Mouse Model Of Infection Research Articles

Arenicolide Family Macrolides Provide a New Therapeutic Lead Combating Multidrug-Resistant Tuberculosis.

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) poses a significant threat to health globally. During searching for new chemical entities regulating MDR- and XDR-Mtb, chemical investigation of the black oil beetle gut bacterium Micromonospora sp. GR10 led to the discovery of eight new members of arenicolides along with the identification of arenicolide A (Ar-A, 1), which was a previously reported macrolide with incomplete configuration. Genomic analysis of the bacterial strain GR10 revealed their putative biosynthetic pathway. Quantum mechanics-based computation, chemical derivatizations, and bioinformatic analysis established the absolute stereochemistry of Ar-A and arenicolides D-K (Ar-D-K, 2-9) completely for the first time. Biological studies of 1-9 revealed their antimicrobial activity against MDR and XDR strains of Mtb. Ar-A had the most potent in vitro antimicrobial efficacy against MDR- and XDR-Mtb. Mechanistically, Ar-A induced ATP depletion and destabilized Mtb cell wall, thereby inhibiting growth. Notably, Ar-A exerted a significant antimicrobial effect against Mtb in macrophages, was effective in the treatment of Mtb infections, and showed a synergistic effect with amikacin (AMK) in a mouse model of MDR-Mtb lung infection. Collectively, our findings indicate Ar-A to be a promising drug lead for drug-resistant tuberculosis.

The PdxR-PdxKU locus involved in vitamin B 6 salvage is important for group A streptococcal resistance to neutrophil killing and survival in human blood

ABSTRACT Streptococcus pyogenes (Group A Streptococcus , GAS) is a Gram-positive bacterium that inflicts both superficial and life-threatening diseases on its human host. Analysis of fitness using a transposon mutant library revealed that genes predicted to be involved in vitamin B 6 acquisition are associated with fitness in whole human blood. Vitamin B 6 is essential for all life and is important for many cellular functions. In several streptococcal species, it has been shown that mutants in B 6 acquisition exhibited reduced virulence phenotypes and were attenuated during infection. In GAS, B 6 acquisition is believed to be controlled by the pdxR-pdxKU locus, where PdxR is a positive regulator of pdxKU , which encodes for a B 6 -substrate kinase and permease, respectively. Mutants in the regulator (Δ pdxR ) and salvage machinery (Δ pdxKU ) both exhibited modest growth defects when grown in oxygenated conditions with limited vitamin B 6 precursors. ∆ pdxR and ∆ pdxKU mutants also exhibited an impaired ability to survive when challenged with whole human or mouse blood. This defect was characterized by reduced survival in the presence of human neutrophil-like HL60s, primary polymorphonuclear leukocytes, and antimicrobial peptide LL-37. Promoter analysis showed that PdxR is an autoregulator and activated pdxKU in the absence of B 6 . Interestingly, ∆ pdxR and ∆ pdxKU mutants were not attenuated in mouse models of infection, suggesting a species-specific impact on virulence. Overall, it appears that pdxR-pdxKU is associated with GAS vitamin B 6 metabolism as well as pathogen survival during encounters with the human innate immune system. IMPORTANCE Bacterial pathogens such as Streptococcus pyogenes (Group A Streptococcus , GAS) must be able to obtain needed nutrients in their human host. Vitamin B 6 or pyridoxal 5’ phosphate is essential for all life and is important for many cellular functions. In other streptococcal pathogens, B 6 acquisition has been shown to be important for their ability to cause disease. Here, we show that loss of the putative vitamin B 6 salvage pathway locus pdxR-pdxKU affects GAS pathogenesis when encountering innate immune responses from phagocytic neutrophils and antimicrobial peptides within the host. pdxR- pdxKU may contribute to oxygen tolerance through B 6 ; however, there appear to be other mechanisms for salvaging vitamin B 6 . Overall, pdxR-pdxKU is associated with GAS resistance to the human innate immune response and oxygen tolerance and contributes modestly to B 6 metabolism.

Tracking inflammation resolution signatures in lungs after SARS-CoV-2 omicron BA.1 infection of K18-hACE2 mice

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which can result in severe disease, often characterised by a ‘cytokine storm’ and the associated acute respiratory distress syndrome. However, many infections with SARS-CoV-2 are mild or asymptomatic throughout the course of infection. Although blood biomarkers of severe disease are well studied, less well understood are the inflammatory signatures in lung tissues associated with mild disease or silent infections, wherein infection and inflammation are rapidly resolved leading to sequelae-free recovery. Herein we described RNA-Seq and histological analyses of lungs over time in an omicron BA.1/K18-hACE2 mouse infection model, which displays these latter features. Although robust infection was evident at 2 days post infection (dpi), viral RNA was largely cleared by 10 dpi. Acute inflammatory signatures showed a slightly different pattern of cytokine signatures compared with severe infection models, and where much diminished 30 dpi and absent by 66 dpi. Cellular deconvolution identified significantly increased abundance scores for a number of anti-inflammatory pro-resolution cell types at 5/10 dpi. These included type II innate lymphoid cells, T regulatory cells, and interstitial macrophages. Genes whose expression trended downwards over 2–66 dpi included biomarkers of severe disease and were associated with ‘cytokine storm’ pathways. Genes whose expression trended upward during this period were associated with recovery of ciliated cells, AT2 to AT1 transition, reticular fibroblasts and innate lymphoid cells, indicating a return to homeostasis. Very few differentially expressed host genes were identified at 66 dpi, suggesting near complete recovery. The parallels between mild or subclinical infections in humans and those observed in this BA.1/K18-hACE2 mouse model are discussed with reference to the concept of “protective inflammation”.

Impact of an intranasal L-DBF vaccine on the gut microbiota in young and elderly mice

ABSTRACT Shigella spp. cause bacillary dysentery (shigellosis) with high morbidity and mortality in low- and middle-income countries. Infection occurs through the fecal-oral route and can be devastating for vulnerable populations, including infants and the elderly. These bacteria invade host cells using a type III secretion system (T3SS). No licensed vaccine yet exists for shigellosis, but we have generated a recombinant fusion protein, L-DBF, combining the T3SS needle tip protein (IpaD), translocator protein (IpaB), and the LTA1 subunit of enterotoxigenic E. coli labile toxin, which offers broad protection in a mouse model of lethal pulmonary infection. The L-DBF vaccine protects high-risk groups, including young and elderly mice. Here, we investigated how the gut microbiota of young and elderly mice responds to intranasal L-DBF vaccination formulated in an oil-in-water emulsion (ME). Samples from lungs, small intestines, and feces were collected on day 14 after 2 or 3 doses of L-DBF in ME. 16S rRNA gene sequencing revealed age-dependent changes in gut microbiota post-vaccination. The vaccine-induced changes were more prominent in the elderly mice and were most significant in the intestinal tract, indicating that vaccination by the intranasal route can have a tremendous impact on the gut environment. These findings provide insight into the communication between the intranasal mucosal surface following subunit vaccination and the microbiota at a distant mucosal site, thereby highlighting the impact of vaccination and the host’s microbiome.

Lethal model for respiratory syncytial virus infection using C57BL/6 mice.

A mouse model of respiratory syncytial virus (RSV) infection is useful for fundamental research aimed at developing antiviral drugs. Previous mouse models of RSV infection were unable to adequately mimic the pathophysiology of human patients due to the low amplification efficiency of this virus in the mouse lung. Furthermore, mice other than BALB/C mice are difficult to use for the RSV infectious model. We established a new mouse-adapted RSV strain, MP11. The MP11 virus can cause severe pneumonia in C57BL/6 mice and efficiently replicate and induce inflammation in the lung. Therefore, C57BL/6 mice can be used for RSV infection experiments using MP11 virus. We established a reverse genetics system for the MP11 virus using our mouse model. This system enables detailed analyses of the MP11 virus, such as functional analysis of each viral protein. Our study provides techniques that can advance fundamental research in elucidating the pathogenesis of RSV infections.

Activation of STAT3-mediated ciliated cell survival protects against severe infection by respiratory syncytial virus.

Respiratory syncytial virus (RSV) selectively targets ciliated cells in human bronchial epithelium and can cause bronchiolitis and pneumonia, mostly in infants. To identify molecular targets of intervention during RSV infection in infants, we investigated how age regulates RSV interaction with the bronchial epithelium barrier. Employing precision-cut lung slices and air-liquid interface cultures generated from infant and adult human donors, we found robust RSV virus spread and extensive apoptotic cell death only in infant bronchial epithelium. In contrast, adult bronchial epithelium showed no barrier damage and limited RSV infection. Single nuclear RNA-Seq revealed age-related insufficiency of an antiapoptotic STAT3 activation response to RSV infection in infant ciliated cells, which was exploited to facilitate virus spread via the extruded apoptotic ciliated cells carrying RSV. Activation of STAT3 and blockade of apoptosis rendered protection against severe RSV infection in infant bronchial epithelium. Lastly, apoptotic inhibitor treatment of a neonatal mouse model of RSV infection mitigated infection and inflammation in the lung. Taken together, our findings identify a STAT3-mediated antiapoptosis pathway as a target to battle severe RSV disease in infants.

Efficacy of a saline wash plus vancomycin/tobramycin-doped PVA composite (PVA-VAN/TOB-P) in a mouse pouch infection model implanted with 3D-printed porous titanium cylinders.

The efficacy of saline irrigation for treatment of implant-associated infections is limited in the presence of porous metallic implants. This study evaluated the therapeutic efficacy of antibiotic doped bioceramic (vancomycin/tobramycin-doped polyvinyl alcohol composite (PVA-VAN/TOB-P)) after saline wash in a mouse infection model implanted with titanium cylinders. Air pouches created in female BalBc mice by subcutaneous injection of air. In the first of two independent studies, pouches were implanted with titanium cylinders (400, 700, and 100 µm pore sizes) and inoculated with Staphylococcus aureus (1 × 103 or 1 × 106 colony-forming units (CFU)/pouch) to establish infection and biofilm formation. Mice were killed after one week for microbiological analysis. In the second study, pouches were implanted with 400 µm titanium cylinders and inoculated with S. aureus (1 × 103 or 1 × 106 CFU/pouch). Four groups were tested: 1) no bacteria; 2) bacteria without saline wash; 3) saline wash only; and 4) saline wash plus PVA-VAN/TOB-P. After seven days, the pouches were opened and washed with saline alone, or had an additional injection of PVA-VAN/TOB-P. Mice were killed 14 days after pouch wash. The first part of the study showed that low-grade infection was more significant in 400 µm cylinders than cylinders with larger pore sizes (p < 0.05). The second part of the study showed that saline wash alone was ineffective in eradicating both low- and high-grade infections. Saline plus PVA-VAN/TOB-P eradicated the titanium cylinder-associated infections, as manifested by negative cultures of the washouts and supported by scanning electron microscopy and histology. Porous titanium cylinders were vulnerable to bacterial infection and biofilm formation that could not be treated by saline irrigation alone. Application of PVA-VAN/TOB-P directly into the surgical site alone or after saline wash represents a feasible approach for prevention and/or treatment of porous implant-related infections.

H2S scavenger as a broad-spectrum strategy to deplete bacteria-derived H2S for antibacterial sensitization

Bacteria-derived H2S plays multifunctional protective roles against antibiotics insult, and the H2S biogenesis pathway is emerging as a viable target for the antibacterial adjuvant design. However, the development of a pan-inhibitor against H2S-synthesizing enzymes is challenging and underdeveloped. Herein, we propose an alternative strategy to downregulate the H2S levels in H2S-producing bacteria, which depletes the bacteria-derived H2S chemically by H2S scavengers without acting on the synthesizing enzymes. After the screening of chemically diversified scaffolds and a structural optimization campaign, a potent and specific H2S scavenger is successfully identified, which displays efficient H2S depletion in several H2S-producing bacteria, potentiates both bactericidal agents and photodynamic therapy, enhances the bacterial clearance of macrophages and polymorphonuclear neutrophils, disrupts the formation of bacterial biofilm and increases the sensitivity of bacterial persister cells to antibiotics. Most importantly, such an H2S scavenger exhibits sensitizing effects with gentamicin in Pseudomonas aeruginosa -infected pneumonia and skin wound female mouse models. In aggregate, our results not only provide an effective strategy to deplete bacteria-derived H2S and establish the H2S biogenesis pathway as a viable target for persisters and drug-resistant bacteria, but also deliver a promising antibacterial adjuvant for potential clinical translation.

Asiatic acid inhibits HBV cccDNA transcription by promoting HBx degradation

BackgroundHepatitis B virus (HBV) infection is a persistent global public health problem, and curing for chronic hepatitis B (CHB) through the application of existing antiviral drugs is beset by numerous challenges. The viral protein HBx is a critical regulatory factor in the life cycle of HBV. Targeting HBx is a promising possibility for the development of novel therapeutic strategies.MethodsThe Nano-Glo® HiBiT Lysis Detection System was used to screen the herbal monomer compound library for compounds that inhibit HBx expression. Western blotting was used to examine proteins expression. Southern blotting or Northern blotting were used to detect HBV DNA or HBV RNA. ELISA was performed to detect the HBsAg level. The effect of asiatic acid on HBV in vivo was investigated by using recombinant cccDNA mouse model.ResultsAsiatic acid, an extract of Centella asiatica, significantly reduced the HBx level. Mechanistic studies demonstrated that asiatic acid may promote the degradation of HBx in an autophagy pathway-dependent manner. Subsequently, asiatic acid was found to reduce the amount of HBx bound to covalently closed circular DNA (cccDNA) microchromosomes, and repressive chromatin modifications then occurred, ultimately inhibiting cccDNA transcriptional activity. Moreover, in HBV-infected cells and a mouse model of persistent HBV infection, asiatic acid exhibited potent anti-HBV activity, as evidenced by decreased levels of HBV RNAs, HBV DNA and HBsAg.ConclusionsAsiatic acid was identified as a compound that targets HBx, revealing its potential for application as an anti-HBV agent.

Functional redundancy in Candida auris cell surface adhesins crucial for cell-cell interaction and aggregation

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als4112 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

Emergence of highly virulent and multidrug-resistant Escherichia coli in breeding sheep with pneumonia, Hainan Province, China.

Sheep are a rarely raised livestock in Hainan Island, China, because of the unfavorable tropical marine climate. Here, this article reports a severe pneumonia in the sheep breeding and domestication facility caused acute mortality during the winter 2021-2022. Six sheep were clinically dissected and histopathologically observed. The bacteria were isolated and cultured by traditional methods and identified by 16S rRNA sequencing. The genotypes, serotypes, virulence genes and antimicrobial resistance genes were analyzed by PCR and whole genome sequencing. The pubMLST website was used for phylogenetic analysis of related strains. Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility test. The antimicrobial susceptibility test standard was referred to the Clinical and Laboratory Standards Institute (CLSI). The virulence of bacteria was detected by mouse infection model. Etiology and histopathology examination of the pneumonia reveled pulmonary abscess and alveolar neutrophilia and pulmonary fibrinous exudates. Escherichia coli was the only bacterial species isolated, primarily from the lungs and blood of the six dead or moribund sheep, a total of 29 E. coli strains were isolated. Antimicrobial resistance profiling shows that all the isolates were resistant to six agents (penicillin, ampicillin, cephalothin, neomycin, erythromycin, and vancomycin) belonging to five classes of antibiotics, classifying them as multi drug resistant (MDR). Furthermore, genotyping analysis revealed all strains were common with 11-17 virulence factors indicating high pathogenicity. The lab mice infection model shows that all strains severely affect the health status particularly weight loss, lethargy, pneumonia and shortly lead to death. The molecular epidemiological analysis indicated most strains share the same genotype as previously reported strains in humans and other farmed animals this suggests a high possibility of cross-species transmission (CST) of virulent and MDR isolates. This CST could be from sheep to humans and other farmed animals or from humans and other farmed animals to sheep. Therefore, this study indicates that E. coli is an emerging threat that causes sheep pneumonia in Hainan, and the quarantine of contacts is important to control the spread of virulent E. coli and the transmission of acquired resistance genes between humans and farmed animals such as sheep.

Cryptosporidium lysyl-tRNA synthetase inhibitors define the interplay between solubility and permeability required to achieve efficacy.

Cryptosporidiosis is a diarrheal disease caused by infection with Cryptosporidium spp. parasites and is a leading cause of death in malnourished children worldwide. The only approved treatment, nitazoxanide, has limited efficacy in this at-risk patient population. Additional safe therapeutics are urgently required to tackle this unmet medical need. However, the development of anti-cryptosporidial drugs is hindered by a lack of understanding of the optimal compound properties required to treat this gastrointestinal infection. To address this knowledge gap, a diverse set of potent lysyl-tRNA synthetase inhibitors was profiled to identify optimal physicochemical and pharmacokinetic properties required for efficacy in a chronic mouse model of infection. The results from this comprehensive study illustrated the importance of balancing solubility and permeability to achieve efficacy in vivo. Our results establish in vitro criteria for solubility and permeability that are predictive of compound efficacy in vivo to guide the optimization of anti-cryptosporidial drugs. Two compounds from chemically distinct series (DDD489 and DDD508) were identified as demonstrating superior efficacy and prioritized for further evaluation. Both compounds achieved marked parasite reduction in immunocompromised mouse models and a disease-relevant calf model of infection. On the basis of these promising data, these compounds have been selected for progression to preclinical safety studies, expanding the portfolio of potential treatments for this neglected infectious disease.

Multiplex antimicrobial activities of the self-assembled amphiphilic polypeptide β nanofiber KF-5 against vaginal pathogens

BackgroundVaginal infections caused by multidrug-resistant pathogens such as Candida albicans and Gardnerella spp. represent a significant health challenge. Current treatments often fail because of resistance and toxicity. This study aimed to synthesize and characterize a novel amphiphilic polypeptide, KF-5, and evaluate its antibacterial and antifungal activities, biocompatibility, and potential mechanisms of action.ResultsThe KF-5 peptide was synthesized via solid-phase peptide synthesis and self-assembled into nanostructures with filamentous and hydrogel-like configurations. Characterization by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) confirmed the unique nanostructural properties of KF-5. KF-5 (125, 250, or 500 µg/ml) demonstrated potent antibacterial and antifungal activities, with significant inhibitory effects on drug-resistant Candida albicans and Gardnerella spp. (P < 0.05). In vitro assays revealed that 500 µg/ml KF-5 disrupted microbial cell membranes, increased membrane permeability, and induced lipid oxidation, leading to cell death (P < 0.05). Cytotoxicity tests revealed minimal toxicity in human vaginal epithelial cells, keratinocytes, and macrophages, with over 95% viability at high concentrations. Molecular dynamics simulations indicated that KF-5 interacts with phospholipid bilayers through electrostatic interactions, causing membrane disruption. In vivo studies using a mouse model of vaginal infection revealed that 0.5, 1, and 2 mg/ml KF-5 significantly reduced fungal burden and inflammation, and histological analysis confirmed the restoration of vaginal mucosal integrity (P < 0.01). Compared with conventional antifungal treatments such as miconazole, KF-5 exhibited superior efficacy (P < 0.01).ConclusionsKF-5 demonstrates significant potential as a safe and effective antimicrobial agent for treating vaginal infections. Its ability to disrupt microbial membranes while maintaining biocompatibility with human cells highlights its potential for clinical application. These findings provide a foundation for further development of KF-5 as a therapeutic option for combating drug-resistant infections.

Lytic promoter activity during herpes simplex virus latency is dependent on genome location.

Herpes simplex virus 1 (HSV-1) is a significant pathogen that establishes lifelong latent infections with intermittent episodes of resumed disease. In mouse models of HSV infection, sporadic low-level lytic gene expression has been detected during latency in the absence of reactivation events that lead to production of new viruses. This viral activity during latency has been reported using a sensitive Cre-marking model for several lytic gene promoters placed in one location in the HSV-1 genome. Here, we extend these findings in the same model by examining first, the activity of an ectopic lytic gene promoter in several places in the genome and second, whether any promoters might be active in their natural context. We found that Cre expression was detected during latency from ectopic and native promoters, but only in locations near the ends of the unique long genome segment. This location is significant because it is in close proximity to the region from which latency-associated transcripts (LATs) are derived. These results show that native HSV-1 lytic gene promoters can produce protein products during latency, but that this activity is only detectable when they are located close to the LAT locus.IMPORTANCEHSV is a significant human pathogen and the best studied model of mammalian virus latency. Traditionally, the active (lytic) and inactive (latent) phases of infection were considered to be distinct, but the notion of latency being entirely quiescent is evolving due to the detection of some lytic gene expression during latency. Here, we add to this literature by finding that the activity can be found for native lytic gene promoters as well as for constructs placed ectopically in the HSV genome. However, this activity was only detectable when these promoters were located close by a region known to be transcriptionally active during latency. These data have implications for our understanding of HSV gene regulation during latency and the extent to which transcriptionally active regions are insulated from adjacent parts of the viral genome.

Sensory neurons regulate stimulus-dependent humoral immunity in mouse models of bacterial infection and asthma

Sensory neurons sense pathogenic infiltration to drive innate immune responses, but their role in humoral immunity is unclear. Here, using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma, we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae, sensory neuron depletion increases bacterial burden and reduces B cell numbers, IgG release, and neutrophil stimulation. Meanwhile, during A. alternata-induced airway inflammation, sensory neuron depletion decreases B cell population sizes, IgE levels, and asthmatic characteristics. Mechanistically, during bacterial infection, sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma, sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden, while VIPR1 deficiency increases infection. Similarly, exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice, while substance P deficiency ameliorates asthma. Our data, thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen.

The plasmid-encoded members of paralogous gene family 52 are dispensable to the enzootic cycle of Borrelia burgdorferi.

Lyme disease, the leading vector-borne disease in the United States and Europe, develops after infection with Borrelia burgdorferi sensu lato bacteria. Transmission of the spirochete from the tick vector to a vertebrate host requires global changes in gene expression that are controlled, in part, by the Rrp2/RpoN/RpoS alternative sigma factor cascade. Transcriptional studies defining the B. burgdorferi RpoS regulon have suggested that RpoS activates the transcription of paralogous family 52 (PFam52) genes. In strain B31, PFam52 genes (bbi42, bbk53, and bbq03) encode a set of conserved hypothetical proteins with >89% amino acid identity that are predicted to be surface-localized. Extensive homology among members of paralogous families complicates studies of protein contributions to pathogenicity as the potential for functional redundancy will obfuscate findings. Using a sequential mutagenesis approach, we generated clones expressing a single PFam52 paralog, as well as a strain deficient in all three. The single paralog expressing strains were used to confirm BBI42, BBK53, and BBQ03 surface localization and RpoS regulation. Surprisingly, the PFam52-deficient strain was able to infect mice and complete the enzootic cycle similar to the wild-type parental strain. Indeed, the presence of numerous pseudogenes that contain frameshifts or internal stop codons among the PFam52 genes suggests that they may be subjected to gene loss in B. burgdorferi's reduced genome. Alternatively, the lack of phenotype might reflect the limitations of the experimental mouse infection model.

CD4+ T cell help during early acute hepacivirus infection is critical for viral clearance and the generation of a liver-homing CD103+CD49a+ effector CD8+ T cell subset.

In hepatitis C virus (HCV) infection, CD4+ and CD8+ T cells are crucial for viral control. However, a detailed understanding of the kinetic of CD4+ T cell help and its role in the generation of different CD8+ T cell subsets during acute infection is lacking. The absence of a small HCV animal model has impeded mechanistic studies of hepatic antiviral T cell immunity and HCV vaccine development. In this study, we used a recently developed HCV-related rodent hepacivirus infection mouse model to investigate the impact of CD4+ T cell help on the hepatic CD8+ T cell response and viral clearance during hepacivirus infection in vivo. Our results revealed a specific kinetic of CD4+ T cell dependency during acute infection. Early CD4+ T cell help was essential for CD8+ T cell priming and viral clearance, while CD4+ T cells became dispensable during later stages of acute infection. Effector CD8+ T cells directly mediated timely hepacivirus clearance. An analysis of hepatic CD8+ T cells specific for two different viral epitopes revealed the induction of subsets of liver-homing CD103+CD49a+ and CD103-CD49a+ effector CD8+ T cells with elevated IFN-γ and TNF-α production. CD103+CD49a+ T cells further persisted as tissue-resident memory subsets. A lack of CD4+ T cell help and CD40L-CD40 interactions resulted in reduced effector functions and phenotypical changes in effector CD8+ T cells and a specific loss of the CD103+CD49a+ subset. In summary, our study shows that early CD4+ T cell help through CD40L signaling is essential for priming functional effector CD8+ T cell subsets, including unique liver-homing subsets, and hepacivirus clearance.

Single cell spatial profiling of FFPE splenic tissue from a humanized mouse model of HIV infection

BackgroundLatency remains a major obstacle to finding a cure for HIV despite the availability of antiretroviral therapy. Due to virus dormancy, limited biomarkers are available to identify latent HIV-infected cells. Profiling of individual HIV-infected cells is needed to explore potential latency biomarkers and to study the mechanisms of persistence that maintain the HIV reservoir.MethodsSingle cell spatial transcriptomic characterization using the CosMx Spatial Molecular Imager platform was conducted to analyze HIV-infected cells in formalin-fixed paraffin-embedded sections of splenic tissue surgically obtained from an HIV-infected humanized mouse model. Regulation of over a thousand human genes was quantified in both viremic and aviremic specimens. In addition, in situ hybridization and immunohistochemistry were performed in parallel to identify HIV viral RNA- and p24-containing cells, respectively. Finally, initial findings from CosMx gene profiling were confirmed by isolating RNA from CD4 + T cells obtained from a person living with HIV on antiretroviral therapy following either PMA/Ionomycin or DMSO treatment. RNA was quantified using qPCR for a panel of targeted human host genes.ResultsSupervised cell typing revealed that most of the HIV-infected cells in the mouse spleen sections were differentiated CD4 + T cells. A significantly higher number of infected cells, 2781 (1.61%) in comparison to 112 (0.06%), and total HIV transcripts per infected cell were observed in viremic samples compared to aviremic samples, respectively, which was consistent with the data obtained from ISH and IHC. Notably, the expression of 55 genes was different in infected cells within tissue from aviremic animals compared to viremic. In particular, both spleen tyrosine kinase (SYK) and CXCL17, were expressed approximately 100-fold higher. This data was further evaluated against bulk RNA isolated from HIV-infected human primary CD4 + T cells. A nearly 6-fold higher expression of SYK mRNA was observed in DMSO-treated CD4 + T cells compared to those stimulated with PMA/Ionomycin.ConclusionThis study found that the CosMx SMI platform is valuable for assessing HIV infection and providing insights into host biomarkers associated with HIV reservoirs. Higher relative expression of the SYK gene in aviremic-infected cells from the humanized mouse HIV model was consistent with levels found in CD4 + T cells of aviremic donors.

A chronic Pseudomonas aeruginosa mouse lung infection modeling the pathophysiology and inflammation of human cystic fibrosis.

Host-pathogen interaction studies of Pseudomonas aeruginosa cystic fibrosis (CF) lung infections have been hampered by limitations of mouse infection models. Here we combine synthetic CF sputum medium (SCFM) agar beads and Scnn1b -Tg transgenic mice to model the mucus obstructed airways and complex inflammatory characteristic of the human cystic fibrosis lung environment. In this model, which we name SCFM-Tg-mice, we use SCFM to cause changes in bacterial gene expression consistent with sputum collected from people with CF and the Scnn1b-Tg mice produce excessive airway mucus like people with CF. We show that SCMF-Tg-mice infected with P. aeruginosa have defects in lung function and increased inflammation that is consistent with human CF lung infections. This model can be adapted for other bacterial species and can be used to test hypotheses about bacterial pathogenesis and potential treatments in a CF human-like system.

Notch4 regulatory T cells and SARS-CoV-2 viremia shape COVID19 survival outcome.

Immune dysregulation and SARS-CoV-2 plasma viremia have been implicated in fatal COVID-19 disease. However, how these two factors interact to shape disease outcomes is unclear. We carried out viral and immunological phenotyping on a prospective cohort of 280 patients with COVID-19 presenting to acute care hospitals in Boston, Massachusetts and Genoa, Italy between June 1, 2020 and February 8, 2022. Disease severity, mortality, plasma viremia, and immune dysregulation were assessed. A mouse model of lethal H1N1 influenza infection was used to analyze the therapeutic potential of Notch4 and pyroptosis inhibition in disease outcome. Stratifying patients based on %Notch4+ Treg cells and/or the presence of plasma viremia identified four subgroups with different clinical trajectories and immune phenotypes. Patients with both high %Notch4+ Treg cells and viremia suffered the most disease severity and 90-day mortality compared to the other groups even after adjusting for baseline comorbidities. Increased Notch4 and plasma viremia impacted different arms of the immune response in SARS-CoV-2 infection. Increased Notch4 was associated with decreased Treg cell amphiregulin expression and suppressive function whereas plasma viremia was associated with increased monocyte cell pyroptosis. Combinatorial therapies using Notch4 blockade and pyroptosis inhibition induced stepwise protection against mortality in a mouse model of lethal H1N1 influenza infection. The clinical trajectory and survival outcome in hospitalized patients with COVID-19 is predicated on two cardinal factors in disease pathogenesis: viremia and Notch4+ Treg cells. Intervention strategies aimed at resetting the immune dysregulation in COVID-19 by antagonizing Notch4 and pyroptosis may be effective in severe cases of viral lung infection.