bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptomics approaches reveal neuronal antiviral immune mechanisms
Authors: Negatu Seble G , Vazquez Christine , Bannerman Carl , Saldana Xoshill , Jurado Kellie Ann
Published: August 2023

Arthropod-borne viruses exist worldwide and contribute to many neurological infections. Viral infections in the central nervous system (CNS) can lead to virus- and immune-mediated damage of the brain, which can cause severe physical and cognitive defects, or even death. While antiviral responses (such as Type I interferon) have been characterized in the periphery, it is not well understood how innate immune response are coordinated by the various cell types present in the brain. Certain neurotropic arboviruses, such as La Crosse Virus (LACV), predominantly infect neurons; however, it remains unclear how neurons mediate early innate immune responses in the CNS. Upon infection of primary murine cortical neurons and human IPSC-derived cerebral organoids with LACV, we observed widespread infection. Transcriptomics approaches revealed infection of neurons results in the upregulation of interferon-stimulated genes (ISGs) that are also detectable at the protein level. Notably, we found that bystander uninfected neurons have higher ISG expression than neighboring infected cells. Future directions aim to characterize how infected neurons communicate with uninfected bystander neurons during infection. To address this, I have optimized a ProbeSeq pipeline where I infect primary cortical neurons and further characterize infected and uninfected populations using fluorescence in situ hybridization and downstream RNA-sequencing.

Supported by the Penn Presidential PhD Fellowship.
Neuregulin-1 reduces vascular leak and airway epithelium permeability caused by respiratory viral infection
Authors: Hussain Syed-Rehan A , Santoro Jennifer , Rohlfing Michelle , Peeples Mark E , Grayson Mitchell H
Published: August 2023

Respiratory viruses, such as Respiratory Syncytial Virus (RSV), are associated with significant morbidity and mortality. Mice with pre-existing atopy are protected from mortality to Sendai virus (SeV), a rodent virus related to RSV. We found that lungs of atopic mice had increased levels of neuregulin-1(NRG1), and hypothesized that SeV induces death via lung vascular leak, and that NRG1 might inhibit vascular leakage. Non-atopic C57Bl/6 mice were treated with NRG1 (500ng/30 mcl, i.n) or PBS for five days before infection with SeV (2×105 pfu) or UV-inactivated SeV (UV-SeV) and 8 days post viral inoculation, Evan’s Blue Dye (EBD) was injected i.v with EBD concentration determined spectrophotometrically one hour later in homogenized lungs. Vascular leak in SeV-infected mice was 65.25% and 46.62% more than in UV-SeV-treated or SeV-infected mice pre-treated with NRG1, respectively. We also found that RSV infection of well-differentiated human bronchial epithelial cells (hBEC) grown at the air-liquid interface caused 70KDa FITC-Dextran leakage 8 days post-inoculation. NRG1 pretreatment for 5 days before RSV inoculation reduced epithelial leakage 8 days post-inoculation by 2.5 fold (p<0.001). These results suggest NRG1 has the potential to improve epithelial barrier function in rodent and human systems and may explain the increased survival following a severe respiratory viral infection.

Abigail Wexner Research Institute at Nationwide Children’s Hospital, Start-up fund to MHG
Omicron Breakthrough Infection Elicits Superior Humoral and Mucosal Immunity to SARS-CoV-2 Variants than Booster Doses
Authors: Nantel Sabryna , Sheikh-Mohamed Salma , Chao Gary , Hu Queenie , Kurtesi Alexandra , Wood Heidi , Colwill Karen , Seifried Laurie , Bourdin Benoîte , Brockman Mark , Piccirillo Ciriaco , Quach Caroline , Gingras Anne-Claude , Gommerman Jennifer , Decaluwe Hélène
Published: August 2023

In light of ongoing booster vaccination campaigns, one recurring question is whether individuals infected with SARS-CoV-2 should still receive a booster dose if they were infected after completing their primary series. We aimed to assess the intensity of functional humoral and cellular immune responses induced by breakthrough infections during the B.1.1.529 wave in Canada compared to individuals who received three doses of vaccine. Because hybrid immunity has been shown to induce particularly potent immunity, we hypothesized that breakthrough infections could induce superior immune response to variants than booster doses.

To address this important public health question, we analyzed samples from two cohorts of individuals naïve to SARS-CoV-2 or who were primary infected before vaccination. We compared responses 45 days after a third vaccine dose or an Omicron breakthrough infection. Our results show that mild breakthrough infection induces cell-mediated immune response to variants comparable to a vaccine booster dose. In contrast, breakthrough subjects had higher salivary IgG and IgA levels against the Omicron Spike and enhanced reactivity to the ancestral Spike for the IgA isotype, which also reacted with SARS-CoV-1. Serum neutralizing antibody levels against the ancestral strain and the variants were also higher after breakthrough infection, especially in individuals naïve to the infection prior to vaccination. Altogether our results demonstrate that an Omicron breakthrough infection can substitute a booster dose of vaccine, while also supporting the need for mucosal vaccine boosts to potentially replicate the improvement on mucosal and humoral immunity without the risks associated to being infected with SARS-CoV-2.

Research supported by grants from the Canadian Institutes of Health Research, Public Health Agency of Canada and the Ontario COVID-19 Rapid Research Fund through Ontario Together.
Radiation causes long term changes in virus-specific and virtual memory CD4 T cells
Authors: Kannan Shravan Kumar , Heidarian Mohammad , Swanson Whitney , Griffith Thomas S , Harty John T , Badovinac Vladimir P
Published: August 2023

Threat of nuclear attack and accidents at nuclear power plants increase our risk of radiation exposure. Radiation effects on immune homeostasis are well-studied, but their effect on memory CD4 T cells is poorly defined. To explore that, we adoptively transferred naïve Thy1.1 TCR-Tg SMARTA CD4 T cells specific for LCMV-derived gp61 epitope into naïve Thy1.2 C57BL/6 mice before LCMV-Armstrong infection. At memory time point, infected recipient mice were subjected to 0Gy (non-radiated, control group) or 5Gy of total body radiation. We found that naïve CD4 T cells suffered significant numerical loss post radiation, but they numerically recovered in few weeks through increased proliferative expansion. However, radiated SMARTA and endogenous GP61-specific memory CD4 T cells sustained a prolonged loss in number that fail to recover even after 3 months. Radiation also leads to transient impairment in the ability of memory CD4 T cells to produce cytokines (IFNγ, TNFα) in response to cognate antigen stimulation, or to proliferate upon adoptive transfer into new hosts in response to secondary LCMV infection. Interestingly, the endogenous ‘effector-like’ CD4 T cell numbers recovered faster than SMARTA memory T cells. We hypothesized that virtual memory T cells (CD4 Tvm) are contributing to the recovery of this effector like CD4 T cells in the radiation mediated lymphoreplete conditions. Tetramer enrichment of CD4 T cells specific to the gp66 and 2W1S epitope showed that radiation increases the representation of gp66 specific CD4 Tvm in naïve mice. Together, these data suggest that radiation poses long-lasting defects to virus specific memory CD4 T cell responses, and it facilitates CD4 Tvm proliferation to overcome the lymphopenia.
Disruption of IL-33-mediated costimulation to CD4+ T cells leads to peripheral Treg generation and resolution of T cell-mediated pathology
Authors: Dwyer Gaelen K , Mathews Lisa , Lee Amanda H , Saba Johnny Bou , Beeravolu Harshita , Villegas Vazquez Jose A , Poholek Amanda , Blazar Bruce R , Luther Sanjiv A , Shlomchik Warren D , Turnquist Heth Roderick
Published: August 2023

Stromal signals are becoming appreciated as critical coordinators of immunity. We recently demonstrated that fibroblastic reticular cell (FRC)-derived IL-33 directly stimulates CD4+ T cells during priming to promote IL-12-independent Type 1 T helper cell (Th1) differentiation and expansion. Utilizing TCR transgenic (Tg) T cells lacking the IL-33 receptor, ST2, we established that IL-33 is a costimulatory molecule augmenting TCR signaling to low affinity or concentration antigens. Blockade of costimulatory molecules can lead to peripheral regulatory T cell (pTreg) induction and our RNAseq analysis suggested that the absence of early IL-33 stimulation increased CD4+ T cell expression of IL-10 and Foxp3. Thus, we hypothesized that disruption of the ST2/IL-33 pathway in CD4+ T cells would mediate pTreg generation and the resolution of T cell-mediated pathology. To test this hypothesis, we compared Tg B6 mice allowing inducible ST2 deletion (R26-CreERT2xSt2fl/fl (ST2FL) to wildtype (WT) R26-CreERT2 (ST2WT) as T cell donors in a CD4-driven, lethal Graft-vs-Host Disease (GVHD) model. Irradiated BALB/c recipients received WT CD45.1+ bone marrow and either ST2WT or ST2FL. Recipient mice received tamoxifen (TAM) on days 10–14 post-transplant. Assessment of CD45.1−CD45.2+Kd−CD4+ T cells at day 21 revealed robust ST2 deletion associated with increased Foxp3+ expression in effector populations (CD4+CD44+CD69+CD62L−CCR7− and CD44+CD69−) in the lymphoid organs and tissues. TAM-treated recipients of ST2FL T cells had improved GVHD clinical scores and weight gain compared to ST2WT. Thus, IL-33 is a novel and potent stromal cell-derived costimulatory signal whose targeting may help resolve active CD4+ Th1 cell-mediated pathology.

Supported by grants from NIH R01AR073527, R01HL22489, and R56AI139327 (to HRT); and T32CA082084 and F30AI147437 (to GKD).
PAXBP1 SERVES AS A NOVEL REGULATOR OF T HELPER SPECIFICATION BY RESTRAINING THE TYPE 1 PROGRAM
Authors: Chisolm Danielle Alexandria , Morimoto Yuki , Philips Rachael Laura , Nagashima Hiroyuki , Meremikwu Ijeoma , Meylan Francoise , Yao Chen , Chaitankar Vijender , O’Shea John J , Kanno Yuka
Published: August 2023

To properly extinguish a diverse number of pathogens naïve T cells speciate into several T helper subsets in a highly regulated manner. In order to identify novel regulators of T helper specification we explored single cell RNA-seq datasets of T cells exposed to several different challenges. From this analysis, we found Paxbp1, a poorly described nuclear factor, was highly expressed in Th2 cells with its expression correlating well with Gata3. To understand the role Paxbp1 plays in T helper differentiation we generated a mouse model where Paxbp1 was deleted in T cells using a CD4cre model. Analysis from scRNA-seq reveal that Paxbp1 deficiency leads to a decrease in mature naïve T cells in both the thymus and periphery. Moreover, loss of Paxbp1 results in an aberrant expression of a Type I program, despite being exposed to a Type 2 environment in a reductionist approach; specifically, Paxbp1 deficiency results in the expression of what appears to be a terminal cytotoxic program. During in vivo challenges, T cells from Paxbp1 deficient animals recapitulated these results showing an increase in expression of the Type 1 program. Current work is aimed at determining the function of Paxbp1 by identifying binding partners through mass spectrometry. Additionally, we will use scATAC-seq to define transcription factors with differential activity in Paxbp1 deficient T cells. Altogether, our data suggests that Paxbp1 may be a previously unrecognized player in T helper specification and development that constrains inappropriate expression and accessibility of programs employed by alternative lymphocyte lineages.
CD9 identifies and regulates a highly functional and unique subset of T follicular helper (Tfh) cells necessary for the promotion of antibodies during infection
Authors: Weinstein Jason D , Antao Olivia , Sanchez Gina Marie , Petersen Kyleigh , Francis Rebecca , Mayer Daniel
Published: August 2023

T follicular helper (Tfh) cells comprise a subset of CD4+ T helper (Th) cells phenotypically and functionally distinct from Th1 and Th2 cell subsets. Tfh cells are located within B cell follicles and germinal centers (GC), where they secrete the cytokines IL-21 and interferon-γ (IFN-γ) which aid in B cell proliferation and differentiation. CD9, a tetraspanin, has been shown to accumulate on CD4 T cells at the immunological synapse, playing a role in co-stimulation and stabilization of the synapse via regulation of the integrins LFA-1 and VLA-4. CD9 has been shown to be expressed on activated CD4 T cells, but whether it remains upregulated specifically on Tfh cells is unknown. We identified a distinct population of CD9-expressing Tfh cells (CD9hi) following acute viral infections and examined their role in promoting GC responses. Following acute LCMV infection, CD9hi Tfh cells are found in the follicles and GCs, yet exhibited enhanced proliferation and migratory capacity to the chemokines CXCL12 and CXCL13. Epigenetically, CD9hi Tfh cells demonstrated a distinct transcriptional profile and chromatin architecture from their CD9lo counterpart, with an upregulation of cell cycling, PI3K, and integrin-regulated genes. Functionally, CD9hi Tfh cells had elevated VLA-4 expression and were the predominant producers of IL-21 and IFN-γ, indicating a specific role in B cell maturation and differentiation. Furthermore, deletion of CD9 did not alter Tfh cell development, but lead to their reduction of IL-21 and IFN-γ production and a decrease in overall antibody responses. Therefore, we identified that CD9hi Tfh cells are a highly functional and unique subset necessary for the promotion of antibodies during infection.

R01 AR073912
Regulation of plasmacytoid dendritic cells by ER stress-mediated metabolic responses
Authors: Chaudhary Vidyanath , Ah Kioon Marie Dominique , Hwang Sung-Min , Mishra Bikash , Lakin Kimberly , Kirou Kyriakos , Zhang-Sun Jeffrey , Wiseman Luke , Spiera Robert , Crow Mary K , Gordon Jessica , Cubillos-Ruiz Juan R , Barrat Franck J
Published: August 2023

Plasmacytoid dendritic cells (pDCs) chronically produce type I interferon (IFN-I) in autoimmune diseases, including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). We report that the IRE1α-XBP1 branch of the unfolded protein response (UPR) inhibits IFN-α production by TLR7- or TLR9-activated pDCs. In SSc patients, UPR gene expression was reduced in pDCs, which inversely correlated with IFN-I–stimulated gene expression. CXCL4, a chemokine highly secreted in SSc patients, downregulated IRE1α-XBP1–controlled genes and promoted IFN-α production by pDCs. Mechanistically, IRE1α-XBP1 activation rewired glycolysis to serine biosynthesis by inducing phosphoglycerate dehydrogenase (PHGDH) expression. This process reduced pyruvate access to the tricarboxylic acid (TCA) cycle and blunted mitochondrial ATP generation, which are essential for pDC IFN-I responses. Notably, PHGDH expression was reduced in pDCs from patients with SSc and SLE, and pharmacological blockade of TCA cycle reactions inhibited IFN-I responses in pDCs from these patients. Hence, modulating the IRE1α-XBP1–PHGDH axis may represent a hitherto unexplored strategy for alleviating chronic pDC activation in autoimmune disorders.
Intestinal lamina propria stromal cells function as the target of androgens and microbiota metabolites for the prevention of lupus
Authors: Ma Jing , Harder James W , Alard Pascale , Kosiewicz Michele M
Published: August 2023

The gut microbiota affects autoimmune diseases including lupus. We found that female and male microbiota in NZBxNZWF1 (BWF1) mice differ. Our goal is to understand the relationship between sex, microbiota and disease. Microbiota depletion in females has no impact on disease and female microbiota does not induce disease in males; male microbiota can protect female mice from disease while castrated male microbiota loses this ability (castration induces disease and alters the microbiota in males). Intestinal lamina propria stromal cells (LPSC) can affect microbiota and immunoregulation [via regulating CD103 dendritic cell (CD103DC) development and consequently, peripheral regulatory T cells (pTregs)]. CD103DC function and pTregs are both decreased in female and castrated male mice. Endogenous androgens directly induce genes, RALDH1 and 2 (i.e., retinoic acid production), TGFB and GM-CSF, in LPSC that are critical for CD103 development. Transferred male gut microbiota induce expression of the same genes in LPSC in female recipients that we determined was not due to androgens produced by the male microbiota, since no androgen response genes, ANGTL and CHRD, were induced in highly androgen-sensitive LPSC. The male microbiota metabolomic profile differs from either female or castrated male mice, and includes the RXR agonist metabolite(s), phytol/phytanic acid, that directly induce(s) LPSC to express RALDH1 and 2, TGFB and GM-CSF. We propose that 1) endogenous androgens regulate CD103DC function and microbiota function through the LPSC in males; (2) male-modified microbiota produces metabolite(s) that may mediate protection in female recipients through the LPSC and subsequent restoration of CD103DC function and pTregs.

Funded by a TIL grant from the Alliance for Lupus Research and the NIH (R01AR067188)
Galectin-3 contributes to pathogenesis of IgA nephropathy
Authors: Chen Yi-Liang , Ka Shuk-Man
Published: August 2023

IgA nephropathy (IgAN) is the most common type of autoimmune glomerulonephritis that frequently progresses to end-stage renal disease. However, the molecular pathogenesis underlying IgAN remains largely unknown. This study investigated the role of galectin-3 (Gal-3), a galactoside-binding protein in IgAN pathogenesis. Two complementary mouse IgAN models, a model induced with TEPC-15 hybridoma using Gal-3 knockout (KO) mice, and a spontaneous IgAN model of “grouped” ddY (gddY) mice were employed. Gal-3 expression increased with disease severity in the glomeruli, peri-glomerular regions, and some renal tubules in both the inducible and spontaneous IgAN models. Gal-3 KO in the TEPC-15 hybridoma-induced IgAN mice significantly improved proteinuria and renal function and reduced severity of renal pathology, including neutrophil infiltration and decreased differentiation of Th17 cells from renal-draining lymph nodes, despite increased percentages of regulatory T cells. Gal-3 KO also inhibited the NLRP3 inflammasome, yet it enhanced autophagy and improved renal inflammation and fibrosis. Moreover, administration of 6-de-O-sulfated, N-acetylated low-molecular-weight heparin, a competitive Gal-3 binding inhibitor, restored renal function and improved renal lesions in passive IgAN mice. These results suggest that Gal-3 is critically involved in IgAN pathogenesis by activating the NLRP3 inflammasome and promoting Th17 cell differentiation. Therefore, targeting Gal-3 action may represent a new therapeutic strategy for treatment of this renal disease.

Nil
An essential role of iron in Tfh cell development and effector function
Authors: Scindia Yogesh , Agarwal Annanya , Gong Minghao , Abboud Georges , Mehrad Borna , Morel Laurence
Published: August 2023

Background

T follicular helper CD4+ cells (Tfh) are essential for maintenance of systemic lupus erythematosus (SLE) as they help germinal center B cells generate high affinity auto-antibodies. The activation of STAT3 is critical for generation of Tfh cells as it promotes the transcription of BCL6 (the essential transcription factor for Tfh cell differentiation). It is recognized that activated, proliferating SLE CD4+ T cells, contain more iron than controls. Iron binds CDK1 to accentuate JAK1 phosphorylation and activation of STAT3 in proliferating cells. Whether increased iron content of SLE CD4+ T cells augments JAK1-STAT3 signaling and their differentiation into Tfh cells has not been investigated.

Experiments

RNAseq and qPCR was used to compare iron regulatory pathways in purified Tfh cells from WT and SLE-prone mice. Labile iron (bio-active) content was measured in freshly isolated Tfh cells. The effect of iron chelator on in vitro Tfh differentiation and effector function was evaluated.

Results

Compared to WT, SLE Tfh cells displayed significant increase in genes associated with iron uptake and transmembrane iron transport. Labile iron content and CDK1 gene expression were elevated in SLE Tfh cells. Iron chelation attenuated STAT3 activation, BCL6 expression and IL-21 production in in vitro generated Tfh cells.

Conclusion

Our novel data identify an essential role of iron in Tfh cell differentiation and effector function. Increased iron and CDK1 may amplify JAK1-STAT3 signaling and potentiate Tfh cell differentiation. This can be blocked by restricting free iron availability. Understanding this aspect of Tfh cell biology in SLE may support strategies that control labile iron as potential adjunct therapy.

Supported by grants from Vifor Pharma (P0213104, P0226952) to Yogesh Scindia NIH (R01AI135128) to Borna Mehrad NIH (R01AI128901) to Laurence Morel
IL-4 role in the development of the lupus mouse model induced by non-bilayer phospholipids arrangements
Authors: Baeza Carlos Wong , Reséndiz-Mora Albany , Barrera-Aveleida Giovanna , Sotelo-Rodríguez Anahi , Galarce-Sosa Iván , Isidro-Magallán Elías , Santiago-Hernández Juan Carlos , Wong-Baeza Isabel , Baeza Isabel
Published: August 2023

Non-bilayer phospholipids arrangements (NPAs) are lipid molecular associations that are different from the bilayer and are transient. However, if they become stable, they can trigger a disease in mice that is similar to human systemic lupus erythematosus. NPAs can be stabilized in liposomes formed by cylindrical and conical lipids or in cell membranes by drugs such as procainamide or chlorpromazine. In wild-type BALB/c mice, this lupus model has been developed by administering liposomes with NPAs induced and stabilized with procainamide or chlorpromazine, by administering the drugs alone or by administering the monoclonal antibody H308; these last two procedures stabilize the NPAs in the membranes of mouse cells. Anti-NPAs, anti-cardiolipin, anti-histones and anti-coagulants auto-antibodies have been detected in the serum of these mice; in addition, the mice present facial lesions and alterations in the skin and kidney. In this work, we evaluated the participation of the TH2 response, through IL-4, which is its most characteristic cytokine, in the development of the NPAs-induced lupus model in mice. Wild type or IL-4 knockout BALB/c mice were administered with liposomes bearing drug-induced NPAs, the drugs alone, or anti-NPA H308 antibody to induce the lupus-like disease. IL-4 KO mice showed minor disease manifestations, compared to wild-type mice, with decreased production of anti-NPA IgG antibodies, no anti-cardiolipin, anti-histones and anticoagulant antibodies, and no kidney or skin lesions. In these mice, H308 was the only inducer of anti-NPA IgG antibodies. These findings suggest that IL-4 has a key role in the development of the murine lupus-like disease induced by NPA stabilization.

This study was supported by grants from IPN (SIP20232128, SIP20232132 and SIP20232135).
TIPE proteins control directional migration of human T cells via GPCR and lipid second messenger signaling
Authors: Sun Honghong , Yu Jiyeon , Zamani Ali , Goldsmith Jason , Etwebi Zienab , Pear Warren S , Chen Youhai H
Published: August 2023

Infiltration of specific tissues by circulating leukocytes is a common pathogenic mechanism of inflammatory diseases. G-protein-coupled receptors (GPCR) are essential for sensing chemokine gradients by leukocytes during immune responses. However, how leukocytes integrate site-specific directional cues such as chemokine gradients and make directional decisions in human immune cells is largely unknown. The TNFa-induced protein 8-Like (TIPE or TNFAIP8) family proteins are newly described pilot proteins that control directional migration (chemotaxis) of leukocytes to targeted tissues in mice. Without TIPE proteins, leukocytes become directionless, but the underlying mechanism remains elusive. Using siRNA and biochemical methods, we demonstrated here that two TIPE family members, TNFAIP8 and TIPE2, synergistically controlled directional migration (chemotaxis) in human CD4 T cells: TNFAIP8, through interacting with the Ga subunit of heterotrimeric G-proteins, and TIPE2, through utilizing lipid second messenger signaling by binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3). Using site-directed mutagenesis, we identified the region of Ga that interacts with TNFAIP8, and the amino acids of TIPE2 protein crucial for its interaction with PIP2 and PIP3. We also discovered that TIPE protein membrane translocation was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm of human T cells integrating GPCR and phospholipid signaling pathways to control directional migration of human immune cells. Additionally, our findings have implications for therapeutic targeting of TIPE proteins in human inflammatory and autoimmune diseases.

NIH 5R01AI136945
Extravasation of pathogenic human type 17 Th cells requires both endothelial cell-bound and non-cell bound chemokines
Authors: Parween Farhat , Singh Satya , Zhang Hongwei , Kathuria Noshin , Farber Joshua
Published: August 2023

Extravasation of pro-inflammatory T cells is critical for their effectiveness in host defense and for mediating tissue damage in immune-mediated disease. These cells co-express multiple chemokine receptors and understanding the activities of individual receptors in extravasation is essential for exploiting the chemokine system for therapeutic benefit – either to block or enhance the trafficking of these cells into sites of inflammation. CCR6 is expressed on all human Th17 cells, and we found that CCR6+CCR2+ cells are enriched both in cells that express a pathogenic Th17 signature, including the ability to produce GM-CSF and IFNγ, and are unusually efficient at transendothelial migration (TEM). Ligands for CCR6, CCR5 and CXCR3 were secreted by and bound to endothelial cells activated with TNFα and IFNγ, and these receptors could all mediate firm arrest of CCR6+CCR2+ cells under flow - but could not mediate TEM. TEM required CCR2, whose ligands, CCL2, CCL7 and CCL8, were all secreted by activated endothelial cells but failed to bind to endothelial cell surfaces. Chemokines were secreted from the basal as well as the apical sides of endothelial cells. Pre-treating CCR6+CCR2+ cells with CCL2 or transducing endothelial cells to express a surface-bound CCL2-CXCL9 chimera led to both CCR2-mediated arrest and, importantly, inhibition of TEM. Taken together, our data suggest that while arrest requires endothelial surface-bound chemokine, TEM requires a transendothelial gradient maintained by the rapid dilution of non-bound luminal chemokine by flow. Consequently, mediating arrest versus TEM are not inherent, but nonetheless mutually exclusive activities of receptors that depend, respectively, on binding vs. non-binding chemokines.

NIH
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

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

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

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

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

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

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

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

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

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

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.