bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR4 augments HSV-2 induced NF-κB activity in human cervical epithelial cells (136.28)
Authors: Tang Xianying , Liu Hongya , Wu Xinxing , Li Hui
Published: January 2023

Herpes simplex virus type 2 (HSV-2) infects the genital epithelium and can be transmitted to the central nervous system, establishing lifelong infection. Toll-like receptors (TLRs) have been reported to initiate innate immune responses to microorganisms. It has been shown that TLR2 and TLR9 are involved in recognition of different HSV strains by dendritic cells (DCs). Our previous study has shown that HSV-2 infection leads to dramatic activation of the NF-κB signaling pathway and induction of IL-6 and IFN-beta. We report here that HSV-2 activates NF-κB reporter and TLR4/MD2 augments this activation. The NF-κB reporter construct was co-transfected into human cervical epithelial (HCE) cells together with either vectors or TLR4/MD2 expression plasmids. The transfected cells were then infected with HSV-2 at 3 MOI and luciferase activity was measured and normalized to Renilla luciferase activity. We observed a relatively low, but reproducible induction (approximately 1.5-fold) of reporter activity in HCE cells with HSV-2 infection compared to that in mock-infected cells. Importantly, an augmented induction of the NF-κB reporter was seen while cells were co-transfected with TLR4/MD2 in HSV-2 infected cells. Thus, TLR4 potentially plays a role in mediating its activation. Effect of shRNA against TLR4 on this induction is under active investigation. This work was supported by grants from the National Natural Science Foundation of China (No. 30500465, 30810103052)
Platelet-derived soluble TREM-Like Transcrip-1 (TLT-1) induces human and mouse Dendritic Cell activation through a TLR-4-dependent mechanism. (136.32)
Authors: de la Rosa Gonzalo , Tewary Poonam , Yang De , Gattis James , Lubkowski Jacek , Oppenheim Joost , McVicar Daniel , Washington Valance
Published: January 2023

Triggering Receptor Expressed on Myeloid Cells-Like Transcrip-1 (TLT-1) is a member of the multifunctional TREM family of receptors that also include TREM-1, 2, 3, and TLT-2. TLT-1 is expressed on megakaryocytes and platelets at low levels, but upon activation with thrombin, its membrane expression is up-regulated and shed, yielding a soluble form (sTLT-1). Here we describe for the first time the ability of human sTLT-1 to activate human monocytes and antigen-presenting cells. Treatment of myeloid Dendritic Cells (DCs) with sTLT-1 resulted in an increase of co-stimulatory molecules, induction of pro-inflammatory cytokines production, and their ability to prime lymphocyte proliferation. sTLT-1 failed to activate murine Bone Marrow-derived Dendritic Cells from TLR4-deficient mice, and blocking antibodies against TLR4 reduced sTLT-1-induced cytokine production by human monocytes and DCs, indicating that TLR4 is involved in sTLT-1 signaling. Therefore, human sTLT-1 has the ability to endow APCs with the capacity to initiate adaptive immune responses, and may therefore be a potential target of anti-inflammatory therapies.
Identification of the functional TLR4 TIR dimerization interface using a decoy peptide approach (136.37)
Authors: Toshchakov Vladimir , Szmacinski Henryk , Lakowicz Joseph , Vogel Stefanie
Published: January 2023

Agonist-induced oligomerization of TIR domains of TLRs and TLR adapters initiates intracellular signaling. Identification of TIR domain surface areas that mediate functional TIR-TIR interactions is key to the rational design of therapeutics that specifically target TLR signaling. We have designed a library of 11 cell-permeating decoy peptides each of which represents a non-fragmented patch of the TLR4 TIR surface, such that the library entirely encompasses the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating sequence derived from Antennapedia homeodomain and tested for the ability to inhibit early cytokine mRNA induction and MAPK activation elicited in peritoneal murine macrophages by LPS. Five peptides, 4R1, 4R3, 4BB, 4R9, and 4αE, potently inhibited all manifestations of TLR4 signaling examined. These 5 peptides and a control peptide (CP) were then examined for the ability to bind directly to TLR4 TIR by FRET using time-resolved fluorescence spectroscopy. Peptides were labeled with Bodipy-TMR-X (BTX); the C-terminus of TLR4 was fused to Cerulean fluorescent protein (Cer). BTX-labeled peptides 4R1, 4BB, and 4αE efficiently quenched fluorescence of TLR4-Cer expressed in HeLa cells, indicating efficient FRET. BTX-4R3 exhibits weak quenching. BTX-labeled CP and 4R9 did not change TLR4-Cer fluorescence lifetime. These data suggest that the area between BB loop of TLR4 and its fifth helical region mediate TLR4 TIR dimerization.
Structural studies of Toll like receptor signaling adaptors. (136.45)
Authors: Snyder Greg , Jiang Jiansheng , Chen Kang , Fresquez Theresa , Smith Patrick , Snyder Nathaniel , Luchetti Timothy , Cirl Christine , Miethke Thomas , Tjandra Nico , Xiao Tsan
Published: January 2023

Signaling downstream of the Toll like receptors (TLRs) involves Toll-IL-1R homology (TIR) domains as well as recruitment of critical signaling adaptors MyD88 and TIRAP/Mal. Studies in human patients, dominant negative mutants and knockout mice have shown that mutations in Arg196, the BB loop, DD loop, “Poc” site Ile179 are critical for MyD88 TIR domain signaling and appropriate responses to pathogen stimuli. We previously reported the structure of MyD88 TIR domain that showed a unique conformation for its BB loop, as well as a crystallographic lattice that reveals interactions between the BB-DD and BB-EE loops. We now report additional crystal forms which retain BB-DD and BB-EE interactions as well as extend the resolution to 1.4 Å. A yeast two-hybrid analysis of the domain interface residues observed from the crystal structure lattice shows that key residues including the human Arg196Cys mutation are critically important for homotypic TIR domain binding. Additionally, we report NMR solution studies showing binding between MyD88 and bacterial TIR domain protein TcpC, which has been demonstrated to negatively regulate MyD88-dependent TLR adaptor signaling. Finally, we report the X-ray structure of a second critical TIR adaptor protein, TIRAP/Mal. We observe key residues and loops important for TIRAP/Mal adaptor function. Structural insights from these studies may aid our understanding of the molecular mechanisms by which TLRs and TIR domain adaptors interact and signal.
T. cruzi interference with host cell membrane integrity triggers the release of Plasma Membrane-derived Vesicles: A mechanism for entry into mammalian cells (137.1)
Authors: Ansa-Addo Ephraim , Inal Jameel
Published: January 2023

Trypanosoma cruzi is a flagellated protozoan transmitted by various insects of the Reduviidae family and the causative agent of the debilitating Chagas disease with 50,000 deaths per annum. The lack of an effective treatment for the chronic form means the disease still remains an important health issue in Latin America. Disease transmissions from human-to-human can occur through contact with contaminated blood, during pregnancy or blood transfusion. Moreover, new means of transmission from contaminated food, and sharing of intravenous needles, means the disease now has the potential to be an international threat and cases are being seen in North America, Europe and Japan due to increasing human migration. Parasite entry into cells involves complex processes, not well characterised, and so the rationale of this study was to identify other possible host cell invasion strategies. Here, we show that T. cruzi triggers the release of PMVs from host cells. Parasites then take advantage of the membrane breach to gain entry into cells before lysosome-mediated membrane repair. Furthermore, we show, that T. cruzi triggers release of PMVs through integrins and lipid raft microdomains, which are all present on the plasma membrane. Inhibiting the activation of these receptors by the parasite stops the release of PMVs and this inhibits T. cruzi entry into cells. This study adds to our knowledge on T. cruzi infection strategies and possible pathways that could be therapeutically targeted.
Regulatory T cell suppressive potency dictates the balance between bacterial proliferation and clearance during persistent Salmonella infection (137.11)
Authors: Johanns Tanner , Ertelt James , Rowe Jared , Lai Joseph , Way Sing Sing
Published: January 2023

The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Although regulatory T cells (Tregs) have been widely implicated to control host defense against infection, their role in dictating the natural progression of persistent infection remains incompletely defined. Herein, a sharp dichotomy in infection tempo between early and late time points during persistent Salmonella infection is demonstrated. Early after infection, blunted effector T cell activation and increasing bacterial burden coincide with enhanced Treg suppressive potency. Reciprocally, at later time points, robust effector T cell activation and bacterial clearance occur in parallel with diminished Treg suppression. By enumerating the relative impact of Treg ablation on infection outcome at both early and late time points of persistent infection, the role of Treg suppressive potency in dictating the natural progression of infection was verified. Thus, dynamic control of Treg suppressive potency dictates the tempo of persistent bacterial infection.
Immune Molecular Plasticity Links Viral Virulence to Protective T cell Vaccines (137.2)
Authors: Salek-Ardakani Shahram , Flynn Rachel , Arens Ramon , Yagita Hideo , Smith Geoffrey , Borst Jannie , Schoenberger Stephen , Croft Michael
Published: January 2023

Attenuated non-replicating or weakly replicating viruses are being used as vaccines but they generally elicit poor T cell immunity and might not afford optimal protection. We show that the virulence of a virus leads to altered levels of T cell memory because of the differential use of stimulatory receptors. Persistently replicating virus results in selective engagement of the TNFR family molecules OX40 (CD134) and CD27. These molecules drive enhanced CD8 T cell memory, and determine the protective capacity of a virus when used as a vaccine. Therefore the length of exposure to viral antigen results in the immune system engaging novel protein interactions, which are otherwise inactive. Defining this link between virulence and immunogenicity will aid the design of safe but still effective T cell vaccines.
IFN-γ expression by regulatory T cells in the CNS of mice persistently infected with neurotropic coronavirus (137.27)
Authors: Zhao Jingxian , Perlman Stanley
Published: January 2023

Mice infected with mouse hepatitis virus, strain JHM develop a chronic infection in the CNS characterized by low levels of viral antigen persistence and retention of virus-specific CD4 and CD8 T cells at the site of infection. We have previously demonstrated that T cell maintenance in the infected CNS is a dynamic process, requiring replenishment from peripheral antigen- experienced and naïve T cell pools. Since mice continued to show clinical improvement, these results raised the possibility that Tregs contributed to a quiescent state in the CNS. Here we show a preferential influx of Tregs to the CNS at very early stage of the infection. They are not converted from Foxp3- cells. Intriguingly, not only IL-10 but also IFN-γ producing Tregs are found in the CNS as early as d 5 p.i. While there are much less (<1%) Tregs making IL-10 during chronic infection, a proportion (>15%) of them continue to express IFN-γ. IFN-γ expression is correlated with T-bet upregulation in Tregs. IFN-γ producing Tregs are induced locally in the CNS from both virus-specific and nonspecific Treg pools. While the frequencies of total Tregs decrease dramatically by d 7 p.i. and remain constant thereafter, two thirds of Tregs lose their Foxp3 expression between d 7 and d 28 p.i. in adoptive transfer experiments. We propose that Tregs acquire effector cell phenotype during type 1 inflammation and the maintenance of Tregs in the persistently infected CNS is also dynamic, requiring recruitment of new Tregs.
Downregulation of TACI by Neisseria meningitidis type C polysaccharide vaccine is responsible for its weak immunogenicity (137.4)
Authors: Akkoyunlu Mustafa , Katsenelson Nora , Blake Milan , Kanswal Sunita
Published: January 2023

Antibodies against the capsular polysaccharide (CPS) antigen of Neisseria meningitidis are bactericidal against meningococcus. However CPSs are poorly immunogenic T cell independent type II antigens. The B cell molecule TACI is shown to be essential for the development of antibody response against CPS antigens. TACI is a receptor for the TNF family cytokines BAFF and APRIL. In this study, we have determined that the CPS of N. meningitidis-C (MCPS) inhibits BAFF or APRIL induced plasma cell development and Ig secretion from mouse B cells. Correlating with this, TACI was strongly downregulated in MCPS exposed B cells. BAFF activates both the classical and alternative NF-kB pathway in B cells. Investigation of BAFF mediated signaling demonstrated that MCPS inhibits both the signaling pathways. Previously, we had shown that CpG strongly upregulates TACI expression. We therefore tested whether CpG would block the inhibitory effect of MCPS. The results showed that CpG partially reversed TACI suppression induced by MCPS. Supporting these in vitro results, CpG was not able to augment antibody response against MCPS when a CpG containing MCPS vaccine was injected into mice. Taken together our data demonstrates that CPS mediated downregulation of TACI on B-cell surface could be a mechanism by which meningococcal bacteria evade host immune response. Also, inhibition of B cells through suppression of TACI may explain the poor immunogenicity of CPS based vaccines.
Regulation of virus-specific acute and memory CD8+ T cell response by Galectin-9/TIM-3 interaction (137.5)
Authors: Sehrawat Sharvan , Hirashima Mitsuomi , Rouse Barry
Published: January 2023

We demonstrate that galectin-9 (Gal-9) acts to constrain CD8+ T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with α-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8+ T cell response to HSV. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8+ T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved apoptosis of TIM-3+ Teffectors as well as the promotion of Foxp3+ regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance primary and memory responses.
IL-28(IFN-lambda) inhibits Th2-mediated allergic airway inflammation through the modulation of dendritic cell function (138.11)
Authors: Übel Caroline , Kolsida Ourania , Hausding Michael , Andreakos Evangelos , Finotto Susetta
Published: January 2023

IL-28(IFN-Lambda) is a newly discovered cytokine that is profoundly down-regulated in allergic asthma. Here, we reveal a novel role of IL-28 in suppressing Th2 cell differentiation and Th2-mediated pathology. Inhibition of endogenous IL-28 function in IL-28Ralpha-/- mice exacerbated allergic airway disease by augmenting Th2 responses and IgE levels, and down-regulating Th1 and regulatory T cell responses. Moreover, treatment of wild-type mice with recombinant or adenovirally-expressed IL-28 ameliorated allergic airway disease, suppressed Th2 responses and induced IFN-gamma and CD4+CD25+FoxP3+ regulatory T cells. Central to IL-28 immunoregulatory activity was its capacity to modulate CD11c+ conventional DC function to down-regulate OX40L, up-regulate T-bet and IL-12, and promote Th1 cell differentiation. Consistently, IL-28-mediated protection was absent in IFN-gamma-/- mice and was adoptively transferred by IL-28-treated CD11c+ cells. These data demonstrate a critical role of IL-28 in controlling T cell responses in vivo through the modulation of conventional DC function in models of allergic asthma.
An orexigenic hormone ghrelin enhances LPS-induced IL-10 production in human peripheral blood-derived macrophages. (138.12)
Authors: Baatar Dolgor , Lee Jun Ho , Brill Margaret , Lustig Ana , Carter Arnell , Taub Dennis
Published: January 2023

Ghrelin, a circulating orexigenic hormone, exerts potent therapeutic effect in several models of inflammatory diseases, such as endotoxemic shock, ulcerative colitis and autoimmune encephalomyelitis. Suppression of production of pro-inflammatory cytokines from immune cells has been proposed as a mechanism of this effect of ghrelin. Here we report that ghrelin enhances lipopolysaccharide (LPS)-induced production of the anti-inflammatory cytokine IL-10 from human peripheral blood monocyte-derived macrophages. Interestingly, IL-10 production from LPS-activated monocytes was not affected by ghrelin. Further studies revealed that the ghrelin receptor, namely, growth hormone secretagogue receptor (GHS-R), is significantly upregulated during macrophage differentiation. Moreover, ghrelin enhanced expression of the transcription factor c-Maf essential for IL-10 gene expression in macrophages. Whether induction of c-Maf by ghrelin is responsible for the enhanced IL-10 production and the significance of IL-10 in overall anti-inflammatory action of ghrelin in vivo is currently under investigation.
Simultaneous correlation of cytokine production with Treg and Th17 cell proliferation (138.21)
Authors: Elia Jeanne , Rabenstein Jacob , O'Donnell Erika , Rohrer Jurg
Published: January 2023

Functionally-polarized CD4+ T cell subsets have been identified based on their distinctive patterns of cytokine secretion, transcription factor expression and function. As a signature cytokine, Th17 express high levels of interleukin 17A (IL-17) whereas Treg are characterized by the expression of the Foxp3 transcription factor. In the mouse system TGFb is required for the production of IL-17A, although additional cytokines are produced including Th1 (IL-2, IFNg) and Th2 (IL-4, IL-10 ) cytokines. The goal of the experiment was to evaluate the correlation of cell cycle (VFSE), cytokine production via both CBA and intracellular staining, Foxp3 expression (Treg), and cytokine-induced phosphorylation events. Multiple fixation and permeabilization conditions were used to allow resolution of intracellular cytokines, Foxp3, and phospho-proteins. FoxP3+ CD4 T cells were present at initiation of culture and were shown to divide by appearing on subsequent VFSE peaks. IL-17A began to appear after day 2 and was restricted to newer generations of cells, unlike FoxP3 which was expressed at time 0 and restricted to the older generations. Further analysis showed the IL-17A population coexpressed IL-2 at day 2, but the populations diverged upon maturation, becoming mutually exclusive by day 4. Analysis of Stat5 phosphorylation revealed heterogeneous responses to secreted cytokines, with newer generations of cells showing the most robust phosphorylation responses.
Vagus Nerve Stimulation Produces an Anti-Inflammatory Monocyte Phenotype in Blood. (138.25)
Authors: Valdes-Ferrer Sergio , Rosas-Ballina Mauricio , Olofsson Peder , Chavan Sangeeta , Tracey Kevin
Published: January 2023

Cytokine production and immune cell migration are under the control of the nervous system. Electrical stimulation of the vagus nerve attenuates cytokine production in endotoxemia, and down-modulates neutrophil migration in animal models of inflammation. The effect of vagus nerve stimulation (VNS) on PBMC phenotypes has not been characterized. We performed VNS or sham surgery in mice (n=5 group) and 24 hours later collected blood for analysis by flow cytometry. The total number of CD11b+ expressing cells was not affected, but CD11b+/Ly-6C+ monocytes significantly decreased in frequency (0.5% vs. 3.2%; p=0.005). VNS decreased dendritic cell numbers defined as CD11b+, CD11c+ (0.4% vs. 1.8%; p=0.042), or CD11c+, F4/80+ (1.6% vs. 2.7%; p=0.025). VNS did not affect the total number of circulating T cells (CD3+). VNS did not affect CD11b+ or Ly-6Clow, which are known to be VEGF-producing and anti-inflammatory. The present results indicate that VNS reduced the proportion of circulating pro-inflammatory monocytes and dendritic cells, and decreased the expression of monocytic lineage cells, in particular those expressed by M1 monocytes which are known to secrete TNF, Il-6, IL-12 (and reduced amounts of IL-10). Together, this indicates that direct activation of a neural circuit can down-modulate inflammatory cell phenotypes in blood, producing an anti-inflammatory cellular milieu.
Physiological control of NKT cell activation by extracellular adenosine via A2B adenosine receptor (138.27)
Authors: Kini Radhika , Subramanian Meenakshi , Ohta Akiko , Sitkovsky Michail , Ohta Akio
Published: January 2023

Negative regulatory mechanism of immune responses is essential to prevent excessive inflammatory tissue injury. Adenosine involves in physiological down-regulation of inflammation via A2A adenosine receptor (A2AR). In this study, we provide evidence that A2B adenosine receptor (A2BR) is also important in the physiological control of hepatic inflammation. Acute hepatitis induced by intravenous concanavalin A (Con A) injection was significantly more severe in A2BR-/- mice than in wild-type mice. The exaggerated inflammation in A2BR-/- mice accompanied higher levels of IL-4 from NKT cells suggesting A2BR-mediated suppression of NKT cell activation. In vitro activation of NKT cells by α-galactosylceramide (α-GalCer) showed that stimulation of A2BR is inhibitory to NKT cell activation. Furthermore, injection of α-GalCer induced higher serum cytokine levels and stronger liver damage in A2BR-/- mice. These results show that the lack of A2BR diminished negative regulatory mechanism of NKT cells by endogenously produced adenosine and, therefore, caused more severe acute hepatitis. Indeed, transfer of A2BR-/- NKT cells into RAG2-/- mice resulted in stronger Con A-induced liver injury than wild-type NKT cells. There have been controversial reports regarding the role of A2BR in inflammation. Our results demonstrate that NKT cell-mediated immune responses are under the physiological control of extracellular adenosine through A2BR signaling.
A novel means of immunoregulation: CD46 attenuates effector responses of human conventional and unconventional T cells by distinct mechanisms (138.8)
Authors: Le Friec Gaelle , Cardone John , Vantourout Pierre , Roberts Andrew , Cope Andrew , Hayday Adrian , Kemper Claudia
Published: January 2023

Many studies including human genetics have implicated IL10-producing cells as key in limiting immune pathology during infection and in preventing autoimmunity. Here we show that the complement regulator CD46 regulates IL10 production in an IL2-dependent fashion. More specifically, CD3/CD46-activation of human CD4+ T cells in low IL2 induces strong IFNγ secretion, whereas increasing IL2 induces a ‘switch’ to immunosuppressive Tr1-like cells that are initially IFN-γ+/IL10+, and then IL-10+. We propose that CD46 activation initially promotes antimicrobial Th1 effector responses, while expansion of this response increases local IL2, promoting a CD46-dependent switch to IL10-mediated immunoregulation. The significance of this in vivo is supported by the failure of CD3/CD46-activated CD4+ T cells from rheumatoid arthritis patients to switch to the IL10+ state, producing ≥20-fold more IFNγ than IL10. IL10-switching of CD4+ T cells is mediated by the CYT-1 isoform of the alternatively spliced cytoplasmic tail of CD46. Interestingly, human γδ T cells, whose importance to the early effector phase of immune responses is increasingly apparent, express predominantly the CYT-2 isoform of CD46. Thus, they do not produce IL10 in response to TCR/CD46 co-engagement, but rather significantly decrease their IFNγ and TNFα production. Thus, we propose that CD46 uses distinct molecular mechanisms to regulate unconventional and conventional T cells across the span of the immune response.
IL-12 induces human CD4+CD45RA-CD25hiCD127low/neg regulatory T cells to secrete IFNγ and IL-10 and acquire a non-regulatory effector phenotype (138.9)
Authors: Dominguez-Villar Margarita , Hafler David , Baecher-Allan Clare
Published: January 2023

Regulatory T cells (Treg) play a pivotal role in maintaining a balance between the immune response and peripheral tolerance. Recent evidence indicates that regulatory T cells are more functionally plastic than originally thought. Not only does the cytokine milieu modulate their capacity to suppress, but it can also induce Tregs to secrete cytokines (IL-17). The IL-12 family of cytokines (IL-12, IL-23, IL-27, IL-35) are key players in the regulation of T cell responses. We have studied the distinct effects that these cytokines (IL-12, IL-23 and IL-27) exert on human Treg function and phenotype. RESULTS: Surprisingly, we have found that IL-12 induces memory CD127lo Tregs to express T-bet and produce IFN-gamma and IL-10. Furthermore, in this state, these Tregs not only exhibit a non-regulatory effector phenotype, but they are also markedly less able to suppress the activation of co-cultured CD4 T cells. Importantly, although these IL-12-treated Tregs are less suppressive, their Foxp3 expression is not significantly altered. Additional data indicates that this is a transient state of reduced Treg suppression.. Thus this represents a way for the immune system to inactivate Treg function to allow an immune response to proceed.
Immuno-signature of CD4+ Treg and T inducer cells (139.14)
Authors: Perna Avi , Nosworthy Lara , Yang Xifeng
Published: January 2023

T regulatory cells (Tregs), a subset of well-known suppressive T effector cells, are phenotypically characterized as CD4+CD25high Foxp3+CD39+. A recent report has identified CD4+CD25-CD39+ cells as T “inducer” (Tind) and demonstrated their ability to potentiate proliferation and cytokine production in vitro. In this study, we examined the expression of surface molecules on Tregs and Tind in normal healthy individuals by multicolor flow cytometric analysis utilizing a panel of fluorochrome conjugated mAbs to integrins, ICAMs, chemokine receptors, co-stimulatory molecules, and activation markers. In comparison with conventional CD4+ T cells, both Tregs and Tind cells showed higher expression of integrins (integrin aL, a4, a5, b1) and some chemokine receptors (CCR2, CCR4, CCR6 and CXCR3), but lower of PECAM-1, CCR7 and CXCR4. Additionally, an enhanced expression of activation markers ICOS, CD95(Fas), PD-1 and CD45RO was observed on both cell types. In contrast, the expression of CD7, CD127 and CD38 was at lower levels on Tregs and Tind than conventional CD4+ T cells. Comparison of Tregs and Tind showed the expression levels of most markers were non-differential. However, Tregs expressed less CD127 and CD38 than Tind, whereas Tind expressed more PD-1 than Tregs. These findings suggest Tind and Tregs may possess a functional relationship.
T-bet-independent effects of IL-12 family cytokines on regulation of Th17 responses to infection (139.5)
Authors: Smeltz Ronald , Cobb Dustin , Hambright Derek
Published: January 2023

T-bet is an IFN-γ-inducible transcription factor that promotes Th1 differentiation. Previously we reported that T-bet (Tbx21)-deficient mice develop a robust Th17 response to parasitic infection, despite a normal Th1/IFN-γ response. The purpose of this study was to determine if IFN-γ exerted any influence on antigen-specific Th17 responses to T. cruzi, a parasite and causative agent of Chagas disease. Neutralization of endogenous IFN-γ or addition of recombinant IFN-γ to purified CD4+ T cell cultures from T. cruzi-infected mice had minimal effect on IL-17 production. CD4+ T cells expressed IFN-γRα, thus were capable of responding to IFN-γ. To determine if IFN-γ could act in an APC-dependent manner, we neutralized IFN-γ and IL-12 in splenocyte cultures, since one effect of IFN-γ on APC is induction of IL-12. While anti-IFN-γ modestly increased IL-17 production, addition of anti-IL-12 led to a significant enhancement of antigen-specific IL-17. In contrast, addition of IL-23 to T-bet-deficient cultures led to a striking increase in IL-17 production. These data show that IL-12 and IL-23 can influence Th17 responses in a T-bet-independent manner; and that the effects of IFN-γ are not necessarily related to its ability to induce T-bet expression in T cells. These data further illustrate the synergy between T cell and APC-intrinsic factors in regulation of Th17 responses to infection.
Estrogen related receptor-α is a global metabolic and fate determinant for T lymphocytes (139.8)
Authors: Michalek Ryan , Jacobs Sarah , Chang Ching-yi , Ilkayeva Olga , Maciver Nancie , Jones Russell , Giguere Vincent , McDonnell Donald , Rathmell Jeffrey
Published: January 2023

T lymphocyte activation initiates rapid cell growth and proliferation that is fueled by increased glucose metabolism. Molecular factors contributing to this transition and how metabolism impacts immunity, however, have remained uncertain. We have uncovered a critical role for the orphan nuclear receptor Estrogen Related Receptor-alpha (ERRα) in this metabolic program and T cell differentiation. In the absence of ERRα activated T cells failed to elevate glucose metabolism, resulting in diminished growth, proliferation, and effector function. The generation of inducible regulatory T cells was also selectively inhibited in the absence of ERRα. Metabolomic analysis demonstrated that the loss of ERRα promoted a shift from glycolytic metabolism to lipid oxidation. Importantly, defects in growth and differentiation in the absence of ERRα were overcome by addition of excess lipids as an alternate mitochondrial fuel. Furthermore, induction of lipid metabolism in T cells selectively promoted the generation of regulatory T cells in vitro and in vivo and suppressed the differentiation of effector Th17 cells in vitro. Together, these results demonstrate that ERRα is essential to drive glucose metabolism of activated T cells and that metabolic phenotype can strongly influence T cell differentiation.
Effect of green tea extract and epigallocatechin gallate on bovine neutrophil function (140.10)
Authors: Kimura Kayoko , Palic Dusan , Roth James
Published: January 2023

Neutrophils play an important role as the first line of defense in protecting hosts from microbial invasion, but the delayed migration and prolonged presence of neutrophils can result in sustained inflammation. The aim of this study was to determine effect of green tea extract (GT) and its major anti-inflammatory component, epigallocatechin gallate (EG), on bovine neutrophils in order to decipher possible clinical use of GT and EG to alleviate inflammation. Effects of GT and EG on degranulation, oxidative burst, CD11b expression, and neutrophil apoptosis were investigated at concentrations from 1 to 100 μg/mL (GT) or μM (EG) in different assay conditions. Concurrent, pre, and post treatment of GT and EG with saline or standard stimulant (calcium ionophore or phorbol myristate acetate) significantly reduced degranulation and superoxide production compared to control (no GT/EG). Significant reduction in CD11b expression was observed in EG treated samples, but not all GT samples had similar effect. GT and EG at concentrations of 50 and 100 μg/mL (GT) or μM (EG) triggered neutrophil apoptosis determined by Annex-V cell surface binding and 7 amino-actinoycin intracellular staining. The in vitro reduction of cattle neutrophil function suggests potential of GT and EG to be used in clinical applications as support to anti-inflammatory therapy.
Azole derivative drugs inhibit neutrophil chemotaxis by blocking the Calcium permeant channel TRPM2 (140.12)
Authors: Partida-Sanchez Santiago , Sumoza-Toledo Adriana , Bhagat Harivadan , Lange Ingo , Cortado Hanna , Fleig Andrea
Published: January 2023

Azole derivatives exert direct anti-inflammatory activity in fungal infections and other cutaneous inflammatory conditions. Inhibition of PMN chemotaxis and leukotriene biosynthesis has been suggested to explain this phenomenon, yet the molecular mechanism of action for these drugs remains unknown. Recently we showed that NAD+ derived metabolites, cADPR and ADPR; regulate PMN chemotaxis by inducing extracellular Ca2+ influx via the ion channel TRPM2. Here we investigated whether azole derivatives inhibit PMN chemotaxis in vitro by altering the production of NAD+-derived metabolites or by blocking plasma membrane channel-conducted Ca2+ influx. First, we demonstrated that econazole and clotrimazole significantly inhibited PMN directionality and speed in an EZ-Taxiscan chemotaxis assay. Interestingly, econazole and clotrimazole, as well as FA, another compound proposed to block the TRPM2 channel, blocked fMLP-induced Ca2+ influx, rendering defective Ca2+ responses comparable to those responses observed in fMLP-stimulated TRPM2 KO PMN. Moreover, we found that pre-treatment of neutrophils with econazole or clotrimazole did not alter NAD+ metabolism, but its effect was by blocking a membrane channel with physiological properties similar to TRPM2. Collectively, these data suggest that compounds, which antagonize TRPM2-mediated Ca2+ signaling pathway, may function as effective inhibitors of PMN recruitment, and will likely prove useful in the treatment of inflammatory diseases.
Evaluation of the effects of disease-causing mutations in type I TNF receptor (TNFR1) on neutrophil responses (140.8)
Authors: Pelletier Martin , Bulua Ariel , Kastner Daniel , Siegel Richard
Published: January 2023

Mutations in the type 1 TNF receptor (TNFR1) cause an autosomal-dominantly inherited autoinflammatory disorder called TNF receptor-associated periodic syndrome (TRAPS). The disease is characterized by recurrent fevers with abdominal pain, migratory rash and inflammation. Patients also develop serositis, including peritonitis, as a manifestation of disease flares. TRAPS-associated mutant receptors do not traffic to the cell surface and accumulate in the endoplasmic reticulum, which may lead to abnormal TNF-independent receptor signaling and sustained inflammation. We examined the contents of peritoneal lavage fluid after intraperitoneal injection of LPS in TNFR1 mutant mice. Interestingly, heterozygous TNFR1 mutant mice had increased neutrophil infiltrates into the peritoneal cavity after LPS injection. We have investigated whether the exaggerated infiltration of neutrophils in mice with TNFR1 mutations is due to an intrinsic neutrophil abnormality or the release of more neutrophil-attracting chemokines by macrophages. We have performed chemotaxis experiments using neutrophils isolated from TRAPS patients as well as TNFR1 mutant mice, and measured the migratory responses of neutrophils toward chemoattractants and macrophage-derived supernatants. These results will provide insights into whether the intracellularly retained mutant TNFR1 may function to enhance local neutrophil accumulation in response to inflammatory stimuli in a cell autonomous manner.
Innate imprinting of resident alveolar macrophages by a preceding allergic bronchial inflammation increases the inflammatory reactivity through autocrine IFN-β. (141.18)
Authors: Naessens Thomas , Vander Beken Seppe , De Koker Stefaan , Lienenklaus Stefan , Vakaet Luc , Grooten Johan
Published: January 2023

Macrophages play an important and dynamic role in the initiation, propagation and resolution of inflammation. We investigated the dynamics of resident alveolar macrophages (rAM) and its pathophysiological consequences in experimental asthma. Using CD45 chimeric mice and intratracheal instilled fluorescent microparticles to distinguish resident from elicited AM, we observed a near complete disappearance of rAM during consecutive allergen challenges. As a consequence, after clearance of inflammation, inflammation-experienced rAM replaced the initial population of rAM. Strikingly, these secondary rAM showed a markedly altered responsiveness to LPS stimulation. Especially genes induced by IFN-β showed strongly increased expression in secondary rAM as opposed to their near absent induction in primary rAM. This switch from an IFN-β deficient to an IFN-β adequate phenotype increased the inflammatory sensitivity of secondary rAM as observed with rAM from IFN-β -/- mice. Also the basal differentiation status of secondary rAM was modified, dependent on the nature of the preceding allergic inflammation. M2-biased differentiation was seen following eosinophilic asthma while M1-biased differentiation was present following neutrophilic hypersensitivity pneumonitis. Taken together, these observations indicate innate imprinting of rAM by preceding allergic inflammation in the lungs.
Absence of the Type I IL-4 receptor increases the severity of airway inflammation in a murine model of asthma (141.6)
Authors: Dasgupta Preeta , Smith Elizabeth , Keegan Achsah
Published: January 2023

The TH2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation, eosinophilia, mucus hypersecretion and also drive alternative activation of macrophages (AAM). Although both cytokines share receptor subunits and signaling machinery, it is becoming apparent that IL-4 and IL-13 have differential roles in asthma pathogenesis. While clearly important in TH2 differentiation, the contribution of IL-4 signaling via the Type I receptor in allergic responses remains unclear. Here, we adoptively transferred wild-type ova primed CD4+ T cells into C57BL/6, Rag2KO, or gamma c (γc) KO mice. We found that γc KO mice developed increased inflammation around the airways and blood vessels in the lungs upon ova challenge when compared to WT and Rag2KO mice. Moreover, significantly higher numbers of eosinophils were present in the bronchoalveolar lavage (BAL) and lung tissue in γc KO mice. Immunohistochemical studies showed that all mouse strains expressed characteristic AAM proteins FIZZ1 and YM1 in lung epithelial cells, while macrophages expressed only YM1. Interestingly, γc KO mice had high levels of FIZZ1 protein in their BAL at baseline, which dropped sharply upon ova treatment. These results suggest that the Type I R acts to suppress allergic lung inflammation and, in the presence of TH2 effectors, the Type II R can mediate allergic responses in its absence. Further experiments are required to fully elucidate the role of the Type I IL-4R and γc in asthma.
Type I IFN facilitates the expansion of CD4+CD25+Foxp3+ regulatory T cells through upregulation of GITRL on dendritic cells (143.13)
Authors: Hong Jian
Published: January 2023

Interferon-beta (IFN-β) is a disease-modifying agent used in the treatment for multiple sclerosis (MS). Its mechanisms are complex and still not fully understood. It was observed clinically that the number of CD4+CD25+FOXP3+ regulatory T cells increase in patients treated with IFN-β for over 6 months. Here we demonstrate that IFN-β-induced up-regulation of glucocorticoid-induced TNF receptor ligand (GITRL) on dendritic cells (DC) promotes the proliferation of anergic Treg cells in mice. In vitro treatment of DC with IFN-β increases the expression of GITRL at both mRNA (by ~5 folds in PCR) and protein level (by ~25% in flow cytometry). Purified Treg cells, when stimulated with anti-CD3 and anti-CD28 Abs, remarkably proliferate in the presence of IFN-β pretreated DC. Blockade of GITRL with antibody diminishes the ability of DC to stimulate Treg cell proliferation. IFN-β shows no effect on the TGF-β induced conversion of CD4+CD25- T cells to Foxp3+ Treg cells in mice. DCs of IFNAR-/- mice are less potent in facilitating the proliferation of Treg cells. Treatment of EAE mice with mIFN-β for 2 months results in the upregulation of peripheral Foxp3+ Treg cells. However, in mice depleted with DC by liposomal clodronate treatment, IFN-β failed to induce the increase of Treg cells in vivo. Our findings are not only essential for the understanding of the effect of IFN-β in the treatment for MS but also provide insight into how Treg cells accordingly respond to inflammation.
Collaborative Effects of IL-17 and IL-23 in Deletion of Antigen Stimulated CD4+CD8+ Double Positive Thymocytes (143.17)
Authors: Li Hao , Wu Qi , Yang PingAr , Wang John , Kolls Jay , Hsu Hui-chen , Mountz John
Published: January 2023

Increased levels of IL-17 and IL-23 have been associated with autoimmunity,but their effects on thymocyte selection have not been studied.To test this,normal B6 mice were injected with adenovirus expressing either IL-17 (AdIL17) or IL-23 (AdIL-23).There was dramatic reduction of thymus cellularity and increased apoptosis of DP thymocytes in AdIL-23 injected mice.Coinjection of AdIL-17 potentiated the effects of AdIL-23.Coinjection of anti-IL-17 antibody with AdIL-23 partially corrected the effects of AdIL-23 to deplete DP thymocytes by blocking IL-17 induced recruitment of CD11b+ and CD11c+ cells in the junction between cortex and medulla where they exhibit the maximal interaction with DP thymocytes.AdIL-23 but not AdIL-17 or AdLacZ treatment largely increased the expression of IL-23 receptor in DP thymocytes and further lead to 100 fold increased expression of RORgt which is an important DP thymocyte apoptosis inducing molecule.In vitro coculture of thymocytes from 2D2 TCR transgenic mice with MOG35-55 peptide pulsed dendritic cells showed that there were enhanced apoptosis and a lower proliferative response of thymocytes in the presence of IL-23 but not IL-17.Taken together,our results suggest that IL-17 acts to recruit antigen-presenting cells to interact with DP thymocytes,IL-23 induces deletion of Ag encountering DP thymocytes.Thus,interaction of these two cytokines limits the generation of self-reactive thymocytes and does not lead to autoimmunity in normal mice.
Resistance of Cbl-b-/- Teff to Tregs is infectious (143.21)
Authors: O'Conor Catherine , Housley William , Clark Robert
Published: January 2023

The E3 ubiquitin ligase Cbl-b regulates T cell activation, and dysregulation of this protein in both mice and humans has been associated with autoimmunity. Our lab previously proposed resistance to CD4+CD25+ Tregs as one mechanism for the autoimmunity observed in Cbl-b-/- mice. Recently, we confirmed the in vivo resistance of Cbl-b-/- CD4+CD25- T effectors (Teff) to wild type (WT) Tregs using a model of GVHD. Examining the cells ex vivo, we observed a resistance of Cbl-b-/- Teff to suppression of cytokine production. To further characterize this resistance, WT Teff were examined for their ability to be suppressed by Tregs when mixed with Cbl-b-/- Teff in vitro. We found that WT Teff became resistant to Treg-mediated cytokine suppression when co-cultured with Cbl-b-/- Teff. This is the first evidence that resistance to Tregs is “infectious” and can be transmitted to WT Teff. We next determined that in vitro resistance of Cbl-b-/- Teff is only partially reversed by antibodies to IL-2 and IL-4, suggesting that other mechanisms, in addition to increased production of these cytokines, mediate this resistance. The ability of one subpopulation of Teff to induce Treg resistance in another population may have significant implications for mechanisms underlying autoimmune disease. Finally, using Cbl-b-/- Teff to identify mechanisms of infectious resistance may provide important clues in understanding Treg function and in developing novel strategies for treatment of autoimmune disease.
Knock down of kappa light chain RNA induces receptor editing (143.22)
Authors: Shen Shixue , Manser Tim
Published: January 2023

Light chain receptor editing functions as a critical mechanism in maintaining self tolerance among B lymphocytes. Current models for how this process is induced vary with regard to the type of BCR signaling involved. Here we show that in polyclonal and non-autoreactive B cell populations, genetic knockdown of kappa light chain RNA via a shRNA retrovirus efficiently induces BCR receptor editing to lambda light chain expression both in vitro and in vivo. This correlates with increased level of Rag gene expression and retarded development in the B cell compartment. These data demonstrate that BCR engagement of autoantigen is not required for receptor editing and add support to models suggesting a role for “tonic” BCR signaling in the regulation of this process.
Neuropilin-1 is important for proper suppression of inflammation and autoreactivity (143.23)
Authors: Bynoe Margaret , Solomon Benjamin , Chae Wookjin , Mueller Cynthia , Alabanza Leah
Published: January 2023

The surface molecule Neuropilin-1 (Nrp1), was originally identified for its role in neuronal development. Recently, Nrp1 has been implicated in several aspects of immune function, including maintenance of the immune synapse and development of regulatory T cells (Treg). We provide the first evidence for a role of Nrp1 in the pathogenesis of an immune mediated disease, experimental autoimmune encephalomyelitis (EAE), which serves as an animal model for the central nervous system inflammatory disorder multiple sclerosis (MS). Using a mouse cell type specific deletion system, we observed that the lack of Nrp1 on CD4+ T cells results in increased EAE disease severity. These Nrp1 conditional knockout mice exhibit preferential Th17 lineage commitment. Further, CD4+ T cells expressing Nrp1 can suppress Th17 cell expansion and cytokine production both in vitro and in vivo. These findings suggest that Nrp1 is essential for proper maintenance of peripheral tolerance and its absence can result in unchecked autoreactive responses.
A key role for Fas ligand on B cells in unleashing T cell diabetogenicity by suppression of IL-10-mediated islet tolerance (143.34)
Authors: Xiao Zuoxiang
Published: January 2023

A key role for the Fas pathway in promoting organ-specific-autoimmunity is indicated by resistance of mice bearing the lpr mutation of Fas or gld mutation of Fas ligand (FasL) to several organ-specific autoimmune diseases, including autoimmune diabetes and multiple sclerosis. However, the underlying mechanism remains elusive. Here we show that FasL expressed on B cells plays a key role in facilitating activation of diabetogenic T cells in pancreatic lymph nodes (PLNs) and pancreatic islet accumulation resulting in initiation of insulitis in the autoimmune diabetes-prone NOD mice. Expression of FasL on diabetogenic T cells themselves is not sufficient to mediate its diabetogenic role. By contrast, expression of FasL on B cells leads to deletion of localized IL-10-producing B cells, the presence of which in PLNs and pancreatic islets is associated with suppression of diabetogenic T cell priming and islet accumulation, respectively. Consequently, FasL inactivation by gld mutation or antibody blockade preserves the local IL-10-producing B cells and protects islets from diabetogenic T cells by a mechanism that is reversed by IL-10R blockade in vivo. The results provide novel insights into how FasL orchestrates the pathogenic relationship among B cell subsets in its implications for diabetogenic T cells.