bluuebunny/crossref_metadata_2025 huggingface dataset:
Internalization of T-Cell Receptor Mimic Antibody RL6A by Brain Derived Endothelial Cells Analyzed by Flow Cytometry
Authors: Park Jee Hyun , Hawkins Oriana E. , Chintala Navin K. , Weidanz Jon A. , Bickel Ulrich
Published: January 2023

T-cell receptor mimic monoclonal antibodies (TCRm) recognize peptide-MHC class I complexes. They combine the specificity of T-cell receptors with high binding affinities in the low nanomolar to picomolar range characteristic of monoclonal antibodies. The TCRm RL6A targets YLLPAIVHI peptide-HLA-A2 complexes, where the peptide is derived from the p68 RNA helicase protein. Previous work showed that this target is expressed by brain endothelial cells forming the blood-brain barrier (BBB). Upon binding, RL6A crosses the BBB via a receptor-mediated transcytosis mechanism. Confocal microscopy indicated that, after internalization, RL6A co-localized with the early endosomal marker (EEA1). Here we used conventional flow cytometry and imaging flow cytometry to quantitatively analyze the internalization process. Endothelial cell monolayers formed by hCMEC/D3 cells were incubated with unlabeled RL6A for time periods up to 120 min. Surface bound RL6A was detected by a secondary, PE-labeled antibody. RL6A disappeared in a biphasic pattern from the surface of hCMEC/D3 cells, with an initial half-life (t1/2) of internalization of about 3 min and a second, much slower phase (half life about 3 h) likely reflecting recycling to the membrane. In a complementary approach using imaging flow cytometry (Amnis ImagestreamX) and IDEAS analysis software, the internalization of AlexaFluor488-RL6A could be directly visualized and evaluated. The results confirmed that RL6A internalizes rapidly into hCMEC/D3 cells. In conclusion, flow cytometry is a suitable tool to analyze trafficking of specific peptide-HLA complexes in endothelial cells.
Enhancing CD4+ T cell function as a strategy for improving antibiotic therapy of Mycobacterium tuberculosis infection
Authors: Costa Diego L , Mayer-Barber Katrin D , Andrade Bruno B , Amaral Eduardo P , Maiga Mamoudou , Namasivayam Sivaranjani , Mittereder Lara , Orland Sandra , Barber Daniel L , Sher Alan
Published: January 2023

In order to develop appropriate combined immuno/chemotherapeutic interventions to treat Mtb infection, it is important to understand the interaction between the host immune response and antibiotic therapy of this pathogen. We and others have observed that CD4 T+ cell responses to Mtb decline after conventional chemotherapy. In an attempt to sustain CD4+ T cell responsiveness during antibiotic treatment, we repeatedly immunized mice with the immunodominant peptides (ESAT-64-17 or Ag85B280-294) from the major Mtb antigens ESAT-6 and Ag85B beginning at the start of drug treatment 28days post aerosol infection. When bacillary loads were assayed after 4 weeks of treatment, we found that peptide administration failed to enhance the bacterial clearance observed with chemotherapy alone. Similarly, weekly adoptive transfer of in vitro differentiated Th1 cells specific for ESAT-6 (C7) or Ag85B (P25) during antibiotic treatment failed to improve bacterial clearance despite the successful migration and proliferation of these cells in the lung parenchyma. These findings suggest that therapeutic vaccination by boosting CD4+ T cell responses during the initial phase of Mtb chemotherapy is unlikely to improve the efficacy of treatment. Nevertheless, additional experiments have suggested a major role for endogenous T lymphocytes in drug-mediated control of Mtb infection. Thus, we have observed that antibiotic treatment is ineffective in T cell deficient (TCRα−/−) mice that have received higher dose infections. We are currently evaluating the nature of this T cell requirement and whether it can be targeted to improve the outcome of Mtb therapy.

This work was supported by the intramural research program of the NIAID.
Comparative analysis of signature genes in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-infected porcine monocyte-derived dendritic cells at differential activation statuses.
Authors: Miller Laura C , Sang Yongming , Bayles Darrell O , Blecha Frank
Published: January 2023

Activation statuses of monocytic cells, e.g. monocytes, macrophages and dendritic cells (DCs), are critically important for antiviral immunity. In particular, some devastating viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), are capable of directly infecting these cells to subvert host immunity. Monocyte-derived DCs (mDCs) are major target cells in PRRSV pathogenesis; however, the plasticity of mDCs in response to activation stimuli and PRRSV infection remains unstudied. In this study, we polarized mDCs, and applied genome-wide transcriptomic analysis to compare signature genes involved in mDCs activation and response to PRRSV infection. Our long-term goal is to integrate activation status with antiviral responses in these cells and to functionally modulate them for a prototypic cellular adjuvant/vaccine that is ideal for potentiating antiviral immunity. Porcine mDCs were polarized with mediators for 30 hours, then mock-infected, infected with PRRSV strain VR2332, or a highly pathogenic PRRSV strain (rJXwn06), for 5 h. Total RNA was extracted and used to construct sequencing libraries for RNA-Seq procedures previously optimized. Comparisons were made between each polarized and unpolarized group (i.e. mediator vs. PBS), and between PRRSV-infected and uninfected cells stimulated with the same mediator. Many of the genes showing the most variability were related to cellular structure and innate immune response. The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected mDCs as compared to VR-2332 infected mDCs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.
Transcript analysis of natural killer (NK) cell specific genes in the liver, kidney and spleen tissues of rag1
−/− mutant zebrafish in response to in vivo administration of TLR ligands
Authors: Muire Preeti Judith , Hanson Larry , Yoder Jeffrey , Petrie-Hanson Lora
Published: January 2023

T and B cells mediate specific protective immunity. NK cells can also mediate a memory response to murine cytomegalovirus in T and B cell deficient rag2−/− mice. T and B cell deficient rag1−/− mutant zebrafish mediate protective immunity to intracellular bacteria. NK cells have not been characterized in zebrafish due to a lack of tools to target specific markers. The aim of this study was to evaluate NK cell tissue distribution and transcript response to TLR ligands in rag1−/− mutant zebrafish. We accomplished this by determining the level of expression of ifnγ, t-bet, nitr9, and NK lysin (nkl) a, b, c and d transcripts following exposure to β glucan, Poly I:C, R-848 and VTX-2337. Expression levels were normalized to the basal level of expression and the fold changes were compared to that of endotoxin free PBS (carrier control) injected group. R848 and VTX-2337 exposure changed the gene expression profile in a tissue specific manner. Nitr9 protein expression was analyzed by western blot and detectable levels were found in the liver. Nitr9 expression correlated with the expression of ifnγ in liver and kidney. In another experiment, fish were injected with TLR ligand when vaccinated, were only vaccinated, or not vaccinated. After bacteria challenge, fish that received β glucan or R848 when vaccinated had the highest survival. This suggests the TLR ligands have an adjuvant activity on NK cell protective responses. Our results of NK cell stimulation indirectly demonstrate the presence of NK cells in zebrafish and suggest a possible means of enhancing NK cell based immunity. These findings demonstrate the clinical significance of the rag1−/− mutant zebrafish model.
Functional impairment of CMV-reactive cellular immunity during pregnancy
Authors: Boeckl Katharina , Reuschel Edith , Barabas Sascha , Bendfeldt Hanna , Deml Ludwig , Seelbach-Göbel Birgit
Published: January 2023

Background

Cytomegalovirus (CMV) is the most common congenital viral infection. Mother-to child transmission (MTCT) can cause severe disability of the child. Intact CMV-specific cell-mediated immunity (CMI) has been shown to prevent uncontrolled replication in healthy individuals. In this study we aim to determine whether CMV-specific CMI is impaired in pregnant women, thus increasing the overall risk for active CMV replication and transmission.

Methods

CMV-specific CMI in peripheral blood of 61 pregnant women in the three trimenons of their pregnancy has been determined using T-Track® CMV for detection of IE-1 and pp65-reactive effector cells by IFN-γ ELISpot. Additionally CMV- IgG and IgM titers were examined.

Results

CMV-specific CMI was detected in 70 % of CMV-seropositive pregnant women. Also 4 % of CMV-IgG seronegative women showed IE-1- but not pp65- reactive cells. The overall number of CMV-reactive cells in pregnant women was lower compared to a matched non-pregnant control group. Postpartum the IE-1- but not pp65- specific CMI is still lower than in the non-pregnant control group.

Conclusion

Functional analysis of CMV-reactive immune cells using T-Track® CMV indicates a systemic down-regulation of the CMV-specific CMI in pregnant women.
The IFIH1 A946T autoimmune risk variant exhibits enhanced signaling and promotes T1D pathogenesis
Authors: Gorman Jacquelyn A , Hundhausen Christian , Errett John , Ge Yan , Arkatkar Tanvi , Clough Courtnee , Dai Xuezhi , Khim Socheath , Oukka Mohamed , Concannon Patrick , Gale Michael , Buckner Jane H , Rawlings David J
Published: January 2023

Genome wide association studies have linked SNPs in innate signaling programs to an increased risk for autoimmune disease. One SNP, rs1990760, within the IFIH1 viral sensor (leading to an A to T amino acid change at codon 946) is strongly associated with multiple autoimmune diseases including type 1 diabetes (T1D). The impact of this variant on viral sensing and autoimmune pathogenesis, however, has not yet been determined. Using an overexpression system, we demonstrate that IFIH1-A946T exhibits heightened basal and post-stimulation signaling and increased viral control. Consistent with this finding, primary PBMCs from healthy subjects expressing the variant exhibit heightened signaling. To study IFIH1-A946T in vivo, we generated a novel knock-in murine model. Both heterozygous and homozygous variant mice display enhanced basal Type I IFN mRNA expression and splenomegaly. Moreover, homozygous pups manifest an embryonic survival defect. Further, IFIH1-T946 mice exhibit increased frequency of STZ-induced T1D. Finally, intercross of IFIH1-T946 mice with animals expressing the T1D risk variant in PTPN22 lead to a synergistic increase in T1D development. Together, our data demonstrate that IFIH1-A946T alters antiviral signaling and strongly supports the model that increased Type I IFN production driven by the variant leads to an inflammatory state that promotes autoimmunity, particularly in association with other autoimmune risk alleles.
TAPP adaptors control Akt-dependent metabolic activation of germinal center B cells and suppress autoimmunity via binding to the SHIP product PI(3,4)P2
Authors: Marshall Aaron J , Jayachandran Nipun , Landego Ivan , Hou Sen , Meija Edgard M , Sheikholeslami Kimia , Hatch Grant M
Published: January 2023

The phosphoinositide phosphatase SHIP regulates B cell activation by converting the PI 3-kinase product PI(3,4,5)P3 to PI(3,4)P2. While this activity of SHIP appears critical for generation of effective adaptive immunity while avoiding autoimmunity, the function of PI(3,4)P2 in B cells remains relatively unexplored. Tandem PH domain containing proteins (TAPPs) are adaptor proteins that specifically bind to PI(3,4)P2 via their C-terminal PH domains, targeting them to the plasma membrane. Mice bearing inactivating mutations in the PH domains of both TAPP1 and TAPP2, uncoupling them from PI(3,4)P2, exhibit hypergammaglobulinemia and develop lupus-like characteristics including anti-DNA antibodies and deposition of immune complexes in kidneys. Here we find that TAPP KI mice develop chronic germinal centres (GCs) and age-associated increases in B cell expression of activation, exhaustion and memory markers, all of which are B cell intrinsic and dependent on the co-stimulatory molecule ICOS. TAPP KI B cells exhibit elevated phosphorylation of Akt and its target GSK3, and increased survival. Strikingly, TAPP KI B cells exhibit increased metabolic activity associated with elevated oxygen consumption rate, increased extracellular acidification rate, increased expression of glucose transporter Glut1 and increased glucose transport rate. Together our findings suggest that TAPP-PI(3,4)P2 interaction is important for regulating signalling via Akt, B cell metabolism and development of autoimmunity.
Apoptotic nucleolus is recognized by complement C1q and degraded by C1 proteases
Authors: Lu Jinhua , Cai Yitian , Teo Boon Heng Dennis , Yeo Joo Guan , Chen Junjie
Published: January 2023

In infections, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 hr, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. By affinity chromatography, a number of nuclear proteins were found to bind to C1q including nucleophosmin 1 and nucleolin. In vivo, C1q exists as C1 complex (C1qC1r2C1s2) and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of nucleolar proteins neucleolin and nucleophosmin 1. Cleavage of purified nucleophosin 1 by C1 is studied in details revealing three defined fragments of 11, 17 and 18 kDa, respectively. This was inhibited by C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduce autoimmunity. These findings suggest broader substrate spectrum of C1 proteases and help understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus.
IL-17 drives but can also restrain psoriatic inflammation via distinct, target cell-specific mechanisms
Authors: Siebenlist Ulrich , Ha Hye-lin , Wang Hongshan , Claudio Estefania , Tang Wanhu
Published: January 2023

Psoriasis is a chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory cells. The mechanisms underlying psoriasis in humans and mice are still only partially understood, although IL-17 cytokines are known to make crucial contributions, cytokines that signal via the obligatory adaptor CIKS/Act1. Here we identify critical roles of CIKS/Act1-mediated signaling in imiquimod-induced psoriatic inflammation, a mouse model that shares features with the human disease. We found that IL-17 cytokines/CIKS-mediated signaling into keratinocytes was essential for neutrophilic microabscess formation; in addition, it contributed to hyperproliferation and markedly attenuated differentiation of keratinocytes, at least in part via direct effects. Interestingly, IL-17 signaling into keratinocytes also exerted a negative feedback to eventually delimit further accumulation of IL-17-producing γδ T cells in skin. This stands in contrast to IL-17 cytokines/CIKS-mediated signaling into non-keratinocytes, particularly dermal fibroblasts, which promoted cellular infiltration and enhanced accumulation of IL-17-producing γδ T cells in skin, comprising an initial positive feed-forward mechanism. We will report on recent progress in understanding in particular the mechanism by which signaling into keratinocytes eventually feeds back to prevent excessive IL-17 production. Our data show that CIKS-mediated signaling is dynamic and central to the development of both dermal and epidermal hallmarks of psoriasis, but also activates mechanisms that delimit inflammation.
BCAP inhibits myeloid cell development from hematopoietic progenitors.
Authors: Duggan Jeffrey M , Hamerman Jessica A
Published: January 2023

B cell adaptor for PI3-kinase (BCAP) is a signaling adaptor expressed in hematopoietic cells including macrophages, monocytes, and neutrophils. Here we asked if BCAP plays a role in development of these myeloid cells. We found that BCAP was expressed in bone marrow (BM) hematopoietic progenitors, including LSK (Lin−Sca1+cKit+), CMP (Common Myeloid Progenitor) and GMP (Granulocyte/Macrophage Progenitor) cells, suggesting that BCAP may impact myelopoiesis. BCAP−/− mice had more BM monocytes than WT mice, and in mixed chimeras generated with a 1:1 ratio of WT and BCAP−/− BM, monocytes and neutrophils in the BM, blood and spleen exhibited skewing towards BCAP−/− origin, showing a competitive advantage for BCAP−/− myeloid cells. Thus we hypothesized that BCAP inhibits myeloid development. Consistent with this hypothesis, BCAP-deficient BM LSK, CMP and GMP cells out-competed WT progenitors in mixed chimeras. In an in vitro myeloid colony-forming-unit assay, sorted BCAP−/− progenitors produced more myeloid cells than WT progenitors, supporting a cell-intrinsic role of BCAP in inhibiting myeloid differentiation. Furthermore, BCAP−/− progenitors were more mature than WT progenitors, indicating that BCAP−/− progenitors display accelerated myeloid development. During cyclophosphamide-induced myeloablation or specific monocyte depletion, BCAP−/− mice replenish circulating monocytes earlier than WT mice. Lastly, BCAP−/− progenitors showed increased expression of the myeloid-differentiating transcription factors IRF8 and C/EBPα compared to WT progenitors. Together, these data identify BCAP as an inhibitor of myeloid development from BM hematopoietic progenitors in the steady state and during demand conditions.
Propranolol modulates androgen-expanded myeloid-derived suppressor cells (MDSC) in severely burned mice
Authors: Kobayashi Makiko , Ito Ichiaki , McCalla Claudia T , Herndon David N , Suzuki Fujio
Published: January 2023

Severe burn injuries produce chronic hypermetabolic and hypercatabolic states persisting long after injury. For the improvement of net protein balance and lean body mass, we are trying to use oxandrolone (OX, a synthetic analog of testosterone) in severely burned patients subjected to the standard of care (SOC) including propranolol (PPL, a blocker of β-adrenergic receptors). OX has the potential to exacerbate post-burn immunoparalysis through the expansion of MDSC (Gr-1+CD11b+ cells). Since propranolol accelerates MDSC maturation, the effect of OX on the expansion of MDSC in severely burned mice subjected to SOC was studied. Mice were treated s.c. with OX (0.1 mg/kg, 1 and 12 h post burn) alone or in combination with PPL (3.3 mg/kg, 12 h post burn). MDSC in splenic MNC from these mice were analyzed by flow cytometry. Also, peritoneal Mϕ from these were analyzed for intracellular TNF-α, as an inhibitor of MDSC maturation. In the results, the number of MDSC increased 5.5-fold in burn mice treated with OX, as compared to that of MDSC in burned mice. However, this increase was not seen in burn mice treated with OX and PPL in combination. TNF-α+ cells increased 2.5-fold in the peritoneal Mϕ of burn mice treated with OX, but not in burn mice treated with OX and PPL. These results show that the number of MDSC is not increased by OX in severely burned mice treated with PPL.
NF-κB restricts NLRP3 inflammasome activation via p62-dependent mitophagy which eliminates mitochondrial signals
Authors: Zhong Zhenyu , Umemura Atsushi , Sanchez-Lopez Elsa , Liang Shuang , Shalapour Shabnam , Wong Jerry , He Feng , Boassa Daniela , Perkins Guy , Ali Syed Raza , McGeough Matthew G , Ellisman Mark H , Seki Ekihiro , Gustafsson Asa B , Hoffman Hal M , Diaz-Meco Maria T , Moscat Jorge , Karin Michael
Published: January 2023

NF-κB, a key activator of inflammation primes the NLRP3-inflammasome for activation by inducing pro-IL-1β and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1β-dependent inflammation and enhances macrophage death. Therefore, the “NF-κB-p62-mitophagy” pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair. This pathway, however, is bypassed when NLRP3 is genetically activated.
Elucidation of the NLRP3 interactome
Authors: Fairgrieve Marian , Davis Michael , Gale Michael J
Published: January 2023

Upon pathogen detection, the immune system triggers a variety of signaling pathways. Two key pathways are the proinflammatory NLRP3 inflammasome pathway, which activates the proinflammatory cytokines IL-1β and IL-18 via activation of Caspase-1 (CASP1), and the antiviral IFN pathway, which signals through the JAK-STAT pathway to upregulate key IFN-stimulated genes. Importantly, a growing body of evidence demonstrates that crosstalk between these pathways is required to fine tune the host immune response, and the co-regulation between the pathways can be either positive or negative. For example, upon viral infection, IL-1β synergizes with INF to suppress viral replication. At other times, as in the treatment of multiple sclerosis with INF-β therapy, INF suppresses IL-1β expression and activation. However, the mechanism governing inflammasome-INF crosstalk remains unclear. To address this gap in our understanding, we conducted a series of proteomic studies aimed at identifying novel interacting partners of NLRP3 and CASP1. We employed an affinity purification and mass spectrometry-based approach. Interestingly, we found that components of the JAK-STAT pathway interact with NLRP3. We will present data characterizing this interaction as well as the role of JAK-STAT proteins in regulating inflammasome-IFN crosstalk. These findings will enhance our understanding of how these two cascades act in concert to impact infection and immunity.
Mutant Interferon-γ receptor 1 lacking cytoplasmic domain may transduces signals leading to mycobacterial susceptibility
Authors: Shieh Chi-Chang K , Chiu Darren Jianjhih , Chiang Kuo-Ming , Yen C-L
Published: January 2023

Interferon-γ (IFN-γ) is a crucial cytokine for innate and acquired immunity against infections by intracellular bacteria including mycobacteria. Mutations in the genes involved in the IL-12-IFN-γ pathway cause impaired immunity against lowly virulent mycobacteria and lead to Mendelian Susceptibility to Mycobacterial Disease (MSMD). How the mutations differentially affect leukocyte activation signals and lead to different clinical presentations has not been characterized. We previously analyzed a family with autosomal dominant MSMD and found an 818del4 mutation on the IFN-γ receptor gene IFNGR1 leading to high expression of IFN-γ receptor on leukocytes. While IFN-γ-induced IL-12 and TNF-α secretion from peripheral blood mononuclear cells (PBMCs) were low in the patients. IFN-γ and LPS-induced NADPH oxidase priming and hence reactive oxygen species production was normal. We hence cloned the 818del4 mutant and wildtype IFNGR1 into lentiviral vector and expressed the mutant IFNGR1 on human embryonic kidney 293T cells. We found that stimulation with IFN-γ increased the phosphorylation of Akt and activated p47phox through phosphorylation of Erk1/2 in cells expressing 818del4 mutant IFNGR1. Our results implicated that the truncated IFN-γ receptor may transmit differential signals in leukocytes which alter the toll-like receptor signals by activating the Akt activity and contribute to the severity of immunodeficiency in patients with autosomal dominant MSMD.
Amphibian innate immune mediators protect against human Influenza strains
Authors: Holthausen David J , George Sanil , Jacob Joshy
Published: January 2023

Frogs and toads are incredible reservoirs of biologically active peptides. Amphibians secrete host defense peptides from their skin as part of their innate immune response. This ancient response acts to protect the amphibians against microbes. The quantity and scope of these secreted peptides dwarfs mammalian analogues, accounting for a substantial portion of all known host defense peptides. The non-invasive and non-harmful methods for frog peptide collection, in tandem with the abundance and breadth of these peptides, makes them excellent choices for novel peptide drug therapies. Studies have shown that these peptides can effectively neutralize enveloped viruses, mycobacteria, gram-negative and gram-positive bacteria, fungi, and even cancerous or transformed cells. Because of the nature of antimicrobial peptides, often targeting the most critical, conserved aspects of a micro-organism, they may prove a vital alternative to conventional drugs plagued by pathogen resistance. Given this untapped potential for anti-viral therapies, we assessed novel host defense peptides from the skin of the Indian fungoid frog, Hylarana malabarica. During our analysis, we isolated several peptides from H. malabarica that show anti-viral activity against human influenza viruses. Our studies indicate that peptides from H. malabarica demonstrate anti-influenza activity in vitro, and also show potential as an anti-viral therapy in vivo.
Characterization of the Normal Mouse IGH Antibody Repertoire
Authors: Rettig Trisha A , Bye Bailey , Peacaut Michael J , Chapes Stephen K
Published: January 2023

While the process of antibody generation in response to pathogens and immunization is well understood, the normal antibody repertoire needs characterization to better understand the diversity of the antibody response. The antibody molecule is generated from multiple gene segments; variable (V), diversity (D), and joining (J). These segments rearrange (V, D, and J in the heavy chain, V and J in the light chain) and combine with a constant region, which determines the effector function within in the host. The V, D, and J gene segments are combined to form the complementary determining region 3 (CDR3), providing much of the variability within the B cell receptor repertoire. To address heavy chain rearrangement in the unstimulated antibody repertoire, we pooled and sequenced the total splenic RNA from three groups of normal C57BL/6J. V, D, J, and constant region usage and CDR3 formation were analyzed. We developed a bioinformatic workflow to analyze the most common usage and combinations of the V, D, and J gene segments and their pairings with constant regions. We examined length and composition of CDR3 along with the ontology of the observed antibodies. The preliminary screen identified over 33,000 sequences as possible antibodies; 5,743 were hypothesized to be productive sequences. The largest V-gene family represented was V1, followed by V2 and V6. The average CDR3 length was 11 amino acids and we identified a number of high-occurrence CDR3 amino acid sequences. These data provide a glimpse of the normal C57BL/6J heavy chain response and validate the bioinformatics needed for our future immunization studies. Supported by NASA grants NNX13AN34G and NNX15AB45G and NIH grant GM103418 and the Molecular Biology Core supported by CVM-KSU.
Epidemiologically characterized high and low transmission isolates of Mycobacterium tuberculosis induce distinct infection outcomes in mice
Authors: Verma Sheetal , Bhatt Kamlesh , Rodrigues Rodrigo Ribeiro , Jones-López Edward C. , Palaci Moises , Alland David , Dietze Reynaldo , Ellner Jerrold , Salgame Padmini
Published: January 2023

In a study of household contacts of infectious TB cases (HHC) conducted by us in Brazil, “index” cases were categorized into High (HT) and Low (LT) transmission groups based on the proportion of household contacts with a positive tuberculin skin test. In this study we wanted to determine whether Mycobacterium tuberculosis (Mtb) isolates with different transmission phenotypes diverged in their interaction with the host immune system. Initial in vitro analysis of 15 isolates from each group revealed significantly higher levels of TNF produced by macrophages infected with HT isolates. Two each of the HT and LT isolates were further characterized in vivo in the C3HeB/FeJ mice which develop lesions characteristic of human TB. Through the course of these infections, both the LT isolates exhibited a significantly higher bacterial burden and enhanced cellular recruitment to the lungs. Mice infected with the LT isolates exhibited early pulmonary inflammation that rapidly progressed to widespread consolidation of the granulomatous lesions. In contrast, mice infected with the two HT isolates formed organized cellular aggregates that contained abundant lymphocytes and foamy macrophages. Interestingly, pathological progression of the cellular aggregates into caseous fibrotic lesions, with abundant extracellular bacilli, was observed with one of the HT isolates. Furthermore, elevated levels of the pro-inflammatory cytokines IL-1β, IL-6 and IL-17 were found in the lungs of mice infected with the LT isolates. These results suggest that distinct interactions between microbe and host immunity might lead to differential trajectory in intracellular growth pattern and lung pathology that underlie differences in transmission potential.
Mycobacterium tuberculosis produces small noncoding RNAs during infections to regulate mycobacterial and eukaryotic gene expression
Authors: van Oers Nicolai S , Srivastava Shashikant , Raj Prithvi , Dozmorov Igor , Wakeland Edward K , Gumbo Tawanda
Published: January 2023

Mycobacterium tuberculosis (Mtb) presently infects 1/3 of the World’s population, causing both active and latent pulmonary tuberculosis (TB). The pathogenic mechanisms used by Mtb remain poorly defined. We used a small RNA sequencing strategy on RNA extracted from Mtb-infected THP-1 macrophages to characterize the changes in eukaryotic microRNAs (miRs) and identify any mycobacterially-encoded small RNAs. A large number of mammalian microRNAs were differentially regulated during the infection course. Target prediction programs indicated that the mRNAs regulated by the miRs were involved in immune processes, metabolic pathways, cell communication, and developmental events. In addition, 35 Mtb-encoded small RNAs (18–30 nucleotides, mRs) were discovered. Several of the Mtb-encoded mRs were detected in lung tissue biopsies from TB infected monkeys. The 100–150 nucleotides surrounding the 35 Mtb-encoded small RNAs had extensive secondary RNA folding capabilities, including hairpins and antisense stretches more characteristic of eukaryotic pre-microRNAs. Two such small RNAs, Mtb-mR-1 and Mtb-mR-6 were transcribed in Mycobacterium avium complex and Mtb in a hairpin loop and antisense sequence-dependent manner. These mRs were not expressed in Mycobacterium smegmatis, revealing a restricted expression to pathogenic strains. Mtb-encoded mR-1 and mR-6 modulated both prokaryotic and eukaryotic gene expression. Taken together, our findings reveal potentially novel pathogenic processes utilized by Mtb during infections.
Role of secreted IgM in regulating the host response to Mycobacterium tuberculosis
Authors: Mehta Simren , Xu Jiayong , Wang Yanhua , Chan John
Published: January 2023

B cell aggregates, which display characteristics of germinal center reactions, are a prominent feature in the lungs of Mycobacterium tuberculosis (Mtb)-infected hosts. Using the B cell-deficient mouse strain μMT, we have previously shown that B cells are required for optimal protection against aerosol Mtb infection. μMT mice display exacerbated lung granulomatous inflammation during acute tuberculosis (TB), and this can be ameliorated by adoptive transfer of B cells or treatment with immune serum, suggesting a role for immunoglobulin in modulating the local lung response to Mtb. To investigate the role of antibody in a protective host response to Mtb, we utilized mice deficient in various immunoglobulins. Mice unable to secrete IgM (μS−/−) were more susceptible to aerosol Mtb infection than those lacking all other immunoglobulin isotypes, and displayed increased mortality during chronic TB. Relative to wild-type (WT) mice, Mtb-infected μS−/− mice had fewer lymphoid aggregates and a delayed granulomatous response in the lungs, despite having greater numbers of activated pulmonary CD4+ T cells. Treatment of μS−/− mice with immune serum (derived from Mtb-infected WT mice) during acute infection, led to increased lymphoid aggregates and plasma cells in the lungs, more pulmonary infiltrate, a concomitant decrease in the lung bacterial burden, and ultimately delayed the onset of death of μS−/− mice during chronic TB. These data suggest IgM plays an important role in controlling Mtb infection by orchestrating local granulomatous responses in the lungs, which leads to improved host survival during the chronic phase of infection.
Mycobacterium tuberculosis hijacks alveolar macrophages to translocate from the airway to the lung parenchyma.
Authors: Cohen Sara Beth , Adams Kristin , Urdahl Kevin
Published: January 2023

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a chronic bacterial infection of the lung that affects one third of the world’s population. While lung alveolar macrophages (AM) are thought to be the first host cell infected with Mtb, it remains unclear how bacteria leave AM in the airway and disseminate to the lung parenchyma. Using fluorescent Mtb to identify infected cells and non-surgical intra-tracheal antibody delivery to label airway-resident cells, we found that airway-resident AM are indeed the first cells infected by Mtb. Interestingly, infected AM become increasingly resistant to airway labeling over time, with only 30% of infected AM localizing to the airway by Day 21 post-infection, suggesting that infected AM exit the airway and translocate to the lung parenchyma as infection progresses. In contrast, uninfected AM continue to predominantly label with the airway antibody, suggesting that migration of AM into the lung parenchyma is specific to infected cells. In the absence of the TLR adaptor MyD88, but not IL12R or type I IFN, infected AM are partially impaired in their ability to migrate, implicating MyD88 signaling in this trafficking pathway. Surprisingly, AM infected with the attenuated ΔdrrA strain that lacks the virulence lipid PDIM are impaired in translocation to the parenchyma, identifying a possible role for this lipid in promoting AM migration. Conversely, AM infected with the hypervirulent SA161 strain translocated to the lung parenchyma more rapidly 10 days post-infection. Combined, these studies provide insights into the cellular mechanisms of Mtb entry into the lung parenchyma following aerosol infection, a key step in the pathogenesis of tuberculosis.
Commensal E. coli induced colonization resistance against mucosal Salmonella infection
Authors: Lu Chunchi , Zhao Xiadan , Lai Marvin A , Lopez-Yglesias Americo H , Quarles Ellen K , Lo Chiata , Smith Kelly D
Published: January 2023

Salmonella is the causative agent in Salmonellosis and is a leading cause of gastrointestinal bacterial infections worldwide. During mucosal Salmonella infection Salmonella must first establish a niche in the gastrointestinal tract in order to initiate infection. Colonization resistance (CR), defined as the ability of intestinal microbiota and host defenses to protect against pathogens, is the first line defense against intestinal infection. The mechanism for commensal bacteria mediated CR has been studied for a long time; however, only a few studies have explored how microbiota can induce CR against Salmonella infection. Herein we demonstrate that a mouse commensal E. coli strain mediates CR against mucosal Salmonella Typhimurium infection, and that this CR is independent of the inflammasome, TLR5, MyD88, adaptive immunity, and a complex microbiome. The commensal E. coli strain does not directly compete with Salmonella growth in vitro, and is only capable of inducing colonization resistance in vivo. Gene expression analysis demonstrates that colonization of mice by commensal E. coli induces a large number of host defense genes involved in the regulation of mucosal barriers, innate immune cells and arachidonic acid metabolism. Our studies demonstrate that commensal E. coli interacts with the host through a novel pathway to induce CR against mucosal Salmonella infection and illuminate the complex interactions between microbiota and the host that shape defenses and defend against enteric infections.
Modulation of TRAIL mediated apoptosis signaling by Helicobacter pylori
Authors: Hsu Ping-Ning , Lin Woan-Yu
Published: January 2023

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. However, the mechanisms by which cause disease remain unclear. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through down-regulation of cellular FLICE-inhibitory protein (FLIP). Our study thus demonstrates H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. We further demonstrated that FLIPS expression was consistent with Akt activation. H. pylori induced reduced Akt phosphorylation and led to decreased FLIPS expression in human gastric epithelial cells. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.
Development of Dictyostelium discoideum as a model to identify evolutionarily-conserved virulence factors in Escherichia coli
Authors: Snyder Michelle , Weichseldorfer Matthew , Parker Dominique , Qureshi Mohammed , Phan Philong , White Theresa , Leicht Benjamin , Alkharouf Nadim , Rasko David , Hemm Matthew
Published: January 2023

Escherichia coli are a typical component of mammalian gut microbiota; however, a number of pathogenic strains remain a health concern for developing countries. Identification and disruption of virulence factors that promote infection could lead to reduction in infection. To identify novel bacterial virulence factors, we are using the soil amoeba, Dictyostelium discoideum, which preys upon bacteria, including E. coli, in the soil, as a model for mammalian phagocytic cells. Using random mutagenesis, we have created mutant stocks of MG1655 E. coli. These stocks were fed to D. discoideum, enriching the culture and selecting for mutants that were resistant to killing upon phagocytosis. After several rounds of selection, resistant mutant strains were isolated, and we found that some strains showed up to 100-fold increase in survival over a 2 h period. Once isolated, the genomes of the resistant mutant strains were sequenced and the sequences compared to that of the initial WT strain using Illumina’s MiSeq technology. While analyzing the sequences from these mutant strains, we found mutations in several potential genes of interest and are working to characterize their roles in the cell. We are testing E. coli strains reconstituted with specific mutations to identify particular mutations that are associated with phagocytosis-resistant phenotypes. These studies are aimed at identifying particular bacterial genes that may be involved in regulation of virulence. In addition, given that the development of mechanisms to resist D. discoideum predation may have contributed to the selection and maintenance of bacterial virulence factors against mammalian hosts, these studies may provide insight on the evolution of host-pathogen interactions.
Peripheral Francisella tularensis infection results in neural invasion and pathologic inflammation.
Authors: Ramos Muniz Mireya G , Pon Alexandra , Jones Devin , Spencer Charles T
Published: January 2023

Francisella tularensis infection triggers an overactive inflammatory response by stimulating production of host pro-inflammatory cytokines. Human tularemia, caused by F. tularensis, is a zoonotic disease transmitted by aerosol particles, direct contact, and via arthropod vectors, particularly ticks. Curiously, during peripheral inoculation in a mouse model of tularemia, F. tularensis was unexpectedly found in brain homogenates, though there is no prior evidence suggesting neural tropism. In the periphery, macrophages provide innate immune defense against foreign material, including bacteria. Microglial cells are resident macrophages of the brain and spinal cord and previously have been reported to rapidly respond to pathological changes in the central nervous system, serving a similar function as macrophages. Their rapid activation is an important factor in guarding the neural parenchyma against infectious diseases, inflammation, and neurodegeneration while maintaining and facilitating the return to tissue homeostasis. Therefore, we hypothesized that, having invaded the brain by an unknown mechanism, F. tularensis may be capable of eliciting an immune response following infection of microglial cells. Since macrophage infection leads to elicitation of inflammation, infection of microglia similarly stimulate inflammation leading to neural pathology. Mice were inoculated on the rear flank with fluorescently labeled bacteria. Using fluorescent confocal microscopy, labeled F. tularensis was localized in the brains of infected mice. We analyzed brain homogenates for production of inflammatory cytokines at these sites through Milliplex and qPCR.
Modulation of Gut Inflammation by Dietary Fibers in Murine Chronic Colitis
Authors: Singh Vishal , Yeoh Beng San , Saha Piu , Vijay-Kumar Matam
Published: January 2023

Natural dietary fiber serves as a substrate for gut microbiotal fermentation generating short chain fatty acids, which are thought to offer protection against inflammatory bowel disease (IBD). However, certain fibers are known to cause moderate to severe intestinal side effects in IBD patients. We hypothesize that soluble fibers (inulin & pectin) which are more accessible for microbiotal fermentation may offer pronounced gut protective effects than insoluble fiber (cellulose). To demonstrate this, chronic colitis was induced in dietary cellulose, inulin or pectin fed C57BL/6 mice by administering three weekly injections of α-IL-10R neutralizing antibody. Mice were euthanized two weeks after the last injection and colitis development was examined by serological, biochemical, histological and immunological parameters.

WT mice receiving dietary cellulose along with α-IL-10R mAb exhibited robust colitis as characterized by splenomegaly, colomegaly, elevated colonic MPO activity, pro-inflammatory cytokines (Kc, Il-1β & iNos), systemic and fecal Lcn2 levels (an inflammatory marker) and colon histology score when compared to control mice. Similar results were observed in toll-like receptor 5 knockout (Tlr5KO) mice which are prone to develop microbiota-dependent spontaneous colitis. Surprisingly, prebiotic fiber inulin failed to protect against α-IL-10R neutralization-induced chronic colitis in both WT and Tlr5KO mice. Interestingly, pectin ameliorates colitis development, supporting the notion that not all dietary fibers are created equal in modulating host gut inflammation. Further mechanistic study on how pectin dampens gut inflammation may yield development of novel dietary therapeutics to treat human IBD.
Group 2 innate lymphoid cells collaborate with dendritic cells to potentiate memory Th2 cell responses.
Authors: Halim Timotheus You Fu , Hwang You Yi , Scanlon Seth , Garbi Natalio , Zaghouani Habib , Fallon Padraic G , McKenzie Andrew
Published: January 2023

Rapid memory CD4+ T helper 2 (Th2) cell activation during allergic inflammation requires their recruitment into the affected tissue. Tissue-resident innate immune cells such as dendritic cells (DC) are essential for orchestrating local memory Th2 cell responses. We now know that tissue-resident group 2 innate lymphoid cells (ILC2) are an important innate source of type-2 cytokines in barrier sites. However, it is currently not known if ILC2 remain functionally important after the generation of adaptive Th2 cells. Here we demonstrate that group 2 innate lymphoid cells (ILC2) play a critical role in memory Th2 cell responses. By targeted ILC2 depletion, using ILC2-specific diphtheria toxin receptor expressing iCOS-T mice, we show a profound impairment in Th2 cell localization to the lungs and skin of sensitized mice after allergen re-challenge. Mechanistically, ILC2-derived IL-13 is critical for eliciting IRF4+CD11b+CD103− DCs to produce the Th2 cell-attracting chemokine CCL17. Consequently, the sentinel function of DCs is contingent on ILC2s for the generation of an efficient memory Th2 cell response. These results elucidate a key new innate mechanism in the regulation of the adaptive memory immune response to allergens.
R970, A Selective Activator of Nrf2, is Efficacious in a Murine Model of Multiple Sclerosis
Authors: Owyang Alexander , Masuda Esteban S , Payan Donald G , Park Gary , Lau David , Torneros Allan , Pablo Florentino San , Bhamidipati Somasekhar , Singh Rajinder , Duncton Matthew A. J.
Published: January 2023

The transcription factor Nrf2 (nuclear factor erythroid-derived-2-like 2) is a key mediator of the anti-oxidant response. Nrf2 can be activated by alkylating its chaperone, Keap-1, resulting in a retardation of Nrf2 degradation, and increased Nrf2 in the cytoplasm. Upon translocation to the nucleus, Nrf2 binds an Antioxidant Response Element (ARE), which leads to the transcription of antioxidant gene products, many of which may be cytoprotective. Activation of the Nrf2 pathway may be useful against a wide-range of disorders, such as multiple sclerosis, psoriasis, and Huntington’s Disease, among others. For example, monomethylfumarate (MMF), the active component of the multiple sclerosis drug, dimethylfumarate (DMF), has been shown to activate the Nrf2-pathway at therapeutic concentrations. However, MMF also serves as an agonist of the niacin receptor, which is likely responsible for the flushing side-effect seen in patients taking DMF.

We have discovered a novel class of molecules that are potent activators of Nrf2 and are inactive at the niacin receptor, yet are efficacious in vivo in a model of multiple sclerosis. Lead candidate R970 is an orally-bioavailable small molecule activator of Nrf2 (EC50 = 1 uM; MMF EC50 = 127 uM), which is inactive at the niacin receptor (0% activity at 500 uM; MMF EC50 ca. 1 uM). Additionally, R970 is selective against a range of kinase, G-protein-coupled receptor, ion-channel, and transporter targets. When dosed orally in a murine model of multiple sclerosis, R970 delayed the onset and also suppressed clinical disease. Further exploration with R970 and other selective activators of Nrf2 is ongoing and may yield a next-generation Nrf2-therapeutic, with an improved side-effect profile.