Common Microbial Genital Infections and Their Impact on the Innate Immune Response to HPV in Cervical Cells.

The persistence of high-risk (HR) human papillomavirus (HPV) genotypes is a prerequisite of cervical cancer. It is not clear whether and how bacterial vaginosis (BV) and sexually transmitted infections (STIs) cause higher rates of persistent HPV infection. This study aimed to characterize mucosal innate immunity to HPV, comparing different conditions. Specifically, expression levels of genes coding for Toll-like receptors (TLR)7 and 9, several type III Interferon-related genes (IFNL1, 2, 3, their specific receptor subunit IFNLR1, and the IFN-stimulated gene ISG15). Chemokines CCL5 and CCL20 were measured in cervical cells positive, or not, for HPV, BV, and STIs. HPV DNA was detected in 51/120 (42.5%) enrolled women, two/third were HR-HPV genotypes. More than 50% of samples were BV- and/or STI-positive. HPV-positive women had BV, but not other STIs, more frequently than the HPV-negative. TLR9 and IFNL1 mRNAs were expressed in the LR, but much less in the HR HPV infection. Enhanced levels of TLR9, TLR7, IFNL2, and IFNLR1 were observed in HPV-positive women with BV and STI. TLR9-increased expression was associated with HPV persistence in previous studies; hence, bacterial coinfections may enhance this risk. Prospective measurements of type III IFNs and IFNLR1 are warranted to evaluate whether this response may act as a double-edged sword in infected epithelia.

High prevalence of Merkel cell polyomavirus is associated with dysregulation in transcript levels of TLR9 and type I IFNs in a large cohort of CF patients from the Italian (Lazio) reference center for cystic fibrosis.

Merkel cell polyomavirus (MCPyV) has been detected in respiratory specimens including those from Cystic Fibrosis (CF) patients, raising questions about its immunological and clinical relevance in the respiratory tract. MCPyV might promote an inappropriate antiviral response contributing to a chronic inflammatory response and resulting in detrimental effects in CF. Respiratory samples (n = 1138) were randomly collected from respiratory tract of CF patients (n = 539) during July 2018-October 2019. MCPyV-DNA detection was performed by real time PCR and positive samples were characterized by sequencing of the NCCR genomic region. The transcript levels of Toll-like receptor 9 (TLR9) and type I interferon (IFN-I) genes (IFNα, IFNß and IFNε) were examined by real-time RT-PCR assays. MCPyV-DNA was detected in 268 out of 1138 respiratory specimens (23.5%) without any difference in the prevalence of MCPyV-DNA according to age, gender or bacteriological status of CF individuals. Thirteen out of 137 CF patients remained positive for MCPyV-DNA over the time (a median follow-up period of 8.8 months). Detection of MCPyV-DNA in respiratory specimens was not associated with the occurrence of exacerbation events. Both MCPyV positive adolescents (11-24 years) and adults (≥25 years) had lower mRNA levels of TLR9, IFNß, IFNε and IFNα than the negative patients of the same age group, while MCPyV positive children produced increased levels of TLR9 and IFN-I genes (p < 0.05 for TLR9, IFNß, IFNε) with respect to the negative ones. There were significant differences in TLR9 levels (p < 0.01), but not in those of IFNs, between MCPyV-DNA positive and negative patients with S. aureus, P. aeruginosa or both. Overall, these results indicate that MCPyV-DNA is frequently detected in the respiratory samples of CF patients and might influence the expression levels of IFN-related genes in an age dependent manner. The concomitant detection of MCPyV together with S. aureus and/or P. aeruginosa correlated with alterations in TLR9 levels suggesting that virus-bacteria coinfections might contribute to affect antiviral immunity in CF patients.

KI and WU Polyomavirus in Respiratory Samples of SARS-CoV-2 Infected Patients.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been declared a global pandemic. Our goal was to determine whether co-infections with respiratory polyomaviruses, such as Karolinska Institutet polyomavirus (KIPyV) and Washington University polyomavirus (WUPyV) occur in SARS-CoV-2 infected patients. Oropharyngeal swabs from 150 individuals, 112 symptomatic COVID-19 patients and 38 healthcare workers not infected by SARS-CoV-2, were collected from March 2020 through May 2020 and tested for KIPyV and WUPyV DNA presence. Of the 112 SARS-CoV-2 positive patients, 27 (24.1%) were co-infected with KIPyV, 5 (4.5%) were positive for WUPyV, and 3 (2.7%) were infected simultaneously by KIPyV and WUPyV. Neither KIPyV nor WUPyV DNA was detected in samples of healthcare workers. Significant correlations were found in patients co-infected with SARS-CoV-2 and KIPyV (p < 0.05) and between SARS-CoV-2 cycle threshold values and KIPyV, WUPyV and KIPyV and WUPyV concurrently detected (p < 0.05). These results suggest that KIPyV and WUPyV may behave as opportunistic respiratory pathogens. Additional investigations are needed to understand the epidemiology and the prevalence of respiratory polyomavirus in COVID-19 patients and whether KIPyV and WUPyV could potentially drive viral interference or influence disease outcomes by upregulating SARS-CoV-2 replicative potential.

ACE2 expression is related to the interferon response in airway epithelial cells but is that functional for SARS-CoV-2 entry?

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS-CoV-2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs' activation, ISG15, in children (n = 59) and adults (n = 48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 expression, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs' activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS-CoV-2 infection in the respiratory tract.

SARS-CoV-2 Entry Genes Expression in Relation with Interferon Response in Cystic Fibrosis Patients.

The expression rate of SARS-CoV-2 entry genes, angiotensin-converting enzyme 2 (ACE2), the main viral receptor and the proteases, furin and transmembrane serine protease 2 (TMPRSS2) in cystic fibrosis (CF) individuals is poorly known. Hence, we examined their levels in upper respiratory samples of CF patients (n = 46) and healthy controls (n = 45). Moreover, we sought to understand the interplay of type I interferon (IFN-I) with ACE2, furin and TMPRSS2 by evaluating their gene expression with respect to ISG15, a well-known marker of IFN activation, in upper respiratory samples and after ex vivo IFNß exposure. Lower ACE2 levels and trends toward the reduction of furin and TMPRSS2 were found in CF patients compared with the healthy controls; decreased ACE2 amounts were also detected in CF individuals with pancreatic insufficiency and in those receiving inhaled antibiotics. Moreover, there was a strong positive correlation between ISG15 and ACE2 levels. However, after ex vivo IFNß stimulation of nasopharyngeal cells, the truncated isoform (dACE2), recently demonstrated as the IFN stimulated one with respect to the full-length isoform (flACE2), slightly augmented in cells from CF patients whereas in those from healthy donors, dACE2 levels showed variable levels of upregulation. An altered expression of SARS-COV-2 entry genes and a poor responsiveness of dACE2 to IFN-I stimulation might be crucial in the diffusion of SARS-CoV-2 infection in CF.

Differential toll like receptor expression in cystic fibrosis patients' airways during rhinovirus infection.

OBJECTIVES: Since an inappropriate and sustained activation of TLRs may contribute to a chronic inflammatory response resulting in detrimental effects in cystic fibrosis (CF) patients, we sought to examine whether HRV infection might alter the respiratory expression of TLRs according to the microbiological status of CF patients. METHODS: Respiratory samples were collected from the respiratory tract of CF patients (n = 294) over a period of 12 months. In addition to the usual microbiological investigation, HRV-RNA detection and typing were performed by RT-PCR and sequencing. HRV viral load and TLRs levels were measured by RT-Real Time PCR. RESULTS: HRV-RNA was detected in 80 out of 515 respiratory samples (15.5%) with a similar rate in all age groups (0-10 years, 11-24 years, ≥ 25 years). Patients infected with different HRV A, B and C species exhibited higher levels of TLR2, TLR4 and TLR8 as compared to HRV negative patients. Moreover, the expression level of TLR2, TLR4 and TLR8 correlated with high level of HRV viral load. HRV positive patients co-colonized by Staphylococcus aureus or Pseudomonas aeruginosa showed also enhanced amounts of TLR2 and TLR2/4-mRNAs expression respectively. In the case of presence of both bacteria, TLR2, TLR4, TLR8 and TLR9 levels are elevated in positive HRV patients. CONCLUSIONS: TLRs, especially TLR2 and TLR4, increased in HRV positive CF individuals and varies according to the presence of S. aureus, P. aeruginosa and both bacteria.