bluuebunny/crossref_metadata_2025_split huggingface dataset:
Type I interferon-dependent regulation of granzyme B protein expression by memory CD8+ T cells during the early stages of a respiratory virus challenge (130.13)
Authors: Kohlmeier Jacob E , Miller Shannon C , Cookenham Tres , Roberts Alan D , Woodland David L 
Published: January 2023

Abstract
Memory T cells established following the resolution of influenza and parainfluenza virus infections provide robust protection from a secondary virus challenge. Recently, we demonstrated that the rapid recruitment of circulating memory CD8+ T cells to the lung airways during the early stages of a secondary challenge is crucial for limiting early viral replication. In the course of these studies, we observed that the cytolytic protein granzyme B (gzmB) was highly expressed by virus-specific memory CD8+ T cells recruited to the lung airways. However, the mechanism by which gzmB protein is up-regulated in these cells was unclear. In the current study, we demonstrate that challenge with a disparate virus is sufficient to up-regulate the expression of gzmB protein, suggesting that this process is antigen-independent. To discern the mechanism of gzmB protein expression, we employed a mixed bone marrow chimera model to directly compare wild-type and gene-deficient virus-specific memory CD8+ T cells within the same host. These studies demonstrate that the antigen-independent expression of gzmB protein was dependent on STAT1, and requires signaling through the IFNα/β receptor. Thus, type I interferons act directly on memory CD8+ T cells to increase gzmB protein expression during the early stages of a secondary virus challenge. This work is supported by funds from the NIH and the Trudeau Institute.
Host Immune and Apoptotic Responses to Low Pathogenic Avian Influenza Virus H9N2 in Human Tracheobronchial Epithelial Cells (130.15)
Authors: Xing Zheng , Harper Richart , Danunciacion Jerome , Yang Zengqi , Cardona Carol J 
Published: January 2023

Abstract
Avian influenza virus (AIV) H9N2 subtype has circulated in wild birds and domestic poultry, has successfully crossed the species boundary to transmit to and infect humans and have contributed to the generation of highly pathogenic H5N1 viruses. Little is known about host responses to H9N2 viruses in human airway respiratory epithelium. We used apically differentiated primary human tracheobronchial epithelial (hTBE) cultures to study host antiviral and apoptotic responses to an H9N2 virus strain. Compared to the immortalized bronchial epithelial cells HBE1, differentiated hTBE were less susceptible to the H9N2 virus. Among the antiviral cytokines, IFN-β was the prominent antiviral component in primary cultures. We found that IL-6, TNF-α, IP10 and CCL5 seemed to play important roles in proinflammatory responses while IL-1β and CCL5 were downregulated in primary hTBE cells. The AIV H9N2 virus caused apoptosis in both HBE1 and primary hTBE cultures. In addition to the extrinsic apoptotic pathway, we found that the mitochondria-mediated intrinsic cascade participated in H9N2-induced apoptosis, demonstrated by the cytosolic presence of active caspase 9 and cytochrome c. Further, our data indicated that FLIP and Bcl-2 family members Bax and Bcl-Xs appeared to be involved in the apoptotic regulation. The findings will further our understanding on host defense mechanisms and pathogenesis in human respiratory epithelium infected with AIV H9N2 virus.
A vaccine containing recombinant poxvirus proteins and flagellin promotes protective immunity against vaccinia virus (132.17)
Authors: Delaney Kristen N , Mizel Steven B 
Published: January 2023

Abstract
Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses to a variety of antigens. The vaccinia virus antigens L1R and B5R are highly immunogenic in the context of the parent virus, but recombinant forms of the proteins are only weakly immunogenic. Therefore, we evaluated the response to these antigens when flagellin was used as an adjuvant. We found that flagellin promoted a robust antigen-specific humoral response to poxvirus antigens delivered intranasally and intramuscularly, but intramuscular immunization was more efficient. Flagellin/poxvirus antigen fusion proteins promoted humoral responses that were greater in magnitude than antigen and flagellin as separate proteins. However, there was competition when more than one fusion protein was administered. At least three immunizations with flagellin/poxvirus fusion proteins were required to confer protection in mice against challenge with vaccinia virus. Although mice were protected, they still exhibited significant, but reversible weight loss.
This work was supported by a grant from the National Institutes of Health, P01 AI 60642 (to S.B.M.)
Interfering overlapping epitopes contribute to the subdominance of an HLA-A2-Restricted HIV Gag-specific Epitope (132.18)
Authors: Kan-Mitchell June , Nyakeriga Alice M , Salkowitz Janelle R , Costanzo Margaret C , Sidney John , Sette Alessandro , Ayyavoo Velpandi 
Published: January 2023

Abstract
Recapitulating immunodominant A2-restricted HIV-specific CTLs that do not suppress virus in vivo is unlikely to be an effective vaccine strategy. To detect novel subdominant determinants in HIV-1 Gag, we primed CD8+ T cells from five seronegative donors with autologous dendritic cells transduced to express Gag. T cells were re-stimulated weekly with monocytes pulsed with 123 15-mer overlapping peptides (OLPs) spanning Gag. Responses were identified with OLP matrix pools followed by interrogation at the single OLP level in responsive pools using IFN-γ ELISPOT assays. One OLP (Gag145-159) was recognized by all donors. Of note, this reactivity predominated in three of five T cell cultures. Fine mapping with progressively truncated peptides revealed three overlapping epitopes: RTLNAWVKV (RV9), RTLNAWVKVV (RV10) and TLNAWVKVV (TV9). TV9 is a known epitope that is rarely recognized in vivo. In contrast, RV9 and RV10 are novel. Although the latter bound with lower affinities to HLA-A2 than TV9, RV9- and RV10-cultures were readily generated. RV9- and RV10-T cells were cytotoxic, capable of secreting many cytokines, and suppressive of HIV replication in vitro.
In sum, we report two novel naturally processed and presented epitopes in HIV p24 that are recognized by pre-infection T cell repertoires. Further studies are needed to explain why these reactivities are rare during infections and more importantly, whether this conserved region of the HIV proteome has value as a prophylactic vaccine for A2 individuals.
Multiple antigen encounters decrease memory CD8 T cell-mediated protection against chronic viral infection (132.8)
Authors: Nolz Jeffrey C. , Harty John T. 
Published: January 2023

Abstract
While multiple studies demonstrate the enhanced protective capacity of primary memory compared to naive CD8 T cells, much less is known about the function and properties of memory CD8 T cells that have received multiple antigen exposures through either recurring infections or from booster immunizations. Therefore, we analyzed the ability of primary and secondary memory CD8 T cells to protect against a variety of pathogens expressing a common antigen. Our findings demonstrate that secondary memory CD8 T cells provide better per cell protective capacity than primary memory CD8 T cells against acute infections with both LCMV Armstrong and Listeria monocytogenes. In contrast, while primary memory CD8 T cells were able to effectively prevent a chronic infection with LCMV clone 13, an equal number of secondary memory T cells were unable to clear virus and became functionally exhausted. Delayed re-expression of CD62L on the secondary memory CD8 T cell population affected control of LCMV clone 13, as sorted CD62Lhi primary and secondary memory CD8 T cells were equally capable in preventing chronic infection. Overall, these studies demonstrate that multiple antigen encounters impact the ability of memory CD8 T cells to protect against different pathogens, information that should be considered during vaccine design.
Supported by NIH (RO1-AI42767 to JTH and T32-AI07343 to JCN)
CCR2-mediated signals are dispensable for inflammatory monocyte trafficking into liver during Listeria monocytogenes infection (133.11)
Authors: Shi Chao , Velázquez Peter , Hohl Tobias , Dustin Michael , Pamer Eric 
Published: January 2023

Abstract
Trafficking of leukocytes into peripheral tissues is essential for immune defense. Murine models demonstrate that Ly6Chigh inflammatory monocytes traffic to sites of infection and play a crucial role in innate immunity. The recruitment of inflammatory monocyte is dependent on CCR2, a chemokine receptor that responds to MCP-1 and MCP-3. CCR2-/- mice have decreased number of inflammatory monocytes in the periphery due to their inability to traffic out of the bone marrow. Retention of monocytes in the bone marrow markedly increases the susceptibility to Listeria monocytogenes infection. Although inflammatory monocytes circulating in the bloodstream continue expressing surface CCR2, it remains unclear whether CCR2 is required for monocyte trafficking into and within peripheral tissues. In this study, we investigated the trafficking of inflammatory monocytes to the liver, a major peripheral site of L. monocytogenes infection. By tracing inflammatory monocytes which were adoptively transferred into the bloodstream, we found that CCR2+/+ and CCR2-/- monocytes trafficked similarly into infected livers. Histological and intravital imaging showed that CCR2 was dispensable for monocyte migration within the liver. Our studies demonstrate that CCR2-mediated signals principally facilitate monocyte emigration from the bone marrow and are dispensable for tissue infiltration during bacterial infection.
Encephalitozoon cuniculi (Microsporidia) suppresses apoptosis in human macrophages (133.12)
Authors: Didier Elizabeth S. , Sokolova Yuliya Y. , Alvarez Xavier , Bowers Lisa C. 
Published: January 2023

Abstract
Microsporidia are fungi-related, obligate intracellular parasites. Members of the genus, Encephalitozoon, are clinically important as causes of emerging and opportunistic infections associated with diarrhea and systemic disease. E. cuniculi replicates within macrophages and is believed to disseminate via trafficking monocytes/macrophages. Little is known about how microsporidia survive within macrophages. To address this, the affect of E. cuniculi infection on the ability of macrophages to undergo apoptosis was evaluated. Cultures of human THP-1 macrophages were inoculated with live or dead E. cuniculi (2:1 spores to cells). After 1, 2, 4, and 8 days, the cultures were induced for apoptosis with staurosporine (50 ng/ml) for 4 hours and assessed via TUNEL. Macrophages inoculated with live spores exhibited 1.8-, 3.1- and 3.2-fold less apoptosis than those inoculated with dead spores after 1, 2 and 4 days, respectively. After 8 days, macrophages inoculated with live spores exhibited signs of rupture due to infection and displayed only a 1.64-fold reduction in TUNEL-positive cells compared with macrophages given dead spores. Caspase 3 activity associated with apoptosis was measured on day 4 and was 4.3-fold lower in macrophages treated with live spores compared with those inoculated with dead spores. These early results suggest that E. cuniculi may inhibit apoptosis of macrophages to prolong their survival within these cells. This research was supported by funding from the N.I.H. (RR00164 and AI071778).
Role of Toll-like receptors in the response of human macrophages to Leishmania panamensis infection (133.17)
Authors: Gallego Carolina , Saravia Nancy , Valderrama Liliana , Golenbock Douglas 
Published: January 2023

Abstract
Background: The macrophage is both the host and effector phagocytic cell in the recognition and clearence of L. panamensis parasites. The contribution of Toll-like receptors (TLRs) in the outcome of infection is unknown in American cutaneous leishmaniasis. Objetive: The aim of this study is to determine whether TLRs have a role in the recognition of L. panamensis and identify which TLRs are involved in the response in human macrophages. Methods: TNFα production and cellular expression of TLRs were measured in human primary macrophages from healthy donors in response to TLRs ligands and to the infection with L. panamensis promastigotes. Production of TNFα in TLR positive and TLR knockout murine macrophages was determined in response to promastigotes and amastigotes of L. panamensis. Results and Conclusion: L. panamensis induced a variable production of TNFα by human macrophages from different donors with the same kinetics and similar level as TLR ligands. Expression of TLR1, TLR2, TLR3 and TLR4 in macrophages increased during L. panamensis infection. Activation of wild-type mouse macrophages indicated a possible role of TLRs in the innate recognition of Leishmania. Activation of TLR2 by promastigotes but not by amastigotes suggests a possible role for TLR2 in the recognition and outcome of infection.
The roles of type I IFN receptor and neutrophils in regulating innate immunity to Leishmania parasites (133.26)
Authors: Xin Lijun , Vargas Inchaustegui Diego A. , Raimer Sharon S. , Kelly Brent C. , Hu Jiping , Zhu Leiyi , Sun Jiaren , Soong Lynn 
Published: January 2023

Abstract
Type I IFNs exert diverse effector/regulatory functions in innate and adaptive immune responses to viruses and bacteria; however, their roles in parasitic infections are less clear. It has been reported that parasite-induced type I IFNs are critical for NO-dependent disease control, and that administration of IFN-β has a dose-dependent protective effect in L. major-infected mice. Surprisingly, we found that following infection with L. amazonensis parasites, IFNAR-/- mice developed significantly attenuated disease with low levels of cytokines and sera antigen-specific IgG. The marked reduction in tissue parasite loads and inflammatory responses even at 3 days of infection in IFNAR-/- mice suggests the possibility of neutrophil-mediated parasite clearance. First, although WT and IFNAR-/- mice were efficient in recruiting CD11b+F4/80+Gr1+ peritoneal neutrophils at 6 h post-injection of parasites, knockout mice displayed unique phenotypic changes in Gr1lowCD11bhigh macrophages and Gr1highCD11blow neutrophils at 24 h. Second, in addition to release more neutrophil elastase and myeloperoxidase, IFNAR-/- not STAT1-/- neutrophils had significantly higher rates of spontaneous and infection-induced apoptosis than did their WT controls. Third, while macrophages alone responded comparably to parasites, the interactions between macrophages and IFNAR-/- neutrophils (but not WT and STAT1-/- neutrophils), greatly enhanced parasite killing. Finally, adoptive transfer of WT bone marrow neutrophils into IFNAR-/- mice did enhance tissue parasite loads. This study suggests an important role for type I IFNs in regulating neutrophil turnover and Leishmania infection and provides new information on innate immunity to protozoan parasites.
Role of macrophage scavenger receptor MARCO in innate immunity against Influenza (133.29)
Authors: Ghosh Sanjukta , Kobzik Lester 
Published: January 2023

Abstract
The lung macrophage scavenger receptor MARCO mediates clearance of low doses of bacteria and particulates, but its role in pulmonary defenses against viruses is unknown. We investigated the role of MARCO against infection with influenza virus APR/8/34. We observed that MARCO-deficient mice had increased PMNs in the lungs early after infection compared to wild-type controls (day 2, WT vs KO, percentage of PMNs: 16.46 ± 2.070 vs 36.37 ± 3.063, N=10 per group, p &lt; P&lt;0.0001). MARCO-/- mice showed greater induction of transcripts of acute inflammatory cytokines in the lungs (e.g., CXCL1, MIP-1alpha, RANTES, CD40L, C-Reactive Protein). During this early innate response phase, the viral titer was lower in the lungs of MARCO-/- mice. Notably, the MARCO-/- mice showed greater survival following low dose viral challenge than the WT (80% vs 50%, N=10 per group). In contrast, a higher viral dose led to increased neutrophilia and accelerated mortality. In vitro infection of MARCO-/- or WT macrophages showed no significant difference in virus uptake, although increased expression of CXCL1 was noted. We speculate that MARCO plays a role in the intracellular signaling of virus-infected macrophages leading to a change in its gene expression pattern and enhanced neutrophil chemoattraction in MARCO-/- lungs, responses which are linked ultimately to improved survival.
NIH Grant ES011008-08
Pattern Recognition Receptor Engagement is Necessary for Leishmania Inhibition of Macrophage Phagosome Maturation. (133.35)
Authors: McDowell Mary  Ann , Polando Rachel 
Published: January 2023

Abstract
Leishmania species are obligate intracellular parasites that reside primarily within macrophages (MP), cells whose primary function is to eliminate invading micro-organisms. Leishmania parasites avoid MP destruction by delaying the normal phagosome maturation pathway, allowing the parasites time to develop into a more resistant life cycle form. Leishmania entry into host cells is receptor mediated. These parasites are able to engage numerous cell surface receptors including, mannose receptor (MR), Toll-like receptors (TLR), Fc receptors (FcR), and complement receptor 3 (CR3). Assessing EEA-1 (early marker) and LAMP-1 (late marker) by confocal microscopy, we investigated the role that pattern recognition receptor engagement has on the maturation of Leishmania-containing phagosomes. CR3, TLR-2, and FcR were necessary for the 5 hour phagosome maturation delay characteristic of Leishmania infection. MR deficient macrophages exhibited a unique phagosome maturation pattern not observed for TLR-2, CR3 and FcR deficient MP. Progression to the late phagosome occured at one-hour similar to phagosomes in TLR-2, CR3 and FcR deficient MP; however, at 4 hours phagosomes returned to the early state as marked by EEA1 with late maturation occurring again at 5 hours. Interestingly, pattern recognition receptor deficiency resulted in faster phagosome maturation but had no influence on parasite entry.
Interleukin 17A controls Interleukin 17F production to maintain precise regulation of blood neutrophil counts in mice (133.8)
Authors: von Vietinghoff Sibylle , Ley Klaus 
Published: January 2023

Abstract
Granulocyte colony-stimulating factor (G-CSF), its receptor and interleukin-17 receptor A (IL-17RA) are all required to maintain baseline neutrophil counts in mice. Here, we tested whether IL-17F could compensate and maintain baseline neutrophil counts in the absence of IL-17A. Unlike the reduced neutrophil counts found in IL-17RA deficient mice, neutrophil counts were mildly increased in IL-17A deficient (Il17a-/-) animals. There was no evidence for infection or altered neutrophil function. Plasma G-CSF and IL-17F levels were elevated in Il17a-/- compared to wild-type mice. IL-17F was mainly produced in the spleen and mesenteric lymph nodes, but IL-23 was unaltered in Il17a-/- mice. Instead, Il17a-/- splenocytes differentiated with IL-6, TGF-β and IL-23 ex-vivo produced significantly more IL-17F in response to IL-23 than wild-type cells. Adding recombinant IL-17A to Il17a-/-splenocyte cultures reduced IL-17F mRNA and protein secretion. These effects were also observed in wild-type but not IL-17RA deficient cells. We conclude that IL-17A mediated suppression of IL-17F production and secretion is relevant to maintain the normal setpoint of blood neutrophil counts in vivo.
Modulation of CD1d Expression During MCMV Infection (134.11)
Authors: Reilly Emma C , Brossay Laurent 
Published: January 2023

Abstract
CD1d is an MHC Class I-like antigen presenting molecule that is unique in that it presents lipids rather than peptides to a specific subset of T-cells called invariant Natural Killer T (iNKT) cells. Within hours of stimulation, activated iNKT cells release large quantities of cytokines, promoting cell-mediated immune responses. We examined the expression of CD1d on macrophages, dendritic cells, and B-cells from C57Bl/6 mice and on in vitro cultures both at steady state and during murine cytomegalovirus (MCMV) infection. We found an upregulation of CD1d mainly on macrophages that peaked at day 3 post-infection that was either minimal or not present in the other CD1d expressing cell types that we examined. The cause of this increased expression could potentially be direct (virus-mediated) and/or indirect (due to cytokines or other mediators). To test if CD1d upregulation was direct we used a GFP+ MCMV (clone RVG-102). To test for the roles of the main cytokines produced during MCMV infection we infected IFNαβ R and IL-12 deficient mice. We found that the modulated expression of CD1d on macrophages during MCMV infection is partially dependent of type 1 IFN and the infection itself. We are currently evaluating in more details the molecular mechanism and consequences of the MCMV induced CD1d upregulation. Supported by NIH grant 46709 and GAANN training grant.
NK cell receptors 2B4 and NKp46 utilize FcεRIγ for cell-mediated cytotoxicity (134.18)
Authors: Bida Anya T. , Upshaw Jadee L. , Dick Christopher J. , Schoon Renee A. , Billadeau Daniel D. 
Published: January 2023

Abstract
ITAM-containing adaptors such as CD3ζ, FcεRIγ, and DAP12 are linked to a variety of activating receptors on NK cells and are thus presumed to be important for NK cell cytotoxicity, but whether these ITAM adaptors are unique or functionally redundant in the development of cytotoxicity has remained elusive. Two receptors expressed on NK cells, 2B4 and NKp46, can activate "natural cytotoxicity" against EBV transformed B cells or redirect ADCC. We therefore examined the requirement for ITAM adaptors in 2B4 and NKp46-initiated cytotoxicity using siRNA suppression. Here we provide evidence that FcεRIγ is required for 2B4-initiated cytotoxicity in redirected ADCC assays, whereas CD3ζ and DAP12 are not. Importantly, 2B4 surface levels were not altered by suppression of FcεRIγ, suggesting the possibility that this ITAM adaptor was functioning to costimulate 2B4-initiated killing. Interestingly, FcεRIγ, but not CD3ζ, was important for NKp46-initated cytotoxicity in redirected ADCC assays and siRNA toward FcεRIγ led to a significant reduction in NKp46 surface levels. FcεRIγ is thus an important ITAM adaptor molecule for cytotoxicity initiated by 2B4 through the regulation of NKp46 cell surface levels. FcεRIγ may also be important for killing EBV transformed cells since 2B4 and NKp46 are involved in killing EBV transformed B cells. This work was supported by NCI grant CA47752 to D.D.B. and NIH grant F31-AI75632 to A.T.B.