bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of human ferroportin (SLC40A1) inferred from mass spectrometry restraints
Authors: Parry Christian , Vazquez-meves Guelaguetza , Ivanov Andrey , Lin Xionghao , Kumari Namita , Nekhai Sergei
Published: January 2023

Nearly all organisms require iron for red blood cell manufacture, respiration, metabolism and immunity, and as cofactor in several enzymes. Paradoxically, free iron is toxic from the production of reactive oxygen species which induce cellular injury and damage to DNA. It is essential that iron is tightly regulated. Ferroportin is the only known exporter of cellular iron in mammals. Its function and expression are tightly regulated by hepcidin. Carriers of ferroportin mutation Q248H show reduced sensitivity to hepcidin, have elevated iron stores and high HIV-1 viral load. In HIV-1 infection, there is marked alteration in iron balance indicated by high ferritin levels. As well, iron-regulating peptide hepcidin is greatly elevated. High iron stores correlate with high morbidity, increased opportunistic infections and faster progression to AIDS: high iron content in bone marrow macrophage parallels greater infection, immune dysfunction and poor prognosis. By contrast, other studies show that patients who were being treated for iron overload with iron chelators had delayed AIDS progression and longer survival. In spite of its importance little structural information is available on human ferroportin, and how iron is transported through ferroportin is not understood. We have built the structure of ferroportin using hybrid methods with restraints from mass spectrometry. Our model comprises 12 transmembrane helices. The iron binding site matches what is seen in crystal structures of distant orthologs. We are using this structure along with functional data to answer outstanding questions about the mechanism of ferroportin, iron transport and the importance of the Q248H mutation found in African and black American populations.
Immune Complex (ICs) generate PD1int CD4+ T cells which are Bcl6+IFN-γ+ unlike exhausted PD1high cells
Authors: Chauhan Anil K
Published: January 2023

PD1 is targeted for cancer therapy, however the mechanisms that drive its expression are poorly recognized. Role for Fc-receptors in CD4+ T-cells responses has been ignored in the last decade. We and others have now shown the activation induced expression of CD16a (FcγRIIIa) and CD32 (FcγRIIa) (doi:10389/fimmu.2018.02814). Three groups have now shown that CD32 expression in CD4+ T cells mark HIV-1 provirus enrichment. Thus, we examined whether immune complexes (ICs) by engaging CD16a play a role in PD1 expression and Tfh cell development.

Using flow cytometry, biochemical techniques and RNA-seq, we establish a new subset of CD4+ PD1intCD16a+ Tcells. These cells are present in vivo and can be generated in vitro upon IC signaling. These cells express Bcl6+ and produce IFN-γ, IL-17A, and IL-21. A second population of terminally exhausted PD1highCD16a−Bcl6− do not produce cytokines also exist. CD16a ligation by ICs generates CD4+ T effector cells and relocate endosomal NATLRs to the cell surface for joint signaling with FcRs (JI, 198: 4596, 2017).

CD16a signaling up-regulate Tfh transcription factors c-Maf, FOXO, Bcl6, also lincRNA and microRNA associated with TFH/T cell differentiation i.e. miR155, 17HG, 22 and HOTIP. Upregulation of IL-27 and ICOS via CD16a could induce c-Maf expression. CD16a signal in CD4+ T cells showed up-regulation of ubiquitination, heat shock proteins, proteasome assembly, GPCR signaling genes and 1139 genes from GO: 007062 category of extracellular exosomes.

Our findings establish a new mechanism that drives PD1 expression. Tfh cells are enriched in HIV-1 provirus and expression of FcRs in such cells suggest their role in viral enrichment. Does CD16a+ mark non-exhausted CD4+ Tfh cells remains an open question.
Activation of aryl hydrocarbon receptor with structurally diverse ligands represses class switch to immunoglobulin A
Authors: Costa Alex B , Hirsch Aspen L , DeKrey Gregory K
Published: January 2023

Activation of the aryl hydrocarbon receptor (AhR) with its prototypical agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) represses activation-induced cytidine deaminase expression and B cell class switch recombination (CSR). Structurally diverse AhR agonists have been shown to have differing impacts on T cell differentiation. We hypothesized that AhR activation with structurally diverse agonists would have differential impacts on CSR in B cells. The following agonists were tested: the endogenous ligands cinnabarinic acid (CA), 5,11-dihydroindolo[3,2-b]carbazole-6-carboxaldehyde (FICZ), and kynurenine (Kyn); the pharmaceuticals omeprazole (Omep) and tamoxifen (Tam); and the dietary component indole-3-carbanol (I3C). Mouse splenic CD19+ B cells were cultured with LPS, α-IgD-dextran, and TGF-β1 to stimulate CSR to IgA. IgA expression was measured by flow cytometry. Relative to control cells (10.84% IgA+), exposure to CA, FICZ, I3C, Kyn, Omep, Tam, or TCDD significantly decreased the proportion of IgA+ cells (P < 0.05) to 4.48%, 2.48%, 2.25%, 4.39%, 0.79%, 6.95% and 3.93%, respectively. In contrast, CH-223191, an AhR antagonist, significantly increased the proportion of IgA+ cells to 16.19%. These results suggest that repression of CSR is a common effect of AhR agonists in activated mouse B cells.
E3 ubiquitin ligase Fbw7 regulates B cell survival and T cell-independent responses
Authors: Ramezani-Rad Parham , Leung Charlotte R , Apgar John R , Rickert Robert C
Published: January 2023

B cells continuously populate peripheral organs to maintain a large number of diverse mature B cell clones. These cells have controlled temporal boundaries and await their potential activation in a naïve resting state. Newly emerging B cells are maintained by survival signals while previous B cell generations die through apoptosis. During activation, B cells alter their metabolic machinery to enable categorical fate decisions. The E3 ubiquitin ligase, Fbw7, mandates proteasomal degradation of several oncogenes involved in cellular proliferation, growth, and survival. To elucidate the role of Fbw7 in B cells, we studied the function of Fbw7 in a conditional knockout mouse model. Ablation of Fbw7 during early B cell development diminished the mature recirculating B cell pool in the bone marrow and decreased the B cell population in the spleen. Further, we found a drastic reduction of the B1 cells in the peritoneal cavity. These unexpected results prompted us to generate a novel acute deletion model for Fbw7 to study mature B1 and B2 cells. Acute deletion of Fbw7 confirmed reduction of B cell population within a few weeks. When challenged with T cell-independent antigens the overall antibody responses were reduced. BCR-stimulation of B cells ex vivo showed reduced calcium signaling, poor proliferation potential and upregulation of apoptosis in knockout cells. Ectopic expression of BCL2 in B cells, partially rescued the apoptotic fate of these cells. Our results indicate for the first time that Fbw7 is a critical regulator in B cells, contributing to survival signaling and modulating B cell responses.
Extracellular ST6Gal-1 calibrates B cell IgG production by cell non-autonomous extrinsic sialylation
Authors: Irons Eric Edward , Punch Patrick Raymond , Lau Joseph T.Y.
Published: January 2023

Cell surface glycans are well-positioned to modify receptor interactions and their downstream biological outcomes. Although canonical glycosylation occurs intracellularly, glycosyltransferases also exist in the blood and can catalyze extracellular reactions with platelet-derived sugars. We have recently reported that such extrinsic glycosylation by the sialyltransferase ST6Gal-1 enhances survival and BCR signaling in immature B cells. Here, we report that mice lacking extrinsic ST6Gal-1 had reduced total and antigen-specific IgG after bone marrow transplantation, despite unaltered IgG half-life and reconstitution of mature B cells and plasma cells. Rather, this deficiency was accompanied by reduced IgG+ splenic B cells and impaired per-cell IgG secretion after BCR stimulation. In vitro, extrinsic sialylation of immature B cells with recombinant ST6Gal-1 enhanced expression of IgM, IgD, and CD86, and potentiated IgG production upon stimulation. Surprisingly, this was not associated with increased IgG germline transcripts or isotype switching, but more robust antigen-induced proliferation, generating more IgG-producing cells. In vivo, hydrodynamic transfection with St6gal1 DNA resulted in an increase in total serum IgG. Finally, infusion of recombinant ST6Gal-1 enlarged splenic follicles and the follicular B cell population, highlighting therapeutic potential in modifying B cell development. Currently, vaccine adjuvants rely on mechanical or inflammatory properties to trigger an immune response. Our results suggest the manipulation of a bloodborne sialyltransferase may be a novel B cell-targeted, biological strategy to boost vaccine-induced and overall IgG production.
Investigating Transcriptional Regulators of Memory T Follicular Helper Cells
Authors: Deng Tianda , Shaw Laura , Yu Bingfei , Nguyen Quynh P , Hedrick Stephen M , Goldrath Ananda W
Published: January 2023

Generation of T-cell memory is crucial in conferring vaccine-induced immunity, particularly against pathogens where neutralizing antibodies alone are insufficient at providing long-term protection. While great advances have been made in understanding the generation and maintenance of memory CD8+ T cells and B cells, mechanisms underlying the generation of memory CD4+ T cells have remained relatively elusive. This limitation is in part due to the multi-potency and lineage plasticity exhibited by CD4+ T helper (TH) cells. Using the LCMV viral infection model, we show that the T follicular helper (TFH) subset are the predominant CD4+ memory T cell type based on its relative abundance following pathogen elimination as well as its multi-potent potential upon antigen re-challenge. This multi-potency of TFH memory cells during secondary challenge has also been observed in influenza infection and acute bacterial infection, suggesting that the TFH memory subset is most capable of providing a comprehensive and robust secondary response. From advancements in CD8+ memory T cell studies, it has become clear that transcription factors (TF) serve as crucial arbiters for the cell-fate decisions between short-lived effector and memory. To investigate the transcriptional mechanisms underlying TFH formation, we employed a novel bioinformatics analysis utilizing the PageRank algorithm that combines both RNA-seq and ATAC-seq and have identified putative transcriptional regulators of memory TFH differentiation. Further understanding of TFH memory formation will undoubtedly unveil new insights into CD4+ T cell memory in hopes of improving vaccine-based immunity.
Mechanistic investigations of Granzyme A-mediated γ9δ2 T cell TB protective effects
Authors: Rasi Valerio , Xia Mei , Wood David C , Eickhoff Christopher S , Hoft Daniel F
Published: January 2023

One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb). Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. GzmA when released by TB-specific γδ T cells has been shown to inhibit the intracellular replication of the pathogen. To elucidate the mechanism(s) involved in this inhibition, the lab has purified human GzmA. The effects of this highly purified GzmA on mycobacterial replication inside human macrophages are being investigated using systems biology. A 2D-DIGE proteomic approach of PBMC-adherent cells has been performed to identify putative pathways involved in the control of mycobacterial replication. Our preliminary analysis of two PPD+ volunteers indicates that ER-stress responses (HSPA5, HSP90B1, P4HB, PDIA3) and purinergic channel receptor activation (ATP5B/C1/D/H/O) may be important for GzmA-mediated intracellular mycobacterial inhibition. Verification of these results is being performed to confirm the overall mechanism, and to exclude unrelated signals. Elucidation of the detailed pathways involved in pathogen clearance could lead to the development of novel chemotherapeutic and/or immunotherapeutic approaches for control of Mtb infection.
Circulating Memory is sufficient for protective immunity to secondary infection with Chlamydia
Authors: Labuda Jasmine C , McSorley Stephen J
Published: January 2023

Chlamydia trachomatis is the most prevalent bacterial STI worldwide and yet no vaccine currently exists to protect the public from this reproductive tract pathogen. Recent reports suggest that Tissue Resident Memory (TRM) T cells are essential for vaccine-induced protection against Chlamydia trachomatis in a mouse infection model. TRM T cells have also been implicated in protective immunity to other reproductive tract pathogens, including Herpes Simplex Virus. Here, we examine a requirement for circulating versus resident memory in protection against secondary infection with Chlamydia muridarum (Cm). When FTY720 was used to block memory lymphocyte circulation, protection against secondary infection was unaltered, demonstrating that local resident mechanisms within the female reproductive tract are sufficient for secondary protection. However, parabiosis surgery of memory and naïve mice demonstrated that naïve mice receiving circulating memory were also fully protected from Cm challenge, indicating that TRM are dispensable for protection. Ongoing experiments are examining the protective capacity of both TRM T cells and circulating memory and whether memory lymphocyte cluster (MLC) formation within the reproductive tract is an essential component of naturally acquired or transferred protective memory. Understanding the differential contribution of resident versus circulating memory to protective responses is vital for the development of new vaccines against Chlamydia infection.
Drug treatment of tuberculosis diminishes but does not abolish the protection against secondary M. tuberculosis challenge
Authors: Ganchua Sharie Keanne C , Cadena Anthony M , Hopkins Forrest F , Fortune Sarah , Flynn JoAnne L
Published: January 2023

Early human studies found that individuals with latent M. tuberculosis (Mtb) infection are less likely to develop active tuberculosis (TB) compared to naïve individuals with similar levels of exposure. Using Mtb-barcoded libraries (Lib A - first infection; Lib B - second infection), we showed that an existing Mtb infection in cynomolgus macaques protects against the establishment and progression of a second Mtb challenge. Studies suggest that humans treated for active TB are at higher risk for developing disease following reinfection. Therefore we hypothesized that drug treatment would compromise the protection against a second Mtb infection. To test this hypothesis, we treated macaques infected with Lib A with anti-TB drugs for three months before infecting with Lib B. As a control, a group of macaques were infected with both Lib A and B following the same timeline but without drug treatment. In both groups, the number of granulomas arising from the second infection (Lib B) was significantly fewer than the number of granulomas from the first infection (Lib A). Comparing with the naïve group from our previous study, both drug and non-drug treated groups had significantly fewer live bacteria in the Lib B granulomas albeit the drug treated group had >10x higher bacterial burden than the untreated group. The Lib B granulomas in both groups were smaller and less inflammed (determined by PET CT) compared to granulomas from the naïve animals. No difference in the T cell response in Lib B granulomas was found in both drug and non-drug treated groups. These data suggest that the protection conferred by the first infection to the secondary Mtb challenge remains intact and that bacteria from the second infection were rapidly controlled regardless of treatment.
Resistance to inflammasome-mediated nitric oxide production is involved in Diffuse Cutaneous Leishmaniasis
Authors: Quirino Gustavo Fernando Silva , Santos Leonardo Lima , Borges Valeria Matos , Zamboni Dario Simoes
Published: January 2023

Leishmania amazonensis is one of the etiological agents of Cutaneous Leishmaniasis in Brazil. Infected individuals can present different forms of the disease, such as the Localized Cutaneous Leishmaniasis (LCL), characterized by round papules in the skin and the Diffuse Cutaneous Leishmanisis (DCL), a more severe form of the disease, characterized by multiple nodules in the skin, which are full of parasites. We have reported the protective function of the NLRP3 inflammasome during L. amazonensis infection in mouse, thus we aimed to evaluate if the inflammasome is involved in the determination of the LCL or DCL disease. We observed that skin biopsies of patients with DCL lesions present upregulation of inflammasome genes such as ASC, CASP1 and IL18. However, IL-1b and IL-18 levels in the plasma of LCL and DCL patients were similar. We used in vitro and mouse models to evaluate if the inflammasome is involved in the determination of the LCL or DCL. Macrophages do not differ in their ability to produce inflammasome-dependent IL-1b after infection with LCL and DCL. The inflammasome is involved in the control of LCL proliferation, but DCL isolates bypass the restrictive function of this platform both in vitro and in vivo. Further analysis showed that DCL isolates are resistant to inflammasome-mediated nitric oxide production. These data suggest that the resistance to the inflammasome effector functions may underlie the development of controlled (LCL) or disseminated (DCL) Leishmaniasis. We conclude that parasite-intrinsic factors related to resistance to the inflammasome might determine the outcome L. amazonensis infection.
Alveolar macrophage maturation is required for efficient killing of GBS in the lung
Authors: Lund Sean J , Patras Kathryn A , Yamamura Asami , Hernandez Gilberto , Lakhdari Omar , Nizet Victor , Prince Lawrence S
Published: January 2023

Group B Streptococcus (GBS) is the most common pathogen in neonates but rarely causes disease in adults. GBS causes pneumonia in newborns, suggesting a defect in lung innate immunity at birth. Using a novel neonatal mouse model of GBS pneumonia, we showed that newborn mice have reduced killing of GBS in the lung and more severe and persistent lung injury. Here we test the hypothesis that defects in alveolar macrophage (AM) maturation in newborns leads to GBS susceptibility. Clodronate depletion of AM from adult mice prevented GBS killing, supporting the requirement of AM for GBS protection. AM maturation and function requires GM-CSF, which is abundantly expressed in the lung. While AM maturation occurred on day 4 in WT mice, GM-CSF knockout mice (Csf2−/−) failed to develop AM. Consistent with the role of AM in GBS immunity, neonatal Csf2−/− mice had reduced GBS killing and lung macrophages failed to phagocytose GBS. Csf2−/− macrophages also had reduced expression of multiple Siglecs, cell surface receptors implicated in protection against encapsulated bacteria. Newborn lung macrophages expressed primarily Siglec-E (SigE), whereas expression of other Siglecs were only detected in more mature AM. We used SigE−/− mice to test its role in neonatal GBS pneumonia. GBS killing in SigE−/− neonates was similar to WT. However SigE−/− macrophages appeared to phagocytose GBS more efficiently in vivo, suggesting GBS may employ SigE to avoid detection by neonatal macrophages. Our data show that mature alveolar macrophages are required for protection against pulmonary GBS infection and potentially implicate the developmental regulation of macrophage Siglec expression in neonatal lung immunity.
Exploiting the molecular mimicry of Non-typeable Haemophilus influenzae with ketodeoxynonulosonic acid: Confusing the bacterium in its own game
Authors: Saha Sudeshna , Coady Alison , Choudhury Biswa , Chen Xi , Ram Sanjay , Nizet Victor , Varki Ajit
Published: January 2023

The distinctly human mucosal commensal and opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently responsible for acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults, and causes otitis media in infants. A key tool in this organism’s pathogenic arsenal is its ability to mimic glycans on human cells, which like all vertebrate cells, display diverse glycans with terminal 9-carbon-backbone monosaccharides called sialic acids. NTHi pathogenesis is related to its remarkably efficient uptake of trace amounts of free host N-acetylneuraminic acid (Neu5Ac, the primary sialic acid in humans) allowing “cloaking” of surface glycans of lipooligosaccharides (LOS) with this monosaccharide. This helps evasion from host immune clearance by “self-mimicry”, while facilitating colonization and biofilm formation. Since the mechanism of NTHi sialic acid uptake evolved by convergent evolution and is not selective for Neu5Ac, we propose to exploit this mimicry using another sialic acid called 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn). While NTHi growth was unaffected by Kdn in media, we found glycosidically-bound Kdn in LOS, which made NTHi sensitive to human sera and whole blood killing. The role of sialylation in vivo was further explored using a novel mouse model, with a human-like sialic acid profile. This mouse showed better acute NTHi colonization of the respiratory tract following intranasal challenge compared to wild type mice. Since Kdn is a metabolic byproduct in humans and should be non-toxic, the possibility of using Kdn as a safe intervention against NTHi colonization and subsequent infection is currently being explored using this mouse model.
Probiotic exopolysaccharide protects mice from acute Staphylococcus aureus bloodstream infection, reducing bacterial burden and limiting inflammation
Authors: Paik Wonbeom , Alonzo Francis , Knight Katherine L
Published: January 2023

Staphylococcus aureus causes severe systemic infection with high mortality. We previously showed that the probiotic Bacillus subtilis, produces an exopolysaccharide (EPS) that induces anti-inflammatory M2 macrophages. We used a lethal S. aureus bloodstream infection model, and found that mice treated with EPS prior to infection survived, and had reduced bacterial burden in the liver and spleen, as well as reduced levels of inflammatory cytokines. We showed that the EPS-induced M2 macrophages inhibited the growth of S. aureus in vitro, and by using an inhibitor of reactive oxygen species (ROS), we found that the growth inhibition of S. aureus was mediated by ROS, an M1-like property. However, these macrophages retained the M2-like capacity to inhibit proinflammatory activation of T cells by S. aureus superantigen. To test if the reduction in proinflammatory cytokines in EPS-treated mice during infection is due to anti-inflammatory effects of EPS rather than to the reduced bacterial load, we isolated splenocytes from EPS-treated mice and stimulated them with S. aureus ex vivo. We found that levels of IFN-γ, KC, and IL-6 were decreased, whereas the production of IL-10 was increased, indicating that the decrease in inflammation by EPS is not solely due to reduction in bacterial load. We suggest that the EPS-induced M2 macrophages represent “hybrid” macrophages capable of both M1- and M2-like functions and could be used to protect hosts from lethal S. aureus infection by bolstering anti-bacterial immunity, while simultaneously limiting inflammation.
Adhesion requirements of methicillin-resistant Staphylococcus aureus in bacterial pneumonia and influenza super-infection
Authors: Grousd Jennifer A , Richardson Anthony , Cooper Vaughn , Alcorn John F
Published: January 2023

In the respiratory tract, Staphylococcus aureus is associated with infections ranging from asymptomatic colonization to severe necrotizing pneumonia. While staphylococcal pneumonia has always been highly associated with influenza infections in young children and the elderly, it has emerged as the major cause of pneumonia associated with influenza since the 2009 H1N1 pandemic. Despite the clinical relevance of methicillin-resistant S. aureus (MRSA), few studies have examined bacterial factors of MRSA in pneumonia and how preceding influenza may alter the behavior of MRSA in the lung. Successful establishment of infection by bacterial pathogens requires adhesion to host cells. Staphylococcal species express a broad range of surface proteins that are involved in adhesion to host cells, known as the MSCRAMM family. MSCRAMM proteins have known roles in upper respiratory tract colonization, however their role in the lung is unknown. Preliminary data suggest that the MSCRAMM family members are important within the lung as MRSA lacking all MSCRAMMs (ΔsrtA) has decreased immune infiltrate in influenza super-infection. Additionally, the MSCRAMM family member SdrD has a role in both bacterial adherence as well as epithelial integrity and immunity in MRSA pneumonia and super-infection. Mice infected with MRSA lacking SdrD (ΔsdrD) have lower bacterial burden and increased expression of epithelial antimicrobial peptides cathelicidin and Reg3g with no difference in overall inflammation measured by immune infiltrate and pro-inflammatory cytokine levels. This suggests that MSCRAMM family members play a role in infection within the lung in both MRSA pneumonia and super-infection and can alter the immune response.
ANTI-ASIALO GM1 TREATMENT DURING SECONDARY TOXOPLASMA GONDII INFECTION IS LETHAL AND DEPLETES T CELLS
Authors: Denton Stephen L , Ivanova Daria L , Gigley Jason P.
Published: January 2023

Anti-NK1.1 and anti-Asialo GM1 (ASGM1) antibodies are used to deplete NK cells. Many antibody depletion strategies in vivo can have off-target effects. In a vaccine challenge model of T. gondii infection, we observed treatment of mice with these antibodies resulted in different survival outcomes after lethal secondary infection. Anti-ASGM1 resulted in 100% death and greater parasite burden at the site of infection than anti-NK1.1 treatment. We also found that anti-ASGM1 treatment depleted T cells. While both CD8+ and CD4+ T cells were affected, CD8+ T cells were more susceptible to the anti-ASGM1 treatment. Surface ASGM1 of the T cells was expressed on a higher percentage of CD8+ T cells than CD4+ T cells. Not all CD8+ T cells expressed ASGM1. In immunized mice ASGM1 was highly enriched on the surface of effector memory (Tem) and central memory (Tcm) CD8+ T cells, with a greater effect on Tem cells. After secondary parasite challenge, Tem, Tcm, effector (Tef) and naïve (Tn) CD8+ T cells were positive for ASGM1. Anti-ASGM1 treatment during rechallenge resulted in greater depletion of activated IFNγ+, Granzyme B+, Tem and Tef than Tcm and Tn CD8+ T cells. In addition, anti-ASGM1 depleted IFNγ+ CD4+ T cells. Recombinant IFNγ supplementation prolonged the survival of anti-ASGM1 treated mice, demonstrating that anti-ASGM1 eliminated IFNγ-producing T and NK cells important for control of the parasite. Our results thus highlight that anti-ASGM1 is not an optimal choice for targeting only NK cells and more precise approaches should be used to target them. This study also uncovers ASGM1 as a marker of activated effector T cells and the potential importance of changes in sialylation in lipid rafts for T cell activation during T. gondii infection.
Arginine-polyamine biosynthesis pathway and mediate bacillary survival in macrophages
Authors: Rameshwaram Nagender Rao , Singh Parul , Mukhopadhyay Sangita
Published: January 2023

Macrophages play a major role in the immune system, both as antimicrobial effector cells and as immunoregulatory cells, which induce, suppress or modulate adaptive immune responses. These key aspects of macrophage biology are fundamentally driven by the phenotype of macrophage that is prevalent in an evolving or ongoing immune response. One of the PE family proteins, PE11 (LipX or Rv1169c), demonstrate esterase activity and specific to pathogenic mycobacteria is found to polarize the macrophage towards M2 phenotype. In this study, we report that in macrophages, Msmeg-PE11 down-regulates inducible NO synthase (iNOS) and antimicrobial NO production, but in parallel there is an induction of arginase-I via p38 MAPK activation, and of ornithine decarboxylase (ODC), the first and rate-limiting step in the polyamine synthesis. Furthermore, results revealed that arginase-I regulates Msmeg-PE11 growth directly by affecting the polyamine synthesis in macrophages. These results identify a novel mechanism of immune dysregulation induced by M. tuberculosis lipolytic enzyme PE11 and by targeting PE11 and/or host arginine-polyamine biosynthesis pathway with specific inhibitors could negatively affect M. tuberculosis growth and have a positive impact in the treatment of multi-drug resistant tuberculosis.
Polyfunctional CD4+ T cells specific for Trypanosoma cruzi can be restored after in vitro treatment with IL-7 and IL-27
Authors: Natale Maria Ailen , Minning Todd , Alvarez Maria Gabriela , Lococo Bruno , Albareda Maria Cecilia , Bertocchi Graciela , Tarleton Rick , Laucella Susana
Published: January 2023

Chronically Trypanosoma cruzi-infected subjects bear several features associated with immune exhaustion, including an impairment in the polyfunctionality of T cells over time and alterations in the IL-7/IL-7R and IL-27/IL-27R axis. Since IL-7 and IL-27 have several functions on different immune cells, gene expression analysis was conducted in total PBMCs in response to rhIL-7 and rhIL-27 in vitro. TBX21, EOMES, GZMB and MIG-cytokine-induced gene expression was low in patients with severe Chagas disease compared with those without cardiac involvement. IL-7-induced TBX21 expression positively correlated with the magnitude of T. cruzi-specific T-cell responses. We also assessed whether treatment of PBMCs of chronically T. cruzi infected subjects with IL-7/IL-27 could improve the quality of T. cruzi-specific T-cell responses. Polyfunctionality of CD4+ T cells was improved after treatment with IL-7 and IL-27, by increasing the production of IL-2 on CD4+ T cells producing two or three functions in response to T. cruzi antigens in patients with mild cardiac disease. These findings support that alterations in IL-7/IL-27 pathways in chronic Chagas disease are not only at the signal-transduction level but also at the transcriptional regulation level. In vitro treatment of PBMCs of patients in early clinical stages of chronic Chagas disease can restore T-cell function.
Host Resistance to pulmonary Mycobacterium tuberculosis infection requires CD153 expression
Authors: Foreman Taylor W , Sallin Michelle , Kauffman Keith D. , Riou Catherine , Bruyn Elsa Du , Sakai Shunsuke , Hoft Stella , Nelson Christine , Myers Timothy , Gardina Paul , Sher Alan , Moore Rashida , Wilder-Kofie Temeri , Moore Ian N , Sette Alessandro , Lindestam Arlehamn Cecilia S , Wilkinson Robert , Barber Daniel L
Published: January 2023

Mycobacterium tuberculosis infection (Mtb) is the leading cause of death due to a single infectious agent and is among the top ten causes of all human deaths worldwide. CD4 T cells are essential for resistance to Mtb infection, and for decades it has been thought that IFNγ production is the primary mechanism of CD4 T-cell-mediated protection. However, IFNγ responses do not correlate with host protection, and several reports demonstrate that additional anti-tuberculosis CD4 T-cell effector functions remain unaccounted for. Here we show that the tumour-necrosis factor (TNF) superfamily molecule CD153 (encoded by the gene Tnfsf8) is required for control of pulmonary Mtb infection by CD4 T cells. CD153-deficient mice develop high pulmonary bacterial loads and succumb early to Mtb infection. Reconstitution of T-cell-deficient hosts with Tnfsf8−/− CD4 T cells, similar to Ifng−/− CD4 T cells, fails to rescue mice from early mortality. In Mtb-infected non-human primates, Mtb-specific CD153-expressing CD4 T cells inversely correlates with bacterial loads in granulomas. In Mtb-infected humans, CD153 defines a subset of highly polyfunctional Mtb-specific CD4 T cells that are much more abundant in individuals with latent Mtb infection compared to those with active tuberculosis. Thus, CD153 is a major immune mediator of host protection against pulmonary Mtb infection and CD4 T cells are one important source of this molecule.
B7x expression on the bronchial epithelium delays CD4 T cell priming after Mycobacterium tuberculosis infection
Authors: Sakai Shunsuke , Kauffman Keith D. , Kamenyeva Olena , Zang Xingxing , Barber Daniel L
Published: January 2023

The priming of CD4 T cell responses after Mycobacterium tuberculosis (Mtb) infection is delayed compared to other pathogens. Although it has been thought that the slow replication rate of the bacteria is responsible for the lag in T cell priming, it is possible that negative regulatory pathways that control inflammation in the lung also contribute to this slow T cell response. Here we show that the orphan co-inhibitory ligand B7x contributes to the delay in T cell priming following Mtb infection. We find that B7x is expressed on bronchiolar epithelial cells, and B7x−/− mice display enhanced bacterial control and survive longer than WT mice after pulmonary Mtb infection. The enhanced resistance of B7x−/− mice to Mtb infection is completely dependent on CD4 T cells, but at day 28 post-infection, the peak of the T cell response, there is little difference in the number or function of Mtb-specific effector CD4 T cells. Instead, we find that Mtb-specific CD4 T cells are primed in the mediastinal lymph nodes (mLNs) and accumulate in the lungs much earlier in B7x−/− mice compared to WT mice. B7x blockade is also able to enhance Mtb-specific T cell priming even after inoculation of non-replicating irradiated Mtb into the lungs. Moreover, after ESAT-6/CpG lung immunization, B7x blockade reduces the amount of adjuvant needed to drive T cell expansion in the mLNs by ~50 fold. Collectively, our results indicate the expression of the immune checkpoint molecule B7x by the bronchial epithelium contributes in part to the notorious lag in CD4 T cell responses during tuberculosis by increasing the threshold of innate stimuli needed for the induction of T cell priming.
Increased duration of antibiotic treatment progressively alters gut microbiota and revokes protection from ocular autoimmunity
Authors: Salvador Ryan S. , Horai Reiko , Jittayasothorn Yingyos , Tang Jihong , O’Huigin Colm , Thovarai Vishal , Yuan Wuxing , Caspi Rachel
Published: January 2023

Autoimmune uveitis, thought to be driven by retina-specific T cells, is a major cause of blindness. Using a model of experimental autoimmune uveitis (EAU), we showed that long-term elimination of commensals by oral antibiotic treatment (ABX) did not affect susceptibility to EAU, but others reported that short-term ABX was protective. To reconcile these contradictory findings, we studied whether duration of ABX differentially affected EAU development, gut microbiota and host-immune responses.

B10.RIII mice were treated orally with a broad-spectrum antibiotic cocktail, starting 12-weeks (long-term) or 1-week (short-term) before IRBP immunization for EAU induction, and continuing until the end of the experiment. Short-term ABX delayed the onset of EAU, whereas long-term ABX showed a pattern of EAU kinetics and severity similar to untreated SPF controls. Metagenomic analyses revealed ABX duration-dependent depletion of gut microbial communities. Interestingly, ABX also resulted in progressive depletion of CD4+ and CD4+CD8+ T cells from the gut intraepithelial lymphocyte (IEL) compartment. Notably, these IELs showed some cytotoxic activity in vitro, and are thought to help preserve gut mucosal barrier integrity.

We propose that microbiota play dual roles in uveitis development: they provide a stimulus for uveitogenic effector cells, but also maintain a “regulatory” IEL population, whose progressive loss reverses the temporary protection seen with short-term ABX. Further understanding of the role of IELs in the microbiota-gut-eye axis may lead to novel treatments for autoimmunity through manipulation of the commensal microbiome and gut epithelium.
Control of mucosal barrier functions and gut microbiome homeostasis by DUSP6 in colitis
Authors: KAO CHENG-YUAN , Chang Cherng-Shyang , Liao Yi-Chu , Huang Chih-Ting , Ruan Jhen-Wei , Lin Chiao-Mei , Tan Tse-Hua , Liao Yu-Chieh
Published: January 2023

Inflammatory bowel disease (IBD) is a common intermittent gastrointestinal condition and the incidence and prevalence of IBD are usually high in industrialized countries in North America and Europe. Several gut microbiome studies have been conducted in different IBD cohorts and certain specific taxonomic shifts have been associated with IBD. Although there is a relative escalation in the abundance of Enterobacteriaceae, including Escherichia coli and Fusobacterium, so far no single microbe has been demonstrated to cause IBD. It is plausible that intestinal microbiota dysbiosis may contribute to IBD pathogenesis by loss of health-promoting commensal symbionts or gain of pathobionts. We applied phosphoproteomics profiling and RNA-seq analysis in DUSP6-deficient Caco-2 cells. Our results suggested that DUSP6 could affect gut barrier functions and this is further confirmed with a dextran sulfate sodium (DSS)-induced colitis mouse model. Interestingly, with 16S rRNA gene sequencing we found that the gut/fecal microbiota in Dusp6-deficient mice was more resistant to DSS-induced dysbiosis compared with wild-type mice. In summary, these findings indicate that Dusp6-deficiency is a novel host genetic factor that could modulate gut barrier functions and mucosal immunity and inhibiting DUSP6 might be a novel strategy to shape the homeostasis of microbiota and beat IBD.
PLGA-encapsulated Chlamydia recombinant MOMP enhances serum and mucosal Th1 antibodies including memory and effector T-cells via subcutaneous prime-boost as compared to the intramuscular priming route immunization of mice
Authors: Sahu Rajnish , Verma Richa , Dixit Saurabh , Duncan Skyla , Ipinmoroti Ayodeji , Singh Shree R , Dennis Vida A
Published: January 2023

Vaccine developmental efforts are rapidly targeting biodegradable nanoparticles-based vaccines with encapsulated antigen. We have developed a PLGA 85/15 (poly (lactic-co-glycolic acid) Chlamydia nanovaccine by encapsulating its recombinant MOMP (major outer membrane protein) that induced robust adaptive immune responses and provided enhanced protection against C. muridarum (Cm) challenge in subcutaneously immunized mice. In the current study, we compared the immunogenicity and efficacy of priming routes; subcutaneous (sc) or intramuscular (im) followed by two sc-boosts to evaluate serum and mucosal antibodies [Th1 (IgG2a, IgG2b), Th2 (IgG1)] as well as memory and effector T-cells activation after immunization and Cm challenge of mice. We observed significantly high levels of MOMP-specific serum and mucosal antibodies with high avidity especially for Th1 antibodies. A significant increase in Th1 serum and mucosal antibodies with even higher avidity was observed after genital tract Cm challenge in the sc-prime-boost immunized mice. Evaluation of the Th1/Th2 antibody titer ratio revealed that the nanovaccine evoked a higher Th1 and lower Th2 response, which skewed to a dominated Th1 antibody response after bacterial exposure to sc-prime mice. In addition, CD4+ memory (CD44highCD62Lhigh) and effector (CD44highCD62Llow) T-cells proliferation was about two-fold higher in the sc-prime mice. Herein, we reveal that the subcutaneous prime-boost immunization with the nanovaccine was more effective in enhancing the Th1 antibody response and memory and effector T-cell responses against a chlamydial genital tract challenge of mice.
Effects of high affinity engineered class II-restricted full length and single chain CAR-format T cell receptors specific for a naturally occurring Listeria monocytogenes epitope on T cell activation and specificity
Authors: Johnson Deborah , Myers Sheldon , Magoffin Wyatt , Orton Taylor , Tellez-Frietas Claudia M , Christensen Ken , Weber K. Scott
Published: January 2023

High affinity T cell receptors (TCRs) are absent from the native T cell repertoire. While current hypotheses propose that high affinity TCRs are more sensitive to antigen and have a competitive advantage in immune responses, emerging evidence suggests that higher affinity T cells may reach a threshold where they experience decreased functionality, loss of binding specificity, and become prone to anergy. High affinity TCRs are especially attractive to facilitate immunotherapies as native TCR binding affinity may be too low to effectively initiate activation. However, the effects of increased TCR affinity for CD4+ T cells specific for naturally occurring epitopes is poorly understood. To address this question, we engineered via yeast display several class II-restricted high affinity TCRs specific for a naturally occurring peptide from Listeria monocytogenes protein listeriolysin O. Our high affinity clones have KD values as low as 8.7 nM and half-lives as long as 174 minutes when measured by tetramer dissociation assays. This panel of class II restricted high affinity TCRs specific for a naturally occurring Listeria monocytogenes epitope provides a novel means of testing the role of affinity and CD4+ T cell activation responses in the context of an infection.
Enhancement of antigen-specific responses following protein vaccination by Albumin- Flt3L expanded dendritic cells: Potential implications
Authors: Esquivel Daniel , Lam Brandon Krishna , Lee Brandon , Tan Marietta , Wu T.C. , Hung Chien-Fu
Published: January 2023

Due to the crucial role that dendritic cells (DCs) play in uptaking antigens following vaccination and cross presenting these antigens for the activation of antigen-specific immunity, a strategy that can induce potent expansion and activation of cross-presenting DCs may serve as a potential strategy to enhance the efficacy of therapeutic and prophylactic vaccinations. Flt3L is a cytokine known to expand cross-presenting DCs, but is limited by its global distribution and short half-life in vivo. To overcome these shortcomings, we generated a novel immunostimulatory molecule by fusing Flt3L to Albumin; a protein known for its long half-life and trafficking to lymph nodes. Our results revealed that Albumin-Flt3L (Alb-Flt3L) retains Flt3L’s endogenous capabilities to increase total CD11c+MHCII+, CD11c+MHCII+B220+SiglecH+, and CD11c+MHCII+CD8a+ DC populations in vitro, and in vivo following a single injection, compared to Flt3L. Our data also demonstrates that following vaccination with whole Ovalbumin protein (OVA), Alb-Flt3L treated mice saw an increase in antigen-specific immunity, measured by OVA-tetramer-positive CD8+ T cells, CD4+ and CD8+ cells producing IFN-g following ex vivo restimulation, and serum OVA specific IgG2a. When vaccinated with HPV-associated antigen E7 as a long peptide that requires uptake and cross-presentation to effectively load on MHCI and subsequently activate CD8+ T cells, E7-specific T cell populations significantly increased in Alb-Flt3L treated mice compared to controls. Further investigation is underway to study the ability of Alb-Flt3L to enhance vaccination induced immune responses as well as to study the mechanism by which these phenomena occur.
Oral R848 Reduces Host Susceptibility to Enteric and Systemic L. monocytogenes Challenge
Authors: Kayraklioglu Neslihan , Horuluoglu Begum Han , Elango Madhivanan , Turan Aslihan , Klinman Dennis M
Published: January 2023

Toll-like receptors (TLR) trigger the immune system to mount a rapid innate response capable of protecting the host from a wide variety of bacterial and viral pathogens. There is interest in harnessing TLR agonists to reduce the susceptibility of at-risk populations to infection. Yet the widespread prophylactic use of TLR agonists is compromised by the need to administer most of them by parenteral injection. An exception is the TLR7/8 agonist R848 which can boost GI and systemic immunity when administered orally. This work examines the effect of R848 on host susceptibility to L. monocytogenes in a murine challenge model. Results show that prophylactic administration of R848 significantly reduced susceptibility to infection over the ensuing 1–7 days in Balb/C mice. Oral R848 led directly to B cell stimulation and accelerated the response of T cells to subsequent L. monocytogenes challenge. Oral R848 may thus be of use in reducing the susceptibility of at-risk individuals to infection.
A potently neutralizing site III-specific human antibody prevents human metapneumovirus replication in vivo
Authors: Mousa Jarrod , Diaz Darren , Crabtree Jackelyn , Pena-Briseno Alma , Bar-Peled Yael , Tripp Ralph A.
Published: January 2023

Human metapneumovirus (hMPV) is a leading cause of lower respiratory tract infection in infants and children, yet there is currently no vaccine available for prevention of hMPV disease. hMPV has three surface proteins: the small hydrophobic, the attachment, and the fusion (F) protein. The hMPV F protein is the sole target of neutralizing antibodies. To develop new therapeutics for disease intervention and to inform the development of an effective vaccine, we isolated several new human monoclonal antibodies (mAbs) to the hMPV F protein. A subset of these mAbs were determined to be neutralizing by a plaque reduction assay. The neutralizing mAbs bind to all four subgroups of the hMPV F protein, and neutralize viruses from both the A and B genotypes. We conducted epitope binning assays on the OctetRED384 system to determine the antigenic targets of each mAb. Three of the four mAbs (196, 314, 201) bind near the previously discovered mAb DS7. One mAb, MPV364, competes with the site III mAbs MPE8 and 25P13, yet unlike these mAbs, MPV364 does not cross-react with the respiratory syncytial virus F protein. MPV364 exhibits potent and broadly neutralizing activity below 20 ng/mL. We examined the therapeutic efficacy of MPV364 to prevent replication of hMPV in vivo and found a reduction in lung viral titers. These data suggest antigenic site III on the hMPV F protein elicits potently neutralizing mAbs, and should be incorporated into future vaccine candidates. Furthermore, MPV364 should be further examined for protection from hMPV disease in additional animal models of infection.
Immunoprotection versus immunopathology by type I and III interferon against human metapneumovirus
Authors: Zhang Yu , Xu Jiuyang , Miranda-Katz Margot , Rich Helen E , Manni Michelle L , Tollefson Sharon , Sarkar Saumendra N , Alcorn John F , Williams John
Published: January 2023

Human metapneumovirus (HMPV) is a leading cause of respiratory tract infection in infants and children. The disease outcome of HMPV infection ranges from mild infection to severe bronchiolitis and fatal pneumonia. HMPV was discovered in 2001 and its disease mechanisms are not fully understood.

To study HMPV pathogenesis, we tested nine HMPV clinical isolates in an established C57BL/6 mouse model. HMPV clinical isolates induced variable disease severity ranging from mild to fatal disease. Mice infected by non-virulent strain TN/94-49 did not show significant weight loss, but mice infected with virulent isolate C2-202 showed dramatic weight loss and 40% mortality within 5 days post-infection. These findings were confirmed in other inbred mouse strains. C2-202 infected mice also suffered from impaired pulmonary function post-recovery. Lung virus replication did not correlate with disease severity, suggesting immune-mediated pathogenesis. Further investigation revealed increased type I and III interferons, pro-inflammatory cytokine production, and neutrophil infiltration in C2-202 infected mice. HMPV C2-202 infection also inhibited dendritic cell recruitment and down-regulated MHC-II surface expression on macrophages. Stat1/Stat2 double knockout mice lacking interferon signaling exhibited reduced weight loss, lessened disease severity, and increased dendritic cell recruitment after C2-202 infection.

Our results indicate that severe disease caused by HMPV clinical isolates was due to exuberant interferon response leading to immuno-pathological tissue damage. These data suggest interferon signaling plays an important role in HMPV pathogenesis, and thus serves as a potential therapeutic target.