bluuebunny/crossref_metadata_2025 huggingface dataset:
Oenothein B isolated from Epilobium angustifolium enhances cytokine and pathogen-induced IFNγ production by bovine mononuclear cells (108.1)
Authors: Ramstead Andrew , Kimmel Emily , Schepetkin Igor , Quinn Mark , Jutila Mark
Published: January 2023

Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen that has been implicated in Johne’s disease in cattle and possibly Crohn’s disease in humans. Cells expressing the NK cell activating receptor NKp46 have been shown to play an important role in mycobaterial infections in cattle by producing IFNγ and lysing infected monocytes. Therefore, compounds that enhance the activity of these cells may be useful in the treatment of these types of pathogens. Oenothein B is an immunomodulatory tannin isolated from Epilobium angustifolium plants and shown to have antitumor properties. This compound is known to activate myeloid cells, as well as NK cells, NKT cells, and γδ T cells. Previous work from our laboratory has shown that certain tannins have the ability to enhance the response of innate lymphocytes to secondary stimuli. Here, we report that oenothein B is capable of priming bovine peripheral blood mononuclear cells to produce increased levels of IFNγ in response to the IFNγ-inducing cytokine IL-18. This enhanced response is dependent upon the presence of cells expressing NKp46. Our preliminary results also suggest that oenothein B is able to enhance IFNγ production in response to M. avium subsp. paratuberculosis infected monocytes. This demonstrates that priming of innate lymphocytes by supplementation with specific polyphenols may be a useful strategy to enhance immunity to certain intracellular pathogens.
NKT cells are essential for innate immune control of vaccinia viral infection in vivo (108.3)
Authors: Novy Patricia , Yang Yiping
Published: January 2023

The role of natural killer T (NKT) cells during the innate immune response to pathogen remains incompletely defined. We previously showed that early control of vaccinia virus is critically dependent on the activation of natural killer (NK) cells. Here we show that in CD1d-deficient hosts, early control of vaccinia virus is compromised. This defect in viral clearance corresponds to an insufficient NK cell response, as NK cells from CD1d-deficient hosts have reduced production of the effector molecules IFN-γ and Granzyme B. Furthermore, NK cells from infected CD1d-deficient mice fail to lyse target cells when used in a chromium release assay. These data suggest a role for NKT cells in NK cell activation. In vitro, NK cell activation in response to vaccinia is dependent on both dendritic cells (DC) and NKT cells. We observe that NKT cells produce high levels of IFN-γ in response to vaccinia virus, and the production of IL-12 by DC is dependent on NKT cell presence. Direct contact between NKT cells and DC is required to fully license them to effectively activate NK cells, a phenomenon that may be attributed to upregulation of activating NK ligands RAE-1 and MULT-1 on DC surfaces. Overall, our data show that NKT cells are required for the activation of NK cells during vaccinia infection and suggest that NKT cells license DC to optimally activate NK cells. These data indicate that NKT cells are key players in the early generation of an innate immune response to viral infection.
Spaceflight alters expression of microRNA during T cell activation (109.16)
Authors: Hughes-Fulford Millie , Boonyaratakanakornkit Jim , Chang Tammy , Li Chai-Fei
Published: January 2023

Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab suggesting that immune-suppression is a major barrier to safe, long-term human space habitation and travel. We present definitive evidence that early signal transduction events are inhibited by the microgravity (ug) environment of spaceflight. Human leukocytes were stimulated with T cell mitogens onboard the International Space Station using a 1g centrifuge as the Earth’s 1g control. Microarray analysis demonstrated significant (p< .05) differential expression of 54 genes in ug vs 1g conditions. Fifty percent of the genes most significantly inhibited in ug as compared to 1g showed marked over-representation of NFKB binding sites in their promoter regions. Qrtpcr confirmed that upregulation of key genes in the NFKB pathway were inhibited in ug. Inhibition of the NFKB pathway leads to activation dysfunction during spaceflight. Lack of immune response in microgravity occurs at the cellular level. In our recent spaceflight studies of early T cell activation, we have discovered that the expression of miRNAs in both man and mouse require gravity for appropriate regulation. MiRNAs expressed during activation in normal gravity (g), were down regulated in spaceflight. These data suggest that gravity may regulate T-cell activation by two separate mechanisms; promoter regions and microRNA.
The role of syntaxin11 in cytotoxic T lymphocytes (109.26)
Authors: Halimani Mahantappa , Pattu Varsha , Junker Christian , Marshall Misty , Schwarz Eva , Krause Elmar , Matti Ulf , Hoth Markus , Rettig Jens
Published: January 2023

Cytotoxic T Lymphocytes (CTLs) form immunological synapses (IS) with antigen presenting cells in order to kill them. Killing requires release of lytic granules which occurs via exocytosis, and is dependent on SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. Syntaxin11 is a SNARE protein that is predicted to be involved in the fusion of lytic granules in CTLs, since mutations within the coding region of Syntaxin11 lead to Familial Hemophagocytic Lymphohistiocytosis (FHL4), an immune disorder caused by impaired granule exocytosis. We have investigated the contribution of Syntaxin11 to lytic granule release. We used live cell imaging to observe the trafficking of full length Syntaxin11. Using CD3 specific antibodies as a marker for a functional IS, we show that Syntaxin11 accumulates at the IS after CTL polarization and conjugation with the APC has occurred. Syntaxin11 was also associated with Munc18-2, consistent with the role of Munc18-2 as a syntaxin chaperone and as a docking factor. Using a Syntaxin11 antibody we also confirmed the subcellular localization of Syntaxin11 in fixed CTLs with confocal microscopy. Syntaxin11 was associated with Munc18-2 and CD3 at the IS in fixed CTLs, consistent with our observations in live cell imaging of CTLs. These results support the conclusion that Syntaxin11 is involved in lytic granule exocytosis.
Human CD8β isoforms with distinct cytoplasmic tails differ in modulating T cell response and regulated cell surface expression (109.31)
Authors: Coman Maria , Thakral Deepshi , Bandyopadhyay Arunima , Riley James , Kavathas Paula
Published: January 2023

The CD8αβ coreceptor influences CD8 T cell recognition and responses in anti-tumor and -viral immunity. The ancestor to the human and chimpanzee CD8β gene acquired two additional exons absent in the mouse that lead to the expression of multiple isoforms (M1-M4) as a result of alternative splicing. In humans these isoforms differ in their cytoplasmic tails and in their expression pattern. The M-1 isoform is predominant in naïve T cells whereas M-4 is predominant in effector memory T cells. To study functional differences we are co-transducing CD8α, each CD8β isoform, and MHCI restricted NY-ESO-1 specific TCR into human CD4+ T cells and measuring cytokine production after activation. We have found differences in induction of cytokine producing cells such as the MIP-1β chemokine with different isoforms. The M-4 isoform cytoplasmic tail has unique sorting motifs that regulate its cell surface expression and it is modified by phosphorylation after activation. The cytoplasmic tail of M-4 could associate with ubiquitinated substrates in 293T cells and was itself mono-ubiquitinated. The M-4 isoform has unique properties that likely favored its selection in effector memory T cells.
Lck-mediated tyrosine phosphorylation of Discs Large Homolog 1 at position Y222 is required to coordinate alternative p38 activation in T cells (109.33)
Authors: Crocetti Jillian , Silva Oscar , Humphries Lisa , Round June , Miceli M. Carrie
Published: January 2023

Stimulation through the T cell receptor (TCR) leads to rapid activation of proximal tyrosine kinases, Lck and ZAP70, which couple to distinct downstream signaling cascades and functional outcomes. We have shown that MAGUK family scaffold protein Discs Large Homolog 1 (Dlgh1) juxtaposes activated Lck and ZAP70 with p38 MAP kinase to selectively activate the alternative p38 pathway and downstream NFAT; but not NFκB. Lck, ZAP70 and p38 are known to complex with Dlgh1 in response TCR engagement. However, Lck and ZAP70 binding sites within Dlgh1 and the mechanism by which Dlgh1 positions and activates Lck and ZAP70 allowing p38 activation remain incompletely understood. Knowing YxxL/I is a preferred motif for Lck tyrosine phosphorylation and that SH2 domains of ZAP70 and Lck prefer to bind Lck-phosphorylated substrates; we identified Dlgh1 Y222 as a potential target of phosphorylation and/or binding. We subsequently assessed the potential role of Dlgh1 Y222 phosphorylation in coordinating the alternative p38 pathway. Preliminary studies using rLck, rZAP70 and GST-Dlgh1 Y222F fusion proteins demonstrate that Y222 is a primary site of Lck phosphorylation required for optimal alternative p38 activation. Experiments are underway to assess the role of Dlgh1 Y222 in Lck and ZAP70 binding and alternative p38 activation in primary T cells. These experiments will elucidate molecular mechanisms behind Dlgh1 coordination of the alternative p38 pathway and TCR signal specificity.
Decreased innate interferon production in type 2 polarized macrophages upon human rhinovirus infection (110.1)
Authors: Stanciu Luminita , Nikonova A. , Traub S. , Khaitov M. , Johnston S.
Published: January 2023

The role of M1 and M2 macrophages (derived in a type 1, respectively in a type 2 cytokine milieu) in human rhinovirus (HRV) infections is not known. M2 macrophage numbers are believed to be increased in asthma and could play a role in the increased susceptibility to HRV-infections. We hypothesized that upon HRV infection M2 produce less innate interferons (IFN-α/β/λ) as compared to M1 macrophages. Methods. Monocyte-derived macrophages (MDM) obtained from PBMC were polarized to M2 (by treatment with IL-4) and to M1 (by treatment with IFN-γ&TNF-α) and infected with HRV16. IFN-α/β/λ gene expression was measured by RT-PCR and protein levels by ELISA. Supernatants of HRV16-infected M1& M2 and MDM were titrated on Hela-Ohio cells to ascertain their 50% tissue culture infective dose TCID50/ml. Results. Upon HRV16 infection we found increased IFN-α1, -β and -λ1 gene expression at 8h and IFN-α1 and -λ1 gene expression at 24 h in M1 as compared to M2 macrophages and MDM. Increased production of IFN-β and -λ1 at 8h and for IFN-α, -β and -λ1 at 24 h, 48 h and 72 h was observed in HRV16-infected M1 as compared to M2 macrophages and MDM. Rhinovirus titer was decreased in M1 macrophages. In conclusion, M1 macrophages produce high levels of antiviral IFN-α/β/λ1 and consequently are less susceptible to virus infection. In contrast M2 macrophages produce low levels of IFN-α/β/λ and this could play a role in virus-induced exacerbations of asthma.
Phospholipases are required for Francisella tularensis induction of macrophage prostaglandin E2 synthesis. (110.15)
Authors: Woolard Matthew , Navratil Aaron , Studt Ashley , Bryan Joshua
Published: January 2023

Francisella tularensis is the causative agent of respiratory tularemia, a serious disease with 30-60% mortality in untreated individuals. F. tularensis is capable of inducing the synthesis of prostaglandin E2 (PGE2) from macrophages. F. tularensis induction of PGE2 synthesis during respiratory tularemia inhibits the development of beneficial T cell responses during respiratory tularemia. Signal transduction events in the macrophage that lead to the synthesis of PGE2 in response to F. tularensis are unclear at this time. Phospholipase (PLases) and lipases are enzymes that cleave phospholipids and lipids repectively leading to the generation of secondary messenger molecules that initiate signal transduction events. PLases are critical to LPS induction of PGE2 production; however the role of PLases and lipases during F. tularensis PGE2 induction is unknown. Utilizing phosholipase and lipase inhibitors we have demonstrated that cPLA2 and DAGL activity are required for F. tularensis to induce PGE2 synthesis in macrophages. This is the first study demonstrating involvement of PLases in the macrophage response to F. tularensis. Subsequent studies will clarify the PLases specific aspects PLases play in the PGE2 synthesis.
CD180 restricts replication of Legionella pneumophila in macropahges and is associated with susceptibility to Legionnaire’s Disease (110.25)
Authors: Misch Elizabeth , Arentz Matthew , Verbon Annelies , Sexson Shannon , Skerrett Shawn , Hawn Thomas
Published: January 2023

Legionella pneumophila (Lp) is a gram-negative bacterium that opportunistically infects the human alveoloar macrophage to cause a pneumonic illness known as Legionnaire’s Disease (LD). The role of CD180 (RP105), a non-signalling member of the TLR family that regulates TLR2 and TLR4 function, in Lp pathogenesis is unknown. We hypothesized that CD180 regulates susceptibility to Lp infection in mice and humans. We infected bone-marrow derived macrophages with Lp (Lp02 strain) and observed greater replication (~2-3-fold) in Cd180-/- mice compared to C57Bl/6 controls. This difference was present over a range of MOIs (0.1 to 2.0) at 24, 48, and 72 hours after infection. Enhanced replication of Lp in Cd180-/- macrophages was not associated with increased pyroptotic cell death, as assessed by IL-1β or LDH, or alterations in key innate immune cytokines or effectors, such as IL-6, TNF, or nitric oxide. We next examined whether CD180 polymorphisms were associated with LD with a study of 108 cases and 508 controls in the Netherlands. Four SNPs, rs574464, rs1428469, rs1697143, and rs1803440, were associated with protection from LD, with ODs ranging from 0.27-0.50 (p values: 0.005-0.045, analysis with dominant genetic model). The functional effects of these polymorphisms and the mechanism of CD180 regulation of Lp replication in macrophages are currently being examined. Together, these results suggest that CD180 critically mediates the immune response to Lp in mice and humans.
Naringenin: an immunomodulator of Interleukin (IL)-6, IL-8, and Tumor Necrosis Factor secretion from Chlamydia trachomatis-infected macrophages and epithelial cells (110.28)
Authors: Yilma Abebayehu , Singh Shree , Fairley Stacie , Terrell Kaitlyn , Taha Murtada , Subbarayan Praseetha , Dennis Vida
Published: January 2023

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted infections, is responsible for a myriad of immunopathological changes associated with reproductive health. Chlamydia infects macrophages and epithelial cells, which secrete cytokines such as TNF, IL-6 and IL-8 that are key protagonists of acute inflammation. These cytokines put the patient at risk for major health issues such as pelvic inflammatory disease and infertility if not controlled. Counterbalancing their levels helps mitigate acute inflammation. We have shown that IL-10 regulates inflammatory cytokines elicited by C. trahomatis in innate immune cells. Here we explored the hypothesis that Naringenin, a natural flavonone, with anti-inflammatory properties, when used alone or co-cultured with IL-10 will modulate inflammatory cytokines secreted from C. trachomatis-infected epithelial (HeLa) cells and mouse J774 macrophages. We exposed C. trachomatis-infected cells to Naringenin (100, 10, 1, 0.1, and 0.01 μg/mL) alone or with IL-10 (10 ng/mL). Our results as demonstrated using cytokine ELISA, confocal microscopy and MTT assay, indicate that Naringenin abrogated TNF, IL-8, and IL-6 levels in a dose-dependent fashion. Cytokine levels were reduced further by Naringenin co-cultured with IL-10. The MTT assay showed the anti-inflammatory effect of Naringenin was not due to cell death. The data provides evidence for a novel therapeutic direction in controlling C. trachomatis-induced acute inflammation.
Dendritic cells from burn and septic injured rats modulate CD4+ CD25+ T cells from sham control rats (111.13)
Authors: Fazal Nadeem , Shelip Alla , Siddiqui Erum , Ali Ashraf , Al-Ghoul Walid
Published: January 2023

Dendritic cells (DCs) play a vital role in presenting antigen to CD4+ CD25+ (Reg) T cells. We hypothesize that burn plus sepsis injury adversely affects antigen presenting cells, in particular the DCs, such that these DC are unable to adequately prime Reg T cell; the inadequate stimulation of Reg T cell by DCs may contribute to burn-induced impairment in T cell IL-2 production and proliferation. In this study, we examined the effect of presence of anti-CD3, anti-CD28, and/or IL-2 in the T cell cultures with or without the added APC/DCs. The results show when sham rat Reg T cells were cocultured with DCs or APCs from burn septic rats, IL-2 production and proliferation was decreased, compared to that in sham T cells cocultured with sham DCs or APCs. When exogenous IL-2 was added to sham Reg T cell and burn and sepsis rat APC cocultures, there was an enhancement in Reg T cell proliferation to the level found in cocultures of sham rat T cells with sham rat APCs. Sham rat T cell proliferation was enhanced in the presence of anti-CD3 but not to the level in cocultures with burn plus sepsis rat APCs. Similarly, addition of anti-CD28 produced but a small increase (~20%) in proliferation of sham rat Reg T cell cocultured with rat APCs, compared to the effect of anti-CD28 in cocultures of sham rat T cell with sham rat APCs. These studies indicate a role of burn-derived APC and DCs in modulating a Reg T cell response in burn and sepsis injury.
Bacillus anthracis capsule promotes TNF-α expression and NF-kB activation through activation of TLR2 in phorbol 12-myristate 13-acetate-differentiated THP-1 and human monocyte-derived dendritic cells (111.14)
Authors: Cho Min-Hee , Ahn Hae-Jeong , Shin Youg-Woo , Yoo Cheon-Kwon , Rhie Gi-eun
Published: January 2023

Anthrax is a highly lethal infectious disease caused by the spore-forming Bacillus anthracis. Poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). The treatment of PGA on differentiated THP-1 cells and hMoDCs leads to the specific extracellular release of TNF-alfa in dose-dependent manner as shown by ELISA. Subsequently ELISA results were validated by microarray analysis and RT-PCR. In addition, the increase of TNF-alfa production was inhibited by TLR2-blocking antibody, TL2.1, indicating the enhancement of cytokine productions is TLR2-dependent. Also, we confirmed that PGA-induced TNF-alfa production occurs via activation of transcription factors nuclear factor (NF)-kB through RT-PCR and phosphorylation of IkB by Western blot analysis. These data indicate that PGA of B. anthracis elicite TNF-alfa production through TLR2 pathway via NF-kB activation in PMA-differentiated THP-1 cells and hMoDCs.
Potency of n-3 PUFA anti-inflammatory effects on human and rodent immune cell responses (111.16)
Authors: Kasprowicz Deborah , Ravindran Nair Sangeetha , Gonyon Tom , Gerstein Clare , Fulghum Tim , Carter Phil
Published: January 2023

Pharmaceutical parenteral emulsions consisting of n-6 poly-unsaturated fatty acids (PUFAs) alone (Intralipid®), n-6 PUFAs and n-9 mono-unsaturated fatty acids (MUFAs; e.g. Clinoleic®), n-3 PUFAs alone (Omegaven®), or a combination of n-3, n-6, n-9 and medium chain triglycerides (SMOF®) provide nutritional benefit to critical care patients, as well as influencing immune cell function. The efficacy of these compounds in modulating cell function varies depending on the formulation composition, route of administration, dose, and immune status of the individual. In general while n-9 MUFAs are neutral, n-6 PUFAs promote inflammatory responses. In contrast, n-3 PUFAs have immunosuppressive properties including blocking cell proliferation, cytokine production, and surface protein expression. However, there is limited data assessing the effects of n-3-containing pharmaceutical emulsions on immune cells across different species. In the current work, we determined the anti-inflammatory effects of n-3 PUFA-containing emulsions on an LPS-induced cytokine response in mouse, rat, and human mononuclear cells. We found that while n-3 PUFAs reduced the LPS-induced cytokine response in all species tested, these compounds were less potent in rodent cells compared to human cells. These findings provide insight into differences in the potency of n-3 PUFA anti-inflammatory effects across species and may be relevant for selecting the appropriate model system for assessing n-3 PUFAs efficacy in vivo.
Phenotypic and functional profiling of GM-CSF- and M-CSF-driven macrophages (111.18)
Authors: Wiener Doris
Published: January 2023

Monocyte-derived macrophages(MΦs) are a heterogeneous population of cells. MΦ function is dependent on polarization to M1 (proinflammatory) or M2 (anti-inflammatory) phenotypes. Described inconsistencies in M1 and M1 vs. M2 function may be due to different differentiation protocols. We performed analysis of GM-CSF-driven and M-CSF-driven M1 and M2 cells. Monocyte-to-MΦ differentiation was driven by exposure to GM-CSF or M-CSF for 6 days. GM-CSF-driven MΦs were polarized toward M1(GM-M1) with LPS+IFNγ; alternatively, M-CSF-driven MΦs were polarized toward M1(MC-M1) with LPS+INFγ or toward M2 with IL-4. MΦ phenotypes were determined by flow cytometry, ELISA and qRT-PCR. GM-M1, MC-M1 and M2 cells have distinct morphologies as well as different expression patterns of (1) scavenger receptors (SR-A, LDLR, CD36, SR-B1); (2) surface markers (CD14, CD16, CD36, CD80, CD163); and (3) cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12, TNFα, IFNγ). CD14, CD16, CD36, and CD163 expression was significantly higher in M2 than in MC-M1 or GM-M1 MΦs. CD80 expression was higher in both M1 populations compared to M2 cells. IL-10 expression was higher in MC-M1 than in GM-M1 or M2 cells; IL-12 expression was detected only in GM-M1 cells. Finally, MC-M1 cells had more phagocytic activity than GM-M1 cells. Our results characterize the profiles of GM-CSF and MC-SF driven MΦs. A rigorous analysis of MΦ polarization may facilitate the search for novel therapeutic strategies in disease prevention and treatment.
Alternative macrophage activation and lung host defense against Pneumocystis murina (111.20)
Authors: Nelson Michael , Christmann Benjamin , Metz Allison , Werner Jessica , Steele Chad
Published: January 2023

We have recently reported that Src TKO mice, which demonstrate augmented lung clearance of P. murina (PM) (Infect Immun, May 2009), have enhanced expression of alternative macrophage activation (M2a) markers such as RELM-α and Arg-1 as well as the M2a-associated chemokines CCL17 and CCL22 (J Immunol, February 2011). We have subsequently discovered that M2a development is a specific response to PM lung infection as AMs isolated from PM-infected mice have increased expression of RELM-α and Arg-1 and produce CCL17 and CCL22 ex vivo. Moreover, M2a development during PM lung infection requires CD4 T cells, as their depletion leads to an inability to maintain optimal lung expression of M2a markers. A putative effector function of the M2a response is CCL17/CCL22-mediated recruitment of eosinophils. Analysis of eosinophil peroxidase mRNA expression revealed increased levels in Src TKO mice and decreased levels in CD4-depleted mice, which directly correlated to the magnitude of M2a development in these two models. In preliminary studies, mice deficient in eosinophils demonstrated impaired PM lung clearance. In addition, administration of simvastatin to mice increased AM M2a marker expression and reduced PM lung burden. Collectively, our data provides evidence that M2a development is the natural immune response to PM, a response regulated by SFKs and requires CD4 T cells for maintenance. Moreover, our work supports a role for eosinophils in lung host defense against Pneumocystis.
Activation of NOD2 induces an IL-32 regulated dendritic cell program in leprosy (111.22)
Authors: Schenk Mirjam , Modlin Robert
Published: January 2023

The ability of the innate immune system to recognize and respond to microbial pathogens is determined by the interaction of microbial ligands with host receptors located in specific cellular compartments. For Mycobacterium leprae, tricaylated lipoproteins are recognized by cell surface TLR2/1 and muramyl dipeptide (MDP) by intracellular NOD2. The association of NOD2 polymorphisms with susceptibility to leprosy led us to hypothesize that activation of NOD2 regulates key innate immune host defense pathways in leprosy. We first examined gene expression profiles of monocytes stimulated with NOD2 ligand (MDP) or TLR2/1 ligand (19kD) and compared them to those found in lesions of tuberculoid (T-lep) vs. lepromatous (L-lep) leprosy. NOD2 activation and T-lep lesions are both characterized by DC gene programs. NOD2 activation induces differentiation into CD1b+ DC that are potent antigen presenting cells compared to TLR2/1L-triggered DC. Gene expression analysis and gene knockdown experiments revealed that NOD2-induced DC differentiation is regulated by IL-32. Furthermore, NOD2L activation of monocytes from L-lep patients show reduced induction of IL-32 and CD1b+ DC; however, CD1b expression is rescued by the addition of exogenous IL-32. In conclusion, we provide evidence that NOD2L induces a IL-32-regulated DC program in monocytes, with relevance to the pathogenesis and therapeutic intervention in infectious diseases such as leprosy.
Regulation of FoxO1 by mTORC2 negatively controls Toll-like receptor responses (111.24)
Authors: Wang Huizhi , Brown Jonathan , Suttles Jill , Kinane Denis , Graves Dana , Martin Michael
Published: January 2023

Activation of the Akt pathway plays a pivotal role in regulating the inflammatory response. Rictor is a component of the mTORC2 complex, and its loss has been shown to abrogate the phosphorylation of Akt at serine 473. However, the biological importance of mTORC2 in innate immunity is currently unknown. Here we demonstrate that rictor plays a critical role in TLR-signaling via its ability to regulate FoxO1. Genetic deletion or siRNA-mediated knockdown of rictor enhanced the production of pro-inflammatory cytokines upon LPS-stimulation, whereas the levels of the anti-inflammatory cytokine IL-10 were suppressed. The hyper-inflammatory phenotype was due to a defective Akt signaling axis, since rictor-deficient cells or rictor knockdown DC exhibited attenuated Akt phosphorylation and kinase activity, whereas complementation with a constitutively active Akt ablated the enhanced inflammatory response. Analysis of downstream targets showed phosphorylation of FoxO1 was impaired in rictor-deficient or rictor knockdown cells. The loss of rictor resulted in elevated levels of nuclear FoxO1 and an inability to export FoxO1 into the cytoplasm upon TLR-stimulation, an event that was restored upon complementation with a constitutively active Akt. FoxO1 deletion in DC resulted in an attenuated TLR mediated inflammatory response in which rictor knockdown was unable to rescue. These findings identify a novel signaling pathway by which mTORC2 regulates the TLR-mediated inflammatory response
Mechanism of formation of novel bone forming cells (monoosteophils) from LL-37 treated monocytes. (111.26)
Authors: Zhang Zhifang , Shively John
Published: January 2023

BACKGROUND: Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Recently, our published paper showed that LL-37, the active peptide of human cathelicidin, has an ability to induce monocyte differentiation to a new type of bone forming cells (monoosteophils). We now have investigated the mechanism of monoosteophil formation. RESULTS: Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to monoosteophils. Monoosteophil on osteologic discs built bone-like nodules which consist of calcium and phosphorus. Monoosteophils are identified by up-regulation of integrin a3 and a3b1 and down-regulation of CD14, CD16, CD32, and CD163 on the membrane. Microarry analyses indicated the differential expression of 206 genes in the formation of monoosteophils. Among these genes we found elevated level of CCL22. CONCLUSION: the present study demonstrated that integrin a3 and a3b1, CD14, CD16, CD32, and CD163 can be used as differentiation markers of monoosteophils and that the chemokine CCL22 may be involved in their formation.
Understanding the signaling kinetics that drive dendritic cell maturation (111.28)
Authors: Gonzalez-Lergier Joanna , Hartmann Boris , Sealfon Stuart
Published: January 2023

Dendritic cells are antigen-presenting cells responsible for the initiation of immunity. Immature DCs act as sentinels that, upon recognizing a pathogen, undergo a maturation process; the mature DCs then secrete signals to stimulate the appropriate immune response. Previous studies have shown that the cytokines that are produced by infected DCs during their maturation process can initiate a paracrine signaling program in naïve DCs that subsequently leads to a partial maturation of these cells thereby making them more capable of mounting an efficient response. Further combinatorial studies have shown that three cytokines (TNFα, IL-1β and IFNβ) are primarily responsible for this activation of naïve DCs, Consequently, there must be some convergence in the diverse signaling pathways initiated by these cytokines that is responsible for the syngergistic effect in DC maturation that has been observed. The phosphorylation and translocation profiles of signaling proteins and transcription factors downstream of these three cytokines are being used, in combination with measurements of maturation marker upregulation and ability to stimulate T cells, to built a PLS model in order to identify the key signaling pathways that are responsible to the partial maturation of these DCs. Further in-depth studies can then be carried out in order to identify more precisely the regulation that occurs in these cells that makes them more efficient in their professional-antigen presenting abilities.
Chronic morphine modulates actin polymerization leading to inhibition of Fc-gamma receptor mediated phagocytosis (111.29)
Authors: Ninkovic Jana , Roy Sabita
Published: January 2023

Morphine has been known to modulate innate immune functions, by inhibiting macrophage phagocytosis. However, mechanisms by which morphine inhibits macrophage phagocytic ability remain to be explained. Our results indicate that chronic morphine treatment in vitro and in vivo inhibited Fcγ receptor mediated phagocytosis in murine macrophages by inhibiting actin polymerization. Using fluorescence microscopy and fluorometry, we showed that chronic morphine treatment led to inhibition of actin polymerization resulting in impaired internalization of opsonized dextran beads. Chronic morphine treatment inhibited activation of Rac1-GTPase, therefore inhibiting formation of lamellipodia and membrane ruffling. Morphine’s inhibition of Rac1-GTPase activation was abolished in J774 macrophages transfected with constitutively active Rac1. In addition, chronic morphine treatment led to an increase in intracellular cAMP, which resulted in inhibition of actin polymerization through a PKA dependant pathway. DB-cAMP, by increasing intracellular cAMP, led to suppression of actin polymerization while H89 treatment induced inhibition of PKA and abolished morphine’s inhibitory effect implicating cAMP as the key effector in morphine’s modulation of actin. Together these data indicate that chronic morphine by increasing cAMP, activating PKA which inhibits Rac1 GTP-ase, leads to inhibition of actin polymerization and phagocytosis.
Human plasmacytoid dendritic cells express Dectin-1 as an immune regulatory receptor (111.32)
Authors: Joo HyeMee , Ni Ling , Li Xiao-Hua , Li Dapeng , Duluc Dorothee , Gannevat Julien , Morel Christine , Zurawski Sandra , Banchereau Jacques , Zurawski Gerard , Oh SangKon
Published: January 2023

Dectin-1 is expressed on myeloid-derived antigen-presenting cells and plays an important role in host immunity against microbial infections. We here demonstrate for the first time that human plasmacytoid dendritic cells (pDCs) express functional Dectin-1. Dectin-1 could deliver intracellular signals to activate pDCs through Syk. As results of activation, pDCs upregulated surface expression of HLA-DR and co-stimulatory molecules. However, pDCs expressed lower levels of CD80, CD86, and CCR7 compared to pDCs activated via TLR9. Signaling via Dectin-1 also induced pDCs to secrete cytokines (IL-6, IFNα, and TNFα) and chemokines (IL-1β, IP-10, IL-8, MIP-1α, and MIP-1β). Most notably, pDCs activated via Dectin-1 secreted greater amount of IL-1β than pDCs activated via TLR9. Dectin-1 stimulated pDCs selectively upregulated inflammation-related gene signatures. We further demonstrated that pDCs activated via Dectin-1 could polarize CD8+ T cells toward IL-10-producing regulatory CD8+ T cells. Activation of pDCs, but not mDCs, via Dectin-1 also resulted in enhanced Th2 type CD4+ T cell responses as well as B cell differentiation into plasmablasts, and this was partly dependent on IL-1β secreted from pDCs. Therefore, we conclude that Dectin-1 expressed on human pDCs possesses unique function to control host immune responses.
Phenelzine potentiates production of proinflammatory cytokines via the NF-κB pathway in LPS-activated microglia cells (112.11)
Authors: Kim Hyunseong , Chung Hwan-Suck , Shin Minkyu , Bae Hyunsu
Published: January 2023

Phenelzine is a potent monoamine oxidase (MAO) inhibitor that is widely used and has been shown to be clinically efficacious in patients with depression. It is also well known that nitric oxide (NO) synthase inhibitors show preclinical antidepressant-like properties, which suggests that NO is involved in the pathogenesis of depression. The purpose of this study was to determine if phenelzine affects the production of NO and tumor necrosis factor-alpha (TNF- α) in activated BV-2 microglia cells. Phenelzine increased the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS), as well as the release of TNF- α and IL-6 in BV-2 microglia cells. It is also confirmed that phenelzine increased the levels of NO, TNF-α and IL-6 in LPS-activated primary microglia cells. Phenelzine increased nuclear translocation of NF-κB by phospholylation of IκB-α in LPS-activated microglia cells. Taken together, these findings suggest that high doses of phenelzine could aggravate inflammatory responses in microglia cells that are mediated by NO and TNF-α.
Beta 7 integrin is essential for the induction of oral tolerance (112.15)
Authors: Kadono Takafumi , Ohmatsu Hanako , Sato Shinichi
Published: January 2023

Oral tolerance is one of the hallmarks of intestinal immunity that induces systemic unresponsiveness to orally ingested antigens. Beta 7 integrin is known to mediate lymphocyte migration to gut-associated lymphoid tissues, especially Peyer’s patches. We assume that lack of beta 7 integrin might affect oral tolerance due to defective lymphocyte migration to intestinal tissues. When mice were intraperitoneally immunized with OVA following oral OVA administration, beta 7 integrin deficient mice showed defective oral tolerance measured by OVA-specific IgG and IgM antibody titers, numbers of OVA-specific antibody forming cells, and proliferative responses to OVA. Oral tolerance in L-selectin deficient mice was unimpaired. Moreover, beta 7 integrin/ L-selectin double deficient mice showed defective oral tolerance to a similar degree as beta 7 integrin deficient mice, suggesting no additive effect of L-selectin deficienty. We also measured cytokine secretion from splenocytes following oral tolerance induction and found that whereas IFN gamma secretion was decreased in wild-type mice, this reduction was abrogated in beta 7 deficient mice. These results demonstrate that beta 7 integrin is essential for oral tolerance.
SNX9 as a novel biomarker for chronic inflammation-induced immunosuppression: implications for cancer therapy (112.16)
Authors: Ish-Shalom Eliran , Baniyash Michal
Published: January 2023

Chronic inflammation is the cause for immunosuppression observed in human and mouse with developing tumors; myeloid derived suppressor cells (MDSCs) accumulate and induce T and NK cell dysfunction associated with z down-regulation. Moreover, we discovered that z chain could serve as a biomarker to evaluate the efficacy of a given therapy; whether the tumor regresses or reappears, based on the developing chronic inflammatory environment. Recently, we identified SNX9, which is also expressed in B cells, as an additional biomarker playing a role in a pathway different from that of the z chain, for evaluating the immune status under chronic inflammatory conditions. Using the mouse melanoma model system (B16-F10.9), we found that SNX9 expression was significantly decreased, correlating with the tumor induced inflammatory environment. Our results also identify down-regulation of SNX9 expression in additional pathologies characterized by chronic inflammation such as during autoimmune disorders and infectious diseases, which correlates with elevated levels of MDSCs. Moreover, 5FU chemotherapy treatment, led to the recovery of SNX9 expression levels by eliminating the MDSCs population, indicating that SNX9 down-regulation is a reversible phenomenon and reflects the host’s chronic inflammatory state and immune status. These results may lead to a well-controlled personalized treatment of cancer-bearing hosts’ immune status by combining measurements of both z and SNX9.
Vitamin E isoforms directly bind PKCα and differentially regulate PKCα activity. (112.8)
Authors: McCary Christine , Yoon Youngdae , Cho Wonhwa , Cook-Mills Joan
Published: January 2023

Tocopherol isoforms of vitamin E differentially regulate inflammation and vascular cell adhesion molecule (VCAM)-1 signaling during leukocyte transendothelial migration. During VCAM-1 signaling, tocopherols modulate oxidative activation of protein kinase Cα (PKCα). Although tocopherols are antioxidants, it is not known whether tocopherols also directly regulate PKCα; therefore, we determined whether α-tocopherol and γ-tocopherol directly regulate non-oxidative or oxidative activation of purified PKCα. In a non-oxidative environment, phosphatidylserine (PS)/calcium cofactor-induced PKCα activity was increased by γ-tocopherol but not by α-tocopherol. In contrast, PS-independent oxidative-activation of PKCα was inhibited by α-tocopherol and by 10-fold higher concentrations of γ-tocopherol. In binding studies, lipid vesicles containing α-tocopherol or γ-tocopherol increased binding to the C2 domain (the PS binding domain), but not the C1b domain, of PKCα. To determine direct binding of tocopherols to PKCα in the absence of other lipids, we measured binding of a fluorescent α-tocopherol (NBD-α-tocopherol) to PKCα. Consistent with vesicle binding studies, NBD-α-tocopherol and PS competed for binding to PKCα, indicating that α-tocopherol binds the C2 domain. In summary, α-tocopherol and γ-tocopherol bind the C2 domain of PKCα, but differentially regulate PKCα activity. Therefore, α-tocopherol and γ-tocopherol can function as agonists or antagonists at the C2 domain of PKCα.
Gads is required for the expansion phase of CD8+ T cell-mediated immunity, but not the memory phase (113.27)
Authors: Zhang Elizabeth Yan , Parker Brooks , Yankee Thomas
Published: January 2023

The Gads adaptor protein is critical for TCR-mediated Ca2+ mobilization. We investigated how Gads regulates CD8+ T cell-mediated immunity following infection with an intracellular pathogen. Naïve CD8+ T cells from Gads+/+ OT-I or Gads-/- OT-I mice were adoptively transferred into congenic wild-type mice that were subsequently infected with OVA-expressing Listeria monocytogenes. After infection, expression of the activation markers was impaired in Gads-/- cells. Analysis of the kinetics of the immune response to infection suggested that Gads was not required for the onset of proliferation of OVA-specific CD8+ T cells, but was required for the maximal expansion of them. These data from the infection experiment were in contrast to our in vitro data, which showed Gads-dependent signaling pathways accelerated the exit from G0 phase of the cell cycle. These differences suggest that the inflammatory environment of the infected animals overcomes the need for Gads at early time points. At the peak of the immune response, comparable percentages of Gads+/+ and Gads-/- cells could produce IFN-γ, suggesting Gads-/- cells were functional. We also found that Gads was not required for the differentiation of naïve CD8+ T cells into memory cells, but Gads-/- memory cells did not expand as well as Gads+/+ cells upon re-infection. We conclude that Gads regulates the expansion phase of the primary and secondary immune responses, but not the generation of memory CD8+ T cells following infection.
The licensing of natural killer cells (113.3)
Authors: Rytova Valeria , Takei Fumio , Jiang Boxiang
Published: January 2023

The interaction between inhibitory Ly49 receptors and MHCI is important for the functional maturation of NK cells, known as “NK cell licensing.” NK cells from MHCI-deficient β2microglobulin (β2m)-KO mice are not cytotoxic and cannot efficiently kill their targets, as they are not licensed. Our goal is to determine the mechanism through which β2m-KO NK cells are kept hyporesponsive to otherwise NK-sensitive targets. Comparison through cytotoxicity assays confirmed that ex-vivo NK cells from poly-IC-injected wild type (WT) mice kill the prototypic YAC1 target, whereas NK cells from β2m-KO mice do not. Interestingly, both express similar levels of granzyme B and adhere to immobilized ICAM-1, the ligand for LFA-1. Further, confocal analysis of NK cells interacting with YAC1 showed that WT NK cells aggregate and polarize lytic granules toward their targets, while β2m-KO NK cells are deficient in granule polarization. Once stimulated by plastic beads coated with ICAM-1, both WT and β2m-KO NK cells reorganize their actin cytoskeleton. Stimulation by beads coated with ICAM-1, H60 and CD48 (ligands for stimulatory NKG2D and 2B4 receptors), induces aggregation and polarization of lytic granules in WT but not in β2m-KO NK cells. This suggests that critical components of the cytotoxicity pathway generated by stimulatory receptor interactions are impaired in β2m-KO NK cells, preventing aggregation and polarization of cytotoxic granules, and the release of their contents towards targets.
Investigating the role of 4-1BB and 4-1BBL in dendritic cell immunobiology (113.6)
Authors: Mbanwi Achire , Lin Gloria , Watts Tania
Published: January 2023

The tumour necrosis factor (TNF) receptor family member 4-1BB, and its TNF family ligand, 4-1BBL, have been thoroughly investigated as potent modulators of the CD8 T cell response. During a viral infection, 4-1BBL on APC, binding to 4-1BB on activated T cells mediates survival signaling in activated and memory CD8 T cells. Similarly to T cells, 4-1BB is also induced on dendritic cells (DC) following activation; however, how 4-1BB and 4-1BBL modulate DC biology is poorly understood. We provide evidence that DC express both 4-1BB and 4-1BBL upon activation with a microbial stimulus in vitro, and that these molecules interact on maturing DC. Using an in vivo DC vaccination model, we demonstrate that in the absence of 4-1BBL on the DC, there is a 2-3 fold defect in the ability of these DC to generate influenza-specific CD8 T cells in a WT host. This defect is not observed when 4-1BB-/- hosts are vaccinated with WT DC, suggesting that 4-1BBL on the DC is not acting through 4-1BB in the host, but intrinsically on the DC. Even more striking is that 4-1BB-/- DC do not show this defect in the generation of flu-specific CD8 T cells, raising the possibility of a 4-1BB-independent 4-1BBL mechanism. In that regard, we further show that anti-4-1BBL antibodies can induce Erk activation in WT DC, but not in 4-1BBL-/- DC. These results identify 4-1BBL as a DC modulator and have overall implications for vaccine design.
High mobility group nucleosome-binding protein 1 acts as an alarmin critical for the induction of immune response (113.7)
Authors: Yang De , Postnikov Yuri , Li Yana , Tewary Poonam , de la Rosa Gonzalo , Kliman Dennis , Furusawa Takashi , Bustin Michael , Wei Feng , Oppenheim Joost
Published: January 2023

Alarmins, defined as endogenous mediator(s) capable of promoting the recruitment and activation of antigen-presenting cells (APCs) including dendritic cells (DCs), can potentially promote immunity, however, their essential contribution to the induction immune responses remain to be demonstrated. Here we report the identification of HMGN1 as a novel alarmin that is critical to the induction of antigen-specific immune response. HMGN1 induced DC maturation and recruitment of APCs and activated NF-κB and multiple MAPKs, in a MyD88-dependent manner. HMGN1 promoted antigen-specific immune response upon co-administration with an antigen. Furthermore, knockout of HMGN1 in mice greatly reduced antigen-specific antibody and T cell responses upon intraperitoneal immunization with an antigen using LPS as an adjuvant. The impaired ability of HMGN1 KO mice to mount antigen-specific immune responses was accompanied by both deficient DC recruitment at sites of immunization and reduced production of inflammatory cytokines. Bone marrow chimera experiments revealed that HMGN1 derived from non-leukocytes played a more critical role in the induction of antigen-specific antibody and T cell responses. Thus, HMGN1 acts as a novel alarmin critical for the induction of adaptive immune response.
IL17 autoregulatory system in vitro and in vivo in a mouse model of autoimmune and viral diseases (114.5)
Authors: Nagata Takako , Ghilardi Nico , Kolls Jay
Published: January 2023

In the IL17 family, IL17A and F are known to be primary players. Concanavalin A (Con A) induced hepatitis is a model of autoimmune and viral diseases. We reported the production of IL17A increases by more than two orders of magnitude and IL17F by more than one order in IL17 receptor (IL17R) KO, in vivo and in vitro, compared to wildtype (WT). We have also shown IL17F in vitro downregulates the level of IL17A and F in WT, but doesn’t significantly downregulate either in IL17R KO. Hence, IL17F appears to autoregulate through IL17R. Here, we examine if and how IL17A and F each contribute to this system, in vivo and in vitro, with IL17A KO and IL17F KO. Furthermore, their contributions to liver damage are quantitatively evaluated. Results: IL17F increased around 7-fold in IL17A KO compared to WT both in vitro and in vivo. IL17A levels were similar in IL17F KO and WT in vitro and in vivo. Overall, IL17A level in IL17R KO>>in IL17F KO=in WT and IL17F level in IL17R KO>in IL17A KO>in WT. ALT (alanine aminotransferase) was significantly lower in IL17A KO than in WT. IL17F KO showed similar level to WT. Conclusion: Both IL17A and F appear to autoregulate in vivo and in vitro. However, since IL17A binds IL17R more strongly than IL17F, IL17R would tend to be monopolized by IL17A in WT. This explains why IL17A level in IL17F KO was similar to WT. IL17A appears to be primarily responsible for Con A induced hepatic inflammation.