Infected Mice Research Articles

Pilot Evaluation of S-(3-[18F]Fluoropropyl)-d-Homocysteine and O-(2-[18F]Fluoroethyl)-d-Tyrosine as Bacteria-Specific Radiotracers for PET Imaging of Infection

PurposeThere is currently no ideal radiotracer for imaging bacterial infections. Radiolabelled d-amino acids are promising candidates because they are actively incorporated into the peptidoglycan of the bacterial cell wall, a structural feature which is absent in human cells. This work describes fluorine-18 labelled analogues of d-tyrosine and d-methionine, O-(2-[18F]fluoroethyl)-d-tyrosine (d-[18F]FET) and S-(3-[18F]fluoropropyl)-d-homocysteine (d-[18F]FPHCys), and their pilot evaluation studies as potential radiotracers for imaging bacterial infection.Proceduresd-[18F]FET and d-[18F]FPHCys were prepared in classical fluorination-deprotection reactions, and their uptake in Staphylococcus aureus and Pseudomonas aeruginosa was evaluated over 2 h. Heat killed bacteria were used as controls. A clinically-relevant foreign body model of S. aureus infection was established in Balb/c mice, as well as a sterile foreign body to mimic inflammation. The ex vivo biodistribution of d-[18F]FPHCys in the infected and inflamed mice was evaluated after 1 h, by dissection and gamma counting. The uptake was compared to that of [18F]FDG.ResultsIn vitro uptake of both d-[18F]FET and d-[18F]FPHCys was specific to live bacteria. Uptake was higher in S. aureus than in P. aeruginosa for both radiotracers, and of the two, higher for d-[18F]FPHCys than d-[18F]FET. Blocking experiments with non-radioactive d-[19F]FPHCys confirmed specificity of uptake. In vivo, d-[18F]FPHCys had greater accumulation in S. aureus infection compared with sterile inflammation, which was statistically significant. As anticipated, [18F]FDG showed no significant difference in uptake between infection and inflammation.Conclusionsd-[18F]FPHCys uptake was higher in infected tissues than inflammation, and represents a fluorine-18 labelled d-AA with potential to detect a S. aureus reference strain (Xen29) in vivo. Additional studies are needed to evaluate uptake of this radiotracer in clinical isolates.

Circulating KLRG1+ long-lived effector memory T cells retain the flexibility to become tissue resident.

KLRG1+ CD8 T cells persist for months after clearance of acute infections and maintain high levels of effector molecules, contributing protective immunity against systemic pathogens. Upon secondary infection, these long-lived effector cells (LLECs) are incapable of forming other circulating KLRG1- memory subsets such as central and effector memory T cells. Thus, KLRG1+ memory T cells are frequently referred to as a terminally differentiated population that is relatively short lived. Here, we show that after viral infection of mice, effector cells derived from LLECs rapidly enter nonlymphoid tissues and reduce pathogen burden but are largely dependent on receiving antigen cues from vascular endothelial cells. Single-cell RNA sequencing reveals that secondary memory cells in nonlymphoid tissues arising from either KLRG1+ or KLRG1- memory precursors develop a similar resident memory transcriptional signature. Thus, although LLECs cannot differentiate into other circulating memory populations, they still retain the flexibility to enter tissues and establish residency.

Effect of acetylcholinesterase inhibition on immune cells in the murine intestinal mucosa

The gastrointestinal tract (GI) is the largest immune organ whose function is controlled by a complex network of neurons from the enteric nervous system (ENS) as well as the sympathetic and parasympathetic system. Evolving evidence indicates that cross-communication between gut-innervating neurons and immune cells regulates many essential physiological functions including protection against mucosal infections. We previously demonstrated that following paraoxon treatment, 70 % of the mice were able to survive an oral infection with S. typhimurium, a virulent strain of Salmonella enterica serovar Typhimurium. The present study aims to investigate the effect that rivastigmine, a reversible AChE inhibitor used for the treatment of neurodegenerative diseases, has on the murine immune defenses of the intestinal mucosa. Our findings show that, similar to what is observed with paraoxon, administration of rivastigmine promoted the release of secretory granules from goblet and Paneth cells, resulting in increased mucin layer. Surprisingly, however, and unlike paraoxon, rivastigmine treatment did not affect overall mortality of infected mice. In order to investigate the mechanistic basis for the differential effects observed between paraoxon and rivastigmine, we used multi-color flowcytometric analysis to characterize the immune cell landscape in the intraepithelial (IE) and lamina propria (LP) compartments of intestinal mucosa. Our data indicate that treatment with paraoxon, but not rivastigmine, led to an increase of resident CD3+CD8+ T lymphocytes in the ileal mucosa (epithelium and lamina propria) and CD11b− CD11c+ dendritic cells in the LP. Our findings indicate the requirement for persistent cholinergic pathway engagement to effect a change in the cellular landscape of the mucosal tissue that is necessary for protection against lethal bacterial infections. Moreover, optimal protection requires a collaboration between innate and adaptive mucosal immune responses in the intestine.

Impact of Western Diet on Enterohemorrhagic Escherichia coli Colonization in the Human In Vitro Mucosal Artificial Colon as Mediated by Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) is a major food-borne pathogen that causes human disease ranging from diarrhea to life-threatening complications. Accumulating evidence demonstrates that the Western diet enhances the susceptibility to enteric infection in mice, but the effect of diet on EHEC colonization and the role of human gut microbiota remains unknown. Our research aimed to investigate the effects of a Standard versus a Western diet on EHEC colonization in the human in vitro Mucosal ARtificial COLon (M-ARCOL) and the associated changes in the gut microbiota composition and activities. After donor selection using simplified fecal batch experiments, two M-ARCOL bioreactors were inoculated with a human fecal sample (n = 4) and were run in parallel, one receiving a Standard diet, the other a Western diet and infected with EHEC O157:H7 strain EDL933. EHEC colonization was dependent on the donor and diet in the luminal samples, but was maintained in the mucosal compartment without elimination, suggesting a favorable niche for the pathogen, and may act as a reservoir. The Western diet also impacted the bacterial short-chain fatty acid and bile acid profiles, with a possible link between high butyrate concentrations and prolonged EHEC colonization. The work demonstrates the application of a complex in vitro model to provide insights into diet, microbiota, and pathogen interactions in the human gut.

Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.

Semaglutide outperforms insulin in restoring neutrophil function against implant-related infection in diabetic and obese mice: experimental research.

Bone and joint infections (BJI) are a significant complication after arthroplasty and fracture fixation, particularly challenging in patients with type 2 diabetes mellitus (T2DM) and obesity. Semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), have shown efficacy in managing T2DM and obesity. However, its impact on BJI risk and neutrophil function remains unclear. To investigate whether preoperative semaglutide treatment (1) reduces the risk of BJI in diabetic and obese mice undergoing intra-articular implants, and (2) outperforms insulin in restoring neutrophil function to mitigate implant-related infection. A C57BL/6 mouse model of T2DM/obesity was induced using a high-fat diet (HFD) for 12 weeks. Mice received preoperative insulin or semaglutide therapy for 1-28 days. BJI risk was assessed using an intraarticular-implant model challenged with S. aureus or E. coli. Neutrophil function was evaluated through bactericidal activity, superoxide production, and migration ability. Semaglutide treatment led to a significant and sustained reduction in body weight and improved glucose tolerance in HFD mice. Both insulin and semaglutide therapies significantly reduced BJI risk, with semaglutide showing a more pronounced effect over time. Semaglutide therapy also enhanced neutrophil bactericidal activity, superoxide production, and migration ability compared to insulin therapy. Preoperative semaglutide treatment effectively reduces BJI risk and improves neutrophil function in diabetic and obese mouse models. These findings suggest that semaglutide may be a promising pharmacological intervention to mitigate infection risk in orthopedic patients with T2DM or obesity.

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

Helminth induced monocytosis conveys protection from respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) infection in infants is a major cause of viral bronchiolitis and hospitalisation. We have previously shown in a murine model that ongoing infection with the gut helminth Heligmosomoides polygyrus protects against RSV infection through type I interferon (IFN-I) dependent reduction of viral load. Yet, the cellular basis for this protection has remained elusive. Given that recruitment of mononuclear phagocytes to the lung is critical for early RSV infection control, we assessed their role in this coinfection model. Mice were infected by oral gavage with H. polygyrus. Myeloid immune cell populations were assessed by flow cytometry in lung, blood and bone marrow throughout infection and after secondary infection with RSV. Monocyte numbers were depleted by anti-CCR2 antibody or increased by intravenous transfer of enriched monocytes. H. polygyrus infection induces bone marrow monopoiesis, increasing circulatory monocytes and lung mononuclear phagocytes in a IFN-I signalling dependent manner. This expansion causes enhanced lung mononuclear phagocyte counts early in RSV infection that may contribute to the reduction of RSV load. Depletion or supplementation of circulatory monocytes prior to RSV infection confirms that these are both necessary and sufficient for helminth induced antiviral protection. H. polygyrus infection induces systemic monocytosis contributing to elevated mononuclear phagocyte numbers in the lung. These cells are central to an anti-viral effect that reduces the peak viral load in RSV infection. Treatments to promote or modulate these cells may provide novel paths to control RSV infection in high risk individuals.

Contrasting Disease Progression, Microglia Reactivity, Tolerance, and Resistance to Toxoplasma gondii Infection in Two Mouse Strains.

Our study investigated the innate immune response to Toxoplasma gondii infection by assessing microglial phenotypic changes and sickness behavior as inflammatory response markers post-ocular tachyzoite instillation. Disease progression in Swiss albino mice was compared with the previously documented outcomes in BALB/c mice using an identical ocular route and parasite burden (2 × 105 tachyzoites), with saline as the control. Contrary to expectations, the Swiss albino mice displayed rapid, lethal disease progression, marked by pronounced sickness behaviors and mortality within 11-12 days post-infection, while the survivors exhibited no apparent signs of infection. Comparative analysis revealed the T. gondii-infected BALB/c mice exhibited reduced avoidance of feline odors, while the infected Swiss albino mice showed enhanced avoidance responses. There was an important increase in microglial cells in the dentate gyrus molecular layer of the infected Swiss albino mice compared to the BALB/c mice and their respective controls. Hierarchical cluster and discriminant analyses identified three microglial morphological clusters, differentially affected by T. gondii infection across strains. The BALB/c mice exhibited increased microglial branching and complexity, while the Swiss albino mice showed reduced shrunken microglial arbors, diminishing their morphological complexity. These findings highlight strain-specific differences in disease progression and inflammatory regulation, indicating lineage-specific mechanisms in inflammatory responses, tolerance, and resistance. Understanding these elements is critical in devising control measures for toxoplasmosis.

Upregulation of inflammatory genes and pathways links obesity to severe COVID-19

Obesity is a risk factor for developing severe COVID-19. However, the mechanism underlying obesity-accelerated COVID-19 remains unclear. Here, we report results from a study in which 2–3-month-old K18-hACE2 (K18) mice were fed a western high-fat diet (WD) or normal chow (NC) over 3 months before intranasal infection with a sublethal dose of SARS-CoV2 WA1 (a strain ancestral to the Wuhan variant). After infection, the WD-fed K18 mice lost significantly more body weight and had more severe lung inflammation than normal chow (NC)-fed mice. Bulk RNA-seq analysis of lungs and adipose tissue revealed a diverse landscape of various immune cells, inflammatory markers, and pathways upregulated in the infected WD-fed K18 mice when compared with the infected NC-fed control mice. The transcript levels of IL-6, an important marker of COVID-19 disease severity, were upregulated in the lung at 6–9 days post-infection in the WD-fed mice when compared to NC-fed mice. Transcriptome analysis of the lung and adipose tissue obtained from deceased COVID-19 patients found that the obese patients had an increase in the expression of genes and the activation of pathways associated with inflammation as compared to normal-weight patients (n = 2). The K18 mouse model and human COVID-19 patient data support a link between inflammation and an obesity-accelerated COVID-19 disease phenotype. These results also indicate that obesity-accelerated severe COVID-19 caused by SARS-CoV-2 WA1 infection in the K18 mouse model would be a suitable model for dissecting the cellular and molecular mechanisms underlying pathogenesis.

Application of Theiler's murine encephalomyelitis virus in treatment of multiple sclerosis.

Theiler's murine encephalomyelitis virus (TMEV) infected mice have been often used as an animal model for Multiple sclerosis (MS) due to their similar pathology in the central nervous system (CNS). So far, there has been no effective treatment or medicine to cure MS completely. The drugs used in the clinic can only reduce the symptoms of MS, delay its recurrence, and increase the interval between relapses. MS can be caused by many factors, and clinically MS drugs are used to treat MS regardless of what factors are caused rather than MS caused by a specific factor. This can lead to inappropriate medicine, which may be one of the reasons why MS has not been completely cured. Therefore, this review summarized the drugs investigated in the TMEV-induced disease (TMEV-IDD) model of MS, so as to provide medication guidance and theoretical basis for the treatment of virus-induced MS.

Fasciola gigantica: Ultrastructural localisation of neoblast recruitment in somatic tissues during growth and development in the hepatic parenchyma of experimentally infected mice

Application of ‘omics’ technology, and advances in in vitro methods for studying the growth of Fasciola hepatica, have highlighted the central role of migrating neoblasts in driving forward development and differentiation towards the adult-like form. Neoblast populations present molecular heterogeneity, morphological variation and changes associated with recruitment of these stem cells into their final tissue locations. However, terminal differentiation towards function, has received much less attention than has been the case for the free-living Platyhelminths. An actively replicating neoblast population, comprising cells with heterochromatic nuclei consistent with regulation of gene expression, has been identified in the parenchyma of juvenile Fasciola gigantica migrating in the liver of experimentally infected mice. In some of these cells, early cytoplasmic differentiation towards myocyte function was noted. Neoblasts have also been identified close to, and incorporated in, the subtegumental zone, the gastrodermis and the excretory ducts. In these locations, progressive morphological differentiation towards terminal function has been described. This includes the appearance of specific progenitors of type-1, type-2 and type-3 tegumental cells, the latter possibly contributing to tegumental spine development. ‘Cryptic’ surface molecular differentiation is postulated to account for recognition and ‘docking’ of migrating neoblasts with their final site for terminal differentiation.

Macrophage-targeted versus free calcitriol as host-directed adjunct therapy against Mycobacterium tuberculosis infection in mice is bacteriostatic and mitigates tissue pathology

Host-directed therapy (HDT) with vitamin D in tuberculosis (TB) is beneficial only if the subject is deficient in vitamin D. We investigated pulmonary delivery of 1,25-dihydroxy vitamin D3 (calcitriol) in mice infected with Mycobacterium tuberculosis (Mtb). We made two kinds of dry powder inhalations (DPI)— soluble particles or poly(lactide) (PLA) particles. We compared treatment outcomes when infected mice were dosed with a DPI alone or as an adjunct to standard oral anti-TB therapy (ATT). Mice infected on Day 0 were treated between Days 28–56 and followed up on Days 57, 71, and 85. Neither DPI significantly reduced Mtb colony forming units (CFU) in the lungs. Combining DPI with ATT did not significantly augment bactericidal activity in the lungs, but CFU were 2-log lower in the spleen. CFU showed a rising trend on stopping treatment, sharper in groups that did not receive calcitriol. Lung morphology and histology improved markedly in animals that received PLA DPI; with or without concomitant ATT. Groups receiving soluble DPI had high mortality. DPI elicited cathelicidin, interleukin (IL)-1 and induced autophagy on days 57, 71, and 85. Macrophage-targeted calcitriol is therefore bacteriostatic, evokes innate microbicidal mechanisms, and mitigates pathology arising from the host response to Mtb.

Brain -cyst-driven genes expression in Toxoplasma Gondii Tehran strain: a parasitic-immunogenicity assessment by dint of RNA-Seq.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite of warm-blooded vertebrates. At present, High-throughput RNA sequencing analysis have made it possible to determine the role of effective genes in host immune response. The aim of the present study is to global transcriptome analysis of the brain of mice infected with T. gondii Tehran strain for the first time and also to evaluate the expression of effective genes in the chronic form of infection. RNA was extracted from the samples and the library was prepared and sequenced using the IlluminaNovaSeq 6000 system. After analyzing gene expression changes, the results were confirmed by real-time method. We found 125 genes that were significantly differentially expressed between infected and non-infected samples (p < 0.0005). Gene ontology analysis revealed that the expression of many genes is critical for pathways such as T cell receptor signaling pathway, Natural Killer cell mediated cytotoxicity, Lysosome and Apoptosis of the host. As infection with Tehran strain leads to chronic infection in mice, therefore, we investigated the genes effective in creating the chronic form of Toxoplasma infection. The comparative analysis of genes showed increases in the expression of genes ctla4, ccl4, cd3e, c3, lcn2, gbp5, usp18, cyba, tap1 and samhd1 in the in the infected sample, which highlights their role in causing chronic infection. RNA-seq provides a valuable tool for analyzing host transcriptomes, better understanding the parasite-host interaction, and developing future drug and vaccine targets.

Evolution of the pathogenic mold Aspergillus fumigatus on high copper levels identifies novel resistance genes.

Aspergillus fumigatus is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the A. fumigatus transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved A. fumigatus wild-type and mutant ΔaceA or ΔcrpA strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H+-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type A. fumigatus confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the pma1 mutation L424I preserves Pma1 H+-ATPase activity under high Cu concentrations and that the cpa1 mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of A. fumigatus adaptation to diverse environmental challenges.IMPORTANCEAspergillus fumigatus is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved A. fumigatus under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.

Rapid NETosis Is an Effector Mechanism to Combat Ocular Herpes Infection.

Neutrophils are known mediators of innate immunity, yet their effector function in herpesvirus infections remains poorly understood. Here, we elucidate the mechanistic action and pivotal role of neutrophil extracellular traps (NETs) during herpes simplex virus type 1 (HSV-1) ocular infection. Neutrophils were collected from mice for HSV-1 infection, fluorescence imaging, and immunoblotting assay. Tear samples from healthy subjects and patients with HSV-1 and mice were collected at L. V. Prasad Eye Institute, India, and at the University of Illinois, USA, respectively. For the in vivo study, C57BL/6 mice as well as diversity outbred mice were infected with HSV-1 (McKrae strain) followed by tear fluid collection at various time points (0-10 days). Samples were used for Flow cytometry, ELISA, and immunofluorescence assay. Human transcriptomic profile of keratitis dataset was used evaluate NETosis signaling pathways. We also performed neutrophil depletion studies. Our data revealed a discernible temporal NET formation (NETosis) predominantly in the infected eye, across normal and diversity outbred murine models and human cases of HSV-1 infection. HSV-1 instigates swift NETosis governed by caspase-1 activation and myeloperoxidase secretion. Distinct accumulations of neutrophils, remaining unengaged in NET release in the contralateral eye post-infection, hinting at a proactive defensive posture in the uninfected eye. Moreover, neutrophil depletion accentuated ocular pathology, augmented viral load, and escalated disease scores, substantiating the protective effects of NETs in curtailing viral replication. Our report uncovers a previously unexplored mechanism of NETosis through pro-inflammatory cell death in response to ocular HSV-1 infection, and HPSE up-regulation, identifying new avenues for future studies.

MEK-inhibitor treatment reduces the induction of regulatory T cells in mice after influenza A virus infection.

Regulatory T cells (Tregs) play a crucial and complex role in balancing the immune response to viral infection. Primarily, they serve to regulate the immune response by limiting the expression of proinflammatory cytokines, reducing inflammation in infected tissue, and limiting virus-specific T cell responses. But excessive activity of Tregs can also be detrimental and hinder the ability to effectively clear viral infection, leading to prolonged disease and potential worsening of disease severity. Not much is known about the impact of Tregs during severe influenza. In the present study, we show that CD4+/CD25+FoxP3+ Tregs are strongly involved in disease progression during influenza A virus (IAV) infection in mice. By comparing sublethal with lethal dose infection in vivo, we found that not the viral load but an increased number of CD4+/CD25+FoxP3+ Tregs may impair the immune response by suppressing virus specific CD8+ T cells and favors disease progression. Moreover, the transfer of induced Tregs into mice with mild disease symptoms had a negative and prolonged effect on disease outcome, emphasizing their importance for pathogenesis. Furthermore, treatment with MEK-inhibitors resulted in a significant reduction of induced Tregs in vitro and in vivo and positively influenced the progression of the disease. Our results demonstrate that CD4+/CD25+FoxP3+ Tregs are involved in the pathogenesis of severe influenza and indicate the potential of the MEK-inhibitor zapnometinib to modulate CD4+/CD25+FoxP3+ Tregs. Thus, making MEK-inhibitors even more promising for the treatment of severe influenza virus infections.

In vivo Labeling and Intravital Imaging of Bacterial Infection using a Near-infrared Fluorescent D-Amino Acid Probe.

Bacterial infections still pose a severe threat to public health, necessitating novel tools for real-time analysis of microbial behaviors in living organisms. While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their in vivo uses are often limited. Here, we report a near-infrared fluorescent D-amino acid (FDAA) probe, Cy7ADA, for in situ labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria in vitro and pathogenic Staphylococcus aureus in mice after intraperitoneal injection. Because of Cy7's high tissue penetration and the quick excretion of free probes via urine, real-time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA's capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three-dimensional pathogen imaging in tissue-cleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating near-infrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections in vivo.

Characterization of Unique Pathological Features of COVID-Associated Coagulopathy: Studies with AC70 hACE2 Transgenic Mice Highly Permissive to SARS-CoV-2 Infection.

COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as ~ 3 and ~ 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and inflammatory mediators could be detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio (NLR). Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy. SARS-CoV-2 could induce the release of circulating neutrophil extracellular traps (NETs), along with activated platelet/endothelium marker. Immunohistochemical staining with anti-platelet factor-4 (PF4) antibody revealed profound platelet aggregates especially within blocked veins of the lungs. We showed that acute SARS-CoV-2 infection triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis. Finally, we highlighted the potential role of Annexin A2 (ANXA2) in fibrinolytic failure. ANXA2 is a calcium-dependent phospholipid-binding protein that forms a heterotertrameric complexes localized at the extracellular membranes with two S100A10 small molecules acting as a co-receptor for tissue-plasminogen activator (t-PA), tightly involved in cell surface fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced ANXA2/S100A10 association in infected mice, this protein might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19.

Brain region-specific alterations in gene expression trajectories in the offspring born from influenza A virus infected mice

Influenza A virus (IAV) infection during pregnancy can increase the risk for neurodevelopmental disorders in the offspring, however, the underlying neurobiological mechanisms are largely unknown. To recapitulate viral infection, preclinical studies have traditionally focused on using synthetic viral mimetics, rather than live IAV, to examine consequences of maternal immune activation (MIA)-dependent processes on offspring. In contrast, few studies have used live IAV to assess effects on global gene expression, and none to date have addressed whether moderate IAV, mimicking seasonal influenza disease, alters normal gene expression trajectories in different brain regions across different stages of development. Herein, we show that moderate IAV infection during pregnancy, which causes mild maternal disease and no overt foetal complications in utero, induces lasting effects on the offspring into adulthood. We observed behavioural changes in adult offspring, including disrupted prepulse inhibition, dopaminergic hyper-responsiveness, and spatial recognition memory deficits. Gene profiling in the offspring brain from neonate to adolescence revealed persistent alterations to normal gene expression trajectories in the prefronal cortex, hippocampus, hypothalamus and cerebellum. Alterations were found in genes involved in inflammation and neurogenesis, which were predominately dysregulated in neonatal and early adolescent offspring. Notably, late adolescent offspring born from IAV infected mice displayed altered microglial morphology in the hippocampus. In conclusion, we show that moderate IAV during pregnancy perturbs neurodevelopmental trajectories in the offspring, including alterations in the neuroinflammatory gene expression profile and microglial number and morphology in the hippocampus, resulting in behavioural changes in adult offspring. Such early perturbations may underlie the vulnerability in human offspring for the later development of neurodevelopmental disorders, including schizophrenia. Our work highlights the importance of using live IAV in developing novel preclinical models that better recapitulate the real-world impact of inflammatory insults during pregnancy on offspring neurodevelopmental trajectories and disease susceptibility later in life.