bluuebunny/crossref_metadata_2025 huggingface dataset:
Age‐Dependent Differential Polarization of Lung Dendritic Cells in Response to RSV infection
Authors: Qureshi Mahboob , Durre Kainath , Yang William
Published: June 2021

Bronchiolitis and pneumonia due to RSV infection result in significant number of pediatric hospitalization in the United States. The survivors develop airway hyper‐responsiveness (AHR) and wheezing in later life. A predominant Th2 response prevails in the respiratory tract of these individuals. Development of Th1 vs. Th2 responses largely depends on the nature of antigen and the type of antigen presenting cell (APC) involved. Dendritic cells (DC) are the major APC in the respiratory tract. In the present studies, using a mouse model, we examined whether young infants respond differently to RSV infection than adults, which may explain their post‐RSV‐infection susceptibility to asthma. Flow cytometry revealed that the proportions of myeloid DC (mDCs; CD11blo CD11chi/med CD45R−) to plasmacytoid DCs (pDCs; CD11blo CD11clo CD45R+) are different in the pup and adult lungs. We also determined the differential responses of neonatal and adult DCs to RSV infection in regard to expression of MHC and co‐stimulatory molecules. In addition, production of Th1 cytokines were significantly lower and that of Th2 cytokines were higher in the lungs of pups compared to that in adults in response to RSV infection. Thus, different proportions of mDCs to pDCs in the neonatal lungs and inefficient maturation of neonatal lung DCs may underlie the mechanism of differential responses of neonates and adults to RSV as well as development of AHR and susceptibility to asthma.
PU.1 expression in T cells is required for the development of allergic airway inflammation
Authors: Sehra Sarita , Nguyen Evelyn , Ahyi Ayele‐Nati N , Chang Hua‐Chen , Nutt Stephen L , Kaplan Mark H
Published: June 2021

The well documented Ets family transcription factor, PU.1 is an important regulator of mast cell, granulocyte and B cell development. Previous studies from our laboratory have identified a novel role for PU.1 in regulating Th2 cell cytokine expression. To further define the role of PU.1 in the development of allergic inflammation in vivo, we studied the development of allergic airway inflammation in mice with a conditional deletion of PU.1 in T cells. Our results indicate that allergen sensitized and challenged PU.1 conditional knockout mice demonstrate reduced airway infiltration of eosinophils and T lymphocytes, reduced Th2 and Th17 cytokine expression in the lung and lower total and allergen specific IgE levels in comparison to wild type mice. These results suggest a critical role for T cell expression of PU.1 in the regulation of allergic airway inflammation.
Immune response and clinical reaction to milk protein in an adjuvant‐free mouse model of milk allergy
Authors: Gonipeta Babu , Parvataneni Sitaram , Gangur Venu
Published: June 2021

Milk allergy is a major type of food allergy with potential for life threatening anaphylactic reaction. Mechanism of sensitization to milk protein is incompletely understood at present. Here we tested the hypothesis that transdermal exposure to milk protein, without an adjuvant, is sufficient to sensitize mice for milk allergy. All mice were on a diet (NIH 5K52) free of milk protein. Thus, repeated transdermal exposure of BALB/c mice (n=10 per group) to milk protein (Greer Lab's cow milk protein extract) resulted in a robust IgG1 but a modest IgG2a response. However, IgE response was very modest even after six transdermal exposure to milk protein. Nevertheless, oral challenge of sensitized mice but not saline control mice with milk protein resulted in clinical symptoms of systemic anaphylaxis by 1 hour post‐challenge. Furthermore, oral challenge also resulted in hypothermia as measured by a drop in rectal temperature at 30 minutes post‐challenge. These data demonstrate for the first time that: (i) transdermal exposure to milk protein results in a modest allergic response; and (ii) oral challenge of sensitized mice results in an immediate hypersensitivity reaction characterized by systemic anaphylaxis. Funding: US EPA STAR#R833133
Aspergillus niger conidia induces asthma in mice.
Authors: Porter Paul , Corry David
Published: June 2021

Asthma results from immune tolerance failure. Commonly used asthma models avoid tolerance by injection of antigens. Our group developed a more physiologically relevant asthma model using potent allergen inhalation. These allergens contain proteinase activities which produce and sustain an asthma phenotype. Preliminary work using dust indicated the homes of asthmatic children contain mainly fungal proteinases. Aspergillus niger (AN) was a common dust isolate and produced a strong proteinase activity similar to that observed in dust. Daily intra‐nasal challenge with as few as 50,000 live (but not sterilized) AN conidia in C57/Bl6 mice is sufficient to establish an asthma phenotype including airway hyper‐reactivity, eosinophilia, and elevated interleukin‐4 secretion in the lung. The asthma phenotype is weak in mice exposed to 5,000 and 10,000 conidia, but the combination of conidia and ovalbumin (a benign antigen) increased the asthma phenotype. Viable fungus persisted in the lungs of C57/Bl6 for over seven days after a single exposure of 400,000 conidia. Sterilized AN conidia were not able to induce asthma phenotype. This data lends support for the concept that some asthma could result from persistence of fungal exposure and possibly low grade fungal infection. This research may shed new light onto the underlying cause of asthma and result in the development of better clinical care treatments.
The receptor for complement anaphylatoxin C5a protects against the development of airway hyperresponsiveness in allergic asthma by inhibiting cysteinyl leukotriene pathway
Authors: Sinha Meenal , Drouin Scott , Wetsel Rick
Published: June 2021

We have reported that complement component C5 is protective against airway inflammation in a rodent model of Aspergillus fumigatus induced allergic airway disease. Using mice deficient in the C5a receptor (C5aR) and an antagonist specific for the C5aR, we tested if these responses could be mediated by C5a, an activation‐induced cleavage fragment of C5. Consistent with previous results, C5aR−/− mice and wild‐type (WT) mice treated with a specific C5aR antagonist exhibited exacerbated airway hyperresponsiveness (AHR) compared to the WT mice. We also examined if Th2 responses, that are known to regulate many aspects of allergic airway inflammation including AHR, were elevated in the lungs of C5aR−/− mice as C5a and its receptor are thought to promote Th1 responses. Surprisingly, Th2‐type cytokines IL‐5 and IL‐13 were significantly reduced in the lungs and bronchoalveolar lavage fluid of C5aR−/− mice. However, pretreatment with a specific cysteinyl leukotriene (cysLT) receptor 1 antagonist could abolish AHR in C5aR−/− as well as in WT mice, suggesting that the absence of C5aR leads to increased cysLT signaling in the lung. These data demonstrate a previously unobserved dual role of the C5aR in allergic lung disease, i.e., protection against AHR mediated by cysLT pathway suppression, but elevation in Th2 cytokines.

Supported by N. I. H. Grants AI025011 and HL074333 (to R.A.W.)
Role of Matrix‐metalloprotease‐7 in a Model of Experimental Asthma
Authors: Goswami Sangeeta , Barranco Wade , Greenlee Kendra J , Seryshev Alexander B , Corry David B , Kharadmand Farrah
Published: June 2021

Bronchial asthma is a respiratory tract disease characterized by airway hyper‐responsiveness, mucus hyper‐production and inflammation. Airway epithelial cells are the initial sites where allergens are recognized. However, the role of epithelial cells in modulation of airway inflammation is not fully understood. One critical group of enzymes that regulate inflammatory processes in the lung is Matrix metalloproteases (MMP). We hypothesize that epithelial specific MMP7 modulates asthmatic phenotype by modifying inflammatory mediators produced by airway epithelium. We found that MMP7 null mice had reduced airway hyper‐reactivity in response to allergen challenge as well as fewer eosinophils in broncho‐alveolar lavage (BAL) fluid compared to WT mice. IL‐25 and Eotaxin, the critical mediators for asthma phenotype, were also reduced in MMP7−/− mice. Proteomic analysis of BAL fluid identified that Retinaldehydrogenase‐1 (RALDH‐1), a rate limiting epithelial specific enzyme for retinoic acid production, was up regulated in MMP7−/− asthmatic mice. In vivo inhibition of RALDH‐1, increased eotaxin protein expression in the airway and rescued the asthma phenotype in MMP7 −/− mice. Together, our data supports the role of MMP7 in modifying airway inflammation in asthma. We propose that identifying in vivo substrates for MMP7 may provide novel therapeutics for treatment of asthma.

This work is funded by NIH grant (U19AI070973) to FK.
TSLP is important in the effector phase of allergic skin inflammation
Authors: He Rui , Oyoshi Michiko , Garibyan Lilit , Kumar Lalit , Ziegler Steven , geha Raif
Published: June 2021

TSLP is a cytokine expressed by keratinocytes and is important in allergic inflammation. Allergic skin inflammation elicited by epicutaneous (EC) immunization of mice with ovalbumin (OVA), which shares many characteristics of the skin lesions of atopic dermatitis (AD), was found to be severely impaired in TSLPR−/− mice, as evidenced by decreased infiltration of eosinophils and decreased local expression of Th2 cytokines. Secretion of Th2 cytokines by splenocytes from EC sensitized mice in response to OVA was normal. Skin dendritic cells from TSLPR−/− mice were normal in their ability to migrate to draining LN, express activation markers and induce proliferation and Th2 cytokine production by naïve T cells. T cells from TSLPR−/− mice expressed the skin homing receptor E‐selectin ligand normally, and homed to the skin normally, but failed to transfer allergic skin inflammation to WT recipients. TSLP enhanced Th2 cytokine secretion by targeting TSLPR on antigen specific T cells. Intradermal injection of anti‐TSLP blocked the development of allergic skin inflammation following antigen challenge of intraperitoneally immunized WT mice. These findings suggest that TSLP is essential for antigen driven Th2 cytokine secretion by skin infiltrating T cells and could be a therapeutic target in allergic skin inflammation
Interaction between Surfactant Protein‐A and Toll‐like Receptor‐4 in Lung
Authors: Awasthi Shanjana , Brown Kevin , Wolf Roman , White Gary
Published: June 2021

Toll‐like receptors (TLR) and surfactant protein (SP)‐A, have been characterized as pathogen‐pattern recognition receptors. Both, TLR4 and SP‐A, have been found to interact in vitro, the in vivo evidence has been lacking. In this study, we investigated the binding between TLR4 and SP‐A in baboon lung by immunoprecipitation/immunoblotting and microwell‐based methods. Basal expression of total TLR4 and SP‐A proteins was quantified in fetal and adult baboon lung tissues by western immunoblotting and ELISA using TLR4 and SP‐A‐specific antibodies. We found that the TLR4 and SP‐A are co‐immunoprecipitated from baboon lung tissues. The binding between the two proteins was also confirmed under in vitro condition. Although the expression of TLR4 and SP‐A, alone, was found to be developmentally regulated, there was no significant difference in the co‐immunoprecipitated TLR4 and SP‐A proteins from fetal or adult baboon lung tissues as detected after immunoprecipitation/cross‐immunoblotting. The interaction between these pathogen‐recognition receptors may play a critical role in pulmonary innate immunity.

Research Support: American Lung Association and Presbyterian Health Foundation.
Systemic activation of macrophages by interferon gamma (IFN‐γ) causes consumptive cytopenias and hemophagocytosis
Authors: Zoller Erin , Lykens Jennifer , Filipovich Lisa , Jordan Michael
Published: June 2021

IFN‐γ is an important mediator of immunopathology in a variety of disease states. We have previously demonstrated that IFN‐γ is critical for the development of hemophagocytic lymphohistiocytosis (HLH), a rare but highly fatal disorder characterized by cytopenias and the appearance of macrophages engulfing apparently normal blood cells (‘hemophagocytosis’). In order to determine whether systemic IFN‐γ exposure is sufficient to produce features of this disorder, we infused mice with physiologically relevant amounts of this cytokine. We have found that IFN‐γ infusion causes rapid (within 5 days) and severe anemia in a dose dependent manner. The rapid onset of this anemia, along with the absence of hemorrhage, indicates that red blood cells are being consumed. Furthermore, macrophages in these mice are more likely to take up non‐opsonized red blood cells as well as soluble, fluid‐phase markers, suggesting that this IFN‐γ driven pathology is a non‐specific, macropinocytic‐like process. In order to define the role of macrophages in this process, we developed transgenic mice which express a dominant negative mutant IFN‐γ receptor under a macrophage‐specific promoter (CD68). These mice are largely protected from IFN‐γ ‐induced anemia, indicating that IFN‐γ is acting directly on macrophages to cause consumptive anemia. Supported by grants from USIDNET, Histiocytosis Association of America, and NIH‐R01HL091769.
Bcl‐3 Inhibits Toll‐like Receptor Signaling and Inflammation By Blocking NF‐kB p50 Ubiquitination
Authors: Carmody RuaidhrÃ‐ , Ruan Qingguo , Palmer Scott , Hilliard Brendan , Chen Youhai
Published: June 2021

Toll‐like receptor (TLR) activation triggers the production of inflammatory cytokines and shapes the adaptive and innate immunity to pathogens. However, uncontrolled activation of TLR can lead to severe inflammatory disorders. We report here that B cell leukemia (Bcl)‐3 serves as an essential negative regulator of TLR4 signaling. Bcl‐3‐deficient macrophages are hyper‐responsive to TLR4 activation and are unable to develop tolerance to lipopolysacchrides (LPS). In the absence of Bcl‐3, the inhibitory p50 homodimer of the nuclear factor (NF)‐kB family is ineffective in suppressing gene expression because it is rapidly ubiquitinated and degraded upon binding to DNA. Bcl‐3 effectively inhibits K48 polyubiquitination of the p50 homodimer and stabilizes its interaction with DNA through the formation of a stable Bcl‐3:(p50)2:DNA complex. By controlling stimulatory NF‐kB dimer loading and dimer exchange at the site of gene promoters, the stabilized Bcl‐3:(p50)2:DNA complex confers a state of long‐lasting promoter hypo‐responsiveness (tolerance) to TLR signaling. In addition, Bcl3−/− mice are more susceptible to autoimmune diabetes and display increased chemokine production and defective Th2, but not Th1 responses. These data establish that Bcl‐3 is a key regulator of NF‐kB transcriptional activity during autoimmune diabetes and acute or chronic infections. Supported by grants from NIH, USA.
Toll‐like receptors are critical for protection against West Nile virus infection
Authors: Sabouri Amir H. , Marcondes Maria Cecilia G. , Berger Michael , Xiao Nengming , Beutler Bruce , Fox Howard S. , Sarvetnick Nora E.
Published: June 2021

West Nile virus (WNV) is a neurotropic flavivirus that emerged in the US in 1999. WNV, now endemic throughout North America, has become a frequent global cause of viral encephalitis. Previous studies have established an essential protective role of both innate and adaptive immune response components in WNV encephalitis. Toll‐like receptors (TLR) play an essential role in initiating innate responses by sensing non‐self components. Whether TLR signaling pathways are required for host defense against WNV infection has not been fully explored. In the present work, we evaluated the role of MyD88 and TRIF, two key adaptor molecules of TLR signaling, in WNV susceptibility and spread into the brain. The absence of TLR signaling through MyD88 and TRIF simultaneously resulted in increased vulnerability to WNV infection with a rise in mortality from 68% (wild‐type mice) to 100% (MyD88/TRIF dKO mice), and a decrease in average survival times. MyD88/TRIF dKO mice showed increased virus replication in the brain and within associated lymphoid tissues. Brain‐infiltrating macrophages were more abundant in infected MyD88/TRIF dKO mice, and expressed higher levels of CD86 than in WT mice. However, these cells failed to up‐regulate MHC Class II, suggesting a defective activation pathway. Cytokine production (IFNα, IL‐6 and TNFα) in WNV‐infected peritoneal macrophages was impaired in MyD88/TRIF dKO mice. Interestingly, we observed a defect in WNV‐specific CD8 T cell responses in MyD88/TRIF dKO mice. Our work s suggests a dominant protective role of Toll‐like receptors against WNV infection. NIH Grant AI059619.
Differential regulation of Listeria‐induced DC maturation responses by MyD88 and the type I IFN receptor
Authors: Brzoza Kristina Lynn , Hiltbold Elizabeth M.
Published: June 2021

Dendritic cell (DC) maturation is a key process in the development of protective immunity to intracellular pathogens such as Listeria monocytogenes (L.m.). We have previously reported that cytoplasmic invasion of L.m. enhances maturation of DC and improves their ability to prime CD8+ T cell responses. Both the MyD88 and type I IFN signaling pathways are important in the L.m. response due to the multiple TLR ligands present on an intact bacterium as well as the large amounts of IFN‐β that are produced upon cytosolic entry. Therefore, in order to address the regulation of L.m.‐induced DC maturation, we generated DC from both MyD88−/− and IFNAR−/− mice and infected them with either cytosolic or phagosomally retained (Hly‐) Listeria. We determined that MyD88 has a role in the production of cytokines by both cytosolic and phagosomally retained L.m., with MyD88 being crucial in the absence of cytosolic entry. Additionally, we found that upregulation of costimulatory molecules was only observed on DC infected with wild type Listeria and this induction was mediated through the type I IFN receptor. We also assessed the ability of matured DC to take up fluorescent tracers and determined that IFNAR is important in the shutdown of uptake capacity associated with the process of DC maturation. Thus, the overall DC maturation process induced by bacterial infection relies on signals from TLR as well as cytosolic PRR. (NIH AI057770)
Impaired Priming and Activation of the NADPH Oxidase in Patients with TLR Signaling Defects
Authors: Singh Anjali , Zarember Kol A , Jain Ashish K , Gallin John I
Published: June 2021

The NADPH oxidase of neutrophils (PMN) plays a key role in host defense by producing reactive oxygen species (ROS). Here we examine the molecular basis of priming and activation of the NADPH oxidase in PMN from patients with two types of TLR signaling defects: interleukin‐1 receptor associated kinase‐4 deficiency (IRAK4) and NF‐kB essential modulator deficiency. (NEMO) We observed Impaired superoxide generation by LPS‐primed and fMLP‐activated IRAK4 PMN that correlated with inefficient translocation of cytosolic factors: p47phox, p67phox and Rac2 and the flavo‐cytochrome subunit: gp91phox to the membrane. While IRAK4 PMN produced ≤ 5% of normal ROS levels, NEMO PMN displayed ~50% of normal ROS with normal translocation of regulatory subunits. Compared to LPS‐primed normal PMN, p47phox phosphorylation was significantly lower in IRAK4 PMN. We further observed the down‐modulation of p47phox phosphorylation and membrane translocation by phosphatidylinositol 3‐kinase (PI3K) inhibitor LY2940002 in LPS‐primed normal PMN. In addition the phosphorylated levels of signaling kinases p21‐activated kinase (Paks) (IRAK4 deficiency) and p38 (IRAK4 and NEMO deficiency) were significantly lower in PMN of TLR signaling defects. We, hereby, reveal the role of defective p38‐ PI3K‐ Rac‐Paks signaling pathway in IRAK4 deficiency patient in impairing the NADPH oxidase mediated ROS generation.

Research support: NIH, NIAID
Dendritic cells and natural killer dendritic cells are influenced by stress from thermal injury
Authors: Kawasaki Takashi , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: June 2021

We have previously reported that mice with partial‐thickness burn injuries (PT‐burned mice) are resistant to burn wound infections, while mice with full‐thickness burn injuries (FT‐burned mice) are susceptible. Recently, the contributions of dendritic cells (DC) and natural killer dendritic cells (NKDC: a novel subset of DC with NK cell properties) on host antibacterial innate immunities have been described. Therefore, in this study, the influence of stress from thermal injury on the DC properties and NKDC appearance was examined in 2 different murine models of thermal injuries.

PT‐burned mice (mice with 1 to 2nd degree, 25% TBSA scald burn) and FT‐burned mice (mice with 3rd degree, 25% TBSA flame burn) were prepared as described previously. DC were prepared from spleens of mice 1, 3, 5, 7 and 14 days after burn injury. These cells (1×106 cells/ml) were cultured for 24 hrs with LPS or SAC. Cultured fluids harvested were assayed for CCL2, CCL3, IL‐10 and IL‐12 by ELISA. For NKDC, DC were analyzed for CD11c and DX5 expressions by flow cytometry.

In the results, CCL3‐ and IL‐12‐producing DC were demonstrated in spleens of PT‐burned mice 3 to 5 days after the injury. In contrast, CCL2‐ and IL‐10‐producing DC were obtained from spleens of FT‐burned mice. Also, as compared with DC from normal mice, the marker of NKDC was increased 75% in DC from PT‐burned mice, while these cells in the DC preparation from FT‐burned mice decreased 65%.

These results suggest that NKDC, as well as DC, may play a role on the antibacterial resistance of thermally injured hosts. (Supported by SHC NA #8610.)
Pet stimulates macrophage and epithelial cells MAPKp38 activity within a different time frame
Authors: Benitez‐Guzman Alejandro , Eslava Carlos , Torres‐Marquez M Eugenia
Published: June 2021

Pet belongs to SPATEs toxin family and it is able to trigger several responses on the host. Such consequences to the host or bacteria itself are under aggressive investigation, as to understand how to support host defense mechanisms. NFκB/MAPK p38 mediated is one of the pathways involved in producing pro‐inflammatory interleukins. The kinase seemingly participates in epithelial survival before challenges. Considering that each cellular type conveys different protection functions for the host, the question arises of whether the pattern response induced by Pet is the same. Here, we established the MAPKp38 activation time course in murine macrophages and in HEp‐2 epithelial cells. A biphasic response curve was obtained for both cell types, although one of the activation peaks was different (30 min/12 hours and 30 min/48 h for macrophages and HEp‐2 cells, respectively). The first activity peak is related to external prompting of MAPKp38, while at least in macrophages, the latter response could be associated either with internalized Pet or a response induced by a product of primed macrophages. Studies of Pet internalization pattern for HEp‐2 cells are being carried out.
Estrogen induces the expression of estrogen receptor in human macrophages
Authors: Jensen Amy L , Pioli Patricia A
Published: June 2021

Monocytes and macrophages (Mφs) are exposed to fluctuations in estradiol (E2) during the menstrual cycle. In some cell types E2 has been shown to regulate expression of its own receptor (ER). E2 levels peak pre‐ovulation and during pregnancy when regulation of immune responses is needed to ensure reproductive success. We hypothesized that E2 up‐regulates ER expression to increase sensitivity to E2 and subsequently influence E2‐regulated inflammatory responses. As human monocytes and Mφs express both isoforms of the ER, ERα and ERβ, we determined whether E2 influences ER expression in these cells. We found no gender difference in mRNA or protein levels in monocytes and MØs. E2 treatment augmented ERα mRNA and protein expression in female Mφs and this was at least in part mediated transcriptionally. Intriguingly, E2 had no effect on monocyte ERα mRNA and protein levels. Up‐regulation of ERα during monocyte‐to‐Mφ differentiation correlated with an increased inhibitory effect of E2 on LPS‐induced IL‐8 production. These data show that ERα is differentially expressed and regulated in human monocytes and Mφs, and E2 acts as a positive regulator of ERα in Mφs. Importantly, ERα expression correlates with the ability of E2 to influence Mφ activation. This may be important when E2 levels are high and tolerance of sperm and an allogeneic fetus are imperative.

Supported by NIH AI51877 and NIH T32AR007576 (to ALJ)
Increased NK‐DC co‐operation and resulting adaptive immune response after HSV infection involves TLR signaling
Authors: Woolard Stacie Necole , Nandakumar Subhadra , Kurt‐Jones Evelyn , Kumaraguru Uday
Published: June 2021

The induction of DC maturation by TLR ligands is considered as a pivotal link between innate and adaptive immune responses. This renders the DCs capable of directing the T cell response. NK cells also express some of the TLRs expressed by DCs. It is our hypothesis that HSV orchestrates the early event of NK cell and DC interaction through TLR mediated signaling.

To test this, we used autologous DCs that were stimulated with various TLR ligands described to be associated with HSV infection (TLR 2, 3, and 9) or cytokines or virus. NK cells (generated from NK precursors or isolated from lymphoid organs) were co‐cultured for 18 hours in the presence or absence of these DCs and later analyzed for their phenotypic changes (CD25, CD69, Chemokine receptors CCR7 and CxCR3), cytokine production (IFN‐γ), and cytotoxicity (YAC cells). NK cells’ acquired cytolytic ability was also tested against Daudi target cells which are usually resistant to NK killing. DCs and NK cells generated from bone marrows of MyD88 knockout were used similarly and in mix and match combination.

The DCs were analyzed for upregulation of activation and co‐stimulatory markers (CD80, CD86, MHC class I and II, CD40 etc.). They were also analyzed for their stimulatory activity after osmatically loading of ova protein or HSV gB protein and measuring their ability to induce activation and proliferation of peptide specific T cells.

The results suggest that the initial interaction between the DCs and NK cells are largely due to signaling through the TLR. If the cells lacked MyD88 signaling their productive interaction was greatly diminished.

Acknowledgement: ETSU startup funds to U. Kumaraguru
Purifying selection of MR1 explains it ability to present of an evolutionarily conserved ligand to mucosal associated invariant T cells.
Authors: Huang Shouxiong , Kim Sojung , Lybarger Lonnie , Hansen Ted
Published: June 2021

MR1 is a non‐MHC encoded class Ib molecule that activates/selects mucosal associated invariant T (MAIT) cells. It has been speculated that MAIT cells function as “innate” T cells similar to NKT cells. To understand how evolutionary pressure influences MR1 antigen presentation, we compared the MR1 on different mammalian species ranging from 81–100% aa identity in the a1/a2 domains. Synonymous versus non‐synonymous mutations in seven MR1 homologues from rodents to primates provided clear evidence that MR1 evolved under purifying selection. Functionally, expression of bovine, rat, or mouse MR1 in a human cell line was sufficient to activate mouse MAIT cell hybridomas, demonstrating presentation of a cross‐species conserved ligand and a shared engagement of the TCR on mouse MAIT cells. Furthermore, the failure of human MR1 to activate MAIT cells, was mapped to a single residue Leu155 predicted to interact only with the TCR. Consistent with this striking cross‐species presentation, most species‐variable residues, occuring sat positions in the prototypical MHCI/peptide structure, are unrelated to ligand binding or TCR interaction. This evidence strongly suggests that MR1, similar to CD1, utilizes a highly conserved ligand binding groove to bind conserved ligands for presentation to MAIT cells. Thus these findings provide evolutionary evidence that MAIT cells function as innate T cells.
The antigenic activity of glycosphingolipids with tetrasaccharide sugars from Sphingomonas bacteria to natural killer T cells
Authors: Pei Bo , Kinjo Yuki , Bufali Simone , Imamura Masakazu , Fujio Masakazu , Wong Chi‐Huey , Seeberger Peter , Kronenberg Mitchell
Published: June 2021

Natural Killer T (NKT) cells, which express an invariant V(14 TCR (iNKT cells), regulate various immune responses by rapidly secreting cytokines upon stimulation with glycolipids presented by CD1d, a MHC class I‐like molecule. It has been shown a monosaccharide containing glycosphingolipid (GSL‐1) from Sphingomonas bacteria is a natural antigen for iNKT cells and mice lacking these cells have a reduced bacterial clearance. However Sphingomonas bacteria have tetrasaccharide containing glycolipids as well as monosaccharide‐containing ones. Here, we studied the stimulation of iNKT cells with two synthesized GSL‐4 antigens (4A and 4B) and a variant of GSL‐4A. We show that GSL‐4A but not 4B could stimulate iNKT hybridomas, although the response to 4A was much weaker than that to GSL‐1. There was no requirement for antigen endocytosis because GSL‐4A presentation by cells transfected with wild type CD1d was equivalent to cells expressing a CD1d with a cytoplasmic tail mutation (I/A), in which the mutant CD1d molecules cannot traffick to lysosomes. Using an antigen presenting cell‐free assay, we could confirm that lysosomal antigen processing was not necessary for GSL‐4A stimulation. Our data indicate that glycolipids containing more complex carbohydrates can be antigens for direct recognition by TCRs expressed by iNKT cells, although weaker than GSL‐1.
Mutations of C3 in Atypical Hemolytic Uremic Syndrome (aHUS)
Authors: Miller Elizabeth C , Liszewski M Kathryn , Gupta Ritu , Hauhart Richard , Fremeaux‐Bacchi Veronique , Goodship Timothy , Atkinson John
Published: June 2021

This non‐enteropathic, often familial disease is characterized by a microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. Dysregulation of the complement system predisposes to this life‐threatening thrombomicroangiopathy. Heterozygous mutations in one of three inhibitors (Factor H, MCP, Factor I) that regulate C3 are present in ∼50% of patients. In two large aHUS cohorts, we have now identified mutations in C3 in ∼10% of patients. There were 9 distinct mutations in 14 patients from 11 families. Site directed mutagenesis was performed and the mutant proteins transiently expressed in 293T and COS‐1 cells and quantitated by ELISA. One mutant (nonsense) led to a truncated protein and one (Cys to Trp) was not expressed. The expressed mutants were assessed for their binding to regulators and cofactor activity (CA). Five mutants displayed decreased binding (∼30% of WT) to MCP and a corresponding reduction in CA. Four of these also showed a decrease in binding (∼70% of WT) to FH. Thus, we propose that a primary resistance to CA leads to a secondary gain of function. This is the first description of heterozygous, function altering mutations in C3 and their linkage to human disease. They further establish a key role for excessive complement activation predisposing to aHUS and provide insight into binding sites of C3. This work is supported by T32AI007163, R01A1041592, and R01A1037618.
The parasitic nematode Ascaris suum secretes a hemoglobin that scavenges host derived free radicals and activates dendritic cells through TLR4
Authors: Madala Satish Kumar , Hodges Marcus , Shweta Trivedi , Virgilio Bundoc , Urban Joseph F , Keane‐Myers Andrea
Published: June 2021

Ascaris lumbricoides is the most successful geohelminth since it currently infects >1.5 billion people world‐wide. Here,we characterize a readily available source of soluble products in the pseudo‐coelomic fluid (PCF) from adult A. suum. A 42 kDa Ascaris suum hemoglobin (AsHb)was recognized by sera from infected pigs and was identified in the muscle and intestinal tissues and the excretory/secretory products obtained from third and fourth‐stage larvae (L3/L4) and adult worms. Comparative sequence information indicated sequence homology in parasitic but not free living nematodes. Exposure of BMDC to AsHb induced the maturation markers CD40, CD86, and MHCII. Co‐stimulation of BMDC with AsHb and LPS decreased IL‐12 production and significantly increased IL‐10 compared to stimulation with LPS alone. AsHb‐dependent maturation and IL‐10 production were abrogated in BMDC from TLR4‐deficient mice. In addition,confocal studies demonstrate co‐localization of AsHb with TLR4 in mouse BMDC, followed by internalization of AsHb through a receptor‐mediated endocytic pathway. Finally, AsHb was able to remove extracellular free radical reductants produced by activated swine bronchial‐alveolar cells.
Dendritic Cell Subsets Differentially Sense and Respond to Listeria monocytogenes Infection both In Vitro and In Vivo
Authors: Mitchell Latoya M. , Grayson Jason M. , Hiltbold Elizabeth M.
Published: June 2021

Dendritic cells (DC) bridge the innate and adaptive immune systems and are important for protection against the intracellular, bacterial pathogen Listeria monocytogenes (Lm). DC can be generated in vitro by treating bone marrow with the cytokines GM‐CSF or Flt3L and can be isolated in vivo from tissues such as the spleen. Current studies show that GM‐CSF DC are representative of monocyte‐derived, inflammatory DC found in vivo, while Flt3L DC correlate to three resident, splenic DC subsets – CD8α+/CD24+DC, CD11b+DC, and B220+ DC. Our goal is to determine how these different DC subsets recognize and respond to Lm infection.

Our previous data indicate that Flt3L DC induce surface costimulatory molecules and cytokines in response to both wild‐type and Hly− strains of Lm in contrast to GM‐CSF DC, which mature in response to wild‐type Lm. The response to Hly− Lm in Flt3L DC was dependent on MyD88 but not TRIF or TLR7. Upon isolation of B220+ and CD11b+ DC subsets from Flt3L DC cultures, CD11b+ DC underwent robust maturation and became infected to a higher percentage than the B220+ DC. In vivo data indicate that splenic DC subsets respond differentially to wild‐type Lm infection but lack a response to Hly−. In addition, the peak of costimulatory molecule upregulation was dependent upon the infectious dose. Taken together, our data support differential responses of DC subsets to Lm infection. 1R01AI057770‐01A1
Regulation of Innate Immune Responses by Mycobacterium Tuberculosis Gene Products
Authors: Medeiros Jennifer , Hansen Eric , van Oers Nicolai S.C.
Published: June 2021

Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB), infects 1/3 of the world's population. Many humans cannot effectively clear Mtb, requiring prolonged multi‐antibiotic drug therapies. This problem is compounded with multi‐ and extremely drug resistant strains of TB. We hypothesize that a number of genes in the Mtb genome inhibit innate immune effector cells.

To identify such gene products, we developed an innate immune response colorimetric reporter assay in macrophages and dendritic cells (DCs) infected with a non‐pathogenic strain, Mycobacterium smegmatis (Msm). In this assay, a murine IL‐6 promoter was cloned into an enhanced green fluorescence protein expression vector. DCs and macrophages exposed to Msm exhibited a 5–15 fold increase in GFP expression. Protein kinase G and protein tyrosine phosphatase B did not suppress IL‐6 driven GFP. We are currently cloning a Mtb‐encoded plasmid library into Msm. Recombinant Msm clones are being used to screen for Mtb genes that suppress IL‐6‐driven inflammatory responses. Suppressive clones will be sequenced and characterized for their capacity to suppress innate inflammatory pathways.

The identification of Mtb‐encoded effector molecules that regulate the immune response is vital for the development of novel therapeutic treatments of tuberculosis. Supported by High‐Impact/High‐Risk Award, UT Southwestern Medical Center
Coordinate uptake of mycobacteria sand oxLDL by differential expression of IL‐10 and IL‐15 derived CD209+ macrophage subsets in human leprosy
Authors: Montoya Dennis , Ochoa Maria , Cruz Daniel , Loncaric Anya , Krutzik Stephan , Modlin Robert
Published: June 2021

Macrophage differentiation and function were investigated in the context of leprosy, which presents as a clinical and immunologic spectrum. The disseminated lepromatous (L‐lep) form is filled with lipid‐laden macrophages and numerous mycobacteria, while the localized tuberculoid form has macrophages but no mycobacteria found. Th1 macrophages correspond to T‐lep and can be derived in vitro with IL‐15. We hypothesized that the Th2 cytokine, IL‐10, can lead to CD209+ macrophage differentiation correlating to L‐lep macrophage phenotype and function. IL‐10 derived macrophages (M10) were found to express CD209 and CD163 in contrast to M15 being CD209+CD163−. M10 were determined to be highly endocytic by multiple assays and phagocytose more mycobacteria than IL‐15 derived macrophages (M15). Furthermore, M10 efficiently take in oxidized LDL (oxLDL) and become foam cell macrophages. Macrophage subsets in leprosy lesions were analyzed and we found CD209+CD163+ M10 macrophages in L‐lep lesions, while CD209+CD163− M15 macrophages were found in T‐Lep lesions. Furthermore, CD163+ macrophages in lepromatous leprosy lesions harbored mycobacteria and apolipoprotein B, a component of oxLDL, suggesting that M10 macrophage subset may lead to foam cell formation and mycobacterial uptake the pathogenesis of leprosy.
The roles of MyD88, TLR2, TLR5, and TLR9 in phagocytosis and inflammatory signaling by Borrelia burgdorferi
Authors: Shin Sarah Ok , Behera Aruna , Hu Linden
Published: June 2021

The role of TLR signaling in the phagocytosis of Borrelia burgdorferi is just beginning to be studied. Here, we show that bone marrow derived macrophages (BMDM) from MyD88 deficient mice were unable to efficiently internalize the B. burgdorferi. Transfection of a MyD88 dominant negative plasmid into Raw264.7 cells produced similar results on phagocytosis. Loss of TLR2, TLR5 and TLR9in BMDM produced no defects in phagocytosis of B. burgdorferi. Release of inflammatory cytokines was greatly diminished in MyD88−/−BMDM cells in response to B. burgdorferi, but only partially affected in TLR2−/−BMDM. Cytochalasin D inhibition of phagocytosis reduced inflammatory signaling significantly, but not to the level of the MyD88−/−BMDM. Addition of cytochalasin D to TLR2−/− BMDM inhibited inflammatory responses to B. burgdorferi to the level of MyD88−/− BMDM consistent with contributions from both recognition of external products and lysosomal sampling by TLR2 to inflammatory signaling. TLR5 also contributes to inflammatory signaling in response to B. burgdorferi. Taken together, these results show that MyD88, but not TLR2, TLR5 and TLR9, is important for the uptake of B. burgdorferi and MyD88 affects inflammatory responses both through its effects on phagocytosis and its role in transducing signals from TLR2 and TLR5. (RO1 AI50043)
Prediction of clonal specificity frequencies for hybridomas using affinity partitioning analysis of test sera
Authors: Kaattari Stephen , Vogelbein Mary A.
Published: June 2021

The development of monoclonal antibodies (Mabs) that can differentiate between closely related organic compounds can be exceptionally difficult. Recent studies attempting to produce high affinity antibodies that can differentiate between DNT and TNT have met with success by first screening antisera from candidate mice using an affinity partitioning technique. Although simple ELISA procedures may yield equivalent titers to such closely related molecules, affinity partitioning of the same parent antisera have demonstrated up to a 150‐fold higher affinity for DNT over TNT within a subset of the entire antibody pool. Splenic fusion correspondingly demonstrated that >30% of the hybridomas were specific for DNT. Thus, this procedure enables the investigator to predict the frequency and specificity of future hybridomas, even when no differences can be distinguished simply by ELISA titers. This research was supported by the Office of Naval Research and the Virginia Institute of Marine Science.
Comparative effect of human albumin formulations on functional activities of cryopreserved PBMC
Authors: Moskowitz Keith A , Hickman Scott , Meixell Ron , Jeffrey Lisa , Manak Mark
Published: June 2021

Cryopreservation (CP) of peripheral blood mononuclear cells (PBMC) to remain functional upon thawing is important for their use in immunotherapy studies. Sera are often used for CP, but demonstrate variability and are replete with growth factors that can result in high backgrounds during cytokine assays. We compared two human serum albumins (HSA) [Grifols® (G) and Bayer® (B)] in the formulation of PBMC CP media containing DMSO (CryoMedia™), and monitored post‐thaw recovery, viability, and secretion of interferon‐γ (IFN‐γ) by ELISpot in response to PHA, CMV and CEF peptide pool stimuli. PBMCs from a leukopack were washed then cryopreserved at 1.5×107/ml; viability prior to CP was approx. 98%. After storage in LN2 vapor mean recoveries were 80.4 (95% CI 55.6–105.2) and 68.0 (95% CI 64.4–71.6) for G and B, respectively, (p=0.4). Post‐thaw viabilities were 92% and decreased respectively to 74 % and 79% for G and B after 48 hours. In response to PHA stimuli, mean IFN‐γ ELISpot response was 1024 (95% CI 996–1051) and 436 (95% CI 407–46) SFC/well using PBMC formulated in B and G HSA, respectively (p < 0.001). Differences were also observed in the response to CMV peptide stimuli using B [18.4 (95% CI 15.7–21.2)] and G [15.4 (95% CI 13.3–17.5)], p < 0.05. Response to CEF peptides for both formulations was similar [16.6 (95% CI 13.3–20.0), B] and [17.4 (95% CI 14.7–20.1), G], p = 0.7. Background to media containing no stimuli were <1 SFC/well for both. The data suggest differences in HSA formulations used during CP of PBMC may elicit significantly different cellular immune responses to select peptide antigens. Additional PBMC donors and studies of proliferation and apoptosis are in process to further elucidate the functional effects of specific manufactures’ HSA during PBMC CP.
Exposure to specific components of a diabetes risk behavior prevention program associated with select psychosocial, dietary and anthropometric outcomes
Authors: Gittelsohn Joel , Kumar Mohan , Ho Lara S , Rosecrans Amanda , Rimal Rajiv N , Harris Stewart B , Sharma Sangita
Published: June 2021

We implemented a multi‐institutional diabetes risk behavior prevention program in 7 First Nations in northwestern Ontario using culturally relevant community‐, school‐ and store‐based components. Data collected in intervention communities (n =56 pre‐post respondents) were analyzed to assess the impact of exposure (adjusted for baseline value) on psychosocial outcomes, dietary behaviors and body mass index (BMI). Individual intervention components varied considerably with print media having higher levels of exposure. Exposure to shelf labels had a positive impact on dietary intention (β = 0.217; p=0.017). Dietary intention (β =0.204; p=0.045) and outcome expectations (β =0.224; p=0.019) were also predicted by exposure to the logo. Exposure to flyers was a significant predictor of dietary knowledge (β =0.023; p=0.022) while self‐efficacy was associated with exposure to posters (β =0.0300; p=0.051). Participation in community events was negatively associated with decreased BMI (β = −0.312; p=0.049) after controlling for known predictors. However, overall exposure to the program was not significantly associated with any outcomes, indicating that specific intervention channels are associated with specific outcomes. Intervention programs aimed at changing psychosocial factors and behaviors require greater exposure to more varied intervention components to have a positive impact.
Prevalence of anemia is lower in post‐folic acid fortification period compared to pre‐fortification period
Authors: Ganji Vijay , Kafai Mohammad R
Published: June 2021

It is not known whether improved folate status from folic acid fortification has had any impact on the prevalence of anemia in the US. Trends in indices and prevalence of anemia and macrocytosis with a focus on comparing pre‐fortification data (1988–1994) with post‐fortification data (1999–2004) were investigated (n=26596). In men, from 1988–1994 to 2003–2004, hemoglobin and hematocrit values slightly but significantly improved from 15.1 to 15.5 g/dL (2.6%) (P<.0001) and from 44.5 to 45.7% (2.6%) (P<.0001), respectively. In women, the improvement was from 13.3 to 13.7 g/dL in hemoglobin (3%) (P<.0001) and from 39.5 to 40.4% in hematocrit (2.3%) (P<.0001). There was a modest but significant increase in MCV from 1998–1994 to 2003–2004 in men (P=0.0001). MCV increased significantly in both older (>60 years) men (P=.0105) and women (P=.0141). In women but not in men, prevalence of anemia was significantly lower in 1999–2004 (10.4%) relative to 1988–1994 (7.5%, a 27.9% reduction, P=.0005). In general, prevalence of macrocytosis did not change significantly from 1988–1994 to 2003–2004. In US women, folic acid fortification may be indirectly responsible for the reduction of prevalence of anemia from 1988–1994 to 1999–2004. In the post‐fortification period, increased MCV in non‐target populations such as men and older persons may suggest an adverse effect of folic acid fortification.
Knowledge, attitude toward people living with HIV/AIDS: Comparison between the U.S. and Japanese nutrition students
Authors: Hayakawa Mai , Tam Chick F
Published: June 2021

Objective: The number of HIV/AIDS patients has been increasing both in the U.S. and Japan. This study compared the knowledge of HIV/AIDS and the attitude toward HIV/AIDS population in the U.S. and Japanese nutrition students. Method: A self‐administered questionnaire was assigned to each participant in their respective countries. Questions included basic knowledge of HIV/AIDS, medical nutrition therapy (MNT) for the HIV+ patients and the attitudes toward HIV population. The total score of knowledge section, Score 1 for basic knowledge of HIV/AIDS and Score 2 for MNT questions were determined by counting the number of correct answers. Result: 296 questionnaires (194 from Japan, 102 from the U.S.) were analyzed. Compared with Japanese students, the U.S. students demonstrated higher total score (11.6 ± 3.1 vs. 7.0 ± 2.8, P=0.000), Score 1(7.0 ± 1.5 vs. 4.7 ± 1.6, P=0.000), and Score 2 (4.5 ± 2.1 vs. 2.3 ± 1.8, P=0.000). Regarding to the attitude toward the HIV population, Japanese students were not comfortable when having HIV+ person visiting their home. Conclusion: This study showed that the U.S. nutrition students were more knowledgeable about HIV/AIDS than Japanese students based on the comparison of scores. The score differences are due to culturally different perception of HIV/AIDS, the limited exposure to HIV information and HIV/AIDS patients in Japan.