bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic Measures of Macrophage Chemotaxis
Authors: Tikhonenko Maria , O’Clair Lindy , Roddy Meagan , Schroeder Kirk , Appledorn Daniel
Published: January 2023

Macrophages are crucial regulators of homeostasis, however, their functions can be compromised, leading to a wide variety of diseases such as diabetes, fibrosis, atherosclerosis and tumor metastasis. Macrophage recruitment by cytokines is an essential step of their function. The main obstacles for studying macrophage chemotaxis in vitro include low resident tissue macrophage cell number, lengthy isolation periods, and low percentage of migrating cells. In this report we illustrate a novel, fully automated, kinetic, label-free method for measuring macrophage chemotaxis. This method requires significantly fewer cells compared to traditional methods, thus allowing for increased throughput. Using the human monocyte cell line, THP-1, we first examined the directed migration of untreated vs. PMA-differentiated (macrophage-like) THP-1 cells. Using as few as 5000 cells per well, we observed a significantly different chemotactic response depending on treatment: pre-activated THP-1 had minimal to no response to C5a while differentiated THP-1 had a strong concentration-dependent response. Primary human PBMC-derived Macrophages were also evaluated. Using 2000 cells per well we found that both M1 (pro-inflammatory) and M2 (pro-regenerative) macrophages consistently responded to C5a with minimal, yet significant responses towards Rantes, SDF-1α, CXCL10, MDC and M-CSF. M1 cells exhibited slower response compared to M2 cells. Our data also revealed a significant alteration in kinetic responses depending on C5a concentration, with higher concentrations of C5a eliciting a slower response than lower concentrations, thus illustrating the requirement for kinetic measurements when investigating macrophage chemotaxis.
TSLP acts on neutrophils to drive complement-mediated killing of methicillin-resistant Staphylococcus aureus
Authors: West Erin E , Spolski Rosanne , Kazemian Majid , Kemper Claudia , Leonard Warren J
Published: January 2023

Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections, often presenting as serious skin infections in otherwise healthy individuals, have become a world-wide epidemic problem and warrant attention for therapeutic intervention. Accordingly, defining the mechanisms that govern the activation and regulation of the immune response to MRSA is clinically important and could lead to the discovery of much needed rational targets for therapeutic manipulation. The cytokine thymic stromal lymphopoeitin (TSLP) is most well characterized as a major contributor to Th2-type responses in which it plays a deleterious role in the promotion of allergic responses. However, while TSLP is highly expressed at barrier surfaces, including the skin, its role in host-defense against bacterial infections has not been well elucidated. Unexpectedly, we found that TSLP can play a protective role in the skin by acting directly on mouse neutrophils to increase their killing of MRSA in both an in vitro killing assay and an in vivo skin infection model. Additionally, TSLP also directly acts on purified human blood neutrophils to reduce MRSA burden. Therefore, neutrophils represent previously unrecognized potent responders to TSLP. Surprisingly, we found that TSLP mediates these effects by directly engaging the complement C5 system to modulate reactive oxygen species production by neutrophils. Thus, TSLP increases MRSA killing in a neutrophil- and complement-dependent manner, revealing an unanticipated key connection between TSLP and the innate complement system. These data demonstrate an unrecognized beneficial role for TSLP in the skin, with potentially important clinical implications.
Human neutrophil extracellular trap formation is modulated upon stimulation with Toll-like-receptor ligands
Authors: Singh Ram Raj , Randhawa Darshan , Valera Isela
Published: January 2023

Polymorphonuclear neutrophils are considered to be the first line of defense against infections. One of the many strategies that neutrophils use for removal of foreign material is via the deployment of neutrophilic extracellular traps (NETs). NETs are chromatin fibers decorated with antimicrobial proteins. It is presumed that all NETs produced by neutrophils (mainly through stimulation with PMA) are similar in function and structure. Here, we demonstrate that when neutrophils are stimulated with different Toll-like-receptor (TLR) ligands, they produce NETs which are unique in structure and function with respect to its TLR stimulation. Not only does this demonstrate at the physiological level that NET formation is selective to its TLR Ligand, but it also represents a new layer of neutrophil NETosis modulation. Further elucidation of this layer of modulation of NET formation by endogenous and exogenous TLR ligands in disease states may have important implications for the pathogenesis and intervention in human diseases.
Innate IFNs and pro-inflammatory cytokines in local control of Respiratory Syncytial Virus infection of respiratory epithelial cells.
Authors: Uehling Megan , Hillyer Philippa , Shepard Rachel , Sheik Faruk , Luongo Cindy , Buchholz Ursula , Collins Peter L , Donnelly Raymond P , Rabin Ronald L
Published: January 2023

The host innate response contributes to the severity of disease caused by Respiratory Syncytial Virus (RSV). The primary target of RSV is the airway epithelium. The host becomes infected by inhaling droplets containing viral particles, which then infect small foci of cells. Infected cells then express types I and III interferons (IFN) and pro-inflammatory cytokines to alert neighboring epithelial and myeloid cells of danger. Despite their inherent differences, A549 (carcinoma, type II alveolar epithelium) and BEAS-2B (transformed bronchial epithelium) cells are often used interchangeably to study RSV-epithelial interactions. We compared these two cell lines by infecting them with low MOIs of RSV expressing GFP (rgRSV). We measured expression of types I and III IFNs, interferon stimulated genes (ISGs), pro-inflammatory cytokines and signaling intermediaries. We found that BEAS-2B cells contained rgRSV within foci of ~10–15 cells, but all A549 cells were infected by 48h. Both cell lines highly expressed IFN-β, IFN-λ1 and -λ2, but expression was greater in the A549s. Despite lower levels of IFN expression, BEAS-2Bs expressed higher levels of most classic antiviral ISGs (ISG15, MX1, PKR) and critical TLRs, RLRs and IFN receptors. In contrast, A549 cells expressed higher levels of NF-kB associated genes (CCL2, CCL5, CXCL8). Our data suggests that a balance between expression of NF-kB genes and ISGs may determine local control of RSV infection by respiratory epithelial cells.
A surprising link between the inflammasome and the centrosome
Authors: Davis Michael Alan , Fairgrieve Marian , Gale Michael
Published: January 2023

The NLRP3 inflammasome is a multisubunit protein complex of the innate immune system that senses and responds to a broad range of cell-intrinsic, pro-inflammatory stimuli. Upon activation in non-mitotic cells, NLRP3 aggregates with the inflammasome subunits ASC and Caspase-1 to assemble a single perinuclear superstructure. However, the processes and mechanisms that orchestrate inflammasome assembly and drive pyroptosis are largely unknown. Here, we show that the inflammasome recruits and colocalizes with known centrosome-associated proteins, γ-Tubulin and Centrin, in a Caspase-1 dependent manner. While the NLRP3 inflammasome does not colocalize with the centrosome itself, we show that the inflammasome assembles in close proximity to the centrosome early after NLRP3 activation. Furthermore, inflammasome formation results in a breakdown of the microtubule network as well as decreased γ-Tubulin and Pericentrin staining at the centrosome. These observations are strengthened by our proteomic studies identifying >30 centrosomal interacting with NLRP3 and/or Caspase-1. One protein in particular is Nek7, a serine/threonine kinase required for the recruitment of γ-Tubulin and Pericentrin to the centrosome, suggesting that inflammasome activation phenocopies Nek7 loss. Finally, depletion of Nek7 inhibits pyroptotic cell death. Therefore, these data reveal a novel link between the inflammasome and centrosome, possibly bridged by NEK7, and provide mechanistic insight into inflammasome formation and pyroptosis.
Nek7 is an essential mediator of NLRP3 activation downstream of potassium efflux
Authors: He Yuan , Zeng Melody Yue , Yang Dahai , Motro Benny , Nunez Gabriel
Published: January 2023

Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation remains unknown. We have identified Nek7 as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of Nek7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasome. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-Nek7 complex, which along with ASC oligomerization and ASC speck formation were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed that Nek7 was required for NLRP3 inflammasome activation in vivo. Our studies demonstrate that Nek7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.
Dysregulated type I interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in SARS-CoV-infected mice
Authors: Channappanavar Rudragouda , Fehr Anthony , Vijay Rahul , Jincun Zhao , Meyerholz David K , Perlman Stanley
Published: January 2023

Highly pathogenic human respiratory coronaviruses cause acute lethal disease characterized by an exuberant inflammatory response and severe lung damage. Although fatal outcomes due to immunopathological events following SARS-CoV infection have been well established, factors initiating detrimental inflammatory responses are not well understood. Since persistent elevation of IFN-I suggested a pathogenic role in SARS patients, we explored the possibility that IFN-I was critical in the initiation of events that led to lethal lung immunopathology. Using mice infected with SARS (Severe Acute Respiratory Syndrome)-CoV, we show that robust virus replication accompanied by dysregulated type I interferon (IFN-I) signaling orchestrated exuberant inflammatory responses and lung immunopathology with diminished survival. Delayed IFN-I signaling promoted the accumulation of pathogenic inflammatory monocyte-macrophages (IMMs) resulting in elevated lung inflammatory cytokine/chemokine levels, extensive vascular leakage and impaired virus-specific T cell responses. Genetic ablation of the IFN-αβ receptor (IFNAR) or depletion of IMMs completely protected mice from lethal infection, without affecting viral load. Together, these results demonstrate that IFN-I signaling is detrimental in highly susceptible SARS-CoV-infected BALB/c mice, in large part by promoting the influx of highly pathogenic inflammatory monocytes-macrophages (IMMs) and identify IFN-I and IMMs as potential therapeutic targets in patients infected with severe CoV and perhaps other highly pathogenic respiratory viruses.
Cytokine production in the skin is regulated by metabolites from the microbiome that influence histone acetylation.
Authors: Sanford James Asbury , Huang Chun-Ming , Gallo Richard L
Published: January 2023

Immune functions of the skin, including cytokine production from epidermal keratinocytes, are influenced by the resident microbiota. We hypothesized that a prominent member of the normal skin microbiome, Propionibacterium acnes (P. acnes), can affect cutaneous inflammation through the epigenetic mechanism of inhibiting histone deacetylase (HDAC) activity. GC-MS analysis revealed that P. acnes can generate high levels of the short-chain fatty acids (SCFAs) propionic and valeric acid. These molecules increased levels of histone acetylation as seen by Western blot analysis of H3K9ac and H3K27ac and ChIP-PCR of H3K9ac in the IL-6 promoter. Using qPCR, RNA-sequencing, and multiplex ELISA we found that HDAC inhibition alone had no effect on cytokine production from primary cultures of normal human keratinocytes (NHEK), but greatly increased the production of many cytokines and chemokines (i.e. IL-1β, IL-6, IL-8, TNFα, and CCL5) elicited by ligands of TLR2 or TLR3. In direct contrast, HDAC inhibition suppressed TLR-mediated inductions of these cytokines in monocytes and dendritic cells. The mechanism of inhibition in PBMCs and DCs was consistent with previous findings demonstrating increased expression of Mi-2β and activation of the NuRD co-repressor complex, events that were absent in NHEK. Inhibition of HDAC activity in mouse skin confirmed these responses in vivo. siRNA screening identified a novel mechanism involving HDAC8 and HDAC9 as responsible for these effects in keratinocytes. Together, these results show that the skin local environment and microbe-derived SCFAs influence inflammatory gene expression in a cell type-specific manner, thus explaining selective inflammation in diseases such as acne.
Engineered C-reactive protein with improved protective action against pneumococcal infection
Authors: Ngwa Donald Neba , Gang Toh B , Singh Sanjay K , Agrawal Alok
Published: January 2023

The anti-pneumococcal function of native C-reactive protein (CRP) involves the binding of CRP to phosphocholine molecules present on Streptococcus pneumoniae and subsequent activation of the complement system. However, when pneumococci recruit complement inhibitory protein factor H on their surface, they escape the attack of complement. We have reported previously that CRP, in a non-native conformation, expresses the ability to bind immobilized factor H. Accordingly, we hypothesized that such non-native CRP would bind to factor H on pneumococci in vivo, mask its complement inhibitory activity, and therefore should be able to protect against complement-resistant pneumococci. We performed site-directed mutagenesis of CRP and identified a mutant which bound to factor H-coated pneumococci but not to factor H in fluid phase. Mutant CRP was found to be free in the mouse serum and blood and its rate of clearance was similar to that of native CRP. As reported previously, native CRP protected mice from lethal pneumococcal infection when injected into mice 30 minutes prior to the administration of pneumococci, but did not offer any protection when injected at 24 hours after the administration of pneumococci. In contrast, that was not the case with mutant CRP; mutant CRP protected mice from infection more effectively than native CRP, by increasing the survival and decreasing bacteremia. We conclude that, while native CRP is protective only against early-stage infection, mutant CRP capable of binding to factor H may protect against both early-stage and late-stage infections and thus is superior to native CRP in fighting against the disease.
Characterization of the interactions between fish serum proteins and pathogenic bacteria by using label-free quantitative proteomics
Authors: Dong Miao , Tang Sze Wing , Shen Wei , Au Doris W.T. , Lam Yun Wah
Published: January 2023

Fish blood is a valuable source of antimicrobial proteins which form a crucial part of the innate immune system, but the types and functions of these proteins are still not fully understood. The objectives of this study are to systematically characterize the serum proteins from fish that stably bind to bacteria and to investigate how bacteria respond to these interactions. Serum from turbot, but not the heat-inactivated control, significantly decreased the number of E. tarda bacteria. By conjugating fish serum proteins with fluorescent dye, we showed that E.tarda were coated with multiple labeled proteins. In order to systematically identify these bacteria-binding proteins, we used E.tarda cells to capture turbot serum proteins and subjected the samples to shotgun proteomic analysis, followed by label-free quantitation. A total of 76 fish proteins have been identified, including known antimicrobial proteins such as complement components and Wap65-2. 34 proteins with no previously known immunological function were also identified to bind to E.tarda. This approach also allowed the study of the proteomic changes in E.tarda exposed to turbot serum. Our data indicated a rapid decrease of translation factors and an upregulation of membrane components in response to serum treatment. Interestingly, fish serum induced a rapid suppression of the expression of bacterial antioxidant enzymes like catalase and led to an accumulation of reactive oxygen species, suggesting that fish serum can override a bacterial self-defence mechanism. Taken together, this work offers a comprehensive view of the interactions between fish serum proteins and pathogenic bacteria, and reveals previously unknown factors and mechanisms in fish innate immunity.
Expanded CD4+ CD25− FoxP3+ Regulatory T cell population in HIV-TB patients
Authors: Quiroga M Florencia , Vecchione Maria Belen , Suarez Guadalupe , Laufer Natalia , Ben Graciela , Sued Omar , Angerami Matias T
Published: January 2023

Tuberculosis (TB) and HIV alter the immune system, and coinfected (HIV-TB) individuals usually show deregulations in lymphocyte’s immune functions. We previously observed an increased frequency of “unconventional” Treg (uTreg, CD4+CD25−FoxP3+) during HIV-TB. Therefore, we aimed to explore the phenotype and function of uTreg and conventional Treg subsets (cTreg, CD4+CD25+FoxP3+) among HIV-TB (n=34) patients and healthy donors (HD, n=30). We evaluated the expression of CD39, PD1, GITR and maturation status by flow cytometry in cTreg and uTreg. Also, IL-10, TGF-β, IFN-γ production and suppressive capacity were analyzed in cocultures of Tregs and effector lymphocytes. We found diminished expression of CD39 and higher levels of PD-1 on uTreg compared to cTreg in both HIV-TB (p<0.01) and HD (p<0.001). Nevertheless, GITR expression was increased only in uTreg of HIV-TB patients (p>0.05). In addition, uTreg and cTreg showed differences in maturation status both in HIV-TB and HD groups (p<0.05, p<0.01 respectively), due to Effector Memory uTregs expansion (p>0.05). Interestingly, both HIV-TB and HD showed higher production of IFN-γ only in uTreg population (p<0.01), though no significant differences were observed for IL-10 and TGF-β production between uTreg and cTreg. Moreover, IFN-γ+ cells were restricted to the CD39− uTreg population. Finally, when suppressive capacity was evaluated, both uTreg and cTreg inhibited T cell-proliferation and IFN-γ production in a similar extent (p<0.05). These findings suggest that uTregs, which are expanded during HIV-TB coinfection, are fully functional in terms of inhibition of T cell responses despite of an altered surface expression of characteristic Treg markers and cytokine production.
Environmental bioaerosol antigen Methanosphaera stadtmanae induces a TH17 inflammatory lung response via TLR4 signaling
Authors: Bernatchez Emilie , Gold Matthew J , Langlois Anick , Blais-Lecours Pascale , Duchaine Caroline , Marsolais David , McNagny Kelly M , Blanchet Marie-Renée
Published: January 2023

Bioaerosols in occupational environments are highly associated with the development of inflammatory lung diseases. The archaea specie Methanosphaera stadtmanae (MSS) is found in high concentrations in poultries, dairy farms and swine confinement buildings bioaerosols (up to 108 archaea/m3). MSS induces a strong specie-dependent inflammatory lung response in mice, characterized by T cells, eosinophils, neutrophils, and IgG production. However, the polarity of lung response induced by MSS and the mechanisms underlying this inflammatory response remain unknown. Using a mouse model, we show that MSS induced a weak TH2 (CD4+/IL-13+T cells) strong TH17 (CD4+/IL-17A+T cells) lung response, characterized by IgG1 (but not IgG2a and IgE) production. Moreover, mice did not develop airway hyperresponsiveness following MSS exposure. Interestingly, increasing MSS quantity led to a lower eosinophil count associated with a decreased TH2 response. Using transgenic mice, we found that eosinophils, mast cells and ILC2 cells are not required for the TH2 and TH17 inflammatory response to MSS. However, Tlr4−/− mice (but not Tlr2−/−) had reduced airway inflammation compared to WT mice after exposure to MSS, indicating a crucial role for TLR4 activation in this specific response. Finally, heat denaturation and zymography studies suggested TLR4 activation likely occurs through MSS protein antigen recognition rather than by enzymatic activity and cell damage. We conclude that MSS induces a TH17 inflammatory lung response via TLR4 signaling.
How genetically modified probiotic bacteria can modify human dendritic cell functions?
Authors: Tóth Márta
Published: January 2023

Selected lactic-acid bacterial strains such as Lactobacilli are classified as probiotics with beneficial effects on the human host. It has previously been shown that the group of Lactobacillus rhamnosus/casei/paracasei are able to prevent cytokine-induced apoptosis of intestinal epithelial cells by secreted factors identified as peptidoglycan-hydrolases. To exert their protective and immunomodulatory effects, these bacteria have to interact with the underlying epithelial and immune cells, such as dendritic cells (DCs). DCs recognize Lactobacilli and their components by pattern recognition receptors (PRR) that may induce stimulatory or inhibitory T-lymphocyte responses.

Our results showed that the inflammatory response of human monocyte-derived DCs (moDCs) to bacterial stimuli and the ability to polarize T-lymphocytes to different directions was highly dependent on the intact structure of the bacterial cell wall, while the expression of the cell surface molecules of moDCs was independent on the modification of the peptidoglycan (PG) layer. It was also found that the secreted bacterial cell wall preparations were able to activate moDCs demonstrated by the increased levels of pro – and anti-inflammatory cytokines and elevated expression of cell surface markers independently on the genetic manipulation of the cell wall.

Based on these results we conclude that the genetic modification of the bacterial cell wall is able to modulate the stimulatory potential of moDCs and has an impact on the outcome of T-lymphocyte responses at the level of the whole bacteria. In contrast to the effects of the specific PG extracts, the functions of moDCs are independent on the genetically modified bacteria or their cell wall and its components.
Type I IFN dependent and independent mechanisms of protection in C. difficile infection
Authors: Snyder Deann Teresa , Hedges Jodi F , Robison Amanda , Broadaway Susan C. , Walk Seth T. , Jutila Mark A
Published: January 2023

Clostridium difficile infection (CDI) is increasingly prevalent in individuals in health care settings and in the community. C. difficile is a gram positive, anaerobic bacteria that forms spores resistant to common disinfectants. Upon infection, C. difficile spores germinate, divide and produce damaging toxins resulting in intestinal inflammation. While inflammatory mediators are known to affect disease severity, the role of type I IFN signaling in CDI has not been characterized. We hypothesized that type I IFN signaling was important for innate protection in CDI. IFNAR-deficient mice succumbed to CDI more rapidly than wild type mice. Since type I IFNs can reduce inflammasome activity, we assessed IL-1β expression in multiple tissues, and discovered that IFNAR mice expressed more IL-1β protein, and less insulin-like growth factor (IGF) transcripts in response to CDI. To investigate a potential treatment, we treated mice with the type I IFN-inducing dsRNA mimetic, polyIC. PolyIC treatment delayed onset of symptoms in survival studies. Surprisingly, the protective effect of polyIC was not a result of IFNAR signaling; treatment increased IGF and decreased IL-1β in both wild type and IFNAR-deficient mice. Finally, treating wild type mice with recombinant type I IFN also reduced disease severity. These findings suggest direct type I IFN signaling protects from CDI and treatment with polyIC may protect mice via a pathway independent of IFNAR signaling. Considering that recombinant type I IFN is already used clinically and novel treatment options for CDI are needed, further investigation of innate immune pathways may present new treatment options for this emerging infection.
Increased Treg cells and expression of CD39/CD73 during listeriosis in aging mice
Authors: Cavanaugh Christopher , Gaines Dennis , Pereira Marion , Williams Kristina , Alam M. Samiul
Published: January 2023

Foodborne Listeria monocytogenes (LM) is a cause of serious illness & death in the elderly. Aging is associated with increased susceptibility to pathogens. We investigated the cause of this increased susceptibility of aging mice to LM infection using an oral model. Young & old C57BL/6 mice were gavaged with murinized LM and T-helper (Th) cell responses were evaluated. After infection, tissues were examined for bacterial burden & histology. Isolated splenocytes were analyzed for intracellular Th1/Th2 cytokine expression (TNF-α, IL-10 and IFN-γ) & distribution of Treg (CD4+CD25h) & Th17 cells before & after infection. The expression of adenosine producing enzymes, CD39/CD73, in Th cells was analyzed by FACS & qRT-PCR. Old LM-infected mice lost more body-weight & had a higher bacterial burden in the liver/spleen than young-infected mice. Uninfected old mice showed a higher baseline proinflammatory response when compared to young control mice. After infection, the proinflammatory cytokine, IFN-g, was higher in young mice as compared to old mice. An increase in Th-17 cells was observed in the older mice before & after infection. Increased levels of IL-10 & Treg cells were observed in older mice after infection. Higher expression of CD39/CD73 was observed in the Th cells of the older mice & the Treg expression of CD39 doubled after infection, suggesting increased production of adenosine, an anti-inflammatory mediator. Increased LM infection in aging mice is associated with reduced IFN-g response & increased anti-inflammatory responses from Treg cells with high expression of CD39 in splenocytes. Increased accumulation of splenic Th17 cells in LM-infected old mice can further contribute to increased inflammation leading to LM pathogenesis.
Host response to Neisseria gonorrhoeae infection in female wild type mice is dictated by the dominant sex hormone
Authors: Francis Ian P , Shaik-Dasthagirisaheb Yazdan , Islam Epshita , Gray-Owen Scott D , Wetzler Lee
Published: January 2023

BACKGROUND

Gonorrhea, a sexually transmitted disease (STD) caused by Neisseria gonorrhoeae (GC), causes major sequelae in women. Epidemiology suggests a link between symptomatic gonorrhea and the normal female hormone cycle. The aim of this study was to evaluate the murine gonorrhea-model immune responses to infection during progesterone dominate diestrus, and estrogen dominate estrus.

METHODS

Hormone cycle stage was determined by cytology of vaginal washes. Mice were infected with 107 bacteria in PBS directly placed into a uterine horn. Serum, upper and lower genitourinary (UGU, LGU respectively) tract tissues were collected at 6 or 18 h post infection. Cytokines levels in these tissues were measured by multiplex immuno assay. Gene transcription in UGU and LGU samples was measured by microarray.

RESULTS

Infection during estrus induced a moderate cytokine response at the site of infection. Cytokine levels were much greater when infection occurred during diestrus. Microarray analysis of infected diestrus tissues showed significant enrichment of those pathways associated with host immune response when infection occurred in diestrus.

CONCLUSIONS

GC infection during diestrus was shown to be highly inflammatory. Immune pathways were strongly upregulated as determined by Gene set enrichment analysis (GSEA) of microarray data. The most differentially expressed pathways were associated with pattern-recognition-receptor activation and cytokine response. GSEA analysis of estrus tissue showed very limited enrichment of any immune processes. These data together lead us to conclude that the inflammatory response to GC infection in mice is strongly dependent on the dominant sex hormone at the time of infection.
Gene expression profiles predict treatment outcomes in Brucellosis
Authors: Dufort Matthew J , Hanniffy Sean , Álvarez Raquel Conde , Gorvel Vilma Arce , Altman Matthew C. , Khaenam Prasong , Solera Javier , Gersuk Vivian H. , Bosilkovski Mile , Moriyón Ignacio , Linsley Peter S , Gorvel Jean-Pierre
Published: January 2023

Brucellosis is a zoonotic disease caused by Brucella bacteria, acquired by humans through contact with or consumption of products from infected animals. Treatment outcomes vary widely; some patients recover, others relapse, and others develop chronic symptoms despite therapy. We employed expression profiling using RNA sequencing (RNAseq) to better understand variation in outcomes and investigate disease mechanisms. We performed RNAseq analysis of whole blood samples from 125 Brucellosis patients and 51 healthy controls from Macedonia. Patients were subdivided based on disease history into primary, secondary, and chronic infection. Secondary and chronic cases did not show differences in gene expression from healthy controls or from each other. Primary cases displayed numerous transcriptional changes relative to healthy controls. Up-regulated genes were enriched for response to interferon-gamma, cytolysis, T cell proliferation, and cell cycle; down-regulated genes, for B cell proliferation. Primary cases were further subdivided based on outcome into those who resolved with treatment and those who developed disease relapse during treatment. In comparison to healthy controls, relapse cases showed larger magnitude differences in expression at diagnosis than resolution cases; this suggests that relapse could potentially be predicted based on transcription levels at time of diagnosis. Differences in gene expression observed at diagnosis were no longer present 6–9 months after diagnosis. Our results indicate opportunities to prognostically identify patients that may require more intensive treatment and monitoring, and to better understand the symptomology of and immune response to Brucella infection.
CD4+ T cells from human neonates and infants respond variously against antigens of Staphylococcus aureus, but not against Bifidobacter longum ssp. infantis
Authors: Brunner-Weinzierl Monika C , Pierau Mandy , Schlueter Dirk , Lampe Karin , Arens Christoph , Jorch Gerhard , Katrin Vogel
Published: January 2023

T cells in human neonates, infants, and adults differ dramatically in the initiation, strength, and stability of their responses. In this study, we investigate cellular mechanisms of CD4+ T cells from neonates, infants and adults to show the antigen specific response to Staphylococcus aureus and Bifidobacterium longum ssp. infantis.

T cells from surgically excised adenoids of infants in different ages, cord blood and peripheral blood from healthy donors were characterized by flow cytometry and functional assays. Therefore CD14+ monocytes were incubated with extracts of Staphylococcus aureus and Bifidobacterium longum ssp. infantis to stimulate enriched T cells.

CD4+ CD45RA and CD4+ CD45R0 T cells proliferate and up regulate the activation-associated molecule CD25 in response to Staphylococcus aureus whereas stimulation with Bifidobacterium longum ssp. infantis had no effect. In functional assays, we could show that T-cell proliferation is induced by Staphylococcus aureus via TCR-specific signal transduction. In this line, the antigen-specific response can be reduced by blockade of MHC class II molecules. Surprisingly, an inverse correlation between the percentages of Staphylococcus aureus induced proliferating T cells and age of infants is observed.

In the case of activation with Staphylococcus aureus functional specific T cells are identified in cord blood, adenoids of infants, and adults with age-dependent characteristics.

This work highlights the gap between specific T cell responses of neonates, infants, and adults in terms of quality and quantity. Our findings will help to understand the relationship between pathogens and T cells and to optimize future strategies for therapeutic interventions.
Towards an Understanding of Protective Cellular Immunity - a TCR Transgenic Mouse Reactive with Chlamydia muridarum
Authors: Poston Taylor , Qu Yanyan , Russell Ali , Girardi Jenna , Nagarajan Uma , Darville Toni
Published: January 2023

A major limitation in understanding the development of protective T cell memory against Chlamydia is the difficulty in characterizing low frequencies of antigen-specific T cells. To better understand the kinetics and phenotype of specific CD4 T cells, we developed a C. muridarum-specific T-Cell Receptor (TCR) transgenic (Tg) mouse. Primed T cells from C57BL/6 mice immune to C. muridarum were stimulated with EB/RB for 5 days and fused with BW5147 cells. Specific clones producing IL-2 and IFN-γ were harvested and screened for TCR Vα and Vβ by qPCR. Cloned transgenes were extracted, purified, and co-microinjected into fertilized oocytes. PCR and FACS confirmed integration and expression. Tg T cells were analyzed ex vivo for activation markers and cytokine production after EB/RB stimulation. 90% of peripheral CD4+ T cells express TCR transgenes and exhibited enhanced proliferation and increased expression of IFN-γ, IL-2, and CD69, compared to controls. Naive T cells were adoptively transferred from CD45.2 Tg mice into CD45.1 mice one day before intravaginal inoculation with C. muridarum. Tg cells showed enhanced proliferation and TNF-α, IL-2, and IFN-γ production by day 5 compared to controls. Tg T cells collected from SLOs and oviducts on days 5, 8, 12, 22, 44, and 65 post-infection exhibited enhanced expression of memory and activation markers. Adoptive transfer of Tg or WT CD4 T cells to Rag1−/− mice rescued an otherwise lethal C. muridarum genital infection. WT and Tg CD4 T cells exhibited comparable chlamydial clearance from the genital tract. Adoptive transfer of Chlamydia-specific CD4+ T cells offers a powerful approach to characterize cellular activation, differentiation, and memory development.
Development of a metal-based detection method for simultaneous protein and gene expression analysis in single cells by mass cytometry
Authors: Mavropoulos Anastasia , Majonis Daniel , He Mingxiao , Park Emily , Ornatsky Olga
Published: January 2023

The Fluidigm® CyTOF system is a mass cytometer that uniquely enables high-dimensional single-cell analysis of complex populations. Mass cytometry is based on inductively coupled plasma time-of-flight mass spectrometry used for multiplex proteomic analysis. In this approach, metal-conjugated affinity reagents are used to tag the components of cells. The cells are nebulized and sent to an argon plasma, ionizing the multi-atom metal tags, which are then analyzed by a time-of-flight mass spectrometer.

Gene expression can be finely tuned through the synthesis of RNA and through the control of its stability and location. Misregulation of gene expression can have severe consequences, such as developmental disorders, cancer and autoimmune diseases. The ability to acquire complete spatial-temporal profiles of gene expression is therefore critical for the understanding of disease pathophysiology, medical diagnostics and drug discovery.

In this presentation, we discuss the development of a multiplex method for targeted RNA detection using the Fluidigm CyTOF® and Advanced Cell Diagnostics RNAscope® platforms. This novel assay includes the hybridization of RNA-specific target probes, followed by signal amplification and ending with the binding of amplifier-specific metal-labelled probes. Presently, we are able to detect four different mRNA probes in a single-cell analysis setting. The detection of RNA is compatible with Fluidigm products such as Maxpar® antibodies. Future developments will include high-dimensional detection that enables researchers to investigate the simultaneous expression profile of RNA and protein across millions of cells.
Transcutaneous delivery of protein tyrosine phosphatase ameliorates inflammatory skin diseases
Authors: Kim Won-Ju , Koo Ja-Hyun , Cho Hyun-Jung , Kim Ji-Yun , Lee Sohee , Kim Jong Hoon , Suh Minah , Shin Eui-Cheol , Choi Je-Min
Published: January 2023

Transcutaneous delivery of therapeutic drugs has many advantages of topical treatment over the systemic administration for inflammatory skin diseases such as atopic dermatitis or psoriasis. However, the greatest challenge for transcutaneous drug delivery limits clinical applications due to the skin tissue barrier. Here, we identified a novel transcutaneous delivery peptide, AP, from human neuronal adhesion protein (Astrotactin 1) which could deliver a macromolecule such as a protein into skin tissue. AP-conjugated EGFP protein exhibited significantly higher intracellular transduction efficacy in HaCaT (keratinocyte), NIH3T3 (fibroblast) and Jurkat (T cell) cells compared with control proteins. In addition, AP-EGFP and –dTomato protein was efficiently localized into the dermis as well as epidermis in mouse skin tissue following transcutaneous administration. Next, we generated AP-rPTP protein, which is chimeric protein of AP and phosphatase domain of TC-PTP. AP-rPTP inhibited phosphorylation of STAT1, 3, 5 in mouse splenocytes and T cells and reduced cytokines production by activated splenocyets. To confirm its in vivo relevance, we transcutaneously applied it to dermatitis mouse models. Multiple skin patch administrations of AP-rPTP protein significantly ameliorated tissue inflammation in oxazolone-induced contact dermatitis and imiquimod-induced psoriasis mouse model. Our results collectively demonstrate that AP is a novel human derived transcutaneous delivery peptide and AP-rPTP protein could be a therapeutic biomolecule in inflammatory skin diseases such as allergic dermatitis and psoriasis.
Sinomenine attenuates angiotensin II-induced autophagy via inhibition of p47-phox translocation to the membrane and influences reactive oxygen species generation in podocytes
Authors: Zhang Jingbo , Wang Weili , Cai Juan
Published: January 2023

Background/Aims

Sinomenine, a pure alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, and sinomenine hydrochloride (SN) has been successfully used for the therapy of rheumatoid arthritis (RA) and kidney diseases. Autophagy is a cytoprotective mechanism used by podocytes and other cells to alleviate the effects of oxidative stress, and angiotensin II (Ang II) significantly promotes podocyte autophagy. However, excessive autophagy may lead to cell death and podocyte depletion. The present study evaluated the effect of SN in podocytes induced by Ang II.

Methods

Podocytes were pretreated with graded concentrations (10−8 M ~ 10−4 M) of SN and then stimulated with Ang II. The LC3B protein and the p47-phox membrane fraction were measured by Western blot. Autolysosomes were assessed by transmission electron microscopy. FACS was used to quantify the ROS produced by podocytes. The translocation of p47-phox to the membrane was investigated by immunofluorescence.

Results

The 10−8M ~ 10−4M of SN alone did not effect ROS generation or podocyte autophagy. The 10−8M and 10−6M SN attenuated Ang II-induced autophagy in podocytes. Furthermore, SN decreased the level of ROS generation in Ang II-induced podocytes via inhibition of NOX subunit p47-phox translocation to the membrane.

Conclusion

The appropriate concentration of SN attenuated Ang II-induced podocyte autophagy through ROS generation, at least in part, by regulating NOX subunit p47-phox translocation to the membrane.
Exploiting T cell co-inhibition to delay autoimmune disease recurrence
Authors: Burrack Adam L , Osum Kevin , Pauken Kristen , Fife Brian
Published: January 2023

Type 1 diabetes (T1D) results from T cell-mediated destruction of insulin-producing pancreatic β cells. Individuals with long-term disease are at risk of developing life-threatening complications. β cell replacement is a therapy for T1D but is limited by recurrent autoreactive T cell targeted β cell death. Thus, β cells better equipped to inhibit local T cell responses may survive longer in autoimmune recipients. Programmed-death 1 (PD-1) signaling through its ligand PD-L1 inhibits T cells, and may serve as a prominent defense in T1D. Using flow cytometric analysis, in the absence of T cells in NOD.RAG−/− mice we do not detect β cell PD-L1 expression. However, with T cells, we observed an increased proportion of β cells expressing PD-L1 in female non-obese diabetic (NOD) mice which had not developed diabetes. In addition, the majority of remaining live β cells at diabetes onset in NOD mice continue to express high levels of PD-L1. These three situations suggest that islet β cells may increase PD-L1 expression as a last line of defense to limit infiltrating T cell mediated destruction. To manipulate β cell PD-L1 expression prior to transplantation, we screened a panel of diabetes-related cytokines and found that IFN-γ enhances β cell PD-L1 expression. Unfortunately, islet transplant survival was not prolonged, which we hypothesized was due to enhanced MHC class I expression, facilitating CD8+ T cell-mediated killing. We therefore de-coupled PD-L1 from enhanced MHC I expression. Using this approach, enforced β cell PD-L1 expression delays disease recurrence. These data support our hypothesis that β cells expressing T cell co-inhibitory molecules, like PD-L1, can locally inhibit autoreactive T cells which may prevent transplant destruction.
Prophylactic and therapeutic neutralization of IL-21 bioactivity protects mice from lethality induced by human T cells in a xenogeneic mouse model of graft vs host disease
Authors: Wang Shu , Naiman Brian , Gonzalez Carlos , Herbst Ronald , Kolbeck Roland , Ettinger Rachel
Published: January 2023

IL-21 is a pluripotent cytokine involved in both T and B cell activation/differentiation and plays a major role in Xenogeneic graft vs. host disease (XenoGVHD) where human lymphocytes are transferred into NOD/SCID/gc mice. In order to elucidate the underlying mechanisms of IL-21 action, a neutralizing anti-human (but not anti-mouse) IL-21 mAb, was administered prior to cell transfer and the resultant effects on disease progression determined. Strikingly, IL-21 neutralization halted T cell expansion, and decreased CD4/CD8 T cell ratio resulting in complete protection to wasting and lethality. Loss of IL-21R signaling significantly inhibited several cytokines including, GM-CSF, IL-10, TNF, IFNγ, IL-17A and IL-9, while surprisingly significantly increased IL-5. Both T cell expansion and serum cytokines dramatically increased after discontinuation of anti-IL-21 treatment, resulting in weight loss and death of the mice. Anti-IL-21 was protective when administered 1 or 2 weeks post-graft, where 100% and 40% survival was found, respectively. These data demonstrate that blockade of IL-21 did not simply result in loss of T cell survival, but rather inhibited homeostatic CD4 T cell proliferation and selectively impacted cytokine production switching the response from a Th1 pathogenic to a Th2 protective response. Thus, blockade of IL-21 was not globally eradicating T cell responses, but rather shaping the immune response resulting in protection from GVHD-induced lethality.
Bim identifies tumor-reactive effector CD8+ T cells in cancer patients respond to anti-PD-1 therapy
Authors: Dong Haidong , Dronca Roxana S
Published: January 2023

Endogenous tumor-reactive CD8+ T cell responses to tumors are controlled by immune checkpoints. Immune checkpoint blockade strategy has demonstrated therapeutic effects in some solid cancers. However, only a portion of patients responds to this therapy and there are no biomarkers to help predict and monitor treatment outcomes. We report that Bim is a downstream signaling molecule of the PD-1 pathway that reflects the degree of PD-1 interaction with its ligand B7-H1. B7-H1 protein directly induced Bim protein up-regulation in pre-activated human CD8+ T cells via regulation of AKT activation and phosphorylation of Bim. In mouse models, genetic absence of PD-1 in T cells and B7-H1 deficiency in tumors prevented Bim up-regulation in tumor-infiltrating CD8+ T cells. Among tumor-reactive PD-1+ CD11ahigh CD8+ T cells in the peripheral blood of cancer patients, Bim expression was significantly associated with expression of PD-1 and effector cell markers (T-bet and granzyme B). The frequency of Bim+ PD-1+ CD8+ T cells significantly increased in the peripheral blood of advanced melanoma patients compared to healthy donors. In melanoma patients, high Bim levels were correlated with poor overall survival. Importantly, patients who experienced clinical benefit after anti-PD-1 therapy had higher frequency of Bim+PD-1+CD8+ T cells in the peripheral blood at baseline compared to non-responding patients; however, this cell population decreased significantly after successful anti-PD-1 therapy. Our results indicate that Bim reflects the degree of PD-1 interaction with its ligand PD-L1 in effector CD8+ T cells, and measurement of Bim levels may provide a less invasive method to monitor or predict responses to anti-PD-1 therapy.
Signaling events resulting from synchronous BCR and CD180 activation on murine B cells differ from activating CD180 or the BCR alone
Authors: Roe Kelsey , Shu Geraldine , Draves Kevin E. , Clark Edward A.
Published: January 2023

CD180 (RP105), expressed on dendritic cells and B cells, is a receptor closely related to TLR4. CD180 signaling does not require adaptor proteins like MyD88, instead it resembles B cell receptor (BCR) signaling, including the activation of PI3K and Akt. Recently, others and we have found αCD180 also induces the Pim-1 kinase, but much of the CD180 signaling pathway remains ill defined. We previously found that targeting antigen (Ag) to B cells via CD180, by coupling the Ag to an anti-CD180 antibody (Ag-αCD180), induces a rapid and strong Ag-specific IgG antibody (Ab) response in mice. Furthermore, IgG Ab responses were maintained in immunodeficient mice that lack mature B cells (BAFFR−/−). These studies led us to investigate the signaling events that occur within B cells upon coincident activation of the BCR and CD180. For this study we have employed B1-8hi transgenic mice, which have a BCR that binds to NP-hapten on some splenic B cells, and the K46μm17 murine B cell line, that expresses both CD180 and an NP-specific BCR. By stimulating these NP-specific B cells with αCD180, NP-isotype Ab control or an NP-αCD180 conjugate, we are defining the signaling elements specific to the activation of the BCR and CD180 together vs. single receptor ligation only. Utilizing these models, we are working to characterize the survival and proliferation signals induced, including Pim-1 and Akt, and to phenotype the B cell expansion in mice following NP-αCD180 vaccination. Data will be presented showing how CD180-BCR co-ligation differs from signaling through either receptor alone. These studies will enhance our understanding of how CD180 Ag targeting is able to induce strong humoral responses even in immunodeficient mice. (Supported by NIH grant R01AI44257).