bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a novel antigen presenting cell population modulating anti-influenza type-2 immunity (131.3)
Authors: Yoo Jae-Kwang , Galligan Carole , Burke Daniel , Virtanen Carl , Fish Eleanor N
Published: January 2023

Anti-influenza type 2 (T2) immunity contributes to both immunoprotection and immunopathology, yet the underlying mechanisms modulating T2 immunity remain ill-defined. Here, we describe a novel murine antigen (Ag) presenting cell (APC) population, designated virus activated plasmacytoid (VAP) cells: mPDCA1+CD11c-TcR-β-B220-CD19-CD38+CD44intCD45+Gr1+. VAP cells are bone marrow derived leukocytes. Phenotypic, morphologic and molecular characterization distinguished VAP cells from other immune cell populations. Pulmonary influenza A virus infection activates circulating VAP cells to enter the lungs and produce interleukin (IL)-25, a strong inducer of T2 pulmonary inflammation. Subsequently, VAP cells capture viral Ag in the lungs and migrate to the draining lymph node (DLN) and spleen, with delayed kinetics relative to DCs, where they show APC activity. In the DLN, influenza virus activated VAP cells produce IL-4 and induce T helper (TH) 2 effector cell polarization by modulating GATA-3 expression. Finally, adoptive transfer of influenza virus activated VAP cells enhanced TH2 effector T cell responses in influenza virus infected recipient mice and subsequently, anti-influenza T2 humoral responses including IgG1 and G2b were significantly increased. Collectively, our results highlight the importance of VAP cells as novel immuno-modulators of T2 immunity against influenza A virus infection in mouse.

These studies were supported by a Canadian Institutes of Health Research grant MOP-15094 to E.N.F.

J.K.Y. was supported by a Connaught Scholarship and an Ontario Graduate Scholarship in Science and Technology.
The Pseudomonas aeruginosa toxin Cif reduces MHC class I antigen presentation and viral immunity (131.4)
Authors: Bomberger Jennifer , Ely Kenneth , Bangia Naveen , O'Toole George , Green William , Enelow Richard , Stanton Bruce
Published: January 2023

Pseudomonas aeruginosa is an opportunistic pathogen critical to the pathogenesis of nosocomial infections, chronic obstructive pulmonary disease, ventilator-associated pneumonia, bronchiectasis and Cystic Fibrosis. Patients chronically infected with P. aeruginosa often experience debilitating viral exacerbations, the cause of which is unknown. Previously, we demonstrated that Cif, a bacterial toxin secreted by P. aeruginosa, promotes the degradation of several ABC transporters involved in innate immunity and the drug bioavailability, including CFTR and Pgp. The current study was conducted to test the hypothesis that Cif enhances the degradation of the transporter associated with antigen processing (TAP) in airway epithelial cells, resulting in reduced class I MHC antigen presentation and therefore, diminished immune responses to viral pathogens. Cif inhibited the deubiquitinating enzyme, ubiquitin specific protease 10, resulting in a time-dependent increase in the polyubiquitination and proteosomal degradation of TAP1. The Cif-mediated decrease in TAP1 expression resulted in a decrease in class I MHC expression at the plasma membrane. Furthermore, Cif decreased MHC class I antigen presentation and cytotoxic T lymphocyte (CTL) recognition of influenza virus antigen, as measured by TNF-α secretion, in a time-dependent manner. These observations suggest that P. aeruginosa, by secreting Cif, inhibits the ability of CTL to eliminate viral infections, severely compromising the immune defenses of the lung.
Human plasmacytoid dendritic cells preferentially conjugate with and fuse with acute or chronically HIV-IIIB infected cells. (131.7)
Authors: Jacobs Evan S , George Thaddeus C , Singh Sukhwinder , Wnek Richard , Fischer Paul , Nordone Shila K , Dean Gregg A , Bocarsly Patricia Fitzgerald
Published: January 2023

Plasmacytoid Dendritic Cells (PDC) produce copious amounts of IFN-a in response to TLR-7 or -9 stimulation and serve as a link between the innate and adaptive immune systems. In HIV infection, there is a gradual depletion of PDC as well as a functional deficiency in remaining circulating PDC. The mechanism of PDC depletion is not yet understood. We have demonstrated using imaging flow cytometry (ImageStream®) that PDC preferentially take up membrane and cytoplasm from HSV and Influenza infected cells in an endocytic process called "nibbling" that results in IFN-α production by the PDC. Using acutely HIVIIIB infected CD4 T cells or chronically HIVIIIB infected H9 T cells, PDC preferentially form conjugates with HIVIIIB infected vs uninfected T cells, but, rather than being nibbled, the majority of these encounters lead to PDC/infected T cell fusions. Acutely infected CD4 T cells but not chronically-infected H9 cells stimulated PDC to produce IFN-α, albeit it lower levels than other TLR7/9 inducers. Fusions were blocked with the addition of the specific fusion inhibitors (T-20 or AMD-3100), but there was no increase in conjugate formation or uptake of material. We hypothesize that this successful subversion of PDC nibbling and IFN-α production by HIV-infected cells contributes to loss of PDC and the deficient IFN-α production seen in HIV-infected patients. Supported by AI26806.
[alpha]-GalCer analogues with branched acyl chain enhance humoral and cellular immune responses in a nasal influenza vaccine (132.11)
Authors: Lee Yoon-Sook , Lee Kyoo-A , Lee Jae-Young , Kang Min-Hee , Kim Sang-Hee , Kang Chang-Yuil
Published: January 2023

α-galactosylceramide (α-GalCer) can act as a safe and effective adjuvant for a nasal vaccine that induces protective immune responses against tumors and viral infections. In this study, α-GalCer analogues were designed to have branched acyl chain by modification of their fatty acyl chain based on the CD1d/glycolipid structures. Two α-GalCer analogues with various branched chain lengths; LDH-II-28 and LDH-II-30 were prepared and evaluated for their efficacy as a nasal influenza vaccine adjuvant. These analogues displayed improved solubility over α-GalCer and effectively stained NKT cells both in mouse and human. They also potently stimulated NKT cells to exhibit different cytokine release profiles from α-GalCer in vitro and in vivo. When profiling serum cytokines in vivo, LDH-II-30 provoked both Th1/Th2 cytokines, while LDH-II-28 induced more Th2-biased cytokine release with diminished IFN-γ production. We found that single immunization of inactivated influenza virus A/PR/8/34 (PR8) together with α-GalCer analogues enhanced PR8-specific humoral and cellular immune responses in both systemic and mucosal compartments. Notably, LDH-II-30 exhibited potent adjuvant effects with significantly higher systemic IgG, mucosal sIgA Ab titers and enhanced CTL generation compared to immunization with inactivated PR8 alone, while addition of LDH-II-28 to inactivated PR8 only marginally increased PR8-specific immune responses. These results suggest that α-GalCer analogues with branched acyl chain could be used as an effective mucosal adjuvant for the induction of influenza vaccine-specific humoral and cellular immune responses.
Characterization of the anti-lethal factor (LF) antibody response in Anthrax Vaccine Adsorbed (AVA) individuals (132.15)
Authors: Crowe Sherry R , Engler Renata J M , Ballard Jimmy D , Harley John B , Farris A Darise , James Judith A
Published: January 2023

The currently licensed anthrax vaccine (AVA) has been designed to elicit primarily an anti-protective antigen response. However, animal studies have clearly demonstrated that antibodies to the lethal factor component of the toxin can provide protection against challenge. This study was designed to identify AVA vaccinated individuals that generate anti-LF responses and to determine if these anti-LF antibodies can mediate protection. Plasma was obtained from one thousand vaccinated individuals and thirty unvaccinated controls. Only 69 individuals (6.9%) had significant levels of anti-LF antibodies. The presence of these antibodies did not correlate with in vitro protection against lethal toxin. To determine if antibodies against specific regions of LF could mediate protection, we used overlapping decapeptides to identify six common antigenic regions within those individuals with high levels of anti-LF antibodies and high toxin neutralizing activity. Epitope-specific antibodies directed against three of these common antigenic regions were isolated and select antibodies were able to protect in both in vitro tissue culture and in vivo mouse toxin challenge models. Findings from these studies have important implications for vaccine design and immunotherapeutic development. NIAID: 5U19AI062629
Memory B cells are required to maintain long-lived plasma cells in mice following viral infections (132.16)
Authors: Langley William A , Mueller Scott N , Ahmed Rafi
Published: January 2023

Antibody responses have been found to last for life following lymphocytic choriomeningitis virus (LCMV) infection in mice and have been estimated to have a half-life of up to 200 years for some pathogens in humans. These antibodies are produced by long-lived plasma cells which have been shown to have a half-life of at least 138 days in mice. However, the role of memory B cells in the maintenance of plasma cell numbers remains unclear. We have been attempting to define this role by infecting hCD20 transgenic mice with viral pathogens, treating them with rituximab (an anti-CD20 antibody), and then following the antibody levels in these mice. Rituximab treatment of hCD20 mice selectively depletes naive and memory B cells while leaving plasma cells untouched. During long-term treatment of LCMV immune mice, there is a slow, steady decline of antibody levels and a decrease in the number of LCMV-specific antibody secreting cells in the spleen and bone marrow. This decrease in antibody levels was also observed with influenza and vesicular stomatitis virus (VSV) infected mice and correlates with decreased hemagglutination inhibition titers in influenza infections and decreased neutralization titers in VSV infections. These results suggest a critical role for memory B cells in the maintenance of long-lived plasma cells following viral infections.

This work was supported by NIH grant AI30048.
Efficacy of HIV-1 and FIV subunit vaccines against FIV (132.3)
Authors: Coleman James K , Pu Ruiyu , Yamamoto Janet K
Published: January 2023

Defining the minimum essential vaccine components required for protective immunity to HIV and FIV remains a major challenge. A recent study reported that recombinant HIV-1 capsid p24 vaccines conferred cross-protection against FIV in cats. Current studies were performed to confirm this protection and identify other protective HIV/FIV proteins. Pathogen-free cats were immunized 3X with HIV p24 (CA), HIV p66 (reverse transcriptase RT), FIV p24, FIV p66, FIV p15 (matrix MA), or combination in adjuvant (plus LPS/IL-12), and were challenged 3 wk later with FIV. Infection was monitored by immunoblot for FIV antibodies, and by FIV RT and PCR tests of lymphoid cells. Cellular immunity was monitored by virus-specific IFN-γ ELISPOT and CFSE-based proliferation of T cells. Protection rates were 2/8 cats for HIV p24, 2/8 HIV p24+p66, 3/7 HIV p66, 1/8 FIV p24, 0/3 FIV p15, 2/7 FIV p24+p66, 5/7 FIV p66, 2/4 HIV p24+FIV p66, and 0/8 adjuvant or PBS. Vaccinated cats had strong IFN-γ and CD4/CD8-CFSE responses to HIV or FIV peptides, but developed no virus neutralizing antibodies. Combined protection rates for current and previous studies are 40/64 cats for HIV p24, 4/25 FIV p24, 5/7 FIV p66, 3/7 HIV p66, 0/3 FIV p15, 0/22 adjuvant, and 0/37 PBS. Thus, cross-protective immunity between HIV-1 and FIV is achievable, and selection of vaccine proteins and epitopes based on structural homology that risk viral fitness is an effective strategy.
Severely burned patient peripheral linage– CD34+ cells are inhibitory on the antimicrobial peptide production by human epidermal keratinocytes (133.10)
Authors: Kobayashi Makiko , Jeschke Marc G , Benjamin Debra , Yoshida Shohei , Kawasaki Takashi , Kotani Takuya , Herndon David N , Suzuki Fujio
Published: January 2023

The importance of epidermal keratinocyte-produced antimicrobial peptides in the control of skin infections has been described. However, these peptides are not produced sufficiently in tissues surrounding the burn area. In this study, linage- CD34+ cells that infiltrated into burn area tissues were shown to be inhibitory on the antimicrobial peptide production by human epidermal keratinocytes (NHEK). Peripheral blood mononuclear cells (PBMC) taken from 5 severely burned patients (3rd degree, more than 40% total body surface area burn) enrolled in these experiments. NHEK (1 x 105 cells/ml, lower chamber) were cultured with 1 x 105 cells/ml of healthy donor PBMC or patient PBMC in a dual-chamber transwell. Forty-eight hrs after cultivation, cells in the lower chamber were assayed for human β-defensin-1 (HBD-1) and cathelicidin (CAP-18) mRNAs by RT-PCR. Without any stimulation, mRNAs for HBD-1 and CAP-18 were consistently expressed by NHEK. mRNA expression for HBD-1 and CAP-18 by NHEK was not influenced when peptide producer cells were transwell-cultured with normal donor PBMC. However, the expression of HBD-1 and CAP-18 mRNAs by NHEK was greatly suppressed when they were transwell-cultured with patient PBMC. Suppressor cells in patient PBMC for the peptide production by NHEK were determined to be linage- CD34+ cells. Linage- CD34+ cells isolated from healthy donor PBMC did not have any suppressor cell activities. These results indicate that linage- CD34+ cells isolated from severely burned patient PBMC are suppressor cells on the antimicrobial peptide production by epidermal keratinocytes. This study was supported by SHC NA #8610.
Commensal microflora induce host defense to E.coli pneumonia through toll-like receptors (133.13)
Authors: Chen Lee-Wei , Hsu Ching-Mei
Published: January 2023

The role of commensal microflora in host response to pneumonia has not been defined. This study is to investigate whether commensal microflora in gut increase the host defense against pneumonia through TLRs signaling.We treated C3H/HeN and C3H/HeJ mice with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in intestinal tract. E.coli was injected to trachea to induce E.coli pneumonia.

Commensal depletion induced a 15-fold and a 2.5-fold increase in E.coli pneumonia-induced bacterial counts in lung and blood, respectively and a 30% decrease of lung MPO activity. Commensal depletion induced a suppression of TLR4, TNFα&, KC, and CXCR2 expression; and NF-κB activity in intestine and LPS supplement reversed them. LPS supplement increased lung MPO activity and decreased E.coli pneumonia-induced bacterial counts in WT mice but not in C3H/HeJ mice. Commensal depletion induced a decrease of phagocytic activity of alveolar macrophages, reduced NF-κB activity, and increased KC, MIP-2, and IL-1β mRNA expression of lung in WT mice but not in C3H/HeJ mice. In conclusions, commensal microflora are critical in inducing TLR4 expression and NF-κB activation of intestine, increasing the lung immunity, and decreasing the inflammatory signaling in lung after E.coli pneumonia. Given that the stimulatory effect of LPS supplement on lung immunity is abolished in TLR4-deficient mice, we conclude that both commensal microflora and TLR4 ligand increase host defense against pneumonia through toll-like receptors.
Innate immunity to tuberculosis: a forward genetic approach (133.15)
Authors: Tobin David M. , Ray John P. , Moens Cecilia , Ramakrishnan Lalita
Published: January 2023

We have developed a forward genetic screen in the zebrafish to identify host genes that control resistance and susceptibility to tuberculosis. Using facile live imaging techniques to screen mutant larvae for alterations in the pathogenesis of Mycobacterium marinum, we have identified multiple genetic loci that alter myeloid development and functions, including macrophage microbicidal capacity, migration and aggregation. Through a positional cloning approach, we have identified genes affected by three mutations causing hypersusceptibility to M. marinum. One of these mutations lies in the gene encoding leukotriene A4 hydrolase (LTA4H), the biosynthetic enzyme that produces leukotriene B4 (LTB4). In spite of LTB4's role as a potent leukocyte chemoattractant, the mutant is not compromised in the initial migration of macrophages to the site of infection. Rather mutant macrophages are defective in limiting intracellular bacterial growth and are quickly killed by the infection. Chemical inhibitors of this pathway mimic the mutant phenotype. Finally, we show that the protective effect of LTA4H in limiting mycobacterial growth may be attributable to a role in the regulation of TNF, as mutant fish fail to induce TNF transcription in response to infection.
Interleukin-12 is dispensable for functional interferon (IFN)-gamma producing CD8+ cytotoxic T cells in Toxoplasma gondii infection (133.28)
Authors: Daniel Benjamin J , Liu AiJie , Pandeswara Srilakshmi , Ludwig Sara M , Brumlik Michael J , Church Kristina M , Kious Mark J , Curiel Tyler J
Published: January 2023

Interleukin (IL)-12 is a proinflammatory cytokine generally regarded as a master regulator of type 1 T cell mediated immunity against intracellular pathogens, including T. gondii and cancers. A critical measure of IL-12 activity is its ability to induce IFN-γ production by NK and T cells. Using a transgenic T. gondii expressing ovalbumin (OVA), we show here that by flow cytometry analysis T. gondii-OVA infected BL6 IL-12-/- mice generate OVA-pentamer+CD8+ T cells similar to WT and surprisingly, T. gondii-infected IL-12-/- mice produce more IFN-γ +CD8+ T cells than T. gondii-infected WT mice (avg 9.6% in IL-12-/- vs 3.7% in WT). We confirmed that function of OVA-specific CD8+ T cells in IL-12-/- was comparable to BL6 WT using an in vivo OVA+ target lysis assay. As expected, IFN-γ +CD4+ T cells decreased from an avg 29% to 9% in WT mice compared to IL-12-/- mice respectively when infected with T. gondii. These results demonstrate a novel IL-12-independent pathway to CD8+ CTL that may be less dependent of CD4+ T cell help than previously thought.
HSV-2 infection induces TLRs and NF-κB-dependent cytokines in human cervical epithelial cells (133.41)
Authors: Li Hui , Li Xiaoling , Wei Yun , Tan Yun , Wu Xinxing
Published: January 2023

Herpes Simplex Virus type 2 (HSV-2) is one of the most common sexually transmitted pathogen worldwide. The host immune response induced by viral infection is cell-type specific. Little is known about the innate immune response to this virus in its natural host cells. In this study, we established an in vitro HSV-2 infection model with human cervical epithelial (HCE) cells. The viral infection was sufficient to induce expression of Toll-like receptors (TLRs), and western blot and reporter assays suggest that HSV-2 infection leads to dramatic activation of the NF-κB signaling pathway. The two critical cytokines,IL-6 and IFN-beta,were also induced after HSV-2 infection and maintained high levels during the course of experiment. The presence of NF-κB inhibitor, Isohelenin significantly blocked the production of both IL-6 and IFN-beta in response to HSV-2. Thus, our data provide direct evidence that the activation of NF-κB is required for the production of both IL-6 and IFN-beta induced by HSV-2 in HCE cells. Taken together, our results suggest the potential contributions of TLRs and a critical role of NF-κB in the innate immune response to HSV-2 infection in its natural host cells.

This work was supported by grants from the National Natural Science Foundation of China (No. 30500465,30810103052)
Functional Modulation of Dendritic Cells and Macrophages by Japanese Encephalitis Virus through MyD88 Adaptor Molecule-Dependent and Independent Pathways (133.42)
Authors: Eo Seong Kug , Han Young Woo , Rahman M. M. , Kim Seon Ju , Kim Seong Bum
Published: January 2023

Dendritic cells (DCs) are potent initiator of T cell-mediated immunity and undergo maturation during viral infections. However, few reports describing the interactions of DCs with Japanese encephalitis virus (JEV), which remains the most cause of acute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macrophages. JEV replicated its viral RNA in both cells with different efficiency, and we found that JEV infection of macrophages followed classical activation pathway of upregulation of costimulatory molecules and proinflammatory cytokine production (IL-6, TNF-¥á, and IL-12). On the contrary, JEV-infected DCs failed to upregulate costimulatory molecules (CD40, CD80, CD86, and MHC class II). Of more interest, along with production of proinflammatory cytokines, DCs infected by JEV released anti-inflammatory cytokine IL-10, which was not detected in macrophages. Moreover, signaling through MyD88 molecule, a pan-adaptor molecule of Toll-like receptors (TLRs), and p38 MAPK in JEV-infected DCs was found to have roles in production of cytokines and subversion of primary CD4+ and CD8+ T cell responses. We also found that IL-10 released from JEV-infected DCs mediated the reduced priming of CD8+ T cells but not CD4+ T cells. Our data suggest that JEV induce functional impairment of DCs through MyD88-dependent and independent pathways, which subsequently lead to poor CD4+ and CD8+ T cell responses, resulting in boosting viral survival and dissemination in the body.
Vesicular stomatitis virus infects primary murine microglia and astocytes and induces replication-dependent inflammatory cytokine production (133.47)
Authors: Marriott Ian , Chauhan Vinita S , Sterka David G , Grdzelishvili Valery Z
Published: January 2023

Vesicular stomatitis virus (VSV) is a rhabdovirus that closely resembles its deadly cousin, rabies virus. Intranasal inoculation of mice with VSV leads to infection of the central nervous system (CNS) and an acute encephalitis that results in a high rate of mortality similar to that observed during human rabies infections. Importantly, this encephalitis is accompanied by the rapid activation and proliferation of resident brain cells including microglia and astrocytes, suggesting a role for such cells in the initiation and/or progression of CNS inflammation. Our recent study showed that these glial cells express RIG-I and MDA5, cytosolic receptors for replicating RNA virus motifs. However, it is presently unclear whether microglia and astrocytes can harbor replicating VSV, or if such replication is required for the inflammatory immune responses of these resident CNS cells. In this study we demonstrate that primary murine microglia and astrocytes are permissive for VSV infection and replication. Importantly, active replication appears to be required for robust inflammatory mediator production as such responses were markedly lower in cells exposed to either inactivated or replication defective viral particles. Interestingly, this effect is not limited to neurotropic Mononegaviruses as the paramyxovirus, Sendai virus, can similarly infect glial cells and elicit inflammatory immune responses. Finally, we have confirmed that in vivo intranasal VSV administration results in viral infection and replication within mouse glial cells in situ. This study suggests that viral replication within resident glial cells could play a key role in the development of lethal CNS inflammation following neurotropic Mononegavirus infection.
NADPH oxidase-derived ROS mediates mast cell degranulation induced by secretory products secreted by Trichomonas vaginalis (133.5)
Authors: Min Deulle , Shin Myeong Heon
Published: January 2023

Trichomonas vaginalis is known to be a sexually transmitted protozoan parasite causing vaginitis in women. During infection in vivo, secretory products by T. vaginalis can disrupt the epithelial junctions so that these products gain access to, and alter the function of, underlying cells of the innate immune system. Mast cells are known to play a key role in mucosal immune responses. In this study, we investigated whether NADPH oxidase-derived reactive oxygen species (ROS) are closely associated with mast cell degranulation by Trichomonas-secreted products. When Trichomonas-secreted products were added to human mast cells (HMC-1 cells) in a transwell system (pore size 0.2 um), it caused extensive degranulation of mast cells, which was confirmed by ultrastructural change, increased expression of CD63 at the cell surface, and liberation of granular proteins such as histamine. In addition, mast cells were found to harbor different isoforms of NADPH oxidase. Stimulation with Trichomonas-secreted products induced intracellular accumulation of ROS in mast cells. Next, we investigated whether NADPH oxidase-derived ROS are one of the main signaling molecules responsible for Trichomonas-triggered mast cell degranulation. Pretreatment of cells with NADPH oxidase inhibitors such as DPI or apocyanin strongly inhibited ROS generation and mast cell degranulation induced by Trichomonas-secreted products. These results suggest that secretory products by Trichomonas vaginalis contain signaling molecules to trigger activation of NADPH oxidase in mast cells, which triggers degranulation leading to initiating and regulating mucosal inflammation.
HCV core/gC1q Receptor ligation transduce a signal across the plasma membrane possibly by beta1 integrin complex. (133.51)
Authors: Tosello-Trampont Annie , Tacke Robert , Hahn Young S.
Published: January 2023

Hepatitis C virus (HCV) infection in humans is associated with viral persistence, which leads to the development of liver diseases. HCV core protein has been reported to be detectable in bloodstream of HCV-infected patients, and contains the immunomodulatory function. We have previously demonstrated that the interaction of HCV core with the complement receptor gC1qR inhibits the inflammatory response, particularly the TLR4-mediated production of IL-12 in monocytes. The inhibition of IL-12 production depends on the activation of the PI-3 kinase pathway. However, it is still unknown how this receptor anchors at the cell surface to transduce intracellular signaling since it has no transmembrane or GPI domain. To determine the protein(s) involved in the gC1qR complex responsible for anchoring gC1qR, we examined the association of gC1qR with β1 integrin (a putative transmembrane partner). Here, we show by performing GST-gC1qR pull down assays on human cell lysats, that β1 integrin specifically interacts with gC1qR. Moreover, we examine the cell surface localization of gC1qR and β1 integrin by immunofluorescence microscopy after THP-1stimulation by HCV core. Our data demonstrate that HCV core enhance the formation of gC1qR/β1 integrin complexes on HCV core-treated monocytes. These results suggest that β1 integrin may play the role of anchoring protein to gC1qR on the cell surface.

NIH grant RO1AI057591
T cell receptors mediate docking of cytotoxic granules at the immunological synapse (133.7)
Authors: Qu Bin , Pattu Varsha , Junker Christian , Becherer Ute , Matti Ulf , Rieger Heiko , Schwarz Eva C , Rettig Jens
Published: January 2023

TCR enrichment and cytotoxic granules accumulation at the immunological synapse are necessary for cytotoxic T lymphocyte-mediated killing. Using immunocytochemistry, confocal microscopy, 4D fast live cell imaging and total internal reflection fluorescence microscopy, we analyzed the localization and transport of recycling TCR vesicles and cytotoxic granules in parallel. We found that recycling TCR vesicles are very often connected with cytotoxic granules. Both are co-transported in a paired manner to the IS where recycling TCR enhance the docking and release of cytotoxic granules. Quantitative analysis of vesicluar transport revealed that pairing enables CTL to kill within a few minutes after synapse formation. These data not only highlight an important function of recycling TCR, but also show that pairing of different vesicles is an unexpected mechanism to target vesicles to a defined area within CTL.

This project was funded by the Deutsche Forschungsgemeinschaft (Graduate Research School 1326, DFG grant RE 1092/6-1 to J.R and M.H.) and a HOMFOR grant from the Medical Faculty (to J.R. and M.H).
CD57 defines a functionally unique subset of NK cells in humans (134.1)
Authors: Lopez-Verges Sandra , Milush Jeffrey M , Pandey Suchitra , Norris Philip , Nixon Douglas , Lanier Lewis L
Published: January 2023

CD57 is a marker of terminal differentiation on human CD8+ T cells and possibly on human NK cells. Newborn and fetal NK cells do not express CD57, however the frequency of CD57-bearing NK cells increases with age. Utilizing PBMC obtained from healthy donors we are assessing the phenotypic and functional differences between CD57+ and CD57neg NK cells. CD57+ NK cells express different activation markers and a distinct repertoire of NK receptors suggestive of a more mature phenotype. Preliminary data also indicate that there is a higher frequency of IFNγ CD57+ NK cells compare to CD57neg NK cells when stimulated through their activating receptors, CD57+ NK cells are less sensitive to activation-induced apoptosis, and interestingly, proliferate less when co-cultured with target cells and/or cytokines. The combination of a more activated phenotype and a decreased capacity to proliferate suggests CD57+ NK cells are terminally differentiated. Studies are underway to assess the phenotypic and functional differences of CD57+ and CD57neg NK cells during infection with HIV and other viruses.

Funding: NIH. SLV is a Cancer Research Institute/Irvington Institute Fellow. LLL is an American Cancer Society Professor.
Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse (134.13)
Authors: Sanborn Keri B , Rak Gregory D , Maru Saumya Y , Difeo Analisa , Martignetti John A , Favier Remi , Banerjee Pinaki P , Orange Jordan S
Published: January 2023

NK cell cytotoxicity requires the formation of an actin-rich immunological synapse (IS) with a target cell and the polarization of perforin-containing lytic granules toward the IS. Following the polarization of lytic granules, they traverse through the actin-rich IS to join the NK cell membrane in order for the directed secretion of their contents to occur. We examined the role of myosin IIA in facilitating this pre-final step in lytic NK cell IS function. Lytic granules in and derived from a human NK cell line, or ex vivo human NK cells, were constitutively associated with myosin IIA. When isolated using density gradients, myosin IIA-associated NK cell lytic granules directly bound to F-actin and the interaction was sensitive to the presence of ATP under conditions of flow. In NK cells from patients with a nonsense mutation in myosin IIA, NK cell cytotoxicity, lytic granule penetration into F-actin at the IS, and interaction of isolated granules with F-actin were all decreased. Similarly, inhibition of myosin function also diminished the penetration of lytic granules into F-actin at the IS, as well as their final approach to and their dynamics at the IS. Thus, NK cell lytic granule-associated myosin IIA enables their interaction with actin and final transit through the actin-rich IS to the synaptic membrane, and can be defective in the context of naturally occurring human myosin IIA mutation.

Supported by NIH grants R01 AI067946 (JSO) and T32-GM07229 (KBS).
TNF driven activation of invariant NKT cells favours regulation of combined gut and joint inflammation. (134.22)
Authors: Elewaut Dirk , Jacques Peggy , Van Beneden Katrien , Apostolaki Maria , Lambrecht Bart , Kollias George
Published: January 2023

Invariant natural killer T (iNKT) cells are a subset of glycolipid reactive T cells, implicated in the protection against microbial pathogens and immune regulation. While the pathways leading to iNKT cell activation in the context of infectious diseases have been uncovered, much less is known how iNKT cells become activated in the context of chronic inflammatory diseases. Here, we unveil a novel mode of iNKT cell activation which occurs during chronic TNF driven inflammation through generation of inflammatory dendritic cells. This in turn, leads to a spontaneous activation of iNKT cells by promoting CD1d mediated presentation of endogenous glycolipids. This mode of activation is capable of attenuating combined Crohn's like ileitis and joint inflammation occurring in TNFΔARE mice, and thus may represent a natural counter regulatory mechanism to dampen TNF driven inflammation.
NK cell-mediated, Kupffer cell-dependent inhibition of liver injury attending biliary obstruction and cholestasis (134.25)
Authors: Cheng Chao-Wen , Wintermeyer Philip , Gregory Stephen H.
Published: January 2023

Natural killer (NK) cells are innate immune effector cells that participate in the first line of host defenses. In addition to expressing cytotoxic activity, NK cells produce cytokines and engage other cell populations to modulate adaptive immunity. Compared to other organs, the liver contains a large percentage of NK cells suggesting that NK cells play a unique role in the liver; the function of hepatic NK cells, however, is not well understood. Here we examined the contribution of NK cells to the tissue injury that occurs following bile duct ligation (BDL) in a mouse model of biliary obstruction. The number of activated hepatic NK cells was increased markedly at 18 hours following BDL. NK cells activation was suppressed in mice rendered Kupffer cell-depleted prior to ligation. Higher plasma alanine aminotransferase activity, an indictor of increased liver injury, occurred in NK cell-depleted mice. In contrast, IL-6 mRNA expression and protein production in the livers of BDL, NK cell-depleted mice were suppressed. Purified Kupffer cells derived from NK cell-depleted mice following BDL produced less IL-6 in culture than did Kupffer cells derived from non-depleted animals. Treatment with recombinant murine IL-6 reduced liver injury in BDL, NK cell-depleted mice. Taken together, these data suggest that hepatic NK cells suppress cholestatic liver injury by stimulating IL-6 production by Kupffer cells.
PI3-Kinase Links NKG2D Signaling to a CrkL Pathway Involved in Natural Killer Cell Adhesion, Polarity, and Granule Secretion (134.3)
Authors: Segovis Colin M , Schoon Renee A , Dick Christopher J , Nacusi Lucas P , Leibson Paul J , Billadeau Daniel D
Published: January 2023

The NK cell activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell - target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D coated beads is impaired in the absence of CrkL. Antibody-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras-family GTPase Rap1 is required for NK cell killing and is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization and granule release.
A simple new method for negative enrichment of monocytes from mouse blood and bone marrow (134.39)
Authors: Zaborowska Catherine , Fairhurst Maureen , Fairey Mark , Fogg Darin , Thomas Terry
Published: January 2023

The mononuclear phagocyte system is comprised of tissue macrophages, dendritic cells, blood monocytes and their bone marrow (BM) progenitors. Transgenic mouse models have recently provided insight into the biology of monocytes in vivo. However a general method for isolating monocytes from mice is needed to better study their functions.

We describe a rapid and simple method for the enrichment of monocytes from mouse BM and peripheral blood that does not require a density gradient and yields high purity and recovery. BM was harvested from femurs and tibia by crushing the bones. Blood was collected with heparin and red blood cells were removed by ammonium chloride lysis. The monocytes were then enriched using immunomagnetic, column-free negative selection (EasySep®). Briefly, unwanted cells were specifically labeled with dextran-coated magnetic particles using a cocktail of bispecific tetrameric antibody complexes. The sample was placed in a magnet and the supernatant containing unlabeled monocytes was collected. The separation procedure can be automated with a pipetting robot (RoboSep®). Purity of CD11b+Ly6G- cells as assessed by flow cytometry ranged from 80-93% for BM and 92-98% for blood with recovery of 46 ±11 % (n=38) and 25 ±10 % (n=20) respectively. This protocol will provide easy access to monocytes, enriched from peripheral blood and BM, for further studies of immune and inflammatory responses.
Effect of glycyrrhizin (GL) on β-defensin production in tissues surrounding the burn area of thermally injured mice (134.44)
Authors: Yoshida Shohei , Shigematsu Kenji , Asai Akira , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: January 2023

The decreased production of antimicrobial peptides in tissues surrounding the burn area has been demonstrated in patients and animals with severe burn injury. Previously, sepsis stemming from burn wound infections with Pseudomonas aeruginosa was effectively controlled in burn mice treated with GL. Therefore, in this study, effects of GL on β-defensin production in tissues surrounding the burn area were examined. BALB/c mice with 25% TBSA of 3rd degree flame burn were treated i.p. with GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan) at various schedules after burn injury. Eight punch biopsies (8-mm diameter) per mouse were obtained from skin tissues surrounding the burn area of these mice 24 hrs after burn injury. The homogenates of 8 biopsies were assayed for MBD-1 and MBD-3 by ELISA. In the results, normal mouse skin homogenates consistently contained 0.8 ± 0.4 μg/ml of MBD-1 and 6.9 ± 1.8 μg/ml of MBD-3. However, MBD-1 and MBD-3 were not detected in homogenates of tissues surrounding the burn area. When 10 mg/kg of GL was administrated i.p. to mice 2 and 12 hrs after burn injury, the amounts of MBD-1 and MBD-3 in the homogenates of tissues surrounding the burn area recovered to the level shown in normal mouse tissue specimens. MBD production in skin tissues of normal mice was not influenced by GL treatment. These results indicate that, after treatment with GL, the decreased production of β-defensins in tissues surrounding the burn area is improved in thermally injured mice. This study was supported by SHC NA #8610.
A critical role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response (134.52)
Authors: Gronski Matthew Adam , Kinchen Jason M , Juncadella Ignacio , Franc Nathalie , Ravichandran Kodi S
Published: January 2023

Calcium plays a key role as a second messenger in many cell types, but a role for calcium signaling during apoptotic cell removal is unclear. Using studies in cell lines and in the context of a whole organism, we demonstrate that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that such calcium flux is essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium via CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in C. elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes appeared to affect early signaling events, leading to decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the anti-inflammatory TGF-β response seen in phagocytes during engulfment of apoptotic cells. Taken together, these data point to a previously unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: formation of the phagocytic cup and internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.
Gene expression profiling of enhanced lung inflammation during pregnancy (134.54)
Authors: Fedulov Alexey V , Lamoureux Denise , Yang Zhiping , Kobzik Lester
Published: January 2023

Pregnant mice show sustained lung inflammation after exposure to environmental particles, titanium dioxide (TiO2), which are otherwise 'inert' in non-pregnant individuals (Am J Respir Cell Mol Biol. 2008. 38(1):57-67). We sought to characterize the gene expression program that underlies this inflammation by PCR array analysis. Selection of candidate genes for this array was informed by prior genome-wide chip microarray results, amended with hypothesis-driven candidates. METHODS: Pregnant E14 mice and controls received intranasal instillations of 50 ug/mouse of TiO2 particle suspensions or vehicle alone; n=5/group. After 48 hrs lungs were extracted, total lung RNA was isolated and samples were tested by real-time PCR via a custom 96-well PCR array (SABiosciences). Normalized dCt values were analyzed by pair-wise comparisons (ANOVA) and by main-effects tests (factorial ANOVA). RESULTS: Pregnant, but not control mice, show significant upregulation of ten genes (e.g. Itgax, CD4, Igsf6, Tlr2 and Tlr4) and a trend to upregulation of another ~20 genes, with fold change of 1.5-4.0. There is indication of influx of dendritic cells and T-helpers with some evidence of Th2 polarization. CONCLUSION: PCR array analysis identifies a gene expression signature for the enhanced inflammatory response to TiO2 particles seen in pregnant lungs, and suggests effects on adaptive immune responses. 1K99ES015425.
Activation of mouse complement C3 by human C-reactive protein (134.59)
Authors: Singh Sanjay K , Suresh Madathilparambil V , Hammond David J , Takahashi Kazue , Agrawal Alok
Published: January 2023

Human C-reactive protein (CRP), once bound to pneumococcal C-polysaccharide (PnC), interacts with C1q and activates the classical pathway of complement in human serum. In mouse serum also, PnC-bound CRP activates complement, but not through the classical pathway because human CRP does not interact with mouse C1q. This study was undertaken to determine the pathway through which human CRP activates mouse complement and to generate a CRP mutant incapable of activating mouse complement. We found that human CRP could bind to mouse mannose-binding lectins, indicating that human CRP might activate the lectin pathway of complement in mouse serum. Further experiments to investigate whether human serum activates the lectin pathway of complement in mouse serum are ongoing. We next determined the complement C3-activating potential of two CRP mutants, Y175A and L176Q, which do not bind to human C1q. As expected, both CRP mutants did not activate C3 in human seum. However, in mouse serum, the C3-activating potential of the L176Q CRP, but not of Y175A CRP, was drastically reduced compared to that of wild-type CRP. It has been shown previously that human CRP protects mice from lethality following infection with Streptococcus pneumoniae. The L176Q CRP is a useful mutant of CRP to define the contribution of CRP-mediated complement activation in the anti-pneumococcal function of human CRP in mouse models of infection. NIH R01HL071233
Requirements for the binding of C-reactive protein to complement factor H (134.60)
Authors: Hammond David J , Singh Sanjay K , Suresh Madathilparambil V , Pangburn Michael K , Agrawal Alok
Published: January 2023

Human C-reactive protein (CRP) protects mice from lethality following infection with Streptococcus pneumoniae type 3. Because both CRP and type 3 pneumococci are reported to bind to complement regulatory protein factor H, we propose that the factor H-binding capability of CRP may contribute to anti-pneumococcal function of CRP. In this study, we explored interactions between CRP and factor H with the aim to generate a CRP mutant incapable of binding to factor H. Surprisingly, we found that freshly-purified CRP did not bind human factor H. But, in the presence of phosphoethanolamine (PEt), freshly-purified CRP bound to human factor H efficiently in a dose-dependent manner. In contrast, and as reported previously, when an old preparation of CRP was thawed and used, CRP bound to human factor H even in the absence of PEt. Identical results were obtained with murine factor H. We also found that the mutations at the C1q-binding site on CRP did not affect the binding of PEt-complexed CRP to factor H, indicating separate binding sites on CRP for C1q and factor H. The mechanism of action of PEt in the binding of CRP to factor H is unknown; however, it seems that PEt modifies CRP in a way similar to storage-induced modification of CRP. Our findings raise the possibility that CRP, once in contact with a PEt-containing surface, gains factor H-binding specificity to modulate the complement attack on factor H-bound pneumococci. NIH R01HL071233
Early complement factors in the immunocomplex generated during intestinal ischemia / reperfusion injury (134.65)
Authors: Lee Haekyung , Green Danielle J. , Lai Lawrence , Hou Joan , Liu David , Cheong Cheolho , Park Chae Gyu , Zhang Ming
Published: January 2023

One novel example of the innate autoimmunity occurs in ischemia/reperfusion (I/R) injury, in which reperfusion of ischemic tissues elicits an acute inflammatory response activated by natural IgM (nIgM) binding to ischemia-specific self antigen. To identify the immediate downstream pathway following nIgM-ischemic antigen interaction, we isolated the nIgM-ischemic antigen immunocomplexes from the local tissue of animals treated in intestinal I/R injury model, and examined the presence of initial molecules of three complement pathways. As expected, mannan-binding-lectin (MBL) was detected in the nIgM-ischemic Ag immunocomplex. In addition, C1q was also present on the immunocomplex. However, Factor B was not detected in the immunocomplex. To further examine the role of the alternative pathway in I/R injury, we employed Factor B knockout mice in the intestinal model. Our results showed that Factor B knockout mice were not protected from local tissue injury, and their complement system was activated by nIgM during I/R. These results indicated that the lectin pathway of complement operates immediate downstream of nIgM and ischemic antigen interaction during intestinal I/R. Furthermore, the classical pathway also involves the formation of nIgM-ischemic antigen immunocomplex. Finally, the alternative pathway of complement is not involved in I/R injury induction in the current intestinal model.