bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of host protection against infection with Streptococcus pneumoniae conferred by activation of the aryl hydrocarbon receptor (AhR) (129.11)
Authors: Vorderstrasse Beth A , Akerlund Christine , Wang Tao
Published: January 2023

The AhR is a nuclear receptor whose biological function is under active study. We have found that AhR activation protects mice from infection with S. pneumoniae, a common human pathogen that is a leading cause of death. Specifically, treatment with an AhR agonist lowered pulmonary bacterial burdens in mice and substantially reduced morbidity and mortality. The protective effect was not due to direct toxicity to the bacteria, and did not reflect an enhanced inflammatory response or neutrophil recruitment. The current studies tested the hypothesis that AhR activation within lung epithelial cells inhibited their ability to bind bacteria to reduce invasion. A549 lung epithelial cells were exposed to an AhR agonist (TCDD) and co-cultured with S. pneumoniae. Bacterial adherence was visualized by staining with TTC. The expression of adherence molecules (E-cadherin and PAFR) in lung homogenates was also measured. A second hypothesis was that AhR activation induced the production of a soluble protein that killed bacteria in the lung. Bacteria were cultured in concentrated lung lavage fluid from TCDD-treated infected mice. The results of these experiments did not support the hypothesis that AhR activation within lung epithelial cells altered bacterial adherence or bacterial survival, and future studies will focus on early innate immune cell responses to explain host protection. Supported by NIH/NIEHS and WSU Foundation.
Molecular mechanism of immune evasion by Francisella tularensis through the induction of prostaglandin E2 from infected cells. (129.14)
Authors: Woolard Matthew D. , Fuller James R. , Hensley Lucinda L. , Manoil Colin , Kawula Thomas H. , Frelinger Jeffrey A.
Published: January 2023

Francisella tularensis is a highly virulent gram negative bacterium. Because of the low infectious dose, we investigated possible mechanisms of immune evasion. We have previously demonstrated Francisella tularensis ability to induce the production of prostaglandin E2 (PGE2) from infected cells. PGE2 is a potent regulator of immune responses. Our objective is to identify the F. tularensis product responsible for the induction of PGE2 production. We screened a Francisella transposon mutant library and identified 20 candidate genes. These candidate genes were then expressed in ANA-1s, a macrophage cell line. The expression of open reading frame FTL0119 in ANA-1's caused the macrophage cell line to produce more PGE2 than ANA-1s transfected with other candidate genes. We then generated nonpolar markerless deletions of the FTL0119 ORF in F. tularensis Live Vaccine Strain. As expected, mutant F. tularensis LVS failed to induce the production of PGE2 from bone marrow-derived macrophages. Thus we have identified a gene, FTL0119, which is likely to be directly responsible for the induction of PGE2 from F. tularensis infected macrophages and a contributor to immune evasion.
In MyD88-/- mice infected with Mycobacterium tuberculosis ESAT6-specific CD4 T cells expand normally but display impaired IFN-γ production due to a T cell intrinsic role for the adaptor molecule (129.19)
Authors: Mayer Katrin D , Barber Daniel L , Sher Alan
Published: January 2023

MyD88-/- mice fail to control Mycobacterium tuberculosis (Mtb) replication and rapidly succumb to infection. This phenotype has been attributed to IL-R1 and TLR dependent defects in innate immunity although we have also described impaired Th1 cytokine production in these animals. It is unclear whether the partial defect in Th1 response is due to lack of expansion of antigen (Ag)-specific cells or their failure to differentiate into IFN-γ secreting cells. To address this, we stained with a MHC II tetramer to compare pulmonary I-Ab ESAT61-20 specific CD4 T cells in Mtb infected WT and MyD88-/- mice. Interestingly, expansion of Ag specific CD4 T cells was comparable in the two mouse strains. However, we repeatedly observed an approximate 50% reduction in the frequency of IFN-γ secreting Ag-specific cells in MyD88-/- mice, despite uniform T-bet expression. Non-competitive and competitive mixed BM chimeras were employed to determine whether this IFN-γ production defect reflects a T-cell intrinsic role for MyD88. In both strategies, a significant proportion of Ag-specific MyD88-/- T cells still failed to secrete IFN-γ upon re-stimulation when compared to T cells from WT chimeric controls or MyD88+/+ T cells in the same animal. Thus, in murine Mtb, MyD88 expression is dispensable for expansion but required for optimal IFN-γ secretion by Ag-specific CD4 T cells due largely to a T cell intrinsic function of the adaptor molecule.

Work supported by NIAID Intramural Research Program.
Pathogen-specific regulatory T cells impair protective immunity in tuberculosis (129.25)
Authors: Shafiani Shahin , Heard Glady's T , Jenkins Marc K , Takatsu Kiyoshi , Urdahl Kevin B
Published: January 2023

Following aerosol infection with Mycobacterium tuberculosis (Mtb), Foxp3-expressing regulatory T cells (T regs) proliferate, accumulate at sites of infection, and suppress protective immunity. Here we use TCR transgenic CD4+ T cells specific for an immunodominant Mtb antigen (Ag85B240-254/I-Ab) to investigate the origin and specificity of T regs in tuberculosis. These cells robustly proliferated after transfer into Mtb-infected mice, but did not convert into Foxp3-expressing T regs. Instead, T regs expanded from the pool of Foxp3-expressing cells that existed prior to infection. To further examine the specificity requirements for responding T regs, we used a subset of these transgenic Mtb-specific T cells that co-expressed Foxp3. Only T regs that recognized Mtb antigen proliferated and accumulated at sites of infection and transfer of even small numbers of these cells into infected mice resulted in a higher Mtb burden in the lungs. Using Mtb-specific MHC class II tetramers to track the endogenous pathogen-specific T cell response, Mtb-specific T regs were found to mediate this effect by delaying the appearance of Mtb-specific, IFN-γ-producing CD4+ T cells in the lung, a delay that has been previously associated with impaired resistance to tuberculosis. Our results suggest that individuals containing increased numbers of pathogen-specific T regs will be more susceptible to severe disease.
Novel role for TNF-alpha in memory CD8 T cell-mediated protection against liver stage Plasmodium berghei infection (129.27)
Authors: Butler Noah S. , Schmidt Nathan W. , Harty John T.
Published: January 2023

Malaria results in >1,000,000 deaths per year worldwide. Although no licensed vaccine yet exists, much effort is currently focused on subunit vaccines that elicit CD8 T cell responses directed against liver stage antigens. Multiple T cell effector molecules play a role in anti-microbial immunity mediated by memory CD8 T cells including IFN-gamma, perforin, TRAIL, FasL and TNF-alpha. However, it is not known which effector pathways are required for memory CD8 T cell-mediated immunity to liver stage Plasmodium infection. Here, we used a novel immunization strategy to generate memory CD8 T cells in the wild type and gene-deficient BALB/c mouse model of Plasmodium berghei sporozoite infection to examine the role of these T cell effector molecules in resistance to the liver stage infection. In contrast to previous work examining protection mediated by TCR transgenic effector CD8 T cells, our studies reveal that endogenous memory CD8 T cell-mediated protection is in part IFN-gamma-dependent. Importantly, we also show that neutralization of TNF-alpha in immunized mice markedly reduces memory CD8 T cell-mediated protection. Thus, our studies identify both IFN-gamma and TNF-alpha as important components of the non-cytolytic pathways that underlie memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. Supported by grants from the NIH (to JTH) and an NRSA fellowship (T32AI007260-23 to NSB).
A vaccine containing OprF311-341/OprI/type A- and B-flagellin fusion proteins provides enhanced clearance of nonmucoid Pseudomonas aeruginosa (129.3)
Authors: Weimer Eric , Lu Haiping , Wozniak Daniel , Mizel Steven
Published: January 2023

Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients, there is no approved vaccine for human use against P. aeruginosa. The goal of this study was to establish whether a multivalent vaccine containing type A and B flagellins as well as the outer membrane proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. Intramuscular immunization with flagellins + OprI (separate), or OprI/flagellins fusion proteins generated significant anti-flagellin IgG responses. However, only OprI/flagellins generated anti-OprI IgG. Immunization with OprF311-341/OprI/Flagellins elicited high affinity anti-flagellins, OprI, and OprF-specific IgG. Anti-flagellin, OprI, and OprF IgG promoted deposition of C3 on P. aeruginosa. Although these antibodies exhibited potent antibody-dependent complement-mediated killing of nonmucoid bacteria, they were significantly less effective with mucoid isolates. Mice immunized with OprF311-341/OprI/Flagellins had a significantly lower bacterial burden three days post-challenge and cleared the infection at a significantly faster rate than control mice. Based on our results, OprF311-341/OprI/A- and B-flagellin fusion proteins have substantial potential as a vaccine against nonmucoid P. aeruginosa.

Funded by Cystic Fibrosis Foundation MIZEL0810
Changes in the repertoire of influenza-specific CD8+ T cell responses (130.1)
Authors: Lee Jessica  B , Pipeling Matthew , Oelke Mathias , McDyer John , Ramachandra Lakshmi , Canaday David , Schneck Jonathan
Published: January 2023

Influenza has a high morbidity and mortality rate throughout the world. Geriatric individuals have an increased risk for complications arising from influenza infection and account for over 90% of influenza related deaths. To assess why geriatrics are unable to combat influenza infection as well as adult individuals, we used artificial antigen presenting cells (aAPC) to generate influenza-specific CD8+ T cells ex vivo from healthy donors aged 21-60 and geriatric donors over the age of 65. We found a very diverse response to the subdominant influenza peptides, PB1413-421, NS1123-132, NA231-239, NA75-84, PA46-54, and PA225-233 in the adult donors. While we were able to generate CD8+ T cells against the immunodominant influenza M158-66 peptide in all donors, the geriatric donors lacked the broad, multi-specific response to the subdominant influenza epitopes that we found in the adult donors. These results suggest that aging leads to a hole in the T cell receptor (TCR) repertoire and the presence of CD8+ T cells against subdominant influenza peptides plays a major role in clearance of influenza infection.
Adoptive transfer and pulmonary homing of NK cells rescues NK cell-deficient mice from early lethality during acute influenza A virus infection (130.11)
Authors: Carlin-Brown Lindsey E. , Fullenkamp Colleen A. , McGill Jodi , Legge Kevin L. , Heusel Jonathan W.
Published: January 2023

Natural Killer (NK) cells are vital components of the innate antiviral immune response. NK cells are widely distributed, including the lung (comprising 5-10% of mononuclear cells). We have shown that anti-NK1.1-mediated depletion of NK cells in C57Bl/6 or recombinant-congenic BALB.B6-CT6 mice results in early morbidity and increased mortality to mouse-adapted influenza A virus (IAV), similar to that reported for Ncr1-deficient mice lacking the NKp46 receptor. Adoptive IV transfer of resting CFSE-labeled NK cells prepared from spleen or lung results in pulmonary NK cell reconstitution, with a striking preferential homing of pulmonary NK cells to lung compared to splenic NK cells. NK cell-reconstituted mice are comparable to NK cell-replete controls in morbidity and mortality to acute IAV infection, indicating that adoptive NK cell transfer mediates protection. Future experiments will test the protective capacity of adoptively transferred NK cells deficient in NK cell effector functions (IFN-γ, perforin, TRAIL, or FasL) during acute IAV infection. This novel system will expand our understanding of how NK cells populate the lung and contribute toward early anti-influenza virus immunity.
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

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

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

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

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

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

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

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

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

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

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 < P<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

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

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

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

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.
Thermodynamic effects of mutating hot-spot tyrosine residues in the NKG2D binding site (134.19)
Authors: Maddox Michael K. , McFarland Benjamin J.
Published: January 2023

Immune responses are largely governed by tight, highly specific protein-protein interactions. This study focused on one such interaction between the homodimeric immunoreceptor NKG2D, found on natural killer cells, and its MHC-like ligands, which are expressed on stressed cells. Previous research has shown that the NKG2D-ligand interface structure is dominated by 4 central tyrosine residues at positions 152 and 199. We mutated these residues to smaller and larger amino acids in order to investigate the role of the tyrosines in NKG2D-ligand complexes. Eight tyrosine mutants were tested for binding to MICA, MICB and a high-affinity MICA mutant using surface plasmon resonance. For example, mutating Y199 to phenylalanine had a larger detrimental effect on binding than did Y152F, implying that the former tyrosine is more sensitive to the removal of the hydroxyl. Alanine mutations were most detrimental to complex affinity but we were still able to detect binding. We found the vant Hoff enthalpies of each mutant NKG2D-ligand interaction. We correlated observed binding energies with those predicted by a fixed-backbone RosettaDesign mutation simulation. Even simple fixed-backbone RosettaDesign provided qualitative agreement with results for 14 of 15 mutant NKG2D-ligand interactions, and quantitative agreement for 11 of those. To complete this study we plan to investigate the hydrophobic effect in the center of the site by making an additional 3 mutations (I,L, and V) at each residue.
Identification of a cell surface ligand for NKp30 (134.20)
Authors: Levin Steven D , Brandt Cameron S , Yi Eugene C , Kennedy Jacob , Gao Zeren , Fox Brian , Haldeman Betty , Ostrander Craig , West Robert , Xu Wenfeng
Published: January 2023

We have identified a common gene structure found in all known members of the CD28 family and have used this conserved structure to include the Natural Cytotoxicity Receptor NKp30 as a CD28 family member. Soluble forms of NKp30 specifically bound to the chronic myelogenous leukemia cell-line K562 and inhibited cytolysis by the human NK line NK-92. This suggested the presence of a functional counter-structure for NKp30 on the surface of K562 and we employed a proteomic approach to identify this molecule. Our analysis identified an unannontated gene specifically from K562 lysates, which when transfected into P815 cells conveyed NKp30-specific binding activity and sensitivity to NKp30-mediated cytolysis by NK cells. The predicted protein and gene structure of this molecule suggested it was a member of the B7 family and we therefore designated it B7h6. Expression analysis suggested limited expression on normal tissues but expression was apparent on a subset of tumor lines and expression correlated well with sensitivity to NKp30-mediated cytolysis further supporting the notion that this molecule serves as a functional counter-structure for NKp30.
Acute inflammation with IL-18/IL-12 or α-GalCer treatment induces liver iNKT cell apoptosis and repopulation from peripheral tissues, whereas chronic inflammation ablates systemic iNKT cells with thymic-dependent repopulation (134.24)
Authors: Subleski Jeff J , Hall Veronica L , Scarzello Anthony J , Wolfe Thomas B , Ortaldo John R , Hodge Deborah L , Weiss Jon M , Chan Tim , Wiltrout Robert H
Published: January 2023

We previously showed that IL-18/IL-12 treatment of tumor bearing mice elicited significant anti-tumor activity concurrent with a decrease in the detectable number of invariant NKT (iNKT) cells. We now demonstrate that acute treatment with IL-18/IL-12 initially decreased the number of NK1.1(+) iNKT cells while α-GalCer decreased both NK1.1(+) and NK1.1(-) iNKT cells in the liver. Genomic qPCR analysis of iNKT cell's restricted Vα14Jα18 TCR confirmed the loss of cells at 24hrs in the liver rather than receptor down modulation. A small or no decrease in the genomic levels of Vα14Jα18 from other lymphoid tissues indicates the loss of iNKT cells from IL-18/IL-12 or α-GalCer treatment of mice was regulated by the liver microenvironment. Furthermore, an increase in AnnexinV was detected on liver iNKT cells following treatment with IL-18/IL-12 and an increase in cells that were tunnel positive was observed in the liver following α-GalCer treatment of mice. Although iNKT cells initially (24hrs) decrease in the liver following acute administration of α-GalCer or IL-18/IL-12, these cells subsequently (72hrs) reappear and expand in the liver. To study the expansion of iNKT cells in the liver, we administered BrdU to mice 24hrs after acute treatment with IL-18/IL-12 or α-GalCer and found a high percentage of proliferating liver iNKT cells were recent emigrating NK1.1(-) iNKT cells.
A requirement for early neonatal exposure to vitamin D for iNKT cells (134.26)
Authors: Yu Sanhong , Cantorna Margherita
Published: January 2023

CD1d-reactive invariant natural killer T (iNKT) cells are a unique subset of lymphocytes, which play important roles in immune regulation. We have shown that the vitamin D receptor (VDR) is required for the development of iNKT cells. Here we determined the effect of the vitamin D ligand (calcitriol) on iNKT cells. Like the VDR KO mice fewer iNKT cells were identified in the calcitriol deficient mice. However, ligand deficiency did not affect the function of the remaining iNKT cells. In addition, calcitirol deficiency did not affect the development of iNKT cells in the thymus, while VDR KO iNKT cells were blocked at the CD44+/NK1.1- stage of development. The 1alpha-hydroxylase (encoded by cyp27B1) is an enzyme required for production of calcitriol. Cyp27B1 KO mice are calcitriol deficient because they cannot convert vitamin D to the active form. Cyp27B1 ko/+ breeders were fed vitamin D deficient diets. At three weeks of age WT and Cyp27B1 KO littermates were weaned and fed diets that contain vitamin D for the next 5 weeks. Analysis of iNKT cells from these mice showed that both the WT and Cyp27B1 KO littermates had very few iNKT cells. In fact the percentage of iNKT cells in the liver of vitamin D deficient mice littermates were very low 1.2-1.8% and lower than in the VDR KO mice (6%). Early exposure to calcitriol in utero is required for iNKT cell development and iNKT cells failed to recover in either WT or Cyp27B1 KO littermates that were calcitriol sufficient from the age of 5-8 wks. The data show that there are differences in the role of the VDR and the vitamin D ligand on iNKT cells. In addition, the data suggest a critical role for calcitriol early either during neonatal development or the first 3 weeks post-birth in inducing iNKT cells.
Detection of fungal particles by Dectin-1 triggers reorganization of macrophage membrane proteins to form a “phagocytic synapse” (134.35)
Authors: Goodridge Helen S , Wawrowsky Kolja A , Reyes Christopher N , Ma Jun , Wolf Andrea J , Becker Courtney A , Vasilakos John P , Underhill David M
Published: January 2023

The β-glucan receptor Dectin-1 is a central player in the anti-fungal responses of myeloid phagocytes. Detection of β-glucan-containing particles by Dectin-1, which signals via an ITAM-like motif in its cytoplasmic tail, triggers phagocytosis, the induction of an oxidative burst, and production of a variety of inflammatory mediators by macrophages and dendritic cells. In contrast, soluble β-glucans, which are too small to trigger phagocytosis, fail to activate Dectin-1 signaling. However, soluble β-glucans immobilized on latex beads are capable of inducing Dectin-1-mediated responses. We used immunofluorescence microscopy to study the rearrangement of macrophage surface proteins upon binding of β-glucan particles to Dectin-1, and observed the formation of a synapse analogous to the immunological synapse that forms between a T cell and an antigen presenting cell. Our data indicate that the formation of this "phagocytic synapse" is required to initiate Dectin-1 signaling, and that the inability of soluble β-glucans to trigger such reorganization accounts for their failure to activate Dectin-1 signaling.

This work is supported by grants to DMU from the NIH (AI071116, GM085796) and AHA (0640100N); HSG is a CCFA Research Fellow
Exploring the development of endotoxin tolerance and anti-endotoxin activity of LL-37 in mammalian cells (134.55)
Authors: Pena (Serrato) Olga M , Pistolic Jelena , Falsafi Reza , Gardy Jennifer , Hancock Robert E. W.
Published: January 2023

The innate immune response is the first line of defense against microorganisms. However, its over-stimulation can result in excessive inflammation that can lead to deadly syndromes like sepsis. Initially, septic patients exhibit a high immune response, which later shifts towards an immunosuppressive state. This state may be related with a phenomenon called endotoxin tolerance, which is a reduced capacity to respond to antigens. Recent studies have demonstrated that cationic host defense peptides, such as LL-37, have broad immunomodulatory activities like diminishing inflammatory responses. Therefore, the purpose of these two phenomena may be to reduce the immune response and control excessive inflammation. Although much studied, mechanisms that lead to their development are not clear. In this work, time course analysis by RT-qPCR, western blot, and microarrays on human peripheral blood mononuclear cells and monocytic cell line, have shown similar patterns in the up- and down-regulation of important immune players like pro/anti-inflammatory mediators, and negative regulators during the development of endotoxin tolerance and anti-endotoxin activity of LL-37. Results from this work may not only help clarify how each of these phenomena occur, but also may find a link between them.

This work has been supported by Genome British Columbia and Genome Prairie for the Pathogenomics of Innate Immunity Research Program, and by the Foundation for the National Institutes of Health and Canadian Institutes for Health Research through the Grand Challenges in Global Health Initiative.
Complement components modulate activation of mouse NK cells (134.67)
Authors: Qing Xiaoping , Koo Gloria C , Tomlinson Stephen , Salmon Jane E
Published: January 2023

Activation of TLR pathway and complement cascade are two critical defense mechanisms of the innate immune system recently found to interact and influence effector cell function. Natural killer (NK) cells, essential participants in anti-viral and anti-tumor responses, are activated by TLR-3, -4, -7, and -9 pathways via priming by dendritic cells. It is not known whether products of complement activation influence NK cell responses. To examine this possibility, we analyzed NK cell activation in mice lacking complement components or their receptors. Splenocytes from C3-/- mice had significantly more IFN-ã positive NK cells than C3+/+ mice (2-4 fold higher, P<0.01-0.05) when stimulated with PMA and ionomycin, or with TLR agonists: poly I:C (TLR3), Imiquinoid (TLR7), and LPS (TLR4). Increased NK cell activation by Poly IC was also evident in mice lacking C3a and C5a receptors (C3aR-/- 33%, C5aR-/- 45% vs. BALB/c WT 23% p<0.05). Furthermore, intravenous injection of CR2-Crry, an inhibitor of C3 convertase targeted to the sites of complement activation, markedly enhanced IFN-ã production in NK cells in response to poly I:C (CR2-Crry 35% vs PBS 17%, p<0.01). Our results indicate that NK cell activation was negatively regulated by complement components. Blockade of complement activation is a potential therapy to amplify NK cell responses and promote enhanced host defense against viral infections and tumors.

This study is supported by NIH grant R01AI055007 awarded to Dr Jane Salmon.
Mechanism of M-CSF pathway-mediated innate resistance to tuberculosis (134.76)
Authors: Yang Chao-Tsung , Swaim Laura E , Ramakrishnan Lalita
Published: January 2023

The outcome of mycobacterial infections is determined by interactions between host and bacterial factors yet only very few host factors that affect the susceptibility to the infections are known. In particular, there has been an increasing appreciation that both innate immunity and hematopoietic growth factors such as M-CSF play important roles in modulating tuberculosis.

The Ramakrishnan laboratory has established a model for mycobacterial pathogenesis using Mycobacterium marinum (Mm) in the optically transparent and genetically tractable zebrafish. This model allows us to dissect the host cellular and genetic mechanisms that regulate macrophage function during infection. We have found that zebrafish with mutations in the M-CSF-receptor, fms, are hypersusceptible to Mm. M-CSF has pleitropic effects on macrophage development and function. To determine its mechanism of resistance in infection, we performed a step-wise comparison of infection in wildtype and fms-mutant fish. We found that initial macrophage production, as well as macrophage migration to infection sites, phagocytosis and microbicidal capacity were all normal in the fms mutants. Hypersusceptibility became apparent only after granulomas had formed. We are currently testing the hypothesis that the M-CSF pathway is required for macrophage replenishment during infection to maintain granuloma integrity and retain the bacteria within a more restrictive intracellular niche. This study may shed light on the genetics of macrophage homeostasis during mycobacterial infection.