bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycolysis is critical for granulocytic myeloid-derived suppressor cell (G-MDSC) activity during Staphylococcus aureus biofilm infection
Authors: Horn Christopher Michael , Heim Cortney E , Kielian Tammy L
Published: January 2023

Staphylococcus aureus (S. aureus) is a leading cause of prosthetic joint infection (PJI). These infections are often intractable due to biofilm formation, which are complex bacterial communities that adhere to biotic and abiotic surfaces. Within a biofilm, there is considerable heterogeneity in bacterial metabolism and gene expression that serves to create an anti-inflammatory milieu at the site of infection. This is due, in part, to the preferential recruitment of granulocytic myeloid-derived suppressor cells (G-MDSCs), a population of pathologically activated immature leukocytes with immunosuppressive features. Our prior work revealed that G-MDSCs are integral for promoting biofilm persistence via the production of interleukin-10 (IL-10) in response to biofilm-derived lactate. However, the mechanism responsible for G-MDSC anti-inflammatory activity in the context of biofilm infection is unknown. Using a mouse model of S. aureus PJI, time-course single-cell RNA sequencing (scRNA-seq) revealed a significant enrichment in glycolytic and hypoxic transcriptional signatures in infiltrating G-MDSCs. G-MDSCs significantly increased their glycolytic activity when co-cultured with S. aureus biofilm in vitro and inhibiting glycolysis during PJI in vivo with 2-DG nanoparticles significantly attenuated G-MDSC suppressive activity concomitant with a significant reduction in bacterial burden. This study demonstrates that glycolysis is required for G-MDSC-mediated immunosuppression and biofilm persistence during S. aureus PJI. Furthermore, our results demonstrate that G-MDSC metabolism can be targeted to improve infection outcomes.

Supported by funding from NIH (2P01 AI083211) & UNMC (UNMC Graduate Fellowship)
Identifying innate immune receptors causing production of inflammatory cytokines by infection of Francisella tularensis.
Authors: Gutierrez Stephanie K , Spencer Charles T , Setzu Nicole
Published: January 2023

Francisella tularensis is a pathogenic species and a causative agent of tularemia. This bacterium is incredibly infectious and often lethal in that F. tularensis induces the overproduction of pro-inflammatory cytokines. This study aims to identify the innate immune receptors responsible for the production of inflammatory cytokines in response to F. tularensis infection. The study uses an immortalized C57BL/6 Bone Marrow Macrophage (BMMac) cell line as the control and knockout BMMac deficient in various innate immune receptors. The cells are passaged and seeded before infecting them with F. tularensis for 2 hours. The cells were treated with a high dose of gentamicin for 2 hours, followed by a low dose of gentamicin for an incubation period of 24 hours. The supernatant from these cells is then collected and analysis processed by ELISA, an enzyme-linked immunosorbent assay, to measure the production of inflammatory cytokines. The results were compared with the positive control of wild-type BMMac and the negative control of uninfected BMMac cells. Analysis of these mutants reveals the involvement of innate immune receptors in triggering the pro-inflammatory cytokine storm in response to F. tularensis infection that leads to the host’s death.

Supported by NIH (SC1 GM122699-01A1)
Merocytophagy contributes to dissemination of Francisella tularensis and is enhanced by SYK signaling in macrophages
Authors: Deobald Kelly N , Steele Shaun P , Kawula Thomas H
Published: January 2023

Merocytophagy is a phagocytic process characterized by the transfer of cytosolic content between macrophages and the formation of a double-membraned vesical within recipient cells. Studies conducted in our laboratory have demonstrated that proteins and intracellular pathogens can be transferred between cells by this contact-dependent mechanism. In particular, Francisella tularensis, a highly infectious pathogen that spreads from cell to cell by hijacking the phagocytic pathway in macrophages, has been shown to replicate within cells that receive cargo via merocytophagy. While these observations may point to an alternative route of F. tularensis dissemination in vivo, it is also possible that this mechanism of bacterial spread could amplify immune responses. Ongoing studies aim to characterize molecular and cellular players in merocytophagy to gain a better understanding of this phenomenon and to develop strategies to assess merocytophagy in vivo in the future. In cell-to-cell contact studies, inhibition of spleen tyrosine kinase (SYK) expression and activity showed decreased cargo transfer by merocytophagy. Pharmacologic inhibition of SYK was also associated with decreased surface expression of several integrins, giving evidence that cell-to-cell contact is stabilized by integrins during merocytophagic transfer in macrophages.

Supported by grants from NIH and NIGMS (R56 AI139476 and T32 GM008336).
UBX Domain Protein 6 Positively Regulates JAK-STAT1/2 Signaling
Authors: Harrison Andrew G , Ketkar Harshada , Wang Penghua
Published: January 2023

Type I/III IFNs induce expression of hundreds of IFN-stimulated genes through the JAK/STAT pathway to combat viral infections. Although JAK/STAT signaling is seemingly straightforward, it is nevertheless subjected to complex cellular regulation. In this study, we show that an ubiquitination regulatory X (UBX) domain-containing protein, UBXN6, positively regulates JAK-STAT1/2 signaling. Overexpression of UBXN6 enhanced type I/III IFNs–induced expression of IFN-stimulated genes, whereas deletion of UBXN6 inhibited their expression. RNA viral replication was increased in human UBXN6-deficient cells, accompanied by a reduction in both type I/III IFN expression, when compared with UBXN6-sufficient cells. Mechanistically, UBXN6 interacted with tyrosine kinase 2 (TYK2) and inhibited IFN-β–induced degradation of both TYK2 and type I IFNAR. These results suggest that UBXN6 maintains normal JAK-STAT1/2 signaling by stabilizing key signaling components during viral infection.

Supported by R01AI132526 and R21AI155820 to Penghua Wang
CD138 contributes to plasma cell dynamics and competition in the bone marrow
Authors: Park Rosa , Benet Zachary , Fooksman David R
Published: January 2023

Plasma cells (PCs) are terminally-differentiated, non-proliferative immune cells that mainly reside in the bone marrow (BM) and specialize in antibody secretion. Previous work in the laboratory has shown that CD138, the canonical PC surface marker, mediates PC survival in a cell-intrinsic manner and is required for long-term humoral immunity. Since CD138 surface expression levels are heterogeneous among PCs, we hypothesized that they regulate PC function and survival. We co-transferred WT and CD138+/− B cells into WT or CD138+/− recipient mice, immunized them, and tracked WT versus CD138+/− PCs. Consistent with their genotypes, donor-derived WT PCs express higher CD138 levels than CD138+/− PCs. In WT recipient BM, donor-derived WT PCs outnumber CD138+/− PCs. Yet in CD138+/− recipient BM, donor-derived WT and CD138+/− PC frequencies are similar, suggesting that PC competition affects survival in the BM and is mediated by CD138 expression. Our previous intravital imaging studies had shown that BM PCs are motile and organize into clusters, and these behaviors are regulated by adhesion molecules and cytokines. Since CD138 interacts with an array of adhesion ligands and cytokines, CD138 may enable PCs to efficiently scavenge for survival factors and assemble into clusters that may control survival or function. To test this idea, we compared movement and cluster formation among WT, CD138+/−, and CD138−/− PCs in the BM using intravital imaging. We also characterized surface marker and gene expression profiles of WT, CD138+/−, and CD138−/− PCs to gain further insight into how CD138 levels affect PC function. These studies will improve our understanding of factors governing PC function and survival, with implications for vaccine development.

Supported by grants from NIH (R01 HL141491, T32 GM007288)
SHIP-2 inhibits human microglia-like cell function in a TREM2 independent manner
Authors: Ramakrishnan Gautham S , Humphrey Mary Beth
Published: January 2023

Microglia are a crucial component of the immune response in neurodegenerative diseases including Alzheimer’s Disease (AD). The Triggering Receptor Expressed on Myeloid Cells #2 (TREM2) mediates microglial phagocytosis and migration, and promotes metabolic homeostasis. Src homology domain-containing inositol 5-phosphatase 1 (SHIP-1) inhibits TREM2 in myeloid cells like macrophages and osteoclasts. SHIP-2, a paralog of SHIP-1, inhibits other myeloid cell receptors including Macrophage Colony-Stimulating Factor (M-CSF). We hypothesize that SHIP-2, inhibits TREM2-mediated microglial functions. To test our hypothesis, we studied the effect of pharmacological inhibition of SHIP-2 (and SHIP-1) in the function of wild-type (HMC3WT) and Trem2−/− human microglia-like cells (HMC3T2KO), which predominantly express SHIP-2 compared to SHIP-1. HMC3WT displayed superior phagocytic ability of oligomerized amyloid β and apoptotic neurons, have a larger lysosomal compartment, and have increased mitochondrial activity compared to HMC3T2KO. Treatment with a pan SHIP-1/2 inhibitor (SHIPi) K161 significantly improved the phagocytic uptake of fluorescently tagged oligomerized Aβ42 in both HMC3WT and HMC3T2KO. K161 treatment resulted in an enlarged lysosomal compartment and increased mitochondrial activity in both HMC3WT and HMC3T2KO. Our data suggests that, in cases of low SHIP-1 expression, pan SHIP inhibition improves microglia-like cell functions independently of TREM2. Further studies to look for SHIP2-TREM2 interactions and the effect of SHIP-1 expression in combination with SHIPi treatment in HMC3 cell function are underway. SHIPi are non-toxic in HCM3 cells and may offer potential treatment options for AD.

Supported by grants from NIH (1RF1AG059717-01)
C3aR dampens intracellular cAMP to control TRIF-mediated inflammation in activated macrophages
Authors: Corcoran Jesse , Hickman James , Napier Brooke
Published: January 2023

Recent studies from our lab and others have shown that complement components can enhance cell-intrinsic inflammatory signaling pathways. Specifically, we have found that activation of complement receptor C3aR enhances TLR4 signaling in lipopolysaccharide (LPS)-treated macrophages via enhanced p-38 phosphorylation. We found C3aR−/− mice treated with systemic LPS have decreased inflammatory cytokines (IL-6 and TNF) compared to wild-type (WT) mice. Our results corroborate previous studies showing C3aR−/− mice display decreased systemic cytokines (IL-6 and TNF) and increased bacterial burden during Gram-negative (GN) infection compared to WT mice. These results suggest C3aR is a critical regulator of TLR4 signaling. TLR4-TRIF signaling leads to activation of IRF3 and expression of interferon-regulated genes. We find that primary bone marrow-derived macrophages (BMDMs) from C3aR−/− mice show significantly reduced TRIF-dependent ifnb expression and IFNb secretion following 24h LPS exposure. Additionally, WT BMDMs treated with a C3aR antagonist (C3aRi) show a similar reduction in ifnb expression after LPS-treatment. These data demonstrate C3aR is critical for TRIF-dependent signaling downstream of TLR4 activation. Here we find WT BMDMs treated with C3aRi and LPS show increased intracellular cAMP. These findings demonstrate C3aR activation leads to depleted cAMP, which is essential for TRIF-mediated signaling downstream of TLR4. We hypothesize that C3aR is working as an inhibitory GPCR to regulate inflammation via intracellular cAMP and enhancing TRIF signaling in LPS-treated macrophages. Our findings are the first to suggest C3aR reduces cAMP to control TRIF-mediated inflammation in activated macrophages.
IL-17 cytokines preferentially act on naive CD4+ T cells with IL-17AF inducing the greatest transcriptomic changes.
Authors: Crawford Michael Patrick , Borcherding Nicholas , Karandikar Nitin
Published: January 2023

CD4+ T-helper 17 (Th17) T cells are important in the regulation of protective immunity during infection and in self-tolerance/autoimmunity. Th17 cells, through the secretion of IL-17, act in promotion of inflammation and are thus a major potential therapeutic target in autoimmune disorders. Recent reports show that the IL-17 family cytokines, IL-17A, IL-17F and IL-17AF, can directly act on CD4+ T-cells, inducing functional changes in these cells. Here we show that functional and transcriptomic changes were preferentially induced in naïve CD4+ T-cells, but not in memory CD4+ T-cells. As early as 48 hours post-IL-17 exposure, naïve cells showed transcriptome changes, whereas memory cells remained unaffected as late as 7 days. The functional differences occurred despite similar IL-17RC and IL-17RA receptor expression on these subsets. Also, the differential IL-17-mediated changes in naïve, but not memory, cells occurred even in transwell/co-culture systems, with each subset maintaining its respective functional phenotype. Notably, there were differences in downstream transcriptional signaling by the three IL-17 cytokines. The IL-17AF heterodimer conferred both the greatest transcriptional change and most altered functional consequences. Detailed analysis of the transcriptome provides important insights into the genes and pathways that are altered as a result of IL-17-induced signaling and may serve as targets of future therapies.

This work was supported, in part, by grant awards from the NIH to N.J.K. (R01 AI121567) and N.B. (F30 CA29655).
Loss of the nuclear receptor PPARγ primes CD8+ T cells for exhaustion
Authors: Shah Rushil , Christofides Anthos , Aksoylar Halil-Ibrahim , Pal Rinku , Boussiotis Vassiliki A , Patsoukis Nikolaos
Published: January 2023

Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to nuclear hormone superfamily, with three distinct types identified: PPARapha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). The role of PPARγ in the immune system has been mainly studied in macrophages, where it is known to mediate M2 polarization. Synthetic ligands for activating PPARγ ameliorate disease outcomes in various models of inflammation and autoimmunity. Here, we investigated how PPARγ affects antigen-specific effector and memory responses of CD8+ T cells using OT-I TCR transgenic mice and OT-I mice with T cell-specific PPARγ deletion. During CD8+ T effector differentiation in vitro by IL-2 culture following stimulation with cognate antigen, PPARγ deletion resulted in a rapid and enhanced T effector cell expansion and activation. However, PPARγ deficient OTI cells were more prone to exhaustion. This effect coincided with decreased mitochondrial fitness and spare respiratory capacity, high ROS levels and a significant increase in oxidized lipids. During generation of T effector cells in vivo, PPARγ deletion resulted in similar outcomes. At the memory phase there were no numerical differences in the T memory subsets in vivo, but upon rechallenge in vitro, PPARγ-deficient antigen-specific T cells had impaired capacity for effector differentiation. Our findings uncover a new role of PPARγ in CD8+ T responses and indicate that PPARγ has an indispensable role in regulating metabolism-mediated cues that imprint T cell differentiation and long-term effector capacity.

Supported by RO1CA212605, RO1CA229784-01, RO1CA238263 (to VAB)
Optimal CD8+ T cell effector function requires costimulation-induced RNA-binding proteins that reprogram the transcript isoform landscape
Authors: Karginov Timofey , Menoret Antoine , Vella Anthony T
Published: January 2023

Boosting T cell activation through costimulation directs defense against cancer and viral infections. Despite many studies targeting costimulation in clinical trials, the increased potency and reprogramming of T cells endowed by costimulation is poorly understood. Canonical dogma states that transcription mediates T cell activation. Here, we show that the spliceosome, controlling post-transcriptional alternative splicing and alternative polyadenylation, is the most significantly enriched pathway in T cells after CD134/CD137 costimulation. Costimulation of CD8+ T cells significantly increased expression of 29 RNA-binding proteins (RBPs) while RNA-seq uncovered over 1000 differential alternative splicing and polyadenylation events. Using in vivo mouse and in vitro human CRISPR models, we demonstrate that RBP Tardbp, among multiple other RBPs, is required for effector cytokine production, CD8+ T cell clonal expansion, and isoform regulation after costimulation. The prospect of immune response optimization through reprogramming of mRNA isoform production offered herein opens new avenues for experimentally and therapeutically tuning the activities of T cells.

Supported by NIH grants AI136955 and R21AI139891.
The role of Cdk5/p35 kinase activity in natural killer cell cytotoxicity
Authors: Wong Derek P , Parameswaran Reshmi
Published: January 2023

Natural killer (NK) cells and T cells are immune cells with cytotoxic function, but while the distinct role of Cyclin-dependent kinase 5 (Cdk5) in the activation and effector function of T cells has been explored, its role in NK cells is unknown. Here, we show that both Cdk5 and its obligatory co-activator p35 protein are expressed in NK cells. We also detect Cdk5 kinase activity in NK cells, and kinase inhibition via p35 shRNA results in increased NK cell cytotoxicity. Overexpression of p35, which increases Cdk5 kinase activity, results in decreased NK cell cytotoxicity. Exogenous expression of a kinase-dead Cdk5 mutant that is still capable of binding p35 also increases NK cell cytotoxicity. Meanwhile, p35 knockout mouse NK cells exhibit significantly higher cytotoxicity against murine melanoma cells compared to wild-type NK cells. Based on our preliminary data, we conclude that Cdk5/p35 kinase activity negatively regulates NK cell cytotoxicity, with implications for the potential improvement of NK cell-based cancer immunotherapies.

Supported by grants from the NIH (R21 CA246194, T32 GM007250) and the St. Baldrick’s Scholar Award.
TL1A promotes a multi-cytokine Th9 cell phenotype
Authors: Chu Michelle , Kaplan Mark H
Published: January 2023

The TNF superfamily member TL1A is a costimulatory molecule that signals through its receptor DR3 on T lymphocytes. The Th9 subset of T lymphocytes secretes the pleiotropic cytokine IL-9 which has functions in allergic airway disease, helminth infections, and tumor immunity. TL1A signaling has been shown to increase IL-9 production by Th9 cells. However, its role in regulating other functions of Th9 cells is unknown. Here we demonstrate that TL1A increases expression of IL-9 and IL-13 as well as the frequency of IL-9 and IL-13 co-expressing cells in Th9 cell cultures through flow cytometric analyses. We also show that the Il9 and Il13 promoter and enhancer regions are differentially accessible in response to TL1A over a five-day culture period through chromatin accessibility assays. At the Il9 locus, TL1A enhances binding of IRF4, BATF, and PPARg. Mechanistically, this is linked to decreased H3K9 tri-methylation and increased H3K4 tri-methylation at Il9 enhancer region CNS2. At the Il13 locus, TL1A enhances binding of BATF at the Il13 promoter alongside decreased H3K9 tri-methylation and increased H3K4 tri-methylation. Ongoing experiments define the function of TL1A-induced multi-cytokine producing cells. Together, these data indicate that TL1A contributes to heterogeneity of IL-9-secreting T cell populations.

Supported by NIH Combined Adult and Pediatrics Pulmonary Research Training Program T32
Dietary iron deficiency impairs the functionality but not generation or establishment of memory T cells following influenza infection
Authors: Bradley Marissa C , Idzikowski Emma , La Carpia Francesca , Gray Joshua I , Rybkina Ksenia , Guyer Rebecca S , Pethe Kalpana , Hod Eldad A , Connors Thomas J
Published: January 2023

Protection from viral respiratory tract infections (VRTIs) is dependent on the activation, differentiation, and maintenance of antigen specific memory T cells. Iron is essential for fundamental metabolic processes in the human body, with iron deficiency, the most prevalent micronutrient deficiency worldwide, resulting in increased morbidity and mortality to VRTIs. The role of iron in the establishment and maintenance of memory T cells is not fully defined. Using a murine model of dietary iron modulation, we define the impact of iron deficiency on anti-viral T cell memory formation, functionality, and resultant protection during primary and heterosubtypic influenza infections. Multi-parameter flow cytometry and stimulation assays were performed on T cells isolated from lung and spleen at effector and memory time points following primary (X31) infection, as well as 5 days post heterosubtypic (PR8) challenge. Strikingly, there was no significant difference in the numbers or proportions of CD4+ and CD8+ influenza specific memory T-cells at any time point when comparing iron deficient to iron replete mice. However, memory T cells generated by iron deficient mice displayed impaired production of key effector cytokines, most notably IFN-γ and IL-17a. Importantly, following heterosubtypic challenge, iron deficient mice exhibited increased morbidity, as defined by increased weight loss, and significantly diminished IFN-γ production by T cells across all sites. This work reveals that distinct iron profiles did not interfere with the generation or maintenance of influenza specific T cells but iron deficiency significantly impacts memory T cell anti-viral functionality resulting in impaired protective responses.

Supported by -K23 AI141686 -UL1 TR001873
Helpless CD8 T cell memory explained: prolonged antigen presentation drives a temporal rather than terminal defect
Authors: van der Heide Verena , Davenport Bennett , Jhun Kevin , Homann Dirk
Published: January 2023

CD4 T cell help is critical for long-term antiviral CD8 T cell immunity. Despite seemingly normal effector properties, “helpless” CD8 memory T cells (TM) generated in the absence of CD4 T cells during initial priming exhibit a substantially blunted recall response. Yet, how lack of CD4 help specifically impairs CD8 TM formation in the context of acute viral infections remains elusive. Here, we demonstrate that in transiently and constitutively CD4 T cell-deficient mice “helpless” antiviral CD8 TM to acute lymphocytic choriomeningitis virus (LCMV) exhibit a wide array of homeostatic, phenotypic, and functional defects that are broadly consistent with delayed CD8 TM maturation. Mechanistically, “helpless” CD8 T cell memory is not grounded in altered CD8 effector T (TE) cell differentiation in the absence of CD4 T cells but in exposure of post-effector CD8 T cells to protracted viral antigen presentation. Conversely, efficient generation of “helped” CD8 T cell memory is contingent on CD40L-mediated activities by LCMV-specific CD4 T cells that promote timely clearance of viral antigen. Importantly, we show that “helpless” memory-precursor CD8 TE display comparable memory potential to their “helped” counterparts, and that, once viral antigen is cleared, complete resolution of “helpless” CD8 memory is achieved over a period of ~1.5 years during which “helpless” CD8 TM eventually acquire mature TM properties including fully restored recall capacities. Collectively, our data redefine “helpless” CD8 T cell memory as a temporal rather than terminal defect, with implications for our general conceptualization of CD8 T cell memory as well as for the development of preventive and therapeutic strategies to improve protective CD8 TM function.

Supported by grant NIH R01AI093637.
TRM and mucosal tissue sites are protected from age-associated phenotypic changes in secondary lymphoid organs
Authors: Lam Nora , Caron Daniel P , Davis-Porada Julia , Jensen Isaac J , Lee YoonSeung , Morrison-Colvin Rory , Poon Maya M L , Szabo Peter A , Ural Basak B , Wells Steven B , Kubota Masaru , Matsumoto Rei , Farber Donna L
Published: January 2023

The persistence and maintenance of immunological memory is critical for long-term immunity generated in response to infection and vaccination. Aging of memory T cells in humans has predominantly been studied in the peripheral blood, and considerably less is known about the aging of memory T cells within tissues. Using tissues obtained from human organ donors, we investigated how tissue localization, expression of the CD103 integrin, and age influence the phenotype of tissue resident memory T cells (TRM). Using multi-parameter flow cytometry, we characterized diverse tissue sites from donors ages 9 to 93 years old and demonstrate that age-associated changes in the T cell compartment exhibit tissue- and subset- specific dynamics. CD8 T cells in blood-rich tissues spleen and lung have elevated expression of replicative senescence markers CD57 and KLRG1 relative to lung-associated lymph nodes (LLN), jejunum, and mesenteric lymph nodes (MLN). CD103+ TRM have the lowest expression of CD57 and KLRG1 across all tissue sites relative to both CD103− TRM and circulating TEM. CD8 T cells in lymphoid tissues spleen, LLN, and MLN exhibit the largest age-associated increases in expression of CD57, KLRG1, CD244, and PD-1. Together, these results demonstrate that age-associated phenotypic changes are most prominent in secondary lymphoid organs, while TRM and mucosal sites exhibit few senescent changes.

Supported by the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) and grants from NIH (P01 AI106697, U19 AI057266)
The Rv2623-Rv1747 interaction influences phosphatidyl-myo-inositol levels on the cell envelope of Mycobacterium tuberculosis
Authors: Schami Alyssa , Ke Wei , Allué-Guardia Anna , Olmo-Fontánez Angélica M , Chan John , Torrelles Jordi B
Published: January 2023

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), has a complex cell envelope that provides a barrier of protection to various environments. Peripheral lipids on the M.tb cell envelope act as virulent factors influencing the host immune response to infection. Of these, some phosphatidyl-myo-inositol mannosides (PIMs) and their associated lipoglycans (lipomannan; mannose-capped lipoarabinomannan) influence M.tb-host interactions by increasing phagocytosis, blocking the maturation of M.tb-containing phagosomes, and increasing the anti-inflammatory response of infected macrophages. However, there is limited knowledge regarding how M.tb regulates PIMs levels, and how this regulation influences infection outcomes. In our previous studies, we hypothesized that Rv2623 and Rv1747 work together to regulate PIMs on the M.tb cell surface. Indeed, deleting the universal stress protein Rv2623 increases PIMs levels, growth, and virulence in vivo; while deleting the ABC transporter Rv1747 decreases PIMs levels. Here we investigate the mechanistic regulation of M.tb PIMs levels and its impact on growth and pathogenesis in vitro by assessing multiple M.tb strains with Rv2623 or Rv1747 mutated at different amino acids. Depending on the mutation, Rv2623 interacts with Rv1747 to modulate M.tb cell envelope PIMs levels at different degrees. Mutants with increased PIMs levels have increased growth in macrophages, and PIMs levels on the M.tb cell envelope may also correlate to resistance to certain anti-TB drugs. Overall, our data indicate that Rv2623 negatively regulates Rv1747 to modulate PIMs levels on the M.tb cell envelope, ultimately influencing growth and M.tb-host interactions in vitro and in vivo.

Supported by grants from NIH/NIAID (1R01AI146340-01A1)
Gut microbiome regulates serotonin production in the neonatal intestine to promote immune tolerance in early life
Authors: Sanidad Katherine Z , Rager Stephanie L , Ananthanarayanan Aparna , Callaghan Ryann , Li Tingting , Jin Jenny C , Amir Mohammed , Luo Ryan , Silver Randi , Artis David , Guo Chun-Jun , Krumsiek Jan , Inohara Naohiro , Zeng Melody Y
Published: January 2023

The gut microbiome in neonates is vastly different from that in adults due to dietary changes and gut maturation. However, the neonatal gut metabolome, largely shaped by gut bacteria, remains largely undefined. Using high-throughput metabolomics, we compared >500 metabolites in the ileum of specific pathogen-free (SPF) wild-type neonatal and adult mice. We found that most metabolites at significantly higher levels in neonates were neurotransmitters, including serotonin (5-HT), a neurotransmitter critical in the regulation of gut motility and mood stabilization. SPF neonates had significantly higher 5-HT levels in the ileum compared to both SPF adults and germ-free (GF) neonates, suggesting the gut microbiome as a key driver of generation in the neonatal intestine. Gut bacteria in neonates modulate the enzymes TPH-1 and MAO-A involved in the synthesis and breakdown of 5-HT. In addition, unlike in the adult intestine where 5-HT is produced primarily by enterochromaffin cells, gut bacteria are major producers of 5-HT in the neonatal intestine. The immunomodulatory role of 5-HT in the neonatal intestine has not been explored. We demonstrate that 5-HT directly promotes the differentiation regulatory T cells while diminishing interferon gamma and interleukin-17 produced in CD4 T cells, ex vivo and in vivo, in the neonatal intestine. Oral gavage of 5-HT into neonatal mice enhanced immune tolerance toward both commensal bacteria and dietary antigens. Our study has elucidated unique mechanisms by gut microbiota to regulate 5-HT generation in the neonatal intestine, and a novel immunomodulatory role for intestinal 5-HT to promote immune tolerance towards both dietary antigens and commensal bacteria during the early developmental stage.
Age associated differential innate immune response to strains of Escherichia coli in neonatal sepsis model
Authors: Muske Josey , Greenfield Kara G , Harlow Olivia , Knoop Kathryn
Published: January 2023

Bloodstream infections, causing late-onset neonatal sepsis (LOS), have an incidence rate of 10% in very low birth weight (<1500 grams) infants. Despite the prevalence in neonates, the current understanding of bloodstream infections and the resulting immune response is predominately based on research in adults, which is often a complication of surgery. In contrast, sepsis in infants is often a result of the dissemination of bacteria from the gut. Additionally, observations have shown differences in the neonatal immune response, such as a decrease in gene expression associated with TLR signaling pathways, compared to adults. One question remaining in the field is the age dependent differences in pathogen detection by innate cells, including dendritic cells and macrophages. Use RAW-Blue reporter cells, and splenocytes cultured from mice at different ages, we have found disparate responses to gut-resident E. coli dependent on pathogen status of the bacteria. Specifically, we have observed differential increases in IL-6, TNFα, and IL-1β, along with NFκB activation that is dependent on live bacteria. Additionally, changes in this response were associated with age, suggesting differences in the innate immune response throughout early life. Future directions include measuring RNA expression of dendritic cells and macrophages between the age groups and exploring how the differences contribute to infection outcomes in a model of sepsis.

Supported by grants from NIH (DK122187).
Assessing mucosal antibody responses to SARS-CoV-2 in humans and mice.
Authors: Isho Baweleta , Abe Kento , Zuo Michelle , Jamal Alainna J , Cao Eric , Chao Gary , Li Zhijie , Rini James M , Tan Darrell , McGeer Allison J , Gingras Anne-Claude , Gommerman Jennifer L
Published: January 2023

COVID-19 is a respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We previously developed an enzyme-linked immunosorbent assay (ELISA) protocol to profile antibody responses to the SARS-CoV-2 Spike protein and its receptor-binding domain (RBD) in the saliva of patients with COVID-19. Anti-SARS-CoV-2 antibody responses were readily detected in saliva, peaking at 16–30 days post-symptom onset. While anti-SARS-CoV-2 IgM/IgA were found to decay, IgG responses were more long-lived, persisting up to 105 days. Now we wish to know (1) if anti-SARS-CoV-2 antibodies are generated locally in the oral cavity, (2) the relative kinetics of IgA versus IgG appearance in the oral cavity, and (3) whether salivary antibodies have the capacity to neutralize SARS-CoV-2. To answer these, I have adapted our ELISA to detect secretory component-associated SARS-CoV-2-specific antibodies. I found that 33.3% and 26.9% of COVID-19 patients were positive for secretory component-associated antibodies to Spike and RBD, respectively. Secretory component-associated antibodies correlated with antigen-specific IgA levels, particularly for the Spike antigen. Moreover, using saliva from contact-traced subjects, I found that anti-Spike/RBD IgM/IgA are detected in the saliva 7 days post-exposure, prior to IgG. Lastly, I detected Spike-specific antibody-secreting cells in lymphoid tissues draining the upper respiratory tract upon intranasal infection of K18-hACE2 mice with a non-lethal dose of SARS-CoV-2. In summary, I have provided evidence in humans and mice that a local antibody response that quickly class switches to IgA occurs in the oral cavity and draining lymph nodes upon infection with SARS-CoV-2.
Decoding atopic dermatitis: patient heterogeneity and genetic variation contributing to immune-mediated dermatologic disease in the UK Biobank
Authors: Truong Dongnhu T , Molineros Julio , Chen Yanqing , Guan Meijian , Hart Amy , Keyes Bryce , Seiber Karsten B , Li Shuwei , Waterworth Dawn , Black Mary Helen
Published: January 2023

Background

Dupilumab is the only approved biologic used to treat moderate to severe atopic dermatitis (AD) but has a non-response rate of 55–60%. This suggests underlying heterogeneity in AD etiology, requiring alternate therapies for patients.

Study Objective

Identify biomarkers and disease conditions in the UK Biobank that stratify AD patients into homogenous subgroups for genetic characterization.

Methods

We identified 13,137 AD cases and 13,136 age and sex-matched controls using hospital ICD9/10 and primary care codes in the UK Biobank. A total of 94 biomarkers and 1,596 disease PheCodes were used to isolate traits that vary in AD patients relative to controls; K-means (quantitative) and K-modes (binary) clustering was performed to identify subgroups within AD patients. Genetic analysis was performed for subgroups vs. controls using REGENIE.

Results

We observed enrichment of dermatologic, gastrointestinal, and rheumatologic conditions in AD patients, as well as differences in eosinophil percentage, Cystatin C, C-reactive protein, and erythrocyte distribution width relative to controls. There were eight subgroups of AD patients including groups with co-occurring psoriasis, asthma and gastrointestinal problems, penicillin allergy, and AD with no co-morbidities. Genetic analyses revealed novel HLA and non-HLA associations with subgroups, while also replicating known associations with FLG.

Conclusion

There were eight distinct AD subgroups based on co-occurring disease states. Genetic analyses show subgroup-specific factors can contribute to disease pathology—signaling potential utility for differential therapies and highlight mechanisms for targeted therapeutic development in the treatment of AD.
The effect of induced lymphatic circulation on lymphangiogenesis and inflammatory mediators in rats with adjuvant induced arthritis
Authors: Volin Michael V , Blucher Kelly , Zanotti Brian , Incrocci Ryan , Del Toro Rosalinda Monroy , Jain Shreya , Weber Daniel , Gober Calley , Swanson-Mungerson Michelle
Published: January 2023

Rheumatoid arthritis (RA) is a chronic inflammatory disease involving dysfunctional lymphatic circulation leading to edema. The adjuvant-induced arthritis (AIA) rat model was used to study the effect of lymphatic pump treatment (LPT) on the expression levels of inflammatory cytokines and lymphangiogenesis within the draining popliteal lymph nodes (PLN). PLN from both control and LPT treatment groups were harvested, and their cDNA were analyzed by qPCR to determine the levels of cytokines, VEGF-C, and VEGFR-3. Additionally, immunohistochemistry was used to visualize the expression of VEGF-C and VEGFR-3 and flow cytometry was used to quantify leukocyte cell types in the PLN. The LPT group showed a decrease in ankle circumference and arthritis grade a few days after the initiation of treatment. Flow cytometry of popliteal lymph nodes showed an increase in CD8, CD4, and CD3 cells in arthritic rats, however cell populations were not significantly different between treatment groups. The qPCR data indicated that the LPT group had a slight reduction in levels of IL-17a, IL-4, IL-6, and IL-1β. IHC showed that the PLN from the LPT group had the highest expression area of VEGF-C and the lowest expression area of VEGFR-3 compared to control. In conclusion, The LPT group showed a reduction in ankle circumference and arthritis score, suggesting that LPT helped alleviate edema and inflammation. When compared to control, the LPT group had lower expression of pro-inflammatory cytokines and elevated levels of VEGF-C, an important lymphatic vessel growth factor, in PLN. Thus, the data suggests that LPT may elevate the swelling and inflammation in rat AIA through the reduction of inflammatory cytokines and the elevation of a lymphatic growth factor.
Deficiency in B Cell Maturation Antigen reveals sex differences in Experimental Autoimmune Encephalomyelitis.
Authors: Kumar Gaurav , Ko Rose M , Bhatt Neal , Axtell Robert C
Published: January 2023

Multiple sclerosis (MS) is immune mediated demyelinating disease of the central nervous system (CNS) leading to neuronal damage. The disease is heterogeneous with respect to onset and clinical course, being more prevalent in women then in men with a ratio of 3:1. Further sex differences have been observed with males having a more severe and debilitating disease course compared to females. The complex interplay of immune cells, sex hormones or genetic differences could be contributing to these clinical phenomena, which remains to be elucidated. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, several reports have demonstrated no sex differences in disease onset or severity in C57BL/6 mice. Herein we report that deficiency in B Cell Maturation Antigen (BCMA) in C57BL/6 mice reveals a sex difference in EAE. BCMA plays a prominent role in B cell function, however the role BCMA plays in MS and EAE is unknown. Our initial goal of this project was to determine the impact BCMA deficiency has on EAE. In doing so we confirmed that EAE disease was not different in males and females from BCMA+/+ mice. Strikingly in the BCMA−/− mice, there was a significant increase in EAE disease severity in males compared to females. The increased EAE in the BCMA−/− male mice was associated with increased demyelination and infiltration of inflammatory T cells and macrophages into CNS. Also, we observed decreased splenic transitional B cells in male BCMA−/− mice compared to females. Castration of mice significantly reduces EAE in BCMA−/− mice as compared to sham controls. Altogether, our data demonstrate that male hormones exacerbates EAE in BCMA−/− mice by augmenting inflammatory responses compared to females leading to increased neuro-inflammation.

Supported by NIH (R01AI137047 and R01EY027346)
The fate and function of NK cells in the suppressive tumor microenvironment
Authors: Liu Xia , Ma Feiya , Zhang Yuanqin , Tao Yan , Hoft Daniel F , Peng Guangyong
Published: January 2023

Natural killer (NK) cells are an important cell subset of the innate immune system. Accumulating evidence indicates that NK cells are dysfunctional in the tumor microenvironment during cancer development. However, the dynamic changes in phenotypes and functional interactions within the tumor microenvironment during tumor development and progression are unknown. Here, we used both the mouse E0771 breast cancer and B16F0 melanoma tumor models to mimic different clinical stages of human cancers and characterized the dynamic distributions and qualities of NK cells in different organs and tumors associated with tumor progression. We found that NK cells are dynamically involved in the immune responses to cancer with different distributions and phenotypic profiles in tumor sites and other peripheral organs during the course of tumor development and progression. In the early stages of tumor development, NK cells exhibit effector properties. In the later cancer stages, NK cells have impaired cytotoxic capacities and dysfunctional states. Importantly, we found that melanoma and breast cancer cells promote NK cells to become senescent NK cells in vitro and in vivo in tumor microenvironments. These studies provide a better understanding of the dynamic and functional role of NK cells in anti-tumor immunity, which may facilitate the development of novel immunotherapies targeting NK cells for cancer treatment.

the Melanoma Research Alliance, American Cancer Society, and the National Institutes of Health.
Isolation of alpaca-derived single-domain antibodies against zika virus non-structural proteins to interrogate viral replication
Authors: Weinstein Jules B , Tafesse Fikadu G
Published: January 2023

Zika virus (ZIKV) is a re-emergent flavivirus that triggered a global health emergency from 2015 to 2017. ZIKV and other impactful flaviviruses, including dengue virus, lack the antiviral treatments and vaccines needed to diminish their threat to communities world-wide. This is in part due to gaps in our understanding of the flavivirus lifecycle; despite numerous investigations into flavivirus non-structural (NS) protein function. I hope to forge a deeper understanding of crucial NS protein functions and interactions throughout the ZIKV replication cycle. I will use alpaca-derived variable heavy-chain-only (VHH) antibody fragments, isolated from an alpaca immunized with ZIKV NS proteins, to perturb the replication cycle and therefore identify key NS protein epitopes used during replication. I individually purified NS1, NS2b, NS3, NS4b, NS5 for use in alpaca immunization, phage display panning, and enzyme-linked immunosorbent assays (ELISAs). From the alpaca peripheral blood I isolated RNA and amplified VHH genes, enriched for NS-protein binders with iterative phage display, and screened potential high binders via ELISA. I have isolated VHH sequences that interact with the ZIKV helicase, NS3, and the RNA-dependent RNA polymerase, NS5. I plan to purify each VHH and express them in ZIKA-permissive A549 cells as stable lines. Using purified VHH, I will analyze their binding ability in-vitro by ELISA and bio layer interferometry, and quantify in-vitro inhibition of NS3 protease and helicase activity for NS3 binders, and NS5 polymerase and methyltransferase activity. With the stable cells lines I will investigate each VHHs ability to impede ZIKV virus production and formation of replication complexes in infected cell lines.

Support from training grant “Interactions at the Microbe-Host Interface” T32AI007472
RhCMV/SIV tropism modulation programs unconventional CD8+ T cell priming and vaccine efficacy
Authors: Hancock Meaghan , Hansen Scott , Malouli Daniel , Marshall Emily , Hughes Collette , Randall Kurt T , Morrow David , Ford Julia , Gilbride Roxanne , Selseth Andrea , Trethewy Renee Espinosa , Bishop Lindsey , Oswald Kelli , Shoemaker Rebecca , Berkemeier Brian , Bosche William , Hull Michael , Nekorchuk Michael , Busman-Sahay Kathleen , Estes Jacob , Axthelm Michael , Smedley Jeremy , Shao Danica , Edlefsen Paul , Lifson Jeffrey , Fruh Klaus , Nelson Jay , Picker Louis J
Published: January 2023

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens demonstrate a vaccine efficacy where 50–60% of vaccinated rhesus macaques are protected from SIV challenge. Intriguingly, RhCMV/SIV vectors elicit CD8+ T cells recognizing epitopes presented by MHC-II and MHC-E instead of MHC-Ia. We are studying how these unconventional T cell responses are elicited and contribute to the efficacy against SIV challenge. Here we utilize host microRNA (miRNA)-mediated vector tropism restriction to show that MHC-II- and MHC-E-restricted responses are primed by directly infected, non-overlapping cell types in rhesus macaques. Targeting essential RhCMV genes with myeloid cell-selective miR-142-3p eliminated MHC-E-restricted CD8+ T cell priming, yielding an exclusively MHC-II-restricted response, whereas endothelial cell-selective miR-126-3p targeting eliminated MHC-II-restricted CD8+ T cell priming, yielding an exclusively MHC-E-restricted response. Incorporation of both restriction elements reverts CD8+ T cell responses back to conventional MHC-Ia restriction. Using these otherwise isogenic vectors we show that although they demonstrate similar overall immunogenicity, only the vectors programmed to elicit MHC-E-restricted CD8+ T cell responses provided protection against SIV challenge. The MHC-E-only RhCMV/SIV vaccine efficacy did not exceed that of the parental 68-1 RhCMV/SIV vectors (that elicits both MHC-II and MHC-E responses) indicating that while the MHC-II-restricted CD8+ T cell responses are neutral to overall vaccine efficacy, an additional component of 68-1 RhCMV/SIV-induced immunity contributes to overall vaccine efficacy.

This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) grants UM1 AI124377 and U19 AI128741 to LJP; the Oregon National Primate Research Center Core grant from the National Institutes of Health, Office of the Director (P51 OD011092); contracts from the National Cancer Institute (# HHSN261200800001E) to JDL; and the Bill and Melinda Gates Foundation grant OPP1107409.
T cell programming by the cytomegalovirus MHC class I homologue UL18
Authors: Taher Husam , Malouli Daniel , Hansen Scott G , Mansouri Mandana , Iyer Ravi , Papen Courtney , Schell John B , Cleveland-Rubeor Hillary , McArdle Matthew R , Hughes Colette M , Randall Kurt T , McNett Alisha , Picker Louis J , Früh Klaus
Published: January 2023

Rhesus cytomegalovirus (RhCMV) strain 68-1 vectored vaccines elicit MHC-II-and MHC-E-, but not classical, MHC-Ia-restricted CD8+ T cell responses. This is due to the presence of a viral MHC-E ligand encoded by Rh67 (UL40 in HCMV) and the combined deletion or inactivation of Rh157.5/.4 (UL128/UL130 in HCMV) and Rh158–Rh161 (members of the UL146 family of CXC chemokines). Importantly, the ability to elicit MHC-E-restricted CD8+ T cell responses correlates with the efficacy of RhCMV-vectored vaccines to control and clear infection by highly virulent simian immunodeficiency virus (SIV). To investigate whether HCMV encodes additional modulators of unconventional CD8+ T-cell priming that are not conserved in non-human primate CMVs, we inserted non-conserved HCMV genes into 68-1 and monitored the induction of the unconventional CD8+ T cells. This screen revealed that the UL18 protein of HCMV prevented the induction of MHC-II and MHC-E restricted CD8+ T cells resulting in a classical, MHC-Ia-targeted T cell response. UL18 is a MHC-Ia homolog and associates with cellular β2 microglobulin and peptides. However, unlike host MHC-Ia, this trimeric complex does not engage with the T cell receptor, but binds with high affinity to the inhibitory receptor LIR-1 widely expressed on leukocytes. Indeed, 68-1 recombinants expressing UL18 mutants lacking LIR-1-binding no longer interfered with the induction of MHC-II and MHC-E restricted CD8+ T cells. Thus, HCMV UL18 appears to prevent unconventional T cell priming by binding to LIR-1. In the event that this mechanism is conserved in the human host, UL18 could interfere with HCMV/HIV vaccine efficacy as the ability to elicit MHC-E-restricted CD8+ T cells is likely required for protection against HIV.