bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of the gut microbiota by antibiotics exposure during early life promotes spontaneous Th2 responses and loss of tolerance to dietary antigens
Authors: Gustafsson Jenny K , McDonald Keely , Newberry Rodney
Published: January 2023

Allergic disorders, manifest by T helper 2 (Th2) type immune responses to environmental antigens, are rapidly increasing in children. Regulatory T cells (Tregs) limit Th2 responses, and Treg deficiencies are seen in children with allergic disorders, implying that Treg deficiencies or dysfunction underlie the pathogenesis of Th2 mediated disease. What incurs Treg deficiency and its relationship to the increasing incidence of allergy is unknown, however, studies have shown that the intestinal microbiota plays an important role in regulating Treg differentiation during early life. With this in mind we explored whether oral exposure to specific antibiotics in early life altered Treg populations and predisposed to Th2 responses. We found that exposure of mice to a combination of broad spectrum oral antibiotics between days 10 and 20 of life abrogated tolerance to a dietary antigen, ovalbumin (Ova). Exposure of mice to single antibiotics of varying spectra between days 10 and 20 of life had a range of effects from abrogation of oral tolerance to the spontaneous development of Th2 responses manifested by increased levels of IL-13 in the serum in the absence of challenge. Ongoing studies are evaluating the effect of the respective antibiotic regimens on the intestinal microbial community using 16S bacterial DNA sequencing, with the aim to link alterations in the microbial composition to the immunological profile of the respective treatment groups. In summary, our results show that disruption of the intestinal microbiota by exposure to specific antibiotics during a period in early life results in loss of Tregs, promotes Th2 immunity, and impair establishment of oral tolerance to dietary antigen.
The role of type 2 innate lymphoid cells in RSV-induced asthma exacerbation.
Authors: Warren Kristi J , Pavlik Jacqueline A. , DeVasure Jane M. , Dickinson John D. , Sisson Joseph H. , Wyatt Todd A.
Published: January 2023

Respiratory viral infections cause devastating airway inflammation and remodeling in asthmatic children and adults. Type 2 innate lymphoid cells (ILC2) have been characterized individually in both asthma and viral infections. We hypothesize that ILC2 are activated by IL-33 in our model, and thereby enhance the asthmatic phenotype in the lung. Asthma induction was performed in eight week old, female BALB/c mice using ovalbumin (OVA) sensitization and airway challenge. After the last airway challenge, mice were infected with RSV (3,250 TCID50 U/mouse). Lungs were collected for ILC2 sorting, immune cell quantitation, and analysis of IL-4, IL-13, IL-33 and IL-25 on days 1–4 of infection. A subset of lungs were collected and fixed for histological analysis. Results show that mucus production (PAS+ cells) and lymphocytic recruitment were enhanced with RSV infection and asthma. The expression of MUC5AC, CLCA3, IL-25 and IL-33 mRNA levels were increased at least two-fold in the lungs of OVA+RSV mice compared to RSV-infected alone. We detected comparable numbers of ILC2 in BAL fluid from both OVA+ and OVA+RSV mice, yet an evaluation of total lung lymphocytes yielded more ILC2 in the OVA+RSV mice as compared to OVA alone. ILC2 from OVA+RSV mice were sorted and treated with IL-33 for 24 hours. These cells produced greater IL-4 mRNA than cells isolated from OVA only mice. Our results describe a potential role for ILC2 in the exacerbation of asthma with RSV infection. Future studies are planned to determine the exact contribution of ILC2 to type 2 immunity and pulmonary function in our asthma-viral infection model.
Dual Blockade of OX40 and CD30 Signaling Reduces Memory Th2-driven Lung Inflammatory Responses to House Dust Mite Allergen
Authors: Gracias Donald Tom , Mehta Amit Kumar , Yagita Hideo , Croft Michael
Published: January 2023

Allergic asthma is an inflammatory disease that is induced by immune responses to airborne allergens in the lungs. Several studies have identified T cells as being important players in asthma, with Type 2 helper T cells (Th2) being a critical subset. The selective removal of pre-existing pathogenic memory Th2 cells could be a key process leading to enhanced tolerance. We have attempted to identify signaling molecules in the TNF superfamily that regulate memory Th2 cells in a house dust mite (HDM) model of allergic lung inflammation. C57BL/6J mice up-regulated OX40L to higher levels than other TNF family members measured in the lungs in response to HDM exposure. We then tested whether OX40-OX40L interactions were required for memory Th2 cell responses driven by HDM. While OX40-deficient mice displayed strongly reduced overall lung inflammation and Th2 development when challenged with HDM, therapeutic blocking of OX40L at the time of memory T cell reactivation did not result in similar inhibition of allergic responses, implying OX40L may have been important for initiation but not the memory response. Similar negative results were gained by targeting another TNF superfamily protein, CD30L, that was also up-regulated by HDM exposure. However, we observed significant reduction in airway inflammation when OX40L was simultaneously neutralized with CD30L. Dual blockade was found to target memory Th2 cell expansion and effector function but did not prevent accumulation of pTreg cells. This suggests that persisting memory responses to complex allergens are controlled by several co-stimulatory interactions, and only abrogation of signaling through multiple TNF superfamily members might promote airway tolerance.
Inducible Bronchus-Associated Lymphoid Tissue (iBALT) attenuates pulmonary Th2 responses
Authors: Hwang Ji Young , Southworth Kristen , Rangel-Moreno Javier , de la Luz Garcia-Hernandez Maria , Carragher Damian M , Randall Troy D
Published: January 2023

Inducible bronchus-associated lymphoid tissue (iBALT) is an ectopic lymphoid tissue formed in the lung after pulmonary infection or inflammation. This local lymphoid tissue is structurally similar to conventional secondary lymphoid organs (SLOs), with separated B and T cell areas, specialized stromal cells and lymphoid dendritic cells (DCs). The presence of iBALT is typically associated with pulmonary pathology and advanced lung disease, particularly in patients with chronic obstructive pulmonary disease (COPD), rheumatoid lung disease, pulmonary fibrosis and chronic infections, suggesting that iBALT functions to exacerbate local immune responses and pathology. Here, we tested whether the presence of iBALT affected the pulmonary immune responses to allergens. We induced iBALT formation in neonatal mice by pulmonary administration of lipopolysaccharide (LPS), and when the mice were adults, sensitized and challenged them intranasally with ovalbumin (OVA). We found that mice with iBALT exhibited reduced Th2 responses, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production. The presence of iBALT delayed the accumulation of Th2 cells and eosinophils in the lung following challenge and altered the spatial distribution of T cells in the lung. We also found that the more organized iBALT structure was, the fewer eosinophil infiltrates were observed in house dust mite (HDM)-induced allergic inflammation. Although Th2 cells are generated in mice with iBALT, they are concentrated in iBALT area and more dilute in lung parenchyma.

Overall, our studies expanded the field of iBALT from observation of what happens in pulmonary diseases to determining the machinery involved in the responses.
Adeno-associated viral vector-mediated anti-inflammasome gene transfer alleviates asthmatic airway inflammation in OVA-sensitized mice model
Authors: Kuo Ming-Ling
Published: January 2023

Bronchial asthma is a chronic inflammatory disorder of the airway characterized by airway hyperresponsiveness (AHR), pulmonary eosinophilia, elevated serum IgE, and dysregulated T helper (Th) 2 cytokines. It is becoming urgent to identify target molecules for the preventive and therapeutic purpose. ATP accumulation in bronchoalveolar lavage fluid (BALF) of asthmatic subjects indicates that extracellular ATP and the downstream responses are involved. ATP is an activator of NLRP3 inflammasomes and tightly regulated by CD39, a nucleoside triphosphate diphosphohydrolase encoded by entpd1. Upregulated BALF ATP levels and decreased CD39 RNA were accompanied with asthmatic cardinal features in ovalbumin (OVA)-sensitized mice. Recombinant adeno-associated virus (rAAV) has been developed to carry exogenous genes with safe and low toxic therapeutic potential. The different serotypes and tropisms of AAVs benefit tissue specific gene targeting. This study aims for determining whether lung-targeting rAAV carrying CD39 (AAV/CD39) is able to reduce extracellular ATP in BALF and inhibit the OVA-induced allergic inflammation in an asthmatic murine model. The mice were sensitized and challenged with OVA. rAAV vectors were administrated into the lungs of mice 3 days before the first OVA exposure. The data indicated that ATP levels in BALF were downregulated accompanied with rescued CD39 expression. The administration of AAV/CD39 was able to suppress AHR, pulmonary eosinophilia, and the production of IL-5 from OVA-activated mediastinal lymph nodes (MLNs) cells. The treatment of AAV/CD39 attenuated the asthmatic airway inflammation and AHR. Collectively, ATP could be a potential target for gene therapy on asthma.
The Role of Regulator of G-Protein-Signaling 4 (RGS4) in Control of Airway Hyper-responsiveness in Asthma
Authors: Madigan Laura , Gordon Elizabeth , Ganesan Sundar , Levine Stewart , Druey Kirk M
Published: January 2023

Asthma is a common respiratory disease characterized by airway hyper-responsiveness (AHR), inflammation, and airway obstruction. In most patients, this disease can be managed with steroids and bronchodilators. However, these treatments are not effective for the 15%-25% of patients with severe asthma who may develop fixed airway obstruction due to increased airway smooth muscle (ASM) mass.

ASM cells control airway patency through G-protein coupled receptors (GPCRs). Namely, GPCRs can activate the IP3 pathway to induce calcium release and airway contraction. Regulators of G-protein signaling (RGS) are a family of proteins that are known to negatively regulate the activity of GPCRs. In ASM cells, RGS4 is relatively abundant compared to other RGS4 proteins. In people with asthma, increased RGS4 expression is observed in the group with greatest impairment in lung function.

We hypothesize that aberrant RGS4 expression and/or function contributes to AHR through dysregulated GPCR signaling in ASM cells. We studied bacterial artificial chromosome (BAC) transgenic (Tg) mice, which overexpress both RGS4 and eGFP under the control of endogenous RGS4 promoter. In models of acute asthma, Tg mice exhibited reduced AHR as assessed by measurements of lung resistance in live, anesthetized mice and contraction of precision cut lung slices. These findings suggest that RGS4 is an important controller of bronchial tone in asthma through its effects on contraction signaling in ASM cells.
Transgenic expression of H-2Db class I molecules influences the development of brain atrophy during Picornavirus infection
Authors: Huseby April M , Atanga Pascal A , Gamez Jeffrey D , Cumba-Garcia Luz M , Macura Slobodan I , Johnson Aaron J
Published: January 2023

Brain atrophy is a common feature of numerous neurological diseases in which the role of neuroinflammation remains poorly defined. We have previously demonstrated the development of brain and spinal cord atrophy in the Theiler’s murine encephalomyelitis virus (TMEV) model of multiple sclerosis. However, cellular and molecular mechanisms of brain atrophy remain poorly understood. We therefore evaluated the contribution of major histocompatibility (MHC) class I molecules in atrophy development during TMEV infection. To accomplish this, we created a novel transgenic FVB/NJ mouse by introducing the H-2Db (Db) class I molecule. Expression of Db class I molecule confers resistance to persistent TMEV infection and demyelination in the normally susceptible FVB/NJ strain (H2-Dq class I haplotype). Next, we compared the development of brain atrophy, assessed by volumetric analysis of T2-weighted MRIs, in FVB/Db mice to wild-type FVB/NJ mice following viral infection. FVB/NJ mice did not significant brain develop atrophy over 4 months, whereas significant brain atrophy was observed in the FVB/Db mice. FVB/Db mice also developed a significant increase in the overall number of CNS infiltrating CD8 T cells and a strong CD8 T cell response towards an immunodominant Db TMEV epitope, VP2121-130. A number of CD8 T cells were also observed in close proximity to virus infected neurons. We therefore propose a hypothesis that class I restricted CD8 T cell responses promote the development of brain atrophy. This model provides a unique opportunity to analyze the contribution of immune cells to neuronal loss and brain atrophy in a system where persistent virus infection and demyelination are not confounding variables.
Investigation of immune cell maintenance and function in MBL-sufficient and MBL-deficient individuals
Authors: Dean Melinda M , Luimstra Jolien , Rooks Kelly , Flower Robert L
Published: January 2023

Background and Aims

Mannan-binding lectin (MBL) is a mediator of innate immunity, deficiency is associated with increased severity of infection. Pathogen recognition and complement activation functions are well characterised, however, mechanisms behind the reduced overall response to pathogens in of MBL-deficient (MBL-D, defined as <0.5μg/mL) individuals remains poorly understood.

Methods

Absolute counts of NK cells, neutrophils, monocytes, myeloid DC, plasmacytoid DC, lymphocyte subsets (including Tregs), and the concentrations of 12 inflammatory cytokines were assessed on freshly collected peripheral blood (healthy volunteers) and following 6 and 24 hours in vitro for MBL-sufficient (MBL-S, n=10) and MBL-D (n=11) individuals (unpaired T-test, 95% CI). Mixed lymphocyte response (MLR) infection models (LPS, zymosan) as well as intracellular staining were used to study the maintenance and function of T and B lymphocytes.

Results

We observed few differences between MBL-S and MBL-D individuals in immune cell maintenance and function. Of note, we found a significantly higher proportion of CD4+CD25+Foxp3+ Tregs in MBL-D individuals compared to MBL-S individuals following 3 days autologous MLR in both unstimulated and zymosan-stimulated cultures.

Conclusions

This study supports the hypothesis that in association with immune challenge MBL deficiency can result in poorer patient outcomes. Our finding of increased Tregs in MBL-D individuals warrants further investigation in the clinical setting, as a potential mechanism underpinning increased disease susceptibility and severity of infection in MBL-D individuals.
Distinct features of human CD8 T cell TCR repertoire specific to influenza A virus matrix protein M1
Authors: Chen Guobing , Yang Xinbo , Ko Annette , Gao Mingming , Zhang Yongqing , Shi Alvin , Sun Xiaoping , Mariuzza Roy A. , Weng Nan-Ping
Published: January 2023

CD8 T cells play a vital role in the immune response to viral infection. Diversity of the T cell antigen receptor (TCR) is believed to be essential for the host to survive in this microorganism-rich world. However, there is limited information regarding the characteristics of the TCR repertoire to defined viral antigens in humans. Here, we report a comprehensive analysis of CD8 TCR repertoire diversity (size, V and J gene usage), the consensus binding motifs in CDR3, affinity, and structure of selected a/b paired TCRs to influenza A virus matrix protein M1 (GILGFVFTL, GIL) on HLA-A2+ healthy individuals. Using high-throughput sequencing coupled with the unique molecular identifier (UMI) method, we identified several hundred GIL-binding TCRa and TCRb. TRAV13-1 (63.2%) and TRAJ42 (72.5%) were most frequently found in alpha chain whereas TRBV19 (94.9%) and TRBJ2-5 (54.8%) were frequently found in beta chain. Furthermore, the length of the CDR3 amino acid region was highly restricted with over 70% of alpha (10) and of beta (11). CDR3 sequence analysis revealed shared motifs among different TCRs (a and b chains). We isolated paired expressed TCRa/b from single cells for further structural analysis. The affinity between TCRs and GIL-HLA-A2 complexes showed a wide range (1.8–191 mM). Finally, we resolved two crystal structures of TCRs-GIL-HLA-A2 complexes and found that the TCRs exhibited similar patterns of interactions with the antigen, even though the V, J, and CDR3 lengths were different. Together, our findings revealed the size of the antigen-specific CD8 TCR repertoire, the consensus binding motifs in CDR3, binding affinity of TCR and pMHC, and shared patterns of TCR-pMHC interactions.
Regulation of macrophage anti-microbial functions by the histone methyltransferase MLL1
Authors: Carson William F , Cavassani Karen , Soares Elyara , Hirai Soichiro , Kittan Nicolai , Joshi Amrita , Schaller Matthew , Gallagher Katherine , Matsukawa Akihiro , Aronoff David M , Dou Yali , Kunkel Steven L
Published: January 2023

Mixed-lineage leukemia 1 (MLL1) is a chromatin-modifying enzyme responsible for histone modifications thought to activate gene transcription. Previous studies have linked MLL1-dependent histone methylation to gene expression in response to inflammatory stimuli. To determine if MLL1 plays a role in driving immune responses during inflammatory responses, mice deficient in a single copy of MLL1 (MLL1+/−) were subjected to an experimental model of polymicrobial peritonitis. MLL1+/− mice exhibited increased survival as compared to control animals, correlating with a decreased bacterial burden and decreased levels of proinflammatory chemokines and cytokines in the peritoneal lavage. Bone-marrow derived macrophages from MLL1+/− mice exhibited increased antimicrobial activity in vitro, including increased phagocytosis of bio-particles and increased killing of Group A Streptococcus. Similar results were observed in vitro with MLL1 knockout macrophages (Lys2-Cre MLLfx/fx), with Cre+ bone marrow-derived macrophages exhibiting increased killing of Group A Streptococcus as compared to Cre− littermate-derived macrophages. Gene chip analysis of RNA expression in Cre− and Cre+ macrophages indicated numerous genes whose expression patterns were modulated as a result of MLL1 deficiency in response to classical activation stimuli. Expression pattern changes included both expected decreases in proinflammatory genes, and also surprisingly increases in genes associated with interferon responses and antimicrobial defense. These results implicate MLL1 as a key epigenetic regulator of the complex inflammatory and anti-microbial functions of macrophages.
Cholesterol crystals activate the lectin complement pathway via ficolin-2 and MBL - implications for the progression of atherosclerosis
Authors: Pilely Katrine , Rosbjerg Anne , Genster Ninette , Gál Péter , Pál Gábor , Stahl Gregory L. , Mollnes Tom Eirik , Espevik Terje , Garred Peter
Published: January 2023

Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques by inducing inflammation and by functioning as an endogenous danger signal. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. In this study we hypothesized that pattern recognition molecules (PRM) from the lectin pathway bind CC and functions as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC using recombinant proteins, specific inhibitors as well as deficient and normal sera. Binding was assessed by flow cytometry and microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium dependent manner while ficolin-2 binding was calcium independent. No binding was observed for ficolin-1 or ficolin-3. Moreover, we demonstrated that IgM, but not IgG bound to CC and that C1q binding was facilitated by IgM. In conclusion our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.
The Effect of Hepsin in Interferon Induction Pathways
Authors: Liu Helene Minyi , Hsin Fu
Published: January 2023

The induction of type I interferon (IFN) is critical for antiviral innate immune response. During virus infection, Retinoic acid-inducible gene I (RIG-I) recognizes the viral RNA and activates Mitochondrial antiviral-signaling protein (MAVS). MAVS further triggers downstream signaling to activate Interferon regulatory factor 3 (IRF3) to induce type I IFN. Many viral proteases cleave adapter proteins in the IFN induction pathway, such as HCV NS3/4A cleaving MAVS, as a strategy to evade anti-viral innate immunity. However, it is not clear whether host protease may also regulate IFN induction pathway to control the steadily low expression of IFN in uninfected cells. Here we report that Hepsin (HPN), which belongs to type II transmembrane serine protease family, could inhibit the production of type I IFN during RNA virus infections in several cell-lines. Hepsin expression could be detected in Huh7 and HepG2 hepatocytes but not ubiquitously in all cell types. Knocking-out Hepsin in mouse embryonic fibroblasts resulted in higher expression of IFNβ and interferon response genes (ISGs). Besides, when co-transfected with N-RIG, which is constitutively activating IFNβ expression, Hepsin could decrease IFNβ promoter activity. However, under the expression of IRF3-5D, Hepsin could not reduce IFNβ promoter activity, suggesting that Hepsin might target certain adaptor proteins downstream of RIG-I and upstream of IRF3. We showed an inverse correlation with wild-type Hepsin expression but not the protease-deficient mutant Hepsin in the IFNβ promoter activities . Consequently, these results reveal a novel role of Hepsin in regulating the type I IFN induction pathway in hepatocytes.
Emerging roles of an innate immune regulator TAPE in Toll-like receptors, RIG-I-like receptors, and beyond
Authors: LING PIN , Chen Kuan-Ru , Kao Chen-Chu , Chuang Huai-Chia , Tan Tse-Hua
Published: January 2023

Pattern-recognition receptors (PRRs) trigger innate immune defenses against pathogen infection via downstream signaling pathways linking to inflammation and cell-autonomous immunity like phagocytosis and autophagy. IKK family kinases, IKKα and IKKβ, function to relay PRR signals to proinflammatory cytokine production to amplify innate immune responses. TBK1, a non-canonical IKK kinase, links nucleic acid sensors to type I interferon induction against viral infection and also regulates the autophagic clearance of intracellular bacteria. TBK1-Associated Protein in Endolysosomes designated TAPE, also known as CC2D1A, is an innate immune regulator acting upstream of Trif to regulate the TLR3 and TLR4 pathways, or upstream of MAVS to regulate the cytosolic RIG-I-like receptor (RLR) pathways. To our best knowledge, TAPE is the first regulator implicated in both the endosomal TLR and cytosolic RLR pathways at such an early step. We are thus interested in investigating in vivo roles of TAPE in innate immunity and molecular mechanisms by which TAPE regulates TLRs, RLRs, and possibly other PRRs. TAPE conditional knockout (cKO) mice, in which TAPE is selectively disrupted in immune cells, have been generated for our study. Our results showed that upon influenza A virus infection, TAPE cKO mice exhibited a more severe mortality than wild type mice. Further, TAPE cKO mice were shown to be more susceptible to Salmonella Typhimurium infection but more resistant to LPS-induced septic shock. Notably, ex vivo results showed that TAPE was critical for the autophagic clearance of Salmonella Typhimurium. Together, our data support a critical role for TAPE in regulating innate immune defenses through TLRs, RLRs, and autophagy.
EHD1 as a Positive Regulator of Macrophage CSF1R
Authors: Cypher Luke R , Bielecki Timothy Alan , Iseka Fany M , Band Vimla , Band Hamid
Published: January 2023

Macrophage biology is regulated via receptor tyrosine kinase (RTK) CSF-1 receptor (CSF1R). Anomalous macrophage biology has been shown to be hallmark in diseases such as cancer and atherosclerosis. Although CSF1R signaling has been studied, little is known about the mechanisms which govern the endocytic trafficking of the receptor. EHD1 is known to regulate endocytic recycling of receptors, but the role of EHD1 in macrophages is unstudied. In this study, we discovered EHD1 knockout (KO) murine bone marrow-derived macrophages (BMDMs) had markedly reduced total and surface CSF1R levels and a corresponding decreased downstream functional output due to decreased CSF1R signaling. EHD1 KO led to perinuclear CSF1R accumulation which co-localized with the Golgi (GM130). These findings reveal a novel and essential role for EHD1 in transporting newly synthesized CSF1R to the cell surface. Our study expands our understanding of RTK trafficking which has potentially dramatic implications for designing new targeted therapies for devastating human diseases.
The C-terminal tail of TRIM56 dictates antiviral restriction of influenza A and B viruses by impeding viral RNA synthesis
Authors: Liu Baoming , Li Nan L , Shen Yang , Bao Xiaoyong , Fabrizio Thomas , Elbahesh Husni , Webby Richard J. , Li Kui
Published: January 2023

Accumulating data suggest that tripartite motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae (yellow fever virus, dengue virus and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Herein, we show TRIM56 puts a check on replication of influenza A and B viruses in cell culture, but does not inhibit Sendai virus or human Metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza activity was independent of the E3 ligase activity, B-box, or coiled-coil domains. Rather, deletion of a 63-residue long, C-terminal tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Consistent with this, a fraction of TRIM56 was re-distributed to the nucleus after IAV infection. Together, these data reveal novel antiviral activity of TRIM56 against influenza A and B viruses and provide novel insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. These findings raise the possibility that small molecules mimicking the C-terminal portion of TRIM56 could be developed into broad influenza antivirals.
Plasmid electrotransfer activates DNA pattern recognition receptors and induces production of proinflammatory molecules
Authors: Heller Loree , Bosnjak Masa , Znidar Katarina , Cemazar Maja
Published: January 2023

Electroporation or electrotransfer is an effective delivery system for introducing plasmid DNA into cells and tissues. During this process, pDNA primarily enters the cell via endocytosis, although some DNA reaches the cytosol. Endosomal, cytosolic, and nuclear DNA-specific pattern recognition receptors (PRRs) in many cell types have been described. Activated PRRs induce the production of proinflammatory molecules and programmed cell death.

In B16.F10 mouse melanomas, electrotransfer of empty backbone plasmid induced the production of several proinflammatory cytokines and chemokines including IFNβ, implicating the binding and activation of intracellular DNA-specific PRRs. Histologically, tumor necrosis independent of caspase-3 was observed. The purpose of this study was to investigate whether tumors and cells express cytosolic DNA sensors and whether these sensors respond to pDNA electrotransfer.

Although the mRNAs for several DNA sensors were detected in tumors, none was significantly upregulated. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated after DNA electrotransfer. The mRNAs for several DNA sensors were present in these cells and DAI, DDX60, and p204 mRNAs were significantly upregulated after pDNA electrotransfer. DDX60 protein levels were coordinately upregulated. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival.

Taken together, increased IFNβ and DNA sensor expression accompanied by cell death and tumor necrosis indicate that DNA electrotransfer activates intracellular DNA sensors in B16.F10 cells and tumors, producing both in vitro and in vivo effects.
CoCr microparticles induce a robust type 2 innate immune response that is dependent on SYK and BTK signaling
Authors: Palma Mark J , Mishra Pankaj Kumar , Millman Ariel , Hallab Nadim J , Beebe Kathleen , Benevenia Joseph , Gause William C
Published: January 2023

Cobalt chrome (CoCr) is a major component of orthopaedic implants and a source of metallic wear debris, which may contribute to harmful inflammation and implant failure. Intra-peritoneal inoculation of CoCr induces a robust innate type 2 response with increases in M2 macrophages, eosinophils, and neutrophils and this response is dependent on Spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK) signaling, but not TLR, Caspase-1, or Cyclooxygenase signaling. We have also shown that this robust response is independent of T and B cell signaling. To examine this response in a more clinically relevant murine model, bilateral intra-articular knee injections of CoCr microparticles were given to C57BL/6 mice and changes in different innate immune cells were assessed from knee synovial cell isolates. Administration of the SYK inhibitor BAY 61-3606, or the BTK inhibitor Ibrutinib, abrogated innate immune cell recruitment and M2 macrophage polarization which is otherwise observed 2 days after inoculation with CoCr microparticles. The presence of M2 macrophages (CD68+, CD206+, and CD163+) is also observed in human peri-prosthetic tissue samples obtained from patients undergoing revision joint arthroplasty. Taken together, our findings suggest that BTK signaling is required for the type 2 inflammatory response to CoCr microparticles, providing a potential novel target for control of harmful inflammation leading to fibrosis and aseptic loosening.
zVAD-induced necroptosis in TLR3/4-stimulated macrophages involves TRIF-dependent interferon signaling pathway and ROS-dependent autophagy
Authors: Lin Wan Wan
Published: January 2023

Depending on the cell types, caspase inhibition might lead to autophagy. In macrophages, necroptosis and autophagic cell death involving reactive oxygen species (ROS) production can be induced by co-treatment with lipopolysaccharide (LPS) and a cell-permeable pan-caspase peptide inhibitor zVAD. Here we elucidated the signaling pathways and molecular mechanisms underlying this process. First we confirmed that primary bone marrow-derived macrophages treated with LPS plus zVAD or polyIC plus zVAD display cell death with necrotic and autophagic features. Although zVAD did not affect LPS- or polyIC-induced IKK, JNK and p38 activation, it enhanced IRF3 and STAT1 activation as well as type I IFN gene and protein expression. Interestingly these enhancing actions of zVAD were inhibited by necrostatin-1. Our data further support the involvement of autocrine IFNs action in ROS-dependent necroptosis, as cell death induced by TLR ligands with zVAD co-treatment was prevented by ROS scavenger BHA, neutralizing antibody targeting IFNR and JAK inhibitor AZD1480. Consistently, LPS plus zVAD-elicited ROS production was inhibited by necrostatin-1, IFNR antibody and AZD1480. Accordingly, both cell death, LC3II accumulation and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. Taken together, we propose a new molecular basis for TLR-mediated necroptosis under caspase inhibition in macrophages, which involves the enhanced TRIF-RIP1-IRF3 signaling pathway and autocrine IFNs-IFNR-JAK-STAT1 action, leading to ROS-dependent autophagic cell death.
Comparison of the TLR7 and TLR9 innate immune signaling pathways in african green monkeys and rhesus macaques
Authors: Cox Kara S. , Wang I-Ming , Nefedov Alexey , Zhang Xiaohua Douglas , Dubey Sheri , Bett Andrew , Casimiro Danilio , Mandl Judith , Feinberg Mark B.
Published: January 2023

Innate immunity is an evolutionary mechanism which is involved in the initial detection of pathogens and stimulates the first line of host defense. The Toll-like receptor (TLR) family has been the most extensively studied innate immune pathway and it has been shown that TLRs can be activated in response to virtually any microbe which invades the host. These innate immune responses play a critical role in the development of pathogen-specific adaptive immune responses and disease pathology and are targeted by the use of adjuvants in modern vaccines. Importantly, divergent TLR7 and TLR9 signaling pathways between rhesus macaques and sooty mangabeys have been shown to distinguish between pathogenic and nonpathogenic AIDS virus infections in those species. Therefore, it is important to understand the TLR signaling pathways of non-human primate (NHP) species that are currently used for infectious disease and vaccine research. In the present study, we administered defined TLR7 and TLR9 agonists to two distinct NHP species, african green monkeys and rhesus macaques. We evaluated B cell, T cell, NK cell, monocyte, plasmacytoid dendritic cell and myeloid dendritic cell activation at 0, 24, 72 and 168 hours after dosing. Gene expression profiling and full pathway analysis was also evaluated to illuminate fundamental aspects of innate immune responses that underlie the mechanism of action of TLR-agonist adjuvants in these two NHP species.
Catalase deficiency in thymic stromal cells promotes key stromal functions in the young, steady state mouse thymus
Authors: Semwal Manpreet Kaur , Xiao Yangming , Venables Thomas , Griffith Ann V
Published: January 2023

T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells directs the progenitors along a well-characterized program of differentiation. However, the biology of the stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen (ROS) levels in this population, eventually leading to thymic atrophy. Here, we address the possibility that high ROS levels serve physiological functions in stromal cells in the young, steady state thymus. Our preliminary data indicate that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function are diminished in young mice relative to non-transgenic littermates. We find evidence of diminished tissue-restricted antigen (TRA) expression and autophagy in mCAT Tg mice, both of which are critical for establishing a self-tolerant T cell repertoire. We propose that a high ROS environment in stromal cells promotes promiscuous gene expression and autophagy critical for stromal induction of T cell tolerance in the young thymus, while accumulated oxidative damage ultimately undermines healthy thymus function.
Alteration of T cell Repertoire in insulin-sensitive tissues following the onset of Type 2 diabetes
Authors: Hernandez Jeniffer B , Yoon Young-Sil , Montminy Marc
Published: January 2023

The CREB/CRTC2 pathway functions importantly in the liver, where it promotes fasting glucose balance by upregulating the gluconeogenic program. More recently CREB has emerged as an important regulator of immune function. We have shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of the pro-inflammatory T cell, Th17. We are curious to determine if CRTC2 plays a role in the chronic inflammatory state during obesity that contributes to the development of insulin resistance. One of the consequences of insulin resistance is hyperglycemia, therefore we set out to determine what the effect of culturing T cells under high glucose concentrations would be. Interestingly, T cells cultured under high glucose conditions had a decreased ability to differentiate into Th17 cells compared to T cells cultured under normal glucose conditions. CRTC2-deficient T cells had a greater decrease in their ability to differentiate into Th17 cells in high glucose concentrations compared to wildtype T cells. To determine if the T cell repertoire is altered in insulin-sensitive tissues following diet induced Type 2 diabetes, wildtype mice were fed a normal chow or high fat diet for 20 weeks. Consistent with our in vitro findings, lean mice had a greater number of IL17+ T cells compare to obese mice. Our current findings suggest that IL17+ T cells may have a beneficial role instead of a role in promoting the chronic inflammatory state during obesity that contributes to the development of insulin resistance.
CCR7+ naive iNKT serve as precursors for iNKT effector subsets and distinct mechanisms are involved in their thymic retention
Authors: Wang Haiguang , Lee You Jeong , Jameson Stephen C. , Hogquist Kristin A.
Published: January 2023

Invariant natural killer T cells (iNKT) are CD1d restricted, lipid recognizing αβ T cells that modulate immunity under various contexts. iNKT are comprised of distinct effector subsets, NKT1, NKT2 and NKT17, characterized by long term retention in the thymus. However, the developmental steps of iNKT cell effector differentiation have been controversial, and mechanisms underlying their thymic retention are unclear. Specifically, the markers historically used to define stage 1 and 2 precursors also include mature effector NKT2 and NKT17 cells. Using RNAseq, we identified CCR7 as a candidate marker for undifferentiatied or naïve iNKT precursor. We demonstrate here that CCR7+ iNKT cells, distinguished from stage 0 iNKT by low CD24 expression, migrate from thymic cortex to medulla to give rise to iNKT effector subsets. CCR7+ iNKT express higher Rag2GFP, indicating they are “younger” than other thymic iNKT cells. Moreover, iNKT recent thymic emigrants in the periphery, identified by intrathymic labeling, are predominantly CCR7+ iNKT that express both Rag2GFP and KLF2, and are Qa2 negative. Busulfan chimeras confirmed that CCR7+ naive iNKT emigrate from thymus well before the accumulation of thymic effector iNKT cells. Type I and II interferon signals synergistically up-regulate CD69 in NKT1, and contribute to their retention in thymus. NKT17 highly express CD103 that might be critical for their thymic retention. Although it remains unclear how NKT2 are retained, their selective expression of IL-4 and RANK ligand influence effector CD8 T cell, as well as thymic DC and medullary epithelial cells (mTEC) development. Our findings provide novel insights into the developmental pathway of iNKT cells and the mechanisms of their thymic retention.
TRAF1 negatively regulates C-type lectin receptor-induced proinflammatory response to fungal infection
Authors: Xiao Hui , Deng Zihou , Watts Tania H
Published: January 2023

Fungal infections pose serious health threat worldwide, causing severe mucosal and systemic candidiasis in elderly people, AIDS patients and organ recipients. Through sensing fungal cell-wall components b-glucan and mannan, C-type lectin receptors (CLRs) dectin-1 and dectin-2/3 play pivotal role in the induction of anti-fungal innate and adaptive immune responses. However, the regulatory mechanisms of CLR signaling remain to be better understood. Indeed, our previous work demonstrated that the protein tyrosine phosphatase SHP-2 acts as a positive regulator of CLR-induced signaling, and thus plays a critical role in DCs to promote anti-fungal Th17 response. In this study, we found that fungus-elicited CLR signals are also stringently controlled by negative regulation. Upon C. albicans infection, TRAF1 was highly induced in skin, lung and kidney. Elevated TRAF1 expression was also detected in macrophages and DCs stimulated by dectin-1 and dectin-2/3 ligands, respectively. Mechanistically, TRAF1 acted as a feed-back negative regulator critically controlling the induction of proinflammatory genes, such as Cxcl1 and Tnf, in response to fungal infection. Consistently, TRAF1-deficient mice exhibited increased neutrophil-infiltration, highly efficient fungal eradication and ameliorated tissue damage, culminating on improved host defense and better survival. Taken together, this study identified a new feed-back regulatory mechanism by which CLRs regulate anti-fungal proinflammatory response.
The life cycle stages of the opportunistic fungal pathogen Pneumocystis murina provoke a differential immune response in antigen-presenting cells
Authors: Evans Heather Marie , Garvy Beth A
Published: January 2023

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. The life cycle of Pneumocystis species consists of a trophic stage and an ascus-like cystic stage. Our lab has published data demonstrating that cysts stimulate NF-κB translocation in alveolar macrophages, but the trophic form does not. Here, we demonstrate that infection of wild-type adult mice with a normal mixture of trophic and cystic P. murina life forms elicits a more robust innate immune response in the alveolar spaces and lung parenchyma than infection with the trophic form alone. To determine the pattern recognition receptors that bind trophozoites and cysts, we examined expression of C-type lectin receptors on lung CD11c+ dendritic cells in vivo. Infection with a mixture of trophozoites and cysts resulted in the upregulation of dectin-1 and mannose receptor, while infection with trophozoites alone resulted in upregulation of dectin-2, DEC-205 and mannose receptor. In addition, we demonstrate that bone marrow-derived dendritic cells stimulated with a mixture of trophozoites and cysts produced the proinflammatory cytokines IL-1β and IL-6. In contrast, exposure to trophozoites suppressed β-glucan-induced IL-1β, IL-6, and TNFα production by bone marrow-derived dendritic cells. A targeted gene expression assay is being used to evaluate gene expression in dendritic cells following stimulation with trophozoites alone or a mixture of trophozoites and cysts. We propose that trophozoites suppress β-glucan-signaling, but are sufficient for stimulating protective responses in immunocompetent individuals.
Examining the function of chemokine receptor deficient-monocytes during infection with Nippostrongylus brasiliensis
Authors: Barnes Mark A , Wang Spencer , Lo David , Nair Meera G
Published: January 2023

Nippostrongylus brasiliensis (Nb) is a murine helminth that acutely infects the lungs and the small intestine, and induces a strong Th2 immune response, which includes contributions from innate immune cells such as monocytes. CCR2, the receptor for MCP1, which is expressed primarily on monocytes, was predictably important for monocyte recruitment during Nb infection. However, it was also shown to be necessary to control hemorrhaging as CCR2−/− mice had more blood in the bronchoalveolar lavage fluid compared to wild-type (WT) mice. In addition, CCR2−/− mice produced higher egg burden and more viable worms based on parasite ATP assays. Signaling through CX3CR1, another receptor expressed on monocytes provoked the opposite phenotype. CX3CR1 was not crucial for cellular recruitment after infection, but Nb from CX3CR1−/− mice produced less eggs and were less viable than Nb from WT mice. To investigate potential mechanisms by which monocytes influence worm viability, infective stage Nb (L3) were co-cultured with sorted Ly6c+ monocytes from CX3CR1 WT or knockout (ko) mice. After 5 days in culture, Nb incubated with ko Ly6c+ cells were less motile than Nb cultured with WT Ly6c+ cells. Taken together, these data indicate that CCR2-dependent infiltrating monocytes control inflammation and promote worm killing, while CX3CR1 signaling in monocytes promotes worm viability potentially by influencing worm motility.
Anti-Fungal Functional Tc17 Cells Persist as Long-Lasting Memory Without Plasticity Towards IFNγ
Authors: Nanjappa Somashekarappa Gowda , McDermott Andrew , Fites Scott , Klein Bruce
Published: January 2023

Memory homeostasis of Th1/Tc1 cells is well characterized, whereas memory Th17-cell homeostasis, persistence and plasticity are debated. We have shown that IL-17A-producing CD8+ T-cells (Tc17) are necessary for vaccine-induced fungal immunity in CD4+ T-cell compromised hosts. Here, we systematically evaluated persistence, fidelity, plasticity and functional role in immunity of anti-fungal Tc17 cells using IL-17A fate-mapping reporter mice. We show that Tc17 cells stably maintained as functional bona fide IL-17A producing memory cells, with little plasticity towards IFNγ, even in the absence of vaccine antigen. Memory Tc17 cells stably and progressively improved in their production of multiple Tc1 cytokines but not IFNγ, and contributed for IL-17A-dependent vaccine resistance. Memory Tc17 cells were canonical CD8+ T cells portraying a set of phenotypic attributes that were distinct from Tc1 cells while remaining Ror(γ)hi, T-betlo and EOMESlo. Interestingly, in contrast to Tc1, memory Tc17 cells expressed lower levels of anti-apoptotic factors Bcl-1, Bcl-xL and MCL-1. However, basal homeostatic proliferation of memory Tc17 cells was significantly higher than Tc1 cells. Finally, we unraveled a dichotomous role of HIF-1α for effector vs memory Tc1 and Tc17 cells, where HIF-1α was essential for expression of IL-17A in memory cells while it was required for expression of IFNγ in effector cells.
TRAF6 in dendritic cell plays a key role to control immune response to dietary antigens
Authors: Han Daehee , Choi Yongwon , Surh Charles D
Published: January 2023

TRAF6 is critical for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). Recently, by studying mice DC-specific deletion of TRAF6 (TRAF6ΔDC) we have reported that TRAF6 expression in DCs is important to prevent a spontaneous Th2-associated gut inflammatory disease. To formally demonstrate an involvement of commensal microbiota, we have generated TRAF6ΔDC bone marrow chimeras under germ-free (GF) conditions. Unexpectedly, the Th2-associated disease in GF TRAF6ΔDC chimeras was exacerbated compared with TRAF6ΔDC chimeras generated under conventional conditions. Finally, to test whether the Th2-associate disease is driven by dietary antigens (Ags), we established TRAF6ΔDC bone marrow chimeras in “Ag-free” mice, in which GF mice are raised with a chemically-defined elemental diet devoid of macromolecules. Strikingly, the induction of a spontaneous intestinal Th2-associated inflammatory disease was dramatically averted in “Ag-free” TRAF6ΔDC chimeras, indicating that TRAF6 expression in DCs is essential to establish and/or maintain oral tolerance to dietary Ags.