Effects of Typeable and Non-typeable Haemophilus influenzae on Human CD4⁺ T Cell Proliferation and Production of Th1 and Th2 Cytokines

Introduction

Haemophilus influenzae bacterium inhabits the upper respiratory tract and may cause local and systemic diseases (1). Haemophilus influenzae strains are classified into capsulate or typeable H. influenzae (THi) and acapsulate or non-typeable H. influenzae (NTHi) based on the expression of a polysaccharide capsule (1). Typeable H. influenzae strains include six serotypes (a–f), with type b being the most virulent of them all (2). Typeable H. influenzae strains can cause severe diseases, including septicemia and meningitis (3,4). Non-typeable H. influenzae strains are characterized by the absence of a polysaccharide capsule, which distinguishes them from typeable strains (5). These strains may colonize the respiratory tract and are often implicated in exacerbation of chronic obstructive pulmonary disease (COPD). Non-typeable H. influenzae is the most prevalent bacterial cause of recurrent otitis media during childhood (6) and COPD exacerbations in humans (7) and mice (8). Furthermore, NTHi promote biofilm formation and exhibit a strong resistance to the immune system's antimicrobial defenses, which enables them to survive in the COPD lung and cause persistent infections that recur (9).

The initial step in bacterial colonization is attaching to specific receptors on human mucosal epithelial cells. Outer membrane proteins (OMPs) P2 and P5 are involved in the binding of NTHi to mucin, whereas binding to mucosal epithelial cells is mediated by pili (10), high-molecular-weight surface proteins (HMW1 and HMW2) (11), H. influenzae adhesin (12), Haemophilus adhesion and penetration (Hap) protein (13), OMP P5 (14), and the opacity-associated (Opa) protein A (15).

Bacterial colonization may induce host damage and trigger specific immune responses that can eradicate the bacteria. T lymphocytes detect microbial antigens in conjunction with major histocompatibility complex molecules. In the presence of costimulatory signals, T cells become activated and produce cytokines that regulate immune responses. T helper-1 (Th1) cells are generated by T-bet expression through STAT1 and STAT4 upon T cell receptor (TCR) stimulation in the presence of interferon-gamma (IFN-γ) and interleukin-12 (IL-12), respectively. T helper-2 cells are stimulated by activation of TCR-stimulated T cell factor 1 and IL-2 and IL-4 signaling (16).

Mucosal CD4⁺ T cell priming occurs in mucosa-associated lymphoid tissue following bacterial invasion, leading to the production of effector and memory T cells (17). Activated CD4⁺ T cells produce both Th1 cytokines, such as TNF-α and IFN-γ, and Th2 cytokines, including IL-5 and IL-10. Th1 cytokines activate cell-mediated immunity, whereas Th2 cytokines stimulate humoral immunity (18,19). The sequel of infectious diseases is largely dependent on the balance between Th1 and Th2 cytokines (20).

Bacteria can inhibit lymphocyte proliferation and cytokine production, while others can stimulate the production of pro-inflammatory cytokines. Failure to evoke a strong proinflammatory response from immune cells might impair host clearance of pathogens and prolong colonization. Haemophilus influenzae utilizes capsular polysaccharide for immune evasion, resulting in a deficiency of co-stimulatory signals that impairs robust lymphocyte activation and proliferation (21). Also, it can directly trigger programmed cell death in lymphocytes, effectively shutting down the adaptive immune response before it can fully develop (22).

Previous studies highlighted the importance of lymphocyte responses in the pathogenesis of NTHi infection. The incidence of COPD exacerbations due to NTHi suggests that the immunological defense mechanisms against these bacteria are hindered. It has been demonstrated that exacerbations of COPD are associated with a decrease in the proliferation of T cells in response to the outer membrane lipoprotein P6 of NTHi (23). Additionally, CD4⁺ memory T cells specific for NTHi were present at low rates in the peripheral blood of COPD patients and healthy controls (24). Furthermore, COPD due to NTHi was associated with Th2 cytokines and decreased expression of CD40 ligand (1). However, the immune responses of CD4⁺ T cells to H. influenzae are not clearly characterized. This study aimed to investigate the effects of live variants of single NTHi and THi strains on CD4⁺ T cell proliferation and production of Th1 and Th2 cytokines in vitro.

Materials and Methods

Bacterial Strains

Variants of a single strains of THi (132b⁺, 132b^-, 132b^-p5^-) and NTHi (A950002, A950002p5^-, A850052, d1, d3) were used in this study. Bacterial strains 132b^-, A950002, and d1 express P5 protein whereas P5 deficient strains include 132b^-p5^-, A950002p5^-, and d3. Haemophilus ­influenzae strains were cultivated on brain heart infusion agar enriched with 5% blood. 

Isolation of Primary CD4+ T Cells

Peripheral blood (18 mL) was collected from healthy individuals and placed in a Falcon tube containing 2 mL sodium citrate as an anticoagulant. The blood was carefully layered over 20 mL of Histopaque®-1077 (Sigma-Aldrich, Dorset, UK) in 50 mL conical tubes. Following centrifugation, peripheral blood mononuclear cells (PBMCs) were extracted from the buffy coat layer. CD4⁺ T cells were then isolated from the PBMCs using negative selection, adhering to the protocol provided with the immunomagnetic CD4⁺ T cell isolation kit (Miltenyi Biotec, Surrey, UK). The cells were subsequently cultured in RPMI 1640 medium (Sigma Aldrich, Dorset, UK), enriched with 10% inactivated fetal bovine serum, 20 mM HEPES buffer (Sigma-Aldrich, Dorset, UK), 1% L-glutamine (Sigma-Aldrich, Dorset, UK). Cell culture was kept in a humidified incubator at 37°C with 5% CO2. For adhesion, cell proliferation, and cytokines assays, the T cells were infected with live strains of H. influenzae at a multiplicity of infection (MOI) of 100:1. This MOI was chosen based on a previously published study (25).

Bacterial Adhesion Assay

To upregulate carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) on the surface of CD4⁺ T cells, IL-2 (200 U/mL) was added to the cell cultures for 48 hours. After washing, the cells were infected with live strains of THi and NTHi at MOI of 100:1 and incubated for 3 hours at 37°C in a humidified atmosphere of 5% CO2. Cells and bacteria were transferred to a 5 μm Transwell filter (Costar, Corning, NY, USA). To remove nonadherent bacteria, the filters were washed six times with Hanks' balanced salt solution (HBSS; Sigma-Aldrich, Dorset, UK). The filters were then transferred to new wells, and 200 μL of 1% saponin was added per filter for 30 minutes to release cell-associated bacteria. These bacteria were harvested by four 200 μL washes with HBSS, and suspensions of bacteria were plated after appropriate dilutions to estimate the colony-forming units (CFU). The bacterial adhesion assay was conducted in duplicate, and adhesion was quantified by counting the number of bacterial CFUs attached to the CD4⁺ T cells, as previously described (25).

T Cell Proliferation Assay and Th1/Th2 Cytokine Analysis

To evaluate the impact of live bacteria on CD4⁺ T cell proliferation, cells were seeded at a density of 2 × 10⁵ cells/well in 24-well plates pre-coated with anti-CD3 antibody (OKT3) at 1 µg/mL. Simultaneously, the cells were infected with bacteria at MOI of 100:1. Gentamicin (50 µg/mL) was added 3 hours post-infection and maintained throughout the experiment to stop bacterial overgrowth. On day 3, cell proliferation was evaluated by determining the [³H]-thymidine incorporation, as outlined in prior studies (26). Briefly, 100 µL aliquots of each CD4⁺ T cell culture were added in triplicate to a 96-well plate, pulsed with [³H]-thymidine (1 µCi/well), and incubated for 6 hours in a humidified atmosphere of 5% CO2 at 37°C. Radioactivity was quantified by a liquid scintillation beta counter (1450 Microbeta; LKB Wallac, Turku, Finland). The results were expressed as the average counts per minute (CPM) of duplicate cultures. Additionally, 50 µL samples of cell supernatants were collected in duplicate on day 3 for cytokine analysis. The levels of IFN-γ, TNF-α, IL-5, and IL-10 were measured using a human Th1/Th2 cytokine cytometric bead array (CBA) kit (BD Biosciences, Oxford, UK), following the manufacturer’s guidelines. Cytokine concentrations were determined based on fluorescent intensities (FL2), and average values were computed using CBA software (BD Biosciences, Oxford, UK). The Th1/Th2 CBA assay is a reliable and sensitive method for simultaneously quantifying multiple cytokines (27). 

Statistical Analysis

All data were analyzed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). Statistical comparisons were performed using one-way analysis of variance (ANOVA) with Tukey's and Dunnett's multiple comparison tests, and unpaired t-tests. The statistical significance was set as p<0.05. In figures, significance is indicated as follows: * = p≤0.05, ** = p≤0.01, *** = p ≤0.001, **** = p≤ 0.0001, ns = p>0.05. 

Results

Adhesion of Haemophilus influenzae to CD4+ T Cells

Variants of single strains of THi and NTHi were used to assess the adhesion of H. influenzae to CD4⁺ T cells. Carcinoembryonic antigen-related cell adhesion molecule 1 was upregulated on CD4⁺ T cells by stimulation with IL-2 before infection with bacteria. As shown in Figure 1, both THi and NTHi strains are bound to CD4⁺ T cells to varying degrees. Typeable H. influenzae (132b⁺ and 132b^-) bind more to CD4⁺ T cells compared to NTHi A850052 (p=0.0004) and d1 (p=0.0196 and 0.0219), respectively. However, there was no significant difference between THi (132b⁺ or 132b^-) and NTHi A950002 (p=0.3812 and 0.3325), respectively.

Figure 1:

Quantitative analysis of the binding of typeable Hi (THi) and non-typeable Hi (NTHi) to CD4⁺ T cells. The CD4⁺ T cells were infected with live single strains of THi (132b⁺, 132b^-) and NTHi (A950002, A850052, d1) at a multiplicity of MOI of 100:1. The bacterial binding was assessed by viable count assays. Data are representative of at least two independent experiments. A one-way ANOVA followed by Tukey's multiple comparisons test was used to compare NTHi and NTHi. THi (132b⁺ and 132b^-) vs NTHi A850052 (p=0.0004) and THi (132b⁺ and 132b^-) vs d1 (p=0.0196 and 0.0219) respectively. THi (132b⁺ or 132b^-) vs NTHi A950002 (p=0.3812 and 0.3325), respectively.

Figure 1:

Quantitative analysis of the binding of typeable Hi (THi) and non-typeable Hi (NTHi) to CD4⁺ T cells. The CD4⁺ T cells were infected with live single strains of THi (132b⁺, 132b^-) and NTHi (A950002, A850052, d1) at a multiplicity of MOI of 100:1. The bacterial binding was assessed by viable count assays. Data are representative of at least two independent experiments. A one-way ANOVA followed by Tukey's multiple comparisons test was used to compare NTHi and NTHi. THi (132b⁺ and 132b^-) vs NTHi A850052 (p=0.0004) and THi (132b⁺ and 132b^-) vs d1 (p=0.0196 and 0.0219) respectively. THi (132b⁺ or 132b^-) vs NTHi A950002 (p=0.3812 and 0.3325), respectively.

As shown in Figure 2, the presence of P5 protein in P5⁺ strains (132b^-, A950002, and d1) increased the binding to CD4⁺ T cells significantly compared to P5-deficient strains (132b^-p5^-, A950002p5^-, and d3), with p value less than 0.05 [132b^- vs. 132b^-p5^- (p=0.0009), A950002 vs. A950002p5- (p=0.0039), d1 vs. d3 (p=0.0014)]. However, P5-deficient strains can still bind to T cells. These findings suggest that the P5 protein contributes to H. influenzae binding to T cells in a significant but non-essential way.

Figure 2:

Quantitative analysis of the binding of P5+ or P5- H. influenzae to active CD4+ T cells. Black bars represent P5+ strains (132b-, A950002, d1) and grey represent P5 deficient strains (132b-p5-, A950002p5- and d3). The bacterial binding was assessed by viable count assays. Data are representative of at least two independent experiments. p=0.0009 for 132b^- vs. 132b^-p5^-, p=0.0039 for A950002 vs. A950002p5^-, p=0.0014 for d1 vs. d3.

Figure 2:

Quantitative analysis of the binding of P5+ or P5- H. influenzae to active CD4+ T cells. Black bars represent P5+ strains (132b-, A950002, d1) and grey represent P5 deficient strains (132b-p5-, A950002p5- and d3). The bacterial binding was assessed by viable count assays. Data are representative of at least two independent experiments. p=0.0009 for 132b^- vs. 132b^-p5^-, p=0.0039 for A950002 vs. A950002p5^-, p=0.0014 for d1 vs. d3.

Effect of Haemophilus influenzae on CD4⁺ T Cell Proliferation

CD4⁺ T cells were stimulated by immobilized anti-CD3 (iCD3) antibody. The cells were infected with live THi or NTHi strains in the presence of iCD3 antibody. Figure 3 shows that THi (132b^-, 132b^-p5) and NTHi strains (A850052, d1, d3) caused marked inhibition of CD4⁺ T cell proliferation (p<0.0001), whereas THi strain 132b⁺ and NTHi strain A950002 induced moderate inhibition compared to uninfected controls (p=0.0004, p=0.0079), respectively. In contrast, NTHi strain A950002p5^- increased proliferation slightly (p=0.0064). The overall pattern of CD4⁺ T cell proliferation was not significantly influenced by the P5 protein.

Figure 3:

Effect of H. influenzae on CD4⁺ T cell proliferation. The cells were stimulated with anti-CD3 antibody and then either left uninfected (control) or infected with THi (132b⁺, 132b^-, 132b^-p5^-) or NTHi (A950002, A950002p5^-, d1, d3) at MOI of 100:1. The unstimulated cells were cultured without iCD3 or bacteria. CD4 proliferation was determined by [³H]-thymidine incorporation on day 3. Results are represented as the means of duplicate samples. Data are representative of at least two independent experiments.  A one-way ANOVA followed by Dunnett's multiple comparisons test was used to compare all H. influenzae strains with the control. THi strains (132b^-, 132b^-p5) and NTHi strains (A850052, d1, d3) inhibit CD4⁺ T cell proliferation markedly (p<0.0001). THi strain 132b⁺ and NTHi strain A950002 induced moderate inhibition (p=0.0004, p=0.0079), respectively. NTHi strain A950002p5^- increased proliferation slightly (p=0.0064).

Figure 3:

Effect of H. influenzae on CD4⁺ T cell proliferation. The cells were stimulated with anti-CD3 antibody and then either left uninfected (control) or infected with THi (132b⁺, 132b^-, 132b^-p5^-) or NTHi (A950002, A950002p5^-, d1, d3) at MOI of 100:1. The unstimulated cells were cultured without iCD3 or bacteria. CD4 proliferation was determined by [³H]-thymidine incorporation on day 3. Results are represented as the means of duplicate samples. Data are representative of at least two independent experiments.  A one-way ANOVA followed by Dunnett's multiple comparisons test was used to compare all H. influenzae strains with the control. THi strains (132b^-, 132b^-p5) and NTHi strains (A850052, d1, d3) inhibit CD4⁺ T cell proliferation markedly (p<0.0001). THi strain 132b⁺ and NTHi strain A950002 induced moderate inhibition (p=0.0004, p=0.0079), respectively. NTHi strain A950002p5^- increased proliferation slightly (p=0.0064).

Effect of Haemophilus influenzae on Th1 and Th2 Cytokine Production

The production of Th1 (IFN-γ and TNF-α) and Th2 (IL-5, and IL-10) cytokines by CD4⁺ T cells were assessed using a cytometric bead array to assess Th1 and Th2 immunological responses to H. influenzae infection (Figure 4). IFN-γ production was significantly inhibited by both THi and NTHi strains (except A950002p5^- strain), with marked suppression observed for NTHi strains (A850052, d1, d3) and the THi strains 132b^- and 132b^-p5^- (p<0.0001). TNF-α production was significantly inhibited by132b- strain (p=0.0002), A950002 strain (p=0.01), A850052, d1and d3 strains (p<0.0001). In contrast, THi strain 132b⁺ and NTHi strain A950002p5 significantly increased TNF-α production (p<0.0001). IL-5 production was reduced by all THi and NTHi strains (p<0.0001), while all THi and NTHi (A850052, d1, d3) decreased IL-10 production (p<0.0001).

Figure 4:

Effects of H. influenzae on the production of Th1 cytokines (IFN-γ and TNF-α) and Th2 cytokines (IL-5 and IL-10) by CD4⁺ T cells. The cells were stimulated with anti-CD3 antibody and then either left uninfected (control) or infected with THi (132b⁺, 132b^-, 132b^-p5^-) or NTHi (A950002, A950002p5^-, d1, d3) at MOI of 100:1. The unstimulated cells were cultured without anti-CD3 or bacteria.  The cell supernatants were analyzed on day 3 for Th1 and Th2 cytokines by cytometric bead array, and the average of the calculated cytokine values was calculated by CBA software. One-way ANOVA Dunnett’s multiple comparisons test was used to compare all H. influenzae strains with the control. *p=0.0101, **p=0.0016, ***p=0.0001 and 0.0002, ****p<0.0001.

Figure 4:

Effects of H. influenzae on the production of Th1 cytokines (IFN-γ and TNF-α) and Th2 cytokines (IL-5 and IL-10) by CD4⁺ T cells. The cells were stimulated with anti-CD3 antibody and then either left uninfected (control) or infected with THi (132b⁺, 132b^-, 132b^-p5^-) or NTHi (A950002, A950002p5^-, d1, d3) at MOI of 100:1. The unstimulated cells were cultured without anti-CD3 or bacteria.  The cell supernatants were analyzed on day 3 for Th1 and Th2 cytokines by cytometric bead array, and the average of the calculated cytokine values was calculated by CBA software. One-way ANOVA Dunnett’s multiple comparisons test was used to compare all H. influenzae strains with the control. *p=0.0101, **p=0.0016, ***p=0.0001 and 0.0002, ****p<0.0001.

Discussion

This study demonstrates that both THi and NTHi strains of H. influenzae bind to CD4⁺ T cells at varying degrees. The presence of the P5 protein enhanced binding in both strain types; however, they can still bind to T cells in its absence, likely due to the presence of other adhesins on the bacterial surface. Activated human CD4⁺ T cells express CEACAM1 (28), β1 integrins (29), and Toll-like receptor (TLR) 2 and TLR4 (30). It has been demonstrated that the OMP-P2 and P6 interact with TLR2 (22,31,32), and the P5 protein interacts with CEACAM1 (33,34). Additionally, NTHi strain d1 expresses P5 protein bound strongly to Chinese hamster ovary (CHO)-CEACAM1 cells, whereas P5-deficient d3 strain did not (34). Moreover, H. influenzae expresses a range of adhesins that bind to host cells, such as Haemophilus surface fibrils that bind to vitronectin (35), and pili, which adhere to fibronectin and heparin-binding matrix proteins (36,37).

Immunity to microbial infection is organized by Th cells. Th1 cells are essential for immunity to intracellular pathogens via the production of IFN-γ, which activates macrophages, and IL-2, which induces lymphocyte proliferation. On the contrary, Th2 cells produce IL-4, IL-5, IL-10, and IL-13, which support humoral immunity and play a crucial role in eliminating extracellular pathogens (38). Antimicrobial Th1 and Th2 responses are typically linked to resistance and susceptibility to infectious diseases, respectively. This was demonstrated in intracellular pathogens Leishmania major and Mycobacterium leprae (18,39). The interaction of H. influenzae with mucosal CD4⁺ T cells is a significant factor in determining the outcome of H. influenzae infection. Herein, the results of this study have shown that certain live strains of NTHi and THi inhibited T cell proliferation and markedly reduced Th1 and Th2 cytokine production. This immunosuppressive effect may represent a mechanism by which the adaptive immune response is evaded. 

Bacteria can manipulate the inhibitory signaling to avoid host defense. IgA proteases and phase variation of lipopolysaccharide enable NTHI to evade mucosal immune mechanisms and invade respiratory epithelial cells, allowing it to live intracellularly (3). On the contrary, H. influenzae can stimulate CD4⁺ T cell immune responses (1). In response to the NTHi antigen, the activated Th (CD4+CD69+) cells produced Th1 cytokines (IFN-γ and IL-2) in healthy controls (1). However, the bronchiectasis group had predominant Th2 cytokines such as IL-4 and IL-10 (1). In murine models, nasal immunization with NTHi antigen induced specific Th1 and Th2 responses (40). Furthermore, stimulation with P6 protein induced CD4⁺ T cell proliferation in P6-immunized mice, and these cells upregulated mRNA for Th2 cytokines (41). The findings presented in this study may appear to be different from previous reports. Unlike prior studies that used bacterial antigens (1,40,41). The bacterial strains used in this study are well-characterized, specific live strains, which may explain the difference.

The inhibition of T-cell proliferation can be explained by several mechanisms. Haemophilus ducreyi-reactive CD4⁺ T cell proliferation was markedly increased by CD25⁺CD4⁺ T cell depletion, underscoring the function of regulatory T (Treg) cells in regulating the immunological response to bacterial infection (42). Regulatory T cells can suppress the immune responses through cellular interactions and/or release of IL-10 and TGF-β (43). It has been shown that IL-10 released by activated B lymphocytes in humans inhibited CD4+CD25- T-cell proliferation in vitro (44). Furthermore, Foxp3⁺ CD25⁺ Treg cells may produce suppressor cytokines, such as TGF-β and IL-10, induce apoptosis or granzyme-mediated cytolysis, or compete with effector T cells for IL-2. After Treg activation, the cells may express galectin-1, which may arrest the cell cycle when interacting with receptors on effector T cells (45). 

This study demonstrated the immunosuppressive properties of certain H. influenzae strains in vitro.

Conclusion

The present study demonstrates that certain strains of H. influenzae inhibit the proliferation of primary human CD4⁺ T cells and suppress the production of Th1 and Th2 cytokines. This immunosuppressive effect may contribute to the ability of H. influenzae to evade the adaptive immune response and establish respiratory colonization and persistence of the infection. The molecular mechanisms behind H. influenzae's immunosuppressive effect on CD4⁺ T cells require more research.