bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4 memory T cells regulate mediators of lung damage during respiratory infections (130.8)
Authors: Jelley-Gibbs Dawn M , McKinstry K. Kai , Dibble John P , Roberts Alan D , Woodland David L , Swain Susan L
Published: January 2023

Activated T cells play a central role in controlling respiratory virus infections. However, these T cells also mediate considerable pulmonary immunopathology and lung damage. The level of immunopathology tends to be much less in secondary compared to primary infections, but the underlying reasons for this difference are unclear. To determine if CD4 memory T cells regulate pulmonary immunopathology, we transferred influenza-specific CD4 memory T cells into naïve mice challenged with influenza virus and assessed (i) inflammation, (ii) antigen presentation, and (iii) de novo T cell stimulation in the lung environment. The presence of CD4 memory T cells during influenza infection greatly reduced the expression of pro-inflammatory cytokines and chemokines in the lungs, as well as the ability of alveolar macrophages and pulmonary dendritic cells to stimulate naïve CD4 and CD8 T cell responses directly ex vivo. CD4 memory T cells also inhibited the accumulation of newly primed CD4 and CD8 T cells in the lungs, without reducing virus titers or de novo T cell responses in the periphery. We propose that CD4 memory T cells are capable of limiting pulmonary immunopathology by preventing the influx and retention of newly primed T cells in the lungs during respiratory infections. We are currently assessing whether the effective induction of CD4 memory populations through vaccination protects against excessive pulmonary immunopathology and secondary bacterial pneumonia. Funded by NIH AI057158 NBC-Lipkin and Trudeau Institute.
Cationic Liposome-DNA Complex Adjuvant (JVRS-100) Combined with Heat-Inactivated Whole Virion Influenza Vaccine Confers Protection against Homotypic and Heterosubtypic Lethal Challenge (132.1)
Authors: Chang Stella , Lay Marla , Callejo Bernadette , Liang Lily , Fairman Jeff
Published: January 2023

Background: Current vaccine strategies for influenza are highly dependent on matching of hemagglutinin, which is susceptible to periodic antigenic drift. A vaccine that elicits a robust antibody and T-cell response would be less susceptible to primary vaccine failure. Methods: Balb/c mice were vaccinated with heat-inactivated PR/8/34 (H1N1) or HKx31 (H3N2) with or without the JVRS-100 adjuvant. Mice were assessed for antibody and T-cell responses as well as challenged with a lethal dose of PR/8/34. Mice were monitored for clinical signs of infection, changes in body weight, and mortality. Results: Mice vaccinated with heat-inactivated virus combined with JVRS-100 adjuvant, showed increased HAI (450 vs. 30, P=0.007) and T-cell responses based on splenocyte restimulation (7700 vs. 30 pg/ml, P<0.001) when compared with unadjuvanted virus. Mice vaccinated with homologous virus with adjuvant exhibited no weight loss at six days post infection (+5.4% vs. -15.3%) compared with unadjuvanted virus following 6xLD50 challenge with PR/8/34. Mice vaccinated with JVRS-100-adjuvanted heterologous virus also showed less weight loss (-7% vs. -26.7%, P=0.004) compared with unadjuvanted virus following 6xLD50 challenge with PR/8/34. Conclusion: Heat-inactivated whole influenza virus adjuvanted with JVRS-100 showed increased immunogenicity and greater protection from homotypic and heterosubtypic lethal challenge. Adoptive transfer studies suggest that the cross-protection is due at least in part to a cross-reactive antibody response.
Topical patch vaccines target antigen to cutaneous dendritic cells efficiently inducing potent cell mediated immune responses (132.19)
Authors: Erdos Geza , Toussaint Helene , Williams Melissa , Ozdoganlar Burak O. , Falo, Jr. Louis D.
Published: January 2023

The skin contains a high density of accessible dendritic cells making it an attractive target for vaccine delivery. However, the remarkable barrier function of the skin continues to present a formidable physical obstacle to cutaneous vaccine design. To selectively overcome this obstacle, we evaluated the capacity of dissolving microneedle arrays (MNAs) to deliver protein vaccines to the skin and skin DCs. In initial experiments, biocompatible solid MNAs were fabricated with integrated particulate tracers. Image analysis demonstrated that these MNAs effectively penetrated both mouse and human skin. Further, following patch immunization labeled particulates were identified in dendritic cells in human and murine skin, and in the draining lymph nodes of immunized animals. To evaluate immunogenicity, we incorporated OVA as a soluble (sOVA) or particulate (pOVA) model protein antigen into fabricated patches, and evaluated CTL responses in immunized animals. Patch delivery of low doses of either sOVA or pOVA efficiently induced potent CTL responses comparable to those generated by optimized gene gun based immunization. Taken together, these results demonstrate the feasibility of antigen delivery for cutaneous immunization, and support the development of topical patch vaccines as an efficient and potent strategy for protein antigen based immunization. Funded by NIH.
Protection from acute viral disease by memory CD8+ T cells of a single specificity (132.23)
Authors: Chhann Sanda S , Sigal Luis J
Published: January 2023

Ectromelia virus (ECTV), the causative agent of mousepox, is a highly lethal mouse pathogen of the Orthopoxvirus (OPV) genus which also includes vaccinia virus (VACV) and variola virus. Mouse strains such as C57Bl/6 (B6) are naturally resistant to ECTV infection while others such as BALB/c and DBA2/J are highly susceptible. We have previously shown that resting memory CD8+ T cells can protect BALB/c mice from mousepox. To establish a model in the B6 background, we tested B6.D2(D6Mit149-D6Mit15)Lus/J (B6D2-D6) mice, a B6 congenic strain with the NK complex from DBA2/J (where a susceptibility gene to ECTV maps). We found that B6D2-D6 mice are sensitive to ECTV and can be protected by adoptive transfer of multivalent memory CD8+ T cells obtained from convalescent mice. To test whether memory CD8+ T cells of a single specificity can protect, we immunized B6D2-D6 mice with dendritic cells pulsed with B8R20-27, the dominant VACV/ECTV determinant in the B6 background. Strikingly, all the mice immunized with DC-B8R20-27 but not control peptide resisted the disease. Thus, memory CD8+ T cells of a single specificity are sufficient for protection. Because it would be of interest to know whether subdominant determinants can also be targets of protective memory cells, we used intracellular cytokine staining and in vivo cytotoxicity assays to identify additional VACV determinants conserved in ECTV. Interestingly, several determinants classified as subdominant based on their low IFN-γ production were found to be good targets of killing by effector CD8+ T cells. Future work will determine whether memory cells of these subdominant specificities also protect.

This work was supported by NIH grant R01AI065544 to LJS.
NK cells inability to produce IFN gamma in MyD88-/- mice contributes to the delayed clearance of Chlamydia muridarum primary genital infection (133.2)
Authors: Nagarajan Uma M , Sikes James D , Prantner Daniel , Goodwin Anna , Darville Toni
Published: January 2023

We have previously shown that MyD88-/- mice have delayed clearance of C. muridarum genital infection in comparison to wild type (WT) mice. The goal of the present study was to investigate the specific mechanisms by which MyD88 protects against C.muridarum infection. A 10-fold higher bacterial load was observed in cervical tissues of MyD88-/- mice at day (d) 4-post infection, suggesting impairment in the early innate response. Further, IFNγ levels were undetectable in the genital secretions of MyD88-/- mice until d4-d5 post infection, though they were restored to normal levels by d7 post infection. Interestingly, recruitment of inflammatory cells such as PMNs, macrophages and NK cells to mouse cervix remains mostly unchanged between WT and MyD88-/- mice at d4 post infection. To address this conundrum, NK cells from the cervix of infected mice at d4 post infection were FACS sorted and the levels of IFNγ mRNA assessed. Overall, MyD88-/- NK cells induced 16 fold less IFNγ than WT NK cells. Surprisingly, this defect in IFN gamma production did not translate to T cells isolated at d7 post-infection from iliac nodes of MyD88-/- mice, suggesting cell type specific regulation. The NK cells dysfunction in MyD88-/- mice also led to ineffective suppression of Th2 response, a plausible explanation for the delayed clearance of infection in MyD88-/- mice. Interestingly, MyD88-/- mice resolve a challenge infection of C. muridarum as effectively as WT mice, suggesting that an adaptive memory response is not impaired in the absence of MyD88 signaling.
Effect of caspase inhibitors on Salmonella typhimurium induced apoptosis of chicken macrophages (133.27)
Authors: Babu Uma S , Gaines Dennis , Raybourne Richard B , Fonseca Maira , Williams Kristina , Bigley Elmer , Pereira Marion
Published: January 2023

Salmonella typhimurium SR11 (ST) induced apoptosis of chicken macrophage cell line (HD11 cells) was assessed with or without caspase inhibitors. Apoptosis was measured upon the uptake of green fluorescent protein-labeled bacteria using flow cytometry. Dual staining of HD11 cells with annexin V PE and 7-amino-actinomycin-D (7-AAD) allowed differentiation of dead and damaged cells from those undergoing apoptosis. Our results reflect the late apoptotic cells that were positive for annexin V PE and 7-AAD staining. HD11 cells were infected for up to 48 hours and apoptosis was determined at baseline, 24 and 48 hours post-infection. We tested the effect of caspase inhibitors including, general, 1, 2, 3, 4, 6, 8, 9, 10 and 13 at100 and 200 μM concentrations on ST mediated apoptosis. Our data showed that ST induced significant apoptosis at 24 and 48 hours post-infection compared to uninfected cells. In addition, most of the caspase inhibitors inhibited apoptosis at very similar levels however 10 and 13 were the most effective at 24 and 48 hours. At 48 hours, most of the cells were in the necrotic state but incubation with caspase inhibitors 10 and 13 resulted in higher number of viable cells.
Feedback inhibition of macrophage responses to IFNγ during bacterial infection is mediated by IFNαβ (133.32)
Authors: Lenz Laurel L , Rayamajhi Manira , Andreasen Karl , Humann Jessica
Published: January 2023

We previously showed that increased IFNγ production by natural killer (NK) cells correlates with increased susceptibility to infection with Listeria monocytogenes. Here, we report that L. monocyogenes suppresses macrophage responsiveness to IFNγ and describe a mechanism contributing to this suppression. Macrophages infected with L. monocytogenes produced a soluble factor that reduced IFNGR1 transcripts and cell surface expression of both IFNGR1 and IFNGR2 proteins on infected and bystander macrophages and on certain other immune cells. We identify this factor as IFNβ or a substance induced by type I IFN, since (1) only L. monocytogenes strains that induce IFNβ caused IFNGR downregulation, (2) IFNGR downregulation was not seen in macrophages lacking a functional type I IFN receptor (IFNAR1-deficient mice), (3) IFNGR-downregulation was partially dependent on STAT1 and was induced by treatment of cells with recombinant IFNβ or IFNαβ -inducing toll like receptor (TLR) agonists. Our findings demonstrate that IFNβ induced during intracellular bacterial infection feeds back through IFNAR to inhibit IFNGR expression and IFNγ-dependent gene expression. These findings offer a common mechanistic explanation for the ability of IFNβ to enhance host susceptibility to intracellular bacterial infection and to suppress inflammation in the context of diseases such as multiple sclerosis.
Multiplex paracrine signaling leads to antiviral activated DC State (133.40)
Authors: Hartmann Boris M , Borderia-Giner Antonio V , Moran Thomas M , Sealfon Stuart C
Published: January 2023

Infection of immature dendritic cells (DCs) by virus stimulates their maturation into antigen presenting cells (APC). Infected DCs secrete TNFα, IFNα, IFNβ, MIP1α, RANTES, IL-6 and IL-8 which expose uninfected DCs to this complex cytokines/chemokines milieu. Recently, we showed that this paracrine signaling from infected DCs induced an antiviral-primed DC state referred to as antiviral activated DCs (AVDCs, J Immunology 2008 PMID: 18981106). AVDCs are relatively resistant to virus infection in comparison with naïve DCs and achieve accelerated and augmented levels of co-stimulatory molecule expression with virus infection. Here we investigate the factors reponsible for AVDC formation. No single factor induces a similar surface marker pattern in DCs as observed in AVDCs. The effects of all possible double combinations on naive DCs have been evaluated by RNA expression pattern, surface marker expression, as well as imaging flow analysis of rate of phagocytosis and DC-T cell interaction. The results illuminate how DCs integrate multiple paracrine signals to modulate their antiviral state.
Primary human astrocytes functionally express RIG-I, a member of the retinoic acid-inducible gene-I family of viral pattern recognition receptors (133.48)
Authors: Chauhan Vinita S , Furr Samantha R , Grdzelishvili Valery Z , Marriott Ian
Published: January 2023

Astrocytes are ideally positioned to detect and respond to invading pathogens of the CNS and previous studies have demonstrated the ability of these cells to produce key inflammatory mediators following exposure to members of the Mononegavirales order of viruses that are the causative agents of lethal human CNS diseases including rabies. However, the mechanisms by which resident cells of the CNS perceive such viral challenges have not been defined. Recently, several cytosolic DExD/H box RNA helicases have been described that can bind early viral replicative intermediates leading to the realization that retinoic acid-inducible gene I (RIG-I)-like helicases, such as RIG-I, function as intracellular receptors for RNA viruses. In the present study, we demonstrate that cultured primary human astrocytes constitutively express RIG-I receptor protein. Interestingly, such expression is rapidly elevated following exposure to vesicular stomatitis virus (VSV), a neurotropic rhabdovirus that closely resembles rabies virus. Evidence for the functional nature of RIG-I expression in human glial cells comes from the observation that this molecule associates with its downstream effector molecule, interferon promoter stimulator-1 (IPS-1), following VSV infection as assessed by co-immunoprecipitation techniques. Furthermore, we have confirmed that this molecule functions as an intracellular pattern recognition receptor in astrocytes by demonstrating that a putative ligand for RIG-I can elicit inflammatory cytokine production by this cell type. These findings raise the exciting possibility that RIG-I plays an important role in the detection of viral CNS pathogens and the initiation of protective immune responses or, alternatively, the progression of damaging inflammation within the brain.
Targeting natural killer cell potassium channels for selective immunomodulation (134.14)
Authors: Hu Xueyou , Moore Rebecca V. , Pennington Michael W. , Beeton Christine
Published: January 2023

Natural killer (NK) lymphocytes play roles in the innate immune defense against tumors and viruses but also participate in graft-versus-host disease and organ transplant rejection. Selective immunomodulation of NK cell subsets would therefore prove beneficial.

We have found that human NK cells express two types of potassium channels: Kv1.3 and KCa3.1. Interestingly, NK cells adherent to plastic in the presence of cytokines up-regulate KCa3.1 channels while non-adherent NK cells up-regulate Kv1.3 channels. In going with these results, we found that selective blockers of Kv1.3 channels preferentially inhibited natural cytotoxicity, antibody-mediated cytotoxicity and Fas-mediated cytotoxicity by non-adherent NK cells while selective blockers of KCa3.1 channels preferentially targeted adherent NK cells. We observed the same dichotomy when studying the effects of potassium channel blockers on the ability of adherent and non-adherent NK cells to proliferate and produce cytokines. We have successfully used selective Kv1.3 channel blockers to inhibit autoreactive T lymphocytes for the treatment of autoimmune diseases without inducing generalized immunosuppression. Targeting NK cell potassium channels may prove beneficial for the selective immunomodulation of NK cell subsets.

Funded by the American Heart Association and Baylor College of Medicine.
Deficiency in Sphingolipids Has No Effect on Self-antigen Presentation to Invariant NKT Cells (134.17)
Authors: Pei Bo , Speak Annie , Platt Frances , Cerundolo Vincenzo , Kronenberg Mitchell
Published: January 2023

NKT cells with an invariant TCR (iNKT cells) represent a unique subset of T lymphocytes sharing properties of both natural killer cells and T lymphocytes. iNKT cells regulate immune responses by rapidly secreting large amount of cytokines. Their development and maturation is dependent on self-Ags presented by CD1d, a MHC class I-like antigen-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids. Here, we used LY-B, a sphingolipid-deficient cell line, to study the nature of the endogenous lipid Ags that stimulate iNKT cells. With only 10% glycosphingolipid level of wild type cells, and these predominantly ganglioside GM3, mouse CD1d transfectants of LY-B still showed comparable ability to stimulate iNKT cell hybridomas. The self-Ag presentation was CD1d-dependent, because a specific antibody against mouse CD1d could block it. The finding was further confirmed by a cell-free Ag presentation assay. Surprisingly, alkaline digestion, which specifically hydrolyzes acyl fatty acids from phospholipids, dramatically reduced the antigenic activity of a cell sonicate while αGalCer was not affected. Sphingolipid ceramide N-deacylase, which specifically frees acyl fatty acids from sphingolipids, eliminated the activity of αGalCer without any effect on the activity in the cell sonicate. These data show that the endogenous Ags stimulating iNKT cells need not be glycosphingolipids.
Low T Cell Function in Preterm Neonates (PN) as Analyzed by Cylex ImmuKnow Assay (ICF) May be Responsible for a Higher Rate of Infection (134.47)
Authors: Reddy Sivapriya Thumati , Ge Shili , Puliyanda Dechu , Pao Andy , Jordan Stanley C. , Simmons Charles F. , Toyoda Mieko
Published: January 2023

Background: Cell mediated immunity is a critical defense mechanism against various pathogens. Lymphocyte number and function, and their links to risk for infection in neonates are not well understood. ICF measures immune function by assessing ATP levels in mitogen stimulated CD4+ cells. We investigate CD4+ cell function in neonates and the implication for infections. Methods: Heparinized cord blood from 30 term neonates (TN) and 3 PN (25-28 weeks) were submitted for ICF. Results: ATP levels were significantly lower in PN than TN (221 + 49 vs. 588 + 293 ng/ml, p<0.02). All 3 PN were exposed to maternal chorioamnionitis and treated postnatally for clinical sepsis, while none of the TN showed evidence of infection. In TN, the 18 vaginally delivered (VD) had significantly higher ATP levels than 12 born by cesarean (CS) (731±255 vs. 373±206 ng/ml, p<0.001). Of the 12 TN born by CS, 3 were exposed to labor and 9 were not, and showed similar ATP levels (277±110 vs. 405±225 ng/ml, NS). All 3 PN (1 CS) exposed to labor had low ATP levels. Conclusions: 1) CD4+ cell function is significantly lower in PN than TN, 2) VD in TN, but not CS w/ or w/o labor, increases ATP levels, suggesting that delivery via the birth canal is stressful for the TN and stimulates immune response, 3) ATP levels are not elevated in PN despite VD, suggesting that immune response in PN is poor, and may be partly responsible for a higher rate for infection in this patient population.

Research Sponsors: Dept. of Pediatrics & Transplant/Immunology Lab, Cedars-Sinai Medical Center, Los Angeles, CA
IL-15 and DAP-10 mediated IL-22 production by Natural Killer cells in human Mycobacterium tuberculosis-infection. (134.7)
Authors: Dhiman Rohan , Mohanalxmi Indramohan , Barnes Peter , Paidipally Padmaja , Vankayalapati Ramakrishna
Published: January 2023

We studied the mechanisms of IL-22 production by NK cells in human Mycobacterium tuberculosis (M. tb) infection. CD3-CD56+ NK cells from 17 healthy donors produced IL-22 upon culturing with autologous monocytes and irradiated M. tb (417 ± 99 vs 52 ± 22 pg/ml, p < 0.001). In 14 healthy donors anti-IL-15 and anti-IL-23 reduced IL-22 production by NK cells from 494 ± 110 pg/ml to 193 ± 50 pg/ml (p = 0.01) and 256.1 ± 64.7 pg/ml (p = 0.03) respectively. In contrast, anti-IL-12 and anti-IL-18 had no effect. Recombinant IL-15 induced IL-23 receptor expression (18.7 ± 5.1% vs 6.7 ± 1.9%, p =0.03). Recombinant IL-15 induced IL-22 production by NK cells (4294 ± 1627 vs 43.50 ± 29 pg/ml, p=0.04), but recombinant IL-12 and 1L-18 had no effect. To identify signaling pathways involved in IL-15 mediated IL-22 production, we cultured NK cells from healthy donors with recombinant IL-15 for 48 hrs. In 5 healthy donors IL-15-stimulated NK cells expressed 12 times more DAP10 mRNA compared to control NK cells. In 8 healthy donors DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells, compared to control siRNA-transfected NK cells (346 ± 121 vs 1248 ± 465 pg/ml, p =0.05). Studies are underway to determine the effect of IL-22 on intracellular survival of M. tuberculosis in macrophages. We conclude that IL-15 and DAP-10 are involved in IL-22 production by NK cells in human M. tb infection.
Identification of a novel IκB family member and its role in innate immune responses (134.73)
Authors: Yamauchi Shumpei , Ito Hiroaki , Miyajima Atsushi
Published: January 2023

The transcription factor NF-κB is a key mediator for inducible transcription in the innate immune system. While the signaling pathways from cell surface receptors to NF-κB activation have been extensively studied, the regulatory mechanisms of NF-κB transcriptional activity still remain elusive. To address this question, we performed microarray analysis of bone marrow derived dendritic cells and found a novel protein that is structurally related to the IκB family proteins. We provisionally named this molecule as IκBη and further characterized its expression and functions. IκBη was widely expressed in antigen presenting cells and predominantly localized in the nucleus. Overexpression of IκBη in NIH3T3 cells upregulated the NF-κB transcription activity. In contrast, knockdown of IκBη by siRNA in Raw264.7 cells resulted in severely impaired production of IL-6 and TNF-α in response to LPS. Biochemical study also indicated the interaction between IκBη and NF-κB on the IL-6 promoter. Taken together, it is suggested that IκBη modulates the production of proinflammatory cytokines by positively regulating the NF-κB transcription activity in innate immune responses.
Modulation of TLR4 signaling by TLR2 in the macrophage response to Mycoplasma pneumoniae (135.10)
Authors: Lai Jen-Feng , Zindl Carlene L. , Duffy Lynn B. , Atkinson Thomas P. , Chaplin David D
Published: January 2023

Several clinical reports suggest that Mycoplasma pneumoniae (Mp) can contribute importantly to asthma pathogenesis. As a foundation for understanding interactions between Mp and asthma, we are first studying the mechanisms by which the host normally eliminates Mp. We showed previously that macrophages are critical for elimination of Mp from the lungs of mice, and that MyD88-NFκB signaling is needed for this response. Here, we focus on the role of TLR signaling in the macrophage response to this microbe. Several mycoplasma lipoproteins have been shown to activate macrophages via TLR2, the majority through the TLR2/6 heterodimer. Interestingly, we found enhanced clearance of Mp from TLR2-/- mice, but reduced clearance of Mp from TLR1-/- or TLR6-/- mice. The enhanced clearance in TLR2-/- mice is dependent on both TLR4 signaling and the presence of macrophages. These data suggest interactions between TLR2 and TLR4 in the anti-Mp response. We extended these studies in vitro by infecting bone marrow-derived macrophages (BMM) with Mp. After infection with Mp, TLR2-/- BMM showed increased phosphorylation of IRF3 and NFκB, and up-regulation of IFN-β and IL-6 mRNA expression, compared to Mp-infected wild type BMM. These effects were TLR4-dependent. Altogether, TLR2 plays an inhibitory role in the regulation of TLR4 signaling in the macrophage response to Mp. Supported by NHLBI HL073907.
Neisserial Lipooligosaccharides (LOS) Differentially Modulate Innate Immune Responses in Human Monocytes throughTLR4 Signaling (135.17)
Authors: Liu Mingfeng , Jarvis Gary A
Published: January 2023

Neisseria gonorrhoeae and N. meningitidis engage innate immune receptors TLR4 and TLR2, as well as TREM1 and TREM2. In this study, we report differential innate immune activation by Neisserial LOS in monocytes. LOS differentially induced proinflammatory mediators TNF-alpha, IL-1 beta, IL-6 and IL-8 in THP1 monocytic cells as well as IL-10, IL-12, IFN-alpha, MIP-1 alpha, MIP-1 beta, and RANTES in human monocytes. Interestingly, while gonococcal LOS was not able to induce GM-CSF in monocytes, GM-CSF expression was enhanced by meningococcal LOS in both THP1 cells and monocytes. In THP1 cells, the induction of TNF-alpha by LOS was inhibited by anti-TLR4 and anti-CD14 antibody, but not by anti-TLR2, suggesting that the TLR4, but not TLR2, was responsible for the induction of cytokines. The ability of LOS to directly activate the TLR4 pathway was demonstrated by using TLR4-MD2 transfected HEK reporter cells, the activation of which was blocked by lipid IVa, a TLR4-MD2 antagonist. In monocytes, lipid IVa also inhibited the induction of all cytokines as well as CD80 expression by gonococcal LOS, but it only inhibited the activation of TNF-alpha, IL-10 and GM-CSF by meningococcal LOS. These results suggest that Neisserial LOS differentially activate innate immunity in monocytes through the TLR4 pathway, but that the induction of innate immune responses by meningococcal LOS may rely on the engagement of additional pattern recognition receptors.
Essential role of albumin in CD14-independent activation of MD-2/TLR4 by endotoxin (135.20)
Authors: Esparza Greg A , Teghanemt Athmane , Zhang De-Sheng , Gioannini Theresa , Weiss Jerrold
Published: January 2023

Host response to endotoxin (E) depends on the ordered interactions of LBP, CD14, MD-2 and Toll-Like Receptor (TLR) 4. CD14 is required for efficient transfer of E monomers to MD-2 (/TLR4) and for induction of TRIF-dependent signaling by activated TLR4. At high E concentrations, MD-2/TLR4-dependent cell activation by E is possible without CD14 through an unknown mechanism. We now show that incubation of purified E aggregates (Mr = 20 million) in PBS with = 0.1% albumin in the absence of the divalent cations Ca2+ and Mg2+, yields monomeric E:albumin complexes (Mr ~70,000). E monomers transfer from E:albumin to MD-2 and to MD-2/TLR4 directly and induce MD-2/TLR4-dependent, CD14-independent cell activation. Our findings suggest for the first time a mechanistic basis for CD14-independent cell activation by E. E:CD14 and E:albumin complexes should provide valuable reagents for future studies of MyD88-dependent (CD14-independent) and TRIF-dependent (CD14-dependent) TLR4 signaling by E.

Work supported by NIH/NIAID #AI59372.
Increased inflammation, impaired bacterial clearance, and metabolic disruption after Gram-negative sepsis in Mkp-1 deficient mice (135.23)
Authors: Liu Yusen , Wang Xianxi , Frazier W. Joshua , Meng Xiaomei , Nelin Leif D
Published: January 2023

MAPKs are crucial for TNF-α and IL-6 production by innate immune cells in response to TLR ligands. MAPK phosphatase (Mkp)-1 deactivates p38 and JNK MAPKs, abrogating the inflammatory response. We have previously demonstrated that Mkp-1-/- mice exhibit exacerbated inflammatory cytokine production and increased mortality in response to challenge with LPS and heat-killed Staphylococcus aureus. However, the function of Mkp-1 in host defense during live Gram-negative bacterial infection remains unclear. We infected Mkp-1+/+ and Mkp-1-/- mice with live E. coli i.v., and examined the effects of Mkp-1 deficiency on animal survival, bacterial clearance, metabolic activity and cytokine production. We found that Mkp-1 deficiency predisposed animals to accelerated mortality and was associated with more robust production of TNF-α, IL-6 and IL-10, greater bacterial burden, altered inflammatory mediator activities, and substantial changes in the mobilization of energy stores. Treatment with the bactericidal antibiotic gentamicin, given 3 h post E. coli infection, protected Mkp-1+/+ mice from septic shock yet had no effect on Mkp-1-/- mice. Thus, during Gram-negative bacterial sepsis Mkp-1 not only plays a critical role in the regulation of cytokine production but also acts to orchestrate the bactericidal activities of the innate immune system and control the metabolic response to stress.

The Research was supported by grants from NIAID (AI57798 and AI68956).
Bordetella pertussis filamentous hemagglutinin induces nuclear translocation of RelB/p52 in U-937 macrophages (135.24)
Authors: Abramson Tzvia , Kedem Hassya , Relman David
Published: January 2023

Filamentous hemagglutinin (FHA) is a dominant adhesin expressed by the respiratory pathogen Bordetella pertussis that also triggers inflammatory as well as immunosuppressive functions in the host. Given the role of NF-κB transcription factor family members in these functions, we examined the effects of FHA on NF-κB activation at early and late time points in two cell types relevant to natural infection: human bronchial epithelial (BEAS-2B) cells and human macrophage-like (U-937 derived) cells. We recently showed that FHA blocks proteasomal activity in both, the bronchial epithelial cell line and in monocyte-derived macrophages, resulting in accumulation of IκBα at 2 hours or greater incubation times. RelB and alternative NF-κB pathways regulate monopoiesis and monocyte-derived dendritic cell subset development. We report here that incubation of U-937 cells with FHA for more than 4 hours results in reduced levels of cytosolic RelB and increased levels of nuclear RelB and p52, as shown by Western blot analysis. No such effects were observed in bronchial epithelial cells (BEAS-2B). These data suggest that FHA induces nuclear translocation of RelB /p52 and activation of the alternative NF-κB pathway in U-937 cells, but not in BEAS-2B cells, and offer a molecular mechanism by which FHA might affect development and maturation of myeloid host cells.
Regulation of cell cycle and survival by type-I IFN and TLR7 in B-lymphocytes (135.43)
Authors: Priest Stephen O , Baumgarth Nicole
Published: January 2023

Innate signaling is an essential part of the anti-viral immune response. One such signal induced during an infection is type-I interferon (IFN), an anti-viral cytokine with immune modulatory effects on many cell types, including B cells. It also affects cell cycling and survival. Other signals are delivered via innate toll-like receptors (TLR) that can induce B cell proliferation in response to select microbial products, independent of, or in addition to B cell receptor signaling. Previous studies showed that these two innate stimuli affect the magnitude and quality of the local influenza virus-specific B cell response, and the sensitivity of B cells to non-specific activation via self-antigens. The precise mechanisms by which these innate TLR and type-I IFN signals exert their effects on the antiviral B cell responses, however, remain unclear. We show that type-I IFN regulates steady-state expression of innate viral recognition receptors TLR3 and TLR7 in B cells and their induction in local B cells following infection with influenza virus. IFN also induced several other factors regulating cell cycling and survival, notably cyclin D2 and Bcl-2, in these cells. Furthermore, stimulation by both TLR7 and type-I IFN, but not TLR3, strongly modulated cell cycle entry and survival of B cells. Our data identify the control of cell cycle and survival as key effects of TLR7 and type-I IFN on B cell responses. Supported by NIH AI51354.
CpG oligodeoxynucleotide incorporated basement membrane extract facilitate wound repair in mice (135.5)
Authors: Sato Takashi , Shimosato Takeshi , Klinman Dennis M
Published: January 2023

Wound healing involves complex processes including re epithelialization and wound contraction. Recent reports showed that the innate immune pathway of TLR4-mediated MyD88 signaling can affect the healing process by modulating epithelial cell migration and the production of angiogenesis factors. However, it has not been elucidated whether the TLR9 pathway could promote wound healing.

Synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs mimic the ability of bacterial DNA to stimulate an innate immune response via TLR9. We incorporated CpG ODN into a vehicle (basement membrane extract, BME) that slowly released bioactive ODN to wound sites. In vivo studies using an excisional skin biopsy model showed that CpG ODN treatment significantly accelerated wound repair in normal mice (p < 0.0001). The same effect was observed in aged mice (whose wound healing was otherwise delayed). Consistent with these effects being mediated by CpG signaling via TLR9, CpG ODN did not accelerate wound healing in TLR9 knock out mice.

Histologic studies indicate that CpG treatment promoted the local accumulation of macrophages, increased the production of VEGF, and promoted neovascularization, resulting in accelerated granulation and wound closure (p < 0.01). These findings suggest that CpG ODN may represent a promising novel therapy for wound repair.
CD180 is a positive regulator of TLR signals in B cells (135.64)
Authors: Chaplin Jay W , Kasahara Shinji , Clark Edward A , Ledbetter Jeffrey A
Published: January 2023

CD180 is a Toll-Like Receptor (TLR) that regulates the response of B cells and Dendritic Cells (DC) to TLR ligands. In the absence of CD180, B cell responses to TLR4/2 ligands are low, whereas LPS-stimulated DC produced more inflammatory cytokines. We examine the relationship of CD180 and TLR signals by in vitro B cell proliferation and cytokine production. Proliferation induced by anti-CD180 agonist antibody did not depend upon TLR signals and synergized with LPS in B cell proliferation; this synergy was mediated by LPS signals through MyD88 but not through TRIF. Anti-CD180 did not directly induce cytokine production by B cells but augmented IL-6, IL-10, and TNF-alpha production in combination with multiple TLR ligands. We also examine CD180 signals by in vivo injection of anti-CD180, which produced a greater than two-fold transient increase in splenic B cells without detectable toxicity. The B cell expansion peaked at day 3 and returned to normal levels by day 14. Proliferation of B cells isolated from injected mice was diminished at day 7 for CD180, LPS, and antigen signals. Responsiveness to CD180 and LPS returned to normal levels by day 14 while antigen signals remained attenuated. We conclude that CD180 signals interact with multiple TLR signals and can induce both transient B cell expansion and desensitization without overt toxicity.
A role of IgM antibodies in monosodium urate crystal formation and associated adjuvanticity (135.71)
Authors: Kanevets Uliana , Sharma Karan , Dresser Karen , Shi Yan
Published: January 2023

Uric acid is a product of purine metabolism that is secreted from injured or dying cells and can act as an endogenous adjuvant. However, uric acid must crystallize to monosodium urate (MSU) before it can activate dendritic cells and cause gouty inflammation. At normal serum uric acid levels, crystallization of uric acid is not observed in vitro. The crucial question therefore is how the crystals form and cause inflammation. It has been reported that serum from rabbits immunized with MSU, increased uric acid precipitation rates. When C57BL6 or Balb/c mice were immunized with MSU, they produced serum factors which bind to MSU crystals, a phenomenon absent in mu chain deficient IgH (or muMT) mice. These observations show that antibodies may be involved in MSU crystallization. Antibodies produced via B cell hybridomas, were observed to precipitate uric acid in vitro, with the variable region acting as the binding domain. Most importantly, the presence of antibody was instrumental in significantly increasing the basal level of inflammation and mediating the adjuvant effect of uric acid in antigen dependent T cell cytotoxicity in B cell deficient mice. Therefore, we have identified a factor in determining uric acid precipitation and possibly its ability to function as an endogenous adjuvant. This finding suggests a new mechanism of the pathogenesis of gouty arthritis and uric acid induced immune activation.
NLRP12 REGULATES TLR2/DECTIN-1 FUNGAL RECEPTOR SIGNALING THROUGH ASSOCIATION WITH CARD9 (135.74)
Authors: Lord Christopher , Goto Hisatsugu , Thomas Joseph M. , Mukherjee Sambuddho , Nambu Hiroya , Cramer Robert A. , Lin Xin , Wright Jo Rae , Williams Kristi
Published: January 2023

The NLR (nucleotide-binding domain, leucine-rich repeat) proteins are recently recognized as important intracellular regulators of host defense and immunity. One NLR gene, NLRP12/Monarch-1, has emerged as an important inhibitor of inflammatory gene expression in myeloid cells via regulation of TLRs and TNFR signal transduction. In contrast to its known role in regulating bacterial induced TLR activation of downstream genes, little is known about the role of NLRP12 in regulating the immune response to fungal pathogens. Here we show that NLRP12 modulates innate immune cellular responses to A. fumigatus by regulating TLR2/Dectin-1 fungal receptor signaling through its association with downstream signaling proteins CARD9 and Bcl10, but not Malt1. NLRP12, consisting of a pyrin, a nucleotide binding and a leucine rich repeat domain associates with the caspase recruitment domain containing proteins CARD9 and Bcl10 via the adaptor protein ASC. The most severe form of the NLRP12 disease associated mutant protein lacks the ability to associate with ASC, CARD9 and Bcl10 resulting in the lack of NLRP12's normal negative regulatory function. These data demonstrate the important role played by NLRP12 in modulating the innate immune response to fungal pathogens via its regulation of the TLR2/Dectin-1 induced CARD9 signaling pathway.

This work was supported by NIH grants HL-30923 and HL-68072 (JRW), NIH COBRE grant RR020185 (RAC) a SERCEB award and funding from CCBVP to KLW.
The promoter for the long isoform of mouse NKG2D is a B1 retrotransposon with a functional GABP site (136.24)
Authors: Lai Benjamin Chiehmin , Zhang Ying , Rogers Sally L , Mager Dixie L
Published: January 2023

The mouse gene for the natural killer (NK) cell activating receptor NKG2D produces two protein isoforms, NKG2D-S and NKG2D-L, which differ by 13 amino acids at the N-terminus and have different signaling capabilities. These two isoforms are produced through the use of alternative promoters but the regulation of these promoters has not been investigated. In this study, we show that rat NKG2D has the same splicing pattern as that of the mouse and we mapped transcriptional start sites in both species. We also show that the NKG2D-L specific promoter, active in both resting and stimulated NK cells, is derived from a rodent B1 retrotransposon that inserted before mouse-rat divergence. This B1 insertion is associated with loss of a nearby splice acceptor site that subsequently allowed creation of the short NKG2D isoform found in mouse but not human. Reporter assays suggest that the B1 promoter has no inherent lymphoid tissue-specificity. Finally, we have identified different binding sites for the ETS family member GABP within both the mouse and rat B1 elements that are necessary for high promoter activity.

This work was supported by the Canadian Institutes of Health Research and the Cancer Research Society.
An Allergy-Associated Polymorphism in a Novel Regulatory Element Enhances IL13 Expression (136.27)
Authors: Kiesler Patricia , Shakya Arvind , Tantin Dean , Vercelli Donata
Published: January 2023

The cytokine IL13 is a critical effector of T helper 2 (Th2)-mediated responses and allergic inflammation. We previously characterized the function of common IL13 variants that show strong, replicated associations with allergy-related phenotypes. We also demonstrated that during CD4+ Th cell differentiation, chromatin at the IL13 locus is extensively remodeled and becomes hypersensitive to nucleases at multiple sites. Among these, HS4 in the distal promoter is constitutively present in naïve and differentiated T cells, and spans a common allergy-associated SNP, IL13-1512AC (rs1881457). Here we characterize HS4 as a novel cis-regulatory element that upregulates IL13 transcription in Th2 cells and we show that the rs1881457 risk allele significantly alters HS4 activity and enhances IL13 promoter activity by creating a binding site for the transcription factor Oct-1. Experiments in mice heterozygous for an Oct-1 null mutation demonstrated that physiologic levels of Oct-1 are required for increased expression of the IL13-1512C promoter allele. Our results illustrate how a functional variant in a regulatory element may modulate susceptibility to a common complex disease.
Children with autism spectrum disorders (ASD) characterized by frequent viral infection and subsequent loss of cognitive skills differ in mRNA expression of peripheral blood monocytes from other ASD children (136.7)
Authors: Jyonouchi Harumi , Geng Lee , Streck Deanna , Toruner Gokce
Published: January 2023

We have reported presence of an ASD subset distinguished by recurrent loss of previously acquired cognitive skills with worsening behavioral symptoms triggered by infection (typically viral), which may be associated with altered TLR responses important for neuro-immune interactions involving IL-1β. This study assessed gene expression of peripheral blood (PB) monocytes in this ASD subset (ASD-test group, N=14, median 7.8 yr) in comparison with control ASD (N=11, median 6.8 yr) and normal children (N=5, median 8yr). Both the ASD groups regressed at the onset of ASD and had high frequency of non-IgE mediated food allergy. The ASD test group also had lower IL-10 production with recall antigens than 24 normal controls. Microarray analysis revealed difference of mRNA expression (two fold or more) of 1593 genes in the ASD-test group and 2704 genes ASD-controls as compared to normal controls. IL-1 pathway related genes were over-represented in the ASD-test group (p=0.002) while IL-2, IL-3, IL-9, α-integrin (all p<0.05), and TNF-α pathway (p=0.001) genes were over-represented in the ASD-control group (GeneSpring GX). Among 844 genes belonging to both the ASD groups, IL-2 and IL-3 pathway related genes were enriched (p<0.05). Our results indicate notable difference in the ASD test and control groups at the level of mRNA expression in PB monocytes, supporting our previous results. Partly funded by Jonty Foundation and ARI.
Characterization of naive CD4+ T cells with specificity to self-antigen (137.21)
Authors: Moon James J , Jenkins Marc K
Published: January 2023

During thymic development, T cells with specificity to self-antigens are deleted, thereby ensuring that the peripheral repertoire is devoid of potentially autoreactive clones. However, the ultra-low frequency of naive epitope-specific T cells has made the direct test of this theory technically unfeasible in the past. Using peptide:MHC (pMHC) multimers coupled with magnetic bead enrichment, we have directly studied endogenous polyclonal populations of naive CD4+ T cells with specificity for an I-Ab-restricted model antigen, 2W1S, that is foreign in C57BL/6 mice (B6) or self in mice transgenically expressing the model antigen (Act-2W). Act-2W mice contained a population of 2W1S-specific T cells that was about 25% the size of that in B6 mice, demonstrating that thymic deletion of self-antigen specific T cells is incomplete. Nonetheless, these cells were tolerant to 2W1S, as they did not develop autoimmune responses or respond to large doses of exogenous 2W1S antigen administered in vivo. Antigen receptors expressed by these cells exhibited features of reduced pMHC ligand binding affinity, indicating a preferential loss of high-affinity 2W1S-specific clones during thymic selection. Moreover, a greater proportion of 2W1S-specific T cells expressed FoxP3 in Act-2W mice than in B6 counterparts, suggesting that regulatory T cell development may also function as a mechanism of T cell tolerance to self-antigens.

This work was funded by NIH grants PO1-AI35296 and F32-AI063793.
Characterization of Sema4D and Sema7A mRNA Expression in Developing C57BL/6 Mouse Thymocytes (138.24)
Authors: Chatterjea Devavani , Linder Grace Elizabeth , Matthes David J
Published: January 2023

Semaphorins are a family of membrane-bound and secreted signaling molecules with active roles in nearly every organ system in processes ranging from heart morphogenesis to tumor development. We are investigating the potential roles of these molecules during T cell development in the thymus. Using Real Time PCR, we have characterized the mRNA expression of Sema4D and Sema7A, semaphorins with known peripheral immune system activity, in subsets of developing thymocytes. While Sema4D and Sema7A mRNA is expressed by all subsets of thymocytes, there appears to be no differential expression of these transcripts between CD4-CD8-, CD4+CD8+, CD4+, and CD8+ populations. While Sema7A expression has previously been described in the human thymus, this is the first time Sema4D and Sema7A mRNA expression has been reported in the murine thymus.
Increases in miRNA Expression in Aged Thymocytes (138.25)
Authors: Thoman Marilyn L , Virts Elizabeth L
Published: January 2023

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression during cellular processes such as differentiation and development. Alterations in miRNA expression have been linked to age-related changes in mouse liver and Alzheimer disease in the human brain. We decided to investigate a possible role for miRNA activity in age-related thymic decline. To address this question, we isolated lin- triple negative (TN) thymocytes from young (4 months) and aged (24 months) mice, and compared the miRNA profile of 625 miRNAs using qPCR. Thirty-two percent of the miRNAs (199) exhibited a 3-fold or greater increase in the aged animals while only 5% (34 miRNAs) were down-regulated by 3-fold or more. The magnitude of the differences in miRNA expression between young and aged thymocytes was unexpected. The TN population encompasses at least four identifiable subpopulations, representing distinct steps in differentiation. It is likely that these subpopulations differ in miRNA profile. These TN populations are certainly present in varying frequency in young and aged murine thymus. Therefore, some of the overall miRNA expression differences may reflect compositional differences in the TN populations used for RNA isolation. This possibility is being examined by analysis of individually sorted subsets. More intriguing is the possibility that the large number of highly expressed miRNAs found in the aged TN population is indicative that protein expression in these cells is rigidly controlled. We are investigating this possibility by identifying gene targets for a subset of the most highly expressed mRNA. These data provide us with essential information concerning miRNA expression changes that may be associated with the age-specific decline in thymopoiesis.