bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment with TLR7/8 agonist compromises intestinal epithelium integrity in a lupus prone mouse model.
Authors: Terrell Morgan Lee , Elshikha Ahmed Samir , Kanda Nathalie , Brown Josephine , Zeumer-Spataro Leilani , Morel Laurence
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies leading to chronic inflammation and subsequent organ damage. Both spontaneous and induced mouse models have been central to the dissection of disease mechanisms. Mounting evidence suggests that increased gut permeability, or leaky gut, and the resulting translocation of pathobionts or their metabolites can impact SLE pathogenesis. Resiquimod (R848), a TLR7/8 agonist has been shown to induce lupus-like autoimmunity in various non-autoimmune mouse strains. Here, we examined the effect of the epicutaneous administration of R848 in the B6.Sle1.Sle2.Sle3 triple congenic (TC) spontaneous mouse model of lupus as compared to their non-autoimmune B6 controls. R848 induced a much stronger autoimmune activation in pre-autoimmune TC mice than in B6 controls. Unexpectedly, R848 decreased gut barrier integrity in TC but not in B6 mice. Immunofluorescence staining of the ileum showed a reduced expression of the tight junction protein claudin-1 and an increased infiltration of immune cells in R848-treated TC as compared to either R848-treated B6 or untreated control mice. The reduced intestinal epithelial integrity was functionally confirmed with a FITC-dextran intestinal permeability assay. These results demonstrate for the first time that TLR7/8 activation, which plays an essential role in autoimmune activation, also induces a leaky gut in lupus-prone mice. Ongoing studies are expected to unveil the causal relationships between TLR7/8 activation, gut barrier integrity, and microbial dysbiosis in SLE.

Supported by R01 AI143313 from the NIH to LM
The TLR7 agonist imiquimod mediates immune activation in primary Sjögren’s syndrome
Authors: Punnanitinont Achamaporn , Kasperek Eileen M , Kramer Jill M
Published: January 2023

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by chronic inflammation of exocrine tissue, resulting in loss of tears and saliva. Patients also experience many extra-glandular disease manifestations. Treatment for pSS is palliative, and there are currently no treatments available that target disease etiology. Previous studies in our lab demonstrate that Myd88 is crucial for pSS pathogenesis in the NOD.B10Sn-H2b (NOD.B10) pSS mouse model, although the way in which Myd88-dependent pathways become activated in disease remains unknown. Based on its importance in other autoimmune diseases, we hypothesized that TLR7 activation accelerates pSS pathogenesis. We administered the TLR7 agonist Imiquimod (Imq) to pre-disease NOD.B10 females 3 times a week for 6 weeks (n = 8). Parallel experiments were performed in which age and sex-matched controls received a base cream lacking Imq (n = 9). Saliva was collected following pilocarpine injection before and after treatment. Animals were euthanized, spleens and cervical lymph nodes (cLNs) harvested, and flow cytometry was performed. Imq-treated animals exhibited lymphadenopathy, splenomegaly, and decreased salivary flow. Both cLNs and splenocytes from Imq-treated animals demonstrated an increase in the percentage of activated/memory T cells (CD4+, CD44+, CD62L−) and TLR7 expressing B cells (B220+, CD69+, TLR7+). The percentage of B and CD4+ T cells expressing TLR7 was increased in spleen and cLNs following Imq administration. Aged-associated B cells (ABCs) (B220+, CD23−, CD21−, T-bet+) were also expanded in the spleens of Imq-treated mice. In summary, activation of TLR7 mediates adaptive immune activation and promotes expansion of the ABC subset in pSS.

Supported by NIH/NIDCR R01DE29472
HLA Class II Polymorphisms Influence Gut Microbiota Composition and Modulate Disease in Transgenic Mice Model of Multiple Sclerosis
Authors: Shahi Shailesh K , Ali Soham , Lehman Peter , Ghimire Sudeep , Guseva Natalya V , Mangalam Ashutosh K
Published: January 2023

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with unknown etiology. The interaction of both genetic and environmental factors plays an important role in MS pathogenesis. Recently, gut microbiota has emerged as a potential environmental factor in MS pathology due to its ability to modulate host immune response. Yet, it is unknown whether genetic factors, such as HLA class II gene(s), can influence gut microbiota which then can modulate MS through regulation of the host immune response. In the present study, we investigated the involvement of gut microbiota in HLA class II genes mediated susceptibility and resistance to MS/experimental autoimmune encephalomyelitis-EAE (an animal model of MS). Previously, we showed that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to proteolipid protein-PLP91–110 induced EAE, whereas AE-KO.HLA-DQ8 transgenic mice were resistant to PLP91–110. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed increased EAE severity compared with HLA-DR3 mice. Distinct microbiota in HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 transgenic mice compared to AE-KO mice suggested an important role of HLA class-II gene in shaping the gut microbiota composition. We also observed that gut microbiota of HLA-DQ8 mice was more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of HLA-DQ8 on an HLA-DR3 background increases disease severity, our data suggest that HLA-DQ8 restricted microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota may contribute to EAE susceptibility or resistance in a transgenic animal model of MS.

The authors acknowledge funding from the National Multiple Sclerosis Society (RG 5138A1/1T), NIAID/NIH (1R01AI137075-01), a Carver Trust Medical Research Initiative Grant, and the University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH (P30 ES005605). SA was supported by the Emory Warner Fellowship, which provides medical students at the Carver College of Medicine the opportunity to take a full year out of their medical school curriculum to work in a laboratory in the University of Iowa Department of Pathology.
Risk factors for development of severe gastrointestinal dysmotility in patients with systemic sclerosis
Authors: Jakhar Ishaan , Singh Medha , Singh Annapoorna , Sahil Suman , Singh Daulath
Published: January 2023

Background

Systemic Sclerosis (SSc) is an autoimmune condition of unknown etiology. We aim to investigate incidence, characteristics, and risk factors for gastrointestinal (GI) dysmotility in patients diagnosed with SSc using Cerner HealthFacts, a national longitudinal database representing approximately 69 million patients.

Methods

All patients over 18 years of age with SSc were included in our analysis. Incidence of GI dysmotility was identified using ICD coding. Patient characteristics were evaluated as potential risk factors for development of GI dysmotility using multivariable regression built using all variables with p<0.1 on univariable analysis.

Results

Overall, 0.4% (58/13,930) of patients with SSc developed GI dysmotility. Mean age was 57.23±15.22 years and 16% (2,228/13,930) were male and 86.1% (11,502/13,930) were female. In terms of race, 70.15% (9,772/13,930) were white and 14.82% (2,064/13,930) were African American. 92.51% (12,886/13,930) received care at an acute care facility and 81.51% (11,354/13,930) received care at an urban facility. The variables which conferred the highest odds of developing GI dysmotility included presence of hepatic comorbidities (OR 2.35), digestive comorbidities (OR 6.8), muscular dystrophy (OR 569.2), and Raynaud (OR 1.98). In univariable analysis and multivariable regression with 95% CI; 4 variables were predictive for GI dysmotility, and none were protective against GI dysmotility.

Conclusions

In this longitudinal national database study of patient with diagnosed SSc - hepatic, digestive comorbidities, muscular dystrophy, and Raynaud were noted to be associated with high risk of developing GI dysmotility. Careful evaluation and follow up is advised for SSc patients.

Supported by None
Bi-allelic TTC21A mutations in common variable immunodeficiency patients
Authors: Zhang Zinan , Lenardo Michael J , Smith Kenneth G C
Published: January 2023

Very little is known about the gene TTC21A – it was first described as a gene in the AP20 sub-region of chr3p.21.3 in which deletions were associated with cancer. Initially, TTC21A mRNA expression was only found in the testis, consistent with a recent study linking homozygous TTC21A mutations to decreased fertility due to structural defects in sperm flagella. Interestingly, the gene is also expressed in B lymphocytes with several smaller predicted protein coding transcripts enriched particularly in naïve and memory B cells. Here we report four individuals of one kindred with common variable immunodeficiency (CVID) with two different sets of rare biallelic TTC21A variants. Based on whole genome sequencing analysis, TTC21A was the only candidate gene that co-segregated with disease across three generations and eight individuals of this family with an autosomal recessive mode of inheritance. The affected individuals had low serum IgG and a significant defect in class-switching. In-depth B cell immunophenotyping revealed that, relative to healthy family controls, the patients had significant skewing of increased transitional (CD27− IgD+ CD24+ CD38+) B cells and markedly reduced terminally differentiated plasmablasts (IgD− CD20low CD38+). The patients with the rare homozygous variant also had significantly reduced expression of the B cell specific TT21A variant relative to the family controls and patients with the compound heterozygous variants. Initial immunization experiments of Ttc21a CRISPR knock-in mice mirrored the patients’ phenotype with decreased class switching; however subsequent repeats have been inconclusive. Additional work is necessary to further elucidate the immune function of TTC21A in B cells.

Supported by the NIH Oxford-Cambridge MD/PhD Scholarship
T cell extrinsic mechanics explaining the T cell hyperactivation seen in Activated PI3K δ Syndrome
Authors: Bier Julia , Brink Robert , Tangye Stuart , Deenick Elissa K
Published: January 2023

Activated PI3K Delta Syndrome (APDS) is a rare condition caused by heterozygous gain of function mutations in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many surface receptors expressed by T cells and has been implicated in the control of CD4+ T cell helper differentiation, activation, and proliferation. APDS patients have increased PI3K activity leading to several immune manifestations with the some of the most common features being lymphoproliferation, respiratory tract infections, Th2-related pathologies, and impaired antibody responses.

We previously analysed a CRISPR/Cas9 mouse model (Pik3cdE1020K). This showed intrinsic changes impacting on T cell function (increased Th2 cytokines and poor Tfh function) but also extrinsic factors driving a decrease in frequency of naïve and a concurrent increase in memory T cells and Tfh cells, indicating T cell hyperactivation. Here we investigated these extrinsic components further. We found that PI3K B cells are driving Tfh differentiation. We also explored the role of PI3K in maintaining Treg function and identity and determined if Treg dysregulation was driving T cell hyperactivation.

These studies reveal an important role of PI3K in modulating T cell differentiation, activation, and function through both T cell intrinsic and extrinsic mechanisms. An improved understanding of T cell hyperactivation can provide insight into the lymphoproliferation that affects most APDS patients. Overall, these results reveal the potential to dissect the mechanisms underlying disease, providing grounds to define new drug targets and ultimately improve the clinical management of these patients.
22q11.2 Deletion Syndrome Causes a Thymus Hypoplasia Corrected by Mesenchymal Cell Replacement
Authors: van Oers Nicolai S , Bhalla Pratibha , Moses Angela , Kumar Ashwani , Xing Chao , Wysocki Christian , Cleaver Ondine , Markert M Louise , de la Morena M Teresa
Published: January 2023

Thymus hypoplasia occurs in several clinical conditions including 22q11.2 deletion syndrome (22q11.2DS). 22q11.2DS is the most common human microdeletion disorder, affecting 1/4000. Thymuses from 60–70% 22q11.2DS patients are small and produce fewer T cells than normal. For individuals with a severe hypoplasia, an allogenic thymus tissue graft is needed to restore thymopoiesis. To determine the molecular mechanisms contributing to a small thymus in 22q11.2DS, we compared the development of the thymus in embryos from various 22q11.DS mouse models, normal controls and Foxn1 mutant mice. Reaggregate fetal thymic organ culture assays reveal that replacing mesenchymal cells from 22q11.2del hypoplastic lobes with normal ones restores tissue expansion and thymopoiesis. Thymic epithelial cells used as substitutes cannot. This is distinct from the Foxn1 mutant mice, wherein defective thymic epithelial cell functions lead to thymus hypoplasia/aplasia. Single cell RNA sequencing of normal and hypoplastic thymus lobes revealed differential expression of transcripts that primarily impacted 5 distinct mesenchymal cell subsets in 22q11.2DS. These transcripts are involved in cell-cell interactions, collagen deposition and growth. Elevated levels of collagen are present in the hypoplastic thymus tissues, suggesting a structural restriction. Mesenchymal and epithelial cell differentiation/expansion assays reveal a selective reduction in mesenchymal tissue expansion due to 22q11.2DS

Supported by grants from the National Institutes of Health (R01AI114523, R21AI144140) and the Jeffrey Modell Foundation (MdlM, CAW)
Characterization of Human FOXN1 Mutations Uncovers both Loss- and Gain-of-Function Outcomes
Authors: van Oers Nicolai S , Moses Angela , Bhalla Pratibha , Wysocki Christian , Seroogy Christine , Markert M Louise , de la Morena M Teresa
Published: January 2023

Thymus hypoplasia is reported for individuals with autosomal recessive, compound heterozygous and single allelic FOXN1 mutations. FOXN1 is the master transcriptional regulator of thymus epithelial cell development. Autosomal recessive mutations in FOXN1 lead to a Nude/SCID phenotype due to the ensuing T cell lymphopenia and impaired hair follicle extrusion. Targeted exome and whole genome sequencing for patients with low TRECs (measure of T cell output) has increased the number of diverse FOXN1 mutations to over 40. The consequence of these FOXN1 mutations on patients is varied and somewhat complicated by some individuals having only a transient delay in T cell development that corrects over time. We compared the functions of the FOXN1 mutants with luciferase reporter assays and nuclear localization experiments. For selected FOXN1 mutations, mice were developed to genocopy these to assess impacts on thymopoiesis. We identify partial and complete loss-of-function mutations along with gain-of-function and dominant negatives. Comparative analyses of murine thymopoiesis reveal some compound het Foxn1 mutations cause a transient thymus hypoplasia while others cause a permanent small thymus. Taken together, our findings establish FOXN1 genotype-phenotype relationships and suggest rapid functional screening approaches can be used to define the impact of different mutations of clinical relevance.

Supported by grants from NIH (R01AI114523, R21AI144140) and the Jeffrey Modell Foundation (MdlM, CAW)
Unraveling the mechanism of sepsis-mediated multiple organ dysfunction syndrome by RNA sequencing analysis in the pediatric population
Authors: Bermudez Marie , Yuki Koichi , Koutsogiannaki Sophia
Published: January 2023

Sepsis is a significant healthcare burden, with high morbidities and mortalities and no specific therapy is yet available. More than 42,000 children develop severe sepsis each year in the U.S and 67% of those patients develop multiple organ dysfunction syndrome (MODS). In this study we aim to elucidate the mechanism of sepsis-mediated MODS by RNAseq analysis in PBMCs from the peripheral blood of pediatric patients. Comparison between control and septic patients revealed that transcriptomic signatures associated with receptor-mediated endocytosis (Iglc3, Igkv3d-20), anaphase-promoting complex (APC)-dependent catabolic process (Pttg1, Cdc20) heme-metabolism (Alas2, Slc25a39) and oxygen transport (hemoglobulin pathway) (Hbb, Hba2, Hbq1) were upregulated in sepsis. Heme/hemoglobulin related genes were downregulated in the recovery phase of those patients. In addition, neutrophil chemotaxis (CCL3L1, IL1B, CCL4L2, CCL3), phagocytosis (IGLL5, IGHG1, IGHG2), and interleukin-1 receptor binding (IL1RN, IL1B) pathways were upregulated in the recovery phase, while T cell activation (CD2, ITK, ZAP70, CD8A, TREML2) and differentiation (LCK, IL7R), and negative regulation of NIK/NF-kappaB signaling (SPAN6, CPNE1, ADIPOR1) pathways were downregulated. Many of those genes upregulated in the septic patients are associated with lower survival probability in the adult population, while genes upregulated in the recovery phase are associated with higher survival probability, based on the Biological Information Database of Sepsis (BIDOS). Given data on the pediatric population are scarce, our study reveals unique gene signatures of PBMCs associated with pediatric sepsis that could be novel targets for therapeutic intervention.

Supported by R21HD099194-01A1, NICHD The Anesthesia Research Distinguished Trailblazer Award, Boston Children’s Hospital
CEACAM1 is an IL-2R-dependent biomarker in patients with multiple autoimmune-diseases undergoing low-dose IL-2 therapy
Authors: Yu Aixin , Moro Alejandro , Rosenzwajg Michelle , Tchitchet Nicolas , Klatzmann David , Malek Thomas R
Published: January 2023

Low-dose (LD) IL-2 is a promising strategy to boost Tregs in patient with autoimmune diseases. This therapeutic strategy would benefit by identifying biomarkers that are highly and selectively responsive to IL-2R signaling in Treg and Teff cells. To initially address this point, RNA-seq was used to screen for genes that are induced in CD4+ Foxp3+ Treg and CD4+ CD45RO+ T effector/memory (TEM) cells from normal subjects in vitro by IL-2R, but not TCR/CD28, signaling. CEACAM1 was identified as a candidate gene in Tregs 4 and 24 hours post-stimulation. However, CEACAM1 was optimally upregulated in Treg and TEM cells after an extended 5 day culture with anti-CD3/CD28 and IL-2. Notably, anti-IL-2 essentially completely inhibited this upregulation, indicating that CEACAM1 expression was strictly IL-2 dependent. Tregs were shown to express the long isoform of CEACAM1 with inhibitory ITIM domains and the ITIMs may be functionally active as most in vitro activated CEACAM1+ Tregs did not express Ki67. To assess CEACAM1 as an IL-2-dependent biomarker during LD-IL-2 therapy, CD25 and CEACAM1 were evaluated at the protein and RNA levels for patient samples from eight distinct autoimmune diseases. In all samples, CEACAM1 was upregulated after a 5-day induction course of LD-IL-2 in Tregs but not TEM cells. This finding correlated with the effect of LD-IL-2 on CD25. CEACAM1 levels returned to baseline 14 days post-LD-IL-2 treatment. After the induction course of LD-IL-2, most CEACAM1+ Tregs were Ki67−, consisting with an inhibitory role for CEACAM1. Collectively, our findings indicate that CEACAM1 is a biomarker for LD-IL-2 therapy whose expression is highly dependent on IL-2R signaling and suggest that CEACAM1 may function to regulate Treg homeostasis.

Supported by R01AI131643
Bacillus anthracis peptidoglycan alters human M2-like macrophage phenotype and efferocytic function in the presence of human serum
Authors: Mytych Josh S , Pan Zijian , Lopez-Davis Charmaine , Lawrence Christina , James Judith , Popescu Narcis , Coggeshall Mark , Farris Darise
Published: January 2023

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen associated molecular pattern contributing to anthrax pathology including coagulopathy in late stage disease. Lymphocyte apoptosis is an established phenomenon in bacterial sepsis, including fulminant anthrax. Clearance of apoptotic cells (efferocytosis) is thought to be mediated by tissue-resident macrophages (MF), modeled in vitro as M2-like MF. Circulating innate cells need serum opsonization of PGN for cellular activation, but opsonization requirements for human MF activation have not been reported. Herein, we tested the hypothesis that PGN requires human serum opsonization for MF activation and inhibits human M2-like MF efferocytosis of apoptotic human neutrophils.

MF were differentiated in vitro with M-CSF and polarized to M2 phenotype with dexamethasone. Cytokine and soluble receptor levels were assessed by ELISA and Luminex assays. Cell surface markers and fraction of MF containing fluorescently-labeled apoptotic human neutrophils were assessed by flow cytometry. M2-like MF recognition of PGN was enhanced in the presence of human but not bovine serum, noted by increased secretion of TNF-a and IL-10 in PGN-treated culture supernatants. Efferocytosis, and cell surface expression of MerTK, Tyro3, Axl, αVβ5, CD36 and Tim-3, all pro-efferocytic receptors, was significantly downregulated by pre-treatment of MF with PGN. Increases in soluble pro-efferocytic receptors were detected in PGN-treated MF culture supernatants.

We conclude that PGN impairs efferocytosis by human M2-like macrophages, likely by down-regulating the expression of cell surface receptors known to signal apoptotic cell engulfment.

Supported by NIH (U19AI062629).
IL-27 modulates T cell cytokines in the T cell-osteoclast crosstalk during HIV infection
Authors: Li Tong , Hadigan Colleen , Kumar Jai , Kumar Princy , Catalfamo Marta
Published: January 2023

In People with HIV (PWH), chronic immune activation and systemic inflammation are associated with increased risk to develop comorbidities including bone loss. IL-27 has been shown to modulate osteoclastogenesis and T cell function, however, the roles of IL-27 and T cell activation in the setting of HIV infection are largely unknown. In the present study, we evaluated the effects of IL-27 in modulating T cell function during T cells:Osteoclasts (OCs) crosstalk in PWH and healthy controls. We developed an in vitro coculture system of T cells with OCs from PWH and healthy controls. T cells and autologous OCs were isolated from frozen PBMCs of PWH (n= 26) and healthy controls (n= 10). We found that the stimulation of T cells from PWH and healthy controls cultured in the presence of OCs significantly enhanced T cell secretion of pro- (IFN-γ, TNF-α, IL-17) and anti-inflammatory (IL-10) cytokines. IL-27 down-regulated secretion of IL-17 (p< 0.001) and IL-10 (p= 0.003) from PWH but not in healthy controls and had a weak effect reducing RANKL surface expression in CD4 T cells (p= 0.047) when T cells were cultured with OCs. In addition, the level of IFN-γ secreted by stimulated T cells cultured alone was negatively associated with T score in absence (R= −0.59, p= 0.013) or in presence of IL-27 (R= −0.64, p= 0.007). All together these results suggest that in the context of HIV infection IL-27 may promote a dominant Th1 response (IFN-γ and TNF-α secretion) generating a “pro-osteoclastic” environment.

Supported by NIH R01AI145549-02
SARS-CoV-2 infection reduces the frequencies of angiogenic CD4 and CD8 T cells (Tang)
Authors: AbdusSamad Maryam , Li Tong , Timpone Joseph , Kumar Jai , Kumar Princy , Hunt Ryan C , Catalfamo Marta
Published: January 2023

SARS-CoV-2 is one of the leading causes of mortality worldwide with rising death toll over 5.4 million. The severe presentation of the disease is associated with systemic inflammation, activation of the coagulation cascade and immunological dysfunction. Numerous studies implicate endothelial activation and dysfunction as playing an important role; however, the pathways involved are not well-understood. Endothelial injury and damage trigger a mechanism of vascular repair mediated by bone marrow derived endothelial progenitor cells and recently discovered a type of T cells with angiogenic properties. In this study, we hypothesize that SARS-COV-2 alters this pathway. To address this hypothesis, we determined changes in the frequency and phenotype of these two cell types, the circulating endothelial progenitor (EPC) (LIN4− CD45− CD34+) and Tang cells (CXCR4+ CD31+) using PBMCs from hospitalized COVID-19 positive (n = 10) and healthy (n = 11) donors. Our results indicate a decrease in the frequency of CD4 and CD8 Tang cells compared with healthy controls (p=0.003; p=0.01). In addition, Tang cells showed a significant increase in the expression of progenitor marker (CD49f) (p<0.001; p=0.016), and no changes were observed in the frequency and phenotype of the LIN4− CD45− CD34+. Moreover, a negative association was observed between Tang cells and plasma levels of the procoagulant PAI-1 biomarker (r=−0.806, p=0.007; r=−0.818, p=0.006), and CD8 Tang cells were inversely correlated with the levels of MMP9 and TNFa, (r=−0.879, p=0.002; r=−0.709, p=0.027). Altogether these data suggest that SARS-CoV-2 infection reduces the frequency of Tang cells and promote activation and may interfere with their vascular repair function.
T-cell infiltrates and microglia adopt long-term gene signature changes leading to age-specific responses to traumatic brain injury in mice
Authors: Chen Zhangying , Islam Mecca , Davis Booker , Schwulst Steven
Published: January 2023

Aged traumatic brain injury (TBI) patients suffer increased mortality and long-term neurocognitive/neuropsychiatric morbidity than younger patients. Microglia, the resident macrophages of the brain, are complicit in both. We hypothesized that aged microglia would fail to return to a homeostatic state after TBI and adopt a long-term, injury-associated state within aged brains compared to young brains after TBI. Young and aged male C57BL/6 mice underwent TBI via controlled cortical impact vs. sham injury and were sacrificed four months post-TBI. We utilized single-cell RNA sequencing to examine age-associated cellular responses after TBI. Brains were harvested with CD45+ cells isolated via florescence-activated cell sorting. cDNA libraries were prepared via the 10x Genomics Chromium Single Cell 3’ Reagent Kit, followed by sequencing on a HiSeq 4000 instrument and computation analyses. Post-injury, aged mice demonstrated a proportional decrease in homeostatic microglia, and greater increased infiltrating T cells compared to young-adult mice. Of note, aged mice post-injury had a subpopulation of age-specific, immune-inflammatory microglia resembling gene profiles of neurodegenerative disease-associated microglia with enriched pathways involving in leukocyte recruitment. Contrastingly, post-injury, aged mice demonstrate a heterogenous T-cell infiltration with gene profiles corresponding to CD8 effector memory, CD8 native-like, CD4, and double-negative T cells and enriched pathways such as macromolecule synthesis. Taken together, our data showed that age-specific gene signature changes in the T-cell infiltrates and the microglial subpopulation contribute to increased vulnerability of the aged brain to TBI.

Supported by R01 GM130662
Neural Dysfunction Following Francisella tularensis Infection
Authors: Tena Anahis , Hightower Rebekah T , Sanchez Guillen Michelle A , Spencer Charles T
Published: January 2023

Francisella tularensis is a gram-negative intracellular coccobacillus that is the causative agent of tularemia. Local, acute inflammation is beneficial since it can clear bacterial infections, remove cellular damage and promote the healing of tissue. However, F. tularensis is capable of bypassing the immune system and causing an over-activation of inflammation, known as a “cytokine storm”. This cytokine storm increases host cell damage, nutrient availability and serves an immune distraction allowing the bacterium to replicate. Other infections that cause cytokine storms include Ebola, pandemic influenza, hantavirus, and HIV, all BSL3 pathogens.

The routes of transmission for F. tularensis are many and intertwined. Infection could be the result of inhaling air droplets containing infectious bacteria, coming into direct contact with an infected animal or being bitten by an infected arthropod such as a tick, flea or mosquito. Eating and drinking contaminated meat and water can also lead to infection and different disease manifestations occur depending on the route of transmission.

While extensive research has documented infection of the spleen, liver and lung, there hasn’t been any reports of F. tularensis productively infecting the brain, until now. This work focuses on characterizing the effects of F. tularensis infection of the brain. We hypothesize that damage caused by F. tularensis neural infection will have a negative effect on behavior. This work aims to identify these negative effects based on the anatomical location of the bacteria foci, regions containing numerous bacteria. With the use of brain mapping and behavioral assays, we seek to determine the impact of F. tularensis infection on behavior and cognition.
Outside-in IL1b-HMGB1 signaling axis may drive astrocytic response in drug resistant epilepsies.
Authors: Clarkson Ben D S , Lafrance-Corey Reghann , McCarthy Christina , Johnson Renee , Shrestha Ramila Barun , Howe Charles L
Published: January 2023

We recently reported normalization of CSF cytokine levels following successful treatment of drug-resistant epilepsy (DRE) with recombinant interleukin-1 receptor antagonist (rIL1RA). To further understand the mechanism of this therapeutic outcome, we interrogated the ability of serum and CSF factors that are elevated in drug resistant-seizure disorders to promote inflammatory responses in astrocytes and human brain microvessel endothelial cells (HBMVECs). We report that CCL2, TNFa, IL6, and CXCL8/1 were strongly induced in 1) astrocytes and HBMVECs stimulated with IL1β (blocked by rIL1RA) and 2) astrocytes but not HBMVECs in response to DRE patient serum but not control serum (partially blocked by rIL1RA). This suggested that other serum factors contribute to astrocyte activation in this DRE. HMGB1 and IL18 were also dysregulated in DRE patient serum; however, IL18 failed to stimulate cytokine secretion from astrocytes. In contrast, HMGB1 induced cytokine secretion from astrocytes, which was blocked by the TLR4 inhibitor TAK242. Remarkably, TAK242 also prevented cytokine production by astrocytes in response to serum from some patients with DRE. Finally, IL1β induced the polarized uptake and secretion of HMGB1 across transwell inserts harboring confluent monolayers of HBMVECs suggesting that IL1b signaling may cause endothelial cells to transport serum HMGB1 into the perivascular space—promoting TLR4-dependent cytokine release from astrocytes. This translation of an “outside” inflammatory signal into an “inside” inflammatory response may underlie seizure disorders that are resistant to anti-seizure drugs.
Trained Immunity in Neurodegenerative Diseases
Authors: Bossu’ Paola , Sireno Laura , Lembo Micaela , Toppi Elisa
Published: January 2023

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are complex and progressive neurodegenerative disturbances leading to cognitive or motor dysfunctions and lacking effective therapy. A deregulation of innate immune response and inflammation appears to be deeply involved in both illnesses, though with still unclear mechanisms. Evidence in animals attributes a neurodegeneration-modulating role to mechanisms of innate memory, but human data on this topic are still scarce. In this study, we investigated whether -possibly due to concomitant factors including enhanced infectious burden, microbial dysbiosis and genetic risk factors linked to immunity-the innate immune cells of AD and PD patients show long-term functional reprogramming characterized by either potentiated or tolerant responses, which could ultimately contribute to disease development. Thus, following repeated in vitro stimulation with either heat-inactivated Candida albicans or LPS, we measured the cytokine response of CD14+ cells from AD and PD patients, both in comparison with those of age- and gender-matched healthy control subjects and in association with symptom severity. We observed different cytokine patterns associated with tolerant or potentiated responses among the three groups of subjects, pointing to different and disease-dependent trained immune status in patients. Our results would provide insights about how previous triggering of innate immunity can influence neuroinflammation and disease progression, offering new knowledge on trained immunity impact in neurodegenerative diseases.
Effect of BPA on cytokine expression and regeneration in larval zebrafish
Authors: Walser-Kuntz Debby , Wang Hongshen , Alvarez Luis , Intasin Pear
Published: January 2023

Bisphenol-A (BPA) binds to estrogen receptors ER-α and β and is used in the production of polycarbonate plastic and epoxy resins. BPA presents a potential environmental challenge to the immune system of both humans and wildlife due to its use in food containers and thermal receipts and its resulting presence in surface water and wastewater runoff. We observed that BPA exposure downregulates the expression of the chemokine that functions to recruit neutrophils in both mice (KC) and zebrafish (cxcl8) and that it decreases neutrophil recruitment to the injury site in MPO::GFP zebrafish. Neutrophils are essential for the removal of damaged tissue and invading microbes, thus facilitating tissue regeneration of the tail fin. To investigate BPA’s effects on larval zebrafish tail regeneration, we induced a tail injury at day 3 post fertilization (dpf) and measured tail fin growth at day 1 and 3 post injury (dpi). Fish exposed to 100 ng/ml BPA regenerate a smaller tail fin area at 3 dpi (p-value < 0.01). We hypothesized that BPA exposure and altered neutrophil recruitment could affect regeneration by directly altering the expression of regeneration-related genes wnt10a and il-10 or their downstream genes and/or indirectly by regulating inflammatory genes including il-1 and il-6. The expression of il-1, il-6, il-10, and wnt10a were detected in zebrafish embryos from 2–6 dpf using qPCR. BPA exposure reduces both il-1 and il-6 expression 3 hours post-injury, suggesting that BPA reduces the inflammatory response. To determine whether BPA alters gene expression of wnt10a, il-10, or their downstream-regulated genes qPCR will be performed at 24- and 72-hours post-injury.

Supported by Carleton College.
The N-glycosyltransferase ST6GAL1 plays a key role in M2 macrophage anti-inflammatory functions
Authors: Barnes Rob , Dolatshahi Sepideh
Published: January 2023

Macrophages are effector cells of the innate immune system, and play distinct roles depending on their polarization state. Glycosylation is a ubiquitous post translational modification made to proteins that plays a large role in protein biophysical properties and function. Through computational analysis of a publicly available macrophage transcriptome data set, we demonstrated that glycosylation changes dramatically across different macrophage polarizations. Specifically, we identified the gene coding for the glycosyltransferase ST6GAL1, which terminally sialylates protein glycans, as significantly differentially expressed across macrophage polarities. ST6GAL1 was downregulated in macrophages polarized by M1 polarizing signals TNFa and TNFa+IFNg and upregulated by M2 polarizing signals IL-4 and IL-13. We hypothesized that changes in terminal sialic acid frequency during glycosylation play a role in M2 macrophage immunosuppressive actions. An analysis of SIGLEC signaling pathways revealed that increasing ST6GAL1 expression was concordant with increasing CD52 expression, a ligand for SIGLEC10. SIGLEC proteins contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) which inhibits immune action upon activation. SIGLEC10 expression increased in both M1 and M2 polarized macrophages, suggesting a possible mode of immunosuppression regulated by ST6GAL1. The increased frequency of terminally sialylated glycans and CD52 on M2 surfaces may increase the frequency with which M2 macrophages bind to and activate SIGLEC proteins on other immune cells. To test this hypothesis, we will knockout and amplify M2 macrophage ST6GAL1 expression to determine its influence on M2 immunosuppressive capacity.
Notch2, Bcl6, and IRF2 govern differentiation and survival of murine nonclassical monocytes.
Authors: O’Connor Kevin W , Liu Tiantian , Kim Sunkyung , Murphy Theresa , Murphy Kenneth M
Published: January 2023

Nonclassical (patrolling) monocytes are a population of monocytes which monitor the vasculature and participate in tissue repair. Though nonclassical monocytes are thought to be developmentally derived from classical monocytes, the mechanisms that control the differentiation of classical monocytes into nonclassical monocytes are still not completely understood. Signaling through the Notch2 receptor appears to trigger development of nonclassical monocytes. Also, the transcription factors C/EBPβ and Nur77 (Nr4a1) are required for nonclassical monocyte development in vivo. We have now identified additional transcription factor requirements for the differentiation and/or survival of nonclassical monocytes in vivo. We find that mice with conditional deletion of Bcl6 in myeloid cells or germline IRF2 deficiency show a severe loss of nonclassical monocytes. Both Bcl6 and IRF2 are induced during the transition from classical to nonclassical monocyte. In vitro culture of myeloid progenitors on stromal cells expressing DLL1, a Notch2 ligand, recapitulates aspects of nonclassical monocyte development seen in vivo, confirming Notch signaling as a key initiating event in the development of nonclassical monocytes. This system allows for the identification of the downstream targets of Notch2 signaling that drive nonclassical monocyte development, as well as the analysis of the impact of other transcription factors on the differentiation and survival of these cells in vivo. We present a model of the steps required for development of murine nonclassical monocytes.

Supported by NIH (RO1 AI150297, RO1 CA248919)
Nuclear soluble cGAS senses DNA virus infection
Authors: Wu Yakun , Song Kun , Hao Wenzhuo , Wang Lingyan , Li Shitao
Published: January 2023

The DNA sensor cGAS detects cytosolic foreign DNA from pathogens or self-DNA from cellular damage and instigates type I interferon (IFN) expression. Recent studies find that cGAS also localizes in the nucleus and tethers to the chromatin. Despite how cGAS is inhibited in the nucleus is well elucidated, whether nuclear cGAS participates in DNA sensing is not clear. Here, we report that herpes simplex virus 1 (HSV-1) infection causes the release of cGAS from the chromatin into the nuclear soluble fraction, and the free nuclear cGAS senses viral DNA to induce innate immune response. We first found that the infection of several HSV-1 strains led to cGAS release from the chromatin in several types of cells, including in RAW264.7 macrophages, THP-1 monocytes, and bone marrow-derived macrophages. DNA damage agent cisplatin failed to induce nuclear soluble cGAS, suggesting that cGAS release from the chromatin is not due to DNA damage or apoptosis. Interestingly, inhibition of HSV-1 replication blocked the release of nuclear soluble cGAS. To determine the role of nuclear cGAS in DNA virus infection, we reconstituted a Dox-induced nuclear-only cGAS mutant into cGAS knockout RAW264.7 cells. After induction, only chromatin-bound cGAS was found in the reconstituted cells. Similarly, HSV-1 infected also induced nuclear soluble cGAS in this cell line. More importantly, the nuclear-only cGAS stable cells limited HSV-1 infection by producing cGAMP and inducing mRNA expression of type I IFN and interferon-stimulated genes. Taken all together, our study demonstrates that nuclear soluble cGAS is a DNA sensor inside the nucleus.

This research was funded by the National Institutes of Health (R21AI137750 and R01AI141399 to S.L.).
Lipopolysaccharide pre-treatment prevents viral induced death by priming macrophages for a robust anti-viral immune response
Authors: Resiliac Jenny , Rohlfing Michelle , Santoro Jennifer , Hussain Syed-Rehan A , Grayson Mitchell H
Published: January 2023

Rationale

Respiratory viruses are an increasing public health burden. Using Sendai virus (SeV, murine parainfluenza virus type 1), previous data indicate that pretreatment of C57BL6 wild-type (WT) mice with 0.1μg of lipopolysaccharide (LPS) prevented mortality from a normally lethal SeV infection. However, the mechanism by which LPS pre-treatment confers this survival advantage is unknown. LPS is a potent activator of macrophages. We hypothesized that LPS prevents viral induced death by stimulating the recruitment of macrophages, which could drive a more robust anti-viral immune response and subsequent survival. Therefore, we examined the change in lung macrophage subsets with LPS pretreatment of SeV infected mice.

Methods

WT mice were inoculated with 30 μL of 0.1 μg LPS or PBS i.n. 24 hours before SeV (2e6 pfu). After LPS sensitization, lungs were harvested at 6 hours, 24 hours, and days 1,3,5, and 7 post SeV infection (PI SeV); single lung cell suspensions isolated and stained for various innate immune cells by flow cytometry to assess the immune cells recruited by LPS.

Results

Mice pre-treated with LPS had a significant increase of alveolar macrophages (GR1- Siglec F+ CD11c+) at days 1 and 3 PI SeV (p=0.080 and p=0.0026, respectively; n≥6). Additionally, interstitial macrophages (GR1- Siglec F− CD11b+ CD11c+ F4/80+ MHCII+) were significantly increased at days 0 and 3 PI SeV (p=0.025 and p=0.019 respectively; n≥6). The LPS effect was no longer significant by day 5 PI SeV.

Conclusion

. LPS pretreatment of mice increases macrophages early during the SeV infection. Further studies will be needed to determine if the increase in these macrophages provides the protection from viral induced mortality seen in our model and the cytokines produced.

This work was supported by the Abigail Wexner Research Institute at Nationwide Children’s Hospital (MHG).
The impact of lipid nanoparticles (LNP) on anti-viral pathways and immune function in aged individuals
Authors: Connors Jennifer R , Cusimano Gina , Kim Kenneth , Alameh Mohamad-Gabriel , Weissman Drew , Kutzler Michele , Haddad Elias K
Published: January 2023

Aging leads to increased morbidity and mortality posing a big challenge to societies as the world population shifts to an increased proportion of aged individuals. Immunosenescence, a condition of chronic increased level of basal inflammation and immune system dysregulation, is suspected to decrease vaccine efficacy and enhance COVID-19 complications in older individuals. Current vaccination efforts like mRNA vaccines utilize lipid nanoparticle (LNP) platforms that elicit the immune response however, little is known about how LNP adjuvantation affects anti-viral pathways and immune function in aged individuals. We have previously shown that type I IFN production including the phosphorylation of IRF7 and TBK-1 in the TLR 7/8, RIG-I or cGAS-STING pathways and phagocytosis is dysregulated in innate immune cells (i.e., cDC1s, cDC2s, and CD14dimCD16+ monocytes) from healthy older adults (n=11). Our preliminary data utilizing LNP show that in response to LNP stimulus at 4 and 24 hours, older adults have decreased IFNy output. Thus, we further hypothesize that cDC1, cDC2, and CD14dim CD16+ monocytes from healthy, older adults (n=11) will have dysregulated anti-viral signaling and antigen presentation in response to LNP stimulation as measured by the defect in type I IFN production, phosphorylation of IRF7, TBK-1, and immune function like phagocytosis and antigen presentation via flow cytometry. These data support our previous reports of a delayed/decreased proinflammatory output following stimulation and demonstrate potential targets of antiviral pathways that can resolve dysfunctions of the aging immune system for an effective vaccination response.

Supported by NIH (U19 AI128910-04S1)
Astrocytic RIPK3 confers protection against deleterious neuroinflammation during Zika virus infection
Authors: Lindman Marissa , Angel Juan , Newman Kimberly , Atkins Colm , Daniels Brian
Published: January 2023

This study aims to identify the function(s) of RIPK3 signaling in astrocytes following Zika virus infection. Previous work found that RIPK3 signaling in Zika virus-infected neurons activates inflammatory transcription factors such as NFκB and IRF1, leading to the upregulation of inflammation-associated transcripts. We were thus interested in determining the role of RIPK3 signaling in astrocytes, which are critical regulators of neuroinflammation. Using mice with an astrocyte-specific conditional Ripk3 deletion, we found that intracranial Zika virus infection was significantly more lethal in mice deficient in astrocytic Ripk3 than in littermate controls. To identify mechanisms underlying this difference, we isolated and infected primary fore- and hind-brain astrocytes with Zika virus to determine the transcriptional consequences of genetic Ripk3 ablation. Surprisingly, we found increased expression of several chemokines, cytokines and ISGs in Ripk3−/− hindbrain astrocytes, in contrast to our previous findings in neurons. Subsequent leukocyte profiling from the brains of Zika virus-infected mice revealed increased numbers of CD4+ and CD8+ T cells, natural killer cells, and monocytes in mice deficient in astrocytic Ripk3 compared to those found in littermate controls. As previous work has demonstrated that astrocytic type I interferon signaling in the hindbrain is responsible for downregulating proinflammatory molecules to prevent lethal neuroinflammation, our data suggest that synergistic signaling between type I IFN and RIPK3 in hindbrain astrocytes suppresses deleterious neuroinflammation and promotes host survival in the setting of Zika virus encephalitis.

Supported by a grant from NIH (R01 NS120895).
Effector-mediated subversion of proteasome activator (PA)28αβ enhances lysosomal pathogen targeting within cytokine-activated macrophages
Authors: Shames Stephanie R
Published: January 2023

Macrophages activated by proinflammatory cytokines, such as tumor necrosis factor (TNF) and interferon (IFN)-γ, are potently and broadly microbicidal. Legionella pneumophila causes Legionnaires’ Disease via replication within host macrophages using an arsenal of hundreds of translocated virulence factors termed effector proteins. Effectors are critical for intracellular replication but can also enhance pathogen clearance in mammalian hosts via effector-triggered immunity. The effector LegC4 confers a fitness disadvantage on L. pneumophila in mouse models of Legionnaires’ Disease and uniquely potentiates the antimicrobial activity of macrophages activated with either TNF or IFN-γ. We found that LegC4 binds proteasome activator (PA)28α, a subunit of the PA28αβ (11S) proteasome regulator, and that the LegC4 restriction phenotype is abolished within PA28αβ-deficient macrophages. PA28αβ facilitates ubiquitin-independent proteasomal degradation of oxidant-damaged proteins. Impaired proteasome activity results in compensatory upregulation of lysosomal degradation pathways to relieve oxidative proteotoxic stress. We found that LegC4 impairs the resolution of oxidative proteotoxic stress and enhances phagolysosomal fusion with the Legionella-containing vacuole. PA28αβ has been traditionally associated with antigen presentation and adaptive immunity; however, our data support a model whereby suppression of PA28αβ by LegC4 impairs resolution of oxidative proteotoxic stress, which culminates in lysosomal killing of L. pneumophila within activated macrophages. This work provides a solid foundation on which to evaluate induced proteasome regulators as mediators of cell-autonomous immunity.

This work is supported by NIH grant P20GM130448.