bluuebunny/crossref_metadata_2025 huggingface dataset:
Type I IFN induces vascular permeability delivering iron binding proteins to the site of Cryptococcosis gattii infection resulting in local nutritional immunity and contained infection
Authors: Davis Michael J , Pinheiro Giovana , Hoke Liz , Chang Yun , Kwon-Chung June
Published: January 2023

While Cryptococcus neoformans primarily infect AIDS patients, Cryptococcus gattii (Cg) causes deadly mycosis in mostly non-HIV patients even in Cg endemic areas with high rates of AIDS. Previous work found that type I IFN (t1IFN), which is induced by HIV infection, protects mice from Cg. Thus, we sought to examine the mechanisms mediating t1IFN protection from Cg. Previous data ruled out cellular immunity, so we examined soluble immune factors. We found that induction of t1IFN by stabilized poly(I:C) (pICLC) induced the expression of serum proteins into the lung airspace fluids. PICLC induced the leak of intravenously injected FITC-dextran into lung airspace fluids suggesting that serum proteins reach the lung airspace via t1IFN-induced vascular permeability. The pICLC-mediated expression of serum proteins into the lung airspace was dependent on t1IFN but not IL-6, IL-1 or TNF-α indicating that this effect is mediated by t1IFN and not by other cytokines characterized to affect vascular permeability. Transferrin and ferritin were among the leaked serum proteins found in the lung airspace and were directly inhibitory to cryptococcus in vitro so we hypothesized that the presence of these proteins in the lung airspace may result in local iron deficiency. Iron supplementation reversed pICLC-protection while iron chelation mediated similar protection as pICLC. Together these data suggest that t1IFN induces vascular permeability in the lung which delivers iron binding proteins to the Cg niche. These host proteins restrict iron to Cg and limit infection. In addition to explaining the pICLC-induced protection from Cg, these studies illustrate a novel connection between t1IFN and vascular permeability that may be significant to other systems.
Mda5/MAVS are essential for host resistance against Aspergillus fumigatus
Authors: Wang Xi , Caffrey Alayna Katherine , Espinosa Vanessa , Cunha Cristina , Croteau Walburga , Lacerda João , Campos António , Lagrou Katrien , Maertens Johan , Rivera Amariliz , Carvohlo Agostinho , Cramer Robert A , Obar Joshua J
Published: January 2023

Type I and III interferons act as important activators of antifungal neutrophil response in the lungs. RIG-I like receptors (RLR) are cytosolic RNA sensors that signal through the MAVS adaptor in order to activate interferon responses against viruses. Whether this pattern-recognition receptor family has broader effects on host immunity against other pathogen families remains to be fully explored. Herein we demonstrate that Mda5/MAVS signaling was essential for host resistance against pulmonary Aspergillus fumigatus challenge through the regulation of antifungal leukocyte responses. Interesting, induction of type I interferons after A. fumigatus challenge was only partially dependent on Mda5/MAVS signaling, while type III interferon expression was entirely dependent on Mda5/MAVS signaling. Ultimately, type I and III interferon signaling drove the expression of CXCL10, which is critical in resistance against invasive aspergillosis in transplant patients. Importantly, we found that polymorphisms in Ifih1 and Mavs were associated with the incidence of invasive pulmonary aspergillosis in a human HSCT cohort. Moreover, polymorphisms in the Ifih1 gene alter the inflammatory response induced in patients with invasive pulmonary aspergillosis, which include interferon-responsive chemokines. In conclusion, our data broaden the role of the RLR family in the regulating innate immunity during infection to include a role in immunity against Aspergillus fumigatus in both mice and humans.
Innate immune responses are manipulated by a streptococcal communication system
Authors: Rahbari Kate , Federle Michael
Published: January 2023

Group A Streptococcus (GAS) employs numerous systems to regulate genetic programs, and infection causes a spectrum of outcomes in humans ranging from commensalism to superficial disease (e.g. pharyngitis) or invasive disease (e.g. necrotizing fasciitis). Quorum sensing (QS) is a mechanism of cell-to-cell communication used by bacteria to synchronize genetic programs across populations. We previously determined that activation of the Rgg2/3 QS system in GAS results in bacterial surface alterations, leading us to hypothesize that QS could alter interactions with host cells. To test this, macrophages were infected with mutant strains of GAS containing either constitutively activated (QS-ON) or repressed (QS-OFF) Rgg2/3, and pro-inflammatory cytokine responses were measured. Macrophage responses to QS-ON GAS were attenuated, resulting in decreased NFκB activity and decreased production of TNFα, IL-6, and IFNβ compared to infection with QS-OFF GAS. Macrophage responses to mixed QS populations remained attenuated, suggesting the effect of QS-ON GAS is dominant. Furthermore, QS-ON GAS suppressed TNFα responses induced by various TLR agonists, suggesting an active mechanism of repression occurs downstream in these pathways. Together, these data suggest that GAS utilizes Rgg2/3 QS to manipulate innate immune responses. Disabling bacterial communication using a QS-inhibitor restored pro-inflammatory responses against the bacteria, highlighting its potential application as an alternative treatment strategy.
The Function of Brain Th1-like regulatory T cells in Suppressing Lethal Immune Pathology and Neurological Deterioration during Cryptococcus neoformans Meningoencephalitis
Authors: Xu Jintao , Zhao Jessica , Ivey Michael , Lopez Rafael , McClellan Brandon , Do Steven , Malepati Akanksha , Kroon Samantha , Mysel Alexa , Olszewski Michal A
Published: January 2023

Infection-induced inflammation in the brain frequently drives severe immunopathology, resulting in lasting neurological sequelae or high mortality. Cryptococcus neoformans meningoencephalitis (CM) is one of the leading causes of neurological death worldwide. The pathology of CM is increasingly recognized to be driven by brain inflammation, especially in immunocompetent or immune reconstituted patients. The function of FOXP3+ regulatory T (Treg) cells in maintaining immune balance under these conditions is still controversial.

Using a murine CM model, we found that Tregs massively accumulate in the mouse brain, suppressing lethal immune pathology and neurological deterioration during CM. Brain-infiltrating Tregs showed a CD44highCD62Llow activated status similar to effector T cells. Intriguingly, brain Tregs acquire a Th1-like phenotype, expressing transcription factor T-bet, IFN-γ, and chemokine receptor CXCR3. This Th1-like phenotype is critical for Treg accumulation in the brain during CM. Treg depletion using an anti-CD25 antibody intensifies CNS inflammation and results in deteriorated neurological symptoms and increased mortality. Mechanistically, we showed that CNS Tregs regulate activation of recruited monocyte-derived cells as well as cytokine production of T effector cells, both of which promote brain immunopathology during CM. Interestingly, Treg depletion only showed a minor effect on glia activation. Therapeutically, we showed that selectively enhancing Treg frequency through low-dose IL-2 treatment improves mice survival and neurological functions during CM. Our findings suggest that Tregs and their products may provide therapeutic opportunities for neuronal protection during CM.
Bacterial Extracellular Vesicle Mediated Host-Pathogen Interactions in Pneumococcal Infections
Authors: Yerneni Saigopalakrishna S , Werner Sarah , Azambuja Juliana H , Ludwig Nils , Aggarwal Surya , Eutsey Rory , Lucas Peter C , Campbell Phil G , Hiller Luisa
Published: January 2023

Extracellular vesicles (EVs) represent a highly sophisticated cell-to-cell mailing system across all biological kingdoms. EVs have long been characterized from many Gram-negative species and, recently from Gram-positive bacteria, including the major respiratory pathogen Streptococcus pneumoniae (pneumococcus).

Our studies reveal that pneumococcal-derived vesicles can be internalized by macrophages, T cells, and epithelial cells. In vitro, EVs induce cytokine signaling in macrophages, including dose-dependent NF-kB signaling in murine RAW 264.7 and human primary macrophages. When administered systemically into a mouse, pneumococcal EVs result in splenomegaly and induced a sepsis-like cytokine storm. When immobilized in a hydrogel implant for local administration into a mouse, pneumococcal EVs recruited lymphocytes and macrophages. Moreover, pneumococcal lipoproteins are major contributors to NF-kB signaling and inflammatory responses, as these phenotypes were substantially reduced with EVs from a lipoprotein deficient strain (Δlgt) as compared to EVs from the wildtype strain. Taken together, in vivo studies suggest that pneumococcal vesicles alone are sufficient to induce inflammatory responses and tissue damage in mammalian hosts.

Overall, our data suggest that pneumococcal EVs display potent immunomodulatory effects on host immune cells highlighting their pivotal role during the infectious process, either by manipulating host responses or by triggering host-defense systems. Thus, pneumococcal EVs are virulence determinants and may be effective tools for vaccine development.
An unconventional T cell population is critical for protection from repeat pulmonary infection with Klebsiella pneumoniae
Authors: Smith Catherine Diane Morffy , Wasbotten Rachel , Twentyman Joy , Rosen David A.
Published: January 2023

Due to emerging antibiotic resistance, the nosocomial pathogen Klebsiella pneumoniae is an increasing public health threat. The extent to which survivors are protected against re-infection, or potential correlates of such protection, are poorly understood. We have explored the immunological mechanisms required for protection against K. pneumoniae re-infection using a nonlethal murine model of pulmonary K. pneumoniae challenge. Mice that survive K. pneumoniae TOP52 infection are protected from subsequent re-infection by an adaptive immune response. Genetically modified mice lacking mature lymphocytes or mature T cells were equally susceptible to primary and secondary infection, suggesting that T cells are required for protection. Intriguingly, neither CD4+ nor CD8+ T cells were absolutely required for either the establishment or execution of a protective memory response as depletion of CD4+ and/or CD8+ T cells during either primary or secondary infection did not impair protection against re-infection. However, we observe an expansion of γδ T cells following primary exposure to K. pneumoniae and a profound amplification of this population following secondary infection. Experiments using genetically modified mice deficient in γδ T cells or intact mice treated with γδ T cell depleting antibodies suggest that this T cell subpopulation is important for protection against re-infection. Thus, we hypothesize that K. pneumoniae exposure drives the development of a resident memory-like population of γδ T cells that are maintained in the lung and contribute to the efficient control of secondary K. pneumoniae infection.
Neonatal Deficiencies in NK cell-Activating Cytokines in the Context of Bordetella pertussis Infection
Authors: Jordan Cassandra Martinez , Mitchell Ashley E , Carbonetti Nicholas H
Published: January 2023

Whooping cough, caused by Bordetella pertussis, has seen a resurgence in recent years, in part due to the switch from the whole cell to acellular vaccine. Although both adults and infants can suffer from severe paroxysmal cough, infants can experience a fatal health decline. Disease progression in infant and adult mouse models show some parallels to human disease. Adult mice exhibit severe lung pathology, but overcome infection, while infant mice experience mild lung pathology, develop leukocytosis, bacterial dissemination, and succumb to infection. We hypothesize that this discrepancy in response to infection is due to a deficiency in neonatal Interferon gamma (IFN-y) production by Natural Killer (NK) cells, which can activate protective T-helper 1 (Th1) cells. We further hypothesize that this deficiency is in part due to the inability of neonatal antigen presenting cells to produce the NK cell-activating cytokines Interleukin (IL) 12 and IL18 when exposed to B. pertussis. To test this hypothesis, we isolated splenocytes from infected and uninfected infant and adult mice, incubated with heat killed B. pertussis, and analyzed for mRNA expression of cytokines via qRT-PCR. While adult splenocytes responded robustly with upregulated expression of IL12 and TNF-α, infant splenocytes failed to upregulate expression of these cytokines in response to bacterial stimulation. Moreover, analysis of lung tissue from infected mice revealed robust upregulation of IL12, IL18 and TNF-α in adult mice but not in infant mice. These results indicate that infant mouse cells fail to upregulate expression of NK cell-activating cytokines in response to B. pertussis infection, leading to a lack of protective IFN-y responses.
Staphylococcus epidermidis facilitates intracellular pathogen clearance through upregulation of antimicrobial protein perforin-2 (P-2) in the human skin gamma delta T cells
Authors: Strbo Natasa , O’Neill Katelyn E. , Head Cheyanne R. , Padula Laura , Stojadinovic Olivera , Pastar Irena , Tomic-Canic Marjana
Published: January 2023

Staphylococcus epidermidis (SE), nonvirulent Gram-positive (G+) bacterium, is a member of the normal human skin microbiota with beneficial relationship with the host. Gamma delta (GD) T cells as the major T cell population in epithelial tissues have been implicated in maintaining tissue integrity, regulating inflammation and defending against pathogens. Perforin-2 (P-2) is a recently described antimicrobial protein responsible for clearance of intracellular G+ and G− bacteria. We examine the relationship between SE and P-2 expression in the human skin.

Healthy human skin tissue was used for flow cytometric and bacterial infection analyses. We analyzed P-2 expression at a single cell resolution using an amplified fluorescence in situ hybridization (FISH) technique for detection of P-2 mRNA in combination with immune-phenotyping. Methicillin resistant Staphylococcus aureus (MRSA) intracellular killing assay was performed on the skin cells that were pretreated with S. epidermidis for 24 h.

We found increase in the frequency of skin GDT cells and induction of P-2 transcripts in GDT cells after S. epidermidis infection. Incubating skin cells with S. epidermidis for 24h prior infection with MRSA resulted in rapid intracellular clearance of MRSA. Our findings reveal a novel P-2 mediated mechanism by which skin commensal bacteria may exert their beneficial role in modulating host innate immune response. In contrast, establishment of S. aureus biofilm resulted in suppression of P-2 expression, revealing a mechanism by which S. aureus escapes cutaneous immunity to cause persistent biofilm wound infections.
IgM+IgD− B cells in Human GALT Harbor Characteristics of B1 B cells
Authors: Bukhari Ameera M
Published: January 2023

During embryogenesis, B cell lymphopoiesis occurs in fetal liver (FL) and bone marrow (BM). B cells derived from these organs differ in their Ig VH gene usage, their morphological and functional characteristics, and they home to different organs in the mature fetus. Although in mice, FL-B cells are known to home to the peritoneum and GALT, little is known of these cells in humans. We identified a FL-like B cell population representing 10–20% of total B cells in human GALT (appendix and tonsil), but only 1–2% in peripheral blood. These cells express IgM, but not IgD on their surface, spontaneously secrete antibodies in culture, and preferentially class-switch to IgA upon LPS stimulation, three characteristics of FL-B cells. The GALT IgM+IgD− B cells are negative for immature B cell markers CD10 and CD5, plasma cell markers CD38hi and CD138, the activation marker CD43 and the transitional B cell marker CD24hi as expected for FL B cells. In infants and children less than 10 years of age, most (70%) of these IgM+IgD− cells are CD27- non-memory B cells. These data support the hypothesis that the GALT IgM+IgD− B cells are of FL origin. Consistent with this idea, approximately 50% of the B cells in an appendix from a 1-day-old neonate were IgM+IgD−. Deep BCR sequence analysis of these cells will establish their developmental origin.
Role of Granzyme B in mucosal immune responses
Authors: Hoek Kristen L , Nazmi Ali , Singh Kshipra , Piazuelo M. Blanca , Wilson Keith , Olivares-Villagómez Danyvid
Published: January 2023

Granzyme B is a serine protease initially described in cell-mediated cytotoxicity. Recently, granzyme B has been found to be produced by non-cytotoxic cells, and elevated levels of granzyme B or cells expressing it have been implicated in many disease states. To investigate the role of granzyme B in mucosal immune responses, we utilized two models of intestinal inflammation: infection with Citrobacter rodentium, a colon-specific bacterium that induces intestinal inflammation in mice; and a T cell transfer model of colitis. Following infection, C. rodentium colonization was observed in the colon of both wt and grzB−/− animals at 14d post-infection. While wt mice displayed no overt signs of infection, severe weight loss as well as disease-associated changes in appearance and colon pathology were observed in grzB−/− mice at 10–14d post-infection. Similarly, adoptive transfer of grzB−/− CD4+CD45RBhi naïve effector T cells into rag2−/− recipients led to severe weight loss and disease-associated changes in appearance by 21d post-transfer, while rag2−/− recipients of wt CD4+CD45RBhi T cells were relatively unaffected at this early time point. Interestingly, disease in recipients of grzB−/− T cells corresponded with increased IL-17+ CD4+ T cells in the mesenteric lymph nodes and colon lamina propria relative to recipients that received wt T cells. These data suggest that granzyme B functions in a protective manner during intestinal inflammation, possibly playing a role in T cell differentiation by holding generation of IL-17-producing T cells in check.
Th17 immunity in the colon is controlled by two novel subsets of colon-specific mononuclear phagocytes
Authors: Huang Hsin-I , Youssef Nourhan , Jewell Mark , Huang Min-Nung , Hammer Gianna
Published: January 2023

Immune responses in the intestine are coordinated by specialized populations of mononuclear phagocytes. Although the functions of intestine-resident mononuclear phagocytes and the different cell subsets that constitute these populations are thought uniform across the entire intestine, it is clear that the anatomy, microbes, and immunological demands are distinct for small and large intestine. Whether these distinctions also include organ-specific mononuclear phagocyte populations, or that these have organ-specific roles in immunity are unknown. Here we identify two novel subsets of colon-specific mononuclear phagocytes, a dendritic cell (DC) subset and a macrophage subset, both of which are exclusive to the colon and are not found in small intestine. These colon-specific DCs and macrophages co-expressed CD14 and CD24, markers which are generally singularly expressed by intestinal DCs and macrophages and commonly used to distinguish between these two cell types. Colon-specific CD14+CD24+ DCs and macrophages failed to express macrophage markers CX3CR1, F4/80, CD64 and CD88, and surprisingly, both the DC and macrophage subset were dependent on the transcription factor IRF4. We further find that colon and small intestine have distinct antigen presenting cell (APC) requirements for Th17 immunity and that while colon-specific CD14+CD24+ APCs were essential for Th17 immunity in the colon, this role in small intestine was mediated by different APC subsets. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.
The role of JAML-CAR costimulation in gamma delta T cell-mediated repair of the intestinal epithelium
Authors: Johnson Margarete Daisy , Witherden Deborah , Havran Wendy L
Published: January 2023

Tissue-resident γδ T cells are key players in the maintenance of the body’s epithelial barrier surfaces and perform an essential role in promoting tissue repair. Although γδ IELs are important coordinators of repair in the intestinal epithelium, relatively little is known about how these cells recognize and respond to damage. Recognition of skin wounds by γδ T cells requires costimulation through the junctional adhesion molecule-like protein (JAML) by ligation of coxsackie and adenovirus receptor (CAR), raising the possibility that these molecules may have a similar function in the gut. Here we evaluated the role of JAML-CAR costimulation in the activation and function of γδ IELs using the dextran sulfate sodium (DSS) model of intestinal injury and repair. Following DSS treatment we found that γδ IELs upregulated their surface expression of JAML and observed a shift in the isoforms of CAR expressed by intestinal epithelial cells. In addition, JAML−/− mice exhibited more severe weight loss and epithelial damage than wildtype animals in response to DSS treatment. While we saw that JAML−/− mice had lower numbers of IELs in the colon, this difference did not account for the more severe response to DSS-induced injury in these animals. Importantly, the activation of γδ IELs following DSS treatment was reduced in the absence of JAML-CAR costimulation. In addition, we found evidence of impaired γδ IEL production of IL-17, a cytokine known to protect against DSS-induced epithelial injury. Taken together our results suggest that JAML-CAR costimulation may be needed for the activation of tissue repair functions in γδ T cells across barrier tissues and supports targeting this pathway for therapeutic approaches for the acceleration of healing.
Analysis of tissue specific mechanisms that control the maintenance of Foxp3+ regulatory T cells in the intestine
Authors: Morales Elisa Cruz , Hart Andrew , Laufer Terri M
Published: January 2023

Our lab studies the tissue specific pathways implicated in the maintenance of intestinal Foxp3+ regulatory T cells (Tregs). Previously we used K14 transgenic mice lacking peripheral TCR-MHCII to show that the small intestine lamina propria (siLP) is a niche for natural Tregs that can be filled and maintained independently of Ag-specific signals. K14 siLP Tregs display an effector phenotype (CD62loCD44hiCD103+ICOS+) similar to WT. Effector Tregs are known to be largely peripheral tissue-resident cells that require continued TCR and costimulatory signals for their maintenance. In contrast, we find that in K14 mice the blockade of the CTLA4/CD28—but not ICOS—pathway reduces the proliferation of siLP Tregs while preserving their effector phenotype (CD62loCD44hi). Thus, our results suggest that a population of thymically derived effector Tregs that depend on costimulatory signals can be maintained in the siLP independently of Ag-specific signals. B7-1/B7-2 and CD28/CTLA4 are cell surface molecules that interact through cell-cell contact. By microscopic analyses we show that a population of siLP Tregs localize inside of organized lymphoid structures known as isolated lymphoid follicles (ILF). B cells are the largest lymphocyte population within ILFs. Interestingly, our preliminary data suggest that B cells play a role in maintaining siLP Tregs in steady state, as the depletion of B cells with Rituximab reduces Treg numbers in the intestine of huCD20 mice. Additional imaging studies show ILF Tregs in intimate contact with B cells, suggesting that B cells might influence Tregs numbers by a cell-cell contact dependent mechanism. Thus, our data highlight the intricate signaling network that regulates intestinal Treg homeostasis.
Biological Evaluation of the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A for Disease Profiling of 48 Immune Factors
Authors: Keith Robert H , Lokshin Anna , Prosser Denise , Pepin Danielle , Xiao Qiang
Published: January 2023

Cytokine, chemokine and growth factor research plays a significant role in achieving a deeper understanding of the immune system and disease. We recently developed a MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A multiplex assay for simultaneous measurement of picogram levels of 48 human immune factors using Luminex® xMAP® technology in small volumes of serum, plasma or cell culture samples. Distinctly different stimulant-dependent cytokine responses were observed for challenged human PBMCs. Both LPS and Con-A induced similarly the secretion of 13 analytes, whereas LPS preferentially induced 10 analytes, including TNFα, IL-1α and IL-1β. Con-A preferentially induced 16 analytes including Th17 cell cytokines IL-17A and IL-17F. Plasma samples, from active (n=20) and benign (n=18) ovarian cancer patients (Dr. Richard Moore, U of Rochester Medical Center) were tested at UPMC. Fourteen analytes were significantly elevated in active patients vs. benign patients including: cytokines TNFα, IL-5, M-CSF; chemokines MCP-3, Fractalkine; Growth Factors FLT-3L, PDGF-BB (p<0.05); cytokines IL-6, IL-18 (p<0.02); cytokines IL-7, IL-27, IL-10, IL-13 (key role in cancer pathogenesis); chemokine MIP-1β (p<0.01). Residual serum and plasma sepsis patient samples (n=16) were compared to those from healthy controls (n=20) and 7 analytes were significantly elevated in sepsis patients: cytokines IL-1RA, IL-6 (p<0.05), IL-7, IL-27, MIP-1β (p<0.02), M-CSF; chemokine MIG (p<0.01). Twelve other analytes trended higher in sepsis samples. Thus, the Human Cytokine/Chemokine/Growth Factor Panel A is a useful tool for profiling 48 immune factors both in cell culture or blood samples for the study of human disease and inflammation.
Using super-resolution microscopy to detect and resolve the distribution of biomarkers that are relevant in a range of immuno-oncology studies
Authors: Burns Sheila , Senior Michael
Published: January 2023

Super-resolution imaging is a novel, Nobel prize-winning technology that can count individual receptors on the membranes of tumour cells and immune cells. It is ultra-sensitive, able to detect as few as 10 molecules per cell. Moreover, super-resolution provides the molecular coordinates of these receptors with nanometre precision, allowing unprecedented accuracy to investigate the interaction between multiple protein species.

We will highlight the ability of super-resolution to detect and resolve the distribution of biomarkers that are relevant in a range of immuno-oncology studies. Recently developed tools allowed us to parameterize the molecular distribution of a target such as checkpoint inhibitors. We further revealed the intermolecular distances between two targets of a novel bispecific proteins. These methods are more relevant than ever before, as ONI’s Nanoimager has packaged the necessary tools into a plug-and-play, desktop-compatible solution. This system shows dynamic imaging of live cell-cell interactions to immune synapse profiling at single molecule detection with nanometre resolution imaging that applies to fixed cells and tissue sections.
EVALUATION OF A MULTIPLEX ARBOVIRUS SEROLOGY ASSAY AS A TOOL FOR SEROSURVEILLANCE
Authors: Venkateswaran Neeraja , O’Brien Thomas W , Nelson Wiilam Max , Venkateswaran Kodumudi S
Published: January 2023

Arthropod-borne viruses (Arboviruses) constitute a serious threat to global health by causing significant mortality and morbidity. Recent years have seen the emergence of arboviruses due to climatic changes, the adaptation of mosquitoes, and other arthropods to a newer geographic area, urbanization, and increased global travel and trade. Currently, most serosurveillance programs focus on single pathogen testing that may miss the prevalent pathogen or misdiagnose due to the presence of cross-reacting antibodies from closely related arboviruses. In the wake of the 2015–2016 Zika virus outbreaks, we developed a multiplex suspension array for the detection of IgG antibodies and avidity to multiple analytes of arboviruses on the Luminex platform to overcome these limitations. We used optically coded magnetic microspheres to immobilize antigens from 10 different arboviruses, namely, Zika (ZIKV), Dengue all four serotypes (DENV 1–4), West Nile (WNV), Japanese Encephalitis (JEV), Yellow Fever (YFV), Tick-Borne Encephalitis (TBEV), and Chikungunya (CHIKV. This 18-plex assay also includes five different internal controls that assure the assay and reagent performance. Initial characterization of the platform was performed using experimentally infected primate samples collected longitudinally before and after infection with ZIKV. In this paper, we describe the results of the evaluation of this multiplex platform using over 400 well-characterized healthy and diseased state human serum and plasma samples from various commercial and collaborating resources. We conclude that this assay provides a relevant tool for efficient monitoring of population based arboviral antibodies in endemic areas and also for detection of outbreaks.
Evaluation of intrahepatic macrophages in patients with non-alcoholic steatohepatitis using multispectral imaging
Authors: Saldarriaga Omar A , Jiao Jingjing , Beretta Laura , Freiberg Benjamin , Krishnan Santhoshi , Rao Arvind , Booth Adam L. , Stevenson Heather L.
Published: January 2023

Patients with non-alcoholic steatohepatitis (NASH) have different rates of fibrosis progression. We hypothesized that this is due to intrahepatic macrophages. Kupffer cells (KCs) can affect the development of fibrosis by recruiting systemic macrophages that activate stellate cells. Liver biopsies were collected from patients with NASH and either minimal (n=6) or advanced (n=5) fibrosis. We used spectral imaging to evaluate KCs (CD68+), monocyte-derived (Mac387+), pro-fibrogenic (CD163+), pro- (CD14+), and anti- inflammatory (CD16+) macrophages. NanoString evaluated gene expression profiles in the same biopsy. t-distributed stochastic neighbor embedding (t-SNE) plots and phenotype matrices were used to visualize differences between groups. Mean normalized counts from ‘cell seg’ data were used to determine the percentage of phenotypes present. Results showed that patients have variable macrophage phenotypes in the liver depending on their propensity to develop hepatic fibrosis. Patients with minimal fibrosis showed increased CD14+ macrophages and more protective gene expression profiles. Patients with advanced fibrosis had significantly increased numbers of pathogenic pro-inflammatory/pro-fibrotic macrophages (MAC387+, CD163+) and increased expression of genes associated with mixed macrophage activation (IDO1, CD86, HLADR, IL1β, TNFα, COX-2, Dectin-1, CD209, PI3K/AKT3, IL10R, TGFB1, IRF4, CLEC7A/DECTIN-1). However, when we analyzed each patient’s liver biopsy on an individual basis, subtle variations were observed between the patients in each group. Thus, the various phenotypes of macrophages present in patients with NASH is complex and is only partially associated with fibrosis stage.
Gain a Better Understanding of Immunology using Single-Molecule Localization Microscopy
Authors: Burns Sheila
Published: January 2023

Studying protein organization and function at molecular level is a major technical challenge that limits our understanding from both a diagnostic and treatment perspective. Single-molecule localization microscopy (SMLM) is a Nobel Prize winning technique that allows quantitative imaging below the diffraction limit and can be applied to significantly improve our understanding of many biological phenomena. We provide a versatile imaging platform with unrivalled stability and flexibility to work on a lab bench. The data presented was acquired using two and three-color, super-resolution imaging of EVs and TCR alpha and beta receptors and reveal their spatial organization at 20 nm resolution. Additional experiments to show levels of expression on the cell surface, yielding the characterization of receptor distribution and ultimately activity and signaling. Experiments will show the wide-ranging applicability of our instrument as a tool for multiple biological analyses encompassing many fields within immunology.
Whole-body immunoPET imaging to monitor immune activation status in situ
Authors: Gibson Heather , Hackett Justin , White Jordan , Mandella Josh , Zeglis Brian , Keinanen Outi , Kalluri Jhansi , Barr Stephen , McCarthy Claire , Wiesend Wendy , Viola Nerissa
Published: January 2023

Advances in immune modulation for cancer and inflammation warrant parallel development of imaging technologies to gauge in situ immune activity. We have developed a toolbox of full-length monoclonal antibody (mAb) positron emission tomography (immunoPET) tracers by desferrioxamine conjugation and 89Zr radiolabeling. Tracer uptake is reported as % injected dose/gram (%ID/g) tissue. Because mAbs to cell surface molecules often facilitate depletion, we tested enzymatic removal of the Fc glycan of CD8 mAb 2.43 to disrupt Fc:FcR interaction and prevent depletion. In the CT26 tumor model, CD8 immunoPET showed similar uptake between WT and deglycosylated tracers. However, unlike WT 2.43, the deglycosylated tracer did not deplete peripheral CD8 T cells, supporting this approach. In addition to surface markers, tracers that define cell function may have great utility. Checkpoint blockade therapy (anti-PD-1/anti-CTLA-4) in CT26-bearing mice shows IFNg immunoPET tracer uptake in 5/5 treated (16.5 ± 3.6 %ID/g) and 2/5 untreated (12.1 ± 4.8 %ID/g) tumors, possibly indicating baseline CT26 immunogenicity. To evaluate antigen presenting cell activation, an IL-12-specific tracer was also developed. Mouse mammary tumor TUBO-bearing mice showed enhanced tracer uptake after intratumoral injection of adenovirus encoding GM-CSF (2.6 ± 0.7 %ID/g) versus controls (0.4 ± 0.4 %ID/g). IL-12 PET was also tested for applications outside of cancer by mimicking inflammation with i.m. LPS injection. LPS-treated mice showed localized, elevated uptake at the site of injection (3.7 ± 0.7 %ID/g) versus controls (0.7 ± 0.1 %ID/g). These results support the use of immunoPET imaging for non-invasive monitoring of immune activity in situ. NIH R37 CA220482.
In silico mouse model of infection and immunity
Authors: Jonas Daniel , Kirby Michael , Schenkel Alan
Published: January 2023

An organism’s immune system tries to protect it by identifying the presence of pathogens and attempting to eliminate them. The defense is twofold: innate immune cells mobilize rapidly, while acquired immune cells slowly develop into pathogen-killing specialists. These responses incur collateral tissue damage, which anti-inflammatory mediators seek to control. This system of checks and balances is responsible for host survival. Experimental research has demonstrated how vastly complex these interactions are, indicating a place for theoretical and computational study. In this work we develop a comprehensive differential equation model of the immune system by considering interactions between immune system components in the presence of pathogen or tissue trauma. Through this step-by-step construction we explore the dependence of the anti-inflammatory mediators on pathogen levels, and also how they temper the immune response at the end of infection. We then challenge the “virtual mouse” with typical pathogens of varying virulence and observe the outcomes via model simulation. We find that anti-inflammation can downregulate the activation and proliferation of immune cells or promote apoptosis as cessation mechanisms, suggesting the need for in vivo experiments. Bifurcation theory describes how the outcomes of infection depend on model parameters, from which we conclude that initial insult and pathogen growth rate allow us to predict whether or not the in silico mouse overcomes the disease in a deterministic framework.
Molecular patterns expressed by a human gut commensal strain of Lactobacillus suppress neuroinflammation
Authors: Sanchez John Michael Sison , Doty Daniel Jason , DePaula-Silva Ana Beatriz , Libbey Jane E. , Fujinami Robert S
Published: January 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that affects 2.5 million people worldwide. Growing evidence suggests that perturbation of the gut microbiota, a dense collection of microorganisms that colonize the gut, plays a functional role in MS. Utilizing experimental autoimmune encephalomyelitis (EAE), the most commonly used murine model of MS, we previously identified a significant negative correlation between the relative abundance of gut Lactobacillus and the severity of EAE, mirroring what is seen in MS patients. We now report that a human gut-derived commensal strain of Lactobacillus paracasei (Lb) can suppress preclinical murine models of MS with both prophylactic and therapeutic administration. Surprisingly, we found that heat-killed Lb was as effective as live Lb in protecting animals against EAE, as measured by disease score and demyelination. Heat-killed Lb-treated animals had reduced numbers of CNS-infiltrating leukocytes and this was associated with a decrease in select chemokines in the serum. The beneficial effect of heat-killed Lb was dependent on host expression of Toll-like receptor 2 (TLR2), which senses major components of the Lb cell wall, as EAE is not suppressed by heat-killed Lb in TLR2-deficient mice. Thus, Lb-associated molecular patterns may be an important signal to prevent aberrant peripheral immune cell infiltration into the CNS and warrant further investigation as a novel therapy for MS patients.
B Cell derived IL-4 promotes IL-10 secreting TIM-1+ regulatory B cell expansion and regulates Th2 T cell responses
Authors: Zheng Leting , Song Zhixing , Mohib Kanishka , Rothstein David M.
Published: January 2023

TIM-1+ B cells are enriched for IL-10+ B regulatory cells (Bregs). However, TIM-1+ B cells are also enriched for IL-4+ B effector 2 cells (Be2). Tolerogenic α-TIM-1 (RMT1-10) prolongs islet allograft survival (GS) by expanding (IL-10+) Bregs. Interestingly, IL-4KO and IL-4Rα-KO mice are deficient in Bregs and are not responsive to αTIM-1. To establish the importance of IL-4R signaling, and the source of IL-4 for Breg induction, we made tamoxifen (TAM) inducible BALB/c hCD20Cre.ERT2+/− X IL-4Rαfl/fl (B-IL-4RαKO) and hCD20Cre.ERT2+/− X IL-4/13fl/fl mice (B-IL-4KO) to respectively delete IL-4Rα or IL-4/IL-13 in B cells. After TAM, αTIM-1 treatment of alloimmunized B-IL-4RαKO mice had ↓ TIM-1+ B cells (50%), Be2 (50%) and Bregs (40%) vs. IL-4Rαfl/fl littermate controls and a concomittant 33%↑ in IFNγ+ and 33% ↓ in IL-10+ CD4 T cells, and a 20%↑ in IFNγ+ CD8 T cells vs. controls. Finally, α–TIM-1 led to >100d GS in 75% of control mice, but failed to improve GS in B-IL-4RαKO mice (MST 20d, p=0.03). Similarly, TAM treated B-IL-4KO mice had a 3x ↓ in Be2 and Bregs vs. IL-4fl/fl controls, and a 6x drop in IL-4+ CD4 T cells. Importantly, B-IL-4KO mice rejected islets more rapidly (MST 20d) than controls (MST 31d, p=0.04), and α–TIM-1 failed to improve B-IL-4KO GS (MST 17d) vs. controls (MST >100d). In contrast, in Th2 dominant OVA-induced allergic airway disease (AAD), B-IL-4KO mice had 3x decrease in total lymphocyte infiltrate, 5x decrease CD4 T cells and 8x decrease eosinophils in BAL fluid vs. controls. CD4+ T cells from whole lung tissue produced 2x less IL-4 and 3x less IL-5 vs. controls. Hence, IL-4 production by B cells is important for both Breg induction and Th2 polarization and absence of B cell IL-4/IL-13 promotes allograft rejection yet reduces AAD.
Salvia miltiorrhiza inhibits allospecific memory CD8+ T cell generation and allograft rejection in mice
Authors: Dai Zhenhua , Liu Huazhen , Qiu Feifei , Chen Yuchao
Published: January 2023

It’s well known that memory T cells pose a threat to long-term allograft survival. The objective of this study is to suppress generation of donor-specific memory T cells via short-term treatment with a natural and non-cytotoxic herb. Previous studies have demonstrated that Chinese herb Salvia miltiorrhiza (Danshen) regulates immune responses. In this study, B6 mice were transplanted with BALB/c islets and treated with Danshen and/or low doses of rapamycin (0.25 mg/kg/day). To generate allospecific memory T cells, Thy1.1+ CD8+CD44low T cells were sorted by FACSAria and injected into B6 recipients one day before transplantation. Graft-infiltrating T cell proliferation was determined by BrdU staining while the memory cells were enumerated via FACS.

We found that Danshen alone significantly extended islet allograft survival (MST = 28 vs. 13 days, n=9, P<0.05). Interestingly, administration of both Danshen and low doses of rapamycin further prolonged islet allograft survival (MST = 83 versus 28 days), with 44% of recipients achieving long-term islet graft survival (>100 days). Moreover, Danshen suppressed generation of memory CD8+ T cells compared to control two months after transplantation (Thy1.1+CD8+CD44high cell numbers: 1.3±0.2 vs. 2.5±0.3, ×104/LN, P<0.05 and 4.3±0.6 vs. 8.5±0.9, ×105/spleen, P<0.05) while Danshen plus rapamycin further reduced CD8+ memory T cells compared to Danshen alone (0.7±0.1 vs. 1.3±0.2/LN, P<0.05 and 2.6±0.4 vs. 4.3±0.6,/spleen, P<0.05). Finally, Danshen alone inhibited graft-infiltrating T cell proliferation at one week (52±4 vs. 35±3 %, P<0.05). Thus, Danshen inhibited donor-specific memory CD8+ T cell generation and promoted induction of long-term murine allograft survival.
Peroxiredoxin 1 controls β-catenin activity and Epstein-Barr Virus-encoded MicroRNA in Nasopharyngeal Carcinoma
Authors: Noh Minhye , Gong Eun-Yeung , Akinleye Temitope Michael , Rajbongshi Lata , Kim Yeong Seok , Hur Dea Young
Published: January 2023

BACKGROUND

Nasopharyngeal Carcinoma(NPC) is a rare tumor in the world, but high in some countries such as southern China, Mediterranean Africa and Middle East. According to studies, Epstein-Barr virus is affected more aggressive in NPC. In NPC, EBV-miR-BARTs have been demonstrated, especially EBV-miR-BART17 have relation to β-catenin activity but the studies are still unclear. Peroxiredoxin 1 (Prdx1) is an antioxidant enzyme and is highly susceptible to oxidative stress.

METHODS

To study the roles of Prdx1 and EBV-miR-BART17-5p in NPC C666-1 and HK1 cells, it is transfected to siRNA of Prdx1 and then it is measured using RT-PCR for RNA level and western blot for protein level. Colony forming assay and Wound-healing assay were used to detect the cell proliferation in NPC cells.

RESULT

EBV-miR-BART17-5p expression was higher in C666-1 than HK1 cells. The expression of Prdx1 is different between C666-1 and HK1 cells because of EBV genes. EBV-miR-BART17 expression was found to relate to cell proliferation through targeting NF-κB signaling pathway.

CONCLUSION

These data suggested that Prdx1 and EBV-miR-BART17-5p play roles in inducing β-catenin activity in NPC cells. Our findings suggest that the regulating of Prdx1 is an important therapeutic target for NPC.
Nanoparticles as potential therapeutics for acute myeloid leukemia
Authors: Simmet Thomas , Gaiser Ann Kathrin , Schmiech Michael , Syrovets Tatiana
Published: January 2023

Acute myeloid leukemia (AML) is associated with an unfavourable prognosis. Hence, new therapeutic agents with higher specificity and lower general cytotoxicity are urgently required.

We synthesized biocompatible engineered nanoparticles and used various physico-chemical methods for their characterization. Cell viability was assessed by analysis of activities of mitochondrial dehydrogenases and colony-formation assay. Intracellular targets were identified by Western immunoblotting, fluorescent microscopy, flow cytometry, and various functional tests.

We have previously shown that amino-functionalized polystyrene nanoparticles (PS-NH2) induce autophagy in leukemia cells and are selectively cytotoxic to leukemia cells compared to human macrophages and PBMC. We created now biocompatible amino-functionalized nanoparticles with an inert metallic gold core, Au-NH2, which were selectively highly cytotoxic towards different AML cell lines and PBMC from acute myeloid leukemia patients, but not to PBMC from healthy donors. We show that in leukemia cells, Au-NH2 treatment induces BID cleavage to tBID, increases mitochondrial superoxide levels, inhibits activity of complexes I, III, IV of the mitochondrial electron transport chain, and induces loss of the mitochondrial membrane potential as well as cytochrome c release from the mitochondrial intermembrane space and caspase 3 activation. Likewise, Au-NH2 treatment induces a rapid decrease in mTOR pathway activity, induces lysosomal rupture in THP-1 and HL-60 leukemia cells but not so in macrophages and PBMC. Thus, this engineered material holds great promise as a novel nanotherapeutic for treatment of acute myeloid leukemia independent of cytogenetic profile.
The Ikaros1, IKZF1, risk locus for Acute Lymphoblastic Leukemia (ALL) at chromosome 7p12.2 regulates the expression of the FIGNL1 gene
Authors: Laurynenka Viktoryia , Ho Danielle , Huang Yongbo , Parameswaran Sreeja , Kottyan Leah C. , Weirauch Matthew T. , Perentesis John , Kaufman Kenneth M. , Mizukawa Benjamin , Harley John B.
Published: January 2023

ALL, the most common pediatric cancer, originates from progenitor B-cells in ~85% of cases. IKAROS Family Zinc Finger 1 (IKZF1) is an important locus for ALL. Our goal was to identify the functional variants, thereby nominating a potential causal mechanism for ALL.

We evaluated all 27 genetic polymorphisms in disequilibrium at r2>0.8 with the tag, the most highly associated risk variant at IKZF1 locus. They map to intron 7, 3′ UTR and 3′ flanking sequence of IKZF1. Fidgetin-like protein 1 (FIGNL1) is 45,524 bases from the tag variant and is nominated by eQTL and DNA looping data along with IKZF1 as possible targets. Epstein-Barr virus (EBV) transformed GM12878 cells were transduced with lentiviral vectors for stable expression of fusion proteins dead (d) Cas9-VP64 and activator MS2-p65-HSF1 (synergistic activation mediator (SAM) system). We designed 27 single guide (sg) RNAs, one for each of the 27 variants and evaluated expression perturbation in cells transfected with pools and individual sgRNA. Expression of FIGNL1, but not IKZF1, was convincingly altered in this system. The reduction of FIGNL1 expression by ~50% was isolated to two neighboring variants separated by 1 kb. The risk allele at these variants increases FIGNL1 expression in wild type cells, however in EBV transformed B-cells FIGNL1 expression is decreased. Other data show that the EBV encoded DNA binding co-factor EBNA3C binds at or very near this locus in EBV transformed B cells.

Our results nominate two closely located SNPs in the last intron of IKZF1 as potentially causal variants, controlling the expression of FIGNL1, which ordinarily participates in homologous recombination during DNA double-strand break repair, maintenance of genomic stability and prevention of cancer.