Infection Of Epithelial Cells Research Articles

Olive Leaf Extract (OLE) as a Novel Antioxidant That Ameliorates the Inflammatory Response in Cystic Fibrosis.

The deletion of phenylalanine at position 508 (F508del) produces a misfolded CFTR protein that is retained in the ER and degraded. The lack of normal CFTR channel activity is associated with chronic infection and inflammation which are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. Moreover, LPS-dependent oxidative stress downregulates CFTR function in airway epithelial cells. Olive leaf extract (OLE) is used in traditional medicine for its effects, including anti-oxidant and anti-inflammatory ones. We found that OLE decreased the intracellular ROS levels in a dose-response manner in CFBE cells. Moreover, OLE attenuates the inflammatory response to LPS or IL-1β/TNFα stimulation, mimicking the infection and inflammatory status of CF patients, in CFBE and primary nasal epithelial (HNE) cells. Furthermore, we demonstrated that OLE restored the LPS-mediated decrease of TrikfaftaTM-dependent F508del-CFTR function in CFBE and HNE cultures. These findings provide strong evidence of OLE to prevent redox imbalance and inflammation that can cause chronic lung damage by enhancing the antioxidant activity and attenuating inflammation in CF airway epithelial cells. Additionally, OLE might be used in combination with CFTR modulators therapy to improve their efficacy in CF patients.

Infection of ferrets with wild type-based recombinant canine distemper virus overwhelms the immune system and causes fatal systemic disease.

Canine distemper virus (CDV) causes systemic infection resulting in severe and often fatal disease in a large spectrum of animal host species. The virus is closely related to measles virus and targets myeloid, lymphoid, and epithelial cells, but CDV is more virulent and the infection spreads more rapidly within the infected host. Here, we aimed to study the pathogenesis of wild-type CDV infection by experimentally inoculating ferrets with recombinant CDV (rCDV) based on an isolate directly obtained from a naturally infected raccoon. The recombinant virus was engineered to express a fluorescent reporter protein, facilitating assessment of viral tropism and virulence. In ferrets, this wild type-based rCDV infected myeloid, lymphoid, and epithelial cells, and the infection resulted in systemic dissemination to multiple tissues and organs, especially those of the lymphatic system. High infection percentages in immune cells resulted in depletion of these cells both from circulation and from lymphoid tissues. The majority of CDV-infected ferrets reached their humane endpoints within 20 d and had to be euthanized. In that period, the virus also reached the central nervous system in several ferrets, but we did not observe the development of neurological complications during the study period of 23 d. Two out of 14 ferrets survived CDV infection and developed neutralizing antibodies. We show for the first time the pathogenesis of a non-adapted wild type-based rCDV in ferrets. IMPORTANCE Infection of ferrets with recombinant canine distemper virus (rCDV) expressing a fluorescent reporter protein has been used as proxy to understand measles pathogenesis and immune suppression in humans. CDV and measles virus use the same cellular receptors, but CDV is more virulent, and infection is often associated with neurological complications. rCDV strains in current use have complicated passage histories, which may have affected their pathogenesis. Here, we studied the pathogenesis of the first wild type-based rCDV in ferrets. We used macroscopic fluorescence to identify infected cells and tissues; multicolor flow cytometry to determine viral tropism in immune cells; and histopathology and immunohistochemistry to characterize infected cells and lesions in tissues. We conclude that CDV often overwhelmed the immune system, resulting in viral dissemination to multiple tissues in the absence of a detectable neutralizing antibody response. This virus is a promising tool to study the pathogenesis of morbillivirus infections.

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells.

The strong contribution of RAS-related protein 1b (Rap1b) to cytoskeleton remodeling determines intracellular and extracellular physiological activities, including the successful infection of viruses in permissive cells, but its role in the HSV-1 life cycle is still unclear. Here, we demonstrated that the HSV-1 immediate early (IE) gene ICP4 inhibits protein kinase A (PKA) phosphorylation to induce Rap1b-activation-mediated viral infection. Rap1b activation and membrane enrichment begin at the early stage of HSV-1 infection and remain active during the proliferation period of the virus. Treating the cells with Rap1b small interfering RNA (siRNA) showed a dose-dependent decrease in viral infection levels, but no dose-dependent increase was observed after Rap1b overexpression. Further investigation indicated that the suppression of Rap1b activation derives from phosphorylated PKA and Rap1b mutants with partial or complete prenylation instead of phosphorylation, which promoted viral infection in a dose-dependent manner. Furthermore, the PKA agonist Forskolin disturbed Rap1b activation in a dose-dependent manner, accompanied by a decreasing trend in viral infection. Moreover, the HSV-1 IE gene ICP4 induced PKA dephosphorylation, leading to continuous Rap1b activation, followed by cytoskeleton rearrangement induced by cell division control protein 42 (CDC42) and Ras-related C3 botulinum toxin substrate 1 (RAC1). These further stimulated membrane-triggered physiological processes favoring virus infection. Altogether, we show the significance of Rap1b during HSV-1 infection and uncover the viral infection mechanism determined by the posttranslational regulation of the viral ICP4 gene and Rap1b host protein.

Spectrum, Clinicopathologic Profile, and p16 Expression Pattern of Nonmalignant Cervical Tissues in Enugu, South-East Nigeria

Abstract Background p16 is a marker for p16-induced transformation of high-risk human papilloma virus (hrHPV)-infected cervical epithelium. HPV is a known etiologic agent of cervical cancer. Persistent hrHPV infection of cervical epithelium causes transformation of the infected epithelial cells resulting in increased proliferative potential of the cells and subsequent progression to frank malignancy. Early detection of transformation in cervical cells is crucial in reducing morbidity and mortality associated with cervical cancer. Materials and Methods We aimed to study the clinicopathologic profile of nonmalignant cervical lesions and their p16 staining pattern. Histopathology requisition forms, blocks, and slides of cases containing cervical tissue with nonmalignant diagnosis received into the morbid anatomy department of University of Nigeria Teaching Hospital, Enugu, from 2009 to 2018 were studied. Fresh sections from the blocks were immunohistochemically stained with p16 and examined. Results One-hundred and ninety-one cases were studied. Majority of the cases are normal cervical epithelium. Chronic nonspecific cervicitis was the major non-neoplastic lesion present and accounted for 33.3% of the biopsy and the mean age was 50.5 years. Other lesions were nabothian cyst (8.4%), cervical polyp (10.5%), low-grade and high-grade squamous intraepithelial lesion (LSIL and HSIL; 6.3 and 2.6%, respectively). The mean age for LSIL was 40.3 years, while that of HSIL was 45.2years. Four LSIL, two HSIL, one polyp, one chronic nonspecific cervicitis, and one lobular endocervical hyperplasia stained positively with p16. Conclusion The most common benign lesion of the cervix is cervicitis. Chronic cervicitis is negative to p16 immunoreactivity. There are more low-grade cervical intraepithelial neoplasia (CIN) than high-grade CIN. The low-grade CIN overexpress p16 in one-third of cases. There are more cases of p16-negative high-grade CIN in this study.

Human beta defensin-3 mediated activation of β-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5.

Respiratory Syncytial Virus (RSV) is a non-segmented negative-sense RNA virus belonging to the paramyxovirus family. RSV infects the respiratory tract to cause pneumonia and bronchiolitis in infants, elderly, and immunocompromised patients. Effective clinical therapeutic options and vaccines to combat RSV infection are still lacking. Therefore, to develop effective therapeutic interventions, it is imperative to understand virus-host interactions during RSV infection. Cytoplasmic stabilization of β-catenin protein results in activation of canonical Wingless (Wnt)/β-catenin signaling pathway that culminates in transcriptional activation of various genes regulated by T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. This pathway is involved in various biological and physiological functions. Our study shows RSV infection of human lung epithelial A549 cells triggering β-catenin protein stabilization and induction of β-catenin mediated transcriptional activity. Functionally, the activated β-catenin pathway promoted a pro-inflammatory response during RSV infection of lung epithelial cells. Studies with β-catenin inhibitors and A549 cells lacking optimal β-catenin activity demonstrated a significant loss of pro-inflammatory chemokine interleukin-8 (IL-8) release from RSV-infected cells. Mechanistically, our studies revealed a role of extracellular human beta defensin-3 (HBD3) in interacting with cell surface Wnt receptor LDL receptor-related protein-5 (LRP5) to activate the non-canonical Wnt independent β-catenin pathway during RSV infection. We showed gene expression and release of HBD3 from RSV-infected cells and silencing of HBD3 expression resulted in reduced stabilization of β-catenin protein during RSV infection. Furthermore, we observed the binding of extracellular HBD3 with cell surface localized LRP5 protein, and our in silico and protein-protein interaction studies have highlighted a direct interaction of HBD3 with LRP5. Thus, our studies have identified the β-catenin pathway as a key regulator of pro-inflammatory response during RSV infection of human lung epithelial cells. This pathway was induced during RSV infection via a non-canonical Wnt-independent mechanism involving paracrine/autocrine action of extracellular HBD3 activating cell surface Wnt receptor complex by directly interacting with the LRP5 receptor.

Dicoumarol is an effective post-exposure prophylactic for SARS-CoV-2 Omicron infection in human airway epithelium

Repurposing existing drugs to inhibit SARS-CoV-2 infection in airway epithelial cells (AECs) is a quick way to find novel treatments for COVID-19. Computational screening has found dicoumarol (DCM), a natural anticoagulant, to be a potential SARS-CoV-2 inhibitor, but its inhibitory effects and possible working mechanisms remain unknown. Using air-liquid interface culture of primary human AECs, we demonstrated that DCM has potent antiviral activity against the infection of multiple Omicron variants (including BA.1, BQ.1 and XBB.1). Time-of-addition and drug withdrawal assays revealed that early treatment (continuously incubated after viral absorption) of DCM could markedly inhibit Omicron replication in AECs, but DCM did not affect the absorption, exocytosis and spread of viruses or directly eliminate viruses. Mechanistically, we performed single-cell sequencing analysis (a database of 77,969 cells from different airway locations from 10 healthy volunteers) and immunofluorescence staining, and showed that the expression of NAD(P)H quinone oxidoreductase 1 (NQO1), one of the known DCM targets, was predominantly localised in ciliated AECs. We further found that the NQO1 expression level was positively correlated with both the disease severity of COVID-19 patients and virus copy levels in cultured AECs. In addition, DCM treatment downregulated NQO1 expression and disrupted signalling pathways associated with SARS-CoV-2 disease outcomes (e.g., Endocytosis and COVID-19 signalling pathways) in cultured AECs. Collectively, we demonstrated that DCM is an effective post-exposure prophylactic for SARS-CoV-2 infection in the human AECs, and these findings could help physicians formulate novel treatment strategies for COVID-19.

Tryptophan-like side chain holding aptamers inhibit respiratory syncytial virus infection of lung epithelial cells

Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between its pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep pace with the continuously evolving pathogens.

Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host.

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems.

Anti-SARS-CoV-2 Activity of Polysaccharides Extracted from Halymenia floresii and Solieria chordalis (Rhodophyta)

Even after hundreds of clinical trials, the search for new antivirals to treat COVID-19 is still relevant. Carrageenans are seaweed sulfated polysaccharides displaying antiviral activity against a wide range of respiratory viruses. The objective of this work was to study the antiviral properties of Halymenia floresii and Solieria chordalis carrageenans against SARS-CoV-2. Six polysaccharide fractions obtained from H. floresii and S. chordalis by Enzyme-Assisted Extraction (EAE) or Hot Water Extraction (HWE) were tested. The effect of carrageenan on viral replication was assessed during infection of human airway epithelial cells with a clinical strain of SARS-CoV-2. The addition of carrageenans at different times of the infection helped to determine their mechanism of antiviral action. The four polysaccharide fractions isolated from H. floresii displayed antiviral properties while the S. chordalis fractions did not. EAE-purified fractions caused a stronger reduction in viral RNA concentration. Their antiviral action is likely related to an inhibition of the virus attachment to the cell surface. This study confirms that carrageenans could be used as first-line treatment in the respiratory mucosa to inhibit the infection and transmission of SARS-CoV-2. Low production costs, low cytotoxicity, and a broad spectrum of antiviral properties constitute the main strengths of these natural molecules.

Cathelicidin-derived antiviral peptide inhibits herpes simplex virus 1 infection.

Herpes simplex virus 1 (HSV-1) is a widely distributed virus. HSV-1 is a growing public health concern due to the emergence of drug-resistant strains and the current lack of a clinically specific drug for treatment. In recent years, increasing attention has been paid to the development of peptide antivirals. Natural host-defense peptides which have uniquely evolved to protect the host have been reported to have antiviral properties. Cathelicidins are a family of multi-functional antimicrobial peptides found in almost all vertebrate species and play a vital role in the immune system. In this study, we demonstrated the anti-HSV-1 effect of an antiviral peptide named WL-1 derived from human cathelicidin. We found that WL-1 inhibited HSV-1 infection in epithelial and neuronal cells. Furthermore, the administration of WL-1 improved the survival rate and reduced viral load and inflammation during HSV-1 infection via ocular scarification. Moreover, facial nerve dysfunction, involving the abnormal blink reflex, nose position, and vibrissae movement, and pathological injury were prevented when HSV-1 ear inoculation-infected mice were treated with WL-1. Together, our findings demonstrate that WL-1 may be a potential novel antiviral agent against HSV-1 infection-induced facial palsy.

Oridonin Inhibits Mycobacterium marinum Infection-Induced Oxidative Stress In Vitro and In Vivo.

Prior to the COVID-19 pandemic, tuberculosis (TB) was the leading cause of death globally attributable to a single infectious agent, ranking higher than HIV/AIDS. Consequently, TB remains an urgent public health crisis worldwide. Oridonin (7a,20-Epoxy-1a,6b,7,14-tetrahydroxy-Kaur-16-en-15-one Isodonol, C20H28O6, Ori), derived from the Rabdosia Rrubescens plant, is a natural compound that exhibits antioxidant, anti-inflammatory, and antibacterial properties. Our objective was to investigate whether Ori's antioxidant and antibacterial effects could be effective against the infection Mycobacterium marinum (Mm)-infected cells and zebrafish. We observed that Ori treatment significantly impeded Mm infection in lung epithelial cells, while also suppressing inflammatory response and oxidative stress in Mm-infected macrophages. Further investigation revealed that Ori supplementation inhibited the proliferation of Mm in zebrafish, as well as reducing oxidative stress levels in infected zebrafish. Additionally, Ori promoted the expression of NRF2/HO-1/NQO-1 and activated the AKT/AMPK-α1/GSK-3β signaling pathway, which are both associated with anti-inflammatory and antioxidant effects. In summary, our results demonstrate that Ori exerts inhibitory effects on Mm infection and proliferation in cells and zebrafish, respectively. Additionally, Ori regulates oxidative stress by modulating the NRF2/HO-1/NQO-1 and AKT/AMPK-α1/GSK-3β signaling pathways.

WS18.06 The impact of cytomegalovirus on airway epithelial gene expression

Introduction: The pathogenicity of cytomegalovirus (CMV) has predominantly been recognized in immunosuppressed populations & newborns. However, a recent study in persons with CF observed that CMV seropositive individuals died from lung disease 8 years sooner than those who were seronegative. Further, CMV DNA was identified in those individuals’ sputum (Parkins 2019, ERJ). This observation aligns with increasing evidence associating CMV infection with worse outcomes in many inflammatory conditions, and a higher overall mortality. To date, models of CMV infection of human bronchial epithelial (HBE) cells have not been established, limiting our ability to study its impact in CF airways.

25-hydroxycholesterol inhibits human papillomavirus infection in cervical epithelial cells by perturbing cytoskeletal remodeling.

Persistent high-risk human papilloma virus (HR-HPV) infection is the main risk factor for cervical cancer, threatening women's health. Despite growing prophylactic vaccination, annual cervical cancer cases are still increasing and show a trend of younger onset age. However, therapeutic approaches towards HPV infection are still limited. 25-hydrocholesterol (25HC) has a wide-spectrum inhibitory effect on a variety of viruses. To explore efficient interventions to restrict HPV infection at an early time, we applied different pseudoviruses (PsV) to evaluate anti-HPV efficacy of 25HC. We tested PsV inhibition by 25HC in cervical epithelial-derived HeLa and C-33A cells, using high-risk (HPV16, HPV18, HPV59), possibly carcinogenic (HPV73), and low-risk (HPV6) HPV PsVs. Then we established murine genital HPV PsV infection models and applied IVIS to evaluate anti-HPV efficacy of 25HC in vivo. Next, with the help of confocal imaging, we targeted 25HC activity at filopodia upon HPV exposure. After that, we used RNA-seq and Western blot analysis to investigate (1) how 25HC disturbs actin cytoskeleton remodeling during HPV infection and (2) how prenylation regulates the cytoskeletal remodeling signaling pathway. Our findings suggest that 25HC perturbs F-actin rearrangement by reducing small GTPase prenylation. In this way, the phenomenon of HPV virion surfing was restricted, leading to failed infection.

Potential mechanisms mediating PM2.5-induced alterations of H3N2 influenza virus infection and cytokine production in human bronchial epithelial cells

Exposure to particulate matter (PM) has been associated with increased hospital admissions for influenza. Airway epithelial cells are a primary target for inhaled environmental insults including fine PM (PM2.5) and influenza viruses. The potentiation of PM2.5 exposure on the effects of influenza virus on airway epithelial cells has not been adequately elucidated. In this study, the effects of PM2.5 exposure on influenza virus (H3N2) infection and downstream modulation of inflammation and antiviral immune response were investigated using a human bronchial epithelial cell line, BEAS-2B. The results showed that PM2.5 exposure alone increased the production of pro-inflammatory cytokines including interleukin-6 (IL-6) and IL-8 but decreased the production of the antiviral cytokine interferon-β (IFN-β) in BEAS-2B cells while H3N2 exposure alone increased the production of IL-6, IL-8, and IFN-β. Importantly, prior exposure to PM2.5 enhanced subsequent H3N2 infectivity, expression of viral hemagglutinin protein, as well as upregulation of IL-6 and IL-8, but reduced H3N2-induced IFN-β production. Pre-treatment with a pharmacological inhibitor of nuclear factor-κB (NF-κB) suppressed pro-inflammatory cytokine production induced by PM2.5, H3N2, as well as PM2.5-primed H3N2 infection. Moreover, antibody-mediated neutralization of Toll-like receptor 4 (TLR4) blocked cytokine production triggered by PM2.5 or PM2.5-primed H3N2 infection, but not H3N2 alone. Taken together, exposure to PM2.5 alters H3N2-induced cytokine production and markers of replication in BEAS-2B cells, which in turn are regulated by NF-κB and TLR4.

SARS-CoV-2 infection impairs NK cell functions via activation of the LLT1-CD161 axis.

Natural killer (NK) cells plays a pivotal role in the control of viral infections, and their function depend on the balance between their activating and inhibitory receptors. The immune dysregulation observed in COVID-19 patients was previously associated with downregulation of NK cell numbers and function, yet the mechanism of inhibition of NK cell functions and the interplay between infected cells and NK cells remain largely unknown. In this study we show that SARS-CoV-2 infection of airway epithelial cells can directly influence NK cell phenotype and functions in the infection microenvironment. NK cells were co-cultured with SARS-CoV-2 infected epithelial cells, in a direct contact with A549ACE2/TMPRSS2 cell line or in a microenvironment of the infection in a 3D ex vivo human airway epithelium (HAE) model and NK cell surface expression of a set of most important receptors (CD16, NKG2D, NKp46, DNAM-1, NKG2C, CD161, NKG2A, TIM-3, TIGIT, and PD-1) was analyzed. We observed a selective, in both utilized experimental models, significant downregulation the proportion of CD161 (NKR-P1A or KLRB1) expressing NK cells, and its expression level, which was followed by a significant impairment of NK cells cytotoxicity level against K562 cells. What is more, we confirmed that SARS-CoV-2 infection upregulates the expression of the ligand for CD161 receptor, lectin-like transcript 1 (LLT1, CLEC2D or OCIL), on infected epithelial cells. LLT1 protein can be also detected not only in supernatants of SARS-CoV-2 infected A549ACE2/TMPRSS2 cells and HAE basolateral medium, but also in serum of COVID-19 patients. Finally, we proved that soluble LLT1 protein treatment of NK cells significantly reduces i) the proportion of CD161+ NK cells, ii) the ability of NK cells to control SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells and iii) the production of granzyme B by NK cells and their cytotoxicity capacity, yet not degranulation level. We propose a novel mechanism of SARS-CoV-2 inhibition of NK cell functions via activation of the LLT1-CD161 axis.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

The role of infected epithelial cells in Chlamydia-associated fibrosis.

Ocular, genital, and anogenital infection by the obligate intracellular pathogen Chlamydia trachomatis have been consistently associated with scar-forming sequelae. In cases of chronic or repeated infection of the female genital tract, infection-associated fibrosis of the fallopian tubes can result in ectopic pregnancy or infertility. In light of this urgent concern to public health, the underlying mechanism of C. trachomatis-associated scarring is a topic of ongoing study. Fibrosis is understood to be an outcome of persistent injury and/or dysregulated wound healing, in which an aberrantly activated myofibroblast population mediates hypertrophic remodeling of the basement membrane via deposition of collagens and other components of the extracellular matrix, as well as induction of epithelial cell proliferation via growth factor signaling. Initial study of infection-associated immune cell recruitment and pro-inflammatory signaling have suggested the cellular paradigm of chlamydial pathogenesis, wherein inflammation-associated tissue damage and fibrosis are the indirect result of an immune response to the pathogen initiated by host epithelial cells. However, recent work has revealed more direct routes by which C. trachomatis may induce scarring, such as infection-associated induction of growth factor signaling and pro-fibrotic remodeling of the extracellular matrix. Additionally, C. trachomatis infection has been shown to induce an epithelial-to-mesenchymal transition in host epithelial cells, prompting transdifferentiation into a myofibroblast-like phenotype. In this review, we summarize the field's current understanding of Chlamydia-associated fibrosis, reviewing key new findings and identifying opportunities for further research.

Role of the cGAS/STING pathway in the control of Brucella abortus infection acquired through the respiratory route.

Despite the importance of the respiratory route for Brucella transmission, the lung immune response to this pathogen is scarcely characterized. We investigated the role of the cGAS/STING pathway of microbial DNA recognition in the control of respiratory Brucella infection. After in vitro B. abortus infection, CFU numbers were significantly higher in alveolar macrophages (AM) and lung explants from STING KO mice than in samples from wild type (WT) mice, but no difference was observed for cGAS KO samples. CFU were also increased in WT AM and lung epithelial cells preincubated with the STING inhibitor H151. Several proinflammatory cytokines (TNF-α, IL-1β, IL-6, IP-10/CXCL10) were diminished in Brucella-infected lung explants and/or AM from STING KO mice and cGAS KO mice. These cytokines were also reduced in infected AM and lung epithelial cells pretreated with H151. After intratracheal infection with B. abortus, STING KO mice exhibited increased CFU in lungs, spleen and liver, a reduced expression of IFN-β mRNA in lungs and spleen, and reduced levels of proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and lung homogenates. Increased lung CFU and reduced BALF cytokines were also observed in cGAS KO mice. In summary, the cGAS/STING pathway induces the production of proinflammatory cytokines after respiratory Brucella infection, which may contribute to the STING-dependent control of airborne brucellosis.

Tibetan medicine Jiuwei Qingpeng San could inhibit influenza A virus induced lung injury via regulating the expression of IFN-γ and its signaling pathway

IntroductionThe Tibetan formula Jiuwei Qingpeng San (JWQPS) has been applied for severe respiratory infection with a centuries-old history and contemporarily applied for influenza induced pneumonia. Although it is empirically effective, its antiviral efficacy and the underlying mechanisms against influenza A (IAV) virus induced lung injury have not been elucidated yet. MethodsThe in vitro anti-viral activity of JWQPS against H1N1 was reflected by the detection of cell viability of PR8 infected BEAS-2B cells. A replication competent PR8 carrying Gaussia luciferase (PR8-Luci) was further utilized to infect BEAS-2B cells to determine the inhibitory activity of virus replication by detection of the intensity of bioluminescence that correlates with viral load. Transcriptome was utilized to analyze the differences of the gene expression profiles in BEAS-2B cell line between the groups with or without JWQPS treatment after PR8 infection. BALB/c mice were intranasally inoculated with 60 or 150 p.f.u. of PR8 and intragastric administered with JWQPS (400 mg/kg/day, 200 mg/kg/day, 100 mg/kg/day). The mice infected with lethal dosages of 60 p.f.u. were monitored and weighed daily for 14 days and the survival rates were calculated. Concentrations of Nucleoprotein (NP) of IAV in BALF that stand for viral load were determined at 3 or 6 d.p.i. by enzyme linked immunosorbent assay (ELISA). Interferons and lymphotactin in the BALF collected at 3 d.p.i. were detected with the corresponding ELISA kits. The level of pro-inflammatory cytokines in BALF collected at 6 d.p.i. were detected using Th1/Th2 Cytokine 11-Plex Mouse ProcartaPlex Panel. The numbers of leukocyte in BALF were counted by an automated blood cell counter. The lung tissues of mice that were not subjected to BALF collection were harvested at 6 d.p.i. to check lung lesions by hematoxylin-eosin staining and lung coefficient calculation. ResultsJWQPS inhibited IAV replication in vitro, down regulated NP in BALF of H1N1 infected BALB/c mice and protected the mice from IAV caused death. The elevated level of lymphotactin, IFN-γ and lymphocytes in BALF were observed 3 days post infection after JWQPS administration. The improved lung lesions, the alleviated leukocytes infiltration, and the decreased pro-inflammatory levels were observed 6 days post infection. Transcriptomic analysis of H1N1 infected lung epithelial cell line revealed JWQPS treatment could dramatically regulate the genes involved in defense response to virus and interferon associated signaling pathways. DiscussionOur study indicated that JWQPS could promote the chemotactic effect of lymphocytes towards site of infection to eliminate the pathogen at early phase of infection and alleviate the development of inflammation thereafter by reducing the occurrence of neutrophils and lymphocytes into the pulmonary alveoli by a rather complicated regulatory network in immune responses.

Viral Protein Accumulation of Zika Virus Variants Links with Regulation of Innate Immunity for Differential Control of Viral Replication, Spread, and Response to Interferon.

Asian lineage Zika virus (ZIKV) strains emerged globally, causing outbreaks linked with critical clinical disease outcomes unless the virus is effectively restricted by host immunity. We have previously shown that retinoic acid-inducible gene-I (RIG-I) senses ZIKV to trigger innate immunity to direct interferon (IFN) production and antiviral responses that can control ZIKV infection. However, ZIKV proteins have been demonstrated to antagonize IFN. Here, we conducted in vitro analyses to assess how divergent prototypic ZIKV variants differ in virologic properties, innate immune regulation, and infection outcome. We comparatively assessed African lineage ZIKV/Dakar/1984/ArD41519 (ZIKV/Dakar) and Asian lineage ZIKV/Malaysia/1966/P6740 (ZIKV/Malaysia) in a human epithelial cell infection model. De novo viral sequence determination identified amino acid changes within the ZIKV/Dakar genome compared to ZIKV/Malaysia. Viral growth analyses revealed that ZIKV/Malaysia accumulated viral proteins and genome copies earlier and to higher levels than ZIKV/Dakar. Both ZIKV strains activated RIG-I/IFN regulatory factor (IRF3) and NF-κB pathways to induce inflammatory cytokine expression and types I and III IFNs. However, ZIKV/Malaysia, but not ZIKV/Dakar, potently blocked downstream IFN signaling. Remarkably, ZIKV/Dakar protein accumulation and genome replication were rescued in RIG-I knockout (KO) cells late in acute infection, resulting in ZIKV/Dakar-mediated blockade of IFN signaling. We found that RIG-I signaling specifically restricts viral protein accumulation late in acute infection where early accumulation of viral proteins in infected cells confers enhanced ability to limit IFN signaling, promoting viral replication and spread. Our results demonstrate that RIG-I-mediated innate immune signaling imparts restriction of ZIKV protein accumulation, which permits IFN signaling and antiviral actions controlling ZIKV infection. IMPORTANCE ZIKV isolates are classified under African or Asian lineages. Infection with emerging Asian lineage-derived ZIKV strains is associated with increased incidence of neurological symptoms that were not previously reported during infection with African or preemergent Asian lineage viruses. In this study, we utilized in vitro models to compare the virologic properties of and innate immune responses to two prototypic ZIKV strains from distinct lineages: African lineage ZIKV/Dakar and Asian lineage ZIKV/Malaysia. Compared to ZIKV/Dakar, ZIKV/Malaysia accumulates viral proteins earlier, replicates to higher levels, and robustly blocks IFN signaling during acute infection. Early accumulation of ZIKV/Malaysia NS5 protein confers enhanced ability to antagonize IFN signaling, dampening innate immune responses to promote viral spread. Our data identify the kinetics of viral protein accumulation as a major regulator of host innate immunity, influencing host-mediated control of ZIKV replication and spread. Importantly, these findings provide a novel framework for evaluating the virulence of emerging variants.