bluuebunny/crossref_metadata_2025 huggingface dataset:
Resp-Aid in Ova-challenged mice decreased airway inflammatory cell infiltration, AHR and IL-13 cytokine expression levels
Authors: Nascimento Amanda AN , Sandini Thaisa Meira , Thompson Brooke , Schneberger David , Bernardi Maria Martha , Singh Baljit , Kirychuk Shelley
Published: January 2023

Introduction

Asthma is an inflammatory disease caused by repeated immediate phase hypersensitivity and late-phase allergic reactions in the lungs. Resp-Aid is a formula composed of 9 medicinal herbs that supports in the resolution of respiratory disorders (Coltsfoot, Marshamallow, Plantain, Mullein, Horehound, Licorice, Lobelia, Ginger, and Myrrh). These herbs, alone, have shown positive effects in reducing symptoms associated with asthma. However, the effects of the combination of these herbs on asthma is not well understood. Therefore, we conducted a series of studies to explore the mechanism of action of Resp-Aid.

Material and Methods

C57Bl/6 male mice (n = 7 each group) were challenged with or without ovalbumin (OVA). After the challenge, they were divided into 4 groups where 2 groups received 1 drop of Resp-Aid twice daily for 7 days and 2 groups acted as control. On day 5 of the treatment with Resp-Aid the airway hyperresponsiveness (AHR) was measured. After the treatment with Resp-Aid, lungs and bronchoalveolar lavage (BAL) fluid were collected and the cytokines (IL-4, IL-5, IL-13, IL-17a, IFNy), MPO and EPO were quantified.

Results

All OVA-challenged mice had increased AHR, leukocytes and cytokine levels compared to their respective controls showing the development of an asthmatic response. The Resp-Aid asthmatic groups had decreased AHR, IL-13, neutrophils and EPO levels compared to asthmatic mice.

Conclusion

This data show that Resp-Aid changed the immune response related to asthma, and it may be a potential target for therapeutic intervention in allergic lung disease or asthma.
Paneth cells regulate allergic sensitization in the gastrointestinal tract
Authors: Kim Eunsoo , Burnett Drew J. , Joldrichsen Marisa R , Mori-Akiyama Yuko , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

The gut immune homeostasis plays a major role in controlling sensitization to food allergens and the development of allergic responses in the gastrointestinal tract, but also to skin or lung. Epithelial cells sensing of commensal microbes is crucial for the maturation of the gut immune system and antibiotic treatment during early life is believed to have contributed to the increased incidence of food allergy. Paneth cells secrete antimicrobial products and cytokines in the crypts of the small intestine, but their role in food allergy remains elusive. We found that SOX9ΔIEC mice, which lack of Paneth cells, display dysbiosis with increased microbial diversity. Interestingly, Paneth cell deficiency have increased numbers of ILC2 in the intestinal lymphoid tissues. Upon oral sensitization with a model food antigen in the presence of cholera toxin, SOX9ΔIEC mice developed significantly higher allergen-specific serum IgE responses, and enhanced signs of allergic responses (i.e., swelling, hypothermia, impairment of lung functions) after nasal allergen challenge. Transplantation of fecal materials from SOX9ΔIEC mice was able to transfer the hyper-allergenic phenotype to control wild-type mice and bacteria-free fecal extract from SOX9ΔIEC mice increased the number of IgE producing B cells than fecal extract from control mice with treatment of anti-CD40 and IL-4 in vitro. Ongoing studies are assessing the metabolites present in the intestinal lumen of SOX9ΔIEC mice and their effect of intestinal epithelial cells and myeloid cells to elucidate the mechanisms by which impaired or defective anti-microbial functions of Paneth cells regulate allergic sensitization of the GI tract.
Tracking the dynamics of the peanut-specific CD4+ T cell response in humans reveals novel disease-associated subsets
Authors: Lozano-Ojalvo Daniel , Berin M. Cecilia
Published: January 2023

Peanut allergy is associated with the presence of CD154+ Th2A and Tfh13 cells. The use of a single activation marker such as CD154 at a single time-point after stimulation, may bias the detection of terminal effector cells. Our objective was to track the dynamics of T cell in response to peanut to obtain a more comprehensive picture of allergen-specific immunity associated with disease.

PBMCs from peanut allergic (n=65), food allergic but peanut tolerant (n=5), and healthy controls (n=3) were incubated with a peanut extract for 6–48 h. Cell activation was monitored by conventional and high dimensional flow cytometry (30-marker panel) and cytokine production by cytokine capture assay. Analysis was performed by 2-D gating and unsupervised clustering (FlowSOM).

Peanut induced an upregulation of CD154/CD69 on mature effector memory cells that was observed at 6h and maintained for 48h. Cytokine capture showed secretion of IL-2, -4, -13, and -5 from these cells, which peaked at 6 h. The use of additional timepoints and activation markers identified two additional subsets specifically associated with disease: CD137+ and OX40/CD25 on other two cell subsets after 24 h. CD137+ cells secreted IFN-g, while OX40/CD25 cells secreted none of the cytokines tested. OX40/CD25 cells were reduced by IL-2 neutralization and likely include bystander activated cells. Clustering by FlowSOM analysis confirmed the identity of these three unique clusters, and provided additional phenotypic markers for CD154+ (CCR4+/CCR6+), CD137+ (CXCR3+), and CD25/OX40 (Foxp3+CD27−).

Analysis of the dynamics of peanut-induced T cell activation revealed novel subsets including Th1 and regulatory cells co-existing with previously described highly differentiated Th2 cells.
Protease allergens induce attenuated T follicular helper responses compared to aeroallergens resulting in low-affinity IgE production
Authors: Tharakan Anuj , Martin Rebecca
Published: January 2023

Anaphylaxis is a type of severe allergic reaction that is initiated by cross-linking of high-affinity IgE on mast cells leading to mast cell degranulation. Recent evidence demonstrates that T follicular helper cells (Tfh) producing both IL-4 and IL-13, termed Tfh13s, are necessary for the generation of anaphylactic IgE. The mechanisms regulating the polarization of these T cells, however, remain unclear. Parasite infection, which induces strong IgE responses, fails to induce Tfh13 polarization. We therefore hypothesized that papain, a cysteine protease that resembles parasitic CA family peptidases, would similarly fail to induce Tfh13 polarization compared to the aeroallergen Alternaria due to differential action on dendritic cells (DCs). We found that intranasal immunization with LPS+OVA and Alternaria+OVA induced a 2-fold Tfh expansion in the draining lymph node compared to papain+OVA. Additionally, Alternaria stimulated Tfh13 polarization and high-affinity IgE production while papain only induced Th2 responses and non-specific IgE. Analysis of DC migration by immunization with fluorescently tagged OVA alongside these adjuvants revealed markedly different migration kinetics following each stimulus. However, numbers of Ag+ DCs, amount of antigen uptake, and proportions of Ag+ migratory vs. resident DCs in the mediastinal lymph nodes were similar between all groups. Thus, we demonstrate that papain, in contrast to Alternaria, is unable to induce Tfh13 polarization and anaphylactic IgE. We propose that the differential effects of these adjuvants are likely mediated by alternative mechanisms of DC licensing and distinct DC transcriptional profiles.
A novel bovine beta-lactoglobulin-induced passive systemic anaphylaxis model using humanized NOG hIL-3/GM-CSF Tg mice
Authors: Ito Ryoji , Katano Ikumi , Otsuka Iyo , Takahashi Takehi , Ito Mamoru , Simons Peter
Published: January 2023

Background

Food allergy is a common disease that is caused by intake of allergen-containing foods such as milk, eggs, peanuts, and wheat. Systemic anaphylaxis is a severe hypersensitive allergic reaction resulting from degranulation of mast cells or basophils after crosslinking of their surface high affinity IgE receptors (Fce-RI) by allergen-specific IgE and allergens. In this study, we developed a novel human mast cell/basophil-engrafted mouse model that recapitulates systemic anaphylaxis by milk’s major allergen beta-lactoglobulin (BLG).

Method

Human CD34+ hematopoietic stem cells were transferred into NOG (nonTg) or NOG hIL-3/GM-CSF Tg mice. After 14–16 weeks, mature human basophils and mast cells increased in Tg mice, and bovine BLG specific human IgE was intravenously injected in these humanized mice, followed by either intravenous or oral bovine BLG protein exposure, 1 day later. Subsequently, allergic reactions were monitored by measuring body temperature, serum histamine level, and anaphylaxis scoring. Furthermore, the adrenaline agent, ‘epinephrine’ was used to investigate whether the allergic symptoms could be suppressed.

Results

After bovine BLG exposure, body temperature in Tg mice, but not in nonTg mice, gradually decreased within 10 minutes and 80 % of Tg mice died within 60 minutes. Serum histamine levels and anaphylaxis scores in Tg mice were markedly increased compared to nonTg mice after 30 minutes BLG challenge. These allergic symptoms were significantly inhibited by epinephrine treatment in Tg mice.

Discussion

The current hIL-3/GM-CSF Tg mouse model could possibly be used to develop novel anaphylaxis drugs in food allergy.
Selective Serotonin Reuptake Inhibitors Suppress IL-33-Mediated Mast Cell Function
Authors: Kee Sydney Ann , Haque Tamara , Ryan John J
Published: January 2023

Interleukin-33 (IL-33) is an inflammatory cytokine that promotes allergic disease by activating ILC2, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Therefore, clinical interventions targeting IL-33 may be beneficial for allergic disease. We have sought ways of re-purposing FDA-approved drugs for use in allergic inflammation. Because drugs in the selective serotonin reuptake inhibitor class (SSRIs) have documented anti-inflammatory effects, we tested the hypothesis that SSRIs can attenuate mast cell function. We show that the SSRI fluoxetine inhibits IL-33-mediated cytokine production in mouse bone marrow derived mast cells in vitro and reduces IL-33-induced peritonitis in vivo. These data indicate that fluoxetine may be an effective therapeutic for IL-33- and mast cell-associated diseases.
Evidence for sexual dimorphism in mast cell-mediated innate immune response against Group B Streptococcus
Authors: Piliponsky Adrian Martin , Gendrin Claire , Quach Phoenicia , Guga Eric , Bhise Gauri , Saha Siddhartha , Brokaw Alyssa , Moeser Adam , Rajagopal Lakshmi
Published: January 2023

Mast cell response to stimuli was recently described to be sexually dimorphic with female mast cells exhibiting increased ability to produce and release mediators upon activation. This suggests that female mast cells may have evolutionary advantages and an important role in innate immunity. In fact, sexual dimorphism of mast cell innate immune function may be particularly relevant to our understanding of mast cell contribution to host immunity against Group B Streptococcus, a bacterial pathogen associated with pre-term birth. This hypothesis is supported by our published studies, which indicate that mast cells contribute to host defense against systemic GBS infection and our observations that female mice are more efficient in their ability to limit systemic GBS infection than male mice. The contribution of individual sexually dimorphic factors to the immune response against GBS is unknown.

Here, we show that female mice produce higher amounts of pre-formed coagulation factor XIIIA (FXIIIA) when compared to corresponding male mice, and that female bone marrow-derived cultured mast cells contain higher amounts of FXIIIA than male mast cells. We used FXIII-deficient mice to show that FXIIIA decreases the severity of systemic GBS infections. Moreover, our studies indicate that FXIIIA limits GBS dissemination by enhancing bacteria entrapment within fibrin clots.

Together, these studies have the potential to provide novel mechanistic understanding into how female mast cells contribute towards defense against GBS infection and promote successful pregnancies via the release of FXIIIA.
Blocking of mast cell-mediated passive anaphylaxis to peanut using covalent heterobivalent inhibitor in a humanized mouse model
Authors: Alakhras Nada Salem , Shin Jaeho , Smith Scott S. , Bilgicer Basar , Kaplan Mark H.
Published: January 2023

Anaphylaxis to peanut allergen is a severe hypersensitive allergic reaction characterized by life-threatening symptoms. Anaphylaxis is mediated by IgE-dependent mast cell activation that leads to the release of inflammatory mediators. In a previous report, we described the design of a covalent heterobivalent inhibitor (cHBI) that selectively forms covalent bonds with allergen specific-IgE (sIgE) on mast cells and abates allergic reactions in ex-vivo peanut challenges. Here, we utilized a humanized mouse model to recapitulate key features of anaphylaxis to peanut in humans, and we examined the efficacy of cHBI in blocking the anaphylactic reaction in this model. NSGS (NOD-scid Il2rgnull IL-3/GM-CSF/SCF) mice engrafted with human hematopoietic stem cells were treated with an allergen-specific human IgE monoclonal antibody and then challenged with a peanut allergen. We found that the peanut-challenged humanized mice can develop rapid anaphylaxis and mast cell degranulation following challenge. The anaphylactic reaction was characterized by a substantial decline of core body temperatures and upregulation of human mast cell degranulation markers. Additionally, we found that the severity of the anaphylactic reaction is hIgE dose-dependent. Most importantly, cHBI protected the mice from developing an anaphylactic reaction demonstrating the ability of cHBI to block IgE-mediated allergic reactions in the humanized mice. Overall, our results show that the humanized mouse model is a novel model to study human mast cell IgE-mediated allergic reactions and demonstrate that cHBI is an effective potential therapeutic inhibitor for food allergy.
IFNγ-induced mechanisms restrict intracellular replication of the lysosome-adapted bacterial pathogen, Coxiella burnetii
Authors: Ganesan Sandhya , Andrews Jenna M. , Alvarez Natalie N. , Baskerville Maia J. , Roy Craig R.
Published: January 2023

Coxiella burnetii is an intracellular bacterial pathogen that causes flu-like illness called Q-fever. C. burnetii extensively subverts host-cell vesicle traffic to create an acidic, lysosome-derived vacuole to support its intracellular replication. This is a unique evolutionary adaptation since lysosomal environment is hostile to most microbes. IFNγ signaling is essential for host protection against C. burnetii, but the underlying immune defense strategies are not well-defined. Using siRNAs to deplete IFNγ-induced genes, we identified that restriction of intracellular C. burnetii requires atleast two host factors, Indoleamine Dioxygenase 1 (IDO1) and Syntaxin 11 (STX11). IDO1 activity depletes tryptophan availability by catabolizing cellular tryptophan. This mechanism renders C. burnetii metabolically inactive since it is a tryptophan auxotroph, but is not sufficient to clear the infection. STX11 is a SNARE protein (Soluble NSF Attachment Protein Receptor) that promotes fusion of lysosome-derived organelles with target membranes. Bacterial replication as well as size of the C. burnetii-containing vacuole (CCV) are significantly increased in STX11-deficient cells. These cells also exhibited increased extracellular release of lysosomal proteins. STX11 over-expression reverses these phenotypes. Furthermore, STX11 accumulated in the CCV during infection. We hypothesize that STX11 regulates CCV-associated cargo-sorting and vesicle fusion events, leading to a sub-optimal vacuolar environment for C. burnetii. These findings demonstrate that, IFNγ-induced nutrient deprivation and altered intracellular niche can synergistically and effectively restrict lysosome-adapted pathogens.
Preoperative exercise protects the liver from surgical stress by altering the immune environment
Authors: Zhang Hongji , Xia Yujia , Onuma Amblessed , He Jiayi , Tsung Allan , Huang Hai
Published: January 2023

Introduction

Inflammation after liver surgery can lead to post-operative liver dysfunction, significant morbidity and mortality. Preoperative exercise has been associated with reduced postoperative complications. However, the mechanisms behind remain to be elucidated. We hypothesize that preoperative exercise can alter the hepatic immune microenvironment(HIM) to allow protection from liver injury.

Methods

8-week-old mice were randomly divided into exercise and sedentary groups. The exercise group ran on a motorized treadmill at a speed of 12.5 m/min for 60 min/day, 5 days/week for 4 weeks whereas sedentary mice were exposed to the same treadmill but held motionless. Both groups were then subjected to surgical stress as modeled by 70% partial liver ischemia/reperfusion (I/R). Single-cell(SC)RNA sequencing was performed on hepatic CD45+ cells isolated from both groups.

Results

The exercise group had significantly lower serum alanine transaminase compared to sedentary mice after liver I/R. SC-RNA analysis of the hepatic immune cells revealed distinct macrophage subpopulations that correlated with cells derived from both groups. Among these macrophages, the expression of genes related to anti-inflammatory cytokines were significantly higher in exercised mice compared to sedentary mice, whereas pro-inflammatory cytokines and chemokines were significantly lower suggesting that exercise modulates the transcriptomic change in macrophages towards an anti-inflammatory phenotype.

Conclusion

Our study showed that preoperative exercise alters the HIM which offers a rationale for preoperative exercise therapy for patients undergoing liver surgery thereby decreasing post-operative inflammation and morbidity.
Distinct contributions of Type I and Type III IFNs in innate fungal immunity
Authors: Espinosa Vanessa , Dutta Orchi , Mc Elrath Constance , Du Peicheng , Chang Yun-Juan , Cicciarelli Bryan , Pitler Amy , Whitehead Ian , Obar Joshua J , Durbin Joan , Kotenko Sergei , Rivera Amariliz
Published: January 2023

Type I (IFN-α/β) and Type III (IFN-λ) interferons are important mediators of antiviral immunity but whether they are required for antifungal defense is unclear. We report that mice defective in Type I and/or Type III IFN receptor expression are highly susceptible to infection with the opportunistic human fungal pathogen Aspergillus fumigatus (Af). Type I IFN (IFNAR) and Type III (IFNLR1) receptor expression on hematopoietic cells was required for antifungal defense while their expression on non-hematopoietic was dispensable. Consistently, mice with gene-specific deletion of IFNLR1 or STAT-1 on granulocytes were highly susceptible to Af. These data demonstrate a requisite and non-redundant role for Type I and Type III IFNs for the activation of antifungal immunity. In addition, we found that Type III IFNs act as important activators of reactive oxygen species generation in antifungal neutrophils, and require early expression of Type I IFN for optimal induction. Importantly, CCR2+ monocytes act as an important source of Type I IFN, and are required for the optimal expression of Type III IFN and fully functional neutrophil antifungal activity. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2+ monocytes or by exogenous administration of IFN-a and IFN-l. Altogether, our data identified Type III IFNs as major regulators of neutrophil activation, and Type I IFNs as early activators of IFN-λ expression and response. Innate antifungal defense is thus coordinated by the actions of Type I and Type III IFNs, a finding that suggests a broader therapeutic potential for these cytokines as activators of antifungal immunity.
CD3 downregulation on T-cells is concomitant with arginase upregulation on myeloid cells in the synovial fluid of patients with juvenile idopathic arthritis
Authors: Giacalone Vincent D , Cammarata-Mouchtouris Alexandre , Moncada Diego , Shenoy Sreekala , Ponder Lori , Gergely Talia , Vega-Fernandez Patricia , Manos Cynthia , Flanagan Elaine , Prahalad Sampath , Tirouvanziam Rabindra
Published: January 2023

Rationale

Juvenile idiopathic arthritis (JIA) is an inflammatory autoimmune disorder driven by dysfunction of the joint tissue and abnormal immune responses. As deep phenotyping of synovial fluid (SF) might yield new targetable mechanisms of inflammation in JIA patients, we initiated the Emory Multi Omics JIA Immunology (EMOJI) project.

Methods

Patients with JIA between the ages of 6 and 18 were consented and enrolled for collection of SF from the knee and venous blood. Blood and SF leukocytes were analyzed by flow cytometry. Platelet-free plasma and debris-free SF supernatant were obtained by dual centrifugation and a 20-plex chemiluminescent assay (Meso Scale Discovery) was used to quantify cytokines.

Results

We report data on blood and SF collected at the same visit from 7 JIA patients. JIA subtypes represented were psoriatic (N=1), oligoarticular extended (N=1), oligoarticular persistent (N=2) and polyarticular (N=3) arthritis; all rheumatoid factor-negative. CD3 expression was decreased on SF compared to blood T-cells. Concomitantly, expression of the CD3-inhibitory enzyme arginase was increased on SF compared to blood neutrophils and macrophages. However, arginase-rich primary granules were not significantly exocytosed by SF neutrophils. Finally, we measured high levels of neutrophil chemoattractants (IL-8, G-CSF, and IL-1α) in SF compared to plasma.

Conclusions

Neutrophils are recruited to the joints of JIA patients and accumulate arginase on their surface, presumably released from tertiary, but not primary, granules. Arginase activity in the SF may lead to CD3 downregulation and T-cell inhibition, potentially in an effort to limit autoimmune complications.
-Brucella induction of RIDD activity promotes intracellular parasitism by subverting BLOS1-controlled immune defense
Authors: de Figueiredo Paul , Wells Kelsey , He Kai , Pandey Aseem , Cabello Ana-Lucia , Gomez Gabriel , Rice-FIcht Allison , Sze Sing-hoi , Qian Xiaoning , Ficht Thomas A
Published: January 2023

The intracellular bacterial pathogen and biothreat agent Brucella is the causative agent of brucellosis, a global zoonotic disease that is associated with acute and chronic symptoms in humans and animals. The mechanisms by which the pathogen colonizes host cells remain obscure. Here, we demonstrate that infection activates the regulated IRE1-dependent decay (RIDD) of mRNA encoding BLOS1 (biogenesis of lysosome-related organelles 1 subunit 1), a protein that promotes endosome-lysosome fusion. RIDD-deficient host cells as well as mice harboring a RIDD-incompetent variant of IRE1a displayed resistance to infection. Moreover, host cells carrying a RIDD resistant variant of BLOS1 displayed enhanced trafficking of the pathogen to lysosomes, and increased resistance to intracellular parasitism. Finally, cells harboring mutations in BLOS1 displayed increased susceptibility. Taken together, the data demonstrate that host RIDD activity on BLOS1 transcripts regulate Brucella intracellular parasitism by disrupting BLOS1-directed lysosomal trafficking activity, thereby promoting maintenance of the pathogen’s replicative niche in the endoplasmic reticulum.
Clearance of Plasmodium falciparum-infected red blood cells by NK cells and monocytes
Authors: Sekar Padmapriya , Arora Gunjan , Long Eric O
Published: January 2023

Antibodies naturally acquired during repeated exposure to the parasite Plasmodium falciparum are essential for control of blood-stage malaria. NK cells are innate immune cells with an important role in containing viral infections and tumor growth, mainly by direct cytotoxicity and inflammatory cytokine production. Recently, we reported that primary human NK cells lyse Plasmodium falciparum-infected red blood cells (iRBCs) via antibody-dependent cellular cytotoxicity (ADCC) in presence of IgG isolated from adults living in a malaria-endemic region. This NK-mediated lysis results in inhibition of parasite growth in vitro. Live imaging showed release of parasitophorous vacuoles (PV) from iRBCs after incubation with NK cells. Many of these PVs gave propidium iodide-positive signals, suggesting loss of viability of P. falciparum inside the PVs after NK-mediated iRBC lysis. I am interested to know whether and how PVs are eliminated and postulate that monocytes play a role in clearance of PVs, thereby reducing the inflammatory response. To observe the interaction of NK cells and monocytes with PVs, I isolated PVs from iRBCs by mild saponin treatment and mechanical disruption of RBC membranes. Purified PVs bind IgG isolated from adults living in a malaria-endemic region, showing that adults develop antibodies against Plasmodium antigens at the surface of PVs. Preliminary live imaging experiments show phagocytosis of PVs by monocytes. Currently I am testing the contribution of Fc receptors on monocytes to phagocytosis of PVs and the interaction of NK cells with PVs.
Bcl3 dependent regulation of Dendritic Cell function in Toxoplasma gondii infection
Authors: Guha June , Claudio Estefania , Jankovic Dragana , Siebenlist Ulrich
Published: January 2023

The atypical IκB family member Bcl-3 associates with p50/NF-κB1 or p52/NF-κB2 homodimers in nuclei, thereby either positively or negatively modulating transcription in a context-dependent manner. Bcl-3 is critical for host resistance to Toxoplasma gondii and Bcl-3-deficient mice succumbed within 3–5 weeks after infection, correlating with an apparently impaired Th1-type adaptive immune response rather than a defect in the innate immune response. Surprisingly, mice lacking Bcl-3 in CD11c+ dendritic cells (DC) were as susceptible as mice globally deficient for Bcl-3, demonstrating that Bcl-3 is required in these cells to prime protective T-cell-mediated immunity to T. gondii. It remains to be determined however whether conventional or monocyte derived DCs are critical mediators of a successful T cell response against the invading pathogen.

We generated mice specifically lacking Bcl-3 in conventional DCs (Zbtb46 cre knockout [KO]) or in monocyte derived cells, including monocyte derived DCs (LysM cre KO). Surprisingly, both conditional KO strains were as susceptible to T. gondii infection as CD11c+ conditional or total KO mice. Interestingly, we detected the presence of a unique subpopulation of conventional DCs that expressed the monocytic markers CD64 and Ly6C in infected spleen and various tissues including lung. Furthermore, this population was reduced in the absence of Bcl3. This hybrid DC population may have a crucial role in the T cell responses after infection, in particular in the generation of inflammatory cytokines such as IFNg, a cytokine which is indispensable for host protection.
IκBζ regulates NLRP3 inflammasome activation
Authors: Ahn Huijeong , Lee Geun-Shik
Published: January 2023

Inflammasome activation induces the maturation and secretion of interleukin (IL)-1β and -18, and is dependent on NF-κB signaling to induce the transcription of the inflammasome components, called the priming step. This study elucidated the role of IκBζ, an atypical IκBs (inhibitor of κB) and a coactivator of NF-κB target genes, on the activation of inflammasome. Bone marrow-derived macrophages (BMDMs) that originated from IκBζ-encoding Nfkbiz gene depletion mice presented a defect in NLRP3 inflammasome activation. In addition, the Nfkbiz+/− and Nfkbiz−/− mice significantly attenuated serum IL-1β secretion in response to a monosodium urate injection, a NLRP3 trigger, when compared with Nfkbiz−+/+ mice. The lack of IκBζ in BMDMs produced a disability in the expression of Nlrp3 and pro-Il1β mRNAs during the priming step. In addition, ectopic IκBζ expression enhanced the Nlrp3 promoter activity. Overall, IκBζ controlled the activation of NLRP3 inflammasome by upregulating the Nlrp3 gene during the priming step.
Tyrosine Phosphorylation of NLRP3 by Src Family Protein Tyrosine Kinase Lyn Suppresses NLRP3 Inflammasome Activation
Authors: Xiao Yizhi , Tang Juan , Lin Guoxin , Guo Hui , Zhang Jian
Published: January 2023

The NLRP3 inflammasome is a multi-protein complex that triggers the activation of inflammatory caspase-1 and the maturation of IL-1β in response to microbes and danger signals in host cells. However, how the NLRP3 inflammasome is regulated is not fully understood. Here, we show that NLRP3 is tyrosine phosphorylated upon activation of the NLRP3 inflammasome, and that NLRP3 tyrosine phosphorylation correlates with its ubiquitination. Further, we identified Lyn as the protein tyrosine kinase that phosphorylates NLRP3 at Tyr 918, which facilitates its ubiquitination and proteasome-mediated degradation. Consistent with these data, NLRP3 tyrosine phosphorylation and ubiquitination is abrogated in macrophages lacking Lyn, which correlates with heightened IL-1 production. Therefore, our data demonstrate that Lyn-mediated tyrosine phosphorylation of NLRP3 is a prerequisite for its ubiquitination, thus dampening NLRP3 inflammasome activity.
Au nanorod-induced NLRP3 inflammasome activation is mediated by ER stress
Authors: Vandebriel Rob , Remy Sylvie , Vermeulen Jolanda , Hurkmans Evelien , Bastus Neus , Pelaz Beatriz , Puntes Victor , Parak Wolfgang , Pennings Jeroen , Nelissen Inge
Published: January 2023

The widespread and increasing use of engineered nanomaterials (ENM) increases the risk of human exposure, generating concern that ENM may provoke adverse health effects. In this respect, their physicochemical characteristics are critical. The immune system may respond to specific ENM properties by inflammatory reactions. Inflammasome activation has drawn significant attention since inflammasomes, especially NLRP3 respond to a wide range of stimuli including nanoparticles, and their activation is associated with various inflammatory diseases, including lung fibrosis, obesity and type-2 diabetes. Endoplasmic reticulum (ER) stress has been reported as one of the mechanisms underlying NLRP3 inflammasome activation.

In this study, PEGylated Au ENM of ≈60 nm, having different shapes (stars, spheres and rods) were extensively characterized, and tested for possible LPS contamination. PMA-activated THP-1 cells were exposed to these ENM, and cell viability and IL-1β production were measured to assess NLRP3 activation. In addition, the exposed cells were subjected to gene expression analysis using microarray analysis to investigate related signalling pathway regulation.

PEGylated Au nanorods (NR), but not nanostars or nanospheres, showed NLRP3 inflammasome activation. Cells deficient in the NLRP3 scaffold or ASC adaptor did not show this effect. Only NR-exposed cells showed down-regulation of ER-associated cholesterol metabolism.

This finding may suggest that ER stress mediates NLRP3 inflammasome activation by Au NR.

Supported by the EU funded project FutureNanoNeeds (Grant agreement N° 604602).
Coronary artery bypass grafting surgery differentially modulates the leucocyte gene expression profile
Authors: Perros Alexis J , Chong Fenny , Engkilde-Pedersen Sanne , Esguerra-Lallen Arlanna , Rooks Kelly , Faddy Helen M , Hewlett Elise , Naidoo Rishendran , Tung John-Paul , Fraser John F , Tesar Peter , Ziegenfuss Marc , Smith Susan , O’Brien Donalee , Flower Robert L , Dean Melinda M
Published: January 2023

Coronary artery bypass grafting (CABG) surgery triggers a systemic inflammatory response activating leucocyte subsets and cytokine secretion. Changes to the patient immune profile post-surgery may contribute to adverse outcomes such as infection. Investigating patient immune modulation at the molecular level may improve our understanding of the underlying mechanisms associated with adverse outcomes post-surgery. This study assessed changes to the leucocyte gene expression profile of CABG patients (n=50) at 5 time-points (admission, intra-operative, ICU, day 3, day 5). RNA was isolated from freshly collected whole blood for cDNA synthesis, pre-amplification and RT-PCR of 48 genes using the TaqMan Gene Expression Array Cards (Applied Biosystems). Gene expression at each time-point was compared to admission (repeated-measures one-way ANOVA with Dunnett’s post-test; P<0.05). Antigen presentation capacity was decreased throughout the post-operative period, evidenced by reduced expression of key regulator genes TAP1, TAP2, HLA-DRA and CD40. CABG surgery increased gene expression of cytokines and chemokines including IL-12A, IL-6 and TNF before decreasing throughout the post-operative period. CABG also modulated the expression of signalling molecules including JAK1, RELA and STAT3, however expression returned towards baseline levels post-surgery. This study demonstrated CABG significantly impaired the patient immune response and key immune pathways at the molecular level. Understanding how CABG alters the patient immune profile assists in elucidating underlying mechanisms of adverse outcomes.
Leukotriene B4 receptors have roles in the development of endotoxic shock
Authors: Park Donghwan , Kwak DongWook , Kim Jae-Hong
Published: January 2023

Sepsis is the most common disease in patients treated in the intensive care units. Endotoxic shock, the most critical form of sepsis, is caused by gram-negative bacterial infection. However, the detailed mechanism of endotoxic shock remains unclear. In the present study, we observed that the productions of leukotriene B4 and 12(S)-hydroxyeicosatetraenoic acid, inflammatory lipid mediators acting on LTB4 receptors BLT1/2, were highly up-regulated in peritoneal lavage fluid and serum of LPS-induced endotoxic shock mouse model. Furthermore, we found that BLT1/2-dependent signaling pathways mediate the expression of IL-17, IL-6, and IL-1β, key cytokines for the development of endotoxic shock, via NF-κB activation in the LPS-induced mouse model. We also found that the inhibition of BLT1/2 significantly attenuates inflammation and tissue damages induced by endotoxic shock, and enhances the survival rate of this inflammatory complication. These results suggest that leukotriene B4 receptors play a critical mediatory role in the development of endotoxic shock. Our findings would contribute to the discovery of therapeutic targets for endotoxic shock.
Single Receptor reciprocity as message encrypted in CD40-ligand
Authors: Saha Bhaskar , Ghosh Soumyakanti , Sarode Aditya
Published: January 2023

Interaction between CD40-Ligand (CD40L) and CD40 results in cellular signaling that regulates immune responses against pathogens, autoantigens, tumors and transplantation antigens. The 115–155 amino acids stretch is responsible for CD40L functions and mutations therein results in XIgM syndrome. We hypothesize that each of these amino acids of CD40L encodes specific message. These messages are decoded by CD40 signaling and are expressed as a specific profile of functions. Each single substitution in the XIgM-related amino acids in the 115–155 41-mer peptides in CD40L selectively altered CD40 signaling and effector functions- cytokine productions, HMGCoA reductase, ceramide synthase, inducible nitric oxide synthase and arginase expression, and control of Leishmania infection. There were sets of peptides that triggered reciprocal functions from the same receptor CD40. These results suggest residue-specific encryption of a distinct set of messages. These messages collectively frame CD40L pleiotropy and offer a result-driven ligand manipulation strategy for generating antagonists or superagonists as immunotherapeutics.
Suppression of Inflammasome Activation by IRF8 and IRF4 in cDCs is Critical for T cell Priming
Authors: McDaniel Margaret , Pasare Chandrashekhar
Published: January 2023

Activation of inflammasome leads to pyroptotic cell death thereby eliminating the replicative niche of virulent pathogens, a process integral to innate immunity. While inflammasome-associated cytokines such as IL-1β and IL-18 have an established role in T cell function, whether inflammasome activation in dendritic cells (DCs) is critical for T cell priming is not clear. Here, we discover that inflammasome activation in conventional DCs (cDCs) is detrimental to the induction of adaptive immunity. We found that lymphoid organ resident cDCs actively suppress inflammasome activation to avoid pyroptotic cell death. This protection from inflammasome-induced cell death is critical for both CD4 and CD8 T cell priming. Transcription factors IRF8 and IRF4, in cDC1s and cDC2s respectively, mediate this suppression of inflammasome activation by limiting the expression of inflammasome-associated genes. We show that reduced expression of IRF8 leads to aberrant inflammasome activation in cDC1s which hampers subsequent CD8 T cell priming. Conversely, overexpression of either of IRF4 or IRF8 is sufficient to inhibit inflammasome activation in macrophages. These results uncover the molecular mechanism of inflammasome suppression in cDCs, and ascribe a novel post-developmental role for IRF4 and IRF8 in cDC function.
A high throughput screen to identify small molecule activators of IRF3-mediated antiviral apoptotic pathway
Authors: Chattopadhyay Saurabh , Glanz Anna , Chawla Karan , Rahman Tia , Walters Dean , Varghese Merina , Steimle Grace , Taylor Travis
Published: January 2023

The transcription factor interferon (IFN) regulatory factor 3 (IRF3) provides cellular antiviral response by expressing IFN and antiviral genes. In addition to the transcriptional activation, IRF3 triggers a direct pro-apoptotic pathway, RIPA, in virus-infected cells. The pathway of IRF3 activation in RIPA is independent of and distinct from the transcriptional activation of IRF3 (Chattopadhyay et al. Immunity, 2016, J Virol, 2013, 2011, EMBO J, 2010). Using knock-in mice expressing transcriptionally-inactive, but RIPA-competent, IRF3 mutant, we demonstrated that RIPA contributes to the antiviral response of the host. Because RIPA is a cellular antiviral pathway, we hypothesized that RIPA-promoting small molecules would act as antiviral agents. To test this, we conducted a high throughput screen of a library of 1,200 FDA-approved compounds to identify RIPA activators. Our screen isolated twenty-four primary candidates with diverse therapeutic activities, e.g. anti-cancer, anti-inflammatory, anti-bacterial, etc. We screened these primary candidates for their antiviral activities against RNA (vesicular stomatitis virus) and DNA (herpes simplex virus-1) viruses. The antiviral screen revealed a small subset of antiviral compounds, which specifically promote RIPA but not the transcriptional pathway of IRF3. We further validated their antiviral activities against additional viruses, e.g. respiratory syncytial virus and vector-borne flaviviruses. Therefore, we report a high throughput screen, which identified novel antiviral compounds that are specific activators of RIPA branch of IRF3. Currently, we are investigating the mechanisms of these RIPA-promoting antiviral compounds.
B cell subsets in pericardial adipose tissues and their role in cardiovascular health
Authors: Wu Lan , Van Kaer Luc
Published: January 2023

We have recently reported that murine pericardial adipose tissues house a large number of lymphoid clusters that have been referred to as fat-associated lymphoid clusters. We have further shown that these tertiary lymphoid tissues are enriched with B cells, in particular B-1a cells. These cells continuously produce a low level of IL-10 under unchallenged conditions, and further enhance the production of this cytokine upon stimulation. Following acute myocardial infarction (MI), these cells significantly expand in pericardial adipose tissues, and they accumulate in the infarcted heart during the resolution phase of MI-induced inflammation where they play a protective role by facilitating the termination of MI-induced inflammation. In our efforts to target B cells for the maintenance of cardiac health, we have now performed a comprehensive characterization of B cell subsets in murine pericardial adipose tissues focusing on cytokines. Using cytokine profiling, RNA sequencing and single-cell RNA sequencing analyses, we have found that subsets of B cells in pericardial adipose tissues are fundamentally biased. Whereas B-2 cells are equipped to produce more pro-inflammatory cytokines, B-1a cells are biased towards producing anti-inflammatory cytokines. These results have paved our way in designing effective therapeutic strategy to target subsets of B cells for the treatment of cardiovascular diseases.
Induction of T cell-independent hypermutated and protective IgG and IgA antibody responses by engagement of TLR4 or TLR5 and BCR
Authors: Rivera Carlos Eduardo , Chupp Daniel P , Yan Hui , Im John , Fernandez Maria , Zan Hong , Xu Zhenming , Casali Paolo
Published: January 2023

Somatic hypermutation (SHM) and class-switch DNA recombination (CSR) together with plasma cell and memory B cell differentiation are central to the maturation of the antibody response. Prompted by our finding that CSR and plasma cell differentiation can be induced in vitro by a TLR ligand together with BCR crosslinking, we hypothesized that TLR and BCR co-engagement can induce mature T-independent antibody responses in vivo. To test this hypothesis, we injected Tcrβ−/−Tcrδ−/− mice, which are devoid of T cells, with NP-LPS (co-engaging NP-specific BCR and B-cell TLR4) or Salmonella flagellin (co-engaging flagellin-specific BCR and B-cell TLR5). These mice readily generated class-switched B cells, plasma cells and NP- or flagellin-specific IgG antibodies. The response to NP-LPS, which was also elicited in NSG mice (as defective in most innate immune elements in addition to the absence of T and B cells) grafted with purified B cells, evoked frequent VH region mutations, including the Trp33Leu mutation that would underpin the production of high-affinity anti-NP antibodies even in the absence of sustained germinal center formation, as well as the generation of long-lived memory B cells. These, together with the strong in vivo bactericidal activity of Salmonella flagellin-specific IgG and IgA elicited in Tcrβ−/−Tcrδ−/− mice, demonstrated that TLR/BCR co-engagement can induce mature, anamnestic and protective antibody responses, thereby providing the basis for new TLR-based B cell-targeted vaccine strategies to overcome T cell deficiencies.
IL-27-producing B cells are key regulators of anti-viral immunity and antibody responses
Authors: Pratumchai Isaraphorn , Huang Zhe , Zak Jaroslav , Teijaro John R
Published: January 2023

Emerging evidence indicates that B lymphocytes regulate immunity by producing antibodies, presenting antigens and secreting cytokines that provide signals to other cells to modulate both humoral and cellular immunity. Here, we show that B cells produce IL-27 upon activation by TLR4 and CD40 in the presence of IL-21 in vitro. Using Lymphocytic choriomeningitis virus (LCMV) Clone 13 as a model of a persistent viral infection, we identify plasma B cells as major source of IL-27. Further, we show that IL-27-producing B cells are key players in the regulation of immunity. Mice with conditional B cell–specific IL-27p28 knockout were unable to control persistent LCMV infection and failed to respond to anti-IFNAR1 immunotherapy. The impaired immunity found in these mice was associated with reduced virus-specific IgG2a/2c. In addition, these mice displayed reduced virus-specific CD4+ T cells and T follicular helper (Tfh) cells, suggesting that B-cell-derived cytokines regulate immunity by providing signals to T cells. The effects of Il27ra deletion from T cells mirrors the phenotypes observed in mice lacking B-cell-derived IL-27. In addition, IL-27ra deficient (KO) Tfh cells lost their ability to produce IL-21, resulting in aberrant production of virus-specific antibodies to LCMV clone 13 in IL-27ra KO mice. Finally, RNA-sequencing of virus-specific CD4+ T cells identified IL-27R-dependent genes during persistent LCMV infection. Collectively, our data reveal a central role of plasma cell-derived IL-27 in the regulation of T cell functions. Importantly, our study describes cellular interactions between T and B cells and highlights the importance of virus-specific IgG2a/2c during persistent LCMV infection.
B cell-mediated activation and proliferation of NK cells : crossroads of adaptive and innate immunity
Authors: Im Kyungtaek , Kim Kwanghee , Kim Ji-young , Lee Kyung-mi
Published: January 2023

Belonging to innate immunity, natural killer (NK) cells constitute of circulating lymphocytes which provide a body’s first line of defense against infection and cancer. In the spleen,, NK cells are likely to interact with B cells, but the functional consequence of this interaction remains uncertain. To address this question, we co-cultured primary NK cells with freshly isolated B cells from the spleen and investigated the influence of B cells on NK cells. Here we show that co-presence of B cells in cultures facilitated proliferation of NK cells, as measured CFSE dilution assays. CFSE results showed that proliferation of NK cells by B cells need direct and in direct stimulation. This correlated with microarray data showing that expression of genes involved in the regulation of cell proliferation was significantly increased in NK cells in NK-B co-cultures. NK cells cultured with B cells exhibited higher expression of CD69, B220, CD25 activation markers, suggesting that B cells drastically facilitated NK cell activation. Lack of B cells in mice weakened anti-tumor effect as well as anti-pathogen response, suggesting that NK cells exhibited higher anti-tumor activity in the presence of B cells. The synergistic effect of B cells on NK cell functions also occurred in humans. Given the fact that NK cells can exert a regulatory role in cancer or infection, co-presence of B cells can potentiate the role of NK cells by promoting their proliferation and cytotoxicity.
Characterizing region-specific lymphocyte migration within the post-stroke brain
Authors: Torres Vanessa O. , Poinsatte Katie , Ortega Sterling B , Kong Xiangmei , Plautz Erik J , Ramirez Denise M.O. , Ajay Apoorva , Meeks Julian P , Goldberg Mark P , Stowe Ann M
Published: January 2023

Background

Studies in our lab show that while B cell-depleted mice exhibit motor deficits, impaired neurogenesis, increased anxiety, and spatial memory deficits after stroke, CD8 T cell depletion reduces post-stroke motor deficits. Therefore, we hypothesized that B cells migrate into brain regions outside the infarct to promote post-stroke motor and cognitive recovery, unlike pro-inflammatory CD8 T cells that migrate to the area of injury.

Methods

Naïve donor B or CD8 T cells were labeled with e450 dye and injected i.v. into recipient mice at 7, 24, 48 and 72h after receiving a 60-minute transient middle cerebral artery occlusion (tMCAo). Peripheral and brain lymphocyte migration was assessed using flow cytometry and serial two-photon tomography (STPT) 96h after tMCAo.

Results

e450+ lymphocytes were detected in the periphery 96h after stroke. STPT co-registered with the Common Coordinate Framework (Allen Institute) created a 3D brain reconstruction that identified lymphocyte diapedesis. B cells were found bilaterally in areas outside the ischemic penumbra, such as the olfactory areas (p<0.05) and dentate gyrus (p<0.05), whereas CD8 T cells migrated into ipsilesional areas including the somatosensory cortex (p<0.01) and brainstem (p<0.01).

Conclusions

B cell diapedesis differs distinctly from that of CD8 T cells throughout the ischemic brain. While B cells migrate bilaterally into brain regions mediating cognitive deficits and neurogenesis, CD8 T cells exhibit greater unilateral migration into the injured hemisphere, including motor and sensory-related areas. Future directions will determine if the spatial location of CD8 T cells or B cells mediates post-stroke pathology or supports functional recovery, respectively.