Introduction:Haemophilus influenzae type a (Hia) has recently emerged as a cause of invasive disease in North American Indigenous children. Factors determining the outcomes of exposure to the pathogen, from asymptomatic carriage to fatal disease, are poorly understood. The role of innate immune activation in the pathogenesis of invasive Hia disease remains unexplored. We used clinical Hia isolates to determine whether innate immune responses depended on the presence of the capsule, strain genetic background, and abilities to cause invasive disease. Methods: Differentiated THP-1 cells and HL-60 neutrophil-like cells were stimulated with four Hia strains (invasive or noninvasive; encapsulated or nonencapsulated), in comparison to 1 invasive and 1 noninvasive non-typeable H. influenzae. Surface expression of ICAM-1 and CD64 and release of pro-inflammatory cytokines TNF-α and IL-1β were quantified. Results: In vitro Hia infection resulted in robust activation of inflammatory responses in terms of expression of ICAM-1 and release of TNF-α and IL-1β, irrespective of the presence or absence of the capsule, or abilities to cause invasive disease. Inhibition of TLR4 decreased TNF-α release by THP-1 cells stimulated by Hia. Conclusion: Powerful activation of pro-inflammatory responses induced by Hia may contribute to the pathogenesis of invasive Hia disease. As the activation of macrophages and neutrophils did not depend on encapsulation or source of Hia isolation, the functional abilities of phagocytic cells unlikely represent a limiting factor in host defenses. The development of invasive versus noninvasive disease may depend on the functional abilities of the adaptive immune system.

Haemophilus influenzae is a human-restricted Gram-negative pathogen able to cause severe invasive infections such as sepsis and meningitis [1]. The most virulent strains express a polysaccharide capsule that protects the bacteria against immune defense mechanisms. The capsules of various strains differ in chemical structure: six serotypes (a-f) are distinguished based on the antigenic properties of capsular polysaccharides; nonencapsulated H. influenzae strains are termed non-typeable (NTHi) [1]. Experimental studies have established that among all H. influenzae, the serotype b (Hib) is the most virulent followed by serotype a (Hia); NTHi strains are less virulent than encapsulated ones [2, 3]. Prior to the introduction of the pediatric Hib-conjugate vaccines in the late 1980s to the early 1990s, Hib was the most common cause of meningitis in children globally [4]. Immunization against Hib resulted in a dramatic decrease of the incidence rates of invasive Hib disease [4]. However, in the post-Hib vaccine era, NTHi and non-type b encapsulated H. influenzae became the major causes of invasive disease in industrial countries with universal pediatric immunization programs [5].

Over the last two decades, Hia became the significant cause of severe invasive disease in several North American regions with a high proportion of Indigenous populations, particularly in the Arctic [6]. In some Indigenous populations, incidence rates of invasive Hia disease are close to invasive Hib prior to the introduction of immunization against Hib, with young children being most affected [7]. The invasive Hia disease can manifest as very severe clinical forms, such as meningitis, pneumonia, or septic arthritis, with high case-fatality rates [6, 8]. Yet, Hia infection is characterized by a wide clinical spectrum as Hia can cause local infections as well as asymptomatically colonize the upper respiratory tract [9‒11]. Our recent studies found that over 9% of healthy Indigenous children carried Hia in the nasopharynx [12].

Factors determining the outcomes of exposure of young children to the pathogen, in the range of asymptomatic carriage to fatal disease, are poorly understood. Molecular genetic studies of encapsulated H. influenzae have identified a strong association of bacterial virulence with mutations increasing the amount of capsular material, such as the IS1016-bexA deletion in the cap locus [13, 14]. Analysis of clinical Hib and Hia isolates revealed that bacteria exhibiting such mutations caused extremely severe inflammatory responses in vivo [15, 16], suggesting that the capsule may contribute to inflammation via innate immune activation. However, the mechanisms mediating an enhanced inflammation caused by mutant capsular forms of H. influenzae are unknown.

Although the role of adaptive immunity in host defense against encapsulated H. influenzae has been established, the role of innate immunity is still incompletely understood [17]. It is known that recognition of H. influenzae by the innate immune system involves certain pattern recognition receptors (PRRs), such as Toll-like receptors (TLR) 2 and 4 [18, 19]. H. influenzae, which is a typical extracellular pathogen, can nevertheless enter the cytosol and consequently reach intracellular NOD-like receptors [20‒22]. Some studies have demonstrated that TLR2, TLR4, and NOD1 are all required for mucosal clearance of capsulated (Hib), but not of nonencapsulated strains [23], suggesting that the capsule is recognized by PRRs, and the resulting signaling is essential for innate immune responses. However, the role of H. influenzae capsule in the activation of innate immune responses remains unexplored.

In this study, we used Hia strains isolated from patients with various forms of clinical disease, to determine whether in vitro innate immune responses depend on the presence of the capsule, strain genetic background, and abilities to cause invasive versus noninvasive disease. To normalize the outcome of the study, we applied standardized methods of stimulating two human cell lines with several bacterial strains under the same conditions.

Cell Culture Conditions

The human THP-1 monocytic leukemia cell line (ATCC, Manassas, VA, USA) was cultured in RPMI-1640 medium (Sigma-Aldrich, Oakville, ON, Canada) supplemented with 20% fetal bovine serum (FBS) (R&D Systems, Inc., Minneapolis, MN, USA) and 0.4% antibiotic-antimycotic (Gibco, Eugene, OR, USA) at 37°C and 5% CO2. The cells were passaged every 3–5 days, to maintain an approximate concentration of 1 × 106 cells/mL. Macrophage differentiation was induced adding 20 ng/mL phorbol myristate acetate (Sigma-Aldrich, Oakville, ON, Canada) to 2.5 × 106 cells/mL in 6-well plates (Corning Incorporated, Corning, NY, USA) followed by incubation for 24 h at 37°C and 5% CO2. The medium was then removed, cells were washed with RPMI-1640 medium, and 2 mL of RPMI-1640 supplemented with 10% FBS was added for another 48 h incubation at 37°C and 5% CO2. Macrophage differentiation was confirmed by examination under microscope.

The human HL-60 myeloblastic leukemia cell line (ATCC, Manassas, VA, USA) was cultured in RPMI-1640 medium supplemented with 20% FBS and 1% antibiotic-antimycotic at 37°C and 5% CO2. Cells were passaged every 2–3 days to maintain a concentration between 1 × 105 and 1 × 106 cells/mL. Differentiation was initiated by seeding in T-25 tissue culture flasks at a concentration of 1 × 105 cells/mL in RPMI-1640 medium supplemented with 20% FBS, 1.25% dimethyl sulfoxide (Fisher BioReagents, PA, USA), and 1% antibiotic-antimycotic. Medium was replaced every 2 days and cell concentration was maintained below 1 × 106 cells/mL. Differentiation was complete 9 days after initiation and confirmed microscopically.

H. influenzae Strains and Culture Conditions

H. influenzae serotype a (Hia) strains 11-139, 14-61, 13-0074, and 13-240 were provided by Dr. Raymond Tsang (National Microbiology Laboratory, Winnipeg, MB, Canada). Noninvasive NTHi 375, isolated from the middle ear of a child [24], and invasive NTHi 08-254 isolated from blood of a pediatric patient (Dr. Tsang’s collection) were also used (Table 1). Bacteria were grown on brain heart infusion (BHI) agar plates containing 10 μg/mL hemin chloride and 5 μg/mL nicotine adenine dinucleotide for 16 h at 37°C and 5% CO2. Isolated colonies were transferred to BHI broth containing growth factors and grown to mid-log phase at 37°C with shaking. Prior to their use for stimulation of differentiated THP-1 and HL-60 cells, bacterial suspensions were diluted with BHI broth containing growth factors to reach an OD600 of 0.1.

Table 1.

Characteristics of H. influenzae strains

SerotypeLabelST1EncapsulationInvasivenessSource of isolation
11-139 23 yes yes From blood of an adult patient [25
14-61 23 yes no From the middle ear of a pediatric patient [25
13-0074 23 no yes From blood of a pediatric patient (R. Tsang, personal communication) 
13-240 yes yes From blood of a pediatric patient [25
NTHi2 375 no no From the middle ear of a pediatric patient [24
NTHi2 08-254 599 no yes From blood of a pediatric patient (R. Tsang, personal communication) 
SerotypeLabelST1EncapsulationInvasivenessSource of isolation
11-139 23 yes yes From blood of an adult patient [25
14-61 23 yes no From the middle ear of a pediatric patient [25
13-0074 23 no yes From blood of a pediatric patient (R. Tsang, personal communication) 
13-240 yes yes From blood of a pediatric patient [25
NTHi2 375 no no From the middle ear of a pediatric patient [24
NTHi2 08-254 599 no yes From blood of a pediatric patient (R. Tsang, personal communication) 

1Determined by the multilocus sequence typing [25].

2Non-typeable H. influenzae.

Stimulation with H. influenzae

Immediately prior to stimulation, the culture medium in the wells containing differentiated THP-1 macrophages was replaced with 2 mL fresh RPMI-1640 medium supplemented with 10% FBS. Bacterial suspensions with OD600 of 0.1, washed twice and resuspended in sterile PBS, were used to achieve a multiplicity of infection (MOI) of 0.1, 1, 10, or 100. Cell number and viability were determined with a Bright-Line Hemacytometer (Hausser Scientific, Horsham, PA, USA) using a 1:1 dilution factor with 0.4% Trypan blue solution (Sigma-Aldrich, St. Louis, MO, USA). THP-1 cells were stimulated with Hia strains at indicated MOI, using sterile PBS for the negative control, and Escherichia coli lipopolysaccharide (LPS) (Invitrogen, Carlsbad, CA, USA) for the positive control. In some experiments, cells were stimulated with Hia lipooligosaccharide (LOS) isolated from Hia 11-139 (provided by Dr. Andrew Cox, The National Research Council, Ottawa, ON, Canada [25]). The quantity of LOS corresponding to the number of bacterial cells used for stimulation was calculated as previously described [26]. The plates were incubated at 37°C and 5% CO2 for 1 h, then 100 μg/mL gentamycin (United States Biological, Salem, MA, USA) was added, and incubation continued for an additional 17 h at 37°C and 5% CO2.

To determine the role of LOS in cellular responses to Hia, THP-1 cells were treated with TLR4 inhibitors using two methods: (1) the treatment with 1 μg/mL LPS from Rhodobacter sphaeroides (LPS-RS) (InvivoGen) immediately prior to stimulation with bacteria, with an equivalent volume of sterile PBS used as control; (2) pretreatment with 5 or 10 μg/mL anti-TLR4 mouse IgG (InvivoGen) 1 h prior to stimulation with bacteria, using mouse control IgG1 (InvivoGen) as an isotype control.

Following stimulation of THP-1 cells, supernatants were collected for use in ELISA and stored at −80°C. Cells were collected through cold shock and manual scraping and immediately used for immunostaining and flow cytometry analysis.

For HL-60 cell stimulation, differentiated cells were plated in 6-well plates at a concentration of 2.5 × 106 cells/mL in RPMI-1640 medium supplemented with 20% FBS. The cells were stimulated with Hia at MOI of 1, 10, or 100 as described above, using PBS as negative control and E. coli LPS as positive control. Plates were incubated at 37°C and 5% CO2 for 1 h, then 100 μg/mL gentamycin was added, and plates were incubated for an additional 71 h at 37°C and 5% CO2. Following incubation, both adherent and nonadherent cells were collected for immediate use in flow cytometry analysis.

Enzyme-Linked Immunosorbent Assays

Enzyme-linked immunosorbent assays (ELISA) were completed to quantify TNF-α and IL-1β in supernatants. Human TNF-α and human IL-1β-uncoated ELISA kits were used according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Primary antihuman TNF-α or IL-1β and secondary (biotin-conjugated antihuman TNF-α or IL-1β) antibodies, standards, the enzyme (streptavidin-HRP concentrate), and substrate (tetramethylbenzidine) were provided in the ELISA kits. Supernatants from stimulation experiments were diluted 40× in the diluent provided in the ELISA kits, to achieve a range within the limits of the assay. Samples were run in duplicate on the ELISA plates and the means were used for analysis. Plates were read at 450 nm and 570 nm for wavelength subtraction.

Flow Cytometry Analysis

Collected cells were centrifuged and resuspended in sterile PBS supplemented with 10% FBS for immunostaining with 1 μg/mL phycoerythrin-conjugated mouse-antihuman ICAM-1 (intercellular adhesion molecule 1) (BD Biosciences, Mississauga, ON, Canada) or CD64 (Invitrogen) for 1 h at 4°C in the dark, then washed twice, resuspended with sterile PBS, and placed on ice. Immediately prior to flow cytometry analysis, cells were stained with 1 μg/mL propidium iodide (PI) (BD Biosciences) to test for cell death. Flow cytometry was completed on the SONY SA3800 spectral cell analyzer with SA3800 Software (SONY Corporation, CA, USA). The desired population was gated based on light scattering properties; 10,000 gated events were collected. Gates for PI were created to obtain the % PI-negative cells and acquire the mean fluorescence intensity (MFI) for the ICAM-1 or CD64-positive population. Phycoerythrin-conjugated isotype control (Invitrogen) was used to exclude nonspecific antibody binding from analysis.

Statistical Analysis

Data were expressed as a mean of at least 3 independent experiments. Statistical difference was determined with a one-way analysis of variance (ANOVA) with a Tukey post hoc test or Student’s t test for paired comparisons. A p value of <0.05 was reported as statistically significant. Statistical analysis was performed using Graph-Pad Prism 9 (GraphPad Prism Software Inc., San Diego, CA, USA).

Stimulation of differentiated THP-1 cells with genetically and phenotypically diverse clinical Hia isolates resulted in a similar increase in cell surface expression of ICAM-1 and in TNF-α and IL-1β release. For the optimization experiment, we used Hia 11-139 (ST23), an encapsulated invasive clinical isolate, which was utilized for the production of the capsular polysaccharide in the development of a new Hia polysaccharide-protein conjugate vaccine [27]. Stimulation of differentiated THP-1 cells with Hia 11-139 for 18 h (the first hour without and 17 h with antibiotics) resulted in a dose-dependent increase in the surface expression of ICAM-1 (Fig. 1a, b). The stimulation/PBS control ratio varied from 7.9 ± 1.3 (MOI 0.1) to 12.3 ± 1.9 (MOI 10), p = 0.02 (online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000540582). The highest ICAM-1 expression was found on cells stimulated at MOI 10, and this MOI was used in the following experiments for comparison between different H. influenzae strains. ICAM-1 expression at MOI 10 was significantly higher compared to the LPS control (p = 0.04). In these experiments, cellular viability, determined by the Trypan blue exclusion assay, varied between 91% and 99% (data not shown).

Fig. 1.

Cell surface expression of ICAM-1 on differentiated THP-1 cells stimulated with Hia clinical isolate Hia 11-139 at MOI 0.1, 1, 10, 100, or with E. coli LPS at 100 ng/mL for 18 h (flow cytometry analysis). a ****p < 0.0001, statistical significance between MOI of 0.1, 1, 10, 100, or LPS and control (PBS) (n = 4 independent experiments). b Histograms showing ICAM-1-positive events in the PI-negative gate, with MFI shown (results of one representative experiment). PE, phycoerythrin; MFI, mean fluorescence intensity.

Fig. 1.

Cell surface expression of ICAM-1 on differentiated THP-1 cells stimulated with Hia clinical isolate Hia 11-139 at MOI 0.1, 1, 10, 100, or with E. coli LPS at 100 ng/mL for 18 h (flow cytometry analysis). a ****p < 0.0001, statistical significance between MOI of 0.1, 1, 10, 100, or LPS and control (PBS) (n = 4 independent experiments). b Histograms showing ICAM-1-positive events in the PI-negative gate, with MFI shown (results of one representative experiment). PE, phycoerythrin; MFI, mean fluorescence intensity.

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Stimulation of differentiated THP-1 cells with diverse Hia strains, including encapsulated (11-139, 14-61, 13-240) and nonencapsulated mutants (13-0074), invasive (11-139, 13-0074, 13-240) or noninvasive (14-61) isolates at MOI 10, resulted in a similar significant increase in ICAM-1 cell surface expression (Fig. 2). The effect of two nonencapsulated NTHi strains (noninvasive 375 and invasive 08-254) was comparable to Hia. No difference in cellular viability following stimulation with different bacterial strains was noticed (data not shown). Under the same conditions, stimulation of cells with 106 ng of Hia 11-139 LOS (48 ng/mL) that was equivalent to the amount of LOS present in 5 × 106 bacterial cells (MOI 10) induced slightly lower ICAM-1 expression in comparison to the effect of Hia 11-139, p > 0.05 (Fig. 2).

Fig. 2.

Cell surface expression of ICAM-1 on differentiated THP-1 cells stimulated with Hia clinical isolates 11-139 (n = 5), 14-61 (n = 6), 13-0074 (n = 7), 13-240 (n = 7), non-typeable (NTHi) 375 (n = 3) and 08-254 (n = 3) at MOI 10, Hia LOS at 48 ng/mL (n = 2), or E. coli LPS at 100 ng/mL (n = 12) for 18 h as described in the Methods section (flow cytometry analysis). ****p < 0.0001, statistical significance between treatments and PBS; ##p < 0.01, between bacterial stimulation and LPS. MOI, multiplicity of infection; MFI, mean fluorescence intensity ± SD.

Fig. 2.

Cell surface expression of ICAM-1 on differentiated THP-1 cells stimulated with Hia clinical isolates 11-139 (n = 5), 14-61 (n = 6), 13-0074 (n = 7), 13-240 (n = 7), non-typeable (NTHi) 375 (n = 3) and 08-254 (n = 3) at MOI 10, Hia LOS at 48 ng/mL (n = 2), or E. coli LPS at 100 ng/mL (n = 12) for 18 h as described in the Methods section (flow cytometry analysis). ****p < 0.0001, statistical significance between treatments and PBS; ##p < 0.01, between bacterial stimulation and LPS. MOI, multiplicity of infection; MFI, mean fluorescence intensity ± SD.

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Under the same conditions of stimulation, we observed a considerable release of pro-inflammatory cytokines TNF-α and IL-1β into cell culture supernatants, with no significant differences observed between different Hia and NTHi strains (Fig. 3a, b). In comparison to the effect of LPS, all the bacterial strains induced a significantly higher release of TNF-α, whereas the concentration of IL-1β was higher in supernatants from stimulation with Hia 14-61, 13-0074, and 13-240, but was not noticeably different from stimulation with Hia 11-139 or either NTHi strain (Fig. 3a, b).

Fig. 3.

Cytokine release from differentiated THP-1 cells stimulated with Hia clinical isolates 11-139, 14-61, 13-0074, 13-240, non-typeable (NTHi) 375 and 08-254 at MOI 10, or E. coli LPS at 100 ng/mL for 18 h as described in the Methods section. Concentrations of TNF-α (a) and IL-1β (b) were measured by ELISA (pg/mL). Data represent mean cytokine concentrations ± SEM (n = 7–11 independent experiments). *p < 0.05, ****p < 0.0001, statistical significance between treatments and PBS; #p < 0.5, ###p < 0.001, ####p < 0.0001, between bacterial stimulation and LPS.

Fig. 3.

Cytokine release from differentiated THP-1 cells stimulated with Hia clinical isolates 11-139, 14-61, 13-0074, 13-240, non-typeable (NTHi) 375 and 08-254 at MOI 10, or E. coli LPS at 100 ng/mL for 18 h as described in the Methods section. Concentrations of TNF-α (a) and IL-1β (b) were measured by ELISA (pg/mL). Data represent mean cytokine concentrations ± SEM (n = 7–11 independent experiments). *p < 0.05, ****p < 0.0001, statistical significance between treatments and PBS; #p < 0.5, ###p < 0.001, ####p < 0.0001, between bacterial stimulation and LPS.

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Immunostimulatory Effect of Hia Partially Depended on LOS

Stimulation of differentiated THP-1 cells with different concentrations of purified Hia 11-139 LOS or E. coli LPS demonstrated the ability of both compounds to increase the expression of ICAM-1 (Fig. 4a, b) as well as TNF-α and IL-1β release (online suppl. Fig. 1) in a dose-dependent manner. To assess the impact of LOS-dependent signaling on pro-inflammatory responses to H. influenzae, we treated THP-1 cells with specific TLR4 inhibitors and quantified the release of TNF-α and IL-1β following stimulation with Hia 11-139. Using mouse monoclonal antibody specific to TLR4, we observed its dose-dependent inhibitory effect on the release of TNF-α induced by Hia 11-139 and Hia 11-139 LOS. Both 5 and 10 μg/mL anti-TLR4 concentrations had a significant inhibitory effect on TNF-α induced by E. coli LPS, without any impact of the isotype control antibody (Fig. 5a). Under the same conditions, no significant effect of anti-TLR4 on IL-1β release by Hia-stimulated cells was noted, while inhibition of TLR4 decreased the release of IL-1β induced by both LOS and LPS although in a smaller degree as compared to TNF-α (Fig. 5b).

Fig. 4.

Cell surface expression of ICAM-1 on differentiated THP-1 cells stimulated with Hia LOS at 1 ng/mL, 10 ng/mL, or 100 ng/mL (n = 3) (a) or E. coli LPS at 1 ng/mL, 10 ng/mL, or 100 ng/mL (n = 4) (b) for 18 h as described in the Methods section (flow cytometry analysis). **p < 0.01, ****p < 0.0001, statistical significance from 1 ng/mL treatment. MFI, mean fluorescence intensity ± SD.

Fig. 4.

Cell surface expression of ICAM-1 on differentiated THP-1 cells stimulated with Hia LOS at 1 ng/mL, 10 ng/mL, or 100 ng/mL (n = 3) (a) or E. coli LPS at 1 ng/mL, 10 ng/mL, or 100 ng/mL (n = 4) (b) for 18 h as described in the Methods section (flow cytometry analysis). **p < 0.01, ****p < 0.0001, statistical significance from 1 ng/mL treatment. MFI, mean fluorescence intensity ± SD.

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Fig. 5.

Cytokine release from differentiated THP-1 cells stimulated with Hia clinical isolate 11-139 at MOI 1, E. coli LPS, or Hia 11-139 LOS at 10 ng/mL for 18 h following inhibition of TLR4 as described in the Methods section. Concentrations of TNF-α (a, c) and IL-1β (b, d) were measured by ELISA (pg/mL). Data represent mean cytokine concentrations ± SEM (n = 3–13 independent experiments). Statistical significance was determined using one-way ANOVA with a Tukey post hoc test. a, b THP-1 cells were pretreated with 5 (LB) or 10 μg/mL (HB) mouse anti-TLR4 IgG 1 h prior to stimulation; IgG Control, mouse control IgG1 at a concentration of 10 μg/mL. *p < 0.05, **p < 0.01, ***p < 0.001, ****p ≤ 0.0001 statistical significance between treatments and IgG control. c, d THP-1 cells were treated with 1 μg/mL LPS from R. sphaeroides (LPS-RS) immediately prior to stimulation, with an equivalent volume of sterile PBS used as control. *p < 0.05, ***p < 0.001, ****p ≤ 0.0001, statistical significance between LPS-RS treatment and control (PBS).

Fig. 5.

Cytokine release from differentiated THP-1 cells stimulated with Hia clinical isolate 11-139 at MOI 1, E. coli LPS, or Hia 11-139 LOS at 10 ng/mL for 18 h following inhibition of TLR4 as described in the Methods section. Concentrations of TNF-α (a, c) and IL-1β (b, d) were measured by ELISA (pg/mL). Data represent mean cytokine concentrations ± SEM (n = 3–13 independent experiments). Statistical significance was determined using one-way ANOVA with a Tukey post hoc test. a, b THP-1 cells were pretreated with 5 (LB) or 10 μg/mL (HB) mouse anti-TLR4 IgG 1 h prior to stimulation; IgG Control, mouse control IgG1 at a concentration of 10 μg/mL. *p < 0.05, **p < 0.01, ***p < 0.001, ****p ≤ 0.0001 statistical significance between treatments and IgG control. c, d THP-1 cells were treated with 1 μg/mL LPS from R. sphaeroides (LPS-RS) immediately prior to stimulation, with an equivalent volume of sterile PBS used as control. *p < 0.05, ***p < 0.001, ****p ≤ 0.0001, statistical significance between LPS-RS treatment and control (PBS).

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In addition, treatment of THP-1 cells with LPS from the photosynthetic bacterium R. sphaeroides (LPS-RS), with reported abilities to act as TLR4 antagonist [28], resulted in a similar effect, i.e., decreasing TNF-α but not IL-1β release by Hia-stimulated cells (Fig. 5c, d). Interestingly, although LPS-RS completely inhibited both TNF-α and IL-1β induced by E. coli LPS stimulation, its inhibitory effect on TNF-α induced by LOS was much smaller (Fig. 5c). Moreover, LPS-RS increased the amount of IL-1β released by cells stimulated with LOS (Fig. 5d), suggesting that LPS-RS may interact with LOS via yet unidentified mechanisms.

Considering the significance of neutrophils as the major type of innate immune cells, in addition to the analysis of macrophage responses, we studied the expression of pro-inflammatory markers by differentiated neutrophil-like HL-60 cells stimulated with H. influenzae. Besides ICAM-1, we analyzed the surface expression of CD64, the high-affinity receptor for the Fc portion of IgG (FcγRI), following the stimulation with Hia 11-139. The MFI of ICAM-1 and CD64 significantly increased in a dose dependent manner from MOI 1 to MOI 100, with a higher expression level of ICAM-1 as compared to CD64 (Fig. 6a, b).

Fig. 6.

Cell surface expression of ICAM-1 (a) and CD64 (b) on differentiated HL-60 cells stimulated with Hia clinical isolate Hia 11-139 at MOI 1, 10, or 100 for 72 h (flow cytometry analysis). ***p < 0.001, **p < 0.01 compared to all other treatments (one-way ANOVA with a Tukey post hoc test). MFI, mean fluorescence intensity (n ≥ 3 independent experiments).

Fig. 6.

Cell surface expression of ICAM-1 (a) and CD64 (b) on differentiated HL-60 cells stimulated with Hia clinical isolate Hia 11-139 at MOI 1, 10, or 100 for 72 h (flow cytometry analysis). ***p < 0.001, **p < 0.01 compared to all other treatments (one-way ANOVA with a Tukey post hoc test). MFI, mean fluorescence intensity (n ≥ 3 independent experiments).

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When the expression of ICAM-1 was compared between THP-1 and HL-60 cells, the increases in MFI with increasing MOI followed the same pattern, with highest MFI detected at MOI 100. However, the MFI values of ICAM-1 on HL-60 cells were about one order of magnitude lower than the corresponding values on THP-1 cells (Fig. 1, 6a, Fig. 7a, b). For example, stimulation of HL-60 cells with Hia 11-139 induced a 2.5- and 4.3-fold increase in ICAM-1 MFI over unstimulated cells at MOI 1 and 100, correspondingly (online suppl. Table 2). Under the same conditions of bacterial stimulation, ICAM-1 expression on THP-1 cells increased by 10.2- and 11.7-fold (online suppl. Table 1).

Fig. 7.

a ICAM-1 ell surface expression on differentiated HL-60 cells stimulated with Hia clinical isolates 11-139, 14-61, 13-0074, 13-240 (n = 3) and non-typeable H. influenzae (NTHi) 375 (n = 4) at MOI 1 and 100, E. coli LPS at 100 ng/mL (n = 20) and 1 μg/mL (n = 3) for 72 h. Flow cytometry analysis, ratio of stimulation (MFI)/PBS control (MFI) ± SD. Statistical significance between MOI 1 and MOI 100 for each isolate, and between E. coli LPS at 100 ng/mL and 1 μg/mL displayed (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); one-way ANOVA with a Tukey post hoc test. b Histograms showing ICAM-1 expression on differentiated HL-60 cells stimulated with Hia 11-139 or LPS; flow cytometry. MFI, mean fluorescence intensity indicated (results of one representative experiment); PE, phycoerythrin.

Fig. 7.

a ICAM-1 ell surface expression on differentiated HL-60 cells stimulated with Hia clinical isolates 11-139, 14-61, 13-0074, 13-240 (n = 3) and non-typeable H. influenzae (NTHi) 375 (n = 4) at MOI 1 and 100, E. coli LPS at 100 ng/mL (n = 20) and 1 μg/mL (n = 3) for 72 h. Flow cytometry analysis, ratio of stimulation (MFI)/PBS control (MFI) ± SD. Statistical significance between MOI 1 and MOI 100 for each isolate, and between E. coli LPS at 100 ng/mL and 1 μg/mL displayed (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); one-way ANOVA with a Tukey post hoc test. b Histograms showing ICAM-1 expression on differentiated HL-60 cells stimulated with Hia 11-139 or LPS; flow cytometry. MFI, mean fluorescence intensity indicated (results of one representative experiment); PE, phycoerythrin.

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No significant difference in ICAM-1 expression on HL-60 cells stimulated by different isolates was noted at MOI 1. At MOI 100, NTHi 375 showed the highest stimulatory effect compared to each other isolate (p < 0.0001); Hia 13-240 had a higher immunostimulatory effect than Hia 11-139 (p < 0.05), Hia 14-61 (p < 0.05), and Hia 13-0074 (p < 0.01) (Fig. 7a).

In this study, we have used two human cell lines, THP-1 cells differentiated to macrophages and HL-60 neutrophil-like cells, in an in vitro infection model to determine whether innate immune responses to clinical Hia isolates depend on their characteristics. We selected 4 genetically and phenotypically diverse Hia strains isolated from patients with different forms of clinical infection. These strains have been thoroughly characterized with regard to their molecular-genetic features, using whole-genome sequencing; their sequence types (STs) and phylogenetic relations based on amplification and sequencing of 7 enzyme genes (adk, atpG, frdB, fucK, mdh, recA) and detection of the superoxide dismutase gene (sodC) have been established [29]. In addition, the prevalence of various Hia STs in different geographic regions and populations as well as their association with invasive disease has been described [30]. We were interested in comparing cellular responses of encapsulated Hia bacteria to an Hia mutant which was able to cause an invasive disease despite the lack of the polysaccharide capsule, the major virulence factor of encapsulated H. influenzae [31]. In addition, we tested cellular responses to two nonencapsulated NTHi strains with different capabilities of causing invasive disease.

As a readout of the cellular responses, we have studied the cell surface expression of ICAM-1 and the release of two pro-inflammatory cytokines, TNF-α and IL-1β, assessed post one-hour long stimulation of innate immune cells with live bacteria followed by additional incubation in the presence of an antibiotic to kill extracellular bacteria. ICAM-1 is a transmembrane glycoprotein, member of the immunoglobulin superfamily, which mediates leukocyte-endothelial cells and leukocyte-leukocyte interactions crucial in the process of cellular trafficking during inflammatory responses. In addition, ICAM-1 expressed on antigen-presenting cells interacts with LFA-1 on T cells in the process of immunological synapse formation providing essential co-stimulatory signals for T-cell activation and hence represents a critical molecule involved in both innate and adaptive immune responses [32]. Previous studies identified ICAM-1 as a receptor for H. influenzae on airway epithelial cells mediating bacterial adhesion through the outer membrane protein P5 and the type IV pilus of NTHi [33, 34]. However, to the best of our knowledge, no studies assessed the role of ICAM-1 in interactions of encapsulated H. influenzae with leukocytes or identified any bacterial structures involved in this process. In the context of bacterial infections, most published studies addressed ICAM-1 expression on epithelial or endothelial cells, yet it was recently demonstrated that LPS stimulation induced ICAM-1 expression in macrophages that resulted in an enhanced phagocytosis [35]. When we tested whether stimulation of macrophage and neutrophil-like cell lines with Hia made an impact on ICAM-1 expression, we found a dose-dependent upregulation of this molecule on both cell lines. Although an increase in ICAM-1 expression on neutrophils was much smaller than on macrophages, the data suggested that both cell types became activated that could result in their enhanced functions including phagocytosis and antigen-presenting capacities. In addition, H. influenzae infection induced a significant release of TNF-α and IL-1β by THP-1 cells, further supporting their activation. The production of both cytokines and ICAM-1 is regulated by the transcription factor NF-κB, as a result of the pro-inflammatory signaling initiated by the recognition of pathogen-associated molecular patterns (PAMPs) by PRRs [36]. However, no differences in ICAM-1 expression or cytokine release induced by encapsulated versus nonencapsulated H. influenzae or invasive versus noninvasive clinical isolates were noted: all the tested strains produced similar responses. Intriguingly, in HL-60 cells, Hia 13-240 (ST4) exhibited a higher immunostimulatory effect than any ST23 Hia strains (Fig. 6a). The ST4 belongs to a genetically distinct H. influenzae clonal complex as compared to ST23 [29], and ST4 isolates have been associated with enhanced virulence and severe inflammation [15]. Although our observations call for further mechanistic studies, one may speculate that an excessive neutrophil activation caused by ST4 can contribute to the severity of inflammatory responses in invasive Hia disease [37]. To further study pro-inflammatory responses of neutrophil-like cells, we quantified the surface expression of the high-affinity receptor for the Fc portion of IgG (FcγRI) following infection of HL-60 cells with a representative invasive strain, Hia 11-139. At the higher bacterial load (MOI 100), the FcγRI expression level significantly rose over the baseline, indicating an increase in cellular capacities of binding IgG. In the real-life infectious process, the engagement of FcγRI via binding Hia-specific IgG antibodies would initiate immunoreceptor tyrosine-based activation motif-mediated intracellular signaling that would in turn result in enhanced neutrophil functional abilities, i.e., phagocytosis, production of superoxide and pro-inflammatory cytokines, all essential factors in host defense against H. influenzae infection [38, 39].

The role of the polysaccharide capsule as the major virulence factor in infections caused by encapsulated bacteria has been well established by earlier studies. It has been demonstrated that the capsule protects Hib against phagocytosis and the bacteria can only be effectively destroyed by phagocytes in the presence of opsonizing antibodies specific to the capsular antigens [31]. Whether H. influenzae capsule can activate innate immune responses or be recognized by PRRs remains uncertain, although the immunostimulatory effect of the capsule of another Gram-negative bacterium, Klebsiella pneumoniae, has been reported [40]. In this study, using 3 encapsulated Hia strains in comparison to a nonencapsulated Hia mutant, as well as 2 nonencapsulated NTHi, we found that the pro-inflammatory effect of H. influenzae did not depend on the presence or absence of polysaccharide capsule. Moreover, the development of invasive versus noninvasive clinical disease did not appear to directly depend on the presence of the capsule or the degree of the activation of pro-inflammatory responses. As further discussed below, our results indicated that LOS, rather than the polysaccharide capsule, had the major role in the activation of pro-inflammatory responses to H. influenzae infection.

Although NTHi less commonly causes invasive disease than encapsulated H. influenzae, and hence it is considered to be less virulent, recent studies suggest that the invasive capabilities may depend on some factors unrelated to the presence of the capsule. Indeed, encapsulated H. influenzae are commonly isolated from asymptomatic carriers, as well as from individuals with noninvasive infections; for example, in this study, the source of encapsulated Hia 14-61 isolation was the middle ear. We identified Hia as a cause of noninvasive disease, such as otitis media, in Northwestern Ontario, Canada [11]. In the same geographic region, Hia was isolated from the nasopharynx in 9% of healthy children [12]. High prevalence of Hia carriage among healthy individuals has been reported by other researchers; for example, in Alaska, 43% of individuals having contact with cases of Hia invasive disease were asymptomatically colonized by Hia [41]. Although biological characteristics associated with the ability of encapsulated H. influenzae to cause invasive rather than noninvasive disease have not been determined, in the case of NTHi, recent findings shed light on some potential mechanisms. Invasive NTHi isolates were found to be more resistant to complement-mediated killing than noninvasive NTHi; the resistance was associated with the capacity of bacteria to evade binding of IgM [42]. In addition, particular structural LOS characteristics due to phase-variation of certain LOS biosynthetic genes were found to be associated with abilities of NTHi to cause invasive versus noninvasive disease [43].

LOS is the major virulence factor of both encapsulated and nonencapsulated H. influenzae. Unlike LPS typical for most Gram-negative bacteria, LOS lacks the repeating O-antigen, but includes the tri-heptose oligosaccharide backbone covalently attached to a 3-deoxy-d-manno-oct-2-ulosonic acid moiety, that is the core region [44]. The core region is covalently linked to the lipid A, a TLR4 ligand, which has powerful abilities to activate innate immune and inflammatory responses [45]. In our experiments, exposure of THP-1 cells to a neutralizing TLR4 antibody [46] or TLR4 antagonist LPS-RS [28] resulted in a decrease in TNF-α production induced by Hia 11-139 or LOS isolated from Hia 11-139. These findings showed the significance of TLR4-mediated signaling in macrophage pro-inflammatory responses to H. influenzae. However, under the same experimental conditions, inhibition of TLR4 did not affect the release of IL-1β by cells stimulated with Hia. In contrast to the regulation of TNF-α expression, which is directly mediated by the TLR4 downstream signaling followed by the activation of NF-κB [47], IL-1β production depends on a 2-signal mechanism [48]. Whereas the primary signal, largely mediated by the NF-κB activation, leads to the expression of pro-IL-1β, the secondary signal is required for the activation of the inflammasome complex, being an essential factor for mature IL-1β production [49]. Therefore, the activation of LOS-induced pro-inflammatory signaling in the process of H. influenzae infection would be insufficient for IL-1β release. Yet, the ability of H. influenzae to activate the NLRP3 inflammasome has been demonstrated in an in vitro model of NTHi infection [50]. Indeed, H. influenzae express a plethora of immunostimulatory molecules recognized by innate immune receptors including those implicated in the inflammasome formation [51]. Considering that inhibition of TLR4 did not result in complete abrogation of TNF-α release following Hia stimulation, the role of H. influenzae immunostimulatory molecules beyond LOS could be considerable. Immunostimulatory effect of H. influenzae could be mediated by the recognition of peptidoglycan, porin, and DNA, by NOD1, TLR2, and TLR9, correspondingly [22, 52, 53]. In our infection model, we used stimulation with live H. influenzae followed by incubation with killed bacteria likely providing a sufficient RNA signal, which belongs to the special class of viability-associated PAMPs as described by Sander et al. [54].

Limitations

The major limitation in this study is the use of an in vitro model of bacterial infection. Such a model inevitably underestimates complex interactions among various innate immune cells and between innate and adaptive immune mechanisms. To accurately assess the mechanisms of host-pathogen interactions, including the role of different virulence factors in the development of the infectious process, it would be beneficial to use in vivo experimental models although any animal model would not necessarily reproduce the complexity of human immune responses. As using cell lines does not completely represent the response of circulating leukocytes, further studies on primary human monocytes and neutrophils would help to clarify the role of innate immune cell activation in the pathogenesis of invasive H. influenzae disease. While we selected several clinical isolates on the base of their distinct features, they do not represent the whole spectrum of H. influenzae strains, which cause various forms of clinical disease, especially in the case of NTHi, characterized by remarkable genetic heterogeneity.

We have established that in vitro infection of human innate immune cells with Hia leads to the powerful activation of pro-inflammatory responses that may potentially contribute to the pathogenesis of invasive Hia disease. However, presence or absence of the polysaccharide capsule was not associated with the strength of the pro-inflammatory responses. Likewise, abilities to cause invasive or noninvasive disease were not directly related to the immunostimulatory capacities of H. influenzae strains we used in this study. These findings suggest that the development of invasive versus noninvasive clinical disease may depend on the abilities of the adaptive immune system to control rapidly replicating bacteria via the action of antibodies specific to capsular and noncapsular antigens that will opsonize H. influenzae for phagocytosis. It appears that the functional abilities of phagocytic cells can be activated by PAMPs of H. influenzae regardless of the presence of the capsule, with LOS being the major factor responsible for the innate immune activation.

We thank Dr. Raymond Tsang (National Microbiology Laboratory, Winnipeg, MB, USA) for his kind donation of H. influenzae clinical isolates and Dr. Andrew Cox (National Research Council, Ottawa, ON, USA) for donation of Hia 11-139 LOS.

The work was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. This study is exempt from ethical committee approval as all work was performed in vitro using cell lines and no primary human samples were used. According to the Tri-Council Policy Statement “Ethical Conduct for Research Involving Humans” (Government of Canada, TCPS 2, 2018), the reuse of de-identified human somatic cell lines does not require REB review. According to the Tri-Council Policy Statement “Ethical Conduct for Research Involving Humans” (Government of Canada, TCPS 2, 2018), written informed consent is not required for research that relies exclusively on the reuse of de-identified human somatic cell lines where the researcher does not know or have access to the identity of the participant.

The authors declare no conflicts of interest.

This work was supported by the National Science and Engineering Research Council (NSERC) Discovery Grant (Ref. 1464208), the Canadian Immunization Research Network (Grant CT20ON31), and NOSM University Faculty Association Research Development Grant (MU). The funders had no role in the design, data collection, data analysis, and reporting of this study.

Marina Ulanova conceived and designed the study and wrote the manuscript. Brenda Huska, Courtney Ferris, and Zaid Shahid carried out the experiments and analyzed the data. All the authors reviewed the manuscript prior to submission.

All data generated during this study are included in this article and its online supplementary material files available without any restrictions. Further inquiries can be directed to the corresponding author.

1.
Murphy
TF
.
Haemophilus species (including H. influenzae and Chancroid)
. In:
Mandell
GL
,
Bennett
JE
,
Dolin
R
,
Mandell
D
, editors.
Bennett’s principles and practice of infectious diseases
. 7th ed.
Philadelphia
:
Churchill Livingstone Elsevier
;
2010
. p.
2911
9
.
2.
Zwahlen
A
,
Kroll
JS
,
Rubin
LG
,
Moxon
ER
.
The molecular basis of pathogenicity in Haemophilus influenzae: comparative virulence of genetically-related capsular transformants and correlation with changes at the capsulation locus cap
.
Microb Pathog
.
1989
;
7
(
3
):
225
35
.
3.
Langereis
JD
,
de Jonge
MI
.
Unraveling Haemophilus influenzae virulence mechanisms enable discovery of new targets for antimicrobials and vaccines
.
Curr Opin Infect Dis
.
2020
;
33
(
3
):
231
7
.
4.
Peltola
H
.
Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 Years after the use of the polysaccharide vaccine and a decade after the advent of conjugates
.
Clin Microbiol Rev
.
2000
;
13
(
2
):
302
17
.
5.
Slack
MPE
,
Cripps
AW
,
Grimwood
K
,
Mackenzie
GA
,
Ulanova
M
.
Invasive Haemophilus influenzae infections after 3 decades of Hib protein conjugate vaccine use
.
Clin Microbiol Rev
.
2021
;
34
(
3
):
e0002821
.
6.
Ulanova
M
,
Tsang
RSW
.
Haemophilus influenzae serotype a as a cause of serious invasive infections
.
Lancet Infect Dis
.
2014
;
14
(
1
):
70
82
.
7.
Tsang
RSW
,
Ulanova
M
.
The changing epidemiology of invasive Haemophilus influenzae disease: emergence and global presence of serotype a strains that may require a new vaccine for control
.
Vaccine
.
2017
;
35
(
33
):
4270
5
.
8.
Plumb
ID
,
Lecy
KD
,
Singleton
R
,
Engel
MC
,
Hirschfeld
M
,
Keck
JW
, et al
.
Invasive Haemophilus influenzae serotype a infection in children: clinical description of an emerging pathogen-Alaska, 2002-2014
.
Pediatr Infect Dis J
.
2018
;
37
(
4
):
298
303
.
9.
Holdaway
MD
,
Turk
DC
.
Capsulated Haemophilus influenzae and respiratory-tract disease
.
Lancet
.
1967
;
1
(
7486
):
358
60
.
10.
Nolen
LD
,
Tiffany
A
,
DeByle
C
,
Bruden
D
,
Thompson
G
,
Reasonover
A
, et al
.
Haemophilus influenzae serotype a (Hia) carriage in a small Alaska community after a cluster of invasive Hia disease, 2018
.
Clin Infect Dis
.
2021
;
73
(
2
):
e280
6
.
11.
Ulanova
M
,
Tsang
RSW
,
Nix
EB
,
Kelly
L
,
Shuel
M
,
Lance
B
, et al
.
Epidemiology of invasive Haemophilus influenzae disease in northwestern Ontario: comparison of invasive and noninvasive H. influenzae clinical isolates
.
Can J Microbiol
.
2023
;
69
(
6
):
219
27
.
12.
Ulanova
M
,
Tsang
RS
,
Nix
EB
,
Tan
B
,
Huska
B
,
Kelly
L
, et al
.
Carriage of Haemophilus influenzae serotype A in children: Canadian Immunization Research Network (CIRN) study
.
J Assoc Med Microbiol Infect Dis Can
.
2024
;
9
(
1
):
20
31
.
13.
Kroll
JS
,
Moxon
ER
,
Loynds
BM
.
An ancestral mutation enhancing the fitness and increasing the virulence of Haemophilus influenzae type b
.
J Infect Dis
.
1993
;
168
(
1
):
172
6
.
14.
Kroll
JS
,
Moxon
ER
,
Loynds
BM
.
Natural genetic transfer of a putative virulence-enhancing mutation to Haemophilus influenzae type a
.
J Infect Dis
.
1994
;
169
(
3
):
676
9
.
15.
Kapogiannis
BG
,
Satola
S
,
Keyserling
HL
,
Farley
MM
.
Invasive infections with Haemophilus influenzae serotype a containing an IS1016-bexA partial deletion: possible association with virulence
.
Clin Infect Dis
.
2005
;
41
(
11
):
e97
103
.
16.
Lima
JBT
,
Ribeiro
GS
,
Cordeiro
SM
,
Gouveia
EL
,
Salgado
K
,
Spratt
BG
, et al
.
Poor clinical outcome for meningitis caused by Haemophilus influenzae serotype A strains containing the IS1016-bexA deletion
.
J Infect Dis
.
2010
;
202
(
10
):
1577
84
.
17.
Sadarangani
M
.
Protection against invasive infections in children caused by encapsulated bacteria
.
Front Immunol
.
2018
;
9
:
2674
.
18.
Lorenz
E
,
Chemotti
DC
,
Jiang
AL
,
McDougal
LD
.
Differential involvement of toll-like receptors 2 and 4 in the host response to acute respiratory infections with wild-type and mutant Haemophilus influenzae strains
.
Infect Immun
.
2005
;
73
(
4
):
2075
82
.
19.
Wang
X
,
Moser
C
,
Louboutin
J-P
,
Lysenko
ES
,
Weiner
DJ
,
Weiser
JN
, et al
.
Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung
.
J Immunol
.
2002
;
168
(
2
):
810
5
.
20.
Swords
WE
,
Buscher
BA
,
Ver Steeg Ii
K
,
Preston
A
,
Nichols
WA
,
Weiser
JN
, et al
.
Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor
.
Mol Microbiol
.
2000
;
37
(
1
):
13
27
.
21.
Girardin
SE
,
Boneca
IG
,
Carneiro
LAM
,
Antignac
A
,
Jéhanno
M
,
Viala
J
, et al
.
Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan
.
Science
.
2003
;
300
(
5625
):
1584
7
.
22.
Ratner
AJ
,
Aguilar
JL
,
Shchepetov
M
,
Lysenko
ES
,
Weiser
JN
.
Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria
.
Cell Microbiol
.
2007
;
9
(
5
):
1343
51
.
23.
Zola
TA
,
Lysenko
ES
,
Weiser
JN
.
Mucosal clearance of capsule-expressing bacteria requires both TLR and nucleotide-binding oligomerization domain 1 signaling
.
J Immunol
.
2008
;
181
(
11
):
7909
16
.
24.
Mell
JC
,
Sinha
S
,
Balashov
S
,
Viadas
C
,
Grassa
CJ
,
Ehrlich
GD
, et al
.
Complete genome sequence of Haemophilus influenzae strain 375 from the middle ear of a pediatric patient with otitis media
.
Genome Announc
.
2014
;
2
(
6
):
e01245
14
.
25.
Nix
EB
,
Choi
J
,
Anthes
C
,
Gaultier
GN
,
Thorgrimson
J
,
Cox
AD
, et al
.
Characterization of natural bactericidal antibody against Haemophilus influenzae type a in Canadian first nations: a Canadian Immunization Research Network (CIRN) clinical trials Network (CTN) study
.
PLoS One
.
2018
;
13
(
8
):
e0201282
.
26.
Gaultier
GN
,
Colledanchise
KN
,
Alhazmi
A
,
Ulanova
M
.
The immunostimulatory capacity of nontypeable Haemophilus influenzae lipooligosaccharide
.
Pathog Immun
.
2017
;
2
(
1
):
34
49
.
27.
Cox
AD
,
Williams
D
,
Cairns
C
,
St Michael
F
,
Fleming
P
,
Vinogradov
E
, et al
.
Investigating the candidacy of a capsular polysaccharide-based glycoconjugate as a vaccine to combat Haemophilus influenzae type a disease: a solution for an unmet public health need
.
Vaccine
.
2017
;
35
(
45
):
6129
36
.
28.
Golenbock
DT
,
Hampton
RY
,
Qureshi
N
,
Takayama
K
,
Raetz
CR
.
Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes
.
J Biol Chem
.
1991
;
266
(
29
):
19490
8
.
29.
Tsang
RSW
,
Shuel
M
,
Ahmad
T
,
Hayden
K
,
Knox
N
,
Van Domselaar
G
, et al
.
Whole genome sequencing to study the phylogenetic structure of serotype a Haemophilus influenzae recovered from patients in Canada
.
Can J Microbiol
.
2020
;
66
(
2
):
99
110
.
30.
Shuel
M
,
Knox
N
,
Tsang
RSW
.
Global population structure of Haemophilus influenzae serotype a (Hia) and emergence of invasive Hia disease: capsule switching or capsule replacement
.
Can J Microbiol
.
2021
;
67
(
12
):
875
84
.
31.
Moxon
ER
,
Kroll
JS
.
The role of bacterial polysaccharide capsules as virulence factors
.
Curr Top Microbiol Immunol
.
1990
;
150
:
65
85
.
32.
Bui
TM
,
Wiesolek
HL
,
Sumagin
R
.
ICAM-1: a master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis
.
J Leukoc Biol
.
2020
;
108
(
3
):
787
99
.
33.
Avadhanula
V
,
Rodriguez
CA
,
Ulett
GC
,
Bakaletz
LO
,
Adderson
EE
.
Nontypeable Haemophilus influenzae adheres to intercellular adhesion molecule 1 (ICAM-1) on respiratory epithelial cells and upregulates ICAM-1 expression
.
Infect Immun
.
2006
;
74
(
2
):
830
8
.
34.
Novotny
LA
,
Bakaletz
LO
.
Intercellular adhesion molecule 1 serves as a primary cognate receptor for the type IV pilus of nontypeable Haemophilus influenzae
.
Cell Microbiol
.
2016
;
18
(
8
):
1043
55
.
35.
Zhong
H
,
Lin
H
,
Pang
Q
,
Zhuang
J
,
Liu
X
,
Li
X
, et al
.
Macrophage ICAM-1 functions as a regulator of phagocytosis in LPS induced endotoxemia
.
Inflamm Res
.
2021
;
70
(
2
):
193
203
.
36.
Medzhitov
R
,
Horng
T
.
Transcriptional control of the inflammatory response
.
Nat Rev Immunol
.
2009
;
9
(
10
):
692
703
.
37.
Leliefeld
PHC
,
Wessels
CM
,
Leenen
LPH
,
Koenderman
L
,
Pillay
J
.
The role of neutrophils in immune dysfunction during severe inflammation
.
Crit Care
.
2016
;
20
:
73
.
38.
Daëron
M
.
Fc receptors as adaptive immunoreceptors
.
Curr Top Microbiol Immunol
.
2014
;
382
:
131
64
.
39.
Wang
Y
,
Jönsson
F
.
Expression, role, and regulation of neutrophil fcγ receptors
.
Front Immunol
.
2019
;
10
:
1958
.
40.
Hua
K-F
,
Yang
F-L
,
Chiu
H-W
,
Chou
J-C
,
Dong
W-C
,
Lin
C-N
, et al
.
Capsular polysaccharide is involved in NLRP3 inflammasome activation by Klebsiella pneumoniae serotype K1
.
Infect Immun
.
2015
;
83
(
9
):
3396
409
.
41.
Hammitt
LL
,
Hennessy
TW
,
Romero-Steiner
S
,
Butler
JC
.
Assessment of carriage of Haemophilus influenzae type a after a case of invasive disease
.
Clin Infect Dis
.
2006
;
43
(
3
):
386
7
.
42.
Dudukina
E
,
de Smit
L
,
Verhagen
GJA
,
van de Ende
A
,
Marimón
JM
,
Bajanca-Lavado
P
, et al
.
Antibody binding and complement-mediated killing of invasive Haemophilus influenzae isolates from Spain, Portugal, and The Netherlands
.
Infect Immun
.
2020
;
88
(
10
):
e00454
20
.
43.
Phillips
ZN
,
Brizuela
C
,
Jennison
AV
,
Staples
M
,
Grimwood
K
,
Seib
KL
, et al
.
Analysis of invasive nontypeable Haemophilus influenzae isolates reveals selection for the expression state of particular phase-variable lipooligosaccharide biosynthetic genes
.
Infect Immun
.
2019
;
87
(
5
):
e00093
19
.
44.
Schweda
EKH
,
Richards
JC
,
Hood
DW
,
Moxon
ER
.
Expression and structural diversity of the lipopolysaccharide of Haemophilus influenzae: implication in virulence
.
Int J Med Microbiol
.
2007
;
297
(
5
):
297
306
.
45.
Lee
MS
,
Kim
Y-J
.
Signaling pathways downstream of pattern-recognition receptors and their cross talk
.
Annu Rev Biochem
.
2007
;
76
:
447
80
.
46.
Lévêque
M
,
Simonin-Le Jeune
K
,
Jouneau
S
,
Moulis
S
,
Desrues
B
,
Belleguic
C
, et al
.
Soluble CD14 acts as a DAMP in human macrophages: origin and involvement in inflammatory cytokine/chemokine production
.
FASEB J
.
2017
;
31
(
5
):
1891
902
.
47.
Dentener
MA
,
Bazil
V
,
Von Asmuth
EJ
,
Ceska
M
,
Buurman
WA
.
Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages
.
J Immunol
.
1993
;
150
(
7
):
2885
91
.
48.
Dinarello
CA
.
Overview of the IL-1 family in innate inflammation and acquired immunity
.
Immunol Rev
.
2018
;
281
(
1
):
8
27
.
49.
Franchi
L
,
Muñoz-Planillo
R
,
Núñez
G
.
Sensing and reacting to microbes through the inflammasomes
.
Nat Immunol
.
2012
;
13
(
4
):
325
32
.
50.
Rathinam
VAK
,
Vanaja
SK
,
Waggoner
L
,
Sokolovska
A
,
Becker
C
,
Stuart
LM
, et al
.
TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria
.
Cell
.
2012
;
150
(
3
):
606
19
.
51.
Rotta Detto Loria
J
,
Rohmann
K
,
Droemann
D
,
Kujath
P
,
Rupp
J
,
Goldmann
T
, et al
.
Nontypeable Haemophilus influenzae infection upregulates the NLRP3 inflammasome and leads to caspase-1-dependent secretion of interleukin-1β: a possible pathway of exacerbations in COPD
.
PLoS One
.
2013
;
8
(
6
):
e66818
.
52.
Galdiero
M
,
Galdiero
M
,
Finamore
E
,
Rossano
F
,
Gambuzza
M
,
Catania
MR
, et al
.
Haemophilus influenzae porin induces toll-like receptor 2-mediated cytokine production in human monocytes and mouse macrophages
.
Infect Immun
.
2004
;
72
(
2
):
1204
9
.
53.
Mogensen
TH
,
Paludan
SR
,
Kilian
M
,
Ostergaard
L
.
Live Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis activate the inflammatory response through toll-like receptors 2, 4, and 9 in species-specific patterns
.
J Leukoc Biol
.
2006
;
80
(
2
):
267
77
.
54.
Sander
LE
,
Davis
MJ
,
Boekschoten
MV
,
Amsen
D
,
Dascher
CC
,
Ryffel
B
, et al
.
Detection of prokaryotic mRNA signifies microbial viability and promotes immunity
.
Nature
.
2011
;
474
(
7351
):
385
9
.