Biology subjects

Johnson, E. E.

Publications and source records attributed to Johnson, E. E..

9 recordsLinked to original sources

bioRxiv2026

Epithelial NCAPD3 expression protects against stress-induced intestinal injury in mice

Intestinal epithelial cells are central players in mucosal barrier integrity and host-microbe interactions. Genetic studies have revealed that epithelial dysfunction is a key contributor to the pathogenesis of inflammatory bowel disease. Non-SMC condensin II complex subunit D3 (NCAPD3) is essential for chromatin organization and stability. NCAPD3 also promotes antimicrobial defense and autophagy responses in vitro. NCAPD3 expression is decreased in intestinal epithelial cells from patients with ulcerative colitis; however, it is not known whether loss of NCAPD3 expression drives intestinal barrier dysfunction or is a result of disease-associated inflammation. To investigate this relationship in vivo, a tissue-specific approach was required, as global constitutive knockout of NCAPD3 is embryonic lethal. Therefore, a transgenic mouse line with doxycycline-inducible expression of a short hairpin RNA targeting NCAPD3 restricted to villin-expressing cells was generated (NCAPD3KD mice) to enable the study of NCAPD3 function in the intestinal epithelium. Treatment of NCAPD3KD mice with 9-tert-butyl doxycycline resulted in [~]75% reduction of NCAPD3 protein in EpCAM+ intestinal cells. Short-term epithelial NCAPD3 knockdown did not induce spontaneous colitis but was associated with increased serum amyloid A and a trend towards increased intestinal permeability. Upon dextran sodium sulfate or Salmonella enterica serovar Typhimurium {Delta}AroA challenge, NCAPD3KD mice exhibited exacerbated weight loss, higher disease activity, increased histopathological damage, abnormal colonic cytokines and chemokines, and significantly increased intestinal permeability. These results indicate that NCAPD3 expression in the intestinal epithelium is required for optimal barrier maintenance and antimicrobial defense under chemical or microbial stress. These findings support prior in vitro observations and solidify NCAPD3 as a regulator of intestinal epithelial barrier function and mucosal host defense. Author SummaryNCAPD3 is a multifunctional protein with established roles in chromatin organization, genome stability, mitochondrial function, and antimicrobial defense. Dysregulated NCAPD3 is implicated in human diseases, such as inflammatory bowel disease (IBD) and microcephaly; however, due to its essential role in cellular division, determination of whether NCAPD3 loss drives these pathologies in vivo has been lacking. Using a new transgenic mouse model that selectively reduces NCAPD3 expression in intestinal epithelial cells, our study establishes NCAPD3 as an epithelial regulator of the mammalian intestine that enhances epithelial barrier resilience and antimicrobial defense during stress. Although dispensable for short-term basal homeostasis, NCAPD3 function becomes critical during epithelial injury and enteric infection. Reduced NCAPD3 expression may therefore lower the threshold for inflammatory disease by weakening barrier integrity, amplifying inflammatory cascades, and impairing antimicrobial defenses. These findings position NCAPD3 as a potential modulator of IBD susceptibility and highlight chromatin organization as an important, previously underappreciated layer of intestinal epithelial regulation.

cell biology↗

bioRxiv2026

Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

Borrelia burgdorferi causes over 470,000 infections annually [1]. Following a tick bite, Borrelia spirochetes replicate in human skin and disseminate through tissues, including subcutaneous adipose tissue. Adipose tissue is a complex organ containing non-immune and immune cells that play a significant role in the immune response [2], yet little is known about adipose tissue signaling after Borrelia infection. We investigated the landscape of immune signaling within adipose tissue-resident cells during Borrelia infection in human tissue (skin and adipose tissue) ex vivo. Immune pathways overall were downregulated in adipose tissue during Borrelia infection. Despite this, adipose stem/progenitor cells exhibited increased pro-inflammatory and extracellular matrix (ECM)-related signaling (IL-6, MIF, collagen, laminin, fibronectin), positioning them as key hubs of intercellular communication during infection. Myeloid lineage cells showed cluster-specific upregulation of immune genes such as IL1B and TNF. Network analyses highlighted laminin, and strong outgoing MIF signals in all adipose-resident cell clusters. Among the identified genes modulated after Borrelia infection, we observed reduced expression levels of serum amyloid A in adipose tissue-resident cells, a marker typically elevated in Lyme disease patient serum during acute infection. Here, we show that human serum amyloid A1 and serum amyloid A2 directly bind B. burgdorferi spirochetes, using flow cytometry. Functionally, serum amyloid A2 reduces spirochete viability in vitro in a dose-dependent manner and enhances opsonization and phagocytosis by macrophages. Together, these findings show that adipose tissue is a site of active immune signaling in the setting of Borrelia infection and identifies SAA as a host defense factor against Borrelia.

immunology↗

bioRxiv2025

CONSERVED LIFE HISTORY PHENOTYPES AMONG HISTORICALLY ISOLATED CLADES IN THE CHIRAL LIVEBEARER FISH OF LOWER CENTRAL AMERICA

Lower Central America contains some of the highest freshwater fish diversity on the planet. Yet, species by species, we still know remarkably little about factors that have created this diversity and the threats that could put it at risk. The chiral livebearer (Xenophallus umbratilis) exemplifies these deficits. This freshwater livebearing fish species primarily occupies habitats along the high-elevation fringes of large volcanoes in the central cordillera of Costa Rica. Previous work suggests that current populations have been isolated by repeated marine incursions beginning around 5 mya. Genetic data point to four putative clades that could be considered evolutionarily distinct. Unfortunately, beyond this we know little about adaptive evolution of demographically relevant traits among these clades, making it difficult to determine the conservation status of this species overall. Moreover, a recent IUCN assessment of this species was limited to only a portion of its geographic range and lacked population level information from many locations where it occurs. To address these gaps, here we describe the life history of this species, taken from 23 collections made across its geographic range from locations where this fish is locally abundant. This allowed us to determine if life history traits differ among populations coincident with known population structure. Despite long periods of isolation among clades, we found only modest evidence for divergence in life history phenotypes, and this was limited to just two of the four clades. These data suggest that life history traits are mostly conserved among populations, data that are critical to future conservation planning.

ecology↗

bioRxiv2025

DMEM and EMEM are suitable surrogate media to mimic host environment and expand leptospiral pathogenesis studies using in vitro tools

Pathogenic Leptospira species can survive and thrive in a wide range of environments. Distinct environments expose the bacteria to different temperatures, osmolarities, and amounts and sources of nutrition. However, leptospires are mostly cultured, in a laboratory setting under in vitro conditions that do not reflect natural environments. This constraint on laboratory cultures limits the applicability of in vitro studies to the understanding of even simple pathogenic processes. Here we report, investigate, and identify a medium and conditions that mimic the host environment during leptospirosis infection, expanding the available in vitro tools to evaluate leptospiral pathogenesis. We quantified genome-wide gene expression of pathogenic Leptospira interrogans cultured in different in vitro media compositions (EMJH, DMEM, EMEM, and HAN). Using EMJH as standard, we compared gene expression in these compositions to genome-wide gene expression gathered in a host environment: whole blood (WB) of hamsters after infection with pathogenic leptospires. Leptospires cultured in DMEM and EMEM media shared 40% and 47% of all differentially expressed genes (DEGs) of leptospires present within WB (FDR<0.01), while leptospires cultured in HAN media only shared 20% of DEGs with those from WB. Furthermore, gene and pathway expression of leptospires cultured on DMEM and EMEM media exhibited a better correlation with leptospires grown in WB, including promoting expression of a similar leptospiral lipid A profile to the one identified directly in host tissues. Taken together, these results indicate that commercial cell-culture media EMEM or DMEM are better surrogates for in vivo pathogenic studies than EMJH or HAN media in Leptospira. These alternative culture conditions, using media that are a standard supply worldwide, provide a reproducible and cost-effective approach that can accelerate research investigation and reduce the number of animal infections necessary for basic research of leptospirosis.

microbiology↗

bioRxiv2022

Inhibition of pyrimidine synthesis in murine skin wounds induces a pyoderma gangrenosum-like neutrophilic dermatosis accompanied by spontaneous gut inflammation

Pyoderma gangrenosum (PG) is a debilitating skin condition often accompanied by inflammatory bowel disease (IBD). Strikingly, [~]40% of patients that present with PG have underlying IBD, suggesting shared but unknown pathogenesis mechanisms. Impeding the development of effective treatments for PG is the absence of an animal model that exhibits features of both skin and gut manifestations. This study describes the development of the first experimental drug-induced mouse model of PG with concurrent intestinal inflammation. Topical application of pyrimidine synthesis inhibitors on wounded mouse skin generates skin ulcers enriched in neutrophil extracellular traps (NETs) and pro-inflammatory cellular as well as soluble mediators mimicking human PG. The mice also develop spontaneous intestinal inflammation demonstrated by histologic damage. Further investigations revealed increased circulating immature low-density IL-1{beta} primed granulocytes that undergo enhanced NETosis at inflamed tissue sites supported by increase in circulatory citrullinated histone 3, a marker of aberrant NET formation. Granulocyte depletion dampens the intestinal inflammation in this model, further supporting the notion that granulocytes contribute to the skin-gut crosstalk in PG mice. We anticipate that this novel murine PG model will enable researchers to probe common disease mechanisms and identify more effective targets for treatment for PG patients with IBD.

immunology↗

bioRxiv2022

N-phosphonacetyl-L-aspartate enhances type I interferon anti-viral responses through activation of non-canonical NOD2 signaling

Type I interferon production and the expression of interferon-stimulated genes (ISGs) are key components of an innate immune response to many microbial pathogens. Dysregulation of this response can result in uncontrolled infection, inflammation, and autoimmune disease. Understanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights into infection control strategies and autoimmune disease therapies. Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor that acts as both a bacterial sensor protein and a mediator of antiviral responses. Antibacterial functions of NOD2 are enhanced by treatment with the small molecule inhibitor of pyrimidine biosynthesis N-phosphonacetyl-L-aspartate (PALA), though how this might function in the host antiviral response remains unknown. Therefore, we tested the ability of PALA to enhance NOD2-dependent antiviral responses. Alone, PALA treatment of macrophages was not sufficient to induce interferon {beta} (IFN{beta}) production or ISG expression. Instead, PALA synergized with IFN{beta} stimulation to enhance expression and activation of interferon-stimulated gene factor 3 (ISGF3) and induce the upregulation of a subset of ISGs in co-treated cells. Furthermore, PALA treatment of epithelial cells resulted in impaired viral replication of the herpesvirus, human cytomegalovirus. Induction of the PALA-enhanced antiviral response required activation of non-canonical NOD2 signaling mediated by mitochondrial antiviral-signaling protein (MAVS) and interferon response factor 1 (IRF1), rather than the classical receptor-interacting serine/threonine protein kinase 2 (RIP2) pathway or other IRFs previously reported to mediate NOD2 antiviral responses. These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses and antiviral activity. Significance StatementUnderstanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights to improve infection control strategies and autoimmune disease therapies. This work demonstrates that the pyrimidine synthesis inhibitor N-phosphonacetyl-L-aspartate synergizes with type I interferon to enhance antiviral responses through activation of a non-canonical NOD2 signaling pathway. These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses.

molecular biology↗

bioRxiv2021

Development of a Reproducible Porcine Model of Infected Burn Wounds

Severe burns are traumatic and physically debilitating injuries with a high rate of mortality. Bacterial infections often complicate burn injuries, which presents unique challenges for wound management and improved patient outcomes. Currently, pigs are used as the gold standard of pre-clinical models to study infected skin wounds due to the similarity between porcine and human skin in terms of structure and immunological response. However, utilizing this large animal model for wound infection studies can be technically challenging and create issues with data reproducibility. We present a detailed protocol for a porcine model of infected burn wounds based on our experience in creating and evaluating partial thickness burn wounds infected with Staphylococcus aureus on six pigs. Wound healing kinetics and bacterial clearance were measured over a period of 27 days in this model. Enumerated are steps to achieve standardized wound creation, bacterial inoculation, and dressing techniques. Systematic evaluation of wound healing and bacterial colonization of the wound bed is also described. Finally, advice on animal housing considerations, efficient bacterial plating procedures, and overcoming common technical challenges is provided. This protocol aims to provide investigators with a step-by-step guide to execute a technically challenging porcine wound infection model in a reproducible manner. Accordingly, this would allow for the design and evaluation of more effective burn infection therapies leading to better strategies for patient care. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/456568v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d00970org.highwire.dtl.DTLVardef@11bb8caorg.highwire.dtl.DTLVardef@10110d0org.highwire.dtl.DTLVardef@f6d89d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2019

Differential sperm motility mediates the sex ratio drive shaping mouse sex chromosome evolution

SummaryThe search for morphological or physiological differences between X- and Y-bearing mammalian sperm has provoked controversy for decades. Many potential differences have been proposed, but none validated, while accumulating understanding of syncytial sperm development has cast doubt on whether such differences are possible even in principle. We present the first ever mammalian experimental model to trace a direct link from a measurable physiological difference between X- and Y-bearing sperm to the resulting skewed sex ratio. We show that in mice with deletions on chromosome Yq, birth sex ratio distortion is due to a relatively greater motility of X-bearing sperm, and not to any aspect of sperm/egg interaction. Moreover, the morphological distortion caused by Yq deletion is more severe in Y-bearing sperm, providing a potential hydrodynamic basis for the altered motility. This reinforces a growing body of work indicating that sperm haploid selection is an important and underappreciated evolutionary force.

developmental biology↗

bioRxiv2019

A conserved requirement for Fbxo7 during male germ cell cytoplasmic remodelling

Summary statementFbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here we show that in addition to the previously-known Parkinsonian and haematopoietic phenotypes, Fbxo7-deficient male mice are completely sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the cells undergo cytoplasmic remodelling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7-deficient males. Mutation of the Drosophila Fbxo7 orthologue, nutcracker (ntc) was previously shown to cause sterility at a similar stage of germ cell development, indicating that the requirement for Fbxo7 is conserved. The ntc phenotype was attributed to proteasome mis-regulation via an interaction with the proteasome regulator, DmPI31. Our data suggest rather that in mice, the requirement for Fbxo7 is either independent of its interaction with PI31, or relates specifically to cytoplasmic proteasome activity during spermiogenesis.

developmental biology↗