bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying molecules that “fine-tune” T cell antigen receptor signaling during thymocyte selection. (36.40)
Authors: Pinkhasov Julia , Lee Jan , Li LiQi , El-khoury Dalal , Lesourne Renaud , Hwang SuJin , Zhang Yongqing , Weng Nan Ping , Love Paul
Published: January 2023

The prevailing view of thymocyte selection is that the fate of developing cells is dictated exclusively by the affinity/avidity of the expressed TCR for self-MHC/self-ligand. We hypothesize that there is significant plasticity in the signaling processes that control T cell development and that the ability to ‘fine-tune’ the TCR signaling response, through the regulated expression of molecules that positively or negatively impact the TCR signaling response operates to maximize the TCR repertoire. One example of a ‘fine-tuning’ molecule that was previously characterized is the cell surface protein CD5. We found that CD5 is an inhibitory tuning receptor, and its surface expression is directly and proportionately regulated by the affinity of the TCR for selecting ligand. In order to identify additional candidates for ‘TCR-tuning’, we employed a TCR transgenic experimental system where TCR expression was fixed on all developing thymocytes and where the affinity of the positively selecting TCR/self-MHC/self-ligand interaction could be altered by changing the MHC haplotype. Thymocytes that successfully underwent selection under conditions of relatively low or relatively high avidity interactions were analyzed by microarray. These experiments identified CD5 as well as several additional potential tuning molecules not previously thought to play a role in ‘TCR-tuning’. Our results provide important new insights into the genetic and molecular processes that control thymocyte selection.
Alterations in Bone Marrow Stroma with Aging (36.5)
Authors: Bilwani Fareena , Knight Katherine
Published: January 2023

B lymphopoiesis arrests in rabbits by 16 weeks of age, and data suggest that the bone marrow (BM) environment is responsible for this. We hypothesize that this arrest is due to differences in the BM stroma of young and adult rabbits. Using limiting dilution analysis to quantify the number of CFU in BM of 2 to 6 wk-old rabbits, we found an 8-10 fold decrease in the number of stromal progenitors by 6 wk-of-age. This decrease in stromal progenitors is reflective of the age-related decline in progenitor B cells in BM. Histology of BM sections reveals trabecular bone formation in young rabbits but not in adults; in contrast, BM from adults shows large amounts of adipose tissue. We tested the capability of stromal progenitors to differentiate into adipocytes or osteoblasts in vitro , and found that stromal progenitors from BM of young rabbits displayed a higher propensity to form oseoblastic colonies than do stromal progenitors from BM of adults, which more readily formed adipocytic colonies. We conclude that with aging, the number of BM stromal progenitors decreases dramatically, and further, that these progenitors from adults more readily differentiate into adipocytes than into osteoblasts. We suggest that these changes in BM are responsible for the arrest of B lymphopoiesis.
Analysis of signals that regulate the fate of early T cell precursors (36.53)
Authors: Al-Kuhlani Mufadhal , Ciriza Jesús , Peiris Tanuja , Ramos Joseph , Bañuelos Daniel , García-Ojeda Marcos
Published: January 2023

The differentiation process of Hematopoietic Stem Cells (HSC) to Common Lymphoid Progenitors (CLP), that give raise to dendritic, NK, B and T cells, depends on various cues received from their microenvironment. Notch-1 is a transmembrane receptor that plays a crucial role on CLP commitment towards T cell lineage at the expense of B cell fate. The role of the transcription factor GATA-3, as well as its regulatory interaction with Notch-1 signals during early T cell development, is poorly characterized. In vitro studies show that GATA-3-deficient (GATA-3-/-) T cell progenitors do not develop beyond the CD44+CD25+ double negative 2 (DN2) stage. Remarkably, GATA-3-/- DN2 cells generate B cells in the presence of constant Notch-1 signals, which normally block B cell development. These GATA-3-/- DN2 cell express elevated levels of Deltex-1, a Notch-1 regulator. Therefore, we hypothesize that GATA-3 transcriptional activity controls the expression of Deltex-1 and possibly other Notch-1 regulators. The expression levels of various regulators of Notch-1 in wild type and GATA-3-/- T cell progenitors will be evaluated using quantitative PCR. The effect of GATA-3 deficiency on the expression level of various genes involved in Notch signaling will be evaluated by qPCR array. Revealing the interaction mechanism of GATA-3 with Notch-1 will contribute to our understanding of how early lymphocyte progenitors become committed toward the T cell lineage.
Propagation of common lymphoid progenitors in OP9-DL1 co-culture results in generation of cells incapable of β-selection (36.54)
Authors: Van De Wiele C. Justin , Marino Julie , Davis Brenda , Tan Chibing , Taylor Ashlee , Teague T. Kent
Published: January 2023

Common lymphoid progenitors derived from murine bone marrow have previously been demonstrated to be capable of long-term propagation in OP9-DL1 co-cultures. Cultures of this type initially display a wave of differentiation to the double positive stage of T cell development that is observable at three to four weeks post-culture initiation. Continued propagation eventually produces a heterogeneous population of cells that collectively resemble thymocytes derived from animals incapable of β-selection. As TCRβ gene rearrangement occurs at this stage in normal thymocytes, we evaluated the rearrangement status of the co-culture derived cells. Sequential, weekly analysis revealed that TCRβ rearrangements occurred as expected in cultures that differentiated to the DP stage. After the initial wave of DP production however, TCRβ rearrangements were no longer detectable. Further analysis revealed that portions of the CD3 complex were expressed on the derived cells at levels equivalent to or higher than wild-type thymocytes at a similar maturational stage while pre-Tα levels were found to be expressed at a lower level. Finally, by using a novel in vitro β-selection assay, we demonstrate that the cultured cells still do not initiate differentiation past the DN3a stage. These data indicate that while ex vivo derivation and maintenance of T cell precursors is possible, standard OP9-DL1 culture conditions lead to development of cells resistant to induction of the β-selection program.
Thymocyte autoreactivity and altered thymic epithelial development in the absence of CD28-CD80/86 and CD40-CD40L interactions (36.69)
Authors: Williams Joy , Zhang Jingjing , Klug David , Nitta Takeshi , Kruhlak Michael , Sharrow Susan , Granger Larry , Adams Anthony , Gress Ronald , Takahama Yousuke , Hodes Richard
Published: January 2023

CD28-CD80/86 and CD40-CD40L interactions are critical to the activation of mature T cells and also have significant effects on T cell development and repertoire selection. Notably, the effects of these costimulatory interactions can be bi-directional, affecting both CD28- and CD40L-expressing T cells and the B7- and CD40-expressing cells with which these T cells interact. We have therefore analyzed development of a self-tolerant T cell repertoire as well as the development of thymic cortico-medullary structure in mice deficient for both the CD28-CD80/86 and CD40-CD40L costimulatory pathways. We find that CD4 thymocytes from CD40/CD80/CD86 KO mice respond vigorously to syngeneic antigen presenting cells, in contrast to the weak responses of thymocytes from either CD40 KO or CD80/CD86 KOs. Interestingly, we also find that the thymic medullary epithelial compartment (mTEC) is uniquely disrupted in CD40/CD80/CD86 deficient mice. The profound reduction in thymic medullary epithelial cells in CD40/CD80/CD86 deficient mice is accompanied by a significant decrease in CD4 SP thymocyte expression of several TNF family members including LTa, LTb, and CD30L. Expression of RANK-L, previously shown to be critical for development of mTEC in the embryonic and adult thymus, is unaffected in CD40/CD80/CD86 deficient thymocytes. These results indicate that thymocyte-mTEC crosstalk mediated either directly or indirectly through CD40 and B7 pathways is critical to thymic stromal development.
Asp299Gly TLR4 polymorphism impairs sensing of Escherichia coli, Rickettsia akari and LPS-mediated activation of MyD88- and TRIF-dependent signaling pathways (37.17)
Authors: Medvedev Andrei , Song Chang , Quevedo-Diaz Marco , Qiu Fu , Figueroa Leandra
Published: January 2023

TLR4 is a key sensor of Gram negative bacteria and LPS. The Asp299Gly (D299G) and Thr399Ile (T399I) SNPs of TLR4 have been associated with increased risks or severity of certain bacterial infections and Gram negative sepsis. This study employed transfection-based complementation of TLR4-negative HEK293T cells to elucidate the role of TLR4 SNPs in sensing Gram-negative bacteria and LPS, and to determine their effect on LPS-elicited activation of MyD88- and TRIF-dependent signaling pathways. Overexpression of wild-type YFP-TLR4 led to marked activation of the NF-κB-dependent pELAM-luciferase reporter in HEK293/CD14/MD2 transfectants exposed to heat-killed E. coli, R. akari and LPS, whereas the D299G (but not T399I) TLR4 exhibited signaling deficiency. Likewise, LPS-mediated activation of TRIF-dependent pRANTES-luciferase and p125 (IFN-beta)-luciferase reporters was significantly lower in HEK293/CD14/MD-2 cells expressing D299G YFP-TLR4. Immunoprecipitation and immunoblot analyses revealed similar total expression levels of wild-type and mutant YFP-TLR4 proteins, and similar LPS binding was observed in HEK293/CD14/MD-2 cells expressing WT or mutant TLR4 species, as detected by FACS. Our data indicate that the D299G SNP impairs TLR4-elicited activation of MyD88- and TRIF-dependent signaling pathways in response to heat-inactivated Gram negative bacteria and LPS not due to lower expression levels of mutant TLR4 or impaired LPS recognition by D299G TLR4/MD2/CD14 complexes.
Blockade of triggering receptor expressed on myeloid cells (TREM) signalling during a primary influenza infection reduces susceptibility to secondary bacterial pneumonia (37.21)
Authors: Xin Gang , Wissinger Erika , Hussell Tracy
Published: January 2023

TREM-1 is a cell surface receptor, first described in neutrophils and monocytes, which act synergistically with Toll-like receptors and Nod-like receptors to enhance inflammation. In addition, soluble TREM-1 (sTREM-1) has also been detected in mouse and human sera, where it regulates TREM-1 signals by competing for binding to the as-yet-unknown natural TREM-1 ligand. sTREM-1 has been used as a predictive biomarker due to the fact that it is detected at high levels during severe pneumonia and sepsis. TREM-1 signalling has been manipulated experimentally in mouse models of septic shock using a synthetic peptide (LP-17) which blocks the interaction between TREM-1 and its ligand. LP-17 treatment significantly reduces the inflammatory responses associated with sepsis and improves the outcome of a lethal secondary Streptococcus pneumoniae infection in mice undergoing a primary influenza infection. Treatment with LP-17 also reduced bacterial colonization of the nasopharyngeal cavity and the airway. These data support the idea that manipulating TREM-1 is a suitable therapeutic strategy to control secondary bacterial pneumonias.
The expression of NODs and inflammatory cytokines in tissues of mice infected with Mycobacterium leprae (37.24)
Authors: Kang Tae Jin , Kim Tae Hoon , Lee Geum Seon , Kim Se-Kon , Jin Song Hou , Kim Dojin , Kim Kyungjae , Chae Gue-Tae
Published: January 2023

The nucleotide-oligomerization domain (NOD) is an important molecule involved in host defense. RICK/RIP2 contains a caspase recruitment domain and has been implicated in the activation of the transcriptional factor NF-kB downstream of Toll-like receptors and Nod-like receptors. In order to determine the function of Nod1 and Nod2 in M. leprae infection, we measured the Nod1 and Nod2 mRNA level in tissues of nu/nu mouse infected with M. leprae. The expression of NOD1 and NOD2 mRNA was induced in tissues of nu/nu mice infected with M. leprae. In addition, the expression of inflammatory cytokines such as TNF-α and IL-1β was increased in tissues of mice infected with M. leprae, compared to those of non-infected mice. We also established HEK 293 cells to express Nod1 and Nod2 cDNA. RICK expression is, interestingly, regulated by Nod2-dependent pathway in M. leprae infection. These results suggest that NOD family proteins may have a role in the immune response to M. leprae.
IRF5 deficiency severely impairs the development of T helper 1 responses following Leishmania donovani infection. (37.3)
Authors: Stager Simona , Paun Andrea , Joshi Trupti , Pitha Paula
Published: January 2023

The transcription factor Interferon Regulatory Factor 5 (IRF5) has been shown to be involved in the induction of proinflammatory cytokines in response to viral infections and TLR activation and to play an essential role in the innate inflammatory response. In this study, the experimental model of visceral leishmaniasis was used to investigate the role of IRF5 in the generation of Th1 responses and subsequent formation of Th1-type liver granulomas in Leishmania donovani infected mice. Here we show that IRF5 deficiency severely impairs the development of Th1 responses to L.donovani leading to the formation of Th2-type-like granulomas. These granulomas are characterized by a strong IL-4 response, upregulation of arginase 1 and Fizz1 mRNA and concomitant low iNOS expression. This study identifies IRF5 as a critical molecular switch for the development of Th1 immune responses following L.donovani infections and as an important molecule in the regulation of arginase 1 and iNOS expression.
Shaping the immune response to Francisella tularensis by the first infected cell (37.33)
Authors: Steele Shaun , Buntzman Adam , Hensley Lucinda , Kawula Thomas , Bixby Lisa , Frelinger Jeffrey
Published: January 2023

Francisella tularensis, the causative agent of tularemia, induces a host response that is dependent on the route of infection. Intranasal inoculations are more virulent and require fewer bacteria initially than an intradermal inoculation to produce a lethal infection (10^3 organisms in an intranasal inoculation versus 10^6 organisms in an intradermal inoculation). However, one day post infection, the bacterial load is similar in the spleen and lung regardless of the route of infection. This indicates that host response is specific to the infection site and that this response occurs within hours post infection. To test this hypothesis, 6-10 week old C57B6/J mice are given either intradermal or intranasal inoculations of live vaccine strain F. tularensis or fully virulent Schu S4 and, after four hours, the cell types are sorted by flow cytometry and examined to determine which cell populations are initially infected. Our initial results in the lung show that essentially all cells infected by four hours are alveolar macrophages, to the exclusion of other myeloid cells and lung parenchyma. To determine the important cytokines shaping the immune response, the alveolar macrophages are infected with F. tularensis in vitro and the cytokines the cells produce are analyzed. This will allow for targeted pharmacological alterations of the cytokine response and should influence the course of infection in vivo.
Borrelia burgdorferi elicits dysregulated IL-10 production by macrophages via a TLR2-dependent, yet distinct, signaling pathway than those involved in pro-inflammatory responses (37.40)
Authors: Chung Yutein , Wells Daniel , Waldman Joshua , Arden Jessica , Wooten R. Mark
Published: January 2023

Borrelia burgdorferi (Bb) is a tick-borne spirochete that causes Lyme disease. Bb efficiently evade host defenses and persist in host tissue despite eliciting strong immune responses that involve Toll-like receptor 2 (TLR2)-mediated events. Virulent Bb are known to rapidly elicit substantial IL-10 production by skin-resident antigen presenting cells (APCs) such as macrophages (MΦs), and blocking IL-10 significantly enhances Bb clearance and APC immune functions. We hypothesize that the mechanisms involved in the dysregulated IL-10 response are initiated rapidly and distinct from those involved in proinflammatory responses. To address this, APCs were pretreated with cytochalasin D (block Bb phagocytosis) and/or antibodies that specifically neutralize TLR2 function, thus allowing delineation of early responses to Bb. MΦs produced significant IL-10 levels in response to Bb independent of phagocytosis events, whereas most proinflammatory mediators required phagocytosis. IL-10 produced in response to intact Bb was only partially reduced after TLR2-blockage, while IL-10 elicited by the Bb lipoprotein OspA was completely abrogated by TLR2-blockage. Studies using chemical inhibitors indicated that IL-10 production was dependent on both PI3 kinase and MAPK p38 activity, while most Bb-elicited proinflammatory mediators were not. These studies suggest that dysregulated IL-10 responses to Bb involve a TLR2-associated, but distinct pathway from those utilized in proinflammatory responses.
NK cells are required for defense against pulmonary K. pneumoniae infection in mice and mediate optimal production of protective cytokines, including IL-22. (37.41)
Authors: Xu Xin , Weiss Ido , Wilson Mark , Farber Joshua
Published: January 2023

Among the products of Th17 cells are IL-17A and IL-22, which are required for protection against extracellular pathogens, such as K. pneumoniae. Although published data suggest that T cells are the source of these cytokines during pulmonary infection with K. pneumoniae, we found that Rag2-/- mice did not differ from wild-type mice in survival, bacterial counts or levels of IL-22 in the lungs after intratracheal infection with K. pneumoniae. By contrast, K. pneumoniae-infected Rag2-/-γc-/- mice failed to produce IL-22 and had higher bacterial counts as compared with wild-type controls. Together, these data suggested a possible role for NK cells in host defense, and in the production of IL-22, during K. pneumoniae infection. Lung NK cells showed a conventional surface phenotype and lacked NKp46 and CCR6, markers for IL-22-producing (“NK22”) NK cells. Moreover, we found no increase in lung NK cells during infection. However, as compared with control mice, mice depleted of NK-cells using anti-Asialo GM1 showed decreased survival and higher bacterial counts, as well as increased dissemination of K. pneumoniae to blood and liver. In addition, NK cell depletion led to decreased production of IL-22 and other cytokines important for protection, including IL-12, TNF-α, and IL-6. Together, our data show that NK cells are required for defense against pulmonary K. pneumoniae infection and optimal production of protective cytokines, including IL-22. Supported by the IRP, NIH, NIAID.
Alternatively activated macrophages kill the parasitic nematode Strongyloides stercoralis in conjunction with neutrophils through two unique mechanisms. (37.44)
Authors: Bonne-Annee Sandra , O’Connell Amy , Hess Jessica , Abraham David
Published: January 2023

Previous studies have demonstrated that protective adaptive immunity in mice against the parasitic nematode Strongyloides stercoralis requires Th2 cytokines, antibody and complement activation. In addition, neutrophils function in both the innate and adaptive immune responses. The goal of this study was to determine the role of macrophages (MΦ) in the protective immune response against S. stercoralis. Bone marrow-derived MΦ were either 1) cocultured with neutrophils and larvae in Transwell plates or 2) placed in diffusion chambers with larvae and implanted subcutaneously into naïve or immunized mice. Larval killing did not occur when either MΦ or neutrophils were cultured independently with larvae in vitro. Larval killing did occur when both cell types were in contact with the worms. In addition, MΦ with soluble help from neutrophils or neutrophils with soluble help from MΦ could also kill the worms. MΦ killed larvae in both naïve and immunized mice when implanted with diffusion chambers. MΦ were stimulated ex-vivo with IL-4 to induce alternatively activated macrophages (AAMΦ) or IFN-γ/LPS to induce classically activated macrophages CAMΦ). AAMΦ and not CAMΦ 1) killed larvae in vitro and 2) provided soluble help to neutrophils to kill the larvae. AAMΦ and not CAMΦ killed larvae in vivo during the innate and adaptive immune responses. We conclude from these studies that AAMΦ can directly and indirectly, in conjunction with neutrophils, kill the nematode S. stercoralis.
Differential expression and regulation of inflammatory mediators in macrophages from Lyme disease-resistant C57BL/6J and disease-susceptible C3H/HeN mice (37.6)
Authors: Gautam Aarti , Embers Monica , Dixit Saurabh , Singh Shree , Morici Lisa , Gautam Rajeev , Dennis Vida
Published: January 2023

C3H/HeN (C3H) mice develop severe arthritis whereas; C57BL/6J (C57) mice develop mild arthritis after experimental infection with the Lyme disease spirochete Borrelia burgdorferi. We hypothesize that these host responses may depend on differences in the expression and regulation of inflammatory mediators induced by spirochetes during the early stage of Lyme disease. In the present study, bone marrow-derived macrophages from C3H and C57 mice were stimulated with live B. burgdorferi or with its lipoprotein outer surface protein A; supernatants were collected and subjected to proteome analysis using multiplex ELISA. Both stimulants induced significantly higher levels of IL-6, TNF-α, IFN-γ, KC, IL-9, MCP1/CCL2, IL-5, GM-CSF, and IL-12p70 in cells of C3H than in those of C57 mice. In contrast, MIP1α/CCL3, MIP1β/CCL4, RANTES/CCL5, IP-10/CXCL10, MIP2/CXCL2, IL-17, IL-1α, IL-1β, LIX/CXCL5, G-CSF, CXCL9/MIG and IL-10 levels were significantly greater in C57 than in C3H mice. Exogenous IL-10 abrogated the expression levels of inflammatory mediators. Neutralization of endogenous IL-10 resulted in enhanced production of mediators; the effect was more evident in C57 mice. Our data show differential expression and regulation of inflammatory mediators in B. burgdorferi-stimulated macrophages of C57 and C3H mice. The potent action of endogenous IL-10, especially in C57, suggests a key role played by IL-10 to abrogate inflammation during the early stage of Lyme disease in this mouse strain.
Evaluation of West Nile virus IgY polyclonal antibodies (38.10)
Authors: Hunter SheRae , Konschak Sarah , Adkins Steven , Schiltz James , Petell James , Bradley David
Published: January 2023

Previously, we have demonstrated that goose-derived anti-West Nile Virus (WNV) antibodies are both protective against WNV infection in avian and mammalian subjects, and specific for a broad spectrum of WNV proteins. West Nile Virus (WNV), a single stranded positive sense RNA virus from the family Flaviviridae, is capable of causing severe disease in humans including meningitis, encephalitis, and/or poliomyelitis and may ultimately lead to permanent paralysis or death. The natural reservoir for WNV is wild birds, but the virus can be spread through a mosquito vector to mammals. The epidemiology of WNV infections indicates that a population-wide vaccine is neither necessary nor cost-effective. However, an effective passive therapeutic antibody treatment would provide an excellent alternative for individuals with WNV infection involving the CNS. Characterization of the protective WNV-specific goose antibodies indicates a polyclonal response predominately of the IgY isotype with diverse specificities, including WNV proteins E, pre-M and NS3. Further evaluation indicates that multiple epitopes are recognized on the E, M, NS1, and NS3 proteins. This diverse reactivity suggests enhanced recognition of and protection against various replicative and envelope components within the virus, across WNV strains, and potentially for other flaviviruses.
The Y-chromosome translocation, Yaa, decreases murine susceptibility to herpes simplex encephalitis (38.16)
Authors: Richardson Angela , Kreutzberger Christine , Jennings Stephen , Blankenhorn Elizabeth
Published: January 2023

The C57BL/6J.YSB consomic males bear a Y chr with a translocation (Yaa) from X bearing TLR7. The translocation reduces susceptibility to EAE, a mouse model of multiple sclerosis. We hypothesized that B6.Yaa mice would also show decreased susceptibility to herpes simplex encephalitis (HSE), another neuroinflammatory condition, compared to the consomic partner, C57BL/6J (B6). Four week-old B6 and B6.Yaa male mice were inoculated with neurovirulent HSV-1. B6.Yaa mice had significantly increased survival (MST = 8 days; 40% survival) compared to B6 (MST = 6 days; 0% survival) by log-rank test. Surprisingly, the brains of the less susceptible B6.Yaa mice had a significantly greater number of viral genome copies than B6 mice (p = 7 x 10-9), as measured by quantitative real-time PCR. We analyzed expression levels of known markers of susceptibility to HSE in the two strains (including cell surface markers, cytokines, and cell-signaling molecules) by real-time PCR assays of RNA from draining lymph nodes and infected tissues. Brains of infected B6.Yaa mice demonstrated significantly increased expression of CD4, IL-1α, IL-1β, IL-6, and TNFα compared to B6 mice on dpi 5. Cluster analysis of expression levels from the 26 transcripts studied (including 21 host transcripts and 5 viral transcripts) in the brains of B6 and B6.Yaa mice demonstrated distinct patterns of gene expression. Thus the Yaa translocation alters the adaptive immune response to HSV-1 and increases survival in these mice.
Immune evasion protein mK3 defines the physiological importance of non-lysine ubiquitination of MHCI substrates (38.8)
Authors: Wang Xiaoli , Herr Roger , Rabelink Martijn , Hoeben Rob , Wiertz Emmanuel , Hansen Ted
Published: January 2023

Ubiquitin modification of proteins plays a prominent role in regulation of multiple cell processes including ER-associated degradation. Until recently ubiquitination of substrates was thought to occur only via isopeptide bonds, typically to lysine residues or less frequently to the N-terminus of a substrate. We and others recently found that certain viral MARCH E3 ligases can promote ubiquitin conjugation to non-lysine residues via ester or thiolester bonds. The molecular basis for these novel modifications, however, remained undetermined. To probe the mechanism and importance of non-lysine ubiquitination, we have studied the viral immune evasion protein mK3, a RING-CH type E3 ligase encoded by γHV68. We recently identified Ube2j2 as the primary cellular E2 required for mK3-induced MHCI heavy chain ubiquitination and degradation in the proteasome. Surprisingly, Ube2j2-mK3 preferentially ubiquitinates serine residues via ester bonds even when lysine residues are present on wildtype substrates. Furthermore, Ube2j2-mK3 not only adds the initial ubiquitin on the substrates but also promotes assembly of a lysine 48-linked polyubiquitin chain. These findings demonstrate for the first time the physiological important of this novel ubiquitination mechanism and that the same E2-E3 complex can catalyze the coupling of ubiquitin to substrates via an ester bond and assemble a specific polyubiquitin chain via isopeptide bonds.
Aberrant activation of STAT3 in primary human endothelial cells by the latent KSHV protein kaposin B (39.18)
Authors: King Christine , Gillis Laura , McCormick Craig
Published: January 2023

The proto-oncogene STAT3 is a latent transcription factor that upon activation drives the expression of a number of genes involved in cell proliferation, survival, and immune responses. Canonical STAT3 activation occurs via phosphorylation of Y705, dimerization, and nuclear translocation, followed by phosphorylation of S727 for maximal transcriptional activity. Kaposi’s sarcoma associated herpesvirus (KSHV) is the infectious cause of several AIDS-related cancers including Kaposi’s sarcoma (KS). Here we show that latent KSHV infection of primary human endothelial cells (ECs) activates STAT3, and identify a key latency protein, kaposin B, that contributes to this activation. Kaposin B expression in ECs causes STAT3 phosphorylation at S727, relocalization to punctate structures within the nucleus known as ‘STAT3 bodies’, and enhanced expression of STAT3 target genes. Recent work shows that the tripartite motif-containing protein 28 (TRIM28, a.k.a. TIF-1β, KAP-1) negatively regulates STAT3 by recruiting transcriptional silencing complexes. Here, we demonstrate for the first time that TRIM28 is a bona fide target of the host cell kinase MK2 and show that KSHV infection and kaposin B expression results in MK2 activation and concomitant phosphorylation and inactivation of TRIM28. Taken together, our studies reveal an important new regulatory mechanism governing STAT3 activation in endothelial cells and suggest novel protein targets for the treatment of KS.
Continual cell cycle progression during migration permits rapid CTL division at sites of viral infection (39.24)
Authors: McGavern Dorian , Kang Silvia , Herz Jasmin , Kim Jiyun , Dustin Michael
Published: January 2023

Following virus infection, cytotoxic T lymphocytes (CTL) divide rapidly to eradicate the pathogen and prevent the establishment of persistence. The magnitude of an anti-viral CTL response is thought to be controlled by the initiation of a cell cycle program within lymphoid tissues. However, it is presently not known whether this division program proceeds during migration or is influenced locally at sites of viral infection. Here we demonstrate that anti-viral CTL remain in cell cycle while transiting to infected tissues. One third of virus-specific CTL within blood were found to be in various stages of cell cycle following infection with lymphocytic choriomeningitis virus (LCMV). Using two-photon microscopy, we revealed that effector CTL divided within the virus-infected central nervous system (CNS) as well as meningeal blood vessels. Within the CNS, MHC I-dependent interactions with antigen presenting cells influenced the division program by pushing effector CTL into cell cycle. These results demonstrate that CTL are poised to divide in transit and that their numbers can be influenced at the site of infection through interactions with local APCs.
RSV Strain 2-20 Induces More Severe Disease in BALB/c Mice than RSV Strain 3-12 (39.25)
Authors: Stokes Kate , Lee Sujin , Chi Michael , Sturkie Carla , Peebles R. , Moore Martin
Published: January 2023

Respiratory syncytial virus (RSV) is the chief cause of bronchiolitis and viral pneumonia in infants. Mucus production contributes to morbidity and mortality in RSV disease. The TH2 cytokine IL-13 is a mediator of mucus production. RSV strain differences can play a role in differential immunologic phenotypes. The pathogenesis of RSV clinical isolate strains was examined in BALB/c mice. RSV strain 2-20 induced significant weight loss, lung IL-13 levels, and bronchiolitis in BALB/c mice. In contrast, RSV 3-12 exhibited a less severe phenotype similar to the RSV A2 laboratory strain. Mucin expressing cells in the lungs of BALB/c mice infected with RSV A2, 2-20, or 3-12 were quantified with a digital scanning microscope. RSV 2-20 induced greater mucin expression than RSV 3-12 and RSV A2. T cell and IFN-γ-expressing T cells were enumerated by flow cytometry. RSV 2-20 infection resulted in fewer IFN-γ-expressing T cells and lower overall T cell numbers in the lungs of BALB/c mice compared to RSV 3-12 and RSV A2 infection. These data correlate the heighted virulence and mucogenicity of RSV 2-20 with a diminished T cell response. RSV strain differences have an impact on RSV immunopathogenesis.
A model of natural oral Salmonella infection without the need for force-feeding (40.19)
Authors: McCarra Joshua , O'Donnell Hope , McSorley Stephen
Published: January 2023

The current murine model for Salmonella infection uses oral gavage, a methodology that forces large quantities of Salmonella into the stomach after pre-treatment with sodium bicarbonate. This methodology differs substantially from natural oral infections where small quantities of bacteria are delivered in contaminated food or water. In this study, we examined whether simply adding Salmonella to the drinking water of C57BL/6 mice could result in productive infection. Mice administered water bottles containing 1x10(9) cfu/ml of salmonella became sick and eventually succumbed to infection. While sodium bicarbonate treatment increased bacterial loads it was not required for successful infection. Mice could be productively infected with Salmonella using doses as low as 8x10(6) cfu/ml. We are currently examining the kinetics of bacterial infection and mucosal T cell activation using this new model of oral infection.
The compositional organization of Mycobacterium tuberculosis granulomas is analogous to ectopic lymphoid organs (40.2)
Authors: Slight Samantha , Rangel-Moreno Javier , Lin Yinyao , Selman Moises , Randall Troy , Khader Shabaana
Published: January 2023

Secondary lymphoid organs (SLOs) are integral in generating an effective adaptive immune response. Homing of cells to SLOs is a result of homeostatic chemokines, such as CXCL13. CXCL13, the ligand for CXCR5, is responsible for the recruitment of germinal center cells including B cells, T follicular helper (Tfh) cells and follicular dendritic cells (FDCs). Recently, CXCL13 has been reported in the lungs of mice following infection with Mycobacterium tuberculosis (Mtb). We found CXCL13 to be localized within the granuloma in both mice and human lungs as early as day 22 post infection in mice. In addition, we found Tfh cells, B cells, and FDCs localized within the granuloma of mice and humans. Recruitment of Tfh cells was seen in the draining lymph nodes by day 15 with accumulation in the lung by day 21 in Mtb infected mice. Infiltration of Tfh cells in the lung coincided with an increase in mRNA for known Tfh factors such as ICOS, BCL-6, IL-6 and IL-21, which followed upregulation of CXCL13 at day 18. Interestingly, another CXCR5+ population infiltrated the lung prior to accumulation of Tfh cells. These cells accumulate at day 12 and express surface markers suggestive of a monocyte lineage. Further, CXCR5-/- mice are more susceptible to Mtb infection by day 21. Our data suggest that the Mtb granuloma may function as an ectopic lymphoid organ, which may require involvement of CXCR5+ cells to elicit protective immunity.
Role of host lymphopenia in the immune reconstitution disease triggered by CD4 T cell transfer into mycobacterial infected T cell deficient mice (40.20)
Authors: Barber Daniel , Mayer-Barber Katrin , Antonelli Lis , Sher Alan
Published: January 2023

Approximately 10-30% of HIV patients develop a paradoxical inflammatory disease shortly after beginning anti-retroviral therapy (ART), termed Immune Reconstitution Inflammatory Syndrome (IRIS), that is emerging as a major problem in the clinical management of the HIV pandemic. Patients with the lowest CD4 counts, the most rapid drop in viral loads after ART and an AIDS defining illness, such as mycobacterial infection, are at highest risk. This indicates that T cell reconstitution itself may be pathogenic rather than beneficial when CD4 T cells suddenly repopulate a lymphopenic host harboring an opportunistic infection. Here we demonstrate that a rapidly fatal wasting occurs in mice when CD4 T cells are transferred into Mycobacterium avium infected TCRαKO animals. This reconstitution disease was dependent on IFNγ and TNFα but not IL-4 or IL-17A. Non-specific T cells induced no disease, but transfer of T cells specific for bacterial or endogenous antigens was rapidly lethal. Importantly, M. avium infected TCRαKO and ovalbumin-specific TCR Tg mice were susceptible hosts, whereas M. avium-specific TCR Tg mice were resistant to reconstitution disease. These data demonstrate that during M. avium infection of lymphopenic hosts the lack of bacteria-specific T cells, rather than the lymphopenic environment per se, predisposes the host to develop reconstitution disease when repopulating CD4 T cells encounter Ag. This work was supported by the NIAID intramural research program.
Predominant IFN-γ-mediated expression of CXCL9, CXCL10, and CCL5 proteins in the brain during chronic infection with Toxoplasma gondii in BALB/c mice resistant to development of toxoplasmic encephalitis (40.21)
Authors: Suzuki Yasuhiro , Kudo Tomoya , Wen Xiangshu , Payne Laura , Wang Xisheng , Rodgers Laurel
Published: January 2023

We examined the role of IFN-γ in expression of chemokine mRNA and proteins in the brain during chronic infection with Toxoplasma gondii using BALB/c and BALB/c-background IFN-γ knockout (IFN-γ-/-) mice. BALB/c mice are genetically resistant to development of toxoplasmic encephalitis and establish a latent, chronic infection in the brain through IFN-γ-mediated immune responses. Amounts of mRNA for CXCL9, CXCL10, CXCL11, CCL2, CCL3, and CCL5 significantly increased in the brains of wild-type mice after infection. CXCL9, CXCL10, and CCL5 mRNA were most abundant among these chemokines. An increase in amounts of mRNA for CXCL10, CCL2, CCL3, and CCL5 was also observed in the brains of IFN-γ-/- mice after infection, although CXCL10 and CCL5 mRNA levels in infected IFN-γ-/- mice were significantly lower than those of infected wild-type animals. Amounts of mRNA for CXCL9 and CXCL11 remained at the basal levels in infected IFN-γ-/- mice. When amounts of the chemokine proteins were examined in the brain homogenates of uninfected and infected mice of both strains, large amounts of CXCL9, CXCL10, and CCL5 were detected only in infected wild-type animals. These results indicate that CXCL9, CXCL10, and CCL5 are the chemokines predominantly induced in the brains of genetically resistant BALB/c mice during chronic infection with T. gondii, and their expression is dependent on IFN-γ.
Promotion of Th1/Th2 immunity with anti-TGF-β treatment protects mice against Neisseria gonorrhoeae infection and induces immune memory (40.22)
Authors: Liu Yingru , Russell Michael
Published: January 2023

Infection with N. gonorrhoeae triggers an intense inflammatory response characterized by an influx of neutrophils, yet natural infection does not induce effective specific immunity or immune memory. Our previous studies have demonstrated in a murine model of vaginal gonococcal infection that innate immunity governed by Th17 cells was the mainstay of the immune responses elicited by this pathogen. Herein we show in vitro that N. gonorrhoeae could specifically inhibit Th1 and Th2 responses of mouse CD4 T cells, but enhance Th17 response. We found that N. gonorrhoeae increased production of TGF-β by mouse lymphocytes in vitro, and concomitantly suppressed Th1/Th2 cytokines. In mouse vaginal explant cultures, the high constitutive production of TGF-β strengthened the suppressive effect of N. gonorrhoeae on Th1/Th2 responses. Anti-TGF-β, but not anti-IL-17, could divert the immune response to N. gonorrhoeae from Th17 towards Th1/Th2 modes. ELISA, microarray and flow cytometry studies indicated that when mice infected with N. gonorrhoeae were treated with anti-TGF-β, the host Th17 immune response was diminished, while Th1/Th2 responses were enhanced. Anti-TGF-β treatment led to faster clearance of infection and induced protection against secondary challenge, which did not occur in control-treated mice. Our results suggest that N. gonorrhoeae suppresses Th1/Th2-mediated adaptive immune response, and that this effect can be manipulated to enhance specific protective immunity.
TNF-α and Th17 collaborate to induce airway neutrophilia during persistent fungal infection in a MyD88 and Dectin-1-dependent fashion (40.23)
Authors: Fei Mingjian , Bhatia Shikha , Oriss Timothy , Khare Anupriya , Yarlagadda Manohar , Kolls Jay , Iwakura Yoichiro , Ray Prabir , Ray Anuradha
Published: January 2023

Aspergillus fumigatus is a fungus that induces allergic bronchopulmonary aspergillosis (ABPA), a disease exhibiting both airway eosinophilia and neutrophilia. The disease is acquired through the inhalation of spores, which are cleared efficiently in healthy humans but not in patients with cystic fibrosis or asthma. The impaired clearance and sustained inflammation contribute to the pathogenesis of ABPA. Neutrophils are important in host defense, but uncontrolled neutrophilia causes lung pathology in ABPA. We have developed an animal model of ABPA that mimics persistent fungal stimulation in ABPA. Interestingly, the model produced different outcomes in BALB/c and C57BL/6 mice with high neutrophilia in the former but low neutrophilia and high eosinophilia in the latter, which provided us with an opportunity to study the mechanisms underlying excessive airway neutrophilia. Our results show that IL-17A, which has been associated with neutrophilia in many models, was induced at similar levels in both strains. However, the BALB/c mice expressed high levels of TNF-α and collaboration between IL-17A and TNF-α was essential in inducing the high neutrophilia in BALB/c mice. Our investigations in MyD88-/- and Dectin-1-/- mice show an important contribution of both pathways in inducing high TNF-α and IL-17A responses in BALB/c mice. Taken together, we identify a novel mechanism involving the cooperation of TNF-α and Th17 for the regulation of airway neutrophilia in Aspergillus infection.
Nasopharyngeal colonization by Streptococcus pneumoniae elicits long-lasting immune responses that confer protection from invasive lung infections. (40.25)
Authors: Gerstein Rachel , Maung Nang , Goodwin Aaron , Cloherty Erin , Muñoz-Elías Ernesto , Camilli Andrew , Leong John
Published: January 2023

Streptococcus pneumoniae can cause life-threatening pulmonary and systemic infection. However, an apparent paradox is that the bacterium resides, usually harmlessly, in the nasopharynx of many, if not most people. Humoral immunity is thought to be the primary defense against serious pneumococcal infection, and we hypothesized that nasopharyngeal colonization results in the generation of an antibody response that provides long-term protection against lung infection. In fact, we found that intranasal colonization resulted in a pneumococcal-specific antibody response and protection from a subsequent high-dose S. pneumoniae lung challenge that is lethal in naïve mice. This protection lasted many weeks and seemed to be independent of the pneumococcal capsular serotype, suggesting that natural infection might trigger an immune response to a conserved antigen (or antigens) that provides protection from a broad range of S. pneumoniae strains. We found that T cell-deficient (TCR δβ-/-) mice survived both the intranasal inoculation and lung challenge better than B cell-deficient (μMT-/-) mice. Our data suggest that nasopharyngeal colonization elicits an antibody response that protects against lethal S. pneumoniae lung infection and this protective response might consist of both T-cell dependent and T-cell independent B cells.
Investigating mechanisms for limited CD8 T-cell expansion in experimental visceral leishmaniasis. (40.5)
Authors: Bankoti Rashmi , Stager Simona
Published: January 2023

Leishmania donovani, a causative agent of visceral leishmaniasis, establishes chronic infections in the spleen in humans and in mice. Thus far, the only cells that are known to be able to reduce the splenic parasite burden following immunotherapy in mice are antigen-specific CD8+ T-cells. Yet, L. donovani appears to be able to evade this defense by limiting the expansion and effector functions of CD8+ T-cell responses. Since a strong clonal expansion of antigen-specific CD8+ T-cells has been shown to be essential for the control of several pathogens, we are particularly interested in dissecting the mechanisms by which L. donovani interferes with the expansion of Leishmania-specific CD8+ T-cells. We first investigated possible inhibitory pathways that may be involved in the suppression of expansion of CD8+ T-cells. Surprisingly, we found that blocking either B7H1 or LAG-3 did not affect the early expansion and functionality of CD8+ T cells. We next evaluated the possible role of IL-10 in limiting the expansion of CD8+ T cells. IL-10R blockade resulted in increased expansion and greater cytotoxic capacity of parasite-specific CD8+ T-cells. However, depletion of Tregs and/or NK cells, which are thought to be the major sources of IL-10 during acute infection, did not improve the magnitude of expansion nor the ability of CD8+ T cells to produce Granzyme B and IFN-γ. Other IL-10 producing cell populations and inhibitory pathways will be discussed.
Tracking the Re-emerging Traits of Tuberculosis (41.9)
Authors: Mottley Hana , Salgame Padmini , Bhatt Kamlesh , Uzelac Aleksandra , McBride Amanda
Published: January 2023

Tuberculosis (TB) is a re-emerging health concern infecting nearly one third of the world’s total population and causing 1.5 million human deaths. These bacilli bacteria express multiple favorable adaptations including numerous antibiotic resistant strains. During my summer laboratory experience, I worked on a research team within the Emerging and Re-emerging Disease laboratory. The focus of the research is to develop baseline concentration numbers of cytokine messengers released by mouse cells after mice are infected with a new lab strain of TB and to test cells for the presence of four different cytokine types: TNF α, P40, P70, and INF γ in order to understand which TB strain induces which cytokines type. ELISA tests determine the type and concentration of cytokines while PCR testing determines what type of proteins are synthesized within infected cells due to the signals induced by the tested TB strain. I have adapted my laboratory experience into an educational unit which demonstrates how the KO mice that are used in the TB research laboratory are created for my students. This will provide my students with a real-world view into the biotechnological world.
Caspase-1 independent IL-1β Production is critical for MyD88-mediated host resistance to Mycobacterium tuberculosis (42.13)
Authors: Mayer-Barber Katrin , Barber Daniel , Shenderov Kevin , White Sandra , Wilson Mark , Cheever Allen , Kugler David , Sutterwala Fayyaz , Sher Alan
Published: January 2023

Host resistance to Mycobacterium tuberculosis (Mtb) requires the TLR/IL-1R adaptor MyD88. To investigate the contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mtb, we compared the outcome of Mtb infection in MyD88, TRIF/MyD88, IL-1R1 and IL-1β-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burdens and necrotic lung pathology suggesting a major role for IL-1β signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1β expression, displayed increased cytokine levels relative to WT animals, arguing that MyD88 regulates host resistance to Mtb at the level of IL-1β/IL-1R1 signaling rather than TLR-mediated triggering of cytokine production. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome mediated IL-1β processing, showed unimpaired IL-1β production and importantly, were considerably less susceptible to infection than IL-1β deficient mice. Thus, our data argue that the production of mature host-protective IL-1β during Mtb infection can occur independently of caspase-1 activation and ASC-containing inflammasomes. Together our findings reveal a critical role for IL-1β in acute host resistance to Mtb and indicate that during this bacterial infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1 cleavage.