bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel gene expression profile in blood cells from cattle experimentally exposed to Mycobacterium avium subsp. paratuberculosis (164.24)
Authors: Thirunavukkarasu Shyamala , Purdie Auriol , Plain Karren , de Silva Kumudika , Whittington Richard
Published: January 2023

Mycobacterium avium subspecies paratuberculosis (Mptb) is the causative agent of the chronic debilitating condition called Johne’s (JD) disease that affects ruminants and is a suspected causative agent in Crohn’s disease in humans. Identifying and characterising the different strategies employed by Mptb to alter the host immune system in its favour and thereby persist intracellularly could hold the key to fighting this disease. Affymetrix Genechip Bovine Gene microarrays were performed using the RNA prepared from blood samples collected from exposed and non-exposed control cattle from the controlled experimental model of bovine JD. Bio-informatics tools and data mining were applied to identify relationships between genes and physiological functions. Genes related to lipid pathways and anti-bacterial defence mechanisms were identified and validated in a larger cohort at different time-points post-infection by quantitative polymerase chain reaction. Several novel gene pathways were shown to be differentially regulated in bovine JD linking lipid pathways and anti-bacterial defence mechanisms. Downregulation of the cellular cholesterol homeostasis mechanism could act as a countermeasure to infection. A novel model was developed that explores how these could impact the host immune response in a double edged sword manner that leads to the progression of pathogenesis.
Reprogrammed Foxp3+ regulatory T cells provide essential CD40L- mediated help that is synergistic with blockade of indoleamine 2,3-dioxygenase . (165.1)
Authors: Sharma Madhav , Munn David
Published: January 2023

We have previously shown that large numbers of FoxP3+ Treg cells undergo rapid reprogramming into activated T helper cells after vaccination with antigen plus Toll-like receptor 9 (TLR-9) ligand. Helper activity from converted Treg cells provided essential help during initial priming of CD8+ T cells to a new cross-presented antigen. We now show that help from Treg cells is delivered via CD40L, and (unlike help from conventional non-Treg CD4+ cells) did not require preactivation or prior exposure to antigen. In hosts with established tumors, Treg reprogramming was suppressed by tumor-induced IDO. Therapeutically, there was fundamental synergy between blocking IDO-dependent suppression and driving CD40-mediated DC activation. When IDO was blocked, required CD40L signal could derive from pre-activated memory conventional CD4+ helper cells (if available), reprogrammed Tregs, or exogenous anti CD40 agonist antibody. Agonist antibody and the IDO inhibitor drug 1-methyl-tryptophan showed potent synergy. Chemotherapy allowed extensive reprogramming of Tregs in tumor draining lymph nodes in response to anti-tumor vaccine, but only if IDO was blocked with 1MT. The effects of pharmacologic IDO inhibitor on Treg reprogramming was fully recapitulated by blocking the CTLA4 pathway. Thus reprogrammed Tregs participate as previously unrecognized contributors early CD8+ T cell responses against tumors.
Infection with the helminthic parasite Heligmosomoides bakeri alters B cell function and impairs vaccine induced antibody responses (166.1)
Authors: Wroblewska Joanna , Iweala Onyinye , Kline Austin , Urban Joseph , Nagler Cathryn
Published: January 2023

Worldwide endemic infection with gastrointestinal helminths is a potential challenge to successful vaccination in underdeveloped regions. In the current study we demonstrate that infection with the natural murine pathogen H. bakeri significantly reduced serum antibody responses to both parenteral immunization with ovalbumin (OVA) adsorbed to alum and oral vaccination with an attenuated OVA-expressing Salmonella strain. The reduction in serum antibody responses correlates with a helminth-mediated increase in the proportions of IL-10 secreting CD4+FoxP3+Tregs and CD4+FoxP3-Tr1 cells. ImageStream analysis showed that the vaccine was predominantly detectable in macrophages and dendritic cells in the mesenteric lymph node (MLN) of uninfected mice. Salmonella vaccine-carrying CD19+ B cells were increased, and macrophages were reduced, in the MLN of H. bakeri infected mice. Helminth infection also altered the effector function of CD19+ B cells, which express high amounts of TGF-β after infection, and can act as antigen presenting cells to induce IL-10 secreting T cells in vitro. Taken together, these data suggest that helminth infection alters vaccine efficacy by multiple complementary mechanisms, including inducing an immunosuppressive T cell environment, altering antigen presentation, and modulating B effector function.
Ag-specific recall of cytokine producing CD4 and CD8 T cells in humans immunized with genetically attenuated Plasmodium falciparum sporozoite vaccine (166.12)
Authors: Zarling Stasya , Spring Michele , Ehrler Lindsey , Chalom Isaac , Funk Theresa , Cowman Alan , Ockenhouse Christian , Speake Cate , Heppner D. , Duffy Patrick , Kappe Stefan , Krzych Urszula
Published: January 2023

A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by deletion of 2 pre-erythrocytic genes, was administered to 6 subjects by bites from Anopheles mosquitoes. Previous studies of immune responses elicited by recombinant malaria vaccines show that protection is linked to Ag-specific T cells producing IFN-γ, TNF, and/or IL-2. In this study we asked if Pf GAP vaccine induced Ag-specific T cell responses characterized by inflammatory cytokines. We analyzed pre- and post vaccination PBMC for Ag-specific T cell responses to well characterized pre-erythrocytic and erythrocytic P. falciparum protein and peptide Ags. Additionally, we examined responses to several novel Pf liver-stage Ag (Pf LSA) because Pf LSA-dependent immunity might contribute to GAP-induced protection. IFN-γ and TNF responses to multiple antigens were significantly enhanced post vaccination; IFN-γ responses were primarily attributed to CD8+ T cells while TNF was secreted primarily by CD4+ T cells. Notably, a subject with a breakthrough peripheral parasitemia did show positive IFN-γ responses to erythrocytic-stage Ags, but no responses were recalled with the novel Pf LSA. In summary, two exposures to a Pf GAP vaccine induced persisting T cell responses specific for Pf LSA in humans. Future trials that assess the efficacy of Pf GAP vaccines should examine the role of LSA-specific responses in protection.
Identification of live attenuated vaccine candidates for tularemia prophylaxis (166.20)
Authors: Bakshi Chandra Shekhar , Mahawar Manish , Metzger Dennis , Malik Meenakshi
Published: January 2023

Francisella tularensis (Ft) is the causative agent of a fatal human disease, tularemia. Ft was used in bioweapon programs in the past and is now classified as a category A agent owing to its possible use as a bioterror agent. Despite over a century since its discovery, an effective vaccine is yet to be developed. A live attenuated vaccine strain (LVS) exists; however, an unknown cause of attenuation and adverse effects in immunized individuals eluded its licensing as a vaccine. This study identified potential vaccine candidates for prophylaxis of respiratory tularemia. Five mutants of Ft LVS [FTL_0057, FTL_0291, FTL_0304, FTL_0325 and superoxide dismutase B (SodB)] were tested for their protective efficacy against Ft SchuS4 challenge. BALB/c mice were immunized intranasally (i.n.) with these mutants. Doses as high as 5x10(3) CFU (for SodB) or 1x10(7) CFU (for other mutants) did not cause any adverse reaction in immunized mice. A kinetic experiment revealed that the immunized mice cleared the mutants by days 14-21 post-immunization (PI). I.n. challenge with 100 CFU of Ft SchuS4 30 days PI protected 100% of mice immunized with SodB, FTL_0057, FTL_0291 and FTL_0325 mutants, while all the mice immunized with FTL_0304 mutant succumbed to infection. Mice that survived the challenge cleared Ft SchuS4 by day 45 post-challenge. This study has identified superior, safer and genetically defined live attenuated vaccine candidates for prevention of respiratory tularemia.
Overcoming the immune inhibitory environment associated with Staphylococcus aureus biofilms by activated macrophage transfer (166.6)
Authors: Heim Cortney , Hanke Mark , Kielian Tammy
Published: January 2023

Staphylococcus aureus (S. aureus) can establish biofilm infections on natural and artificial surfaces, which are typically recalcitrant to antibiotic therapy. Our laboratory has shown that S. aureus biofilms evade macrophage (MΦ) anti-microbial activity, further complicating biofilm eradication. To overcome the local immune inhibitory environment created by S. aureus biofilms, we have examined whether local administration of IFN-γ and peptidoglycan activated MΦs facilitates biofilm clearance in vivo using two treatment paradigms. In a prophylactic approach, mice received activated MΦs at 12, 24, and 48 h post-infection, whereupon bacterial burdens on infected catheters and surrounding tissues were quantitated at days 3 or 14 post-infection to evaluate the longevity of MΦ-mediated biofilm clearance. For established biofilms, activated MΦs were introduced at days 7 and 9 post-infection. In both treatment paradigms, activated MΦs led to significant reductions in bacterial burdens on infected catheters and catheter-associated tissues compared to vehicle controls. In addition, many proinflammatory mediators were significantly elevated in MΦ-treated animals in both models, indicating that MΦ administration is capable of overcoming the local immune inhibitory environment created during S. aureus biofilm formation. Collectively, these findings demonstrate that the local administration of activated MΦs enhances the host immune response to facilitate S. aureus biofilm clearance.
CD300a binds to phosphatidylethanolamine and phosphatidylserine and modulates the uptake of dead cells. (167.2)
Authors: Borrego Francisco , Andersen John , Calvo Eric , Choi Seung-Chul , Coligan John , Simhadri Venkateswara
Published: January 2023

CD300a is an immuno-receptor tyrosine based inhibitory motif (ITIM) containing molecule that belongs to the CD300 family of paired activating/inhibitory receptors clustered in human chromosome 17. It has been shown that its ligation inhibits activation signals on cells of both myeloid and lymphoid lineages. To identify the ligand of CD300a, we generated an Fc fusion protein containing the extracellular domain of CD300a (CD300a-Ig). We show that CD300a-Ig specifically binds to dead cells that are evolutionary apart, such as human and insect cells, suggesting that the ligand has to be conserved. Using ELISA, ultracentrifugation, surface plasmon resonance, and reporter cell assays we identified phosphatidylethanolamine (PE) and phosphatidylserine (PS), two phospholipids that translocate to the outer leaflet of the plasma membrane of dead cells, as the ligands for CD300a. Our mutational and structural modeling studies identified residues that are involved in the binding of CD300a to PE and PS and that form a cavity where the hydrophilic heads of PE and PS can penetrate. Ectopic expression of CD300a, in the otherwise CD300a negative L929 cells, down-regulated the phagocytosis of dead cells. Furthermore, we also show that CD300a inhibits the uptake of apoptotic cells by human macrophages. Collectively, our results indicate that PE and PS are ligands for CD300a and that these interactions may play an important role in the removal of dead cells by phagocytes.
NDRG2 expression suppresses osteoclast differentiation by down-regulating PU.1 (167.6)
Authors: Lim Jong-Seok , Kang Kyeongah , Yang Young , Lee Myeong-Sok
Published: January 2023

N-myc downstream-regulated gene 2 (NDRG2) has been characterized as a regulator of dendritic cell differentiation. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with LPS, TNF-α, 1α,25-dihydroxyvitamin D3 (1,25D3), or a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (RANKL). U937 cells stably expressing NDRG2 display reduced tartrate-resistant acid phosphatase (TRAP) activity, as well as defects in F-actin ring formation and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial to the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, cathepsin K, αv integrin, and MMP-9. Activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with by down-regulation of the expression of the PU.1 transcription factor. Finally, NDRG2 expression inhibited the differentiation of bone marrow cells and macrophages into osteoclasts, as judged by the failure of these cells to develop giant F-actin rings and resorption pits. Taken together, our results suggest that expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis.
miR146a facilitates DENV2 replication by targeting TRAF6 and inhibiting interferon production (168.19)
Authors: Huang Xi , Wu Siyu , Zhang Ping
Published: January 2023

microRNAs (miRNAs) are involved in the regulation of multiple cellular processes and have recently been indicated to modulate the innate immune response. However, the role of miRNAs in dengue virus (DENV) infection remains unknown. Our study test the expression of several innate immune-related miRNAs, including miR-9, 21, 132, 146a and 155, in the monocytic cells after infection with DENV serotype 2 (DENV2). Among these miRNAs, miR-146a expression was significantly increased by DENV2 infection, and its induction was impaired by inhibition of IKK, NF-κB, p38 or JNK mitogen-activated protein kinase (MAPK), or by silencing of interferon-γ promoter stimulator 1 (IPS-1). In addition, our data revealed that miR-146a facilitated DENV2 replication, accompanied by a significantly downregulation of TNFR-associated factor 6 (TRAF6), interferons (IFNs), as well as IFN-stimulated genes. Finally, knockdown of TRAF6 enhanced the DENV2 replication and reduced the IFN production, while overexpression of TRAF6 successfully reversed the proviral effect mediated by miR-146a, suggesting that miR-146a exerts its functions through targeting TRAF6, thereby destroying the IFN and the antiviral state. These findings suggest that miR-146a might be a promising therapeutic target for dengue disease.
Human metapneumovirus impairs the capacity of dendritic cells to prime antigen-specific naïve T cells (168.22)
Authors: Céspedes Pablo , González Pablo , Kalergis Alexis
Published: January 2023

Human metapneumovirus (hMPV) is a respiratory virus that mainly affects infants, the immunocompromised and the elderly. Worldwide, it has been estimated that hMPV is the second cause of upper and lower acute respiratory tract infection in infants causing significant economic burden. Similar to other respiratory viruses, hMPV can repeatedly re-infect the host without major antigenic changes. These observations suggest that hMPV may have evolved molecular mechanisms to interfere with the onset of an adequate host immune response, preventing the establishment of a long-lasting immunological memory. Recent in vitro studies have shown that hMPV infects human dendritic cells (DCs) and decreases their capacity to activate memory CD4+ T cells. However, the mechanisms underlying this reduced T cell activation remain elusive. Here we show that in spite hMPV-infected mouse DCs underwent maturation in response to hMPV, they are rendered inefficient at activating antigen-specific naïve CD4+ T cells. Decreased T cell activation was partially mediated by soluble factors secreted by hMPV-infected DCs and was virus strain-dependent. However, our results suggest inhibition of T cell activation by hMPV-infected DCs is mainly mediated by a cell contact-dependent mechanism. Taken together, our data suggest that hMPV interferes with DC function by soluble and membrane-bound determinants that likely affect T cell activation and the anti-viral adaptive immunity in susceptible hosts.
Human plasmacytoid dendritic cells and myeloid Dendritic cells fuse with chronically HIV-infected T cells (168.23)
Authors: Descalzi-Montoya Dante , Dai Jihong , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

Our laboratory has previously shown that plasmacytoid dendritic cells (pDC) fuse with chronically-HIV infected H9 T cells and also with acutely infected CD4+ primary T cells; a process mediated by virus/CD4 and CXCR4 interactions. In the present study, using the Amnis ImageStream, we observed that fusions can also form between chronically-HIV infected H9 T cells and myeloid dendritic cells (mDC). MDC showed a tendency to form more fusions than pDC. Although the fusion of mDC with HIV-infected H9 T cells was prevented by the CXCR4 inhibitor AMD3100, it was not affected by the CCR5 inhibitor Tak779 or the fusion inhibitor T20. This result indicates gp41 conformational changes may not be involved in this process, but rather it is mediated by CXCR4 interactions. On the other hand, pDC-T cell fusion formation is indeed inhibited by T20 and AMD3100 in our system, indicating that fusion in mDC and pDC may occur via distinct mechanisms. Moreover, there was no difference in induced fusions between pDC and acutely HIV-MN- infected H9 T cells or pDC with AT2-inactivated HIV-MN stimulated T cells, suggesting viral replication is not required for fusion. In conclusion, our observation that non-infectious HIV promotes fusion formation contributes to the notion that HIV bound to the host cell membrane surface may mediate this process. More importantly, in vivo, mDC and pDC fusion with HIV-infected T cells could contribute to the depletion of blood mDC and pDC in HIV-1 infected individuals.
Mouse CMV m153 glycoprotein binds a ligand on dendritic cells (168.28)
Authors: Revilleza Maria Jamela , Mans Janet , Natarajan Kannan , Mage Michael , Carey Rosalene , Boyd Lisa , Margulies David
Published: January 2023

A valuable model for studying virus-host interactions is mouse cytomegalovirus (MCMV), which encodes the m145 family of glycoproteins with predicted MHC-I structure. m153, expressed independently of beta-2 microglobulin and peptide, and is found on the surface of infected cells by flow cytometry. We constructed a reporter cell to identify tissues expressing the ligand of m153 by preparing the DNA encoding the extracellular domains of m153 linked to human zeta chain, and introducing this construct into 43.1 cells that fluoresce green on activation. We identified CD11c+ splenic dendritic cells (DC) and bone marrow DC as potent stimulators. Further characterization of these cells via multi-color staining with an m153 tetramer, and DC markers and antibody blocking experiments suggest a possible candidate for the m153 ligand. This may contribute to our understanding of the steps in virus lifecycle, and shed light on the evolution of viral immunoevasins.
Mechanisms of TRAIL expression by human plasmacytoid dendritic cells (168.29)
Authors: Serei Virian , Dai Jihong , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

One major mechanism of CD4+ T cell decline during HIV infection is due to the death ligand-mediated apoptosis of uninfected CD4+ T cells. TNF-Related Apoptosis Inducing Ligand (TRAIL) is one death ligand involved in this process and is expressed by a number of peripheral blood mononuclear cells upon HIV infection, including plasmacytoid dendritic cells (pDC). The mechanisms of TRAIL expression by pDC still need to be elucidated. To better characterize the expression of TRAIL, pDC were stimulated with TLR7 and TLR9 ligands with or without the addition of brefeldin A (BFA), a Golgi inhibitor. We directly stained pDC for surface TRAIL, and also stained for intracellular TRAIL by permeabilizing pDC with 0.1% Triton-X after overnight fixation with 2% PFA. Flow cytometry analysis revealed that with TLR7 and TLR9 agonists, there was an increase in TRAIL expression on the surface of pDC, while with BFA addition, there was a decrease in surface TRAIL expression accompanied with an increase in intracellular TRAIL expression. Using Amnis Image Stream, we were also able to visualize the location of intracellular and surface TRAIL expression on each pDC. This study showed that upon TLR7 and TLR9 stimulation, pDC upregulate TRAIL expression, and with the addition of BFA, TRAIL is prevented from reaching the surface, accumulating in the cytoplasm of pDC.
Co-stimulatory receptors CD28 and CTLA4 are required for optimal NK cell effector function and memory formation during MCMV infection (168.3)
Authors: Karo Jenny , Sun Joseph
Published: January 2023

Natural Killer (NK) cells are generally classified as a component of the innate immune system. However, recent evidence in mice and humans suggests that NK cells have properties of both innate and adaptive immunity. Optimal T cell activation requires both T cell receptor ligation, a co-stimulatory signal provided by CD28, and regulation by the inhibitory receptor CTLA-4. Although NK cells have been shown to express CD28, the importance of co-stimulation in NK cell responses in vivo has not been previously investigated. Using mice deficient in CD28, and receptor-blocking antibodies, we investigated the role of this receptor on NK cells using a mouse model of cytomegalovirus (MCMV) infection. After primary infection with MCMV, we found that CD28 is necessary for early viral control of MCMV by antigen-specific Ly49H+ NK cells and consequently memory NK cell formation. In addition, we have new evidence that CTLA-4 is highly expressed on activated NK cells during MCMV infection. Surprisingly, like CD28, expression of CTLA-4 was also required for optimal proliferation and function of Ly49H+ NK cells during MCMV infection. Manipulation of these co-stimulatory signals within the immune system is an emerging strategy of effective vaccination and tumor therapy. Therefore, understanding the molecular mechanisms behind effective NK cell responses to pathogens will be critical in our attempts to modulate the immune system against these ailments.
Immune regulation by natural killer cells through lysis of activated T cells (168.4)
Authors: Waggoner Stephen , Welsh Raymond
Published: January 2023

We have previously shown that natural killer (NK) cells can influence viral pathogenesis either by directly lysing virus-infected cells or through lysis of activated CD4 T cells. The latter phenomenon contributed to an inhibition of anti-viral T cells that favored viral persistence and host survival rather than lethal pathology during lymphocytic choriomeningitis virus (LCMV) infection. Infections with unrelated viruses, including Pichinde virus (PV), or inoculation with the type I IFN inducer, polyI:C, also triggered NK cell lysis of activated CD4 T cells. This lysis was coincident with poor control of PV infection and diminished PV-specific memory T cell responses, suggesting that such NK cell immunoregulation is a general principle of virus infection. LCMV infection was unable to induce NK cell lysis of activated T cells in IFN receptor (IFNAR)-deficient mice, implicating type I IFN in stimulation of this NK cell activity. Expression of the CD48, the ligand for the inhibitory NK cell receptor 2B4/CD244, was greater on NK cell-resistant CD8 T cells than their CD4 counterparts. However, genetic deletion of 2B4 facilitated lysis of activated CD8 T cells, and was associated with impaired clearance of both acute and chronic virus infections. Thus, type I IFN expression during virus infection or vaccination stimulates a 2B4/CD48-regulated, NK cell-mediated lysis of anti-viral T cells which impairs viral control and immune memory.
In vivo delivery of lipidoid nanoparticles containing PD-L1 siRNA enhances NK and CD8+ T cell-mediated hepatic antiviral immunity (168.6)
Authors: Dolina Joseph , Sung Sun-Sang , Novobrantseva Tatiana , Hahn Young
Published: January 2023

Effective clinical application of anti-viral immunotherapies necessitates enhancing the functional state of NK and CD8+ T cells. Persistence of viral infections in the liver is established by activation of the PD-1/PD-L1 inhibitory pathway. The expression of PD-1 ligand (PD-L1) is widespread and found on multiple cell types including Kupffer cells, liver sinusoidal endothelial cells, non-resident macrophages, dendritic cells, NK cells, T cells, and hepatocytes. To examine the role of PD-L1 expression by myeloid cells, we determined the magnitude and quality of antiviral immune responses by administering PD-L1 siRNA encapsulated in lipidoid nanoparticles (LNP) in mice prior to or after viral infection. Our studies indicate that Kupffer cells preferentially engulfed PD-L1 LNP leading to specific silencing of Pdl1 during adenovirus and MCMV infection. Treatment of PD-L1 LNP enhanced NK cell and CD8+ T cell intrahepatic accumulation, and depending upon timing of PD-L1 LNP treatment, either NK cell or CD8+ T cell effector function, as assessed by IFN-γ and granzyme B production, were enhanced. Importantly, CD8+ T cell-mediated viral clearance was enhanced through targeted blockade of the PD-1/PD-L1 inhibitory pathway. Our results demonstrate that PD-L1 knockdown on Kupffer cells is central in determining the outcome of liver viral infections. The implications of this research also extend into other diseases including cancer where reversal of CD8+ T cell tolerance is critical.
Therapeutic effect of a CXCR2 ligand acetylated Pro-Gly-Pro against sepsis (169.13)
Authors: Bae Yoe-Sik , Kim Sang Doo , Lee Ha Young , Kim Hak Jung , Baek Suk-Hwan
Published: January 2023

Acetylated Pro-Gly-Pro (Ac-PGP) is an endogenous degradation product of extracellular collagen that binds to leukocyte-expressed chemoattractant receptor CXCR2. Although certain agents that block CXCR2-mediated signaling protect against experimental sepsis, the roles of Ac-PGP and CXCR2 in sepsis are unclear. In this study we investigated the role of Ac-PGP and its receptor, CXCR2, in murine models of cecal ligation and puncture (CLP)-induced polymicrobial sepsis and organ injury. The activation of CXCR2 by Ac-PGP dramatically improved survival in three experimental sepsis models. Ac-PGP elicited bactericidal activity via the generation of hydrogen peroxide, inhibited lung inflammation, and reduced immune cell apoptosis. Fluorescein isothiocyanate-labeled PGP bound directly to CXCR2, and the protective effect of Ac-PGP in sepsis was abolished in CXCR2-deficient mice. Ac-PGP treatment enhanced the production of type 1 cytokines (IFN-γ and IL-12) but inhibited the production of proinflammatory cytokines (tumor necrosis factor [TNF]-α, IL-1β, and IL-6) in vivo. In vitro, Ac-PGP directly increased IFN-γ production and decreased the LPS-stimulated production of TNF-α by mouse splenocytes and human leukocytes. Furthermore, direct treatment of LPS-stimulated splenocytes with IFN-γ resulted in diminished secretion of TNF-α and IL-6. CXCR2 and Ac-PGP are thus novel target and starting molecules, respectively, for the development of therapeutic agents against sepsis.
Acute inflammasome activation and downregulation after severe burn injury in humans. (169.23)
Authors: Maile Rob , Maggin Jeremy , Zihni Ahmed , Sparks Carilyn , Ting Jenny , Cairns Bruce
Published: January 2023

Cytokine activation and the systemic pro-inflammatory shock response following burn injury results in multiple organ failure. We hypothesized that the NLRP3 inflammasome (a multiprotein complex constructed in macrophages following stimulation by danger signals), and related genes are elevated following burn injury in humans. We enrolled 18 patients: 7 with large burns (>30% total body surface area burn, TBSA) and 11 with small burns (< 30% TBSA). Blood was collected at 24, 24-48 and 48-72 hours after burn. Mononuclear cells were harvested, total RNA isolated and levels of NRLP3, Caspase-1, IL-1β, Pyrin and TNFα measured using quantitative real time PCR (qtPCR) and compared to normal pooled mRNA. Serum protein levels of Caspase-1, TNFα and IL-1β were quantified by ELISA and compared to normal serum. At all time points and %TBSA, the relative copy number of Caspase-1 mRNA was significantly elevated but IL-1β mRNA was decreased compared to normal. NLRP3, Pyrin and TNFα mRNA copy numbers were unchanged. Protein levels of active IL-1β, TNFα and caspase-1 were significantly increased at all time points and %TBSA. These data indicate that inflammasome component caspase-1 expression is elevated after burn, cleaving pro-IL1β into the active form. Concurrent downregulation of IL-1β gene expression occurs to counter an overt inflammatory response. This is the first transcriptional evidence of inflammasome activation and homeostatic downregulation in burn patients.
TNFR25SF AND GALECTIN-9 COMBINATION THERAPY REDUCES HERPES SIMPLEX VIRUS-1 INDUCED STROMAL KERATITIS BY EXPANDING REGULATORY T CELLS AND REDUCING PATHOGENIC TH1 CELLS (170.17)
Authors: Jagadeesh Reddy Pradeep Babu , Suryawanshi Amol , Schreiber Taylor , Rajasagi Naveen , Hirashima Mitsuomi , Podack Eckhard , Rouse Barry
Published: January 2023

Ocular infection with HSV-1 results in a chronic immunoinflamammtory reaction in the cornea, which is primarily orchestrated by CD4 T cells. Hence, targeting proinflammatory CD4 T cells or increasing the representation of cells that regulate their function are relevant therapeutic strategies. In the present report, we use an agonistic mAb (4C12) to TNF receptor 25 (TNFR25) that selectively expands numbers of regulatory T cells (Tregs). Treatment early after ocular HSV infection with 4C12 caused a three fold increase in the Treg numbers in the cornea along with a consequent reduction in SK lesion severity. However, 4C12 treatment was less effective if given 6 days after infection since it expanded proinflammatory T effectors that also express TNFR25. To suppress the latter population, galectin-9 (Gal-9) was used which causes apoptosis of Th1 cells. When therapy with both 4C12 and gal-9 was used in combination, the recipients showed significantly reduced angiogenesis and SK lesions over single treatment controls. The beneficial outcome of the combination therapy was attributed to the expansion of the Treg population that expressed CD103 needed for trafficking to inflammatory sites along with a marked reduction in the infiltration of pathogenic CD4 T cells. Levels of several proinflammatory cytokines and chemokines were also reduced. Our results demonstrate that combination therapy may be a promising approach to control HSV induced SK lesions, a common cause of human blindness.
Innate immune response contributes to virus-mediated neuronal death through activation of SARM (170.2)
Authors: Peterson Karin , Woods Tyson , Mukherjee Piyali
Published: January 2023

Neuronal cell death is a hallmark of various neurological diseases including virus infection. The innate immune response in the CNS contributes to neuronal damage through cytokine and neurotoxin production as well as the recruitment of inflammatory cells. However, the role of the innate immune response in the induction of neuronal death is poorly understood. La Crosse virus (LACV), which is a major cause of pediatric encephalitis in the United States, predominantly infects neurons and is associated with neurodegenerative changes. Infection results in neuronal apoptosis. In the current studies we identified sterile alpha and HEAT/Armadillo motif containing 1 (SARM1) protein, a negative regulator of TLR signaling, as a moderator of LACV induced neuronal apoptosis. SARM 1 was induced in neurons following LACV infection and SARM1 knock down studies in neurons demonstrated increased viability during LACV infection as compared to wild-type controls. Direct TLR stimulation of neurons induced SARM 1 which correlated with neuronal apoptosis. Thus, SARM induced as a response to innate immune activation appears to contribute to neuronal death. We are currently investigating the mechanisms underlying SARM induced neuronal death following LACV infection.
HIV-1 Nef limits communication between LAT and Slp-76 to reduce formation of Slp-76 signaling microclusters (170.20)
Authors: Abraham Libin , Bankhead Peter , Pan Xiaoyu , Engel Ulrike , Fackler Oliver
Published: January 2023

Signal transduction through the T cell receptor (TCR) triggers actin rearrangements, receptor clustering and dynamic organization of signaling microclusters (SM) to form a stable Immunological synapse (IS). Nef, a pathogenicity factor of Human Immunodeficiency Virus (HIV), disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption we employed quantitative microscopy to analyze the effects Nef exerts on formation and composition of TCR proximal SM. Despite marked inhibition of actin remodeling and cell spreading, the induction of SM containing the TCR itself or Zap70 was not affected by Nef. However, Nef potently inhibited the subsequent formation of SM that contained Slp-76 to reduce microcluster density in Nef expressing or HIV-1 infected T cells. Using a Nef mutant selectively defective in inhibition of actin remodeling (Nef F195I), we found that Nef-mediated disruption of actin remodeling and Slp-76 SM are independent activities. Strikingly, the effects of Nef on Slp-76 SM correlated with trans-golgi network targeting of LAT, an adaptor protein that couples Zap-70 to Slp-76. Nef-induced disruption of SM formation and composition depended on its ability to disrupt both, actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus disrupts early TCR signaling by limiting the communication between LAT and Slp-76 and preventing the dynamic formation of Slp-76 SM.
Regulatory T cells decreased HIV transmission from DC to conventional T cells (170.21)
Authors: Moreno-Fernandez Maria , Chougnet Claire
Published: January 2023

HIV infection of CD4+ T cells is quite efficient by transfer of the virus from dendritic cells (DC) to CD4+ T cells. Activation levels of both cells play a crucial role in the efficiency of the infection. Regulatory T cells (Treg) are known to control the activation levels of both CD4+ T cells and DC. Treg frequency is increased in mucosal tissues of HIV-infected individuals and Treg can reduce HIV infection in conventional CD4+ T cells (Tcon) and macrophages. We therefore hypothesize that Treg can also control DC: Tcon HIV transmission. Monocyte derived DC were infected with the HIV R5 strains BaL and cultured for 1 day with autologous Tcon in the presence or absence of autologous Treg. Formation of Tcon:DC clusters and the percentage of infection in these clusters were determined by multicolor flow cytometry and Imaging flow cytometry (ImageStream). Culture with Treg resulted in a significant decreased of Tcon:DC cluster infection (p=0.025). Looking at the underlying mechanism we found that blockage of CTLA-4 significantly reduces Treg inhibitory function. Treg-derived cAMP also appears to be involved in Treg suppressive activity, as pharmacological decrease of cAMP levels in Treg abolished Treg suppression of HIV transmission. Our data suggest a complex role of Treg during HIV infection: they inhibit specific immune responses, but their inhibition of HIV transmission at several levels may also play a beneficial role during early HIV transmission events
Exercise modulates the immune response to influenza infection in aged mice. (170.28)
Authors: Naik Shibani , Gallup Jack , Yu Shan , Sim Young-Je , Yoon Kyoung-Jin , Nettleton Daniel , Kohut Marian
Published: January 2023

Exercise training prior to influenza virus infection in aged mice decreases illness severity, reduces lung viral load, and decreases inflammatory cytokine/chemokine levels in bronchoalveolar lavage (BAL) fluid. We hypothesized that an exercise-associated increase in anti-viral responses (Type I IFN and related genes, pentraxins) may contribute to reduced viral load. Exercise may also increase the rate of cell turnover in the lungs to limit the spread of virus. METHODS: Old mice (18mos) were exercised moderately 5d/wk for 10 wks, rested for 24h, and then infected with influenza A/PR/8/34 H1N1. Non-exercised old mice and young mice (2mos) were controls. Lungs were collected several times post-infection (p.i.); mRNA was detected in lungs using RT qPCR. Cytokines in BAL were measured by ELISA. Formalin Fixed Paraffin Embedded (FFPE) lungs were used to detect the presence of virus and Caspase 3 by IHC. RESULTS: After exercise training, interferon-inducible antiviral gene expression (Oas1b, B2m, BST2 & GBP1) was increased, but at 12-24 hr p.i., exercise was associated with decreased BAL IFNα & IFNβ, and reduced lung mRNA of IFNα4 and Oas1a. Exercise did not alter viral attachment or apoptosis 6hr p.i. The results suggest that exercised basal level expression of several interferon-related genes which may contribute to the early reduction in viral load.
Enhancing protection against highly pathogenic H5N1 avian influenza A viruses (170.31)
Authors: McGargill Maureen , Keating Rachael , Lukens John , Hertz Tomer , Harris Tarsha , Edwards Benjamin , Creasy Blaine , Doherty Peter , Thomas Paul
Published: January 2023

Highly pathogenic H5N1 avian influenza A viruses pose a serious threat for a deadly pandemic. Vaccination is the most effective strategy for controlling influenza, however it is not possible to predict which H5N1 variants may acquire the ability to transmit efficiently between humans, and conserved epitopes between different subtypes do not induce sufficient immunity. Thus, a strategy to increase the efficacy of universal vaccines is essential for preventing a deadly pandemic. Others demonstrated that the immunosuppressive drug, rapamycin, paradoxically increased the number of memory T cells during LCMV infection. Thus, we examined whether rapamycin increases the number of memory T cells during influenza infection, and consequently enhances protection against a secondary infection with a heterosubtypic H5N1 strain. Mice were immunized with HKx31 (H3N2), treated with rapamycin for 30 days, and challenged with a modified H5N1 virus, ΔVn1203. We found rapamycin protected mice from a lethal ΔVn1203 infection, but not in the absence of a primary infection. Interestingly, rapamycin increased the number of influenza-specific CD8 T cells after the primary and secondary infection. However, memory CD8 T cells were not required for protection. Rather, CD4 T cells and B cells were critical for the enhanced survival following rapamycin treatment. These data suggest that rapamycin protects against a highly pathogenic, heterosubtypic influenza infection by altering the antibody response.
Regulatory T cells control the clinical expression of viral immunopathology by acting at both the lesion site and draining lymphoid tissue (170.9)
Authors: Veiga Parga Tamara , Sparwasser Tim , Rouse Barry
Published: January 2023

Ocular herpes simplex virus infection can cause a blinding CD4+ T cell orchestrated immunoinflammatory lesion in the cornea called Stromal Keratitis (SK). Lesion severity can be influenced by the balance of CD4+ T effectors and Foxp3+ regulatory T cells (Treg) at the onset of infection. We have evaluated the role of Treg during the ongoing clinical phase of the disease by taking advantage of transgenic mice, which express a diphtheria toxin receptor enhanced GFP fusion protein under the control of the Foxp3 locus, permitting Treg depletion. Depleting Treg during the clinical phase of the disease resulted in significantly more severe lesions. This outcome was explained both by Treg influencing the activity of inflammatory T cells at the lesion site as well as effects in the lymphoid tissue that reduced trafficking of T cells and neutrophils to the eye, as well as the activation state of T effectors. Our results demonstrate that Treg can beneficially influence the impact of ongoing tissue damaging responses to a virus infection and imply that therapies that boost Treg function hold promise as a modality to control a lesion that is an important cause of human blindness.
Ectopic expression and presentation of diabetogenic antigens in the lymph nodes of NOD mice. (171.10)
Authors: Creusot Remi , Yip Linda , Fathman C. Garrison
Published: January 2023

Ectopic expression and presentation of peripheral tissue antigens (PTAs) in lymph node stromal cells has been shown to contribute to the maintenance of peripheral tolerance. During disease progression in non-obese diabetic (NOD) mice, at 12 weeks of age (the onset of destructive insulitis), expression of genes encoding many PTAs (including insulin and chromogranin A) as well as the function of the transcription regulator Deaf1, are diminished in the pancreatic lymph nodes (PLNs). We observed a defect in insulin presentation in NOD mice both in vivo (in adoptive transfer experiments) and in vitro (using purified stromal cells) compared to 12-wk old diabetes-resistant (NOD x NOD.B10) F1 mice. We identified two stromal cell subsets within the gp38+ CD31- fibroblastic reticular cells and the gp38- CD31- double negative (DN) population that can express and present insulin to CD4 T cells. Data generated to date suggest that Deaf1 controls the expression of insulin at least in DN cells. Inflammation can induce the splicing of Deaf1 mRNA into a dominant negative isoform in the PLNs. Preliminary data suggest that a substantial proportion of T cells stimulated in vivo or by stromal cells ex vivo induce Foxp3 expression. Thus, diminished PTA expression and presentation in 12-wk old NOD mice may impair the maintenance of peripheral tolerance and may explain the subsequent progression from peri-insulitis to destructive insulitis and hyperglycemia.
The Effects of Manzamine A (MZA) on Synovial Fibroblast Adhesion (173.32)
Authors: Mc Dermott Sarah , Klosowska Karolina , Zanotti Brian , Volin Michael , Woods James
Published: January 2023

Isolated from sponges on the ocean floor, MZA has anti-inflammatory properties which might be beneficial in treating rheumatoid arthritis (RA) or osteoarthritis (OA). Preliminary data suggested that MZA causes RA fibroblast-like synoviocytes (FLS) to detach from gelatin. We hypothesized that MZA decreased the adhesive properties of synovial fibroblasts isolated from RA, OA, or normal joints. All fibroblasts were isolated from tissues collected from patients with IRB approval. Photos of cells were taken at various time points (1, 2, 4, 8, 12, and 24 hrs) and counted for adherence after treatment with various concentrations of MZA or vehicle on gelatin, collagen, or laminin substrates. Flow cytometry was used to determine α1 integrin subunit expression on various cell types, since this subunit is important in binding to adhesive substrates. RA FLS on gelatin appeared most sensitive to MZA, in that 1 µM could significantly detach cells in as little as 8 hrs (n=4, p<0.05). RA FLS were more adherent to laminin and most adherent to collagen IV. In contrast, normal or OA fibroblasts on gelatin required incubation with 10 µM MZA for 12 hrs to obtain significant detachment. Flow cytometry did not demonstrate differences in α1 integrin expression between MZA treated cell types (RA FLS, OA fibroblasts, or normals) and controls. We conclude that MZA is exerting both a concentration and time dependent effect on cell adhesion and that α1 integrin may not be involved.
Characterization of synapse-kinapse balance in CD8 T cells (173.35)
Authors: Mayya Viveka , Neiswanger Willie , Blair David , Wiggins Chris , Dustin Michael
Published: January 2023

T cells form stable junctions, referred to as ‘Immunological Synapses’ (IS), and also moving junctions, referred to as ‘Immunological Kinapses’ (IK), with surfaces presenting relevant adhesion and activating ligands from antigen-presenting cells (APCs). We have observed that the naïve CD8 T cells are biased towards IK, whereas antigen-experienced CD8 T cells are biased toward IS. We have also observed that naïve CD8 T cells show multiple inter-conversions between IS and IK states as previously described for naïve CD4 T cells. Further, the naïve CD8 population exhibits considerable heterogeneity in IS-IK states. Inhibition of PKCθ, myosin II, and rhoA GTPase activity promotes the IS state. The memory CD8 T cells show dispersed staining of phosphorylated ERM proteins (pERM), indicating that pERM is distributed throughout the cytoplasm. In contrast, the naïve CD8 T cells have polarized accumulation of pERM proteins, with a predominant banding pattern at one end of the cell periphery above the plane of the immunological synapse. Interestingly, the polarized accumulation of pERM is lost upon inhibition of PKCθ. These results provide a mechanistic insight into the role of PKCθ in promoting IK state in naïve CD8 T cells.
Neutrophil activities are required with hapten-primed CD8 T cell production of IFN-γ to mediate the edema of contact hypersensitivity (173.9)
Authors: Kish Danielle , Fairchild Robert
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Effector CD8 T cell recruitment into the hapten challenged skin parenchymal tissue to elicit CHS requires prior CXCL1-directed neutrophil infiltration within 3-6 h after challenge. Depletion of neutrophils at the time sensitized mice are challenged with hapten ablates CD8 T cell infiltration into the challenge site indicating the requirement for this initial neutrophil infiltration. The current study tested whether the CHS response to 2, 4-dinitrofluorobenzene required neutrophils at times the effector CD8 T cells had infiltrated and were activated in the hapten challenged skin tissue parenchyma. Neutrophil depletion 8 h following hapten challenge of sensitized mice eliminated CHS swelling responses 16 h later despite flow cytometry and immunohistochemistry analyses of the skin challenge site indicating the presence of infiltrating T cells. ELISPOT analyses also indicated the presence of hapten-specific T cells producing IFN-γ and IL-17 in the skin challenge site of sensitized mice depleted of neutrophils 8 h after challenge and IFN-γ mRNA expression levels in the skin challenge site were similar in sensitized mice with or without the neutrophil depletion. These results indicate that neutrophil-mediated activities are required at the time of hapten-primed CD8 T cell activation in the skin challenge site parenchyma to mediate the tissue edema of the CHS response.
Catalytic inhibition of TYK2 exerts selective effects on human cytokine responses (174.13)
Authors: Sohn Sue , Barrett Kathy , Chang Christine , Kohli Pawan , Liang Jun , Blair Wade , Magnuson Steven , Wu Lawren
Published: January 2023

TYK2 is a JAK family protein tyrosine kinase activated in response to multiple cytokines, including Type I IFNs, IL-6, IL-10, and IL-12/23. Genetic mutation at the tyk2 locus has been linked to broad-range immune deficiency and dysregulation in a human patient, however in murine strains that lack TYK2 expression, only select pathways are compromised. We have utilized a panel of TYK2-selective small molecule inhibitors with varying degrees of selectivity against other JAK kinases to address the requirement for TYK2 catalytic function in cytokine response pathways in human cells. Our results suggest that, similar to the effects of TYK2 deficiency in murine cells, the scope of biological processes requiring TYK2 catalytic function is much more narrow and selective in human cells than previously appreciated.