Sex Differences in the Response to Viral Infections: TLR8 and TLR9 Ligand Stimulation Induce Higher IL10 Production in Males

BACKGROUND: Susceptibility to viral infections as well as their severity are higher in men than in women. Heightened antiviral responses typical of women are effective for rapid virus clearance, but if excessively high or prolonged, can result in chronic/inflammatory pathologies. We investigated whether this variability could be in part attributable to differences in the response to the Toll-Like Receptors (TLR) more involved in the virus recognition. METHODS: Cytokine production by peripheral blood mononuclear cells (PBMCs) from male and female healthy donors after stimulation with Toll-like receptors (TLR) 3, 7, 8, 9 ligands or with viruses (influenza and Herpes-simplex-1) was evaluated. RESULTS: Compared to females, PBMCs from males produced not only lower amounts of IFN-α in response to TLR7 ligands but also higher amounts of the immunosuppressive cytokine IL10 after stimulation with TLR8 and TLR9 ligands or viruses. IL10 production after TLR9 ligands or HSV-1 stimulation was significantly related with plasma levels of sex hormones in both groups, whereas no correlation was found in cytokines produced following TLR7 and TLR8 stimulation. CONCLUSIONS: Given the role of an early production of IL10 by cells of innate immunity in modulating innate and adaptive immune response to viruses, we suggest that sex-related difference in its production following viral nucleic acid stimulation of TLRs may be involved in the sex-related variability in response to viral infections

Characterization of cervico-vaginal microbiota in women developing persistent high-risk Human Papillomavirus infection

Abstract Changes in cervico-vaginal microbiota with Lactobacillus depletion and increased microbial diversity facilitate human papillomavirus (HPV) infection and might be involved in viral persistence and cancer development. To define the microbial Community State Types (CSTs) associated with high-risk HPV−persistence, we analysed 55 cervico-vaginal samples from HPV positive (HPV+) women out of 1029 screened women and performed pyrosequencing of 16S rDNA. A total of 17 samples from age-matched HPV negative (HPV−) women were used as control. Clearance or Persistence groups were defined by recalling women after one year for HPV screening and genotyping. A CST IV subgroup, with bacterial genera such as Gardnerella, Prevotella, Megasphoera, Atopobium, frequently associated with anaerobic consortium in bacterial vaginosis (BV), was present at baseline sampling in 43% of women in Persistence group, and only in 7.4% of women in Clearance group. Atopobium genus was significantly enriched in Persistence group compared to the other groups. Sialidase-encoding gene from Gardnerella vaginalis, involved in biofilm formation, was significantly more represented in Persistence group compared to the other groups. Based on these data, we consider the CST IV-BV as a risk factor for HPV persistence and we propose Atopobium spp and sialidase gene from G. vaginalis as microbial markers of HPV−persistence

Differences in Inflammatory Response Induced by Two Representatives of Clades of the Pandemic ST258 <i>Klebsiella pneumoniae</i> Clonal Lineage Producing KPC-Type Carbapenemases

<div><p>ST258-<i>K</i>. <i>pneumoniae</i> (ST258-KP) strains, the most widespread multidrug-resistant hospital-acquired pathogens, belong to at least two clades differing in a 215 Kb genomic region that includes the cluster of capsule genes. To investigate the effects of the different capsular phenotype on host-pathogen interactions, we studied representatives of ST258-KP clades, KKBO-1 and KK207-1, for their ability to activate monocytes and myeloid dendritic cells from human immune competent hosts. The two ST258-KP strains strongly induced the production of inflammatory cytokines. Significant differences between the strains were found in their ability to induce the production of IL-1β: KK207-1/clade I was much less effective than KKBO-1/clade II in inducing IL-1β production by monocytes and dendritic cells. The activation of NLRP3 inflammasome pathway by live cells and/or purified capsular polysaccharides was studied in monocytes and dendritic cells. We found that glibenclamide, a NLRP3 inhibitor, inhibits more than 90% of the production of mature IL-1β induced by KKBO1 and KK207-1. KK207-1 was always less efficient compared to KKBO-1 in: a) inducing NLRP3 and pro-IL-1β gene and protein expression; b) in inducing caspase-1 activation and pro-IL-1β cleavage. Capsular composition may play a role in the differential inflammatory response induced by the ST258-KP strains since capsular polysaccharides purified from bacterial cells affect NLRP3 and pro-IL-1β gene expression through p38MAPK- and NF-κB-mediated pathways. In each of these functions, capsular polysaccharides from KK207-1 were significantly less efficient compared to those purified from KKBO-1. On the whole, our data suggest that the change in capsular phenotype may help bacterial cells of clade I to partially escape innate immune recognition and IL-1β-mediated inflammation.</p></div

Differential Th17 response induced by the two clades of the pandemic ST258 <i>Klebsiella pneumoniae</i> clonal lineages producing KPC-type carbapenemase

<div><p>The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of <i>Klebsiella pneumoniae</i> (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (<i>cps</i>) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using <i>ex vivo</i>-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4⁺IL17⁺ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent <i>K</i>. <i>pneumoniae</i> reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host.</p></div

Effect of capsular polysaccharides from ST258 KP strains on caspase-1 activation.

<p>Monocytes were cultured at 2x10⁶/ml with purified 5 μg/ml of capsular polysaccharides from ST258-KP strains for 7 hours. Cells were lysed and immunoblotted with antibodies specific caspase-1 (p20) and tubulin. Results from one representative experiment out of three performed are shown. Histograms show the results of densitometric analysis (mean ± SE) from three different experiments.</p

IFN-α production by PBMCs following TLR stimulation or infection with influenza virus or HSV-1.

<p>Panel A: PBMCs from males (n = 20) or from females (n = 40) were stimulated with Imiquimod as TLR7 ligand, ssRNA40 as TLR8 ligand or infected with influenza virus (PR8). Panel B: PBMC from males or females were stimulated with Poly(I:C) as TLR3 ligand, ODN 2006 as TLR9 ligand or infected with HSV-1. IFN-α was measured in culture supernatants by immunoplex array. Data for each donor are calculated as mean IFN-α production in stimulated cultures – mean IFN-α production of unstimulated cultures. Data are presented as box-and-whisker plots, with boxes extend from the 25^th percentile to the 75^th percentile, with a horizontal line at the median while whiskers extend to the lowest and highest data point. Black stars indicate mean values. Statistical analysis was performed by two-tailed non parametric Mann-Whitney test. Significance levels were fixed at p<0.05. IFN-α concentrations in unstimulated or mock-infected cultures ranged between 2 and 30 pg/ml (mean 9.2±1.4). No significant differences between the groups were detected.</p

IL10 production by PBMCs following TLR9 stimulation or infection with HSV-1 and sex hormones-correlation.

<p>Panel A: IL10 production by PBMCs from males (n = 20) and females (n = 40) following stimulation with ODN 2006 as TLR9 ligand. Panel B: Spearman rank correlation analysis between IL10 production and DHS levels in the male group. Panel C: Spearman rank correlation analysis between IL10 production and E2 levels in the female group. Panel D: IL10 production by PBMCs from males (n = 20, mean age, years 47±14, range 22–62, females in post menopausal age (n = 20, mean age 59±7, range 48–71) and females in reproductive age (n = 20, mean age 33±9 range 25–54), following stimulation with CpG-ODN 2006 as TLR9 ligand. Panel E: IL10 production by PBMCs from males, females in post-menopausal age (n = 20, mean age 59±7, range 48–71), and females in reproductive age (n = 20, mean age 33±9, range 25–54) following infection with HSV-1. Data of IL10 production are calculated as mean IL10 production in stimulated cultures – mean IL10 production of unstimulated cultures. Data are presented as box-and-whisker plots, with boxes extend from the 25^th percentile to the 75^th percentile, with a horizontal line at the median while whiskers extend to the lowest and highest data point. Black stars indicate mean values. Statistical analysis was performed by two-tailed non-parametric Mann-Whitney test. Significance levels were fixed at p<0.05. Analysis of linear trends was performed using Spearman rank correlation. The level of statistical significance was set at p<0.05. IL10 concentrations in unstimulated or mock-infected cultures ranged between 2 and 68 pg/ml (mean 28.3±1.6). No significant differences between the groups were detected.</p

IL-1β production by monocytes and MDC cultured with strains of <i>K</i>.<i>pneumoniae</i>-KPC with different capsular phenotype.

<p>IL-1β production by monocytes and MDC cultured with strains of <i>K</i>.<i>pneumoniae</i>-KPC with different capsular phenotype.</p

IL10 production by PBMCs following TLR8, TLR7, TLR3 stimulation or infection with influenza virus.

<p>PBMCs from males (n = 20) or from females (n = 40) were stimulated with ssRNA40 as TLR8 ligand, Imiquimod as TLR7 ligand, Poly(I:C) as TLR3 ligand (Panel A) or infected with PR8 influenza virus (Panel B). IL10 was measured in culture supernatants by immunoplex array. Data for each donor are calculated as mean IL10 production from stimulated cultures – mean IL10 production of unstimulated cultures. Data are presented as box-and-whisker plots, with boxes extend from the 25^th percentile to the 75^th percentile, with a horizontal line at the median while whiskers extend to the lowest and highest data point. Black stars indicate mean values. IL10 concentrations in unstimulated or Mock-infected cultures ranged between 2 and 68 pg/ml (mean 28.3±1.6). Statistical analysis was performed by two-tailed non parametric Mann-Whitney test. Significance levels were fixed at p<0.05.</p

Effect of ST258 KP strains on NLRP3 in inflammasome activation.

<p><b>Panel A/B: Effect of ST258-KP strains on pro-IL-1</b>β <b>cleavage and caspase-1 activation.</b> Monocytes or MDC from 3 different donors were cultured at 2x10⁶/ml with live ST258 KP strains for 7 hours at 1:1 cell ratio. Cells were lysed and immunoblotted with antibodies specific to IL-1β and caspase-1 (p20). Results from one representative experiment out of three performed are shown. Histograms show the results of densitometric analysis (mean ± SE) from three different experiments. <b>Panel C: Effect of glibenclamide on cytokine production by monocytes or MDC.</b> Monocytes or MDC from four different donors were cultured at 10⁶ cells/ml with live bacterial cells at 1:1 ratio in the presence (grey columns) or absence (white columns) of 100 μM glibenclamide. Cytokines were measured by Immunoplex array in conditioned media collected after 7 hours of culture.</p