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Mutations ofCystic Fibrosis Transmembrane conductance Regulator(CFTR) lead to Cystic Fibrosis (CF), but the substantial phenotypic variations are determined by non-CFTRallelic diversity. To map novel disease phenotypes in a CF mouse model, we used Collaborative Cross (CC) mice, a highly genetically diverse mouse resource population.ΔF508-Cftrhomozygosity produced a fully penetrant lethal phenotype by eight weeks in two CC lines. The lethality of CC006ΔF508/ΔF508was fully prenatal while CC037ΔF508/ΔF508showed either prenatal or postnatal lethality. Novel phenotypes of CC037ΔF508/ΔF508were revealed early in life including respiratory and systemic inflammatory profiles, and blood, bone marrow, pancreas, heart, and reproductive tract pathologies. Severe intestinal blockage was observed as common in other CF mouse models. These results suggest that the exploration of CF disease phenotypes in a mouse population with diverse genetic profiles is needed to map the genetic origin of currently unidentified disease traits and their potential translation to humans.

Aim Obesity is a risk factor for COVID-19, but the underlying mechanisms are unclear. We investigated the role of adiponectin (an anti-inflammatory adipokine), leptin (a pro-inflammatory adipokine) and their ratio (Adpn/Lep) in this context. Design Single-centre, prospective observational study. Methods. Adiponectin and leptin were measured in 60 COVID-19 patients with mild (not hospitalised, n=11), moderate (hospitalised but not requiring intensive care, n=25) and severe (admission to the intensive care unit [ICU] or death, n=24) disease. Results Adiponectin and leptin levels were similar across severity groups, but patients with moderate severity had the highest Adpn/Lep ratio (1.2 [0.5; 2.0], 5.0 [1.6; 11.2], 2.1 [1.0; 3.6] in mild, moderate and severe disease; P = 0.019). Adpn/Lep, but not adiponectin or leptin alone, correlated with systemic inflammation (C reactive protein, CRP: Spearman's rho 0.293, P = 0.023). When dividing patients into Adpn/Lep tertiles, adiponectin was highest, whereas leptin was lowest in the third (highest) tertile. Patients in the highest Adpn/Lep tertile had numerically lower rates of obesity, diabetes and hypertension, and lower rates of death or admission to ICU versus other tertiles. At linear regression in the whole cohort, CRP significantly predicted Adpn/Lep (β 0.291, P = 0.022), while female gender (β -0.289, P = 0.016), diabetes (β -0.257, P = 0.028), and hypertension (β -239, P = 0.043) were negative predictors. Conclusions We speculate that the rise in Adpn/Lep, due to increased adiponectin and reduced leptin, is a compensatory response to systemic inflammation. In patients with worse cardiometabolic health (e.g. diabetes, hypertension) this mechanism might be blunted, possibly contributing to higher mortality.

Nontypeable Haemophilus influenzae (NTHi) is commonly isolated from airways of patients suffering from chronic respiratory diseases, such as COPD or cystic fibrosis (CF). However, to what extent NTHi long-term infection contributes to the lung inflammatory burden during chronic airway disease is still controversial. Here, we exploited human respiratory samples from a small cohort of CF patients and found that patients chronically infected with NTHi had significantly higher levels of interleukin (IL)-8 and CXCL1 than those who were not infected. To better define the impact of chronic NTHi infection in fuelling inflammatory response in chronic lung diseases, we developed a new mouse model using both laboratory and clinical strains. Chronic NTHi infection was associated with chronic inflammation of the lung, characterised by recruitment of neutrophils and cytokine release keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), granulocyte colony-stimulating factor (G-CFS), IL-6, IL-17A and IL-17F) at 2 and 14 days post-infection. An increased burden of T-cell-mediated response (CD4+ and γδ cells) and higher levels of pro-matrix metalloproteinase 9 (pro-MMP9), known to be associated with tissue remodelling, were observed at 14 days post-infection. Of note we found that both CD4+IL-17+ cells and levels of IL-17 cytokines were enriched in mice at advanced stages of NTHi chronic infection. Moreover, by immunohistochemistry we found CD3+, B220+ and CXCL-13+ cells localised in bronchus-associated lymphoid tissue-like structures at day 14. Our results demonstrate that chronic NTHi infection exerts a pro-inflammatory activity in the human and murine lung and could therefore contribute to the exaggerated burden of lung inflammation in patients at risk., The pathological impact of long-term infection by nontypeable Haemophilus influenzae (NTHi) is still debated. Chronic NTHi infection fuels lung inflammation in human samples and in a new mouse model of bacterial long-term persistence. https://bit.ly/3lvyvge

Background and aims Patients infected with SARS-CoV-2 range from asymptomatic, to mild, moderate or severe disease evolution including fatal outcome. Thus, early predictors of clinical outcome are highly needed. We investigated markers of neural tissue damage as a possible early sign of multisystem involvement to assess their clinical prognostic value on survival or transfer to intensive care unit (ICU). Methods We collected blood from 104 patients infected with SARS-CoV-2 the day of admission to the emergency room and measured blood neurofilament light chair (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and total tau protein levels. Results We found that NfL, GFAP, and tau were significantly increased in patients with fatal outcome, while NfL and UCH-L1 in those needing ICU transfer. ROC and Kaplan–Meier curves indicated that total tau levels at admission accurately predict mortality. Conclusions Blood neural markers may provide additional prognostic value to conventional biomarkers used to predict COVID-19 outcome. Supplementary Information The online version contains supplementary material available at 10.1007/s00415-021-10595-6.

In viral diseases T cells exert a prominent role in orchestrating the adaptive immune response and yet a comprehensive assessment of the T-cell repertoire, compared and contrasted with antibody response, after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is currently lacking. A prior population-scale study of the municipality of Vo’, Italy, conducted after the initial SARS-CoV-2 outbreak uncovered a high frequency of asymptomatic infected individuals and their role in transmission in this town. Two months later, we sampled the same population’s T-cell receptor repertoire structure in terms of both diversity (breadth) and frequency (depth) to SARS-CoV-2 antigens to identify associations with both humoral response and protection. For this purpose, we analyzed T-cell receptor and antibody signatures from over 2,200 individuals, including 76 PCR-confirmed SARS-CoV-2 cases (25 asymptomatic, 42 symptomatic, 9 hospitalized). We found that 97.4% (74/76) of PCR confirmed cases had elevated levels of T-cell receptors specific for SARS-CoV-2 antigens. The depth and breadth of the T-cell receptor repertoire were both positively associated with neutralizing antibody titers; helper CD4+ T cells directed towards viral antigens from spike protein were a primary factor in this correlation. Higher clonal depth of the T-cell response to the virus was also significantly associated with more severe disease course. A total of 40 additional suspected infections were identified based on T-cell response from the subjects without confirmatory PCR tests, mostly among those reporting symptoms or having household exposure to a PCR-confirmed infection. Taken together, these results establish that T cells are a sensitive, reliable and persistent measure of past SARS-CoV-2 infection that are differentially activated depending on disease morbidity.

•AbstractNontypeable Haemophilus influenzae (NTHi) is commonly isolated from airway of cystic fibrosis (CF) patients. However, to what extent NTHi persistence contributes to the lung inflammatory burden during CF chronic airway disease is controversial. Here, we aimed at determining the pathological role of NTHi persistence in a cohort of CF patients and in a newly generated mouse model of NTHi persistence.In our study cohort, we found that CF patients chronically colonized by NTHi had significantly higher levels of IL-8 and CXCL1 than those who were not colonized. To better define the impact of NTHi persistence in fuelling inflammatory response, we developed a new mouse model using both laboratory and CF clinical strains. NTHi persistence was associated with chronic inflammation of the lung, characterized by recruitment of neutrophils and cytokine release (KC, G-CFS, IL-6 and IL-17A) at 2 and 14 days postinfection. An increased burden of T cell mediated response (CD4+ and γδ cells) and higher levels of matrix metalloproteinase 9, known to be associated with tissue remodelling, were observed at 14 days post-infection. Of note we found that both CD4+IL-17+ cells and levels of IL-17 cytokine were enriched in mice at advanced stage of NTHi chronic infection. Moreover, by immunohistochemistry we found CD3+, B220+ and CXCL-13+ cells localized in bronchus-associated lymphoid tissue-like structures at day 14.Our results demonstrate that NTHi persistence exerts a pro-inflammatory activity in the human and murine lung, and could therefore contribute to the exaggerated burden of lung inflammation in CF patients.

Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection., Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression.

Understanding the significance of human genetic diversity in modulating host susceptibility to opportunistic infections is an emerging challenge in the field of respiratory illnesses. While it is recognized that diverse bacterial strains account for differential disease manifestations, emerging data indicate that host genetic diversity is an important determinant factor that influences the severity of opportunistic infections. With particular regard to respiratory illnesses mediated by the gram-negative bacterium Pseudomonas aeruginosa, diverse genetic background is also emerging as a key contributor. Human-genome-wide association studies are a common approach for determining the inter-individual genetic variation associated with variability of the pulmonary infections. Historically, diverse murine inbred mouse strains and ex-vivo cellular models were considered complementary to human studies for establishing the contribution of genetic background to P. aeruginosa respiratory infections. More recently, the development of a new mouse model of infection, mirroring human airway diseases, combined with innovative murine resource populations, modelling human genetic variation, provides additional insights into the mechanisms of genetic susceptibility. In this review, we cover the recent state of the art of human and animal studies and we discuss future potential challenges in the field of P. aeruginosa respiratory infections.

Cystic Fibrosis (CF) lung disease is associated with dysregulation of host defence systems, which ultimately disrupts the balance between inflammation and resolution and leaves the host susceptible to repeated infection. However, the mechanisms underlying these defects are complex and continue to garner significant interest among the CF research community. This review explores emerging data on novel aspects of innate host defence with promising biomarker and therapeutic potential for CF lung disease. Improved understanding of inflammation and host defence against pathogens in patients and animal models during the progression of CF lung disease is pivotal for the discovery of new therapeutics that can limit and/or prevent damage from birth.

Pulmonary infections caused by Mycobacterium abscessus (MA) have increased over recent decades affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of MA pulmonary infection. To overcome this challenge we refined the method of agar beads to establish MA chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after MA infection. In this model, MA was able to establish a persistent lung infection up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of neutrophils, lymphocytes and aggregates of vacuolated foamy cells, mimicking the damage observed in humans. Furthermore, MA lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of MA pathogenesis and the efficacy of novel treatments.

Several chronic respiratory diseases are characterized by recurrent and/or persistent infections, chronic inflammatory responses and tissue remodeling, including increased levels of glycosaminoglycans which are known structural components of the airways. Among glycosaminoglycans, heparan sulfate (HS) has been suggested to contribute to excessive inflammatory responses. Here, we aim at (i) investigating whether long-term infection by Pseudomonas aeruginosa, one of the most worrisome threat in chronic respiratory diseases, may impact HS levels, and (ii) exploring HS competitors as potential anti-inflammatory drugs during P. aeruginosa pneumonia. P. aeruginosa clinical strains and ad-hoc synthesized HS competitors were used in vitro and in murine models of lung infection. During long-term chronic P. aeruginosa colonization, infected mice showed higher heparin/HS levels, evaluated by high performance liquid chromatography-mass spectrometry after selective enzymatic digestion, compared to uninfected mice. Among HS competitors, an N-acetyl heparin and a glycol-split heparin dampened leukocyte recruitment and cytokine/chemokine production induced by acute and chronic P. aeruginosa pneumonia in mice. Furthermore, treatment with HS competitors reduced bacterial burden during chronic murine lung infection. In vitro, P. aeruginosa biofilm formation decreased upon treatment with HS competitors. Overall, these findings support further evaluation of HS competitors as a novel therapy to counteract inflammation and infection during P. aeruginosa pneumonia.

The pathophysiological mechanisms driving exaggerated inflammation and tissue damage associated to un-resolved airway infections during chronic lung diseases remain to be elucidated. Recent reports...

Infection is one of the leading causes of human mortality and morbidity. Exposure to microbial agents is obviously required. However, also non-microbial environmental and host factors play a key role in the onset, development and outcome of infectious disease, resulting in large of clinical variability between individuals in a population infected with the same microbe. Controlled and standardized investigations of the genetics of susceptibility to infectious disease are almost impossible to perform in humans whereas mouse models allow application of powerful genomic techniques to identify and validate causative genes underlying human diseases with complex etiologies. Most of current animal models used in complex traits diseases genetic mapping have limited genetic diversity. This limitation impedes the ability to create incorporated network using genetic interactions, epigenetics, environmental factors, microbiota, and other phenotypes. A novel mouse genetic reference population for high-resolution mapping and subsequently identifying genes underlying the QTL, namely the Collaborative Cross (CC) mouse genetic reference population (GRP) was recently developed. In this chapter, we discuss a variety of approaches using CC mice for mapping genes underlying quantitative trait loci (QTL) to dissect the host response to polygenic traits, including infectious disease caused by bacterial agents and its toxins.

Infection is one of the leading causes of human mortality and morbidity. Exposure to microbial agents is obviously required. However, also non-microbial environmental and host factors play a key role in the onset, development and outcome of infectious disease, resulting in large of clinical variability between individuals in a population infected with the same microbe. Controlled and standardized investigations of the genetics of susceptibility to infectious disease are almost impossible to perform in humans whereas mouse models allow application of powerful genomic techniques to identify and validate causative genes underlying human diseases with complex etiologies. Most of current animal models used in complex traits diseases genetic mapping have limited genetic diversity. This limitation impedes the ability to create incorporated network using genetic interactions, epigenetics, environmental factors, microbiota, and other phenotypes. A novel mouse genetic reference population for high-resolution mapping and subsequently identifying genes underlying the QTL, namely the Collaborative Cross (CC) mouse genetic reference population (GRP) was recently developed. In this chapter, we discuss a variety of approaches using CC mice for mapping genes underlying quantitative trait loci (QTL) to dissect the host response to polygenic traits, including infectious disease caused by bacterial agents and its toxins.

Patients with P. aeruginosa airways infection show markedly variable clinical phenotypes likely influenced by genetic backgrounds. Here, we investigated the cellular events involved in resistance and susceptibility to P. aeruginosa chronic infection using genetically distinct inbred mouse strains. As for patients, different murine genotypes revealed variable susceptibility to infection. When directly compared, resistant C3H/HeOuJ and susceptible A/J strains revealed distinct immune responsiveness to the pathogen. In C3H/HeOuJ resistant mice, IL17-producing cells rapidly and transiently infiltrated the infected lung, and this was paralleled by the acute accumulation of alveolar macrophages, bacterial clearance and resolution of infection. In contrast, A/J susceptible mice revealed a more delayed and prolonged lung infiltration by IL17+ and IFNγ+ cells, persistence of innate inflammatory cells and establishment of chronic infection. We conclude that the host genetic background confers diverse immunoreactivity to P. aeruginosa and IL17-producing cells might contribute to the progress of chronic lung infection.

Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a−/− or IL-17ra−/− mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection.

Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies.

Background P. aeruginosa is one of the top three causes of opportunistic human bacterial infections. The remarkable variability in the clinical outcomes of this infection is thought to be associated with genetic predisposition. However, the genes underlying host susceptibility to P. aeruginosa infection are still largely unknown. Results As a step towards mapping these genes, we applied a genome wide linkage analysis approach to a mouse model. A large F2 intercross population, obtained by mating P. aeruginosa-resistant C3H/HeOuJ, and susceptible A/J mice, was used for quantitative trait locus (QTL) mapping. The F2 progenies were challenged with a P. aeruginosa clinical strain and monitored for the survival time up to 7 days post-infection, as a disease phenotype associated trait. Selected phenotypic extremes of the F2 distribution were genotyped with high-density single nucleotide polymorphic (SNP) markers, and subsequently QTL analysis was performed. A significant locus was mapped on chromosome 6 and was named P. aeruginosa infection resistance locus 1 (Pairl1). The most promising candidate genes, including Dok1, Tacr1, Cd207, Clec4f, Gp9, Gata2, Foxp1, are related to pathogen sensing, neutrophils and macrophages recruitment and inflammatory processes. Conclusions We propose a set of genes involved in the pathogenesis of P. aeruginosa infection that may be explored to complement human studies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2676-4) contains supplementary material, which is available to authorized users.

Trimethoprim-sulfamethoxazole (SXT) is a possible alternative for the treatment of community- and hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) due to the susceptibility of most MRSA strains to the drug. However, after long-term treatment with SXT, thymidine-dependent (TD) SXT-resistant small-colony variants (SCVs) emerge. In TD-SCVs, mutations of thymidylate synthase ([TS] thyA ) occur. Until now, it has never been systematically investigated that SXT is triggering the induction and/or selection of TD-SCVs. In our study, we performed induction, reversion, and competition experiments in vitro and in vivo using a chronic mouse pneumonia model to determine the impact of SXT on the emergence of TD-SCVs. SCVs were characterized by light and transmission electron microscopy (TEM) and auxotrophism testing. Short-term exposure of S. aureus to SXT induced the TD-SCV phenotype in S. aureus SH1000, while selection of TD-SCVs with thyA mutations occurred after long-term exposure. In reversion experiments with clinical and laboratory TD-SCVs, all revertants carried compensating mutations at the initially identified mutation site. Competition experiments in vitro and in vivo revealed a survival and growth advantage of the Δ thyA mutant under low-thymidine availability and SXT exposure although this advantage was less profound in vivo . Our results show that SXT induces the TD-SCV phenotype after short-term exposure, while long-term exposure selects for thyA mutations, which provide an advantage for TD-SCVs under specified conditions. Thus, our results further an understanding of the dynamic processes occurring during SXT exposure with induction and selection of S. aureus TD-SCVs.

Host genotype is an important determinant of susceptibility to infections. Among opportunistic pathogens, Pseudomonas aeruginosa is a major cause of respiratory infections with markedly variable clinical outcomes in individuals at risk. Comparing susceptibility to P. aeruginosa acute infection in nine inbred mouse strains, we recently found that the host genetic makeup is responsible for diverse cell-mediated immunity and bacterial replication. Most recently we adopted a P. aeruginosa chronic infection model to identify cellular events correlating with infection outcome, i.e. resistance or susceptibility. C3H/HeOuJ and A/J mice were infected with P. aeruginosa embedded in agar beads to mimic chronic infection. We report that by day 2 of infection, resistant C3H/HeOuJ mice show similar neutrophils and macrophages recruitment and phagocytic capacity compared to susceptible A/J mice. However, resistant mice had lower markers of acute inflammation (IL-6, GCSF, IL12, MCP-1, MIP1a and MIP1b) and significantly higher ratio IL17/IFN-gamma-producing CD4 + T cells when compared to susceptible ones; this correlated with lower bacterial counts and faster body weight recovery. By day 14, the frequency of IL-17-producing cells had declined in resistant mice, which resolved the infection, while cells had accumulated in susceptible ones, which showed marked signs of lung pathology. We suggest that the host genetic background might shape the kinetic of Th17-dependent responses, and by that determine susceptibility or resistance to P. aeruginosa airway infection.

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.

Objectives CF is characterized by loss of pulmonary function and tissue injury. As the disease progresses, P. aeruginosa adapts to the host and dramatically modifies its phenotype; however, it is still unclear if and how bacterial adaptation influences pathogenesis and disease development. Methods Initially-acquired and CF-adapted P. aeruginosa clonal variants were challenged in C57Bl/6 and CF ko mice after their inclusion in agar beads. Mice were evaluated for bacterial count, lung histopathology, markers of inflammation and tissue damage. Patho-adaptive traits of P. aeruginosa isolated from CF patients airways were correlated with clinical scores. Results Different to P. aeruginosa initially-acquired strains, CF-adapted variants caused long-term chronic infection in mice by shaping the immune response and activating pathways relevant to tissue damage and remodelling. At an advanced stage of a P. aeruginosa infection, murine airways displayed hallmarks of remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several markers of tissue damage. In mice, these pathologic traits are only partially dependent on the CFTR genetic background. In CF patients, we observed correlation between P. aeruginosa patho-adaptive traits and inflammation, remodelling processes and lung functions suggesting exploitable disease markers. Conclusion We conclude that bacterial adaptation may be a potential risk factor in the progression of airway disease and a critical success determinant for reproducing the human disease in mice. Supported by Italian CF Research Foundation.

Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology., IMPORTANCE Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact on S. aureus virulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy. To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response. Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection.

The mechanisms governing the epidemiology dynamics and success determinants of a specific healthcare-associated methicillin-resistant S. aureus (HA-MRSA) clone in hospital settings are still unclear. Important epidemiological changes have occurred in Europe since 2000 that have been related to the appearance of the ST22-IV clone. Between 2006 and 2010, we observed the establishment of the ST22-IV clone displacing the predominant Italian clone, ST228-I, in a large Italian university hospital. To investigate the factors associated with a successful spread of epidemic MRSA clones we studied the biofilm production, the competitive behavior in co-culture, the capacity of invasion of the A549 cells, and the susceptibility to infection in a murine model of acute pneumonia of the two major HA-MRSA clones, ST22-IV and ST228-I. We showed that persistence of ST22-IV is associated with its increased biofilm production and capacity to inhibit the growth of ST228-I in co-culture. Compared to ST228-I, ST22-IV had a significantly higher capacity to invade the A549 cells and a higher virulence in a murine model of acute lung infection causing severe inflammation and determining death in all the mice within 60 hours. On the contrary, ST228-I was associated with mice survival and clearance of the infection. ST22-IV, compared with ST228-I, caused a higher number of persistent, long lasting bacteremia. These data suggest that ST22-IV could have exploited its capacity to i) increase its biofilm production over time, ii) maintain its growth kinetics in the presence of a competitor and iii) be particularly invasive and virulent both in vitro and in vivo, to replace other well-established MRSA clones, becoming the predominant European clone.

Pseudomonas aeruginosais an opportunistic pathogen and causes a wide range of acute and chronic infections.P. aeruginosainfections are kept in check by an effective immune surveillance in the healthy host, while any imbalance or defect in the normal immune response can manifest in disease. Invasive acute infection in the immunocompromised patients is mediated by potent extracellular and cell bound bacterial virulence factors. Life-threatening chronic infection in cystic fibrosis patients is maintained by pathogenic variants that contribute to evade detection and clearance by the immune system. Here, we reviewed the molecular basis of receptor-mediated recognition ofP. aeruginosaand their role in initiating inflammation and the colonization. In addition, the consequence of theP. aeruginosagenetic adaptation for the antibacterial defence and the maintaining of chronic infection are discussed.

The pathophysiological mechanisms driving exaggerated inflammation and tissue damage, associated to un-resolved airway infections, in the context of chronic lung diseases remain to be elucidated. In the host-pathogen interplay, two-component defense responses, resistance and tolerance, are increasingly described. Recent reports prompt the hypothesis that chronic lung disease associated to persistent infections, such as Pseudomonas aeruginosa , may be mediated by IL-17 immunity. Thus, we dissected the IL17 pathway during long term chronic infection by P. aerginosa in murine models. When C57Bl/6 mice were infected with P. aeruginosa embedded in agar beads for one month, we found that IL-17A is sustained and secreted by CD4+ T cells at the advanced phases. To directly address the role of IL-17, we infected IL-17a − / −, IL-17ra − / − and congenic controls comparing their host response. Deficiency of IL-17A/IL-17RA axis was associated with: (i) increased incidence of chronic infection and bacterial burden indicating its role in the host resistance to P. aeruginosa ; (ii) reduced markers of tissue damage (pro-MMP-9, elastin degradation, TGF- β 1), cytokines levels (KC, IL-33) and tissue innate infiltrating cells, proving alteration of host tolerance, through tissue damage control. Blockade of IL-17 activity, by an anti-IL-17A mAb starting from ten days post-infection did not affect host resistance in terms of bacterial load and incidence of infection, but increase mainly host tolerance, reducing exaggerated neutrophils infiltration, and levels of KC, pro-MMP-9, TGF- β 1. Overall, the IL-17A cytokine represents a potential host-based intervention to ameliorate lung physiology without compromising host resistance against pathogens during chronic airway infection.

Introduction Cystic Fibrosis (CF) lung disease is characterized by transient airway P. aeruginosa infections and excessive neutrophil-dominated inflammation early in life followed by permanent chronic infection that causes persistent respiratory symptoms and decline in lung functions.As the disease progresses, strain variants can be distinguished from the strain initially acquired.However, the contribution of P. aeruginosa adaptation to lung disease is not completely understood.Comparing sequential strains isolated from a CF patient in multihost pathogenesis system, including non-mammalian and mammalian hosts, our previous works showed that P. aeruginosa early strains were lethal, while late adapted clonal isolates were attenuated in acute virulence but retained their capacity to persist in murine lungs 1,2,3 . Methods To dissect the contribution of P. aeruginosa patho-adaptive strains during the progression of lung disease, we report the host-response in murine models of acute and chronic P. aeruginosa infection.As a model system in studying long term and persistent CF infections, the agar beads mouse model was used in a time course for up to three months in C57Bl/6 mice and Cftrtm1UNCTgN (FABPCFTR) mice.Bacterial count, cytokines profile, assessed by bioplex assay, and lung pathology, assessed by H& E, Masson’s and AB-PAS staining, were evaluated. Results During acute infection, cytokines and chemokines amounts in the whole lungs were lower after infection with late P. aeruginosa patho-adaptive strains compared with early strain, confirming that late strains have evolved the capacity to reduce innate immune system detection.After one month of chronic infection with P. aeruginosa late strains, cytokines/chemokines involved in leukocytes recruitment (IL-1 β , IL-6, KC, MIP-1 α and MIP-2) were lower than after acute infection.Impressively, MCP-1, IL-17A, IFN- γ and TNF- α , correlated to tissue damage control, were maintained during acute and chronic infection, indicating a continuous and elevated triggering of specific pathways.Furthermore, high TGF- β production were found in the whole lung, confirming that late adapted strains may modulate tissue damage control during chronic infection.Preliminary results in CF mice showed similar trends to wt mice.Moreover cytokines mainly correlated with Th17 lymphocytes pathways, like CD40L, IL-17F, IL-23p19, IL-22, MIP-3 α and IL-21, were tested in the lung homogenates by bioplex.Results show that these markers are activated after one month of chronic infection in CF and wt mice, indicating that late adapted strains may modulate tissue damage control through this pathway.Immune cellular phenotype of lymphocytes involved in this chronic model of infection is under investigation.In addition, histopathological examination of lung tissue sections showed that late adapted strains induced mucin-positive goblet cells metaplasia, collagen deposition and apoptosis, typical hallmarks of damage in the airway of CF patients. Conclusion Our findings suggest that during chronic infection, P. aeruginosa patho-adaptive variants attenuate cytokines involved in innate immune system detection, while modulate those mainly linked to tissue damage control, favoring bacterial survival and persistence. Supported by Italian CF Research Foundation (FFC#20_2011)