (encoding zinc fingers) was deleted by tamoxifen treatment. Thus, this novel mouse model will allow us to separate GATA3-deficient “Th2” cells and “ILC2s” during in vivo immune responses. We are now using this reporter system to explore the role of GATA3 in various cells types generated in vivo by comparing GATA3-sufficient and GATA3-deficient reporter positive cells.