The Role of TLR2 and Bacterial Lipoprotein in Enhancing Airway Inflammation and Immunity

Non-typeable Haemophilus influenzae (NTHI) colonizes the lower respiratory tract of patients with chronic obstructive pulmonary disease and also causes exacerbations of the disease. The 16-kDa lipoprotein P6 has been widely studied as a potential vaccine antigen due to its highly conserved expression amongst NTHI strains. Although P6 is known to induce potent inflammatory responses, its role in the pathogenesis of NTHI infection in vivo has not been examined. Additionally, the presence of an amino-terminal lipid motif on P6 serves to activate host Toll-like receptor 2 (TLR2) signaling. The role of host TLR2 and NTHI expression of the lipoprotein P6 on the induction of airway inflammation and generation of adaptive immune responses following chronic NTHI stimulation was evaluated with TLR2-deficient mice and a P6-deficient NTHI strain. Absence of either host TLR2 or bacterial P6 resulted in diminished levels of immune cell infiltration within lungs of mice exposed to NTHI. Pro-inflammatory cytokine secretion was also reduced in lungs that did not express TLR2 or were exposed to NTHI devoid of P6. Induction of specific antibodies to P6 was severely limited in TLR2-deficient mice. Although mice exposed to the P6-deficient NTHI strain were capable of generating antibodies to other surface antigens of NTHI, these levels were lower compared to those observed in mice exposed to P6-expressing NTHI. Therefore, cognate interaction between host TLR2 and bacterial P6 serves to enhance lung inflammation and elicit robust adaptive immune responses during NTHI exposure. Strategies to limit NTHI inflammation while simultaneously promoting robust immune responses may benefit from targeting the TLR2:P6 signaling axis

Recombinant Sialyltransferase Infusion Mitigates Infection-Driven Acute Lung Inflammation

Inappropriate inflammation exacerbates a vast array of chronic and acute conditions with severe health risks. In certain situations, such as acute sepsis, traditional therapies may be inadequate in preventing severe organ damage or death. We have previously shown cell surface glycan modification by the circulating sialyltransferase ST6Gal-1 regulates de novo inflammatory cell production via a novel extrinsic glycosylation pathway. Here, we show that therapeutic administration of recombinant, bioactive ST6Gal-1 (rST6G) mitigates acute inflammation in a murine model mimicking acute exacerbations experienced by patients with chronic obstructive pulmonary disease (COPD). In addition to suppressing proximal neutrophil recruitment at onset of infection-mediated inflammation, rST6G also muted local cytokine production. Histologically, exposure with NTHI, a bacterium associated with COPD exacerbations, in rST6G-treated animals revealed consistent and pronounced reduction of pulmonary inflammation, characterized by smaller inflammatory cuffs around bronchovascular bundles, and fewer inflammatory cells within alveolar walls, alveolar spaces, and on pleural surfaces. Taken together, the data advance the idea that manipulating circulatory ST6Gal-1 levels has potential in managing inflammatory conditions by leveraging the combined approaches of controlling new inflammatory cell production and dampening the inflammation mediator cascade

Lipid Motif of a Bacterial Antigen Mediates Immune Responses via TLR2 Signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2−/− DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively; our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen

Lipid motif on P6 augments T cell proliferation and cytokine production.

<p>WT mice were immunized s.c. with 40 µg of native P6 emulsified in CFA and IFA one week later. (<b>A</b>) Proliferation of CD3⁺ cells isolated from draining lymph nodes was measured following 4 day co-culture with syngeneic irradiated BMDCs pulsed with 0.25, 0.12, and 0.06 µg/ml native P6 (▪) or non-lipidated rP6 (•). Media alone control (▴) was performed for background proliferation of T cells. Thymidine was added to the wells for the last 16 hrs of incubation. (<b>B</b>) Splenocytes from the same animals were co-cultured overnight with 0.06 µg/ml antigen pulsed irradiated BMDCs in ELISPOT plates coated with anti-cytokine mAb. Plates were developed and spots enumerated microscopically. *p<0.01 1way ANOVA with Bonferroni post-test comparison of native P6 to non-lipidated rP6.</p

Enhanced endocytosis of lipid-expressing P6 by BMDCs.

<p>BMDCs from WT mice were incubated overnight with Cy3-conjugated native P6 or non-lipidated rP6. Cells were incubated with 100 µM LysoTracker Red for 30 min prior to harvest and then stained with anti-CD11c FITC. (<b>A</b>) Representative image analysis of Cy3-conjugated endocytosis and co-localization with LysoTracker for bacterial P6 (top row) and non-lipidated rP6 (bottom row). (<b>B</b>) Percent of CD11c⁺ BMDCs that endocytosed Cy3-conjugated antigen and co-localize with LysoTracker. (<b>C</b>) Amount of endocytosed antigen present in Cy3⁺ cells as determined by Cy3 mean fluorescent intensity (MFI). (<b>D</b>) Antigen uptake by BMDCs determined by two-color flow cytometry (FITC vs Cy3). Percent of CD11c⁺ cells that have endocytosed Cy3-conjugated antigen over unstimulated BMDCs are provided in each plot, in addition to MFI.</p

TLR2 expression is important for antibody and recall cytokine responses against P6.

<p>WT (black bar) and TLR2^−/− (gray bar) mice were immunized i.p. with 40 µg of native P6 emulsified in CFA, IFA, and PBS. (<b>A</b>) Anti-P6 Ig levels were measured in pre-immune and post-immune sera by ELISA. (<b>B–D</b>) Frequency of cytokine secreting T cells in spleens from the same animals after 16 weeks were measured by ELISPOT. Splenocytes were assayed directly <i>ex vivo</i> and after 3 day restimulation with BMDCs pulsed with native P6. Plates were developed and spots enumerated microscopically. *p<0.05 2way ANOVA with Bonferroni post-test comparison of WT and TLR2^−/−.</p