bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoglobulin G and M Detection for Leprosy Diagnosis (129.18)
Authors: Duthie Malcolm S , Goulart Isabela M B , Reed Steven G , Lobato Janaina , Goulart Luiz R
Published: January 2023

Leprosy is a curable disease that requires better diagnostic and prognostic tools to accompany preventive and therapeutic strategies. Circulating IgM antibodies against the M. leprae phenolic glycolipid-I (PGL-I) antigen have previously been used to assess humoral immunity. The presence of elevated titers of anti-PGL-I IgM reflects total bacterial load in the body; these antibodies, however, are generally low or absent in paucibacillary patients. Our objective was to compare 3 new recombinant antigens, ML0405, ML2331, and LID1 (comprising critical regions from ML0405 and ML2331), which recognize serum IgG, in comparison and in combination with PGL-I IgM. Serum samples were collected from 105 patients across the leprosy spectrum at the initial diagnosis and again after treatment. Patient positivity for LID1, ML0405, ML2331 and PGL-I tests was 67%, 62%, 65%, and 76%, respectively. A combination of the LID1 and PGL-1 antigens gave a positivity of 80%. After treatment, the ML0405 and PGL-I assays decreased their ELISA index values for all clinical forms, except for the LL form. Importantly, both antigens have been positively correlated with the bacillary load, clinical forms and the operational classification at diagnosis. Our results suggest that the combination of LID1 and PGL-I antigens, recognizing the IgG and IgM response, respectively, could be employed as an auxiliary tool for leprosy diagnosis.

This work was funded in part by American Leprosy Mission.
TgMAPK1 is an important virulence determinant in Toxoplasma gondii affecting stage differentiation. (129.23)
Authors: Brumlik Michael James , Ludwig Sara M , Pandeswara Srilakshmi , Jeansonne Duane P , Livi Carolina B , Daniel Benjamin J , Church Kristina M , Kious Mark J , Curiel Tyler J
Published: January 2023

Toxoplasma gondii is a medically important, obligate intracellular protozoan parasite that causes significant morbidity and mortality in hosts with defective cellular immunity. Using antisense RNA knockdown, we generated T. gondii tachyzoites expressing 5 - 10-fold less TgMAPK1, a stress-response mitogen-activated protein kinase. In infection, T. gondii converts from the rapidly-dividing tachyzoite to the latent bradyzoite form. We show here that T. gondii tachyzoites with reduced TgMAPK1 display distinctive bradyzoite characteristics, including expressing Bag1 (a bradyzoite-specific antigen) and significantly reducing Sag1 (a tachyzoite-specific antigen). Although knockdown (KD) tachyzoites were dramatically less sensitive to the anti-proliferative effects of IFN-γ than wildtype (WT), these parasites were nonetheless significantly less virulent than WT in BL6 mice, based on: (i) KD parasites achieved only a fraction of the parasite burden in tissues of infected mice relative to control parasites, (ii) mice infected with KD produced substantially less IFN-γ, and (iii) the majority of mice challenged with KD were able to survive lethal challenge. These results show that TgMAPK1 is an important virulence determinant affecting stage conversion, proliferation, and replication. This work was supported by funds from NIH AI060424 and Johnson & Johnson Focused Giving.
Characterization of immune responses to Yersinia pestis infection: resistant versus susceptible mice(129.29)
Authors: Lambert Nathaniel D , Nilles Matthew L , Bradley David S
Published: January 2023

We have previously reported that inbred B10.T(6R) mice are resistant to Yersinia pestis infection, compared to susceptible strains, e.g. Swiss Webster mice; the mean LD50's for B10.T(6R) females is 14000 CFU's, compared to Swiss Webster females, at 22 CFU's. Interestingly, B10.T(6R) mice demonstrated a more robust pro-inflammatory response early in infection, and a lesser anti-inflammatory response later on, when compared to susceptible Swiss Webster mice. Significantly higher levels of TNF-alpha were detected in B10.T(6R) on day 1 while TNF-alpha responses were delayed in Swiss Webster mice. Conversely, IL-10 levels were not changed in B10.T(6R) mice while IL-10 levels increased significantly in Swiss Webster mice. Yersinia outer membrane proteins (YOP) are translocated into the host cell's cytoplasm via a type three secretion system. After contact with a eukaryotic cell, YOPs then interact with cellular components to disrupt the host's immune response and induce the production of suppressive cytokines, e.g. IL-10. We postulate that the resistant B10.T(6R) strain of mice are able to overcome initial infection with Y. pestis through higher levels of pro-inflammatory cytokines and decreased levels of immune suppressive cytokines later in infection.
Influenza-specific memory CD4 T cells regulate acute inflammation independent of innate immune recognition (130.10)
Authors: Strutt Tara  M. , McKinstry K. Kai , Dutton Richard W. , Swain Susan L.
Published: January 2023

It is generally accepted that the flow of immunologically relevant information during the early stages of responses against pathogens is one-way, - that inflammation induced upon pattern recognition by highly conserved receptors of the innate immune dramatically impacts subsequent antigen-specific T and B cell responses. We asked if the reverse occurs, and if cells of the adaptive immune system can influence the character and magnitude of innate inflammatory responses. We show here that resting, antigen-specific memory CD4 T cells can dramatically alter innate inflammatory responses within 36 hours of viral infection in a manner independent of other T cells and TLR signaling. Virus-specific memory CD4 T cells transferred to naïve mice that are then challenged with influenza induce greater expression of multiple inflammatory mediators both at the site of infection and systemically upon cognate recognition of antigen in an IFN-gamma independent fashion. Our results show that the adaptive immune system can profoundly influence the character of inflammation following pathogen challenge, demonstrating a new role for memory CD4 T cells in controlling virus titers during protective immune responses.
Coxsackievirus B3 has profoundly different inhibitory effects on the MHC class I and class II antigen presentation pathways (131.2)
Authors: Kemball Christopher C , Harkins Stephanie , Whitmire Jason K , Feuer Ralph , Flynn Claudia T , Whitton J Lindsay
Published: January 2023

Coxsackievirus B (CVB) infections cause considerable morbidity and mortality, and strategies to treat or prevent CVB-associated disease may benefit from a clearer understanding of the host immune response to virus infection. CVB3 induces minimal endogenous naïve CD8 and CD4 T cell responses, possibly because viral antigen (Ag) presentation is extremely limited. Several CVB3 proteins are known to cooperatively downregulate MHC class I on infected cells in vitro. In this study, we used transgenic (Tg) CD4 and CD8 T cells as sensors to evaluate viral Ag presentation by the MHC class I and class II pathways in vivo. Our analysis revealed a striking difference in Tg T cell responses: CD4 Tg T cells proliferated in CVB3-infected mice whereas CD8 Tg T cells failed to divide. Moreover, virus infection generated multi-functional memory CD4 T cells, which expanded dramatically following challenge infection, and rapidly differentiated into secondary effector Th1 cells. Although naïve CD8 Tg T cells were unresponsive to CVB3, they persisted in the host and responded vigorously when stimulated by a different virus encoding their cognate Ag. These data suggest that CVB3 utilizes a powerful immune evasion strategy in vivo that differentially affects the two Ag presentation pathways, and that this difference regulates the host's capacity to mount CD4 and CD8 T cell responses. This work was supported by NIH R01 AI42314 and T32 NS41219.
Engineering Immunity against HIV - a Stem Cell Vaccine (132.12)
Authors: Yu Kenneth K , Tsai Jonathan , Yang Lili , Baltimore David
Published: January 2023

An effective vaccine against HIV is urgently needed. We are pursuing a novel gene therapy approach to combat HIV by engineering HSCs to produce human B cells programmed to make anti-HIV broadly neutralizing antibodies (bNAbs). We accomplish this by using lentiviral vectors to modify the long-lived HSCs with engineered bNAb genes. We hypothesize the transplantation of the engineered HSCs will confer life-long HIV-specific immunity; in effect, a "Stem Cell Vaccine" for HIV. In nature the soluble Ab and membrane BCR are produced by complex alternative splicing events difficult to implement in the context of a lentiviral vector. We have overcome this obstacle by developing a novel "Synthetic-Switch" antibody gene that mimics the function of the membrane-bound BCR and secretory antibody. Furthermore, the Synthetic-Switch approach enables us to produce "isotype-switched" anti-HIV B cells without the need for complex DNA rearrangement. The Synthetic-Switch approach thus successfully captures the essential functions of the roughly 2-Mbp immunoglobulin loci in a compact 10kb lentiviral vector. Herein we present this successful first step toward an anti-HIV stem cell vaccine.
How the strength of peptide-MHC and TCR interaction impacts CD8 T cell responses (132.24)
Authors: Zehn Dietmar , Bevan Michael J.
Published: January 2023

The pool of naïve T cells specific for any foreign peptide/MHC-I epitope is diverse and contains cells that differ in their functional avidity. Theoretical considerations imply that there are likely to be more low than high avidity T cells specific for any foreign antigen. We studied to what extent lower avidity T cells participate in an immune response and we investigated how the dominance of high avidity T cells evolves over the course of an infection. To control the strength of stimulation that CD8 T cells receive during an immune response, we used a model system in which OT-1 TCR transgenic CD8 T cells were stimulated during a Listeria monocytogenes infection either by their native ligand SIINFEKL or by different SIINFEKL-derived altered peptide ligands. Using this system we observed that even very weak TCR-ligand interactions are sufficient to induce rapid T cell proliferation and to generate functional effector and memory CD8 T cells. However, the strength of the TCR-ligand interaction impacts when expansion stops and contraction begins and when the cells exit lymphoid organs, i.e. strongly stimulated T cells contract and exit lymphoid organs later than do weakly stimulated cells. Our data challenges the prevailing view that strong TCR ligation is a prerequisite for CD8+ T cell activation. Instead, very weak interactions are sufficient for activation, but strong TCR ligation is required to sustain T cell expansion.
Pre-clinical testing of T cell responses to influenza vaccine/adjuvant combinations in older adults (132.5)
Authors: Ma Kewei , Haynes Laura , Wilschut Jan , McElhaney Janet E.
Published: January 2023

Objective: The purpose of this experiment was to determine whether inflammatory cytokines or TLR ligands could be combined with influenza vaccine in older adult PBMC to enhance the response to live influenza virus challenge.

Methods: PBMC from five older adults (>65 years old) and six young (20-38 years old) adults were obtained at six months post-vaccination and cultured for five days with split-virus vaccine (SVV) and the TLR3 ligand, poly I:C, or a cocktail of pro-inflammatory cytokines (TNF-α, IL-1, IL-6; T/1/6). Stimulated PBMC were then challenged with live influenza virus for 20 hours and supernatants and lysates prepared for assays of IL-1, IL-6, TNF-α in 5-day supernatants, and the IFN-γ:IL-10 ratio and granzyme B activity in 20-hour virus-challenged PBMC.

Results: We observed an increased response to SVV in younger compared to older adults, but a dose-response relationship between poly I:C (added to a fixed dose of SVV) and the IFN-γ:IL-10 ratio and granzyme B levels improved the response in older adult PBMC. This response was associated with a dose-response increase in pro-inflammatory cytokine levels in SVV/poly I:C-stimulated PBMC. However, a cocktail of these inflammatory cytokines (T/1/6) added to the PBMC culture suppressed rather than enhanced the T cell response in older adults.

Conclusion: Poly I:C combined with influenza vaccine was shown to enhance the T cell response to influenza in aged PBMC by mechanisms that cannot be replicated by the simple addition of inflammatory cytokines to the vaccine.
Characterization of the diversity of the CD8+ T cell repertoire against the same epitope induced by bacterial or viral immunization (132.9)
Authors: Rudd Brian D , Venturi Vanessa , Davenport Miles P , Smithey Megan , Alzhanova Dina , Way Sing Sing , Nikolich-Zugich Janko
Published: January 2023

Prevention of infectious diseases is most efficiently accomplished by vaccination, and most vaccines rely on generation of protective antibodies. On the other hand, elicitation of CD8+ T cells plays a key role in immune protection against many intracellular pathogens. Despite the widespread success of many vaccines, the underlying basis of these protective immune responses is still poorly understood. While vaccine development has historically focused on the magnitude of the response, recent advances in immunology suggest that sufficient diversity of CD8+ T cell immune responses is also critical in controlling various infections and such information may be especially critical in the rational design of vaccines against chronic and persistent infections. To address these issues, we are currently investigating whether CD8+ T cell repertoire against a protective epitope can be influenced by the infectious/inflammatory context in which the epitope is encountered. In an effort to assess the extent of TCR diversity that results from different infectious vectors, CD8+ T cells specific for the immunodomant epitope (gB 498-505) were sequenced from mice infected with HSV-1 or with Vaccinia virus (VV) or Listeria monocytogenes (LM) expressing the immunodominant peptide. By comparing CD8+ T cell repertoires induced by different pathogens, we hope to understand how and to what extent the CD8+ T cell repertoire is able to shaped.

NIH grant #7R01AI066096-05
Memory immune mechanisms against parasitic tissue-dwelling Heligmosomoides polygyrus larvae (133.25)
Authors: Kreider Timothy , Gaydo Andrew G , Urban, Jr. Joseph F , Gause William C
Published: January 2023

The nematode parasite H. polygyrus has both a parasitic intestinal tissue-dwelling larval phase and a lumen-dwelling adult phase in its life cycle. Primary inoculation of naïve mice resulted in chronic infection while secondary inoculation after drug-cure to clear adult worms triggered a protective Th2-type memory response, characterized by a distinct Th2-type granuloma that developed by 4 days after secondary but not primary inoculation. We investigated the effects of this memory response on early larval development. As early as day 4 after inoculation, parasitic larvae in the intestine were fewer in number, exhibited shorter lengths, and were more widely distributed across the small intestine in the memory compared to the primary response. Mice administered DMFO, which inhibits ornithine decarboxylase, after secondary inoculation exhibited significantly increased larval numbers and lengths, but no change in parasite distribution, suggesting an important role for polyamines in sustaining a host-protective memory response. In addition, B cell-deficient mice contained larvae with increased lengths and altered distribution in the intestine after secondary inoculation that was comparable to the response observed after a primary inoculation. The early host-protective response appeared to be TSLP-independent because it was not impaired in TSLPR-/- mice. These studies suggested that memory Th2-type responses impaired tissue-dwelling parasite migration and development as early as four days after infection, and that this response was partly dependent on polyamine production and B cells.
Macrophages are significant producers of IL-1beta during C. muridarum genital tract infection and IL-1beta secretion is dependent on chlamydial viability but not growth (133.3)
Authors: Prantner Daniel , Brade Helmut , Rank Roger , Nagarajan Uma
Published: January 2023

Recent findings have implicated secretion of the proinflammatory cytokine interleukin-1β as a crucial player in female genital tract pathology during chlamydial infection. However, the major cellular source of IL-1β during an in vivo infection and mechanistic details of its release have yet to be determined. In the present study, flow cytometry was used to isolate highly enriched cell populations from the genital tracts of C. muridarum infected mice at the peak of IL-1β secretion. Purified F4/80+ macrophages and Ly-6G+ neutrophils, but not CD45- epithelial cells, expressed high levels of both IL-1β mRNA and protein. Macrophages infected with C. muridarum in vitro secrete low amounts of IL-1β, however they can be induced to secrete high amounts when prestimulated with E. coli LPS prior to infection. Prestimulation with purified LPS from C trachomatis L2 was also sufficient to amplify IL-1β secretion, suggesting that prestimulation by chlamydial ligands could account for the high levels of IL-1β observed in vivo. Using LPS prestimulated macrophages as a model system, it was further determined that IL-1β secretion was dependent on activation of the protease caspase-1 in a manner that required both potassium efflux and the activity of serine proteases. Additionally, chlamydial-induced IL-1β secretion was blocked when chlamydiae were inactivated by UV irradiation or heat treatment but occurred normally during treatment with chloramphenicol, rifampin, and INP0007, illustrating that only bacterial viability and not bacterial growth is necessary for this response. This study was supported by NIH grant #AI067678.
SLAM controls killing of bacteria through regulation of phosphatidylinositol-3-phosphate in phagosomes (133.31)
Authors: Berger Scott B , Romero Xavier , Wang Ninghai , Faubion William A , Terhorst Cox P
Published: January 2023

Macrophages eradicate pathogens by phagosomal reactive oxygen species, nitric oxide and by exposure to phagolysosomal enzymes. Whereas a number of surface receptors are requisite for recognition and entry of bacteria into the phagosome, their role in bacterial killing is largely unknown. Here we show that the surface receptor SLAM (Slamf1, Cd150) enters the macrophage phagosome together with Gram-negative, but not with Gram-positive bacteria. In the bacterial phagosome, SLAM regulates production of phosphatidylinositol-3-phosphate (PI3P), which is a key factor in the assembly of the active NADPH-oxidase (Nox2) enzyme complex, as well as in phagosome maturation. Consequently, these two bactericidal processes are defective in the absence of SLAM, resulting in an impaired clearance of attenuated S. typhimurium sseB- in SLAM-deficient mice. This is the first example of a surface receptor, which is a co-stimulatory molecule in T cell responses, also governing distinct functions in intracellular vesicles of phagocytic cells.

This work was supported by a grant from the NIH (AI-15066)
Role of galectin-3 in macrophage response to Listeria monocytogenes infection (133.34)
Authors: Weng I-Chun , Hsu Daniel K , Liu Fu-Tong
Published: January 2023

Galectin-3 is a β-galactoside-binding animal lectin expressed in various immune cell types including macrophages. It has been implicated as a positive regulator of macrophage phagocytosis and as a negative regulator of LPS-mediated inflammation. However, its role in macrophage antibacterial function has not been fully elucidated. The aim of this study was to investigate the role of galectin-3 in macrophage antibacterial function against Listeria monocytogenes (LM) infection by studying bone marrow-derived macrophages (BMM) from galectin-3-deficient (gal3-/-) and wild-type (gal3+/+) mice. After in vitro infection, gal3-/- BMM contained significantly fewer viable intracellular bacteria than gal3+/+ BMM. Consistently, immunofluorescent staining of infected BMM revealed a significantly lower percentage of cytosolic LM escaped from phagosomes in gal3-/- BMM compared to gal3+/+ BMM. Higher levels of reactive nitrogen intermediate nitric oxide (NO) but lower levels of inflammatory cytokine IL-1β were detected in LM-infected gal3-/- BMM compared to gal3+/+ cells. In addition, we noted elevated levels of NAD(P)H in gal3-/- BMM which may contribute to higher amounts of NO in gal3-/- BMM. We conclude that galectin-3 plays a regulatory role in macrophage antibacterial function by interfering with the production of NO, thereby facilitating the survival and replication of intracellular bacteria.
Rapid deletion of antigen-specific CD4+ T cells following infection represents a strategy of immune evasion and persistence for Anaplasma marginale (133.36)
Authors: Han Sushan , Norimine Junzo , Wendy Brown C.
Published: January 2023

We reported the rapid and permanent deletion of immunization-induced bovine CD4 T cell responses to Anaplasma marginale major surface proteins (MSPs) after infection. To determine if antigen-specific T cells were anergic, sequestered, or deleted, CD4 T cells specific for a MSP1a T cell epitope were enumerated with a DRB3*1101 tetramer. The pre-challenge frequency of tetramer+ CD4+ cells of 0.025% dropped rapidly at the peak of infection to control levels of 0.0003%, paralleling loss of antigen specific T cell responses. Background frequencies of tetramer+ CD4+ T cells in spleen and lymph node indicated lack of sequestration. We hypothesize that this rapid and long-term deletion of specific T cells is driven by high antigen load during acute and persistent infection. To test this, cattle immunized with MSP1a and MSP2 of A. marginale will be challenged with A. marginale or A. centrale, a subspecies in which MSP2 but not MSP1a epitopes are conserved with those in A. marginale. We predict deletion of CD4+ T cells specific for MSP1a and MSP2 after infection with A. marginale, but only deletion of MSP2-specific T cells after infection with A. centrale, as MSP1a-specific T cells will not be exposed to high antigen load in the latter cattle. This experiment will determine if infection-mediated CD4+ T cell depletion is dependent on the presence of specific antigen in the infecting pathogen.
ATP plays a role as a danger signal that activates macrophages during adenoviral infection (133.38)
Authors: Lee Benjamin H , Kushwah Rahul , Bertics Paul , Hu Jim
Published: January 2023

We have previously shown that adenovirus infection of epithelial cells and macrophages co-culture resulted in synergistic induction of pro-inflammatory mediators such as cytokines, nitric oxide (NO), and reactive oxygen species (ROS) and caused cytotoxic responses. Here, we investigated whether ATP plays a role during the inflammatory response against adenovirus using the co-culture system. We found that pre-treatment of the co-culture with oxidized-ATP inhibited induction of NO and ROS and reduced cytotoxicity during adenoviral infection. Furthermore, we demonstrated that ATP signalling through P2X7 receptor is required in generation of NO and ROS using P2X7 deficient macrophage cell line. These results indicate that ATP plays a role as a danger signal to regulate macrophage activation during inflammatory response against adenoviral infection.
Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1 (133.49)
Authors: Cole Alexander M , Venkataraman Nitya , Ruchala Piotr , Waring Alan J , Lehrer Robert I , Stuchlik Olga , Pohl Jan
Published: January 2023

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins - 18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature stop codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature stop codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. Since retrocyclins are broadly antiviral, the ability to reawaken retrocyclin genes from their 7 million years of slumber could provide a novel way to secure enhanced resistance to several major viral pathogens. AI052017; AI065430
Predictors of human norovirus infection and immunity (133.52)
Authors: Leon Juan S , Fernandez Marina L , Ailes Elizabeth C , Lindesmith Lisa C , Seitz Scot R , Smith Emily R , Baric Ralph S , Moe Christine L
Published: January 2023

Noroviruses (NoV) are the main cause of epidemic gastroenteritis worldwide. We have previously shown that both an individual's secretor status (genetic) and induction of Norwalk virus-specific salivary IgA (immune) are important predictors of infection with Norwalk virus (NV). We still do not understand the factors that mediate the immune response, and infection, to Norwalk virus. To address this need, we have extended our analysis of immunological, genetic, and demographic parameters of 109 volunteers challenged with various doses of Norwalk virus through univariate and multivariate regression analyses. We found that gender, ethnicity, levels of pre-challenge NV-specific serum IgG or levels of pre-challenge NV-specific salivary IgA were not significant predictors of infection or development of a NV-specific antibody response. The presence but not magnitude of pre-challenge serum NV-specific IgG and secretor positive status were significantly associated with infection. Dose of inoculum received and age of the individual may also be predictors of infection and development of an NV-specific antibody response. We observed significant collinearity between the age of the volunteer and the presence of pre-challenge serum NV-specific IgG. Lastly, we found a strong association between levels of salivary and serum NV-specific antibody levels, after adjusting for covariates. Taken together, these data will be used to better understand the immune response to NV infection and design effective NoV vaccines to protect at risk populations including children, food handlers, and the elderly.
B and T lymphocyte attenuator tempers early infection immunity (133.6)
Authors: Brown Nicholas K. , Sun Yonglian , Miller Mendy , Fu Yang-Xin
Published: January 2023

Co-inhibitory pathways are thought to act in later stages of an adaptive immune response, but whether co-inhibition contributes to early innate immunity is unclear. We show that engagement of the newly discovered co-inhibitory receptor B and T lymphocyte attenuator (BTLA) by herpesvirus entry mediator (HVEM) is critical for negatively regulating early host immunity against intracellular bacteria. Both HVEM-/- and BTLA-/-, but not LIGHT-/-, mice are more resistant to listeriosis compared to wild type mice, and blockade of the BTLA pathway promotes, while engagement inhibits, early bacterial clearance. Differences in bacterial clearance were seen as early as 1 day post-infection, implicating the initial innate response. Therefore, innate cell function in BTLA-/- mice was studied. We show that innate cells from BTLA-/- mice have a lower threshold for activation, and secrete significantly more pro-inflammatory cytokines upon stimulation with listeria. These results provide the first evidence that a co-inhibitory pathway plays a critical role in regulating early host immunity against infection. (Supported by CA115540, AI062026, and DK58897 to YXF and T32AI007090 to NKB)
Decreased IL-15 Production and Impaired Natural Killer Cell Activity in Influenza Infected C57BL/6 Aged Mice (133.9)
Authors: Nogusa Shoko , Ritz Barry W , Gardner Elizabeth M
Published: January 2023

The role of Natural Killer (NK) cells during the innate immune response to primary influenza infection was investigated in young and aged mice. Young (2~6mon) and aged (22mon) C57BL/6 mice were infected intranasally with influenza A (H1N1, PR8) and NK cell cytotoxicity was assessed in lung and spleen during the first 3 days of infection. Aged mice demonstrated both decreased influenza-inducible NK cytotoxicity and reduced percent and number of NK cells relative to young mice. To establish a role of NK cells in controlling influenza infection, NK cells were depleted in young mice by injecting anti-NK1.1 antibody prior to infection. NK-depleted young mice exhibited increased weight loss and elevated lung virus titers compared to intact young or aged mice. These data indicate that NK cell function is impaired in aged mice, and more importantly, indicate the essential role of NK cells in controlling virus titers in lung, regardless of age. Notably, NK cells from young and aged mice had similar lytic efficiencies. However, aged mice demonstrated reduced plasma IL-15 levels relative to young mice. These results suggest that the NK cell residing environment is compromised in aged mice and, that decreased IL-15 in aged mice may be associated with decreased NK cell proliferation or survival, resulting in decreased NK cell number and an impaired total NK cell response to influenza infection. Supported by NIH AG029637-01.
Auto-antigenic stimulation potentiates NKT cells for subsequent innate IFN-gamma secretion (134.23)
Authors: Wang Xiaohua , Gumperz Jenny
Published: January 2023

NKT cells produce protective immune mediators at early stages of infections, preceding the responses of MHC-restricted T cells, and thus are thought a key bridge between innate and adaptive immune responses. Early IFNγ secretion by NKT cells during bacterial infections can result from autoantigenic activation with co-stimulation from IL-12&IL-18. However, the cellular processes underlying the rapid responsiveness of NKT cells remain poorly understood. Here we show that auto-antigenic stimulation of NKT cells potentiates them to secrete IFNγ in response to subsequent exposure to IL-12&IL-18. Rested NKT cells did not respond to IL-12&IL-18, however, NKT cells pre-exposed to CD1d+ APCs subsequently showed a transient ability to transcribe IFNγ mRNA and to secrete IFNγ protein in response to these cytokines without concurrent TCR stimulation. The potentiation effect was dependent on TCR signaling during pre-exposure. The subsequent cytokine response was p38 dependent and calcium signaling independent. The potentiation effect was transient, which distinguishes it from classical Th1 differentiation. The window of responsiveness was not due to regulation of cytokine receptor expression on NKT cells. Thus, we hypothesize that autoantigenic activation may induce epi-genetic changes in NKT cells that transiently confer an innate-like ability to respond to cytokines without concurrent TCR signaling.
Role of CCL2-producing PMN on the conversion of naive monocytes to M2bMonocytes (134.28)
Authors: Asai Akira , Shigematsu Kenji , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: January 2023

CCL1-producing monocytes with immunosuppressive properties (M2bMonocytes) have been demonstrated in peripheral blood of severely burned patients. Also, these burned patients are shown to be carriers of CCL2-producing PMN (PMN-II). Neither M2bMonocytes or PMN-II has been demonstrated in peripheral blood of healthy donors. In this study, the role of PMN-II on the conversion of naive monocytes to M2bMonocytes was investigated. PMN-II were induced from immuature PMN after stimulation with 0.0075% SAC and 10-6 M terubutaline. Immature PMN were obtained from peripheral blood of healthy volunteers 3 hrs after 1-hr of programmed exercise, as previously described. PMN-II (upper chamber) were transwell-cultured with naive monocytes (syngeneic to PMN-II, lower chamber) for 24 hrs. Cells harvested from the lower chamber were re-cultured for 24 hrs for the production of CCL1 (a biomarker of M2bMonocytes), and culture fluids obtained were assayed for CCL1 by ELISA. Naive monocytes did not produce CCL1 even when they were transwell-cultured with healthy donor PMN (normal PMN). However, CCL1 was detected in culture fluids of naive monocytes that were previously cultured with PMN-II. Anti-CCL2 mAb inhibited the appearance of M2bMonocytes in transwell-cultures between naive monocytes and PMN-II. Also, M2bMonocytes were generated from naive monocytes after they were cultured with recombinant CCL2. These results indicate that PMN-II, or CCL2 produced by PMN-II, play an important role on the generation of M2bMonocytes commonly demonstrated in burn patient peripheral blood. This study was supported by SHC NA #8840.
IL-17-induced apoptosis correlates positively with the release of myeloperoxidase in isolated neutrophils from mice (134.32)
Authors: Silverpil Elin , Glader Pernilla , Lindén Anders
Published: January 2023

BACKGROUND

Interleukin (IL)-17 can recruit neutrophils (PMNs) through indirect mechanisms and thereby contribute to pulmonary host defense. IL-17 may induce PMN apoptosis as well; and this through a direct mechanism (Silverpil et al, EAACI 2007). We now determined whether IL-17-induced PMN apoptosis is associated with increased bactericidal activity.

METHODS

Blood PMNs from Balb/c mice were isolated and cultured in vitro (48 hrs) in medium with mouse recombinant IL-17 protein (1-100 ng/mL) or in medium alone (control). Apoptosis and necrosis of the PMNs were measured on a flow cytometer targeting Annexin V and 7AAD, whereas the bactericidal activity was assessed by measuring extracellular myeloperoxidase (MPO) in the conditioned medium using ELISA. Data shown as (mean [SEM]), n=4-6.

RESULT

IL-17 increased PMN apoptosis (Annexin V+, 7AAD-) from 34 [1] % in the control group to a maximum of 55 [8] % (p< 0.05, Mann-Whitney U-test), but it did not affect PMN necrosis (Annexin V+, 7AAD+, not shown). The MPO concentration correlated positively with PMN apoptosis (p<0.01, Spearman's rank correlation).

CONCLUSION

This study demonstrates that IL-17-induced PMN apoptosis correlates with MPO release in vitro. This finding suggests that IL-17 is involved in terminating the innate component of pulmonary host defense in a controlled manner, while at the same time maintaining bactericidal activity.
Macrophages express a TCRαβ-based variable immunoreceptor (134.34)
Authors: Puellmann Kerstin , Beham Alexander W , Fuchs Tina , Kzhyshkowska Julia , Gratchev Alexei , Laird Rebecca , Wessels Johannes T , Neumaier Michael , Ganser Arnold , Kaminski Wolfgang E
Published: January 2023

Immunological dogma holds that variable immunoreceptors are restricted to lymphocytes. However, recent studies demonstrate that a subpopuation of human neutrophils expresses the TCRαβ (PNAS 103:14441, 2006) and mouse thymic granulocytes exhibit rearrangement of their TCR loci (Nature 452:764, 2008). Here, we provide evidence that a subpopulation of circulating human monocytes, monocyte-derived macrophages and tissue macrophages constitutively express a TCRαβ-based variable immunoreceptor.

TCRαβ + monocyte/macrophage subpopulations were assessed using with laser scanning cytometry and repertoire diversities were determined by CDR3 spectratyping. We find that human macrophages express individual-specific TCR Vαβ repertoires. Th1 or Th2 cytokine dependent in vitro differentiation triggers expression of distinct Vαβ repertoires in macrophages. Moreover, Th1 polarized macrophages show enhanced secretion of the major monocyte chemoattractant MCP-1 in response to costimulation with αCD3/CD28 .

The presence of a variable immunoreceptor in macrophages together with our previous identification of the TCR in a subset of neutrophils challenge the traditional dichotomy of the vertebrate immune system into non-specific/myeloid and antigen-specific/lymphoid immunity. They unravel a much closer kinship between both lineages than commonly assumed.

Supported by DFG grants KA 1078/4-1, BE 1768/5-1
Macrophage phagocytosis and lysozyme-based digestion of peptidoglycan are required for IL-1β production in response to S. aureus (134.38)
Authors: Underhill David M. , Shimada Takahiro , Park Bong Goo , Goodridge Helen S. , Wolf Andrea J. , Becker Courtney , Reyes Christopher , Götz Friedrich , Liu George Y.
Published: January 2023

IL-1β produced by phagocytes is important for protection against Staphylococcus aureus, a bacterium that causes serious skin and invasive diseases and is contributing to a current public health crisis due to the emergence of methicillin-resistant strains. Secretion of IL-1β requires both activation of a transcriptional signal to stimulate production of pro- IL-1β, (usually provided by TLR signaling) and a second signal to stimulate processing by inflammasome complexes and release of the mature cytokine. We show here that phagocytosis and lysozyme-based degradation of bacterial cell walls are functionally coupled to activation of Nalp3 inflammasomes and secretion of IL-1β in response to live S. aureus and to S. aureus peptidoglycan. Further, a S. aureus peptidoglycan-modifying enzyme that makes its peptidoglycan resistant to lysozyme strongly suppresses inflammasome activation. This is the first demonstration of a case whereby a bacterium avoids IL-1β secretion through chemical modification of its cell wall peptidoglycan, and we hypothesize that this reduced IL-1β production is key to successful mucosal colonization and infection.

This research is supported by grants from NIGMS and the American Heart Association.
Regulation of Murine Splenic B Cell Complement Receptor 3 Expression by Complement Component C3 (134.57)
Authors: Jacobson Amanda  C , Weis Janis J , Weis John H
Published: January 2023

Complement component C3 has established roles in both innate and adaptive immune responses. C3 cleavage products function in B cell activation through the complement receptors CD21/35. Phenotypes involving antibody production between CD21/35-/- and C3-/- mice are not always congruent, implying additional roles for C3 in B cell responses. To further characterize complement and complement receptors, we have identified a role for C3 in the regulation of CR3 on splenic B cells. Splenic B2 cells are not defined as expressing CR3 (as opposed to peritoneal cavity B1 B cells) yet the analysis of splenic B cells from C3KO animals demonstrated cell surface expression of CR3. B cells from WT and C3KO animals both express the Cr3 gene indicating that the lack of CR3 on WT B cells is not due to a deficiency of the transcript/protein. C3KO and WT animals have similar B cell sub-populations as well as identical CR3 expression on macrophages and neutrophils. The C3-deficient environment was responsible for altered CR3 expression as WT splenic B cells transferred into C3KO animals expressed cell surface CR3 within 48hrs while transfer of C3KO splenic B cells into WT animals extinguishes surface expression of CR3. Furthermore, transfer of C3-producing splenic macrophages into C3KO mice depressed CR3 expression by resident B cells. These data suggest a rapid turnover of CR3 on splenic B cells due to C3-bound complexes and the loss of CR3 on such cells may alter the function or localization within the splenic architecture.

NIH-AI-2458 to JHW and NIH-AI-32223 to JJW
Inactivation of the MiRNA-processing enzyme Dicer impairs NK cell responses in vitro and in vivo (134.6)
Authors: Bezman Natalie , McManus Michael T , Lanier Lewis L
Published: January 2023

Natural killer (NK) cells are specialized lymphocytes of the innate immune system that can recognize and eliminate transformed and virus-infected cells. Although the transcriptional and signaling networks that modulate activation of NK cells have been intensely studied, less is known about the post-transcriptional mechanisms that regulate NK cell function. MicroRNAs (miRNAs) have recently emerged as critical regulators of the differentiation and function of many cell types. We hypothesize that miRNA-mediated gene regulation is critical for the activation and function of NK cells. To this end, we generated a mouse strain with tamoxifen-inducible disruption of Dicer, the endonuclease responsible for miRNA processing. Tamoxifen administration efficiently induced Cre-mediated recombination of Dicer in various lymphocyte lineages, including NK cells. Our studies suggest that lack of miRNAs following Dicer inactivation leads to reduction in the number of mature NK cells in spleen and liver. Freshly isolated Dicer-deficient NK cells exhibited reduced proliferation in response to IL-2 and showed impaired cytokine production after stimulation of the activating receptors NK1.1, Ly49H, and Ly49D. Ongoing studies investigate whether miRNAs are necessary for NK cell activation and expansion to MCMV infection in vivo.
Peripheral blood monocytes express surface C1q and its orientation may dictate early events of monocyte to dendritic cell differentiation (134.62)
Authors: Hosszu Kinga K , Santiago-Schwarz Frances , Peerschke Ellinor I.B. , Ghebrehiwet Berhane
Published: January 2023

C1q is a collagen-like glycoprotein comprising two functionally distinct regions–collagen tail (cC1q) and globular heads (gC1q). Our earlier experiments showed that C1q regulates monocyte to DC differentiation by suppressing DC development at the earliest stages. This suggested that C1q may be expressed on monocytes, and through interactions with C1qRs, could regulate early signals that lead to differentiation. Using ELISA and flow cytometric analysis, we confirmed the surface expression of C1q on fresh monocytes, and the level could be sustained beyond day 3 when monocytes were cultured with a mono-macrophage specific growth factor (M-CSF). In the presence of DC growth factors (GM-CSF+IL-4) however, surface expression rapidly declined coinciding with firm commitment to the DC lineage on day 3. Anti-gC1q IgG bound only to macrophages but not to monocytes or immature DCs, suggesting that C1q is bound to monocytes via gC1q, whereas on macrophages it is anchored via cC1q. Thus the orientation of surface C1q may dictate regulation of the early processes that signal progression from the monocyte lineage (innate) towards the DC lineage (adaptive immunity). More importantly, since M-CSF is a normal constituent in the blood, the expression of C1q on fresh monocytes may reflect an M-CSF/C1q coupled regulatory process operating to maintain steady state. [Supported by NIH Grant R01 AI-060866 (to BG) and R03 AR-05396 (to FSS)]
HCV Core/gC1qR Interaction Regulates LPS-Mediated MAPK Activation (134.66)
Authors: Tacke Robert S , Waggoner Stephen N , Tosello-Trampont Annie , Hahn Young S
Published: January 2023

Hepatitis C Virus (HCV) is a dangerous human pathogen that is able to establish chronic infection in its host. Studies of the immune response to the virus reveal a weak and severely delayed T and B cell response. HCV core protein is the first viral protein made following infection within hepatocytes. In addition to forming the viral capsid, HCV core protein is secreted from infected hepatocytes and is present at high levels in the peripheral blood of infected individuals. Previous data from our laboratory has shown that extracellular HCV core protein binds complement receptor gC1qR. This interaction results in the suppression of LPS-mediated IL-12 production in a PI3K dependant manner in human monocytes. Here we show that signaling through the gC1qR results in the suppression of LPS-mediated MAPK activation in a PI3K-dependent fashion. Further, we show that gC1qR-mediated signaling activates AKT, and that AKT is able to phosphorylate GSK-3β and MAPKKK family protein ASK1 at serines 9 and 83 respectively. Phosphorylation at these sites results in reduced activity of both kinases. Interestingly, both GSK-3β and ASK1 are important for LPS-mediated IL-12 production. These results suggest that HCV core protein binds gC1qR on the surface of human monocytes resulting in the activation of the PI3K/AKT pathway. AKT then inhibits LPS-mediated GSK-3β and ASK1 activation resulting in impaired MAPK activation and IL-12 production. NIH Grant RO1AI057591 supported this research.
C5b-9 inhibits myelin gene expression in oligodendrocyte progenitor cells (134.68)
Authors: Tegla Cosmin A , Ito Takahiro , Cudrici Cornelia , Rus Horea
Published: January 2023

Active demyelinating multiple sclerosis (MS) lesions can be subdivided into four neuropathological patterns, based on the presence of immunoglobulins and C5b-9 or evidence of primary oligodendrocyte pathology. Following inflammatory demyelination only partial remyelination occurs and is mediated by oligodendrocyte precursor cells (OPCs). No systematic studies were performed to localize the OPCs in relation to the C5b-9 deposits in MS brains. OPCs we identified by immunohistochemistry using antibodies against NG2, an integral membrane chondroitin sulfate proteoglycan. In addition we examined the deposition of C5b-9 and the presence of inflammatory cells. We detected NG2+ cells in both perivascular and parenchymal areas of MS plaques and normal adjacent white and gray matter areas. A subpopulation of NG2-positive cells also presented C5b-9 deposits and suggests that the terminal complement complex might be involved in OPCs differentiation. To test this hypothesis we investigated in vitro the effect of sublytic C5b-9 on OPC differentiation and expression of myelin genes. Sublytic C5b-9 significantly reduced the accumulation of mRNA encoding proteolipid protein and myelin basic protein. These data are the first to implicate the C5b-9 in the limited remyelination seen in MS lesions. Thus, C5b-9 assembly may represent a potential target for therapeutic intervention in MS.
Impact of NADPH Oxidase Inhibition on Influenza A Virus-induced Inflammation (134.80)
Authors: Patel Jenish R. , Christoph Bradley T. , Hussain Sakina F. , Vora Keyur P. , Ranjan Priya , Sambhara Suryaprakash , Gangappa Shivaprakash
Published: January 2023

Activation of innate immunity pathways in susceptible cell types is critical for host defense against influenza-A viruses (IAV). Based on studies signifying a role for NOX1 in inflammation, we hypothesized that IAV of seasonal and pandemic potential may induce different isoforms of NOX enzymes in the respiratory tract, and that attenuation of virus strain-specific NOX enzymes can be exploited to prevent and/or treat severity of disease. Using three respiratory tract-relevant cell lines (epithelial/A549, endothelial/HULEC, and monoctytic/THP1) and three strains of IAV (H1N1/PR8, H3N2/X31, H1N1/WSN), we found significant upregulation of NOX1 in all three cell lines (e.g.-A549: PR8/55 fold; X31/35 fold; WSN/12,250 fold). Furthermore, in contrast to the epithelial cell line showing NOX1 expression by 4h with peak levels at 16h, the monocytic cell line showed delayed (8h) and sustained (48h) levels of NOX1. Interestingly, inhibition of NOX-isoforms in IAV-infected epithelial cells by diphenyleneiodonium (DPI), at a concentration (25μM), which did not impact cell death, showed significant decrease (75%) in virus triggered NOX1 levels. In addition, NOX-inhibition led to significant decrease in virus-induced chemokines (MCP1, MIP3, MIP1β, RANTES, and IP10) and proinflammatory cytokines (IL1α, IL6, IL8, and TNFα). Furthermore, decline in NOX1 correlated with a modest increase in viral NS1. Taken together, in addition to showing definitive evidence for upregulation of NOX1 in response to subtypes of IAV, our results strongly support a role for NOX1 in IAV-driven inflammation.