bluuebunny/crossref_metadata_2025 huggingface dataset:
Th17 inflammation in the lungs is sustained by multiple exposures to house dust and is dependent on TLR4 and conventional dendritic cells
Authors: Shalaby Karim H , Lyons-Cohen Miranda , Thomas Seddon Y , Whitehead Gregory S , Nakano Hideki , Cook Donald N
Published: January 2023

Microbial products can prime antigen-specific T helper 17 cell responses to inhaled allergens. However, once established, it is unclear how pathogenic lung Th17 responses are sustained. To study this, we sensitized mice to inhaled ovalbumin (OVA)together with various environmental adjuvants, including house dust extract(HDE). Animals were then chronically challenged with OVA alone, or OVA plus the adjuvant previously used during sensitization. Challenge with OVA plus HDE elicited greater airway neutrophilia, lymphocytosis and IL-17A than did challenge with OVA alone. HDE exposure with OVA also promoted greater expansion of adoptively transferred, OVA-specific, IL-17A-fate mapping Th17 cells. These effects were independent of enzymatic activity in HDE, but required TLR4 in recipient mice. Analysis of Tlr4 bone marrow chimeric mice and conditional TLR4 knockout mice indicated that Th17 proliferation is sustained by HDE via TLR4 signaling predominantly in hematopoietic cells, including CD11c+ cells, rather than in airway epithelial cells. IL-23 contributes to the maintenance of these lung Th17 cells. Use of genetically modified mice lacking distinct lung myeloid cell subsets, and co-culture of various lung antigen-presenting cell types with mature Th17 cells, revealed that conventional dendritic cells (either CD103+ or CD11b+), rather than lung macrophages or B cells, support the revival of Th17 clonal expansion and activation. Thus, maintenance of Th17-dependent inflammation in the lung requires persistent innate immune responses, TLR4, and the presence of conventional dendritic cells.
Quantification of hTERT alternatively spliced isoforms in resting and activated human T cell subsets
Authors: Patrick Michael S. , Cheng Nai-Lin , An Jie , Weng Nan-ping
Published: January 2023

The activity of telomerase is regulated at multiple levels of the reverse transcriptase (hTERT) subunit. In T cells, it is well documented that telomerase activity is not detected in resting cells while it is high in activated cells. However, it is unclear whether full-length (FL) hTERT mRNA and AS products (ASPs) are expressed in resting T cells and how they affect T cell differentiation and function. Here, we analyzed hTERT mRNA (full-length and 3 types of ASPs) in CD4 and CD8 T cell subsets (naïve, central and effector memory) isolated from 66 healthy human adults. We detected hTERT FL mRNA in resting CD4 and CD8 T cell subsets and found that the level of hTERT FL mRNA decreased from naïve (N) to central memory (CM) to effector memory (EM) cells. Furthermore, hTERT FL mRNA levels were higher in CD4 than in corresponding CD8 subsets and undetectable in CD8 EM of most donors. In vitro stimulation with anti-CD3/CD28 antibody-coated beads induced a high level of hTERT mRNA in all subsets without changing the order of hTERT abundance in CD4 and CD8 T cell subsets. Finally, we measured telomerase activity in these T cell subsets and confirmed that resting T cells did not have detectable telomerase activity, but activated T cells did and the levels of telomerase activity concurred with expression levels of hTERT FL mRNA. In conclusion, quantifying hTERT FL and ASPs in T cell subsets confirmed no telomerase activity in the presence of FL hTERT mRNA in resting T cells and revealed a decrease of hTERT levels with T cell differentiation from naïve to memory. Currently, we are assessing which transcription factors are responsible for regulating hTERT expression levels in T cell differentiation.
LPS-elicited TSLPR expression enriches a functionally discrete subset of human CD14+ CD1c+ monocytes
Authors: Borriello Francesco , Iannone Raffaella , Di Somma Sarah , Vastolo Viviana , Petrosino Giuseppe , Visconte Feliciano , Raia Maddalena , Scalia Giulia , Loffredo Stefania , Varricchi Gilda , Galdiero Maria Rosaria , Granata Francescopaolo , Del Vecchio Luigi , Portella Giuseppe , Marone Gianni
Published: January 2023

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor complex, a heterodimer composed of TSLP receptor (TSLPR) and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLP receptor complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is ill-defined. We demonstrate that TSLP enhances human CD14+ monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14+ CD16− monocytes (TSLPR+ mono) expresses TSLP receptor complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional and transcriptomic analysis revealed specific features of TSLPR+ mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e. GAS6, ALOX15B, FCGR2B, LAIR1). Strikingly, TSLPR+ mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14+ CD1c+ cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared to healthy controls. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14+ CD16− monocytes and prompt further ontogenetic and functional analysis of CD14+ CD1c+ and LPS-activated CD14+ CD1c+ TSLPR+ monocytes.
Binding of small molecule to ASC leads to the suppression of NLRP3 inflammasome and acute gout symptoms
Authors: Lee Hye Eun , Yang Gabsik , Lee Joo Young
Published: January 2023

The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, which is composed of NLRP3, the adaptor protein ASC, and caspase-1, is closely linked to the pathogenesis of metabolic diseases. We investigated whether caffeic acid phenethyl ester (CAPE), an anti-inflammatory phytochemical, could modulate the activation of NLRP3 inflammasome and have beneficial effects on the related disease such as acute gout. In primary mouse macrophages, CAPE blocked NLRP3 inflammasome activation induced by monosodium uric acid (MSU) crystals. In a mouse air pouch inflammation model and an acute gout model, oral administration of CAPE suppressed MSU-induced caspase-1 activation and interleukin (IL)-1b production, correlating with the attenuation of inflammatory symptoms. CAPE inhibited inflammasome activation induced by ASC, but not NLRP3 nor caspase-1. CAPE directly associated with ASC, resulting in the blockade of NLRP3-ASC interaction. These results demonstrate a novel inhibitory mechanism by which small molecules regulate NLRP3 inflammasome targeting ASC, suggesting the therapeutic strategy for NLRP3-related inflammatory diseases.
Composition of the myddosome during the innate immune response
Authors: Gillen Joseph G , Nita-Lazar Aleksandra
Published: January 2023

Critical for the innate immune response, the myddosome is a complex of proteins characterized by the presence of myeloid differentiation factor 88 (Myd88). The myddosome acts to transfer signals from the toll-like receptor (TLR) proteins to TNF receptor-associated factor 6 (TRAF6). In addition to Myd88, the myddosome contains multiple copies of the interleukin-1 receptor associated kinases (IRAK) 2 and 4 and is theorized to form following stimulation of the TLR proteins. Using affinity purification – mass spectrometry analysis (AP-MS), we identified the Myd88-associated proteins in mouse immortalized bone marrow-derived macrophages before and after lipopolysaccharide (LPS) treatment. Prior to LPS treatment, we found the stable association of Myd88 to IRAKs 2 and 4 along with the inhibitory factors IRAK3 and Sterile alpha and TIR motif-containing protein 1 (Sarm1). Following LPS treatment, the inhibitory factors were lost and Myd88 associated to the downstream effector proteins, including TRAF6. In contrast to the model, our results suggest that the cellular myddosome is preassembled but only activates upon stimulation. Because of this, we are examining the mechanism(s) that regulate the activity of the myddosome. We are currently using AP-MS followed by phosphopeptide enrichment with MS/MS analysis to identify possible phosphorylation sites. Using these data, we will evaluate the role of phosphorylation on myddosome activity. In addition, we will use SILAC with AP-MS to examine myddosome assembly and protein phosphorylation during a time course. This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.
Caspase-11-mediated HMGB1 release from the liver drives immune cell pyroptosis in sepsis.
Authors: Deng Meihong , Scott Melanie , loughran Patricia , Wang Qingde , Lu Ben , Billiar Timothy R
Published: January 2023

High mobility group box 1 (HMGB1) was identified as a damage associated molecular pattern in sepsis. Neutralization of the HMGB1 even 24 hrs after the onset of sepsis is protective in experimental models. However, the sources of extracellular HMGB1 in sepsis and its function are not known. To address these questions, we generated myeloid (Lyz-creX HMGB1loxp/loxp) and hepatocyte (HC)-specific (Alb-creX HMGB1loxp/loxp) mouse strains and subjected these mice to cecal ligation and puncture (CLP) sepsis. Whereas CLP led to significant and sustained increases in HMGB1 levels in wild type and myeloid-specific HMGB1−/− mice, the levels were 75% lower in HC-specific HMGB1−/− mice at 18 hrs. This indicates that the liver is dominant source of HMGB1 during sepsis. The HC-specific knockout mice also showed significantly higher survival rates, as well as decreased immune cell pyroptosis in the peritoneum, spleen, and gut. Interestingly, HMGB1 level increased in HC culture media after LPS stimulation, which was not associated with cell death. Furthermore, using Liquid chromatography–mass spectrometry, we found that the HMGB1 released from HC are mainly acetyl form, suggesting that HC actively releases HMGB1 after LPS stimulation. We also found that the release of HMGB1 from cultured LPS-treated HC was TLR4 and caspase-11-dependent. Co-culture of wild type but not HMGB1−/− HC with bone marrow-derived macrophages led to increased pyroptosis of the cultured macrophages in response to LPS in a caspase-11-dependent manner. Our findings indicate that HC release HMGB1 during sepsis in a process that caspase-11-dependent and that liver-derived HMGB1 drives immune cell pyroptosis in severe sepsis.
NOD2 deficiency exacerbates imiquimod-induced, psoriasis-like dermatitis
Authors: Singh Tej Pratap , Cipolla Ellyse , Singh Satya P , Farber Joshua M
Published: January 2023

Nucleotide-binding oligomerization domain containing protein 2 (NOD2) is a CARD domain-containing pattern recognition protein in which inactivating mutations can cause Crohn’s disease. Mechanisms that have been suggested to explain the inflammation in Crohn’s disease associated with loss of NOD2 function include inadequate intestinal antibacterial defense or, on the other hand, diminished inhibition of toll-like receptor (TLR) signaling. Psoriasis is a common autoimmune skin disease for which recent data show significant up-regulation of NOD2 in keratinocytes of the lesional skin. We investigated a role for NOD2 in a TLR-driven mouse model of psoriasis-like dermatitis in which IL-17A and IL-22 are critical contributors. Following topical application of the TLR7/8 agonist, imiquimod, as compared with wild-type controls, Nod2−/− mice showed increases in epidermal thickness, epidermal neutrophils, and numbers of α/β and γ/δ T cells capable of producing IL-17A and IL-22 as well as expression of mRNAs for IL-17 and IL-22 in the treated skin. In addition, we found increased levels of mRNAs for IL-1β, and for several IL-1-inducible proteins, including COX2 and the neutrophil chemoattractants CCL3, CXCL2, and CXCL3, and decreased levels of mRNA for IL-1RA in the imiquimod-treated skin of the Nod2−/− mice. Taken together, these data suggest that NOD2 may be limiting skin inflammation in psoriasis by regulating IL-1/TLR signaling, supporting an important immunomodulatory role for NOD2 in inflammatory disease.
Long noncoding RNA (lncRNA) Negative Regulator of Interferon Response (NRIR) is induced along with other Interferon Induced mRNA in primary NK cells of uninfected and HCV infected persons.
Authors: Zebrowski Elizabeth , Barriocanal Marina , Fortes Puri , Valadkhan Saba , Anthony Donald D
Published: January 2023

Background

Natural Killer cells are thought to participate in control of HCV during the early phase of infection (first 6 months) and during IFN containing HCV therapy. At the same time, IFN resistance underlies wide variability in therapeutic response to IFN containing HCV therapy. We have previously described the lncRNA NRIR to be induced in hepatocytes upon IFNa exposure, and expression of NRIR was found to be responsible for down-modulation of the IFNa response. Whether NRIR is expressed within primary NK cells or participates in the IFN response within these cells has not been known.

Methods

Here we evaluated freshly isolated and IFNa treated primary human NK cells from 8 uninfected donors and 8 chronic HCV infected donors for NRIR expression by qPCR.

Results

We found NRIR expression in 100% of freshly isolated NK cells of uninfected donors and 40% of HCV infected donors. Upon IFNa exposure NRIR was greatly induced in 100% of uninfected donor and 67% of HCV infected donor NK cells.

Conclusions

These data indicate NRIR mediated regulation of the IFN response may be operative in primary NK cells of humans with or without HCV infection. Further studies are needed to determine the effect of NRIR on IFN induced NK cell function.
TLR2 and Adenosine Receptor A2a Induce ERK1/2 and c-FOS in Oral Squamous Carcinoma Cells
Authors: Kurago Zoya B , Palani Chithra , Ramanathapuram Lalitha , Lam-ubol Aroonwan
Published: January 2023

Squamous cell carcinoma (SCC) is a tenacious cancer prevalent at mucosal and skin surfaces. The level of oral (O)SCC differentiation does not correlate with prognosis, while tumor colonization by microorganisms and chronic inflammation may well contribute to OSCC pathogenesis. Microbial pattern recognition toll-like receptors (TLR) in immune cells induce inflammatory mechanisms, as they activate NF-kB and MAPK ERK1/2 pathways. Activated immune cells downregulate adenosine receptors (AR) A1 and A3, but upregulate inhibitory AR A2a and A2b, which also engage ERK1/2 when stimulated by adenosine. Malignant squamous cells express functional TLR2 and TLR4 as well, but their roles in OSCC cells are poorly understood. To address this gap, we evaluated TLR and AR expression and function in OSCC cells and keratinocytes. Five out of six OSCC lines expressed more TLR2 than TLR1, 4 or 6. TLR2, but not TLR4 stimuli induced AR A2a upregulation, while A2b did not increase. AR A1 and A3 mRNA was not detected in any oral squamous cells. TLR2 and A2a were also expressed in SCC cells and in dysplastic epithelial cells of all 17 archival oral specimens evaluated (use of specimens was approved by AU IRB). TLR2/1, TLR2/6 and AR stimuli induced ERK1/2 phosphorylation and accumulation of c-FOS in TLR2-responsive OSCC, which was similar to the effect of epidermal growth factor (EGF) on these cells. TLR2 and AR stimuli in responsive OSCC also stimulated the expression of the proliferation marker Ki-67. We conclude that products specific for TLR2 and A2a, such as bacterial lipopeptides and adenosine/inosine present in the tumor microenvironment, may contribute to OSCC progression via direct effects on malignant cells.
Comparison of the immunomodulatory effects of 3,4-dichlorophenyl-propanamide (DCPA) and N-(3,4-dichlorophenyl)-N-methylpropanamide (N-MeDCPA)
Authors: Barnett John B , Bergdorf Kensey N , McCall Jamie L.
Published: January 2023

Glioblastoma multiforme (GBM) are stage IV brain tumors for which there is no curative treatment. GBM depend on Orai1 calcium channel signaling for migration, proliferation, and the stability of various proteins. Thus, blocking Orai1 channels may provide a target for chemo-ablation of the tumor. DCPA and N-MeDCPA block Orai1 channels; however, N-MeDCPA has less in vivo toxicity. N-MeDCPA inhibits proliferation and migration in the U251MG glioblastoma cell line in a dose-dependent manner by 1.8% to 98.6% and 12% to 48%, respectively. The well-known role of the Orai1 channel in T cell activation may affect its usefulness as a drug. At high concentrations, DCPA inhibits T cell cytokine production; therefore, a direct comparison of the immunomodulatory effects of DCPA and N-MeDCPA on T cell activity and phenotype ex vivo are in progress. Future experiments will determine if N-MeDCPA can modulate the anti-tumor immune responses at concentrations that would directly inhibit the tumor. These results will be key in experiments using a mouse xenograft model of GBM to determine direct tumoricidal effects. If N-MeDCPA inhibits inflammatory and T-cell responses that promote tumor progression, in addition to its direct antitumor effects, it may have a future role in the treatment of GBM.
Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9
Authors: Tursi Sarah , Lee Ernest , Lee Michael , Mederios Nicole , Wilson Paul , Gallucci Stefania , Wong Gerard C , Tukel Cagla
Published: January 2023

Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is amyloid curli which forms immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation type I interferons (IFNs) and anti-double stranded (ds) DNA autoantibodies. DNA complexed with curli stimulates Toll-like receptor (TLR) 9 through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface TLR2, enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to TLR9, inducing production of type I IFNs. Upon stimulation of macrophages with curli-DNA complexes, only 5% of TLR2−/− macrophages harbored intracellular curli, suggesting that TLR2 drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization led to a significant decrease in Ifnβ expression. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils results in the formation of ordered curli-DNA immune complexes resulting in optimal spacing to TLR9. Production of anti-dsDNA autoantibodies and type I IFNS in response to curli-DNA fibers was attenuated in the TLR2−/−, TLR9 mutant, and TLR2−/− - TLR9 mutant mice compared to wildtype mice, suggesting that TLR2 and TLR9 are critical for the autoimmune immune response. Overall, our results identify a pivotal series of events that leads to the severe pro-autoimmune effects of amyloid-expressing bacteria.
Sand fly salivary proteins-parasite-skin interaction
Authors: De Castro Waldione , Serafim Tiago Donatelli , Oliveira Luiz Fabiano , Kamhawi Shaden , Valenzuela Jesus
Published: January 2023

Leishmaniasis is a vector-borne disease transmitted by infected sand flies. During infection, sand fly salivary proteins are inoculated into the host dermis together with Leishmania parasites where proteins from the extracellular matrix are present. In this context, we hypothesize that sand fly salivary proteins interact with the parasite and with proteins from the extracellular matrix. Our preliminary findings show that proteins from sand fly salivary gland homogenate bind to the surface of promastigote forms of the parasite Leishmania infantum. Using recombinant sand fly salivary proteins we identified two major salivary proteins, LJL143 and LJL04, as the main salivary proteins binding Leishmania parasite surface. Furthermore, by a Surface Plasmon Resonance assay we tested the interaction between salivary recombinant proteins with different extracellular proteins. From all the recombinant proteins tested, only LJL04 interacts with Fibrinogen, Fibronectin, Collagen I, Multimeric VTNC and Laminin. Our plans are to confirm the salivary protein-parasite-extracellular matrix biding in vivo, firstly by exposing Balb/c mice ears to uninfected L. longipalpis bites and by detecting the presence of salivary proteins in the ear of mouse dermis post-sand fly bites. As future direction we are also planning to use L. infantum (transgenic fluorescent parasite)-infected sand flies bites to verify the possible co-localization of parasite and sand fly salivary proteins in the skin. Here, for the first time we show interaction between sand fly salivary proteins and Leishmania parasites and sand fly salivary proteins and extracellular matrix proteins.
Anti-alpha-toxin immunoprohylaxis reduces disease severity against a Staphylococcus aureus full-thickness skin wound infection in immunocompetent and diabetic mice
Authors: Ortines Roger Vargas , Cheng Lily , Cohen Taylor S , Gami Abhishek , Dillen Carly A , Ashbaugh Alyssa G , Miller Robert J , Wang Yu , Tkaczyk Christine , Sellman Bret R , Miller Lloyd S
Published: January 2023

Staphylococcus aureus infection is a major complication of acute and chronic skin wounds that delays wound closure and healing. These infections are particularly problematic in diabetic foot ulcers as the infection can become invasive, leading to osteomyelitis that often necessitates limb amputation. Prior reports in S. aureus skin infection models have indicated that neutralizing S. aureus alpha-toxin decreases disease severity by reducing skin lesion size and restoring an appropriate host immune response, however whether alpha-toxin neutralization impacts S. aureus-infected skin wounds is not clear, especially in the setting of diabetes. Herein, we evaluated the efficacy of MEDI4893* a neutralizing human anti-alpha-toxin monoclonal antibody (mAb) in a S. aureus skin wound infection model. Three full-thickness cuts were made on the back skin of wildtype (wt) C57BL/6 and diabetic (TallyHo) mice and the cuts inoculated with a bioluminescent methicillin-resistant S. aureus (MRSA) strain. In both WT and diabetic mice, prophylaxis with MEDI4893* resulted in a significant reduction in skin lesion sizes, decreased bacterial burden, improved re-epithelialization, and a decreased percentage of neutrophils and an increased percentage of macrophages in the skin compared with control mAb treatment. Thus, in S. aureus-infected skin wounds, anti-alpha-toxin mAb treatment reduced disease severity by not only inhibiting dermonecrosis but also by resolving neutrophilic inflammation.
Dectin-2 as a Primary Receptor for NLRP3-inflammasome Activation in Dendritic Cell Responses to Histoplasma capsulatum
Authors: Wu-Hsieh Betty , Chang Tzu-Hsuan , Huang Juin-Hua
Published: January 2023

Inflammasome is an intracellular protein complex that serves as cytosolic pattern recognition receptor (PRR) to engage with pathogens and to process cytokines of the interleukin-1 (IL-1) family into bioactive molecules. It has been established that interleukin-1β (IL-1β) is important to host defense against Histoplasma capsulatum infection. However, the detailed mechanism of how H. capsulatum induces inflammasome activation leading to IL-1β production has not been studied. Here, we showed in dendritic cells (DCs) that H. capsulatum triggers caspase-1 activation and IL-1β production through NLRP3 inflammasome. By reciprocal blocking of Dectin-1 or Dectin-2 in receptor-deficient DCs, we discovered that while Dectin-2 operates as a primary receptor, Dectin-1 serves as a secondary one for NLRP3 inflammasome. Both receptors trigger Syk-JNK signal pathway to activate both signal 1 (the transcription of Il1b) and signal 2 (activation of caspase-1). While both K+ efflux and cathepsin B (but not ROS) function as signal 2, viable H. capsulatum triggers profound lysosomal rupture leading to cathepsin B release. Our study demonstrates the role of Dectin-2 and Dectin-1 in host defense against H. capsulatum infection through induction of NLRP3 inflammasome activation and IL-1b production in DCs.
Pulmonary Infection with Multiple Strains of Aspergillus fumigatus Reveals a Critical Role for IL-1α Signaling in Host Resistance Against Strains that Rapidly Germinate in the Lungs
Authors: Obar Joshua J , Caffrey-Carr Alayna Katherine , Kowalski Caitlin , Beattie Sarah , Blaseg Nathan , Upshaw Chanell , Tang Yi-Wei , Hohl Tobias , Cramer Robert
Published: January 2023

Aspergillus fumigatus strain diversity is reflected in heterogeneous virulence and inflammatory responses. Mechanisms underlying A. fumigatus strain specific immune responses and how this affects disease outcome is unresolved. We observe that the CEA10 strain is able to rapidly efficiently within the airway environment and induces greater lung damage, vascular leakage, and IL-1α release compared to the Af293 strain. Fungal clearance is dependent on IL-1α in mice inoculated with the CEA10 strain, while clearance of AF293 does not require IL-1α. Importantly, both swollen conidia and germlings of the CEA10 strain induce robust IL-1α secretion from macrophages in vitro, whereas only germlings of Af293 induce strong production of IL-1α. Our finding that early fungal germination in the airways drives greater pathology and IL-1α dependent inflammation is further supported by similar results with additional strains. First, serial passage of a non-airway germinating strain (Af293) in low-oxygen conditions results in a strain (EVOL20) that germinates more efficiently in the airways leading to enhanced lung damage, vascular leakage, and IL-1α dependent inflammation. Second, using primary clinical and environmental isolates of A. fumigatus that are able to rapidly germinate in airway conditions follows the same trend. Clinically, our data support the idea that A. fumigatus strain phenotypic variation significantly contributes to disease outcomes. Understanding why different A. fumigatus strains induce distinct immune pathology can reveal novel immunotherapeutic and antifungal targets for the treatment of invasive aspergillosis.
Th17 immunity to tuberculosis requires CD40-CD40L interaction and is enhanced by CD40 engagement on Mycobacterium tuberculosis-infected dendritic cells
Authors: Sia Jonathan Kevin , Madan-Lala Ranjna , Rengarajan Jyothi
Published: January 2023

Mycobacterium tuberculosis (Mtb) impairs dendritic (DC) cell functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper 17 (Th17) cells have emerged as important for protecting against tuberculosis (TB), but the mechanisms involved in generating antigen-specific Th17 responses during Mtb infection are not well defined. We previously reported that Mtb restricts Th17 responses and impairs CD40 expression on dendritic cells (DCs). We now demonstrate that generating IL-17 responses to Mtb requires CD40-dependent costimulation. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced generation of antigen specific IL-17 responses; cytokines were unable to compensate for this reduction. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that targeting the CD40-CD40L pathway represents a novel strategy to enhance early Th17 responses and improves adaptive immunity to TB.
Conditional silencing of the H-2Db class I molecule on CD11c+ Dendritic Cells inhibits the generation of antiviral CD8 T Cells during acute Theiler’s Murine Encephalomyelitis Virus (TMEV) infection in the CNS
Authors: Willenbring Robin , Settell Megan L , Malo Courtney Sarah , Goddery Emma N , Hansen Michael J , Pavelko Kevin D , Pease Larry R , Johnson Aaron J
Published: January 2023

The generation of an effective antiviral CD8 T cell response is required to protect C57BL/6 mice from Theiler’s murine encephalomyelitis virus (TMEV) induced demyelinating syndrome. Our group previously defined the VP2 121-130 peptide presented in the context of the H-2Db class I molecule as the immunodominant antigen recognized by the majority of brain infiltrating CD8 T cells during acute TMEV infection. Our group also determined that this Db:VP2 121-130 epitope specific CD8 T cell response was required for virus clearance. However, the mechanism by which virus antigen-specific CD8 T cells are generated in the brain remained undefined. Therefore, our laboratory generated a novel transgenic mouse system that enables conditional ablation of the H-2Db class I gene in specific antigen presenting cell types (APCs). Previously, analysis of class I-restricted antigen presentation required genetic disruption or depletion of entire APC subsets. We used this novel Cre-Lox transgenic mouse system to define the APC required to generate the immunodominant Db:VP2 121-130 epitope specific CD8 T cell response during acute TMEV infection. In the course of this analysis, we determined that silencing of class I-restricted antigen presentation on CD11c+ dendritic cells (DCs) did not affect thymic development of CD8 T cells. However, loss of Db expression by CD11c+ DCs completely abolished the generation of Db:VP2 121-130 epitope-specific CD8 T cell responses during acute TMEV infection. These findings demonstrate the importance of DCs in generating CD8 T cell responses against a neurotropic virus. We also provide a novel transgenic mouse for future studies involving class I-restricted antigen presentation.
Type I interferon signaling attenuates Regulatory T cells function in LCMV infection
Authors: Gangaplara Arunakumar , Martens Criag , Metidji Amina , Schmidt Rachel , Kanakabandi Kishore , Glass Deborah , Gokhale Ameya S , Lopez-Ocasio Maria , Porcella Stephen F , Shevach Ethan M
Published: January 2023

Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute and chronic infectious diseases remains unclear. We recently demonstrated that IFN-α/β receptor (IFNAR) signaling promotes Treg function in autoimmunity. To dissect the functional role of IFNAR-signaling in Tregs during acute and chronic viral infection, we infected Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice with LCMV Armstrong and Clone-13. In both models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced expression of Treg associated activation antigens. The enhanced activated phenotype was also seen when we compared the transcriptomes of IFNARfl/flxFoxp3YFP-Cre and wild type (WT) Tregs by RNA-Seq on day 25-post Clone-13 infection. LCMV-specific CD8+ T cells from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral IFNγ and TNFα in both acute and chronic LCMV. In the chronic model, the numbers of anti-viral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs than WT mice during chronic infection. Thus, type I IFN signaling in Tregs is context-dependent, resulting in enhanced suppressor function in some models of autoimmunity, but decreased suppressor function in acute and chronic viral infection.
Dichotomous regulation of progenitor-like CD8 T cells during chronic viral infection by TCF1 and type I interferon
Authors: Wu Tuoqi , Ji Yun , Moseman E Ashley , Xu Haifeng , Manglani Monica , Lang Philipp A , Gattinoni Luca , McGavern Dorian B , Schwartzberg Pamela L
Published: January 2023

T cells develop into a dysfunctional state, called T-cell exhaustion, during chronic viral infections and cancer. However, how antigen-specific T cells persist in these situations is poorly understood. Here, we show that CD8 T cells differentiate into a less exhausted TCF1highTim3low subset and a more exhausted TCF1lowTim3high subset in chronic viral infection as well as human and mouse solid tumors. The TCF1highTim3low CD8 T cells, generated early after chronic lymphocytic choriomeningitis virus (LCMV) infection, persist better and mount a far stronger secondary response than TCF1lowTim3high cells. Importantly, TCF1highTim3low cells act as progenitor cells that are capable of both self-renewal and terminally differentiating into TCF1lowTim3high cells. We have further shown that cell-intrinsic TCF1 signaling is necessary and sufficient to program these progenitors early after infection. Cell-intrinsic TCF1 deficiency led to a progressive loss of anti-viral CD4 and CD8 T cell responses during the chronic phase of infection. Several pro-exhaustion factors, including Tim3, Cish, and Blimp1, are suppressed by TCF1. Conversely, TCF1 up-regulates Bcl6, which strongly enforces the progenitor fate. Moreover, cell-intrinsic type I interferon signal suppresses the differentiation of progenitor-like CD8 T cells, and antibody-mediated blockade of IFNAR1 enhances the differentiation of these CD8 T cells in a TCF1-dependent manner. Thus, our study not only identifies a CD8 progenitor population, which is critical for long-term persistence of anti-viral CD8 responses during chronic viral infection, but also uncovers both positive and negative regulators of the early programming of this population.
Tissue-specific requirements for IL-10 in maintaining homeostasis in response to chronic virus infection
Authors: Clambey Eric T , Kimball Abigail K. , Baessler Andrew , Oko Lauren M. , Niemeyer Brian F , Van Dyk Linda F
Published: January 2023

Herpesviruses have co-evolved with their hosts for million of years, establishing an intimate balancing act of lifelong infection with minimal damage to the host. Interleukin (IL)-10 is a major inhibitory cytokine that has been usurped by many herpesviruses to facilitate infection. Here we tested the consequence of IL-10 deficiency on the outcome of murine gammaherpesvirus 68 (γHV68) infection, a small animal model of γHV pathogenesis. While IL-10 deficiency had minimal impact on the control of virus infection, IL-10 deficient mice showed hyper-activation of the immune response with increased levels of T cell activation, effector T cells and prolonged neutrophilia in the infected lung. IL-10 further had a non-redundant role in limiting intestinal inflammation with chronically infected mice showing a prolonged failure to thrive following infection, associated with enhanced colonic Th1 responses. CD4 T cells were a prominent source of IL10 during acute and chronic infection, with IL10+ interferon-gamma+ FoxP3- type 1 regulatory CD4 T cells as the major early source of IL10. High-dimensional mass cytometric analysis of IL10 expressing cells, and IL10 responsive cells, identified a constellation of phenotypes that correlate with robust IL10 expression, and further identified direct and indirect targets of IL10. In total, these data demonstrate the context dependent role of IL10 in constraining the antiviral host response and identify IL10-dependent and –independent regulatory pathways in regulating chronic herpesvirus infection.

This research was funded by grants from the American Heart Association, Crohn’s and Colitis Foundation of America and the UC Department of Anesthesiology (ETC).
The pursuit of better vaccines: uncovering the functional capacity of human cytomegalovirus-induced subsets of NK cells
Authors: Gyurova Ivayla E , Bernstein David , Schlums Heinrich , Bryceson Yenan , Waggoner Stephen N
Published: January 2023

Cytomegalovirus infection (CMV) poses a considerable burden to public health due to significant morbidity and mortality in immunocompromised patients and babies born with the infection, thus highlighting the priority of developing a successful CMV vaccine. Whereas previous CMV vaccination strategies have aimed to induce T- and B-cell responses, we speculate that natural killer (NK) cells hold the key to protective immunity based on the evidence that patients lacking NK cells suffer recurrent, severe infections with multiple herpesviruses, including CMV. Notably, CMV infections in both mice and humans triggered accumulation of unique adaptive NK-cell subsets that putatively possess enhanced effector functions against CMV. This suggests that a pioneering approach to creating an efficacious CMV vaccine would include stimulation of the accumulation and long-term maintenance of these CMV-reactive NK cells. It remains unknown, however, whether vaccines can be designed to stimulate the expansion of these NK-cell subsets. Here, using a unique set of samples collected during a CMV vaccine trial, we aim to determine whether vaccination promotes accumulation of these NK cells. Moreover, we examined the relationship between the activity of NK cells and stimulation of virus-specific T and B cell responses during immunization and in cell culture. Preliminary analysis revealed a supportive role for NK cells in stimulation of CMV-specific T cells during in vitro culture with peptide which correlates to the presence of adaptive NK cells. Our studies represent a key step in understanding the potential role of subsets of human NK cells in primary CMV infection and vaccine-induced immune responses against CMV.
IFN-γ-induced MHCII+ inflammatory monocytes play a role for down-regulating CD8 T cell responses in acute LCMV infection
Authors: Shin Kwang-Soo , Park Young-Jun , Koh Choong-Hyun , Bae Eun-Ah , Kim Il-Kyu , Song Boyeong , Seo Hyungseok , Jeon Insu , Kang Chang-Yuil
Published: January 2023

During viral infection, monocytes play a protective role for clearance of pathogens. However, little is known about the role of recruited monocytes after effector phase, especially as regard to their effects to T cells. Here, we have investigated the phenotypes, the origin and inducing factor, and the effects to CD8 T cells of inflammatory monocytes in virus-infected mice. During Lymphocytic choriomeningitis virus (LCMV)-Arm infection, CCR2+ inflammatory monocytes were accumulated in lymphoid organs and reached their peak at day 8 post infections. Inflammatory monocytes of infected mice expressed high levels of MHCII and low levels of CD11c, which overlap with phenotypes of DC and those of macrophage, respectively. We found that IFN-gamma (IFN-γ) is a factor that differentiates MHCII+ monocytes from BM progenitor cells. Among the BM progenitor cells, common monocyte progenitors (cMoPs) were rapidly converted to MHCII+ inflammatory monocytes in response to GM-CSF and IFN-γ in vitro. In addition, cMoPs that were adoptively transferred to infected recipients were differentiated to MHCII+ inflammatory monocytes. We also found that MHCII+ monocytes-primed CD8 T cells could not produce inflammatory cytokine and granzymeB efficiently, implicating their lowered effector functions, despite their comparable proliferative capacity to conventional DC-primed CD8 T cells. Overall, our data suggest that IFN-γ-induced MHCII+ inflammatory monocytes during acute LCMV infection would play a role in down-regulating CD8 T cell responses.
The role of Runx2 in CD8+ T cell memory during acute LCMV Armstrong infection
Authors: Schutten Elizabeth Anne , Nayar Ribhu , Saikumar Priya , Lu Yu Jung , Abramo Kristin , Berg Leslie J
Published: January 2023

During an acute viral infection, CD8+ T lymphocytes with T cell receptors (TCRs) specific for pathogen epitopes undergo clonal expansion and clear viral infection. After infection is cleared, the majority of the pathogen-specific CD8+ T cells undergo apoptosis, leaving a small population of memory CD8+ T cells that rapidly proliferate upon reinfection of the same antigen. How differentiation between the terminal effector cell population (TECs) and the memory cell population (MPECs) is regulated is not completely understood. Our research has focused on the role of the runt-related transcription factor Runx2 in CD8+ T cell memory formation during acute LCMV Armstrong infection. We have found using a mouse line with a T cell-specific deletion of Runx2 that loss of Runx2 leads to a defect in the formation of MPECs. We have also found that this defect is intrinsic to virus-specific CD8+ T cells. To further elucidate the upstream signaling pathways regulating Runx2 we have examined TCR signaling, cytokine signaling, and transcription factor regulation. These studies indicate that IRF4 levels inversely correlate with Runx2 expression in in vitro culture systems. We have also examined downstream pathways regulated by Runx2 in T cells, including gene expression and migration. This analysis has not revealed Runx2-dependent changes in gene expression in T cells isolated from LCMV-infected mice at D6 post-infection, nor in T cells stimulated in vitro; furthermore, we do not observe altered homing properties of Runx2-deficient T cells. Current studies are focused on examining Runx2 phosphorylation during memory CD8+ T cell differentiation. Overall, our data indicate an important role for Runx2 in CD8+ T cell memory during LCMV Armstrong infection.
Entolimod, a clinical-stage TLR5 agonist, activates antitumor T cell immunity against liver and lung metastases through distinct mechanisms
Authors: Brackett Craig M , Kojouharov Bojidar , Greene Kellee , Trageser Nicholas , Gollnick Sandra , Burdelya Lyudmila , Abrams Scott I , Gudkov Andrei
Published: January 2023

Innate immune modulators can generate an antitumor T cell response. However, significant toxicities associated with systemic administration have significantly limited their clinical use. The natural TLR5 agonist flagellin is unique amonginnate immune modulators because the tissue specificity of TLR5 expression induces a uniquely safe profile of cytokines following systemic TLR5 activation. Entolimod, a pharmacologically optimized flagellin derivative, was initially developed to treat and prevent acute radiation syndrome. Systemic administration of entolimod also showed antitumor effects in multiple preclinical mouse models mimicking clinically occurring liver metastasis. Entolimod suppresses liver metastasis through activation of NF-κB-, AP-1-, and STAT-3-driven immunomodulatory pathways in hepatocytes and a highly coordinated CD4+ T cell independent NK-DC-CD8+ T cell response. Although these studies characterized entolimod as a liver tropic immunotherapy, entolimod also suppresses spontaneous mammary lung metastasis. In contrast to the liver metastatic model, CD4+ T cells are required for the antitumor NK and CD8+ T cell response, indicating that the etiology of the cancer may explain the difference in the therapeutic effects ofTLR5 agonists. These results further strengthen that TLR5 agonists are a safe, effective and broadly applicable immunotherapeutic agent against metastases, which are currently a major cause of cancer-associated mortality. Recent completion of a phase I trial of entolimod in patients with advanced metastatic solid tumors has provided the rationale to test that the efficacy demonstrated in animal cancer models can be translated into immunotherapy of human tumors.
Engineering of novel Class II Transactivator gene delivery systems as molecular adjuvants to improve genetic immunotherapies by inducing de novo MHC II expression in human cells
Authors: de Lucena Palma Mariana , Duangkhae Parichat , Douradinha Bruno , Dhalia Rafael , Mailliard Robbie B , Barratt-Boyes Simon , Oshiro Telma Miyuki , de Azevedo Marques Ernesto Torres , da Silva Duarte Alberto José
Published: January 2023

Class II Transactivator (CIITA) induces transcription of MHC class II genes. This protein can potentially be used to improve genetic immunotherapies by converting non-immune cells into cells capable of presenting antigens to CD4+ T helper cells. However, CIITA expression is complex, tightly controlled and remains unclear whether distinct non-immune cells differ in the regulation of this transcription factor. In the present study, we describe a strategy to develop gene delivery systems capable of promoting the efficient expression of CIITA in non-immune cell lines and in primary human cells in an ex vivo skin explant model. A DNA plasmid and a lentiviral vector were produced, both carrying the human CIITA DNA sequence in silico designed to avoid cis-regulatory elements, and genetically optimized for expression efficacy in human cells. Different human cell types undergoing CIITA overexpression presented high-level de novo expression of MHC II molecules, validating the delivery systems as suitable tools for the evaluation of CIITA potential as a molecular adjuvant for genetic immunizations. Further, we directly compared different non-immune cells according to exogenous CIITA transcriptional activity, protein expression levels and proteasome degradation. Here we show for the first time that distinct types of non-immune cells differentially regulate the transcription factor, and ultimately the cell surface expression of MHC II, through a cell type-specific control of CIITA proteasomal degradation. Our findings contribute to the understanding of the CIITA post-translational regulation by non-immune cells, which can greatly influence the use of this regulator of MHC II genes as a vaccine adjuvant.
HER-2+ breast cancer cells expressing GPI-anchored cytokines induce long lasting antitumor memory response
Authors: Munoz Luis E , Bommireddy Ramireddy , Margaroli Camilla , Shafizadeh Yalda , Bozeman Erica N , Menon Ashwathi P , Kumar Jayanth , Lakshmanan Ramana , Selvaraj Periasamy
Published: January 2023

Breast cancer is the leading cause of death among female cancer patients. Human epidermal growth factor receptor 2 (HER-2) overexpression is associated with poor prognosis in 15–20% of breast cancer patients. Anti-HER-2 antibody (Herceptin) prolongs the survival of the HER-2+ breast cancer patients. However, resistance to Herceptin is a cause for relapse of aggressive metastatic cancer in these patients. Our earlier studies have demonstrated that PD-L1 blockade enhances the efficacy of HER-2+ breast cancer whole cell vaccine by increasing infiltration of T cells into the tumor. The goal of the present study is to determine the duration of protective anti-tumor memory response to HER-2+ breast cancer. Here, we demonstrate the beneficial effect of GPI-anchored cytokine molecules as adjuvants for generating long lasting memory response against HER-2+ as well as HER-2 negative breast cancer in syngeneic tumor models. Female BALB/c mice were challenged with D2F2/E2 (HER-2 transfected D2F2) cells or D2F2/E2 transfected with GPI-IL-12 or GPI-GM-CSF. While the wild-type challenged mice developed tumors, the mice challenged with transfected D2F2/E2 cells were protected. Protected mice were re-challenged with D2F2/E2 cells 3 months later and D2F2 cells 4 months later. All the mice challenged with D2F2 or D2F2/E2 were protected. We have observed strong antibody response against HER-2 and D2F2 cells in these mice. Our results show that long lasting protective anti-tumor memory response against D2F2 and D2F2/E2 is generated by vaccination with D2F2/E2 cells expressing GPI-anchored immunostimulatory molecules.