Biology subjects

Boehm, D.

Publications and source records attributed to Boehm, D..

6 recordsLinked to original sources

bioRxiv2025

Characterization of a Novel Transmembrane Activating STING Agonist using Genetically Humanized Mice

STING is a pattern recognition receptor that activates type I interferon and proinflammatory responses in addition to unrelated molecular processes following exposure of DNA to the cytosol. Its pharmacologic stimulation enhances vaccine potency and generates effective antitumor responses but clinical trials evaluating STING agonists have not led to approval for human use. STING activation can occur through ligand engagement of either cytosolic or transmembrane protein domains, processes to which distinct cellular phenotypes are attributed. However, the only transmembrane STING agonist identified is human selective and in vivo testing in conventional models is not feasible. Here we describe synthesis of novel STING agonists efficacious against allelic variants of the protein. We also describe genetically humanized STING mice and demonstrate their suitability as a model to evaluate in vivo responses following exogenous administration of human-selective agonists. Experiments demonstrate that the lead molecule (termed INI3069) functions through binding to the STING transmembrane region and its comparison with conventional agonists reveals significant differences in molecular and immune effects. INI3069 can also enhance antibody responses to co-administered antigens and antitumor responses. This work both represents the first in vivo examination of the effects of transmembrane STING agonism and demonstrates efficacy of a potential novel vaccine adjuvant and oncological therapeutic.

immunology↗

bioRxiv2025

Comparative Molecular, Innate, and Adaptive Impacts of Chemically Diverse STING Agonists

Pharmacologic activation of the innate immune response is being actively being pursued for numerous clinical purposes including enhancement of vaccine potency and potentiation of anti-cancer immunotherapy. Pattern recognition receptors (PRRs) represent especially useful targets for these efforts as their engagement by agonists can trigger signaling pathways that associate with phenotypes desirable for specific immune outcomes. Stimulator of interferon genes (STING) is an ER-resident PRR reactive to cyclic dinucleotides such as those synthesized endogenously in response to cytosolic dsDNA. STING activation leads to transient generation of type I interferon (IFN-I) and proinflammatory responses that augment immunologically relevant effects including antiviral responses, antigen presentation, immune cell trafficking, and immunogenic cell death. In recent years engineered cyclic dinucleotides and small molecules have been discovered that induce STING and safely confer clinically useful outcomes in animal models such as adjuvanticity of anti-microbial vaccines and tumor clearance. Unfortunately, clinical trials examining the efficacy of STING agonists have thus far failed to satisfactorily recapitulate these positive outcomes and this has prevented their translational advancement. A likely relevant yet perplexingly under investigated aspect of pharmacologic STING activation is the diversity of molecular and immune responses that associate with chemical properties of the agonist. Based on this, a comparative survey of these was undertaken using unrelated STING-activating molecules to characterize the molecular, innate, cellular, and immune outcomes they elicit. This was done to inform and direct future studies aimed at designing and selecting agonists appropriate for desired clinical goals. This revealed demonstrable differences between the agonists in potency, transcriptomes, cytokine secretion profiles, immune cell trafficking, and antigen-directed humoral and cell mediated immune responses. As such, this work illustrates that phenotypes deriving from activation of a protein target can be linked to chemical properties of the engaging agonist and thus heightened scrutiny is necessary when selecting molecules to generate specific in vivo effects.

immunology↗

bioRxiv2024

Renal proximal tubules are sensitive to metabolic acidosis

Patients suffering from distal renal tubular acidosis (dRTA) are sometimes diagnosed with proximal tubule dysfunction with leaks of phosphate, uric acid, amino acids, and low-molecular-weight proteins, also known as Fanconi-like syndrome. The underlying molecular basis is largely elusive. We previously reported on Atp6v0a4 knockout (KO) mice, which exhibit severe metabolic acidosis in combination with proximal tubule dysfunction as evidenced by phosphaturia and proteinuria. Here, we show that Rab7, a key regulator of endo-lysosomal trafficking and lysosomal biogenesis, is strongly diminished in proximal tubules of Atp6v0a4 KO mice, while the number of abnormal Ist1-labelled Lamp1-positive vesicles is increased. This is accompanied by the accumulation of autophagosomes, autolysosomes and autophagic substrates. Importantly, correction of metabolic acidosis with bicarbonate therapy resolves proximal tubule dysfunction and trafficking defects in Atp6v0a4 KO mice. Acid-challenged wildtype mice also show trafficking defects with Rab7-downregulation and an increase in Ist1-labeled Lamp1-positive vesicles and develop proximal tubule damage in the long-term. Similar acidosis-induced alterations also occur in human kidney organoids. Altogether, our data provide insights, why patients suffering from severe dRTA may develop a Fanconi-like syndrome, which may contribute to the progression of chronic kidney failure. Translational StatementPatients with renal acidosis caused by impaired proton secretion in the collecting duct (distal renal tubular acidosis - dRTA) sometimes show unexplained symptoms of proximal tubule dysfunction such as proteinuria and phosphaturia. Here, we show that proximal tubules are particularly sensitive to acidosis as evidenced by impaired trafficking, lysosomal damage and accumulation of autophagic substrates. We also show that early treatment of dRTA by alkali supplementation can prevent proximal tubule dysfunction. Because metabolic acidosis represents a well-known risk factor for the progression of chronic kidney disease (CKD), our findings highlight the potential clinical importance of early alkali supplementation to delay disease progression.

molecular biology↗

bioRxiv2021

Re-investigating the coughing rat model of pertussis to understand Bordetella pertussis pathogenesis

Bordetella pertussis (Bp) is a highly contagious bacterium that is the causative agent of whooping cough (pertussis). Currently, acellular pertussis vaccines (aP; DTaP; Tdap) are used to prevent pertussis disease. However, it is clear that the aP vaccine efficacy quickly wanes, resulting in the re-emergence of pertussis. Furthermore, recent work performed by the CDC suggest that current circulating strains are genetically distinct from strains of the past. Emergence of genetically diverging strains combined with waning aP vaccine efficacy call for re-evaluation of current animal models of pertussis. In this study, we used the rat model of pertussis to compare two genetically divergent strains Tohama 1 and D420. We intranasally challenged seven-week-old Sprague-Dawley rats with 108 viable Tohama 1 and D420 and measured the hallmark signs/symptoms of Bp infection such as neutrophilia, pulmonary inflammation, and paroxysmal cough using whole body plethysmography. Onset of cough occurred between 2-4 days after Bp challenge averaging five coughs per fifteen minutes, with peak coughing occurring at day eight post infection averaging upward of thirteen coughs per fifteen minutes. However, we observed an increase of coughs in rats infected with clinical isolate D420 through 12 days post challenge. The rats exhibited increased bronchial restriction following Bp infection. Histology of the lung and flow cytometry confirm both cellular infiltration and pulmonary inflammation. D420 infection induced higher production of anti-Bp IgM antibodies compared to Tohama 1 infection. The coughing rat model provides a way of characterizing disease manifestation differences between Bp strains.

immunology↗

bioRxiv2020

FOXO1 promotes HIV Latency by suppressing ER stress in T cells

Quiescence is a hallmark of CD4+ T cells latently infected with HIV-1. While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. Here we report that FOXO1, a key regulator of T-cell quiescence, promotes latency and suppresses productive HIV infection. In resting T cells, FOXO1 inhibition induces ER stress and activates two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of PKR-like endoplasmic reticulum kinase (PERK), a known link between ER stress and ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppress HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a novel link between FOXO1, ER stress, and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.

immunology↗

bioRxiv2017

Development Of RNAseq Methodologies To Profile The In Vivo Transcriptome Of Bordetella pertussis During Murine Lung Infection

Bordetella pertussis is an obligate human respiratory pathogen that causes the disease whooping cough. A whole cell vaccine (DTP) was developed in the 1940s and was subsequently replaced in the 1990s with a protein-based subunit acellular vaccine (DTaP; tdap). Today, we are observing a resurgence of whooping cough due to evolution of the pathogen and waning vaccine immunity. The use of vaccines decreased the need for basic research on this pathogen. As a result, numerous questions on the basic pathogenesis of B. pertussis remain to be answered. Microarrays and more recently, RNA sequencing (RNAseq), have allowed the field to describe the in vitro gene expression profiles of the pathogen growing in both virulent and avirulent phases; however, no published studies have described an in vivo transcriptome of the pathogen. To address this need, we have designed and evaluated workflows to characterize the in vivo transcriptome of B. pertussis during infection of the murine lung. During our initial studies, we observed that only 0.014% of the ~100 million 2x50bp illumina reads corresponded to the pathogen, which is insufficient for analysis. Therefore, we developed a simple protocol to filter the bacteria out of the tissue homogenates and separate bacterial cells from the host tissue. RNA is then prepared, quantified, and the B. pertussis to host RNA ratio is determined. Here, we present the protocol and discuss the uses and next directions for which this RNAseq workflow can be applied. With this strategy we plan to fully characterize the B. pertussis transcriptome when the pathogen is infecting the murine lung in order to identify expressed genes that encode potential new vaccine antigens that will facilitate the development of the next generation of pertussis vaccines.

microbiology↗