Biology subjects

Crossland, N. A.

Publications and source records attributed to Crossland, N. A..

13 recordsLinked to original sources

bioRxiv2026

Deep Learning-Based Spatial Immunoprofiling of Multiplex Immunofluorescence Images Distinguishes Tuberculosis Disease States in Diversity Outbred Mice

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a major global health challenge, with approximately 10.8 million new cases and 1.25 million deaths reported in 2023. Human responses to M.tb are heterogeneous with clinical outcomes including bacterial clearance, asymptomatic latent M.tb infection, and severe fatal pulmonary TB. Here, we aim to address knowledge gaps in the organization of M.tb granulomas by identifying cell-based spatial features indicating asymptomatic lung infection. To address this gap, which cannot be directly studied in humans, we used lung sections from M.tb infected Diversity Outbred mice with acute pulmonary TB, asymptomatic M.tb infection, or chronic pulmonary TB that were stained for T cells, B cells, macrophages, and bronchiolar epithelial cells by multiplexed immunofluorescence. We first developed a new, accurate model to automatically segment lung granulomas, detect/quantify the cell types within granulomas, and extract the location of immune cells in granulomas for each disease state. Analysis of model-derived results show that lung granulomas from asymptomatic mice have a characteristic spatial profile consisting of higher CD4+ and CD8a+ T cell densities, closer B cell proximity to bronchiolar epithelium, and increased T cell-macrophage proximity in asymptomatic M.tb infection. Next, we propose a second novel approach to utilize a large language model (LLM) to independently decode complex cellular patterns within granulomas, and distinguished key immunological signatures: balanced immune expression in asymptomatic M.tb mice, dysfunctional responses with low cellularity in acute TB, and highest immune cells in chronic TB. Overall, the results from this study show that lung granulomas in asymptomatic infection are characterized by increased T cell density, increased numbers of peribronchiolar B cells, and T cells closer to macrophages as compared to acute and chronic TB. These methods and results help establish an automated pipeline to extract and analyze data from multiplexed fluorescence images and provide a foundation to better understand how granuloma architecture varies by disease state.

immunology↗

bioRxiv2025

A Mouse Model of SARS-CoV-2-Driven Acute Maladaptive Responses and Chronic Systemic Diseases

Our understanding of SARS-CoV-2 acute and post-acute pathogenesis is hindered by the lack of adequate small animal models. We present RAB/6N, a mouse model prone to severe disease after exposure to SARS-CoV-2 clinical isolates, with lethal cases showing no widespread brain infection typical of the widely used K18-hACE2 mouse model. Lung viral replication in RAB/6N mice remains steady for several days before a decline in viral titers. Delayed initiation of infection clearance is marked by increased lung T-cell extravasation and type-2 immune responses, leading to maladaptive lung consolidation. While systemic antiviral cytokine responses only correlate with SARS-CoV-2 brain infection in K18-hACE2 mice, they are concomitant with pulmonary immune dynamics in infected RAB/6N mice. Convalescent RAB/6N mice display systemic inflammation and decreased antibody titers against SARS-CoV-2 spike RBD, persistent viral RNA and prolonged lymphoid infiltration in the lungs. These animals also exhibit signatures of multi-organ dysfunction, cognitive impairment, cardiac inflammation, hyper- immunoglobulin production, and various autoimmune disorders, illuminating the molecular correlates of various pathologies associated with post-acute sequelae of COVID-19 (PASC). RAB/6N mice pave the way for dissecting the molecular drivers underlying SARS-CoV-2-induced acute maladaptive responses and subsequent post-acute systemic diseases. This preclinical platform also opens opportunities for the exploration of therapeutic interventions against systemic PASC and for anticipating the emergence of PASC-associated comorbidities. One-sentence summaryWe generated a hACE2-transgenic mouse model that develops maladaptive lung immune responses upon acute SARS-CoV-2 infection, leading to fatal outcomes or post-acute systemic disease syndromes in convalescent animals.

microbiology↗

bioRxiv2025

Sub-phenotypes of pneumonia defined by pulmonary histopathological features

Establishing sub-phenotypes of pneumonia based on distinct host processes will be a step towards using host-directed therapies (to complement microbe-directed therapies) more rationally and precisely. Although pneumonia is a pulmonary pathophysiology, histological changes within the lungs have not been leveraged for sub-phenotyping. We addressed this by scoring 18 histopathology features (e.g., type 2 cell hyperplasia or necrosis) across rapid autopsy lung samples from 276 elderly subjects with pneumonia. Machine learning algorithms segregated subjects into seven different sub-phenotypes of pneumonia with distinct histopathology signatures. Quantitative immunofluorescence demonstrated associations of macrophages, neutrophils, T cells, and B cells with select histology features and pulmonary pathology sub-phenotypes. Mouse models revealed corollary sub-phenotypes, although some histology features observed in human lungs were never observed in mice. By illuminating this spectrum of histopathologies and discriminating discrete sub-phenotypes of pneumonia, a foundational framework emerges for developing and using host-directed therapies for subsets of pneumonia patients.

immunology↗

bioRxiv2025

Protocol for Developing a Mouse Model of Post Primary Pulmonary Tuberculosis after Hematogenous Spread in Native Lungs and Lung Implants

This protocol describes a mouse model of post-primary pulmonary tuberculosis (PTB) that develops after hematogenous spread from the primary lesion in native lungs and subcutaneous lung implants. It demonstrates that virulent Mycobacterium tuberculosis (Mtb) disseminates to lymphoid tissue in many organs, but selectively damages the lungs. This approach demonstrates a particular vulnerability of the lung tissue to virulent Mtb independent of the route of infection and provides a robust platform for examining lung-specific mechanisms driving TB pathology. HighlightsO_LIMouse model for studying mechanisms driving post-primary pulmonary TB progression in immune hosts C_LIO_LIModels a hematogenous spread of virulent Mycobacterium tuberculosis to the lungs from a primary site of infection C_LIO_LIAllows for the investigation of lung-specific mechanisms of TB susceptibility using lung tissue implants. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/640830v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@1ce5384org.highwire.dtl.DTLVardef@cd3b88org.highwire.dtl.DTLVardef@1ff7ce7org.highwire.dtl.DTLVardef@614598_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure 1) to properly sequester intracellular iron and 2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled Ifn{beta} superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

immunology↗

bioRxiv2024

Resolution of SARS-CoV-2 infection in human lung tissues is driven by extravascular CD163+ monocytes

While human autopsy samples have provided insights into pulmonary immune mechanisms associated with severe viral respiratory diseases, the mechanisms that contribute to a clinically favorable resolution of viral respiratory infections remain unclear due to the lack of proper experimental systems. Using mice co-engrafted with a genetically matched human immune system and fetal lung xenograft (fLX), we mapped the immunological events defining successful resolution of SARS-CoV-2 infection in human lung tissues. Viral infection is rapidly cleared from fLX following a peak of viral replication, histopathological manifestations of lung disease and loss of AT2 program, as reported in human COVID-19 patients. Infection resolution is associated with the activation of a limited number of hematopoietic subsets, including inflammatory monocytes and non-canonical double-negative T-cells with cytotoxic functions, which are highly enriched in viral RNA and dissipate upon infection resolution. Activation of specific human fibroblast and endothelial subsets also elicit robust antiviral and monocyte chemotaxis signatures, respectively. Notably, systemic depletion of human CD4+ cells, but not CD3+ cells, abrogates infection resolution in fLX and induces persistent infection, supporting evidence that peripheral CD4+ monocytes are important contributors to SARS-CoV-2 infection resolution in lung tissues. Collectively, our findings unravel a comprehensive picture of the immunological events defining effective resolution of SARS-CoV-2 infection in human lung tissues, revealing markedly divergent immunological trajectories between resolving and fatal COVID-19 cases.

immunology↗

bioRxiv2022

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

microbiology↗

bioRxiv2022

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1Omicron

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date1-7. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes3,8. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

microbiology↗

bioRxiv2022

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

immunology↗

bioRxiv2021

Macrophages govern antiviral responses in human lung tissues protected from SARS-CoV-2 infection

The majority of SARS-CoV-2 infections among healthy individuals result in asymptomatic to mild disease. However, the immunological mechanisms defining effective lung tissue protection from SARS-CoV-2 infection remain elusive. Unlike mice solely engrafted with human fetal lung xenograft (fLX), mice co-engrafted with fLX and a myeloid-enhanced human immune system (HNFL mice) are protected against SARS-CoV-2 infection, severe inflammation, and histopathology. Effective control of viral infection in HNFL mice associated with significant macrophage infiltration, and the induction of a potent macrophage-mediated interferon response. The pronounced upregulation of the USP18-ISG15 axis (a negative regulator of IFN responses), by macrophages was unique to HNFL mice and represented a prominent correlate of reduced inflammation and histopathology. Altogether, our work shed light on unique cellular and molecular correlates of lung tissue protection during SARS-CoV-2 infection, and underscores macrophage IFN responses as prime targets for developing immunotherapies against coronavirus respiratory diseases. HIGHLIGHTSO_LIMice engrafted with human fetal lung xenografts (fLX-mice) are highly susceptible to SARS-CoV-2. C_LIO_LICo-engraftment with a human myeloid-enriched immune system protected fLX-mice against infection. C_LIO_LITissue protection was defined by a potent and well-balanced antiviral response mediated by infiltrating macrophages. C_LIO_LIProtective IFN response was dominated by the upregulation of the USP18-ISG15 axis. C_LI

microbiology↗

bioRxiv2021

Dissemination and progression of pulmonary Mycobacterium avium infection in mouse model are associated with type 2 macrophage activation

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are increasing worldwide and a growing public health concern. Pulmonary granulomas are the hallmark of MAC lung infection, yet reliable correlates of granuloma progression and susceptibility in immunocompetent hosts are poorly defined. The development of mouse models that recapitulate the diversity of granulomas seen in MAC pulmonary disease in humans is crucial to study mechanisms of susceptibility in humans and for preclinical evaluation of therapeutics. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. These mice became instrumental in pre-clinical testing of novel interventions. In this study we tested whether the B6.Sst1S that carries the sst1 mutant allele on standard B6 background develop more advanced pulmonary infection with NTM M. avium spp. hominissuis (M.av). To assess pulmonary disease progression, we utilized traditional semi-quantitative histomorphological evaluation and fluorescent multiplex immunohistochemistry (fmIHC) in combination with whole slide imaging and digital image analysis. After infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12 - 20 weeks post infection, although we did not observe the formation of necrotic granulomas during this interval. Using fmIHC, we determined that the disease progression was associated with a steadily increasing proportion of mycobacteria infected Arg1+ and double positive iNOS+/Arg1+ macrophages. The B6.Sst1S granulomas had a greater proportion of Arg1+ and double positive iNOS+/Arg1+ macrophages, and decreased T cell density, as compared to wild type B6 mice. Thus, the genetic composition of the B6.Sst1S mice renders them more susceptible to pulmonary M.av infection. In combination with more virulent clinical isolates of M.av these mice could provide an improved mouse model that recapitulates more severe pulmonary disease in humans. The Arg1 macrophage expression in this model combined with automated fmIHC could serve as a sensitive biomarker for the unbiased assessment of medical countermeasures against NTM infection.

pathology↗

bioRxiv2021

Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses

Globally there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals weight loss post-infection and decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology and prevents viral pneumonia. Combined with the marked stability and low production cost, this novel therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.

microbiology↗

bioRxiv2020

SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.

microbiology↗