bluuebunny/crossref_metadata_2025 huggingface dataset:
House dust mite extract directly regulates the alternative activation of macrophages by a TLR4-independent mechanism. (141.3)
Authors: Keegan Achsah , Qi Xiulan , Vogel Stefanie , Shirey Kari , Heller Nicola
Published: January 2023

Asthma is a clinical disorder characterized by episodes of airway narrowing, inflammation, and mucus production. House dust mites (HDM) are a major source of allergens that contribute to allergy and asthma. HDM contains a complex mixture of multiple agonists of pattern recognition receptors including bacterial-derived LPS, and multiple allergen groups. A recent report demonstrated a role for epithelial-cell expressed TLR4 in the Th2 response to nasal challenge with HDM. We and others have shown that macrophages activated by IL-4/IL-13 differentiate into alternatively activated macrophages (AAM) and participate in the recruitment of eosinophils during Type 2 inflammation. To determine whether HDM has a direct effect on macrophages, we treated bone marrow-derived macrophages (BMM) with HDM in the presence or absence of IL-4. HDM extract induced expression of YM1/2 (2-fold) and Mac 3, but not arginase I. IL-4 induced expression of AAM proteins including arginase I, YM1/2 (8-fold), and Mac3. Interestingly, HDM extract strongly induced the expression of a 39 kDa protein (9-fold) also recognized by the anti-YM1 antibody; this protein was modestly induced by IL-4. Together, HDM and IL-4 strongly induced YM1/2 and the 39 kDa protein. The HDM-induced responses were TLR4-independent. These results suggest that molecular patterns found in HDM extract regulate alternative macrophage activation by a TLR4-independent mechanism. Supported by AI059775 (ADK) and AI18797 (SNV)
The novel role of downstream of kinase-1 in the ovalbumin-induced murine model of asthma (141.4)
Authors: Park Yeong-Min , Lee Chang-Min , Park Jin Wook , Lee Chun Geun
Published: January 2023

The downstream of kinase (DOK)-1, a 62 kDa adaptor protein negatively regulates the protein tyrosine kinase (PTK) pathway in mast cells, but the role of DOK-1 in a murine model of asthma has not been defined. This study provides experimental evidence demonstrating that DOK-1 is a key regulator for aiway inflammation in a murine model of asthma. Here, we have found that the markable reduction of pulmonary CD4+ T cell population and Th2 cytokines leads to the inhibition of pulmonary CD4+ T cell differentiation into Th2 cells in DOK-1 overexpression mice. DOK-1 overexpression in STAT-6 knockout (KO) mice increased the expression of STAT-4 and T-bet, which is transcription factor for Th1 differentiation. Conversely, Th2 regulatory molecules, STAT-6 and GATA-3 are impaired by DOK-1 overexpression in STAT-4 KO mice. Taken together, DOK-1 regulates Th1/Th2 immune balance through STAT4- and STAT6-dependent pathways, our findings provide additional insight into the immunological function of DOK-1 in Th2 inflammation and suggest a new potential therapeutic target for the treatment of asthmatic patients.
Early expression of IL-19 in asthmatic airways promotes Th2 response (141.5)
Authors: Hung Li Yin , Nguyen Tiffany , Huang Fei , Chen Yin , Wu Reen
Published: January 2023

IL-19 is a proinflammatory cytokine that belongs to the IL-10 cytokine family. In addition to its known connection with psoriasis, previous work in our lab along with others, have also shown its association with asthma. Using cultured primary mouse airway cells, we demonstrated that Th2 cytokines such as IL-4 and IL-13 can induce the expression of IL-19 mRNA. In a mouse OVA-induced asthma model, we showed that IL-19 expression is increased in lung tissue during early disease stages. Additionally, we observed that other IL-10 family members such as IL-20, IL-22, or IL-24, were not induced. We further identified that airway epithelial cells, rather than the alveolar macrophage, were the source of IL-19 in the asthma model. Although we show that IL-19 can promote Th2 cytokine production by activated T cells, restimulation with IL-19 does not further increase Th2 cytokine production on memory cells. This phenomenon correlates with a down-regulation of IL-20R2 when cells are differentiated in the presence of IL-19, indicating a negative feedback regulatory mechanism. These results demonstrate that IL-19 is produced by airway epithelial cells during the early stage of the asthmatic response, and may further amplify the Th2 response by acting on activated T cells recruited to the lung during later disease stages by inducing Th2 cytokine production.
Invariant NKT cells in pig lung: a potential asthma model (141.8)
Authors: Gourapura Renukaradhya , Manickam Cordelia , Dwivedi Varun
Published: January 2023

Biomedical research in pigs is critical for better human health, as they are immunologically and physiologically more similar to humans, and are an excellent animal model to alleviate human asthma. Natural Killer T (NKT) cells with an invariant TCR are called "invariant NKT" (iNKT). iNKT cells are activated by CD1d-bound glycolipids with a unique α-anomerically linked sugar (e.g. α-GalCer). For the first time in pigs, we detected iNKT cells and CD1d expression. The pig iNKT cells proliferated and secreted both Th1 and Th2 cytokines when cultured in the presence of α-GalCer. In particular, iNKT cells from lung, and lung draining lymph nodes proliferated to higher numbers (<1% to >30%), with the secretion of more cytokines, compared to iNKT cells from other tissues. Recently, “many pivotal roles” played by iNKT cells in asthma pathogenesis have been reported. Direct activation of pig lung iNKT cells using α-GalCer resulted in airway hyperreactivity (AHR), and clinically those pigs had dyspnea with elevated body temperature. The AHR induced pig lung had petechial hemorrhages with increased mucus secretion in the airway. The α-GalCer received pigs had three-fold increase in cytokines, IL-6, IL-12 and IL-10, and two-fold increase in CD1d expression on the dendritic cells and macrophages at post-24 of inoculation. A moderate increase in iNKT cells in lungs of AHR induced pigs was also detected, compared to controls. This work is supported by USDA and NPB grants to RJG.
Helios is a Marker for Human Thymic-derived FOXP3+ Regulatory T Cells (143.1)
Authors: Tran Dat , Thornton Angela , Shevach Ethan
Published: January 2023

RATIONALE: FOXP3+ regulatory T cells (Tregs) are central to the maintenance of self-tolerance and the prevention of autoimmunity. Since they are generated both in the thymus and the periphery, the ability to distinguish these two populations would be critical to understand their development, particularly in autoimmune and inflammatory conditions. METHODS: Peripheral blood of healthy adult donors and patients with systemic lupus erythematosus as well as cord blood were analyzed for FOXP3 and Helios expression within the CD4+ T cell population by flow cytometry. Cytokine secretion and suppressive function were studied in the Tregs and correlated with Helios expression. RESULTS: Helios is expressed selectively in the FOXP3+ Tregs and is decreased with age. Helios identifies thymic-derived, cytokine non-producing FOXP3+ Tregs and not peripheral or TGFbeta-induced FOXP3+ T cells. Knockdown of Helios with siRNA does not alter the cytokine profile and suppressor function of Tregs, suggesting that Helios is not critical for their anergic and suppressive properties. CONCLUSIONS: Helios is expressed uniquely in human FOXP3+ Tregs and distinguishes the thymic over the peripheral derived Tregs. While it is expressed predominately in the Tregs, it does not appear to play a critical role in their anergic and suppressive functions.
Exclusion of cytoplasmic fragments in flow cytometric analysis of lymphoma samples using DyeCycle Violet (144.1)
Authors: Modiano Jaime , Ito Daisuke , O’Brien Timothy
Published: January 2023

Cytoplasmic fragments that originate from fragile lymphoid cells and are commonly seen in lymphoma fine needle aspirate samples. These cytoplasmic fragments can interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membrane. The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments in lymphoma samples analyzed by flow cytometry. Tumor cells prepared from lymph node biopsy and fine needle aspirate samples of dogs with lymphoma were stained using DyeCycle Violet (DCV, a membrane-permeable DNA-binding fluorescent dye), antibodies against cell surface antigens, and 7-amino-actinomycin D (7-AAD, a membrane impermeable, DNA-binding fluorescent dye). Multi-parameter flow cytometry was used to exclude antigen-positive cytoplasmic fragments for analysis based on the selective uptake and fluorescence of these dyes. Both cytoplasmic fragments, which are DCV-negative and CD45-positive, and dead cells, which are 7-AAD-positive, could be efficiently separated from tumor cells in lymphoma samples. DCV staining is a useful method to improve the gate setting and quantitative analyses of lymphoma samples by flow cytometry. DI and JFM contributed equally to this work
An automatable device for high content chemotaxis assays in the presence of a stable gradient (144.13)
Authors: Vu Elizabeth , Meyvantsson Ivar , Worzella Tracy , Skoien Allyson , Echeverria Victoria , Lamers Casey , Echeverria Daniella , Hayes Steve
Published: January 2023

Current approaches to study chemotaxis are limited by gradient instability, low throughput, cumbersome manual manipulations, high cell number requirements, inability to acquire high-content data, and/or poor data quality and reproducibility. We present here a new microfluidics-based approach to study leukocyte chemotaxis. The device features strong miniaturization of the use of cells (<12,000 cells per data point). The microfluidic channels in the device are arrayed in 96 well format and can be operated by standard liquid handling robotics or by handheld pipettors. Cells migrate along a surface in the presence of a stable gradient of chemoattractant that is maintained for at least three hours. We show the ability to obtain consistent dose response curves to chemoattractants as well as chemotactic inhibitors with the device. Because cells are microscopically accessible, the information content is high. Not only can the number of cells migrated be assessed, but also migration distances of individual cells, and even scoring of cell activation and polarization.
Bio-inspired controlled release for regulatory T cell recruitment in vivo (144.3)
Authors: Jhunjhunwala Siddharth , Raimondi Giorgio , Hall Sherri , Thorne Stephen , Thomson Angus , Little Steven
Published: January 2023

Increased frequency of regulatory T cells (Treg) in organs affected by autoimmunity and in transplants is associated with suppression of autoimmune reactions and graft rejection, respectively. However, to the best of our knowledge very few (if any) effective therapeutic strategies exist to increase local numbers of Treg. In this context, it is noteworthy that tumors evade the immune system through recruitment of Treg using the chemokine CCL22. Our hypothesis was that, if we can mimic this tumor evasion strategy by creating a biological gradient of CCL22 at a local site using controlled release vehicles, we will be able to recruit Treg to that particular site. Here we report the fabrication of CCL22 containing microparticles using a degradable, biocompatible, FDA approved polymer. We programmed these CCL22 particles to release the chemokine continuously over a 3 week period, and designed them such that they do not move from the site of injection in vivo. Importantly, in a murine model for chemotaxis we are able to demonstrate recruitment of adoptively transferred Treg specifically towards the CCL22 microparticles using non-invasive live animal imaging. We envisage that this technology could potentially be useful for the suppression of adverse inflammatory reactions associated with autoimmunity and transplant rejection; a therapeutic application that we are currently testing.
Macrophages Fascilate Chronic Cardiac Rejection through a Monocyte-Induced by IFNγ (MIG) Mediated Process (145.14)
Authors: Moffatt-Bruce Susan , Bobek Daniel , Meyerson Joseph , Wang Jiao Jing , Hadley Gregg
Published: January 2023

Introduction Chronic rejection (CR) in cardiac transplantation leads to allograft dysfunction and transplant related heart failure. We investigated the role of macrophages-associated chemokines, monocyte-induced by IFNγ (MIG) and IFN-inducible protein 10 (IP-10) and their receptor CXCR3, in chronic cardiac allograft rejection. Methods BALB/c (H-2d) hearts were transplanted into C57BL6 (H-2b) recipients (n=6). Group AR had no treatment. Group CR had 3 mg i.p. of GK1.5 (antiCD4). The hearts were analyzed using qRTPCR and IHC. The spleens underwent FACS analysis. CXCR3 transgenic(Tg) and CXCR3KO mice were transplanted with BALB/c hearts. Results In group AR, hearts rejected at day 7. In group CR, the functioning hearts were harvested at day 30. Using ICH group CR allografts had significantly more macrophages compared to both AR allografts and isografts. FACS analysis of splenocytes in group CR detected macrophages to be more abdundant compared to group AR (p<0.05). qRTPCR analysis detected a significant increase in allograft MIG and IP-10 in the group AR,(p<0.05)but only MIG was significantly upregulated in group CR relative to both group AR and isografts(p<0.05). CXCR3Tg and CXCR3KO transplants rejected at day 7. Conclusions Chronic cardiac allograft rejection may be a macrophage dependent process, potentially mediated through MIG. However, our results suggest that MIG and its receptor are likely to only be one component of a highly complicated process.
SA-FasL-Induced Localized Allotolerance to Pancreatic Islets Is Mediated by Phagocytes/TGF-β Axis (145.6)
Authors: Bandura-Morgan Laura , Yolcu Esma , Zhao Hong , Madireddi Shravan , Shirwan Haval
Published: January 2023

We recently showed that pancreatic islets engineered to display on their surface a recombinant SA-FasL protein induce robust localized tolerance. Tolerance was initiated by FasL-induced alloreactive T cell apoptosis and sustained by Treg cells. We herein tested if tolerance induction involves phagocytes engulfing T cells apoptotic bodies as a means of regulation. C57BL/6 mice were transplanted with BALB/c SA-FasL-islets with the transient use of rapamycin given on the day of Tx daily for 15 days. The role of phagocytes and TGF-β in the induced tolerance was tested by using clodronate-loaded liposomes depletion and an Ab to TGF-β. Depletion of phagocytes using clodronate-loaded liposomes one day prior to Tx resulted in prompt rejection of SA-FasL-engineered islets (n=7). Similarly, in vivo blockade of TGF-β early (day 0, n=5), but not late (>100 days, n=5), postTx resulted in acute rejection of long-term allogeneic SA-FasL-islets. Immunohistochemical analysis revealed that only SA-FasL-engineered islets scored positive for TGF-β and have more macrophages localized in the periphery of islet as compared with SA control. In in vitro cultures, we demonstrated that T cells undergoing SA-FasL-mediated apoptosis expressed membrane-bound TGF-β and these cells were engulfed by macrophages. These data suggest that phagocytosis of apoptotic bodies by phagocytes leads to the secretion of TGF-β, which in turn may facilitate the induction of Treg cells that maintain tolerance.
PARP-1 regulates pro-inflammatory cell death (34.13)
Authors: Malireddi RK Subbarao , Kanneganti Thirumala-Devi
Published: January 2023

PARP-1(poly (ADP-ribose) polymerase 1), an enzyme involved in DNA repair, is a nuclear chromatin associated multifunctional protein and belongs to a large family of enzymes that can synthesize long branched polymers of ADP-ribose units by using NAD+( Nicotinamide adenine dinucleotide) as the substrate. DNA strand nicks and breaks activate PARP-1 and the activated enzyme makes branched chains of up to 200 ADP-ribose units and adds covalently to various nuclear proteins such as histones and especially PARP-1 itself. PARP-1 has been shown to play a major role in a number of pathophysiological situations. In this study, we show that cytokine and chemokine profiles from PARP-WT and -KO BMDM showed no significant differences. However, IL1-β secretion is reduced in PARP-1 deficient macrophages, suggesting a role for PARP-1 in pro-inflammatory signaling. PARP-1 deficient macrophages had displayed prominent protection from pro-inflammatory cell death in response to LPS+ATP stimuli. Together, these results demonstrate that PARP-1 contribute to the pathophysiology by regulating the pro-inflammatory cytokine profiles.
Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells (35.10)
Authors: Vimala Raveendran Vineesh , Tan Xiaoyu , Levant Beth , Stechschulte Daniel , Dileepan Kottarappat
Published: January 2023

Histamine (HIS) is a well-recognized modulator of vascular inflammation. We have shown that HIS, acting via H1 receptors (H1R), synergizes LPS-induced production of IL-6, IL-8, PGE2 and PGI2 by endothelial cells. The synergy between HIS and LPS was partly attributed to HIS-induced expression of TLR4. In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) and enhances HIS responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in 2- to 4-fold increases in H1R mRNA expression in a time and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. LPS-induced H1R expression was maximum at 4h after LPS treatment which returned to the basal level by 8h. The LPS-induced H1R gene expression was mediated through TLR4 because gene silencing by TLR4-siRNA inhibited LPS effect. The specific binding of [3H]-pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was 3-fold higher than the untreated cells. When HCAEC were pretreated with LPS for 24h, washed and subsequently challenged with HIS, 15-, 17- and 10-fold increases in IL-6, PGI2 and PGE2 production, respectively, were noted. HIS-induced syntheses of IL-6, PGE2 and PGI2 by LPS-primed HCAEC were completely blocked by the H1R antagonist cetirizine. These results demonstrate that LPS upregulates the expression and function of H1R in HCAEC leading to agonist-induced increase in IL-6, PGI2 and PGE2 production.
Transcriptional regulation of innate αβ CD8+ T cell development in Itk deficient mice (36.35)
Authors: Kraus Zachary , Horai Reiko , Schwartzberg Pamela
Published: January 2023

Inducible T cell kinase (Itk) deficient mice develop a large population of αβ CD8+ innate-like T cells that possess a memory-like phenotype and are dependent on IL-15. Similar to other innate T cell lineages, these cells are positively selected by hematopoietic cells instead of thymic stroma in a SAP dependent manner that involves the interaction of signaling activation molecule (SLAM) family members. While much work has been done to characterize these cells, very little is known about the molecular pathways that prevent (Wt) or initiate (Itk-/-) the expansion of innate CD8+ T cells. One clue came from work examining mice with a T-cell specific deletion of CREB binding protein (CBP) driven from the Lck proximal promoter. These mice revealed a similar expansion of CD8 T cells with an innate phenotype. CBP is a transcriptional co-activator, which possesses histone acetyl transferase (HAT) activity and is known to associate with a wide range of transcription factors, most notably the cAMP response element binding protein (CREB). We find that TCR stimulation induces robust phosphorylation of CREB that is impaired in Itk-deficient T cells. We are currently further evaluating how Itk-deficiency affects CREB/CBP activation, and the impact of these pathways on transcriptional programs leading to innate T cell development.
Hematopoietic stem and progenitor cell defects in the Ts65Dn mouse model of Down Syndrome: implications for the role of oxidative stress in immune dysfunction (36.58)
Authors: Lorenzo Laureanne , Chen Haiyan , Clark Sarah , Yarowsky Paul , Williams Mark
Published: January 2023

Down Syndrome (DS) is a genetic disease caused by trisomy 21. In addition to cognitive defects, it is characterized by hematologic disorders. Previous studies have linked oxidative stress to the pathology of DS. Using the Ts65Dn mouse model of DS, we found increased levels of reactive oxygen species (ROS) in the hematopoietic progenitor cells (HPC) of these mice. We hypothesize that increased oxidative stress in HPC leads to functional changes that contribute to hematologic disorders in DS patients. Analysis of bone marrow cells (BMC) showed that Ts65Dn mice have fewer hematopoietic stem cells (HSC) and common lymphoid progenitors (CLP). Competitive reconstitution studies in vivo and colony-forming assays in vitro were consistent with functional changes in these populations. Hematopoietic defects were also observed in the thymus. A decrease in both thymic cellularity and the percentage of pro-T cells was observed. Decreased expression of the IL-7Rα chain may be a mechanism for defects in CLP and thymic populations. The IL-7Rα chain is part of the receptor for IL-7, a cytokine essential for lymphoid development and homeostasis. Consistent with the pro-survival and proliferative effects of IL-7, ex vivo expression of Bcl-2 and measures of in vivo proliferation were diminished in thymocytes from Ts65Dn mice. The data suggest that HPC defects underlie the immune dysfunction in DS and that oxidative stress may be involved in the hematologic abnormalities observed in Ts65Dn mice.
Normal and Pathological Regulation of DNA Damage Response to T-cell Receptor Gene Rearrangement (36.61)
Authors: Livak Ferenc , Hathcock Karen , Hodes Richard , Bowen Steven
Published: January 2023

The T-cell receptor (TCR) is encoded by tandem arrays of variable (V), diversity (D), and joining (J) gene segments that are somatically rearranged during thymic development. During this process the RAG recombinase introduces transient DNA double strand breaks (DSBs) which are repaired by non-homologous end joining (NHEJ). Additionally, the DSBs are detected by components of the cellular DNA damage response (DDR) which bind to the chromatin within 1 megabase of the break forming foci. One component of DDR, Ataxia Telangiectasia mutated (ATM), plays a major role in activating downstream effectors involved in apoptosis, cell cycle arrest and DNA repair. ATM-deficiency is characterized by reduced rearrangement at the TCR-α locus in CD4/CD8 double positive (DP) thymocytes, high frequency of thymic lymphomas carrying translocations at the TCR-α locus, and increased frequency of TCR-β/TCR-γ trans-rearrangements. Since TCR-β, γ and δ recombination occurs in CD4/CD8 double negative (DN) cells, we examined the effect of ATM deficiency on these rearrangements. We found that rearrangement is impaired at the TCR-β, γ and δ loci. We also demonstrate increased frequency of TCR-β/TCR-γ trans-rearrangements in ATM deficient cells. Such trans-rearrangements may be the result of concurrent recombination at multiple TCR loci in DN cells. We are currently testing this hypothesis using combined immunofluorescence detection of DDR foci and fluorescent in situ hybridization of TCR loci.
Identification and characterization of zebrafish Rpl22 and its role in T cell development in vivo (36.66)
Authors: Zhang Yong , Sun Xiaoli , Rhodes Jennifer , Wiest David
Published: January 2023

Mutations in a number of ribosomal proteins (RP) disrupt hematopoiesis in such syndromes as Diamond Blackfan Anemia; however, RP mutations had not previously been reported to disrupt T lymphocyte development. We recently reported that germline ablation of Rpl22 in mice caused a selective block in development of αβ lineage T cells. To gain insight into the basis by which Rpl22 mutations disrupt T cell development, we are exploiting the zebrafish model system. We identified the zebrafish ortholog of rpl22 (zgc:123327). Whole mount in situ hybridization showed that zebrafish rpl22 is expressed both maternally and zygotically throughout early development, with relative enrichment in the aorta-gonad mesonephros (AGM) and intermediate cellular mass (ICM) which are the sites of primitive (erythroid and myeloid) and definitive (lymphoid) hematopioesis in zebrafish. Despite being highly expressed in the sites of primitive and early definitive hematopoiesis, knocking down Rpl22 using antisense morpholinos did not block either of these processes. However, morpholino knockdown of Rpl22 did block T cell development. Therefore, unlike other ribosomal protein (e.g., Rps19) whose loss primarily impairs erythropoiesis, Rpl22 function appears to be dispensable for erythroid development and is instead selectively important for T cell development. Efforts to exploit the zebrafish system to address the mechanism underlying rpl22 function in T cell development and thymopoiesis are ongoing.
iNKT cell subsets are associated with distinct clinical manifestations of pulmonary sarcoidosis. (37.16)
Authors: Koth Laura , Woodruff Prescott , Nixon Douglas , Snyder-Cappione Jennifer
Published: January 2023

Sarcoidosis is an inflammatory disease of unknown etiology that presents with a wide range of clinical courses, from complete recovery to irreversible lung fibrosis. Invariant Natural Killer T (iNKT) cells are implicated in the progression of many diseases, including sarcoidosis. Given the known phenotypic heterogeneity of this unique T cell subset, we hypothesized that the iNKT cell compartment of individuals with sarcoidosis will have a skewed subset distribution that may track with disease outcome. We used flow cytometry to compare both total iNKT frequencies and distribution of CD4, CD8, CD56, and CD161 subsets in the PBMC of sarcoidosis subjects with a wide range of lung disease severity and healthy controls. We found the lowest iNKT frequencies in subjects with fibrotic lung disease and worst prognosis (Stage IV). This group was also characterized by a higher percentage of CD4/CD8 double-positive (DP) and a lower percentage of CD161+ iNKT cells, a marker profile indicative of recent thymic emigration. In contrast, the percentage of CD56+ iNKT cells correlated directly with a favorable clinical outcome (Stage I), as well as highest total iNKT frequencies among diseased individuals. In summary, we found that distinct iNKT cell subsets tracked with lung disease severity, and may possess different functional properties that directly influence sarcoidosis disease course.
Activation of TLR signalling by Salmonella causes erythropoesis. (37.22)
Authors: Jackson Amy , McSorley Stephen
Published: January 2023

Salmonella infection causes splenomegaly that is often assumed to be due to the increased number of infected phagocytes in the spleen. However, we infected mice with Salmonella and determined that erythrocytes accounted for the largest cell population that increased in the spleen. These splenic erythrocytes were not recruited from peripheral blood and were at an immature stage of development, indicating erythropoesis due to infection. Indeed, erythropoietin (Epo) was induced within days of Salmonella infection and was directly responsible for the observed erythropoiesis. Production of Epo was almost completely inhibited in the absence of TLR signaling indicating that erythropoesis occurs a consequence of early innate activation. Our data suggest that erythropoesis may be a key feature of systemic bacterial infection and we are currently examining whether this is important for the disregulation of splenic architecture or inhibition of adaptive immune responses during Salmonellosis
HCV core/gC1qR ligation activates STAT3 in human innate immune cells via an IL-6 autocrine pathway. (37.23)
Authors: Tacke Robert , Nguyen Virginia , Tosello-Trampont Annie , Mullins David , Hahn Young
Published: January 2023

Hepatitis C virus (HCV) infection in humans is highly efficient in the establishment of persistent infection. Persistent HCV infection can lead to the development of chronic liver disease and hepatocellular carcinoma. T cells in persistently infected individuals exhibit impaired function, and fail to clear virus. HCV core protein secreted from infected hepatocytes, has been reported to inhibit T cell function by inhibiting the pro-inflammatory response of APCs (i.e. monocytes, macrophages, and dendritic cells). To further characterize the molecular basis for APC regulation by extracellular HCV core, we examined the ability of HCV core to induce STAT3 activation. STAT3 plays a pivotal role in regulating the inflammatory response and differentiation of APCs. In this study we report the activation of STAT3 following ligation of HCV core with complement receptor, gC1qR, as well as with a gC1qR agonistic monoclonal antibody. HCV core-mediated STAT3 activation is dependant on the activation of the PI3K/AKT pathway. Additionally, transcription and translation are required for gC1qR mediated STAT3 activation. Lastly, we show that multi-functional cytokine IL-6 is essential for HCV core-induced STAT3 activation. These results suggest that HCV core-mediated STAT3 activation may contribute to alteration of inflammatory responses by monocytes, macrophages, and dendritic cells, leading to impaired T cell responses during HCV infection.
Insight into AhR-mediated increases in neutrophilia during infection: iNOS as a potentially new AhR target gene (37.26)
Authors: Head Jennifer , Begy Beth , Lawrence B. Paige
Published: January 2023

There are many known factors that can affect immune responses to respiratory viral infections such as influenza A virus. Indeed, recent epidemiological studies indicate that in addition to host genetics, age and pathogen sub-types, a variety of environmental factors also impact the immune response to infections. Many of these environmental factors, including diesel exhaust and cigarette smoke, contain ligands for the aryl hydrocarbon receptor (AhR), an environment-sensing transcription factor shown to affect multiple facets of the immune system. Our laboratory has shown that activation of the AhR by the persistent environmental contaminant dioxin exacerbates neutrophil recruitment and increases the expression of inducible nitric oxide synthase (iNOS) in the lungs of influenza virus-infected mice. We show that depleting neutrophils during viral infection does not abrogate AhR-mediated increases in iNOS expression, demonstrating that neutrophil recruitment is downstream of elevated iNOS. We also show that AhR activation in iNOS-deficient mice infected with influenza virus does not affect neutrophil recruitment to the lung. Thus, these findings suggest that these AhR-mediated events are linked, and that increased iNOS expression could be a novel mechanism regulating neutrophil migration the infected lung. Additionally these studies implicate iNOS as a novel AhR target gene and provide new insights into the mechanisms underlying leukocyte recruitment during viral infections.
Induction of distinct neurological disease manifestations during Relapsing fever requires T lymphocytes (40.11)
Authors: Liu Hongqi , Fitzgerald Denise , Gran Bruno , Leong John , Alugupalli Kishore
Published: January 2023

Relapsing fever (RF) borreliosis is a multi-systemic infection characterized primarily by bacteremia but can extend to the central nervous system (CNS). The incidence of CNS disease manifestations in humans depends on the infecting RF Borrelia species. In the murine model of Borrelia hermsii infection we found distinct signs of CNS disease that ranged from a flaccid tail to complete paralysis of hind limbs. Infiltration of large number of T cells into the spinal cord of B. hermsii-infected mice and the up-regulation of MHC-II and CD80 on infiltrating macrophages and on microglial cells indicated a role for T cell and antigen-presenting cell interactions in this pathogenesis. Indeed, B. hermsii infection did not induce CNS disease manifestations in T cell-deficient mice (TCR-βxδ-/-), although it resulted in bacteremia comparable to wildtype level. Moreover the infiltration of immune cells into the spinal cord of TCR-βxδ-/- mice was reduced and the resident microglial cells were not activated. Histopathological analysis of lumbar sections of the spinal cord confirmed severe inflammation in wildtype but not in TCR-βxδ-/- mice. This study demonstrates that distinct neurological disease manifestations develop during RF and that T cells play a critical role in the induction of neuropathogenesis.
Strain-dependent requirement for IFN-γ for respiratory control and immunotherapy in murine gammaherpesvirus infection (42.1)
Authors: Tsai Ching-Yi , Zhang Weijun , Usherwood Edward
Published: January 2023

IFN-γ and perforin are important effector mechanisms used by CD8 T cells to clear virus-infected cells. Previous research has shown that either IFN-γ or pfp was non-essential for viral control after intranasal infection of mice with MHV-68. In this study, we use IFN/pfp double knockout mice to address if these two effector molecules play redundant roles in the control of acute infection with MHV-68 in Balb/C mice. Pfp KO mice and WT mice cleared infectious virus from the lungs, even following high dose infection. However IFN-γ KO and IFN/pfp DKO groups had higher virus titers in the lungs at day 10 post-infection and both groups had higher mortality rates. In IFN/pfp DKO mice, the virus titer and mortality rate were significant higher than IFN-γ KO mice, indicating a role for pfp in protection from disease. WT mice given IFN-γ blocking Ab also showed significant higher viral titers. In contrast, IFN-γ KO mice on a B6 background controlled respiratory infection comparably to WT mice. These data showed that pfp plays a redundant role in the control of virus replication but IFN-γ plays an essential role in Balb/C mice infected with MHV-68. We conclude that there is a marked strain-dependent difference in the effector mechanisms needed to control acute MHV-68 infection between B6 and Balb/C mice. In addition we show that immune therapy that re-establishes viral control after spontaneous reactivation in CD4-deficient mice depends upon pfp in B6 mice but IFN-γ in Balb/C mice.
MAP kinase phosphatase (MKP)-1 in the innate immune response to influenza infection (42.4)
Authors: Wancket Lyn , de Oliveira Stephanie , Liu Yusen
Published: January 2023

MKP-1 is a critical negative regulator of p38 and JNK MAP kinases. While loss of Mkp-1 function enhances cytokine production after bacterial challenge, little is known about the role of Mkp-1 in the innate immune response to viral infections. In this study we examined the role of Mkp-1 in the pulmonary immune response during influenza infection in mice. Mkp-1+/+ and Mkp-1-/- mice were intranasally inoculated with 1,000 plaque-forming units of the mouse-adapted H1N1 A/WSN/33 strain of influenza. Mice were monitored up to 6 days post inoculation and evaluated for altered host physiology, viral replication, and host immune response. Compared to Mkp-1+/+ animals, Mkp-1-/- mice exhibited accelerated weight loss and hypothermia. cDNA array analysis indicated that over 50 immune response-related genes were more robustly expressed in the lungs of Mkp-1-/- mice than in the Mkp-1+/+ mice upon influenza infection. The levels of IL-6, IL-10, and IFN-β were also higher in bronchoalveolar lavage fluid from Mkp-1-/- mice. Mechanistically, p38 and JNK activation was prolonged in Mkp-1 deficient cells treated with agents mimicking viral RNA. Together, these data support the model that Mkp-1 plays an important role in the feedback control of p38 and JNK, thus limiting harmful innate immune response and preventing severe clinical disease during pulmonary influenza infection.
Ad5-vectored FMDV vaccine elicits antigen-specific CTL responses in swine. (43.10)
Authors: Patch Jared , Pedersen Lasse , Moraes Mauro , Grubman Marvin , Golde William
Published: January 2023

Foot-and-mouth disease virus (FMDV) is a highly infectious picornavirus that causes disease in cloven-hoofed animals. Neutralizing antibodies are thought to play a primary role in FMDV immunity, and their induction forms the basis for current vaccine approaches. However, the role of cytotoxic T-lymphocytes (CTLs) in recovery and protection has remained largely unexplored. Immune evasion strategies of FMDV are predicted to impair the development of functional CTL assays. Specifically, virus encoded proteases inhibit expression of Class I MHC molecules by infected cells. In order to overcome these difficulties, we have analyzed the response to a recombinant, human Adenovirus 5 (huAd5) vectored, FMDV vaccine. Swine homozygous for MHC were vaccinated with a replication-defective huAd5 expressing the unprocessed capsid polyprotein of FMDV, strain A24. An MHC-matched cell line was used for antigen stimulation and as a source of target cells in cellular cytotoxicity assays. PBMCs from vaccinated animals were incubated with Ad5-infected target cells for 3-5 days. CD6+ T cells were isolated and mixed with Ad5-FMDV infected target cells. Antigen-specific killing was detected relative to control target cells. This suggests that vaccination with adenovirus vectored vaccine constructs can induce a functional CTL response against epitopes of FMDV in a MHC restricted manner.
Comparative and applied studies of virulence and immuno-escape of an emerging ranavirus pathogen in the amphibian Xenopus model (43.12)
Authors: Chen Guangchun , Li Zhenghui , Cruz Luna Tanya , Ward Brian , Chinchar V. , Robert Jacques
Published: January 2023

Ranaviruses (RV), family Iridoviridae, have become increasingly prevalent pathogens of amphibians around the world, and are possibly involved in the worldwide decline of amphibian populations. Nonetheless, RVs have not yet been studied as intensively as other large DNA viruses. We have established the frog Xenopus as an important experimental model to study viral pathogenicity and host defenses against RVs such as Frog virus 3 (FV3), and evaluate the contribution of immunocompromised animals in the dissemination of the diseases. To evaluate the role of putative viral genes involved in virulence and immune evasion, we are currently developing a method to systematically knockout (KO) putative virulence genes by site-specific integration of a selectable fluorescent marker into the FV3 genome. Susceptible Xenopus larvae will provide an ideal model to evaluate the impact of KOs on in vivo virus load, host mortality and the induction of pro-inflammatory genes. Normal and immunocompromised adult frogs will be used to investigate pathogenicity and ability of KOs to evade adaptive immunity. These studies will help elucidate the interplay between virulence genes and host immune defenses and may reveal original mechanisms of host-pathogen interaction that may be relevant not only to amphibians and other lower vertebrates, but also to humans.
Two-photon imaging kinetic analysis of Bacillus anthracis spore uptake in the respiratory tract. (45.17)
Authors: Quesnel-Hellmann Anne , Fiole Daniel , Mathieu Jacques , Tournier Jean-Nicolas
Published: January 2023

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the upper respiratory tract and transported to the lymph nodes in the early steps of infection. To investigate the role of dendritic cells (DCs) in spore uptake and transport, we infected intranasally CX3CR1 +/gfp mice with Alexa 647-labeled spores. Nasal mucosa, nasal-associated lymphoid tissue (NALT), trachea, lungs and the draining lymph nodes explants were analyzed at different time-points after infection by confocal and/or two photon (2P)-microscopy. We demonstrated that DCs present along the respiratory tract are involved in spore uptake. However, differences in the kinetics of spore phagocytosis between the organs were observed. As soon as 2 hours after infection, DCs bearing spores were found in the thoracic lymph nodes whereas no uptake was observed in the NALT. The role of the NALT in inhalational anthrax will be discussed, as well as some results on lung tissue capture in vivo. Our results demonstrate that DCs play a critical role in spore transport rapidly after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.
Development of Epigallocatechin Gallate as an HIV-1 Microbicide Agent: Impact on Dendritic Cell Innate Immune Responses (46.12)
Authors: Nance Christina , D'Souza Melinda , McMaster Sean , McMullen Ashley , Rajut Zak , Tran Daisy , Siwak Edward , Shearer William
Published: January 2023

Langerhans cells (LC) and monocyte-derived dendritic cells (MDDC), the first line of defense against viral infection, initiate innate immune responses via toll-like receptors (TLR). Previously, we showed inhibition of HIV-1-gp120 binding to CD4 on T cells by the green tea catechin, epigallocatechin gallate (EGCG). We present evidence of EGCG inhibition of the mucosal route of HIV-1 without alteration of dendritic cell (DC) innate immune responses. Methods utilized were CD34+ hematopoietic stem cells and CD14+ cells differentiated to LC and MDDC, respectively, isolated from peripheral blood of HIV-1 seronegative donors, flow cytometry of HIV-1-gp120 binding and TLR profiling with RT-PCR, radioisotope assays for antigen (Ag) studies, MTT assay for cytotoxicity, p24 ELISA for HIV-1 infectivity studies and statistical significance by Student’s t test. EGCG significantly inhibited HIV-1 infectivity in LC in a dose-dependent manner; 100%(50uM;p<0.001), 67%(25uM;p<0.01), 45%(10uM;p<0.05). EGCG significantly inhibited HIV-1-gp120 binding to CD4 in a dose-dependent manner on LC by 95%(50uM;p<0.001), 70%(25uM;p<0.01) and 27%(10uM;p<0.05). Similar results on MDDC. Control catechin did not alter HIV-1-gp120 binding nor infectivity. Neither DC function, as measured by Ag uptake/presentation, nor viability was significantly altered by EGCG. The inhibitory effect on HIV-1-gp120 binding and infectivity of mucosal cells by EGCG may be beneficial in the development of an HIV-1 microbicide.
NSG-hu mice generate HIV specific human immune responses after gp96 vaccination (46.14)
Authors: Gonzalez Louis , Strbo Natasa , Podack Eckhard
Published: January 2023

NOD.SCID common gamma chain (NSG) knockout mice have no functional adaptive immune system of their own; they readily accept human CD34+ hematopoietic stem cell xenografts (NSG-hu). Therefore, NSG-hu mice present a model system in which to study human immunology. Previously, we developed a cell based vaccine combining secreted heat shock protein gp96 and HIV gag protein (293-gp96Ig-HIVgag). Here we use NSG-hu mice vaccinated with 293-gp96Ig-HIVgag to study human immunological responses. Flow cytometry analysis of peripheral blood in pre-vaccination (8-9 week old) animals revealed human engraftment (mCD45 vs. hCD45) ranging from 25%-50%. In addition, human CD45+ cells were detected in specific tissues (spleen, PEC, IEL and MLN); CD4+, CD8+ T cells and NK cells were found within secondary murine lymphoid organs. Post vaccination (12-13 week old) human engraftment of peripheral blood was observed ranging from 15%-79%. In an ELISPOT for IFN-γ, PECs from NSG-hu vaccinated with 293-gp96Ig-HIVgag i.p. generated an HIV specific immune response when stimulated with SLYNTVATL peptide, producing an average of 778 spots per 2x105 cells plated whereas mice vaccinated with 293 cells alone generated an average of 88.5 spots per 2x105 cells plated (p<0.05) We conclude that NSG-hu mice are able to establish and maintain stable hematopoiesis from human derived cells CD34+ cells. Furthermore, NSG-hu mice are capable of mounting a human HIV-specific immune response after gp96 vaccination.
Fibronectin feedback loops and their relationship with inflammatory skin diseases (48.11)
Authors: Kimber Ian , Dearman Rebecca , Basketter David , McFadden John
Published: January 2023

There are several forms of the extracellular matrix protein fibronectin (Fn), such as the alternatively spliced embryonic form that bears an Extra Domain A (FnEDA). This molecule is produced by both T helper (Th) 1 cells and hyperproliferating keratinocytes and binds toll-like receptor (TLR) 4 and induces psoriatic keratinocyte (PK) proliferation. We hypothesise that there are discrete FnEDA feedback loops that impact directly or indirectly on the behaviors of various skin cells and thereby influence skin disease processes. TLR4 stimulation leads to cytokine, chemokine and adhesion molecule expression resulting in Th1/Th17 cell recruitment. Production of FnEDA by recruited Th1 cells will lead to an “inflammatory” FnEDA positive feedback loop. Proliferating PK also release FnEDA in an autocrine fashion (“keratinocyte” loop) and hyperproliferative keratin, a potential autoantigen. An autoimmune reaction would lead to Th1/Th17 cell recruitment with further FnEDA production (“autoimmune” loop). In contrast, in atopic dermatitis (AD) IL-4, IL-13 and vasoactive intestinal peptide down regulate TLR4 and Th2 cells do not produce FnEDA. Therefore, the inflammatory FnEDA loop appears to be actively suppressed in AD, allowing for Th2 dominance. Thus, the inflammatory FnEDA loop is central to Th1/Th17 driven skin inflammatory diseases such as allergic contact dermatitis, whereas there is active down-regulation of this loop in AD and all three FnEDA loops feature in psoriasis.
Cervical mucosal CD8 T cells are more predictive of HPV lesion regression than systemic HPV-specific response (48.13)
Authors: Trimble Connie , Clark Rachael , Hanson Nicole , Tassello Jodie , Frosina Denise , Teague Jessica , Jiang Jenny , Barat Nicole , Kos Ferdynand , Thoburn Christopher , Jungbluth Achim
Published: January 2023

Virtually all squamous cervical cancers and their dysplastic precursor lesions (CIN2/3), are caused by infection with human papillomavirus (HPV), most commonly, HPV16. Although 20-25% of HPV16+ CIN2/3 undergo complete regression, HPV16-specific T cell immunity in blood is only marginally detectable, and does not correlate with lesion regression. To better understand immunity within the cervical microenvironment, we studied T cells and the expression of vascular adhesion receptors in normal and dysplastic human cervical mucosa. The majority of CIN2/3 CD8+ T cells had an activated memory phenotype, and nearly all expressed α4β7. Regression was predicted by lesional intraepithelial CD8 T cells, whereas in persistent disease, CD8 T cells were restricted to the lesional subepithelial stroma. Expression of VCAM-1 and MAdCAM-1, two ligands that support the entry of α4β7+ T cells into tissues, correlated with CD8 infiltrates. Our data provide evidence that reduced adhesion receptor expression on CIN2/3 vasculature may restrict the entry of CD8 T cells into dysplastic mucosa, thereby preventing immunologic clearance. Immune therapies for cervical dysplasia should generate CD8 T cells that express α4β7, and include local manipulation to induce vascular expression of adhesion receptors that can support the entry of these cells into lesional epithelium.