bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of pro-inflammatory Th17 responses during T. cruzi infection by IL-12-family cytokines (164.3)
Authors: Cobb Dustin , Smeltz Ronald
Published: January 2023

Previously we reported that the transcription factor T-bet (Tbx21) regulates Th17 responses to Trypanosoma cruzi infection in an IFN-γ-independent manner. In an effort to further understand this regulation, we examined the development and plasticity of Th17 cells during T. cruzi infection. Th17 cells recovered from infected Tbx21-/- mice were amenable to the inhibitory effects of T-bet, as ectopic expression of T-bet reduced IL-17 expression. We subsequently addressed the role of IL-12 family cytokines IL-12 and IL-27. We report that IL-12p35-/- mice infected with T. cruzi exhibited a significant increase in Th17 cells and peripheral blood neutrophilia, as well as reduced T-bet expression. Ex vivo culture of these cells with IL-12 led to a dramatic reduction in IL-17 production and concomitant increase in IFN-γ. Importantly, the ability of IL-12 to suppress IL-17 was independent of IFN-γ. Surprisingly, IL-27 had no observable effect on Th17 responses to T. cruzi, as infected Ebi-3-/- mice failed to show a significant increase in T. cruzi-specific IL-17 or neutrophilia. Furthermore, IL-27 could not compensate or synergize with IL-12 to suppress IL-17 production ex vivo. Thus, we have established that IL-12, not IL-27, is critical for regulating Th17 responses to T. cruzi.
Cytokine production and cardiac fibrosis in mice reinfected with Trypanosoma cruzi strains (164.8)
Authors: Machado Juliana , Silva Crislaine , Silva Marcos , Borges Diego , Castellano Lúcio , Reis Marlene , Lazo Javier , Ramirez Luis , Rodrigues Virmondes , Rodrigues Denise
Published: January 2023

Introduction: In areas of active transmission of Chagas' disease may occurs reinfection due to repeatedly infections with different T.cruzi strains, but little is known about the immune response induced by reinfections. Objectives: Evaluate the cytokine production and cardiac fibrosis in mice infected and reinfected with T.cruzi strains. Methods: C57BL/6 mice were infected with 3000 trypomastigote forms of T.cruzi (Y strain and Colombian strain). After 90 days, mice from each group were reinfected with the Y strain or with the Colombian strain. A further group were euthanized with only the first infection. After 21 days of the second infection mice were euthanized. The spleen cells were cultured with crude T.cruzi antigen and the cytokines was determined by CBA in culture supernatants. The hearts were removed for measurement of fibrosis. Results: Mice infected with Colombian strain and reinfected with the Y strain showed significantly higher levels of IL-4, IFN-γ and fibrosis. TNF-α was significantly higher in animals infected with the Colombian strain. IL-2 and IL-5 did not show significant difference. Conclusion: The reinfection of mice with the homologous or heterologous T.cruzi strains is associated with modulation of the immune response, as measured by cytokine production by spleen cells in response to antigens of T.cruzi. Modulation of immune response due to reinfection may also interfere on fibrosis formation in the heart tissue in these model of chagas' disease.
Chimeric antigen receptor (CAR) T cell cytokines shape myeloid cell function within the tumor microenvironment and activate host lymphocytes for tumor elimination. (165.4)
Authors: Spear Paul , Barber Amorette , Rynda-Apple Agnieszka , Sentman Charles
Published: January 2023

The infiltration of suppressive myeloid cells into the tumor microenvironment restrains anti-tumor immunity. However, T cell effector cytokines can shape the function of the myeloid lineage to support tumor rejection, regulating the balance between pro- and anti-tumor immunity. In this study, it is shown that effector cytokines secreted by adoptively transferred T cells expressing a chimeric antigen receptor (CAR) shape the function of myeloid cells to promote endogenous immunity and tumor destruction. C57BL/6 mice bearing the ID8 ovarian tumor were treated with effector T cells transduced to express a chimeric NKG2D receptor. GM-CSF produced by the adoptively transferred T cells recruited peripheral F4/80loLy-6C+ cells to the tumor site in a CCR2-dependent fashion. T cell IFN-γ and GM-CSF activated tumor-associated macrophages, increased IL-12p40 production, and augmented antigen presentation by host macrophages to antigen-specific host T cells. In addition, host F4/80+ cells secreted the chemokines CXCL9 and CXCL10 and host T cells were recruited to the tumor site in a CXCR3-dependent manner. Endogenous CD8+ T cells, but not CD4+ T cells, were required for complete tumor elimination. However, host CD4+ T cells were critical for the development of a tumor-specific memory T cell response. These studies show that host macrophages activated by CAR-bearing T cell-derived cytokines inhibited ovarian tumor growth by directly lysing tumor cells and promoting host T cell immunity.
In vivo administration of a novel synthetic GMCSF and IL7 fusion cytokine (GIFT7) leads to transient thymic hyperplasia by driving double-negative (DN), single-positive CD8 (SPCD8) and gamma-delta (γδ) thymocyte proliferation (165.7)
Authors: Hsieh Jeremy , Galipeau Jacques
Published: January 2023

T-cell deficiency secondary to cytotoxic agents, viral assaults or ageing predisposes to defective adaptive immunity. Therefore, therapeutic approaches to enhance thymic output leading to T cell reconstitution are of clinical importance. We develop a novel synthetic fusion cytokine between Granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin 7 Fusion Transgene (GIFT7). In vitro, GIFT7 acts as a hyperagonist for the IL7 receptor (IL7R) leading to hyperphosphorylation of STAT5. To test the effect of GIFT7 on IL7R-expressing primary thymocytes, we dissociated normal thymii from C57BL/6 mice and exposed unfractionated thymic cells in vitro to GIFT7 for 5 days. We observed a marked anti-apoptotic and proliferative response in CD3+CD4-CD8+ single-positive CD8 (SPCD8) and CD4-CD8-CD44+CD25- double negative 1 (DN1) T cell precursors in both αβ and γδ T cell lineage. In vivo, intravenous administration of recombinant GIFT7 at 5 µg/kg daily for 2 days into non-irradiated mice leads to a significant 2 fold increase in thymic DN1 population after 7 days. After 14 days, there was a subsequent 100% increase in total thymocyte number which returned to baseline at day 35. We propose the use of GIFT7 as a novel IL7R targeted thymopoietic growth factor for the treatment of catastrophic human immune deficient ailments characterized by thymic involution such as congenital or HIV-mediated acquired immunodeficiency, post-chemotherapy/radiation therapy and ageing.
A novel synthetic GMCSF and IL7 fusion cytokine (GIFT7) overcomes T cell exhaustion leading to central memory T cell formation and promoting peripheral T cell expansion in a PD1-low state (165.8)
Authors: Hsieh Jeremy , Micci Lucca , Silvestri Guido , Paiardini Mirko , Galipeau Jacques
Published: January 2023

T-cell based immunotherapy represents a promising strategy for chronic viral infections or malignancies. However, its efficacy depends on the infused T cell fitness in vivo. Indeed, T cell exhaustion via the upregulation of PD-1 reflects the maladaptive immune response as the root cause of effector cell failure. To address this issue, we report the development of a novel synthetic fusion cytokine between Granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin 7 Fusion Transgene (GIFT7). We show that GIFT7 leads to a hyperagonistic response manifest by increased pSTAT5 and proliferation in CD3+/CD127+ cells. GIFT7 also selectively expand a CD8+ subset from pre-activated T cells with a Central Memory phenotype defined as CD8+CD44+CD62L+CCR7+KLRG-CD27+PD1-, hereafter GIFT7CD8. Adoptive transfer of OTI-derived GIFT7CD8 into OVA+EG7-bearing mice leads to a significant anti-tumor effect. Furthermore, GIFT7CD8 persist long-term whilst IL2-actiaved CD8 T-cells are undetected. We also demonstrate the unique expansive effect of the human ortholog of GIFT7 on peripheral blood mononuclear cells (PBMC) derived from non-human primates (NHP) and humans, in that GIFT7 stimulation of pre-activated PBMC leads to T cell proliferation without exhaustion (Ki67hi and PD1low) in CD4+, CD8+, and CD4-CD8- compartments. This observation supports the use of GIFT7 primed T-cells for chronic viral infections or malignancies where an inadequate T cell response is a common feature.
Nanoparticle vaccines enhance humoral responses to a malaria antigen by expanding Tfh cells and inducing germinal center formation (166.10)
Authors: Moon James , Suh Heikyung , Li Adrienne , Yadava Anjali , Irvine Darrell
Published: January 2023

Nanoparticles (NP) are promising delivery vehicles for vaccines that promote humoral and cellular immunity in concert. We recently developed a novel lipid-based NP vaccine system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs). ICMVs efficiently entrap and co-deliver antigens and molecular adjuvants to lymphoid tissues, initiating potent cellular and humoral immune responses. ICMVs were synthesized by fusing liposomes containing antigen into multilamellar vesicles, followed by bilayer-to-bilayer crosslinking of lipid bilayers within the vesicle walls to stabilize the particles. ICMVs carrying ovalbumin and the TLR agonist MPLA induced robust cytotoxic T lymphocyte responses in mice, achieving a peak ~30% tetramer+ cells in the CD8+ T-cell population after 3 immunizations. Mice vaccinated with ICMVs containing a candidate Plasmodium vivax antigen raised high-titer, high-avidity antibody responses against the antigen lasting > 1 yr, at 10-fold lower dose of antigen than conventional adjuvants such as alum or Montanide. Compared to vaccination with alum, immunization with ICMVs prolonged antigen accumulation at the draining lymph nodes, enhanced germinal center formation, and increased the expansion of antigen-specific follicular helper T-cells, partially explaining the enhanced antibody response. These results suggest that antigen/adjuvant-carrying ICMVs may serve as a platform to elicit potent cellular and humoral immune responses against infectious pathogens.
Generation of the Recombinant M. smegmatis and M. bovis (BCG) Vaccine with genes of Enteropathogenic E. coli bfpA and intimin. Dias da Silva, W, 1, 3, Vasconcellos, H.L.F. 1,2,3 ; Scaramuzzi, K.1,3; Rodriguez, D.2 ; Nascimento, I.P.2; 1 Laboratório de Imunoquímica, Instituto Butantan São Paulo, SP, Brazi. 2 Laboratório de Biotecnologia, Instituto Butantan São Paulo, SP, Brazil. 3 Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil. (166.15)
Authors: daSilva Wilmar , Vasconcelos Halyka , Scaramuzzi Karina , Nascimento Ivan , Junior Jorge , Pizza Rochane , Kipnis André
Published: January 2023

Escherichia coli (EPEC) is important cause of diarrhea in children. EPEC adhere the intestinal epithelium causing the attaching and effacing (A/E) lesions. Recombinant M. smegmatis (MS) and BCG were constructed to express bfpA or intimin. Briefly, the whole bfpA gene and the intimin portion were amplified by PCR primers from genomic DNA (EPEC), and inserted into pMIP12 at the BamH I/KpnI sites. The vectors pMIP_bfpA and pMIP _intim were introduced separately into MS and BCG. Recombinants clones were selected based on resistance to kanamycin and named rSmeg_pMIP_(bfpA or intimin) and rBCG_pMIP_(bfpA or intimin). The expression of bfpA and intimin were detected by Western blotting using polyclonal anti-BfpA or anti-Intimin as first antibodies. The immunogenicity of these proteins was assessed in C57BL/6 mice. IgA and IgG antibodies anti-BfpA and anti-Intimin were detected in feces or in serum. TNF-α and INF-γ were produced in vitro by spleen cells from mice immunized with recombinant BfpA and recombinant Intimin. Adhesion of EPEC (E2348/69) on Hep-2 target cells was blocked by IgA or IgG from mice immunized with recombinant BfpA or Intimin, but not by non-immune antibodíes. Immunogenic non-infective vectors containing relevant EPEC virulence genes are promising vaccines.
Enhancement of the long term efficacy of BCG vaccine against experimental tuberculosis in mice through rapamycin induced modulation of CD4 and CD8 T cell functions (166.17)
Authors: Bakhru Pearl , Soudani Emily , Khan Arshad , Yuan Daniel , Lewis Dorothy , Hunter Robert , Jagannath Chinnaswamy
Published: January 2023

BCG vaccine protects children but does not induce long-term immunity against tuberculosis. Rapamycin has been found to directly act on MTOR of T cells and extend the longevity of CD8 T cell response to viral infection in mice. We hypothesized that rapamycin co-administration during BCG vaccination may alter the quality of CD8 function to enable long-term immunity. Methods: C57Bl/6 mice were given one dose of BCG vaccine (1 million per mouse; s.c.) or BCG with doses of rapamycin (75 microgram/mg given daily for 30 days) followed by aerosol infection with virulent Mycobacterium tuberculosis (Mtb). At 4 and 8 weeks, antigen specific CD8 T cells, memory specific markers and tetramers specific for Mtb antigens-ESAT6 and TB10.4 were analysed using flow cytometry. Log10 decline of Mtb counts in lungs was determined to correlate T cell phenotype with protection after vaccination. Results: Rapamycin coadministration with BCG enhanced the expansion of antigen-specific CD8 T cells in mice that correlated with a decline in the counts of Mtb in lungs and spleens of mice. Rapamycin increased both CD8 and CD4 T cells in the lungs and spleens and enhanced central memory (Tcm) CD8 T cells in lymphoid organs. Tcm levels, in turn, correlated with an increase in expression of Eomes transcription factor by CD8 T cells. Since Eomes is a determinant of Tcm lineage, we suggest that the long-term efficacy of BCG vaccine can be enhanced through modulation of MTOR dependent pathways of T cells.
Protective B cell epitopes of Francisella tularensis O-polysaccharide targeted by mouse and human antibodies (166.22)
Authors: Lu Zhaohua , Rynkiewicz Michael , Madico Guillermo , Roche Marly , Wang Qi , Hui Julia , Perkins Hillary , Seaton Barbara , Zaia Joseph , Costello Catherine , Sharon Jacqueline
Published: January 2023

Francisella tularensis (Ft), the gram-negative bacterium that causes tularemia, is considered a potential bioterrorism agent. Antibodies to Ft lipopolysaccharide (LPS) are protective against respiratory tularemia in mouse models, and we have previously described mouse monoclonal antibodies (MAbs) to non-overlapping terminal and internal epitopes of the Ft LPS O-polysaccharide (OAg). In the current study, we used competition ELISA with oligosaccharides of defined OAg repeat length, x-ray crystallography and docking studies to map the epitope of the terminal-binding MAb FB11 to one OAg tetrasaccharide repeat at the non-reducing end, and the epitope of the internal-binding MAb Ab52 to six sugar residues, spanning two OAg repeats, each repeat contributing three residues. Both FB11 and Ab52 conferred or prolonged survival in a mouse model of respiratory tularemia against Ft type B live vaccine strain (LVS) or the highly virulent Ft type A strain SchuS4, respectively. The two MAbs were successfully used as reporters, in competition ELISA, to demonstrate the presence of antibodies to both terminal and internal Ft OAg epitopes in the sera of tularemia patients. These results indicate that two Ft OAg repeats are necessary and sufficient for antibody binding to both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularemia. Furthermore, the FB11 and Ab52 MAbs could be used to monitor protective B cell responses in tularemia vaccine trials.
Probiotics and lactogenic immune factors affect neonatal B cell responses to rotavirus vaccine (166.26)
Authors: Chattha Kuldeep , Vlasova Anastasia , Esseili Malak , Kandasamy Sukumar , Siegismund Christine , Rajashekara Gireesh , Saif Linda
Published: January 2023

Breast milk (milk/colostrum [cols]) components and gut commensals play important roles in neonatal immune maturation, establishment of gut homeostasis and immune responses to oral vaccines. We investigated the impact of lactic acid bacteria colonization (Lactobacillus rhamnosus strain GG and Bifidobacterium lactis Bb12, probiotics) with/without mother’s milk/cols on B cell responses to an attenuated human rotavirus (RV) vaccine in a neonatal gnotobiotic pig model. Milk/cols did not affect probiotic colonization in RV vaccinated pigs. However unvaccinated pigs fed milk/cols shed higher fecal titers of probiotic bacteria than non-cols/milk fed colonized controls (p<0.05). In RV vaccinated pigs, milk/cols feeding with probiotic treatment resulted in a trend for higher mean serum IgA RV antibody titers and intestinal IgA antibody secreting cell (ASC) numbers compared to milk-fed, non-probiotic colonized pigs. Probiotic colonization alone did not affect IgA RV antibody titers in vaccinated pigs, but serum IgG RV antibody titers (p<0.05) and gut IgG ASC numbers were lower, suggesting that certain probiotics impact RV vaccine responses. In vaccinated pigs, probiotic or milk/cols supplementation did not enhance total intestinal B cell numbers above that induced by attenuated RV vaccine alone. Our findings suggest that probiotics and milk/cols components (soluble mediators), alone or synergistically, modulate neonatal immune responses to oral RV vaccines, requiring further evaluation.
A vaccine formulated with the Chlamydia trachomatis (Ct) recombinant major outer membrane protein (rMOMP) and TLR2 and TLR9 agonists as adjuvants elicits strong protection against an intranasal challenge (166.4)
Authors: Cheng Chunmei , Pal Sukumar , Tifrea Delia , de la Maza Luis
Published: January 2023

Ct is the most common sexually transmitted bacterial pathogen in the World and there is an urgent need for a vaccine to prevent these infections. To enhance the immunogenicity of a vaccine formulated with the Ct mouse pneumonitis rMOMP, we used combinations of Pam2CSK4 (TLR2) + CpG1826 (TLR9) and Montanide ISA 720 + CpG1826 as adjuvants. Neisseria gonorrhoeae recombinant porin B was used as antigen control with the same adjuvants. Female BALB/c mice were primed twice in the nares (i.n.) or in the colon (cl.) and were boosted twice by the intramuscular plus subcutaneous (i.m.+s.c.) routes. Based on the IgG2a/IgG1 ratio in sera, mice immunized with rMOMP+Pam2CSK4+CpG1826 showed a strong Th2 response while animals vaccinated with rMOMP+Montanide+CpG1826 had a Th1 response. These mice also developed robust Ct-specific T cell proliferation and high levels of IFN-γ. Four weeks after the last immunization, the mice were challenged i.n. with 104 inclusion-forming units (IFU) of Ct. Using changes in body weight, weight of the lungs and number of IFU recovered from the lungs at 10 days post-challenge as parameters of protection, the mice immunized i.n.+i.m.+s.c. with rMOMP+Pam2CSK4+CpG1826 were better protected against the i.n. chlamydial infection than the other groups. In conclusion, a combination of Pam2CSK+CpG1826 elicits strong humoral and cellular immune responses and enhances the protective ability of a Ct-rMOMP vaccine delivered by the mucosal and systemic routes.
Role of Dectin-2 in the serotype-specific antibody production and host protection against Streptococcus pneumoniae infection caused by pneumococcal polysaccharide vaccine (166.8)
Authors: Miyasaka Tomomitsu , Akahori Yukiko , Miyamura Namiko , Toyama Masahiko , Saijo Shinobu , Iwakura Yoichiro , Kinjo Yuki , Oishi Kazunori , Kawakami Kazuyoshi
Published: January 2023

Anti-pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific Ab including IgG2 in spite of its feature of thymus-independent antigen. It remains to be clarified how PPV causes a class-switching from IgM to IgG. Recently, C-type lectin receptors (CLR) sensing polysaccharides have garnered much attention in the host defense to fungal infection. In our recent study, Dectin-2, a CLR involved in the recognition of mannose-rich polysaccharides, has been found to play an important role in the host defense to pneumococcal infection. Therefore, in this study, we addressed how Dectin-2 was involved in the vaccine effect of PPV using a mouse model. The serum levels of anti-serotype 3 pneumococcal capsular polysaccharide (PPS-3) two weeks after vaccination were significantly reduced in Dectin-2 knockout (KO) mice compared to wild-type (WT) mice. Weight loss and hypothermia of mice caused by infection with Streptococcus pneumoniae (URF918) were improved in WT mice transferred with spleen cells from vaccinated WT mice compared to those from vaccinated Dectin-2KO mice. Similar tendency was observed in the survival of infected mice. In in vitro experiments, IL-12p40, TNF-α and IL-6 production by bone marrow-derived dendritic cells (BM-DCs) stimulated with PPV was abrogated in Dectin-2KO. These results suggest that Dectin-2 is involved in the PPV-induced serotype-specific Ab production and host protection through sensing the pneumococcal polysaccharide antigens.
Modulation of immune cell function by an early life experience (167.11)
Authors: Smith Susan , Williamson Lauren , Sholar Paige , Bilbo Staci
Published: January 2023

Pro-inflammatory IL-1 beta cytokine production within the hippocampus (HP) is required for normal learning. However, excessive IL-1 beta levels in the brain can interfere with memory consolidation, resulting in cognitive deficits and neurodegeneration. We have identified the cellular source of IL-1 beta during learning as microglia, a CNS-resident macrophage cell type. Intriguingly, microglia from adult rats that suffered an immune challenge early in life are “primed” for exaggerated IL-1 beta responses, which is maladaptive because it can interfere with normal learning and memory. Similar to control cells, primed microglia do not produce cytokines constitutively. Rather, exaggerated cytokine responses in primed microglia likely result from an altered pattern of transcriptional regulation that was initiated during an early life infection and is propagated into adulthood via specific epigenetic markings. We have found that HP microglia from neonatally infected rats have a global reduction in dimethylated histone H3K9 compared to control (mock-infected) rats. The specific gene loci involved in the regulation of microglial cytokine expression from primed and control rats are now under investigation. Thus, our model of early life infection demonstrates a role for brain-resident immune cells in mediating the “programming” of a later cognitive dysfunction via intrinsic differences in their epigenetic status.
The role of splenic CD103+CD8α+cDCs in the induction of CTL responses during respiratory virus infection (168.11)
Authors: Morimoto Junko , Nakayama Yosuke , Danzaki Keiko , Kajino Kiichi , Uede Toshimitsu
Published: January 2023

We have previously reported that large numbers of virus-specific CD8+ T cells were detected in the spleen following intranasal influenza virus infection. This finding does not seem simply due to the migration of cells from the draining mediastinal lymph nodes (mLN), but rather indicates that spleen can be a site for the induction of primary CTL responses. Although the integrin αEβ7 (CD103)+ migratory DCs have been known as a major DC subset which prime naïve influenza virus-specific CD8+ T cells in the mLN, what kind of DC subset is responsible for the induction of CTL responses in the spleen remains unclear. In this study, we found that increased numbers of CD103+DCs were detectable in the spleen following influenza virus infection, and the majority of CD103+DCs expressed CD8α. Although both CD103+CD11chi DCs (CD103+DCs) and CD103-CD11chi DCs (CD103-DCs) showed activated phenotype evidenced by the expression of co-stimulatory molecules at 9 days post infection, PDL-1 expression pattern was different between these two subsets. Immunohistochemical analysis showed that CD103+DCs localized in the T cell area (also in the B cell area) and purified CD103+DCs produced large amounts of IL-12p40 compared with CD103-DCs. These data suggest that splenic CD103+DCs may play a critical role in the development of CTL responses during influenza virus infection. We are currently investigating how efficiently splenic CD103+DCs act as antigen presenting cells.
Effects of zidovudine on murine viral infection-induced peripheral neuropathy and cytokine responses (168.12)
Authors: Cao Ling , Butler Michael , Draleau Kyle
Published: January 2023

LP-BM5, a murine retroviral isolate, causes a severe acquired immunodeficiency syndrome in C57BL/6 mice, similar to that caused by HIV infection, thus the term murine AIDS (MAIDS). Previously, we have shown that C57BL/6 mice infected with LP-BM5 developed significant hind paw mechanical hypersensitivity, behavioral sign of peripheral neuropathy. To test whether profound inflammatory responses in the nervous system contribute to the development of LP-BM5-induced peripheral neuropathy, we first determined the levels of selected cytokines (IL-1β, IL-6, TNFα, IFNγ, and IL-12) in the lumbar spinal cord and dorsal root ganglia (DRG) by qRT-PCR and in the hind paw skin with FlowCytomix multiplex assay post-LP-BM5 infection (p.i.). In the lumbar spinal cord, mRNA levels of all these cytokines trended upwards over time p.i., whereas in the DRG, IL-1β and IFNγ mRNA levels elevated throughout the time p.i. In the hind paw skin, all cytokines measured (except TNFα that was not detectable) increased transiently and peaked at 4-6 weeks p.i.. Further, administration of the anti-retroviral agent zidovudine (AZT) via drinking water (0.2 mg/ml) significantly reduced LP-BM5-induced mechanical hypersensitivity and cytokine responses in the lumbar spinal cord and DRG at 12 weeks p.i.. Altogether, data indicate that tissue-specific cytokine responses are associated with the development of LP-BM5-induced peripheral neuropathy and these responses can be inhibited by AZT treatment.
Limited Type I IFNs Play a Role in the Pathogenesis of Respiratory Syncytial Virus in Neonatal Mice (168.17)
Authors: You Dahui , Lee Greg , Shrestha Bishwas , Shen Li , Cormier Stephania
Published: January 2023

Respiratory syncytial virus (RSV) remains a major cause of bronchiolitis in infants. Infants developing severe RSV disease have increased risk of developing asthma; the causes of which are unknown. We and others have previously demonstrated that RSV infection results in a skewed Th2 response, which may be responsible for this phenomenon. Here, we show that neonatal mice (< 7 d) expressed limited amounts of type I IFNs in the lung, which was associated with low numbers of pulmonary plasmacytoid dendritic cells (pDCs; a major source of type I IFNs during viral infections) following RSV infection. When neonatal mice were pre-treated with IFNa, the typical Th2 skewed response was decreased compared to untreated control mice. Neonatal mice treated with IFNa demonstrated decreased pulmonary Th2 (IFNg+CD3+CD4+) and Tc2 (IFNg+CD3+CD8+) cells, decreased eosinophils and mucus hyperproduction in the airways, and decreased inflammation in the lung parenchyma upon reinfection. More importantly, pulmonary function of the IFNa treated mice was significantly improved compared to the control mice. We conclude that the limited amount of type I IFNs in the neonates, possibly due to an inchoate pulmonary pDC response, may be partially responsible for the Th2 skewed immune response resulting from neonatal RSV infection. Our study pinpoints IFNa as a possible therapeutic target during infant RSV infection.
Changing expression of and access to STATs in NK cells during viral infections (168.2)
Authors: Kallal Lara , Biron Christine
Published: January 2023

NK cells can respond to viral infections with heightened killing, cytokine production, and proliferation. These functions are influenced by cytokines produced during infections that signal through STAT molecules. The laboratory has previously shown that NK cells express high basal levels of STAT4 and that type 1 IFNs can access STAT4 under these conditions. However, during infections of mice with lymphocytic choriomeningitis virus (LCMV), high STAT1 expression is induced to result in type 1 IFN signaling through STAT1 over STAT4 and subsequent immunoregulatory effects. Here, studies were extended to include STAT3, a known pro-survival and pro-proliferative molecule, and infections with murine cytomegalovirus (MCMV). In contrast to LCMV, MCMV elicits a more complex innate response with high induction of IL-12 as well as type 1 IFN. STAT1 and STAT4 levels were similar in NK cells between both infections, but NK cell STAT3 was increased earlier following MCMV infection. This difference had consequences for NK cell access to the STATs. During both MCMV and LCMV infections, IFN-alpha could activate STAT1, and IL-12 could activate both STAT3 and STAT4. However, IFN-alpha could access STAT3 only during MCMV infection. Ongoing studies are characterizing the pathway to STAT3 induction and the role for this molecule in shaping NK cells during viral infections. Overall, the work is elucidating the pathways for conditioning innate immune responses.
Toll-like receptor 3 signaling inhibits double-stranded RNA-induced apoptosis of macrophages (168.21)
Authors: Ge Baoxue , Chen Jian-Xia , Liu Haipeng , Deubel Vincent , Si-Tahar Mustapha , Alexopoulou Lena , Qin Chengfeng , Liang Guo-Dong , Flavell Richard
Published: January 2023

Most viruses produce dsRNA during their infection. Recognition of dsRNA by TLR3, PKR, RIG-I, or MDA-5 initiates innate immune responses, RIG-I and MDA-5 are sensors for dsRNA to induce proinflammatory cytokines or type I interferon, whereas PKR mediates dsRNA-induced apoptosis. Here, we reported that TLR3 deficiency enhances apoptosis of macrophages in response to poly(I:C) as well as Japanese encephalitis virus (JEV). Unexpectedly, TLR3 deficiency markedly increased the phosphorylation of PKR, and inhibition of PKR by RNAi or a PKR-specific inhibitory oligonucleotide (ION) reduced poly(I:C)-induced apoptosis of TLR3-/- macrophages. Stimulation of TLR3 by poly(I:C) activates PI3K and NF-κB signaling pathways, and inhibition of either PI3K or NF-κB enhanced the poly(I:C)-induced apoptosis of macrophages. Further analysis revealed that TLR3 deficiency, as well as inhibition of PI3K or NF-κB, markedly induced p58IPK, a PKR inhibitor, and inhibition of p58IPK by RNAi reduced the apoptosis of poly(I:C)-induced TLR3-/- macrophages. Lastly, TLR3-/- mice are more resistant to JEV infection, but inhibition of apoptosis by caspase inhibitors reduces their resistance to lethal poly(I:C) stimulation or JEV infection. Together, our findings suggest that stimulation of TLR3 by viral dsRNA may activate PI3K and NF-κB, which induces the p58IPK to suppress the phosphorylation of PKR, thus protecting dsRNA-stimulated or virus-infected cells from apoptosis to facilitate the virus propagation.
Examining the Role of Neutrophils in Respiratory Syncytial Virus (RSV) Infection (168.26)
Authors: Stokes Kate , Moore Martin
Published: January 2023

The inflammatory process in the respiratory tracts of respiratory syncytial virus (RSV)-infected infants is not well characterized. Respiratory syncytial virus infects bronchial epithelial cells, resulting in epithelial activation and damage and an increased neutrophil response. However, the role of neutrophils in RSV pathogenesis is not known. To examine their role, we used a monoclonal antibody to Ly6G to deplete neutrophils in BALB/c mice infected with an RSV clinical isolate. Mice were then examined for weight loss, viral load, cytokine production, mucus production, and lung pathology. Administration of anti-Ly6G resulted in depletion of neutrophils from RSV-infected mice in blood and lungs. Neutrophil-depleted, RSV-infected animals lost more weight than infected isotype-control treated animals. There was, however, no significant effect on viral load. Neutrophil-depleted, RSV-infected mice produced less mucin than isotype-treated, RSV-infected mice. Neutrophil depletion during RSV infection also resulted in decreased TNF-α, IL-13, IL-4 and MIP-1α levels in the lung. Neutrophil depletion during RSV infection resulted in greater bronchiolar and alveolar edema, perivascular cuffing, alveolar hemorrhage, and vasculitis compared to control-treated, RSV-infected mice. This data shows that neutrophils play a major immune modulation role in RSV infection, promoting TH2 cytokine expression and airway mucus expression but also protecting against lung damage and disease severity.
Mass spectrometry-based global profiling of eicosonoid expression in response to influenza A virus infection (168.32)
Authors: Hilmer Kimberly , Graham Amy , Zickovich Julianne , Hilmer Jonathan , Obar Joshua
Published: January 2023

Understanding the innate inflammatory pathways activated during influenza A virus infection is vital to developing effective therapeutics. Our results demonstrate that the inflammatory cascades induced by different influenza A virus strains are not identical. Mice infected with A/WSN/33 show less airway hyper-reactivity than mice infected with A/PR/8/34, likely due to decreased expression of the IL-33/IL-13 axis of inflammation. In addition, we have examined the lipidomic expression profile during flu infections from each of these strains. Lipid extracts were prepared from whole lungs, and specific lipid mediators were both identified and quantified using UHPLC-MS. This approach provides an automated, high-throughput, and high-sensitivity technique to characterize the lipidomic fingerprint of the infection resulting from various influenza A strains. The compounds differentially expressed between strains will ultimately be used to develop targeted therapeutics to lessen both the morbidity and mortality of the seasonal flu.
RON Receptor Tyrosine Kinase, a Negative Regulator of Inflammation, is Decreased During SIV Associated Central Nervous System Disease. (168.33)
Authors: Cary Daniele , Clements Janice , Henderson Andrew
Published: January 2023

Expressed on tissue resident macrophages, the receptor tyrosine kinase, RON, functions to maintain inflammation homeostatisis by activating genes that promote wound repair and resolve inflammation; while repressing genes that perpetuate tissue damage. Chronic HIV infection is associated with dysregulated inflammation that contributes to organ diseases, and we hypothesize that altered RON expression contributes to the development of HIV associated CNS disease. We developed a tractable in vitro model using macrophages isolated from human tonsil; these cells express CD14, CD16, CD206, CCR5, and RON. Treatment with RON ligand induced arginase and inhibited HIV and IL-12p40. Following infection, RON expression was down-regulated. We utilized a well characterized SIV model in macaques to explore the temporal regulation of RON in the brain. Following prolonged SIV infection, RON expression decreased. RON was maintained in animals that did not develop CNS disease and was reduced in macaques that demonstrated CNS disease symptoms. Arginase expression was reduced during late infection whereas expression of the inflammatory genes, interleukin 12p40 and TNFα was elevated. Our data support a model in which during acute SIV infection in the brain, active inflammation is controlled by RON, however, following prolonged infection, RON expression is decreased, genes that quell inflammation are repressed, and inflammatory mediators are induced to directly promote CNS disease.
Inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells (168.34)
Authors: Cao Ling , Malon Jennifer , Vaughn James , Birch Julie
Published: January 2023

We have observed that C57BL/6 (B6) mice infected with a murine retroviral isolate, LP-BM5 developed peripheral neuropathy, along with an elevated expression of calcitonin gene-related peptide (CGRP), in addition to glial activation in the spinal cord. To investigate how these spinal cord changes might contribute to the development of peripheral neuropathy, we examined the effects of CGRP on LP-BM5-induced glial cytokine responses in vitro. Mixed spinal cord glia were prepared from adult B6 mice and infected with various doses of LP-BM5 (0, 1, 10, 100, and 1000 pfu/ml), with or without CGRP co-treatment (1ug/ml). Resulting levels of selected cytokines (IL-1β, IL-6, TNFα, IFNγ, IL-12, and CCL2) in the culture supernatants were measured 1, 4, and 7 days post-treatment via ELISA. Viral loads in both the cells and supernatants were determined at day 7 post-treatment using plaque assay. While little virus was detected in the supernatants, CGRP treatment appeared to reduce viral loads in the infected glial cells. This reduction was correlated with the decrease of LP-BM5 induced TNFα production. IL-6 and CCL2 production, however, were enhanced by CGRP treatment. Further, CGRP did not affect the levels of IL-12p40, and IL-1β, IFNγ. IL-12p70 was not detectable following any treatment. Altogether, the neuropeptide CGRP appears to inhibit viral infection of glial cells. Further study will help to understand the role of CGRP during retroviral infection-induced neurological defects.
Varicella zoster virus can reactivate and infect cerebral adventital fibroblasts, suppressing Jak/STAT and causing stroke. (168.35)
Authors: James Stephanie , Choe Alexander , Traktinskiy Igor , Nagel Maria , Gilden Donald
Published: January 2023

Primary varicella zoster virus (VZV) infection causes varicella (chickenpox), after which virus becomes latent in ganglionic neurons. A declining cell-mediated immunity to VZV leads to virus reactivation, manifesting as zoster (shingles). Zoster is often complicated by chronic pain (post herpetic neuralgia) and other serious neurological disorders such as meningoencephalitis and myelitis. VZV vasculopathy results in transient ischemic attacks and stroke, and is the result of productive virus infection in cerebral arteries. More than 90% of the 6.75 billion world population harbors latent VZV. Adults with zoster have a 30% increased risk of stroke within the following year. Although the exact incidence of stroke caused by VZV is unknown, more than 900,000 people in the United States will develop zoster annually; thus, the magnitude of stroke caused by VZV is considerable. In a study of 3 VZV stroke patients, cerebral arteries were positive for VZV antigen and had intimal layer thickening. Previous experiments with VZV infected skin fibroblasts have shown that VZV inhibits the expression of STAT1α and Jak2, resulting in decreased expression of Interferon α and β (IFNα/β). Here we show that in VZV infected cerebral adventitial fibroblasts STAT1α and Jak2 are suppressed, which is not observed in controls. Thus, we postulate that viral infection of the adventitial fibroblasts suppresses the Jak/STAT pathway which allows for viral persistence, and potential stroke.
Pulmonary NK cell recruitment during influenza A virus infection (168.5)
Authors: Carlin Lindsey , Heusel Jonathan , Legge Kevin
Published: January 2023

Natural Killer (NK) cells are vital components of the antiviral immune response, but their contributions in defense against influenza A virus (IAV) are not well understood. To better understand the contribution of NK cells to IAV immunity, we examined the magnitude and kinetics of the NK cell response during high- and low-dose IAV infections. While an increased accumulation of NK cells in the lung in high-dose versus low-dose infections was observed, the NK cell response in the lymph nodes (LN) was blunted in high-dose infections. Using a dual labeling method to determine the rate of proliferation of pulmonary NK cells, our results show that while some local NK cell proliferation occurs during IAV infection, it is not enough to account for the increase in NK cells observed, suggesting that NK cells are instead largely recruited to the lungs. Therefore, we next determined the roles of the chemokine receptors CCR5 and CXCR3 in NK cell trafficking to the lung and LN using receptor blocking and knock out models. All together, our results demonstrate that NK cells are actively recruited to the lungs during IAV infection, suggesting differential NK cell recruitment could modulate the immune response to IAV during high- and low-dose IAV infections.
Role of lipoxygenase enzymes in RSV-induced differentiation of alternatively activated macrophages (AA-Mϕ) (168.7)
Authors: Shirey Kari Ann , Lai Wendy , Pletneva Lioubov , Blanco Jorge , Vogel Stefanie
Published: January 2023

Severe RSV-induced bronchiolitis has been associated with a mixed “Th1” and “Th2” cytokine storm. Arachidonic acid is the substrate for both COX-2 and 5-LO. In mice, TLR4, IFN-β, and IL-4/13 are required for RSV-induced differentiation of AA-Mϕ that counteract COX-2-induced lung pathology. We hypothesized that there is a reciprocal relationship between induction of COX-2 and AA-Mϕ genes during RSV infection. RSV infection of WT peritoneal macrophages resulted in the induction of 5-LO gene expression early in infection as well as activation of 5-LO enzyme activity later in infection. RSV-infection of 5-LO-/- macrophages and mice showed a loss of AA-Mϕ gene expression, while enhancing COX-2 mRNA and increasing the lung pathology in vivo. Blocking 5-LO or 15-LO with enzyme-specific inhibitors (AA-861 and PD-146176, respectively) also blocked differentiation of RSV-induced AA-Mϕ. RSV-infected cotton rats treated with saline or the COX-2-specific inhibitor, paracoxib, exhibited enhanced RSV-induced 5-LO mRNA expression. These data greatly extend our knowledge of the complex signaling that occurs during RSV infection as well as suggest novel treatment approaches for RSV that promote AA-Mϕ differentiation.
Viral induced encephalitis initiates distinct and functional CD103+ CD11b+ brain dendritic cell populations within the olfactory bulb. (168.8)
Authors: D'Agostino Paul , Kwak Changsoo , Vecchiarelli Haley , Gal Toth Judit , Miller James , Masheeb Zahrah , Bulloch Karen
Published: January 2023

Dendritic cells (DC) are antigen presenting cells found in both lymphoid and non-lymphoid organs, including the brain (bDC) of Cd11c/eyfp transgenic C57Bl/6 mice. Using an intranasal vesicular stomatitis virus infection, we demonstrated that EYFP+ cells amass in areas associated with viral antigens, take on an activated morphology, and project their processes into infected neuronal tissue within the olfactory bulb. These bDC separated into three EYFP+ CD45+ CD11b+ populations: EYFP+ CD45int CD11bhi (P1), EYFP+ CD45hi CD11bhi (P2) and EYFP+ CD45hi CD11bint (P3) populations. All but the P1 population were able to functionally promote both T lymphocyte proliferation and TH1 cytokine production using an OT transgenic antigen presentation assay. Using radiation chimeras, P1 was shown to emanate from the brain, while P2 was established to be peripherally derived. Interestingly, the P3 population was of indeterminate origin, being both radiosensitive and not replenished by donor bone marrow. Finally, each EYFP+ population contained CD11b+ CD103+ sub-populations, and could be distinguished in terms of M-CSFR, Gr-1, and Ly-6C expression. Such phenotypic analysis point to P1 as an activated sub-population of microglia, while P2 was more typical of mucosal DC subsets and P3 appeared to be similar to monocyte-derived DC lineages.
Altered human γδ peripheral blood lymphocyte homeostasis and impaired influenza A viral response in obesity (168.9)
Authors: Costanzo Anne , Taylor Kristen , Han Peggy , Fujioka Ken , Jameson Julie
Published: January 2023

Obesity is an independent risk factor for severe and fatal influenza infection. Little is known about how obesity impacts influenza-specific T cell responses in humans. In healthy adults, γδ T cells are the major circulating population of T cells that rapidly respond to influenza virus infected APCs. The majority express a Vγ9Vδ2 TCR that recognizes phosphoantigens expressed by infected or transformed cells. To determine whether this anti-viral T cell population was impacted by obesity, human peripheral blood mononuclear cells were isolated from non-obese and obese donors and analyzed for homeostatic T cell numbers and function. We found the percentage of Vγ9Vδ2 T cells negatively correlates with increased body mass index (BMI), suggesting the severity of obesity impacts γδ T cell homeostasis. Furthermore, Vγ9Vδ2 T cells exhibit a skewed maturation, with a higher propensity toward a unique γδ T effector CD45RA+ memory cell population. We have identified that obese patients show reduced expression of the homing molecule CCR5 on γδ T cells as well as reduced IFN-γ and TNF-α production in response to influenza infection. This blunted Vγ9Vδ2 T cell response in obese patients is T cell intrinsic as function can be rescued using a strong TCR agonist. Our data show that one arm of the antiviral immune response to influenza is compromised by obesity. These studies support the concept that therapies should be developed to restore immune homeostasis in obese patients.
Influenza A virus pathogenesis: Identification of host factors that contribute to severe respiratory distress (169.18)
Authors: Le Thuc-vy , Steel John , Compans Richard
Published: January 2023

The Center for Disease Control estimates that influenza virus infections contribute to 3,000-49,000 deaths in the United States annually. This broad range in deaths associated with influenza virus infections underscores the wide range in pathogenic effects that is observed following infection with influenza virus. While several factors contribute to pathogenesis, the mechanisms that govern how some infections result in severe respiratory distress, while others are resolved inconsequentially, are only partially defined. To explore the host cellular components that contribute to influenza virus pathogenesis, we utilized two genetically related strains of the 2009 H1N1 pandemic influenza virus A/Netherlands/602/2209 and A/California/07/2009. Although these viruses share nearly 100% sequence identity, mice that are infected with the different viruses exhibit great disparity in the severity of disease symptoms as measured by body weight, mortality and viral replication in the respiratory tracts. Our study reveals several key components that are critical in the balance between an immune response against influenza virus in which the virus is rapidly cleared and damage to the host is moderate, versus a response resulting in severe pathogenic effects that is marked by respiratory distress.
Regulatory Role of HMGB1 on complement activation via the classical pathway (169.3)
Authors: Kim Sook Young , Lee Sang Eun , Kwak Man Sup , Shin Jeon-Soo
Published: January 2023

Serum complement proteins play an important role in resistance to infection. The complement system is strongly activated in sepsis and the complement-activated products of anaphylatoxins have biological activities leading to inflammation. The nuclear protein, high mobility group box 1 protein (HMGB1) is released by inflammatory stimuli and plays a key role as a pro-inflammatory cytokine-like molecule. To investigate whether HMGB1 can induce complement activation, we have looked at the relationship between HMGB1 and complement system in this study. We first demonstrated the involvement of HMGB1 in binding to C1q for classical pathway activation. We observed the activation of complement cascade components such as C4b, C3b, C3c, and the membrane attack complex after C1q activation by HMGB1 in a dose-dependent manner. Moreover, complement activation could be observed in serum from mice after the intravenous injection of HMGB1. We then investigated the involvement of the A and B domain of HMGB1 during complement activation. HMGB1 B box, a well-known pro-inflammatory domain, was able to activate the complement system like wild type HMGB1, while HMGB1 A box showed no activation. The possible role of HMGB1 A box as an inhibitory domain in complement activation is now under investigation. In conclusion, the classical pathway of complement activation could be activated by the interaction of HMGB1 and C1q, showing the novel role of HMGB1 in the pathogenesis of HMGB1-induced shock.