Transcriptome Analysis Reveals Novel Inflammatory Signalings to Glaesserella parasuis Infection.

Glaesserella parasuis (GPS) can cause severe systemic inflammation in pigs, resulting in huge economic losses to the pig industry. At present, no effective method is available for the prevention and control of GPS infection. Molecular breeding for disease resistance is imminent, but disease-resistance genes have not been identified. To study the mechanism of systemic acute inflammation caused by GPS, we established three in vitro infection models (3D4/21 cells, PK15 cells, and PAVEC cells) according to its infection path. There was no significant difference in apoptosis among the three kinds of cells after 12 h of continuous GPS stimulation, while inflammatory factors were significantly upregulated. Subsequent transcriptome analysis revealed 1969, 1207, and 3564 differentially expressed genes (DEGs) in 3D4/21 cells, PK15 cells, and PAVEC cells, respectively, after GPS infection. Many of the DEGs were predicted to be associated with inflammatory responses (C3, CD44, etc.); cell proliferation, growth and apoptosis; gene expression; and protein phosphorylation. Key signaling pathways, including S100 family signaling, bacteria and virus recognition, and pathogen-induced cytokine storm signaling, were enriched based on Ingenuity Pathway Analysis (IPA). Furthermore, a total of three putative transmembrane receptors and two putative G-protein-coupled receptors, namely F3, ICAM1, PLAUR, ACKR3, and GPRC5A, were identified by IPA among the three types of cells. ACKR3 and GPRC5A play pivotal roles in bacterial adhesion, invasion, host immune response and inflammatory response through the S100 family signaling pathway. Our findings provide new insights into the pathological mechanisms underlying systemic inflammation caused by GPS infection in pigs, and they lay a foundation for further research on disease-resistance breeding to GPS.

Expression profiles of miRNAs in the lung tissue of piglets infected with Glaesserella parasuis and the roles of ssc-miR-135 and ssc-miR-155-3p in the regulation of inflammation.

MicroRNAs (miRNAs) have been shown to play an important regulatory role in the process of pathogenic infection. However, the miRNAs that regulate the pathogenic process of G. parasuis and their functions are still unknown. Here, high-throughput sequencing was used to quantify the expression of miRNA in piglet lung tissue after G. parasuis XX0306 strain infection. A total of 25 differentially expressed microRNAs (DEmiRNAs) were identified. GO and KEGG pathway enrichment analysis showed that many of the functions of genes that may be regulated by DEmiRNA are related to inflammatory response and immune regulation. Further studies found that ssc-miR-135 may promote the expression of inflammatory factors through NF-κB signaling pathway. Whereas, ssc-miR-155-3p inhibited the inflammatory response induced by G. parasuis, and its regulatory mechanism remains to be further investigated. This study provides a valuable reference for revealing the regulatory effects of miRNAs on the pathogenesis of G. parasuis. DATA AVAILABILITY: The datasets generated during the current study are not publicly available due to this study is currently in the ongoing research stage, and some of the data cannot be made public sooner yet, but are available from the corresponding author on reasonable request.

Molecular epidemiology and antimicrobial resistance of Haemophilus influenzae isolates from otitis media in Bulgaria.

Haemophilus influenzae is one of the main bacteria responsible for otitis media (OM) among children worldwide. We aimed to estimate the distribution of encapsulated and non-capsulated variants (NTHi), biotypes, antibiotic susceptibility, and molecular epidemiology of H. influenzae isolates recovered from pediatric OM cases in Bulgaria.Capsule detection was done by PCR for bexB gene, absent in NTHi. All encapsulated strains were subjected to PCR serotyping. MIC susceptibility testing was performed according to the criteria of EUCAST. MLST was conducted for all 71 OM isolates.The capsule detection and PCR - serotyping disclosed a predominance of NTHi (90.1%) and a few "a", "f", and "c" types. Biotype I was the most widespread (42.3%). ß-lactam resistance was found in 35.2% of the isolates. MLST represented heterogenic population structure, whereas the most represented clonal complexes belonged to ST-3, ST-57, ST-105, and ST-1426. 42.3% of the STs showed relatedness to globally represented clones, and 11.3% displayed affiliation to international type 2.Most of the H. influenzae isolates recovered from children with otitis media were non-typable strains from biotype I. The examined population structure was genetically diverse, with a predominance of international type 2 isolates.

Use of trans-complementation method to determine the effects of various ftsI mutations on ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains.

Haemophilus influenzae is a causative agent of serious infections, especially among children. ß-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, ß-lactam resistance is due to the presence of ß-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for ß-lactams (BLNAR-ß-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from ß-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced ß-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased ß-lactam susceptibility in H. influenzae.

Transcriptome profiling identifies immune response genes against porcine reproductive and respiratory syndrome virus and Haemophilus parasuis co-infection in the lungs of piglets.

BACKGROUND: Co-infections of the porcine reproductive and respiratory syndrome virus (PRRSV) and the Haemophilus parasuis (HPS) are severe in Chinese pigs, but the immune response genes against co-infected with 2 pathogens in the lungs have not been reported. OBJECTIVES: To understand the effect of PRRSV and/or HPS infection on the genes expression associated with lung immune function. METHODS: The expression of the immune-related genes was analyzed using RNA-sequencing and bioinformatics. Differentially expressed genes (DEGs) were detected and identified by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and western blotting assays. RESULTS: All experimental pigs showed clinical symptoms and lung lesions. RNA-seq analysis showed that 922 DEGs in co-challenged pigs were more than in the HPS group (709 DEGs) and the PRRSV group (676 DEGs). Eleven DEGs validated by qRT-PCR were consistent with the RNA sequencing results. Eleven common Kyoto Encyclopedia of Genes and Genomes pathways related to infection and immune were found in single-infected and co-challenged pigs, including autophagy, cytokine-cytokine receptor interaction, and antigen processing and presentation, involving different DEGs. A model of immune response to infection with PRRSV and HPS was predicted among the DEGs in the co-challenged pigs. Dual oxidase 1 (DUOX1) and interleukin-21 (IL21) were detected by IHC and western blot and showed significant differences between the co-challenged pigs and the controls. CONCLUSIONS: These findings elucidated the transcriptome changes in the lungs after PRRSV and/or HPS infections, providing ideas for further study to inhibit ROS production and promote pulmonary fibrosis caused by co-challenging with PRRSV and HPS.

Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae.

Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens.IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.

Gene Polymorphisms of TLR4 and TLR9 and Haemophilus influenzae Meningitis in Angolan Children.

Bacterial meningitis (BM) is a severe disease caused by various bacterial pathogens. Toll-like receptors (TLRs) protect humans from invading pathogens. In this study, we determined whether single nucleotide polymorphisms (SNPs) of TLR4 and TLR9 are associated with susceptibility to and outcome of BM in Angolan children. Samples were taken from 241 patients and 265 age-matched ethnic controls. The SNPs TLR4 rs4986790 (896A > G) and TLR9 rs187084 (-1486T > C) were determined by high-resolution melting analysis (HRMA). The frequency of variant genotypes in TLR4 was significantly higher in patients with Haemophilus influenzae meningitis than controls (odds ratio (OR), 2.5; 95% confidence interval (CI), 1.2-5.4; p = 0.021), whereas the frequency of variant genotypes in TLR9 was significantly lower in patients with H. influenzae meningitis than controls (OR, 0.4; 95% CI, 0.2-0.9; p = 0.036). No such differences were found with other causative pathogens, such as Streptococcus pneumoniae and Neisseria meningitidis. At the time of discharge, patients with meningitis caused by Gram-negative bacteria who were carriers of variant TLR4 genotypes had a higher risk of ataxia (OR, 12.91; 95% CI, 1.52-109.80; p = 0.019) and other neurological sequelae (OR, 11.85; 95% CI, 1.07-131.49; p = 0.044) than those with the wild-type TLR4 genotype. Our study suggests an association between H. influenzae meningitis and genetic variation between TLR4 and TLR9 in Angolan children.

TLR4 Polymorphism, Nasopharyngeal Bacterial Colonization, and the Development of Childhood Asthma: A Prospective Birth-Cohort Study in Finnish Children.

We aimed to explore the role of TLR4 (rs4986790) polymorphism in the nasopharyngeal (NP) bacterial colonization and its consequent impact on the development of childhood asthma. A semi-quantitative culture of NP swabs was performed on 473 children at 2 months of age and on 213 children at 13 months of age. TLR4 polymorphism was analyzed for 396 children. Children were followed from birth to the age of 7.5 years and the final outcome was physician-diagnosed asthma. The associations between TLR4 genotype, bacterial colonization, and asthma were analyzed. Children with TLR4 AG or GG genotype were more often colonized with Moraxella catarrhalis at 2 months of age (p = 0.009) and Haemophilus influenzae at 13 months of age (p = 0.018). Children who were colonized with H. influenzae at 13 months of age had a significantly higher risk of later development of asthma (p = 0.004). M. catarrhalis or H. Influenzae colonization at 2 months of age or TLR4 genotype Asp299Gly were not associated with the development of childhood asthma. TLR4 Asp299Gly polymorphism was associated with an increased risk of colonization of M. catarrhalis and H. influenzae in children. The colonization with H. influenzae at 13 months of age was associated with a higher risk of later development of childhood asthma.

Pellino-1 Regulates Immune Responses to Haemophilus influenzae in Models of Inflammatory Lung Disease.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

Seasonality of antimicrobial resistance rates in respiratory bacteria: A systematic review and meta-analysis.

BACKGROUND: Antimicrobial resistance (AMR) rates may display seasonal variation. However, it is not clear whether this seasonality is influenced by the seasonal variation of infectious diseases, geographical region or differences in antibiotic prescription patterns. Therefore, we assessed the seasonality of AMR rates in respiratory bacteria. METHODS: Seven electronic databases (Embase.com, Medline Ovid, Cochrane CENTRAL, Web of Science, Core Collection, Biosis Ovid, and Google Scholar), were searched for relevant studies from inception to Jun 25th, 2019. Studies describing resistance rates of Streptococcus pneumoniae and Haemophilus influenzae were included in this review. By using random-effects meta-analysis, pooled odd ratios of seasonal AMR rates were calculated using winter as the reference group. Pooled odd ratios were obtained by antibiotic class and geographical region. RESULTS: We included 13 studies, of which 7 were meta-analyzed. Few studies were done in H. influenzae, thus this was not quantitively analyzed. AMR rates of S. pneumoniae to penicillins were lower in other seasons than in winter with pooled OR = 0.71; 95% CI = 0.65-0.77; I2 = 0.0%, and to all antibiotics with pooled OR = 0.68; 95% CI = 0.60-0.76; I2 = 14.4%. Irrespective of geographical region, the seasonality of AMR rates in S. pneumoniae remained the same. CONCLUSION: The seasonality of AMR rates could result from the seasonality of infectious diseases and its accompanied antibiotic use.

Recurring large deletion in DRC1 (CCDC164) identified as causing primary ciliary dyskinesia in two Asian patients.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a relatively rare autosomal recessive or X-linked disorder affecting ciliary function. In the set of causative genes, however, predominant pathogenic variants remain unknown in Asia. METHOD: A diagnosis of PCD was made following a modern comprehensive testing including genetic analysis; targeted resequencing for screening variants, and Sanger sequencing for determination of the breakpoints, with an additional review of databases to calculate the deletion frequency. A multiplexed PCR-based detection method has also been developed. RESULTS: We ascertained a 50-year-old Japanese male who had been diagnosed with diffuse panbronchiolitis (DPB), but refractory to macrolide therapy. We reevaluated the case and identified a large homozygous deletion spanning exons 1 to 4 of the DRC1 and determined the breakpoints (NM_145038.4: c.1-3952_540 + 1331del27748-bp). In the PCD cohort at the University of North Carolina, we found a female PCD patient of Korean descent harboring the same homozygous deletion. From the Invitae testing cohort, we extracted four carriers of the same deletion among 965 Asian individuals, whereas no deletion was found in the 23,951 non-Asians. CONCLUSION: We speculate that the DRC1 deletion is a recurrent or perhaps founder mutation in Asians. The simple PCR method could be a useful screening tool.

Analysis of Invasive Nontypeable Haemophilus influenzae Isolates Reveals Selection for the Expression State of Particular Phase-Variable Lipooligosaccharide Biosynthetic Genes.

Nontypeable Haemophilus influenzae (NTHi) is a major human pathogen, responsible for several acute and chronic infections of the respiratory tract. The incidence of invasive infections caused by NTHi is increasing worldwide. NTHi is able to colonize the nasopharynx asymptomatically, and the exact change(s) responsible for transition from benign carriage to overt disease is not understood. We have previously reported that phase variation (the rapid and reversible ON-OFF switching of gene expression) of particular lipooligosaccharide (LOS) glycosyltransferases occurs during transition from colonizing the nasopharynx to invading the middle ear. Variation in the structure of the LOS is dependent on the ON/OFF expression status of each of the glycosyltransferases responsible for LOS biosynthesis. In this study, we surveyed a collection of invasive NTHi isolates for ON/OFF expression status of seven phase-variable LOS glycosyltransferases. We report that the expression state of the LOS biosynthetic genes oafA ON and lic2A OFF shows a correlation with invasive NTHi isolates. We hypothesize that these gene expression changes contribute to the invasive potential of NTHi. OafA expression, which is responsible for the addition of an O-acetyl group onto the LOS, has been shown to impart a phenotype of increased serum resistance and may serve as a marker for invasive NTHi.

Airway infection with Nontypeable Haemophilus influenzae is more rapidly eradicated in vitamin D deficient mice.

Chronic obstructive pulmonary disease (COPD), which is characterized by an excessive inflammatory response of the airways, is often complicated by exacerbations. Vitamin D deficiency has been associated with an increased risk for COPD and may predispose COPD patients to a higher exacerbation rate, particularly during smoking. In the current study, we investigated the effect of vitamin D deficiency and cigarette smoke (CS)-exposure on lung inflammation and bacterial clearance after an acute infection with Nontypeable Haemophilus influenzae (NTHi). Vitamin D deficient or sufficient mice were exposed to nose-only CS or ambient air for 6 weeks and oropharyngeally instilled with 106 NTHi. Residual viable NTHi were measured at different time points post-infection. Mechanisms of bacterial clearance (e.g. phagocytosis, pattern recognition receptors, antimicrobial peptides, surfactant proteins and mucin) and lung remodeling (e.g. metalloproteinases, MMP's) were assessed. Although smoking resulted in reduced phagocytosis capacity of macrophages and neutrophils, bacterial clearance was similar to control mice. By contrast and independent of smoking, bacterial clearance was significantly accelerated in vitamin D deficient mice already from 24 h post-infection (p = 0.0087). This faster and complete eradication was associated with a more rapid resolution of cytokines and neutrophils 72 h post-infection and dominated by an upregulation of cathelicidin-related antimicrobial peptide (CRAMP) mRNA during infection (p = 0.026). However, vitamin D deficiency also resulted in more MMP12 protein in broncho-alveolar lavage and a shift in mRNA expression of MMP12/TIMP1 (p = 0.038) and MMP9/TIMP1 (p = 0.024) ratio towards more protease activity. Overall, vitamin D deficient mice resolved NTHi infection faster with a faster resolution of local lung inflammation, possibly through upregulation of CRAMP. This was associated with a disruption of the protease/anti-protease balance, which may potentially scale towards a higher extracellular matrix breakdown.

Comparative genomic and methylome analysis of non-virulent D74 and virulent Nagasaki Haemophilus parasuis isolates.

Haemophilus parasuis is a respiratory pathogen of swine and the etiological agent of Glässer's disease. H. parasuis isolates can exhibit different virulence capabilities ranging from lethal systemic disease to subclinical carriage. To identify genomic differences between phenotypically distinct strains, we obtained the closed whole-genome sequence annotation and genome-wide methylation patterns for the highly virulent Nagasaki strain and for the non-virulent D74 strain. Evaluation of the virulence-associated genes contained within the genomes of D74 and Nagasaki led to the discovery of a large number of toxin-antitoxin (TA) systems within both genomes. Five predicted hemolysins were identified as unique to Nagasaki and seven putative contact-dependent growth inhibition toxin proteins were identified only in strain D74. Assessment of all potential vtaA genes revealed thirteen present in the Nagasaki genome and three in the D74 genome. Subsequent evaluation of the predicted protein structure revealed that none of the D74 VtaA proteins contain a collagen triple helix repeat domain. Additionally, the predicted protein sequence for two D74 VtaA proteins is substantially longer than any predicted Nagasaki VtaA proteins. Fifteen methylation sequence motifs were identified in D74 and fourteen methylation sequence motifs were identified in Nagasaki using SMRT sequencing analysis. Only one of the methylation sequence motifs was observed in both strains indicative of the diversity between D74 and Nagasaki. Subsequent analysis also revealed diversity in the restriction-modification systems harbored by D74 and Nagasaki. The collective information reported in this study will aid in the development of vaccines and intervention strategies to decrease the prevalence and disease burden caused by H. parasuis.

Patients with Chronic Obstructive Pulmonary Disease harbour a variation of Haemophilus species.

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.

Expression of surfactant Protein-A in the Haemophilus influenzae-induced otitis media in a rat model.

The objective of this study was to investigate the expression and the role of surfactant protein A (SP-A) in the middle ear (ME) mucosa in response to bacterial infection in a rat model. Otitis media (OM) was induced by surgical inoculation of non-typeable Haemophilus influenza (NTHi) into the ME cavity of Sprague-Dawley rats. The rats were divided into an NTHi-induced OM group and a phosphate-buffered saline-injected control group. The NTHi-induced OM and control groups were subdivided into sets of 6 rats, one for each of the 6 time points (0, 1, 2, 4, 7, and 14 days post-inoculation), at which point the rats were euthanized after inoculation. The concentrations of SP-A in the ME effusion were determined by an enzyme-linked immunosorbent assay (ELISA). Tissue expression of SP-A, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in infected ME mucosa was assessed by immunohistochemical staining. For mRNA expression quantification, RNA was extracted from the ME mucosa and SP-A expression was monitored and compared between the control and OM groups using quantitative polymerase chain reaction (PCR). Expression of IL-1ß, IL-6, and TNF-α in the ME mucosa was also evaluated. SP-A expression was evaluated in the effusion of pediatric OM patients (70 ears) who received ventilation-tube insertion by ELISA. SP-A was detected in normal rat ME mucosa before bacterial inoculation. SP-A expression was up-regulated in the NTHi-induced OM group (p = 0.046). Immunohistochemical staining revealed increased SP-A expression on post-inoculation day 1, 2, and 4 in the OM group. Expression of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) in the ME also increased significantly on post-inoculation day 1, 2, and 4 in the OM group. It correlated with changes in SP-A expression. Expression of SP-A was also identified in the ME effusion of humans. SP-A exists in the ME of the rat and was up-regulated in the ME of NTHi-induced OM. Expression of IL-1ß, IL-6, and TNF-α was increased in the ME of the bacteria-induced OM in the rat model. The results suggest that SP-A may play a significant role in the early phase of OM induction and subsequent recovery from it.

Transcriptional Profiling of Host Cell Responses to Virulent Haemophilus parasuis: New Insights into Pathogenesis.

Haemophilus parasuis is the causative agent of Glässer’s disease in pigs. H. parasuis can cause vascular damage, although the mechanism remains unclear. In this study, we investigated the host cell responses involved in the molecular pathway interactions in porcine aortic vascular endothelial cells (PAVECs) induced by H. parasuis using RNA-Seq. The transcriptome results showed that when PAVECs were infected with H. parasuis for 24 h, 281 differentially expressed genes (DEGs) were identified; of which, 236 were upregulated and 45 downregulated. The 281 DEGs were involved in 136 KEGG signaling pathways that were organismal systems, environmental information processing, metabolism, cellular processes, and genetic information processing. The main pathways were the Rap1, FoxO, and PI3K/Akt signaling pathways, and the overexpressed genes were determined and verified by quantitative reverse transcription polymerase chain reaction. In addition, 252 genes were clustered into biological processes, molecular processes, and cellular components. Our study provides new insights for understanding the interaction between bacterial and host cells, and analyzed, in detail, the possible mechanisms that lead to vascular damage induced by H. parasuis. This may lead to development of novel therapeutic targets to control H. parasuis infection.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

The carriage of interleukin-1B-31*C allele plus Staphylococcus aureus and Haemophilus influenzae increases the risk of recurrent tonsillitis in a Mexican population.

The aim of the present study was to estimate the relative contribution of immunogenetic and microbiological factors in the development of recurrent tonsillitis in a Mexican population. Patients (n = 138) with recurrent tonsillitis and an indication of tonsillectomy (mean age: 6.05 years ± 3.00; median age: 5 years, female: 58; age range: 1-15 years) and 195 non-related controls older than 18 years and a medical history free of recurrent tonsillitis were included. To evaluate the microbial contribution, tonsil swab samples from both groups and extracted tonsil samples from cases were cultured. Biofilm production of isolated bacteria was measured. To assess the immunogenetic component, DNA from peripheral blood was genotyped for the TNFA-308G/A single-nucleotide polymorphism (SNP) and for the IL1B -31C/T SNP. Normal microbiota, but no pathogens or potential pathogens, were identified from all control sample cultures. The most frequent pathogenic species detected in tonsils from cases were Staphylococcus aureus (48.6%, 67/138) and Haemophilus influenzae (31.9%, 44/138), which were found more frequently in patient samples than in samples from healthy volunteers (P < 0.0001). Importantly, 41/54 (75.9%) S. aureus isolates were biofilm producers (18 weak and 23 strong), whereas 17/25 (68%) H. influenzae isolates were biofilm producers (10 weak, and 7 strong biofilm producers). Patients with at least one copy of the IL1B-31*C allele had a higher risk of recurrent tonsillitis (OR = 4.03; 95% CI = 1.27-14.27; P = 0.013). TNFA-308 G/A alleles were not preferentially distributed among the groups. When considering the presence of IL1B-31*C plus S. aureus, IL1B-31*C plus S. aureus biofilm producer, IL1B-31*C plus H. influenzae or IL1B-31*C plus H. influenzae biofilm producer, the OR tended to infinite. Thus, the presence of IL1B-31*C allele plus the presence of S. aureus and/or H. influenzae could be related to the development of tonsillitis in this particular Mexican population.