Biology subjects

Gertje, H. P.

Publications and source records attributed to Gertje, H. P..

9 recordsLinked to original sources

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

3D Multiplexed Immunohistochemistry Using Opal-TSA Amplification for Enhanced Imaging of Pulmonary TB Lesions

Mycobacterium tuberculosis hijacks the host immune system and persists for several years before the onset of active disease. Spatial characterization of epithelial compartments, immune cell populations and bacteria simultaneously within tissue specimens provide significant information about host pathogen interactions. Here, we present a protocol to detect multiple protein markers using Opal-TSA conjugated fluorescent dyes in free floating 10% neutral buffered formalin fixed thick tissue sections (50-100 m), with minimal additional tissue processing not requiring specialized equipment. Use of thick sections provides more information as compared to classic thin microtomy sections (3-10 m), including the capacity for Z stacking and three-dimensional image rendering. Importantly, reduced tissue processing of samples with this method preserves endogenous fluorescent reporter signal. Use of Opal-TSA conjugated fluorescent dyes enhances the sensitivity of low expressing proteins and supports the use of primary antibodies raised in the same species. Before you beginThis protocol describes the specific use of Mtb infected mouse lungs, but is applicable to any tissue type and species of origin. Institutional permissionsObtain institutional permission to perform animal studies and collect tissues under an approved Institutional Animal Care and Use Committee (IACUC) or Institutional Review Board protocol. Our protocol was approved by Boston Universitys Institutional Animal Care and Use Committee (IACUC protocol number PROTO201800218). MiceB6J.C3-Sst1C3HeB/Fej Krmn and B6. Sst1S, ifnb-YFP mice were developed in our laboratory (available from MMRRC stock # 043908-UNC). HighlightsO_LICompatible with Opal-TSA conjugated fluorescent dyes resulting in enhanced signal sensitivity with low background noise C_LIO_LIReduced tissue processing preserves endogenous fluorescent reporter signals C_LIO_LICompatible with primary antibodies raised in the same species C_LIO_LIDoesnt require specialized automated instruments C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/619885v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@3e3439org.highwire.dtl.DTLVardef@1d6ba85org.highwire.dtl.DTLVardef@6f9595org.highwire.dtl.DTLVardef@1c573ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery

Modification of RNA with N6-methyladenosine (m6A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m6A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m6A writers Mettl3 and Mettl14, nor the m6A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro. To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

molecular biology↗

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↗

bioRxiv2023

Aberrant macrophage activation and failed regeneration of pulmonary epithelium promote tuberculosis progression uniquely in lung tissue

Pulmonary tuberculosis (PTB) represents 85% of the disease burden caused by Mycobacterium tuberculosis (Mtb) and promotes aerosol transmission infecting about a quarter of people globally. Most Mtb infections are effectively limited within primary granulomatous lesions. Containment failures lead to hematogenous spread and the formation of post-primary destructive PTB lesions. Factors that favor Mtb survival and replication in the lungs after hematogenous spread despite systemic immunity represent appealing targets for host-directed TB therapies, but are currently unknown. We developed a novel mouse model that mimics progression of chronic post-primary PTB in humans: wherein PTB lesions form after hematogenous spread from a remote primary lesion in immunocompetent but TB-susceptible B6.Sst1S mice. The B6.Sst1S mice developed PTB lesions featuring granulomatous pneumonia, bronchogenic expansion and broncho-occlusion closely resembling post-primary PTB in humans. Using spatial transcriptomic and fluorescent multiplexed immunochemistry, we demonstrated the expansion of myeloid cell populations with the appearance of alternatively activated macrophages, dissolution of initial lymphoid follicles, and accumulation of de-differentiated lung epithelial cells in the advanced PTB lesions. To determine whether lung parenchymal cells or lung oxygenation were necessary for the pulmonary TB progression, we implanted lung and spleen fragments subcutaneously to serve as potential targets for hematogenous spread. The lung (but not spleen) implants displayed characteristic organized granulomas with necrosis and Mtb replication demonstrating that deleterious interactions of aberrantly activated macrophages with the inflammation-injured lung resident cells, and possibly hypoxia, not oxygenation, are critical determinants of PTB progression in immunocompetent hosts. Necrotic TB lesions also developed in subcutaneous implants of human lung tissue in mice with human immune system after respiratory infection. These animal models may serve to further dissect the lung-specific mechanisms of host susceptibility to virulent Mtb and for testing therapeutic interventions targeting these mechanisms.

immunology↗

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↗

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↗