bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic helminth infection protects mice from type III hypersensitivity (HYP6P.277)
Authors: Evans Holly , Mitre Edward
Published: January 2023

In this study we evaluated the effects chronic helminth infection has on type III hypersensitivity, an allergic reaction driven by antigen/antibody immune complex deposition. Mice were sensitized to ovalbumin (OVA) and 4 weeks later infected with Litomosoides sigmodontis for 10 weeks. Chronically infected animals exhibited significantly reduced ear swelling 12-24 hours after intradermal ear challenge with OVA. Although total IgG levels were higher in sensitized + infected mice than sensitized mice, OVA-specific IgG1 and IgG2a were all significantly reduced in mice that were chronically infected. Immune complexes in the ear tissue were detected by fluorescence microscopy and were found to be larger and more focal in infected animals. Histological analysis of infected mice showed reductions in edema, necrosis, and cellular recruitment compared to sensitized animals. Furthermore, a chemokine array performed on ear tissue 6 hours after OVA challenge showed that infected animals had reduced production of neutrophil-specific chemokines MIP-2 and KC. These data demonstrate that a 10 week L. sigmodontis infection protects against type III hypersensitivity responses. Future studies aim to further elucidate the immunological pathways mediating protection against type III hypersensitivity, and to evaluate whether worm-derived therapies can be useful in protection against diseases in which immune complex deposition is involved such as lupus nephritis.
A distinct immune response in steroid-refractory severe asthma and its modeling in mice (HYP7P.288)
Authors: Raundhal Mahesh , Morse Christina , Oriss Timothy , Khare Anupriya , Krishnamoorthy Nandini , Kolls Jay , Wenzel Sally , Ray Anuradha , Ray Prabir
Published: January 2023

Severe asthma (SA) constitutes only 5-10% of all asthmatics but accounts for 50% of the healthcare costs and morbidity associated with the disease. SA patients remain symptomatic despite treatment with high doses of corticosteroids (CS), the mainstay of asthma therapy. The presence of neutrophils in asthma has been correlated with refractoriness to CS, fatal exacerbations and severity. To date, our understanding of the pathological mechanisms central to neutrophil-prominent SA is minimal primarily because of the lack of a suitable animal model, limiting the development and testing of new therapeutics. Studies in our lab have shown that unlike patients with milder disease, SA patients consistently have a mixed Th1/Th2/Th17 immune phenotype in their airways despite high dose CS treatment. With the association of viral and bacterial infections with severity and frequency of asthma exacerbations, we hypothesized that the combination of an allergen with agents that mimic infection would induce a SA phenotype in mice. As in human severe asthmatics, the immune response in this mouse model elicits mixed granulocytic airway inflammation and airway hyperresponsiveness that are poorly responsive to CS. Using RNA sequencing and bioinformatics analysis tools, we have identified molecules that may play a central role in the pathogenesis of SA that can be targeted for developing novel therapies for this disease.
The addition of CpG to alum-adjuvanted vaccines provides a bystander effect that reduces the severity of peanut allergy in mice. (HYP7P.311)
Authors: Johnson-Weaver Brandi , Burks A. , Staats Herman
Published: January 2023

Rationale: The “hygiene hypothesis” predicts that a “clean” environment increases the risk of developing allergic disease while endotoxin exposure is protective. The vaccine adjuvant alum may alter immune responses to enhance the induction or severity of allergic disease. A mouse model of peanut allergy was utilized to determine if endotoxin or alum exposure influence the severity of allergic disease. Methods: C3H/HeN mice were housed in high or low endotoxin environments. Mice received three biweekly ovalbumin (Ova) immunizations adjuvanted with alum or alum combined with MPL or CpG 1826. Peanut sensitization was achieved by gastric gavage of peanut and cholera toxin weekly for 4 weeks beginning one day after immunization with Ova + adjuvants. One week after the last immunization all mice were challenged with peanut and monitored for hypothermia and allergy clinical symptoms. Results: Mice immunized with Ova + Alum + CpG exhibited mild allergic symptoms after peanut challenge (-2.0°C and 1.8 score) that did not statistically differ from challenged unsensitized mice (0.68°C and 0 score). All other immunization groups exhibited severe allergic symptoms after challenge (< -2.5°C and >2.5 score) and were significantly different when compared to challenged unsensitized mice. Conclusion: Endotoxin and alum exposure did not increase the severity of peanut allergy. The addition of CpG 1826 to the Ova + Alum vaccine reduced the severity of peanut-specific allergic disease.
Repeated allergen challenge provokes mechanical sensitivity, hyper-innervation and mast cell accumulation in the vulvar tissue of mice (HYP7P.319)
Authors: Chatterjea Devavani , Swanson Linnea , Ashbaugh Alyssa , Martinov Tijana , Tonc Elena , Benck Charles , Daughters Randy
Published: January 2023

Epidemiological evidence suggests a link between a history of environmental allergies and the risk of developing vulvodynia - a chronic provoked vulvar pain condition affecting up to 20% of women in populates assessed. We have previously shown that acute labiar exposure to a hapten allergen provokes increased mechanical sensitivity in the vulvar tissue of sensitized mice. Here, we show repeated challenge with contact hypersensitivity allergen oxazolone produces sustained mechanical hyperalgesia in the vulvar region of previously sensitized female mice for 10-14 days after final allergen exposure. Acute inflammatory responses including labiar neutrophil influx, T and B cell amplification in the draining iliac lymph nodes, labiar expression of inflammatory cytokine genes (IL-6, IL-1β and Cxcl-2) are all resolved within 5 days after final allergen exposure while increased densities of both peptidergic nerve fibers and mast cells in the labiar tissue are observed. Our findings provide the first evidence of the induction of measurable and sustained pain persisting after the resolution of overt inflammatory events following an allergic response in the labiar/vulvar tissue in mice. We have established the first model of chronic allergy-provoked vulvar pain in mice that can be used to elucidate the underlying etiology of subsets of vulvar pain that present without accompanying inflammation and are associated with histories of allergic skin responses.
Roles of scaffolding protein Gab1 in regulation of myeloid dentritic cell-mediated allergic asthma (HYP7P.281)
Authors: Zhang Yun , Xu Yun , Cen Dong , Xu Jia , Zhang Xue , Ke Yue
Published: January 2023

Scaffolding proteins Gab1 is ubiquitously expressed and belongs to the GAB family (GABs), which integrates multiple signal events. This protein family has recently attracted interest that genetic variants of human GAB1 are considered a risk factor for asthma. Our previous research has revealed a novel function of Gab2 in mediating mucin gene expression and Goblet cell hyperplasia, which is an important process in asthma. However, the function of Gab1 in asthma remains unclear. In asthma exacerbated patients, we found a prominent elevated level of Gab1 in peripheral blood monouclear cells compared to that of convalescence patients. Next, we generated conditional Gab1 deletion mice in myeloid cells (Gab1 KO mice) to determinate physiological the role of Gab1 in asthma. Lung histology and mucus metaplasia indicated that disruption of Gab1 in dentritic cells ) leads to alleviated asthmatic inflammation. Future investigation showed that loss of Gab1 results in impaired DC migration to lymph node, while has no influences on DC maturation, antigen uptake and presentation. Mechanically, Gab1 is essentially involved in CCL19-mediated AKT activation. Together, these finding has characterized a novel of Gab1 in mediating DC migration and that deregulation of Gab1 signal in lungs predisposes asthmatic inflammation.
Live two-photon imaging reveals distinct contribution of neutrophilic and stromal Toll-like receptor 4 in hyper-acute bone marrow response to systemic lipopolysaccharide insult (INC5P.324)
Authors: Huang Alex , Myers Jay
Published: January 2023

Produced in the bone marrow (BM) in large quantities, neutrophils are rapidly mobilized and released into circulation in response to inflammation and infection. How BM neutrophils are released as a result of CXCL12 and CXCL1/CXCL2 gradients have been studied at a long time scale (hours to days) following lipopolysaccharide (LPS) challenge. However, little is known about how neutrophil and BM stroma react during the hyper-acute (minutes to hours) phase. We performed intravital microscopy on mice that express GFP under the Lysozyme M (LysM) promoter. BM chimeras were generated to differentiate TLR4-mediated responses in hematopoietic and radio-resistant stroma. We observed a 2.5-fold increase in the speed and the formation of dynamic cellular clusters in 50% of BM neutrophils 30-60 minutes after LPS injection. The neutrophils achieved a peak average speed of 8.4 um/min, accompanied by their disappearance into circulation as evidenced by a 40% reduction in the GFP fluorescence after 90 minutes. BM chimera experiments revealed that while intra-marrow migration and cellular clustering are dependent on TLR4 signaling in neutrophils, their release into systemic circulation is dependent on stromal TLR4 signaling. Blocking CXCR2 signaling had the same effect as chimeras expressing TLR4-defective stroma, further revealing that BM stroma modulate TLR4-mediated neutrophil mobilization and release via CXCL1/CXCL2 chemokine signaling during the hyper-acute phase of marrow response to LPS.
Binge ethanol exposure alters alveolar macrophage activation in burn-injured mice (INC5P.329)
Authors: Ippolito Jill , Curtis Brenda , Ramirez Luis , Kovacs Elizabeth
Published: January 2023

Alcohol intoxication is involved in 50% of all burn injuries, resulting in an increased risk of lung infection and failure, relative to burn alone. Our work has shown that in a mouse model of episodic binge ethanol exposure followed by burn injury there is prolonged pulmonary inflammation, relative to burn alone. The magnitude of pulmonary inflammation is mediated in part by alveolar macrophages (AMs), thus we examined the effect of ethanol on AM activation. Thirty minutes after binge ethanol exposure, mice were anesthetized and given a 15% total body surface area dorsal scald injury. At 24 and 72 hrs post-injury bronchoalveolar lavage AM granularity was assessed by flow cytometry using side scatter (SSC). At 24 hrs, 12% of AMs from burn alone had high SSC relative to 1.5% of AMs from sham groups (p<0.05). Only 6% of AMs from combined injured had high SSC. By 72 hrs, 3% of AMs from both burn groups had high SSC. Anti-inflammatory M2 phenotype of high SSC AMs was assessed using M2 marker CD206. At 24 hrs, there was no change in CD206. At 72 hrs, AMs from burn alone had a 43% increase in CD206 relative to sham groups. Giving ethanol prior to burn further elevated the expression of this marker by 46% over burn alone. These data indicate ethanol exposure prior to burn injury can differentially alter AM phenotype at early and later time points.
Memory T cell-derived IFN-gamma instructs potent innate cell activation for protective immunity (INC6P.349)
Authors: Lauvau Gregoire , M'Homa Soudja Saidi , Chandrabos Ceena , Yakob Ernest , Veenstra Mike , Palliser Deborah
Published: January 2023

Cells of the innate immune system are essential for host defenses against primary microbial pathogen infections, yet their involvement in effective memory responses of vaccinated individuals has been poorly investigated. Here we show that memory T cells instruct innate cells to become potent effector cells in a systemic and a mucosal model of infection. Specifically, we found that memory T cells control phagocyte, dendritic cell and NK/NK T cell mobilization and induction of a strong program of differentiation which include their expression of effector cytokines and microbicidal pathways, all of which are delayed in non-vaccinated hosts. Disruption of IFN-gamma-signaling inside Ly6C+ monocytes, dendritic cells and macrophages impairs these processes and the control of pathogen growth. These results reveal how memory T cells, through rapid secretion of IFN-gamma, orchestrate extensive modifications of host innate immune responses that are essential for effective protection of vaccinated hosts.
The immunomodulatory drug lenalidomide augments NK cell IFN-γ secretion and lowers the threshold for NK cell activation (INC6P.351)
Authors: Lagrue Kathryn , Chopra Rajesh , Davis Daniel
Published: January 2023

The immunomodulatory drug lenalidomide is widely used for the treatment of multiple myeloma. NK cells can kill tumour cells in early stage myeloma but there is a decline in NK cell cytotoxicity as the disease progresses. Previous studies have shown that lenalidomide increases antibody-dependent cellular cytotoxicity, yet little is known about its effects on cytokine secretion. Here, we show that lenalidomide increases IFN-γ secretion from primary human NK cells stimulated by NK-sensitive target cells. Moreover, after ligation of NK activating receptors CD16 and NKG2D, lenalidomide caused a 2-fold increase in the proportion of primary NK cells producing IFN-γ, and a 10-fold increase in the amount of IFN-γ produced per cell. This implies that lenalidomide acts downstream from early membrane-proximal signalling, which is distinct for these two receptors. In addition, lenalidomide-treated NK cells displayed a 2-fold decrease in the amount of ligand required to reach 50% of maximal activation, establishing that lenalidomide lowers the threshold for NK cell activation. Finally, using super resolution microscopy, we found that lenalidomide treatment increases the periodicity of the actin meshwork at the immune synapse during cytokine secretion, a cytoskeletal rearrangement likely to be important for its mechanism of action. Taken together, these results establish that lenalidomide influences cytokine secretion and lowers the threshold for NK cell activation.
Therapeutic effects of a natural compound isolated from garlic (Allium sativum L.), against experimental polymicrobial sepsis (INC6P.335)
Authors: Ok Chang Youp , Lee Sung Kyun , Park Yoo Jeong , Jung Young Su , Lee Ha Young , Kim Sang Doo , Jung Won Jung , Kim Do Yeon , Choi Young Whan , Bae Yoe-Sik
Published: January 2023

Sepsis is a serious infectious life threatening disease. In this study, we demonstrate that LII-34, a natural compound isolated from garlic (Allium sativum L.), markedly enhances survival rate, by inhibiting lung inflammation in cecal ligation and puncture (CLP) experimental polymicrobial sepsis model. LII-34 strongly reduced bacterial colony unit from peritoneal fluid in CLP mice by stimulating generation of reactive oxygen species. Lymphocyte apoptosis from spleen in CLP mice was also markedly decreased by LII-34 administration. LII-34 also significantly inhibits the production of proinflammatory cytokines such as TNF-α, interleukin-1β (IL-1β) and IL-6 in CLP mice. Lipopolysaccharide-stimulated the production of TNF-α, CCL2, IL-6, and IL-10 are also strongly inhibited by LII-34 in mouse bone marrow-derived macrophages. Taken together, our results indicate that LII-34 shows therapeutic effect against polymicrobial sepsis by enhancing microbial killing and blocking cytokine storm. Chang Youp Ok and Sung Kyun Lee are equally contributed to this work.
The kidney myeloid cell compartment is perturbed in diabetic BTBR ob/ob mice (INC6P.338)
Authors: Fink Lisbeth , Gotfredsen Carsten , Rosendahl Alexander , Pedersen Maiken
Published: January 2023

The BTBR ob/ob mouse is gaining interest as a model of diabetic nephropathy, because it develops progressive albuminuria and, as opposed to the db/db diabetic mouse, is considered to develop fibrotic kidney lesions. The mouse kidney contains multiple types of resident mononuclear phagocytes and neutrophils. In the Akita type 1 diabetes mouse model, macrophage recruitment has been shown to be a key driver of the diabetic nephropathy, and human diabetic nephropathy kidney specimens display increased macrophage infiltration. We aimed to study the myeloid cell populations locally in the BTBR ob/ob kidney. The major myeloid cell population, the macrophage-like cells (CD68+F4/80+), was increased in diabetic, proteinuric BTBR ob/ob mice compared to normo-glycemic BTBR ob/+ mice, whereas monocyte-like cells (CD68+F4/80-) were decreased. In addition, kidney neutrophil counts were reduced in the diabetic mice. Consistent with the overall increase in myeloid cell numbers caused by the increase in macrophages, ICAM-1 expression on endothelial cells was significantly increased. In conclusion, proteinuria in the BTBR ob/ob mouse model of diabetic nephropathy is accompanied by a modulated inflammatory state in the kidney myeloid cell compartment, potentially contributing to the glomerulosclerosis and interstitial fibrosis observed in this model.
Transient receptor potential melastatin 2 protects mice against polymicrobial sepsis by enhancing bacterial clearance (INC6P.342)
Authors: Fang XiangMing
Published: January 2023

Background: Recent studies suggest that transient receptor potential melastatin 2 (TRPM2) channel plays an important role in inflammation and immune response. However, the role and mechanism of TRPM2 in polymicrobial sepsis remains unclear. Methods: We explored the effects of genetic disruption of TRPM2 on mortality, bacterial clearance, organ injury, and systemic inflammation during cecal ligation and puncture (CLP)-induced sepsis. Electrophysiology, immunoblot, bacterial clearance experiment, and quantitative real-time polymerase chain reaction were used to explore the role and mechanism of TRPM2 in sepsis. Results: After CLP, Trpm2-KO mice had increased mortality compared with WT mice. The increased mortality was associated with increased bacterial burden, organ injury, and systemic inflammation. Disruption of TRPM2 decreased HO-1 expression and increased bacterial burden in bone marrow-derived macrophages (BMDMs). Pretreatment of Trpm2-KO mice with HO-1 inducer reversed the susceptibility of Trpm2-KO mice to sepsis by enhancing bacterial clearance. In addition, septic patients with lower monocytic TRPM2 and HO-1 mRNA levels had a worse outcome compared with septic patients with normal monocytic TRPM2 and HO-1 mRNA levels. TRPM2 levels correlated with HO-1 levels in septic patients. Conclusions: Our data demonstrate a protective role of TRPM2 in controlling bacterial clearance during polymicrobial sepsis, possibly by regulating HO-1 expression.
Caspase-1-independent interleukin-1β is required for clearance of bordetella pertussis infections and whole-cell vaccine-mediated immunity (INM6P.413)
Authors: Place David , Muse Sarah , Kirimanjeswara Girish , Harvill Eric
Published: January 2023

Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1) response, which is required for the clearance of B. pertussis. We show that IL-1β but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1β and IL-1R deficient mice have normal but persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.
Activation of AIM2 inflammasome triggers mitochondrial damage and blocks mitophagy (INM6P.409)
Authors: Yu Jiujiu , Murakami Tomohiko , Nagasu Hajime , Horng Tiffany
Published: January 2023

The AIM2 inflammasome is critical for host defense against certain intracellular bacteria and DNA viruses. It is a cytoplasmic complex consisting of the regulatory subunit AIM2, the adaptor ASC, and the effector subunit caspase-1. Stimulus dependent assembly of the complex activates the proteolytic activity of caspase-1, which is required for the processing and production of inflammatory cytokines IL-1β and IL-18. Here we demonstrate that mitochondrial damage is a consequence of activation of the AIM2 inflammasome. We address the underlying mechanisms, thus providing insight into regulation of a key inflammasome effector activity.
POP3 is a novel inhibitor of ALR inflammasomes and controls host defense to DNA virus infections (INM6P.411)
Authors: Stehlik Christian , Khare Sonal , Ratsimandresy Rojo , de Almeida Lúcia , Cuda Carla , Rellick Stephanie , Misharin Alexander , Wallin Melissa , Gangopadhyay Anu , Forte Eleonora , Gottwein Eva , Perlman Harris , Reed John , Greaves David , Dorfleutner Andrea
Published: January 2023

The innate immune system responds to infections and tissue damage by activating cytosolic sensory complexes called inflammasomes. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the adaptor protein ASC through PYRIN domain (PYD)-PYD interaction, links ALRs to the activation of caspase-1 and subsequent maturation of the proinflammatory cytokines IL-1β IL-18 and induction of pyroptotic cell death. A controlled inflammasome response is crucial for maintaining homeostasis, whereas excessive and uncontrolled cytokine production contributes to autoinflammatory diseases. However, ALR inflammasome regulation is poorly understood. Here, we identified the type I interferon (IFN)-inducible PYD-only protein 3 (POP3) as a binding partner of ALRs. POP3 competes with ASC for recruitment to ALRs and thereby inhibits DNA virus-induced ALR inflammasome activation and IL-18 and IFN-γ-dependent host defense in vivo. A mouse model with macrophage-specific POP3 expression recapitulates global AIM2 deficiency and thus, emphasizes the importance of ALR inflammasome regulation in the monocytic/macrophage lineage. Collectively, we show that POP3 represents one of the type I IFN response factors that regulate human and mouse ALR inflammasomes in macrophages within a type I IFN-regulatory loop.
Macrophage anti-viral responses to diabetogenic encephalomyocarditis viruses are mediated by oxidative stress (INM7P.426)
Authors: Burg Ashley , Tse Hubert
Published: January 2023

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic β-cells. Both human and rodent studies suggest that viral infections trigger this process. Proinflammatory cytokines and reactive oxygen species (ROS) produced during an anti-viral response are also considered drivers of T1D. We recently demonstrated that Non-Obese Diabetic (NOD) mice deficient in NADPH oxidase (NOX)-derived ROS (NOD.Ncf1m1J) were highly resistant to T1D, and bone marrow-derived macrophages (BMMΦ) from these mice had reduced anti-microbial responses to LPS and poly(I:C), a viral dsRNA mimic. Therefore, we hypothesize that loss of NOX-derived ROS will reduce the diabetogenicity of viral infections by dampening the innate immune anti-viral response. To this end, we infected BMMΦ with diabetogenic (EMCV-D) or non-diabetogenic (EMCV-B) Encephalomyocarditis viruses. NOD.Ncf1m1J BMMΦ showed significant decreases in TNFα upon EMCV-D (1.2-fold, p<0.004) and EMCV-B (1.5-fold, p<0.0001) infection compared to NOD. This was concomitant with lower protein expression of the viral sensor, RIG-I. Interestingly, only EMCV-D elicited an IL-1β response, which was dramatically reduced (1.9-fold, p<0.0001) in NOD.Ncf1m1J BMMΦ. Our results highlight a crucial role for NOX-derived ROS in the innate response to EMCV infection. Future studies will define the impact of ROS on human anti-viral responses to diabetogenic viruses.
IL22 and CD160 play additive roles in the mucosal host response to Clostridium difficult infection in mice (INM8P.435)
Authors: Sadighi Akha Amir , McDermott Andrew , Carlson Paul , Frank Charles , McDonald Roderick , Falkowski Nicole , Bergin Ingrid , Huffnagle Gary
Published: January 2023

We have previously shown the significant up-regulation of Il22 and increased phosphorylation of STAT3 as part of the mucosal response to C. difficile VPI 10463 infection. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes IL22- and CD160-mediated components. The current study sought to determine the potential role(s) of IL22 and/or CD160 in the mucosal response to C. difficile infection. Infection of mice with C. difficile 630 led to the significant induction of STAT3 phosphorylation and a very similar set of genes in the colon as we had reported previously. C. difficile-infected mice treated with anti-IL22, anti-CD160 or both showed significantly reduced STAT3 phosphorylation in comparison to C. difficile-infected mice that had received neither antibody. In addition, C. difficile-infected mice treated with anti-IL22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile-infected mice. These included pro-inflammatory chemokines and cytokines and anti-bacterial peptides. In addition, mice treated with anti-IL22/CD160 had a lower influx of neutrophils than the other 3 infected groups. These data demonstrate that IL22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL22 and CD160 in mediating both the pro-inflammatory and pro-survival aspects of the mucosal response in this infection.
Participation of the transient receptor potential melastatin 2 ion channel in oxidative stress response and host defense during Helicobacter pylori infection (INM8P.448)
Authors: Algood Holly , Beceiro Susana , Chatuverdi Rupesh , Horvath Dennis , Cortado Hanna , Wilson Keith , Partida-Sanchez Santiago
Published: January 2023

Helicobacter pylori (Hp) is a dominant member of the gastric microbiota in a majority of the world’s population. Hp colonization has been attributed to detrimental outcomes including gastritis and gastric cancer. The immune response to Hp leads to elevated levels of reactive oxygen species and reactive nitrogen species within the gastric mucosa. The redox-sensitive cation channel TRPM2 is activated by direct binding of ADP ribose and under conditions of oxidative and metabolic stress by H2O2 and H+. TRPM2 is strongly expressed in immune cells of the phagocytic lineage, themselves professional oxidants-generators. Here we report that Hp infection of TRPM2-deficient macrophages resulted in higher rates of bacterial killing and increased levels of proinflammatory cytokines and NOX2 compared to wild-type macrophages. Consequently, TRPM2-deficient macrophages displayed significantly higher ROS production during Hp infection, suggesting an increased NADPH oxidase activity. In vivo, when TRPM2-deficient mice were infected orogastrically with Hp their gastric tissues exhibited an increased NOX2 expression and myeloperoxidase activity compared to wild-type mice. Furthermore, enhancement of NADPH oxidase activity in TRPM2-deficient animals increased inflammation and reduced bacterial burden when compared with wild-type mice. Taken together, these data indicate that TRPM2 establishes a negative feedback of ROS production through phagosomal NADPH oxidase during Hp infection.
ApoE3 blocks HCV entry inhibitor ficolin-2 (INM8P.437)
Authors: Zhang Xiao-Lian , Yushan Ren Yushan , Zhao Yinglan
Published: January 2023

Human ficolin-2 is a lectin-complement pathway activator that is present in normal human plasma and is associated with infectious diseases; however, little is known regarding the roles and mechanisms of ficolin-2 during chronic Hepatitis C Virus (HCV) infection. In this study, we found that ficolin-2 could inhibit the entry of HCV at the early stage of virus infection, regardless of the viral genotype. Ficolin-2 neutralized and inhibited the initial attachment and infection of HCV by binding to the HCV envelope surface glycoproteins E1 and E2, blocking HCV attachment to low-density lipoprotein receptor (LDLR) and scavenger receptor (SR-B1), and weakly interfering with CD81 receptor attachment. However, no interference with claudin-1 and occludin receptors attachment was observed. The C-terminal fibrinogen domain of ficolin-2 was identified as the critical binding region for the HCV E1-E2 N-glycans, playing a critical role in the anti-HCV activity. Our data suggest that the HCV entry inhibitor ficolin-2 is a novel and promising antiviral innate immune molecule and the immune escape mechanism mediated by ApoE3 during chronic HCV infection. HCV may be neutralized using compounds directed against the lipoprotein moiety of the viral particle and that ApoE3 may be a new target to combat HCV infection.
Endomembrane structures utilized by pathogens are targeted for disruption by the ubiquitin-like conjugation systems of autophagy (INM8P.439)
Authors: Hwang Seungmin , Choi Jayoung , Biering Scott , Park Sunmin , Virgin Herbert
Published: January 2023

Autophagy is an evolutionarily conserved pathway that sequesters cytoplasmic materials in double membrane bound autophagosomes and degrades the cargo through the fusion between the autophagosome and lysosome. Degradative autophagy has been shown to play crucial roles in immune defense against intracellular pathogens as well as its conventional roles in recycling and remodeling. We found that the ubiquitin-like conjugation systems of the autophagy pathway involved in the conjugation of microtubule associated protein 1 light chain 3 (LC3) to phosphatidylethanolamine on autophagosome membrane, including E1 Atg7, E2 Atg3, and E3 Atg12-Atg5-Atg16L1 complex, play crucial roles in the control of Murine Norovirus and Toxoplasma gondii both in vitro and in vivo. Pharmacologic modulation of the degradative autophagy pathway or genetic deletion of the other essential autophagy genes had no significant effects on controlling the replication of the pathogens by interferon-gamma (IFNγ). Rather the ubiquitin-like conjugation system was required for targeting of LC3 and IFNγ induced effectors onto the endomembrane structures utilized by the pathogens for their replication and consequent disruption of the structures. These data suggest that the ubiquitin-like conjugation systems that reorganize endomembranes during canonical autophagy have evolved to target immune effectors to the endomembrane structures utilized by pathogens as a form of cell autonomous immunity.
Host innate immune responses are different for high and low pathogenicity influenza A virus subtypes (INM8P.443)
Authors: Gupta Goutam , Pardington Paige , Chaudhary Anu , Zeytun Ahmet , Harris Jennifer , Ribeiro Ruy
Published: January 2023

Different influenza A virus subtypes display different pathogenicity during host infection. It is our hypothesis that the host innate immune defense inversely correlates with the viral pathogenicity, i.e., the higher the viral pathogenicity the lower is the host innate immune defense and vice versa. In order to test our hypothesis, we monitored infection of normal human bronchial epithelial cells due to high pathogenicity H1N1 and low pathogenicity H3N2 subtypes during the early stages (0 to 24 hours) of infection. Specifically, we performed whole genome microarray analysis of the infected cells, identified the genes significantly altered in their expression, and validated the expression patterns of these genes by real-time qPCR. Of particular importance were the inducer and effector genes in the IFN-α/β pathway, a prominent innate immune response against viral infection. The prominent inducers include: (i) the OAS genes involved in the production of small viral RNA, (ii) the viral RNA sensors RIG-I, MDA-5, and TLR3, and (iii) the IRF transcription factors involved in the expression of IFN-α/β. The prominent effectors include: (i) ISG15, which activates RIG-I by ubiquitination, (ii) RSAD2 which inhibit the budding of influenza A virus, and (iii) GBPs, which inhibit viral replication. We validate our hypothesis by demonstrating that the release and expression of inducers and effectors of IFN-α/β are lower in high pathogenicity H1N1 than those in low pathogenicity H3N2. .
TERRA enhances production of senescence-associated inflammatory cytokines IL-6 and IL-8 (IRC5P.452)
Authors: Wang Pu
Published: January 2023

Aged or senescent cells develop a complex senescence-associated secretory phenotype (SASP). However, the mechanisms underlying the induction of the SASP are unclear. Telomeres are critical for genome stability and aging. Telomeres are transcribed into long non-coding telomeric repeat-containing RNA (TERRA) molecules. We demonstrate that TERRA and inflammatory cytokines were significantly elevated during senescence. Over expression of TERRA significantly upregulated IL-6 and IL-8, but IL-6 and IL-8 did not modulate TERRA expression. The results show that TERRA expression is related to senescence and increases production of SASP. Our findings expand the biological function of TERRA in senscence-assosciated inflammatory responses.
RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance (IRC5P.465)
Authors: Xiao Yanping , Yu Sanhong , Zhu Baogong , Bedoret Denis , Bu Xia , Francisco Loise , Hua Ping , Duke-Cohan Jonathan , Umetsu Dale , Sharpe Arlene , DeKruyff Rosemarie , Freeman Gordon
Published: January 2023

We report that programmed death ligand 2 (PD-L2), a known ligand of PD-1, also binds to repulsive guidance molecule b (RGMb) which was originally identified in the nervous system as a co-receptor for bone morphogenetic proteins (BMPs). PD-L2 and BMP-2/4 bind to distinct sites on RGMb. Normal resting lung interstitial macrophages and alveolar epithelial cells express high levels of RGMb mRNA, while lung dendritic cells express PD-L2. Blockade of the RGMb:PD-L2 interaction markedly impaired the development of respiratory tolerance by interfering with the initial T cell expansion required for respiratory tolerance. Experiments with PD-L2 deficient mice showed PD-L2 expression on non-T cells was critical for respiratory tolerance but expression on T cells was not required. Since PD-L2 binds to both PD-1, which inhibits anti-tumor immunity, and to RGMb, which regulates respiratory immunity, targeting the PD-L2 pathway has therapeutic potential for asthma, cancer and other immune-mediated disorders. Understanding this pathway may give insights into how to optimally modulate the PD-1 pathway in cancer immunotherapy while minimizing adverse events.
A commensal microbial molecule confers protective anti-inflammatory responses employing both innate and cognate interactions (IRC5P.468)
Authors: Dasgupta Suryasarathi , Kasper Dennis
Published: January 2023

The microbiota is critical in shaping the mammalian host’s immune system. Polysaccharide A, the archetypical immunomodulatory microbial molecule of the gut commensal Bacteroides fragilis, induces regulatory T cells to secrete the anti-inflammatory cytokine interleukin 10. We show, in a model of colitis, that PSA requires both innate and adaptive immunity to generate protection. Dendritic cells mediate PSA’s effect on IL-10 production. Unlike conventional DCs, plasmacytoid DCs exposed to PSA do not produce the proinflammatory cytokines tumor necrosis factor alpha and IL-12, but PDCs do specifically stimulate IL-10 secretion by CD4+ T cells and efficiently mediate PSA-mediated immunoprotection. PSA induces and preferentially ligates Toll-like receptor 2 on PDCs but not on CDCs. Compared with other TLR2 ligands, PSA better enhances PDC expression of co-stimulatory molecules that are required for protection against colitis. PDCs orchestrate beneficial immunoregulatory interaction of commensal microbial molecules with CD4+ T cells through both innate and adaptive immunity.
Macrophage polarization drives granuloma outcome during Mycobacterium tuberculosis infection (IRC5P.475)
Authors: Marino Simeone , Cilfone Nicholas , Mattila Joshua , Flynn JoAnne , Linderman Jennifer , Kirschner Denise
Published: January 2023

Lung granulomas are the pathologic hallmark of tuberculosis (TB), where different immune cell types and bacteria co-localize. Macrophages are the most abundant cells in a granuloma. They are both critical effector and target cells for Mycobacterium tuberculosis (Mtb, the causative agent of TB) infection, and serve as regulators of inflammation and innate immune responses by producing a variety of pro- and anti-inflammatory cytokines. Overall, macrophage function, diversity and location within a granuloma environment are still poorly understood. In this study we used a systems biology approach to better characterize how macrophages are polarized by their microenvironment to mount either a pro- or an anti-inflammatory phenotype. We built on recently published experimental studies on non-human primates that suggest how a continuous dynamic balance between pro and anti-inflammatory signals is achieved to control pathology and restrain bacterial growth within a granuloma. Here we investigated how this balance translates into macrophage polarization and plasticity, and how the relative contribution of these macrophages, broadly classified as ‘M1’ and ‘M2’, evolves over time and leads to an effective immune response, at the granuloma scale. Our in silico granulomas suggest that temporal dynamics of granuloma polarization ratios can be predictive of granuloma outcome. We also find that a signature common to properly formed and functioning granulomas is increased NFkB degradation.
Transferred allogenic S1P3 deficient dendritic cells (DCs) interact with recipient splenocytes to prevent kidney ischemic-reperfusion injury (IRI) (IRC5P.459)
Authors: Bajwa Amandeep , Huang Liping , Ye Hong , Lobo Peter , Okusa Mark
Published: January 2023

The plasticity of DCs permits phenotypic modulation ex-vivo by gene expression or pharmacological agents. Sphingosine-1-phosphate (S1P), a sphingolipid is the natural ligand for S1Pr1-5s. We tested the ability of S1P3-/-(3KO) DC to suppress kidney IRI. BMDCs from B6 WT (B6DC) or 3KOB6DC were adoptively transferred into (->) Balb/c 1d/7d prior to IRI. Naïve Balb (noDC) had higher plasma creatinine (PCr; mg/dl) compared to sham (1.2±0.17;0.2±0.004,p≤0.01). 3KOB6DCs->Balb/c were protected from IRI (PCr:0.3±0.07,p≤0.01). 3KOB6DCs->Balb/c had higher Treg and less CD4 cells compared to naïve or B6DC mice in spleen. The protection afforded by 3KOB6DCs->Balb/c mice required the recipient mouse to have a spleen; Tregs, CD11c+ DC and T/B cells; Splnx Balb, use of Rag1-/- mice or depletion of recipient Tregs (PC61) or DCs (CD11c-DTR) rendered adoptively transferred 3KODCs ineffective. PKH-26 labeled DCs from WT or 3KO->Balb/c were located in the marginal zone (MZ) of recipient spleen with close proximity to Tregs. WT and 3KO mice injected with latex beads to demonstrate clearance efficiency showed disruption of MZ in 3KO. Adoptive transfer of 3KOB6DCs prior to IRI attenuates injury through interaction with splenocytes, recipient DCs resulting in induction of Tregs through possible disruption of MZ macs. We conclude that genetically induced deficiency or pharmacological blockade of allogenic S1P3DCs could serve as a useful therapeutic approach to IRI or DGF associated with Transplant.
IL-33 stimulates dendritic cell secretion of IL-2 that promotes selective expansion of ST2+Foxp3+ regulatory T cells (IRC5P.460)
Authors: Matta Benjamin , Lott Jeremy , Mathews Lisa , Rosborough Brian , Blazar Bruce , Turnquist Heth
Published: January 2023

IL-33 is a pleiotropic IL-1 family cytokine that signals via ST2 and expands ST2+Foxp3+ regulatory T cells (Treg) in vivo. As ST2+ Treg show poor expansion by direct IL-33 stimulation, we sought to define mechanisms mediating their expansion. IL-2 signaling promotes ST2 expression on CD4+ T cells, and dendritic cells (DC) express ST2 (able to respond to IL-33), and are a potential source of IL-2. Thus, we examined if IL-33 mediates ST2+ Treg expansion by stimulating DC IL-2 production. CD11c+ wild type (WT) or IL-2 knockout (KO) bone marrow DC were exposed to IL-33 or LPS. DC phenotype was evaluated by multi-color flow cytometric analysis. The influence of IL-33 DC on T cell function was evaluated in MLR with CD4+ T cells, and T cell proliferation and phenotype were determined by flow analysis. Cytokines were quantitated by ELISA. We found that unlike LPS, IL-33 does not influence DC surface phenotype or induce pro-inflammatory cytokine production compared to controls. However, IL-33 induces a 5-fold increase IL-2 production by DC. In MLR, IL-33 DC selectively expand an activated subset of ST2+Foxp3+ Treg that are CD44hiICOShi. IL-33 DC-derived IL-2 is critical since IL-33-exposed IL-2 KO DC fail to expand ST2+ Treg. In summary, IL-33 licenses DC to selectively expand a subset of activated Treg through production of IL-2, in the absence of classical DC activation. These findings may be harnessed to aid the development of novel therapeutics aimed at promoting immune tolerance.
Signaling by ADAP via the Carma1-TAK1 axis is critical for pro-inflammatory cytokine production but not for cytotoxicity in NK cells. (IRM7P.477)
Authors: Rajasekaran Kamalakannan , Kumar Pawan , Schuldt Kristina , Peterson Erik , Vanhaesebroeck Bart , Dixit Vishva , Thakar Monica , Malarkannan Subramaniam
Published: January 2023

‘Cytokine release syndrome’ (CRS) is a serious side effect of NK or T cell-mediated immunotherapy to treat cancer. A strategy to contain CRS without affecting the cytotoxic potential of NK and T cells is highly warranted. A thorough understanding of the signaling pathways will help to identify molecules that exclusively regulate inflammatory cytokine production but not cytotoxicity. Here, we define an ADAP-dependent signaling axis downstream of Fyn that exclusively regulated cytokine production but not cytotoxicity in NK cells. The adapter protein, ADAP is critical for organizing the Carma1-Bcl10-MALT1 signalosome, which is essential for the activation of TAK1. The initiation of these signaling events is critical for the production of Interferon-γ and other pro-inflammatory cytokines. The defect in cytokine production correlated with a decreased nuclear translocation of c-Fos and c-Jun despite a significantly increased phosphorylation of ERK1/2 and JNK1/2 in the ADAP deficient NK cells. Nuclear translocation of NFκB was also decreased in the ADAP deficient NK cells. Our findings indicate that ADAP is indispensible for activating a signaling cascade that leads to inflammatory cytokine production, whereas it is not essential for those signaling events that lead to cytotoxicity. Our findings demonstrate the feasibility of targeting the functions of unique signaling molecules in order to regulate the production of inflammatory cytokines.
Natural killer T cells mediate the protective effects of 1,25-dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis in mice (IRM7P.478)
Authors: Waddell Amanda , Zhao Jun , Cantorna Margherita
Published: January 2023

Active vitamin D (1,25(OH)2D3) completely blocks the development of experimental autoimmune diseases. However, the molecular and immunobiological mechanisms underlying vitamin D’s anti-inflammatory properties are not fully understood. In the present study, we have employed a murine model of experimental autoimmune encephalomyelitis (EAE) in order to determine the role of natural killer T (NKT) cells in vitamin D-mediated protection from EAE. WT mice or mice deficient in all NKT cells (CD1d-/-) or invariant NKT (iNKT) cells (Jα18-/-) were fed control or 1,25(OH)2D3 (50 ng/day) supplemented diets. All mice fed control diet developed severe EAE. 1,25(OH)2D3treatment protected WT mice, but not CD1d-/- or Jα18-/- mice, from developing EAE. Development and severity of EAE were associated with insufficient suppression of the MOG-specific IL-17 response by 1,25(OH)2D3 treatment in CD1d-/- mice compared to WT mice. Activation of iNKT cells with α-galactosylceramide (α-GalCer) or 1,25(OH)2D3 treatment did not protect IL-4-/- mice from developing EAE. Furthermore, 1,25(OH)2D3 treatment of splenocytes in vitro resulted in decreased α-GalCer-induced IL-17 and increased IL-4 production. These studies demonstrate that NKT cells are important mediators of vitamin D-induced protection from EAE in mice and NKT cell-derived IL-4 may be an important factor in providing this protection.
Peroxisome proliferator-activated receptor delta (PPARδ) limits Th1 inflammation through effects on interferon (IFN)-γ transcription and lymphocyte homeostasis (IRM7P.480)
Authors: Zhao Fei , Drohomyrecky Paulina , Dunn Shannon
Published: January 2023

PPARδ is a nuclear receptor that functions as a transcriptional regulator of various processes including metabolic homeostasis, cell proliferation and differentiation. We previously reported that mice deficient in PPARδ (KO) exhibit more severe experimental autoimmune encephalomyelitis than wild type (WT) mice. This more severe inflammation correlated with an increased expansion of myelin-reactive IFNγ-producing cells in the spinal cord, suggesting that PPARδ may act to limit Th1 inflammatory responses. However, the mechanism of how PPARδ accomplishes this is not clear. Characterization of the spleen and thymus compartments revealed that KO mice exhibit reduced spleen and thymus cellularity as compared to WT mice. The resultant lymphopenia in KO mice associated with a higher frequency of memory CD4+ T cells in the periphery. Radiation bone marrow chimeras were generated and the defect in thymocyte development was found to map to the radio-resistant compartment, suggesting that PPARδ may operate in stromal cells to regulate thymocyte development. This was furthered confirmed by the finding that mice with T cell-restricted deficiency of PPARδ did not exhibit defective thymocyte homeostasis. Furthermore, we observed that naïve CD4+ T cell from both KO and T cell-restricted PPARδ-deficient mice had a higher propensity to produce IFNγ upon stimulation. Thus PPARδ appears to limit Th1 inflammation both through the control of lymphocyte homeostasis and IFNγ production.