Biology subjects

More, S.

Publications and source records attributed to More, S..

At least 19 recordsLinked to original sources

bioRxiv2026

An endolysin-expressing probiotic attenuates Clostridioides difficile infection in germ-free and dysbiotic microbiota-associated mice

Disruption of the intestinal microbiota increases susceptibility to Clostridioides difficile infection (CDI), creating an opportunity for prophylactic interventions before symptomatic disease develops. We investigated whether Escherichia coli Nissle 1917 (EcN) engineered to express the bacteriophage-derived endolysin CD27L could enhance pathogen inhibition and attenuate CDI. Using a plasmid-cured EcN chassis and pMUT2-derived vectors, we generated strains expressing CD27L under constitutive or anaerobically regulated promoter control. The constitutive strain, BioEcN1, reduced viable C. difficile recovery relative to parental EcN and was selected for animal studies. BioEcN1 remained recoverable from monoassociated mice without in vivo antibiotic selection, with similar recovery on selective and nonselective media supporting construct retention. Prophylactic administration reduced clinical disease, body-weight loss, and intestinal histopathological injury in germ-free mice. Observed survival was 100% in BioEcN1-treated challenged mice versus 50% in untreated challenged mice during the monitored period. Protection was accompanied by lower fecal and cecal pathogen burdens. In mice harboring a human-derived dysbiotic microbiota, prophylactic BioEcN1 also reduced clinical and stool-consistency scores, with lower early fecal pathogen recovery and less colon shortening. These findings establish preclinical proof of concept for prophylactic administration of an endolysin-expressing probiotic to attenuate CDI.

microbiology↗

bioRxiv2026

Macrophage-instructed GSDME couples glioblastoma cell-state plasticity with inflammatory cell death

Glioblastoma (GBM) cell states reflect spatial microenvironmental interactions. Here, using COMET spatial proteomics and RNAscope across multiregional human GBM tissue, spanning tumor cores with pseudopalisading regions, infiltrative margins and peripheral regions, together with multiplex spatial profiling of 202 specimens from 33 patients with matched primary and recurrent tumors, we identify full-length gasdermin E (GSDME-FL) as a macrophage-instructed regulator of malignant cell plasticity. Integration with single-cell transcriptomics and functional perturbation shows that GSDME-high tumor cells localize to macrophage-rich perivascular niches and are associated with delayed recurrence and longer patient survival. Mechanistically, macrophage-derived S100A4 activates EGFR-Sp1 signaling to induce GSDME-FL in neighboring GBM cells. GSDME-FL restrains hypoxia-associated mesenchymal plasticity and shapes macrophage-induced tumor state transitions independently of caspase activation. During immunogenic cell death (ICD), cleaved GSDME promotes pre-lytic swelling, early ATP efflux, and the release of canonical ICD-associated cytokines and chemokines. NLRP3 signaling further supports ATP release and licenses macrophage phagocytosis of dying GBM cells, whereas NINJ1-dependent membrane rupture enables terminal HMGB1 release. Vaccination with ICD-treated glioma cells elicits tumor rejection in a prophylactic intracranial challenge model. Notably, co-expression of GSDME and NINJ1 in macrophage-rich perivascular niches provides a spatial correlate of pathway convergence in patient tumors. Thus, our findings define how a spatial macrophage niche induces GSDME-FL, thereby coupling malignant cell state plasticity to the inflammatory properties of GBM cell death.

cancer biology↗

bioRxiv2025

Nitric Oxide-Releasing Thixotropic Hydrogels as Antibacterial and Hemocompatible Catheter Locks

Catheters are indispensable medical tools for accessing blood vessels, hollow organs, and body cavities to facilitate medication delivery and fluid drainage. However, they also serve as major entry points for bacterial contamination and trigger foreign body responses, necessitating locking strategies that are both bactericidal and biocompatible. This study introduces the first gel-based catheter lock, in contrast to conventional liquid locks. The gel is a poloxamer-based hydrogel formulated with 2-hydroxypropyl -cyclodextrin (HP-CD). HP-CD forms supramolecular complexes with the poloxamer to enhance gelation, and with the nitric oxide (NO) donor to modulate NO release kinetics. This thixotropic gel can be injected into the catheter lumen when the catheter is not in use and withdrawn when vascular access is needed. The gel matrix provides a physical barrier that slows bacterial migration and minimizes drug loss. Simultaneously, the released NO functions as a broad-spectrum antimicrobial agent, effectively preventing biofilm formation on both the internal and external surfaces of the catheter. The NO-releasing hydrogel also demonstrates excellent hemocompatibility and reduces clot adhesion. Together, the gel-based lock offers a promising strategy for more effective catheter maintenance and represents a new application of hydrogels.

bioengineering↗

bioRxiv2025

An autoimmunity-associated allele of PTPN22 enhances the innate immune response to promote protection against acute coronavirus infection

Allelic variation can impact viral clearance and disease severity. Still, our understanding of the effect of the autoimmunity-associated allelic variant of Ptpn22 (PEP-R619W) on antiviral immunity remains incomplete, as previous reports have only focused on chronic Lymphocytic Choriomeningitis virus (LCMV) infection. This research defines how the loss of Ptpn22 (PEP-null) and PEP-R619W changes antiviral immunity during an acute coronavirus infection. We address the hypothesis that CRISPR/Cas9-generated PEP-null and PEP-R619W mice have enhanced antiviral immunity over PEP-WT mice during coronavirus infection. Following Mouse Hepatitis Virus (MHV) A59 infection, we interrogated pathology, cytokine production, and cellular responses in the blood, spleen, and liver of PEP-WT, PEP-null, and PEP-R619W mice. Key findings show that PEP-R619W mice have reduced viral titer and weight loss, increased survival, and more mature natural killer (NK) cells in the liver and spleen compared to PEP-WT mice. Interestingly, protection against disease in PEP-null mice was inoculation-dose-dependent, whereas PEP-R619W conferred immunity regardless of infection dose. Further, Rag1-/- PEP-R619W mice had increased survival and reduced viral titer over Rag1-/- PEP-WT mice. PEP-R619W mice also had higher concentrations of IFN{gamma} and enhanced IFN{gamma} production by mature NK cells in the liver at 3 days post-infection. Finally, NK cell depletion elevated PEP-R619W viral titer to similar levels as PEP-WT mice. This is one of the first studies investigating the role of Ptpn22 within NK cells and demonstrates that the Ptpn22 allelic variant augments NK cell function and is beneficial during coronavirus infection. Significance StatementApproximately 5-15% of the North American population has the PTPN22 1858C>T allele, which has been linked with numerous autoimmune diseases and is considered the highest non-HLA risk allele for autoimmunity. Due to this link, the PTPN22 1858C>T allele is often considered pathologic or detrimental. However, recent studies have demonstrated the benefit of this allele in protection from chronic virus infection and some cancers. Yet, a significant research gap remains in understanding how the PTPN22 1858C>T allele impacts the immune response during acute, moribund infections. Using mice that are homozygous for the equivalent, autoimmunity-associated allele in Ptpn22, we demonstrate that this allele uniquely augments innate immunity and enhances Natural Killer cell function to protect against coronavirus infection.

immunology↗

bioRxiv2025

TL1A overexpression in Crohn's Disease and mice alters Paneth cells and microbiota promoting ileal inflammation

Paneth cells regulate host-microbial homeostasis and defects in autophagy and host defense pathways have been associated with inflammatory bowel diseases (IBD). Genetic variants in TL1A (TNFSF15) and its receptor DR3 (TNFRSF25) have been associated with IBD. TL1A expression is increased in IBD patients, particularly in TL1A risk allele carriers. However, effects of TL1A on Paneth cells, resident microbiota, and development of ileitis remain unknown. TL1A overexpression in mice induces Paneth cell hyperplasia and morphological abnormalities preceding the development of ileitis. In Crohns disease (CD) patients, ileal TL1A expression was associated with abnormal Paneth cell phenotypes. We confirmed direct effects of TL1A on Paneth cells in human iPSC-derived human intestinal organoids and mouse Paneth cell-enriched organoids. Resident microbiota was required for TL1A-mediated Paneth cell dysfunction, and ileitis. Tl1a-tg mice were enriched in short chain fatty acid-producing bacteria and the metabolite acetate. Acetate supplementation in WT or Tl1a-tg mice caused ileal inflammation, suggesting that acetate is sufficient to cause ileitis. DR3-deficiency in Paneth cells resulted in Paneth cell abnormalities and microbiome composition changes. Our findings provide a mechanistic link between overexpression of TL1A in CD patients, Paneth cell dysfunction, and enrichment of acetate-producing bacteria and acetate that promotes ileal inflammation. Brief SummaryOverexpression of TL1A drives Paneth cell dysfunction in Crohns Disease and mice leading to microbial and metabolomic changes that promote small bowel inflammation.

immunology↗

bioRxiv2025

Exploiting the Angiotensin-Converting Enzyme Pathway to Augment Endogenous Opioid Signaling

Angiotensin Converting Enzyme (ACE) impacts hemodynamics by regulating the conversion of angiotensin I to the vasoconstricting angiotensin II. We recently identified a non-canonical central role of ACE in the degradation of enkephalin heptapeptide, Met-enkephalin-Arg-Phe (MERF). Enkephalins are short-lived, endogenous opioid peptides that mediate the bodys intrinsic analgesic response. Here we identify chemically diverse ACE inhibitors using an optimized high throughput screening assay to boost endogenous opioid signaling. Our primary hits (thiorphan, D609, and raloxifene) were selected for dose-response characterization, in vitro enkephalin release, in vivo analgesic potency, and in silico analysis. Intracerebroventricular administration of these compounds significantly attenuated pain response, alone and in combination with MERF, which was reversed by opioid receptor antagonist naloxone. Molecular docking provided additional insight into the active site interactions of these scaffolds, which could be exploited further for creation of more potent inhibitors. These results showcase the potential of central ACE inhibitors to modulate endogenous MERF signalling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/639161v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1b8ce53org.highwire.dtl.DTLVardef@1f1cbd3org.highwire.dtl.DTLVardef@17cca3eorg.highwire.dtl.DTLVardef@1c1b35c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Inhibition of the angiotensin-converting enzyme N-terminal catalytic domain prevents endogenous opioid degradation in brain tissue

Angiotensin-converting enzyme (ACE) regulates blood pressure by cleaving angiotensin peptides in the periphery, and can also regulate endogenous opioid signaling by degrading enkephalin peptides in the brain. ACE has two catalytic domains, located in the N-terminal or C-terminal region of the protein, but little is known about the roles that these two catalytic domains play in regulating endogenous opioid degradation in brain tissue. Using acute brain slice preparations from mice of both sexes, we developed methods to study degradation of Met-enkephalin-Arg-Phe (MERF) by ACE, and investigated the role of each ACE catalytic domain in MERF degradation. Using mutant mouse lines with functional inactivation of either the N-terminal domain or the C-terminal domain, we incubated acute brain slices with exogenous MERF at a saturating concentration. The degradation MERF to produce Metenkephalin was only reduced by N-terminal domain inactivation. Additionally, application of a selective N-terminal domain inhibitor (RXP407) reduced degradation of both exogenously applied and endogenously released MERF, while a selective C-terminal domain inhibitor (RXPA380) had no effect. Taken together, our results suggest that the ACE N-terminal domain is the primary site of MERF degradation in brain tissue, and that N-terminal domain inhibition is sufficient to reduce degradation of this specific endogenous opioid peptide. Our results have exciting implications for the development of novel pharmacotherapies that target the endogenous opioid system to treat psychiatric and neurological disorders.

neuroscience↗

bioRxiv2024

Loss of the systemic vitamin A transporter RBPR2 affects the quantitative balance between chromophore and opsins in visual pigment synthesis

The distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and for generating visual pigments for photoreceptor cell function. ROL circulates in the blood bound to the retinol binding protein 4 (RBP4) as RBP4-ROL. Two membrane receptors, RBPR2 in the liver and STRA6 in the eye are proposed to bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Here, we present a longitudinal investigation towards the importance of RBPR2 and influence of the diet on systemic retinoid homeostasis for visual function. Age matched Rbpr2-KO (Rbpr2-/-) and wild-type (WT) mice were fed either a vitamin A sufficient (VAS) or a vitamin A deficient (VAD) diet. At 3- and 6-months, we performed retinoid quantification of ocular and non-ocular tissues using HPLC analysis and complemented the data with visual physiology, rhodopsin quantification by spectrophotometry, and biochemical analysis. At 3-months and compared to WT mice, Rbpr2-/- mice fed either vitamin A diets displayed lower scotopic and photopic electroretinogram (ERG) responses, which correlated with HPLC analysis that revealed Rbpr2-/- mice had significantly lower hepatic and ocular retinoid content. Interestingly, with the exception of the liver, long-term feeding of Rbpr2-/- mice with a VAS diet promoted all- trans retinol accumulation in most peripheral tissues. However, even under VAS dietary conditions significant amounts of unliganded opsins in rods, together with decreased visual responses were evident in aged mice lacking RBPR2, when compared to WT mice. Together, our analyses characterize the molecular events underlying nutritional blindness in a novel mouse model and indicate that loss of the liver specific RBP4-ROL receptor, RBPR2, influences systemic retinoid homeostasis and rhodopsin synthesis, which causes profound visual function defects under severe vitamin A deficiency conditions.

cell biology↗

bioRxiv2024

Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

pharmacology and toxicology↗

bioRxiv2024

Mutation of highly conserved residues in loop 2 of the coronavirus macrodomain demonstrates that enhanced ADP-ribose binding is detrimental to infection

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication. AUTHOR SUMMARYThe conserved CoV macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases by removing ADP-ribose from target proteins and is critical for CoV replication and pathogenesis. Mac1 is a potential therapeutic target for CoV disease and several groups are actively developing Mac1 inhibitors. However, we lack a basic knowledge of how many of the key residues in the Mac1 ADP-ribose binding pocket contribute to its biochemical and virological functions. In this study, we engineered alanine mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and recombinant viruses for both MERS-CoV and SARS-CoV-2 to determine their importance in both Mac1 biochemical functions and CoV infection. Interestingly, an isoleucine-to-alanine mutation in loop 2 of both MERS-CoV and SARS-CoV-2 Mac1 proteins enhanced ADP-ribose binding. But surprisingly, that proved to be detrimental to virus infection, indicating that this isoleucine functions to control Mac1 ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding to promote infection and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.

microbiology↗

bioRxiv2023

Klebsiella pneumoniae Co-infection Leads to Fatal Pneumonia in SARS-CoV-2-infected Mice

SARS-CoV-2 patients have been reported to have high rates of secondary Klebsiella pneumoniae infections. Klebsiella pneumoniae is a commensal that is typically found in the respiratory and gastrointestinal tracts. However, it can cause severe disease when a persons immune system is compromised. Despite a high number of K. pneumoniae cases reported in SARS-CoV-2 patients, a co-infection animal model evaluating the pathogenesis is not available. We describe a mouse model to study disease pathogenesis of SARS-CoV-2 and K. pneumoniae co-infection. BALB/cJ mice were inoculated with mouse-adapted SARS-CoV-2 followed by a challenge with K. pneumoniae. Mice were monitored for body weight change, clinical signs, and survival during infection. The bacterial load, viral titers, immune cell accumulation and phenotype, and histopathology were evaluated in the lungs. The co-infected mice showed severe clinical disease and a higher mortality rate within 48 h of K. pneumoniae infection. The co-infected mice had significantly elevated bacterial load in the lungs, however, viral loads were similar between co-infected and single-infected mice. Histopathology of co-infected mice showed severe bronchointerstitial pneumonia with copious intralesional bacteria. Flow cytometry analysis showed significantly higher numbers of neutrophils and macrophages in the lungs. Collectively, our results demonstrated that co-infection of SARS-CoV-2 with K. pneumoniae causes severe disease with increased mortality in mice.

immunology↗

bioRxiv2023

An autophagy program that promotes T cell egress from the lymph node controls responses to immune checkpoint blockade

Lymphatic endothelial cells (LECs) lining the lymphatic vessels of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady state and tumor-bearing conditions is lacking. Using intravital and high-resolution imaging combined with immune phenotyping, we show that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs, by increasing the availability of the main egress signal S1P. Single-cell RNA-sequencing of tumor-draining LNs from WT and ATG5LEC-KO mice unveils that loss of ATG5 remodels niche-specific LEC phenotypes, involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC-autophagy prevents recruitment of tumor-infiltrating T cells and NK cells and abrogates tumor regression in response to anti-PD-1 or anti-CTLA4-based immunotherapy. Thus, a unique LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/549282v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1a4bfe1org.highwire.dtl.DTLVardef@1317fe2org.highwire.dtl.DTLVardef@c2fa20org.highwire.dtl.DTLVardef@b35dba_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell and tissue specific manner

ADP-ribosyltransferases (ARTs) mediate the transfer of ADP-ribose from NAD+ to protein or nucleic acid substrates. This modification can be removed by several different types of proteins, including macrodomains. Several ARTs, also known as PARPs, are stimulated by interferon, indicating ADP-ribosylation is an important aspect of the innate immune response. All coronaviruses (CoVs) encode for a highly conserved macrodomain (Mac1) that is critical for CoVs to replicate and cause disease, indicating that ADP-ribosylation can effectively control coronavirus infection. Our siRNA screen indicated that PARP12 might inhibit the replication of a MHV Mac1 mutant virus in bone-marrow derived macrophages (BMDMs). To conclusively demonstrate that PARP12 is a key mediator of the antiviral response to CoVs both in cell culture and in vivo, we produced PARP12-/- mice and tested the ability of MHV A59 (hepatotropic/neurotropic) and JHM (neurotropic) Mac1 mutant viruses to replicate and cause disease in these mice. Notably, in the absence of PARP12, Mac1 mutant replication was increased in BMDMs and in mice. In addition, liver pathology was also increased in A59 infected mice. However, the PARP12 knockout did not restore Mac1 mutant virus replication to WT virus levels in all cell or tissue types and did not significantly increase the lethality of Mac1 mutant viruses. These results demonstrate that while PARP12 inhibits MHV Mac1 mutant virus infection, additional PARPs or innate immune factors must contribute to the extreme attenuation of this virus in mice. IMPORTANCEOver the last decade, the importance of ADP-ribosyltransferases (ARTs), also known as PARPs, in the antiviral response has gained increased significance as several were shown to either restrict virus replication or impact innate immune responses. However, there are few studies showing ART-mediated inhibition of virus replication or pathogenesis in animal models. We found that the CoV macrodomain (Mac1) was required to prevent ART-mediated inhibition of virus replication in cell culture. Here, using knockout mice, we found that PARP12, an interferon-stimulated ART, was required to repress the replication of a Mac1 mutant CoV both in cell culture and in mice, demonstrating that PARP12 represses coronavirus replication. However, the deletion of PARP12 did not fully rescue Mac1 mutant virus replication or pathogenesis, indicating that multiple PARPs function to counter coronavirus infection.

microbiology↗

bioRxiv2023

Toll-like receptor 7 (TLR7)-mediated antiviral response protects mice from lethal SARS-CoV-2 infection

SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses cause fatal pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and lethal inflammatory responses in-vivo are not well defined. CoVs possess single-stranded RNA (ssRNA) genome that is abundantly produced during infection and stimulates both antiviral interferon (IFN) and inflammatory cytokine/ chemokine responses. Therefore, in this study, using wild-type control and TLR7 deficient BALB/c mice infected with a mouse-adapted SARS-COV-2 (MA-CoV-2), we evaluated the role of TLR7 signaling in MA-CoV-2-induced antiviral and inflammatory responses and disease outcome. We show that TLR7-deficient mice are more susceptible to MA-CoV-2 infection as compared to infected control mice. Further evaluation of MA-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral type I (IFN/{beta}) and type III (IFN{lambda}) IFNs, IFN stimulated genes (ISGs, ISG15 and CXCL10), and several pro-inflammatory cytokines/chemokines in TLR7 deficient compared to control mice. Reduced lung IFN/ISG levels and increased morbidity/mortality in TLR7 deficient mice correlated with high lung viral titer. Detailed examination of total cells from MA-CoV-2 infected lungs showed high neutrophil count in TLR7 deficient mice compared to control mice. Additionally, blocking TLR7 activity post-MA-CoV-2 infection using a specific inhibitor also enhanced disease severity. In summary, our results conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite robust inflammatory response, TLR7-mediated IFN/ISG responses likely protect the host from lethal disease. Given similar outcomes in control and TLR7 deficient humans and mice, these results show that MA-CoV-2 infected mice serve as excellent model to study COVID-19.

immunology↗

bioRxiv2023

TUMOR ENDOTHELIAL CELL AUTOPHAGY IS A KEY VASCULAR-IMMUNE CHECKPOINT IN MELANOMA

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh/AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy. Congruently, patients responding to immunotherapy exhibit an increased presence of inflamed vessels, interfacing with infiltrating CD8+ T cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, autophagy is a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

cancer biology↗

bioRxiv2023

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in mice

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions ({Delta}Mac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV {Delta}Mac1 BACs, SARS-CoV-2 {Delta}Mac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 {Delta}Mac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, {Delta}Mac1 was quickly cleared causing minimal pathology without any morbidity. {Delta}Mac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. {Delta}Mac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target. SIGNIFICANCEAll CoVs, including SARS-CoV-2, encode for a conserved macrodomain (Mac1) that counters host ADP-ribosylation. Prior studies with SARS-CoV-1 and MHV found that Mac1 blocks IFN production and promotes CoV pathogenesis, which has prompted the development of SARS-CoV-2 Mac1 inhibitors. However, development of these compounds into antivirals requires that we understand how SARS-CoV-2 lacking Mac1 replicates and causes disease in vitro and in vivo. Here we found that SARS-CoV-2 containing a complete Mac1 deletion replicates normally in cell culture but induces an elevated IFN response, has reduced viral loads in vivo, and does not cause significant disease in mice. These results will provide a roadmap for testing Mac1 inhibitors, help identify Mac1 functions, and open additional avenues for coronavirus therapies.

microbiology↗

bioRxiv2023

A systematic comparison of VBM pipelines and their application to age prediction

Voxel-based morphometry (VBM) analysis is commonly used for localized quantification of gray matter volume (GMV). Several alternatives exist to implement a VBM pipeline. However, how these alternatives compare and their utility in applications, such as the estimation of aging effects, remain largely unclear. This leaves researchers wondering which VBM pipeline they should use for their project. In this study, we took a user-centric perspective and systematically compared five VBM pipelines, together with registration to either a general or a study-specific template, utilizing three large datasets (n>500 each). Considering the known effect of aging on GMV, we first compared the pipelines in their ability of individual-level age prediction and found markedly varied results. To examine whether these results arise from systematic differences between the pipelines, we classified them based on their GMVs, resulting in near-perfect accuracy. To gain deeper insights, we examined the impact of different VBM steps using the region-wise similarity between pipelines. The results revealed marked differences, largely driven by segmentation and registration steps. We observed large variability in subject-identification accuracies, highlighting the interpipeline differences in individual-level quantification of GMV. As a biologically meaningful criterion we correlated regional GMV with age. The results were in line with the age-prediction analysis, and two pipelines, CAT and the combination of fMRIPrep for tissue characterization with FSL for registration, reflected age information better.

neuroscience↗

bioRxiv2023

The role of intestine in metabolic dysregulation in murine Wilson disease

Background and aimsMajor clinical manifestations of Wilson disease (WD) are related to copper accumulation in the liver and the brain, and little is known about other tissues involvement in metabolic changes in WD. In vitro studies suggested that the loss of intestinal ATP7B could contribute to metabolic dysregulation in WD. We tested this hypothesis by evaluating gut microbiota and lipidome in two mouse models of WD and by characterizing a new mouse model with a targeted deletion of Atp7b in intestine. MethodsCecal content 16S sequencing and untargeted hepatic and plasma lipidome analyses in the Jackson Laboratory toxic-milk and the Atp7b null global knockout mouse models of WD were profiled and integrated. Intestine-specific Atp7b knockout mice (Atp7b{Delta}IEC) was generated using B6.Cg-Tg(Vil1-cre)997Gum/J mice and Atp7bLox/Lox mice, and characterized using targeted lipidome analysis following a high-fat diet challenge. ResultsGut microbiota diversity was reduced in animal models of WD. Comparative prediction analysis revealed amino acid, carbohydrate, and lipid metabolism functions to be dysregulated in the WD gut microbial metagenome. Liver and plasma lipidomic profiles showed dysregulated tri- and diglyceride, phospholipid, and sphingolipid metabolism in WD models. When challenged with a high-fat diet, Atp7b{Delta}IEC mice exhibited profound alterations to fatty acid desaturation and sphingolipid metabolism pathways as well as altered APOB48 distribution in intestinal epithelial cells. ConclusionCoordinated changes of gut microbiome and lipidome analyses underlie systemic metabolic manifestations in murine WD. Intestine-specific ATP7B deficiency affected both intestinal and systemic response to a high-fat challenge. WD is a systemic disease in which intestinal-specific ATP7B loss and diet influence phenotypic presentations.

molecular biology↗