bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of a high fibre diet on immune cells and the multiple sclerosis animal model
Authors: Sen Monokesh Kumer , Ge Anjie , Liao Eileen , Gard Harrison , Ni Duan , Macia Laurence , Piccio Laura
Published: August 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Studies demonstrated the association between inadequate dietary fibre intake and dysregulation of the immune response, the impact of which in MS remains controversial. Using experimental autoimmune encephalomyelitis (EAE) animal model of MS, this study investigated how dietary fibre regulates adaptive immune cells, gut-microbial composition, and metabolite secretion. Following EAE induction, the high fibre (HF) group showed an earlier onset, higher disease incidence and severity compared to the zero fibre (ZF) group. Immune profiling of peripheral blood cells and splenocytes after 5-weeks on the HF diet revealed a pro-inflammatory immune phenotype, with increased numbers of pro-inflammatory Ly6Chi monocytes and decreased numbers of anti-inflammatory IL-10+CD4+ T-cells and T-regs the spleen compared to the ZF-fed mice. Short chain fatty acids (SCFAs) measurements at 5-weeks on the diet, showed a global increase of acetate, propionate and butyrate concentrations in the HF group compared to ZF cohorts. After EAE induction, Ly6Chi monocytes were decreased peripherally, while there was an increased number of dendritic cells and infiltration of monocytes in CNS in the HF compared to the ZF group. In conclusion, a HF diet was associated with a more pro-inflammatory systemic profile and a worse EAE clinical course. This is likely due to the complex biology of SCFAs, which could have pro- or anti-inflammatory effects depending on the context of immune cell activation. Further research is needed to clarify these mechanisms.

Starter funding support
Antioxidative and anti-inflammatory effect of ethanol extracts from Magnolia kobus DC on LPS-induced in vitro and in vivo model
Authors: Kim Yeyoung , Kim Sanghyun
Published: August 2023

Magnolia kobus DC (M. kobus) has biological effects such as antioxidant, anti-inflammatory and antibacterial properties in its flower, bud, and stem bark extracts. Despite of diverse effectiveness of M. kobus, the pharmacological effects supporting inhibition of oxidation and inflammation on acute lung inflammation states have not been studied yet. The purpose of this study was to evaluate the pharmacological activity of M. kobus leaf ethanol extract (MkDE) by antioxidant and anti-inflammatory effects on acute lung inflammatory diseases. The antioxidant and anti-inflammatory effects of MkDE was evaluated using ROS detection assay, NO assay, qPCR, ELISA, and Western blot on LPS-induced macrophages. The therapeutic effect of MkDE was investigated in vivo oxidative and inflammatory responses by histological observations, qPCR, ELISA, and Western blot using the LPS-induced acute lung inflammation mouse model. MkDE decreased ROS production and protein of Nrf2. MkDE showed a protective effect against H2O2 as well as a decrease in NO production. MkDE reduced the levels of pro-inflammatory cytokines and chemokine, the activation of COX-2 and iNOS, and NF-κB translocation in LPS-induced macrophage. The intraperitoneal injection of MkDE showed to decrease the lung/body weights, infiltrated inflammatory cells into the lung alveoli, pro-inflammatory mediators, and serum released proteins. Furthermore, MkDE inhibited the activation of Nrf2, COX-2 and NF-κB in LPS-induced acute lung tissue. MkDE exhibits the pharmacological effect by reducing oxidative and inflammatory mediators on LPS-induced macrophages and acute lung inflammation mouse model. Taken together, MkDE might be used to treat acute lung disease.
The inhibitory effects of Bakuchicin on ovalbumin-induced allergic airway inflammation via regulating M2 macrophage polarization
Authors: Lee Soyoung , Kim Yeon-Yong , Kim Sang-Hyun
Published: August 2023

Asthma is an airway inflammatory disease caused by the activation of many immune cells, including macrophages. Bakuchicin (BKC), a furanocoumarin, has anti-inflammatory properties. This study aimed to evaluate the effects of BKC on allergic airway inflammation and demonstrate the mechanisms of macrophage activation and differentiation. We assessed the effects of BKC by evaluating inflammatory markers in an ovalbumin (OVA)-induced airway inflammation mouse model and macrophage cells. BKC treatment alleviated histological changes, mast cell infiltration, and collagen deposition, and reduced the levels of myeloperoxidase, eosinophil peroxidase, β-hexosaminidase, and transforming growth factor-β in the bronchoalveolar fluid (BALF) and the level of immunoglobulins in serum. BKC ameliorated inflammatory symptoms, including the level of inducible nitric oxide synthase (iNOS) in the lung and the expression and secretion of pro-inflammatory cytokines in serum. BKC treatment reduced not only the level of Th2 cytokines but M2 macrophage marker expression and population in the BALF. In vitro, BKC inhibited LPS-stimulated production of nitric oxide and expression of pro-inflammatory cytokines through inhibition of iNOS, COX-2, Akt, MAPKs, IκBα, and NF-κB in J774 A.1 mouse macrophages. Moreover, BKC suppressed IL-4-induced M2 macrophage polarization by inhibiting arginase-1, YM-1, and Fizz-1 and regulating sirtuin 2 levels in bone marrow derived macrophages. Collectively, BKC may help in the prevention and treatment of airway inflammation by regulating macrophage activation and polarization.
Unfolded Protein Response Factor ATF6 Augments T Helper Cell Responses and Promotes Mixed Granulocytic Airway Inflammation
Authors: Wu Dandan , Zhang Xing , Zimmerly Kourtney M , Wang Ruoning , Wang Chunqing , Liu Meilian , Yang Xuexian
Published: August 2023

The unfolded protein response (UPR) plays a critical role in many diseases, including airway inflammation. Several recent studies demonstrated a pathogenic role of Activating transcription factor 6a (ATF6a or ATF6), one of the essential arms of UPR, in airway structural cells. However, its role in T helper (TH) cells has not been well examined. In this study, we found that ATF6 was selectively induced by STAT6 and STAT3 in TH2 and TH17 cells, respectively. ATF6 upregulated UPR genes and promoted the differentiation and cytokine production of TH2 and TH17 cells. T cell-specific Atf6-deficiency impaired TH2 and TH17 responses in vitro and in vivo and attenuated mixed granulocytic experimental asthma. ATF6 inhibitor Ceapin A7 suppressed ATF6 downstream gene expression and TH cell cytokine expression in both murine and human memory CD4+ T cells. Administration of Ceapin A7 lessened both TH2 and TH17 responses in vivo, leading to alleviation of both airway neutrophilia and eosinophilia. Thus, our results demonstrate a novel role of ATF6 in TH2 and TH17 cell-driven mixed granulocytic airway disease, suggesting a novel option to combat steroid-resistant mixed and even T2-low endotypes of asthma through targeting ATF6.

This work was supported in part by NIH grants HL148337 and AI142200 (X.O.Y.), and DK110439 and DK132643 (M.L.). We acknowledge the University of New Mexico Comprehensive Cancer Center Flow Cytometry Core supported by NIH CA118100 and the Autophagy, Inflammation, and Metabolism in Disease Center Inflammation and Metabolism Core supported by NIH GM121176
Macrophages promote survival of granulosa cells
Authors: Brodeur Tia Y , Bonney Elizabeth A , McGee Elizabeth , Krementsov Dimitry
Published: August 2023

The ovary contains numerous macrophages, and macrophage depletion in mice leads to dysfunctional ovulation. Macrophage function in the ovary is presumed to be related efferocytosis, however, the absence of macrophages also disrupts follicular development and corpus luteum development. Granulosa cells (GC) are critical to ovarian follicle development and survival, and we hypothesized that macrophage-GC crosstalk promoted GC function. To determine whether macrophage-GC interaction has an anti-apoptotic effect on GCs, BMDM were polarized to proinflammatory phenotype with LPS (M1), alternative activation phenotype with IL-4 (M2), or not stimulated (M0). After removing stimuli, primary mouse GCs were co-cultured with macrophages and serum-starvation was used to induce apoptosis of GCs. The cells were analyzed by flow cytometry, assessing viability by exclusion of propidium iodide and Annexin V. Co-culture with macrophages protected GCs from serum-starvation induced cell death. In co-cultures with quiescent BMDM, 60% of GCs were late apoptotic/early necrotic compared to 81% without co-culture, (p=0.0031). Unexpectedly, LPS-treated macrophages promoted survival of GC slightly more effectively than IL-4-treated or quiescent macrophages (45% versus 60% and 53% apoptotic, respectively p=0.0230). Both quiescent and inflammatory macrophages promote survival of GCs. This result is unexpected, as LPS-treated macrophages secrete TNF-α, a cytokine that is known to induce death in GCs. This study elucidates the role of macrophages in ovarian function by supporting GC survival and may also have implications for the promotion of GC tumors by tumor-associated macrophages.

Supported by grant from the NIH 3P30GM118228-05S3
A computational analysis demonstrating vitamin E nicotinate promoting anti-inflammatory signals
Authors: Suzuki Dante I , Marcocci Lucia , Suzuki Yuichiro J
Published: August 2023

Vitamin E nicotinate is an ester of tocopherol (vitamin E) and niacin (vitamin B3). Niacin is linked to the hydroxyl group of active vitamin E, blocking free radical scavenging properties. While this ester can be chemically synthesized, whether vitamin E nicotinate is formed from vitamin E and niacin in the biological system is unclear. Metabolomics analysis performed in our laboratory demonstrated that the level of vitamin E nicotinate is 30-fold lower in rats suffering from an inflammatory state. The rat diet contained vitamin E acetate and niacin separately (but not in the form of vitamin E nicotinate), and only vitamin E nicotinate (but not other forms of vitamin E) was decreased, suggesting that vitamin E nicotinate is formed in the biological system. To test if vitamin E nicotinate functions independently from simply serving as a source of active vitamin E, we also performed metabolomics to compare the effects of vitamin E nicotinate on cultured cells to those of vitamin E acetate + niacin added separately. Results revealed that cells treated with vitamin E nicotinate for 10 min had significantly higher levels of molecules with anti-inflammatory properties including anandamide, virodhamine, palmitamide, oleamide, and oleoylethanolamide. Vitamin E nicotinate (but not vitamin E acetate + niacin) phosphorylated and activated extracellular signal-regulated kinase (ERK), which also serves as an anti-inflammatory signal. These results suggest a new biological function of vitamin E nicotinate as an anti-inflammatory molecule through non-direct free radical scavenging effects.

Supported by grants from NIH (R21 AG73919, R03 AG71596)
Neutrophil extracellular trap (NET) degradation attenuates acute lung injury and protects mice from ricin-induced acute respiratory distress syndrome (ARDS)
Authors: Sapoznikov Anita , Gal Yoav , Israeli Ofir , Aftalion Moshe , Evgy Yentl , Erez Noam , Falach Reut
Published: August 2023

ARDS is a life-threatening respiratory condition associated with pulmonary vascular hyperpermeability and loss of aerated lung tissue. Ricin is a plant-toxin derived from the seeds of Ricinus communis, irreversibly inactivates ribosomes and arrests protein synthesis. Pulmonary exposure to ricin represents a bona fide model for ARDS, is characterized by pulmonary inflammation with massive neutrophil recruitment and severe disruption of the alveolar-capillary barrier leading to pulmonary edema, compromised gas exchange and respiratory failure. We have shown that upon ricin exposure, neutrophil infiltration into lungs, a hallmark of ARDS, mediate proteolysis of junction proteins. Recently, there is an increasing concern about the deleterious effect of NETs in sterile inflammation, such as in ricin case. During NETosis, PAD4 drives histone citrullination and chromatin decondensation. Decondensed chromatin decorated with citrullinated histones (citH3) and cytoplasmic granular enzymes, such as MPO is expelled from the neutrophil. Herein, we show increasing levels of PAD4, citH3 and MPO in the BALF of ricin exposed mice. BALF neutrophils efficiently produced NETs which were associated with MPO and citH3. Immunohistology of lung sections upon ricin exposure demonstrated propagating NETosis. Treatment of ricin exposed mice with recombinant DNase, targeted against NET-associated extracellular DNA, administrated with anti-ricin antibodies, dramatically decreased the level of extracellular DNA and proinflammatory cytokines in the lungs and conferred higher protection levels to mice in comparison to sole administration of antitoxin. This data highlights the potential clinical benefit of NET inhibiting drugs in ARDS.
Fc modification to transform a pathogenic antibody into a potential therapeutic for thromboinflammatory disease
Authors: Sarkar Amrita , Gollomp Kandace , Khandelwal Sanjay , Arepally Gowthami , Cines Douglas B , Kowalska M Anna , Rauova Lubica , Poncz Mortimer
Published: August 2023

KKO is a HIT (heparin-induced thrombocytopenia)-like mouse monoclonal antibody (moAb) that binds to platelet factor 4 (PF4) when complexed to polyanions like heparin and neutrophil extracellular traps (NETs). Human (h) PF4 protected NETs from DNases and was further enhanced by KKO as it activates platelets and neutrophils via both FcgRIIA and complement, causing prothrombotic state in transgenic mice expressing FcgRIIA and hPF4. We posited through that if Fc activity of KKO were eliminated then it may stabilize NETs and potentially be therapeutic by stabilizing NETs without FcgRIIA and complement activation. We deglycosylated Fc region of KKO to make DGKKO, and tested if we can avoid detrimental effects without interfering with KKO protection of PF4:NET complexes. We also speculated that DGKKO may bind to hPF4:polyanions and prevent them from inducing HIT. Binding efficacy of DGKKO to PF4-heparin complexes was studied by ELISA and dynamic light scattering. Platelet and complement activation were also studied for DGKKO, and we found that DGKKO still bind to PF4-polyanion complexes, with no platelet activation and significantly lower complement activation. DGKKO also enhanced NET nuclease resistance to improve microbial capture by PF4-NETs, preventing thrombocytopenia and improving survival in murine sepsis models. Also, our humanized in vitro microfluidics studies as well as HIT mice studies showed that infusion of DGKKO prevented both thrombocytopenia and thrombosis in a murine model of HIT. These results showed that Fc-modified KKO has protective effects in at least two models of thromboinflammation. Further studies are underway to examine the role of NET stabilization by modified KKO in other thromboinflammatory disorders.

R35 to Mortimer Poncz and CHOP Foerderer Grant to Amrita Sarkar
Biological scaffold implants in a murine volumetric muscle loss trauma model cause time dependent CD62L and CD101 upregulation on “regulatory like” eosinophils
Authors: Lokwani Ravi , Josyula Aditya , Fertil Daphna , Sadtler Kaitlyn
Published: August 2023

Introduction

Previously, we reported a strong increase in the recruitment of eosinophils at injured muscle when treated with a biologic scaffold implant. As such, we sought to characterize the surface marker expression of these eosinophils to understand their role at the site of injury.

Method

Volumetric muscle loss (VML) model was performed on C57BL/6 mice and was subsequently treated with a biologic scaffold (decellularized extracellular matrix). After 7 and 21 days, post-injury, implanted material along with injured tissue was dissected and converted to single cell suspension for flow cytometry.

Results

Compared to uninjured mice, at both 7 and 21 days time point, mice that received biological scaffold featured significantly increased presence of eosinophils at the site of injury. These eosinophils had a ring-shaped nucleus which has been previously associated with regulatory eosinophils. Eosinophils at the site of injury showed time dependent upregulation of CD101 (Mean fold change from uninjured muscle after 7 days post implant was 2.73 and the same was 9.90 after 21 days, (p<0.0001)), and CD62L (Mean fold change from uninjured muscle after 7 days post implant was 3.65 and the same was 11.64 after 21 days(p<0.0001)) expression. Interestingly, the same pattern was not observed with blood eosinophils except that the blood eosinophils at 3-week timepoint were featuring a significant increase in CD62L expression compared with 1 week time point, suggesting potential return of homeostasis in circulatory compartment.

Conclusion

Here, we describe time-dependent transformation in eosinophil phenotype in response to biologic scaffold implantation, including upregulation of adhesion molecule CD62L and a regulatory marker CD101.
UDP-glucose Regulates Dendritic Cell Mitochondrial Respiration via a Nitric Oxide-dependent Mechanism
Authors: Vagher Bay L , Amiel Eyal
Published: August 2023

Dendritic cells (DCs) are key innate immune cells specialized in fine tuning an immune response. Activation of DCs through Toll-Like Receptors (TLRs) is accompanied by a metabolic switch to increased use of glycolytic metabolism fueled by carbohydrates glucose and glycogen. Our lab has shown that DCs require glucose and glycogen to perform their immune effector functions including: production of cytokines and inflammatory mediators, antigen presentation to T cells, and initiation of the T cell-mediated immune response. Recent studies in inflammatory macrophages showed a secondary metabolite in the glycogen synthetic pathway, uridine diphosphate glucose (UDP-glucose), is upregulated through TLR4 stimulation. UDP-glucose exits the cell and signals in an autocrine manner through its specific purinergic receptor P2Y14. Additionally, P2Y14R signaling modulates the expression of proinflammatory pathways including upregulation of inducible nitric oxide synthase (iNOS). Activated bone marrow-derived DCs (BMDCs) upregulate iNOS and produce nitric oxide (NO), which has pleiotropic effects, including inhibiting mitochondrial respiration. Therefore, we hypothesized that the UDP-glucose/P2Y14R/iNOS axis regulates BMDC metabolism, which has further ramifications on DC cell survival and proinflammatory phenotypes. To investigate the impact of UDP-glucose as an autocrine signaling molecule, we characterized proinflammatory gene transcript and protein levels, as well as the functional outcomes of increased iNOS protein in the cell. These studies showed that DCs use the UDP-glucose/P2Y14R/iNOS axis to bolster inflammatory phenotypes including inhibition of mitochondrial respiration leading to exacerbated cell death.

Supported by grants from NIH (R01 AI158710-01, 1R21AI135385-01A1) and UVM (COBRE VCIID Funding Support and Pilot Project)
Gene-dose-dependent roles of DEFA1A3 in the neutralization of uropathogenic Escherichia coli
Authors: Canas Jorge J , Schwaderer Andrew L , Hains David S
Published: August 2023

Genetic polymorphisms of the human genome provide information about disease susceptibility and autoimmunity. Genetic variations involving the innate immune system have the potential to improve the effectiveness of diagnosis and treatment of urinary tract infections in children and adults. DNA copy number variations (CNVs) of DEFA1A3, which encodes for antimicrobial peptides α-defensin 1–3, have been associated with recurrent UTI risk in children with vesicoureteral reflux (VUR). α-Defensins 1–3 are pleiotropic antimicrobial peptides with bactericidal and immunomodulatory properties. Due to the absence of a gene ortholog in mice, traditional murine models to study the effect of DEFA1A3 CNVs are lacking. Our objective is to explore the gene-dosage pleiotropic effects of the DEFA1A3 locus by utilizing a UTI-challenged transgenic mouse model expressing variable copies of the human gene. To model murine UTIs, uropathogenic E. coli (UPEC) pyelonephritis strain (CFT073) or vehicle volume was injected transurethrally into DEFA4/4, DEFA4/0, and DEFA0/0 mice. Urinary tract organs were analyzed for quantifiable bacterial growth, immune cell frequencies, and antimicrobial response gene expression array. Kidney bacterial clearance effects are proportional to gene-dosage; gradually decreasing colonization in mice between DEFA4/4 (71%), DEFA4/0 (57%), and DEFA0/0 (43%) genotypes. The kidney immune cells displayed differential mRNA expression of pro-inflammatory genes Il1β and Il6 genes as well as phagocytic genes Rac1 and Lyz2 between the infected mice genotypes. Our findings support pleiotropic gene-dosage protective roles of the human DEFA1A3 gene in a murine model of UTI-induced pyelonephritis.

Supported by the grants from NIH (R01 DK117934 & R01 DK106286)
IL-33 affects the IL-4-induced development of group 2 innate lymphoid cells in neonatal lungs
Authors: Huang Hui-Yu , Shieh Chi-Chang
Published: August 2023

Abundant IL-33 is produced by alveolar type 2 cells during lung alveolarization phase in the neonatal stage. It promotes Th2 response and may facilitate ILC2 development with IL-4 stimulation. IL-4 has been found to induce IL-10+ ILC2s in the lung environment, however, it is still unknown whether ILC1/2/3 transdifferentiation and IL-10+ ILC2 functions in neonatal lungs are regulated by IL-33. Therefore, we aimed to investigate the regulation role of IL-33 in lung ILC development during neonatal period. To identify the key cytokines under IL-33 regulation in neonatal lungs, we used antibody staining to characterize ILC1/2/3 and measured the cytokines relevant to ILC2 functions in WT and IL-33−/− mice. We found that while lung ILC2s increased from postnatal day (PND) 14 to 28 in WT mice, ILC1s and ILC3s decreased in the same period. However, we found that there were significantly decreased ILC2s on PND 21 in the lungs of IL-33−/− mice when compared with WT mice. We also found that increase in IL-33, IL-4, IL-10 on PND 28 in the lungs of WT mice, which was not apparent in IL-33−/− mice. The sorted neonatal lung ILC2s may secrete more IL-10 after stimulated with IL-4 and IL-33 when compared with adult lung ILC2s from either WT or IL-33−/− mice. Moreover, in vitro cultured ILC populations from IL-33−/− mice after stimulated with IL-4 and IL-33 may enhance ILC1/3 transdifferentiate into IL-10+ ILC2s. Our results showed that IL-33 contributes to ILCs differentiate towards IL-10+ ILC2 through IL-4 and IL-33 stimulation during lung alveolarization phase. As IL-10+ ILC2s have been found to play a regulatory role in allergy, the development of IL-10+ ILC2s during neonatal period may be critical for asthma prevention.
Dysregulation of fetal-derived cochlear resident tissue macrophages mediates CMV-associated sensorineural hearing loss
Authors: Otsuka Kelly , Scott Boston , Park Albert , Beaudin Anna E
Published: August 2023

Cytomegalovirus (CMV) is the most common congenital viral infection in the developed world and the leading cause of non-genetic sensorineural hearing loss (SNHL) in children. Congenital CMV causes progressive SNHL long after acute infection has resolved, suggesting a lasting effect on the developing host immune response in the cochlea. Using fate-mapping models, we investigated the contribution of fetal-derived resident tissue macrophages (RTMs) to normal cochlear development and function, and their role in mediating CMV-associated SNHL. We uncovered dual contribution of fetal-derived cochlear RTMs (F4/80hiCD11blo) from yolk sac (YS) and fetal liver (FL) hematopoiesis at birth. YS-derived RTMs naturally recede postnatally after cochlea development, but remain in key tissues, such as the spiral ligament and stria vascularis, through adulthood. Depletion of fetal-derived RTMs by anti-CSF1R blocking Ab led to SNHL and impairments to cochlear architecture. Similarly, embryonic deletion of YS-derived RTMs by induced deletion of CSF1R leads to SNHL at high frequencies. During acute CMV infection, RTMs expand and become transcriptionally dysregulated in developmental programs, including tissue remodeling, chemorepulsion, otic vesicle morphogenesis, and FGF signaling. Importantly, analysis of CSF1R-Cre labeling suggests YS-derived RTMs retract in response to CMV infection, suggesting RTM expansion is caused by infiltration of BM-derived macrophages. Ongoing work examines spatial distribution of fetal-derived cochlear RTMs, transcriptional differences between YS, FL, and BM-derived RTMs in response to CMV, and how replacement of fetal-derived RTMs with BM-derived RTMs affects tissue function and response to CMV.

Supported by grants from NIAID (T32 5T32AI138945) and University of Utah molecular medicine and immunology, inflammation and infectious disease (3i) initiative
Pseudomonas aeruginosa pulmonary infection results in S100A8/9-dependent cardiac dysfunction
Authors: Rajaram Murugesan , Kumar Naresh , Pestrak Matthew , Wu Qian , Ahumada Omar , Saljoughian Noushin , Shukla Rajnikant , Mitchem Cortney , Nagareddy Prabhakara , William Lafuse , Ganesan Latha , Wozniak Daniel J
Published: August 2023

Pneumonia in ICU patients carries a bleak prognosis with high risk for cardiac dysfunction, largely due to cardiac infection and inflammation. Pseudomonas aeruginosa (P.a.) infection accounts nearly 20% of all cases of hospital acquired pneumonia with mortality rates >30%. P.a. infection induces a robust inflammatory response, which ideally enhances bacterial clearance. Unfortunately, excessive inflammation can also have negative effects, and often leads to cardiac dysfunction with associated morbidity and mortality. However, it remains unclear how P.a. lung infection causes cardiac dysfunction. Using a murine pneumonia model, we found that of P.a. infection of lungs led to severe cardiac left ventricular dysfunction and electrical abnormalities. More specifically, we found that neutrophil recruitment and release of S100A8/A9 in the lungs activates the TLR4/RAGE signaling pathways, which in turn enhance systemic inflammation and subsequent cardiac dysfunction. Paradoxically, global deletion of S100A8/A9 did not improve but aggravated cardiac dysfunction and immortality likely due to uncontrolled bacterial burden in the lungs and heart. Our results indicate that P.a. infection induced release of S100A8/9 is double-edged, providing increased risk for cardiac dysfunction yet limiting P.a. growth.

Funding: NIH grants R01-AI 146252 and R21-AG 127783

Funding: NIH grants R01-AI 146252 and R21-AG 127783
Disease associated monocytes contribute to the inflammatory milieu in hidradenitis suppurativa and have dysregulated expression of transposable elements
Authors: Dimitrion Peter Michael , Hamzavi Iltefat , Yin Congcong , Loveless Ian , Toor Jugmohit , Sulbedi Kalpana , Khalasawi Namir , Miller Angela , Huggins Richard , Adrianto Indra , Veenstra Jesse , Vellaichamy Gautham , Hans Aakash , Daveluy Steven , Athar Mohammad , Liao Wilson , Lim Henry , Ozog David , Zhou Li , Mi Qing-Sheng
Published: August 2023

Hidradenitis suppurativa (HS), an inflammatory skin-disease, manifests as painful lesions in intertriginous skin. Despite their central role in innate immunity the role of monocytes in HS is largely unknown. Here we utilized cytometry by time-of-flight (CyTOF), imaging CyTOF (IMC), cellular indexing of transcriptomes and epitopes by sequencing (CITEseq), spatial transcriptomics (ST), and our recently developed database (HSOmicsDB) to characterize the systemic and cutaneous immune response in patients with HS. CITEseq found differential genes in circulating immune cells from HS patients enriched for cellular activation and extravasation. CyTOF immune profiling of fresh whole-blood samples found decreased frequency of classical Mos (C.Mos), which had increased expression of skin homing chemokine receptors. IMC found C.Mos infiltrated lesional dermis and tunnel regions, but not perilesional (PL) skin. Patients with HS showed increased proportions of activated C.Mo subclusters and a population of SOD2+ C.Mos expressing genes downstream of viral sensing pathways. SOD2+ C.Mos expressed high levels of neutrophil attracting chemokines. ST found SOD2+ C.Mos localized to regions of active inflammation and dermal tunnels. HSOmicsDB showed Mos and Macs expressed SOD2+ C.Mo genes, which were enriched in lesions, but not PL skin. Finally, we show that SOD2+ C.mos in HS have dysregulated expression of transposable elements (TEs), which may be activating viral sensing mechanisms in patients with HS. Together our data shows monocytes in HS function to recruit neutrophils to lesional HS skin, and provides the first evidence implicating TEs as disease promoting factors in HS by leading to C.Mo dysregulation.

Supported By NIH/NIAMS, R01AR078688-01A1 and R21AR079089-01A1
Neonatal bacilllus Calmette-Guerin (BCG) administration did not limit influenza A virus replication or clinical disease in pigs
Authors: Loving Crystal L , Hill Dana , Byrne Kristen
Published: August 2023

A potential immunomodulation mechanism for improving disease resilience is innate training, which relies on epigenetic modifications of immune cells for a heightened (ie, trained) response upon repeated microbial stimulation. Prior experiences indicate bacillus Calmette-Guerin (BCG) exposure increases cytokine production upon secondary exposure to microbe-associated molecular patterns, and epidemiological data suggest improved disease resistance in humans administered neonatal BCG. Neonatal piglets received BCG or mock inoculum via intravenous (IV) route and were subsequently exposed to influenza A virus (IAV) inoculated piglets. Body temperature was recorded and nasal swabs collected through the acute phase of viral infection. At necropsy, gross lung pathology was assessed. In addition, prior to IAV challenge, peripheral blood mononuclear cells (PBMC) and monocytes were isolated from mock and BCG inoculated animals and stimulated in vitro with LPS to evaluate in vivo induction of a trained phenotype via cytokine production and gene expression. PBMC were stimulated with PPD antigen to assess antigen-specific T cell responses to BCG inoculation. PPD-specific IFNγ production by PBMCs was minimal; yet upon in vitro LPS exposure, monocytes isolated from IV BCG treated piglets produced more IL-1β and TNF cytokine than monocytes from mock pigs (ie, trained phenotype). However, no significant differences in clinical presentation or nasal and lung IAV titers were detected. BCG administration induced a trained phenotype in porcine peripheral monocytes, it did not translate into a significant alteration in respiratory viral infection.

USDA-ARS-NIFA 2019-07511
Sex differences in ILC2 function in influenza result from imprinted donor sex intrinsic factors independent of host sex in competitive female-male bone marrow chimeras
Authors: Williams Abigael Parrish , Turner Sean , Chlebicz Magdalena , Miller Reegan , Roe Mandi , Kadel Sapana , Kovats Susan
Published: August 2023

Historical influenza pandemics and the SARS-CoV-2 pandemic revealed gender differences in morbidity of respiratory virus infections. In human or murine influenza A virus (IAV) infection, females of reproductive age generate stronger innate and adaptive antiviral responses leading to immunopathology, while elderly males often experience more severe disease. Sex hormone levels play a role in this disparity, yet little is known about cell intrinsic regulation of immune cells by estrogen or androgen receptors (AR). Lung resident type 2 innate lymphocytes (ILC2s) express high levels of AR, a nuclear hormone receptor that regulates chromatin via epigenetic modifications. While female lungs contain higher ILC2 numbers in homeostasis, we showed that ILC2s in females were preferentially functionally suppressed during IAV infection, whereas ILC2s in males retained robust type 2 function, including IL-5 production. Here, we determined if sex differences in pulmonary ILC2s result from extrinsic factors present in host environments or from imprinted donor-intrinsic factors. We set up competitive bone marrow chimeras by transferring a mix of female and male lineage-depleted bone marrow cells into either male or female hosts. Female hosts harbored more pulmonary ILC2s than male hosts, suggesting the extrinsic sex hormone environment regulates ILC2 numbers. Upon IAV infection, an increased fraction of ILC2s from male donors retained high IL33R expression and produced IL-5, regardless of host sex, suggesting donor sex-intrinsic factors regulate the canonical ILC2 functional response. We are now testing the hypothesis that androgens/AR activity epigenetically imprint genes regulating type 2 function in male ILC2s upon sexual maturity.
Single-cell cloning of NK cells from rhesus macaques: a new methodology for in-depth analysis of MHC-E-dependent adaptive functions
Authors: Rascle Philippe M , Jones Rhianna , Lee Esther , Terry Karen , Jost Stephanie , Reeves R Keith
Published: August 2023

Natural killer (NK) cells provide rapid and robust responses against a wide range of viral infections. More recently, multiple studies have shown that subsets of NK cells are also capable of antigen-specific recall to eliminate infected cells. In humans and nonhuman primates (NHP), antigen-specific memory NK cell responses can at least be partially delineated by expression of NKG2C, a ligand for MHC-E. Due to MHC-E limited polymorphism, MHC-E-presented viral antigens may induce universally antigen-specific memory NK cells.

To study antigen-specific NK cells in NHP, we developed a new in vitro method to clonally expand single rhesus macaque NK cells (rhNKCL) from blood and spleen. To define the effector functions of rhNKCL, we used a calcein acetoxymethyl ester (CAM) cytotoxicity assay and intracellular staining assay in combination with K562 cell lines or autologous BLCL as targets. We also identified, by in silico methodology, peptides from influenza, SIV, and SARS-CoV-2 viruses predicted to display strong binding to MHC-E.

Our procedure showed for the first-time clonal expansion of single rhesus macaque NK cells. We observed a conserved NHP NK cell phenotype of CD3− NKG2A/C+ CD16+ CD56+ following expansion with expression of additional NK cell surface receptors CD8, NKp80, and NKG2D. Upon stimulation with K562 cells, rhNKCL effector functions (cytotoxicity, CD107a, IFNγ, TNFα) were like primary NK cells.

Altogether, this new method of single-cell cloning in rhesus macaques could provide key information about antigen-specific NK cells, including epigenetic, metabolic, transcriptional, and phenotypic profiles, which could be exploited to further enhance antiviral NK cell activity in future vaccine strategies or cell therapies.

Supported by grants from NIH (R01 AI158516, R01 DK130472, R01 AI161010, NIH-funded BEAT-HIV Martin Delaney Collaboratory to cure HIV-1 infection by Combination Immunotherapy UM1 AI164570).
The Immunoregulatory Role of CMKLR1 within the CNS During WNV Encephalitis
Authors: Kwok Keven , Durrant Douglas M
Published: August 2023

West Nile Virus (WNV)-induced neuroinvasive disease (WNND) has been a public health concern in North America since its introduction in 1999. In recent years, severe neurological illness has been reported much more frequently, together with neuromuscular manifestations. Previous studies have shown that dendritic cells (DCs) within the central nervous system (CNS) are required to restrict WNV replication and immunopathology by regulating anti-viral T-cell responses. In this study, we examined the role of CMKLR1 within the CNS during WNV encephalitis. CMKLR1-expressing microglial cells and CNS-infiltrating myeloid DCs have been shown to have an inflammatory role during autoimmune neurological disorders, yet their contribution to WNND remains unknown. In addition, chemerin, the ligand for CMKLR1, was upregulated in the CNS of WNV-infected mice. To evaluate the role of CMKLR1, we infected CMKLR1-deficient (CMKLR1−/−) and wild-type (WT) mice with WNV. WNV-infected CMKLR1−/− mice exhibited increased susceptibility to WNV, loss of virologic control, and more severe clinical signs of disease. Proinflammatory chemokine expression, including CCL5, CXCL9, and CXCL10, was significantly decreased in the absence of CMKLR1 suggesting reduced leukocyte trafficking into the CNS. For future studies, we will determine the numbers and type of infiltrating leukocytes and their role in virologic control within the WNV-infected CNS. Taken together, these data will help clarify the immunoregulatory role of CMKLR1 during WNV encephalitis.

NIH-SC3GM130472
The entry receptor LDLRAD3 is critical for Venezuelan equine encephalitis virus pathogenesis and neuroinvasion in mice
Authors: Kafai Natasha M , Janova Hana , Alippe Yael , Muraro Stefanie , Diamond Michael S
Published: August 2023

Alphaviruses are enveloped positive-stranded RNA viruses of the Togaviridae family. Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus responsible for epidemics in equine and human populations across the Americas. Its potential for use as an aerosolized bioterrorism weapon and the demonstrated risk it poses in research settings highlight the need for countermeasures. VEEV pathogenesis is characterized by a systemic inflammatory response and central nervous system (CNS) infection, which can result in severe neurological complications and sequelae. Our laboratory identified a novel entry receptor for VEEV, low-density lipoprotein receptor class A domain containing 3 (LDLRAD3). Ldlrad3−/− mice demonstrate reduced, but not abrogated, VEEV infection in target tissues and are resistant to disease and lethality. Here we define a critical role for LDLRAD3 in controlling VEEV tissue tropism and pathogenesis by studying the kinetics of infection in wild-type and Ldlrad3−/− mice. Using detailed analyses of viral RNA levels, immunohistochemistry, flow cytometry, and primary neuronal cultures, we observed that VEEV infects LDLRAD3-expressing cell types as early as 6 h after inoculation and once in the CNS, preferentially targets neurons, and not glial cells, in a LDLRAD3-dependent manner. Overall, our studies define the role of LDLRAD3 in VEEV tropism and how LDLRAD3-dependent infection of LDLRAD3 contributes to the host inflammatory responses against VEEV. Finally, our studies demonstrate LDLRAD3-independent infection in Ldlrad3−/− mice, which suggests the existence of alternate receptors for VEEV. These findings may be relevant for the generation of novel receptor-targeted antiviral therapies.

NIAID F30AI164842
The mutation PTPN6Ala455Thr causing pulmonary emphysema affects B cell populations and causes spontaneous pulmonary tertiary lymphoid tissue formation
Authors: Pineault Marie , Bouffard Gabrielle , Milad Nadia , Tremblay Félix , Lechasseur Ariane , Routhier Joanie , Aubin Sophie , Beaulieu Marie-Josée , Bossé Yohan , Maltais François , Morissette Mathieu C
Published: August 2023

Rationale

A new mutation causing severe pulmonary emphysema was recently discovered in a French-Canadian family. The mutation is located in the PTPN6 gene (PTPN6Ala455Thr) leading to a reduction in the activity of SHP-1, a phosphatase that regulates pathways associated to B cells. We aimed to characterize B cell abnormalities associated with aging in both humans and mice carrying this mutation.

Methods

Circulating B cell populations in humans carrying the PTPN6Ala455Thr mutation were analyzed by flow cytometry and immunoglobulin levels were measured in serum. In mice carrying the same mutation (Ptpn6Ala457Thr) aged up to 12 months, B cell populations were analyzed by flow cytometry and lung tissue histology performed. Mice were immunized with the T cell-independent antigen and NP-specific antibodies were measured in plasma.

Results

Human carriers of the PTPN6Ala455Thr mutation had reduced IgG1 and IgG4, and increased IgG3 levels as well as a lower ratio of naive B cells/ isotype switched memory B cells compared to controls. Aging Ptpn6Ala457Thr mice developed spontaneous pulmonary tertiary lymphoid tissues and exhibited higher proportion of B-1 cells, age-associated B cells and germinal center B cells along with lower B-2 cells in the lung and spleen. Ptpn6Ala457Thr mice had lower levels of NP-specific IgG1 antibodies compared to the wild-type group.

Conclusion

SHP-1 appears to affect aging of B cells as the PTPN6Ala455Thr mutation leads to changes in B cell populations associated with age, tertiary lymphoid tissue formation and an alteration of immunoglobulin levels. The link between B cell abnormalities, aging and emphysema requires further investigation.

Funding:CIHR, FRQS

CIHR, FRQS
The divergence of PD-1+CXCR5+CD4+ T cell differentiation and stage-specific regulation of GC-Tfh cells
Authors: Zhu Fangming , McMonigle Ryan J , Schroeder Andrew R , Xia Xianyou , Figge David , Greer Braxton D , González-Avalos Edahí , Sialer Diego O , Wang Yin-Hu , Chandler Kelly M , Getzler Adam J , Brown Emily R , Xiao Changchun , Kutsch Olaf , Pipkin Matthew W , Hu Hui
Published: August 2023

T follicular helper (Tfh) cells were discovered as CD4+ T cells in the B cell follicles that express CXCR5 and help germinal center (GC) B cell responses. However, it is not clear which PD-1+CXCR5+Bcl6+CD4+ T cells will differentiate into PD-1hiCXCR5hiBcl6hiGC-Tfh cells while the others have a different fate, nor is it clear how GC-Tfh cell differentiation is regulated. Here we report that the sustained Tigit expression in PD-1+CXCR5+CD4+ T cells marked the precursor Tfh (pre-Tfh) to GC-Tfh transition, whereas Tigit−PD-1+CXCR5+CD4+ T cells upregulated IL-7Ra to become CXCR5+CD4+ T memory precursor/memory cells—with or without a CCR7+ (or CCR7+CD62L+) central memory phenotype. Our study further shows that pre-Tfh cells undergo substantial further differentiation at the transcriptome and chromatin accessibility levels to become GC-Tfh cells. Mechanistically, we find that c-Maf exerted an important function in the pre- to GC-Tfh transition, and we have identified c-Maf downstream factor Plekho1 as a novel regulator that played a stage-specific role in GC-Tfh cell differentiation.
Covalent TCR-pMHC Interactions: A New Mechanism for T Cell Antigen Recognition and T cell Activation
Authors: Zareie Pirooz , Szeto Christopher , Wirasinha Rushika , Zhang Justin Boxuan , Nguyen Andrea , Riboldi-Tunnicliffe Alan , La Gruta Nicole , Gras Stephanie , Daley Stephen
Published: August 2023

Noncovalent bonds typically mediate interactions between a T cell receptor (TCR) and a peptide-major histocompatibility complex (pMHC) ligand. In this study, we describe a novel mechanism involving a covalent TCR-pMHC interaction through a cysteine-cysteine disulfide bond. By introducing cysteine resodues into the TCR CDR3 and into the peptide of a known TCR-pMHC combination, we show that this disulfide bond formation does not require structural rearrangement and can still occur even when the initial affinity of the TCR-pMHC interaction is low. This mechanism strongly activates TCR signaling and has potential applications in basic immunology to further our fundamental understanding of T cell activation/differentiation as well as applied clinically for improving the efficacy and sensitivity of adoptive T cell therapies. Our findings shed new light on T cell activation and provide additional avenues for the development of modern immunotherapy.
DNA demethylation fine-tunes IL-2 production during thymic development and promotes Regulatory T cell differentiation
Authors: Issuree Priya D , Teghanemt Athmane , Heath Jace , Thurman Andrew , Pezzulo Alejandro
Published: August 2023

Regulatory T (T reg) cells developing in the thymus are essential to maintain tolerance and prevent fatal autoimmunity in mice and humans. Expression of the T reg lineage-defining transcription factor FoxP3, is critically dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that ten-eleven translocation (Tet) enzymes, which are DNA demethylases, are required early during double positive (DP) thymic T cell differentiation and prior to the upregulation of FoxP3 in CD4 single-positive (SP) thymocytes, to promote Treg differentiation. We show that Tet enzymes selectively control the development of CD25− FoxP3lo CD4SPTreg cell precursors and are critical for optimal TCR-dependent IL-2 production, which drive chromatin remodeling at the FoxP3 locus as well as other Treg-effector gene loci in an autocrine/paracrine manner. Together, our results demonstrate a novel role for DNA demethylation in regulating the TCR response and promoting Treg cell differentiation. These findings highlight a novel epigenetic pathway to promote the generation of endogenous Treg cells for mitigation of autoimmune responses.
Sex disparities in pathological features and autoimmunity in high fat diet associated lupus mouse model
Authors: Zhang Xin , Meng Juan , Lobo Gitanjali , Patel Ronak , Ray Alicia Nicole , Quinet Robert , Davis William , Zakem Jerald , Hayat Samina , You Zongbing
Published: August 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is more common in women than men. SLE has been reported with diverse clinical phenotypes of varying severity, which may be caused by sex hormones and environmental factors. Our recent studies showed that high-fat diet (HFD) exacerbated lupus development in MRL/lpr lupus-prone mice. Here we explored the gender difference in lupus progress and autoimmune response to HFD.

Twenty male and twenty female MRL/lpr mice were evenly grouped and fed with a regular diet (RD, 10% fat) or HFD (60% fat) for 14 weeks. Body weights were recorded weekly. SLE progression was monitored by skin lesions, urine protein, titers of anti-dsDNA antibody in serum. Kidney and skin from the dorsum of the neck were embedded for H&E, PAS, and Masson’s staining quantified as kidney index and skin score. Immune cells in the spleens were identified by immunofluorescence staining and flow cytometry.

HFD induced a greater weight gain in female mice than male mice (p<0.01). Skin rash showed up as early as week 6 in female HFD group with a greater histopathological skin score (p<0.01). Splenomegaly, proteinuria, and anti-dsDNA level were increased only in male HFD group. Kidney pathological changes were more severe in male HFD mice with significant increase of kidney index (p<0.05). Significant increases of germinal center B cells, plasma cells, and T follicular helper cells were observed in HFD mice (p<0.05).

SLE progression is sexually dimorphic in lupus-prone mice in response to HFD. Our results parallel many known clinical lupus phenotypes and sexual dimorphism in which male patients are more likely to have severe disease (such as nephritis) than female lupus patients who may have a broader range of lupus symptoms.

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