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Cellular content plays a crucial role in Non-typeable Haemophilus influenzae infection of preinflamed Junbo mouse middle ear.

ABSTRACT: Non-typeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The relationship between the cellular content of the middle ear fluid (MEF) during AOM and infection of NTHi is poorly understood. Using the Junbo mouse, a characterised NTHi infection model, we analysed the cellular content of MEF and correlated the data with NTHi titres. The MEF of the Junbo mouse was heterogeneous between ears and was graded from 1 to 5; 1 being highly serous/clear and 5 being heavily viscous/opaque. At seven-day post-intranasal inoculation, NTHi was not found in grade-1 or 2 fluids, and the proportion of MEF that supported NTHi increased with the grade. Analyses by flow cytometry indicated that the cellular content was highest in grade-4 and 5 fluids, with a greater proportion of necrotic cells and a low-live cell count. NTHi infection of the middle ear increased the cell count and led to infiltration of immune cells and changes in the cytokine and chemokine levels. Following NTHi inoculation, high-grade infected MEFs had greater neutrophil infiltration whereas monocyte infiltration was significantly higher in serous noninfected low-grade fluids. These data underline a role for immune cells, specifically monocytes and neutrophils, and cell necrosis in NTHi infection of the Junbo mouse middle ear.

SUBMITTER: Vikhe PP

PROVIDER: S-EPMC6491974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cellular Immune Response against Nontypeable Haemophilus influenzae Infecting the Preinflamed Middle Ear of the Junbo Mouse.

Project description:Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The pathology of AOM increases during long-term infection in the middle ear (ME), but the host cellular immune response to bacterial infection in this inflamed environment is poorly understood. Using the Junbo mouse, a characterized NTHi infection model, we analyzed the cellular response to NTHi infection in the Junbo mouse middle ear fluid (MEF). NTHi infection increased the total cell number and significantly decreased the proportion of live cells in the MEF at day 1, and this further decreased gradually on each day up to day 7. Flow cytometry analysis showed that neutrophils were the dominant immune cell population in the MEF and that NTHi infection significantly increased their proportion whereas it decreased the monocyte, macrophage, and dendritic cell proportions. Neutrophil and macrophage numbers increased in blood and spleen after NTHi infection. The T-cell population was dominated by T-helper (Th) cells in noninoculated MEF, and the effector Th (CD44+) cell population increased at day 2 of NTHi infection with an increase in IL-12p40 levels. Sustained NTHi infection up to 3?days increased the transforming growth factor ? levels, decreasing the effector cell population and increasing the T-regulatory (T-reg) cell population. In the preinflamed ME environment of the Junbo mouse, neutrophils are the first responder to NTHi infection followed by T-reg immune suppressive cells. These data indicate that sustained NTHi infection in the ME induces the immune suppressive response by inducing the T-reg cell population and reducing immune cell infiltration, thus promoting longer-term infection.

| S-EPMC6867859 | biostudies-literature

Phasevarion of non-typeable Haemophilus influenzae R2866

2015-09-24 | GSE73349 | GEO

Phasevarion of non-typeable Haemophilus influenzae R2866

Project description:H. influenzae R2866 contains a phase-variable DNA methylase modA10, by virtue of 16 tetrameric repeats present after the start codon. Gain or loss of repeats during replication by slipped-strand mispairing can result in a non-functional reading frame. Microarray analyses of wild-type and insertionally inactivated modA were performed to determine genes affected by phase variable modification. Three replicate cultures were grown in either supplemented brain heart infusion broth (R2866 and R3763) or chemically defined media (R3790 and R3791) to an optical density at 600nm of 0.3 to 0.4 (mid-log phase). RNA was isolated by the method of Jahn et al.

2015-09-24 | E-GEOD-73349 | biostudies-arrayexpress

Selection for phase variation of LOS biosynthetic genes frequently occurs in progression of non-typeable Haemophilus influenzae infection from the nasopharynx to the middle ear of human patients.

Project description:Surface structures in Haemophilus influenzae are subject to rapid ON/OFF switching of expression, a process termed phase variation. We analyse tetranucleotide repeats controlling phase variation in lipo-oligosaccharide (LOS) genes of H. influenzae in paired isolates from both the nasopharynx and middle ears of paediatric patients with chronic or recurrent otitis media. A change in expression of at least one of the seven phase variable LOS biosynthesis genes was seen in 12 of the 21 strain pairs. Several strains showed switching of expression in multiple LOS genes, consistent with a key role for phase variable LOS biosynthetic genes in human infection.

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Mathematical model comparisons of potential non-typeable Haemophilus influenzae vaccine effects.

Project description:Vaccines to prevent acute otitis media (AOM) caused by non-typeable Haemophilus influenzae (NTHi) are under development. Because NTHi is highly variable and colonization rates are high, special vaccine characteristics and trial designs might be needed. We examined in mathematical models the equilibrium NTHi-caused AOM rate given hypothetical vaccines that generated immunity identical to corresponding maximal naturally acquired immunity. Vaccines were examined with single effects and combinations of immunity affecting (1) AOM rates given colonization (pathogenicity), (2) susceptibility to colonization, and (3) contagiousness given colonization. Percent reductions in AOM across all preschool children were (1) 34%, (2) 31%, (3) 9%, (1 and 2) 57%, (2 and 3) 50%, and (1, 2, and 3) 75%. Effects on children in daycare vs. not in daycare were (1) 18 vs. 48%, (2) -1 vs. 57%, (3) 13 vs. 5%, (1 and 2) 30 vs. 79%, (2 and 3) 33 vs. 60%, and (1, 2, and 3) 64 vs. 85%. Pure pathogenicity effects (1 alone) will need to be supplemented by transmission effects. The effects of susceptibility (2 alone) are diminished or negative because children protected against colonization have lower levels of immunity to (1) and (3) than unvaccinated children. For trials to predict population effects, both colonization and AOM outcomes must be studied and all three effects must be evaluated. This need arises because, unlike H. influenzae type B, high NTHi exposure diminishes cumulative vaccine effects and high colonization rates generate rapid accumulation of natural immunity that alters the indirect effects of vaccine immunity on transmission differently by age and daycare status.

| S-EPMC1852514 | biostudies-literature

Pneumococcal vaccine impacts on the population genomics of non-typeable Haemophilus influenzae.

Project description:The implementation of pneumococcal conjugate vaccines (PCVs) has led to a decline in vaccine-type disease. However, there is evidence that the epidemiology of non-typeable Haemophilus influenzae (NTHi) carriage and disease can be altered as a consequence of PCV introduction. We explored the epidemiological shifts in NTHi carriage using whole genome sequencing over a 5-year period that included PCV13 replacement of PCV7 in the UK's National Immunization Programme in 2010. Between 2008/09 and 2012/13 (October to March), nasopharyngeal swabs were taken from children <5 years of age. Significantly increased carriage post-PCV13 was observed and lineage-specific associations with Streptococcus pneumoniae were seen before but not after PCV13 introduction. NTHi were characterized into 11 discrete, temporally stable lineages, congruent with current knowledge regarding the clonality of NTHi. The increased carriage could not be linked to the expansion of a particular clone and different co-carriage dynamics were seen before PCV13 implementation when NTHi co-carried with vaccine serotype pneumococci. In summary, PCV13 introduction has been shown to have an indirect effect on NTHi epidemiology and there exists both negative and positive, distinct associations between pneumococci and NTHi. This should be considered when evaluating the impacts of pneumococcal vaccine design and policy.

| S-EPMC6202451 | biostudies-literature

Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa.

Project description:ObjectivesWe sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response.MethodsChinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription-polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism.ResultsSequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM.ConclusionsBased on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.

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The DmsABC Sulfoxide Reductase Supports Virulence in Non-typeable <i>Haemophilus influenzae</i>.

Project description:Although molybdenum-containing enzymes are well-established as having a key role in bacterial respiration, it is increasingly recognized that some may also support bacterial virulence. Here, we show that DmsABC, a putative dimethylsulfoxide (DMSO) reductase, is required for fitness of the respiratory pathogen Haemophilus influenzae (Hi) in different models of infection. Expression of the dmsABC operon increased with decreasing oxygen availability, but despite this, a Hi2019Δd msA strain did not show any defects in anaerobic growth on chemically defined medium (CDM), and viability was also unaffected. Although Hi2019Δd msA exhibited increased biofilm formation in vitro and greater resistance to hypochlorite killing compared to the isogenic wild-type strain, its survival in contact with primary human neutrophils, in infections of cultured tissue cells, or in a mouse model of lung infection was reduced compared to Hi2019WT. The tissue cell infection model revealed a two-fold decrease in intracellular survival, while in the mouse model of lung infection Hi2019Δd msA was strongly attenuated and below detection levels at 48 h post-inoculation. While Hi2019WT was recovered in approximately equal numbers from bronchoalveolar lavage fluid (BALF) and lung tissue, survival of Hi2019Δd msA was reduced in lung tissue compared to BALF samples, indicating that Hi2019Δd msA had reduced access to or survival in the intracellular niche. Our data clearly indicate for the first time a role for DmsABC in H. influenzae infection and that the conditions under which DmsABC is required in this bacterium are closely linked to interactions with the host.

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Project description:Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood-brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood-cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.

| S-EPMC7461124 | biostudies-literature

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