bluuebunny/crossref_metadata_2025_split huggingface dataset:
Double knock-in reporter mice: a novel tool to study age-associated NF-κB dynamics in primary microglia
Authors: Aqdas Mohd , Songkiatisak Preeyaporn , Shah Md Toufiqur Rahman , Oh Kyu-Seon , Sung Myong-Hee 
Published: August 2023

Abstract
Microglia are immune sentinels in the brain that are capable of orchestrating potent inflammatory responses in aging and neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases. NF-κB signaling pathway is commonly recognized as a significant regulator of inflammation and aging. Mapping the spatiotemporal complexity of NF-κB signaling is crucial to understand its impact and function in vivo, but the lack of tools to directly monitor NF-κB protein components has hindered such efforts. Our lab has generated reporter mice with the endogenous RelA (p65) and c-Rel labeled with distinct fluorescent proteins and a double knock-in line with both labeled subunits. To understand how aging affects microglial function, we isolated primary microglia from young and old animals. We cultured them in vitro and validated by P2RY12+, CD11b+ and CD45int. Interestingly, we observed two different microglial populations, one motile, and the other forming clusters. Microglia from aged animals showed a higher prevalence of motile, free-roaming microglia. We also observed these distinct microglial populations in brain slices from the mice ex-vivo. Live-cell imaging of NF-kB dynamics in primary microglia from young and old mice revealed a shift towards c-Rel in old brains. Further, we stained for Ki67 as a proliferation marker to functionally characterize these microglia populations. We observed only the free-roaming microglia to be proliferative in nature. Furthermore, we are investigating the senescence pattern between the two populations to better understand the molecular mechanisms associated with age-dependent and inflammation-derived changes in young and old brains.
Determining the mechanistic basis for Irgm1 mediated control of Mycobacterium tuberculosis infection
Authors: Naik Sumanta Kumar , Nehls Michael , Mreyoud Yassin , Kinsella Rachel L , Smirnov Asya , Chowdhury Chanchal S , Mckee Sam , Dubey Neha , Kreamalmeyer Darren , Stallings Christina L 
Published: August 2023

Abstract
Irgm1 is a 47kDa IFN-γ inducible GTPase that has been shown to be essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. It was originally shown that Irgm1 is recruited to Mtb containing phagosomes in infected macrophages where it was proposed to be involved in autophagy-mediated clearance of Mtb. However, we have previously discovered that autophagy is not required in macrophages to control Mtb replication. In addition, it has since been reported that Irgm1 does not colocalize with phagosomes containing Mycobacterium bovis BCG in IFN-γ treated cells, suggesting that Irgm1 must have non-autophagic roles in TB infection that require detailed investigation. Irgm1−/− mice were shown to have higher levels of type I interferon signaling, as well as CD4+ T cell survival defect, which could explain the susceptibility to Mtb infection, but this has yet to be determined.
We infected Irgm1−/− mice with Mtb and found that they succumbed early to infection with higher bacterial burden, higher numbers of neutrophils, higher numbers of CD19+ B cells, and lower numbers of CD4+ T-lymphocytes in the lungs compared to wild-type (WT) mice. In addition, we found significantly increased expression of interferon-stimulated genes (ISGs) during Mtb infection of Irgm1−/− mice compared to WT. We also observed that T cell proliferation and lymph node responses were severely defective following Mtb infection in Irgm1−/− mice. Bone marrow chimera and transfer of Mtb-specific activated T lymphocytes into Irgm1−/− mice reverses the susceptibility to Mtb infection, indicating that a defect in T cell responses contributes to the susceptibility of Irgm1−/− mice to Mtb infection.
This work was supported by Stephen I. Morse fellowship to Sumanta Kumar Naik and the Project supported by NIH AI142784 and AI132697 and BWF PATH Award to Dr. Christina Stallings.
Dendritic epidermal T cells (DETC) are not required for control of skin Staph infections
Authors: Garrett-Sinha Lee Ann , Battaglia Michael , Sunshine Alex , Luo Wei , Miller Lloyd , Sinha Satrajit , Wohlfert Elizabeth 
Published: August 2023

Abstract
Staphylococcal bacteria are a common cause of skin infections in mice and humans. γδ T cells and the cytokine IL-17 are important in controlling these infections. To test roles for IL-17 in autoimmune Ets1 knockout (KO) mice, we generated double knockout (DKO) mice lacking both Ets1 and IL-17RA. We found that DKO mice develop worse autoimmune disease than Ets1 KO mice and are also susceptible to spontaneous Staphylococcus xylosus skin infections. While IL-17RA KO mice also develop skin lesions, these lesions are found at a higher rate and increased severity in DKO mice. Furthermore, exogenous infection of DKO skin with bioluminescent Staphylococcus aureus results in a failure to clear the bacteria, while IL-17RA KO mice are able to clear. Given that γδ T cells are important in controlling Staph skin infections, we examined these populations and found that both Ets1 KO and DKO mice lack the dendritic epidermal T cells (DETC), while other γδ T cell subsets are present. Further analysis showed that DETC progenitors are present in the fetal thymus of Ets1 KO mice, but DETC are missing from the skin of P5 and adult mice lacking Ets1. We hypothesized that loss of DETC (due to absence of Ets1) might cooperate with loss of IL-17 signaling (due to loss of IL-17RA) to create susceptibility to skin infections. To determine if DETC have a specific contribution to the skin immune response to Staphylococcal bacteria, we depleted DETC from wild-type mice using antibody against TCR Vγ5 followed by S. aureus infection. We found that DETC are not required to clear skin Staph infections, since depleted mice cleared with the same kinetics as non-depleted mice. Studies are in progress to further examine the roles of other γδ T cell populations in clearance of skin Staph infections.
Supported by grant from NIH (R01 AI122720)
Suppression of adaptive immunity by Borrelia burgdorferi in the murine host
Authors: Williams Megan T , Zhang Yan , Kilgore Rebecca , Berg Rance , Allen Michael 
Published: August 2023

Abstract
Borrelia burgdorferi (Bb), the spirochetal agent of Lyme disease, utilizes a variety of strategies to evade and suppress the host immune response, which enable it to persist chronically. These strategies include complement inhibition, antigenic variation, and suppression of adaptive immunity. Previous studies have shown that lymph node (LN) germinal centers generated in response to Bb collapse one-month post-infection. The resulting immune response is characterized by unusually strong IgM production and a lack of long-lived immunity. Here we aimed to better characterize how Bb manipulates host immunity, ultimately resulting in failure to clear the infection. Mice infected with Bb and concomitantly immunized with recombinant SARS-CoV-2 spike protein had an abrogated antibody response to the immunization. To test how long this humoral immune suppression lasts, mice were immunized at 2-, 4-, and 6-weeks post Bb infection. Suppression of host antibody production against SARS-CoV-2 spike protein peaked at 2 weeks post Bb infection but continued for all timepoints measured. We also examined changes to gene expression in murine LNs 15 days post-infection using RT-qPCR and found that live Bb, but not heat-killed Bb, broadly suppressed many genes related to T cell homing and function. Genes which play a critical role in the establishment and maintenance of T cell zones within the LNs, Ccl19 and Ccr7, were significantly downregulated. In addition, both Il2 and Il2ra were downregulated, which is typically observed during the resolution of infection and indicates a potential lack of antigenic restimulation. These data suggest Bb may disrupt T cell responses, which could play a role in the failure to mount an appropriate humoral immune response.
Funding for this work was provided by UNTHSC.
Multiparameter cellular and humoral immunoprofiling to differentiate progressive from nonprogressive non-tuberculous mycobacterial lung disease (NTM-LD)
Authors: Pathakumari Balaji , Marty Paige , Vadiyala Mounika Reddy , Cox Thomas , Van Keulen Virginia , Shah Maleeha , Peikert Tobias , Patricio Escalante 
Published: August 2023

Abstract

Background
Biomarkers that predict the patients most likely to develop progressive NTM-LD are urgently needed. We hypothesize flow cytometric (FC) detection of T cell markers after ex vivo antigen challenge in PBMC can differentiate patients with progressive vs. nonprogressive NTM-LD. We also measured the serum anti-glycopeptidolipid core IgA antibody by commercial ELISA kit and compared with FC immunoprofiling results.

Methods
PBMCs were isolated from patients with NTM-LD and stimulated with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides). We have performed ELISA and FC T-cell phenotypic assays to measure diagnostic accuracy of these assays by ROC and non-parametric statistics analysis.

Results
A total of 29 NTM-LD patients were studied and classified into 15 non-progressive and 14 progressive NTM-LD based on radiological and clinical evaluation. Patients with progressive NTM-LD showed a statistically significantly higher frequency of PPD-specific CD3+/CD8+ IFN-γ+HLA-DR+ subsets and high diagnostic accuracy compared with non-progressors: Sensitivity = 80% specificity = 71.4% with the ROC of 0.72. There were no other statistically significant differences in other antigen-stimulated T-cell subsets but a trend was observed with HLA-DR+TNF-α+, CD25+CD134+ and CD25+PDL1+ T-cell subsets in progressors. The anti-GPL IgA levels did not show a significant difference and might not be useful in distinguishing progressive from non-progressive NTM-LD.

Conclusion
Our study findings suggest that PPD-specific CD8+ IFN-γ+HLA-DR+ can distinguish progressive and nonprogressive NTM-LD patients, which can identify those patients at risk of disease progression.
CD301b+ DCs orchestrate type 2 immune responses to helminth infection in the lung
Authors: Kumamoto Yosuke , Davila-Pagan Alejandro , El-Fenej Jihad , Chen Fei , El-Naccache Darine , Gause William C 
Published: August 2023

Abstract
Helminth parasites induce potent host type 2 immune responses (T2I) that promote parasite clearance and prevent reinfection. The parasite-induced T2I involve both innate and adaptive immune cell components, but how the activation of different cell types is coordinated during the initiation of T2I is incompletely understood. Dendritic cells (DCs) are best known for their role in bridging innate and adaptive immunity by sensing immunological insults and presenting antigens to T cells. We previously found that CD301b+DCs, a major subset of migratory type 2 conventional DCs in peripheral organs, are specifically required for the differentiation of T helper type 2 (Th2) cells in the skin- and lung-draining lymph nodes following infection with Nippostrongylus brasiliensis (Nb) in mice, but their overall role in host immune responses against Nb infection remains unclear. Here, we show that the depletion of CD301b+DCs results in reduced T2I in the lung and impaired worm clearance. The reduction in type 2 cytokine production and eosinophil influx was observed as early as 2 days post-infection before the onset of adaptive immunity, as well as in CD4T cell-depleted mice, suggesting that CD301b+DCs play a critical role in the induction of innate T2I independently of their role in Th2 cell priming. Moreover, CD301b+DC-depleted mice exhibited ameliorated lung hemorrhage with reduced neutrophil infiltration and IL-17 production early after infection. These data suggest the role of CD301b+DCs in co-regulating innate type 2 and type 3 inflammation mechanisms during the early phase of Nb infection in the lung.
This work was supported by NIH grant R01AI132576.
AIDμS double knockout mice but not AID single knockout mice are highly susceptible to Pneumocystis Infection
Authors: Curran Shelly , Bishop Lisa , Kovacs Joseph 
Published: August 2023

Abstract
Pneumocystis pneumonia can be a life-threatening disease to individuals who are immunocompromised due to infection, such as HIV, receiving immunotherapy drugs including corticosteroids or due to genetic defect such as hyper IgM syndrome. B cells are important to the defense and clearance of Pneumocystis infection; however, the exact role has yet to be elucidated. Prior studies have demonstrated that B cell antigen presentation and priming of CD4+ T cells plays a major role in Pneumocystis clearance, while passive transfer of immune serum can prevent Pneumocystis infection in mice. To better understand the B cell role in control and clearance of Pneumocystis infection, using a natural exposure model, we compared the kinetics of Pneumocystis murina infection in activation-induced cytidine deaminase (AID−/−), AIDμS−/− double knockout, and wild-type C57BL/6 mice. By 5 – 6 weeks post initial exposure all three murine strains were infected with Pneumocystis murina, detected by qPCR of the single copy gene dihydrofolate reductase (DHFR). However, after 10 weeks of infection only the AIDμS−/− double knockout mice continued to have high levels of Pneumocystis infection. These data suggests that deficiency of B cells to class switch, such as in the AID−/− strain, is not in itself enough to confer increased susceptibility to Pneumocystis. However, in combination with μS deficiency, which inhibits the secretion of antibodies, it is enough to debilitate the immune response resulting in uncontrolled Pneumocystis infection.
Intramural Research Program of the NIH Clinical Center
Failed induction of lncRNA NeST underlies deficient IFN-γ production by CD8 T cells in sepsis survivors
Authors: Crispin Jose C , Cisneros-Segura Jose Alejandro , Albarrán-Godínez Adrián , García-González H Benjamín , Rodríguez-Rodríguez Noé , Madera-Salcedo Iris Karina , Valdés-Ferrer Sergio Iván , Rosetti Florencia 
Published: August 2023

Abstract
Sepsis is a complex condition of inflammatory and immune dysregulation, triggered by severe infection. In survivors, chronic inflammation and immune dysregulation linger, facilitating the emergence of viral infections. CD8 dysfunction contributes to this immunosuppression. We devised an animal model that enabled us to identify which sepsis-induced defects were intrinsic to CD8 T cells and we dissected an underlying mechanism.
We adoptively transferred CD45.1 CD8 OT-I T cells into CD45.2 congenic mice and subjected them to cecal ligature and puncture, to induce abdominal sepsis. In this model, survivors develop a chronic inflammatory response. One month later, we isolated the transferred CD8 cells. Surface marker expression confirmed they had not been activated through their TCR. CD8 OT-I T cells isolated from septic (or sham operated) mice were then transferred to second recipients which were challenged with ovalbumin-expressing L. monocytogenes (LM-OVA). We compared effector capacities between OT-I cells exposed to chronic inflammation and control cells.
Mice who received OT-I cells exposed to sepsis had higher bacterial burden and a shorter survival when challenged with LM-OVA. OT-I cells isolated from septic mice produced less IFN-γ, but had conserved activation, proliferation, and cytotoxic function. The IFN-γ producing defect was established at the transcriptional level. We evaluated chromatin accessibility and found that, contrary to control cells, cells exposed to sepsis had diminished local accessibility. This was associated with absent expression of the lncRNA NeST, a local regulator of epigenetic landscape. These results identify a mechanism through which chronic inflammation affects CD8 T cell function.
This work was funded by the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico.
Loss of FOXP3+ CD25+ Tregs is independent of type I interferon signaling in lupus-prone mice
Authors: Nguyen Ngoc Thuy , Valencia Julio Cesar , Ramba Meredith , Young Howard A 
Published: August 2023

Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by deposition of autoantibodies that leads to tissue damage and inflammation. Our lab has generated the ARE mice strain that has low, chronic levels of IFNg and lupus-like disease. Regulatory T cells (Tregs) play an important role in preventing autoimmunity by suppressing T cell activation, making them an important therapeutic target. In addition, type I interferon (IFN) has also been broadly implicated in SLE but its specific effect on Tregs remain unknown. To address the impact of a type I IFN signature on the dynamics of regulatory T cells (Tregs), we investigated marker expression on Tregs in type I interferon receptor (IFNAR)-deficient ARE mice.
Single cell suspensions harvested from spleens and kidneys of ARE−/−, IFNAR−/−, and IFNAR−/−/ARE−/− female mice between 150 to 200 days old were stained with a panel of markers associated with Treg function for analysis by flow cytometry.
In the spleen, the proportion of CD4+ FOXP3+ expressing CD25 (IL2Ra) decreased in ARE−/− and IFNAR−/−/ARE−/−, but not IFNAR−/−, compared to wild type (wt). Thus, the disease progression in ARE mice may be due to the loss of bona fide Treg population (FOXP3+ CD25+), as IL-2 contributes to Tregs’ expansion and function. Furthermore, the kidneys of ARE−/− and IFNAR−/−/ARE−/− experienced increased CD4 infiltration as well as a similar decrease in FOX3+ CD25+ Tregs, which might explain the increased inflammation of the kidneys.
IFNg-mediated chronic inflammation appears to downregulate the population of FOXP3+ CD25+ Tregs, independent of type I IFN signaling. Future experiments aim to perform in vitro assays to compare the suppressive ability of Tregs with and without CD25.
TAM receptor tyrosine kinase MERTK as a therapeutic target in the tumor immune microenvironment in a murine Kras mutant lung cancer model
Authors: Yan Dan , Wang Xiaodong , Frye Stephen , Earp H Shelton , DeRyckere Deborah , Graham Douglas K 
Published: August 2023

Abstract
TAM receptor tyrosine kinase MERTK is involved in apoptotic cell clearance to maintain tissue homeostasis. During efferocytosis, MERTK activation also promotes an immunosuppressive phenotype, which can be hijacked by tumor cells to escape immune attacking. Here, we determined the impact of MERTK inhibition on immune cell subsets in the tumor microenvironment in mice with subcutaneous murine CMT167 (Kras-G12V) lung cancer grafts. Although MERTK expression was not detected on tumor associated dendritic cells (DCs), there was a significant increase in the incidence of DCs in tumors from Mertk knockout (KO) mice compared to wild type mice by day 5 after inoculation of tumor cells. Characterization of tumor-associated macrophages by flow cytometry revealed three distinct populations expressing different levels of MHCII (CD11b+MHCIIhigh, CD11b+MHCII+ and CD11bhighMHCIIneg). Tumors from Mertk KO mice had significantly greater numbers of MHCIIhigh macrophages by day 10 after tumor cell inoculation and reduced numbers of MHCIIneg macrophages by day 19. Together, the increased incidence of dendritic cells and the shift toward a more antigen-presenting macrophage phenotype are expected to promote anti-tumor immunity in Mertk KO mice. Indeed, CMT167 tumor growth was significantly reduced in Mertk KO mice and this effect was abrogated in Mertk KO scid mice, which lack functional B and T cells. Treatment with MRX-2843, a MERTK-selective inhibitor currently in Phase 1 clinical trials, also reduced tumor growth in mice. Together, these data demonstrate a critical role for MERTK as a mediator of tumor progression in the tumor immune microenvironment and implicate MRX-2843 as an effective strategy to exploit this finding in the clinic.
The research was supported by the National Cancer Institute of the National Institutes of Health (Emory Lung Cancer Spore, Award Number P50CA217691). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
MAIT cell-mediated inhibition of colorectal tumor growth is associated with increase in IL-5 and accumulation of eosinophils in the tumor microenvironment
Authors: Cheng Olivia J , Lebish Eric J , Jensen Owen , Jacenik Damian , Trivedi Shubhanshi , Beswick Ellen J , Leung Daniel T 
Published: August 2023

Abstract
Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes with cytotoxic and pro-inflammatory function and are enriched in mucosal tissues such as the colon. With studies demonstrating both pro- and anti-tumor capability in various cancer models, the role of MAIT cells in colorectal cancer remains unclear. Our study uses a mouse model of colorectal cancer to show that adoptive transfer of MAIT cells from C57BL/6 donors into MC38-bearing RAG1−/− mice leads to a decrease in tumor growth compared to sham-transfer control. Using qRT-PCR and multiplex cytokine analysis, we show that extracted tumors from MAIT adoptive transfer group have a higher expression of eosinophil markers, Siglecf and Ccr3, as well as higher levels of eosinophil-activating cytokines, such as EOTAXIN-1, IL-5, GM-CSF, suggesting an association between tumor inhibition and eosinophil prevalence and activation. Consistent with this, flow cytometry analysis of human peripheral leukocytes shows increases in IL-5 expression in MAIT cells and activation of eosinophils upon stimulation with MAIT ligand, 5-A-RU, suggesting that MAIT cell activation could modulate eosinophil function. Together, these data suggest that the anti-tumor effect of MAIT cells may involve IL-5- and eosinophil-dependent mechanisms. This study provides evidence of the ability of MAIT cells to modulate the tumor microenvironment and immune cell profile, specifically the potential to recruit and activate eosinophils.
BAFF and APRIL signaling through BCMA regulates microglial cells in the central nervous system
Authors: Kumar Gaurav , Massey Kaylea , Ko Rose M , Axtell Robert C 
Published: August 2023

Abstract
B cell Maturation Antigen (BCMA) is a receptor that engages the cytokines BAFF and APRIL. BCMA is known to play a critical role in regulating B cell proliferation, survival, and differentiation into plasma cells. However, the effect of BCMA on non-B cells has been understudied. We have previously shown that BCMA regulates EAE, where BCMA−/− mice have exacerbated disease compared to BCMA+/+ mice. The increased disease in the BCMA−/− mice was associated with increased infiltration of B cells, T cells and inflammatory macrophages in the central nervous system (CNS). In this study, we assessed the expression patterns of BCMA on different immune cell types infiltrating the CNS of mice with EAE by flow cytometry. We found BCMA is expressed on plasma cells, microglia and inflammatory macrophages that are in the CNS, but not T cells or neutrophils. Next, we compared the numbers and phenotype of different myeloid cells in the CNS and lymph nodes in healthy BCMA−/− and BCMA+/+ mice. We found that BCMA−/− mice have an increased number of resident microglial cells with low CD86 expression in the brain compared to BCMA+/+ mice. Also, we found elevated numbers of macrophages with M1 (measured by the expression of iNOS) phenotype in the brain of BCMA−/− mice compared to BCMA+/+ mice. In lymph nodes, we found an increased number of M1 type macrophages in the BCMA−/− in comparison to BCMA+/+ mice with low CD86 expression. These data suggest a novel role for BCMA in regulating the phenotype and function of microglial and macrophages in CNS and in the peripheral immune tissues. Our data points towards a new pathway regulated by BAFF and APRIL signaling through BCMA and provides clues behind the immunopathogenesis of neuroautoimmune diseases.
This study was supported by grants from the NMSS (RG-1602-07722), the NIH (R01AI137047 and R01EY027346) awarded to R.C. Axtell.