bluuebunny/crossref_metadata_2025 huggingface dataset:
Maintenance and homeostatic proliferation of regulatory CD4 T cells occur independent of the thymus and of secondary lymphoid organs (104.7)
Authors: Camirand Geoffrey , Lakkis Fadi , Rothstein David
Published: January 2023

In vivo manipulations that influence regulatory Foxp3+CD4+ T cells (Treg) homeostasis represent a valuable therapeutic mean for balancing immunity and regulation. However, the signals and the anatomical sites of Treg homeostasis are not completely defined. Therefore, we investigated the role of the thymus, and of secondary lymphoid organs (SLO) on Treg homeostasis in steady state. The absolute numbers of conventional CD4 T cells (Tconv) and Treg in spleen, peripheral lymph nodes, bone marrow, blood, liver, and lungs were determined by flow cytometry in euthymic or thymectomized adult wt B6 mice and in mice lacking SLO (splenectomized Lymphotoxin receptor β knockout). Proliferation was measured by EdU incorporation over a period of 3 days prior to analysis. We found that, in wt B6 mice, Treg exhibited a much higher rate of homeostatic proliferation (HP) than Tconv in both lymphoid and non-lymphoid compartments. Thymectomy of adult B6 mice reduced Tconv numbers in all compartments by 30-60%, while Treg numbers remained primarily unaffected (d30 and d60). In absence of SLO or both thymus and SLO, Treg accumulated in non-lymphoid organs, in blood, and in bone marrow in higher numbers and displayed similar rates of HP compared to wt mice. Our data show that Treg homeostasis occurs independently of thymic production and of SLO, suggesting an intrinsic Treg ability to respond to antigens in non-lymphoid tissues.
Intrahepatic inflammatory profiles in HCV/HIV coinfection versus HCV infection (105.10)
Authors: Hu Sishun , Yu Andy , Amet Tohti , Byrd Daniel
Published: January 2023

We currently have a poor understanding of the immunopathogenesis of accelerated liver damage in HCV/HIV coinfection. Recent studies suggest that ongoing chronic liver inflammation is responsible for the faster liver damage. We therefore used a real-time PCR-based array to study intraheptic inflammation in the liver biopsy specimens from HCV-infected (n=20), HCV/HIV coinfected (n=8) and healthy (n=8) donors. A total of 84 inflammation-related genes encoding inflammatory cytokines, chemokines and their receptors were analyzed. We found that genes encoding CCL19 and CXCR3-associated chemokines (CXCL9, CXCL10, and CXCL11) were significantly up-regulated in both HCV infection and HCV/HIV coinfection when compared with that of liver biopsies from healthy donors. CXCR3-associated chemokine genes were further up-regulated in HCV/HIV coinfection. Meanwhile, 3 genes (CCL25, CRP, and CXCL2) were significantly down-regulated in HCV infection and HCV/HIV co-infection, and CCL25 was further down-regulated in HCV/HIV coinfection. These data were confirmed by in vitro studies using human hepatocyte cell line Huh7.5. Studies of the molecular mechanisms in regulation of these gene activation showed that LPS and viral proteins played significant roles in the changes of intraheptic inflammatory profiles. Our results suggest that the CXCR3-associated chemokines, particularly CXCL10, may play an important role in the immunopathogenesis of accelerated liver damage in HCV/HIV coinfection.
Exhausted CD8 T cells downregulate the IL-18 receptor and become unresponsive to inflammatory cytokines and bacterial coinfections (105.15)
Authors: Zajac Allan , Ingram Jennifer
Published: January 2023

During many chronic infections virus-specific CD8 T cells succumb to exhaustion as they lose their ability to respond to antigenic activation. Combinations of IL-12, IL-18, and IL-21 have been shown to induce the antigen-independent production of IFN-γ by effector and memory CD8 T cells. In this study we have investigated whether exhausted CD8 T cells are sensitive to activation by these cytokines. We show that effector and memory, but not exhausted, CD8 T cells produce IFN-γ and upregulate CD25 following exposure to certain combinations of IL-12, IL-18, and IL-21. This unresponsiveness is associated with downregulation of the IL-18R on exhausted T cells. To evaluate the consequences of IL-18R downregulation on CD8 T cell fate, we tracked IL-18R-/- cells in chronically infected mixed bone marrow chimeras and discovered that the IL-18R-/- cells were preferentially lost during the contraction phase. The antigen-independent responsiveness of exhausted CD8 T cells was also investigated following co-infection with Listeria monocytogenes, which induces the expression of IL-12 and IL-18. Although IL-18R-high memory cells upregulated CD25 and produced IFN-γ, the IL-18R-low exhausted cells failed to respond. Collectively, these findings indicate that as exhausted T cells adjust to the chronically infected environment, they lose their susceptibility to antigen-independent activation by cytokines, which compromises their ability to detect bacterial co-infections.
The role of long-lived plasma and memory B cells in response to challenge with homologous and heterologous West Nile viruses (105.16)
Authors: Purtha Whitney , Bhattacharya Deepta , Diamond Michael
Published: January 2023

Memory B cells (MBC) and long-lived plasma cells (LLPC) are generated after infection, but the significance of each population in conferring protection against secondary challenge remains unclear. Using a mouse model of West Nile virus (WNV) infection, we demonstrate that MBC possess a more cross-reactive repertoire to viral neutralization escape mutants than do LLPC. Through clonal analysis, we found that a high frequency of both MBC and LLPC generated by WNV infection recognize domain III (DIII) of the viral envelope, whereas a substantial fraction of MBC, but not LLPC, continue to recognize DIII with mutations at residues that comprise the neutralizing epitope (DIII-K307E, T330I). Accordingly, a variant virus carrying this mutation (WNV-K307E) was neutralized poorly relative to its wild type counterpart (8.3-fold reduction in EC50 value) by immune serum maintained by LLPC from WNV-immune mice. In contrast, adoptively transferred MBC readily generated antibodies upon WNV-K307E challenge that were not restricted to the wild type neutralizing epitope. These studies have implications on the analysis of protective immunity and evaluation of effective vaccines for viruses that vary at key neutralizing epitopes.
Identification of a conserved self-antigen bearing the 2F5 neutralizing epitope of the HIV-1 gp41 Membrane Proximal External Region (105.2)
Authors: Yang Guang , Holl T. , Liu Yang , Li Yi , Lu Xiaozhi , Nicely Nathan , Kepler Thomas , Alam S. , Liao Hua-xin , Spicer Leonard , Haynes Barton , Kelsoe Garnett
Published: January 2023

Accumulating evidence suggests that the efficient generation of neutralizing HIV-1 antibody may be proscribed by immunological tolerance. The majority of monoclonal human antibodies, including 2F5, that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian cells. Introduction and expression of the 2F5 VDJ rearrangement in knock-in mice results in a B-cell developmental blockade that is characteristic of immunological tolerance. If immunological tolerance does prevent regular expression of neutralizing 2F5-like antibody, then discrete autoantigens that structurally mimic the HIV-1 2F5 epitope are present in mammals. Here, we demonstrate that the 2F5 antibody precipitates a small number of proteins from mouse and human cells, including a major component with a molecular mass of about 50 kDa. We have identified this 50 kDa protein as kynureninase (KYNU), a conserved component of tryptophan metabolism. Remarkably, the conserved H3 domain of mammalian KYNU contains the core 2F5 epitope (ELDKWA), and mutation within this region greatly diminishes 2F5 binding. The 2F5 antibody and its inferred, unmutated precursor bind KYNU with high affinity and specificity. Indeed, recombinant human KYNU is an effective inhibitor of 2F5 binding to HIV-1 envelope. The nature of KYNU as autoantigen was previously unknown and our study suggests that mimicry of self-antigens may represent a general evolutionary strategy of viral pathogens.
Anti-Ebola GP monoclonal antibodies protect mice and guinea pigs against lethal ebola virus challenge (105.29)
Authors: Qiu Xiangguo , Lisa Fernando , Alimonti Judie , Feldmann Heinz , Kobinger Gary , Jones Steven
Published: January 2023

Introduction: Ebola virus (EBOV) causes acute hemorrhagic fever in humans and nonhuman primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (mAbs) against Ebola glycoprotein in a mouse and guinea pig model. Methods: Healthy mice or guinea pigs were given mAbs i.p. in various doses individually or as pools of 3-4 mAbs to test their protection against a lethal dose of mouse- or guinea pig-adapted EBOV. Results: Each of the 8 mAbs (100 microgram) protected mice from a lethal EBOV challenge when administered 1 day before or after challenge. Seven mAbs were effective 2 days post-challenge (dpc), with 1 mAb demonstrating partial protection 3 dpc. Furthermore, 4 mAbs achieved 70-100% protection with a single dose of 12.5 microgram at 1 dpc. In the guinea pig model each mAb showed partial protection 1 dpc, however the mean time to death was significantly prolonged compared to the control group. Moreover, treatment with pools of 3-4 mAbs completely protected the majority of animals, while administration 2-3 dpc achieved 50-100% protection. Conclusions: Our data suggests the mAbs generated are capable of protecting both animal models against a lethal challenge of Ebola virus. These results indicate that mAbs particularly when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of EBOV infection.
HIV Env-mediated, CCR5-dependent depletion of human Vgamma2Vdelta2 T cells (105.33)
Authors: Li Haishan , Mei Bing , Pauza C. David
Published: January 2023

Human Vgamma2Vdeta2 T cells are depleted during HIV infection. The majority of cells in this population are CD4-negative and do not support productive HIV infection; the mechanism for depleting them remains unknown. We noted recently that macaques immunized with an HIV R5-tropic gp120, also experienced severe Vgamma2Vdelta2 T cell depletion. A subsequent in vitro study showed that several different R5-tropic gp120, including those from Bal, ADA, YU2 and CN54 strains, induced human Vgamma2Vdelta2 T cell death that was inhibited by a CCR5 binding antagonist (Maraviroc) and a Caspase 8 inhibitor. The Vgamma2Vdelta2 may be especially vulnerable to cell death because they express high levels of CCR5 that could be modulated by Rapamycin treatment with a consequent reduction in Env-mediated cell killing. We have observed R5-tropic gp120 induced Vgamma2Vdelta2 T cell depletion both in vivo and in vitro, which was mediated by CCR5 receptor and Caspase 8 signaling pathway. We have identified a potential mechanism for Vgamma2Vdelta2 T cell depletion in HIV disease.
Immunogenicity of novel consensus-based DNA vaccines against Hepatitis B core Antigen. (106.1)
Authors: Obeng-Adjei Nyamekye , Yan Jian , Choo Daniel , Weiner David
Published: January 2023

Although effective vaccine against hepatitis B virus (HBV) has existed for over 3 decades, it still continues to be a major epidemic, especially among people of Asian and African descent. There are about 370 million people chronically infected and at risk of developing liver cirrhosis and liver cancer. The only therapies available for chronic infected individuals are interferon-α and nucleoside, which unfortunately do not control viral replication in most patients. This failure in protection with current HBV vaccines in 15% of individuals and the difference in response to treatment in the chronic infected are due, but not limited, to the genetic divergence among different HBV genotypes and to the immune escape mutations that often occur within the surface antigen (HBsAg). To circumvent these issues, we propose a novel consensus-based approach to vaccine design for HBV, using DNA vaccine strategy. A plasmid was designed based on HBV core (HBcAg)-specific consensus sequence from (which genotypes of HBV) with modifications such as codon and RNA optimization for maximum expression. We show that the construct was able to induce a strong antigen-specific T cell response systematically and in the liver of C57BL/6 mice. In addition, a high titer antibody response capable of recognizing a native HBcAg was observed in mice systematically immunized with this construct.
Adjuvanting influenza virus-like particles with glycophosphatidylinosital-anchored immunostimulatory molecules by protein transfer to enhance antiviral immune responses (106.14)
Authors: Patel Jaina , Song Jae Min , Shashidharamurthy Rangaiah , Compans Richard , Kang Sang-Moo , Selvaraj Periasamy
Published: January 2023

The effortless nature through which influenza, a bilayered lipid enveloped virus, can spread via an aerosol route and cause mortality is distressing. The use of virus-like particles (VLPs), that consist of a virus’ capsid proteins but not the viral genome required for replication, as vaccine candidates proves beneficial due to their safe nature and structural similarity with the live virus. In this study, we show that the incorporation of GPI-anchored immunostimulatory molecules onto H5 influenza VLPs can be accomplished through a quick, easy and stable protein transfer method. Affinity chromatography was used to purify the GPI-proteins (ICAM-1 and IL-12) from CHO-K1 cell lysates and incorporation onto the VLPs was determined by western blot analysis. To verify that incorporation occurred specifically via the GPI-anchor, PI-PLC treatment before and after incorporation and inhibition by fatty-acid binding proteins was carried out. Finally, electron microscopy was used to determine whether the structural integrity of the VLPs was altered following protein transfer. Our results indicate that incorporation of both GPI-proteins onto influenza H5 VLPs occurred following a 2 hour incubation at 37°C without disturbing the structural integrity of the VLPs. Therefore, this protein transfer method can be used to enhance the immunogenicity of VLPs easily and quickly to then be used as a potential influenza vaccine.
Therapeutic potential of zoledronic acid in enhancing immune function in aged mice (106.3)
Authors: Ventevogel Melissa , Riebe Kristina , Barefoot Brice , Ramsburg Elizabeth , Sempowski Gregory
Published: January 2023

Hematopoietic stem cells originate in the bone marrow and give rise to immune cells. Zoledronic acid (ZA) is a bisphosphonate shown to modulate skeletal and immune systems. Hip fracture patients treated with ZA had a significant increase in bone density and increased longevity over placebo. We hypothesize ZA treatment will promote bone density in aged mice which in turn will enhance immunity. Young/aged mice were treated with saline or ZA (IV 100ug/kg) and euthanized at 1, 3 and 6 months to determine the impact of ZA on immune homeostasis. Compared to control, ZA-treated young mice showed significant improvement in bone volume. ZA-treated aged mice showed a qualitative improvement in bone volume. Frequency of DN thymocytes and sjTREC were increased over control in ZA-treated aged mice, suggesting improved thymopoiesis. Potential of ZA for improving immunity was also explored. Mice were treated with ZA (3 mo), immunized with flu HA protein (IN, d0,7,14) and then challenged with PR8 influenza (d42). HA-specific Ab response and survival was assessed. No difference in response was observed with ZA treatment in young mice. ZA-treated aged mice however had a stronger HA-specific response over control, suggesting ZA enhanced host response to immunization. Survival of ZA-treated aged mice was also higher than control, suggesting a single infusion of ZA enhances immunity. Given these data, further investigation of ZA as a potential therapeutic for immune senescence is warranted.
Expression of ligands for the NKG2D activating receptor are linked to proliferative signals (108.2)
Authors: Jung Heiyoun , Raulet David
Published: January 2023

NKG2D is an activating receptor expressed on NK cells, CD8 T cells, and portions of NKT cells, gd T cells and CD4 T cells. The ligands for NKG2D receptor are surprisingly diverse, and emerging evidence suggests that different ligands are regulated by distinct cellular stress pathways, and in some cases a specific ligand is regulated by a several stress pathways acting in concert. In this study, we have identified a novel mechanism of regulation of a Rae1 family NKG2D ligand by proliferation stress. In proliferating B6 fibroblasts, Rae1e is induced at both the mRNA and protein levels, and in a manner that is not dependent on the DNA damage response or oxidative stress. Induction and maintenance of Rae1 expression is inhibited by growth factor starvation or any of numerous inhibitors of cell proliferation. Run-on transcription assays demonstrated that regulation is at the level of transcription. Ongoing studies suggest the role of specific cell cycle regulators as essential for Rae1 expression, and have probed their role in this process. This pathway implicates proliferation stress that occurs early in tumorigenesis and infections as a danger signal that induces the innate immune response.
A new subset of CD57+NKG2Chi NK cells is observed after HCMV infection infection (108.4)
Authors: Lopez-Verges Sandra , Milush Jeffrey , Schwartz Brian , Jarjoura Jessica , York Vanessa , Pandey Suchitra , Houchins J. , Miller Steve , Kang Sang-Mo , Norris Phillip , Nixon Douglas , Lanier Lewis
Published: January 2023

During human cytomegalovirus (HCMV) infection, there is a preferential expansion of Natural Killer (NK) cells expressing the activating CD94-NKG2C receptor complex, implicating this receptor in the recognition of HCMV-infected cells. We recently showed that CD57+ NK cells within the CD56dimCD16+ NK cell compartment are highly mature. We hypothesized that human NK cells previously expanded in response to viruses or other pathogens will be marked by expression of CD57. Indeed, we observed in HCMV+ donors that NK cells expressing the activating NKG2C receptor are preferentially CD57+. These CD57+NKG2Chi NK cells have down-regulated the inhibitory NKG2A receptor and some inhibitory KIRs. These CD57+NKG2Chi NK cells respond better to activation through the NKG2C receptor. Furthermore, CD57+NKG2Chi NK cells expressed high amounts of LIR-1, another receptor that recognizes HCMV. Finally, in solid organ transplant recipients with symptomatic HCMV infection the percentage of CD57+NKG2Chi NK cells among the total NK cell population increased as the HCMV plasma viral load decreased. In these patients the NKG2C+ NK cells expanded and proliferated before becoming NKG2Chi and acquiring CD57 expression. Together, these results suggest that during HCMV infection, NKG2C+ NK cells expand to fight the infection, and after several divisions acquire CD57. In humans CD57 might provide a marker of "memory" or "terminally differentiated" NK cells that have been expanded in response to infection.
Role of the receptor-like protein tyrosine phosphatase CD148 in the T cell-independent antibody response (109.12)
Authors: Skrzypczynska Katarzyna , Zhu Jing , Weiss Arthur
Published: January 2023

Efficient elimination of a pathogen is dependent on both rapid and sustained antibody production by B cells. In contrast with conventional (B2) B cells, which produce antigen-specific antibody later in infection, B1 B cells generate natural antibodies and contribute to the T-cell independent (TI) antibody response. B cell activation and subsequent antibody secretion involve tyrosine phosphorylation, which is tightly regulated by protein tyrosine kinases (PTKs) and receptor-like protein tyrosine phosphatases (RPTPs). Previous studies from our lab have demonstrated that the RPTPs CD148 and CD45 have redundant positive regulatory roles in the development and antigen receptor signaling of B2 B cells by acting on on Src PTKs. However, the role of RPTPs in signal transduction in B1 B cells is not well understood. We have found that mice with a targeted deletion of the transmembrane domain of CD148 have an impaired IgM response when challenged with a TI antigen. This suggests that CD148 has a unique role in the TI response that cannot be compensated by CD45. Here, we use the mb1-cre transgenic mouse model as well as adoptive transfer experiments and in vitro analysis of B cell activation to characterize the function and specificity of CD148 in the B1 B cell response to TI antigen. These studies describe a previously unappreciated cell-autonomous role for CD148 in modulating signaling pathways that are important for the generation of TI antibody responses by B1 B cells.
Ghrelin stimulates cell proliferation in T cells (109.22)
Authors: Lee Junho
Published: January 2023

Ghrelin is a 28 amino acid acylated peptide, which is produced and secreted by X/A-like enteroendocrine cells of the stomach. We have recently demonstrated that ghrelin is expressed by various immune cells including T- and B-lymphocytes, monocytes and neutrophils. These immune cells have also been shown to express the ghrelin receptor, namely the growth hormone secretagogue receptor (GHS-R). In the present study, we have investigated the effects of ghrelin on T cell proliferation and its signaling through its cell surface receptor. We have found that ghrelin induces a modest increase in the proliferation of murine CD4+ T cells and T-cell lines. Western blot experiments have demonstrated that GHS-R ligation activates the external receptor activated kinases (ERK1/2) and Akt signaling pathways in a dose-dependent fashion. Moreover, we have also revealed the involvement of the PKC pathway in the phosphorylation of ERK1/2. The pro-proliferative effects of ghrelin through ERK1/2 appears to be mediated through the regulation of the expression of cell cycle proteins, cyclin D1, CDK4, cyclin E and CDK2 as well as Rb. Together, these data suggest that ghrelin promotes the cell proliferation of T cell via the activation of the PKC, PI-3-K, Akt and Erk1/2 signaling pathways and that this GHS-R signal appears to be essential but not sufficient for cell cycle progression.
SLP-2 deficiency in T cells is associated with decreased respiratory complex I levels and impaired TCR-dependent responses (109.27)
Authors: Christie Darah , Lian Dameng , Wang Hao , Hatch Grant , Madrenas Joaquín
Published: January 2023

We have previously reported that stomatin-like protein 2 (SLP-2) regulates human T cell activation through the antigen receptor. In human lymphoid tissues, SLP-2 is widely expressed in the thymus, lymph nodes and tonsils, and its expression is further up-regulated in response to lymphocyte activation. In T cells, SLP-2 is found in two pools: the most abundant is associated with mitochondria while a smaller pool is associated with the plasma membrane. To further investigate the role of SLP-2, we generated T cell-specific SLP-2 deficient mice. These mice had normal numbers of thymocytes and mature T cells. However, SLP-2 deficient T cells showed significantly less T cell activation in response to T cell stimulation in vivo and in vitro. Although SLP-2 deficient T cells did not show significant alterations in mitochondrial biogenesis, they had decreased levels of the NDUSF3, NDUFB8 and NDUFA9 subunits of complex I of the respiratory chain, suggesting a possible role for SLP-2 in regulating the stability of proteins involved in mitochondrial electron transport. SLP-2 deficient T cells also had less cardiolipin within detergent-insoluble microdomains, which may be required for proper respiratory chain assembly and function. Based on these results, we conclude that SLP-2 contributes to T cell activation, in part by regulating stability of complex I of the respiratory chain and sustaining the metabolic requirements for T cell activation.
Role of TRAF3 in TCR signaling (109.30)
Authors: Yi Zuoan , Stunz Laura , Bishop Gail
Published: January 2023

Adaptor molecules play critical roles during signal transduction. TNF receptor associated factor 3 (TRAF3) is involved in a variety of signaling pathways in both innate and adaptive immunity. Recently, we demonstrated that TRAF3-deficient T cells exhibit severely impaired function, that TRAF3 associates with the TCR complex, and TRAF3 plays essential roles in TCR signaling pathways. Here, we find that TRAF3 affects diverse signaling events downstream of the TCR. Upregulation of activation markers in TRAF3-deficient T cells is slower than in Wt T cells, but TCR internalization is not affected. Furthermore, the deficiency of TRAF3 does not affect the recruitment of ZAP70 and Lck after TCR stimulation, but phosphorylation of ZAP70 is decreased. This result suggests that TRAF3 facilitates the interaction between Lck and ZAP70, and without TRAF3, Lck does not phosphorylate ZAP70 efficiently. Additional studies on how TRAF3 interacts with the TCR complex are ongoing.
The ERM family protein Ezrin associates with the JNK signaling complex to regulate its activation and antigen-dependent B cell survival. (109.7)
Authors: Parameswaran Neetha , Liggett Luther , Bagheri Nayer , Gupta Neetu
Published: January 2023

Antigen binding to the B cell antigen receptor (BCR) results in initiation of a highly orchestrated series of enzymatic reactions and protein-protein interactions that transmit the signals for B cell survival and proliferation. The highly conserved Ezrin-Radixin-Moesin (ERM) family members are thought to regulate lymphocyte activation via their membrane-actin crosslinking property, which is in turn regulated by phosphorylation of a conserved threonine residue at their C-terminus. Here, we report a novel signal transduction axis that links antigen-dependent tyrosine phosphorylation of ezrin with activation of the c-Jun N-terminal kinase (JNK). BCR ligation induces phosphorylation of Y353 in ezrin, a residue that is not conserved in other ERM proteins. Phosphorylation of Y353 is dependent on Src and Syk family kinase activity, occurs preferentially on the open conformer of ezrin, and correlates with the strength of BCR signaling in mature B cell subsets. Tyrosine phosphorylated ezrin associates with components of the JNK signaling complex in response to BCR stimulation. A non-phosphorylatable (Y353F) mutant of ezrin acts in a dominant negative manner to specifically impair antigen-induced JNK activation, and reduces B cell survival. Our results demonstrate a unique and novel signaling role for ezrin in regulating antigen-dependent JNK activation and B cell survival.
Discovery and characterization of novel distal regulatory elements involved in the regulation of CIITA promoter three. (109.9)
Authors: Lohsen Sarah , Boss Jeremy
Published: January 2023

Major histocompatibility complex class II (MHCII) molecules on B cells display antigenic peptides that trigger CD4 T cells specific for the same antigen to activate naïve B cells to differentiate into plasma cells. The MHCII transactivator, CIITA, is highly expressed in B cells (corresponding to MHCII expression), but silenced in transition to mature plasma cell. Understanding CIITA regulation can elucidate details of the generation of humoral immunity. Work has been done describing the role of proximal elements in the CIITA locus and their contribution to the regulation of CIITA. Recent experiments have shown that there are elements involved in CIITA regulation in non-immune cells that are much more distal to the promoter than previously thought, up to 50kb from the active promoter. However, no data on these or other elements in B cells and plasma cells were described. Using a medium throughput PCR-based DNaseI hypersensitivity assay to screen across the CIITA locus, regions of accessible chromatin were characterized that distinguish between the active and silenced state of CIITA expression. Computational methods reveal key regions of cross-species homology and transcription factor binding. These methods together identify novel distal regulatory elements that will be confirmed using a Bacterial Artificial Chromosome (BAC) system.
Macrophages differentiation confers resistance to HIV-Vpr induced apoptosis (110.10)
Authors: Busca Aurelia , Kumar Ashok
Published: January 2023

Macrophages represent major viral reservoirs during HIV infection, because they are long lived and resistant to HIV cytopathic effects. We have evaluated the role of Bcl2 and IAPs (inhibitors of apoptosis), the main families of antiapoptotic proteins, in macrophage resistance during HIV infection by using the HIV accessory protein Vpr as a study model for apoptosis. Our results show that human monocytes are sensitive to Vpr but develop resistance following differentiation into macrophages. Vpr efficiently down-regulated cIAP1 and Bcl2 in sensitive monocytes but not in macrophages, suggesting that these molecules could be implicated in the enhanced resistance of macrophages to HIV-Vpr apoptosis. However, Bcl2 inactivation with the HA14-1 inhibitor was able to induce cell death on its own, with no impact on susceptibility to Vpr apoptosis. By using siRNA to knock down the expression of IAP1 and IAP2 proteins and SMAC mimetic to induce their degradation, macrophages could be sensitized to Vpr-induced cell death. Our results suggest that IAPs and not Bcl2 family confer a resistant phenotype that is relevant for macrophage survival in the context of HIV infection. Modulation of the apoptotic pathway during monocyte differentiation provides insight into the possible mechanisms for viral reservoirs persistence in macrophages and into potential therapeutic approaches to target antiapoptotic genes.
A possible mechanism for maintenance of the deleterious allele of human CASAPSE-12 (110.17)
Authors: Hermel Evan , Klapstein Kevin
Published: January 2023

In humans, a functional CASPASE-12 (CASP12) gene has been identified only in persons of African and Southern Indian heritage. CASP12 has been suggested to play a regulatory role in response to bacterial pathogens and in promoting and increased susceptibility to sepsis by down-regulating the production of inflammatory cytokines such as interleukin-1 (IL1). The existence of a gene whose effect is deleterious, and which has been the subject of extensive negative selection in the rest of the human population, implies the simultaneous presence of some selective benefit for persons having CASP12. Given the importance of inflammatory immune responses in controlling the initial stages of infection, and the role that CASP12 plays in down-regulating inflammation, we hypothesize that pathogens which exploit the inflammatory response are restrained by an active CASP12 gene product. A number of candidate pathogens can be ruled out on the basis that IL1 is required for their clearance, while others may select for CASP12 by how they utilize the IL1 response as a part of their pathogenesis.
TGF-RbII signifies the molecular pathway for CD11c+DC-mediated osteoclastogenesis & bone loss in vivo (110.23)
Authors: Liu Yen-Chun Grace , Wang Yan-Shong , Teng Andy Yen-Tung
Published: January 2023

Bone loss is associated with osteoclasts (OC). We reported the development of OC from CD11c+dendritc cells (DDOC) via RANKL & Aggregatibacter actinomycetemcomitans (Aa) stimuli [J.Immunol/JBMR]. We aimed to study: i) proliferation, frequency & osteoclastogenic potential of DDOC in-vivo; ii) identify regulators of DDOC-development. CFU & limiting-dilution-analyses showed that CD11c+DDOC did not manifest detectable proliferation with an estimated OCp frequency 3+0.06% based on TRAP+multinucleated-OC. To assess their physiological relevance, CFSE-CD11c+DC were transferred onto NOD/SCID calvarias with Aa+IFA. By q-IHC, higher numbers of CFSE+CD11c+TRAP+OC were detected, in association with more bone-erosion in mice receiving DC, suggesting that CD11c+DC become TRAP+functional-OC under inflammation in-vivo. Moreover, the number of CD11c+TRAP+ multinucleated-OC was highly correlated with bone-loss on sub-chondral bone of collagen-induced arthritic joints in DBA-mice & Aa-induced periodontitis. Micro-arrays, RT-PCR & flow-cytometry were employed to identify regulator(s) in DDOC-development, demonstrating a (+)-correlation with TGF-RbII expression. Neutralization of TGF-b abolished DDOC development, based on TRAP & resorptive-pit assays in-vitro/in-vivo. These data support that: i) CD11c+DC subset(s) can act as OCp during inflammation; ii) TGF-RbII is essential for DDOC-development, an integral mechanism underlying inflammation-induced osteoclastogenesis at osteo-immune interface.
Cholinergic regulation of atrial natriuretic peptide in spleen during sepsis (110.4)
Authors: Huston Jared , Fritz Jason , Gersch Robert
Published: January 2023

Introduction: Cardiac atrial natriuretic peptide (ANP) regulates blood volume and pressure. ANP is also produced in spleen and reduces pro-inflammatory cytokine production in macrophages. The cholinergic anti-inflammatory pathway targets spleen to inhibit pro-inflammatory cytokines and organ damage in sepsis. We hypothesized that cholinergic regulation of systemic inflammation requires modulation of splenic ANP. Methods: Male 8-12 wk old BALB/c mice received LPS (15 mg/kg, i.p.), and were euthanized after 60 m. Nicotine (2 mg/kg, i.p.) or vehicle was given 1-2 h before LPS. TNF and ANP were measured using ELISA. Other mice received cecal ligation and puncture +/-splenectomy, and were euthanized at 24 h. One-way ANOVA or the Student’s t test was used to compare experimental groups. P-values < 0.05 are significant. Results: Nicotine significantly reduces systemic and splenic TNF. Nicotine significantly reduces systemic ANP and significantly increases splenic ANP. Nicotine fails to regulate TNF or ANP after splenectomy. Splenectomy significantly reduces systemic TNF and ANP (p < 0.07). Splenectomy significantly improves survival and decreases ANP after CLP. Conclusions: Cholinergic stimulation requires spleen to modulate systemic pro-inflammatory cytokines and ANP. Cholinergic stimulation increases splenic ANP and decreases systemic ANP. The cholinergic anti-inflammatory pathway may regulate systemic inflammation by limiting ANP release and enhancing splenic ANP signaling.
Scytonemin inhibits lipopolysaccharide-induced production of nitric oxide, tumor necrosis factor-α and interleukin-1β by blocking NF-κB/Rel signaling in macrophages (112.13)
Authors: Kang Jong Soon , Cho Sun Ah , Han Sang-Bae , Lee Kiho , Kim Hwan Mook
Published: January 2023

Scytonemin, an ultraviolet sunscreen pigment isolated from the extracellular sheath of cyanobacteria, has been reported to exert anti-inflammatory effect. In this study, we examined the effect of scytonemin on lipopolysaccharide (LPS)-induced production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in macrophages and investigated the molecular mechanisms responsible for its effects. Scytonemin significantly inhibited NO production and inducible nitric oxide (iNOS) mRNA expression in LPS-stimulated macrophages. The secretion and mRNA expression of TNF-α and IL-1β were also suppressed by scytonemin treatment in RAW 264.7 cells. Further study demonstrated that scytonemin attenuated LPS-induced NF-κB/Rel activation in RAW 264.7 cells. In addition, scytonemin suppressed phorbol ester-induced skin inflammation in BALB/c mice. These results suggest that scytonemin inhibits the expression of inflammatory mediators, at least in part, by blocking NF-κB/Rel activity and might be a potential therapeutic agent for the treatment of inflammatory diseases.
The role of PKCθ in T cell motility (112.22)
Authors: Cannon Judy , Brown Ivy , Sperling Anne
Published: January 2023

T cell trafficking is critical to immune responses as naïve T cells constantly recirculate into and out of lymph nodes to survey for cognate antigen presented on dendritic cells. While many cell surface receptors including selectins, chemokine receptors, and integrins have been demonstrated to be crucial for T cell motility, less is known about the intracellular mediators of T cell motility. We recently found that PKCθ localizes to the T cell uropod, suggesting a previously unknown role for PKCθ in regulating T cell migration. Consistent with this hypothesis, we now show PKCθ-deficient T cells do not migrate as well as wild type T cells in response to the chemokine CCL21, showing that PKCθ plays a role downstream of the chemokine CCR7. We further show that PKCθ affects the localization of proteins to the uropod, suggesting that one mechanism by which PKCθ affects T cell migration is by regulating the T cell uropod. These data reveal a novel role for PKCθ in regulating T cell migration and suggests that PKCθ signals downstream of CCR7 to regulate T cell motility.
Podoplanin is involved in Th17 cell polarization (113.17)
Authors: Zhang Hong
Published: January 2023

Podoplanin is a mucin-type transmembrane glycoprotein containing extensive O-glycosylation in extracellular domain and two phosphorylation sites in a short cytoplasmic tail. It is expressed in kidney glomerular epithelial cells, lymphatic endothelium and subcapsular epithelial cells in murine thymus. Recently, podoplanin was found on F4/80 positive peritoneal macrophages, indicating that podoplanin is involved in immune response. However, the physiologic function of podoplanin is still unknown. To study whether podoplanin is involved in adaptive immunity, we measured its expression on lymphocyts. The data showed that podoplanin was not detectable on freshly isolated T and B lymphocytes from peripheral blood, spleen and thymus, its expression is also non-inducible after stimulation with PMA, Concanavalin A or LPS. Surprisingly, we found that the podoplanin expression on CD4+ cells was dramatically increased after 3 days culture with Th17 polarization medium compose of immobilized CD3/CD28, IL-6, IL-4, TGFb, IFN-r and IL-23, which showed more than 60% cells were podoplanin positive. However, its expression is not able to be induced on Th1 and Th2 polarized cells. By detecting IL-17 production, we interestingly found that the IL-17+ cells can be divided into podplaninhigh and podoplaninlow populations. The significance of the differential expression on Th17 cells needs to be further investigated.
Lung and blood Th1 and Th17 responses against mycobacterial antigens in patients with pulmonary sarcoidosis. (114.9)
Authors: Ostadkarampour Mahyar , Eklund Anders , Moller David , Grunewald Johan , Wahlström Jan
Published: January 2023

Sarcoidosis is an inflammatory disorder characterized by granulomas most commonly affecting the lungs. The presence of mycobacterial antigens, and recently a specific protein, M. tuberculosis catalase-peroxidase (mKatG), in sarcoidosis tissue has been reported. Th1 cell responses against mKatG have been observed in sarcoidosis lung and peripheral blood cells. IL-17 and IFNγ were evaluated by ELISPOT after stimulation of bronchoalveolar lavage (BAL) fluid and peripheral blood cells with mKatG and M. tuberculosis PPD proteins. Two groups of sarcoidosis patients were compared: HLA-DR3 positive (good prognosis) versus HLA-DR3 negative (bad prognosis). We also did dual-color Fluorospot assay and immunohistochemistry for further characterization. Both mKatG and PPD stimulation of BAL cells resulted in higher frequencies of cells producing IFNγ compared to IL-17. We did not detect any significant difference between BAL and blood regarding IL-17 secretion after stimulation with mKatG, while significantly more BAL cells produced IFNγ in comparison to blood after stimulation with mKatG (p<0.05). Preliminary data do not indicate any difference between patient subgroups regarding IL-17 production. The existence of IL-17+ cells in the granulomas also supports the role of IL-17 in sarcoidosis. The observed Th17 responses against mycobacterial antigens could contribute to the inflammation in sarcoidosis, although they generally occur at lower frequencies than corresponding Th1 responses.
IL17A switches from a pathogenic to a protective function in experimental autoimmune myocarditis in the absence of IFNγ. (115.14)
Authors: Barin Jobert , Baldeviano G. , Talor Monica , Zheng Dongfeng , Cihakova Daniela , Rose Noel
Published: January 2023

Deviation to autoaggressive Th17 responses has been reported to account for the protective effect of IFNγ in certain animal models of autoimmune disease. Building on our previous findings that IL17 signaling was critical for the progression of experimental autoimmune myocarditis (EAM) to dilative heart failure, we sought to address the question of whether IL17 mediated the severe form of EAM observed in IFNγ-deficient mice. To our surprise, we found that IFNγ-/-IL17A-/- double-knockout (DKO) mice had greater mortality compared to IFNγ-/- mice, starting at day 14 and reaching 50% by day 21 of EAM. DKO hearts were severely infiltrated, indicating acute inflammatory heart failure as the mode of death in these mice. We have found evidence of aberrant eosinophilic Th2-deviated infiltration in DKO mice, implicating eosinophils in mediating the enhanced morbidity. Production of IL5 by CD4+ cells was increased in DKO hearts, as well as cardiac production of CCL11/eotaxin. Heart-infiltrating DKO CD4+ cells showed evidence of Th2 deviation. These data point to a novel collaboration between IFNγ and IL17A in controlling eosinophil recruitment by autoaggressive Th2 cells. Moreover, they indicate that myocarditis is not restricted to a single CD4+ subset - but instead reflects redundancy among Th1, Th2, and Th17 populations to elicit qualititative and quantitative variations in disease phenotype and progression.
Novel roles of gp96 and CNPY3 complexes in the biogenesis of Toll-like receptor 9 (116.10)
Authors: Liu Bei , Hong Feng , Wu Shuang , Li Zihai
Published: January 2023

TLR9 is an endolysosomal sensor for DNA, which plays important roles in sensing both self DNA and microbial DNA. Tight regulation of TLR9 biology is important to ensure proper anti-microbe response and yet avoid self-destructive response against self DNA. We have observed that TLR9 is crucially dependent on gp96 (also known as grp94 and HSP90b1) and CNPY3 (also known as PRAT4A), two ubiquitous endoplasmic reticulum chaperones for folding and transport to the endolysosomes. However, the detailed roles of gp96 and CNPY3 in TLR9 biogenesis remain poorly understood. In this study, we found that TLR9 forms a multimolecular complex with gp96 and CNPY3, and the binding of TLR9 to either molecule requires the presence of the other, suggesting that CNPY3 is a co-chaperone for gp96 in folding TLR9. We also examined the roles of other canopy family members in TLR9 folding. CNPY4 (also called PRAT4B) shares 38% amino acid sequence identity to CNPY3 and it has 6 conserved cysteine residues in the mature protein. However, genetic knockdown of CNPY4 only decreased TLR4 signaling, but did not affect TLR9 signaling. Finally, since gp96 also participates in general protein homeostasis and in the ER unfolded protein response (UPR), we hypothesize that gp96 and CNPY3 are the critical link between UPR and TLR responsiveness to protein-toxic stress. Ongoing experiments are carried out to determine if protein-toxic stress and inflammation converge on gp96 and CNPY.
CREB is an essential transcription factor for prostaglandin E2 induced IL-23p19 (116.18)
Authors: Kocieda Virginia , Adhikary Sabina , Emig Fran , Ganea Doina
Published: January 2023

We reported previously that PGE2 promotes IL-23p19 expression in dendritic cells (DC) treated with LPS. The effect is mediated through EP4 through the induction of cAMP. This study is focused on the signaling pathways involved in PGE1OH (an EP4 agonist)-induced p19 expression. EP4 activates adenylate cyclase and increases intracellular cAMP which leads to activation of PKA and EPAC. We found that adenylate cyclase, cAMP, and EPAC play a role in IL-23p19 induction. Using chemical inhibitors of PKA, PI3K, MAPK, and GSK3, we did not see a decrease in IL-23p19 expression. Previous studies reported the involvement of NFkB/c-Rel, ATF-2, SMAD-3 and AP-1 in p19 expression. Since two CRE-binding sites have been identified in the p19 promoter, we focused on CREB as a potential transcription factor activated by PGE1OH. Luciferase experiments including vectors containing mutated CRE sites and expressing dominant negative versions of CREB demonstrated the essential role CREB plays in IL-23p19 expression. In a transcription factor assay we found increased CREB binding activity in nuclear extracts from cells treated with PGE1OH. In addition, a ChIP assay confirmed CREB binding to the p19-promoter in the presence of PGE1OH. Overall, we identified CREB as a new transcription factor required for PGE1OH induction of IL-23p19 expression.
Complement Factor MASP-2 is activated after acute myocardial ischemia in human (116.28)
Authors: Zhang Ming , Hou Yunfang , Cavusoglu Erdal , Lee Daniel , Steffensen Rudi , Yang Liming , Jensenius Jens , Marmur Jonathan , Schwaeble Wilhelm , Ko Wilson , Shevde Ketan
Published: January 2023

Acute blockage of coronary artery results in myocardial injury manifested in clinical situations such as myocardial infarction (MI). Following ischemia, inflammation is provoked and may be related to the clinical outcomes. We studied the acute inflammatory response immediately following myocardial ischemia in human. Our results showed that in acute MI patients circulation levels of complement factor MASP-2 were significantly reduced comparing with those of healthy individuals or of coronary artery disease (CAD) patients without acute MI. This reduction was not due to genetic polymorphism of MASP-2 in acute MI patients. We hypothesized that MASP-2 was activated after acute myocardial ischemia, and tested this in cardiac patients undergoing surgically induced transient global heart ischemia. Our results showed that MASP-2 was significantly reduced in coronary circulation after global heart ischemia and was independently correlated with the post-operative increase of the myocardial injury marker, cardiac Troponin I. These data suggested that MASP-2 is activated in human acute heart ischemia and associated with myocardial injury.
alpha7 nicotinic acetylcholine receptor and cAMP response element binding protein are essential for prolonged monocyte deactivation by vagus nerve signaling (116.37)
Authors: Olofsson Lars Peder , Tusche Mike , Reardon Colin , Rosas-Ballina Mauricio , Parrish William , Levine Yaakov , Gregersen Peter , Diamond Betty , Chavan Sangeeta , Mak Tak , Tracey Kevin
Published: January 2023

The inflammatory reflex is a neural circuit that regulates innate immune responses to endotoxin and other stimulating agents. It confers protection against damaging inflammation by inhibiting synthesis and release of TNF and other cytokines. Action potentials transmitted in the vagus nerve stimulate acetylcholine release, a neurotransmitter which inhibits cytokine production by interacting with a7nAChR expressed on macrophages and other cytokine-producing cells. We studied the effects of electrical stimulation of the vagus nerve, and observed that a brief period of neural stimulation resulted in suppression of TNF release that persisted for several days. To deconvolute this effect, we implanted vagus nerve stimulators in ambulatory canines, and found that brief electrical pulses significantly inhibited endotoxin-induced TNF release for up to 9 days. Exposure of isolated monocytes and macrophages to acetylcholine reduced TNF production for 24 h via signal transduction through a7nAChR. a7nAChR interaction with type 6 adenylate cylcase upregulated cAMP levels, and enhanced phosphorylation of CREB. Transfection of monocytes with a dominant negative CREB blocked the prolonged deactivation of monocytes exposed transiently to acetylcholine or a7nAChR agonists. Thus, signals originating as action potentials in the vagus nerve produce an a7nAChR - CREB dependent deactivated phenotype in monocytes, which has significant implications for understanding the regulation of innate immunity.